Synthesis and structure-activity relationship of aminoarylthiazole derivatives as correctors of the chloride transport defect in cystic fibrosis

Article abstract of DOI:10.1016/j.ejmech.2015.05.030

Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can be targeted by small molecules called generically correctors and potentiators, respectively. Aminoarylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now designed and synthesized a library of novel compounds in order to establish an initial SAR that may provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The new compounds were tested as correctors and potentiators in CFBE41o-expressing F508del-CFTR using a functional assay. A dual active compound, AAT-4a, characterized by improved efficacy and marked synergy when combined with the corrector VX-809 has been identified. Moreover, by computational methods, a possible binding site for AATs in nucleotide binding domain NBD1 has been detected. These results will direct the synthesis of new analogues with possibly improved activity.

Full text of DOI:10.1016/j.ejmech.2015.05.030

European Journal of Medicinal Chemistry 99 (2015) 14e35
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and structureeactivity relationship of aminoarylthiazole
derivatives as correctors of the chloride transport defect in cystic
fibrosis
,
, c,
Emanuela Pesce a 1, Marta Bellotti b 1, Nara Liessi b, Sara Guariento d,
Gianluca Damonte b c, Elena Cichero d, Andrea Galatini e, Annalisa Salis c,
,
Ambra Gianotti a, Nicoletta Pedemonte a, Olga Zegarra-Moran a, Paola Fossa d,
Luis J.V. Galietta a, Enrico Millo b
, c, *
a Istituto Giannina Gaslini, via Gerolamo Gaslini 5, 16147 Genoa, Italy
b Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132 Genoa, Italy
c Center of Excellence for Biomedical Research (CEBR), University of Genoa, Viale Benedetto XV 5, 16132 Genoa, Italy
d Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, 3,
16132 Genoa, Italy
e Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso 31, 16146 Genoa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the
membrane of epithelial cells. Mutations affecting the CFTR gene cause cystic fibrosis (CF), a multi-organ
severe disease. The most common CF mutation, F508del, impairs the processing and activity (gating) of
CFTR protein. Other mutations, like G551D, only cause a gating defect. Processing and gating defects can
be targeted by small molecules called generically correctors and potentiators, respectively. Amino-
arylthiazoles (AATs) represent an interesting class of compounds that includes molecules with dual
activity, as correctors and potentiators. With the aim to improve the activity profile of AATs, we have now
designed and synthesized a library of novel compounds in order to establish an initial SAR that may
provide indications about the chemical groups that are beneficial or detrimental for rescue activity. The
new compounds were tested as correctors and potentiators in CFBE41o-expressing F508del-CFTR using a
functional assay. A dual active compound, AAT-4a, characterized by improved efficacy and marked
synergy when combined with the corrector VX-809 has been identified. Moreover, by computational
methods, a possible binding site for AATs in nucleotide binding domain NBD1 has been detected. These
results will direct the synthesis of new analogues with possibly improved activity.
Received 15 January 2015
Received in revised form
18 May 2015
Accepted 19 May 2015
Available online 28 May 2015
Keywords:
AminoArylthiazole
Corrector
SAR
Cystic fibrosis
CFTR
Docking
© 2015 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
affecting CFTR, a gene coding for a chloride channel expressed in
epithelial cells of different organs, particularly in lungs, pancreas,
Cystic fibrosis (CF) is a genetic disease caused by mutations
and testis [1]. CFTR protein has five domains: two nucleotide
binding domains (NBD1 and 2), two transmembrane domains
(MSD1 and MSD2), and one regulatory domain (R) [1]. So far, a large
number of CF-causing mutations have been identified. CF muta-
tions are classified in 5 classes according to the mechanism of CFTR
loss-of-function [2]. The most common genetic defect, occurring in
70e90% of CF patients, is the deletion of phenylalanine 508
(F508del), located in NBD1. F508del-CFTR, which is unstable and
incorrectly folded, remains trapped in the endoplasmic reticulum
and rapidly degraded by the ubiquitin/proteasome system.
F508del-CFTR also shows an altered channel activity. If the mutant
Abbreviations: CF, cystic fibrosis; CFTR, CF transmembrane conductance regu-
lator; AATs, aminoarylthiazoles; HS-YFP, halide-sensitive yellow fluorescent pro-
tein; SAR, structure-activity relationship; NBD, nucleotide binding domain; HBTU,
O-(Benzotriazol-1-yl)-N,N,N0,N'-tetramethyluronium hexafluorophosphate; DIPEA,
N,N-Diisopropylethylamine.
* Corresponding author. Center of Excellence for Biomedical Research (CEBR),
University of Genova, Viale Benedetto XV 5, 16132 Genoa, Italy.
E-mail address: enrico.millo@unige.it (E. Millo).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.05.030
0223-5234/© 2015 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
15
protein is targeted to the plasma membrane with rescue maneu-
1.1. Chemistry
vers (e.g. incubation of cells at low temperature), it shows a reduced
open channel probability. The deficit of CFTR channel function in
the plasma membrane has a major impact on the airways and the
respiratory system, since CF patients produce a thick mucus that
cannot be correctly cleared and show impairment of innate defense
mechanisms against bacteria [3]. The resulting pulmonary in-
fections cause a progressive loss of respiratory function. At the last
stage of the disease, most patients require lung transplant as life-
saving intervention.
The lead structure can be split into three parts: the central
aminothiazole, the substituent at C-4, and the aryl substituent at N-
2 (Fig. 1). Based on our previous study [10], in which we described
the strict requirement of the N-2 substituents for activity, we chose
to initially build a series of C-4 substituted analogues. To prepare
the compounds we applied a known synthetic route with minor
modifications. The desired target molecules were synthesized in a
range of variable yields via the classic Hantzsch thiazole synthesis
refluxing the appropriate haloketone with an equimolar amount of
the respective N-substituted thiourea (a-f) in absolute ethanol
[13,14] (Scheme 1).
The substituted thioureas were synthesized by condensation of
the appropriate aniline derivative with benzoyl isothiocyanate
followed by saponification to remove the benzoyl group [15]
(Scheme 1). In the search for a simple and effective means of
obtaining aminothiazole derivatives, we have studied the possi-
bility of selective monobromination of ketones. All the bromoke-
tones that were not commercially available were readily prepared
from the corresponding ketones utilizing N-bromosuccinimide
(NBS), catalyzed by trimethylsilyltrifluoromethanesulfonate (TMS-
OTf) in acetonitrile [16]. This method is useful for the side-chain
bromination of heteroaromatic carbonyl compounds without the
ring brominations.
The primary defects caused by CFTR mutations can be treated
with drug-like small molecules. In particular, the folding and the
channel activity defect of F508del can be addressed with com-
pounds named correctors and potentiators, respectively. Correctors
improve the trafficking of mutant CFTR to the plasma membrane,
an effect that requires several hours of treatment to be detected.
Instead, potentiators have a fast effect consisting of an increase in
the open probability of mutant CFTR channels present in the
plasma membrane. While potentiators probably act by directly
interacting with CFTR protein, the mechanism of action of correc-
tors is unknown. They could directly bind to CFTR improving its
folding and stability or regulate the expression/function of other
proteins involved in CFTR processing and trafficking [4]. Different
approaches have been pursued to discover correctors and poten-
tiators: a) in silico screenings; b) high-throughput screenings of
large chemical compound libraries; c) hypothesis-driven studies
based on the present knowledge of CFTR protein homeostasis
[5e7]. With such approaches, a large number of potentiators and
correctors have been identified so far including tetrahy-
drobenzothiophenes, benzofurans, pyramidinetriones, dihy-
dropyridines, anthraquinones, bithyazoles (corr-4a), quinazolines
(VRT-325), and phenylhydrazones [8]. In general, potentiators have
a very high efficacy and a molecule, VX-770, has been recently
approved for the treatment of patients with G551D, a mutation that
causes a severe channel gating defect. This potentiator is very
effective in improving the clinical condition of patients with at least
one copy of G551D mutation. In contrast, all correctors, which are
specifically developed to target the F508del mutation, are charac-
terized by a partial efficacy. Recent studies reveal that the F508del
mutation causes multiple defects to the stability and folding of
CFTR protein [9]. Therefore, only combinations of correctors having
different mechanisms of action are expected to result in a large
rescue of F508del-CFTR. For example, one of the most effective
correctors available so far, VX-809, improves the interaction of
NBD1 with the fourth intracellular loop of CFTR, but does not in-
crease the stability of F508del. Therefore, there is an urgent need to
identify novel correctors that, in combination with VX-809 or
similar compounds, generate an additive/synergic effect.
On the contrary, the ketone of the compounds 4i, 9d, 20j, and
21c was brominated with a solution of copper (I1) bromide in ethyl
acetate [17] (Scheme 1).
The analogues 1c and 1n were prepared by nucleophilic sub-
stitution of 3-methylthioaniline and substituted 2-halothiazole
using sodium hydride in tetrahydrofuran (THF) [10], while 17c
and 17d were prepared with the same reaction in ethanol respec-
tively with piperidine and cyclohexylamine. The same 3-
methylthioaniline
was
conjugated
with
6-bromo-2-
chlorobenzothiazole to furnish 11a in high yield. The compound
2f was synthetized starting from 3, 4, 5 trihydroxy acetophenone
that was first completely acetylated with acetic anhydride in
anhydrous dichloromethane and then brominated with NBS as
previously described. At last, the thiazolic derivative was obtained
in moderate yield by the condensation of N-substituted thiourea a
with bromoketone in refluxing ethanol. Compound 2d was syn-
thetized starting from 2-chloro-30, 4’-dihydroxyacetophenone that
In a previous study, we found that compounds belonging to the
class of aminoarylthiazoles (AATs) show a dual activity as correctors
and as potentiators [10]. Among AATs, the compound EN277I was
particularly able to increase the function of F508del-CFTR at
micromolar concentrations.
With the aim to improve the activity profile of the amino-
arylthiazoles (AATs), we have now designed and synthesized a li-
brary of novel AATs to investigate their effect on mutant CFTR. The
design phase has been assisted by molecular modeling studies
performed using the X-ray data of the human nucleotide binding
domains NBD1, since recent literature has strongly suggested this
domain as a target for small molecules acting on CTFR [11,12].
Compounds were tested with a functional assay in the presence
and absence of VX-809 [11]. Our study has provided novel SAR
information on AATs as correctors and led to the identification of a
molecule, 4a, with a particular ability to rescue F508del-CFTR.
Fig. 1. Sites for modifications of aminoarylthiazole derivatives and structure of the hit
compound EN277I.

16
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
Scheme 1. Reagents and conditions (a) NBS, TMS-OTf, acetonitrile, 40ꢀ, 3e24 h or (b) CuBr2, EtOAc, 60ꢀ, 5 h-12 h (c) anhydrous THF, 25ꢀ, ON (d) EtOH absolute, NaOH 1M, 40ꢀ
8e10 h (e) EtOH absolute, reflux, 2 h-24 h. For complete structure see Tables 1e4.
was functionalized with a reaction with of tert-butylbromoacetate
in anhydrous N, N dimethylformamide (DMF) and subjected to
hydrolysis in acidic ambient to liberate the carboxyl group [18]. This
intermediate reacted with the N-substituted thiourea a to give the
required thiazole (Scheme 1).
previously described and conjugated with N-substituted thiourea a
in the classical condition. In the case of 4k, the thiazole was then
subjected to hydrolysis in acidic ambient to obtain the carboxyl
group. The compound 14e was synthetized from a particular N-
substituted thiourea e obtained from the reaction of 2-ethyl 3-
methyl thioaniline with benzoyl isothiocyanate followed by sap-
onification to remove the benzoyl group as previously described
[15]. The 2-ethyl 3-methyl thioaniline derived from the reaction of
4-ethylnitrobenzene with chlorosulfonic acid and subsequent
reduction with tryphenylphosphine [20,21]. The nitroderivative
was indeed methylated on the thiol group and subsequently
reduced to aniline with Zn/saturated NH4Cl [22] (Scheme 2).
The 3-aminobenzenethiol was alkylated respectively with
iodopropane or iodoethane on the thiol group to obtain particular
substituted amine. The aniline derivatives and the same 3-
aminobenzenethiol were then transformed in the respective thio-
ureas (b, c, d) in the same experimental conditions and conjugated
with bromoketone to obtain the three different thiazoles 14a, 14b
and 14c. To introduce the keto group on the thiophene ring, the
solution of 3-n-butilthiophene was subjected to Friedel and Kraft
acylation with acetyl chloride and aluminium trichloride in anhy-
drous methylene chloride as previously published [23]. A com-
pound corresponding to 1-(4-butylthiophen-2-yl) ethanone was
isolated and then brominated with NBS as previously described.
The bromoketone was the conjugated with the thiourea a to furnish
the final thiazole 21a.
The compounds 15a and 15b were obtained with a simple re-
action of the precursor 4-(3-chlorophenyl)-N-(3-(methylthio)
phenyl) thiazol-2-amine with methyl iodide and benzyl chloride in
anhydrous DMF.
An alternative protocol was developed to build the compound
17a, as the classic reaction by nucleophilic substitution of amine
and substituted 2-halothiazole did not lead to any product. Starting
from 2 aminothiazole derivative, a solution of benzyl chloride was
added to form as main product N-benzyl-4-(4-bromophenyl)thia-
zol-2-amine 17a and as byproduct N,N-dibenzyl-4-(4-
bromophenyl)thiazol-2-amine 17b as previously described [24].
The synthesis of amide derivatives of 2-aminothiazoles were
achieved by condensation reaction of 2-amino-4(-4'bromophenyl)
thiazole with the appropriate carboxylic derivatives with HBTU/
DIPEA activation in anhydrous DMF. This strategy allowed us to
prepare the compounds 16a-16d in high yield and purity. The
introduction of a methyl substituent on thiazole ring in position 5
was obtained with the condensation of the appropriate propio-
phenone: 2-bromo for 18c or 2, 4’-dibromo in the case of the
derivate 18b, with N-substituted thiourea a in absolute ethanol. A
phenyl group was instead introduced in a simple procedure from
the precursor 2-bromo-4'-chloro-2-(4-methylphenyl) acetophe-
none by treatment with the same thiourea (18a). The particular
derivative 18d with a phenacyl group in position 5 of the thiazole
ring derived from the reaction between 2-bromo-1,3-
diphenylpropane-1,3-dione and the thiourea a. The compound
12b was obtained in a three step procedure from the condensation
between ethyl 3-chloropropanoate and thiourea a in refluxing
ethanol, hydrolysis in basic environment, and subsequent reaction
of carboxylic group so obtained with aniline in the presence of a
HBTU/DIPEA [19].
The compound N, 4-bis (3-(methylthio) phenyl) thiazol-2-
amine (4b) was prepared starting from the reduction of 3-
acetylbenzene-1-sulfonyl chloride to 1-(3-mercaptophenyl) etha-
none according to the published procedure [20]. Afterwards, the
ketone was methylated with iodomethane at thiol portion,
brominated with NBS and conjugated with 1-(3-(methylthio)
phenyl) thiourea a.
Thiazole 4i was built starting from 1-(3-aminophenyl) ethanone
that was first N-acetylated with acetic anhydride in anhydrous
dichloromethane and then brominated with CuBr2 in ethyl acetate.
The bromoketone was indeed refluxed with an equimolar amount
of the N-substituted thiourea a to produce the final product. The
compounds 4j and 4k were synthesized from a common precursor,
the 1-(3-hydroxyphenyl) ethanone that was modified with the
introduction of an apolar group on the hydroxyl moiety. The reac-
tion was obtained with the use of tert-butylbromoacetate in
anhydrous DMF [18]. The ketone was then brominated with NBS as
2. Results and discussion
In a previous work, we synthesized a panel of AATs in which
various substituents, in particular aniline and pyridine derivatives,

