Discovery of novel VX-809 hybrid derivatives as F508del-CFTR correctors by molecular modeling, chemical synthesis and biological assays

Article abstract of DOI:10.1016/j.ejmech.2020.112833

Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. It is caused by different mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene, with F508del being the most common. During the last y

Full text of DOI:10.1016/j.ejmech.2020.112833

European Journal of Medicinal Chemistry 208 (2020) 112833
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of novel VX-809 hybrid derivatives as F508del-CFTR
correctors by molecular modeling, chemical synthesis and biological
assays
,
,
Alice Parodi a 1, Giada Righetti b 1, Emanuela Pesce c, Annalisa Salis d, Bruno Tasso b,
Chiara Urbinati e, Valeria Tomati c, Gianluca Damonte a d, Marco Rusnati e,
,
Nicoletta Pedemonte c, Elena Cichero d **, Enrico Millo a
,
, d, *
a Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132, Genoa, Italy
b Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, 3,
16132, Genoa, Italy
c UOC Genetica Medica, IRCCS Istituto Giannina Gaslini, Genova, Italy
d Center of Excellence for Biomedical Research (CEBR), University of Genoa, Viale Benedetto XV 9, 16132, Genoa, Italy
e Department of Molecular and Translational Medicine, University of Brescia, 25123, Brescia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. It is
caused by different mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene, with
F508del being the most common. During the last years, small-molecule therapy chosen to contrast CF
relied on compounds that correct CFTR misfolding and ER retention (correctors such as VX-809), or
defective channel gating (potentiators such as VX-770). Combination therapy with the two series of
drugs has been applied, leading to the approval of several multi-drugs such as Orkambi.
Despite this, this treatment proved to be only partially effective making the search for novel modu-
lators an urgent need to contrast CF. Recently, we reported compound 2a as reference compound of a
series of aminoarylthiazole-VX-809 hybrid derivatives exhibiting promising F508del-CFTR corrector
ability. Herein, we report exploring the docking mode of the prototype VX-809 and of 2a in order to
derive useful guidelines for the rational design of novel optimized analogues. To demonstrate experi-
mentally their effective F508del-CFTR-binding and rescuing potential, the most promising derivatives
had been synthesized and evaluated in biological assays including YFP functional assay on F508del-CFTR
CFBE41o-cells, trans epithelial electrical resistance (TEER) and surface plasmon resonance (SPR). This
multidisciplinary strategy led to the discovery of a second series of hybrids including 7j and 7m endowed
with higher potency than the prototype.
Received 16 June 2020
Received in revised form
4 September 2020
Accepted 5 September 2020
Available online 12 September 2020
Keywords:
Cystic fibrosis
F508del-CFTR
CFTR Corrector
Aminoarylthiazole
Docking
Surface plasmon resonance
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
inherited mutations in a gene that encodes the CF transmembrane
conductance regulator (CFTR) protein, an ATP-binding cassette
Cystic Fibrosis (CF) is a lethal monogenic disease caused by
(ABC) transporter that functions as a Clꢀ channel with complex
Abbreviations: CF, cystic fibrosis; CFTR, Cystic Fibrosis transmembrane conductance regulator protein; AAT, aminoarylthiazole; DMAP, 4-(N,N-dimethylamino)pyridine;
DIPEA, N,N-Diisopropylethylamine; HATU, Hexafluorophosphate azabenzotriazole tetramethyl uronium; BOC2O, di-tert-butyl carbonate; HPLC, high performance liquid
chromatography; ESI-MS, electrospray mass spectrometry; HS-YFP, halide-sensitive yellow fluorescent protein; TEER/PD, Trans-epithelial electrical resistance and potential
difference measurements; SPR, Surface Plasmon Resonance.
* Corresponding author. Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132, Genoa, Italy.
** Corresponding author. Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genoa, Viale Benedetto
XV, 3, 16132, Genoa, Italy.
E-mail addresses: cichero@difar.unige.it (E. Cichero), enrico.millo@unige.it (E. Millo).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2020.112833
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
regulation [1]. These mutations cause improper localization and
functioning of this chloride channel in lung, pancreas, and intestine
thus affecting the normal fluid homeostasis [1]. In CF the lungs are
the most affected organ due to the accumulation of thick mucus,
which results into bacterial load and associated chronic inflam-
mation [2].
More than 2000 mutations have been identified in the CFTR
gene, most of them being very rare, although a pathogenic effect
has been demonstrated only for about 350 variants. By far the most
common disease-causing mutation is F508del, the deletion of the
phenylalanine residue at position 508 of the CFTR amino acid
sequence [3].
a library of compounds synthesized coupling 1-(2,2-difluorobenzo
[d] [1,3]-dioxol-5-yl)cyclopropanecarboxylic acid with various
amine monomers, as described by Wang et al. [28].
Maintaining the benzodioxole group appears a recurrent strat-
egy also taking into account the results obtained with GLPG-2222
[28] and with other correctors described by AbbVie and Gal-
apagos companies, in which this effective substituent was tethered
to the chroman ring or to the tetrahydropyran ring [29].
Our in-house set of compounds has been defined “hybrid
compounds” since their structures were really thought of as a
hybrid between the AAT and the VX-809 scaffolds. Among them, we
identified a number of analogues featuring EC50 values in terms of
Although F508del affects only one residue located on the surface
of the first nucleotide-binding domain (NBD1) [4], structural
studies reveal that the absence of this crucial amino acid could
cause structural changes to the ABC-alpha subdomain of NBD1 [5].
Indeed, loss of Phe-508 breaks up domain-domain interactions
critical for correct assembly and function of CFTR [6e11]. F508del-
CFTR is recognized as abnormal by the endoplasmic reticulum
quality control machinery, leading to degradation by the protea-
some [12,13]. Thus, F508del mutation principally disrupts the
processing and intracellular transport of CFTR protein. Neverthe-
less, any F508del-CFTR that escapes to the plasma membrane can
present two further problems: reduced stability [14] and altered
channel gating [15]. As a consequence, novel state-of-the-art
therapies are needed to address all these problems.
To overtake the defective expression and function of CF mutants,
administering CFTR modulators has been applied. In detail, a CFTR
modulator is a pharmaceutical agent that targets a specific defect in
the CFTR protein. This modulator does not correct mutations in the
gene but rather acts to solve the errors that occur post transcrip-
tionally, either during protein folding, trafficking up to the plasma
membrane, or CFTR functioning. The CFTR modulators can be
classified in different classes following their different mechanisms
of action.
In particular, two classes of small molecules have been devel-
oped named CFTR correctors and CFTR potentiators [12].
CFTR correctors (eg Lumacaftor; VX-809) [16] allow the mutant
proteins to traffic to the plasma membrane while CFTR potentiators
(e.g. Ivacaftor; VX-770) [17] strongly increase channel gating. Since
neither Lumacaftor nor Ivacaftor, by themselves, seem to have
clinical benefit for CF patients with the F508del mutation [18,19],
combination therapy with the two drugs has been applied. This
improved lung function and disease stability [20], leading to the
approval of lumacaftor-ivacaftor combination therapy (named
Orkambi). Dual-combination therapies such as Orkambi and Sym-
deko (containing the corrector Tezacaftor, i.e. VX-661, an analogue
of VX-809, together with Ivacaftor) have been approved for healing
lung function to a modest degree in F508del homozygous patients
[21]. Triple combinations using two distinct correctors and a
potentiator showed good clinical benefit in patients with a
F508del-CFTR mutation on at least one allele [22e24]. However, the
effectiveness of these combinations through the multitude of ge-
notypes remains to be evaluated.
CFTR rescue ability comprised between 0.09 and 0.2
mM, with
compound 2a being the most active (EC50 ¼ 0.087
m
M) (Fig. 1) [25].
On this basis, herein we discuss the design and discovery of a
new library of optimized hybrids, displaying ameliorated potency
values (EC50 values spanning from 0.02 to 0.1 mM), which have been
obtained by modifying some groups on the main core of the
compounds.
In particular, we proceeded with molecular docking studies
focused on the previously identified hybrid prototype 2a, in order
to gain more information about the putative binding mode of the
corrector to the CFTR NBD1 domain. This allowed us to better
explore the structure-activity relationship (SAR) within the hybrid
series and to point out novel beneficial chemical substitutions to
decorate the thiazole ring. In addition, the selection of the most
promising compounds to be synthesized has been performed also
taking into account the information and mathematical prediction
of the QSAR model we previously built and published [25].
The following synthetic efforts, combined with the biochemical
evaluation of the compounds as effective F508del-CFTR binders
through surface plasmon resonance (SPR) and their subsequent
biological evaluation as F508del-CFTR correctors, validated the
effectiveness of the hybrid core in the search for novel potent CFTR
modulators. The overall results have provided novel SAR informa-
tion on hybrids as correctors and led to the identification of mol-
ecules with a particular ability to rescue F508del-CFTR.
2. Results and discussion
2.1. Rational design of novel hybrids as F508del-CFTR correctors
In our previous work, we reported QSAR analyses on different
series of correctors reported in the literature [25]. This allowed us
to identify eight descriptors explaining the corrector ability range
of the library, being five of them bi- and three-dimensional de-
scriptors (2D and 3D). These included parameters related to atoms
and bound counts (b_single, a_IC and a_nH) and surface area de-
scriptors (Vsurf_DD12 and Vsurf_W8). The connectivity-based
descriptor chi1, the so-called distance matrix parameter wei-
nerPol and the potential energy descriptor E_nb were also retained
[25].
As we discussed, these promoted the design of branched scaf-
folds enriched with flexible substituents and quite polar moieties
within overall limited and bulky conformations. Accordingly, we
designed and synthesized a number of hybrids endowed with a
promising CFTR rescue ability, with 2a being the most potent.
Beyond QSAR prediction, herein we proceeded with preliminary
docking studies in order to gain further useful information to guide
the rational design of new 2a analogues. These calculations have
been performed taking into account the theoretical studies so far
disclosed in the literature concerning the reference corrector VX-
809. Indeed, a number of in silico studies deeply investigated the
putative binding mode of VX-809 to different portions of the CFTR
protein, providing support to multiple sites involved in the
In the search for new drugs for CF treatment, in our previous
works we reported the rational design, chemical synthesis and
biochemical characterization of a novel library of aminoarylthiazole
(AAT) and VX-809 hybrid derivatives, exhibiting interesting prop-
erties as F508del-CFTR correctors [25].
This kind of substitution proved also to be effective as shown by
other analogues of VX-809 reported in the literature, such as the
two highly related congeners ALK-809 and SUL-809 featuring an
amide and an acylsulfonamide function instead of the carboxylic
moiety of the prototype (Fig. 1) [26], VX-661 (Tezacaftor), suc-
cessfully evaluated on patients in combination with VX-770 [27], or
2

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Fig. 1. Chemical structure of VX-809, ALK-809, SUL-809 and of the hybrid 2a. The structural variation experienced by 2a and by ALK-809, SUL-809 with respect to the prototype VX-
809 are highlighted in cyan and green, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
corrector mechanism of action [30].
benzodioxole core. The negatively-charged carboxylic group of the
corrector is H-bonded to the backbone of E656. The overall posi-
tioning recognized as POS2 allowed VX-809 to occupy the same
binding pocket described for POS1, since the aromatic rings and all
the substituents are placed in proximity to the previously cited
G461, V603, A655 and N659.
Accordingly, while data reported in the literature surmised VX-
809 directly targeting the full-length F508del-CFTR protein, with a
possible binding site at the NBD1-ICL4 interface [31], other findings
suggested that VX-809 may stabilize an N-terminal domain in CFTR
that contains membrane-spanning domain 1 [32].
On this basis, several virtual screening approaches moved the
discovery of novel F508del correctors exploring the NBD1:NBD2
interface so as the MSD: NBD1, supporting a fundamental role
played by NBD1 at the corrector binding site [33].
Conversely, docking calculations performed on ALK-809 and
SUL-809 revealed a unique putative binding mode in agreement
with the previously named POS2 of VX-809. As shown in Fig. 3, both
compounds display one H-bond between the (sulfon)amide moiety
and the backbone of E656 while the oxygen atoms of the benzo-
dioxole ring feature polar contacts with K464 and T465. Finally, the
amide group linked at the main core of the correctors is projected
towards N659 with H-bonds. Since VX-809 as well as the two an-
alogues ALK-809 and SUL-809 exhibit comparable potency as CFTR
correctors, it could be inferred that POS2 represents the bioactive
conformation of VX-809 analogues.
Docking calculations performed around our first series of hy-
brids proved to be in agreement with POS2 proposed for the
aforementioned VX-809 analogues and also allowed us to better
explain the SAR within this series of promising correctors.
As shown in Supporting information S2 for 5-unsubstituted
AATs, the introduction of a p-substituted phenyl ring linked at
position 4 of the main thiazole was preferred leading to compounds
featuring higher potency values than those of the m-substituted
analogues.
Conversely, an opposite trend can be observed when a methyl
group or ester moiety decorates position 5 of the AAT, making the
m-substitution more effective. In addition, a perspective of all the
results underlines a comparable behavior exhibited by those
compounds showing the benzodioxole ring instead of the fluori-
nate analogue, and a beneficial role played by concomitant sub-
stitutions at positions 4 and 5 of the thiazole main core [25]. In
particular, derivatives 2a-2c displayed higher potency values
within this series of correctors, with 2b-2c being characterized by
an ester moiety linked to position 5 of the thiazole. The most active
analogue 2a displayed a benzoyl group at the same position.
Docking calculations of these compounds revealed that for all of
them the carboxamide moiety is involved in one H-bond with
N659, while the two oxygen atoms of the benzodioxole ring were H
In this context, our research group recently reported surface
plasmon resonance (SPR) studies on F508del-NBD1 performed
with the corrector VX-809 and molecular docking and dynamics
simulations, highlighting a statistically significant correlation be-
tween the observed binding capability to F508del-NBD1 and the
results of the computational methods. On the whole, these results
demonstrated a strong agreement between the theoretical pre-
diction and the experimental SPR-based data, supporting a key role
played at the NBD1 level of CFTR by the corrector VX-809 to exert
any CFTR rescue ability [34].
Thus, in order to better investigate the putative mechanism of
action of this corrector at the biological target, we applied molec-
ular docking studies around the human F508del-CFTR NBD1
domain, taking VX-809 and two analogues ALK-809 and SUL-809 as
reference compounds (as scoring parameters, the predicted energy
values of the NBD1-corrector complexes were listed in Supporting
information S1) [26]. In particular, these two congeners are char-
acterized by an amide and a sulfonamide function instead of the
carboxylic moiety of the prototype, lacking the possibility to exhibit
a negatively charged group.
Based on docking calculations, two putative binding positioning
for the corrector VX-809, namely POS1 and POS2, can be described.
As shown in Fig. 2A (POS1), the oxygen atom of the carboxamide
group of VX-809 is involved in one H-bond with the N659 side-
chain, while the carboxylic group is engaged in ionic contacts with
K464 and H-bonds with T460 and G461. The aromatic rings of the
corrector occupy the protein pocket delimited by S573, G576, V603
and A655, exhibiting van der Waals contacts while the benzo-
dioxole core is projected towards Y577 and E656.
Concerning POS2 (Fig. 2B), two H-bonds with the key residues
K464 and T465 are formed by the two oxygen atoms of the
-bonded to K464 and T465. In the case of 2b (EC50 ¼ 0.30
mM) and
3

