From High-Throughput Screening to Target Validation: Benzo[ d]isothiazoles as Potent and Selective Agonists of Human Transient Receptor Potential Cation Channel Subfamily M Member 5 Possessing in Vivo Gastrointestinal Prokinetic Activity in Rodents

Article abstract of DOI:10.1021/acs.jmedchem.1c00065

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine, and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.

Full text of DOI:10.1021/acs.jmedchem.1c00065

pubs.acs.org/jmc
Article
From High-Throughput Screening to Target Validation:
Benzo[d]isothiazoles as Potent and Selective Agonists of Human
Transient Receptor Potential Cation Channel Subfamily M Member 5
Possessing In Vivo Gastrointestinal Prokinetic Activity in Rodents
Alessio Barilli,* Laura Aldegheri, Federica Bianchi, Laurent Brault, Daniela Brodbeck, Laura Castelletti,
Aldo Feriani, Iain Lingard, Richard Myers, Selena Nola, Laura Piccoli, Daniela Pompilio,
Luca F. Raveglia, Cristian Salvagno, Sabrina Tassini, Caterina Virginio, and Mark Sabat
Cite This: J. Med. Chem. 2021, 64, 5931−5955
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a nonselective monovalent cation
channel activated by intracellular Ca2+ increase. Within the gastrointestinal system, TRPM5 is expressed in the stoma, small intestine,
and colon. In the search for a selective agonist of TRPM5 possessing in vivo gastrointestinal prokinetic activity, a high-throughput
screening was performed and compound 1 was identified as a promising hit. Hit validation and hit to lead activities led to the
discovery of a series of benzo[d]isothiazole derivatives. Among these, compounds 61 and 64 showed nanomolar activity and
excellent selectivity (>100-fold) versus related cation channels. The in vivo drug metabolism and pharmacokinetic profile of
compound 64 was found to be ideal for a compound acting locally at the intestinal level, with minimal absorption into systemic
circulation. Compound 64 was tested in vivo in a mouse motility assay at 100 mg/kg, and demonstrated increased prokinetic activity.
The TRPM subfamily members are broadly expressed in a
variety of cells and tissues, such as sensory ganglia, pancreatic β
cells, immune cells, tongue, heart, and kidneys, and are critical
for sensory physiology, insulin release, magnesium homeostasis,
ischemic injury, and inflammatory responses.6
TRP cation channel subfamily M member 5 (TRPM5) has
been reported to be expressed in mammalian taste buds, in a
subset of cells that coexpress receptors for bitter, sweet, and
umami tastes.7,8 Using a mouse in which the TRPM5 promoter
drives the expression of GFP9 or direct antibody labeling,
TRPM5 expression has been detected in distinct subsets of
olfactory neurons and in the vomeronasal organ, in the upper
and lower gastrointestinal tract, and in the respiratory
system.10,11 TRPM5 is also expressed in pancreatic islets,
INTRODUCTION
■
Transient receptor potential (TRP) cation channels have been
described as polymodal cell sensors,1−4 activated by a vast array
of stimuli in the environment, ranging from temperature, natural
chemicals and toxins, to mechanical stimuli. TRP cation
channels also respond to endogenous agents and messengers
produced during tissue injury and inflammation. They are
grouped into six subfamilies: (i) TRPC for “canonical”
(TRPC1−7), (ii) TRPV for “vanilloid” (TRPV1−6), (iii)
TRPM for “melastatin” (TRPM1−8), (iv) TRPP for “poly-
cystin” (TRPP2, TRPP3, and TRPP5), (v) TRPML for
“mucolipin” (TRPML1−3), and (vi) TRPA for “ankyrin”
(TRPA1).
The TRP family gene products are intrinsic membrane
proteins with six transmembrane-spanning domains (S1−S6)
and a cation-permeable pore region between S5 and S6. The
length of the intracellular amino (N) and carboxy (C) termini
and structural domains varies significantly between members of
the TRP channel subfamilies.5 The cytoplasmic domains are
involved in the regulation and modulation of channel function
and trafficking. Functional TRP channels consist of four
identical or similar TRP subunits.
Received: January 14, 2021
Published: April 23, 2021
https://doi.org/10.1021/acs.jmedchem.1c00065
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 5931−5955
5931

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. HTS workflow.
where it is localized to insulin-secreting β-cells and mediates
depolarizing currents after glucose stimulation.12,13 Recently, it
has been reported that potentiation of TRPM5 with steviol
glycosides stimulates glucose-induced insulin release,14 there-
fore TRPM5 modulators may potentially be useful as insulin
secretagogue modulators. TRPM5 is also expressed in tuft cells,
which are taste-chemosensory epithelial cells of the small
intestine and are suggested to be critical for protection during
enteric infections and inflammatory responses.15 Chemosensory
tuft cells are involved in type-2 immune response initiation
through secretion of the interleukin (IL)-25 by a TRPM5-
dependent mechanism and mice lacking TRPM5 showed a
significant reduction in IL-25 production compared to wild-type
(WT), resulting in the inability to mount an immune response
against helminths.16
TRPM5 is voltage-sensitive, monovalent cation-selective, and
activated by the intracellular Ca2+ increase, producing a transient
depolarizing current.17 Currently, the TRPM5 pharmacology is
poorly understood and a very limited number of compounds
that selectively interact with this ion channel are known.
TRPM5-modulating molecules range from steviol glycosides
such as stevioside, rebaudioside A, and their aglycon steviol,
which potentiates the activity of TRPM5.14 Flufenamic acid is
described to reduce TRPM5 currents but in the micromolar
range of concentrations18 and is active on other ion channels
such as calcium-activated chloride channels and potassium,
calcium, and sodium channels,19 and displays a 10-fold higher
potency for TRPM4.20 Nicotine inhibits TRPM5 currents with
an effective inhibitory concentration in the millimolar range.21
Several azole-based antifungal compounds (clotrimazole,
ketoconazole, econazole, and miconazole) inhibit TRPM5
activity in the micromolar range18 and miconazole and
ketoconazole strongly inhibit the cardiac potassium and calcium
channels in the micromolar range corresponding to clinically
relevant concentrations.22
assay.25 Fluorescent Ca2+ detection with precise temperature
manipulation such as heat for TRPV1 and cold for TRPM826
and fluorescent dye-based assay measuring the fluorescence
quench of fura-2 by TRPM7-mediated Mn2+ influx were
utilized.27
TRPM5 channel activity cannot be assessed with a Ca2+-based
screening method because it is selective for monovalent cations.
Membrane potential dye28 and thallium flux-based assay using a
fluorescent thallium dye were reported.18 Herein, we decided to
use a membrane potential assay technology, followed by
automated patch clamp methods to identify new selective
TRPM5 activators.
RESULTS AND DISCUSSION
■
In the search for novel chemical matter to be utilized as a starting
point for the development of potent and selective agonists of
hTRPM5, a HTS approach was selected. More specifically, we
decided to screen a diversity-based compound library with a
fluorometric imaging plate reader (FLIPR) membrane potential
(FMP) assay protocol. According to the protocol developed, the
test compound is first added to CHO-K1 cell lines stably
expressing hTRPM5 at a concentration of 40 μM to see if it is
able to open the TRPM5 channel resulting in depolarization of
the cell membrane. In a second addition, 200 nM adenosine 5′-
triphosphate (ATP) (EC20) is added to increase the intracellular
Ca2+ concentration, activating the channel by 20% of the
maximal achievable activation. The EC20 concentration of ATP
was selected according to preliminary experiments, and in each
plate 200 nM ATP was added as second addition also in the wells
where, as first addition, was used the vehicle corresponding to
the 0% control. With this double addition protocol, both direct
agonists and potentiators (i.e., compounds increasing depola-
rization only if the channel is partially activated in the presence
of ATP) may be identified.
The HTS campaign was conducted on a library of 300,000
compounds (Figure 1) resulting from the combination of part of
the Aptuit screening collection (250,000 compounds) and a
subset of compounds selected from Takeda internal library
(50,000 compounds). This HTS campaign afforded 4066
positive hits (compounds with >20 or >50% response at the
first or second addition, respectively), including both agonists
and potentiators. Hits were progressed to hit confirmation (at
single concentration, 40 μM in duplicate) and to two counter
assays: an assay protocol measuring Ca2+-flux in CHO-
With the aim of identifying new selective TRPM5 activators
that would help in understanding the physiological and
pathological role of this channel, a high-throughput screening
(HTS) was run. TRP channels in general have been screened
using different methods such as a Ca2+-sensitive fluorescent dye
for TRPA123 and a membrane potential dye for TRPC.24
TRPV1 has been screened applying different technologies such
as the Ca2+-sensitive fluorescent dye, membrane potential dye,
and atomic absorption spectroscopy-based rubidium flux
5932
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
hTRPM5, to determine if potential agonists act on TRPM5
directly, or whether the observed activity was indirect, due to an
intracellular Ca2+ release, and an FMP assay using CHO-WT
cells to identify compounds, which elicit a membrane potential
change independent of TRPM5. The aim of these counter assays
is to identify and exclude false positive compounds.
Table 1. Hit Expansion/Validation by Commercially
Available Compounds
While a good overlap was generally observed between hit
identification and hit confirmation, a large proportion of
compounds active in agonist mode (1st addition) were found
to be also active in CHO-WT cells (false positives) and were,
therefore, discharged. A selection of compounds for follow up
with concentration−response curve (CRC) measurements was
made based on the following criteria: (i) TRPM5 activity in the
FMP assay [>30% for compounds active in the first addition or
>50% for compounds active in the potentiator mode (second
ATP addition) and a difference in activity in the FMP assay
between TRPM5 and WT >30% for both classes of hits], (ii) low
activity in the Ca2+-flux assay (<20% for agonist mode hits or
<50% for potentiator mode hits), and (iii) low activity on the
CHO-WT in the FMP assay (<30%). A total of 350 compounds
were selected for follow-up in CRCs and hTRPM4 selectivity
(FMP assay). For all test samples subjected to CRC, compound
identity and purity were assessed by liquid chromatography
(LC)−mass spectrometry (MS) analysis.
The 73 most promising compounds in terms of hTRPM5
activity and selectivity were selected to be further profiled in
electrophysiology using a QPatch assay, the vast majority of
them (63 out of 73) confirming activity. No difference in the
electrophysiology behavior could be highlighted between
agonists and potentiators and, because it was decided to utilize
electrophysiology as the primary screening assay in the following
hit to lead phase, no further differentiation was made between
these two classes. However, compounds tested in nominally free
intracellular calcium did not show activity (data not shown), and
therefore could be classified as potentiators. The hits were then
clustered and further prioritized based on the calculated
properties such as ligand efficiency (LE)29,30 and lipophilic LE
(LLE)31 to give 16 chemical series, which were further explored
by confirming the activity from solid samples and performing
structure−activity relationship (SAR) expansion mainly through
compound acquisition.
a
In this article, we will describe in detail the optimization of one
of these chemical series, which is represented by compound 1.
This analogue emerged as a moderate activator of hTRPM5
showing a pEC50 = 5.22 when tested in QPatch and pEC50 = 5.33
when tested in SyncroPatch (LE = 0.43 and LLE = 2.91). During
the following optimization process and after a validation phase,
compounds were tested only with a SyncroPatch instrument,
which allowed a higher throughput. The validation phase
showed a good correlation between the QPatch and
SyncroPatch data, although a shift of potency to higher values
for the SyncroPatch assay was observed, likely due to the higher
intracellular calcium concentration used (data not shown).
To perform the initial SAR expansion, the examples shown in
Table 1 were selected by means of substructure and similarity
searches on commercially available databases. As could be
expected, the initial potency of the analogues was very low, with
activity mostly between 1 and 10 μM, but these encouraging first
results, notably the activity of compound 2 (pEC50 6.48, LE =
0.49 and LLE = 4.56), prompted us to further explore this
chemical class through synthesis. At first, three regions were
identified in this scaffold for exploration: a right hand side,
consisting of the imidazole moiety, a left hand side, an aryl
Reported values are the means of at least two independent
experiments. All data were obtained using the SyncroPatch 384
Patch Engine except where stated otherwise.
b
moiety, and a third region was identified in the linker between
the right and the left hand sides. The screening of the first
analogues of hit compound 1 suggested some preliminary
considerations, which we wanted to further explore. In the left
hand side region, electron-withdrawing groups (EWG) seemed
to be more tolerated than electron-donating groups as can be
observed comparing compounds 3 and 4 (pEC50 <4.5 and 4.64,
respectively) with compounds 1, 5, and 7 (pEC50, respectively,
5.33, 5.60, and 5.29). The presence of a hydrogen atom on the
nitrogen in the linker did not seem crucial for activity as can be
observed in compound 6 (pEC50 5.30), where the substitution
on the linker was incorporated in a cycle, and compound 7
(pEC50 5.29). So far, little information could be acquired on the
right hand side region of the molecule: the presence of the
chlorine atom on the imidazole did not appear crucial for activity
(compound 5, pEC50 5.60) and a major steric hindrance in that
part of the molecule was detrimental for activity (compound 8,
5933
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 2. Exploration of Benzofused Moieties on the Left
Hand Side
Table 3. Exploration of the Right Hand Side Moiety
ab
,
compd
X
Y
Z
TRPM5 pEC50
2
9
S
O
NH
NMe
S
O
NH
NMe
N
N
N
N
CH
CH
CH
N
CH
CH
CH
CH
N
N
N
N
6.48 0.16
5.52 0.17
5.22 0.18
5.28 0.16
4.87 0.05
4.79 0.19
<4.5
10
11
12
13
14
15
<4.5
a
Reported values are the means of at least two independent
b
experiments. All data were obtained using the SyncroPatch 384
Patch Engine.
pEC50 < 4.5). The most interesting analogue discovered up to
this point was the benzo[d]isothiazole derivative 2 (pEC50
6.48), which was further explored.
A brief exploration of potential alternatives for the benzo[d]-
isothiazole ring is described in Table 2. The replacement of the
sulfur atom of compound 2 with an oxygen or a nitrogen atom
led to less-active compounds, with benzo[d]isoxazole (com-
pound 9, pEC50 5.52) being slightly more active than the
corresponding indazoles (compound 10, pEC50 5.22 and
compound 11, pEC50 5.28). The isomers of the corresponding
bicyclic systems (compounds 12 and 13) showed lower potency,
while compounds 14 and 15 were completely inactive.
The exploration of the imidazole moiety (right hand side) is
detailed in Table 3. In this case, the removal of the chlorine atom
(compound 18, pEC50 5.29) resulted in a 10-fold loss of activity.
The removal of the methyl group (compound 16, pEC50 < 4.5)
and the removal of one nitrogen atom to give a pyrrolo derivative
(compound 19, pEC50 < 4.5) resulted in complete loss of
activity. The insertion of an additional chlorine atom on the
heteroaromatic ring was not tolerated (compound 17, pEC50
<
4.5). The only substitution that retained most of the activity was
in compound 20 (pEC50 6.34), where the chlorine atom was
replaced by a bromine. The most interesting result was that the
thiazole derivative 25 (pEC50 7.05, LE = 0.57 and LLE = 4.31)
was found to be more active than compound 2. Following this
result, some more exploration of this new heterocyclic ring was
performed. Removing the nitrogen atom on the five membered
ring to give thiophene 26 (pEC50 5.56) resulted in loss of
activity, as was the case of the corresponding imidazole−pyrrole
pair shown in Table 3 (compounds 2 and 19), indicating the
importance of a nitrogen atom in that position. The substitution
of the chlorine atom with another EWG such as −CF3 was
tolerated (compound 27, pEC50 6.31), as was the substitution of
the chlorine atom with a methyl group (compound 28, pEC50
6.24). Interestingly, shifting the position of the methyl group led
to an inactive compound (compound 30, pEC50 < 4.5). While
the electronic contribution of the substituent seemed to be of no
particular importance, compound 29 (pEC50 < 4.5) showed that
a more hindered substituent such as the isopropyl led to an
inactive compound, suggesting that the allowed space around
this region of the molecule was somewhat limited. This was
confirmed when the benzo-fused derivative 33 was tested.
a
Reported values are the means of at least two independent
b
experiments. All data were obtained using the SyncroPatch 384
Patch Engine.
At this stage, we wanted to acquire more information on the
most promising analogues discovered so far, and the results for
compounds 2 and 25 are summarized in Table 4. Both
compounds showed activity on the orthologue mouse channel
similar to that shown for the human channel and very good
selectivity versus TRPM8, TRPV1, TRPV4, TRPA1, and
TRPM4. Kinetic solubility measured from 10 mM stock
solution in dimethyl sulfoxide (DMSO) showed a difference
between compounds 2 and 25. The imidazole moiety of
compound 2 seemed beneficial for solubility (215 μM), while
the thiazole 25 analogue showed very low solubility (50 μM).
