Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach ?

Article abstract of DOI:10.1021/acs.jmedchem.9b01416

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.

Full text of DOI:10.1021/acs.jmedchem.9b01416

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Article
Multi-Target CFTR Modulators Endowed with
Multiple Beneficial Side Effects for Cystic Fibrosis
Patients: Toward a Simplified Therapeutic Approach
Sabrina Tassini, Emily Langron, Leen Delang, Carmen Mirabelli, Kristina Lanko, Emmanuele
Crespan, Miroslava Kissova, Giulia Tagliavini, Greta Fontò, Simona Bertoni, Simone Palese,
Carmine Giorgio, Francesca Ravanetti, Luisa Ragionieri, Claudio Zamperini, Arianna
Mancini, Elena Dreassi, Giovanni Maga, Paola Vergani, Johan Neyts, and Marco Radi
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01416 • Publication Date (Web): 15 Nov 2019
Downloaded from pubs.acs.org on November 15, 2019
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Multi-Target CFTR Modulators Endowed with
Multiple Beneficial Side Effects for Cystic Fibrosis
Patients: Toward a Simplified Therapeutic
Approach
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Sabrina Tassini, Emily Langron, Leen Delang, Carmen Mirabelli, Kristina Lanko,
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Emmanuele Crespan, Miroslava Kissova, Giulia Tagliavini, Greta Fontò, Simona Bertoni,
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Simone Palese, Carmine Giorgio, Francesca Ravanetti, Luisa Ragionieri, Claudio
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Zamperini, Arianna Mancini, Elena Dreassi, Giovanni Maga, Paola Vergani, Johan Neyts,
and Marco Radia,*
This article is dedicated to the memory of prof. Maurizio Botta, a mentor and friend who
inspired everyone who knew him with his unique vision for research and innovation; his legacy
will live on.
a
Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Viale
delle Scienze, 27/A, 43124 Parma, Italy
b
Laboratory of Virology and Experimental Chemotherapy, Rega Institute for Medical
Research, KU Leuven, Minderbroedersstraat 10, 3000, Leuven, Belgium
c
Department of Neuroscience, Physiology and Pharmacology, University College London,
Gower Street, WC1E 6BT London, UK
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Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
Dipartimento di Scienze Medico-Veterinarie, Università degli Studi di Parma, Via del Taglio
10, 43126 Parma, Italy
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Lead Discovery Siena S.r.l., via Vittorio Alfieri 31, Castelnuovo Berardenga, 53019 Siena,
Italy.
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Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, 53100
Siena, Italy
KEYWORDS: F508del-CFTR, cystic fibrosis, multi-target, PI4KIII, broad-spectrum
antivirals, enterovirus.
ABSTRACT
Cystic fibrosis (CF) is a multi-organ disease caused by mutations of the cystic fibrosis
transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to
mucus accumulation, viruses and bacteria triggers acute pulmonary exacerbations, accelerating
disease progression and mortality rate. Treatment complexity increases with patients’ age and
simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently
reported the discovery of multi-target compounds able to “kill two birds with one stone” by
targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as correctors and broad-
spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein
a hit-to-lead optimization and multi-dimensional SAR study that led to compound 23a. This
compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy
with lumacaftor and a promising in vitro ADME profile. It was well tolerated in vivo with no
sign of acute toxicity and histological alterations in key biodistribution organs.
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INTRODUCTION
Cystic fibrosis (CF) is a common lethal autosomal recessive disease caused by mutations
affecting the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated
anion-selective channel expressed in various epithelia such as those of airways, intestinal tract,
pancreas ducts, testes and sweat glands. About 90% of cystic fibrosis patients are either
heterozygous or homozygous for a CFTR protein lacking phenylalanine 508 (ΔF508-CFTR).1
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The ΔF508 mutant is largely unable to exit the endoplasmic reticulum. Only a small fraction
of the synthesized protein reaches the plasma membrane (trafficking defect). However, when
targeted to the plasma membrane, the ΔF508 protein also shows an accelerated rate of
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internalization and an intrinsic low channel activity compared to wild type CFTR. Low anion
flow due to mutated CFTR, causes reduced fluid secretion and accumulation of abnormally
thick mucus that clogs the ducts and favors the development of bacterial/viral infections. CF
epithelial cell vulnerability and dysregulation of the local inflammatory responses lead to more
severe viral infections and appear to render the airway even more prone to bacterial infection,
leading to pulmonary exacerbations that accelerate disease progression, impair quality of life,
increase hospitalization and mortality rate.5-7 To reduce symptoms and slow organ
deterioration, patients receive multiple treatments (antibiotics, steroids, mucolytics,
bronchodilators, pancreatic enzymes) and undergo a daily physical exercise regimen including
physiotherapy to loosen and remove airway mucus. This complex regimen, combined with
patients’ comorbidities, makes therapeutic burden and drug-drug interactions important issues
in CF care.8-11 Over the years, there has been an intensive effort to find drugs acting on the root
cause of the disease: lumacaftor (VX-809) and tezacaftor (VX-661) are pharmacological
chaperones that correct the folding defect and improve trafficking of the mutant, while
ivacaftor (VX-770) is a small molecule acting as a potentiator of the channel’s conductance.12-
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However, F508del-CFTR patients do not benefit from monotherapy with these agents,
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providing only ~4% improvement in lung function measured as improvement in FEV1
percentage predicted. On the other hand, the combination of a corrector (lumacaftor) with a
potentiator (ivacaftor), marketed as Orkambi™, has provided minimal health benefits for
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people ages 12 and older, which were homozygous for the F508del-CFTR mutation. In
addition to the poor effectiveness of Orkambi™ in F508del-CFTR bearing patients, lumacaftor
negatively interacted with ivacaftor, increasing its metabolism and thus requiring a higher dose
of the latter when used in combination as Orkambi, while Ivacaftor reduces F508del-CFTR
surface expression.16-18 Recently, the positive effect of combination treatments in correcting
F508del-CFTR has been highlighted by Carlile et al. by showing that a triple combination of
correctors (MCG1516A, RDR1 and VX-809) binding to different pockets on CFTR, yielded a
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functional expression of the channel that was approximately 90% of wild-type’s. Even if drug
combinations seem to be the right approach for optimal F508del-CFTR functional expression,20
drug-drug interactions (e.g. lumacaftor-ivacaftor, macrolides-lumacaftor/ivacaftor) represent a
serious risk and must be taken into account for a safe and efficient CF therapy.16
Considering the poor efficacy of current therapeutic treatments for F508del-CFTR, the CF
therapeutic burden, the severity of viral infections in CF and their role in facilitating bacterial
colonization,21,22 our research group has recently approached this complex disease by
developing multi-target compounds able to simultaneously act as correctors of the F508del-
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CFTR folding defect and as broad-spectrum antivirals. Here we report the synthesis and
biological profiling of new bithiazole derivatives to elucidate the structural requirements
needed to improve both F508del-CFTR correction and antiviral potencies. Among the
synthesized compounds, 23a-c showed a dual effect on F508del-CFTR, increasing the
membrane expression (in a pHTomato assay) and channel function. They had a corrector effect
additive to VX-809’s, increasing the F508del-CFTR membrane density three-fold more
thanVX-809 alone. The most interesting compound 23a showed a promising in vitro ADME
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profile, including human plasma and microsomal stability, acceptable passive permeation and
no membrane retention. It was also well tolerated in acute toxicity studies conducted on
C57BL/6 mice, with no sign of histological alterations of gut, lung and liver parenchyma.