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Fig. 2. Selected docking poses of VX-809 within the CFTR hNBD1 domain as POS1 (A) and POS2 (B). The most important residues are labelled.
Fig. 3. Selected docking poses of ALK-809 (C atom; pink) (A) and SUL-809 (C atom; purple) (B) within the CFTR hNBD1 domain, both in agreement both POS2 of VX-809 (C atom;
green). The most important residues are labelled. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
2c (EC50 ¼ 0.55
m
M), the presence of the quite flexible ester group at
the thiazole core. In addition, structural variations involving the
substituent linked to position 4 of the thiazole ring could be
managed in an attempt to mimic the methyl group of corrector VX-
809.
Herein we report the chemical synthesis and biological evalu-
ation of 2a analogues focusing on variations of the benzoyl core
(compounds 7a-7z, 12a-12d, 24a-24c), whose potency profile in
terms of CFTR rescue ability have been predicted by means of the
QSAR model we previously built prior to synthesis (Supporting
information S4).
position 5 of the thiazole guides the positioning of the compounds
based on the concomitant m- or p-substituted phenyl ring tethered
to position 4 of the AAT with H-bonding of the hydroxyl group of
the Y577 sidechain or the E656 backbone (Supporting information
S3) via the oxygen atom of the carbonyl group.
However, based on the promising potency of both correctors, it
is thought that H-bonding to N659, K464 and T465 represents a key
requirement of correctors, while interacting with E656 or Y577
could efficiently stabilize the modulator within the hNBD1 domain.
The most probable docking mode of 2a (EC50 ¼ 0.087
mM)
proved to be in agreement with those of VX-809 (POS2) and SUL-
809, as shown in Fig. 4.
2.2. Chemistry
The carboxamide moiety and the benzodioxole ring of the
hybrid highly simulate the binding mode proposed for the proto-
type VX-809 and for the related congener SUL-809, showing the
same contacts. On the whole, the thiazole ring proved to be a good
bioisoster of the pyridine one maintaining the previously
mentioned interactions with the biological target.
Notably, hydrophobic interactions displayed by the benzoic
group of VX-809 and by the sulfonamide moiety of SUL-809 are
experienced by the two aromatic rings connected to the 2a thiazole
core while the oxygen atom of the benzoyl group interacts with the
E656 side-chain, as reported for the two reference compounds.
This information opened the possibility to explore the role
played by different substitutions on the benzoyl moiety of 2a in
order to clarify the relevance of hydrophobic and polar contacts
especially with Y577, and the possibility of modifying the carbonyl
function with other H-bond acceptor moieties properly tethered to
To synthesize all the compounds we applied a known synthetic
route with appropriate modifications [25]. To obtain the benzo-
dioxole substituted portion we use the well-known cyclo-
propanation of active methylene compounds as previously
published [25].
The substituted aminoarylthiazoles are synthesized in different
ways depending of the substituents at position 5 of the thiazole
ring.
For the general structures of (2-amino-4-arylthiazol-5-yl) (aryl)
methanone derivatives a convergent synthesis was developed
(Scheme 1) in accord with the Wang protocol with minor modifi-
cations [35]. To obtain a selective introduction of the substituent
groups on the thiazole ring a protected carbamothioyl amide was
conjugated with substituted
a-bromoketone. A mixture of 1-[4-
(methoxy)phenyl]methanamine 1 and 4-(methoxy)benzaldehyde
2 was heated in refluxing methanol to obtain bis((4-(methoxy)
4

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Fig. 4. Docking mode of 2a (C atom; blue) in comparison with that of VX-809 (C atom; light green) (A) and of SUL-809 (C atom; purple) (B) at the hNBD1 domain of the F508del-
CFTR mutant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
phenyl)methyl)amine 3.
Protected carbamothioyl amide
condensation of benzoylisothiocyanate and bis ((4-(methoxy)
carboxylic acid to its uronium salt using standard peptide coupling
conditions and its subsequent treatment with aniline, in the pres-
ence of diisopropylethylamine in N, N-DMF afforded the anilide
derivative 10, which, on further deprotection of the BOC-protecting
group in trifluoroacetic acid afforded the aminothiazole 11.
The final compound 12a was then obtained by condensation
with the carboxylic group of cyclopropanecarboxylic acid deriva-
tive 6 as previously described.
To prepare 12b a different approach was used. N-(2-amino-4-(4-
methoxyphenyl)thiazol-5-yl)benzamide was prepared through a
two-step synthesis starting from alfa-aminoacetophenone deriva-
tive 14 (Scheme 3). The starting reagent was prepared from 4-
methoxyphenacyl bromide 13 following Delepine reaction (hexa-
4 was then obtained by
phenyl) methyl) amine 3 in high yield.
Condensation of protected carbamothioyl amide and
a-halo
ketone followed by deprotection, afforded the thiazolic derivatives
5, which were then further condensed with 1-(benzo[d] [1,3]
dioxol-5-yl)cyclopropanecarboxylic acid 6 to produce the desired
analogues 7a-7z.
This last synthetic step was achieved by reaction of 2-amino-
thiazole with the carboxylic group of cyclopropane carboxylic acid
derivatives with uronium salt activation in anhydrous solvents [25].
For compound 12a a different route was applied (Scheme 2).
Starting from ethyl 2-amino-4-aryllthiazole-5-carboxylate 8, t-Boc
protection at the aminic group was conducted to minimize decar-
boxylation of the thiazole ring. The protection with t-Boc also
provided a high solubility of the molecule in organic solvents,
allowing for high reactivity of the carboxyl group.
methylenetetramine
in
diethyl
ether)
as
4-
methoxybenzoylmethylammonium chloride salt. Acylation of the
amino functionality with benzoyl chloride proceeded efficiently to
provide N-(2-(4-methoxyphenyl)-2-oxoethyl)benzamide 15. Its
bromination and treatment with the thiourea, following the general
Hantzsch cyclocondensation pattern, was tried but without success.
The very low yield of this procedure is due to the difficulty of
selectively monobromination of derivative 15 as well as isolating the
To prepare the carboxamide derivative 10 the appropriate 2-
amino-thiazole-5-carboxylate 8 was treated with di-tert-butyl
carbonate in the presence of 4-(N,N-dimethylamino)pyridine in
tetrahydrofuran to form the corresponding tert-butyl carbamate
that upon basic hydrolysis afforded the acid 9. Conversion of the
respective
unstable
intermediate
N-(1-bromo-2-(4-
methoxyphenyl)-2-oxoethyl)benzamide.
Scheme 1. Reagents and conditions: (a) methanol, reflux, 3 h; (b) NaBH4, 0 ꢁC to rt, 10 h; (c) Benzoylisothiocyanate, acetone, 0 ꢁC, 1 h; (d) RCOCH2Br, N,N-DMF, 85 ꢁC, 3 h; (e) TFA,
80 ꢁC, 36 h; (f) HATU, DIPEA, N,N-DMF, 50 ꢁC, 18e24 h. For R and complete structures see Table 2.
5

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Scheme 2. Reagents and conditions: (a) BOC2O, DMAP, THF, rt, 4 h; (b) KOH 6 N, THF:EtOH ¼ 1:1.5, 55 ꢁC, 4 h; (c) HATU, DIPEA, N,N-DMF, rt, 5 min; (d) PhNH2, 40 ꢁC, 2 h; (e)
TFA:DCM ¼ 1:1, 0 ꢁC, 4 h; (f) 6, HATU, DIPEA, N,N-DMF, 50 ꢁC, 24 h.
There was a significant increase in yield with the one pot
condensation of N-(2-(4-methoxyphenyl)-2-oxoethyl)benzamide
with thiourea and iodine using triethylamine as catalyst under mild
conditions as reported in the literature [36].
The synthesis of amide derivatives of 2-aminothiazole was
achieved by condensation reaction of the 2-amino-thiazole with
the carboxylic group of cyclopropanecarboxylic acid derivative 6 as
previously described.
relationships as correctors of F508del-CFTR (Tables 1e3).
By using the YFP functional assay on F508del-CFTR CFBE41o-
cells, we tested the compounds after 24 h incubation at different
concentrations to extrapolate the EC50 values of the compounds as
correctors of mutant CFTR (Tables 1e3). Activity, after incubation
with these hybrids, of F508del-CFTR in the plasma membrane was
determined by measuring the rate of HS-YFP quenching caused by
iodide influx as previously published [25]. Activity was then
compared to that of cells treated with vehicle alone (DMSO) or with
For the synthesis of the compounds 12c and 12d, our synthetic
strategy hinged on a reaction between a substituted thiazole in
position 5 and cyclopropanecarboxylic acid derivative. The func-
tionalized thiazoles used for the substitution step were obtained as
shown in Scheme 4 via adaption of reported thiazole synthesis [37].
Thiourea 17 was quantitatively activated as bis-thiazadiene 18 with
excess of commercially available N,N-dimethylformamide dime-
thylacetal in methanol. The thiazole ring was formed after addition
of the corresponding alfa-bromoketones 19a and 19b in THF. In-
termediates 20a-20b, which were not isolated, were deprotonated
in situ by addition of triethylamine. The second imine, used here as
a protecting group, was subsequently removed in situ with
methylamine in water to form the expected products 21a and 21b
in good yield.
The final products 12c and 12d were obtained by condensation
of the 2-amino-thiazole with the carboxylic group of cyclo-
propanecarboxylic acid derivatives as previously described.
To synthesize derivatives 24a, 24b and 24c we applied the same
synthetic route used in Scheme 1 but starting from aliphatic, het-
eroaromatic and heterocycle haloketones 22a, 22b and 22c
(Scheme 5).
the known corrector VX-809 (1 mM).
Based on our previous study [25], in which we had modified the
thiazolic ring at different positions, we have identified that the
most active compound 2a (EC50 ¼ 0.087
mM) was characterized by a
substitution at position 5 of the thiazole ring with benzoyl group
and phenyl ring linked at the thiazole position 4 (Fig. 1).
In addition, we have already demonstrated that substitution at
position 5 of the thiazole ring was necessary for a good activity
since the most active derivatives of the previous series all contained
a substituent at position 5.
In fact, the presence of a bulky substituent (benzoyl or ethyl
acetate) at C-5 might be useful in terms of improving some phar-
macokinetic characteristics, while the derivatives that contain no
substitution led to a decrease in corrector activity [25].
First, removal of the keto group present in the hit compound or
the substitution with the amidic one led to a weak decrease in
activity with respect to the benzoyl analogues: compare 12a
(EC50 ¼ 1.26
m
M) and 12b (EC50 ¼ 1.76
mM) vs. 2a (Table 1).
Furthermore, we have demonstrated that the C-4 aryl group of
the 2-aminothiazole is crucial for activity, as its removal [12c
(EC50 ¼ 2.57
m
M) and 12d (EC50 ¼ 9.3
m
M)] led to decreasing activity
M) (see Table 1).
with respect to the progenitor (EC50 ¼ 0.087
m
2.3. Effect of compounds as correctors of F508del-CFTR trafficking
defect
Thus, we chose to initially build a series of substituted analogues
Table 2, keeping intact, for comparative purpose, the phenyl ring at
position 4.
First, we investigated substitutions of the phenyl ring at position
5 starting from the para position.
Our goal was to identify and characterize potent and selective
correctors of F508del-CFTR trafficking defect.
To verify our considerations all designed compounds were then
synthesized and tested to investigate the structure-activity
A
number of different electron-withdrawing groups were
Scheme 3. Reagents and conditions: (a) hexamethylenetetramine, diethyl ether, rt, 2 h; (b) HCl conc, EtOH, reflux, 3 h; (c) C6H5COCl, NaHCO3, THF, rt, 3 h; (d) thiourea, I2, EtOH, TEA,
80 ꢁC, overnight; (e) 6, HATU, DIPEA, N,N-DMF, 50 ꢁC, 24 h.
6