Another difference between the imidazole and the thiazole series
was the plasma protein binding. For compound 2, the fraction
5934
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 4. In Vitro Characterization of 2 and 25
compound 2
compound 25
7.05 0.10
ab
,
hTRPM5 pEC50
mTRPM5 pEC50
6.48 0.16
ab
,
6.50 0.05
109
446
219, 1.03
7.07 0.08
193
462
126, 0.97
50
<4.8/<4.8/<4.8/5.5/<4.2
c
MiC (human) μL/min/mg protein
MiC (mouse) μL/min/mg protein
c
c
Caco-2 permeability efflux ratio (nm/s)
c
solubility pH 7.4 (μM)
215
a d
,
hTRPM8/hTRPV1/hTRPV4/hTRPA1/hTRPM4 (pEC50)
plasma protein binding (as fraction unbound) human/rat
<4.8/<4.8/<4.8/<4.8/<4.2
10.3/16.5
c
<1/<1
a
b
c
Reported values are the means of at least two independent experiments. All data were obtained using the SyncroPatch 384 Patch Engine. See the
d
Experimental Part section for details. All data were obtained by FLIPR.
Table 5. Exploration of Linkers
address the issue of microsomal clearance, a Met ID experiment
was performed. Two major metabolites were observed, M1
(30%, M/Z = 295) and M2 (10%, M/Z = 151). These two
metabolites suggested an oxidation process on the left hand side
of the molecule (+“O”) and a dealkylation process on the right
hand side region. From this first exploration, some conclusions
could be made: two series were identified, the thiazole series,
which showed greater potency, and the imidazole series, which
showed better overall drug metabolism and pharmacokinetics
(DMPK) parameters for compounds acting as systemic drugs.
The first exploration of the linker entailed the substitution on
the nitrogen atom, as shown in Table 5. The presence of the
hydrogen atom did not seem crucial for the activity as can be
observed by comparing the NH derivative (compound 2, pEC50
6.48) with the corresponding N-methyl derivative (compound
34, pEC50 6.80) on the imidazole series. Because the thiazole
moiety proved to be more active, we thought that the
introduction of a methyl group on the nitrogen of compound
25 could lead to a more potent derivative. However, the thiazole
N-methyl derivative (compound 35, pEC50 6.35) exhibited less
activity than the corresponding thiazole NH derivative
(compound 25, pEC50 7.05). For this reason, the exploration
of potential substituents on the nitrogen atom was performed on
the imidazole series, and the most interesting results are
presented in Table 5. The N-cyclopropylmethyl derivative
(compound 36, pEC50 6.80) retained the same activity as the N-
methyl derivative, but upon increasing the size of the carbon
ring, the activity was completely lost (compounds 38 and 39,
pEC50 < 4.5 for both). An interesting finding was that while the
N-ethyl derivative still showed some activity (compound 40,
ab
,
compd
X
R
TRPM5 pEC50
2
NMe
S
NMe
S
NMe
NMe
NMe
NMe
NMe
NMe
H
H
Me
Me
6.48 0.16
7.05 0.10
6.80 0.36
6.35 0.06
6.80 0.20
5.29 0.09
<4.5
<4.5
6.33 0.09
<4.5
25
34
35
36
37
38
39
40
41
cyclopropylmethyl
i-Pr
cyclohexylmethyl
benzyl
Et
acetyl
a
Reported values are the means of at least two independent
b
experiments. All data were obtained using the SyncroPatch 384
Patch Engine.
unbound was 10.3% in human and 16.5% in rat, while for
compound 25, both values were lower than 1%. For both
compounds, permeability measured with Caco-2 cells was found
to be high (219 nm/s for compound 2 and 126 nm/s for
compound 25). Initially, our goal was to find a compound that
could act as a systemic drug, thus some of our preliminary efforts
were directed toward increasing the metabolic stability, as both
compounds showed very high clearance both in human and
mouse microsomes [for compound 2, MiC (h) = 109 μL/min/
mg protein and MiC (m) = 446 μL/min/mg protein, while for
compound 25, MiC (h) = 193 μL/min/mg protein and MiC
(m) = 462 μL/min/mg protein]. In order to better understand
the possible metabolic pathways of compound 2 and help us
pEC50 6.33), the N-acetyl derivative (compound 41, pEC50
<
4.5) was completely inactive showing that the acetyl group was
not tolerated.
Table 6. Exploration of Linkers
ab
,
c
c
compd
X
R1
R2
H
TRPM5 pEC50
human MiC (μL/min/mg protein)
mouse MiC (μL/min/mg protein)
25
42
43
44
45
NH
NH
NH
NH
O
H
7.05 0.10
<4.5
5.12 0.09
<4.5
193
462
*CO
H
Me
Me
H
Me
H
173
157
250
763
6.08 0.17
a
b
c
Reported values are the means of at least two independent experiments. All data were obtained using the SyncroPatch 384 Patch Engine. See the
Experimental Part section for details.
5935
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
detrimental for the activity confirming that a basic nitrogen is
required (compound 42, pEC50 < 4.5).
Table 7. Exploration of the Benzene Ring
The next exploration was on the benzene ring of the
benzo[d]isothiazole scaffold, and different substitutions were
evaluated on all the available positions. This exploration served
two potential goals: on the one hand, we wanted to further
increase the potency, and on the other hand we wanted to
understand if some of these substitutions could improve the
ADME parameters. As can be observed in Table 7, the most
interesting substitution is at position 6, with the chloro
substituent (compound 47, pEC50 7.61) leading to an increase
in activity compared to the methyl substituent (compound 46,
pEC50 7.41). Both compounds were more active than the
corresponding unsubstituted analogue (compound 25, pEC50
7.05). As can be observed, fluoro substitution did not lead to any
improvement in terms of potency (compound 48, pEC50 7.01)
nor the introduction of the nitrogen atom in the same position
(compound 49, pEC50 6.33). Shifting the substituents on the
other two positions of the benzene ring led to a decrease in
potency, with the analogues substituted in position 4
(compounds 50, 51, 52, and 53) being slightly more potent
than the same analogues substituted in position 7 (compounds
54, 55, 56, and 57). As can be observed in Table 7, microsomal
stability did not improve with any of these substitutions,
indicating that the very high intrinsic clearance measured in vitro
is potentially related to the benzo[d]isothiazole core itself.
As far as potency is concerned, an unexpected result was
observed when the substitution at position 6 was combined with
the methylation of the nitrogen on the linker. We already
described how the N-methyl derivatives are more active than the
NH derivatives on the imidazole series, so it was expected that
the introduction of a substituent on the benzene ring could be
beneficial with these derivatives. However, the -chloro, -fluoro,
and -methyl derivatives (compounds 58, pEC50 5.79; 59, pEC50
5.55; and 60, pEC50 5.28) all exhibited less activity than the
unsubstituted derivative (compound 34, pEC50 6.80), thus
suggesting a possible steric clash between the methyl group on
the nitrogen atom with the ortho substituent on the benzene
ring.
The observation that higher activity could be achieved by
shifting the position of a substituent on the benzene ring
prompted us to explore the best position for the linker on the
benzo[d]isothiazole moiety. This brief exploration (described in
Table 8) showed that activity was completely lost when the
linker was moved to position 4 (see compound 62, pEC50 < 4.5),
was reduced when it was moved to position 7 (see compound
63, pEC50 5.84), and was increased when the linker was moved
to position 6 (see compound 61, pEC50 7.36). In the light of
these results, we wanted to combine the information obtained
on the most active position for the linker with the most
interesting substituents on the benzene ring.
a
Reported values are the means of at least two independent
b
experiments. All data were obtained using the SyncroPatch 384
Patch Engine. See the Experimental Part section for details.
c
This exploration is depicted in Table 9, where the most
interesting ortho substitutions (fluoro, chloro, and methyl) were
applied to both the thiazole and the imidazole series. As can be
observed, all the substitutions led to more active derivatives,
with compound 64 (pEC50 8.10, LE = 0.62 and LLE = 4.8)
bearing a chlorine at position 5, the linker at position 6, and the
thiazole on the right hand side being the most active compound
obtained from this chemical series. Figure 2 shows the details of
the electrophysiology experiment performed on compound 64.
All the previous findings obtained with the linker at position 5
were confirmed with the linker at position 6: the thiazole series
proved to provide more active compounds than the imidazole
Because the Met-ID experiment of compound 2 showed that
one possible path of metabolism entails the dealkylation of the
right hand side moiety, further exploration was performed on the
carbon atom of the linker, mainly to assess if improvement could
be achieved in microsomal stability, as briefly described in Table
6. The introduction of one or two methyl groups on the linker
proved to be detrimental for activity (compound 43, pEC50 5.12,
compound 44, pEC50 < 4.5), and did not give any improvement
in terms of microsomal stability. The replacement of the amine
linker with an oxygen (compound 45, pEC50 6.08) lowered the
potency by 10-fold while increasing clearance. Finally, the
replacement of the methylene group with a carbonyl was
5936
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Table 8. Exploration of the Linker Position
a
b
Reported values are the means of at least two independent experiments. All data were obtained using the SyncroPatch 384 Patch Engine.
The results of the in vivo experiment showed that compound
61 was not suitable for a systemic approach due to the very low
bioavailability. Because the whole chemical series suffered from
a low microsomal stability, we considered to shift from a
systemic to a local approach.
Table 9. Exploration of the Benzene Ring
Compound 64 emerged as the most suitable for this new
approach due to its greater potency (hTRPM5 pEC50 8.10) and
lower microsomal stability [MiC (mouse) = 520 μL/min/mg
protein] compared to compound 61.
a b
,
compd
R1
R2
X
TRPM5 pEC50
61
64
65
66
67
68
69
70
71
72
H
H
H
h
H
H
h
Me
h
h
S
S
7.36 0.14
8.10 0.21
7.95 0.20
7.75 0.15
7.54 0.11
7.42 0.20
7.07 0.16
6.99 0.16
6.53 0.05
6.51 0.13
Cl
Cl
Me
F
Me
H
F
H
Cl
NMe
S
Although a full PK profile is not available, when compound 64
was dosed orally in mouse at 1, 3, and 100 mg/kg, the exposure
in plasma was very limited and could be quantified only at 100
mg/kg at 6 h after administration. However, the exposure
measured in the target organs of the gastrointestinal tract (colon
and ileum) showed a very high exposure at 6 h (colon: 2620 ng/
g; ileum: 7379 ng/g), and some detectable levels at 24 and 48 h,
at the highest dose. These data suggested that compound 64
could be considered as a luminally restricted compound acting at
the intestinal level. See Table 12 for all the relevant data.
In light of this PK behavior, compound 64 was selected as a
compound to evaluate the in vivo effect of a locally acting
TRPM5 agonist at the intestinal level to enhance gastrointestinal
motility in mice. This stimulatory effect is considered clinically
relevant to the management of disorders characterized by
constipation or impaired motility in general. Following the
method described by Zhou et al.,32 we investigated the
stimulatory effect of 64 on gastrointestinal transit in normal
mice, measuring fecal parameters during the time after
administration (Tables 13 and 14, Figures 3 and 4). The
advantage of this model is the possibility to measure the fecal
parameters during the time of the experiment in the same mice;
the use of single cages provided with grid and removable tray on
the bottom permitted to remove feces at the different time
points without disturbing the mice.
S
NMe
NMe
NMe
NMe
NMe
Me
a
Reported values are the means of at least two independent
experiments. All data were obtained using the SyncroPatch 384
Patch Engine.
b
series; the methylation of the nitrogen atom on the linker on the
imidazole series led to a more potent compound (compare
compound 69, pEC50 7.07 with compound 71, pEC50 6.53);
finally, as described for the derivatives in Table 9, the
combination of the N-methylation on the linker with the
ortho substitution on the benzene ring proved to be detrimental
for the activity also with these isomers (compare compound 72
pEC50 6.51 with compound 65 pEC50 7.95).
A full DMPK and selectivity profile for compound 64 is shown
in Table 10, where it is compared with compound 61.
While the whole exploration performed allowed us to obtain a
significant increase in potency, it was clear that the metabolic
stability remained an unresolved issue. In order to confirm if the
in vitro data were predictive of the actual behavior in the animal
model, compound 61 was profiled in an in vivo pharmacokinetic
(PK) experiment following PO and IV administration in mouse,
and the data are summarized in Table 11. The results showed
that compound 61 displays a moderate volume of distribution,
moderate clearance (as predicted from the in vitro experiments),
a short half-life, and a very low oral bioavailability of 1.5% in
mouse.
The test compound was administered orally, in CD-1 mice, at
the doses of 1, 10, and 100 mg/kg and the number and weight of
feces were evaluated 1, 2, 3, and 6 h after treatment with the
tested compound or with the vehicle of formulation. In addition,
the wet feces for each mouse were dried at 105 °C for 24 h to
calculate the percentage of water of feces. At the same time
points, the general health status of mice was monitored for
evaluating the potential side effect on locomotion and general
5937
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. hTRPM5 current recorded in SyncroPatch potentiated by compound 64. (Left) Current traces recorded in the absence (black trace) and in
the presence of increasing concentrations of compound 64 (blue traces, concentrations indicated) obtained on a representative cell. (Right) Current
amplitudes measured in the presence of increasing concentrations of compound 64 and subtracted from the vehicle current amplitude were normalized
to the max value obtained for each cell. Normalized values obtained for the same concentration were averaged and plotted vs the log of concentration.
Vertical bars correspond to the standard error of the mean. The number of averaged values for each concentration ranges from 3 to 12. The estimated
pEC50 value was 8.10. The 95% confidence interval for the fitting is included in the dotted lines.
Table 10. DMPK Characterization of 61 and 64
compound 61
7.36 0.14
7.36 0.07
146
164
106.8, 1.08
511
<4.8/<4.8/<4.8/5.2/<4.2
<1/<1
compound 64
8.10 0.21
7.80 0.15
172
520
41, 1.6
30
<4.8/<4.8/<4.8/5.5/5.5
<1/<1
ab
,
hTRPM5 pEC50
mTRPM5 pEC50
ab
,
c
MiC (human) μL/min/mg protein
MiC (mouse) μL/min/mg protein
Caco-2 permeability efflux ratio (nm/s)
solubility pH 7.4 (μM)
hTRPM8/hTRPV1/hTRPV4/hTRPA1/hTRPM4 (pEC50)
plasma protein binding (as fraction unbound %) human/mouse
c
c
c
ad
,
c
a
b
c
Reported values are the means of at least two independent experiments. All data were obtained using the SyncroPatch 384 Patch Engine. See the
d
Experimental Part section for details. All data were obtained by FLIPR.
a
Table 11. Compound 61 In Vivo PK Studies
compd
adm
dose (mg/kg)
Clobs (mL/min/kg)
31.2 4.98
C
last (ng/mL)
V
ss (L/kg)
t
1/2 (h)
AUClast (μg h/mL)
F %
61
61
IV
PO
1
10
2.99 1.18
2.76 1.24
1.12 0.52
0.81
541 92.6
94.3 19.4
1.54
a
See Experimental Part section for details.
count (p < 0.01) and for the fecal weight (p < 0.01). An increase
of the percentage of water in feces statistically significant at 100
mg/kg was observed, considering the total fecal wet weight
during the 6 h of observation (56.9% with respect to 44.7% of
the vehicle group). The general status of the mice indicated a
normal locomotor and general behavioral condition.
Table 12. Compound 64 In Vivo PK Studies
mean exposure after oral administration in mouse
dose
(mg/kg)
collection time
(h)
plasma
(ng/mL)
colon
(ng/g)
ileum
(ng/g)
a
1
6
24
48
6
24
48
6
24
48
72
BQL
63.8
BQL
BQL
143
BQL
BQL
2620
164
BQL
BQL
BQL
BQL
BQL
BQL
7379
202
BQL
BQL
BQL
BQL
BQL
22.6
BQL
BQL
BQL
The positive results obtained for compound 64 indicate a
potential role for TRPM5 agonists as gastroprokinetic agents.
3
CHEMISTRY
■
100
Compound 2 and the analogues 9−15 were synthesized, as
described in Scheme 1, through reductive amination of the
commercially available heteroaryl amines 73a−h with 5-chloro-
1-methyl-1H-imidazole-2-carbaldehyde.
58
BQL
BQL
BQL
a
BQL = 20 ng/mL for plasma and all tissues.
The derivatives 16−33 bearing various right hand side
moieties were all prepared from benzo[d]isothiazol-5-amine
73a (Scheme 2). Compounds 18 and 25−33 were synthesized
using NaBH4, while for derivatives 16, 17, 19, 20, 22, and 24, the
reductive amination was performed with NaBH(OAc)3. For the
behaviors. Compound 64 demonstrated to increase fecal
parameters in mice, reaching a statistically significant effect at
100 mg/kg between 1 and 2 h after treatment, both for the fecal
5938
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 13. Results of In Vivo Experiment of Compound 64
total number of feces/mouse after treatment (mean SEM)
treatment
1-vehicle
2-compound 64
3-compound 64
4-compound 64
dose (mg/kg)
1 h
0.8
4.3 0.8
3.4 0.8
4.0 0.9
2 h
3 h
6 h
3
5.3 0.7
7.5 1.1
7.3 1.2
9.1 1.1
11.8 1.1
10.3 0.9
13.0 1.6*
20.1 1.4
24.3 1.8
18.5 1.1
16.3 1.7
1
10
100
10.2 1.5**
a
Statistical analysis was performed by Dunnett’s test. N = 12/group. *p < 0.05; **p < 0.01 treated group vs vehicle.
a
Table 14. Results of In Vivo Experiment of Compound 64
total weight (g) feces/mouse after treatment (mean SEM)
treatment
1-vehicle
2-compound 64
3-compound 64
4-compound 64
dose (mg/kg)
1 h
2 h
3 h
6 h
0.08 0.02
0.09 0.02
0.08 0.02
0.12 0.02
0.13 0.02
0.15 0.02
0.18 0.04
0.31 0.05**
0.21 0.03
0.23 0.02
0.23 0.03
0.41 0.05**
0.43 0.05
0.45 0.04
0.39 0.03
0.49 0.05
1
10
100
a
Statistical analysis was performed by Dunnett’s test. N = 12/group. **p < 0.01 treated group vs vehicle.
