Design and synthesis of the first indole-based blockers of Panx-1 channel

Article abstract of DOI:10.1016/j.ejmech.2021.113650

Panx-1 is a membrane channel protein involved in some pathologies such as ischemic stroke, cancer and neuropathic pain, thus representing a promising therapeutic target. We present here a study aimed at obtaining the first class of selective Panx-1 blocke

Full text of DOI:10.1016/j.ejmech.2021.113650

European Journal of Medicinal Chemistry 223 (2021) 113650
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of the first indole-based blockers of Panx-1
channel
,
*
Letizia Crocetti a, Gabriella Guerrini a, Sara Puglioli a, Maria Paola Giovannoni a
,
Lorenzo Di Cesare Mannelli b, Elena Lucarini b, Carla Ghelardini b, Junjie Wang c,
Gerhard Dahl c
a Neurofarba, Pharmaceutical and Nutraceutical Section, University of Florence, Via Ugo Schiff 6, 50019, Sesto Fiorentino, Italy
b Neurofarba, Pharmacology and Toxicology Section, University of Florence, Viale Pieraccini 6, 50139, Florence, Italy
c Department of Physiology and Biophysics, University of Miami School of Medicine, 1600 N.W. 10th Avenue Miami, Florida, 33136, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Panx-1 is a membrane channel protein involved in some pathologies such as ischemic stroke, cancer and
neuropathic pain, thus representing a promising therapeutic target. We present here a study aimed at
obtaining the first class of selective Panx-1 blockers, a new topic for pharmaceutical chemistry, since all
compounds used so far for the study of this channel have different primary targets. Among various
scaffolds analyzed, the indole nucleous emerged, whose elaboration yielded interesting Panx-1 blockers,
Received 19 January 2021
Received in revised form
9 June 2021
Accepted 12 June 2021
Available online 19 June 2021
such as the potent 5-sulfamoyl derivatives 14c and 15b (I% ¼ 100 at 50
mM). In vivo tests performed in the
mouse model of oxaliplatin-induced neuropathy, demonstrated that the hypersensitivity was completely
reverted by treatment with 15b (1 nmol, administered intrathecally), suggesting a relationship between
this effect and the channel blocking ability. Finally, we decided to perform a virtual screening study on
compounds 5b, 6l and 14c using a recently resolved cryo-EM structure of hPanx-1 channel, to try to
relate the potency of our new inhibitors.
Keywords:
Panx-1
Channel blockers
Indole scaffold
Molecular modeling
Neuropathic pain
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
exchange of ions and small molecules between the intra and extra
cellular space [6,7]. The three Pannexin isoforms are physiologically
Pannexins are a family of vertebrate membrane channel pro-
teins consisting of three isoforms, Panx-1, Panx-2 and Panx-3 and
identified initially in 2000 based on their limited homology to the
invertebrate gap junction proteins, the Innexins [1]. Although
belonging to the super family of gap junction proteins, Pannexins
do not function as such [2], but they form channels connecting the
intra and extra cellular spaces and allow the transfer of different
types of ions and molecules, including ATP. Glycosylation of these
proteins even prevents the formation of intercellular gap junction
channels [3e5]. Interestingly, some Innexins, in addition to their
ability to form gap junction channels also provide pores for the
expressed in many parts of our body: Panx-1, the most abundant
and ubiquitous isoform, is found in a wide variety of tissues (heart,
brain, skeletal muscle, testis etc.) [8]; Panx-2 is essentially limited
to the central nervous system [9], while the isoform 3, mainly
expressed in skin and bones, is present in most adult tissues, but in
low concentration [5].
The glycosylation process is instrumental for the expression of
Pannexins on the cell surface and it is involved in the formation of
channels called “Pannexons”. Glycosylation, however, does not
appear to be fundamental for the activity of the channel itself [10].
Through site-directed mutagenesis studies, the specific amino acids
which undergo glycosylation were identified for all three Pannexin
isoforms [3,5]. The glycosylation of Panx-1 occurs on the Asn254 of
the second extracellular loop, while for Panx-2 and Panx-3 it occurs
on Asn86 and Asn71 of the first loop, respectively. Typically, Panx-1
functions as a homo-heteromer. However, immunoprecipitation
experiments suggested that heteromeric Pannexons can also be
constituted by Panx-1-Panx-2 or Panx-1-Panx-3, but never by the
association of Panx-2 and Panx-3 [10,11]. The Pannexins also
* Corresponding author. Department of Neurofarba, University of Florence, Via
Ugo Schiff 6, Sesto Fiorentino, Firenze, 50019, Italy.
E-mail addresses: letizia.crocetti@unifi.it (L. Crocetti), gabriella.guerrini@unifi.it
(G. Guerrini), sara.puglioli@stud.unifi.it (S. Puglioli), mariapaola.giovannoni@unifi.it
(M.P. Giovannoni), lorenzo.mannelli@unifi.it (L. Di Cesare Mannelli), elena.lucarini@
unifi.it (E. Lucarini), carla.ghelardini@unifi.it (C. Ghelardini), jwang1@miami.edu
(J. Wang), gdahl@miami.edu (G. Dahl).
https://doi.org/10.1016/j.ejmech.2021.113650
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
The involvement of Pannexins in a variety of pathological pro-
cesses makes these proteins a very attractive therapeutic target, as
indicated by an abundance of recent literature. Our interest in the
study of Panx-1 blockers was triggered by the fact that a specific
class of Panx-1 inhibitors has not yet been discovered and, as
mentioned above, all compounds used so far for the study of this
channel have a different primary target such as the anti-gout drug
Probenecid, or alternatively are no drugs, as for example the food
dye Brilliant Blue FCF or NPPB.
Abbreviations
Panx
ATP
CNS
BB G
BB FCF
NPPB
CBX
pannexin
adenosine triphosphate
central nervous system
Brilliant Blue G
Brilliant Blue FCF
5-nitro-2-(3-phenylpropylamino)benzoic acid
Carbenoxolone
As approach to this new research, we firstly selected from our
library some poly(hetero)cycle scaffolds, whose synthesis and
elaboration were fast and easily reproducible in order to obtain a
fairly large number of compounds. All these products have been
preliminarily tested on Panx-1 channel, in some case showing a
certain biological activity (data not shown). Among the compounds
exhibiting Panx-1 inhibitory activity, we decided to deep the
investigation on products with indole scaffold since this core is
relatively easy as to synthesize and to manipulate and we thought
that these are basic requirements for the initial phase of a new
project. Therefore, we elaborated the indole scaffold by inserting
the characteristic groups of NPPB and of the drug Probenecid, such
as the nitro group, the sulfonamide function variously substituted,
one or more carboxylic groups, as well as other chemical moieties.
These compounds were screened for effects on Panx-1 using the
Xenopus oocyte expression system. The assay was based on inhi-
bition of Panx-1 mediated currents. Since many inhibitors,
including BBG, Suramin and A438079, do not discriminate between
Panx-1 and P2X7R, candidate compounds were further tested for
effects on P2X7R mediated membrane currents induced by ATP.
Moreover, two selected compounds were tested in vivo in a model
of neuropathic pain and finally, molecular modeling studies were
performed.
PPA
polyphosphoric acid
dimethylformamide
ethanol
tryptophan
arginine
DMF
EtOH
Trp
Arg
Asp
aspartic acid
phenylalanine
Phe
ADME
MW
HBA
HBD
VdW
absorption distribution metabolism elimination
molecular weight
hydrogen bonding acceptor
hydrogen bonding donor
Van der Waals
interact with different protein systems including a-adrenergic re-
ceptors, purinergic receptors, especially P2X7, caspase-1 and
caspase-11 and other components of the inflammasome protein
complex [12,13]. The Panx-1 membrane channel responds to
different stimuli with distinct channel conformations. Most phys-
iological stimuli induce
a large cation- and ATP-permeable
conformation, hence Panx-1 is involved in many processes entail-
ing purinergic signaling. However, when stimulated by positive
membrane potential or by caspase 3 cleavage, the membrane
channel becomes selective for chloride ions, excluding cations and
ATP [14]. To date no one specific and selective Panx-1 blocker is
reported in the literature. However, the availability of drugs such as
Probenecid and Carbenoxolone (CBX), NPPB (5-nitro-2-(3-
phenylpropylamino)benzoic acid) and the food dye Brilliant Blue
FCF (Fig.1), all able to block Panx channels [11,15e17], has been very
useful to understand the functionality and pathophysiology of this
protein. It was recently demonstrated that Pannexins, in particular
Panx-1, are involved in some pathologies such as ischemic stroke,
epilepsy and some types of cancer. Freeman et al. [18] reported high
levels of Panx-1 in human melanomas at different stages of disease
progression associated with their malignant behavior. Moreover,
Panx-1 seems to be implicated in the progression of the Parkinson's
disease by increasing the oxidative stress [19].
Finally, an intriguing therapeutic application of Panx-1 blockers
is neuropathic pain, where the Panx-1 channel seems to be
involved through the purinergic system [20]. ATP and glutamate
play a fundamental role in the transmission of sensory information
from the periphery to the CNS and several lines of evidence have
demonstrated the involvement of ATP and purinergic receptors
such as the P2X family in the regulation of neuropathic pain [21,22].
In the family of P2X receptors, the P2X7 subtype has been identified
as a probably indirect mediator of glutamate release, and its affinity
to high level of ATP causes the formation of a reversible plasma
membrane pore, permeable to hydrophilic solutes up to 900 Da
[23]. One of the P2X7R pathways of pore activation has been
attributed to the opening of Panx-1, whose recruitment on the
plasma membrane and the consequent opening following the
activation of the purinergic system, causes the release of high
amounts of ATP and glutamate, so amplifying the signaling of these
neurotransmitters [16,24].
2. Results and discussion
2.1. Chemistry
The procedures yielding all the new products are reported in
Schemes 1e3 and the structures were confirmed on the basis of
analytical and spectral data. Scheme 1 outlines the synthetic
pathway affording the final compounds 5a,b and 6a-n. The in-
termediates 1a,b (1a [25] and 1b [26]), synthesized according to the
procedure reported in the literature, were treated with the
appropriate alkylaryl halide, commercially available, anhydrous
potassium carbonate (K2CO3) in dry DMF producing the derivatives
2a-n (2a [27] and 2b [28]). The hydrolysis with sodium hydroxide
(NaOH 6 N or NaOH 40%) of the ethoxycarbonyl group at position 2/
3 of the intermediates 2a-n allowed us to obtain the final com-
pounds 6a-n (6b [29]). The intermediates 2a and 2b were also
subjected to catalytic hydrogenation (H2, Pd/C, ethanol) to obtain
the corresponding 5-amino derivatives 3a,b (3b [28]), which in
turn were transformed into the N,N-benzyl derivatives 4a,b. The
final products 5a,b were finally obtained under the same conditions
reported above.
Schemes 2 and 3 depict the synthetic pathways to obtain the final
compounds 13e15 and 20e22, all showing a sulfonamide group at
position 5. Since these products are very similar, differing only for the
alkyl linker and/or the COOH group on the phenyl rings, we decided
to split their synthesis in two schemes, depending on the substituent
at N-1 position: the N-1 benzyl derivatives 13e15 are reported in
Scheme 2, while the N-1 phenethyl derivatives 20e22 in Scheme 3.
The common key intermediate for all the final compounds was the
ethyl 5-sulfamoyl-1H-indole-2-carboxylate 9, described in Scheme 1.
The commercially available 4-sulfonamidophenylhydrazine hydro-
chloride 7 was transformed into the corresponding phenylhydrazone
2