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RESULTS AND DISCUSSION
Over the past decade, the “one-drug, one-gene, one-disease” paradigm has been questioned,
based also on functional genomic studies showing that single-gene knockouts gave a
phenotypic response in a low percentage of cases, due to redundancy and compensatory
signaling of biological networks.24-26 It has been proposed that partial inhibition of multiple
biological targets with a single (multi-target) drug, the so called polypharmacology approach,
is more effective than full inhibition of a single target, reducing target-related toxicity and
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attrition rate in clinical development. This “one-drug, multiple-targets” approach is quite
valuable in the treatment of a complex disease like cystic fibrosis, which is characterized by a
high drug-burden, virus/bacteria-related exacerbations and poor efficacy of current treatments,
especially for patients bearing the F508del-CFTR mutation. Recently, we used a combined
virtual docking/similarity clustering approach to identify, for the first time, single compounds
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acting as broad-spectrum antivirals and correctors of the F508del-CFTR folding defect. We
reasoned that the best way to kill two birds with one stone is if the birds are closed in the same
cage: out of metaphor, we focused on two targets (PI4KIIIβ and F508del-CFTR) that are
localized within the endoplasmic reticulum (ER) to avoid additional problems of delivering the
compound to different locations/compartments. On one side, our multi-target molecule will
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enter the ER and bind F508del-CFTR (like structurally related Corr-4a does) to correct the
folding defect. On the other hand, once inside the ER this molecule will also inhibit PI4KIIIβ
to interfere with the replication of Picornaviruses and may also facilitate restoration of airway
surface liquid (ASL) fluidity by indirectly downmodulating epithelial Na(+) channel (ENaC)
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activity via reduction of PI(4,5)P concentration. In addition, PI4KIIIβ inhibition by the same
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multi-target compound may work as alternative immunosuppressive approach to overcome
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acute cellular rejection (ACR) in CF patients who have received lung transplant. Here we
started by analyzing the structure-activity relationship (SAR) of previously published multi-
target compounds against PI4KIII: as shown in Figure 1a, the central bithiazole scaffold was
fundamental for biological activity, while small aliphatic amide moieties (MeCONH,
EtCONH, t-BuCONH) on the left part improved activity and selectivity. Different substituents
on the right part of the molecule did not much change the activity against PI4KIII, although
only a few modifications were attempted. On the other hand, the SAR of bithiazole derivatives
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(
comprising our multi-target hits) as CFTR correctors has been recently reviewed by Barraja
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and colleagues and is summarized in Figure 1b. Although the binding pocket of this class of
molecules within CFTR has not been identified yet, a few general considerations can be derived
by SAR analysis: i. the central bithiazole scaffold in a planar s-cis conformation is fundamental
for CFTR correction; ii. a small C4-methyl or a C4-C5 cis locked system improve the activity;
iii. functionalization of the left part with Ph, p-EWD-Ph or t-Bu gave the best results; iv.
functionalization of the left part with a 2-MeO-Ph or 2-EtO-Ph improved CFTR correction.
Next, we focused our multi-dimensional SAR analysis on the most promising multi-target
compounds previously developed by us (1-4) in comparison with the known corrector 5 (Corr-
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a) (Figure 1c) which was not further developed because of the high toxic effect and low in
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vivo efficacy. The latter compound was synthesized as described in literature and submitted
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to the same panel of biological assays used for compounds 1-4. As described in Figure 1c,
we analyzed the in vitro activities of the selected compounds i) against a panel of
picornaviruses representative of all major groups; ii) against lipid kinases PI4KIII/ PI4KIII/
PI3K and iii) as F508del-CFTR correctors. Key efficiency metrics were also computed using
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Figure 1. a) PI4KIII 2D-SAR for multi-target inhibitors reported in ref 23. Key PI4KIII-
bithiazole interactions from docking studies are shown: residues giving hydrogen-bond (dotted
lines) or cation- / - interactions (green lines) are shown. PI4KIII residues involved in
hydrophobic interactions are reported with one letter AA abbreviation only. b) CFTR 2D-SAR
for bithiazole molecules derived from ref 31. c) Multi-dimensional SAR of selected multi-
target bithiazoles 1-4 compared to CFTR corrector 5 (Corr-4a). Biological activity was
evaluated against lipid kinases (PI4KIII/ PI4KIII/PI3K), F508del-CFTR correction and
selected picornaviruses: enterovirus 71 (EV71), coxsackievirus B3, poliovirus 1 (PV1), human
rhinovirus 2 (hRV02), human rhinovirus 14 (hRV14). EC and CC values expressed in μM.
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LLE, LELP and cLogP values were calculated using Datawarrior (version 4.6.0).34
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Datawarrior : lipophilic ligand efficiency (LLE), lipophilicity-corrected ligand efficiency
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LELP) and cLogP. As reported by Tarcsay et al., marketed drugs and advanced clinical
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candidates are characterized by LELP<10 and LLE>5. It emerged that small changes in the
functionalization of the bithiazole scaffold may be sufficient to convert the pure corrector 5
into the multi-target compounds 1-4, whose F508del-CFTR correction potency increased with
the bulkiness of the left amide moiety. However, too bulky amide moieties (e.g. substituted
aromatics) decreased the PI4KIII inhibition and consequently all the positive effects
connected with the modulation of this host kinase. The best compromise between lipid kinase
inhibition/selectivity and F508del-CFTR correction efficacy was obtained with the tert-butyl
amide moiety as in compound 2. Looking at the calculated metrics, Corr-4a was predicted to
be very lipophilic and this may account for its toxic effect. Among compounds 1-4, LLE and
LELP parameters indicated compounds 1 and 2 as the most promising starting point for a hit-
to-lead optimization study.
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Considering the incomplete SAR previously obtained by modifying compound 1 on the
right part of the bithiazole scaffold and aiming for less lipophilic compounds, we decided to
replace the right aromatic ring with different aliphatic moieties. To obtain the desired
compounds 9a-d, the bromo-derivative 8, previously prepared,23 was refluxed with the
substituted thioureas 7a-d as described in Scheme 1. The synthesis of 7a-d from the
corresponding primary and secondary amines 6a-d is outlined in the Supporting Info. We also
evaluated the possibility of irreversibly inhibiting PI4KIIIβ, considering that wortmannin is a
potent inhibitor of PI3K isoforms, binding covalently to the ε-amino group of LYS802 in the
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catalytic site. We planned to introduce different electrophilic warheads on the right part of
the bithiazole scaffold that would point towards LYS549 in the PI4KIIIβ ATP-binding pocket.