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Scheme 4. Reagents and conditions: (a) N,N-Dimethyl formamide dimethyl acetal, DCM, reflux, 4 h; (b) THF, TEA, rt to reflux, 18 h for 20a, 20 h for 20b; (c) 33% aq CH3NH2, THF, rt,
24 h; (d) HATU, DIPEA, N,N-DMF, 50 ꢁC, 24 h for 12c, 36 h for 12d.
Scheme 5. Reagents and conditions: (a) N,N-DMF, 85 ꢁC, 2 h; (b) TFA, 80 ꢁC, 36 h; (c) 6, HATU, DIPEA, N,N-DMF, 50 ꢁC, 18 h for 24a, 24 h for 24b and 24c.
certain activity 7c (EC50 ¼ 0.41
m
M) while the carboxylic 7d caused
Table 1
a decrease in activity (EC50 ¼ 7.31
mM). The amidic group, as in
Chemical structure and potency profile of the novel hybrids 12a-12d as F508del
CFTR correctors.
compound 7e, is really deleterious for activity.
To understand if the para position is important for F508del-CFTR
rescue, we introduced electron-donating groups in the same posi-
tion. Incorporation of alkyl amino as in 7f (EC50 ¼ 0.45
in 7g (EC50 ¼ 0.43 M) were not seen to improve the activity; so we
chose to insert a methoxy group like in 7h but no increase of ac-
tivity was observed (EC50 ¼ 0.45 M). A similar result was obtained
M). We tried to
mM), alkyl as
m
m
with a trifluoromethoxy group 7i (EC50 ¼ 0.36
m
increase the hydrophobic portion of the substituent modifying the
length of the alkyl chain like in derivative 7j. Surprisingly the
substitution of methyl group with a propyl one ameliorates the
Compound
X
R
Y
EC50
1.26
(mM)
activity by about five times (EC50 ¼ 0.017
the position of propoxy group from para to meta 7k caused a
M in
Table 2). This characteristic could reveal a striking preference for
hydrophobic groups so we decide to deepen this aspect using steric
hindrance in the structure.
mM). Curiously, changing
CONH
12a
reduction of activity (EC50 ¼ 0.017
m
M vs EC50 ¼ 0.165
m
NHCO
1.76
12b
Three different compounds were designed and built with a
second aromatic ring like thiophene for 7l (EC50 ¼ 1.06
phenyl for 7m (EC50 ¼ 0.07 M) or hetero aliphatic ring 7n
M). As shown in Table 2 compound 7m led to an
increase in activity with an EC50 better than the reference com-
pound 2a.
mM) or
CO
CO
-H
-H
2.57
9.30
m
12c
12d
(EC50 ¼ 0.37
m
Interestingly, compound 7o which contained a phenyl group at
the meta-position showed moderate activity (EC50 ¼ 0.25
mM)
compared with compound 7m where the same group was at the
para position, in line with our expectations.
On the contrary, moving the methoxy group from the para 7h to
the ortho 7p and to the meta position 7q led to a significant increase
anchored at this position such as
(EC50 ¼ 0.10
a
methylthio group 7a
M) but only the first
in activity (EC50 ¼ 0.14
m
M and EC50 ¼ 0.10
mM).
m
M) or bromine 7b (EC50 ¼ 0.53
m
A similar trend was observed using a bromine in the meta po-
was well tolerated and represents one of the most promising hy-
brids of this series. The same considerations can be made with
three different compounds containing ester, carboxylic or amidic
groups. In particular, only the ethyl ester derivative maintains a
sition 7r (EC50 ¼ 0.18
(see 7b EC50 ¼ 0.53
mM) which was better than the para position
mM in Table 2). Changing the bromine to
fluorine in the meta position caused increased potency in 7s
7

Table 2
Table 2 (continued )
Chemical structure and potency profile of the novel hybrids 7a-7z as F508del CFTR
correctors.
Compound
R
EC50
0.07
(mM)
7m
0.37
0.25
7n
7o
Compound
R
EC50
0.10
(mM)
7a
0.14
0.10
0.53
0.41
7p
7q
7b
7c
7.31
0.18
0.13
0.11
7d
7r
7s
7t
NA
7e
7f
0.21
0.40
0.41
0.28
7u
7v
7w
7x
0.43
0.45
7g
7h
0.36
7i
13.70
1.60
0.017
0.16
7j
7k
7y
7z
1.06
7l
0.11
8

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Table 3
mutation, using an electrophysiological technique: the trans
epithelial electrical resistance and potential difference measure-
ments (TEER/PD). We tested 7a and 7m, two of the most potent
derivates described in this study. Epithelia were treated for 24 h
with test compounds at different concentrations: 7a
Chemical structure and potency profile of the novel hybrids 24a-24c as F508del
CFTR correctors.
(5e0.5e0.05 mM), 7m (10 - 1e0.1 mM), VX-809 1 mM (as positive
control) or vehicle alone (DMSO; negative control) and then
assayed. Trans epithelial electrical resistance was measured before
and after stimulation with forskolin (20
to totally activate CFTR. It was then measured after addition of the
CFTR inhibitor PPQ-102 (30 M) to fully block CFTR activity. The bar
mM) plus genistein (50 mM)
m
graphs of Fig. 5 show the delta between the values of electrical
resistance measured before and after CFTR inhibition, converted
into its reciprocal conductance. Long-term treatment of CF primary
bronchial epithelia with 7a and 7m significantly increased both
CFTR-mediated conductance and equivalent short-circuit current.
We then evaluated the rescue of processing defect biochemi-
cally by means of the electrophoretic mobility of CFTR protein. In
Western blots, CFTR protein is detected as two bands, named B and
C, of approximately 150 and 170 kDa, respectively. Band B corre-
sponds to partially glycosylated CFTR residing in the ER, while band
C is the fully processed (mature) CFTR that has passed through the
Golgi. The prevalent form in cells expressing wild-type CFTR is
band C. Lysates of cells expressing F508del-CFTR show primarily
band B, consistent with the severe trafficking defect caused by the
mutation (Fig. 6A). To evaluate the effect of hybrid compounds on
CFTR electrophoretic mobility, we treated F508del-CFTR/HS-YFP
expressing CFBE41o-cells with DMSO (vehicle alone) or test com-
Compound
R
EC50 (mM)
CH3
2.72
0.79
24a
24b
24c
8.50
(EC50 ¼ 0.13
mM).
Given the inclination of the methoxy group for the meta/ortho
substitution, we synthesized two disubstituted analogues which
contain the methoxy group in the ortho position and the second
one in the meta and para position respectively (7t and 7u). Inter-
pounds 7m and 7a (0.5 mM) or VX-809 (1 mM, as positive control).
After 24 h, cells were lysed and lysates were subjected to SDS-PAGE
followed by western blotting. Western blot images were analyzed
with ImageJ software. For each lane, CFTR bands, analyzed as ROI,
were quantified after normalization for GAPDH to account for total
protein loading. Treatment of F508del-CFTR cells with corrector
VX-809 significantly enhanced expression of mature CFTR (band C),
resulting in a change in the C band/B band ratio. Similarly, treat-
ment with the compounds resulted in a significant increase in the C
band/B band ratio, comparable to that obtained following treat-
ment with VX-809 (Fig. 6B).
To further characterize the biological activity of hybrid com-
pounds, we tested 7a, 7j, 7m in combination with VX-809 and VX-
661, in CFBE41o-cells stably expressing F508del-CFTR by means of
the YFP functional assay (Supporting information S5). To this aim,
cells were treated for 24 h with single test compounds or their
estingly, while 4-substitution wasn’t well tolerated (EC50 ¼ 0.4
mM),
inclusion of
(EC50 ¼ 0.11
a
30-OCH3 group afforded potent analogue
mM).
Indeed, we evaluated molecules containing 3, 4-substitued
M).
benzoyl groups like 7v (EC50 ¼ 0.41
m
M or 7w (EC50 ¼ 0.28
m
In these cases, the substitution proved to be tolerated, especially
with regard to the allyl moiety. On the other hand, a high decrease
in activity was observed when one of these substituents was an
amide group as in 7x (EC50 ¼ 13.7
mM) and similarly with 7e. From
the above SAR, it was identified that an amide group was detri-
mental for activity.
Moreover, we decided to test 7y, another derivative containing
20,50-substitued benzoyl groups, considering that compound 7t
containing two methoxy groups in same positions was active
(EC50 ¼ 0.11
The replacement of methoxy groups with chlorine and hydroxyl
M). This may
mM).
displayed about 15-fold loss of potency (EC50 ¼ 1.6
m
indicate that an electron-donating group like eOCH3 at the meta
position was favorable (see 7q and 7t).
A particular compound containing a dioxane moiety 7z was at
last analyzed. The compound exhibited good corrector ability
(EC50 ¼ 0.10
mM) rather similar to that of the lead compound.
All the compounds in the libraries contain at the 5 position only
a benzoyl derivative. We tried to insert a different keto group using
aliphatic like methyl 24a, heteroaromatic like pyridine 24b and
heterocycle, morpholine in this case, 24c in the same position. Only
24b (EC50
¼
0.79
m
M) had
a
certain activity while 24a
M) led to decreasing
(EC50 ¼ 2.72
m
M) and overall 24c (EC50 ¼ 8.5
m
activity with respect to the progenitor (EC50 ¼ 0.087
mM) as
appeared in Table 3.
Fig. 5. Compounds 7a and 7m rescue F508del-CFTR activity in primary bronchial
epithelia. The graph reports the equivalent short-circuit current (calculated from TEER/
PD measurements) in F508del/F508del bronchial epithelia, treated for 24 h with test
compounds at the indicated concentration, or VX-809 (1 mM), or vehicle alone (DMSO).
Data are expressed as means SD, n ¼ 3.
The ability of the novel derivates to rescue F508del-CFTR was
assayed on well-differentiated primary cultures of human bron-
chial epithelial cells from a CF patient homozygous for the F508del
9