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Fig. 1. Examples of Panx-1 blockers found in the literature.
8 [30] with ethyl pyruvate and glacial acetic acid in absolute ethanol;
the further treatment with polyphosphoric acid (PPA) led to the
indole derivative 9. The alkylation reaction carried out with the
appropriate reagent, benzyl bromide or methyl 4-(bromomethyl)
benzoate, and sodium hydride in dry DMF led to a mixture of three
type of N-1 benzyl compounds, but differently alkylated at the sul-
fonamide function: primary sulfonamide derivatives 10a,b
(yield ¼ 34%); tertiary sulfonamide derivatives 11a,b (yield ¼ 27%);
and secondary sulfonamide derivatives 12a,b (yield ¼ 13%) respec-
tively. Moreover, compound 10b was further treated with benzyl
bromide and K2CO3 in anhydrous DMF to obtain the tertiary sul-
fonamide 11c. The hydrolysis in alkaline medium at the reflux
temperature, of esters of type 10, 11 and 12 afforded the final com-
pounds 13e15 bearing a carboxylic function at position 2.
Instead, the alkylation of intermediate 9 with (2-bromoethyl)
benzene (Scheme 3), carried out in the same conditions described
in Scheme 2, afforded a mixture of only two N-1 alkylated com-
pounds, the primary sulfonamide 16 and the secondary sulfon-
amide 17 in 1:2 ratio approximately. Both intermediates 16 and 17
were treated with an excess of benzyl bromide in dry DMF and
K2CO3 to yield compounds 18 and 19. Also, for products 17e19,
hydrolysis with NaOH 6 N at reflux was performed to obtain the
final compounds 20e22, respectively.
The results of the initial screening are reported in Tables 1 and 2
and the first observation is that the most part of our new compounds
is able to block Panx-1 channels at the tested dose. Starting the
discussion of the data from the 5-nitroindoles of type 6 (Table 1),
with the exception of compounds 6i and 6n (I% ¼ 32.3 and 31.7 at
50
mM respectively) all products show a fair activity, exhibiting
percentage inhibition values over 50%. Specifically, the position
bearing the carboxylic group (position 2 or 3) seems not to influence
the efficacy, since all the couples of 2/3-isomers exhibit about the
same value for percent inhibition. Analogously, the lengthening of
the methylene spacer in the derivatives 6a and 6b (compounds 6c-j,
n ¼ 2e5) appears to be of little importance for the inhibition of the
channel. On the contrary, the insertion of an additional carboxyl
group, like in 6k-n (versus 6a-d, respectively) led to different results.
In fact the couple of isomers 6k/6l maintains the same percentage
inhibition levels of the analogues lacking the carboxylic group at the
benzyl 6a and 6b; instead, for the pair of isomers 6m/6n, the position
of the indole nucleus to which the carboxyl group is bound appears
to be important, since compound 6m, showing the COOH at position
3 of the scaffold, exhibits the most high percentage inhibition value
in this series (I% ¼ 73 versus I% ¼ 31.7 for 6n).
The biological data of compounds reported in Table 2, which are
organized not according to the numerical sequence, but taking into
account the order of the discussion, clearly indicates that the
introduction of the sulphonamide group at position 5 simulta-
neously with the insertion of an additional acid group, has
considerably improved Panx-1 blocking efficacy, affording potent
compounds able to completely inhibit Panx-1 channel. In partic-
ular, by analyzing the derivatives 13a, 15a and 14a, we can observe
that there is a modest increase of activity moving from the primary
13a to the secondary (15a)/tertiary (14a) sulphonamides, the latter
exhibiting a similar activity (I% ¼ 29.3, 42.4 and 41, respectively).
The chain elongation, as at position 1 of the indole and/or at the
sulfamoyl moiety (n ¼ 2, compounds 20e22) results in the main-
tenance of modest activity. In any case, the channel inhibition
values remain below 50%, with the exception of secondary
2.2. Biological evaluation
All new products were screened as Panx-1 blockers at the dose
of 50
mM using the Voltage Clamp technique on Xenopus Laevis
oocytes expressing Panx-1 channels. The membrane potential of
the cells was held at ꢀ60 mV and voltage steps to þ60 mV were
applied to induce membrane currents. In each experiment, 100 mM
carbenoxolone (CBX) was applied to verify that the currents were
carried by Panx-1 channels. This concentration of CBX is known to
yield 100% inhibition of Panx-1 activity [31]. If present, leak cur-
rents resistant to CBX were subtracted from the total current to
assess % inhibition mediated by the tested compounds.
3

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Scheme 1. Reagents and Conditions: a) K2CO3, DMF dry, r.t., 300, then Br/Cl-(CH2)n-Ph-R, 80 ꢁC, 4 h; b) EtOH 96%, Pd/C, H2, Parr instrument, 2 h; c) K2CO3, DMF dry, r.t., 30’, then
benzyl bromide, 80 ꢁC, 4 h; d) for 6a-j: NaOH 6 N, EtOH 96%, reflux, 4 h; for 5a,b and 6k-n: NaOH 40%, EtOH 96%, reflux, 2 h.
sulphonamide 22 which shows I% ¼ 57.7.
synthesized the 5-dibenzylamino isomers 5a and 5b, as amino
analogues of the sulphonamide 14a. This modification afforded
very low active or inactive compounds (I% ¼ 19.5 and 0% respec-
tively, Table 2), thus demonstrating that the sulphonamide function
is fundamental for the interaction with the target.
For some selected compounds belonging to both series (6e, 6f,
6l,14c and 15b), dose-response curves were established (Figs. 2 and
3). The protocol was as in the initial screen, except that the com-
pounds were applied successively in increasing concentrations as
shown in Fig. 4 for compound 14c. For reference, CBX was applied
to verify that the currents were indeed carried by Panx1 channels
and did not represent an unspecific leak.
Instead, very interesting results have been obtained by inserting
one or more carboxylic groups in para position of the benzyl frag-
ments in compounds 13a, 14a and 15a, just discussed. The intro-
duction of a COOH at position 4 of the N-1 benzyl in the primary
sulphonamide 13a and in the tertiary sulphonamide 14a afford
compounds 13b and 14c, with very improved potency with respect
to their analogue devoid of the COOH function (I% ¼ 61.7 (13) versus
I% ¼ 29.3 (13a); I% ¼ 100 (14c) versus I% ¼ 41 (14a).
Also the simultaneous insertion of two COOH groups on the
secondary sulphonamide 15a (compound 15b) implicated
a
remarkable increase of potency (I% ¼ 100 at 50
m
M), while the in-
crease in hydrophilicity due to the insertion of an additional
carboxyl group (compound 14b), although producing an interesting
channel blocker potency (I% ¼ 79), is less favourable for the activity.
Finally, with the aim to understand the importance of the sul-
The IC50 values are reported in Table 3 and the most potent
products 14c and 15b, able to completely block Panx-1 channel at
50
and 0.8
(except BB FCF).
m
M, exhibit interesting submicromolar efficacy with IC50 ¼ 0.25
m
M respectively, lower than the reference compounds
phonamide group at position
5 of the indole scaffold, we
4

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Scheme 2. Reagents and conditions: a) Ethyl pyruvate, CH3COOH glacial, EtOH abs, 100 ꢁC, 5 h; b) PPA, 165 ꢁC, 20’; c) DMF dry, NaH, 300, then appropriate Br-(CH2)-Ph-R, reflux, 2 h;
d) DMF dry, anhydrous K2CO3, 30’, then benzyl bromide, reflux, 4 h; e) for 13a, 14a and 15a: NaOH 6 N, EtOH (gt), 100 ꢁC, 4 h; for 13b, 14b,c and 15b: NaOH 40%, 100 ꢁC, 2 h.
To test specificity of the most potent compounds (14c and 15b)
and some with less activity (6e, 13a and 14a), they were applied to
oocytes expressing the P2X7 receptor or a Connexin capable of
forming “hemichannels” in the plasma membrane (Cx32E143) [32]
(Table 4). The rational for this selection of other potential targets is
the observation that many ligands to the P2X7 receptor, irrespective
of whether they are agonists or antagonists, also inhibit Panx-1
channels [33]. On the other hand, Connexins were chosen since
they belong to the superfamily of “gap junction” proteins, together
with Pannexins and Innexins (although Pannexins do not form gap
5

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Scheme 3. Reagents and conditions: a) DMF dry, NaH, 300, then (2-Bromoethyl)benzene, reflux, 2 h; b) DMF dry, anhydrous K2CO3, 30’, then benzyl bromide, reflux, 4 h; c) NaOH
6 N, EtOH (gt), 100 ꢁC, 4 h.
junctions). The results reported in Table 4 indicate that the primary
sulphonamide 13a equally inhibits all three targets (Panx-1, P2X7R
and Connexins), thus demonstrating no selectivity. Also, com-
pounds 14a and the potent 14c show affinity for another target,
beyond Panx-1, in particular 14c exhibits 61.8% inhibition of P2X7R.
Finally, compound 6e and the powerful 15b emerge as selective
Panx-1 blockers since they don't show affinity for Connexin or
P2X7R.
2.3. In vivo studies
The two selective Panx-1 blockers 6e (I% ¼ 69) and 15b (I
% ¼ 100) and the inactive 5b, were chosen for further in vivo studies
and were tested against pain hypersensitivity in the mouse model
of oxaliplatin-induced neuropathy involving Panx-1 channel [34].
Oxaliplatin is a platinum-based chemotherapy drug belonging to
the same family of cisplatin and carboplatin and it is the first line
6