Starting from compound 10, easily prepared by refluxing 8 with thiourea, we introduced
reactive electrophilic moieties on the free amino group by reaction with the proper acyl chloride
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(
Scheme 1). First, by reaction with acryloyl chloride, we introduced an acrylamide Michael
acceptor (compound 9e), commonly found in many covalent kinase inhibitors. Following the
same procedure and using propionyl chloride, we synthesized its unreactive saturated analogue
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(compound 9f), to be used as a reference compound in the evaluation of the mechanism of
action of these covalent inhibitors. Similarly we obtained the bromoacetamide derivative (9g)
and we also added an isopropyl group to the terminal carbon of the acrylamide (9h) to reduce
the electrophilic reactivity of the Michael acceptor. For the synthesis of compound 9h, the acyl
chloride was prepared in situ from the corresponding acid, synthesized following the procedure
3
7
reported in literature. Compound 9i was obtained by reaction of 10 with the commercial 2-
chloroethanesulfonyl chloride in the presence of an excess of base to generate the reactive vinyl
sulfonamide Michael acceptor.38
Scheme 1. Reagents and conditions: i. (a) benzoyl isothiocyanate, DCM, rt, 12 h, (b) method
A (for 7a,b,d) NaOH 1N, THF, reflux, 2-18 h, 80-85%, method B (for 7c) N H H O, rt, 3 h,
2
4
2
7
6%; ii. EtOH, reflux, 1 h, 75-95%; iii. thiourea, EtOH, reflux, 40 min, 90%; iv. (a) method A
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(
for 9e-h) R Cl, DIPEA, THF, rt, 2-6 h, 43-74%, method B (for 9i) 2-chloroethanesulfonyl
chloride, DIPEA, DMAP, THF, rt, 12 h, 40%.
0
1
2
3
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5
6
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8
9
0
1
2
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9
0
Table 1. Activity of synthesized derivatives 9a-i in PI4KIIIβ inhibition assay and in virus-cell-
based enterovirus 71 (EV71) assay.
Cpd
PI4K IIIβ
IC50 (μM)a
EV71
LLE from LELP from
cLogP
IC50
IC50
t = 0 t = 5 t = 20
EC50 (μM)a
CC50 (μM)b
SIc
(PI4KIIIβ) (PI4KIIIβ)
min
min min
9
9
9
9
9
9
9
9
9
1
a
b
c
d
e
f
8.29
14.16
9.52
4.50
2.16
5.39
2.11
-d
-
-
-
-
-
3.88
3.38
3.57
6.22
5.86
5.18
5.57
3.72
8.34
9.55
9.31
3.53
4.90
5.52
5.17
8.72
4.20
1.10 ± 0.16
0.34 ± 0.04
0.88 ± 0.01
12.60 ± 0.02
1.89
25.50 ± 20.70
10.90
23.2
32.2
22.1
26.4
3.2
-
4.47
4.45
2.13
2.97
3.16
3.06
4.03
2.32
4.40
-
19.40 ± 2.08
333.00 ± 2.60
6.01 ± 0.80
-
-
0.57 0.14
5.58 5.21
1.71 1.67
NA
g
h
i
NA
-
-
17.40 4.84 5.58
107.00 ± 94.70 172.00 ± 8.25
1.6
-
NAe
0.48
-
-
-
-
-
-
>290.00
-
4.90
8.60
0.92 ± 2.75
16.50 ± 9.04
17.9
a
b
Values are the mean of at least three independent experiments. CC50 values were assessed by MTS method.
cSelectivity index (SI = CC50/EC50). - = not determined. NA = not active.
d
e
All the synthesized compounds were initially evaluated for their inhibitory potency against
PI4KIIIβ kinase in vitro and for their cell-based antiviral activity against enterovirus 71 (EV71)
using 1 as reference compound. As shown in Table 1, most compounds showed a low
micromolar activity against PI4KIIIβ (column t = 0 min) while only compound 9i was inactive.
Compounds 9e-i were also analyzed to confirm their irreversible inhibition of the PI4KIIIβ
kinase using 9f as a reference compound. For a reversible inhibitor, in a reaction mixture, an
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equilibrium of free enzyme and enzyme in complex with the inhibitor is established ([E] «
[E:I]). As a result, once the equilibrium has been reached, the proportion of enzyme inhibited
by the reversible inhibitor does not change over time. On the contrary, for an irreversible
inhibitor, once the [E:I] complex has formed, dissociation of the enzyme cannot occur. The
amount of [E:I] complex therefore increases over time. Assessing the potency of inhibitors at
different time points, can thus allow us to discriminate between reversible and irreversible
inhibitors. We performed inhibition assays leaving the enzyme in pre-incubation with the
inhibitors for different times [0 min (no pre-incubation); 5 min; 20 min] (Table 1). As expected
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for a reversible inhibitor, the calculated IC of compound 9f did not change over time. On the
50
other hand, compounds 9e and 9h showed IC values that decreased with increasing time of
5
0
pre-incubation. In particular for compound 9e, the IC decreased by about 15 fold from 2.16
5
0
μM (with no pre-incubation) to 0.14 μM (after 20 minutes of pre-incubation). Similarly, the
IC value of 9h decreased by about 3 fold after 20 minutes of pre-incubation with the enzyme.
5
0
In contrast, the bromoacetamide derivative 9g (with stable IC50 values in the same pre-
incubation time frame) did not seem to exhibit a similar irreversible mechanism of action.
Cell-based antiviral activity of compounds 9a-i was evaluated in an EV71-induced CPE-
reduction assay in rhabdosarcoma (RD) cells. The EC and CC values allowed us to calculate
5
0
50
the selectivity index (SI), defined as CC /EC . Compounds 9a-d inhibited EV71 replication
5
0
50
at low micromolar concentrations and with a good selectivity index (>20). However, the
activity of compounds 9b,c was associated with some morphological alterations of RD cells.
On the other hand, both compounds 9a,d showed a good antiviral profile and 9d also had little
effect on the uninfected host cells (minimal cytotoxicity). Compounds 9a-d were also
3
9
evaluated for their CFTR corrector activity using the pHTomato assay : none of the
compounds showed any effect on F508del-CFTR membrane exposure (data not shown).
Among compounds 9e-i, only 9e was able to inhibit EV71 replication at low micromolar
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concentration but this covalent PI4KIIIβ inhibitor had unacceptable levels of cytotoxicity and
no further studies were conducted on these derivatives.
From this first round of evaluations, it was clear that the introduction of aliphatic
moieties on the right portion of the bithiazole scaffold only resulted in a slight loss of affinity
for PI4KIIIβ without a major decrease in antiviral activity (see Table 1). Interestingly, a free
NH on the right side of the bithiazole is not needed for PI4KIIIβ inhibition and antiviral activity
0
1
2
3
4
5
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8
9
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
(Table 1, compound 9a vs 9c) and this observation reveals additional space for chemical
exploration. Finally, an ethanolamine chain on the right resulted in a compound with very low
cytotoxicity (9d), while the absence of CFTR corrector activity may be due to the low steric
bulk on the left of the molecule (C2 acetamido moiety) in line with the previous trend reported
in Figure 1.
Based on the above observations, we planned the synthesis of the new compounds
reported in Schemes 2-4. First, we decided to evaluate the effect of an additional ethoxy chain
on the two most interesting compounds reported in Table 1: the hit 1 and derivative 9a. The
secondary amines 11a,b (the synthesis of 11a is reported in the Supporting Info) were
converted into the corresponding thioureas 12a,b by reaction with ammonium thiocyanate (for
1
2a) or benzoyl isothiocyanate followed by basic deprotection (for 12b). The latter
intermediates were then cyclized by reaction with intermediate 8 in refluxing ethanol to give
the desired compounds 13a,b in good yields (Scheme 2).