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Fig. 6. Biochemical analysis of the F508del-CFTR expression pattern. (A) Electrophoretic mobility of F508del-CFTR in 2 different preparations of CFBE41o-cells, treated for 24 h with
vehicle or test compounds (0.5 M) or VX-809 (1 M). Arrows indicate complex-glycosylated (band C) and core-glycosylated (band B) forms of CFTR protein. (B) The bar graph
reports the densitometric analysis (means SEM, n ¼ 3) of band C/band B ratio normalized to GAPDH. **P < 0.01 versus control (ANOVA with Dunnett’s post hoc test).
m
m
combinations and then assayed. No additive or synergistic effect
was observed when combining 7a, 7j and 7m with VX-809 or VX-
661, supporting the hypothesis that all these compounds possibly
share the same binding site (Supporting information S5).
propoxy group of 7j features hydrophobic contacts with the aro-
matic ring of Y577, being able to reproduce the positioning expe-
rienced by the sulfonamide group of SUL-809.
This kind of docking mode was shared by most of the other
promising hybrids, such as 7a featuring a thiomethyl group onto
the para position of the benzoyl substituent (Supporting infor-
mation S6), while the analogue 7m bearing the biaryl moiety as
bioisoster of the 4-(methylthio)benzoyl group displayed a switched
positioning (Fig. 9). This was in agreement with the docking pose
previously discussed for the first series of hybrids such as for 2b
(Supporting information S3), featuring 7m in any case the recur-
rent previously cited contacts displayed by the benzodioxolane ring
and H-bonds with Y577.
Conceivably, this docking mode is probably due to steric hin-
drance caused by the bulky biaryl substituent selected for this
hybrid. In any case, this positioning allowed maintaining the
aforementioned key contacts with N659, K464 and T465 while
interaction with E656 or Y577 appeared to be both involved in
stabilizing the corrector at the hNBD1 domain (Fig. 9).
2.4. Evaluation of the effective capacity of the compounds to bind
F508del-CFTR
When a compound shows F508del-CFTR rescuing-activity in cell
models, it is important to rule out off-target effects demonstrating
that its mechanism of action corresponds to a direct interaction
with the mutated protein. We then decided to evaluate the effective
capacity of 7j and of 7m to bind to F508del-CFTR exploiting the
biosensor recently described by us in which the mutated protein
was immobilized in membrane-like lipid vesicles as to resemble
the F508del-CFTR environment in vivo [38]. Injection of increasing
concentrations of 7j and of 7m onto the biosensor containing
F508del-CFTR provided saturable and dose-dependent binding
curves (Fig. 7) that permitted the calculation of Kd values equal to
38.2 5.1 and 89.3 35.3 mM for the two compounds respectively.
In the same experimental condition, VX-809, here used as a positive
control, bound F508del-CFTR with similar affinity (Kd value equal
to 72.8 19.5 mM) [33]. Thus, in agreement with docking results, 7j
3. Conclusion
and 7m effectively set up a high affinity complex with F508del-
CFTR that is responsible for the functional rescue of the mutated
channel.
In this work, we successfully applied a multi-disciplinary
approach in order to identify a new series of hybrid compounds
displaying promising F508del-CFTR corrector ability. Preliminary
molecular docking studies performed on VX-809 and 2a revealed
the most substantial key feature involved in corrector binding,
guiding the design of the second series of hybrids featuring struc-
tural variations of the benzoyl group of the prototype 2a. Based on
computational predictions, the most promising compounds had
been synthesized and evaluated as correctors of F508del-CFTR
trafficking defect. SPR results experimentally validated the com-
pound binding to CFTR protein, supporting the effectiveness of 7j
and 7m as optimized F508del-CFTR corrector hybrids. On the
whole, molecular docking of the newly synthesized correctors
revealed H-bonding K464, T465 and N659 through the benzo-
dioxole moiety and the carboxamide function as the necessary
molecular requirements identified within hybrids. However, hy-
drophobic contacts with the protein pocket delimited by Y577 and
E656 allowed improving the corrector potency. A dedicated site-
specific mutagenesis program will represent the prosecution of
this study to validate the results from our multi-disciplinary
approach.
2.5. Molecular docking studies
The results of our docking calculations support the aforemen-
tioned SAR discussion underlining a key role played by the intro-
duction of hydrophobic groups onto the para position of the
benzoyl moiety as highly effective to achieve corrector ability. On
the other hand, the selection of 3,4-substitued benzoyl groups
proved to be well-tolerated, especially in presence of electron-rich
groups (see 7v and 7w). This was motivated by the proper confor-
mation displayed by the corrector, which is able to interact effi-
ciently with E656 and to exhibit contacts with Y577. Accordingly,
the most potent compound 7j displayed a highly similar docking
positioning with those previously discussed for VX-809 and SUL-
809, giving further validation of the relevance of bulky, flexible
and quite lipophilic groups linked at the benzoyl moiety. As shown
in Fig. 8, the benzodioxole moiety of the new corrector is H-bonded
to G461, K464 and T465, while the oxygen atom of the benzoyl
group displays contacts with the backbone of E656. Notably, the
10

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
Fig. 7. Left panels: Blank-subtracted sensorgrams derived from a single cycle analysis of 7j (upper panel) or of 7m (lower panel) both at 9.4, 18.8, 40, 37.5, 75, 150 mM injected on the
F508del-CFTR biosensor. White and black arrows point to the start and end of the injections, respectively. Right panels: Steady-state analysis of 7j (upper panel) or of 7m (lower
panel) injected onto the F508del-CFTR-containing biosensor (same doses as above). The results shown are representative of other three that gave similar results.
Fig. 8. Docking mode of 7j (C atom; orange) in comparison with that of VX-809 (C atom; light green) (A) and of SUL-809 (C atom; purple) (B) at the hNBD1 domain of the F508del
CFTR mutant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9. Docking mode of 7m (C atom; yellow) in comparison with that of VX-809 (C atom; light green) (A) and of SUL-809 (C atom; purple) (B) at the hNBD1 domain of the F508del
CFTR mutant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4. Experimental
Solvent removal was accomplished with a rotary evaporator at
ca.10e50 Torr. The analytical instrument used was an Agilent 1260
high performance liquid chromatography (HPLC). The analytical
4.1. Chemistry
HPLC column was a Phenomenex C18 Luna (4.6 ꢂ 250 mm, 5
mm).
Reagents and solvents were purchased from Sigma Aldrich, Alfa
Aesar, VWR and Zentek used as received unless otherwise
indicated.
The preparative HPLC was Agilent 1260 Infinity preparative
HPLC and the column used for preparative chromatography was a
Phenomenex C18 Luna (21.2 ꢂ 250 mm, 15
mm). The analysis of the
11

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
intermediates and the raw products was performed by liquid
chromatography-electrospray mass spectrometry (HPLC-ESI-MS)
using an Agilent 1100 series LC/MSD ion trap instrument.
HRMS experiments were performed using Q Exactive Orbitrap
instrument by Thermo Scientific.
kept at T ¼ 50 ꢁC until completeness (18e24 h) and then purified by
preparative HPLC. The peak of interest was concentrated to obtain
the title compound as yellow solid with purity of >95% as deter-
mined by HPLC-MS (12.7 mg, 25%).
1H NMR (200 MHz, DMSO‑d6):
d 11.89 (s, broad, 1H, NH);
The nuclear magnetic resonance (NMR) spectrometer was a
Varian Gemini 200 MHz.
The proton spectra were acquired at 200 MHz while carbon
spectra were acquired at 50 MHz, at room temperature. Chemical
7.71e6.57 (m, 12H, arom); 6.02 (s, 2H OCH2O); 2.37 (s, 3H, SCH 3);
1.68e1.42 (m, 2H, CH2, cyclopr); 1.40e1.09 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6):
d 187.3, 165.7, 158.6, 153.9, 146.9,
146.3, 131.8, 130.9, 129.6, 128.9, 128.1, 127.4, 123.1, 114.5, 110.2, 107.8,
100.6, 98.7, 51.8, 30.3, 15.5.
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 with 0.1% formic acid (FOA) and
eluent B was acetonitrile with 0.1% FOA. All analogues submitted for
testing were judged to be of 95% or higher purity based on
analytical HPLC/MS analysis.
HRMS (ESI) calculated for C28H23N2O4S2: [M þ H]þ 515.10992;
found 515.10971.
4.1.2. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-bromobenzoyl)-4-
phenylthiazol-2 yl)cyclopropanecarboxamide (7b)
Compound 7b (17.5 mg, 32%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (4-bromophenyl)methanone (35.9 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown solid.
Compound purity was determined by integrating peak areas of
the liquid chromatogram, monitored at 254 nm.
1H NMR (200 MHz, DMSO‑d6)
d 12.02 (s, 1H, NH), 7.68e6.91 (m,
4.1.1. 1-(Benzo[d][1,3]dioxol-6-yl)-N-(5-(4-(methylthio)benzoyl)-4-
phenylthiazol-2-yl)cyclo propanecarboxamide (7a)
12H, arom), 6.06 (s, 2H, OCH2O), 1.72e1.44 (m, 2H, CH2, cyclopr),
1.38e1.12 (m, 2H, CH2, cyclopr).
A
mixture of (4-methoxyphenyl) methanamine (535
mL,
13C NMR (50 MHz, DMSO‑d6)
d, 187.8, 172.4, 160.2, 154.5, 146.9,
4 mmol) and 4-(methoxy)benzaldehyde (595 L, 4.8 mmol) in
m
146.3, 136.3, 133.6, 131.8, 130.7, 130.6, 129.2, 128.3, 127.4, 125.8,
124.1, 123.3, 110.2, 107.8, 100.6, 30.3, 15.5.
methanol (2 mL) was heated to reflux for 3 h, then cooled to
T ¼ 0 ꢁC, NaBH4 (228 mg, 6 mmol) was added portion wise to the
reaction and the resulting mixture was stirred at room temperature
for about 10 h. Solvent was removed under reduced pressure and
the residue was partitioned between ethyl acetate (EtOAc) and H2O.
The combined organic layers were washed with H2O, and then
dried over anhydrous Na2SO4. After filtration, solvent was removed
in vacuum to afford bis [4-methoxyphenyl]methylamine (962 mg,
94%) as a colorless oil which was used in the next step without
further purification. ESI-MS: m/z 258.0 [MþH]þ.
HRMS (ESI) calculated for C27H20BrN2O4S: [M þ H]þ 547.03271;
found 547.03215.
4.1.3. Ethyl4-(2-(1-(benzo[d][1,3]dioxol-5-yl)
cyclopropanecarboxamido)-4-phenylthiazole-5-carbonyl)benzoate
(7c)
Compound 7c (18.4 mg, 34%) was prepared from ethyl 4-(2-
amino-4-phenylthiazole-5-carbonyl)
benzoate
(35.2
mg,
To a solution of benzoylisothiocyanate (140
mL, 1 mmol) in
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown solid.
acetone (1 mL) cooled at T ¼ 0 ꢁC, bis [4-methoxyphenyl]methyl-
amine (257 mg, 1 mmol) in acetone (1 mL) was added at this
temperature and stirred for an additional 1 h.
1H NMR (200 MHz, DMSO‑d6):
d 12.05 (s, broad, 1H, NH),
The mixture was concentrated under reduced pressure, to afford
N-(bis(4-methoxybenzyl) carbamothioyl)benzamide (398 mg, 95%)
as yellow sticky oil which was used in the next step without further
purification. ESI-MS: m/z 421.0 [MþH]þ.
7.84e6.82 (m, 12H, arom), 6.03 (s, 2H OCH2O), 4.45 (q, J ¼ 7.2, 2H,
OCH2CH3), 1.67e1.48 (m, 2H, CH2, cyclopr), 1.41e1.04 (5H, m, 2H,
CH2, cyclopr þ superimposed t, J ¼ 7.2, 3H, OCH2CH3).
13C NMR (50 MHz, DMSO‑d6):
d 188.2, 172.5, 164.5, 160.5, 155.0,
A
solution of 2-bromo-1-(4-(methylthio)phenyl)ethanone
146.9, 146.3, 141.1, 133.6,131.9, 131.7, 129.5, 128.7, 128.3, 128.2, 127.3,
124.3, 123.2, 110.2, 107.8, 100.6, 60.7, 30.3, 15.5, 13.6.
HRMS (ESI) calculated for C30H25N2O6S: [M þ H]þ 541.14332;
found 541.14314.
(123 mg, 0.5 mmol) and N-(bis(4-methoxybenzyl)carbamothioyl)-
benzamide (210 mg, 0.5 mmol) in N, N-dimethylformamide (DMF)
(3 mL) was stirred at T ¼ 85 ꢁC for 3 h. After cooling to room
temperature, the mixture was partitioned between EtOAc and H2O.
The organic layer was washed with brine and dried over anhydrous
Na2SO4. After filtration, solvent was removed in vacuum and the
residue was stirred in trifluoroacetic acid (TFA) (4 mL) at T ¼ 80 ꢁC
for 24e36 h until complete deprotection. Most of TFA was removed
under reduced pressure. The residue was then neutralized with
NaHCO3 1 N, and then extracted with EtOAc for 3 times. The
combined organic layers were washed with brine and dried over
anhydrous Na2SO4. After filtration, the solution was concentrated
and further crystallized in acetonitrile to afford (2-amino-4-
phenylthiazol-5-yl) (4-(methylthio)phenyl) methanone (127 mg,
78%) as a brown solid. ESI-MS: m/z 328.1 [MþH]þ.
4.1.4. 4-(2-(1-(Benzo[d][1,3] dioxol-5-yl)
cyclopropanecarboxamido)-4-phenylthiazole-5-carbonyl) benzoic
acid (7d)
Compound 7d (9.9 mg, 19%) was prepared from 4-(2-amino-4-
phenylthiazole-5-carbonyl)benzoic acid (32.4 mg, 0.1 mmol) and
benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (20.6 mg,
0.1 mmol) in the same manner as described for 7a as brown solid.
1H NMR (200 MHz, DMSO‑d6):
d 13.18 (s, 1H, broad, COOH);
11.99 (s, broad, 1H, NH); 8.02e6.73 (m, 12H, arom); 6.03 (s, 2H
OCH2O); 1.68e1.43 (m, 2H, CH2, cyclopr); 1.40e1.05 (m, 2H, CH2,
cyclopr).
Benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (20.6 mg,
0.1 mmol) was resuspended in anhydrous DMF (1 mL); HATU
13C NMR (50 MHz, DMSO‑d6):
d 187.7, 171.6, 168.8, 154.6, 146.9,
(38 mg, 0.1 mmol) and DIPEA (35
reaction was vigorously stirred for
phenylthiazol-5-yl) (4-(methylthio) phenyl) methanone (32.7 mg,
0.1 mmol) in anhydrous DMF (500 mL) was added. The reaction was
m
L, 0.2 mmol) were added. The
146.3, 137.0, 131.3, 130.2, 129.4, 128.7, 128.2, 127.5, 123.1, 113.1, 110.2,
107.8, 100.6, 30.3, 15.5.
5
min and (2-amino-4-
HRMS (ESI) calculated for C28H21N2O6S: [M þ H]þ 513.11202;
found 513.11179.
12