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Table 1
Inhibition (%) values of compounds 6a-n.
Compound
R1
R2
R3
Inhibition (%)a
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
CH2-Ph
CH2-Ph
(CH2)2-Ph
(CH2)2-Ph
(CH2)3-Ph
(CH2)3-Ph
(CH2)4-Ph
(CH2)4-Ph
(CH2)5-Ph
(CH2)5-Ph
CH2-Ph-COOH
CH2-Ph-COOH
(CH2)2-Ph-COOH
(CH2)2-Ph-COOH
H
COOH
H
COOH
H
COOH
H
COOH
H
COOH
H
COOH
H
COOH
COOH
H
COOH
H
COOH
H
COOH
H
COOH
H
COOH
H
COOH
H
65c
51.3 5.7 (3)
52.7 0.9 (3)
56.3 1.9 (3)
71 9 (2)c
65.5 4.5 (2)c
65.7 1.2 (3)
NTb
32.3 1.2 (3)
58 10.3 (3)
54 4 (2)
Fig. 2. Quantitative analysis of the % inhibition of Panx1-mediated membrane currents
in oocytes for compounds 6e, 6f and 6l. For reference, inhibition by carbenoxolone at
100 mM, which is known to eliminate membrane currents mediated by Panx-1, was
61 3 (2)c
73 6.5 (3)
31.7 5.3 (3)
assessed for each oocyte at the end of each experiment after the test compounds
induced inhibition. The number of oocytes used is indicated in parentheses. The error
bars in all instances were smaller than the symbol, ranging from 1.0e6.69%.
a
The compounds are tested in triplicate using the Voltage Clamp technique on
Xenopus Laevis oocytes expressing Panx-1 channels (at concentration of 50
m
M).
b
NT ¼ not tested.
c
Dose response curves and IC50 was determined for these compounds.
Table 2
Inhibition (%) values of compounds 13a,b,14a-c, 15a,b, 16, 20e22.
Fig. 3. Quantitative analysis of the % inhibition of Panx1-mediated membrane currents
in oocytes for compounds 14c and 15b. For reference, inhibition by carbenoxolone at
Compound R1
R2
n
R
Inhibition (%)a
100 mM, which is known to eliminate membrane currents mediated by Panx-1, was
13a
15a
14a
16
22
20
H
H
H
1
1
1
2
2
2
2
1
1
1
1
e
e
H
H
H
H
H
H
H
29.3 0.5 (3)
42 4.8 (3)
41 3.7 (3)
NTb
57.7 11.4 (3)
41.3 8 (3)
37.3 3 (3)
assessed for each oocyte at the end of each experiment after the test compounds
induced inhibition. The number of oocytes used is indicated above the data points. The
error bars in all instances were smaller than the symbol, ranging from 1.6e7.9%.
-CH2-Ph
-CH2-Ph
H
-(CH2)2-Ph
-CH2-Ph
-CH2-Ph
H
-CH2-Ph
-CH2-Ph-COOH
-CH2-Ph-COOH CH2-Ph-COOH
-CH2-Ph
H
H
-CH2-Ph
-(CH2)2-Ph
H
-CH2-Ph
H
21
13b
14c
15b
14b
5a
COOH 61.7 3.6 (3)
COOH 100 0 (3)c
COOH 100 0 (3)c
COOH 79 3.7 (3)
COOH
H
H
COOH
e
19.5 5.5 (2)
0 (1)
5b
e
a
The compounds are tested in triplicate using the Voltage Clamp technique on
Xenopus Laevis oocytes expressing Panx-1 channels (at concentration of 50
m
M).
b
NT ¼ not tested.
c
Dose response curves and IC50 was determined for these compounds.
Fig. 4. Membrane current traces of oocytes expressing Panx-1. Oocytes were voltage
clamped at ꢀ60 mV membrane potential. To open Panx1 channels, repetitive voltage
steps 5 s in duration to þ60 mV were applied at a rate of 0.1 Hz. The compound 14c
was applied at increasing concentrations as indicated by the bars. The capacitive
currents appearing with progressive inhibition of the ionic currents were clipped for
clarity.
drug against advanced colorectal cancer [35]. The limitation of
oxaliplatin administration in terms of dose and duration of the
treatment is the neuropathic side effect. In particular the
7

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Table 3
This study represents the first docking experiment on the Panx-1
channel, whose structure has recently been identified [37,38], us-
ing newly synthesized compounds. Indeed, very recent cryo-EM
studies (cryo-electron microscopy) elucidated the architecture of
Pannexin channels and yielded near-atomic-resolution structures
of both human (Homo sapiens) and frog (Xenopus tropicalis) Panx-1.
These studies revealed a heptameric channel architecture, instead
of the hexameric arrangement previously assumed [39,40].
Currently, fourteen cryo-EM structures of Panx-1 are available on
PDB, either of human or Xenopus tropicalis that share 65%
sequence identity; four of these cryo-EM structures (6WBN, 6WBM,
6WBF and 6WBG) have a resolution between 2.83 and 2.97 Å, the
best to date [41].
The human Panx-1 channel, if viewed from the extracellular
entrance, reveals seven Trp74 residues, each belonging to one of the
seven protomers forming the channel. As a matter of convention,
the protomers are named with alphabetic letters A-G in a clockwise
manner if viewed from extracellular domain. The Trp74 residues
lined the channel entry and two main types of interaction are
involved in the assembly of the channel ‘access’. One interaction
embraces the NH pyrrole ring of Trp74 and the benzene ring of the
adjacent residue while the second shows the arginine residue
IC50 values of 6a, 6e, 6f, 6l, 14c and 15b and reference compounds.
Compound
IC50 (m
M)a
6a
6e
6f
6l
14c
15b
NPPB
Probenecid
Carbenoxolone
BB FCF
27.5 3.8, n ¼ 5
12.4 2.1, n ¼ 4
23.1 4.7, n ¼ 3
4.9 1.2, n ¼ 6
0.25 0.1, n ¼ 4
0.8 0.1, n ¼ 5
50b
150b
10c
0.27d
a
IC50 values represent the mean SD of “n” independent ex-
periments (n ¼ 3e6).
b
See reference [15].
See reference [11].
See reference [17].
c
d
involvement of Panx-1 in oxaliplatin-induced neuropathic pain has
become increasingly evident, and antagonists of Panx-1 have been
tested to block the release of ATP and glutamate. Carbenoxolone
(100 mmol/l i.t) and Probenecid (150 mmol/l i.t) resulted to depress
the spinal C-reflex wind-up activity and decrease mechanical
hyperalgesia in neuropathic rats [36]. Moreover, selective antago-
nists, such as 10Panx (10 nmol i.t.) and Brilliant Blue FCF (1 nmol i.t),
are able to revert alterations of the pain threshold in response to
mechanical and thermal stimulation induced by repeated admin-
istration of the antineoplastic agent in the experimental animal
[34].
In our experiments the pain threshold was evaluated by the cold
plate test applying a cold non noxious stimulus (allodynia-like
measure) on the animal's paws. As shown in Fig. 5, oxaliplatin
induced a decrease of the time spent by the animal on the cold
surface before showing clear signs of pain such as paw licking or
jumping. Compounds 6e and 15b were administered intrathecally,
with doses ranging between 0.1 and 1 nmol. 15b induced an
enhancement of pain threshold starting 45 min after injection at
0.3 nmol. Hypersensitivity was completely reverted when 1 nmol
was administered, the effect peaked 60 min after treatment lasting
up to 105 min (Fig. 5, panel A); 6e showed a similar profile with a
slightly lower efficacy. The minimum active dose was 0.3 nmol
(between 60 and 75 min), 1 nmol was able to significantly control
neuropathic pain between 45 and 90 min after administration
(Fig. 5, panel B). To note, the derivative 5b was not efficacy in
relieving neuropathic pain of oxaliplatin-treated animals (Fig. 5,
Arg75 that forms a cation-
and Asp81, respectively.
p interaction and a salt bridge with Trp74
In June 2020 Ruan et a. reported [41] for the first time a cryo-EM
hPanx1 structure co-crystallized with the channel inhibitor CBX.
This drug was found to occupy the extracellular entrance of the
Panx-1 structure. Michalski [42] had already provided a first clue
about how CBX may inhibit Panx-1 channel through modulation of
the extracellular loop, but the identification of a ‘CBX site’ [41]
makes the docking studies possible.
In particular, the entrance of the extracellular domain of the
Panx-1 structure (6WBG) accommodates Carbenoxolone (CBX) that
shows hydrophobic interaction between the steroid-like core and
the aromatic side chain of Trp74; instead, the guanidine group of
Arg75 creates a suitable environment for the polar carboxylic
groups. The mutation of these two amino acids (Trp74Ala and
Arg75Glu) makes the channel insensitive to CBX, reinforcing the
identification of this ‘CBX binding site’ [41]. Thus, starting from
these newly published data, we decided to perform a virtual
screening study (docking analyses performed with AUTODOCK [43]
on the identified ‘CBX binding site’ in Panx-1 channel (PDB code
6WBG), using known inhibitors (CBX, Probenecid, Brilliant Blue FCF
and NPPB) and some of our newly synthesized Panx-1 blockers.
From the poses proposed by AUTODOCK, we have shown, for all
ligands, the possible hydrogen bond (HB) interactions (represented
in the figures as green lines) with a distance Acceptor-Donor (A-D)
<0.35 nm and the possible Van der Waals (VdW) interactions with
a distance atom-atom <0.30 nm (see Table S1, Supplementary
Material).
panel C) in agreement with its in vitro inactivity (I% ¼ 0 at 50
mM).
The new Panx-1 inhibitors appear to provide an effective, innova-
tive approach to chemotherapy-induced neuropathic pain.
2.4. Molecular modeling
Fig. 6 depicts the docking pose of CBX, which shows two
hydrogen bonds involving the carboxylic group and Phe72 and
Arg75 and exhibits a binding energy value of ꢀ10.5 kcal/mol. The
To rationalize the in vitro results, a molecular modeling study
with some representative/interesting compounds, was performed.
Table 4
Selectivity profile of 6e, 13a, 14a, 14c and 15b.
Compounda [10
mM]
P2X7R % inhibitionb
Connexin % inhibition
Panx-1% inhibition
6e
0 (5)
0 (5)
69
13a
14a
14c
15b
15.0 5.4 (4)
0 (3)
61.8 11.1 (5)
0 (5)
15 6.5 (4)
20 (2)
0 (5)
28
50
100
100
0 (6)
a
The compounds were tested at 10
m
M.
b
BzATP (20
mM) was used as stimulus.
8

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Regarding our newly synthesized compounds, the docking ex-
periments on compounds 14c, 6l and 5b, (I% ¼ 100, 61 and 0,
respectively) pointed out differences in type and number of in-
teractions with receptor protein that could explain the biological
results.
In particular, compound 14c, one of the most active compounds
(I% ¼ 100%) shows strong hydrogen bond interaction with Trp74 of
chain D through the 2-carboxy group of the indole scaffold. The
Trp74 residues of chains B and C possibly engage in VdW in-
teractions (see Fig. 7), exhibiting
a binding energy value
of ꢀ10.5 kcal/mol, the same showed by the CBX.
Fig. 8 shows the docking pose of compound 5b. The elimination
of the sulphone group at position 5 of indole scaffold eliminates all
possibilities to form hydrogen bond interaction with those shown
for 14c. The whole molecule adopts a pose where only weak in-
teractions occur. Only a putative VdW interaction with Trp74 chain
C was suggested by AUTODOCK. This fact could explain the com-
plete lack of inhibition activity of 5b (I% ¼ 0).
The complete elimination of the lipophilic moiety (N,N-benzylic
residues) at position 5 of the indole scaffold and the introduction of
a nitro group, as in the compound 6l (Fig. 9) fits the molecule in the
binding site in a different manner. The strong hydrogen bond
engaged by the 2-carboxylic group with the Trp74 of A chain and
the lower binding energy value (ꢀ6.8 kcal/mol) could explain the
appearance of a certain activity for 6l (I% ¼ 61%, IC50 ¼ 4.9
mM),
From these preliminary studies, it could be speculated that the
hydrogen bonds are necessary for the inhibitory activity (14c and
6l) while the presence of only VdW interactions reflect a loss of
activity (5b). Moreover, for the first time, some of the known in-
hibitors (CBX, BBF, probenecid NPPB) are docked in a recognized
site, and this finding could be useful for further studies.
3. Conclusions
Considering that activity of Panx-1 channels is critically
involved in several disease states, the need for inhibitors of this
membrane channel is evident. Although a plethora of compounds is
already known to interfere with Panx-1 channel activity, Panx-1 is a
secondary or tertiary target for most of these compounds [44]. For
example, a reliable Panx-1 channel inhibitor, Probenecid, has been
used to treat gout patients for its ability to inhibit reuptake of uric
acid in the kidney through the urate transporter.
Here we describe the first series of de-novo Panx-1 channel
blockers, developed following a rational approach based on exist-
ing compounds with different primary targets. One of the most
interesting obtained compounds is the Panx-1 specific 15b (100% at
50 mM), which did not interfere with P2X7R or connexin activity and
when administered intrathecally at the dose of 1 nmol was able to
completely revert the hypersensitivity in the mouse model of
oxaliplatin-induced neuropathy.
The specificity of compounds for Panx-1 is essential for diag-
nostic purposes. Thus, it can be assessed if and to what extend
Panx-1 is involved in certain physiological or pathological events.
For therapeutic applications, however, specificity does not need to
be absolute. On the contrary, a compound targeting additional
proteins involved in the same event could be advantageous. For
example, in the innate immune response, both Panx-1 and P2X7R
are intimately involved and both proteins cooperate in the event.
Suppressing both, therefore, may be more effective to constrain an
exaggerated immune response resulting in secondary cell death. On
this basis, it may therefore be worth further study of the new 14c
inhibitor which acts on both Panx-1 and P2X7R.
Fig. 5. Pain relieving effect of A) 15b and B) 6e on oxaliplatin-induced thermal
hyperalgesia in mice. Cold plate test. Oxaliplatin (2.4 mg kgꢀ1) was dissolved in 5%
glucose solution and i.p. administered for 5 consecutive days every week for 14 days.
Starting from day 14, compounds were dissolved in saline solution and intrathecally
administered in a final volume of 5 ml. Pain-related behavior (i.e. lifting and licking of
the hind paw) were observed and the time (s) of the first sign was recorded. C) Lack of
efficacy of 5b tested following the same protocol. ^^P < 0.01 vs vehicle þ vehicle
treated animals; *P < 0.05 and **P < 0.01 vs time the pretest of the same group. Each
value represents the mean
mental sets.
S.E.M. of 10 mice performed in two different experi-
Finally, the molecular modeling studies of newly synthesized
compounds (5b, 6l and 14c), performed on the recognized ‘CBX
binding site’, highlight that the hydrogen bond interactions
docking pose in the ‘CBX binding site’ of the other known inhibitors
are reported in Supplementary Material (see Figs. S1eS3).
9