As shown in Scheme 3, the synthesis of the N-substituted
analogues 34−41 started with the corresponding NH derivatives
2 and 25. While compounds 34 and 35 were prepared using
MeI, the majority of derivatives (36−40) were synthesized
through reductive amination with the corresponding aldehydes
or with acetone (compound 37). Compound 41 was obtained
by reacting compound 2 with acetyl chloride.
The synthetic procedure for the derivatives 42−44 is outlined
in Scheme 4. The reaction of 73a with 5-chlorothiazole-2-
carbonyl chloride generated in situ provided amide 42.35
Derivatives 43 and 44 were obtained by condensation of 73a
with aldehyde 76 or ketone 77, respectively, followed by
addition of MeMgBr.36 5-Chlorothiazole-2-ethanone 77 was
prepared according to the literature.37
The synthesis of the O-linked compound 45 (Scheme 5)
started with the alkylation of benzo[d]isothiazol-5-ol 78, readily
available from the corresponding amine.38
Figure 3. Total number of feces per mouse after treatment with
compound 64. The test compound was administered orally, in CD-1
mice, at the doses of 1, 10, and 100 mg/kg and the total number of feces
was evaluated 1, 2, 3, and 6 h after treatment. Statistical analysis was
performed by Dunnett’s test. N = 12/group. *p < 0.05; **p < 0.01
treated group vs vehicle.
As described in Scheme 6, the substituted derivatives 46−57
and 82−84 were obtained from the commercial ortho-fluoro/
chloro arylaldehydes bearing a nitro group or a bromine atom in
position 5. The isothiazole ring closure was afforded using sulfur
and aq NH4OH39 or with a three-step procedure involving
BnSH, SO2Cl2, and NH3.40,41 Amino intermediates 81a−l were
obtained by nitro reduction or copper-mediated amination42,43
or with halogenation of benzo[d]isothiazol-5-amine 73a. The
subsequent reaction of 81a−l with the appropriate aldehydes
yielded the final products 46−57 and 82−84. Finally,
compounds 58−60 were obtained by reductive N-methylation
of 82−84 using formaldehyde.44
As depicted in Schemes 7 and 8, the variously substituted
derivatives 61−72 were prepared in a similar manner to the
compounds described in Scheme 6.
Figure 4. Total weight of feces (g) per mouse after treatment with
compound 64. The test compound was administered orally, in CD-1
mice, at the doses of 1, 10, and 100 mg/kg and the total weight of feces
(g) was evaluated 1, 2, 3, and 6 h after treatment. Statistical analysis was
performed by Dunnett’s test. N = 12/group. **p < 0.01 treated group vs
vehicle.
CONCLUSIONS
■
In conclusion, the objective of this study was to find a suitable
compound that could selectively interact with the hTRPM5 ion
channel. After a HTS, 73 potential hits were discovered and in
particular in this article the exploration of one of them is
described. After optimization of three distinct regions of hit
compound 1, guided by SAR analysis, compounds 61 and 64
were selected as potentially useful tool compounds for an in vivo
model due to their nanomolar TRPM5 agonist activity and
synthesis of compound 16, the N-trityl-protected aldehyde 75
was prepared according to the known procedures33 and the final
deprotection of the trityl group was accomplished with TFA.34
Derivatives 21 and 23 were obtained reacting 73a with the
appropriate chloromethyl-heterocycles.
5939
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 1. Synthesis of Compounds 2, 9−15
a
Reagents and conditions: (a) (i) 5-chloro-1-methyl-1H-imidazole-2-carbaldehyde, MeOH, 60 °C, 18 h, (ii) NaBH4, 0 °C−room temperature (rt),
2 h, 7−96%, two steps.
a
Scheme 2. Synthesis of Analogues 16−33
a
Reagents and conditions: (a) (i) triphenylmethyl chloride, Et3N, CH2Cl2, rt, 20 h, (ii) n-BuLi, dimethylformamide (DMF), tetrahydrofuran
(THF), −78 °C, 4 h, 57%, two steps; (b) (i) 75, NaBH(OAc)3, CH2Cl2, AcOH cat., rt, 16 h, (ii) TFA, CH2Cl2, rt, 2 h, 42%, two steps; (c) RCHO,
NaBH(OAc)3, CH2Cl2, AcOH cat., rt, 16 h, 28−74%; (d) (i) RCHO, MeOH, 60 °C, 16 h, (ii) NaBH4, 0 °C−rt, 2 h, 16−91%, two steps (e)
RCH2Cl, Et3N, EtOH, 80 °C, 6 h, 15−17%.
a
Scheme 3. Synthesis of N-Substituted Derivatives 34−41
a
Reagents and conditions: (a) NaH, MeI, DMF, rt, 1−16 h, 19−55%; (b) corresponding aldehydes, NaBH(OAc)3, CH2Cl2, AcOH cat., rt, 16 h,
42−59%; (c) acetone, Na(OAc)3BH, CH2Cl2, AcOH cat., rt−reflux, 22 h, 24%; (d) CH3COCl, Et3N, CH2Cl2, rt, 4 h, 74%.
A compound described as a blocker in Patent WO2010/132615 as
example 43 was obtained from Takeda laboratories.
excellent selectivity. In particular, compound 64 was inves-
tigated for its locally acting stimulatory effect on gastrointestinal
transit in mice due to its very low absorption and high
gastrointestinal tract exposure as shown by in vivo PK studies in
mice. Compound 64 showed a statistically significant increase of
the fecal parameters after oral administration at 100 mg/kg. The
positive results obtained for compound 64 indicate a potential
role for TRPM5 agonists as gastroprokinetic agents.
Cell Culture. CHO-K1 cell lines stably expressing human TRPM5
(CHO-hTRPM5) or mouse TRPM5 (CHO-mTRPM5) or human
TRPM4 (CHO-hTRPM4) and the CHO-K1 cell line (CHO) used as
the cell background for the generation of stable cell lines were cultured
in minimum essential medium supplemented with 2 mM L-glutamine
and 8 μg/mL blasticidin for CHO-hTRPM5 or F12-HAM (Ham’s F-12
nutrient mix) supplemented with 250 μg/mL hygromycin B for CHO-
mTRPM5 and CHO-hTRPM4 or F-12-HAM for the CHO cell line. All
media were supplemented with 10% heat inactivated fetal bovine serum
and 1% penicillin−streptomycin. Cells were passaged twice a week and
kept in a humidified incubator at 37 °C and 5% cO2.
QPatch HTX Assay. The QPatch HTX (Sophion Bioscience A/S,
Ballerup, Denmark), an automated electrophysiology platform
described in detail by Mathes et al.,45 was used for confirmation of
selective and specific hits. The extracellular solution was (in mM) NaCl
(137); KCl (4); CaCl2 (1.8); MgCl2 (1); N-(2-hydroxyethyl)-
EXPERIMENTAL PART
■
Materials and Methods. Chemicals were obtained from Sigma-
Aldrich, and cell culture reagents were purchased from Thermo Fisher
Scientific Spa (Italy), GE Healthcare Bio-Sciences (Austria). Cell lines
were purchased from Scottish Biomedical Drug Discovery (Glasgow,
Scotland). Four compounds described as “taste enhancers” in the
Patent WO2008/024364 as examples 2, 3, 1, and 10, respectively, were
synthesized in Aptuit. Compound 10 was used as a positive modulator.
5940
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
concentration calculated with MaxChelator software to control the
intracellular free calcium to obtain 5 μM free concentration.47 Test
compounds were prepared from 6 mM 100% DMSO stocks and dose−
response compound plates were prepared by serially diluting the stock
compound solutions into DMSO, then 200× well plates were obtained
on an Echo 550 (Labcyte) instrument. Assay plates were diluted in
extracellular solution, using a Multidrop dispenser (Thermo Scientific),
and tested in cumulative protocol at final concentrations of 0.03, 0.3,
3.1, and 31.6 μM in 0.5% DMSO, followed by an addition of a positive
modulator at 3 μM and a final addition of a blocker at 10 μM. The
SyncroPatch liquid protocol consists of 180 s for vehicle incubation and
150 s for compound incubation at each concentration tested, followed
by additional 150 s for positive modulator incubation and additional
150 s for blocker incubation. Cells were harvested and resuspended to
0.5 × 106 cell/mL in 30 mL of extracellular solution without BaCl2 and
maintained in a cell hotel reservoir at 10 °C with a shaking speed of 200
rpm for 10 min before starting the experiment. After initiating the
experiment, cell catching, sealing, whole-cell formation, liquid
application, recording, and data acquisition were performed sequen-
tially. Electrophysiological recordings were carried out at rt with
currents recorded on a whole cell configuration patch clamp. Once
whole cell formation was obtained, cells were maintained at a holding
potential of 0 mV and the following voltage protocol was applied at a
frequency of 0.1 Hz: a step to −80 mV for 200 ms, followed by a
depolarizing step to 80 mV for 500 ms and an hyperpolarizing step to
−80 mV for 200 ms, followed by a voltage ramp to 80 mV for 300 ms,
and after additional 300 ms at 80 mV a final step to the holding
potential. No leak subtraction was applied. Current amplitude was
measured at the end of the first depolarizing voltage step.
Data Analysis. The QPatch and SyncroPatch experiments were
performed by measuring the current amplitude in vehicle and in the
presence of compound at the end of each incubation period. The
percentage of current increase was calculated considering the value
recorded in vehicle as 100%. In QPatch data acquisition and analysis
was performed using Sophion QPatch assay software 5.6. In
SyncroPatch data acquisition and analysis was performed using
PatchControl and DataController384 software (Nanion, Inc).
FMP Assay for the hTRPM4 Receptor. The described assay provides
the in vitro agonist potency (pEC50) of compounds at the hTRPM4
receptor stably expressed in CHO cells by measuring the membrane
potential changes using FLIPR (Molecular Devices) and the red
component of the FMP kit (R8126, Molecular Devices). CHO-
hTRPM4 cells were plated at 10,000 cells/well in black, clear-bottom
384-well plates (781092, Greiner Bio-One GmbH) 24 h before the
assay and incubated at 37 °C, 5% CO2. In the assay, cells are washed
twice in Hank’s balanced salt solution (HBSS), 20 mM HEPES, pH 7.4,
leaving 10 μL/well after the final aspiration. Cells are incubated with the
10 μL/well loading solution 2× for 30 min at 37 °C and in 0% CO2.
Compounds were characterized in a concentration-dependent manner
(11 concentrations, 1:3 serial dilutions) for their ability to increase
fluorescence counts and a pEC50 was calculated. 10 μL/well of test
compound solutions, buffer (0% control), and 30 μM A23187 (10 μM
as the final concentration, 100% control) are added to the loaded cells
and membrane potential responses are measured. The final DMSO
concentration in the assay was 0.5%.
Scheme 4. Synthesis of Compounds 42−44
a
Reagents and conditions: (a) (i) 5-chlorothiazole-2-carboxylic acid,
(COCl)2, CH2Cl2, DMF cat., 0 °C−rt, 2 h, (ii) Et3N, CH2Cl2, 0 °C−
rt, 2 h, 53%, two steps; (b) (i) MeMgBr, THF, 0 °C−rt, 1 h, (ii)
Dess−Martin periodinane, CH2Cl2, rt, 1 h, 48%, two steps; (c) (i) 76
(for 43) or 77 (for 44), toluene, reflux, 20 h, (ii) MeMgBr, rt, 3 h, 8−
35%, two steps.
a
Scheme 5. Synthesis of the O-Linked Compound 45
a
Reagents and conditions: (a) 5-chloro-2-(chloromethyl)-1,3-thia-
zole, K2CO3, DMF, rt, 16 h, 62%.
piperazine-N′-ethanesulfonic acid (HEPES) (10); and D-glucose (10);
and the pH was adjusted to 7.4 with NaOH. The intracellular solution
was (in mM) KCl (120); NaCl (10); CaCl2 (x); MgCl2 (4.28),
hydroxyethylethylenediaminetriacetic acid (HEDTA) (10); and
HEPES (10); and the pH was adjusted to 7.2 with KOH. Value x
corresponds to 0.9 mM and it is the concentration calculated with
MaxChelator software to control the intracellular free calcium
concentration and obtain 1 μM free concentration. Test compounds
were prepared in DMSO 100% at a concentration 1000-fold greater
than test concentrations. Stock solutions were diluted in glass vials with
extracellular solution to final perfusion concentrations of 0.1, 1, 10, and
30 suitable for application to cells in the QPatch HTX system. Cells
were detatched with Triple and mixed with the SFM II serum-free
medium supplemented with 25 mM HEPES and then placed in a cell
storage tank in the QPatch machine 5 min before starting the
experiment. Electrophysiological recordings were carried out at rt with
currents recorded in a whole cell configuration patch clamp. Sophion
QPatch assay software was used for cell capture, seal formation, and
obtaining whole cell and data acquisition. Once whole cell formation
was obtained, cells were maintained at a holding potential of 0 mV and
the following voltage protocol was applied at a frequency of 0.1 Hz: a
step to −80 mV for 200 ms, followed by a depolarizing step to 80 mV for
500 ms and an hyperpolarizing step to −80 mV for 200 ms, followed by
a voltage ramp to 80 mV for 300 ms, and after additional 300 ms at 80
mV, a final step to the holding potential. No leak subtraction was
applied. Current amplitude was measured at the end of the first
depolarizing voltage step.
SyncroPatch Assay. The SyncroPatch 384 Patch Engine (Nanion
Technologies, Munich, Germany), an automated multiwell planar
patch-clamp system described in detail by Obergrussberger,46 was used
for focussed library screening and confirmation of selective and specific
hits. Chips with single-hole medium resistance (5−8 MΩ) were used.
The extracellular solution contained (in mM) NaCl (140); KCl (4);
BaCl2 (5); MgCl2 (1); HEPES (10); and D-glucose (5); and the pH was
adjusted to 7.4 with NaOH. The intracellular solution contained (in
mM) K2SO4 (75); KCl (30); NaCl (10); CaCl2 (x); MgCl2 (4.28),
HEDTA (10); HEPES (10); Na-ATP (2); and the pH was adjusted to
7.2 with KOH. Value x corresponds to 2.5 mM and it is the
hTRPM8 FLIPR Calcium Assay. The described assay provides the in
vitro agonist potency (pEC50) and antagonist potency (pIC50) of
compounds at the hTRPM8 channel stably expressed in HEK cells by
measuring changes in the intracellular Ca2+ concentration using FLIPR
technology (Molecular Devices) and the calcium 6 dye (Molecular
Devices, Sunnyvale, CA). HEK-hTRPM8 cells were plated at 20,000
cells/well in black, clear-bottom, poly-D-lysine-coated 384-well plates
(781946, Greiner Bio-One GmbH) 24 h before the assay and incubated
at 37 °C, 5% CO2. On the day of the experiment, the medium was
removed from cells and 30 μL/well loading solution (HBSS buffer with
calcium-6 dye and 2.5 mM probenecid) was added for 1 h at 37 °C and
1 h at rt. A dual addition and read-out FLIPR protocol was applied
allowing for agonist and antagonist characterization. In the first
addition, compounds were characterized in a concentration-dependent
manner (11 point CRC, 1:3 serial dilutions) for their ability to increase
5941
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 6. Synthesis of Derivatives 46−57, 82−84, and 58−60
a
Reagents and conditions: (a) sulfur, NH4OH, DMF, 90 °C, 16 h, 10−69%; (b) (i) BnSH, KOtBu, THF, rt, 2 h, (ii) SO2Cl2, CH2Cl2, rt, 1 h, (iii) 7
M NH3 in MeOH, THF, rt, 1 h, 15−58%, three steps; (c) NaN3, Cu2O, L-proline, DMSO, 100 °C, 1−16 h, 9−78%; (d) Fe, NH4Cl, EtOH, 80 °C,
3 h, 60−77%; (e) NCS, THF, rt, 16 h, 57%; (f) Selectfluor, DMF, rt, 3 h, 11%; (g) 5-chloro-1-methyl-1H-imidazole-2-carbaldehyde or 5-
chlorothiazole-2-carbaldehyde, NaBH(OAc)3, CH2Cl2, AcOH cat., rt, 16 h, 4−67%; (h) (i) 5-chloro-1-methyl-1H-imidazole-2-carbaldehyde or 5-
chlorothiazole-2-carbaldehyde, MeOH, 60 °C, 16 h, (ii) NaBH4, 0 °C−rt, 2−3 h, 18−55%, two steps; (i) HO(CH2O)nH, NaBH(OAc)3, CH2Cl2,
AcOH cat., rt, 24−72 h, 43−56%.
a b
,
Scheme 7. Synthesis of Compounds 61−63
a
b
R = Br, NO2. Reagents and conditions: (a) sulfur, NH4OH, DMF, 90 °C, 16 h, 43−67%; (b) NaN3, Cu2O, L-proline, DMSO, 100 °C, 1−16 h,
50−80%; (c) Fe, NH4Cl, EtOH, 80 °C, 3 h, 40%; (d) (i) 5-chlorothiazole-2-carbaldehyde, MeOH, 60 °C, 16 h, (ii) NaBH4, 0 °C−rt, 2 h, 13−20%,
two steps.
intracellular calcium levels with respect to the standard agonist (−)
menthol (100 μM as the final concentration, 100% control), and if a
concentration-dependent effect was observed, the pEC50 value was
calculated. Immediately after the 5 min recording of this first addition, a
second addition containing 20 μM (−) menthol was performed.