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
17
Scheme 2. Reagents and conditions a) chlorosulfonic acid, 100ꢀ, 2 h (b) PhP3, anhydrous toluene, reflux, 1 h (c) anhydrous DMF, NaH, methyl iodide, 25ꢀ, 3 h (d) anhydrous acetone,
Zn, NH4Cl saturated, 40ꢀ, 2 h (e) anhydrous THF,benzoylisothiocianate, 25ꢀ, ON (f) EtOH absolute, NaOH 1M, 40ꢀ, 8 h (g) 2-bromo-30-chloroacetophenone, EtOH absolute, reflux,
24 h.
were introduced into position 2 at the fixed scaffold of 4-(4bro-
mophenyl)thiazole. One of these derivatives called EN277I (Fig. 1)
showed a significant activity on CFTR processing and gating [10].
In the present work, we have further modified the AAT structure
at different positions, as indicated in Fig. 1, for the identification of
derivatives with improved potency and efficacy. The modifications
were made in one region at a time, to determine the contribution of
a particular structural portion to bioactivity. Such modifications led
to a variable range of activities that helped us in the generation of a
plausible SAR as will be described below.
incubated for 24 h with compounds at three concentrations (2, 10,
and 25 M). After incubation, the activity of F508del-CFTR in the
plasma membrane was determined by measuring the rate of HS-
YFP quenching caused by iodide influx. Activity after incubation
with AATs was compared to that of cells treated with vehicle alone
m
(DMSO) or with the known corrector VX-809 (1 mM). All AATs were
also tested in combination with VX-809 to assess possible additive
effects. Fig. 2 shows data obtained for AATs at 10
informative concentration.
mM as the most
Nearly 120 compounds were synthesized (Tables 1e3) to
investigate the structure-activity relationships of AATs as correctors
of F508del-CFTR. CFBE41o-cells with expression of mutant CFTR
and the halide-sensitive yellow fluorescent protein (HS-YFP) were
2.1. Substitutions at the position 4 of the thiazole ring
The phenyl ring at the C-4 of the 2-aminothiazole is crucial for
activity, as its removal 1a (data, not shown) or substitution with
Table 1
Molecular structures of compounds 1c-o, 2a-f, 3a-c, 4a-l, 5a-c, 6a-f, 8a-b, 19a, 20a-l.
Compound
R
Compound
R
Compound
R
1c
1d
1e
1f
1g
1h
1i
-H
3a
3b
3c
4a
4b
4c
4d
2 eOCH3
2 eCH3
2 -Cl
3 -Cl
3 eSCH3
3 eF
6a
6b
6c
6d
6e
6f
3 eBr; 4 eF
2 eOH; 5 -Br
3 eNO2; 4 -Cl
3 eCH3; 4 -Cl
2,4 -Cl
4 eN(C2H5)2
4 eC5H11
4 eOCH3
4 eOC3H7
4 eSO2CH3
4 eNO2
2,6 -Cl; 3 eF
3 eCF3
8a
1j
4 eCN
4e
3 eI
8b
1k
1l
4 eC2H4Cl
4 eCF3
4 eCH2CF3
4 -Cl
4 eF
2,4 eCH3
2,4 eOCH3
3,4 eOH
3,4,5 eOCH2COOH
3,5 eC(CH3)3; 4 eOH
3,4,5 eOCOCH3
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
3 eNO2
3 eOH
3 eCH3
3 eNHCOCH3
3 eOCH2COOC(CH3)3
3 eOCH2COOH
3 -Br
3 eOH; 5 eCONH2
3,5 eCF3
19a
20a
20b
20c
20d
20e
20f
20g
20h
20i
3 eNO2; 4 -Br
2 eOH; 5 -Cl
2,3 -Cl
2,5 -Cl
3 -Cl; 4 eOCH3
3,4 -Cl
3 -Cl; 4 eF
3 -Cl; 5 eCF3
3 -Cl; 5 eF
3 -Cl; 5 eOCF3
3,5 -Cl; 2 eOH
3,5 -Cl; 4 eOH
3,5 -Cl; 2 eF
1m
1n
1o
2a
2b
2c
2d
2e
2f
2,5 eOCH3
20j
20k
20l

18
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
Table 2
Molecular structures of compounds 1a-b, 7a-h, 9a-g, 10a-o, 12a-b, 21a-c.
Compound
R
Compound
R
Compound
R
1a
-H
9a
10e
1b
7a
-CH3
9b
9c
10f
10g
7b
7c
9d
9e
10h
10i
7d
9f
10j
7e
7f
9g
10k
10l
10a
7g
7h
10b
10c
10m
10n
10d
12a
10o
21a
12b
21b
21c
methyl group (1b) led to loss of activity (Table 2 and Fig. 2).
Furthermore, removal of the bromine present in the hit compound
EN277I led to a decrease in activity with respect to the substituted
analogs (see 1c vs. EN277I). A number of different electron-
donating and electron-withdrawing groups were attached at
various position of the phenyl ring keeping intact, for comparative
purpose, the meta S-methyl substitution at the aniline ring. To
understand the importance of the various positions on the phenyl

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
Table 3 (continued )
19
Table 3
Molecular structures of compounds 13a, 14a-e, 15a-b, 16a-d, 17a-c, 18a-e.1
Compound
R
R1
R2
-H
R3
18a
4 -Cl
18b
18c
18d
4 -Br -CH3
-H
-H
-H
-H
-H
-CH3
Compound
R
R1
R2
R3
13a
14a
4 -Br -H
3 -Cl -H
-H
-H
- H
18e
3 -Cl -CH3
-H
14b
14c
14d
14e
15a
15b
16a
3 -Cl -H
3 -Cl -H
3 -Cl -H
3 -Cl -H
3 -Cl -H
3 -Cl -H
4 -Br -H
-H
-H
ring, different thiazoles were built with different substituents on
the phenyl ring (Table 1).
All electron-donating groups at the para position of the phenyl
ring: alkyl amino (1d), alkyl (1e), alkyloxy (1f, 1g), led to AAT with
no significant activity.
-H
To understand if the para position is important for F508del-CFTR
rescue, we introduced electron-withdrawing groups such as sul-
fonylmethane (1h), nitro and cyano groups (1i, 1j), halopropyl (1k),
and trifluoroalkyl (1l, 1m) but such compounds were inactive or
actually caused a decrease in activity (particularly for 1h) which
could reflect a specific inhibition of F508del-CFTR trafficking or a
more general cell toxicity effect.
As the hit compound contains bromine in para position, we
decided to insert in the same position chlorine (1n) or fluorine (1o).
Surprisingly, this type of replacement in the para position of the
phenyl ring led to a decrease in activity compared to the para-
bromine analog (see Fig. 2). These results are consistent with the
notion that at this position the activity is somehow dependent on
the size of the substituent (bromine is bigger than fluorine) and its
lipophilicity (bromine is more lipophilic than chlorine). Impor-
tantly, addition of further substituents beyond the one in para (2a-
2f) did not evoke any improvement in activity (Fig. 2).
We also investigated the substitutions of the phenyl ring posi-
tions different than para. First, we synthesized a series of de-
rivatives with substituent in the ortho position. The resulting
compounds bearing a 2-methoxy group (3a), a chlorine (3b) or 2-
methyl substitution (3c), did not show any activity.
Importantly, moving the chlorine group from the para (1n) or
the ortho (3c) position to the meta position (4a) led to a significant
increase in activity (Fig. 2). As shown in subsequent sections of the
study, the 4a compound is one of the AATs with the most inter-
esting pharmacological profile in terms of rescue of F508del-CFTR
function and showing improvements with respect to the original
AAT EN277I.
-H
-CH3
-H
16b
16c
16d
4 -Br -H
4 -Br -H
4 -Br -H
-H
-H
-H
-H
17a
17b
17c
4 -Br -H
4 -Br -H
4 -Br -H
We investigated the effect of meta-substitution with an
electron-withdrawing group such as SCH3 (4b), fluorine (4c), CF3
(4d), iodine (4e), and nitro group (4f) or with an electron-donating
group such as hydroxyl (4g), methyl (4h) amidic (4i), and different
oxyacetic group (4j, 4k) but without improvements. Only with
compound 4l containing bromine a weak activity was present.
Introduction of a second substituent in meta (5a and 5b) is also
without effect on activity. Quite particular is the derivative 5c,
where it is somehow surprising the capability of the two methoxy
groups of increasing the efficacy of the molecule if compared to the
-H

20
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
Fig. 2. Activity of AATs as correctors. The bar graphs report F508del-CFTR activity in CFBE41o-cells after treatment with the indicated compounds (10 mM, 24 h). Activity was
measured with the HS-YFP assay. The dotted line indicates the level of activity in cells treated with vehicle alone.
compound 2b which contains the same substituents but in
different places of the ring.
when the second group is a halogen (F), as in the case 6a, we found
a significant activity. In contrast, the presence of a polar group, as in
the derivative 6b was detrimental. It is reasonable to assume that
the introduction of an additional fluorine atom in the molecule
leads to an increase in hydrophobicity with a small bulkiness, a
characteristic likely to be important for activity.
We also found that, when a halogen like chlorine is in the para
position (see 6c, 6d, and 6e) no activity was present independently
of the characteristic of the second substituent (Fig. 2). Moreover,
the activity disappeared completely in the case of three halogens in
the phenyl ring (6f), possibly because of the high steric hindrance.
According to our results, we might assume that, at the meta
We also investigated some compounds with a common feature
like the presence of halogen groups in the ring (Table 1). As
demonstrated with the most effective compounds 4a and EN277I,
the presence of halogens seems fundamental to obtain activity. The
electronic effects of the halogens are ascribed to their inductive
electron attractive properties. Such properties are maximal for
chlorine and bromine, less marked for iodine, and very weak for
fluorine.
To further investigate this issue we tested two derivatives con-
taining bromine in meta position and a second substituent. Only

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
21
position of the phenyl ring, slightly more apolar substituents
guarantee a more favorable interaction with the target binding site,
possibly due to the presence of hydrophobic amino acid residues in
the close proximity. In fact, our docking simulations (see next
sections), suggest that these compounds face P574 and G576 side
chains. In conclusion, the phenyl ring at the C-4 of the 2-
aminothiazole is important for the activity, and a chlorine substit-
uent at the meta position of this ring confers the best result (4a).
Regarding this portion of the molecule, small lipophilic sub-
stituents like bromine are relatively well tolerated, especially in the
meta positions (see 6a), whereas in the ortho position every sub-
stituents led to loss of activity. Moreover, only with bromine and
not with chlorine or fluorine in the para position a fair amount of
activity is achieved.
Our investigation was also directed to assess whether a less
flexible structure could give us more information about the active
conformation at the target binding site, so we synthesized a small
set of derivatives in which the phenyl ring is substituted with a
more hindered group (Table 2). In particular, the phenyl ring was
fused to different structures containing heteroatoms like thiophene
(7a), imidazole (7b), thiazine (7c), oxazole (7d), dioxane (7e),
dioxolane (7f), dioxepane (7g) or devoid of heteroatoms like
cyclohexane (7h).
the phenyl and thiazole-rings by creating other types of derivatives.
In particular, we synthesized compound containing 6-
bromobenzothiazole linked to the methyl thioaniline portion
similarly to EN277I (Table 4). This benzothiazole, named 11a was
devoid of activity.
a
We also speculated if an enhanced activity could be achieved
with the presence of an amidic linker between the thiazole core
and the phenyl ring in position 4. Based upon this assumption, we
decided to separate the thiazolic ring from the substituent in po-
sition 4 through introduction of an amide linkage (Table 2). How-
ever, this type of modification, i.e. insertion of an amide function
between the two rings (12a), produced an inactive compound
(Fig. 2). As the consequent introduction of an additional hydrogen
bond donor was judged undesirable with respect to corrector
properties, the amide-linked analogues like 12a were not further
pursued. With respect to the same rings, we synthesized a deriv-
ative called 12b, with a methylene linker between 4'bromophenyl
and thiazole portions that can be considered the elongated
analogue of EN277I. This modification was not well tolerated, since
the activity decreased significantly. Overall, none of these mole-
cules showed activity as correctors on CFBE41o-cells, suggesting
that proper spacing of the above cited rings is important for
corrector activity, and confirming that the thiazole ring has to be
directly bound to the phenyl moiety.
With the exception of 7a, which showed a modest activity, all
other compounds were inactive (Fig. 2). To confirm these results,
we built two compounds in which two sterically hindered sub-
stituents like thiophene (8a) and pyrrolidine (8b) were introduced
in the para position of the phenyl ring (Table 1). In agreement with
the previous observations, a decrease in activity was observed.
To complete the studies on the position C4 of the thiazole ring,
we synthesized a panel of compounds containing one or two het-
erocyclic rings, replacing the 4-phenyl portion of the thiazole core
with the introduction of the new substituents (Table 2). Saturated
or unsaturated heterocycles at five or six rings were linked to the 4
position of the 2-aminothiazolyl derivative in order to explore the
potential activity of these thiazoles with bulky groups. Accordingly,
we synthesized molecules having a single ring, like pyridine (9a,
9b) furan (9c), thiophene (9d, 9e) and thiazole (9f, 9g), or de-
rivatives with more rings like isoxazole substituted (10a-10c),
thiazolic derivatives (10d-10f), imidazoles (10g, 10h), pyrrole (10i),
triazole (10j), thiophene derivatives (10k), or more hindered like
tetraisoquinoline (10l), isoquinoline (10m), chroman (10n), and
benzofurane (10o).
In conclusion: a) the phenyl ring at the position C4 is important
for the activity and halogen group to the para (bromine) or meta
position (chlorine) confers the best activity b) no bulky groups
(like heteroatomic rings) excluding thiophene are tolerated at the
same position c) phenyl ring must be directly linked to the thia-
zole one.
2.2. Substitutions at the position 2 of the thiazole ring
Our previous data demonstrated that 4-(4'bromophenyl) thia-
zol-2-amine (13a) does not show any activity as corrector (Fig. 2). In
addition, we demonstrated that the presence of different pyridine
derivatives on portion 2 decreases the activity with respect to the
compounds containing the 3-methylthioaniline portion [10].
As 4a is the best compound detected in the first part of our
study, we decided to investigate if its activity could be increased by
modifications in the 3-methylthioaniline portion. The second
round of modifications aimed at improving the activity was made
at the aniline site (Table 3). Therefore, keeping intact the 4-(3-
chlorophenyl) thiazol-2-amine scaffold, small functional groups
were attached at different positions on the ring of aniline portion.
We reasoned that these substituents could affect the hydrogen-
bond donor/acceptor ability of the amino moiety, thereby
improving/decreasing the AATs anchoring at the binding site.
We first modified the 3-methylthioaniline portion by elongation
of the alkyl chain or by removing the methyl group on sulfur atom.
Replacing the methyl group with the ethyl or propyl moiety (14c
and 14b) did not generate activity. Similar negative results were
found for 14a in which the SH group is free. We also synthesized
and tested 14d, containing an S-CF3 group instead of SeCH3. This
product did not show activity, maybe because the higher lip-
ophilicity of the CF3 group with respect to CH3 reduces the inter-
action with the target.
Among them, only 9d showed an effect comparable to that of 4a.
This compound is characterized by a thiophene ring linked to the
thiazole residue, the small size of this molecule (if compared to the
inactive ones) could possibly help the interaction of 9d with the
target.
We examined the importance of the type of connection between
Table 4
Molecular structures of compounds 11a, 17d.
Compound
11a
We investigated the importance of a second substitution at the
aniline ring in ortho positions, with an electron-donating group to
improve the lipophilicity. The compound 14e contains an ethyl
group near the methylthio group. This compound was less effective
than 4a. Our data suggest that the 3-methylthioaniline may be the
best substituent at position 2 of thiazolic ring.
17d
As it is known that often the polar portions of the molecule like
the amino group permit the most specific interactions, we wanted