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Fig. 6. Docking pose of Carbenoxolone (CBX).
Fig. 7. Docking pose of compound 14c in the ‘CBX binding site’.
engaged with Trp74 residues could be responsible for the inhibitory
potency of the active products. For the first time, a molecular
modeling study on the ‘CBX binding site’ on Panx-1 channel is
reported, and the hypothesis that our more active compounds can
block the channel in the extracellular domain as CBX is very plau-
sible. Since to date no other sites on the Panx-1 channel are
10

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Fig. 8. Docking pose of compound 5b.
recognized, this pioneering study could help provide a first rational
design for the development of Panx-1 modulators.
0.01 ppm using solvent as the internal standard. Coupling constants
(J values) are given in Hz and were calculated using ‘TopSpin 1.3’
software rounded to the nearest 0.1 Hz. Mass spectra (m/z) were
recorded on a ESI-MS triple quadrupole (Varian 1200l) system, in
positive ion mode, by infusing a 10 mg/l solution of each analyte
dissolved in a mixture of mQ H2O:acetonitrile 1:1 v/v. All new
compounds possess a purity ꢃ95%; microanalyses indicated by the
symbols of the elements were performed with a Perkine-Elmer 260
elemental analyzer for C, H, and N, and they were within 0.4% of
the theoretical values.
4. Experimental section
4.1. Chemistry
Reagents and starting materials were obtained from commercial
sources. Extracts were dried over Na2SO4, and the solvents were
removed under reduced pressure. All reactions were monitored by
thin layer chromatography (TLC) using commercial plates pre-
coated with Merck silica gel 60 F-254. Visualization was performed
by UV fluorescence (lmax ¼ 254 nm) or by staining with iodine or
potassium permanganate. Chromatographic separations were per-
formed on a silica gel column by gravity chromatography (Kieselgel
40, 0.063e0.200 mm; Merck), flash chromatography (Kieselgel 40,
0.040e0.063 mm; Merck), silica gel preparative TLC (Kieselgel 60
4.2. Synthesis and structural characterization of final compounds
5a,b, 6a, 6c-n, 13a,b, 14a-c, 15a,b, 20-22
4.2.1. General procedure for compounds 5a,b
A mixture of 4a,b (0.53 mmol), NaOH 40% (5 mL) and EtOH 96%
(gt) was stirred at reflux for 2 h. After cooling, the mixture was
acidified with HCl 37% (pH ¼ 2) and the precipitate was recovered
by vacuum filtration and recrystallized with ethanol.
F254, 20 ꢂ 20 cm, 2 mm). Yields refer to chromatographically and
spectroscopically pure compounds, unless otherwise stated. Com-
pounds were named following IUPAC rules, as applied by Beilstein-
Institut AutoNom 2000 (4.01.305) or CA Index Name. All melting
points were determined on a microscope hot stage Büchi apparatus
and are uncorrected. The identity and purity of intermediates and
final compounds were ascertained through 1H NMR, 13C NMR and
TLC chromatography. 1H NMR and 13C NMR spectra were recorded
with Avance 400 instruments (Bruker Biospin Version 002 with
4.2.1.1. 1-Benzyl-5-(dibenzylamino)-1H-indole-3-carboxylic
acid
(5a). Yield ¼ 99%; mp ¼ 204 ꢁC dec. (EtOH). 1H NMR (400 MHz,
MeOD-d4 þ D2O)
d 4.89 (s, 4H, 5-N(CH2-Ph)2) 5.42 (s, 2H, N-CH2-
Ph), 7.14 (d, 2H, Ar, J ¼ 8.0 Hz), 7.19e7.34 (m, 14H, Ar), 7.41 (d, 1H, Ar,
J ¼ 8.8 Hz), 7.84 (s, 1H, Ar), 8.05 (s, 1H, Ar). 13C NMR (100 MHz,
MeOD-d4 þ D2O)
d 52.45 (CH2), 57.52 (CH2), 102.64 (CH), 105.30
SGU). Chemical shifts (
d) are reported in ppm to the nearest
(CH), 108.90 (C), 109.29 (CH), 112.04 (CH), 115.53 (CH), 126.04 (C),
11

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Fig. 9. Docking pose of compound 6l.
125.55 (CH), 127.01 (CH), 127.11 (CH), 127.50 (C), 127.95 (CH), 128.55
(CH), 128.80 (CH), 137.33 (C), 138.71 (C), 150.57 (C), 169.36 (C). ESI-
MS calcd. for C30H26N2O2, 446.55; found: m/z 447.20 [M þ H]þ.
Anal. C30H26N2O2 (C, H, N).
1H NMR (400 MHz, DMSO‑d6)
d 5.58 (s, 2H, CH2-Ph), 7.26e7.34 (m,
5H, Ar), 7.77 (d, 1H, Ar, J ¼ 9.2 Hz), 8.07 (dd, 1H, Ar, J ¼ 9.0 Hz and
J ¼ 1.8 Hz), 8.48 (s, 1H, Ar), 8.87 (d, 1H, Ar, J ¼ 1.6 Hz), 12.59 (exch br
s, 1H, COOH). 13C NMR (100 MHz, DMSO‑d6)
d 50.39 (CH2), 109.60
(C), 112.53 (CH), 117.76 (CH), 118.17 (CH), 126.40 (C), 127.80 (2 x CH),
127.23 (2 x CH), 128.39 (CH), 129.28 (CH), 137.01 (C), 139.33 (CH),
139.69 (C), 142.99 (C), 165.27 (CO). IR
4.2.1.2. 1-Benzyl-5-(dibenzylamino)-1H-indole-2-carboxylic
acid
n
(cmꢀ1): 1506 (NO2), 1595
(5b). Yield ¼ 99%; mp ¼ > 300 ꢁC (190 ꢁC dec.) (EtOH). 1H NMR
(CO), 3333 (OH). ESI-MS calcd. for C16H12N2O4, 296.28; found: m/z
(400 MHz, DMSO‑d6)
d 4.57 (s, 4H, 5-N(CH2-Ph)2) 5.78 (s, 2H, N-
297.08 [M þ H]þ. Anal. C16H12N2O4 (C, H, N).
CH2-Ph), 6.77e6.83 (m, 2H, Ar), 7.02 (d, 2H, Ar, J ¼ 7.6 Hz), 7.13e7.29
(m, 15H, Ar). 13C NMR (100 MHz, DMSO‑d6)
d
47.25 (CH2), 55.62
(CH2), 104.34 (CH), 109.09 (CH), 112.04 (CH), 115.53 (CH), 126.91
(CH), 127.11 (CH), 127.42 (CH), 128.85 (CH), 133.43 (C), 139.51 (C),
139.82 (C), 143.87 (C), 163.76 (C). ESI-MS calcd. for C30H26N2O2,
446.55; found: m/z 447.20 [M þ H]þ. Anal. C30H26N2O2 (C, H, N).
4.2.2.2. 5-Nitro-1-phenethyl-1H-indole-3-carboxylic
acid
(6c).
Yield ¼ 44%; mp ¼ 249e250 ꢁC dec. (2-Methoxyethanol/H2O 3:7).
1H NMR (400 MHz, DMSO‑d6)
d 3.05 (t, 2H, N-CH2CH2-Ph,
J ¼ 7.2 Hz), 4.44 (t, 2H, N-CH2CH2-Ph, J ¼ 7.2 Hz) 7.15e7.23 (m, 5H,
Ar), 7.53 (d, 1H, Ar, J ¼ 9.2 Hz), 7.67 (s, 1H, Ar), 7.88 (dd, 1H, Ar,
J ¼ 9.2 Hz and J ¼ 2.4 Hz), 9.15 (d, 1H, Ar, J ¼ 2.4 Hz). 13C NMR
4.2.2. General procedure for compounds 6a, 6c-n
(100 MHz, DMSO‑d6)
d 36.06 (CH2), 48.22 (CH2), 109.12 (C), 112.23
Compounds 6a-n were obtained following the same procedure
performed for compounds 5a,b using NaOH 6 N (4 h) or NaOH 40%
(2 h) as base and starting from intermediates 2a [27] and 2c-n. After
cooling, the mixture was acidified with HCl 37% (pH ¼ 2), the
precipitate was recovered by vacuum filtration and washed several
time with water and then recrystallized using an appropriate
solvent.
(CH), 117.62 (CH), 117.81 (CH), 126.07 (C), 127.02 (CH), 128.82 (2 x
CH), 129.27 (2 x CH), 138.31 (C), 139.04 (CH), 139.73 (C), 142.75 (C),
165.29 (CO). ESI-MS calcd. for C17H14N2O4, 310.31; found: m/z 311.10
[M þ H]þ. Anal. C17H14N2O4 (C, H, N).
4.2.2.3. 5-Nitro-1-phenethyl-1H-indole-2-carboxylic
acid
(6d).
Yield ¼ 55%; mp ¼ 218 ꢁC dec. (EtOH/H2O 8:2). 1H NMR (400 MHz,
4.2.2.1. 1-Benzyl-5-nitro-1H-indole-3-carboxylic
acid
(6a).
DMSO‑d6)
d
2.98 (t, 2H, N-CH2CH2-Ph, J ¼ 7.2 Hz), 4.82 (t, 2H, N-
Yield ¼ 40%; mp ¼ 252e254 ꢁC dec. (2-Methoxyethanol/H2O 10:1).
CH2CH2-Ph, J ¼ 7.2 Hz), 7.12e7.20 (m, 5H, Ar), 7.49 (s, 1H, Ar), 7.68
12