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
4.1.5. 4-(2-(1-(Benzo[d] [1,3]dioxol-5-yl)
cyclopropanecarboxamido)-4-phenylthiazole-5-carbonyl)-N-
methylbenzamide (7e)
HRMS (ESI) calculated for C28H23N2O5S: [M þ H]þ 499.13276;
found 499.13224.
Compound 7e (12 mg, 11%) was prepared from 4-(2-amino-4-
4.1.9. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(4-phenyl-5-(4-
(trifluoromethoxy)benzoyl)thiazol-2-yl) cyclopropanecarboxamide
(7i)
phenylthiazole-5-carbonyl)-N-methylbenzamide
(68
mg,
0.2 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(41 mg, 0.20 mmol) in the same manner as described for 7a as
brown oil.
Compound 7i (12.5 mg, 23%) was prepared from (2-amino-4-
phenylthiazol-5-yl)
(4-(trifluoromethoxy)phenyl)methanone
1H NMR (200 MHz, DMSO‑d6):
d
12.01 (s, 1H, broad, NH); 11.75
(36 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as pale brown solid.
(s, broad, 1H, NH); 7.71e6.63 (m, 12H, arom); 6.03 (s, 2H OCH2O);
2.43 (s, 3H, CH3N); 1.72e1.46 (m, 2H, CH2, cyclopr); 1.42e1.11 (m,
2H, CH2, cyclopr).
1H NMR (200 MHz, DMSO‑d6):
d 12.03 (s, broad, 1H, NH),
13C NMR (50 MHz, DMSO‑d6):
d
187.8, 172.3, 167.8, 154.1, 146.8,
7.76e6.79 (m, 12H, arom), 6.05 (s, 2H OCH2O), 1.65e1.43 (m, 2H,
CH2, cyclopr), 1.41e1.04 (m, 2H, CH2, cyclopr).
146.3, 136.6, 131.8, 130.3, 129.3, 128.8, 128.1, 127.5, 123.2, 113.5,
110.2, 107.8, 100.6, 38.5, 30.3, 15.5.
13C NMR (50 MHz, DMSO‑d6):
d 187.6, 172.5, 160.4, 154.9, 146.9,
HRMS (ESI) calculated for C29H24N3O5S:[M þ H]þ 526.14366;
found 526.14343.
146.3, 136.3, 133.6, 131.7, 130.8, 129.2, 128.2, 127.2, 124.4, 123.2,
119.8, 110.2, 107.8, 100.6, 30.3, 15.5.
HRMS (ESI) calculated for C28H20F3N2O5S: [M þ H]þ 553.10449;
found 553.10404.
4.1.6. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-(diethylamino)
benzoyl)-4-phenylthiazol-2-yl) cyclopropanecarboxamide (7f)
Compound 7f (12.5 mg, 23%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (4-(diethylamino) phenyl)methanone (35 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
white solid.
4.1.10. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(4-phenyl-5-(4-
propoxybenzoyl)thiazol-2-yl) cyclopropanecarboxamide (7j)
Compound 7j (9.6 mg, 18%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (4-propoxyphenyl) methanone (34 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as pale
yellow solid.
1H NMR (200 MHz, DMSO‑d6):
d 11.94 (s, broad, 1H, NH),
7.79e6.85 (m, 12H, arom), 6.05 (s, 2H OCH2O), 2.83 (q, 4H, J ¼ 6.8,
NCH2), 1.65e1.41 (m, 2H, CH2, cyclopr), 1.40e1.09 (m, 2H, CH2,
cyclopr), 1.04 (t, 6H, J ¼ 6.8, CH3CH2N).
1H NMR (200 MHz, DMSO‑d6):
d 11.94 (s, broad, 1H, NH);
7.65e6.58 (m, 12H, arom); 6.01 (s, 2H OCH2O); 3.93 (t, 2H, J ¼ 7.0,
OCH2CH2); 1.85e1.44 (m, 4H, 2H, OCH2CH2, þ 2H, CH2, cyclopr);
1.41e1.09 (m, 2H, CH2, cyclopr); 0.96 (t, 3H, J ¼ 7.0, CH2CH3).
13C NMR (50 MHz, DMSO‑d6):
d 188.3, 172.3, 163.2, 160.6, 155.2,
146.9,146.3,134.2,133.7,131.9,131.6,129.2,128.7,128.3,128.1, 127.4,
124.3, 123.1, 110.2, 107.8, 100.6, 51.8, 30.3, 15.5, 13.8.
HRMS (ESI) calculated for C31H30N3O4S: [M þ H]þ 540.19569;
found 540.19544.
13C NMR (50 MHz, DMSO‑d6):
d 178.1, 164.7, 156.5, 153.8, 146.9,
146.4,132.2,131.3,129.6,128.9,128.2,127.5, 123.1,113.6,110.2,107.8,
100.6, 99.2, 57.8, 30.3, 22.7, 15.4.
HRMS (ESI) calculated for C30H27N2O5S: [M þ H]þ 527.16406;
found 527.16388.
4.1.7. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-pentylbenzoyl)-4-
phenylthiazol-2-yl) cyclopropanecarboxamide (7g)
Compound 7g (13.5 mg, 25%) was prepared from (2-amino-4-
4.1.11. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(4-phenyl-5-(3-
phenylthiazol-5-yl)
(4-pentylphenyl)methanone
(35
mg,
propoxybenzoyl)thiazol-2 yl)cyclopropanecarboxamide (7k)
3-(Prop-1-yloxy)acetophenone (356 mg, 2 mmol) synthetized
starting from 3-hydroxyacetophenone as previously described [39],
was brominated with CuBr2 using the general procedure previously
described [40] providing 2-bromo-1-(3-propoxyphenyl)ethanone
intermediate (460 mg, 90%). The purity was verified by HPLC-MS
without further purification.
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as pale
yellow solid.
1H NMR (200 MHz, DMSO‑d6):
d 11.90 (s, broad, 1H, NH),
7.61e6.76 (m, 12H, arom), 6.05 (s, 2H OCH2O), 1.65e1.42 (m, 2H,
CH2, cyclopr), 1.41e1.05 (m, 6H, 2H, CH2, cyclopr þ 4H, CH2CH2),
0.98e0.72 (m, 7H, CH2CH2CH3).
A solution of 2-bromo-1-(3-propoxyphenyl)ethanone (128 mg,
0.5 mmol) and N-(bis(4-methoxybenzyl)carbamothioyl)-benza-
mide (210 mg, 0.5 mmol) in DMF (3 mL) was stirred at T ¼ 85 ꢁC for
3 h according to the protocol for compound 7a. After TFA depro-
tection and crystallization in acetonitrile (2-amino-4-
phenylthiazol-5-yl) (3-propoxyphenyl)methanone (118 mg, 70%)
was isolated. ESI-MS: m/z 339.1 [MþH]þ.
13C NMR (50 MHz, DMSO‑d6):
d 188.4, 172.3, 159.5, 174.2, 146.9,
146.3, 134.8, 133.8, 131.8, 128.9, 128.8, 128.0, 127.6, 127.3, 124.3,
123.2, 110.2, 107.8, 100.6, 34.5, 30.3, 30.1, 29.7, 28.5, 21.5, 15.4, 13.4.
HRMS (ESI) calculated for C32H31N2O4S: [M þ H]þ 539.20044;
found 539.20018.
4.1.8. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-methoxybenzoyl)-4-
phenylthiazol-2-yl) cyclopropanecarboxamide (7h)
(2-Amino-4-phenylthiazol-5-yl) (3-propoxyphenyl)methanone
(34
mg,
0.1
mmol)
and
benzo
[1,3]
dioxol-5-
Compound 7h (18.9 mg, 38%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (4-methoxyphenyl)methanone (31 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown solid.
ylcyclopropanecarboxylic acid (20.6 mg, 0.1 mmol) was then con-
jugated in the same manner as described for 7a to furnish, after
HPLC purification, the title compound as pale brown oil (11 mg,
21%).
HRMS (ESI) calculated for C30H27N2O5S: [M þ H]þ 527.16406;
found 527.16385.
1H NMR (200 MHz, DMSO‑d6):
d 11.80 (s, broad, 1H, NH),
7.78e6.92 (m, 12H, arom), 6.04 (s, 2H OCH2O), 3.76 (s, 3H, OCH3),
1.63e1.42 (m, 2H, CH2, cyclopr), 1.41e1.16 (m, 2H, CH2, cyclopr).
4.1.12. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(4-phenyl-5-(4-(thiophen-2-
yl)benzoyl)thiazol-2-yl) cyclopropanecarboxamide (7l)
13C NMR (50 MHz, DMSO‑d6):
d 187.2, 162.5, 159.0, 154.8, 146.9,
146.3, 131.8, 131.2, 129.7, 128.8, 128.1, 127.5, 123.2, 113.1, 110.2, 107.8,
100.6, 97.2, 55.1, 30.2, 15.4.
Compound 7l (11.7 mg, 21%) was prepared from (2-amino-4-
phenylthiazol-5-yl)
(4-(thiophen-2-yl)
phenyl)methanone
13

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
(36.2 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as brown oil.
8.27e6.82 (m, 17H, arom); 6.04 (s, 2H OCH2O); 1.69e1.42 (m, 2H,
CH2, cyclopr); 1.41e1.03 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6):
d 187.9, 171.8, 157.4, 153.3, 146.9,
1H NMR (200 MHz, DMSO‑d6):
d
11.96 (s, 1H, broad, NH);
146.3, 141.7, 137.8, 137.2, 131.3, 129.4, 128.8, 128.1, 127.5, 123.1, 110.2,
107.8, 100.6, 30.3, 15.6.
7.95e6.73 (m, 15H, arom); 6.04 (s, 2H OCH2O); 1.73e1.42 (m, 2H,
CH2, cyclopr); 1.40e1.05 (m, 2H, CH2, cyclopr).
HRMS (ESI) calculated for C33H25N2O4S: [M þ H]þ 515.15349;
found 545.15331.
13C NMR (50 MHz, DMSO‑d6):
d 176.8, 172.1, 159.7, 154.3, 146.9,
146.2,137.6,137.0,133.4,131.7,131.3, 129.6,128.8,128.2,127.5,123.2,
113.4, 110.2, 107.8, 100.6, 30.3, 15.5.
4.1.16. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(2-methoxybenzoyl)-4-
phenylthiazol-2-yl) cyclopropanecarboxamide (7p)
HRMS (ESI) calculated for C31H23N2O4S2: [M þ H]þ 551.10992;
found 551.10971.
Compound 7p (11 mg, 22%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (2-methoxyphenyl) methanone (31 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown oil.
4.1.13. N-(5-([1,10-biphenyl]-4-carbonyl)-4-phenylthiazol-2-yl)-1-
(benzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamide (7m)
Compound 7m (15.3 mg, 28%) was prepared from [1, 10-
biphenyl]-4-yl
(2-amino-4-phenylthiazol-5-yl)methanone
1H NMR (200 MHz, DMSO‑d6):
d 10.97 (s, 1H, broad, NH);
(35.7 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as pale yellow solid.
7.68e6.69 (m, 12H, arom); 6.02 (s, 2H OCH2O); 3.98 (s, 3H, CH3O);
1.68e1.43 (m, 2H, CH2, cyclopr); 1.42e1.03 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6):
d 175.7, 163.1, 158.9, 154.4, 147.0,
1H NMR (200 MHz, DMSO‑d6):
d
12.01 (s, 1H, broad, NH);
146.3, 131.7, 131.1, 129.6, 128.9, 128.1, 127.5, 123.2, 113.8, 110.2, 107.8,
100.6, 97.7, 55.3, 30.3, 15.5.
7.92e6.76 (m, 17H, arom); 6.05 (s, 2H OCH2O); 1.78e1.44 (m, 2H,
CH2, cyclopr); 1.42e1.08 (m, 2H, CH2, cyclopr).
HRMS (ESI) calculated for C28H23N2O5S: [M þ H]þ 499.13276;
found 499.13276.
13C NMR (50 MHz, DMSO‑d6):
d 188.2, 172.2, 156.8, 153.2, 146.9,
146.3,140.7, 137.9,133.2,131.3,129.3,128.9,128.2,127.4,123.1, 110.2,
107.8, 100.6, 30.3, 15.4.
4.1.17. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(3-methoxybenzoyl)-4-
phenylthiazol-2-yl) cyclopropanecarboxamide (7q)
HRMS (ESI) calculated for C33H25N2O4S: [M þ H]þ 545.15349;
found 545.13351.
Compound 7q (10.8 mg, 22%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (3-methoxyphenyl)methanone (31 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as pale
brown oil.
4.1.14. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(4-phenyl-5-(4-(pyrrolidin-
1-yl)benzoyl)thiazol-2-yl) cyclopropanecarboxamide (7n)
Compound 7n (11.5 mg, 21%) was prepared from (2-amino-4-
phenylthiazol-5-yl)
(4-(pyrrolidin-1-yl)phenyl)
methanone
1H NMR (200 MHz, DMSO‑d6):
d 12.04 (s, 1H, broad, NH);
(35 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as pale yellow oil.
7.94e6.76 (m, 12H, arom); 6.04 (s, 2H OCH2O); 4.01 (s, 3H, CH3O);
1.71e1.44 (m, 2H, CH2, cyclopr); 1.40e1.02 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6):
d 177.4, 164.6, 158.4, 154.2, 146.9,
1H NMR (200 MHz, DMSO‑d6):
d
12.08 (s, 1H, broad, NH);
146.2, 131.8, 131.1, 129.7, 128.8, 128.1, 127.4, 123.2, 113.7, 110.2, 107.8,
100.6, 98.1, 55.6, 30.2, 15.4.
7.82e6.75 (m, 12H, arom); 6.01 (s, 2H OCH2O); 2.97 (m, 4H, CH2N);
1.88e1.43 (m, 6H, 4H CH2CH2, þ 2H, CH2, cyclopr); 1.40e1.12 (m,
2H, CH2, cyclopr).
HRMS (ESI) calculated for C28H23N2O5S: [M þ H]þ 499.13276;
found 499.13245.
13C NMR (50 MHz, DMSO‑d6):
d 187.7, 170.3, 160.2, 154.7, 146.9,
146.3,131.8, 131.0, 129.6, 128.7, 128.2,127.5, 123.3, 113.2,110.2, 107.8,
100.6, 48.2, 30.3, 18.6, 15.4.
4.1.18. 1-(Benzo[d][1, 3]dioxol-5-yl)-N-(5-(3-bromobenzoyl)-4-
phenylthiazol-2-yl) cyclopropanecarboxamide (7r)
HRMS (ESI) calculated for C31H28N3O4S: [M þ H]þ 538.18004;
found 538.17987.
Compound 7r (16 mg, 29%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (3-bromophenyl)methanone (35.9 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as pale
brown solid.
4.1.15. N-(5-([1,10-biphenyl]-3-carbonyl)-4-phenylthiazol-2-yl)-1-
(benzo[d][1,3]dioxol-5-yl)cyc lopropanecarboxamide (7o)
1-([1,10-Biphenyl]-3-yl)ethanone (196 mg, 1 mmol) was bromi-
nated with N-bromosuccinimide (NBS) using the general procedure
previously described [25] providing [1,10-biphenyl]-3-carbonyl
bromide intermediate (202 mg, 73%). The purity was verified by
HPLC-MS without further purification.
1H NMR (200 MHz, DMSO‑d6):
d 12.03 (s, 1H, broad, NH);
8.18e6.81 (m, 12H, arom); 6.06 (s, 2H OCH2O); 1.78e1.45 (m, 2H,
CH2, cyclopr); 1.44e1.05 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6):
d 188.3, 172.1, 159.3, 155.3, 146.9,
146.3,137.0,131.2,129.6,129.0,128.2,127.5,123.3,114.2,110.2,107.8,
100.6, 30.3, 15.5.
A solution of [1,10-biphenyl]-3-carbonyl bromide intermediate
(130 mg, 0.5 mmol) and N-(bis(4-methoxybenzyl)carbamothioyl)-
benzamide (230 mg, 0.5 mmol) in DMF (3 mL) was stirred at
T ¼ 85 ꢁC for 3 h according to the protocol for compound 7a. After
TFA deprotection and crystallization in acetonitrile [1,10-biphenyl]-
3-yl (2-amino-4-phenylthiazol-5-yl)methanone (60,5 mg, 34%)
was isolated. ESI-MS: m/z 357.1 [MþH]þ.
HRMS (ESI) calculated for C27H20BrN2O4S: [M þ H]þ 547.03271;
found 547.03219.
4.1.19. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(3-fluorobenzoyl)-4-
phenylthiazol-2-yl) cyclopropanecarboxamide (7s)
Compound 7s (13 mg, 27%) was prepared from (2-amino-4-
[1,10-Biphenyl]-3-yl (2-amino-4-phenylthiazol-5-yl)methanone
(36 mg, 0.1 mmol) and benzo [1,3] dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) was then conjugated in
the same manner as described for 7a to furnish, after HPLC purifi-
cation, the title compound as pale yellow solid (12.5 mg, 23%).
phenylthiazol-5-yl)
(3-fluorophenyl)methanone
(30
mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
white solid.
1H NMR (200 MHz, DMSO‑d6):
d
11.93 (s, 1H, broad, NH);
1H NMR (200 MHz, DMSO‑d6):
d 12.01 (s, 1H, broad, NH);
8.01e6.74 (m, 12H, arom); 6.05 (s, 2H OCH2O); 1.73e1.44 (m, 2H,
14