22
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
to explore whether the hydrogen of the 2-aminothiazole was
important for the activity. The N-methyl derivative of 4a, namely
15a, was not equally effective, thereby indicating that the capability
of being a hydrogen-bond donor is crucial for the activity. This
prompted us to hypothesize that no degree of hindrance was
tolerated in regard to the substitution at this position. Indeed,
introduction of slightly larger groups, such as the benzyl group
(15b) failed to yield active compounds (Fig. 2). Based upon this
assumption, we decided to introduce an amide linkage in the
aminothiazole where in the carbonyl would provide a hydrogen
bond acceptor in place of the 2-aniline moiety. We therefore syn-
thesized a small series of N-2 acyl set of derivatives in which the
amine portion of aniline derivatives is substituted by an amide
group so the polar character of aniline disappears and the new
bond is more rigid while the 4-(4-bromophenyl) thiazole portion is
unchanged.
Four N-acyl substituted aminothiazoles (16a-16d) were syn-
thesized with pyridinyl and phenyl as acyl groups as shown in
Table 3. All synthesized compounds showed no F508del-CFTR
correction (Fig. 2). Regarding the same substituents of EN277I, we
synthesized a derivative called 17a with a methylene linker be-
tween amine group and thiazole portions (Table 3). This linkage
was not well tolerated because the activity decreased dramatically.
Similar negative results were found for 17b in which the aminic
group is totally substituted.
in Table 4) had negligible activity.
These results provide further insights about the interaction
between active AATs and the target: a) aniline must be positioned
at a suitable distance with respect to the thiazole; b) the 2-amino
group of the thiazole cannot be substituted with an amidic
linker; c) no degree of hindrance tolerated in regard to the aminic
group of aniline. Apparently, a certain degree of conformational
freedom of the aromatic ring attached at the 2-amino moiety is
required to confer the desired activity.
2.3. Substitution at the position 5 of the thiazole ring
We finally wanted to investigate whether substitutions at the C-
5 of the 2-aminothiazole were tolerated, and, if so, which charac-
teristics should these substituents possess (Table 3). In fact, the
presence of a bulky substituent at the C-5 might be useful in terms
of improving some pharmacokinetic characteristics. A small group
such as the methyl did not improve the activity compared to the
counterpart (see 18a vs. 1n or 18c vs. 1c) Activity was also absent
when a p-methyl phenyl or benzoyl group was introduced (see 18d
and 18b). Actually, incubation with 18d decreased basal activity
below the value measured in cells treated with vehicle alone
(Fig. 2).
No activity was also found when a methyl group was introduced
at the C5 of the compound 4a as in the molecule (18e). Assessment
of the collocation of these data within the SAR is difficult, but we
might speculate that the phenyl ring, by virtue of its low flexibility,
cannot accommodate into the target binding site in a favorable
manner if the position 5 is taken by a bulky group. In particular,
substitutions at the C-5 position of the 2-aminothiazole cannot be
tolerated thus suggesting that the thiazole ring plays a pivotal role
The derivative 17c does not contain an aromatic amine like
aniline but an aliphatic ring like cyclohexylamine conjugated with
the same scaffold of EN277I. No activity was denoted demon-
strating that the presence of aniline ring is crucial for activity.
Furthermore, we also investigated if the amino moiety could be
embodied into a cycloaliphatic ring. The piperidine derivative (17d
Fig. 3. The position of the two pockets (pocket 1 in red and pocket 2 in blue) on NBD1 as possible binding sites for correctors/potentiators. The amino acid residues that form the
two pockets are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
23
Fig. 5. Potential binding mode of 9d in the pocket 1 of NBD1. C atoms are coloured in
orange, while the residues involved in interactions with the compound are reported
and coloured by the default atom type colour. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Potential binding mode of EN277I, the lead compound, and 4a to pocket 1 in
NBD1. C atoms are coloured in violet and in green respectively, while the residues
involved in interactions with the compounds are reported and coloured by the default
atom type colour. The H-bonds between the ligand and the amino acids are shown in
orange. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
is AAT activity and a modification in that position could cause a
steric hindrance impairing the binding to the target.
2.4. Further investigation on 4a and 9d
With the screening of the first part of synthesized AATs, we
found that the most promising molecules, other than hit compound
EN277I, were 4a and, to a lesser extent, 9d. In these two com-
pounds, it seems that halogen atoms are important for AAT effect,
both in meta and in para positions. Therefore, we decided to first
Fig. 6. Increased F508del-CFTR rescue obtained with corrector combination. Graphs
show F508del-CFTR activity (HS-YFP assay) after treatment of CFBE41o-cells with
EN277I (A) or 4a (B) at different concentrations, in the presence and absence of VX-809
(1
mM). Dotted and dashed lines indicate the level of activity measured with vehicle
(DMSO) or VX-809 alone.

24
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
Table 5
We subsequently considered possible derivatives of 9d. In order
to find if further substitutions of thiophene portion could amelio-
rate its affinity, we synthesized a series of different thiophenes: 21a
and 21b (Table 1). These compounds contain an alkyl chain (butyl
and methyl) in position 3 of the heteroatomic ring with the goal of
increasing the hydrophobic character of the moiety. This type of
modification did not increase the activity. The same position was
investigated with the 21c structure, using a chlorine atom. This
modification caused a loss of effect.
Such data are not conflicting with the previous information
regarding possible substituents in another position: 10k carried a
pyridine ring linked to the thiophene (Table 2) and its lack of ac-
tivity seems to confirm that a small group is needed to avoid loss of
potency.
Potency of active AATs as correctors and/or potentiators.
Corrector
Potentiator
1j
1o
4a
6a
7d
7e
9d
9f
10a
10d
14d
20e
20g
21c
inactive
inactive
5.3
n.d. (*)
inactive
inactive
8.5
inactive
inactive
5.7
inactive
14
34
27
53
inactive
6.4
71
inactive
9.5
58
inactive
32
inactive
inactive
15
n.d. (*)
inactive
Moreover, since preliminary results had confirmed that a
modest activity is obtained when the thiophene ring is linked to the
thiazole scaffold in a different position (see 9d vs. 9e), no other
portion of the thiophene ring was further evaluated.
The Table shows the EC50 values (in mM) obtained by fitting the dose-response of
AATs tested as correctors and potentiators with the HS-YFP functional assay
(CFBE41o-cells with F508del-CFTR). (*) not determined because the compound
showed activity only at one concentration (10 mM).
2.5. Molecular modeling studies
insert a second substituent on the bromophenyl part. Unfortu-
nately, the introduction of a nitro group in ortho position (19a,
Table 1) decreased the corrector activity in comparison to the
progenitor. We then explored the capacity of other substituents to
improve the activity of 4a, keeping intact, for comparative pur-
poses, the meta chlorine in the phenyl ring (Table 1). We first chose
the ortho position by synthesizing a compound (20a) containing a
hydroxyl group. This compound showed a weak activity. Next, at
the same positions, we investigated the effect of the substitution
with an electron-withdrawing group such as chlorine. A chlorine
substituent simultaneously produces an increase in lipophilicity
and an electron-attracting effect. Two different compounds named
20b and 20c with a second chlorine in ortho were synthesized. Such
compounds were inactive.
Then, we investigated the role of a substituent in para. As
evident in the compound 20d, the presence of a methoxy group
abolished the activity while with a second chlorine (20e) a signif-
icant activity was observed although lower than that of the 4a
molecule. Interestingly, the introduction of a fluorine atom as in the
case of 20f in place of chlorine in the para position of the ring led to
a decrease in activity compared to 4a.
The last position to explore for the di-substitution was the meta
one. Three different derivatives were tested. Compound 20h, con-
taining the fluorine atom in meta, was devoid of corrector activity.
As the introduction of second chlorine in meta is quite difficult
because halogen is ortho/para directing, we used the CF3 group that
is comparable in size to chlorine and can replace it when it is placed
in an activated position. The resulting compound 20g was active,
comparable to EN277I, but weaker than 4a. Finally, we investigated
the di-substitution at the phenyl ring in the same position with an
electron-donating group, such as OCF3. The compound named 20i
shows no activity as corrector.
Starting from the hypothesis proposed by Wieczorek and coll
[25]. that VX-809 activity may derive from its binding to CFTR,
specifically to nucleotide binding domains, we performed docking
calculations on the newly synthetized AATs using NBD1 (PDB codes
2PZG and 2PZF, for wild type and mutated protein respectively).
In a first step, the nucleotide binding domains were explored
by means of CastP [26] in order to identify and measure all
surface-accessible pockets as well as interior inaccessible cavities
and thus define all differences which could infer on the ligand
interaction. As it is well known, the wild type NBD1 displays a
different pockets pattern in comparison with the F508del-
protein.
In a second step, a blind docking procedure was applied to
explore the molecular interactions between the newly synthesized
compounds plus VX809, selected as template, and wild type NBD1,
and F508del-NBD1. Results suggested F508del-NBD1 to be the
putative target for these ligands due to the lower energies of
complexes with the mutated protein. Further docking simulations
using Autodock [27] were performed to fully check and validate the
different binding poses that the ligands could assume in complex
with F508del-NBD1, depending on the different moieties/groups
decorating the aminoarylthiazole scaffold. As already published by
Wieczorek and coll. [25], compounds targeting F508del-NBD1
could potentially dock inside three main cavities: one where the
nucleotide binds, plus two pockets, much more flexible in the
mutated protein than in the wild type, named pocket 1 (P1) and
pocket 2 (P2) (Fig. 3).
In agreement with these studies, for all the new AATs com-
pounds, our calculations suggested pocket 1 as principal binding
site on NBD1, while pocket 2 was highlighted as the most probable
site for VX809. The amino acid residues composing pocket 1 are
mainly G458-A462, K464, C491eS495, S549-A554, S557, D572-
L578, E583, V603, S605, S654-E656, R658-S660, L662, and T663.
All the newly synthesized AATs display a common pattern of
interaction involving some of these residues. In particular, each
AAT, according to our calculations, makes one or more hydrogen
bonds with pocket 1 involving S573 and/or R553 and/or F494, plus
In the attempt to identify the more favorable pattern on the ring
maintaining the 3,
5 dichloro substitution (metaemeta di-
substitutions), we tested two derivatives (20j and 20k) containing
as third substituent a polar group (OH). These compounds were
inactive (20j) or weakly active (20k). Another derivative containing
fluorine in ortho (20l), with two chlorines in meta position was not
active in agreement with data obtained with the polyhalogenate
derivative 6f.
A plausible explanation of this result might rely on the steric
hindrance produced by the halogen atoms on the 4-aryl-thiazole,
preventing it from establishing interactions with the surrounding
amino acids at the target binding site.
an additional
pep interaction with the side chain of F494. Addi-
tional polar or, more frequently, van der Waals interactions are
performed by AATs depending on the substitutions on their scaffold
(Figs. 4 and 5).
Interestingly, the three main residues involved in the AATs
binding, have already been described in the literature as key points
in the protein, and mutations on these positions showed to be able