Inhibition of the agonist evoked signal, recorded for 3 min, indicated
antagonist activity of the compound and allowed the calculation of the
compound pIC50 value. The final DMSO concentration in the assay was
0.5%. The assay was performed at rt.
hTRPA1 FLIPR Calcium Assay. The described assay provides the in
vitro agonist potency (pEC50) and antagonist potency (pIC50) of
compounds at the hTRPA1 channel stably expressed in HEK cells by
measuring changes in the intracellular Ca2+ concentration using FLIPR
technology (Molecular Devices) and the calcium 6 dye (Molecular
Devices, Sunnyvale, CA). HEK-hTRPA1 cells were plated at 20,000
cells/well in black, clear-bottom, poly-D-lysine-coated 384-well plates
(781946, Greiner Bio-One GmbH) 24 h before the assay and incubated
at 37 °C, 5% CO2. On the day of the experiment, the medium was
removed from cells and 30 μL/well loading solution (HBSS buffer with
calcium-6 dye and 2.5 mM probenecid) was added for 2 h at 37 °C. A
dual addition and read-out FLIPR protocol was applied allowing for
agonist and antagonist characterization. In the first addition,
compounds were characterized in a concentration-dependent manner
(11 point CRC, 1:3 serial dilutions) for their ability to increase
intracellular calcium levels with respect to the standard agonist (−)
menthol (1 mM as final concentration, 100% control), and if a
concentration-dependent effect was observed, the pEC50 value was
calculated. Immediately after the 5 min recording of this first addition, a
5942
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Scheme 8. Synthesis of Analogues 64−72
a
Reagents and conditions: (a) (i) BnSH, KOtBu, THF, rt, 2 h, (ii) SO2Cl2, CH2Cl2, rt, 1 h, (iii) 7 M NH3 in MeOH, THF, rt, 1 h, 20−53%, three
steps; (b) NaN3, Cu2O, L-proline, DMSO, 100 °C, 16 h, 35−77%; (c) 5-chloro-1-methyl-1H-imidazole-2-carbaldehyde or 5-chlorothiazole-2-
carbaldehyde, NaBH(OAc)3, CH2Cdichloroethane (DCE), AcOH cat., rt−70 °C, 8−36 h, 39−54%; (d) NaH, MeI, DMF, 0 °C−rt, 3 h, 11%; (e)
HO(CH2O)nH, NaBH(OAc)3, DCE, AcOH cat., 40 °C, 48 h, 16%.
second addition containing 100 μM (−) menthol was performed.
Inhibition of the agonist-evoked signal, recorded for 3 min, indicated
the antagonist activity of the compound and allowed the calculation of
the compound pIC50 value. The final DMSO concentration in the assay
was 0.5%. The assay was performed at 37 °C.
hTRPV1 FLIPR Calcium Assay. The described assay provides the in
vitro agonist potency (pEC50) and antagonist potency (pIC50) of
compounds at the hTRPV1 channel stably expressed in CHO cells by
measuring changes in the intracellular Ca2+ concentration using FLIPR
technology (Molecular Devices) and the calcium 6 dye (Molecular
Devices, Sunnyvale, CA).
of the agonist-evoked signal, recorded for 3 min, indicated the
antagonist activity of the compound and allowed the calculation of the
compound pIC50 value. The final DMSO concentration in the assay was
0.5%. The assay was performed at rt.
Fluorescent Membrane Potential Assay for hTRPM5. Cells were
resuspended in the culture medium and seeded onto 384-well assay
plates at 5000 cells/well, and after an incubation of >18 h, they were
washed twice with 50 μL of assay buffer (20 mM HEPES, 1× HBSS, pH
7.4), leaving 10 μL. The FMP dye (FMP kit cat # R8123, Molecular
Devices, Wokingham, UK) dissolved in the assay buffer was then added
(10 μL) and incubated at 37 °C for 30 min. The plates of a batch were
placed in stacker at rt. Compound intermediate plates contained
compound at 3× concentration in the assay buffer with 1.5% DMSO. In
FLIPR, 10 μL of 3× compound or controls were transferred to the cell
plate (first addition) and the relative fluorescence unit (RFU) was
measured for 2 min (40 μM was the final compound concentration;
0.5% DMSO was the 0% control; and 10 μM A23187 was the 100%
control). After such time, 10 μL from 4× ligand plate (200 nM ATP
final concentration, and appropriate controls such as buffer for 0% effect
and 10 μM A23187 as 100% control) were transferred to the cell plate
and RFU was measured for 4 min. The averaged RFU of the baseline
and the max RFU values after first and second addition were exported.
For agonist and positive modulator assessment, the ratio between the
first addition and the baseline and the ratio between second addition
and the baseline were calculated, respectively. Upon treatment of cells
with an agonist of TRPM5, or alternatively with a compound that will
lead to an elevation of intracellular Ca2+ levels, the opening of the
TRPM5 channel will result in a depolarization of the cell membrane and
consequently in a change in the RFU to higher values.
Counter Assays. Two counter assays were set up: an assay measuring
Ca2+-flux in CHO-hTRPM5, to determine if potential agonists act on
TRPM5 directly, or on intracellular Ca2+ release, and a membrane
potential blue dye assay using CHO-K1 cells, to identify compounds
that elicit a membrane potential change independent of TRPM5.
Chemistry. Reagents and solvents were obtained from commercial
sources and used as supplied. The 1H NMR spectra were recorded on
either a Bruker Avance 400, a Bruker Avance III 400, or an Agilent
Direct Drive spectrometer. The spectra were acquired in the stated
solvent, and chemical shifts (δ) are reported in ppm relative to the
residual solvent peak. Chemical shifts are given in parts per million
using conventional abbreviations for designation of major peaks, for
example, s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublets; dt, doublet of triplets; and br, broad. MS was performed using
an ultraperformance LC/mass spectrometry (UPLC/MS) Acquity
system equipped with a photodiode array detector and coupled to a
Waters single quadrupole mass spectrometer. Compound UPLC/MS
retention times (Rt) were obtained using the following methods.
Method 1: Acquity UPLC CSH C18 column (50 mm × 2.1 mm, 1.7 μm
particle size) at 40 °C, a linear gradient from 3 to 100% B over 2 min [A
= 0.1% v/v solution of HCOOH in water, B = 0.1% v/v solution of
CHO-hTRPV1 cells were plated at 10,000 cells/well in black, clear-
bottom 384-well plates (781092, Greiner Bio-One GmbH) 24 h before
the assay and incubated at 37 °C, 5% CO2. On the day of the
experiment, the medium was removed from cells and 30 μL/well
loading solution (HBSS buffer with calcium-6 dye and 2.5 mM
probenecid) was added 1 h at 37 °C and 1 h at rt. A dual addition and
read-out FLIPR protocol was applied allowing for agonist and
antagonist characterization. In the first addition, compounds were
characterized in a concentration-dependent manner (11 point CRC,
1:3 serial dilutions) for their ability to increase intracellular calcium
levels with respect to the standard agonist capsaicin (30 μM as final
concentration, 100% control), and if a concentration-dependent effect
was observed, the pEC50 value was calculated. Immediately after the 5
min recording of this first addition, a second addition containing 100
nM capsaicin was performed. Inhibition of the agonist evoked signal,
recorded for 3 min, indicated antagonist activity of the compound and
allowed the calculation of the compound pIC50 value. The final DMSO
concentration in the assay was 0.5%. The assay was performed at rt.
hTRPV4 FLIPR Calcium Assay. The described assay provides the in
vitro agonist potency (pEC50) and antagonist potency (pIC50) of
compounds at the hTRPV4 channel stably expressed in HEK cells by
measuring changes in the intracellular Ca2+ concentration using FLIPR
technology (Molecular Devices) and the calcium 6 dye (Molecular
Devices, Sunnyvale, CA). HEK-hTRPV4 cells were plated at 20,000
cells/well in black, clear-bottom, poly-D-lysine-coated 384-well plates
(781946, Greiner Bio-One GmbH) 24 h before the assay, in the
presence of 1 μg/mL tetracycline, and incubated at 37 °C, 5% CO2. On
the day of the experiment, the medium was removed from cells and 30
μL/well loading solution (HBSS buffer with calcium-6 dye and 2.5 mM
probenecid) was added for 1 h at 37 °C and 1 h at rt. A dual addition
and read-out FLIPR protocol was applied allowing for agonist and
antagonist characterization. In the first addition, compounds were
characterized in a concentration-dependent manner (11 point CRC,
1:3 serial dilutions) for their ability to increase intracellular calcium
levels with respect to the standard agonist GSK1016790A (100 nM as
the final concentration, 100% control), and if a concentration-
dependent effect was observed, the pEC50 value was calculated.
Immediately after the 5 min recording of this first addition, a second
addition containing 20 nM GSK1016790A was performed. Inhibition
5943
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
HCOOH in acetonitrile (ACN)] and a flow rate of 1 mL/min. Method
2: Kinetex column EVO C18 100 Å (50 mm × 2.1 mm, 1.7 μm particle
size) at 40 °C, a linear gradient from 3 to 100% B over 2 min (A = 10
mM ammonium bicarbonate aqueous solution adjusted to pH 10 with
ammonia, B = ACN) and a flow rate of 1 mL/min. Thin-layer
chromatography analyses were carried out on alumina sheets precoated
with silica gel 60 F254 and visualized with UV light. Flash
chromatography purifications were performed using KP-Sil 32−63
μm 60 Å cartridges. When high-performance liquid chromatography
(HPLC) purification was performed, compounds were purified using a
semipreparative AutoPurification HPLC System from Waters.
AutoPurification System was equipped with a Waters 2525 binary
pump, a Waters 2996 diode array detector (DAD), and a Micromass
ZQ MS detector. A Waters 2767 sample manager fraction collector has
been used for sample injection and fraction recovery. Two different
types of reversed phase methods were used. Method A: XSelect
semipreparative column C-18 CSH (30 × 100 mm, 5 μm, from
Waters), linear gradient from 3 to 100% B over 15 min (A = 0.1% v/v
solution of HCOOH in water, B = 0.1% v/v solution of HCOOH in
ACN), and a flow rate of 40 mL/min; method B: Gemini C-18
semipreparative column (30 × 100 mm, 5 μm, from Phenomenex), a
linear gradient from 3 to 100% B over 15 min (A = 10 mM ammonium
bicarbonate aqueous solution adjusted to pH 10 with ammonia, B =
ACN), a flow rate of 40 mL/min. Purities of all target compounds were
greater than 95% as determined by HPLC analysis. Compounds were
analyzed for and found not to contain pan assay interference
substructural features.
General Procedure for the Synthesis of Compounds 2 and 9−15.
A solution of appropriate heteroaryl amine 73a−h (1 equiv) and 5-
chloro-1-methyl-1H-imidazole-2-carbaldehyde (2 equiv) in MeOH
(0.1 M) was heated at 60 °C for 16 h. After cooling to 0 °C, NaBH4 (3
equiv) was added and the reaction mixture was allowed to warm to rt
and stirred for 2 h. The mixture was treated with a saturated aqueous
NaHCO3 solution and extracted with dichloromethane (DCM). The
organic layer was washed with brine, dried over Na2SO4, filtered, and
evaporated to dryness. The residue was purified by reverse-phase
HPLC or as stated to provide the target compounds.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
isothiazol-5-amine (2). Compound 2 was prepared from benzo[d]-
isothiazol-5-amine 73a (2 mg, 0.013 mmol) according to the general
procedure. The residue was purified by preparative HPLC (method B).
White solid (3.5 mg, 96% yield). 1H NMR (500 MHz, CDCl3): δ 8.77
(s, 1H), 7.75 (d, J = 8.80 Hz, 1H), 7.35−7.19 (m, 1H), 7.02 (dd, J =
8.80, 1.96 Hz, 1H), 6.95 (s, 1H), 4.58 (br s, 1H), 4.41 (d, J = 4.89 Hz,
2H), 3.63 (s, 3H). LC−MS method 2: Rt = 0.79 min. MS-ESI (m/z):
calcd for C12H11ClN4S [M + H]+, 279.0; found, 279.1.
2H), 4.32−4.07 (m, 1H), 4.03 (s, 3H), 3.62 (s, 3H). LC−MS method
2: Rt = 0.69 min. MS-ESI (m/z): calcd for C13H14ClN5 [M + H]+,
276.1; found, 276.1.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
thiazol-5-amine (12). Compound 12 was prepared from benzo[d]-
thiazol-5-amine 73e (51 mg, 0.34 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (0−50% EtOAc/cyclohexane). White solid (48 mg, 51%
yield). 1H NMR (500 MHz, CDCl3): δ 8.93 (s, 1H), 7.73 (d, J = 8.56
Hz, 1H), 7.40 (d, J = 2.20 Hz, 1H), 6.98−6.89 (m, 2H), 4.56 (br s, 1H),
4.41 (d, J = 3.67 Hz, 2H), 3.62 (s, 3H). LC−MS method 1: Rt = 0.48
min. MS-ESI (m/z): calcd for C12H11ClN4S [M + H]+, 279.0; found,
279.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
oxazol-5-amine (13). Compound 13 was prepared from benzo[d]-
oxazol-5-amine 73f (23 mg, 0.17 mmol) according to the general
procedure. The residue was purified by preparative HPLC (method B).
White solid (3 mg, 7% yield). 1H NMR (500 MHz, CDCl3): δ 8.01 (s,
1H), 7.40 (d, J = 8.80 Hz, 1H), 7.06 (d, J = 2.20 Hz, 1H), 6.93 (s, 1H),
6.81 (dd, J = 8.68, 2.32 Hz, 1H), 4.41 (t, J = 3.90 Hz, 1H), 4.36 (d, J =
4.60 Hz, 2H), 3.62 (s, 3H). LC−MS method 2: Rt = 0.68 min. MS-ESI
(m/z): calcd for C12H11ClN4O [M + H]+, 263.1; found, 263.2.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-1H-benzo[d]-
imidazol-5-amine (14). Compound 14 was prepared from 1H-
benzo[d]imidazol-5-amine 73g (23 mg, 0.17 mmol) according to the
general procedure. The residue was purified by preparative HPLC
1
(method B). White solid (38 mg, 85% yield). H NMR (400 MHz,
DMSO-d6): δ 12.05−11.87 (m, 1H), 7.99−7.79 (m, 1H), 7.35−7.18
(m, 1H), 6.91 (s, 1H), 6.95−6.74 (m, 1H), 6.72−6.59 (m, 1H), 6.05−
5.72 (m, 1H), 4.30 (d, J = 5.26 Hz, 2H), 3.58 (s, 3H). LC−MS method
2: Rt = 0.54 min. MS-ESI (m/z): calcd for C12H12ClN5 [M + H]+,
262.1; found, 262.2.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-1-methyl-1H-
benzo[d][1,2,3]triazol-5-amine (15). Compound 15 was prepared
from 1-methyl-1H-benzo[d][1,2,3]triazol-5-amine 73h (47 mg, 0.32
mmol) according to the general procedure. The residue was purified by
preparative HPLC (method B). White solid (54.6 mg, 62% yield). 1H
NMR (400 MHz, CDCl3): δ 7.34 (d, J = 8.88 Hz, 1H), 7.13 (d, J = 1.43
Hz, 1H), 7.01 (dd, J = 8.82, 1.92 Hz, 1H), 6.95 (s, 1H), 4.57 (br t, J =
4.70, 4.70 Hz, 1H), 4.38 (d, J = 4.49 Hz, 2H), 4.25 (s, 3H), 3.63 (s, 3H).
LC−MS method 2: Rt = 0.62 min. MS-ESI (m/z): calcd for
C12H13ClN6 [M + H]+, 277.1; found, 277.2.
Synthesis of 5-Chloro-1-trityl-1H-imidazole-2-carbaldehyde (75).
A solution of 5-chloro-1H-imidazole 74 (2.0 g, 19.5 mmol),
triphenylmethyl chloride (5.44 g, 19.5 mmol), and Et3N (2.7 mL,
19.5 mmol) in DCM (150 mL) was stirred at rt for 20 h. The mixture
was washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness. The residue was dissolved in THF (100 mL), cooled to −78
°C, and n-BuLi (2.5 M in hexanes, 2.55 mL, 6.38 mmol) was added
dropwise. After 1 h at −78 °C, DMF (1.35 mL, 17.4 mmol) was added
dropwise and the reaction mixture was stirred at the same temperature
for 4 h. A saturated aqueous NH4Cl solution was added and the mixture
was allowed to reach rt and extracted with DCM. The organic layer was
washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness to afford 5-chloro-1-trityl-1H-imidazole-2-carbaldehyde 75
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
isoxazol-5-amine (9). Compound 9 was prepared from benzo[d]-
isoxazol-5-amine 73b (23 mg, 0.17 mmol) according to the general
procedure. The residue was purified by preparative HPLC (method B).