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
CH2, cyclopr); 1.42e1.08 (m, 2H, CH2, cyclopr).
1H NMR (200 MHz, DMSO‑d6)
d 12.02 (s, 1H, NH); 9.73 (s, 1H,
13C NMR (50 MHz, DMSO‑d6):
d
188.0, 171.5, 158.7, 156.2, 146.9,
OH); 7.88e6.72 (m, 11H, arom); 6.05 (s, 2H, OCH2O); 5.95e5.61 (m,
1H, CH2CH]CH2); 4.92e4.68 (m, 2H, CH2CH]CH2); 3.18 (d,
J ¼ 15.2, 2H, CH2CH]CH2); 1.77e1.43 (m, 2H, CH2, cyclopr);
1.42e1.14 (m, 2H, CH2, cyclopr).
146.3, 137.0, 133.4, 131.1, 129.7, 128.8, 128.2, 127.6, 123.2, 113.8, 110.2,
107.8, 100.6, 30.3, 15.5.
HRMS (ESI) calculated for C27H20FN2O4S: [M þ H]þ 487.11277;
found 487.11243.
13C NMR (50 MHz, DMSO‑d6)
d 178.4, 170.5, 161.1, 156.2, 152.9,
146.9, 146.3, 145.2, 136.9, 136.5, 131.2, 128.8, 128.3, 123.4, 114.2,
110.2, 107.8, 100.6, 34.9, 30.3, 15.6.
4.1.20. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(2,5-dimethoxybenzoyl)-
4-phenylthiazol-2-yl) cyclopropanecarboxamide (7t)
Compound 7t (13.8 mg, 26%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (2,5-dimethoxyphenyl)methanone (35 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown solid.
HRMS (ESI) calculated for C30H25N2O5S: [M þ H]þ 525.14841;
found 525.14803.
4.1.24. 5-(2-(1-(Benzo[d][1,3]dioxol-5-yl)
cyclopropanecarboxamido)-4-phenylthiazole-5-carbonyl)-2-
hydroxybenzamide (7x)
1H NMR (200 MHz, DMSO‑d6)
d
11.85 (s, 1H, NH), 7.47e6.53 (m,
Compound 7x (12.5 mg, 24%) was prepared from 5-(2-amino-4-
11H, arom); 6.02 (s, 2H, OCH2O); 3.64 (s, 3H, OCH3); 3.47 (s, 3H,
OCH3); 1.72e1.47 (m, 2H, CH2, cyclopr); 1.37e1.16 (m, 2H, CH2,
cyclopr).
phenylthiazole-5-carbonyl)-2-hydroxybenzamide
(34
mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown oil.
13C NMR (50 MHz, DMSO‑d6)
d 172.3, 160.4, 155.2, 152.2, 149.9,
146.9, 146.3, 133.5, 131.7, 128.8, 128.1, 126.7, 123.3, 117.0, 113.5, 111.9,
110.3, 107.8, 100.6, 55.3, 55.2, 30.3, 15.5.
1H NMR (200 MHz, DMSO‑d6)
d 11.89 (s, 1H, NH); 11.38 (s, 1H,
OH); 8.55 (s, 2H, NH2); 7.64e6.68 (m, 11H, arom); 6.05 (s, 2H,
OCH2O); 1.72e1.42 (m, 2H, CH2, cyclopr); 1.42e1.05 (m, 2H, CH2,
cyclopr).
HRMS (ESI) calculated for C29H25N2O6S: [M þ H]þ 529.14332;
found 529.14301.
13C NMR (50 MHz, DMSO‑d6)
d 180.4, 172.6, 168.3, 159.7, 155.5,
4.1.21. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(2,4-dimethoxybenzoyl)-
4-phenylthiazol-2-yl) cyclopropanecarboxamide (7u)
Compound 7u (18.1 mg, 34%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (2,4-dimethoxyphenyl)methanone (34 mg,
0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid
(20.6 mg, 0.1 mmol) in the same manner as described for 7a as
brown solid.
152.8,146.9,146.3,137.1,131.3,128.9,128.2,126.7, 123.4,113.9,110.2,
107.7, 100.6, 30.3, 15.5.
HRMS (ESI) calculated for C28H22N3O6S: [M þ H]þ 528.12292;
found 528.12282.
4.1.25. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(5-chloro-2-
hydroxybenzoyl)-4-phenylthiazol-2-yl) cyclopropanecarboxamide
(7y)
1H NMR (200 MHz, DMSO‑d6)
d 11.98 (s, 1H, NH); 7.62e6.64 (m,
11H, arom); 6.04 (s, 2H, OCH2O); 3.73 (s, 3H, OCH3); 3.62 (s, 3H,
OCH3); 1.70e1.45 (m, 2H, CH2, cyclopr); 1.39e1.09 (m, 2H, CH2,
cyclopr).
Compound 7y (11.2 mg, 11%) was prepared from (2-amino-4-
phenylthiazol-5-yl)
(5-chloro-2-hydroxyphenyl)methanone
(66 mg, 0.19 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (39 mg, 0.19 mmol) in the same manner as
described for 7a as brown oil.
13C NMR (50 MHz, DMSO‑d6)
d 176.4, 170.6, 160.2, 155.3, 152.2,
146.9,146.3,133.8,131.4,128.9,128.2,126.7,123.2,117.8,113.6,110.2,
107.8, 100.6, 55.4, 55.2, 30.3, 15.5.
1H NMR (200 MHz, DMSO‑d6)
d 12.06 (s, 1H, NH); 10.84 (s, 1H,
HRMS (ESI) calculated for C29H25N2O6S: [M þ H]þ 529.14332;
found 529.14298.
OH); 8.01e6.71 (m, 11H, arom); 6.05 (s, 2H, OCH2O); 1.73e1.45 (m,
2H, CH2, cyclopr); 1.43e1.05 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6)
d 185.6, 172.7, 160.9, 155.9, 153.3,
4.1.22. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(3-chloro-4-
methoxybenzoyl)-4-phenylthiazol-2-yl) cyclopropanecarboxamide
(7v)
146.9, 146.2, 141.8, 137.3, 131.1, 128.9, 128.1, 123.2, 117.5, 113.8, 110.2,
107.7, 100.6, 30.3, 15.5.
HRMS (ESI) calculated for C27H20ClN2O5S: [M þ H]þ 519.07814;
found 599.07781.
Compound 7v (14.5 mg, 27%) was prepared (2-amino-4-
phenylthiazol-5-yl)
(3-chloro-4-methoxyphenyl)methanone
(40 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as brown oil.
4.1.26. 1-(Benzo[d][1,3dioxol-5-yl)-N-(5-(benzo[d][1, 3]dioxole-5-
carbonyl)-4-phenylthiazol-2-yl) cyclopropanecarboxamide (7z)
Compound 7z (15.5 mg, 30%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (benzo [d] [1,3] dioxol-5-yl)methanone
(32.4 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as brown oil.
1H NMR (200 MHz, DMSO‑d6)
d 12.14 (s, 1H, NH); 8.03e6.75 (m,
11H, arom); 6.06 (s, 2H, OCH2O); 3.77 (s, 3H, OCH3); 1.78e1.46 (m,
2H, CH2, cyclopr); 1.42e1.10 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6)
d 184.5, 171.5, 162.2, 156.8, 153.1,
146.8, 146.3, 140.5, 137.2, 131.2, 128.8, 128.2, 123.3, 118.1, 113.6, 110.2,
107.7, 100.6, 55.3, 30.3, 15.6.
1H NMR (200 MHz, DMSO‑d6)
d 12.08 (s, 1H, NH); 7.77e6.68 (m,
11H, arom); 6.07 (s, 2H, OCH2O); 6.03 (s, 2H, OCH2O); 1.64e1.41 (m,
2H, CH2, cyclopr); 1.41e1.00 (m, 2H, CH2, cyclopr).
HRMS (ESI) calculated for C28H22ClN2O5S: [M þ H]þ 533.09379;
found 533.09336.
13C NMR (50 MHz, DMSO‑d6)
d 178.8, 171.4, 159.3, 154.8, 151.5,
146.9, 146.3, 137.0, 131.2, 128.8, 128.1, 127.4, 123.2, 114.6, 110.3, 107.8,
100.6, 30.3, 15.4.
4.1.23. N-(5-(3-allyl-4-hydroxybenzoyl)-4-phenylthiazol-2-yl)-1-
(benzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamide (7w)
Compound 7w (10 mg, 19%) was prepared from (3-allyl-4-
HRMS (ESI) calculated for C28H21N2O6S: [M þ H]þ 513.11203;
found 513.11187.
hydroxyphenyl)
(2-amino-4-phenylthiazol-5-yl)methanone
(34 mg, 0.1 mmol) and benzo [1,3]dioxol-5-yl-cyclo-
propanecarboxylic acid (20.6 mg, 0.1 mmol) in the same manner as
described for 7a as brown oil.
4.1.27. 2-(1-(Benzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-
N,4-diphenylthiazole-5-carboxamide (12a)
A solution of ethyl 2-amino-4-phenylthiazole-5-carboxylate
15