Scheme 2. Reagents and conditions: i. method A (for 12a) NH SCN, HCl 1N, 100 °C 12 h,
4
6
5%, method B (for 12b) (a) benzoyl isothiocyanate, THF, reflux, 25 min, (b) LiOH 1M, THF,
reflux, 5 h, 69%; ii. 8, EtOH, reflux, 1 h, 86-89%.
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Next, we decided to modify compound 9d (with low cytotoxicity) by introducing bulkier
moieties on the left part of the bithiazole scaffold, in an attempt to increase CFTR correction
efficacy as suggested by SAR analysis (Figure 1). The condensation of the intermediate 14,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
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0
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0
1
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9
0
1
2
3
4
5
6
7
8
9
0
2
3
previously prepared, with the 1-(2-hydroxyethyl)thiourea 7d gave the common intermediate
1
5, whose hydroxyl group was then protected as t-butyldiphenylsilyl (TBDPS) ether (Scheme
3
). The free 2-amino group on the 4-methylthiazole ring (left part) was then properly acylated
by reaction with different acyl chlorides or anhydrides to obtain compounds 17a-c after TBAF-
deprotection of the crude mixture.
Scheme 3. i.7d, EtOH, reflux, 1 h, 80%; ii. TBDPSCl, imidazole, CH CN, 0 °C, 30 min,
3
1
5
7%; iii. (a) R COCl (for 17a,b) or di-tert-butyl dicarbonate (for 17c), Et N, DCM, rt, 15 h;
3
(
b) TBAF 1.0 M in THF, CH COOH, THF, rt, 18 h, 60-77%.
3
Having previously selected the tert-butyl amide moiety as the best compromise between lipid
kinase inhibition/selectivity and F508del-CFTR correction efficacy, we decided to modify
compounds 13a,b by introducing this bulkier group on the left part of the bithiazole scaffold.
We also decided to introduce aromatic/aliphatic carboxy moieties on the right part of the
molecule that may give profitable interactions with positively charged lysine residues (Lys549,
Lys377) in the binding pocket. Intermediate 18, previously prepared,23 was reacted with
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pivaloyl chloride to give compound 19 which was then brominated in α to the carbonyl, thus
providing the key intermediate 20. The latter compound was finally cyclized by reaction with
thioureas 12a,b or 22a-f (for the synthesis of 22a-f see the Supporting Information) affording
the expected compounds 23a-f (Scheme 4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
0
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0
1
2
3
4
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9
0
1
2
3
4
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6
7
8
9
0
Scheme 4. Reagents and conditions: i. pivaloyl chloride, pyridine, THF, reflux, 12 h, 90%; ii.
Br , 1,4-dioxane, 50 °C, 18 h, 80%; iii. method A (for 22c) NH SCN, HCl 1N, 100 °C 12 h,
2
4
6
5%, method B (for 22d-f) (a) SOCl , MeOH, reflux, 12 h, (b) benzoyl isothiocyanate, DCM,
2
rt, 1 h, (c) NaOH 1M, MeOH, reflux, 2 h, 60-72%; iv. 12a,b, 22c-f, EtOH, reflux, 1h, 68-88%.
The second set of synthesized derivatives (13a-b, 17a-c and 23a-f) was also first assessed
with respect to inhibitory potency against isolated PI4KIIIβ and antiviral activity against EV71.
As shown in Table 2, all compounds inhibited PI4KIIIβ at micromolar and sub-micromolar
concentrations with 13b, 17b and 23c being the most potent kinase inhibitors in vitro. PI4KIIIβ
inhibition correlates well with antiviral effect on EV71 replication with the notable exception
of compounds 13b and 17a. Compounds endowed with the most promising inhibitory potency
against EV71 and higher selectivity index (13a, 17b, 23a, 23c) were also tested against
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additional EVs: coxsackievirus B3 (CVB3), rhinovirus group A (RV02), and rhinovirus group
B (RV14). Results identified compounds 17b, 23a and 23c as the most promising antivirals
against this representative panel of enteroviruses, while the hit compounds 1 and 2 showed
lower SI values in EV71 replication and no activity (1) or high toxicity (2) against hRV02 and
hRV14. Interestingly, the introduction of an ethoxy chain on the right side of the bithiazole
scaffold was able to convert the relatively cytotoxic compound 2 into the less toxic and broad-
spectrum antiviral 23a.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Activity of synthesized derivatives 13a-b, 17a-c, 23a-f in PI4KIIIβ inhibition assay
and against a representative panel of Enteroviruses.
Cpd PI4K
IIIβ
EV71
CVB3
hRV02
hRV14
EC50
EC50
CC50
SIc
EC50
CC50
EC50
CC50
CC50
(μM)a
(μM)b
(μM)a
(μM)b
(μM)a
(μM)b
(μM)a
(μM)b
IC50
(μM)a
13a
13b
0.60
5.80
1.54
NAc
-d
-
-
-
-
-
-
-
4.51 ±
0.52
-
1.10 ±
16.40 ±
1.12
95.50 16.10
20.80 >263.00 >4.51
6.24
-
0
.86
1
1
1
7a
7b
7c
NA
-
-
-
-
-
-
1
.83 ±
361.00 ±
55.80
0
1
.73
.36
113.00 2.30
>293
5.40
41.20
3.80
41.20
-
0.32
1.40 ±
1.30
1
>421
>37.60
-
-
>218
-
-
6.10
-
-
16.80
-
-
>9.1
-
<
0.43
2
8.90 ±
0.50
2
3
.10
.40
(EC90
=
>67.20 6.60
16.80
1
2
2
3a
3b
5.78)
4
.96 ±
4.46 ±
1.85
-
-
-
1.85
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1
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1
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2
2
2
2
2
2
2
2
2
3
3
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5
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0
.42 ±
32.10 ±
8.10
0
.09
53.90 17.00 110.70
9.70
36.30
15.30
36.30
2
3c
0.01
5
.31 ±
9.93
5.46
>282
>271
261
>54.5
>33.1
-
-
-
-
-
-
-
-
-
-
-
-
-
23d
23e
0.26
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6.48 ±
1.88
7.27 ±
0.82
6
0
.98
.48
38.8
-
-
-
-
-
-
-
2
1
2
3f
0.92 ±
2.75
16.50 ±
9.04
17.90
2.17
101.00
± 33.60
15.10
NA
NA
0
.32
0.27 ±
0.50
7.94 ±
1.24
29.20
1.31
2.01 ±
0.05
4.07 ±
1.43
4.85 ±
1.09
3.87 ±
1.62
a
b
Values are the mean of at least three independent experiments. CC50 values were assessed by MTS method.
cSelectivity index (SI = CC50/EC50). - = not determined. NA = not active.
d
e
Next, compounds 13a-b, 17a-c and 23a-f (reported in Table 2) were evaluated for their
CFTR corrector/potentiator activity. As shown in Figure 2A, four compounds (17b, 23a-c)
acted as F508del-CFTR correctors, increasing steady-state levels of F508del-CFTR at the
plasma membrane after 24 h incubation at 10 M. Confirmatory tests on selected derivatives
1
7 and 23, in comparison with hits 3 and 4, were conducted at 3 M concentration of each
molecule. All compounds increased CFTR plasma membrane exposure (Figure 2B), with an
effect that was comparable (for 23a,b) to that of Lumacaftor (VX809). With the exception of
compound 17b, the improvement in CFTR biogenesis caused by all the other compounds also
led to increased anion permeability, estimated from fluorescence quenching of a CFTR-fused
YFP probe following extracellular iodide addition (Figure 2C).