White solid (8.4 mg, 19% yield). 1H NMR (500 MHz, CDCl3): δ 8.59
(s, 1H), 7.46 (d, J = 9.05 Hz, 1H), 7.02 (dd, J = 8.93, 2.32 Hz, 1H),
6.97−6.90 (m, 2H), 4.42 (br s, 1H), 4.37 (d, J = 4.89 Hz, 2H), 3.63 (s,
3H). LC−MS method 2: Rt = 0.73 min. MS-ESI (m/z): calcd for
C12H11ClN4O [M + H]+, 263.1; found, 263.2.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-1H-indazol-5-
amine (10). Compound 10 was prepared from 1H-indazol-5-amine
73c (23 mg, 0.17 mmol) according to the general procedure. The
residue was purified by preparative HPLC (method B). White solid
(17.90 mg, 40% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.61 (br s,
1H), 7.77 (s, 1H), 7.28 (d, J = 8.77 Hz, 1H), 6.92 (s, 1H), 6.89 (dd, J =
8.88, 2.08 Hz, 1H), 6.86 (s, 1H), 5.87 (t, J = 5.48 Hz, 1H), 4.29 (d, J =
5.48 Hz, 2H), 3.58 (s, 3H). LC−MS method 2: Rt = 0.60 min. MS-ESI
(m/z): calcd for C12H12ClN5 [M + H]+, 262.1; found, 262.2.
1
(2.0 g, 57% yield over two steps). White solid. H NMR (400 MHz,
CDCl3): δ 9.22 (s, 1H), 7.32 (m, 9H), 7.12 (m, 6H), 7.10 (s, 1H).
N-((5-Chloro-1H-imidazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (16). A mixture of benzo[d]isothiazol-5-amine 73a (80 mg, 0.52
mmol) and 75 (200 mg, 0.52 mmol) in DCM (15 mL) and catalytic
AcOH was stirred at rt for 30 min. NaBH(OAc)3 (330 mg, 1.56 mmol)
was added and the reaction mixture was stirred at rt for additional 16 h,
then treated with a saturated aqueous NaHCO3 solution and extracted
with DCM. The organic layer was washed with brine, dried over
Na2SO4, filtered, and evaporated to dryness. The residue was purified
by column chromatography on silica gel (0−50% EtOAc/cyclohexane)
to provide N-((5-chloro-1-trityl-1H-imidazol-2-yl)methyl)benzo[d]-
isothiazol-5-amine (190 mg, 72% yield). LC−MS method 1: Rt = 1.48
min. MS-ESI (m/z): calcd for C30H23ClN4S [M + H]+, 507.1; found,
507.1. A solution of N-((5-chloro-1-trityl-1H-imidazol-2-yl)methyl)-
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-1-methyl-1H-
indazol-5-amine (11). Compound 11 was prepared from 1-methyl-
1H-indazol-5-amine 73d (34 mg, 0.23 mmol) according to the general
procedure. The residue was purified by preparative HPLC (method B).
1
White solid (57.40 mg, 90% yield). H NMR (500 MHz, CDCl3): δ
7.82 (s, 1H), 7.26 (d, J = 9.78 Hz, 1H), 6.98−6.88 (m, 3H), 4.36 (s,
5944
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
benzo[d]isothiazol-5-amine (80 mg, 0.16 mmol) in DCM (3 mL) and
TFA (1.5 mL) was stirred at rt for 2 h and then concentrated under
reduced pressure. The residue was purified by column chromatography
on silica gel (20−100% EtOAc/cyclohexane). White solid (17.7 mg,
General Procedure for the Synthesis of Compounds 18 and 25−
33. A solution of benzo[d]isothiazol-5-amine 73a (1 equiv) and the
appropriate aldehyde (1.2−1.5 equiv) in MeOH (0.1 M) was heated at
60 °C for 16 h. After cooling to 0 °C, NaBH4 (3 equiv) was added and
the reaction mixture was allowed to warm to rt and stirred for 2 h. The
mixture was treated with a saturated aqueous NaHCO3 solution and
extracted with DCM. The organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness. The residue was
purified by reverse-phase HPLC or as stated to provide the target
compounds.
1
42% yield over two steps). H NMR (400 MHz, CDCl3): δ 8.73 (s,
1H), 7.77 (d, J = 8.55 Hz, 1H), 7.12 (d, J = 1.97 Hz, 1H), 7.02−6.95 (m,
1H), 6.88 (s, 1H), 4.56 (s, 2H). LC−MS method 1: Rt = 0.66 min. MS-
ESI (m/z): calcd for C11H9ClN4S [M + H]+, 265.0; found, 265.0.
General Procedure for the Synthesis of Compounds 17, 19, 20,
22, and 24. A mixture of benzo[d]isothiazol-5-amine 73a (1 equiv)
and the appropriate aldehyde (1−1.5 equiv) in DCM (0.1 M) and
catalytic AcOH was stirred at rt for 30 min. NaBH(OAc)3 (1.5−3
equiv) was added and the reaction mixture was stirred at rt for
additional 16 h, then treated with a saturated aqueous NaHCO3
solution and extracted with DCM. The organic layer was washed
with brine, dried over Na2SO4, filtered, and evaporated to dryness. The
residue was purified as described for each compound.
N-((4,5-Dichloro-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
isothiazol-5-amine (17). Compound 17 was prepared using 73a (100
mg, 0.68 mmol) and 4,5-dichloro-1-methyl-1H-imidazole-2-carbalde-
hyde (170 mg, 0.95 mmol) according to the general procedure. The
crude material was purified by reverse-phase column chromatography
(5−70% MeCN/0.1% v/v HCOOH in water). Pale yellow solid (60
mg, 28% yield). 1H NMR (500 MHz, DMSO-d6): δ 8.86 (s, 1H), 7.88
(d, J = 8.78 Hz, 1H), 7.27 (s, 1H), 7.10 (d, J = 8.78 Hz, 1H), 6.51 (t, J =
5.35 Hz, 1H), 4.38 (d, J = 5.76 Hz, 2H), 3.62 (s, 3H). LC−MS method
1: Rt = 0.99 min. MS-ESI (m/z): calcd for C12H10Cl2N4S [M + H]+,
313.0; found, 313.0.
N-((1-Methyl-1H-imidazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (18). Compound 18 was prepared from 73a (50 mg, 0.33 mmol)
and 1-methyl-1H-imidazole-2-carbaldehyde (55 mg, 0.5 mmol)
according to the general procedure. The residue was purified by
1
preparative HPLC (method B). White solid (50 mg, 62% yield). H
NMR (500 MHz, DMSO-d6): δ 8.84 (s, 1H), 7.85 (d, J = 8.80 Hz, 1H),
7.33 (d, J = 1.71 Hz, 1H), 7.14 (dd, J = 8.80, 1.96 Hz, 1H), 7.09 (s, 1H),
6.81 (d, J = 0.98 Hz, 1H), 6.41 (t, J = 5.01 Hz, 1H), 4.34 (d, J = 5.38 Hz,
2H), 3.66 (s, 3H). LC−MS method 2: Rt = 0.63 min. MS-ESI (m/z):
calcd for C12H12N4S [M + H]+, 245.1; found, 245.1.
N-((5-Chlorothiazol-2-yl)methyl)benzo[d]isothiazol-5-amine
(25). Compound 25 was prepared from 73a (50 mg, 0.33 mmol) and 5-
chlorothiazole-2-carbaldehyde (73 mg, 0.5 mmol) according to the
general procedure. The residue was purified by preparative HPLC
1
(method B). White solid (15 mg, 16% yield). H NMR (500 MHz,
CDCl3): δ 8.73 (s, 1H), 7.76 (d, J = 8.51 Hz, 1H), 7.56 (s, 1H), 7.17 (d,
J = 1.92 Hz, 1H), 7.00 (dd, J = 8.78, 2.20 Hz, 1H), 4.72−4.65 (m, 3H).
LC−MS method 1: Rt = 1.05 min. MS-ESI (m/z): calcd for
C11H8ClN3S2 [M + H]+, 282.0; found, 282.1.
N-((5-Chloro-1-methyl-1H-pyrrol-2-yl)methyl)benzo[d]-
isothiazol-5-amine (19). Compound 19 was prepared using 73a (100
mg, 0.68 mmol) and 5-chloro-1-methyl-1H-pyrrole-2-carbaldehyde
(100 mg, 0.68 mmol) according to the general procedure. The crude
material was purified by column chromatography on silica gel (50%
EtOAc/cyclohexane). Pale yellow solid (92 mg, 47% yield). 1H NMR
(400 MHz, DMSO-d6): δ 8.85 (s, 1H), 7.85 (d, J = 8.77 Hz, 1H), 7.24
(d, J = 1.97 Hz, 1H), 7.10 (dd, J = 8.77, 2.19 Hz, 1H), 6.28 (t, J = 5.26
Hz, 1H), 6.11 (d, J = 3.51 Hz, 1H), 6.00 (d, J = 3.73 Hz, 1H), 4.24 (d, J
= 5.26 Hz, 2H), 3.53 (s, 3H). LC−MS method 2: Rt = 1.00 min. MS-
ESI (m/z): calcd for C13H12ClN3S [M + H]+, 278.0; found, 278.1.
N-((5-Bromo-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
isothiazol-5-amine (20). Compound 20 was prepared using 73a (150
mg, 0.99 mmol) and 5-bromo-1-methyl-1H-imidazole-2-carbaldehyde
(283 mg, 1.5 mmol) according to the general procedure. The crude
material was purified by column chromatography on silica gel (5−100%
EtOAc/cyclohexane). White solid (238 mg, 74% yield). 1H NMR (400
MHz, CDCl3): δ 8.77 (s, 1H), 7.75 (d, J = 8.77 Hz, 1H), 7.27−7.22 (m,
1H), 7.13−6.95 (m, 2H), 4.60 (br s, 1H), 4.43 (s, 2H), 3.66 (s, 3H).
LC−MS method 1: Rt = 0.54 min. MS-ESI (m/z): calcd for
C12H11BrN4S [M + H]+, 323.0; found, 323.0.
N-((5-Chlorothiophen-2-yl)methyl)benzo[d]isothiazol-5-amine
(26). Compound 26 was prepared from 73a (40 mg, 0.27 mmol) and 5-
chlorothiophene-2-carbaldehyde (59 mg, 0.4 mmol) according to the
general procedure. The residue was purified by preparative HPLC
1
(method B). White solid (62 mg, 81% yield). H NMR (500 MHz,
CDCl3): δ 8.73 (s, 1H), 7.74 (d, J = 8.78 Hz, 1H), 7.18 (d, J = 1.92 Hz,
1H), 6.96 (dd, J = 8.78, 2.20 Hz, 1H), 6.87−6.81 (m, 1H), 6.79 (d, J =
3.84 Hz, 1H), 4.51 (d, J = 5.49 Hz, 2H), 4.30 (br t, J = 4.70, 4.70 Hz,
1H). LC−MS method 2: Rt = 1.06 min. MS-ESI (m/z): calcd for
C12H9ClN2S2 [M + H]+, 281.0; found, 281.2.
N-((5-(Trifluoromethyl)thiazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (27). Compound 27 was prepared from 73a (40 mg, 0.27 mmol)
and 5-(trifluoromethyl)thiazole-2-carbaldehyde (69 mg, 0.32 mmol)
according to the general procedure. The residue was purified by
1
preparative HPLC (method A). White solid (17 mg, 20% yield). H
NMR (500 MHz, CDCl3): δ 8.72 (s, 1H), 8.16−7.97 (m, 1H), 7.77 (d,
J = 8.78 Hz, 1H), 7.14 (d, J = 1.92 Hz, 1H), 7.00 (dd, J = 8.78, 2.20 Hz,
1H), 4.77 (s, 3H). LC−MS method 1: Rt = 1.09 min. MS-ESI (m/z):
calcd for C12H8F3N3S2 [M + H]+, 316.0; found, 316.0.
N-((5-Methylthiazol-2-yl)methyl)benzo[d]isothiazol-5-amine
(28). Compound 28 was prepared from 73a (40 mg, 0.27 mmol) and 5-
methylthiazole-2-carbaldehyde (51 mg, 0.4 mmol) according to the
general procedure. The residue was purified by preparative HPLC
N-((5-Methyl-1,3,4-thiadiazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (22). Compound 22 was prepared using 73a (50 mg, 0.33
mmol) and 5-methyl-1,3,4-thiadiazole-2-carbaldehyde (63 mg, 0.5
mmol) according to the general procedure. The crude material was
purified by column chromatography on silica gel (5−100% EtOAc/
1
(method B). White solid (14 mg, 20% yield). H NMR (400 MHz,
CDCl3): δ 8.72 (s, 1H), 7.74 (d, J = 9.00 Hz, 1H), 7.40 (d, J = 1.17 Hz,
1H), 7.18 (d, J = 1.96 Hz, 1H), 7.00 (dd, J = 8.80, 2.15 Hz, 1H), 4.80−
4.61 (m, 3H), 2.43 (d, J = 0.78 Hz, 3H). LC−MS method 1: Rt = 0.91
min. MS-ESI (m/z): calcd for C12H11N3S2 [M + H]+, 262.0; found,
262.1.
1
cyclohexane). Pale yellow solid (38 mg, 44% yield). H NMR (500
MHz, CDCl3): δ 8.72 (s, 1H), 7.75 (d, J = 8.78 Hz, 1H), 7.19 (s, 1H),
6.99 (dd, J = 8.71, 1.58 Hz, 1H), 4.83 (s, 2H), 4.92−4.66 (m, 1H), 2.74
(s, 3H). LC−MS method 1: Rt = 0.72 min. MS-ESI (m/z): calcd for
C11H10N4S2 [M + H]+, 263.0; found, 263.0.
N-((5-Isopropylthiazol-2-yl)methyl)benzo[d]isothiazol-5-amine
(29). Compound 29 was prepared from 73a (50 mg, 0.33 mmol) and 5-
isopropylthiazole-2-carbaldehyde (51 mg, 0.33 mmol) according to the
general procedure. The residue was purified by preparative HPLC
5-((Benzo[d]isothiazol-5-ylamino)methyl)thiophene-2-carboni-
trile (24). Compound 24 was prepared using 73a (50 mg, 0.33 mmol)
and 5-formylthiophene-2-carbonitrile (56 mg, 0.41 mmol) according to
the general procedure. The crude material was purified by reverse-phase
column chromatography (5−70% MeCN/0.1% v/v HCOOH in
1
(method B). White solid (50 mg, 52% yield). H NMR (400 MHz,
CDCl3): δ 8.73 (s, 1H), 7.74 (d, J = 8.55 Hz, 1H), 7.43 (s, 1H), 7.20 (d,
J = 1.53 Hz, 1H), 7.01 (dd, J = 8.77, 2.19 Hz, 1H), 4.82−4.58 (m, 3H),
3.17 (spt, J = 6.80 Hz, 1H), 1.32 (d, J = 6.80 Hz, 6H). LC−MS method
2: Rt = 0.97 min. MS-ESI (m/z): calcd for C14H15N3S2 [M + H]+, 290.1;
found, 290.1.
N-((4-Methylthiazol-2-yl)methyl)benzo[d]isothiazol-5-amine
(30). Compound 30 was prepared from 73a (40 mg, 0.27 mmol) and 4-
methylthiazole-2-carbaldehyde (51 mg, 0.4 mmol) according to the
1
water). Pale yellow solid (38 mg, 41% yield). H NMR (400 MHz,
CDCl3): δ 8.72 (s, 1H), 7.76 (d, J = 8.55 Hz, 1H), 7.54 (d, J = 3.73 Hz,
1H), 7.13 (d, J = 1.97 Hz, 1H), 7.07 (d, J = 3.73 Hz, 1H), 6.97 (dd, J =
8.66, 2.30 Hz, 1H), 4.67 (d, J = 3.51 Hz, 2H), 4.47 (br s, 1H). LC−MS
method 1: Rt = 1.01 min. MS-ESI (m/z): calcd for C13H9N3S2 [M +
H]+, 272.0; found, 272.0.
5945
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
general procedure. The residue was purified by preparative HPLC
(method B). White solid (18 mg, 25% yield). H NMR (500 MHz,
over Na2SO4, filtered, and evaporated to dryness. The crude material
was purified as described for each compound.
1
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-
methylbenzo[d]isothiazol-5-amine (34). Compound 34 was prepared
using compound 2 (24 mg, 0.09 mmol) according to the general
procedure. The crude material was purified by column chromatography
on silica gel (10−100% EtOAc/cyclohexane). White solid (15 mg, 55%
yield). 1H NMR (400 MHz, CDCl3): δ 8.79 (s, 1H), 7.82 (d, J = 8.78
Hz, 1H), 7.45 (d, J = 2.26 Hz, 1H), 7.30 (dd, J = 8.91, 2.38 Hz, 1H),
6.93 (s, 1H), 4.55 (s, 2H), 3.54 (s, 3H), 2.99 (s, 3H). LC−MS method
1: Rt = 0.58 min. MS-ESI (m/z): calcd for C13H13ClN4S [M + H]+,
293.2; found, 293.2.