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
(248 mg, 1 mmol), di-tert-butyl carbonate (262 mg, 1.2 mmol) and
catalytic 4-(N,N-dimethylamino) pyridine (8 mg, 0.07 mmol) in
anhydrous THF (1 mL) was stirred for 4 h at room temperature. The
mixture was concentrated under vacuum and after extraction with
EtOAc (3 x 3 mL), washed with H2O (3 x 2 mL). The organic phases
were dried over Na2SO4, and concentrated under vacuum. Crys-
tallization in acetonitrile afford ethyl 2-((tert-butoxycarbonyl)
amino)-4-phenylthiazole-5-carboxylate (170 mg, 49%) which was
used in the next step without further purification. ESI-MS: m/z
349.1 [MþH]þ.
pressure to afford the quaternary salt. To the compound, EtOH
(2 mL) and conc. HCl (0.1 mL) were added and the mixture was
refluxed for 3 h. After cooling to room temperature, the solid was
centrifugated, washed with EtOH (3 x 2 mL), and dried under
vacuum to afford pure 4-methoxy20amino-acetophenone hydro-
chloride in near quantitative yield. ESI-MS: m/z 202.6 [MþH]þ.
NaHCO3 (252 mg, 3 mmol) was added to a solution of 4-
methoxy20amino-acetophenone hydrochloride (202 mg, 1 mmol)
in anhydrous THF (2 mL) with stirring at room temperature. Ben-
zoyl chloride (116 ml, 1 mmol) was added to the solution and stirred
A
solution of ethyl 2-((tert-butoxycarbonyl)amino)-4-
for about 3 h at room temperature. The solvent was removed under
reduced pressure to leave a yellow oil. The product was extracted
with dichloromethane (DCM) (3 x 3 mL) and washed with H2O (3 x
2 mL). The organic phase was dried over Na2SO4 and then
concentrated under reduced pressure. Removal of the solvent
produced the N-(2-(4-methoxyphenyl)-2-oxoethyl)benzamide as a
white solid of sufficient purity to be used in the next step (202 mg,
75%). ESI-MS: m/z 269.1 [MþH]þ.
phenylthiazole-5-carboxylate (170 mg, 0.49 mmol) in THF-EtOH
(2.5 mL, 1:1.5) and 6 N aq KOH solution (1 mL) was heated at
T ¼ 55 ꢁC for 4 h. The solution was cooled to T ¼ 0 ꢁC and acidified
with conc. HCl to pH 1. The solution was then concentrated in
vacuum, and after extraction with diethyl ether (3 x 3 mL), washed
with H2O (3 x 2 mL) and dried over anhydrous Na2SO4. After
filtration, the solution was evaporated to obtain 2-[[(tert-butyloxy)
carbonyl]-amino]-4-phenyl-1,3-thiazole-5-carboxylic
acid
N-(2-(4-methoxyphenyl)-2-oxoethyl)benzamide
(107
mg,
(100 mg, 64%) as a brown solid. ESI-MS: m/z 321.1 [MþH]þ.
0.4 mmol) and thiourea (30 mg, 0.4 mmol) were solved in EtOH at
room temperature. To this solution I2 (101 mg, 0.4 mmol) and
The
2-[[(tert-butyloxy)carbonyl]-amino]-4-phenyl-1,3-
thiazole-5-carboxylic acid (75 mg, 0.23 mmol) was resuspended
in anhydrous DMF (1 mL), HATU (87 mg, 0.23 mmol) and DIPEA
triethylamine (TEA) (11 ml, 0.2 mmol) were added. The reactions
were stirred at T ¼ 80 ꢁC overnight. The mixture was concentrated
under vacuum and after extraction with EtOAc (3 x 3 mL), was
washed with H2O (3 x 2 mL). The organic layer was dried over
anhydrous Na2SO4. After filtration, solvent was removed in vacuum
to obtain N-(2-amino-4-(4-methoxyphenyl)thiazol-5-yl) benza-
mide (40 mg, 31%). The thiazole was used without further purifi-
cation. ESI-MS: m/z 325.1 [MþH]þ.
(40
mL, 0.23 mmol) were added. The reaction was vigorously stirred
for 5 min and aniline (23
m
L, 0.24 mmol) in anhydrous DMF (500 L)
m
was added. The reaction was kept at T ¼ 40 ꢁC for 2 h. Solvent was
removed under reduced pressure and the residue was partitioned
between diethyl ether and water. The combined organic layers
were washed with water (3 x 2 mL), and then dried over anhydrous
Na2SO4. After filtration, solvent was removed in vacuum and the
residue was stirred in a mixture of TFA: DCM 1:1 (2 mL) at T ¼ 0 ꢁC
until complete deprotection (4e5 h). Most of TFA was removed
under reduced pressure. The residue was neutralized with NaHCO3
1 N and then extracted with EtOAc for 3 times. Crystallization in
Benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (25 mg,
0.12 mmol) was resuspended in anhydrous DMF (1 mL), HATU
(45 mg, 0.12 mmol) and DIPEA (42
After 5 min, N-(2-amino-4-(4-methoxyphenyl)thiazol-5-yl)benza-
mide (40 mg, 0.12 mmol) in anhydrous DMF (500 L) was added.
mL, 0.24 mmol) were added.
m
acetonitrile
furnishes
2-amino-N,4-diphenylthiazole-5-
The reaction was kept at T ¼ 50 ꢁC until completeness (about 24 h).
The mixture was concentrated under vacuum and after extrac-
tion with EtOAc (3 x 3 mL), washed with H2O (3 x 2 mL). The organic
phase was dried over Na2SO4, concentrated under reduced pressure
and purified by preparative HPLC. The peak of interest was
concentrated to obtain the title compound as brown solid with
purity of >95% as determined by HPLC-MS (11 mg, 18%).
carboxamide (30 mg, 44%). ESI-MS: m/z 296.1 [MþH]þ.
Benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (20.5 mg,
0.1 mmol) was resuspended in anhydrous DMF (1 mL); HATU
(38 mg, 0.1 mmol) and DIPEA (18
mL, 0.1 mmol) were added. After
5 min, 2-amino-N, 4-diphenylthiazole-5-carboxamide (30 mg,
0.1 mmol) in anhydrous DMF (500 mL) was added. The reaction was
kept at T ¼ 50 ꢁC until completeness (about 24 h).
1H NMR (200 MHz, DMSO‑d6):
d 12.19 (s, broad, 1H, NH); 11.59
The mixture was concentrated under vacuum and after extrac-
tion with EtOAc (3 x 3 mL), washed with H2O (3 x 2 mL). The organic
phase was dried over Na2SO4, concentrated under reduced pressure
and purified by preparative HPLC. The peak of interest was
concentrated to obtain the title compound as white solid with
purity of >95% as determined by HPLC-MS (10 mg, 21%).
(s, broad, 1H, NH); 8.02e6.81 (m, 12H, arom); 6.04 (s, 2H OCH2O);
3.86 (s, 3H, OCH3); 1.78e1.44 (m, 2H, CH2, cyclopr); 1.41e1.08 (m,
2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6)
d 180.8, 167.3, 160.5, 151.4, 146.9,
146.4, 137.8, 136.8, 129.2, 128.9, 128.1, 127.5, 121.7, 116.3, 110.8, 107.2,
101.2, 53.7, 30.3, 15.5.
1H NMR (200 MHz, DMSO‑d6):
d
12.08 (s, broad, 1H, NH); 10.63
HRMS (ESI) calculated for C28H24N3O5S: [M þ H]þ 514.14366;
found 514.14359.
(s, broad, 1H, NH); 7.79e6.75 (m, 13H, arom); 6.05 (s, 2H OCH2O);
1.81e1.45 (m, 2H, CH2, cyclopr); 1.43e1.01 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6)
d
183.5, 165.5, 161.9, 151.0, 146.8,
4.1.29. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-methylbenzoyl)
146.3,137.9, 137.0, 129.2,128.9,128.7,128.0,127.5, 121.6, 116.0, 110.8,
107.2, 101.2, 30.3, 15.6.
thiazol-2-yl)cyclopropanecarboxamide (12c)
N,N-Dimethyl formamide dimethyl acetal (1036 mL, 7.8 mmol)
HRMS (ESI) calculated for C27H22N3O4S: [M þ H]þ 484.13309;
found 484.13277.
was added to a suspension of thiourea (198 mg, 2.6 mmol) in DCM
(2 mL). The mixture was heated at reflux for 4 h. The solvent was
removed and the residue was crystallized from diethyl ether to
obtain (1E, 10E)-N0,N00-thiocarbonylbis (N,N-dimethylformimida-
mide) (400 mg, 83%). ESI-MS: m/z 187.1 (M þ H) þ.
4.1.28. N-(2-(1-(Benzo[d][1,3]dioxol-5-yl)
cyclopropanecarboxamido)-4-(4-methoxyphenyl)thiazol-5-yl)
benzamide (12b)
To a stirred solution of 4-methoxyphenacyl bromide (229 mg,
1 mmol) in diethyl ether (2 mL) was added hexamethylenetetra-
mine (140 mg, 1 mmol) all at once and the mixture was stirred for
2 h at room temperature. The resulting solid was centrifugated,
washed with diethyl ether (2 mL), and dried under reduced
(1E, 10E)-N0,N00-Thiocarbonylbis-(N,N-dimethylformimidamide
(100 mg, 0.53 mmol) was suspended in THF (1 mL) and 2-bromo40-
methylacetophenone (113 mg, 0.53 mmol) was added. The mixture
was stirred at room temperature for 15 min and then TEA (147 mL,
1.16 mmol) was added and the mixture was heated at reflux for
18 h. The mixture was concentrated under vacuum and after
16