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
25
Fig. 7. Rescue of F508del-CFTR activity in whole-cell patch-clamp recordings. (A) Representative membrane currents recorded in four cells treated for 24 h with vehicle, VX-809
(1 M), 4a (10 M) plus genistein (50 M), and after inhibition with inh-
M), or 4a þ VX-809. Currents were recorded under resting conditions, after stimulation with forskolin (20
172 (10
M). (B) Summary of currents recorded at þ100 mV in cells treated with indicated compounds.
m
m
m
m
m
to restore the correct folding and activity of F508-del CFTR in vitro
[28e30].
estimated half effective concentration was 5.3 mM (Table 5) and the
maximal effect was equivalent to a nearly two-fold increase
compared to vehicle-treated cells and to 54% of effect of VX-809
Thus, the binding of the AAT, which occurs along the small su-
perficial cavity created by the different rearrangement of NBD1 side
chains caused by the F508 deletion, could be able to correct the
altered shape of the biomolecule, somehow contributing to the
CFTR rescue.
(1 mM), a known corrector that is currently tested in clinical trials.
In comparison, the EN277I was markedly less effective (Fig. 6).
Interestingly, incubation of cell with 4a and VX-809 together
generated a marked synergistic effect, with a five-fold increase in
activity relative to vehicle-treated cells. Under this condition (i.e.
combination with VX-809), EN277I appeared less effective than 4a.
Importantly, activity of 4a, as well as that of EN277I and VX-809,
was totally abolished when the functional assay was performed
in the presence of the CFTR inhibitor 172 (not shown). This result
indicates that AATs increase halide transport by improving the
2.6. Analysis of 4a biological activity
We tested the 4a compound at different concentrations, with
and without VX-809. As shown in Fig. 6, incubation of cells with 4a
generated a dose-dependent increase in halide transport. The

26
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
previous study the EN277I compound required several hours to
fully show activity [10]. Therefore, all AATs were tested after a
30 min incubation (following rescue of F508del-CFTR by low tem-
perature for 24 h). We found that 4a was effective in the potentiator
assay, but its activity was only a small percentage (nearly 25%) of
that of classic potentiators such as genistein and VX-770 (Fig. 9).
AATs with corrector activity, such as 9d, were also modestly
effective as potentiators. Intriguingly, other AATs (7d, 10a, 14d), not
detected as correctors, were markedly active in the potentiator
assay. In particular, the compound 7d showed a maximal effect
comparable to that of genistein and VX-770 and an interesting af-
finity in the low micromolar range (Table 5).
To further confirm the potentiator activity of some AATs, we
tested them on FRT cells expressing G551D-CFTR, the mutant that
displays a severe reduction in gating but not a trafficking defect
(Fig. 9). Interestingly, compound 7d was markedly effective also on
G551D, with a dose-response relationship significantly shifted to
higher concentrations compared to that determined for F508del.
Other compounds, including 4a, 10a, and 14d were instead partially
effective.
3. Conclusion
Fig. 8. Detection of F508del-CFTR protein by western blot. (A) Representative western
blot experiment showing detection of F508del-CFTR in lysates of CFBE41o-cells treated
There is an urgent need to find novel pharmacological correctors
to target F508del, the most frequent mutation among CF patients.
Present correctors, such as VX-809, only elicit a partial rescue of the
mutant protein. It is probable that combination of correctors having
different mechanism of action results in a more marked effect
[31,32]. Further stimulation of F508del-CFTR activity may be ob-
tained by using a potentiator, such as VX-770, that further increases
anion transport by facilitating CFTR channel gating. Our study
started from the observation that EN277I, an aminoarylthiazole,
has an interesting dual activity as corrector and potentiator
(although with modest efficacy and potency). Therefore, we further
investigated this class of molecules by synthesizing novel AAT an-
alogues. The computational studies highlighted for all compounds a
possible common binding mode based on the interaction with
three main residues R553, S573 and F494 located in pocket 1 of
NBD1, and reinforced the hypothesis of an interaction with this
portion of CFTR as site of action for correctors. Long molecular
dynamic simulations, so as to fully evaluate the binding strength of
some selected AATs inside pocket 1, together with some site-
directed mutagenesis experiments will be the further prosecution
of this research.
Interestingly, we found that some AATs have a marked effect as
potentiators, with 7d being the most potent and effective.
Intriguingly, this compound, as well as other strong potentiators
(10a, 14d), were ineffective as correctors. These findings could be
explained with two separate and independent mechanisms of ac-
tion (and possibly binding sites) responsible for improved pro-
cessing and gating by AATs. Some compounds, such as 4a, could
coincidentally show both activities by having a structure compat-
ible with both mechanisms. An alternative hypothesis is that dual
activity by AATs is actually mediated by binding to the same CFTR
site but with a different type of interactions. In particular, it is
possible that maximal efficacy of an AAT as a potentiator implies a
type of interaction and, consequently, of NBD1 conformation that is
detrimental for F508del-CFTR processing. In fact, recent studies
indicate that strongly effective potentiators, such as VX-770, impair
F508del-CFTR stability [33,34]. This mechanism could explain why
it is not possible to obtain dual active AATs with strong potentiator
activity. In this respect, it should be noted that compound 7d, the
strongest potentiator identified in our study, appears to markedly
reduce the trafficking of F508del-CFTR in the corrector assay (Figs. 2
and 9). This issue needs further investigation to assess the
with VX-809 and/or AAT-4a. Arrowheads show the position of bands
B and C.
Expression of Naþ/Kþ-ATPase in the same samples is shown as control. (B) Summary of
all western blot experiments reported as the ratio of the band C intensity to the sum of
band C and band B. Each bar is the mean SEM of 5 separate experiments. *, p < 0.05.
trafficking of F508del-CFTR to the plasma membrane and not by
enhancing the expression and function of another anion channel or
transporter.
To further investigate the effect of 4a, we carried out whole-
cell patch-clamp experiments. After treatment with 4a for 24 h,
cells showed a six-fold increase in the size of cAMP-dependent
currents at all applied membrane voltages in the ꢁ100
to þ100 mV range (Fig. 7). Importantly, these currents have the
typical characteristics associated to CFTR activity, i.e. linear cur-
rentevoltage relationship and lack of voltage-dependent re-
laxations. Furthermore, the currents were abolished by CFTRinh
-
172. As expected, treatment with VX-809 was also effective.
Combined treatment with 4a and VX-809 resulted in additive
effect (Fig. 7).
We also investigated the correcting ability of compound 4a by
analyzing the electrophoretic mobility of F508del-CFTR protein.
Usually, the mutant protein shows a fast mobility due to partial
glycosylation (band B). Following treatment with correctors, the
improved protein trafficking from the endoplasmic reticulum to the
Golgi results in full glycosylation and slower mobility (band C).
Fig. 8 shows that treatment CFBE41o-cells with VX-809 causes, as
expected, an increase in band C intensity relative to band B.
Treatment with AAT-4a alone does not increase band C, possibly
because the method is not sensitive enough as the functional assay.
However, combined treatment with VX-809 and AAT-4a results in a
marked increase of the fully glycosylated F508del-CFTR relative to
cells treated with VX-809 alone (Fig. 8).
2.7. Effect of AATs as potentiators
In a previous study, we found that some AATs have a dual ac-
tivity, as F508del correctors and as potentiator-like molecules.
Interestingly, we found that a significant number of the new AATs
synthesized in our study were also able to stimulate the activity of
F508del-CFTR. Actually, we found that these new compounds are
maximally active even with short-term incubations, whereas in our

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
27
Fig. 9. Activity of AATs as CFTR potentiators. (A, B) Bar graphs show activity of selected AATs in the potentiator (A) and corrector (B) assay. (C) Dose-response relationships of 4a, 7d,
10a, and 14d as potentiators on cells expressing F508del-CFTR or G551D.
possibility to generate compounds with the best combination of
corrector and potentiator activity for mutant CFTR rescue.
The nuclear magnetic resonance (NMR) spectrometer was a
Varian Mercury 300 Plus.
The proton spectra were acquired at 300 MHz while carbon
spectra were acquired at 75 MHz, at room temperature. Chemical
4. Experimental section
shifts are reported in
d units (ppm) relative to TMS as an internal
standard. Coupling constants (J) are reported in Hertz (Hz).
All the raw powders obtained were purified with preparative
HPLC using the following gradient: from 0 to 5 min at 20% eluent B,
then from 5 min to 40 min to 100% eluent B, from 40 to 45 min at
100% eluent B. Eluent A was water þ0.1% formic acid (FOA) and
eluent B was acetonitrile þ 0.1% FOA. All analogues submitted for
testing were judged to be of 95% or higher purity based on
analytical HPLC/MS analysis.
4.1. Chemistry
Reagents and solvents were purchased from Sigma Aldrich, Alfa
Aesar and VWR and used as received unless otherwise indicated.
Solvent removal was accomplished with a rotary evaporator at
ca.10e50 Torr.
The analytical instrument used was an Agilent 1100 high per-
formance liquid chromatograph (HPLC). The analytical HPLC col-
umn was a Waters
preparative HPLC was a Shimadzu SCL-10A and the column used for
preparative chromatography was Phenomenex C18 Luna
Compound purity was determined by integrating peak areas of
the liquid chromatogram, monitored at 254 nm.
mBondapack C18, 3.9 id x 300 mm length. The
a
21.2 ꢂ 250 mm length. The analysis of the intermediates and the
raw products was performed by liquid chromatography-
electrospray mass spectrometry (HPLCeESIeMS) using an Agilent
1100 series LC/MSD ion trap instrument.
4.1.1. General procedure for synthesis of thioureas (a-f)
A solution of benzoyl isothiocyanate (1 eq) in anhydrous tetra-
hydrofuran (THF) was added dropwise to a stirred solution of the
aniline or other amine building block (1 eq) in anhydrous THF

28
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
(1 ml) at room temperature overnight. When the reaction was
completed (controlled with HPLC-MS) the mixture was concen-
trated under vacuum and dissolved in ice cold water (2 ml). After
30 min of stirring, a white pellet appeared. The reaction was
centrifuged at 4000 g for 5 min and the pellet was washed with
water (3 ꢂ 2 ml). Then, the pellet was resuspended in a solution of
ethanol (EtOH) and 1M NaOH 1:1 (2 ml). The reaction was heated to
reflux until the hydrolysis was complete (controlled with HPLC-
MS). After cooling, the reaction mixture was poured into water-
ice (2 ml) and aqueous 1N HCl was added to produce a neutral
solution (pH 6.9e7). The thiourea intermediate typically pre-
cipitates from the neutral solution, and was collected by filtration
and dried. This two-step procedure provided thiourea in-
termediates in 80e95% overall yield, with purities generally>90%
as determined by HPLC-MS.
4.1.5. General procedure for the synthesis of aminoarylthiazole
analogues (Hantzsch synthesis)
Haloketones (1 eq) were conjugated with thioureas (1 eq) in
anhydrous EtOH (1 ml). Reaction was stirred at reflux from 2 to 24 h
until the reaction was judged complete (HPLC-MS). The mixture
was then extracted with ethyl acetate (3 ꢂ 3 ml). The organic
phases were concentrated in vacuum and lyophilized. Products
were verified by HPLC and MS and purified with preparative HPLC.
Relevant fractions were collected and concentrated to afford the
desired product in 40e95% yields, with purity of >95% as deter-
mined by HPLC-MS.
4.1.5.1. 4-(4-bromophenyl)-N-(3-(methylthio) phenyl) thiazol-2-
amine (EN277I). 3-methylthioaniline (12
ml, 0.1 mmol) was dis-
solved in anhydrous THF (500 l) at room temp, and 60% dispersion
m
in mineral oil of NaH (14 mg, 0.33 mmol) was added. When the
bubbling stopped, 2-chloro-4-(4-bromophenyl) thiazole (27 mg,
0.1 mmol) in anhydrous THF (1 ml) was added, and the reaction was
heated to reflux for 24 h. The mixture was then extracted with
diethyl ether (3 ꢂ 2 ml) and washed with water (2 ꢂ 1 ml). The
organic phase was dried under vacuum. The product was purified
by preparative HPLC to afford the title compound.
4.1.2. Synthesis of 1-(3-(methylthio) phenyl) thiourea (a)
A solution of benzoyl isothiocyanate (673
drous THF (2 ml) was added dropwise to a solution of 3-
methylthioaniline (616 l, 5 mmol) in anhydrous THF (2 ml) at
ml, 5 mmol) in anhy-
m
room temperature overnight. When the reaction was completed,
the mixture was concentrated under vacuum and dissolved in ice
cold water. After 30 min of stirring, a white pellet appeared. The
reaction was centrifuged at 4000 g for 5 min and the pellet was
washed with water (3 ꢂ 2 ml). Then the pellet was then resus-
pended in a solution of EtOH and 1M NaOH 1:1 (5 ml). The reaction
was heated to reflux until the hydrolysis was complete (controlled
with HPLC-MS). The mixture was concentrated under vacuum and
a solution of aqueous 1N HCl was added to produce a neutral so-
lution. The reaction mixture was centrifuged at 4000 g for 5 min
and the pellet was washed with water (3 ꢂ 1 ml). The compound
was completely dried and lyophilized to obtain the title compound
as powder and used without further purification.
(24 mg, 65%)
1H NMR (300 MHz, CDCl3):
d
7.73 (dd, J ¼ 7.7, 1.5 Hz, 2H), 7.66
(dd, J ¼ 7.5, 1.5 Hz, 2H), 7.40 (t, J ¼ 2.0 Hz, 1H), 7.24 (t, J ¼ 7.7 Hz, 1H),
7.07 (ddd, J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.95 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H),
6.86 (s, 1H), 2.52 (s, 3H).
13C NMR (75 MHz, CDCl3):
d 160.1, 150.1, 142.6, 137.2, 134, 132.1,
130.5, 128.3, 128.1, 125.2, 123.1, 119.3, 114.5, 113.2, 105.1, 14.9.
ESI-MS m/z ¼ 378.9 [MþH]þ.
4.1.5.2. N-(3-(methylthio)
3-methylthioaniline (12
THF (500 l) at room temp, and 60% dispersion in mineral oil of NaH
phenyl)
thiazol-2-amine
(1a).
ml, 0.1 mmol) was dissolved in anhydrous
m
(880 mg, 88%)
1H NMR (300 MHz, CDCl3)
d 9.65 (s, NH), 7.52 (s, NH2), 7.22 (t,
(14 mg, 0.33 mmol) was added. When the bubbling stopped, 2-
bromothiazole (16.5 mg, 0.1 mmoli) in anhydrous THF (1 ml) was
added, and the reaction was heated to reflux for 16 h. The mixture
was then extracted with ethyl acetate (3 ꢂ 2 ml) and washed with
water (2 ꢂ 1 ml). The product was purified by preparative HPLC to
furnish 1a.
J ¼ 7.6 Hz, 1H), 7.11 (dd, J ¼ 8.0, 7.5 Hz, 1H) 6.93 dd, J ¼ 7.8, 1.5 Hz,
1H), 6.69 (ddd, J ¼ 8.0, 2.4, 0.6 Hz, 1H), 3.52 (s, 3H).
13C NMR (75 MHz, CDCl3)
115.60, 114.73, 15.78.
d 180.9; 139.47,136.35, 129.81,121.17,
ESI-MS m/z:199.3[MþH]þ.
(16 mg,72%)
1H NMR (300 MHz, CDCl3):
d
7.42 (t, J ¼ 2.0 Hz, 1H), 7.27 (t,
4.1.3. General procedure A: preparation of ketones brominated
using N bromosuccinimide
J ¼ 7.7 Hz, 1H),7.19 (dd,J ¼ 7.4 Hz,1H) 7.11 (ddd, J ¼ 8.0, 2.2, 0.9 Hz,
1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.58 (s, 1H), 2.51 (s, 3H).
13C NMR (75 MHz, CDCl3):
119.3, 114.2, 113.3112.2,14.9.
d 159.7, 147.8, 140.6, 137.2, 128.5,
Ketone (1 eq) and N-bromosuccinimide (NBS) (2 eq) were
solved in acetonitrile and trimethylsilyl trifluoromethanesulfonate
(TMS-OTf) (1 eq) was added. The reactions were stirred at T ¼ 40 ꢀC
until completeness, diluted with diethyl ether (2 ml), washed with
H2O (3 ꢂ 2 ml), dried over Na2SO4 and concentrated under reduced
pressure. This procedure provided bromoketones intermediates in
75e90% overall yield, with purities generally>90% as determined
by HPLC-MS. The compounds was used without further
purification.
ESI-MS m/z ¼ 223.0 [MþH]þ.
4.1.5.3. N-(3-(methylthio) phenyl)-4-phenylthiazol-2-amine (1c).
3-methylthioaniline (12 l, 0.1 mmol) was dissolved in anhydrous
THF (500 l) of at room temperature and 60% dispersion in mineral
m
m
oil of NaH (14 mg, 0.33 mmol) was added. When the bubbling
stopped, 2 chloro 4-phenyl thiazole (20 mg, 0.1 mmol) in anhy-
drous THF (1 ml) was added, and the reaction was heated to reflux
for 16 h. The mixture was then extracted with ethyl acetate
(3 ꢂ 2 ml) and washed with water (2 ꢂ 1 ml). The product was
purified by preparative HPLC to furnish 1c.
4.1.4. General procedure B: preparation of ketones brominated
using copper (II) bromide
The ketones were brominated by adding CuBr2 (1.2 eq) in ethyl
acetate (EtOAc) (1 ml) at T ¼ 60 ꢀC to a 1 eq of the appropriate
ketone monitoring the reaction with HPLC-MS. At the end of the
reaction the mixture were washed with water (3 ꢂ 2 ml), dried over
Na2SO4 and the organic phase was concentrated under vacuum.
The dry organic phases were controlled by HPLC and ESI-MS in
order to verify the presence of expected products. These bromo-
ketones intermediates were obtained in 80e90% overall yield, with
purities generally>90% as determined by HPLC-MS.
(26 mg, 86%)
1H NMR (300 MHz, CDCl3):
d
7.79 (ddd, J ¼ 7.7, 1.5, 0.5 Hz, 2H),
7.51 (ddd, J ¼ 7.5, 1.5, 0.5 Hz, 2H), 7.44 (t, J ¼ 2.0 Hz, 1H), 7.39 (tt,
J ¼ 7.5,1.4 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.12 (ddd, J ¼ 8.0, 2.2,
0.9 Hz, 1H), 6.95 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.83 (s, 1H), 2.51 (s,
3H).
13C NMR (75 MHz, CDCl3):
d
160.7, 150.2, 142.6, 137.2, 133.0,
130.8, 129.2, 128.7, 128, 127.5, 125.3, 119.5, 114.5, 113.2, 104.8, 14.8.