N-((5-Chlorothiazol-2-yl)methyl)-N-methylbenzo[d]isothiazol-5-
amine (35). Compound 35 was prepared using compound 25 (29 mg,
0.1 mmol) according to the general procedure. The crude material was
purified by reverse-phase HPLC (method B). White solid (5.8 mg, 19%
yield). 1H NMR (500 MHz, CDCl3): δ 8.78 (s, 1H), 7.81 (d, J = 8.78
Hz, 1H), 7.55 (s, 1H), 7.33 (d, J = 2.47 Hz, 1H), 7.17 (dd, J = 9.06, 2.47
Hz, 1H), 4.78 (s, 2H), 3.18 (s, 3H). LC−MS method 2: Rt = 1.03 min.
MS-ESI (m/z): calcd for C12H10ClN3S2 [M + H]+, 296.0; found, 296.1.
General Procedure for the Synthesis of Compounds 36 and 38−
40. A mixture of compound 2 (50 mg, 0.18 mmol) and the appropriate
aldehyde (0.36 mmol) in DCM (4 mL) and catalytic AcOH was stirred
at rt for 30 min. NaBH(OAc)3 (114 mg, 0.54 mmol) was added and the
reaction mixture was stirred at rt for 16 h, then treated with a saturated
aqueous NaHCO3 solution and extracted with DCM (2×). The
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and evaporated to dryness. The residue was purified by column
chromatography on silica gel (0−50% EtOAc/cyclohexane and then
0−30% MeCN/DCM) affording the target compounds.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-
(cyclopropylmethyl)benzo[d]isothiazol-5-amine (36). Compound
36 was prepared using cyclopropanecarbaldehyde (25 mg, 0.36
mmol) according to the general procedure (25 mg, 42% yield).
White solid. 1H NMR (400 MHz, CDCl3): δ 8.79 (s, 1H), 7.81 (d, J =
8.99 Hz, 1H), 7.57 (d, J = 1.97 Hz, 1H), 7.33 (dd, J = 8.88, 2.30 Hz,
1H), 6.90 (s, 1H), 4.62 (s, 2H), 3.55 (s, 3H), 3.23 (d, J = 6.58 Hz, 2H),
1.15−0.94 (m, 1H), 0.58−0.00 (m, 4H). LC−MS method 1: Rt = 0.73
min. MS-ESI (m/z): calcd for C16H17ClN4S [M + H]+, 333.1; found,
333.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-
(cyclohexylmethyl)benzo[d]isothiazol-5-amine (38). Compound 38
was prepared using cyclohexanecarbaldehyde (40 mg, 0.36 mmol)
according to the general procedure (39 mg, 58% yield). White solid. 1H
NMR (400 MHz, CDCl3): δ 8.77 (s, 1H), 7.78 (d, J = 8.77 Hz, 1H),
7.43 (d, J = 2.19 Hz, 1H), 7.23 (dd, J = 8.88, 2.30 Hz, 1H), 6.90 (s, 1H),
4.59 (s, 2H), 3.49 (s, 3H), 3.23 (d, J = 6.80 Hz, 2H), 1.81−1.62 (m,
6H), 1.33−1.02 (m, 3H), 0.99−0.82 (m, 2H). LC−MS method 1: Rt =
1.07 min. MS-ESI (m/z): calcd for C19H23ClN4S [M + H]+, 375.1;
found, 375.1.
N-Benzyl-N-((5-chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo-
[d]isothiazol-5-amine (39). Compound 39 was prepared using
benzaldehyde (38 mg, 0.36 mmol) according to the general procedure
(29 mg, 44% yield). White solid. 1H NMR (400 MHz, CDCl3): δ 8.75
(s, 1H), 7.80 (d, J = 8.99 Hz, 1H), 7.49 (d, J = 2.19 Hz, 1H), 7.39−7.13
(m, 6H), 6.90 (s, 1H), 4.61 (s, 2H), 4.56 (s, 2H), 3.43 (s, 3H). LC−MS
method 1: Rt = 0.84 min. MS-ESI (m/z): calcd for C19H17ClN4S [M +
H]+, 369.1; found, 369.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-ethylbenzo-
[d]isothiazol-5-amine (40). Compound 40 was prepared using
acetaldehyde (16 mg, 0.36 mmol) according to the general procedure
(32 mg, 59% yield). White solid. 1H NMR (400 MHz, CDCl3): δ 8.78
(s, 1H), 7.81 (d, J = 8.99 Hz, 1H), 7.49 (d, J = 2.19 Hz, 1H), 7.32−7.24
(m, 1H), 6.92 (s, 1H), 4.51 (s, 2H), 3.53 (s, 3H), 3.44 (q, J = 7.02 Hz,
2H), 1.11 (t, J = 7.13 Hz, 3H). LC−MS method 1: Rt = 0.66 min. MS-
ESI (m/z): calcd for C14H15ClN4S [M + H]+, 307.1; found, 307.0.
Synthesis of N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-
isopropylbenzo[d]isothiazol-5-amine (37). A mixture of compound 2
(50 mg, 0.18 mmol) and acetone (26 μL, 0.36 mmol) in DCM (4 mL)
and catalytic AcOH was stirred at rt for 30 min. NaBH(OAc)3 (114 mg,
0.54 mmol) was added and the reaction mixture was stirred at rt for 16
DMSO-d6): δ 8.83 (d, J = 0.82 Hz, 1H), 7.88 (d, J = 8.78 Hz, 1H), 7.13
(d, J = 1.92 Hz, 1H), 7.12−7.07 (m, 2H), 6.91 (t, J = 6.04 Hz, 1H), 4.61
(d, J = 6.04 Hz, 2H), 2.35 (d, J = 1.10 Hz, 3H). LC−MS method 2: Rt =
0.81 min. MS-ESI (m/z): calcd for C12H11N3S2 [M + H]+, 262.0;
found, 262.1.
N-((4,5-Dimethylthiazol-2-yl)methyl)benzo[d]isothiazol-5-amine
(31). Compound 31 was prepared from 73a (50 mg, 0.33 mmol) and
4,5-dimethylthiazole-2-carbaldehyde (61 mg, 0.43 mmol) according to
the general procedure. The crude material was purified by column
chromatography on silica gel (5−100% EtOAc/cyclohexane). White
solid (30 mg, 33% yield). 1H NMR (400 MHz, CDCl3): δ 8.72 (s, 1H),
7.73 (d, J = 8.61 Hz, 1H), 7.18 (d, J = 1.96 Hz, 1H), 6.99 (dd, J = 8.71,
2.25 Hz, 1H), 4.95−4.33 (m, 3H), 2.34 (s, 3H), 2.31 (s, 3H). LC−MS
method 1: Rt = 0.92 min. MS-ESI (m/z): calcd for C13H13N3S2 [M +
H]+, 276.1; found, 276.0.
N-(Thiazol-2-ylmethyl)benzo[d]isothiazol-5-amine (32). Com-
pound 32 was prepared from 73a (40 mg, 0.27 mmol) and thiazole-
2-carbaldehyde (45 mg, 0.4 mmol) according to the general procedure.
The residue was purified by preparative HPLC (method B). White solid
(61 mg, 91% yield). 1H NMR (500 MHz, CDCl3): δ 8.72 (d, J = 0.82
Hz, 1H), 7.79 (d, J = 3.29 Hz, 1H), 7.75 (d, J = 8.78 Hz, 1H), 7.30 (d, J
= 3.29 Hz, 1H), 7.18 (d, J = 1.92 Hz, 1H), 7.02 (dd, J = 8.65, 2.33 Hz,
1H), 4.85−4.66 (m, 3H). LC−MS method 2: Rt = 0.74 min. MS-ESI
(m/z): calcd for C11H9N3S2 [M + H]+, 248.0; found, 248.1.
N-(Benzo[d]thiazol-2-ylmethyl)benzo[d]isothiazol-5-amine (33).
Compound 33 was prepared from 73a (40 mg, 0.27 mmol) and
benzo[d]thiazole-2-carbaldehyde (65 mg, 0.4 mmol) according to the
general procedure. The residue was purified by preparative HPLC
(method B). White solid (16.6 mg, 20% yield). 1H NMR (400 MHz,
CDCl3): δ 8.70 (s, 1H), 8.04 (d, J = 8.22 Hz, 1H), 7.89−7.81 (m, 1H),
7.76 (d, J = 8.61 Hz, 1H), 7.51 (td, J = 7.83, 1.17 Hz, 1H), 7.43−7.34
(m, 1H), 7.21 (d, J = 1.96 Hz, 1H), 7.05 (dd, J = 8.80, 2.15 Hz, 1H),
4.99−4.77 (m, 3H). LC−MS method 1: Rt = 1.06 min. MS-ESI (m/z):
calcd for C15H11N3S2 [M + H]+, 298.0; found, 298.1.
General Procedure for the Synthesis of Compounds 21 and 23.
To a solution of benzo[d]isothiazol-5-amine 73a (1 equiv) and Et3N (2
equiv) in EtOH (0.15 M), the appropriate chloromethyl-heterocycle
(0.8 equiv) was added and the reaction mixture was heated at reflux for
6 h. The mixture was diluted with water and extracted with EtOAc
(2×). The combined extracts were washed with brine, dried over
Na2SO4, filtered, and evaporated to dryness. The residue was purified
by reverse-phase column chromatography (5−80% MeCN/0.1% v/v
HCOOH in water) and then by reverse-phase HPLC (method B) to
provide the target compounds.
N-((5-Chloro-1,2,3-thiadiazol-4-yl)methyl)benzo[d]isothiazol-5-
amine (21). Compound 21 was prepared from 73a (100 mg, 0.66
mmol) and 5-chloro-4-(chloromethyl)-1,2,3-thiadiazole (94 mg, 0.55
mmol) according to the general procedure (23 mg, 15% yield). Pale
yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.76 (s, 1H), 7.75 (d, J =
8.55 Hz, 1H), 7.32 (d, J = 2.19 Hz, 1H), 7.04 (dd, J = 8.77, 2.19 Hz,
1H), 4.85−4.67 (m, 3H). LC−MS method 1: Rt = 0.96 min. MS-ESI
(m/z): calcd for C10H7ClN4S2 [M + H]+, 283.0; found, 283.0.
N-((1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)methyl)benzo-
[d]isothiazol-5-amine (23). Compound 23 was prepared from 73a (90
mg, 0.6 mmol) and 3-(chloromethyl)-1-methyl-5-(trifluoromethyl)-
1H-pyrazole (100 mg, 0.5 mmol) according to the general procedure
(26 mg, 17% yield). Pale yellow solid. 1H NMR (400 MHz, DMSO-d6):
δ 8.84 (s, 1H), 7.85 (d, J = 8.77 Hz, 1H), 7.19 (d, J = 1.97 Hz, 1H), 7.09
(dd, J = 8.77, 2.19 Hz, 1H), 6.81 (s, 1H), 6.43 (t, J = 5.92 Hz, 1H), 4.28
(d, J = 5.92 Hz, 2H), 3.93 (s, 3H). LC−MS method 2: Rt = 1.03 min.
MS-ESI (m/z): calcd for C13H11F3N4S [M + H]+, 313.1; found, 313.2.
General Procedure for the Synthesis of Compounds 34 and 35.
NaH (60% w/w in mineral oil, 2 equiv) was added to a solution of 2 or
25 (1 equiv) in dry DMF (0.1 M) and cooled to 0 °C. The reaction
mixture was stirred at rt for 15 min, then MeI (2 equiv) was added and
stirring was continued for 1 or 16 h, respectively. The mixture was
treated with a saturated aqueous NH4Cl solution and extracted with
EtOAc (3×). The combined extracts were washed with brine, dried
5946
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
h. More acetone (52 μL, 0.72 mmol) was added and the reaction
mixture was heated to reflux for additional 6 h. After cooling to rt, the
mixture was treated with a saturated aqueous NaHCO3 solution and
extracted with DCM (2×). The combined organic layers were washed
with brine, dried over Na2SO4, filtered, and evaporated to dryness. The
residue was purified by reverse-phase HPLC (method B). White solid
(14 mg, 24% yield). 1H NMR (400 MHz, CDCl3): δ 8.78 (s, 1H), 7.78
(d, J = 8.80 Hz, 1H), 7.64 (d, J = 2.20 Hz, 1H), 7.30 (dd, J = 8.80, 2.20
Hz, 1H), 6.83 (s, 1H), 4.43 (s, 2H), 3.91 (quin, J = 6.66 Hz, 1H), 3.58
(s, 3H), 1.22 (d, J = 6.60 Hz, 6H). LC−MS method 2: Rt = 0.95 min.
MS-ESI (m/z): calcd for C15H17ClN4S [M + H]+, 321.1; found, 321.1
[M + H]+.
76 (99 mg, 0.67 mmol) according to the general procedure. The crude
material was purified by reverse-phase HPLC (method B). Yellow solid,
racemic mixture (70 mg, 35% yield). 1H NMR (500 MHz, DMSO-d6):
δ 8.83 (s, 1H), 7.92−7.89 (m, 1H), 7.76 (s, 1H), 7.15−7.08 (m, 2H),
6.82 (d, J = 5.87 Hz, 1H), 4.81 (quin, J = 6.48 Hz, 1H), 1.58 (d, J = 6.85
Hz, 3H). LC−MS method 2: Rt = 0.98 min. MS-ESI (m/z): calcd for
C12H10ClN3S2 [M + H]+, 296.0; found, 296.1.
N-(2-(5-Chlorothiazol-2-yl)propan-2-yl)benzo[d]isothiazol-5-
amine (44). Compound 44 was prepared using 73a (56 mg, 0.37
mmol) and 77 (60 mg, 0.67 mmol) according to the general procedure.
The crude material was purified by column chromatography on silica
gel (0−20% EtOAc/cyclohexane). White solid (9.6 mg, 8% yield). 1H
NMR (400 MHz, CDCl3): δ 8.65 (s, 1H), 7.70 (d, J = 8.77 Hz, 1H),
7.53 (s, 1H), 6.92 (d, J = 1.97 Hz, 1H), 6.85 (dd, J = 8.77, 2.19 Hz, 1H),
4.38 (s, 1H), 1.78 (s, 6H). LC−MS method 1: Rt = 1.17 min. MS-ESI
(m/z): calcd for C13H12ClN3S2 [M + H]+, 310.0; found, 310.0.
Synthesis of 5-((5-Chlorothiazol-2-yl)methoxy)benzo[d]-
isothiazole (45). A mixture of benzo[d]isothiazol-5-ol 78 (50 mg,
0.33 mmol), 5-chloro-2-(chloromethyl)-1,3-thiazole (37 μL, 0.33
mmol), and K2CO3 (91 mg, 0.66 mmol) in dry DMF (2 mL) was
stirred at rt for 16 h. It was treated with a saturated aqueous NH4Cl
solution and extracted with EtOAc (2×). The combined organic layers
were dried over Na2SO4, filtered, and evaporated to dryness. The
residue was purified by column chromatography on silica gel (50%
EtOAc/cyclohexane). Yellow solid (58 mg, 62% yield). 1H NMR (500
MHz, CDCl3): δ 8.83 (s, 1H), 7.88 (d, J = 8.78 Hz, 1H), 7.61 (s, 1H),
7.55 (d, J = 2.47 Hz, 1H), 7.29 (dd, J = 8.78, 2.47 Hz, 1H), 5.38 (s, 2H).
LC−MS method 1: Rt = 1.16 min. MS-ESI (m/z): calcd for
C11H7ClN2OS2 [M + H]+, 283.0; found, 283.1.
General Procedure for the Synthesis of Intermediates
80a,b,d,h,j−l and 86a−c. A mixture of compound 79a,b,d,h,j−l or
85a−c (1 equiv), sulfur (1 equiv), and NH4OH aqueous solution (0.7
M) in DMF (0.7 M) was heated at 90 °C for 16 h. After cooling to rt, the
mixture was treated with a saturated aqueous NH4Cl solution and
extracted with EtOAc (2×). The combined organic layers were dried
over Na2SO4, filtered, and evaporated to dryness. The residue was
purified by column chromatography as described for each intermediate.
6-Methyl-5-nitrobenzo[d]isothiazole (80a). Compound 80a was
prepared using 2-fluoro-4-methyl-5-nitrobenzaldehyde 79a (1 g, 5.46
mmol) according to the general procedure. The residue was purified by
column chromatography on silica gel (0−100% EtOAc/cyclohexane)
(478 mg, 45% yield). LC−MS method 1: Rt = 0.97 min. MS-ESI (m/z):
calcd for C8H6N2O2S [M + H]+, 195.0; found, 195.1.