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
extraction with EtOAc (3 x 3 mL), washed with H2O (3 x 2 mL). The
organic phase was dried over Na2SO4, concentrated under reduced
pressure to obtain (Z)-N’-(5-(4-methylbenzoyl) thiazol-2-yl)-N,N-
dimethylformimidamide (108 mg, 70%). ESI-MS: m/z 273.1
(M þ H) þ.
The mixture was concentrated under vacuum and after extrac-
tion with EtOAc (3 x 3 mL), washed with H2O (3 x 2 mL). The organic
phase was dried over Na2SO4, concentrated under reduced pressure
and purified by preparative HPLC. The peak of interest was
concentrated to obtain the title compound as brown solid with
purity of >95% as determined by HPLC-MS (26.6 mg, 33%).
A 33% (wt./wt.) aqueous methylamine (400 mL) was added to a
solution of (Z)-N’-(5-(4-methylbenzoyl)thiazol-2-yl)-N,N-dime-
thylformimidamide (101 mg, 0,37 mmol) in THF (1 mL) and the
mixture was stirred for 24 h at room temperature. The mixture was
concentrated under vacuum and after extraction with EtOAc (3 x
3 mL), washed with H2O (3 x 2 mL). The organic phase was dried
over Na2SO4, concentrated under reduced pressure to obtain a pale
yellow oil. Crystallization in acetonitrile furnishes (2-aminothiazol-
5-yl) (p-tolyl)methanone (36 mg, 45%). ESI-MS: m/z 219.1 [MþH]þ.
Benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (33 mg,
0.16 mmol) was resuspended in anhydrous DMF (1 mL), HATU
1H NMR (200 MHz, DMSO‑d6):
d 11.87 (s, broad, 1H, NH); 8.08 (s,
1H, arom); 7.79e6.81 (m, 7H, arom); 6.04 (s, 2H OCH2O); 3.89 (s,
3H, CH3); 1.78e1.41 (m, 2H, CH2, cyclopr); 1.40e1.03 (m, 2H, CH2,
cyclopr).
13C NMR (50 MHz, DMSO‑d6)
d 182.7, 171.9, 146.9, 146.3, 137.1,
133.0, 129.7, 128.8, 121.4, 110.8, 107.2, 101.2, 55.9, 30.3, 15.5.
HRMS (ESI) calculated for C22H19N2O5S: [M þ H]þ 423.10146;
found 423.10098.
4.1.31. N-(5-Acetyl-4-phenylthiazol-2-yl)-1-(benzo[d][1,3]dioxol-5-
yl)cyclopropanecarboxamide (24a)
(61 mg, 0.16 mmol) and DIPEA (56
mL, 0.16 mmol) were added. After
5
min, (2-aminothiazol-5-yl) (p-tolyl) methanone (35 mg,
Compound 24a (9.6 mg, 24%) was prepared from 1-(2-amino-4-
phenylthiazol-5-yl) ethanone (22.1 mg, 0.1 mmol) and benzo [1,3]
dioxol-5-yl-cyclopropanecarboxylic acid (20.6 mg, 0.1 mmol) in the
same manner as described for 7a until completeness (18 h) as white
solid.
0.16 mmol) in anhydrous DMF (500 mL) was added. The reaction
was kept at T ¼ 50 ꢁC until completeness (about 24 h).
The mixture was concentrated under vacuum and after extrac-
tion with EtOAc (3 x 3 mL), washed with H2O (3 x 2 mL). The organic
phase was dried over Na2SO4, concentrated under reduced pressure
and purified by preparative HPLC. The peak of interest was
concentrated to obtain the title compound as pale yellow solid with
purity of >95% as determined by HPLC-MS (32.4 mg, 50%).
1H NMR (200 MHz, DMSO‑d6):
d 11.94 (s, broad, 1H, NH);
7.64e6.59 (m, 8H, arom); 6.02 (s, 2H OCH2O); 2.62 (s, 3H, CH3);
1.74e1.43 (m, 2H, CH2, cyclopr); 1.40e1.16 (m, 2H, CH2, cyclopr).
13C NMR (50 MHz, DMSO‑d6)
d 192.5, 180.4, 164.2, 151.0, 146.8,
1H NMR (200 MHz, DMSO‑d6):
d 11.87 (s, broad, 1H, NH); 8.05 (s,
146.3, 137.0, 131.9, 129.2, 128.7, 127.5, 116.0, 110.2, 107.6, 101.2, 30.3,
1H, arom); 7.81e6.78 (m, 7H, arom); 6.04 (s, 2H OCH2O); 2.32 (s,
3H, CH3); 1.81e1.42 (m, 2H, CH2, cyclopr); 1.41e1.00 (m, 2H, CH2,
cyclopr).
29.7, 15.6.
HRMS (ESI) calculated for C22H19N2O4S: [M þ H]þ 407.10655;
found 407.10611.
13C NMR (50 MHz, DMSO‑d6)
d 186.2, 173.6, 146.8, 146.3, 137.0,
133.1, 129.6, 129.1, 121.3, 110.8, 107.2, 101.2, 30.3, 24.6, 15.5.
HRMS (ESI) calculated for C22H19N2O4S: [M þ H]þ 407.10654;
found 407.10608.
4.1.32. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-nicotinoyl-4-
phenylthiazol-2 yl)cyclopropanecarboxamide (24 b)
Compound 24b (16 mg, 34%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (pyridin-3-yl)methanone (28 mg, 0.1 mmol)
and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (20.6 mg,
0.1 mmol) in the same manner as described for 7a until
completeness (24 h) as brown oil.
4.1.30. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(4-methoxybenzoyl)
thiazol-2-yl)cyclopropanecarboxamide (12d)
(1E, 10E)-N0,N00-Thiocarbonylbis-N,N-dimethylformimidamide
(100 mg, 0.53 mmol) was suspended in DCM (1 mL) and 2-bromo-
40-methoxyacetophenone (121 mg, 0.53 mmol) was added. The
mixture was stirred at room temperature for 15 min and then TEA
1H NMR (200 MHz, DMSO‑d6):
d 12.04 (s, broad, 1H, NH);
8.62e8.22 (m, 3H, arom); 7.93e6.76 (m, 9H, arom); 6.06 (s, 2H
OCH2O); 1.73e1.41 (m, 2H, CH2, cyclopr); 1.37e1.02 (m, 2H, CH2,
cyclopr).
(147 mL,1.16 mmol) was added. The mixture was heated at reflux for
about 20 h. At the end of the reaction the solution was concentrated
under vacuum and after extraction with EtOAc (3 x 3 mL), washed
with H2O (3 x 2 mL). The organic phase was dried over Na2SO4,
concentrated under reduced pressure to obtain (Z)-N’-(5-(4-
13C NMR (50 MHz, DMSO‑d6)
d 178.8, 171.5, 164.3, 155.0, 151.7,
146.9, 146.3, 141.8, 138.3, 136.9, 133.2, 129.2, 128.7, 127.5, 124.4,
116.0, 111.8, 107.8, 101.2, 30.3, 15.5.
HRMS (ESI) calculated for C26H20N3O4S: [M þ H]þ 470.11745;
found 470.11708.
methoxybenzoyl)
(108 mg, 70%).
thiazol-2-yl)-N,N-dimethylformimidamide
ESI-MS: m/z 290.1 [MþH]þ.
A 33% (wt./wt.) aqueous methylamine (600
mL) was added to a
4.1.33. 1-(Benzo[d][1,3]dioxol-5-yl)-N-(5-(morpholine-4-
carbonyl)-4-phenylthiazol-2-yl)cyclopropanecarboxamide (24c)
Compound 24c (12.8 mg, 27%) was prepared from (2-amino-4-
phenylthiazol-5-yl) (morpholino)methanone (29 mg, 0.1 mmol)
and benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (20.6 mg,
0.1 mmol) in the same manner as described for 7a until
completeness (24 h) as yellow solid.
solution of (Z)-N’-(5-(4-methoxybenzoyl)thiazol-2-yl)-N,N-dime-
thylformimidamide (108 mg, 0.37 mmol) in THF (1 mL) and the
mixture was stirred for 24 h at room temperature. The solution was
then concentrated under vacuum and after extraction with EtOAc
(3 x 3 mL), washed with H2O (3 x 2 mL). The organic phase was
dried over Na2SO4, concentrated under reduced pressure to obtain
a
yellow oil. Crystallization in acetonitrile furnishes (2-
1H NMR (200 MHz, DMSO‑d6):
d 11.91 (s, broad, 1H, NH);
aminothiazol-5-yl) (4-methoxyphenyl)methanone (46 mg, 53%).
7.72e6.58 (m, 8H, arom); 6.03 (s, 2H OCH2O); 3.72 (t, J ¼ 6.8, 4H,
OCH2); 2.67 (t, J ¼ 6.8, 4H, NCH2); 1.78e1.43 (m, 2H, CH2, cyclopr);
1.41e1.06 (m, 2H, CH2, cyclopr).
MS ESI-MS: m/z 235.1 [MþH]þ.
Benzo [1,3]dioxol-5-yl-cyclopropanecarboxylic acid (39 mg,
0.19 mmol) was resuspended in anhydrous DMF (1 mL), HATU
13C NMR (50 MHz, DMSO‑d6)
d 176.3, 169.8, 162.5, 151.2, 146.9,
(72 mg, 0.19 mmol) and DIPEA (66
m
L, 0.19 mmol) were added. After
146.4, 137.0, 131.1, 129.2, 128.7, 127.5, 116.0, 110.8, 10.2, 101.2, 56.2,
42.8, 30.3, 15.6.
5
min, (2-aminothiazol-5-yl) (4-methoxyphenyl)methanone
(45 mg, 0.19 mmol) in anhydrous DMF (500 mL) was added. The
HRMS (ESI) calculated for C25H24N3O5S: [M þ H]þ 478.14366;
found 478.14327.
reaction was kept at T ¼ 50 ꢁC until completeness (about 36 h).
17

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
4.2. Molecular modeling
electrical resistance (TEER). To test putative correctors, compounds
were added to the basolateral medium for 24 h at 37 ꢁC and 5% CO2,
before measuring the TEER. Control epithelia were treated with
vehicle alone (DMSO). TEER was measured in each well under basal
All the compounds were built, parameterized (Gasteiger-Huckel
method) and energy minimized within MOE using MMFF94
forcefield [MOE: Chemical Computing Group Inc. Montreal. H3A
2R7 Canada. http://www.chemcomp.com]. Docking calculations
were performed taking into account the X-ray structure of the
conditions, after ENaC inhibition with apical amiloride (10
after CFTR stimulation with forskolin (10 M) and genistein (50
on both sides, and after CFTR inhibition with apical PPQ102
(30 M). After each treatment, we waited 10 min before recording
mM),
m
mM)
F508del-CFTR
hNBD1
domain
(pdb
code
¼
4WZ6,
m
resolution ¼ 2.05 Å) [41] by means of LeadIT 2.1.8 software suite
(www.biosolveit.com). In particular, molecular docking studies of
the explored hybrids and reference correctors VX-809, ALK-809 and
SUL-809 were performed considering the putative binding site
within the NBD1 domain as proposed by the site finder module of
MOE software. Then, docking calculations were performed by
means of LeadIT 2.1.8 software suite (www.biosolveit.com) starting
from the best scored hNBD1-VX-809 complex previously derived by
automatic docking with MOE software. The final docking poses
were prioritized by the score values of the lowest energy pose of
the compounds docked to the protein structure. All ligands were
refined and rescored by assessment with the algorithm HYDE,
included in the LeadIT 2.1.8 software. The HYDE module considers
dehydration enthalpy and hydrogen bonding [42,43].
electrical parameters. The TEER values for each well were con-
verted into short-circuit current equivalent by Ohm’s law. The TEER
values were converted into TEEC.
4.3.4. Biochemical analysis of CFTR expression pattern
CFBE41o-cells stably expressing mutant CFTR and HS-YFP were
grown to confluence on 60-mm diameter dishes and treated for
24 h with test compounds or vehicle alone. After 24 h cells were
lysed in RIPA buffer containing a complete protease inhibitor
(Roche). Cell lysates were subjected to centrifugation at
12000 rpm at 4 ꢁC for 10min. Supernatant protein concentration
was calculated using the BCA assay (Euroclone) following the
manufacturer’s instructions. Equal amounts of protein (10 mg) were
separated onto gradient (4e15%) Criterion TGX Precast gels (Bio-rad
laboratories Inc.), transferred to nitrocellulose membrane with
Trans-Blot Turbo system (Bio-rad Laboratories Inc.) and analyzed by
Western blotting. Proteins were detected using monoclonal anti-
CFTR (596, Cystic Fibrosis Foundation Therapeutics, University
of North Carolina, Chapel Hill) or mouse monoclonal anti-GAPDH
(cl.6C5; Santa Cruz Biotechnology, Inc) followed by horseradish
peroxidase (HRP)-conjugated anti-mouse IgG (Abcam), and sub-
sequently visualized by chemiluminescence using the SuperSignal
West Femto Substrate (Thermo Scientific). Chemiluminescence was
monitored using the Molecular Imager ChemiDoc XRS System.
Images were analyzed with ImageJ software (National Institutes of
Health). Bands were analyzed as ROI, normalized against the
GAPDH loading control. Data are presented as mean SEM of in-
dependent experiments.
4.3. Biological evaluations
4.3.1. 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 sup-
plemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL
penicillin, and 100 mg/mL streptomycin.
4.3.2. Fluorescence assay for CFTR activity
CFBE41o-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 compounds and/or VX-809 or
VX-661. After treatment, the culture medium was removed and
cells in each well were stimulated for 30 min at 37 ꢁC with 60 mL
PBS (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
4.3.5. Statistics
Each experimental condition was tested in three independent
experiments, each one performed with three biological replicates
(n ¼ 9). The KolmogoroveSmirnov test was used to evaluate the
assumption of normality. Statistical significance of the effect of
single treatments on CFTR activity or expression was tested by
parametric 1-way ANOVA followed by the Dunnet multiple com-
parisons test (all groups against the control group) as post-hoc test.
In the case of combination of treatments, statistical significance
was verified by ANOVA followed by the Tukey test (for multiple
comparisons) as post-hoc test. Normally distributed data are
expressed as mean SD or mean SEM, as indicated, and signifi-
cances are two-sided. Differences were considered statistically
significant when P was less than 0.05.
(20
mM) and genistein (50 mM).
At the time of assay, microplates carrying CFBE41o-cells were
transferred to a microplate’s reader (FluoStar Galaxy; BMG Labtech,
Offenburg, Germany). The plate reader was equipped with high
quality excitation (HQ500/20X: 500
(HQ535/30 M: 535 15 nm) filters for YFP (Chroma Technology,
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 mL of a modified PBS containing I- instead
of Cl-; final I- concentration in the well: 100 mM). Data were
normalized to the initial background-subtracted fluorescence. To
determine fluorescence quenching rate associated with I- influx,
the final 10 s of data for each well were fitted with an exponential
function to extrapolate initial slope (dF/dt).
10 nm) and emission
4.4. Surface plasmon resonance binding assays
His-tagged human intact F508del-CFTR protein purified as
described [44,45] was provided by Prof. R.C. Ford, Manchester
University, UK. Lipids [synthetic phospholipid blend (Dioleoyl)
DOPC: DOPS [(7:3 w/w)] were from Avant Polar Lipids (Alabaster,
AL). CHAPS and cholesteryl hemi succinate (CHS) Tris salt were
from Sigma-Aldrich (St Louis, MO). Carboxy-methyl dextran CM5
sensor chip, anti-His antibody, 1-ethyl-3-(3-diaminopropyl)-car-
bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS)
were from GE-Healthcare (Milwaukee, WI).
Dose-response relationships from each experiment were fitted
with the Hill equation using the Igor software (WaveMetrics) to
calculate EC50, maximal effect, and Hill coefficient.
4.3.3. Transepithelial electrical resistance (TEER) evaluation
Primary bronchial epithelial cells obtained from one patient
with CF (homozygous for F508del mutation) were seeded at high
density on porous membranes (200.000 cells for 0.33-cm2 mini-
Transwell inserts (Corning, code 3379) for transepithelial
A BIAcore X-100 instrument (GE-Healthcare) was used. The
preparation of the biosensor containing F508del-CFTR in
a
18

A. Parodi, G. Righetti, E. Pesce et al.
European Journal of Medicinal Chemistry 208 (2020) 112833
membrane-like lipid environment was prepared as described [38].
Briefly, anti-His antibody was immobilized on a CM5 sensor chip by
standard amine-coupling chemistry. After sensor chip equilibration
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The manuscript was written through contributions of all au-
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
We wish to thank Prof. Robert C. Ford, University of Manchester,
UK for providing purified intact human F508del-CFTR protein.
This work was supported by Fondazione Italiana Fibrosi Cistica
FFC#6/2017 to EM and EC (with the contribution of Delegazione FFC
di Verona Valpolicella), FFC #10/2019 to MR (with the contribution
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