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
(d,1H, Ar, J ¼ 9.2 Hz), 8.03 (dd,1H, Ar, J ¼ 9.2 Hz and J ¼ 2.0 Hz), 8.68
(m, 2H, N-CH2CH2CH2CH2CH2-Ph), 1.75e1.82 (m, 2H, N-
CH2CH2CH2CH2CH2-Ph), 2.45e2.51 (m, 2H, N-CH2CH2CH2CH2CH2-
Ph), 4.28 (t, 2H, N-CH2CH2CH2CH2CH2-Ph, J ¼ 7.0 Hz), 7.07e7.24 (m,
5H, Ar), 7.77 (d, 1H, Ar, J ¼ 9.2 Hz), 8.07 (dd, 1H, Ar, J ¼ 9.0 Hz and
J ¼ 2.2 Hz), 8.31 (s, 1H, Ar), 8.86 (d, 1H, Ar, J ¼ 2.4 Hz). 13C NMR
(s, 1H, Ar), 13.32 (exch br s, 1H, COOH). 13C NMR (100 MHz,
DMSO‑d6)
d 36.72 (CH2), 46.53 (CH2), 112.18 (CH), 112.59 (CH),
119.23 (CH), 119.94 (CH), 124.93 (C), 126.89 (CH), 128.74 (2 x CH),
129.29 (2 x CH), 132.46 (C), 138.56 (C), 141.36 (C), 141.80 (C), 162.74
(CO). ESI-MS calcd. for C17H14N2O4, 310.31; found: m/z 311.10 [M þ
H]þ. Anal. C17H14N2O4 (C, H, N).
(100 MHz, DMSO‑d6)
d 26.07 (CH2), 29.76 (CH2), 30.86 (CH2), 35.38
(CH2), 46.90 (CH2), 109.18 (C), 112.16 (CH), 117.72 (CH), 117.89 (CH),
126.04 (CH), 126.19 (C), 128.60 (2 x CH), 128.68 (2 x CH), 139.04
(CH),139.75 (C),142.45 (C),142.79 (C),165.32 (CO). ESI-MS calcd. for
4.2.2.4. 5-Nitro-1-(3-phenylpropyl)-1H-indole-3-carboxylic
acid
(6e). Yield ¼ 68%; mp ¼ > 300 ꢁC (EtOH/H2O 8:2). 1H NMR
C
20H20N2O4, 352.39; found: m/z 353.15 [M þ H]þ. Anal. C20H20N2O4
(400 MHz, DMSO‑d6)
d
2.10 (quin, 2H, N-CH2CH2CH2-Ph, J ¼ 7.6 Hz),
(C, H, N).
2.56 (t, 2H, N-CH2CH2CH2-Ph, J ¼ 8.0 Hz), 4.36 (t, 2H, N-CH2CH2CH2-
Ph, J ¼ 7.2 Hz), 7.14e7.25 (m, 5H, Ar), 7.79 (d, 1H, Ar, J ¼ 8.8 Hz), 8.10
(dd, 1H, Ar, J ¼ 8.8 Hz and J ¼ 2.4 Hz), 8.35 (s, 1H, Ar), 8.87 (d, 1H, Ar,
J ¼ 2.4 Hz), 12.51 (exch br s, 1H, COOH). 13C NMR (100 MHz,
4.2.2.9. 5-Nitro-1-(5-phenylpentyl)-1H-indole-2-carboxylic acid (6j).
Yield ¼ 55%; mp ¼ 78e80 ꢁC (EtOH). 1H NMR (400 MHz, DMSO‑d6)
d
1.23e1.30 (m, 2H, N-CH2CH2CH2CH2CH2-Ph), 1.52 (quin, 2H, N-
DMSO‑d6)
d
31.57 (CH2), 32.60 (CH2), 46.76 (CH2), 109.31 (C), 112.13
CH2CH2CH2CH2CH2-Ph,
CH2CH2CH2CH2CH2-Ph,
J
J
¼
7.4 Hz), 1.70 (quin, 2H, N-
7.2 Hz), 2.45e2.51 (m, 2H, N-
(CH), 117.74 (CH), 117.93 (CH), 126.21 (C), 126.40 (CH), 128.62 (2 x
CH), 128.80 (2 x CH), 139.02 (CH), 139.76 (C), 141.29 (C), 142.79 (C),
165.34 (CO). ESI-MS calcd. for C18H16N2O4, 324.34; found: m/z
325.11 [M þ H]þ. Anal. C18H16N2O4 (C, H, N).
¼
CH2CH2CH2CH2CH2-Ph), 4.64 (t, 2H, N-CH2CH2CH2CH2CH2-Ph,
J ¼ 7.2 Hz), 7.10 (d, 3H, Ar, J ¼ 7.6 Hz), 7.20 (d, 2H, Ar, J ¼ 7.4 Hz), 7.33
(s, 1H, Ar), 7.73 (d, 1H, Ar, J ¼ 9.2 Hz), 8.07 (dd, 1H, Ar, J ¼ 9.2 Hz and
J ¼ 2.0 Hz), 8.64 (d, 1H, Ar, J ¼ 2.0 Hz). 13C NMR (100 MHz, DMSO‑d6)
4.2.2.5. 5-Nitro-1-(3-phenylpropyl)-1H-indole-2-carboxylic acid (6f).
d 26.25 (CH2), 30.45 (CH2), 31.14 (CH2), 35.49 (CH2), 44.82 (CH2),
Yield ¼ 84%; mp ¼ > 300 ꢁC (EtOH/H2O 8:2). 1H NMR (400 MHz,
111.49 (CH), 112.01 (CH), 118.95 (CH), 119.74 (CH), 125.20 (C), 126.02
(CH), 128.61 (2 x CH), 128.70 (2 x CH), 134.57 (C), 141.21 (C), 141.70
(C), 142.54 (C), 163.21 (CO). ESI-MS calcd. for C20H20N2O4, 352.39;
found: m/z 353.15 [M þ H]þ. Anal. C20H20N2O4 (C, H, N).
DMSO‑d6)
d
1.97 (quin, 2H, N-CH2CH2CH2-Ph, J ¼ 7.6 Hz), 2.55 (t, 2H,
N-CH2CH2CH2-Ph, J ¼ 8.0 Hz), 4.76 (t, 2H, N-CH2CH2CH2-Ph,
J ¼ 7.2 Hz), 7.00 (s, 1H, Ar), 7.14 (m, 3H, Ar), 7.23 (m, 2H, Ar), 7.53 (d,
1H, Ar, J ¼ 9.2 Hz), 7.96 (dd,1H, Ar, J ¼ 9.2 Hz and J ¼ 2.4 Hz), 8.53 (d,
1H, Ar, J ¼ 2.0 Hz), 12.51 (exch br s, 1H, COOH). 13C NMR (100 MHz,
4.2.2.10. 1-(4-Carboxybenzyl)-5-nitro-1H-indole-3-carboxylic acid
DMSO‑d6)
d
32.54 (CH2), 38.83 (CH2), 44.44 (CH2), 108.39 (CH),
(6k). Yield ¼ 81%; mp ¼ > 300 ꢁC (EtOH). 1H NMR (400 MHz,
111.23 (CH), 117.48 (CH), 118.72 (CH), 125.80 (C), 126.22 (CH), 128.53
(2 x CH), 128.75 (2 x CH), 140.91 (C), 141.13 (C), 141.92 (C), 164.69
(CO). ESI-MS calcd. for C18H16N2O4, 324.34; found: m/z 325.11 [M þ
H]þ. Anal. C18H16N2O4 (C, H, N).
DMSO‑d6)
d
5.68 (s, 2H, N-CH2-Ph), 7.33 (d, 2H, Ar, J ¼ 8.4 Hz), 7.73
(d, 1H, Ar, J ¼ 8.8 Hz), 7.87 (d, 2H, Ar, J ¼ 8.4 Hz), 8.07 (dd, 1H, Ar,
J ¼ 9.2 Hz and J ¼ 2.0 Hz), 8.51 (s, 1H, Ar), 8.88 (d, 1H, Ar, J ¼ 2.0 Hz),
12.79 (exch br s, 2H, 2 x COOH). 13C NMR (100 MHz, DMSO‑d6)
d
50.09 (CH2), 109.80 (C), 112.43 (CH), 117.80 (CH), 118.27 (CH),
4.2.2.6. 5-Nitro-1-(4-phenylbutyl)-1H-indole-3-carboxylic acid (6g).
126.42 (C), 127.74 (2 x CH), 130.29 (2 x CH), 130.81 (C), 139.43 (CH),
139.71 (C), 141.86 (C), 143.05 (C), 165.25 (CO), 167.36 (CO). ESI-MS
calcd. for C17H12N2O6, 340.29; found: m/z 341.07 [M þ H]þ. Anal.
Yield ¼ 62%; mp ¼ 203e205 ꢁC (EtOH). 1H NMR (400 MHz,
DMSO‑d6)
d
1.50 (quin, 2H, N-CH2CH2CH2CH2-Ph, J ¼ 7.6 Hz), 1.78
(quin, 2H, N-CH2CH2CH2CH2-Ph, J ¼ 7.2 Hz), 2.56 (t, 2H, N-
CH2CH2CH2CH2-Ph, J ¼ 7.6 Hz), 4.34 (t, 2H, N-CH2CH2CH2CH2-Ph,
J ¼ 7.0 Hz), 7.12 (d, 3H, Ar, J ¼ 7.6 Hz), 7.22 (t, 2H, Ar, J ¼ 7.6 Hz), 7.80
(d, 1H, Ar, J ¼ 9.2 Hz), 8.08 (dd,1H, Ar, J ¼ 9.2 Hz and J ¼ 2.4 Hz), 8.33
(s, 1H, Ar), 8.87 (d, 1H, Ar, J ¼ 2.0 Hz), 12.50 (exch br s, 1H, COOH).
C17H12N2O6 (C, H, N).
4.2.2.11. 1-(4-Carboxybenzyl)-5-nitro-1H-indole-2-carboxylic acid
(6l). Yield ¼ 83%; mp ¼ > 300 ꢁC (EtOH). 1H NMR (400 MHz,
DMSO‑d6)
d
5.99 (s, 2H, N-CH2-Ph), 7.08 (d, 2H, Ar, J ¼ 8.4 Hz), 7.61
13C NMR (100 MHz, DMSO‑d6)
d
28.39 (CH2), 29.59 (CH2), 34.96
(s, 1H, Ar), 7.75 (d, 1H, Ar, J ¼ 9.2 Hz), 7.83 (d, 2H, Ar, J ¼ 8.4 Hz), 8.12
(dd, 1H, Ar, J ¼ 9.2 Hz and J ¼ 2.0 Hz), 8.76 (d, 1H, Ar, J ¼ 2.0 Hz),
12.95 (exch br s, 2H, 2 x COOH). 13C NMR (100 MHz, DMSO‑d6)
(CH2), 46.77 (CH2), 109.35 (C), 112.13 (CH), 117.76 (CH), 117.90 (CH),
126.20 (CH), 128.71 (4 x CH), 138.99 (CH), 139.74 (C), 142.20 (C),
142.80 (C), 165.34 (CO). ESI-MS calcd. for C19H18N2O4, 338.36;
found: m/z 339.13 [M þ H]þ. Anal. C19H18N2O4 (C, H, N).
d
47.96 (CH2), 112.48 (CH), 113.35 (CH), 120.11 (CH), 120.28 (CH),
125.30 (C), 126.69 (2 x CH), 130.13 (2 x CH), 130.24 (C), 132.04 (C),
141.83 (C), 142.37 (C), 143.34 (C), 162.56 (CO), 167.42 (CO). ESI-MS
calcd. for C17H12N2O6, 340.29; found: m/z 341.07 [M þ H]þ. Anal.
4.2.2.7. 5-Nitro-1-(4-phenylbutyl)-1H-indole-2-carboxylic acid (6 h).
Yield ¼ 92%; mp ¼ 150e153 ꢁC (EtOH). 1H NMR (400 MHz,
C17H12N2O6 (C, H, N).
DMSO‑d6)
d
1.53 (quin, 2H, N-CH2CH2CH2CH2-Ph, J ¼ 7.6 Hz), 1.72
(quin, 2H, N-CH2CH2CH2CH2-Ph, J ¼ 7.2 Hz), 2.56 (t, 2H, N-
CH2CH2CH2CH2-Ph, J ¼ 7.4 Hz), 4.67 (t, 2H, N-CH2CH2CH2CH2-Ph,
J ¼ 6.8 Hz), 7.10e7.16 (m, 3H, Ar), 7.21e2.26 (m, 2H, Ar), 7.52 (s, 1H,
Ar), 7.81 (d, 1H, Ar, J ¼ 9.2 Hz), 8.13 (d, 1H, Ar, J ¼ 9.2 Hz), 8.72 (s, 1H,
Ar), 13.35 (exch br s, 1H, COOH). 13C NMR (100 MHz, DMSO‑d6)
4.2.2.12. 1-(4-Carboxyphenethyl)-5-nitro-1H-indole-3-carboxylic
acid (6 m). Yield ¼ 52%; mp ¼ 255e260 ꢁC dec. (EtOH). 1H NMR
(400 MHz, DMSO‑d6 þ D2O) 3.12 (t, 2H, N-CH2CH2-Ph, J ¼ 7.0 Hz),
d
4.53 (t, 2H, N-CH2CH2-Ph, J ¼ 7.2 Hz), 7.15 (d, 2H, Ar, J ¼ 8.0 Hz), 7.68
(d, 1H, Ar, J ¼ 9.2 Hz), 7.71 (d, 2H, Ar, J ¼ 8.0 Hz), 8.00 (dd, 1H, Ar,
J ¼ 9.0 Hz and J ¼ 2.2 Hz), 8.03 (s, 1H, Ar), 8.86 (d, 1H, Ar, J ¼ 2.0 Hz).
d
28.53 (CH2), 30.28 (CH2), 35.15 (CH2), 44.84 (CH2), 112.33 (CH),
112.85 (CH), 119.59 (CH), 120.19 (CH), 124.98 (C), 126.18 (CH), 128.68
(2 x CH),128.71 (2 x CH),130.44 (C),141.51 (C),141.97 (C),142.30 (C),
162.60 (CO). ESI-MS calcd. for C19H18N2O4, 338.36; found: m/z
339.13 [M þ H]þ. Anal. C19H18N2O4 (C, H, N).
13C NMR (100 MHz, DMSO‑d6)
d 35.88 (CH2), 47.87 (CH2), 111.06 (C),
111.92 (CH), 117.66 (CH), 117.94 (CH), 126.25 (C), 128.93 (2 x CH),
129.75 (2 x CH),133.17 (C),138.24 (CH),139.63 (C),141.78 (C),142.49
(C), 166.07 (CO), 168.84 (CO). ESI-MS calcd. for C18H14N2O6, 354.32;
found: m/z 355.09 [M þ H]þ. Anal. C18H14N2O6 (C, H, N).
4.2.2.8. 5-Nitro-1-(5-phenylpentyl)-1H-indole-3-carboxylic acid (6i).
Yield ¼ 89%; mp ¼ 195e198 ꢁC dec. (EtOH). 1H NMR (400 MHz,
4.2.2.13. 1-(4-Carboxyphenethyl)-5-nitro-1H-indole-2-carboxylic
DMSO‑d6)
d
1.13e1.27 (m, 2H, N-CH2CH2CH2CH2CH2-Ph), 1.49e1.56
acid (6n). Yield ¼ 52%; mp ¼ 294e296 ꢁC (EtOH). 1H NMR
13