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
29
ESI-MS m/z ¼ 299.1[MþH]þ.
intermediate (93 mg, 0.25 mmol) was then conjugated with 1-(3-
(methylthio)phenyl)thiourea a (49 mg, 0.25 mmol) in refluxing
ethanol (1 ml). After 16 h the reaction was complete and the
product was purified by preparative HPLC and the peak of interest
was concentrated to obtain the title compound.
4.1.5.4. 4-(4-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-
amine (1n). 3-methylthioaniline (12
ml, 0.1 mmol) was dissolved in
anhydrous THF (500 l) of at room temp, and 60% dispersion in
m
mineral oil of NaH (14 mg,0.33 mmol) was added. When the
bubbling stopped, 2 chloro 4-(4-chlorophenyl) thiazole (23 mg, 0.1
mmoli), in anhydrous THF (1 ml) was added, and the reaction was
heated to reflux for 24 h. The mixture was then extracted with ethyl
acetate (3 ꢂ 2 ml) and washed with water (2 ꢂ 1 ml). The product
was purified by preparative HPLC to furnish the title product.
(26.5 mg,78%)
(80.3 mg,46%)
1H NMR (300 MHz, CDCl3):
d
7.46 (d, J ¼ 1.5 Hz, 2H), 7.41 (t,
J ¼ 2.0 Hz, 1H), 7.22 (t, J ¼ 7.7 Hz, 1H), 7.11 (ddd, J ¼ 8.0, 2.2, 0.9 Hz,
1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.84 (s, 1H), 2.51 (s, 3H), 2.22
(s, 9H).
13C NMR (75 MHz, CDCl3):
d 170, 169, 168.5, 160.6, 150.2, 146.6,
146.3,142.6,137.2, 136.2,130.6,128.3,119.3, 115.8,115.2, 114.3,113.2,
105.0, 20.3, 20.3.
1H NMR (300 MHz, CDCl3):
d
7.98 (t, J ¼ 7.5, 1.5 Hz, 2H), 7.56 (t,
J ¼ 7.5, 1.5 Hz, 2H), 7.42 (t, J ¼ 2.0 Hz,1H), 7.24 (t, J ¼ 7.7 Hz, 1H), 7.08
(ddd, J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.94 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.87
(s, 1H), 2.51 (s, 3H).
ESI-MS m/z ¼ 473.1[MþH]þ.
4.1.5.7. 4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-
amine (4a). To a
solution of 2-bromo-30-chloroacetophenone
13C NMR (75 MHz, CDCl3):
d
161.3,150.2,142.6,137.2,134.3,131.1,
130.2, 129.3, 128.9, 128.5, 128.3, 119.3, 114.1, 113.3, 105.2, 14.7.
(65 mg, 0.28 mmol) in ethanol absolute (2 ml) 1-(3-(methylthio)
phenyl)thiourea a (56 mg, 0.28 mmol) was added, and the reaction
was heated to reflux for 6 h. The mixture was then extracted with
diethyl ether (3 ꢂ 3 ml) and washed with water (2 ꢂ 1 ml). The
organic phase was dried under vacuum. The product was purified
by preparative HPLC to afford the title compound.
ESI-MS m/z ¼ 333.0[MþH]þ.
4.1.5.5. 2,20,2''((5-(2-((3-(methylthio)phenyl)amino)thiazol-4-yl)
benzene-1,2,3trityl)tris(oxy)) triacetic acid (2d). A solution of tert-
butylbromoacetate (87
1.03 mmol) were added to
m
l, 0.6 mmol) and of K2CO3 (142 mg,
a
solution of 20,30,4'-trihydrox-
(77 mg, 83%)
yacetophenone (25 mg of, 0.15 mmol) in anhydrous DMF (1 ml).
The reaction was stirred at room temperature in the dark for 4 h.
Upon completion, the reaction mixture was extracted with EtOAc
(3 ꢂ 3 ml) and the organic phases were concentrated under vacuum
and resuspended in a solution of trifluoroacetic (TFA)/CH2Cl2 1:1
(1 ml) at room temperature. After 2 h the reaction was controlled in
HPLC-MS and the presence of the product was achieved. The re-
action mixture was extracted with EtOAc (3 ꢂ 3 ml) and the organic
phases were concentrated under vacuum and resuspended in
anhydrous acetonitrile (2 ml). The obtained product, without
further purification, was brominated with NBS following method A.
The reaction mixture was diluted with ethyl acetate (1 ml), washed
with H2O (3 ꢂ 3 ml) and finally concentrated in vacuum to obtain
1H NMR (300 MHz, CDCl3)
d
7.86 (t, J ¼ 1.9 Hz, 1H), 7.71 (dt,
J ¼ 7.5,1.6 Hz,1H), 7.44 (t, J ¼ 2.0 Hz,1H), 7.33 (td, J ¼ 7.7, 0.6 Hz,1H),
7.28 (dt, J ¼ 7.7, 1.8 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.10 (ddd, J ¼ 8.0,
2.2, 0.9 Hz, 1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.87 (s, 1H), 2.53
(s, 3H).
13C NMR (75 MHz, CDCl3):
d 164.1, 150.1, 140.8, 140.5, 136.3,
134.8, 130.0, 129.8, 128.0, 126.4, 124.2, 121.2, 115.6, 114.7, 103.2, 15.8.
ESI-MS m/z ¼ 333.0 [MþH]þ.
4.1.5.8. N, 4-bis (3-(methylthio) phenyl) thiazol-2-amine (4b).
To
a
solution
3-acetylbenzene-1-sulfonyl
chloride
(109 mg,0.5 mmol) in anhydrous toluene (3 mL) in a three-neck
round-bottom flask, reflux condenser, and calcium chloride guard
tube, was added Ph3P (478 mg, 2 mmol) in portions. The reaction
was stirred at reflux for 30 min and then allowed to cool to below
T ¼ 40ꢀ. H2O (1.5 ml) was added and the mixture was stirred for
about 15 min. The aqueous layer was discarded and the organic
layer concentrated to vacuum. The residue was then resuspended
in CH2Cl2 (1 ml) and then washed with saturated NaHCO3
(3 ꢂ 2 ml). The organic layer was dried over Na2SO4 and the solvent
was evaporated under reduced pressure to give 1-(3-
mercaptophenyl) ethanone.
the
2,20,2''-((5-(2-bromoacetyl)benzene-1,2,3-triyl)tris(oxy))tri-
acetic acid. The intermediate (42 mg,0.1 mmol) was then conju-
gated with 1-(3-(methylthio)phenyl)thiourea a (20 mg, 0.1 mmol)
in refluxing ethanol (1 ml). After 24 h the reaction was complete
and the product was purified by preparative HPLC and the peak of
interest was concentrated to obtain the title compound.
(52 mg, 66%)
1H NMR (300 MHz, CDCl3):
d
11.04 (s, 3H), 7.28 (t, J ¼ 2.0 Hz, 1H),
7.26 (t, J ¼ 7.7 Hz, 1H), 7.20 (ddd, J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.97 (ddd,
J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.87 (s, 1H), 6.81 (d, J ¼ 1.5 Hz, 2H), 4.66 (s,
6H), 2.53 (s, 3H).
1-(3-mercaptophenyl)ethanone (50 mg,0.33 mmol) was dis-
solved in anhydrous N,N dimethyl formamide (DMF) (1 ml) and 60%
dispersion in mineral oil of NaH (14 mg, 0.33 mmol) were added at
13C NMR (75 MHz, CDCl3):
d 170.2, 169.9, 168.5, 160.6, 151.4,
152.3, 150.2, 142.6, 139.2, 137.2, 128.3, 127.6, 118.9, 114.2, 113.3,
105.2, 100.7, 98.5, 68.1, 67.8, 67.5, 14.7.
T ¼ 0 ꢀC. After few minutes iodomethane (20
ml, 0.33 mmol) was
added and the reaction was stirred at room temperature overnight.
The ketone was extracted with EtOAc (3 ꢂ 3 ml), organic phase was
concentrated under vacuum and the product was brominated with
NBS (method A) to obtain 2-bromo-1-(3-mercaptophenyl)etha-
none. An ethanolic solution (1 mL) of this ketone (69 mg, 0.3 mmol)
and the 1-(3-(methylthio) phenyl) thiourea a (59 mg, 0.3 mmol)
were heated at reflux for about 5 h until the reaction was judged
complete (HPLC- MS). The reaction mixture was extracted with
EtOAc (3 ꢂ 3 ml), purified by preparative HPLC and the peak of
interest was concentrated to obtain the title compound.
(84.5 mg, 51%)
ESI-MS m/z ¼ 521.1 [MþH]þ.
4.1.5.6. 5-(2-((3-(methylthio)phenyl)amino)thiazol-4-yl)benzene-
1,2,3-triyl triacetate (2f). 20,30,40-Trihydroxyacetophenone (62 mg,
0.37 mmol) was dissolved in dichloromethane (1200
ml) then acetic
anhydride (210 l, 2.22 mmol) was added. The reaction was stirred
m
at room temperature in the dark for 5 h. Upon completion, the
reaction mixture was extracted with EtOAc (3 ꢂ 3 ml) and the
organic phases were concentrated under vacuum and resuspended
in anhydrous acetonitrile (ACN) (2 ml). The obtained product
without further purification was brominated with NBS following
method A. The reaction mixture was diluted with EtOAc (1 ml),
washed with H2O (3 ꢂ 2 ml) and concentrated in vacuum to obtain
1H NMR (300 MHz, CDCl3):
d
7.59 (dt, J ¼ 7.5, 1.6 Hz, 1H), 7.45 (t,
J ¼ 2.0 Hz, 1H), 7.42 (t, J ¼ 7.7 Hz, 1H), 7.40 (t, J ¼ 1.9 Hz, 1H), 7.35
(ddd, J ¼ 7.7, 1.8,0.9 Hz,1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.10 (ddd, J ¼ 8.0,
2.2, 0.9 Hz, 1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.87 (s, 1H), 2.53
the
5-(2-bromoacetyl)benzene-1,2,3-triyl
triacetate.
This