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0
Figure 2. Effects of selected compounds on CFTR biogenesis and function. All treatments
o
were carried out alongside low temperature incubation (30 C), known to improve F508del-
CFTR membrane localization, in order to increase the fluorescence signal. A) F508del-CFTR-
pHTomato present at the plasma membrane was quantified following 24 h incubation in 10 M
of each drug. Incubation with VX809 (Lumacaftor) was assessed in parallel, as a positive
control and results were reported as % of VX809 effect ( %). B) Same as in A conducted on
F
selected compounds 17 and 23 incubated at 3 M in comparison with hit molecules 3 and 4.
C) Anion permeability quantified using a YFP-F508del-CFTR probe39 following 24 h
treatment as in A. D) F508del-CFTR-pHTomato exposed at the plasma membrane, estimated
following 24 h incubation with a lower concentration [3M] of the indicated compounds, with
or without VX-809.
The four most interesting derivatives, 17b, 23a-c, structural analogues of Corr-4a, can be
40
considered as belonging to the so called Class II correctors whose binding site on CFTR (still
unknown) was proved to be distinct from that of Class I correctors (e.g. Lumacaftor, VX-
8
09).41,42 As mentioned in the introduction, combinations of correctors from different classes
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are quite efficient in restoring F508del-CFTR functionality and here we evaluated the
interaction between our best derivatives and VX-809, using the Corr-4a/VX-809 combination
as reference (Figure 2D). All combinations showed an additive effect in improving F508del-
CFTR membrane-localization and the best results were obtained with 23a/VX809 and
0
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3b/VX-809 combinations, which improved the effect of VX-809 and increased the F508del-
CFTR membrane density up to three fold higher than seen with VX-809 alone. This confirms
that our dual-acting molecules, like Corr-4a, are likely to belong to the class-II family of
correctors and target a binding site on the F508del-CFTR that is different from that targeted by
VX-809.
To select the most suitable candidate for subsequent in vivo acute toxicity studies, a multi-
dimensional SAR analysis was conducted by plotting the size-adjusted PI4KIIIβ activity
(
expressed as LELP), antiviral activity (expressed as EV71 EC ), CFTR correction potency
50
(expressed as F508del-CFTR membrane exposure) and cLogP for the synthesized multi-target
inhibitors (9a-i, 13a-b, 17a-c, 23a-f) with respect to those of previously reported hits (1-4) and
reference compound 5 (Corr-4a) (Figure 3).
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Figure 3. A scatter plot of LELP (based on PI4KIIIβ IC ) versus cLogP for all synthesized
5
0
and reference compounds. Colors represent EV71 antiviral activity. Size of the circles
represents CFTR F508del-CFTR membrane exposure expressed as percentage of exposure
obtained following VX-809 incubation. The most representative compounds are labelled.
Dotted blue line follows the evolution of hit 1. Dotted orange line follows the evolution of hit
1
1
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2
. Plotted using Datawarrior (version 4.6.0).34
Lipophilicity-based metrics have been correlated to ADMET properties and may help
identify those compounds endowed with favorable pharmacokinetic and safety properties
(LELP <10) while looking at the most balanced multi-target activity profile. As we initially
mentioned, hit compounds 1 and 2 were chosen as starting points for structure optimization
(
described by dotted lines in Figure 3) because of their better multi-target profile and
LLE/LELP metrics compared to 3 and 4, while Corr-4a is just a pure CFTR corrector with high
lipophilicity. Starting from compound 1 and keeping the acetamido moiety on the left side of
the molecule unchanged, structural modification on the right side (blue dotted circle) led to a
series of derivatives (along the blue dotted line) with good LELP, comparable or lower
lipophilicity than 1 and, in a few cases (9a-d), promising antiviral activity but no effect as
CFTR correctors. On the other hand, starting from compound 2 and keeping the pivaloylamido
moiety on the left side of the molecule unchanged, structural modification on the right side
(orange dotted circle) led to a series of derivatives (along the orange dotted line) with a wide
range of LELP, lower lipophilicity, good antiviral activity and, in a few cases (17b, 23a-c)
improved CFTR corrector properties. Among the latter four compounds, 17b had the best
profile in terms of PI4KIIIβ inhibition, broad-spectrum antiviral activity and LELP but it
showed the lower F508del-CFTR correction potency and additivity but no synergy with
VX809. Compound 23b showed the best F508del-CFTR correction activity, some degree of
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synergy with VX809, moderate PI4KIIIβ inhibition and antiviral activity but high cytotoxicity.
Compound 23c showed the best PI4KIIIβ inhibition potency and was relatively potent/selective
against EV71, showed good F508del-CFTR correction potency but simple additivity with
VX809 and borderline LELP. Compound 23a showed moderate PI4KIIIβ inhibition potency
and some interesting activity against EV71 and CVB3, good F508del-CFTR correction
potency, additivity and possible synergy with VX809 plus borderline LELP with lower
lipophilicity than 23c and Corr-4a. Overall, the multi-target compound 23a showed the most
balanced activity profile and, despite its borderline LELP metrics, it was selected as the best
candidate for the following studies.
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In vitro ADME analysis was conducted on compounds 23a using Corr-4a (5) as a
comparison: aqueous solubility, stability in presence of human liver microsomes (HML),
stability in human plasma, membrane apparent permeability (P ) and binding to human serum
app
albumin (HSA) were determined in parallel for both compounds. As shown in Table 3,
compound 23a showed high human liver microsome (HLM) stability, improved plasma
stability, improved water solubility and acceptable passive permeation (P ) with no
app
membrane retention, coupled with a reduced affinity (K ) and nonsaturable binding (Bmax) to
D
human serum albumin (HSA). These data confirm a borderline ADME profile for compound
2
3a but also a sensible improvement with respect to the reference corrector 5 (Corr-4a).
Table 3. In vitro ADME assays
PAMPAb
Plasma Stabilityd
HSAe
Cpd
Solubility
Metabolic
P
app
Membrane
t1/2 (h)
K
D
(μM)
B
max
(
µM)a
Stability (%)c
-6
(
10 cm/sec) retention (%)
5
0.02
0.1
8.1
1.1
37.9
-
98
98
86.6
11.68
18.49
88.82
81.73
2
3a
579.9
a
b
Aqueous solubility was determined by means of the LC-MS method. Apparent permeability and membrane
c
retention were determined with PAMPA assay; see Experimental Section for details. The human liver microsome
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d
(
HLM) stability is expressed as a percentage of the unmodified parent drug. Stability in human plasma expressed
e
as t1/2, determined by LC-UV-MS (t1/2 = ln 2/Kobs). Binding to human serum albumin (HSA) expressed as ligand
concentration that binds to half the receptor sites at equilibrium (K
D
) and maximum number of binding sites (Bmax)
occupied by the compound .