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
(400 MHz, DMSO‑d6)
d
3.09 (t, 2H, N-CH2CH2-Ph, J ¼ 7.0 Hz), 4.89 (t,
(CH), 130.25 (C), 131.23 (C), 131.75 (C), 140.72 (C), 142.01 (3 x C),
143.66 (C), 162.84 (3 x CO), 167.44 (CO). ESI-MS calcd. for
2H, N-CH2CH2-Ph, J ¼ 7.2 Hz), 7.28 (d, 2H, Ar, J ¼ 8.0 Hz), 7.51 (s, 1H,
Ar), 7.74 (d, 1H, Ar, J ¼ 9.6 Hz), 7.80 (d, 2H, Ar, J ¼ 8.0 Hz), 8.07 (dd,
1H, Ar, J ¼ 9.2 Hz and J ¼ 2.0 Hz), 8.71 (d, 1H, Ar, J ¼ 1.6 Hz). 13C NMR
C
C
33H26N2O10S, 642.64; found: m/z 643.13 [M
33H26N2O10S (C, H, N).
þ
H]þ. Anal.
(100 MHz, DMSO‑d6) d 36.59 (CH2), 46.08 (CH2), 112.29 (CH), 112.77
(CH), 119.38 (CH), 120.05 (CH), 124.97 (C), 129.43 (C), 129.57 (2 x
CH), 129.79 (2 x CH), 132.27 (C), 141.37 (C), 141.91 (C), 143.87 (C),
162.64 (CO), 167.65 (CO). ESI-MS calcd. for C18H14N2O6, 354.32;
found: m/z 355.09 [M þ H]þ. Anal. C18H14N2O6 (C, H, N).
4.2.3.5. 1-(4-Carboxybenzyl)-5-(N,N-dibenzylsulfamoyl)-1H-indole-
2-carboxylic acid (14c). Yield ¼ 73%; mp ¼ 120e123 ꢁC (EtOH). 1H
NMR (400 MHz, DMSO‑d6) 4.23 (s, 4H, SO2N(CH2-Ph)2), 5.96 (s, 2H,
N-CH2-Ph), 6.96e7.01 (m, 4H, Ar), 7.07e7.12 (m, 8H, Ar), 7.52 (s, 1H,
Ar), 7.67 (s, 2H, Ar), 7.83 (d, 2H, Ar, J ¼ 8.4 Hz), 8.31 (s, 1H, Ar), 13.30
4.2.3. General procedure for compounds 13a,b, 14a-c, 15a,b
Compounds 13a,b, 14a-c and 15a,b were obtained following the
same procedure performed for compounds 6a-n using NaOH 6 N
(4 h) or NaOH 40% (2 h) as base and starting from intermediates
10a,b,11a-c and 12a,b. After cooling, the mixture was acidified with
HCl 37% (pH ¼ 2), the precipitate was recovered by vacuum filtra-
tion and washed several time with water and then recrystallized
using an appropriate solvent.
(exch br s, 2H, COOH). 13C NMR (100 MHz, DMSO‑d6)
d 47.73 (CH2),
51.46 (2 x CH2), 112.47 (CH), 112.64 (CH), 113.34 (CH), 123.32 (CH),
123.57 (CH),125.47 (C),126.61 (CH),126.74 (2 x CH),127.78 (2 x CH),
128.54 (2 x CH), 128.60 (CH), 129.06 (CH), 129.53 (CH), 129.84 (C),
130.00 (CH), 130.11 (2 x CH), 130.87 (C), 132.45 (C), 136.40 (C),
136.58 (C), 140.64 (C), 143.67 (C), 162.73 (CO), 167.46 (CO). ESI-MS
calcd. for C31H26N2O6S, 554.62; found: m/z 555.15 [M þ H]þ. Anal.
C
31H26N2O6S (C, H, N).
4.2.3.1. 1-Benzyl-5-sulfamoyl-1H-indole-2-carboxylic acid (13a).
4.2.3.6. 1-Benzyl-5-(N-benzylsulfamoyl)-1H-indole-2-carboxylic acid
Yield ¼ 65%; mp ¼ 233e235 ꢁC dec. (EtOH). 1H NMR (400 MHz,
(15a). Yield ¼ 54%; mp ¼ 213e216 ꢁC dec. (EtOH). 1H NMR
DMSO‑d6)
d
5.91 (s, 2H, N-CH2-Ph), 6.99 (d, 2H, Ar, J ¼ 6.8 Hz),
(400 MHz, DMSO‑d6)
d
3.93 (d, 2H, SO2NH-CH2-Ph, J ¼ 6.4 Hz), 5.92
7.19e7.25 (m, 5H, 3H Ar þ 2H SO2NH2), 7.49 (s, 1H, Ar), 7.71 (s, 2H,
(s, 2H, N-CH2-Ph), 7.03 (d, 2H, Ar, J ¼ 6.8 Hz), 7.13e7.22 (m, 6H, Ar),
7.26 (t, 2H, Ar, J ¼ 7.2 Hz), 7.45 (s, 1H, Ar), 7.63 (dd, 1H, Ar, J ¼ 8.8 Hz
and J ¼ 1.6 Hz), 7.70 (d, 1H, Ar, J ¼ 8.8 Hz), 7.98 (t, 1H, SO2NH-CH2,
J ¼ 6.2 Hz), 8.19 (d, 1H, Ar, J ¼ 1.2 Hz), 13.25 (exch br s, 1H, COOH).
Ar), 8.21 (s, 1H, Ar), 13.21 (exch br s, 1H, COOH). 13C NMR (100 MHz,
DMSO‑d6)
d 47.71 (CH2), 112.09 (CH), 112.39 (CH), 121.39 (CH),
122.53 (CH), 125.05 (C), 126.66 (CH), 127.60 (CH), 128.02 (CH),
128.63 (CH), 129.00 (CH), 130.78 (C), 137.21 (C), 138.66 (C), 140.21
(C), 162.97 (CO). ESI-MS calcd. for C16H14N2O4S, 330.35; found: m/z
331.07 [M þ H]þ. Anal. C16H14N2O4S (C, H, N).
13C NMR (100 MHz, DMSO‑d6)
d 49.65 (CH2), 49.71 (CH2), 110.65
(CH), 114.32 (CH), 124.39 (CH), 126.75 (CH), 126.95 (2 x CH), 127.65
(2 x CH), 128.55 (CH), 128.68 (2 x CH), 128.83 (2 x CH), 128.95 (CH),
129.73 (C), 131.35 (C), 132.58 (C), 137.30 (C), 141.65 (C), 142.60 (C),
162.39 (CO). ESI-MS calcd. for C23H20N2O4S, 420.48; found: m/z
421.12 [M þ H]þ. Anal. C23H20N2O4S (C, H, N).
4.2.3.2. 1-(4-Carboxybenzyl)-5-sulfamoyl-1H-indole-2-carboxylic
acid (13b). Yield ¼ 78%; mp ¼ 288e293 ꢁC dec. (EtOH). 1H NMR
(400 MHz, DMSO‑d6)
d 5.97 (s, 2H, N-CH2-Ph), 7.05 (d, 2H, Ar,
J ¼ 8.0 Hz), 7.21 (exch br s, 2H, SO2NH2), 7.51 (s, 1H, Ar), 7.70 (s, 2H,
Ar), 7.82 (d, 2H, Ar, J ¼ 8.0 Hz), 8.22 (s, 1H, Ar), 12.88 (exch br s, 1H,
COOH), 13.23 (exch br s, 1H, COOH). 13C NMR (100 MHz, DMSO‑d6)
4.2.3.7. 1-(4-Carboxybenzyl)-5-(N-(4-carboxybenzyl)sulfamoyl)-1H-
indole-2-carboxylic acid (15b). Yield ¼ 59%; mp > 300 ꢁC dec.
(EtOH). 1H NMR (400 MHz, DMSO‑d6)
d 3.97 (d, 2H, SO2NH-CH2-Ph,
d
46.71 (CH2), 112.15 (CH), 112.45 (CH), 121.30 (CH), 121.36 (C),
J ¼ 6.0 Hz), 6.08 (s, 2H, N-CH2-Ph), 7.12 (d, 2H, Ar, J ¼ 6.0 Hz), 7.24 (s,
1H, Ar), 7.30 (d, 2H, Ar, J ¼ 6.0 Hz), 7.58 (s, 2H, Ar), 7.68e7.78 (m, 4H,
Ar), 8.00 (t, 1H, SO2NH-CH2, J ¼ 6.0 Hz), 8.11 (s, 1H, Ar), 13.20 (exch
121.92 (CH), 125.17 (C), 126.67 (CH), 120.05 (CH), 130.77 (C), 132.47
(C), 137.23 (C), 143.82 (C), 164.93 (C), 167.46 (CO). ESI-MS calcd. for
C
C
17H14N2O6S, 374.37; found: m/z 375.06 [M
17H14N2O6S (C, H, N).
þ
H]þ. Anal.
br s, 3H, COOH). 13C NMR (100 MHz, DMSO‑d6)
d 46.29 (CH2), 47.35
(CH2), 109.05 (CH), 112.05 (CH), 121.36 (CH), 121.92 (CH), 125.77 (C),
126.59 (CH), 126.90 (CH), 127.88 (2 x CH), 129.64 (2 x CH), 129.93 (2
x CH),130.77 (C),132.47 (C),139.58 (C),143.21 (C),144.03 (C),164.54
(CO), 167.97 (CO). ESI-MS calcd. for C25H20N2O8S, 508.50; found: m/
z 509.10 [M þ H]þ. Anal. C25H20N2O8S (C, H, N).
4.2.3.3. 1-Benzyl-5-(N,N-dibenzylsulfamoyl)-1H-indole-2-carboxylic
acid (14a). Yield ¼ 39%; mp ¼ 184e186 ꢁC dec. (EtOH). 1H NMR
(400 MHz, DMSO‑d6) 4.26 (s, 4H, SO2N(CH2-Ph)2), 5.95 (s, 2H, N-
CH2-Ph), 6.99e7.05 (m, 6H, Ar), 7.12e7.20 (m, 6H, Ar), 7.23 (d,1H, Ar,
J ¼ 7.2 Hz), 7.28 (t, 2H, Ar, J ¼ 7.2 Hz), 7.46 (s, 1H, Ar), 7.70 (d, 1H, Ar,
J ¼ 8.8 Hz), 7.74 (d, 1H, Ar, J ¼ 8.8 Hz), 8.30 (s, 1H, Ar), 13.30 (exch br
4.2.4. General procedure for compounds 20-22
Compounds 20e22 were obtained following the same proced-
ure of hydrolysis performed for compounds 6a-j and 13e15a using
NaOH 6 N (4 h) as base and starting from intermediates 17e19,
respectively. After acidification with HCl 37%, the compounds are
recovered by vacuum filtration and washed several time with water
(for 20 and 22) or by extraction with ethyl acetate (3 ꢂ 15 mL) for
compound 21. The final compounds were purified by crystallization
with ethanol.
s, 1H, COOH). 13C NMR (100 MHz, DMSO‑d6)
d 49.75 (CH2), 53.01
(CH2), 110.65 (CH), 113.35 (CH), 124.31 (CH), 125.78 (CH), 127.00 (2 x
CH), 127.65 (2 x CH), 127.90 (4 x CH), 128.55 (4 x CH), 128.68 (3 x
CH), 129.75 (C), 131.35 (C), 132.50 (C), 136.30 (C), 136.47 (C), 137.65
(C), 142.60 (C), 162.90 (CO). ESI-MS calcd. for C30H26N2O4S, 538.66;
found: m/z 539.20 [M þ H]þ. Anal. C30H26N2O4S (C, H, N).
4.2.3.4. 4,4'-((((2-Carboxy-1-(4-carboxybenzyl)-1H-indol-5-yl)sulfo-
nyl)azanediyl)bis(methylene))
dibenzoic
acid
(14b).
4.2.4.1. 5-(N,N-dibenzylsulfamoyl)-1-phenethyl-1H-indole-2-
Yield ¼ 69%; mp ¼ 280e285 ꢁC dec. (EtOH). 1H NMR (400 MHz,
carboxylic acid (20). Yield ¼ 64%; mp ¼ 175e180 ꢁC (EtOH). 1H NMR
DMSO‑d6)
d
4.36 (s, 2H, SO2NCH2-Ph), 4.54 (s, 2H, SO2NCH2-Ph),
(400 MHz, DMSO‑d6)
d
2.99 (t, 2H, N-CH2CH2-Ph, J ¼ 7.4 Hz), 4.23 (s,
5.99 (d, 2H, N-CH2-Ph, J ¼ 16 Hz), 7.07e7.17 (m, 3H, Ar), 7.32 (d, 1H,
Ar, J ¼ 8.0 Hz), 7.40 (d, 2H, Ar, J ¼ 8.0 Hz), 7.49 (t, 1H, Ar, J ¼ 7.0 Hz),
7.68e7.92 (m, 8H, Ar), 8.01 (s, 1H, Ar), 12.83 (exch br s, 4H, 4 x
4H, SO2N(CH2-Ph)2), 4.83 (t, 2H, N-CH2CH2-Ph, J ¼ 7.4 Hz),
7.04e7.20 (m, 15H, Ar), 7.40 (s, 1H, Ar), 7.65e7.70 (m, 2H, Ar), 8.25
(s, 1H, Ar), 13.20 (exch br s, 1H, COOH). 13C NMR (100 MHz,
COOH). 13C NMR (100 MHz, DMSO‑d6)
d
47.79 (CH2), 52.29 (2 x CH2),
DMSO‑d6) d 29.46 (CH2), 36.79 (CH2), 46.33 (CH2), 51.73 (CH2),
112.24 (CH), 112.78 (CH), 123.27 (CH), 123.75 (CH), 125.55 (C),
126.74 (2 x CH), 128.02 (4 x CH), 128.69 (CH) 129.53 (4 x CH), 130.13
111.17 (CH), 112.46 (CH), 122.68 (CH), 123.39 (CH), 125.18 (C), 126.89
(CH), 127.77 (2 x CH), 128.64 (5 x CH), 128.77 (5 x CH), 129.32 (2 x
14