30
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
(s, 6H).
(3 ꢂ 1 ml). The organic phase was concentrated under vacuum and
13C NMR (75 MHz, CDCl3):
d
191.3, 160.4, 150.5, 142.6, 137.3,
finally dried to obtain the title product.
134.5, 133.2, 129.4, 129.5, 129.2, 125.1, 123.9, 114.2, 113.6, 104.6, 15.7,
(17.5 mg, 95%)
14.6.
1H NMR (300 MHz, CDCl3):
d
7.44 (t, J ¼ 2.0 Hz, 1H), 7.39 (t,
ESI-MS m/z: 345.1 [MþH]þ.
J ¼ 7.5 Hz, 1H), 7.37-7.34 (m, 2H), 7.24 (t, J ¼ 7.7 Hz, 1H), 7.10 (ddd,
J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.94 (ddd,
J ¼ 7.5,1.5,1.5 Hz, 1H), 6.87 (s, 1H), 4.66 (s, 2H), 2.51 (s, 3H), 1.39 (s,
9H).
4.1.5.9. N-(3-(2-((3-(methylthio)phenyl)amino)thiazol-4-yl)phenyl)
acetamide (4 i). 1-(3-aminophenyl)ethanone (50 mg, 0.37 mmol)
was dissolved in CH2Cl2 (600
ml) then acetic anhydride (42
ml,
13C NMR (75 MHz, CDCl3):
d 169.2, 160.6, 158.3, 142.7, 138.2,
0.45 mmol) was added. The reaction was stirred at room temper-
ature upon completeness. The reaction mixture was washed with
0.1N sodium carbonate, the organic phase was concentrated in
vacuum and resuspended in ethyl acetate (1 ml) and CuBr2 (99 mg,
0.3 mmol) was added. The mixture was then stirred at reflux
overnight. The organic phase was washed with H2O (2 ꢂ 2 ml),
concentrated to obtain 1-(3-aminophenyl)-2-bromoethanone. The
bromoketone (64 mg, 0.3 mmol) was resuspended in anhydrous
EtOH (1 ml) and conjugated with 1-(3-(methylthio)phenyl)thio-
urea a (59 mg, 0.3 mmol) as previously described. The final product
was purified by preparative HPLC, the peak of interest was
concentrated to obtain the title compound.
135.5, 128.3, 124.5, 119.3, 114.8, 114.4, 113.5, 113.2, 82.1, 80.3, 63.6,
28.7, 28.4, 27.9,15.1, 14.6.
ESI-MS m/z: 373.1 [MþH]þ.
4.1.5.12. N-(3-(methylthio)phenyl)-4-(thiophen-2-yl)thiazol-2-
amine(9d). 2-acetylthiophene (25 mg, 0.2 mmol) was brominated
according to the general procedure B to obtain 2-bromo-1-(thio-
phen-2-yl)ethanone. This derivative (37 mg, 0.18 mmol) was then
conjugated with 1-(3-(methylthio)phenyl)thiourea a (35.5 mg,
0.18 mmol) in refluxing ethanol (1 ml). After 18 h the reaction was
complete and the product was purified by preparative HPLC and the
peak of interest was concentrated to obtain the title compound.
(47 mg, 78%)
(96 mg, 73%)
1H NMR (300 MHz, CDCl3)
d
7.95-7.89 (m 1H), 7.60-7.50 (d,
1H NMR (300 MHz, CDCl3)
d 7.41-7.37 (m, 2H), 7.30-7.22 (m, 2H),
J ¼ 7.7 Hz, 2H), 7.40-7.29(m, 3H), 7.24 (t, J ¼ 8.0 Hz, 1H), 7.10 (ddd,
J ¼ 8.1, 2.2, 0.8 Hz,1H), 6.93 (ddd, J ¼ 7.8,1.6, 0.9 Hz,1H), 6.83 (s,1H),
2.43 (s, 3H), 2.17 (s, 3H).
7.08-7.03 (m, 2H), 6.97 (ddd, J ¼ 7.8,1.8, 0.9 Hz,1H), 6,70 (s,1H), 2.52
(s, 3H).
13C NMR (75 MHz, CDCl3):
d 164.7, 145.4, 140.6, 140.5, 138.1,
13C NMR (75 MHz, CDCl3)
d
168.5, 164.3, 150.7, 140.9, 140.3,
129.8, 127.86, 125.1, 124.1, 121.3, 115.8, 114.9, 100.7, 15.7.
138.3, 135.2, 129.8, 129.5, 122.1, 121.0, 119.6, 117.6, 115.7, 114.8, 102.7,
ESI -MS m/z ¼ 305.0 [MþH]þ.
24.8, 15.7.
ESI-MS m/z:356.1 [MþH]þ.
4.1.5.13. 6-bromo-N-(3-(methylthio)phenyl)benzo[d]thiazol-2-amine
(11a). At a solution of 6-bromo-2-chlorobenzothiazole (25 mg,0.1
4.1.5.10. tert-butyl 2-(3-(2-((3-(methylthio)phenyl)amino)thiazol-4-
mmoli) in ethanol absolute (1 ml) 3 methylthio aniline (12 ml, 0.1
yl)phenoxy)acetate (4j). A solution of tert-butylbromoacetate
(87 ml, 0.6 mmol) and of K2CO3 (142 mg, 1.03 mmol) were added
mmoli) was added, and the reaction was heated to reflux for 6 h.
The mixture was then extracted with diethyl ether (3 ꢂ 2 ml) and
washed with water (2 ꢂ 1 ml). The organic phase was dried under
vacuum. The product was purified by preparative HPLC to afford
11a.
to a solution of 1-(3-hydroxyphenyl)ethanone (20 mg, 0.15 mmol)
in anhydrous DMF (1 ml). The reaction was stirred at room tem-
perature in the dark for 2 h. Upon completion, the reaction mixture
was extracted with EtOAc (3 ꢂ 3 ml) and the organic phases were
concentrated under vacuum and resuspended in anhydrous
acetonitrile (2 ml). The obtained product, without further purifi-
cation, was brominated with NBS following method A. The reaction
mixture was diluted with ethyl acetate (1 ml), washed with H2O
(3 ꢂ 3 ml) and finally concentrated in vacuum to obtain the tert-
(31 mg, 88%)
1H NMR (300 MHz, CDCl3):
d
8.77 (d, J ¼ 1.5 Hz,1H), 7.75 (dd,
J ¼ 7.5, 1.5, Hz, 1H), 7.64 (d, J ¼ 7.5 Hz, 1H), 7.44 (dt, J ¼ 8.0, 2.0,
2.0 Hz,1H), 7.26 (t, J ¼ 7.7 Hz,1H), 7.10 (td, J ¼ 8.0, 8.0, 2 Hz,1H), 6.97
(ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 2.53 (s, 3H).
13C NMR (75 MHz, CDCl3):
d 174.5, 152.6, 142.5, 137.2, 132.8,
butyl
2-(3-(2-bromoacetyl)phenoxy)acetate.
The
129.3, 128.3, 126, 119.3, 118.8, 117.1, 114.3, 110.2, 14.6.
intermediate(49 mg, 0.15 mmol) was then conjugated with 1-(3-
(methylthio)phenyl)thiourea a (29 mg, 0.15 mmol) in refluxing
ethanol (1 ml). After 2 h the reaction was complete and the product
was purified by preparative HPLC and the peak of interest was
concentrated to obtain the title compound.
ESI-MS m/z ¼ 352.9 [MþH]þ.
4.1.5.14. 2-(2-((3-(methylthio)phenyl)amino)thiazol-4-yl)-N-phenyl-
acetamide (12a). Ethyl 3-chloropropanoate (200 ml,0.15 mmol) was
dissolved in anhydrous THF (2 ml) and 1-(3-(methylthio)phenyl)
thiourea (20 mg, 0.1 mmol) was added. The solution was stirred at
T ¼ 80 ꢀC for 3 h. Then the reaction mixture was extracted with
EtOAc (3 ꢂ 3ml) and the organic phase was resuspended in ace-
tonitrile(ACN) and 1M NaOH 1:1(2 ml). The hydrolysis was com-
plete after 16 h at room temperature. After extraction with EtOAc
(3 ꢂ 2 ml), the organic phase was concentrated under vacuum to
obtain 2-(2-((3-(methylthio)phenyl)amino)thiazol-4-yl)acetic acid
in almost quantitative yield.
(51 mg,79%)
1H NMR (300 MHz, CDCl3):
d
10.98 (s, OH), 7.44 (t, J ¼ 2.0 Hz,1H),
7.40 (t, J ¼ 7.5 Hz, 1H), 7.37-7.34 (m, 2H), 7.28 (t, J ¼ 7.7 Hz, 1H), 7.11
(ddd, J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.95
(ddd, J ¼ 7.5, 1.5, 1.5 Hz, 1H), 6.87 (s, 1H), 4.66 (s, 2H), 2.51 (s, 3H).
13C NMR (75 MHz, CDCl3):
d 169.9, 159.4, 160.6, 150.2, 142.6,
138.1, 134.0, 129.5, 128.6, 119.8, 114.3, 114.5, 113.6, 113.2, 105.4, 64.7,
15.2.
ESI-MS m/z: 429.1 [MþH]þ.
The thiazole was resuspended in anhydrous DMF(1 ml), HBTU
(38 mg, 0.1 mmol) and DIPEA (17
ml, 0.1 mmol) were added, the
4.1.5.11. 2-(3-(2-((3-(methylthio)phenyl)amino)thiazol-4-yl)phe-
noxy)acetic acid (4k). Compound 4j (21.5 mg, 0.05 mmol) was hy-
drolyzed with a solution of trifluoroacetic (TFA)/CH2Cl2 1:1 (1 ml) at
room temperature. After 1 h the reaction was controlled in HPLC-
MS and the presence of the product was achieved. The reaction
mixture was diluted in EtOAc (1 ml) and washed with water
reaction was vigorously stirred for 10 min and then aniline (93
m
l,
0.1 mmol) was added. The mixture was stirred at T ¼ 50 ꢀC over-
night. After extraction with EtOAc (3 ꢂ 3 ml), the organic phase was
concentrated under vacuum. The crude product was then purified
by preparative HPLC, the peak of interest was concentrated in
vacuum and finally lyophilized to obtain the title compound.