6-Chloro-5-nitrobenzo[d]isothiazole (80b). Compound 80b was
prepared using 2-chloro-4-fluoro-5-nitrobenzaldehyde 79b (1 g, 4.9
mmol) according to the general procedure. The residue was diluted in
MeCN (40 mL) and added to a solution of isopentyl nitrite (1.38 mL,
10.25 mmol) and CuCl2 (1 g, 7.68 mmol) in MeCN (46 mL). The
resulting mixture was stirred at 65 °C for 16 h. After cooling to rt, the
mixture was treated with 6 M HCl (aq) and extracted with EtOAc. The
organic layers were dried over Na2SO4, filtered, and evaporated to
dryness. The residue was purified by column chromatography on silica
gel (10% EtOAc/cyclohexane) (529 mg, 48% yield). LC−MS method
1: Rt = 0.99 min. MS-ESI (m/z): calcd for C7H3ClN2O2S [M + H]+,
215.0; found, 215.0.
5-Bromoisothiazolo[5,4-c]pyridine (80d). Compound 80d was
prepared using 2-bromo-5-fluoropyridine-4-carbaldehyde 79d (1.3 g,
6.4 mmol) according to the general procedure. The residue was purified
by column chromatography on silica gel (0−50% EtOAc/cyclohexane)
(136 mg, 10% yield). LC−MS method 1: Rt = 0.81 min. MS-ESI (m/z):
calcd for C6H3BrN2S [M + H]+, 214.9; found, 215.0.
Synthesis of N-(Benzo[d]isothiazol-5-yl)-N-((5-chloro-1-methyl-
1H-imidazol-2-yl)methyl)acetamide (41). Acetyl chloride (13 μL,
0.18 mmol) was added to a solution of compound 2 (50 mg, 0.18
mmol) and Et3N (26 μL, 0.19 mmol) in DCM (5 mL) and cooled to 0
°C. The reaction mixture was allowed to reach rt and stirred for 4 h,
then treated with a saturated aqueous NH4Cl solution and extracted
with DCM (2×). The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and evaporated to dryness. The residue was
purified by column chromatography on silica gel (50−100% EtOAc/
cyclohexane). White solid (42.6 mg, 74% yield). 1H NMR (400 MHz,
CDCl3): δ 8.91 (d, J = 1.00 Hz, 1H), 7.99 (d, J = 8.53 Hz, 1H), 7.87 (d, J
= 1.51 Hz, 1H), 7.30 (dd, J = 8.53, 2.01 Hz, 1H), 6.81 (s, 1H), 5.01 (s,
2H), 3.72 (s, 3H), 1.91 (s, 3H). LC−MS method 1: Rt = 0.55 min. MS-
ESI (m/z): calcd for C14H13ClN4OS [M + H]+, 321.1; found, 321.0.
Synthesis of N-(Benzo[d]isothiazol-5-yl)-5-chlorothiazole-2-car-
boxamide (42). Oxalyl chloride (10 μL, 0.22 mmol) was added to a
solution of 5-chlorothiazole-2-carboxylic acid (25 mg, 0.15 mmol) in
dry DCM (0.7 mL) and catalytic DMF cooled to 0 °C. The reaction
mixture was allowed to reach rt and stirred at rt for 2 h, then the solvent
was evaporated under vacuum. The residue was suspended in dry DCM
(0.5 mL) and added dropwise to a solution of benzo[d]isothiazol-5-
amine 73a (27 mg, 0.18 mmol) and Et3N (42 μL, 0.3 mmol) in DCM
(0.5 mL) and cooled to 0 °C. The reaction mixture was allowed to reach
rt and stirred at rt for 2 h. It was diluted with DCM and washed with
brine. The organic layer was dried over Na2SO4, filtered, and
evaporated to dryness. The residue was purified by reverse-phase
column chromatography (5−80% MeCN/0.1% v/v HCOOH in
1
water). Yellow solid (25 mg, 53% yield over two steps). H NMR
(400 MHz, DMSO-d6): δ 11.16 (s, 1H), 9.14 (s, 1H), 8.77 (d, J = 1.97
Hz, 1H), 8.25−8.17 (m, 2H), 7.99 (dd, J = 8.77, 1.75 Hz, 1H). LC−MS
method 1: Rt = 1.09 min. MS-ESI (m/z): calcd for C11H6ClN3OS2 [M
+ H]+, 296.0; found, 296.0.
Synthesis of 1-(5-Chlorothiazol-2-yl)ethan-1-one (77). MeMgBr
(3 M solution in Et2O, 0.66 mL, 2.0 mmol) was added to a solution of 5-
chlorothiazole-2-carbaldehyde 76 (200 mg, 1.35 mmol) in dry THF (6
mL) and cooled to 0 °C. The reaction mixture was allowed to reach rt
and stirred for 1 h, then treated with a saturated aqueous NH4Cl
solution and extracted with EtOAc. The organic layer was dried over
Na2SO4, filtered, and evaporated to dryness. The residue was purified
by column chromatography on silica gel (0−50% EtOAc/cyclohexane)
affording 1-(5-chlorothiazol-2-yl)ethan-1-ol (220 mg, quantitative
yield). A mixture of 1-(5-chlorothiazol-2-yl)ethan-1-ol (220 mg, 1.35
mmol) and Dess−Martin periodinane (859 mg, 2.03 mmol) in DCM
(5 mL) was stirred at rt for 1 h. The solvent was evaporated and the
residue was purified by column chromatography on silica gel (0−50%
EtOAc/cyclohexane). White solid (120 mg, 48% yield over two steps).
1H NMR (400 MHz, CDCl3): δ 7.29 (s, 1H), 2.68 (s, 3H).
General Procedure for the Synthesis of Compounds 43 and 44. A
mixture of benzo[d]isothiazol-5-amine 73a (1 equiv) and intermediate
76 or 77 (1 equiv) in toluene (0.15 M) was heated at reflux for 20 h.
After cooling to rt, MeMgBr (3 M solution in Et2O, 1.2 equiv) was
added and the reaction mixture was stirred at rt for 3 h. The mixture was
quenched with a saturated aqueous NH4Cl solution and extracted with
EtOAc. The organic layer was dried over Na2SO4, filtered, and
evaporated to dryness. The residue was purified as described for each
compound.
5-Bromoisothiazolo[4,5-b]pyridine (80h). Compound 80h was
prepared using 2-bromo-5-fluoropyridine-4-carbaldehyde 79h (640
mg, 3.2 mmol) according to the general procedure. The residue was
purified by column chromatography on silica gel (0−50% EtOAc/
cyclohexane) (345 mg, 51% yield). LC−MS method 1: Rt = 0.82 min.
MS-ESI (m/z): calcd for C6H3BrN2S [M + H]+, 214.9; found, 214.9.
5-Bromo-7-chlorobenzo[d]isothiazole (80j). Compound 80j was
prepared using 5-bromo-3-chloro-2-fluorobenzaldehyde 79j (400 mg,
1.68 mmol) according to the general procedure. The residue was
N-(1-(5-Chlorothiazol-2-yl)ethyl)benzo[d]isothiazol-5-amine
(43). Compound 43 was prepared using 73a (100 mg, 0.67 mmol) and
5947
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
purified by column chromatography on silica gel (10−80% EtOAc/
cyclohexane) (136 mg, 32% yield). LC−MS method 1: Rt = 1.27 min.
MS-ESI (m/z): calcd for C7H3BrClNS [M + H]+, 247.9; found, 247.9.
5-Bromo-7-fluorobenzo[d]isothiazole (80k). Compound 80k was
prepared using 5-bromo-2,3-difluorobenzaldehyde 79k (200 mg, 0.9
mmol) according to the general procedure. The residue was purified by
column chromatography on silica gel (10% EtOAc/cyclohexane) (145
mg, 69% yield). LC−MS method 1: Rt = 1.16 min. MS-ESI (m/z): calcd
for C7H3BrFNS [M + H]+, 231.9; found, 231.9.
5-Bromoisothiazolo[5,4-b]pyridine (80l). Compound 80l was
prepared using 5-bromo-2-fluoro-3-pyridinecarboxaldehyde 79l (1.3
g, 6.4 mmol) according to the general procedure. The residue was
purified by column chromatography on silica gel (0−50% EtOAc/
cyclohexane) (365 mg, 27% yield). LC−MS method 1: Rt = 0.89 min.
MS-ESI (m/z): calcd for C6H3BrN2S [M + H]+, 214.9; found, 214.9.
6-Bromobenzo[d]isothiazole (86a). Compound 86a was prepared
using 4-bromo-2-fluorobenzaldehyde 85a (10 g, 49.3 mmol) according
to the general procedure. The residue was purified by column
chromatography on silica gel (0−50% EtOAc/cyclohexane) (5.8 g,
55% yield). LC−MS method 1: Rt = 1.08 min. MS-ESI (m/z): calcd for
C7H4BrNS [M + H]+, 213.9; found, 213.9.
4-Bromobenzo[d]isothiazole (86b). Compound 86b was prepared
using 2-bromo-6-fluorobenzaldehyde 85b (500 mg, 2.46 mmol)
according to the general procedure. The residue was purified by
column chromatography on silica gel (0−50% EtOAc/cyclohexane)
(226 mg, 43% yield). LC−MS method 1: Rt = 1.09 min. MS-ESI (m/z):
calcd for C7H4BrNS [M + H]+, 213.9; found, 213.9.
7-Nitrobenzo[d]isothiazole (86c). Compound 86c was prepared
using 2-chloro-3-nitrobenzaldehyde (500 mg, 2.69 mmol) according to
the general procedure. The residue was purified by column
chromatography on silica gel (0−30% EtOAc/cyclohexane) (325 mg,
67% yield). LC−MS method 1: Rt = 0.89 min. MS-ESI (m/z): calcd for
C7H4N2O2S [M + H]+, 181.0; found, 181.0.
General Procedure for the Synthesis of Intermediates 80c,e,i and
89a−c. Benzyl mercaptan (1 equiv) was added dropwise to a solution
of potassium tert-butoxide (1 equiv) in THF (0.15 M). After 5 min at rt,
a solution of substituted benzaldehydes 79c,e,i or 88a−c (1 equiv) in
THF (0.5 M) was added. The reaction was stirred at rt for 2 h and
consumption of starting material was followed by LC−MS. Upon
completion, the reaction mixture was quenched with water, phases were
separated, and the aqueous layer was extracted with EtOAc. The
combined organic layers were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was dissolved in
DCM (0.18 M) and SO2Cl2 (2 equiv) was added dropwise. After
stirring 1 h at rt, the reaction was concentrated under reduced pressure.
The residue was dissolved in THF (0.1 M), cooled to 0 °C, and treated
with ammonia (7 M in MeOH, 1.5 equiv). After stirring 1 h at rt, water
was added and the reaction mixture was extracted with EtOAc (2×).
The combined organic layers were washed with water, dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified as described for each compound.
5-Bromo-6-fluorobenzo[d]isothiazole (80c). Compound 80c was
prepared using 5-bromo-2,4-difluorobenzaldehyde 79c (4 g, 18.4
mmol) according to the general procedure. The residue was purified by
column chromatography on silica gel (20% EtOAc/cyclohexane) (620
mg, 15% yield over three steps). LC−MS method 1: Rt = 1.06 min. MS-
ESI (m/z): calcd for C7H3BrFNS [M + H]+, 231.9; found, 231.9.
5-Bromo-4-methylbenzo[d]isothiazole (80e). Compound 80e was
prepared using 3-bromo-6-chloro-2-methylbenzaldehyde 79e (840 mg,
3.6 mmol) according to the general procedure. The residue was purified
by column chromatography on silica gel (0−10% EtOAc/cyclohexane)
(247 mg, 30% yield over three steps). LC−MS method 1: Rt = 1.16 min.
MS-ESI (m/z): calcd for C8H6BrNS [M + H]+, 227.9; found, 227.9.
5-Bromo-7-methylbenzo[d]isothiazole (80i). Compound 80i was
prepared using 5-bromo-2-fluoro-3-methylbenzaldehyde 79i (672 mg,
3.11 mmol) according to the general procedure. The residue was
purified by column chromatography on silica gel (0−10% EtOAc/
cyclohexane) (413 mg, 58% yield over three steps). LC−MS method 1:
Rt = 1.19 min. MS-ESI (m/z): calcd for C8H6BrNS [M + H]+, 227.9;
found, 227.9.
6-Bromo-5-chlorobenzo[d]isothiazole (89a). Compound 89a was
prepared using 4-bromo-5-chloro-2-fluorobenzaldehyde 88a (655 mg,
2.76 mmol) according to the general procedure. The residue was
purified by column chromatography on silica gel (0−20% EtOAc/
cyclohexane) (360 mg, 53% yield over three steps). LC−MS method 1:
Rt = 1.17 min. MS-ESI (m/z): calcd for C7H3BrClNS [M + H]+, 247.9;
found, 247.9.
6-Bromo-5-methylbenzo[d]isothiazole (89b). Compound 89b was
prepared using 4-bromo-2-fluoro-5-methylbenzaldehyde 88b (600 mg,
2.76 mmol) according to the general procedure. The residue was
purified by reverse-phase column chromatography (5−50% MeCN/
0.1% v/v HCOOH in water) (120 mg, 20% yield over three steps).
LC−MS method 1: Rt = 1.18 min. MS-ESI (m/z): calcd for C8H6BrNS
[M + H]+, 227.9; found, 227.9.
6-Bromo-5-fluorobenzo[d]isothiazole (89c). Compound 89c was
prepared using 4-bromo-2,5-difluorobenzaldehyde 88c (476 mg, 2.15
mmol) according to the general procedure. The residue was purified by
column chromatography on silica gel (0−20% EtOAc/cyclohexane)
(235 mg, 47% yield over three steps). LC−MS method 1: Rt = 1.07 min.
MS-ESI (m/z): calcd for C7H3BrFNS [M + H]+, 231.9; found, 231.9.
General Procedure for the Synthesis of Intermediates 81c−e,h−l,
87a,b, and 90a−c. A mixture of compound 80c−e,h−l or 86a,b or
89a−c (1 equiv), NaN3 (2 equiv), Cu2O (1.1 equiv), and L-proline (1.3
equiv) in DMSO (0.1 M) was heated at 100 °C for 1−16 h.
Consumption of starting material was followed by LC−MS. After
cooling to rt, the mixture was quenched with a saturated aqueous
NH4Cl solution, stirred for 1 h, and then filtered through a pad of
Celite, washing with EtOAc and water. Phases were separated and the
organic one was washed with a saturated aqueous NaHCO3 solution
and brine, dried over Na2SO4, filtered, and evaporated to dryness. The
residue was purified by column chromatography or used in the next step
without any further purification as described for each intermediate.
6-Fluorobenzo[d]isothiazol-5-amine (81c). Compound 81c was
prepared using 80c (230 mg, 0.99 mmol) according to the general
procedure. The residue was used in the next step without any further
purification (130 mg, 78% yield). LC−MS method 1: Rt = 0.67 min.
MS-ESI (m/z): calcd for C7H5FN2S [M + H]+, 169.0; found, 168.9.
Isothiazolo[5,4-c]pyridin-5-amine (81d). Compound 81d was
prepared using 80d (136 mg, 0.64 mmol) according to the general
procedure. The residue was used in the next step without any further
purification (37 mg, 38% yield). LC−MS method 1: Rt = min. MS-ESI
(m/z): calcd for C6H5N3S [M + H]+, 152.0; found, 152.1.
4-Methylbenzo[d]isothiazol-5-amine (81e). Compound 81e was
prepared using 80e (247 mg, 1.08 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (10−50% EtOAc/cyclohexane) (30 mg, 17% yield). LC−MS
method 1: Rt = 0.27 min. MS-ESI (m/z): calcd for C8H8N2S [M + H]+,
165.0; found, 165.0.
Isothiazolo[4,5-b]pyridin-5-amine (81h). Compound 81h was
prepared using 80h (345 mg, 1.61 mmol) according to the general
procedure. The residue was used in the next step without any further
purification (22 mg, 9% yield). LC−MS method 1: Rt = 0.53 min. MS-
ESI (m/z): calcd for C6H5N3S [M + H]+, 152.0; found, 151.9.
7-Methylbenzo[d]isothiazol-5-amine (81i). Compound 81i was
prepared using 80i (413 mg, 1.81 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (10−50% EtOAc/cyclohexane) (131 mg, 44% yield). LC−MS
method 1: Rt = 0.47 min. MS-ESI (m/z): calcd for C8H8N2S [M + H]+,
165.0; found, 165.0.
7-Chlorobenzo[d]isothiazol-5-amine (81j). Compound 81j was
prepared using 80j (413 mg, 1.81 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (10−50% EtOAc/cyclohexane) (19 mg, 19% yield). LC−MS
method 1: Rt = 0.82 min. MS-ESI (m/z): calcd for C7H5ClN2S [M +
H]+, 185.0; found, 185.0.
7-Fluorobenzo[d]isothiazol-5-amine (81k). Compound 81k was
prepared using 80k (145 mg, 0.63 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (10−50% EtOAc/cyclohexane) (19 mg, 19% yield). LC−MS
5948
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
method 1: Rt = 0.71 min. MS-ESI (m/z): calcd for C7H5FN2S [M +
H]+, 169.0; found, 168.9.
chromatography on silica gel (10−30% EtOAc/cyclohexane) (24 mg,
11% yield). LC−MS method 1: Rt = 0.68 min. MS-ESI (m/z): calcd for
C7H5FN2S [M + H]+, 169.0; found, 169.0.