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
CH), 131.20 (C), 131.47 (C), 136.84 (2 x C), 138.71 (C), 140.25 (C),
162.90 (C). ESI-MS calcd. for C31H28N2O4S, 524.64; found: m/z
525.18 [M þ H]þ. Anal. C31H28N2O4S (C, H, N).
4.5. Compounds administration
15b, 6e and 5b were injected i.t in conscious mice as previously
described [46]. Briefly, a 25- l Hamilton syringe connected to a 30-
m
gauge needle was intervertebral inserted between the L4 and L5
region, and advanced 6 mm into the lumbar enlargement of the
spinal cord. Behavioural measurements were every 15 min. Doses
were chosen on the bases of our previous data about known Panx-1
inhibitors [34].
4.2.4.2. 5-(N-Benzyl-N-phenethylsulfamoyl)-1-phenethyl-1H-indole-
2-carboxylic acid (21). Yield ¼ 46%; mp ¼ 168 ꢁC dec. (EtOH). 1H
NMR (400 MHz, DMSO‑d6)
d 2.53 (t, 2H, SO2N-CH2CH2-Ph,
J ¼ 8.0 Hz), 2.98 (t, 2H, N-CH2CH2-Ph, J ¼ 7.2 Hz), 3.18 (t, 2H, SO2N-
CH2CH2-Ph, J ¼ 8.0 Hz), 4.32 (s, 2H, SO2N-CH2-Ph), 4.81 (t, 2H, N-
CH2CH2-Ph, J ¼ 7.4 Hz), 6.95 (d, 2H, Ar, J ¼ 7.2 Hz), 7.09e7.20 (m, 9H,
Ar), 7.22e7.32 (m, 4H, Ar), 7.43 (s, 1H, Ar), 7.62 (d, 1H, Ar, J ¼ 9.2 Hz),
7.67 (d, 1H, Ar, J ¼ 9.2 Hz), 8.25 (s, 1H, Ar), 13.29 (exch br s, 1H,
4.6. Cold plate test
COOH). 13C NMR (100 MHz, DMSO‑d6)
d 32.03 (CH2), 36.61 (CH2),
The animals were placed in
(12 cm ꢂ 20 cm ꢂ 10 cm) with a cold plate as floor. The temperature
of the cold plate was kept constant at 4 ꢁC ꢁC. Pain-related
a
stainless steel box
49.65 (CH2), 53.12 (CH2), 53.40 (CH2), 110.61 (CH), 114.38 (CH),
124.36 (CH), 125.91 (CH), 127.06 (CH), 127.74 (2 x CH), 127.95 (4 x
CH), 128.52 (4 x CH), 128.61 (CH), 128.88 (2 x CH), 129.33 (CH),
129.76 (C), 131.33 (C), 132.54 (C), 136.44 (C), 136.51 (C), 139.42 (C),
145.03 (C), 162.09 (CO). ESI-MS calcd. for C32H30N2O4S, 538.66;
found: m/z 539.20 [M þ H]þ. Anal. C32H30N2O4S (C, H, N).
1
behavior (licking of the hind paw) was observed and the time
(seconds) of the first sign was recorded. The cut-off time of the
latency of paw lifting or licking was set at 60 s [47].
4.7. Statistical analysis
4.2.4.3. 1-Phenethyl-5-(N-phenethylsulfamoyl)-1H-indole-2-
carboxylic acid (22). Yield ¼ 69%; mp ¼ 183e187 ꢁC (EtOH). 1H NMR
Behavioral measurements were performed on 10 mice for each
treatment carried out in 2 different experimental sets. Results were
expressed as mean S.E.M. The analysis of variance of behavioral
data was performed by one way ANOVA, a Bonferroni's significant
difference procedure was used as post-hoc comparison. P values of
less than 0.05 or 0.01 were considered significant. Investigators
were blind to all experimental procedures. Data were analyzed
using the “Origin 9” software (OriginLab, Northampton, USA).
(400 MHz, DMSO‑d6)
d
2.62 (t, 2H, SO2NH-CH2CH2-Ph, J ¼ 7.6 Hz),
2.87 (t, 2H, N-CH2CH2-Ph, J ¼ 7.2 Hz), 2.97 (t, 2H, SO2NH-CH2CH2-
Ph, J ¼ 7.4 Hz), 4.80 (t, 2H, N-CH2CH2-Ph, J ¼ 7.2 Hz), 7.10e7.30 (m,
10H, Ar), 7.53e7.60 (m, 3H, Ar), 8.09 (s, 1H, Ar). 13C NMR (100 MHz,
DMSO‑d6)
d 35.65 (CH2), 36.91 (CH2), 44.66 (CH2), 46.27 (CH2),
110.26 (CH), 111.99 (CH), 121.73 (CH), 122.23 (CH), 125.12 (C), 126.66
(CH), 126.76 (CH), 128.70 (2 x CH), 128.77 (2 x CH), 129.08 (2 x CH),
129.31 (2 x CH), 132.04 (C), 133.52 (C), 138.91 (C), 139.29 (C), 139.71
(C), 163.58 (CO). ESI-MS calcd. for C25H24N2O4S, 448.54; found: m/z
449.15 [M þ H]þ. Anal. C25H24N2O4S (C, H, N).
4.8. Preparation of oocytes
All procedures were approved by the University of Miami
Institutional Animal Care and Use Committee and conducted in
accordance with the Guiding Principles for Research Involving
Animals and Human Beings of the American Physiological Society.
Preparation of oocytes from Xenopus laervis and electrophysiolog-
ical recording were performed as described previously (Dahl and
Pfahnl, 2001). Mouse Panx1 was provided by R. Dermietzel (Uni-
versity of Bochum, Bochum, Germany). Human P2X7R was pro-
vided by A. Surprenant (University of Sheffield, Sheffield, UK). The
generation of Cx32E143 was described previously [32].
4.3. Animals
Male CD-1 albino mice (Envigo, Varese, Italy) weighing
approximately 22e25 g at the beginning of the experimental pro-
cedure, were used. Animals were housed in CeSAL (Centro Stabu-
lazione Animali da Laboratorio, University of Florence) and used at
least 1 week after their arrival. Ten mice were housed per cage (size
26 ꢂ 41 cm); animals were fed a standard laboratory diet and tap
water ad libitum, and kept at 23 1 ꢁC with a 12 h light/dark cycle,
light at 7 a.m. All animal manipulations were carried out according
to the Directive 2010/63/EU of the European parliament and of the
European Union council (22 September 2010) on the protection of
animals used for scientific purposes. The ethical policy of the Uni-
versity of Florence complies with the Guide for the Care and Use of
Laboratory Animals of the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996; University of Florence assur-
ance number: A5278-01). Formal approval to conduct the experi-
ments described was obtained from the Animal Subjects Review
Board of the University of Florence. Experiments involving animals
have been reported according to ARRIVE guidelines. All efforts were
made to minimize animal suffering and to reduce the number of
animals used.
4.9. Electrophysiology
Whole cell membrane current of single oocytes was measured
using a two-electrode voltage clamp and recorded with a chart
recorder. Both voltage-measuring and current-passing microelec-
trodes were filled with 3 M KCl. The recording chamber was
perfused continuously with frog Ringer (OR2) solution (mM: 82.5
NaCl, 2.5 KCl, 1 CaCl2, 1 MgCl2, 1 Na2HPO4, and 5 HEPES, pH 7.5).
Membrane conductance was determined using voltage pulses.
4.10. Molecular modeling
The binding site structure was obtained from hPanx-1 structure
in complex with CBX (PDB ID 6WBG) [41] considering all the amino
acids within a distance of about 2 nm from the structure of the CBX.
The ligands were placed at the binding site through AUTODOCK 4.2
[43]. The main autodock parameters used in this work are:
4.4. Oxaliplatin-induced neuropathic pain model
Mice were treated with oxaliplatin (2.4 mg kgꢀ1) intraperito-
neally administered on days 1e2, 5e9, 12e14 (10 i.p. injections)
[45]. Oxaliplatin was dissolved in 5% glucose solution. Control an-
imals received an equivalent volume of vehicle. Behavioral tests
were performed on day 15.
15