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Acute toxicity studies were conducted on mice treated subcutaneously with 23a: dosage up
to 180mg/kg did not show any changes in motor coordination or sensory/motor reflexes, and
no behavioural alterations were observed up to 15 days after administration (Table S1;
Supporting Information). After fasting, 23a-treated mice recovered initial body weight almost
as quickly as vehicle-treated mice (Figure S1; Supporting Information).
Based on a previous report on the biodistribution of fluorescent bithiazole correctors in mice,43
we analyzed the effect of 23a on the histology of key biodistribution organs: lung parenchyma,
intestine mucosa and liver. At the two time points considered after 23a administration (4h and
7
2h), the histological analysis revealed no visible morphological alteration in all tested organs
(Figure 4), indicating lack of site-specific toxicity for 23a.
In the lung parenchyma, the alveolar microstructure was well maintained, with no sign of
alveolar/interstitial inflammatory infiltrate nor hemorrhage in 23a treated mice. The small
intestine mucosa, after 23a administration, showed a normal columnar lining cell with regular
distribution of Goblet cells, without signs of epithelial hyperplasia and inflammation of the
submucosa. In the liver, a normal hepatic labyrinth of hepatocytes with varying amounts of
vacuolation and basophilic material, representing endoplasmic reticulum, was seen both in
control and 23a-treated animals at both experimental time points. In summary, compound 23a
did not show severe cytotoxicity in vitro, nor were any serious clinical signs observed in the
acute toxicity study.
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Figure 4. Representative Microphotographs of the lung, small intestine and liver of mice
treated with the highest dose of compound 23a and vehicle-treated mice. Scale bar 100 µm.
CONCLUSIONS
The simplification of therapeutic complexity and burden in CF care is one of the top ten
priorities of future CF treatment. To address this issue, we have reported in this work the
optimization of an original polypharmacology approach recently established by our group: the
development of a well-balanced multi-target inhibitor able to simultaneously correct F508del-
CFTR and inhibit the replication of picornaviruses responsible of pulmonary exacerbation.
Chemical exploration of the hit bithiazole scaffolds guided by multi-dimensional SAR allowed
to identify a number of promising compounds able to inhibit PI4KIIIβ (and thus picornavirus
replication), correct the F508del-CFTR folding defect and act additively with the class I
corrector lumacaftor (VX-809). The most promising compound 23a showed a well-balanced
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multi-target activity profile, inhibiting at low micromolar concentration PI4KIIIβ and selected
picornaviruses (EV71, CVB3, hRV02), increasing steady-state levels of F508del-CFTR at the
plasma membrane (around 80% of VX809 effect) and anion permeability (around 50% of VX-
1
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09 effect) and acting additively/synergically with VX-809 to increase F508del-CFTR
membrane density up to three fold higher than VX-809 alone. The multi-target compound 23a
showed, in vitro, improved ADME properties with respect to Corr-4a, including high plasma
and human microsomal stability, improved water solubility and acceptable passive permeation
with no membrane retention (detected for the more lipophilic Corr-4a). Finally, in vivo acute
toxicity studies showed that compound 23a was well tolerated by C57BL/6 mice with no sign
of toxicity and histological alterations in key biodistribution organs up to 180mg/kg.
Compound 23a represent therefore an excellent starting point to better understand the benefits
and potentiality offered by a polypharmacology approach in comparison with standard CF
treatments. Future efforts will be focused on further compounds’ optimization and
quantification of known PI4KIIIβ-mediated beneficial side effects, such as restoration of
airway surface liquid (ASL) fluidity by indirectly downmodulating epithelial Na(+) channel
(ENaC) and prevention of acute cellular rejection that may cause premature graft loss in CF
patients who have received lung transplantation.
EXPERIMENTAL SECTION
Chemistry
General. All commercially available chemicals were purchased from both Sigma-Aldrich
and Alfa Aesar and, unless otherwise noted, used without any previous purification. Solvents
used for work-up and purification procedures were of technical grade. Dry solvents used in the
reactions were obtained by distillation of technical grade materials over appropriate
dehydrating agents. Reactions were monitored by thin layer chromatography on silica gel-
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coated aluminium foils (silica gel on Al foils, SUPELCO Analytical, Sigma-Aldrich) at 254
and 365 nm. Where indicated, products were purified by silica gel flash chromatography on
1
13
columns packed with Merck Geduran Si 60 (40-63 µm). H and C NMR spectra were
0
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0
recorded on BRUKER AVANCE 300 MHz and BRUKER AVANCE 400 MHz spectrometers.
1
Chemical shifts (δ scale) are reported in parts per million relative to TMS. H-NMR spectra
are reported in this order: multiplicity and number of protons; signals were characterized as: s
(singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), bs (broad signal). ESI-mass
spectra were recorded on an API 150EX apparatus and are reported in the form of (m/z).
Elemental analyses were performed on a Perkin-Elmer PE 2004 elemental analyzer. All final
compounds showed chemical purity ≥95% as determined by elemental analysis data for C, H,
and N (within 0.4% of the theoretical values). All final and reference compounds passed the
PAINS filter.44
General Procedure for the Synthesis of Compounds 9a-d.
A solution of intermediate 8 (50 mg, 0.18 mmol) and the proper thiourea 7a-d (0.18 mmol) in
ethanol (2.5 mL) was heated at reflux for 1 h. After cooling to room temperature, saturated
aqueous NaHCO solution, H O and ethyl acetate were added and the aqueous phase was
3
2
extracted three times with ethyl acetate. The combined organic phases were washed with brine,
dried over Na SO and evaporated. The crude was purified by flash chromatography using
2
4
DCM/MeOH (from 97/3 to 95/5) as eluent.
N-(2-(cyclohexylamino)-4'-methyl-[4,5'-bithiazol]-2'-yl)acetamide (9a). Yield 90%. MS
+
1
(ESI) [M + H] : 337.3 m/z. H NMR (DMSO-d 300 MHz): δ 1.08-1.34 (m, 5H), 1.55-1.59 (m,
6
1
H), 1.69-1.73 (m, 2H), 1.91-1.99 (m, 2H), 2.12 (s, 3H), 2.42 (s, 3H), 3.38-3.42 (m, 1H), 6.58
1
3
(
s, 1H), 7.66 (d, 1H, J = 7.5 Hz), 11.98 (s, 1H). C NMR (DMSO-d 100 MHz): δ 17.43, 22.91,
6
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4.86 (2x), 25.78, 32.67 (2x), 53.99, 100.41, 121.13, 142.50, 143.29, 155.29, 167.52, 168.59.
Anal. (C H N OS ) C, H, N.