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
31
(22 mg, 62%).
0.6 Hz, 1H), 7.29 (dt, J ¼ 7.7 1.8 Hz, 1H), 7.24 (t, J ¼ 2.0 Hz, 1H), 7.16 (t,
J ¼ 7.7 Hz, 1H), 6.57 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.89 (s, 1H), 3.41 (s,
1H).
1H NMR (300 MHz, CDCl3):
d
7.61 (d, J ¼ 7.5, 1.5 Hz, 2H), 7.46 (t,
J ¼ 2.0 Hz, 1H), 7.41 (td, J ¼ 7.5, 1.5 Hz, 2H), 7.26 (t, J ¼ 7.7 Hz, 1H),
7.19 (m, 1H), 7.10 (ddd, J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.99 (s, 1H), 6.97
(ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 5.26 (s, NH), 3.85 (s, 2H), 2.53 (s, 3H).
13C NMR (75 MHz, CDCl3):
d 160.6, 150.2, 142.7, 134.8, 133.8,
131.5, 130.5, 129.8, 129.2, 128.7, 125.6, 119.1, 115.3, 113.6, 103.0.
13C NMR (75 MHz, CDCl3):
d
169.9, 159.1, 152.1, 142.6, 138.5,
ESI-MS m/z ¼ 361.0 [MþH]þ.
137.2,128.9,128.5,128.3,128.0,121.6,121.4,119.3,114.2,112.2,104.5,
36.2, 14.8.
4.1.5.17. 4-(3-chlorophenyl)-N-(3-(ethylthio)phenyl)thiazol-2-amine
(14c). Following the same procedure, but using iodoethane (26 ml,
ESI-MS m/z ¼ 356.0 [MþH]þ.
0.33 mmol) instead of iodopropane, 1-(3-ethylthiophenyl) thiourea
d was obtained. The thiourea d (63 mg, 0.3 mmol) was resuspended
in EtOH (1 ml) and conjugated with 2-bromo-30-chlor-
oacetophenone (69 mg, 0.3 mmol) and the reaction was heated to
reflux for 8 h. The crude product was then purified by preparative
HPLC, the peak of interest was concentrated in vacuum and finally
lyophilized to obtain the title compound.
4.1.5.15. 3-((4-(3-chlorophenyl)thiazol-2-yl)amino)benzenethiol
(14a). Commercially available 3-aminobenzenethiol (11
0.1 mmol) was dissolved in anhydrous THF (1 ml). A solution of
benzoyl isothiocyanate (14 l, 0.1 mmol) in anhydrous THF (1 ml)
ml,
m
was added dropwise at room temperature to the reaction mixture
and stirred overnight. When the reaction was complete, the solu-
tion was concentrated under vacuum and diluted in ice cold water
(3 ml). After 20 min of stirring, a white pellet appeared. The re-
actions were centrifuged at 4000 g for 5 min and washed with
water (3 ꢂ 1 ml). Then, the pellet was resuspended in a solution of
EtOH and 1M NaOH 1:1 (2 ml).
The reaction was heated to reflux until the hydrolysis was
complete (controlled by HPLC-MS). The mixture was concentrated
under vacuum and a solution of aqueous 1N HCl was added to
produce a neutral solution. The reaction mixture was centrifuged at
4000 g for 5 min and the pellet was washed with water (3 ꢂ 1 ml).
The compound was completely dried to obtain 1-(3-
mercaptophenyl)thiourea b.
The same thiourea (16.5 mg,0.09 mmol) was resuspended in
EtOH (1 ml) and conjugated with 2-bromo-30-chloroacetophenone
(21 mg, 0.09 mmol) and the reaction was heated to reflux for 8 h.
The crude product was then purified by preparative HPLC, the peak
of interest was concentrated in vacuum to obtain the title
compound.
(72 mg, 67%)
1H NMR (300 MHz, CDCl3):
d
7.87 (dd, J ¼ 1.4, 0.8 Hz, 1H), 7.71
(dt, J ¼ 7.5, 1.5 Hz, 1H), 7.43 (ddd, J ¼ 7.5, 2.0, 0.7 Hz, 1H), 7.35 (dt,
J ¼ 7.7, 1.8 Hz,1H), 7.33 (td, J ¼ 7.7, 0.6 Hz, 1H), 7.28 (t, J ¼ 7.7 Hz,1H),
7.22 (t, J ¼ 0.9 Hz, 1H), 6.73 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.85 (s, 1H),
2.93 (q, J ¼ 8.1 Hz, 2H), 1.31 (t, J ¼ 8.1 Hz, 3H).
13C NMR (75 MHz, CDCl3):
d 160.6, 150.2, 142.6, 136.7, 134.8,
133.5, 129.8, 129.5, 129.1, 128.7, 125.1, 119.3, 113.9, 113.2, 105.4, 29.5,
14.3.
ESI-MS m/z ¼ 347.0 [MþH]þ.
4.1.5.18. 4-(3-chlorophenyl)-N-(3-((trifluoromethyl) thio) phenyl)
thiazol-2-amine (14 d). A solution of 3-((trifluoromethyl)thio)ani-
line (45
ml, 0.3 mmol) in anhydrous THF (1 ml) was conjugated with
benzoyl isothiocyanate (40
m
l, 0.3 mmol) at room temperature
overnight and subsequently hydrolyzed as previously described to
obtain 1-(3-((trifluoromethyl)thio)phenyl)thiourea e.
The thiourea e (70 mg, 0.28 mmol) was dissolved in absolute
EtOH (1 ml) and conjugated with 2-bromo-30-chloroacetophenone
(65 mg, 0.28 mmol) and the reaction was heated to reflux for 3 h.
The product was purified by preparative HPLC and the peak of in-
terest was concentrated to obtain the title compound.
(96 mg, 83%)
(26 mg, 81%)
1H NMR (300 MHz, CDCl3):
d
7.86 (dd, J ¼ 1.4,0.8 Hz,1H), 7.71 (dt,
J ¼ 7.5, 1.6 Hz, 1H), 7.40 (ddd, J ¼ 8.0, 2.1, 0.9 Hz, 1H), 7.35 (td, J ¼ 7.7,
0.6 Hz, 1H), 7.29 (dt, J ¼ 7.7 1.8 Hz, 1H), 7.24 (t, J ¼ 2.0 Hz, 1H), 7.16 (t,
J ¼ 7.7 Hz, 1H), 6.57 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.89 (s, 1H), 3.41 (s,
1H).
1H NMR (300 MHz, CDCl3):
d
7.87 (t, J ¼ 1.9 Hz, 1H), 7.73 (dt,
13C NMR (75 MHz, CDCl3):
d
160.2, 152.2, 143.3, 134.8, 133.8,
J ¼ 7.5,1.6 Hz,1H), 7.44 (t, J ¼ 2.0 Hz,1H), 7.31 (td, J ¼ 7.7, 0.6 Hz,1H),
7.28 (dt, J ¼ 7.7 Hz, 1.8 Hz, 1H), 7.23 (t, J ¼ 7.7 Hz, 1H), 7.11 (ddd,
J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.94 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.89 (s,
1H).
132.5, 130.5, 129.8, 129.3, 128.4, 125.6, 119.1, 115.8, 114.6, 106.1.
ESI-MS m/z ¼ 319.0 [MþH]þ.
4.1.5.16. 4-(3-chlorophenyl)-N-(3-(propylthio)phenyl)thiazol-2-
13C NMR (75 MHz, CDCl3):
d 160.6, 150.2, 142.6, 133.2, 137.2,
amine (14b). 3-aminobenzenethiol (35
m
l, 0.33 mmol) was dis-
136.6,134.4,131.1,129.5,128.8,128.3,124.5,119.3,114.2,113.4,104.8.
solved in anhydrous DMF (1 ml) and 60% dispersion in mineral oil
ESI-MS m/z ¼ 386.9 [MþH]þ.
of NaH (14 mg, 0.33 mmol) was added at T ¼ 0 ꢀC. After few minutes
iodopropane (34
ml, 0.33 mmol) was added and the reaction was
4.1.5.19. 5-((4-(3-chlorophenyl) thiazol-2-yl) amino)-2-
stirred at RT overnight. After extraction with EtOAc (3 ꢂ 1 ml),
ethylbenzenethiol (14 e). A solution of 1-ethyl-4-nitrobenzene
(68 ml, 0.5 mmol) and chlorosulfonic acid (497 ml, 7.5 mmol) was
organic phases were concentrated under vacuum and resuspended
in anhydrous THF(1 ml). A solution of benzoyl isothiocyanate (44
m
l,
heated at T ¼ 100 ꢀC for 2 h. The reaction mixture was suspended in
H2O (3 ml) and extracted with EtOAc (3 ꢂ 3 ml) and the organic
phase was concentrated under vacuum. The product obtained 2-
ethyl-5-nitrobenzene-1-sulphonyl chloride was used for the next
step without further purification (ESI-MS m/z ¼ 249.0).
0.33 mmol) in anhydrous THF (1 ml) was added dropwise at room
temperature to the reaction mixture and stirred overnight.
Following the same procedure of compound 14a, 1-(3-
propylthiophenyl) thiourea c was achieved.
The thiourea c (66 mg, 0.3 mmol) was resuspended in EtOH
(1 ml) and conjugated with 2-bromo-30-chloroacetophenone
(69 mg, 0.3 mmol). The reaction was heated to reflux for 12 h. The
crude product was then purified by preparative HPLC; the peak of
interest was concentrated in vacuum and finally lyophilized to
obtain the title compound.
To
a
solution
of
2-ethyl-5-nitrobenzene-1-sulphonyl
chloride(125 mg, 0.5 mmol) in anhydrous toluene (3 ml) in a
three-neck round-bottom flask, reflux condenser, and calcium
chloride guard tube, was added Ph3P (478 mg, 2 mmol) in portions.
The reaction was stirred at reflux for 1 h and then allowed to cool at
ambient temperature. Deionized H2O (1 ml) was added and the
mixture was stirred for about 30 min. The aqueous layer was dis-
carded and the organic layer concentrated to vacuum. The residue
was then resuspended in CH2Cl2 (1 ml) and then washed with
(55 mg, 46%)
1H NMR (300 MHz, CDCl3):
J ¼ 7.5, 1.6 Hz, 1H), 7.40 (ddd, J ¼ 8.0, 2.1, 0.9 Hz, 1H), 7.35 (td, J ¼ 7.7,
d
7.86 (dd, J ¼ 1.4,0.8 Hz,1H), 7.71 (dt,

32
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
saturated NaHCO3 (2 ꢂ 1 ml). The organic layer was dried over
Na2SO4 and the solvent was evaporated under reduced pressure to
give 2-ethyl-5-nitrobenzenethiol in high yield (ESI-MS m/
z ¼ 183.1).
4.1.5.21. N-benzyl-4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thia-
zol-2-amine (15b). The same protocol was applied with benzyl
chloride (14 ml, 0.12 mmol) instead of methyl iodide. After purifi-
cation with HPLC preparative 15b was obtained.
The arylthiol derivative (58
ml, 0.35 mmol) was dissolved in
(22 mg, 58%)
anhydrous DMF (1 ml) and 60% dispersion in mineral oil of NaH
1H NMR (300 MHz, CDCl3):
d
7.91 (t, J ¼ 1.9 Hz, 1H), 7.71 (dt,
(15 mg, 0.35 mmol) was added at T ¼ 0 ꢀC. After few minutes a
J ¼ 7.5, 1.6 Hz,1H), 7.44 (t, J ¼ 2.0 Hz, 1H), 7.43 (dt, J ¼ 7.7, 0.6, 0.6 Hz,
1H), 7.33 (td, J ¼ 7.5, 7.5, 1.5 Hz, 2H), 7.28 (tt, J ¼ 7.7, 7.7, 1.8 Hz, 1H),
7.26 (t, J ¼ 7.7 Hz,1H), 7.24 (dt, J ¼ 7.5, 2.0, 2.0 Hz, 2H), 7.22 (t, J ¼ 1.4,
1.4 Hz, 1H), 7.10 (ddd, J ¼ 8.0, 2.2, 0.9 Hz, 1H), 6.97 (ddd, J ¼ 7.8, 1.8,
0.9 Hz, 1H), 6.87 (s, 1H), 4.32 (s, 2H), 2.53 (s, 3H).
solution of iodomethane (22 ml, 0.35 mmol) was added and the
reaction was stirred at room temperature for 3 h. After extraction
with EtOAc (2 ꢂ 1 ml), the organic phases were concentrated under
vacuum
and
then
dried
to
obtain
2-ethyl-5-
nitrophenylmethylsulfane (ESI-MS m/z ¼ 197.1).
13C NMR (75 MHz, CDCl3):
d 166.2, 150.2, 149.6, 138.1, 137.2,
The 2-ethyl-5-nitrophenylmethylsulfane (34
m
l, 0.2 mmoli) was
134.8, 134.4,130, 129.5, 128.8, 128.7, 128.5, 127, 126.9, 126, 125.9,
125.6, 117.7, 110.4, 109.9, 105, 56.7, 14.6.
dissolved in acetone (3 ml) and the cooled to T ¼ 0ꢀ. A saturated
solution of NH4Cl (500
ml) was added, followed by addition of zinc
ESI-MS m/z ¼ 423.0 [MþH]þ.
powder (54 mg, 1.8 mmol) over 5 min with vigorous stirring. The
mixture was stirred at room temperature for 2 h and then filtered to
remove the zinc. The solution was then evaporated to vacuum and
dissolved in EtOAc (1 ml). The organic phase was then washed with
saturated NaHCO3 (2 ꢂ 1 ml) and lastly with deionized water
(1 ꢂ 1 ml). The crude product was the purified by preparative HPLC
to obtain 4-ethyl-3-(methylthio) aniline (ESI-MS m/z ¼ 167.1).
The aniline derivative (33 mg,0.2 mmol was then conjugated
4.1.5.22. 2-((4-(4-bromophenyl) thiazol-2-yl) carbamoyl) phenyl ac-
etate (16 a). 2-acetoxybenzoic acid (14 mg, 0.078 mmol) was dis-
solved in anhydrous DMF (500
0.078 mmoli) and DIPEA (14 l, 0.078 mmol) were added. After few
minutes, 2-amino-4(4Br-phenyl) thiazole (10 mg, 0.039 mmol) in
anhydrous DMF (500 l) was added, and the reaction was kept at
ml) at room temp, and HATU (29 mg,
m
m
room temperature for 24 h. The resulting solution was suspended
in CH2Cl2 (1 ml) and washed with H2O (3 ꢂ 2 ml) and finally
concentrated in vacuum. Crude product was purified by preparative
HPLC and the peak of interest was concentrated to obtain the title
compound.
with benzoyl isothiocyanate (27
ml,0.2 mmol) and subsequently
hydrolyzed as previously described to obtain N-(4-ethyl-3-(meth-
ylthio)phenyl)carbamimidothioic acid f (ESI-MS m/z ¼ 226.1).
The thiourea obtained (40 mg, 0.18 mmol) was then conjugated
with 2-bromo-30-chloroacetophenone (34 mg, 0.18 mmol) accord-
ing to the general procedure. The reaction was heated to reflux for
24 h. The final product was purified by preparative HPLC, lyophi-
lized and controlled by ESI-MS to obtain the title compound.
(55 mg, 30%)
(8 mg,49%)
1H NMR (300 MHz, CDCl3):
d
9.08 (s, NH), 7.99 (dd, J ¼ 7.7,1.5 Hz,
1H), 7.87 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.73 (dd, J ¼ 7.7, 1.5 Hz, 2H), 7.69
(ddd, J ¼ 8.0, 7.5, 0.9 Hz, 1H), 7.66 (dd, J ¼ 7.5, 1.5 Hz, 2H), 7.44 (ddd,
J ¼ 7.5, 7.1, 1.5 Hz, 1H), 6.87 (s, 1H), 2.27 (s, 3H).
1H NMR (300 MHz, CDCl3):
d
7.94 (t, J ¼ 1.7 Hz, 1H), 7.71 (dt,
13C NMR (75 MHz, CDCl3):
d 169.2, 164.7, 161.4, 150.2, 148.7,
J ¼ 7.5,1.6 Hz,1H), 7.45-7.42 (m, 2H), 7.28 (d, J ¼ 1.4 Hz,1H), 7.16 (dd,
J ¼ 7.0, 0.9 Hz, 1H), 6.88 (d,J ¼ 7.5 Hz, 1H), 6.97 (s, 1H), 3.43 (s, 1H),
2.60 (q, J ¼ 7.8 Hz, 2H), 1.25 (t, J ¼ 8 Hz, 3H).
133.5, 132.1, 132.1, 130.5, 128.4, 128, 127.4, 125.6, 125.2, 123.5, 123.1,
105.1, 20.3.
ESI -MS m/z ¼ 418.9 [MþH]þ.
13C NMR (75 MHz, CDCl3):
d 162.6, 150.1, 139.8, 134.8, 134.4,
132.2, 130.6, 129.5, 128.6, 126.9, 125.6, 123.4, 116.7, 113.1, 106, 24.9,
4.1.5.23. N-(4-(4-bromophenyl)thiazol-2-yl)-2-hydroxybenzamide
(16b). Following the same procedure, but using 2-hydroxybenzoic
acid (11 mg, 0.078 mmol) in place of 2-acetoxybenzoic acid, 16b
was obtained.
15.1, 14.1.
ESI-MS m/z ¼ 361.0 [MþH]þ.
(6 mg, 41%)
1H NMR (300 MHz, CDCl3):
d
7.79 (ddd, J ¼ 7.7, 1.5, 0.5 Hz, 2H),
4.1.5.20. 4-(3-chlorophenyl)-N-methyl-N-(3-(methylthio)phenyl)
thiazol-2-amine (15a).
7.51 (ddd, J ¼ 7.5, 1.5, 0.5 Hz, 2H), 7.44 (t, J ¼ 2.0 Hz, 1H), 7.39 (tt,
J ¼ 7.5,1.4 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.12 (ddd, J ¼ 8.0, 2.2,
0.9 Hz, 1H), 6.95 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.83 (s, 1H), 2.51 (s,
3H).
4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-amine
(30 mg, 0.09 mmol) was added to a suspension of 60% dispersion in
mineral oil of NaH (14 mg, 0.33 mmol) in dry DMF (1 mL) at T ¼ 0ꢀ.
13C NMR (75 MHz, CDCl3):
d 160.6, 150.2, 142.6, 137.5, 133, 130.8,
After stirring for 15 min, methyl iodide (8
m
l, 0.12 mmol) was added
129.9, 128.7, 128.3, 127.5, 125.3, 119.2, 114.6, 113.3, 104.5, 14.8.
and the reaction mixture was stirred at room temperature over-
night. The mixture was then poured into ice water (3 mL), extracted
with diethyl ether (3 x 3 mL) and the organic layers were washed
with H2O (2 ꢂ 2 ml) and dried (Na2SO4). The product was purified
by preparative HPLC, the peak of interest was collected, concen-
trated to obtain 15 a.
ESI -MS m/z ¼ 376.9 [MþH]þ.
4.1.5.24. N-(4-(4-bromophenyl) thiazol-2-yl) isonicotinamide (16c).
Following the same procedure, but using pyridine-3-carboxylic
acid (9.6 mg, 0.078 mmol) in place of 2-acetoxybenzoic acid, the
title compound was obtained.
(27 mg,87%)
(10 mg,65%)
1H NMR (300 MHz, CDCl3):
d
8.01 (t, J ¼ 1.5 Hz,1H), 7.67 (dt,
1H NMR (300 MHz, CDCl3):
d
9.10 (s, NH), 8.81 (dd, J ¼ 7.7,0.5 Hz,
J ¼ 7.5, 1.5, 1.5 Hz, 1H), 7.45 (dt, J ¼ 7.5, 1.5, 1.5 Hz, 2H), 7.44 (t,
J ¼ 2.0 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.10 (ddd, J ¼ 8.0, 2.2, 0.9 Hz,
1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz, 1H), 6.87 (s, 1H), 3.31 (s, 3H), 2.53
(s, 3H).
2H), 7.81 (dd, J ¼ 7.5, 1.5 Hz, 2H), 7.73 (dd, J ¼ 7.7, 1.5 Hz, 2H), 7.66
(dd, J ¼ 7.5, 1.5 Hz, 2H), 6.87 (s, 1H).
13C NMR (75 MHz, CDCl3):
d 165.7, 164.2, 150.2, 149.7, 147, 140.8,
135.5, 132.6, 130.5, 128.8, 128.3, 123.6, 121.3, 120.5, 105.3.
13C NMR (75 MHz, CDCl3):
d
166.2, 150.2, 149.6, 137.6, 134.8,
ESI-MS m/z ¼ 361.9 [MþH]þ.
134.2, 130.1, 129.5, 128.8, 127, 125.6, 117.7, 110.4, 109.9, 105.4, 40.7,
14.8.
4.1.5.25. N-(4-(4-bromophenyl)thiazol-2-yl)benzamide
Following the same procedure, but using benzoic acid (9.5 mg,
(16
d).
ESI-MS: m/z ¼ 347.0 [MþH]þ.