L. Crocetti, G. Guerrini, S. Puglioli et al.
European Journal of Medicinal Chemistry 223 (2021) 113650
Main AutoGrid parameters
family is homologous to the invertebrate innexin gap junction proteins, Ge-
nomics 83 (2004) 706e716.
[10] S. Penuela, R. Bhalla, K. Nag, D.W. Laird, Glycosylation regulates pannexin
intermixing and cellular localization, Mol. Biol. Cell 20 (2009) 4313e4323.
[11] R. Bruzzone, M.T. Barbe, N.J. Jakob, H. Monyer, Pharmacological properties of
homomeric and heteromeric pannexin hemichannels expressed in Xenopus
oocytes, J. Neurochem. 92 (2005) 1033e1043.
npts 90 90 90
spacing 0.375
gridcenter 123.0 124.0 84.0
# num.grid points in xyz
# spacing(A)
# xyz-coordinates
[12] M. Billaud, A.W. Lohman, A.C. Straub, R. Looft-Wilson, S.R. Johnstone, C.A. Araj,
A.K. Best, F.B. Chekeni, K.S. Ravichandran, S. Penuela, D.W. Laird, B.E. Isakson,
Pannexin1 regulates alpha1-adrenergic receptor-mediated vasoconstriction,
Circ. Res. 109 (2011) 80e85.
Main AutoDock Parameters
ga_pop_size 150
# number of individuals in population
# maximum number of energy evaluations
# maximum number of generations
# do this many hybrid GA-LS runs
[13] W.R. Silverman, J.P. De Rivero Vaccari, S. Locovei, F. Qiu, S.K. Carlsson,
ga_num_evals 2500000
ga_num_generations 27000
ga_run 100
E. Scemes, R.W. Keane, G. Dahl, The pannexin
1 channel activates the
inflammasome in neurons and astrocytes, J. Biol. Chem. 284 (2009)
18143e18151.
[14] G. Dahl, The Pannexin1 membrane channel: distinct conformations and
functions, FEBS Lett. 592 (2018) 3201e3209.
[15] W. Silverman, S. Locovei, G. Dahl, Probenecid, a gout remedy, inhibits pan-
nexin 1 channels, Am. J. Physiol. Cell Physiol. 295 (2008) C761eC767.
[16] R. Iglesias, S. Locovei, A. Roque, A.P. Alberto, G. Dahl, D.C. Spray, E. Scemes,
P2X7 receptor-Pannexin1 complex: pharmacology and signalling, Am. J.
Physiol. Cell Physiol. 295 (2008) C752eC760.
The PDB coordinates files are in Supplementary Material.
Author contribution
[17] J. Wang, D.G. Jackson, G. Dahl, The food dye FD&C Blue No. 1 is a selective
inhibitor of the ATP release channel Panx1, J. Gen. Physiol. 141 (2013)
649e656.
[18] T.J. Freeman, S. Sayedyahossein, D. Johnston, R.E. Sanchez-Pupo, B. O'Donnell,
K. Huang, Z. Lakhani, D. Nouri-Nejad, K.J. Barr, L. Harland, S. Latosinsky,
Crocetti L. and Giovannoni M.P., designed the compounds and
wrote the manuscript; Crocetti L. and Puglioli S. synthesized the
compounds. In vitro studies (inhibition essay) and selectivity test
were performed by Wang J. and Dahl G. Molecular docking and
ADMET assessment were performed by Guerrini G. and Puglioli S.
In vivo studies and related section part were performed by Ghe-
lardini C., Di Cesare Mannelli L. and Lucarini E. All of the authors
have given approval to the final version of the manuscript.
A. Grant, L. Dagnino, S. Penuela, Inhibition of pannexin
1 reduces the
tumorigenic properties of human melanoma cells, Cancers 11 (2019) E102.
[19] E. Ahmadian, A. Eftekhari, M. Samiei, S.M. Dizaj, M. Vinken, The role and
therapeutic potential of connexins, pannexins and their channels in Parkin-
son's disease, Cell. Signal. 58 (2019) 111e118.
[20] Y. Zhang, G. Laumet, S.-R. Chen, W.N. Hittelman, H.-L. Pan, Pannexin-1 up
regulation in the dorsal root ganglion contributes to neuropathic pain
development, J. Biol. Chem. 290 (2015) 14647e14655.
[21] G. Burnstock, Physiopathological roles of P2X receptors in the central nervous
system, Curr. Med. Chem. 22 (2015) 819e844.
Funding
[22] M. Tsuda, H. Tozaki-Saitoh, K. Inoue, Pain and purinergic signalling, Brain Res.
Rev. 63 (2010) 222e232.
[23] S.D. Skaper, P. Debetto, P. Giusti, The P2X7 purinergic receptor: from physi-
ology to neurological disorders, FASEB J 24 (2010) 337e345.
This work was supported by the National Institutes of Health
[R56HL136291].
[24] P. Pelegrin, A. Surprenant, Pannexin-1 mediates large pore formation and
interleukin-1
5071e5082.
b release by the ATP-gated P2X7 receptor, EMBO J. 25 (2006)
Declaration of competing interest
[25] A. Sudhakara, H. Jayadevappa, K.M. Mahadevan, V. Hulikal, Efficient synthesis
of 2-ethoxycarbonyl indoles, Synth. Commun 39 (2009) 2506e2515.
[26] S. Castellano, A. Spannhoff, C. Milite, F. Dal Piaz, D. Cheng, A. Tosco, M. Viviano,
A. Yamani, A. Cianciulli, M. Sala, V. Cura, J. Cavarelli, E. Novellino, A. Mai,
M.T. Bedford, G. Sbardella, Identification of small-molecule enhancers of
arginine methylation catalyzed by coactivator-associated arginine methyl-
transferase 1, J. Med. Chem. 55 (2012) 9875e9890.
[27] P. Levesque, P. Fournier, Synthesis of substituted indole from 2- amino-
benzaldehyde through [1,2]-Aryl shift, J. Org. Chem. 75 (2010) 7033e7036.
[28] B. Hu, J.W. Jetter, J.E. Wrobel, T.M. Antrilli, J.S. Bauer, L. Di, S. Polakowski,
U. Jain, D.L. Crandall, Synthesis and SAR of 2-carboxylic acid indoles as in-
hibitors of plasminogen activator inhibitor-1, Bioorg. Med. Chem. Lett. 15
(2005) 3514e3518.
[29] T. Tanaka, Y. Konishi, D. Kubo, M. Fujino, I. Doi, D. Nakagawa, T. Murakami,
T. Yamakawa, Preparation of (hetero)arylamines having keratinocyte prolif-
eration inhibitory activity, in: PCT Int. Appl., 2014. WO 2014069510 A1
20140508.
[30] L.D. Belli, M.L. Stei, Indoles containing the sulfamoyl group. II. Preparation of
5-sulfamoylindole and derivatives with substituents in the 2- and 3-positions,
Ann. Chim. 51 (1961) 663e674.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113650.
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