1
5
20
4
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
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4
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0
1
2
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N-(4'-methyl-2-((4-methylcyclohexyl)amino)-[4,5'-bithiazol]-2'-yl)acetamide (9b). Yield
+
1
9
1
5%. MS (ESI) [M + H] : 351.3 m/z. H NMR (CDCl 400 MHz): δ 0.91 (d, 3H, J = 5.5 Hz),
3
.04-1.10 (m, 2H), 1.19-1.22 (m, 2H), 1.25-1.27 (m, 2H), 1.74-1.77 (m, 2H), 2.12 (s, 3H), 2.17
1
3
(bs, 1H), 2.51 (s, 3H), 3.21 (bs, 1H), 6.00 (d, 1H, J = 7.1 Hz), 6.41 (s, 1H), 11.60 (s, 1H). C
NMR (DMSO-d 100 MHz): δ 17.00, 22.13, 22.94, 29.70, 31.91 (2x), 33.85 (2x), 55.65,
6
1
01.16, 121.54, 142.17, 143.30, 156.68, 168.21, 168.79. Anal. (C H N OS ) C, H, N.
16 22 4 2
N-(2-(cyclohexyl(methyl)amino)-4'-methyl-[4,5'-bithiazol]-2'-yl)acetamide (9c). Yield
+
1
9
3%. MS (ESI) [M + H] : 351.0 m/z. H NMR (CDCl 400 MHz): δ 1.12-1.18 (m, 1H), 1.37-
3
1.54 (m, 4H), 1.69-1.72 (m, 1H), 1.85-1.87 (m, 4H), 2.23 (s, 3H), 2.58 (s, 3H), 3.00 (s, 3H),
13
3
2
.81 (bs, 1H), 6.42 (s, 1H), 11.74 (s, 1H). C NMR (CDCl 100 MHz): δ 17.18, 23.16, 25.57,
3
5.85 (2x), 29.83 (2x), 32.16, 60.24, 100.55, 122.42, 141.71, 143.91, 156.96, 168.09, 169.99.
Anal. (C H N OS ) C, H, N.
16 22
4
2
N-(2-((2-hydroxyethyl)amino)-4'-methyl-[4,5'-bithiazol]-2'-yl)acetamide (9d). Yield 75%.
+
1
MS (ESI) [M + H] : 299.2 m/z. H NMR (DMSO-d 300 MHz): δ 2.12 (s, 3H), 2.42 (s, 3H),
6
3.31 (q, 2H, J = 5.7 Hz), 3.57 (t, 2H, J = 5.7 Hz), 4.76 (bs, 1H), 6.60 (s, 1H), 7.71 (t, 1H, J =
13
5
1
.7 Hz), 11.97 (s, 1H). C NMR (DMSO-d 100 MHz): δ 17.45, 22.92, 47.65, 59.90, 100.80,
6
21.08, 142.55, 143.16, 155.31, 168.55, 168.63. Anal. (C H N O S ) C, H, N.
1
1
14
4
2 2
General Procedure for the Synthesis of Compounds 9e-h.
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DIPEA (46 μL, 0.29 mmol) was added to a stirred solution of intermediate 10 (50 mg, 0.20
mmol) in dry THF (2 mL) cooled to 0 °C. After 15 minutes a solution of the proper acyl
chloride (0.29 mmol) in dry THF (0.5 mL) was added dropwise. The resulting solution was
0
1
2
3
4
5
6
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8
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warmed to room temperature and stirred for 2-6 h, after which H O and ethyl acetate were
2
added and the aqueous phase was extracted twice with ethyl acetate. The combined organic
phases were washed with brine, dried over Na SO and evaporated. The crude was purified by
2
4
flash chromatography using DCM/MeOH (from 98/2 to 96/4) as eluent to give compounds 9e-
h as white solids.
N-(2'-acetamido-4'-methyl-[4,5'-bithiazol]-2-yl)acrylamide (9e). Yield 43%. MS (ESI) [M +
+
1
H] : 309.2 m/z. H NMR (DMSO-d 400 MHz): δ 2.14 (s, 3H), 2.48 (s, 3H), 5.91 (d, 1H, J =
6
1
0.0 Hz), 6.41 (d, 1H, J = 17.1 Hz), 6.55 (dd, 1H, J = 17.1, 10.0 Hz), 7.26 (s, 1H), 12.08 (s,
1
3
1H), 12.51 (s, 1H). C NMR (DMSO-d 100 MHz): δ 17.41, 22.89, 108.52, 120.27, 129.80,
6
1
30.09, 142.49, 143.40, 155.82, 157.91, 163.52, 168.85. Anal. (C H N O S ) C, H, N.
12 12 4 2 2
N-(2'-acetamido-4'-methyl-[4,5'-bithiazol]-2-yl)propionamide (9f). Yield 74%. MS (ESI) [M
+
1
+
H] : 311.4 m/z. H NMR (DMSO-d 400 MHz): δ 1.10 (t, 3H, J = 7.5 Hz) 2.14 (s, 3H), 2.45
6
1
3
(
q, 2H, J = 7.5 Hz), 2.47 (s, 3H), 7.19 (s, 1H), 12.06 (s, 1H), 12.22 (s, 1H). C NMR (DMSO-d
6
1
00 MHz): δ 9.55, 17.40, 22.89, 28.66, 107.71, 120.42, 142.10, 143.23, 155.77, 158.07, 168.77,
172.76. Anal. (C H N O S ) C, H, N.
1
2
14
4
2 2
N-(2'-acetamido-4'-methyl-[4,5'-bithiazol]-2-yl)-2-bromoacetamide (9g). Yield 68%. MS
+
1
(ESI) [M + H] : 375.2 m/z. H NMR (DMSO-d 400 MHz): δ 2.14 (s, 3H), 2.48 (s, 3H), 4.41
6
13
(
s, 2H), 7.28 (s, 1H), 12.08 (s, 1H), 12.67 (s, 1H). C NMR (DMSO-d 100 MHz): δ 17.36,
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2.88, 42.71, 108.53, 120.13, 142.41, 143.55, 155.85, 157.54, 165.65, 168.96. Anal.
(C H BrN O S ) C, H, N.
11 11 4 2 2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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0
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0
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0
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9
0
(E)-N-(2'-acetamido-4'-methyl-[4,5'-bithiazol]-2-yl)-4-methylpent-2-enamide (9h). Yield
+
1
4
9%. MS (ESI) [M + H] : 351.4 m/z. H NMR (DMSO-d 400 MHz): δ 1.05 (d, 6H, J = 6.7
6
Hz), 2.13 (s, 3H), 2.47 (s, 3H), 2.52-2.51 (m, 1H), 6.20 (d, 1H, J = 15.5), 6.96 (dd, 1H, J =
1
3
1
1
1
5.5, 6.7 Hz), 7.23 (s, 1H), 12.09 (s, 1H), 12.36 (s, 1H). C NMR (DMSO-d 100 MHz): δ
6
7.40, 21.58 (2x), 22.89, 30.85, 108.26, 119.99, 120.37, 142.39, 143.31, 154.24, 155.79,
58.15, 164.02, 168.78. Anal. (C H N O S ) C, H, N.
15 18
4
2 2
Synthesis of N-(4'-methyl-2-(vinylsulfonamido)-[4,5'-bithiazol]-2'-yl)acetamide (9i).