E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
33
0.078 mmol) in place of 2-acetoxybenzoic acid, the title compound
was obtained.
1H NMR (300 MHz, CDCl3):
d
7.78 (dd, J ¼ 7.5, 1.5 Hz, 2H), 7.66
(dd, J ¼ 7.5, 1.5 Hz, 2H), 6.90 (s, 1H), 3.71 (t, J ¼ 7.1 Hz, 4H), 1.59 (m,
(9 mg,64%)
J ¼ 7.2 Hz, 2H), 1.53 (m, J ¼ 7.1 Hz, 4H).
1H NMR (300 MHz, CDCl3):
d
9.13 (s, NH), 8.03 (m, 2H), 7.73 (dd,
13C NMR (75 MHz, CDCl3):
d 157.3, 150.2, 140.1, 132.1, 130, 128.3,
J ¼ 7.7, 1.5 Hz, 2H), 7.70 (td, J ¼ 7.5, 1.5 Hz, 2H), 7.66 (td, J ¼ 7.5,
128.3, 123.1, 105.2, 54.5, 48, 25.8, 25.5, 24.5.
1.5 Hz, 2H), 7.61 (tt, J ¼ 7.4, 1.5 Hz, 1H), 6.83 (s, 1H).
ESI-MS m/z ¼ 325.0 [MþH]þ.
13C NMR (75 MHz, CDCl3):
d 167.7, 164.7, 150.3, 134.5, 133, 132.6,
132, 130.5, 129.5, 128.9, 128.7, 128.5, 127.5, 127.3, 123.1, 105.2.
4.1.5.29. 4-(4-butylthiophen-2-yl)-N-(3-(methylthio)phenyl)thiazol-
2-amine (21a). A solution of 3-n-Butilthiophene (50 mg,
ESI-MS m/z ¼ 358.9 [MþH]þ.
0.33 mmol), acetyl chloride (50
ml, 0.66 mmol) and anhydrous
4.1.5.26. N-benzyl-4-(4-bromophenyl) thiazol-2-amine (17a) and N,
CH2Cl2 (1 ml) was added dropwise with stirring to a mixture of
anhydrous aluminium trichloride (67 mg, 0.5 mmol) in anhydrous
CH2Cl2 (2 ml).
N-dibenzyl-4-(4-bromophenyl)
thiazol-2-amine
(17b).
2-Amino-4-(4-bromophenyl)thiazole (26 mg, 0.1 mmol) was dis-
solved in of anhydrous DMF (1 ml) at room temp, and 60%
dispersion in mineral oil of NaH (8.5 mg, 0.2 mmol) was added at
T ¼ 0 ꢀC. When the bubbling stopped, a solution of benzyl chloride
The solution was stirred at room temperature for 24 h until
completeness. The resulting mixture was then washed with H2O
(3 ꢂ 2 ml). Organic phase was evaporated in vacuum and lyophi-
lized. The obtained product, without further purification, was
brominated with NBS following method A to furnish 2-bromo-1-(4-
butylthiophen-2-yl) ethanone. The bromoketone (78 mg,
0.3 mmol) was then conjugated with 1-(3-(methylthio) phenyl)
thiourea a (60 mg, 0.3 mmol) in refluxing ethanol (1 ml). After 7 h
the reaction was complete and the product was purified by pre-
parative HPLC and the peak of interest was concentrated to obtain
the title compound.
(28 ml, 0.25 mmol), in anhydrous DMF (500 ml) was added and the
reaction was heated to reflux for 12 h. The mixture was then
extracted with diethyl ether (3 ꢂ 2 ml) and washed with water
(2 ꢂ 1 ml). The organic phase was dried under vacuum. The product
was purified by preparative HPLC to afford 17a (24 mg, 69%) and
17b (11.5 mg, 27%).
17a 1H NMR (300 MHz, CDCl3):
d
7.88 (dd, J ¼ 7.5, 1.5 Hz, 2H),
7.64 (dd, J ¼ 7.5, 1.5, Hz, 2H), 7.33 (td, J ¼ 7.5, 7.5, 1.5 Hz, 2H), 7.26 (tt,
J ¼ 7.5, 7.5,1.5,1.5 Hz,1H), 7.23 (dt, J ¼ 7.5,1.5,1.5 Hz, 2H), 6.88 (s,1H),
4.35 (s, 2H).
(79 mg,67%)
1H NMR (300 MHz, CDCl3):
d
7.53 (d, J ¼ 1.5 Hz,1H), 7.44 (t,
13C NMR (75 MHz, CDCl3):
d
164.1, 150.2, 139.4, 134, 132.1, 130.2,
J ¼ 2.0 Hz, 1H), 7.26 (t, J ¼ 7.7 Hz, 1H), 7.10 (ddd, J ¼ 8.0, 2.2, 0.9 Hz,
1H), 7.08 (d, J ¼ 1.5 Hz,1H), 6.97 (ddd, J ¼ 7.8, 1.8, 0.9 Hz,1H), 6.70 (s,
1H),2.65 (t, J ¼ 7.1, 2H), 2.52 (s, 3H), 1.61 (m, J ¼ 7.1 Hz, 2H), 1.31 (m,
J ¼ 7.1 Hz, 2H), 0.90 (t, J ¼ 8 Hz, 3H).
128.9, 128.5, 128.2, 127, 126.9, 126.2, 125.6, 123.1, 105.2, 49.1.
ESI -MS m/z ¼ 345.0 [MþH]þ.
17 b 1H NMR (300 MHz, CDCl3):
d
7.79 (dd, J ¼ 7.5, 1.5 Hz, 2H),
7.66 (dd, J ¼ 7.5, 1.5, Hz, 2H), 7.33 (td, J ¼ 7.5, 7.5, 1.5 Hz, 4H), 7.26 (tt,
J ¼ 7.5, 7.5, 1.5, 1.5 Hz, 2H), 7.23 (dt, J ¼ 7.5, 1.5, 1.5 Hz, 4H), 6.88 (s,
1H), 4.71 (s, 4H).
13C NMR (75 MHz, CDCl3):
d 159.1, 145, 142.5, 140.4, 138.3, 137.2,
128.3, 125.5, 121.7, 119.3, 114.2, 113.2, 105.6, 34.1, 30.9, 22.3, 14.7,14.1.
ESI-MS m/z ¼ 361.1 [MþH]þ.
13C NMR (75 MHz, CDCl3):
d 157.3, 150.2, 140, 138.7, 132.2, 132.1,
131.9, 128.7, 128.5, 128.2, 128.1, 128.1, 128, 127.9, 127.9, 127.8, 127.7,
127, 126.9, 123.3, 105.4, 55.1, 54.3.
4.2. Biological evaluations
ESI-MS m/z ¼ 435.0 [MþH]þ.
4.2.1. Cell culture
The bronchial epithelial cell line CFBE41o-with stable co-
expression of F508del-CFTR and the halide-sensitive yellow fluo-
rescent protein (HS-YFP) was cultured with MEM medium. FRTcells
with co-expression of HS-YFP and G551D-CFTR were instead
cultured with Coon's modified Ham's F-12 medium. Both media
4.1.5.27. 4-(4-bromophenyl)-N-cyclohexylthiazol-2-amine
2-Chloro-4-(4-bromophenyl) thiazole (27 mg, 0.1 mmol) was dis-
solved in anhydrous ethanol (1 ml) at room temperature. A solution
(17c).
of cyclohexylamine (11.5 ml, 0.1 mmoli) was added, and the reaction
was heated to reflux a T ¼ 95o-100ꢀ for 24 h.
were supplemented with 10% fetal calf serum, 2 mM
100 U/ml penicillin, and 100 g/ml streptomycin.
L-glutamine,
Once the reaction was complete, the mixture was extracted with
EtOAc (3 ꢂ 3 ml) and the organic phases were dried under vacuum.
The product was purified by preparative HPLC, the peak of interest
was collected, concentrated to obtain title compound.
(5 mg, 14%)
m
4.2.2. Fluorescence assay for CFTR activity
CFBE41o- and FRT cells with expression of mutant CFTR and HS-
YFP were plated on clear-bottom 96-well black microplates
(Corning Life Sciences, Acton, MA) at a density of 50,000 cells/well
and kept at 37 ꢀC in 5% CO2 for 24 h. For the corrector assay,
CFBE41o-cells were treated for further 24 h with AATs and/or VX-
809. After treatment, the culture medium was removed and cells
1H NMR (300 MHz, CDCl3):
d
7.76 (dd, J ¼ 7.5, 1.5 Hz, 2H), 7.63
(dd, J ¼ 7.5, 1.5, Hz, 2H), 2.57 (m, J ¼ 7 Hz,1H), 1.65-1.56 (m, 4H),1.47
(m, 2H), 1.21-1.18 (m, 4H).
13C NMR (75 MHz, CDCl3):
d 164.7, 150.2, 135, 132.1, 130, 128.3,
127.9, 123.1, 104, 54.4, 32.9, 32.9, 25.7, 25.4, 25.1.
in each well were stimulated for 30 min at 37 ꢀC with 60
(containing 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM
KH2PO4,1 mM CaCl2, and 0.5 mM MgCl2) plus forskolin (20 M) and
ml PBS
ESI-MS m/z ¼ 339.0 [MþH]þ.
m
4.1.5.28. 4-(4-bromophenyl)-2-(piperidin-1-yl)thiazole
(17d).
genistein (50 mM). For determination of potentiator activity on
2-Chloro-4-(4-bromophenyl)thiazole (27 mg, 0.1 mmol) was dis-
solved in of anhydrous ethanol (500 L) at room temp. A solution of
F508del-CFTR, CFBE41o-cells were incubated for 24 h at 27 ꢀC to
allow trafficking of the mutant protein to plasma membrane. Cells
were then stimulated with for 30 min with PBS containing forskolin
m
piperidine (9.8
ml, 0.1 mmol) was added, and the reaction was
heated to reflux a T ¼ 95o-100ꢀ for 6 h.
(20 mM) plus the compound to be tested at the desired concen-
Once the reaction was complete, the mixture was extracted with
EtOAc (3 ꢂ 3 ml) and the organic phases were dried under vacuum.
The product was purified by preparative HPLC, the peak of interest
was concentrated to obtain 17d.
tration. For determination of potentiator activity on G551D-CFTR,
FRT cells were directly stimulated with forskolin plus compound
cocktail without previous incubation at low temperature. At the
time of assay, microplates carrying CFBE41o- or FRT cells were
transferred to a microplates reader (FluoStar Galaxy; BMG Labtech,
(27 mg, 83%)

34
E. Pesce et al. / European Journal of Medicinal Chemistry 99 (2015) 14e35
Offenburg, Germany). The plate reader was equipped with high-
quality excitation (HQ500/20X: 500 10 nm) and emission
(HQ535/30M: 535 15 nm) filters for YFP (Chroma Technology,
Acknowledgements
This work was supported by Ministero della Salute (Ricerca
Finalizzata: GR-2009-1596824; Ricerca Corrente: Cinque per mille),
Telethon Foundation (GGP10026), and Fondazione Italiana Fibrosi
Cistica (FFC#2/2012). Authors would like to thank Dr. Maria Lom-
bardi for her technical assistance in molecular modeling studies.
Brattleboro, VT). The assay consisted of a continuous 14 s fluores-
cence reading with 2 s before and 12 s after injection of an iodide-
containing solution (165 m
l of a modified PBS containing Iꢁ instead
of Clꢁ; final Iꢁ concentration in the well: 100 mM). Data were
normalized to the initial background-subtracted fluorescence. To
determine fluorescence quenching rate associated with Iꢁ influx,
the final 10 s of data for each well were fitted with an exponential
function to extrapolate initial slope (dF/dt).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.05.030.
4.2.3. Patch-clamp recordings for CFTR activity
Whole-cell membrane currents were recorded in CFBE41o-cells
with stable expression of F508del-CFTR. The extracellular (bath)
solution had the following composition: 150 mM NaCl, 1 mM CaCl2,
1 mM MgCl2, 10 mM glucose, 10 mM mannitol, 10 mM Na-HEPES
(pH ¼ 7.4). The pipette (intracellular) solution contained 120 mM
CsCl, 10 mM TEA-Cl, 0.5 mM EGTA, 1 mM MgCl2, 10 mM HEPES,
40 mM mannitol, 1 mM ATP (pH 7.4). During experiments, the
membrane capacitance and series resistance were analogically
compensated using the circuitry provided by the EPC7 patch-clamp
amplifier. The usual protocol for stimulation consisted in 600 ms-
long voltage steps from ꢁ100 to þ100 mV in 20 mV increments
starting from a holding potential of ꢁ60 mV. The waiting time
between steps was 4 s. Membrane currents were filtered at 1 kHz
and digitized at 5 kHz with an ITC-16 (Instrutech) AD/DA converter.
Data were analyzed using the Igor software (Wavemetrics) sup-
plemented by custom software kindly provided by Dr. Oscar Moran.
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