DIPEA (105 μL, 0.60 mmol) and catalytic DMAP (2.4 mg, 0.02 mmol) were added to a stirred
solution of intermediate 10 (50 mg, 0.20 mmol) in dry THF (4 mL). The mixture was cooled
to 0 °C and a solution of 2-chloroethanesulfonyl chloride (30 μL, 0.29 mmol) in dry THF (0.5
mL) was added dropwise. The mixture was stirred at room temperature for 12 hours, after
which H O and ethyl acetate were added and the aqueous phase was extracted twice with ethyl
2
acetate. The combined organic phases were washed with saturated aqueous NH Cl solution,
4
dried over Na SO and evaporated. The crude was purified by flash chromatography using
2
4
+
1
DCM/MeOH (96/4) as eluent. White solid. Yield: 40%. MS (ESI) [M + H] : 345.2 m/z. H
NMR (DMSO-d 300 MHz): δ 2.14 (s, 3H), 2.21 (s, 3H), 5.96 (d, 1H, J = 16.8 Hz), 6.03 (d,
6
1
H, J = 10.3 Hz), 6.89 (dd, 1H, J = 16.8, 10.3 Hz), 8.10 (s, 1H), 8.11 (s, 1H), 12.16 (s, 1H).
1
3
C NMR (DMSO-d 100 MHz): δ 17.11, 22.88, 115.35, 117.46, 127.32, 139.22, 147.96,
6
149.71, 157.63, 169.06, 171.17. Anal. (C H N O S ) C, H, N.
1
1
12
4
3 3
Synthesis of N-(2-amino-4'-methyl-[4,5'-bithiazol]-2'-yl)acetamide (10).
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A mixture of intermediate 8 (500 mg, 1.80 mmol) and thiourea (137 mg, 1.80 mmol) in ethanol
(
8.5 mL) was heated at reflux for 40 minutes. Then saturated aqueous NaHCO solution and
3
ethyl acetate were added to the mixture and the aqueous phase was extracted three times with
ethyl acetate. The combined organic phases were washed with brine, dried over Na SO and
0
1
2
3
4
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9
0
1
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0
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0
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4
concentrated under vacuum. Compound 10 was used in the following step without any further
+
1
purification. Yield 90%. MS (ESI) [M + H] : 255.2 m/z. H NMR (DMSO-d 400 MHz): δ
6
2
.11 (s, 3H), 2.41 (s, 3H), 6.56 (s, 1H), 7.12 (s, 2H), 11.96 (s, 1H).
Synthesis of 1-(3-acetylphenyl)-1-(2-hydroxyethyl)thiourea (12a).
NH SCN (339 mg, 4.46 mmol) was added portionwise to a solution of amine 11a (400 mg,
4
2
.23 mmol) in HCl 1N (3 mL) and the mixture was heated at 100 °C for 12 h. After cooling to
room temperature saturated aqueous NaHCO solution and ethyl acetate were added to the
3
mixture and the aqueous phase was extracted three times with ethyl acetate. The combined
organic phases were washed with brine, dried over Na SO and concentrated under vacuum.
2
4
The crude was purified by flash chromatography using petroleum ether/ethyl acetate (3/7) as
+
1
eluent. Yield 65%. MS (ESI) [M + H] : 239.2 m/z. H NMR (CDCl 400 MHz): δ 2.63 (s, 3H),
3
3
.88 (t, 2H, J = 6.0 Hz), 4.38 (t, 2H, J = 6.0 Hz), 4.70 (bs, 1H), 5.90 (bs, 2H), 7.58 (d, 1H, J =
.8 Hz), 7.61 (t, 1H, J = 7.8 Hz), 7.92 (s, 1H), 8.00 (d, 1H, J = 7.8 Hz).
7
Synthesis of 1-cyclohexyl-1-(2-hydroxyethyl)thiourea (12b).
Benzoyl isothiocyanate (469 μL, 3.49 mmol) was added dropwise to a solution of N-
ethylcyclohexylamine 11b (500 mg, 3.49 mmol) in dry THF (9 mL) and the mixture was
refluxed for 25 minutes. The solvent of reaction was evaporated, the residue was dissolved in
THF/LiOH 1M (1/1, 15 mL) and the mixture was refluxed for 5 h. After cooling to room
temperature, H O and ethyl acetate were added and the aqueous phase was extracted twice with
2
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ethyl acetate. The combined organic phases were dried over Na SO and evaporated. The crude
2
4
was purified by flash chromatography using petroleum ether/ethyl acetate (3/7) as eluent. Yield
+
1
69%. MS (ESI) [M + H] : 203.1 m/z. H NMR (DMSO-d 300 MHz): δ 1.27-1.45 (m, 4H),
6
1
1
1
1
1
1
1
1
1
1
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1
.68-1.94 (m, 6H), 3.50 (m, 2H), 3.52 (bs, 1H), 3.87 (m, 2H), 5.03 (bs, 1H), 6.67 (bs, 2H).
General Procedure for the Synthesis of Compounds 13a,b.
A solution of intermediate 8 (100 mg, 0.36 mmol) and the proper thiourea 12a,b (0.36 mmol)
in ethanol (2.5 mL) was heated at reflux for 1 h. After cooling down to room temperature,
saturated aqueous NaHCO solution, H O and ethyl acetate were added and the aqueous phase
3
2
was extracted three times with ethyl acetate. The combined organic phases were washed with
brine, dried over Na SO and concentrated under vacuum. The crude was purified by flash
2
4
chromatography using DCM/MeOH (96/4) as eluent, to give compounds 13a,b as white solids.
N-(2-((3-acetylphenyl)(2-hydroxyethyl)amino)-4'-methyl-[4,5'-bithiazol]-2'-yl)acetamide
+
1
(
13a). Yield 86%. MS (ESI) [M + H] : 417.3 m/z. H NMR (DMSO-d 300 MHz): δ 2.13 (s,
6
3
H), 2.44 (s, 3H), 2.61 (s, 3H), 3.72 (q, 2H, J = 5.4 Hz), 4.03 (t, 2H, J = 5.4 Hz), 4.92 (t, 1H, J
5.4 Hz), 6.76 (s, 1H), 7.63 (t, 1H, J = 7.8 Hz), 7.83 (d, 1H, J = 7.8 Hz), 7.90 (d, 1H, J = 7.8
=
1
3
Hz), 8.15 (s, 1H), 12.03 (s, 1H). C NMR (DMSO-d 100 MHz): δ 17.40, 22.92, 27.31, 55.78,
6
5
8.26, 102.87, 120.51, 126.54, 126.98, 130.70, 131.61, 138.80, 143.18, 143.74, 145.86, 155.53,
168.65, 168.69, 197.82. Anal. (C H N O S ) C, H, N.
1
9
20
4
3 2
N-(2-(cyclohexyl(2-hydroxyethyl)amino)-4'-methyl-[4,5'-bithiazol]-2'-yl)acetamide
(13b).
+
1
Yield 89%. MS (ESI) [M + H] : 381.4 m/z. H NMR (DMSO-d 300 MHz): δ 1.12-1.34 (m,
6
3
3
H), 1.56-1.64 (m, 3H), 1.79-1.82 (m, 4H), 2.12 (s, 3H), 2.44 (s, 3H), 3.40-3.41 (m, 2H), 3.58-
1
3
.60 (m, 3H), 4.83 (t, 1H, J = 5.4 Hz), 6.71 (s, 1H), 12.00 (s, 1H). C NMR (DMSO-d 100
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