Isothiazolo[5,4-b]pyridin-5-amine (81l). Compound 81l was
prepared using 80l (365 mg, 1.7 mmol) according to the general
procedure. The residue was used in the next step without any further
purification (171 mg, 67% yield). LC−MS method 1: Rt = 0.43 min.
MS-ESI (m/z): calcd for C6H5N3S [M + H]+, 152.0; found, 152.0.
Benzo[d]isothiazol-6-amine (87a). Compound 87a was prepared
using 86a (400 mg, 1.87 mmol) according to the general procedure.
The residue was purified by reverse-phase column chromatography (0−
50% MeCN/water) (226 mg, 80% yield). LC−MS method 1: Rt = 0.54
min. MS-ESI (m/z): calcd for C7H6N2S [M + H]+, 151.0; found, 151.0.
Benzo[d]isothiazol-4-amine (87b). Compound 87b was prepared
using 86b (226 mg, 1.06 mmol) according to the general procedure.
The residue was purified by reverse-phase column chromatography (5−
80% MeCN/water) (80 mg, 50% yield). LC−MS method 1: Rt = 0.55
min. MS-ESI (m/z): calcd for C7H6N2S [M + H]+, 151.0; found, 151.0.
5-Chlorobenzo[d]isothiazol-6-amine (90a). Compound 90a was
prepared using 89a (350 mg, 1.4 mmol) according to the general
procedure. The residue was used in the next step without any further
purification (200 mg, 77% yield). LC−MS method 1: Rt = 0.81 min.
MS-ESI (m/z): calcd for C7H5ClN2S [M + H]+, 185.0; found, 185.0.
5-Methylbenzo[d]isothiazol-6-amine (90b). Compound 90b was
prepared using 89b (120 mg, 0.52 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (0−30% EtOAc/cyclohexane) (30 mg, 35% yield). LC−MS
method 1: Rt = 0.73 min. MS-ESI (m/z): calcd for C8H8N2S [M + H]+,
165.0; found, 165.1.
5-Fluorobenzo[d]isothiazol-6-amine (90c). Compound 90c was
prepared using 89c (223 mg, 0.96 mmol) according to the general
procedure. The residue was purified by column chromatography on
silica gel (0−30% EtOAc/cyclohexane) (80 mg, 50% yield). LC−MS
method 1: Rt = 0.65 min. MS-ESI (m/z): calcd for C7H5FN2S [M +
H]+, 169.0; found, 169.2.
General Procedure for the Synthesis of Intermediates 81a,b and
87c. A mixture of compound 80a,b or 86c (1 equiv), iron powder (3
equiv), and NH4Cl (1 equiv) in EtOH (0.2 M) was heated at reflux for 3
h. After cooling to rt, the mixture was filtered through a pad of Celite,
washing with EtOH, and the filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography on silica
gel (0−10% MeOH/DCM).
General Procedure for the Synthesis of Compounds 46, 49−57,
and 84. A mixture of the appropriate intermediate 81 (1 equiv) and 5-
chloro-1-methyl-1H-imidazole-2-carbaldehyde or 5-chlorothiazole-2-
carbaldehyde (1−1.5 equiv) in DCM (0.1 M) and catalytic AcOH was
stirred at rt for 30 min. NaBH(OAc)3 (2−3 equiv) was added and the
reaction mixture was stirred at rt for 16 h, then treated with a saturated
aqueous NaHCO3 solution and extracted with DCM. The organic layer
was washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness. The residue was purified as described for each compound.
N-((5-Chlorothiazol-2-yl)methyl)-6-methylbenzo[d]isothiazol-5-
amine (46). Compound 46 was prepared using 81a (90 mg, 0.55
mmol) and 5-chlorothiazole-2-carbaldehyde (81 mg, 0.55 mmol)
according to the general procedure. The crude material was purified by
reverse-phase column chromatography (5−60% MeCN/0.1%
1
HCOOH in water). White solid (39 mg, 24% yield). H NMR (400
MHz, CDCl3): δ 8.70 (s, 1H), 7.68 (s, 1H), 7.57 (s, 1H), 7.13 (s, 1H),
4.73 (d, J = 5.70 Hz, 2H), 4.54 (d, J = 5.04 Hz, 1H), 2.40 (s, 3H). LC−
MS method 1: Rt = 1.11 min. MS-ESI (m/z): calcd for C12H10ClN3S2
[M + H]+, 296.0; found, 296.0.
N-((5-Chlorothiazol-2-yl)methyl)isothiazolo[5,4-c]pyridin-5-
amine (49). Compound 49 was prepared using 81d (37 mg, 0.25
mmol) and 5-chlorothiazole-2-carbaldehyde (37 mg, 0.25 mmol)
according to the general procedure. The crude material was purified by
reverse-phase column chromatography (5−50% MeCN/0.1%
1
HCOOH in water). White solid (10 mg, 14% yield). H NMR (400
MHz, CDCl3): δ 8.90 (s, 1H), 8.76 (s, 1H), 7.53 (s, 1H), 6.98 (d, J =
1.10 Hz, 1H), 5.36 (t, J = 5.15 Hz, 1H), 4.84 (d, J = 6.14 Hz, 2H). LC−
MS method 1: Rt = 0.94 min. MS-ESI (m/z): calcd for C10H7ClN4S2
[M + H]+, 283.0; found, 283.0.
N-((5-Chlorothiazol-2-yl)methyl)-4-methylbenzo[d]isothiazol-5-
amine (50). Compound 50 was prepared using 81e (30 mg, 0.18
mmol) and 5-chlorothiazole-2-carbaldehyde (27 mg, 0.18 mmol)
according to the general procedure. The crude material was purified by
column chromatography on silica gel (10−50% EtOAc/cyclohexane).
Yellow solid (17.8 mg, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ
9.09 (d, J = 0.88 Hz, 1H), 7.77 (d, J = 8.77 Hz, 1H), 7.75 (s, 1H), 6.96
(d, J = 8.77 Hz, 1H), 6.26 (t, J = 6.03 Hz, 1H), 4.69 (d, J = 6.14 Hz, 2H),
2.53 (s, 3H). LC−MS method 1: Rt = 1.10 min. MS-ESI (m/z): calcd
for C12H10ClN3S2 [M + H]+, 296.0; found, 295.9.
6-Methylbenzo[d]isothiazol-5-amine (81a). Compound 81a was
prepared using 80a (360 mg, 1.86 mmol) according to the general
procedure (186 mg, 60% yield). LC−MS method 1: Rt = 0.53 min. MS-
ESI (m/z): calcd for C8H8N2S [M + H]+, 165.0; found, 165.0.
6-Chlorobenzo[d]isothiazol-5-amine (81b). Compound 81b was
prepared using 80b (300 mg, 1.40 mmol) according to the general
procedure (158 mg, 77% yield). LC−MS method 1: Rt = 0.82 min. MS-
ESI (m/z): calcd for C7H5ClN2S [M + H]+, 185.0; found, 185.0.
Benzo[d]isothiazol-7-amine (87c). Compound 87c was prepared
using 86c (325 mg, 1.80 mmol) according to the general procedure
(107 mg, 40% yield). LC−MS method 1: Rt = 0.62 min. MS-ESI (m/z):
calcd for C7H6N2S [M + H]+, 151.0; found, 150.9.
4-Chloro-N-((5-chlorothiazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (51). Compound 51 was prepared using 81f (141 mg, 0.76
mmol) and 5-chlorothiazole-2-carbaldehyde (113 mg, 0.76 mmol)
according to the general procedure. The crude material was purified by
column chromatography on silica gel (10−50% EtOAc/cyclohexane)
and by reverse-phase column chromatography (5−60% MeCN/0.1%
1
HCOOH in water). White solid (49 mg, 20% yield). H NMR (400
MHz, DMSO-d6): δ 8.95 (d, J = 0.78 Hz, 1H), 7.95 (d, J = 9.00 Hz, 1H),
7.76 (s, 1H), 7.13 (d, J = 8.61 Hz, 1H), 6.74 (t, J = 6.26 Hz, 1H), 4.77
(d, J = 6.26 Hz, 2H). LC−MS method 1: Rt = 1.20 min. MS-ESI (m/z):
calcd for C11H7Cl2N3S2 [M + H]+, 316.0; found, 316.0.
Synthesis of 4-Chlorobenzo[d]isothiazol-5-amine (81f). N-Chlor-
osuccinimide (178 mg, 1.33 mmol) was added to a solution of
benzo[d]isothiazol-5-amine 73a (200 mg, 1.33 mmol) in THF (10 mL)
and the mixture was stirred at rt for 16 h. The mixture was treated with a
saturated aqueous NaHCO3 solution and extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4, filtered, and
evaporated to dryness. The residue was purified by column
chromatography on silica gel (10−50% EtOAc/cyclohexane) (141
mg, 57% yield). LC−MS method 1: Rt = 0.83 min. MS-ESI (m/z): calcd
for C7H5ClN2S [M + H]+, 185.0; found, 185.0.
N-((5-Chlorothiazol-2-yl)methyl)-4-fluorobenzo[d]isothiazol-5-
amine (52). Compound 52 was prepared using 81g (24 mg, 0.14
mmol) and 5-chlorothiazole-2-carbaldehyde (31.6 mg, 0.21 mmol)
according to the general procedure. The crude material was purified by
column chromatography on silica gel (5% EtOAc/DCM). Yellow solid
(14.4 mg, 34% yield). 1H NMR (400 MHz, CDCl3): δ 8.91 (d, J = 0.66
Hz, 1H), 7.55 (s, 1H), 7.56 (d, J = 8.55 Hz, 1H), 7.07 (dd, J = 8.33, 7.67
Hz, 1H), 4.75−4.84 (m, 1H), 4.73 (d, J = 5.90 Hz, 2H). LC−MS
method 1: Rt = 1.11 min. MS-ESI (m/z): calcd for C11H7ClFN3S2 [M +
H]+, 300.0; found, 300.0.
Synthesis of 4-Fluorobenzo[d]isothiazol-5-amine (81g). Select-
fluor (700 mg, 1.99 mmol) was added to a solution of benzo[d]-
isothiazol-5-amine 73a (200 mg, 1.33 mmol) in DMF (4 mL) and
cooled to 0 °C. The reaction mixture was stirred at rt for 3 h, treated
with a saturated aqueous NaHCO3 solution, and extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4, filtered,
and evaporated to dryness. The residue was purified by column
N-((5-Chlorothiazol-2-yl)methyl)isothiazolo[4,5-b]pyridin-5-
amine (53). Compound 53 was prepared using 81h (22 mg, 0.15
mmol) and 5-chlorothiazole-2-carbaldehyde (21.5 mg, 0.15 mmol)
according to the general procedure. The crude material was purified by
reverse-phase column chromatography (5−90% MeCN/0.1%
1
HCOOH in water). White solid (1.5 mg, 4% yield). H NMR (400
MHz, CDCl3): δ 8.87 (s, 1H), 8.00 (d, J = 8.77 Hz, 1H), 7.51 (s, 1H),
5949
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
6.71 (d, J = 8.99 Hz, 1H), 5.38 (t, J = 5.81 Hz, 1H), 4.94 (d, J = 5.92 Hz,
2H). LC−MS method 1: Rt = 0.92 min. MS-ESI (m/z): calcd for
C10H7ClN4S2 [M + H]+, 283.0; found, 283.0.
2H). LC−MS method 1: Rt = 1.18 min. MS-ESI (m/z): calcd for
C11H7Cl2N3S2 [M + H]+, 315.9; found, 315.9.
N-((5-Chlorothiazol-2-yl)methyl)-6-fluorobenzo[d]isothiazol-5-
amine (48). Compound 48 was prepared using 81c (50 mg, 0.30
mmol) and 5-chlorothiazole-2-carbaldehyde (88 mg, 0.59 mmol)
according to the general procedure. White solid (31.2 mg, 35% yield).
1H NMR (500 MHz, CDCl3): δ 8.70 (s, 1H), 7.59 (d, J = 10.43 Hz,
1H), 7.57 (s, 1H), 7.20 (d, J = 7.68 Hz, 1H), 4.94 (br s, 1H), 4.72 (d, J =
5.76 Hz, 2H). LC−MS method 1: Rt = 1.08 min. MS-ESI (m/z): calcd
for C11H7ClFN3S2 [M + H]+, 300.0; found, 300.0.
6-Chloro-N-((5-chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo-
[d]isothiazol-5-amine (82). Compound 82 was prepared using 81b
(70 mg, 0.38 mmol) and 5-chloro-1-methyl-1H-imidazole-2-carbalde-
hyde (110 mg, 0.76 mmol) according to the general procedure. White
solid (26.2 mg, 22% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.89 (d,
J = 0.66 Hz, 1H), 8.25 (s, 1H), 7.57 (s, 1H), 6.95 (s, 1H), 6.26 (t, J =
5.15 Hz, 1H), 4.49 (d, J = 5.26 Hz, 2H), 3.62 (s, 3H). LC−MS method
1: Rt = 0.66 min. MS-ESI (m/z): calcd for C12H10Cl2N4S [M + H]+,
313.0; found, 313.0.
N-((5-Chlorothiazol-2-yl)methyl)-7-methylbenzo[d]isothiazol-5-
amine (54). Compound 54 was prepared using 81i (131.3 mg, 0.80
mmol) and 5-chlorothiazole-2-carbaldehyde (118 mg, 0.80 mmol)
according to the general procedure. The crude material was purified by
column chromatography on silica gel (10−50% EtOAc/cyclohexane).
Yellow solid (159.2 mg, 67% yield). 1H NMR (500 MHz, DMSO-d6): δ
8.87 (s, 1H), 7.77 (s, 1H), 7.01 (s, 1H), 6.86−6.96 (m, 2H), 4.61 (d, J =
6.04 Hz, 2H), 2.45 (s, 3H). LC−MS method 1: Rt = 1.11 min. MS-ESI
(m/z): calcd for C12H10ClN3S2 [M + H]+, 296.0; found, 296.0.
7-Chloro-N-((5-chlorothiazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (55). Compound 55 was prepared using 81j (19 mg, 0.10 mmol)
and 5-chlorothiazole-2-carbaldehyde (15 mg, 0.10 mmol) according to
the general procedure. The crude material was purified by column
chromatography on silica gel (10−50% EtOAc/cyclohexane) and by
reverse-phase column chromatography (5−60% MeCN/0.1%
1
HCOOH in water). White solid (9.7 mg, 30% yield). H NMR (500
MHz, DMSO-d6): δ 8.96 (s, 1H), 7.78 (s, 1H), 7.23 (d, J = 1.96 Hz,
1H), 7.19−7.22 (m, 2H), 4.66 (d, J = 6.36 Hz, 2H). LC−MS method 1:
Rt = 1.21 min. MS-ESI (m/z): calcd for C11H7Cl2N3S2 [M + H]+, 316.0;
found, 316.0.
N-((5-Chlorothiazol-2-yl)methyl)-7-fluorobenzo[d]isothiazol-5-
amine (56). Compound 56 was prepared using 81k (48 mg, 0.29
mmol) and 5-chlorothiazole-2-carbaldehyde (63 mg, 0.43 mmol)
according to the general procedure. The crude material was purified by
column chromatography on silica gel (7% EtOAc/DCM). White solid
(42 mg, 49% yield). 1H NMR (400 MHz, CDCl3): δ 8.70 (d, J = 4.17
Hz, 1H), 7.56 (s, 1H), 6.98 (d, J = 1.75 Hz, 1H), 6.69 (dd, J = 10.74,
1.75 Hz, 1H), 4.75 (t, J = 5.30 Hz, 1H), 4.63−4.70 (m, 2H). LC−MS
method 1: Rt = 1.14 min. MS-ESI (m/z): calcd for C11H7ClFN3S2 [M +
H]+, 300.0; found, 300.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-6-fluorobenzo-
[d]isothiazol-5-amine (83). Compound 83 was prepared using 81c
(80 mg, 0.48 mmol) and 5-chloro-1-methyl-1H-imidazole-2-carbalde-
hyde (144.5 mg, 0.95 mmol) according to the general procedure. White
solid (25.9 mg, 18% yield). 1H NMR (500 MHz, DMSO-d6): δ 8.82−
8.90 (m, 1H), 7.95 (d, J = 11.25 Hz, 1H), 7.55 (d, J = 7.96 Hz, 1H), 6.93
(s, 1H), 6.35−6.45 (m, 1H), 4.44 (d, J = 5.49 Hz, 2H), 3.61 (s, 3H).
LC−MS method 1: Rt = 0.56 min. MS-ESI (m/z): calcd for
C12H10ClFN4S [M + H]+, 297.0; found, 297.0.
General Procedure for the Synthesis of Compounds 58−60. A
mixture of compounds 82, 83, or 84 (1 equiv) and paraformaldehyde (3
equiv) in DCM (0.05 M) and catalytic AcOH was stirred at rt for 30
min. NaBH(OAc)3 (3 equiv) was added and the reaction mixture was
stirred at rt for 24−72 h. Consumption of starting material was followed
by LC−MS. Upon completion, the mixture was treated with water and
extracted with DCM. The organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness. The crude material
was purified as described for each compound.
6-Chloro-N-((5-chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-
methylbenzo[d]isothiazol-5-amine (58). Compound 58 was prepared
using 82 (50 mg, 0.16 mmol) and paraformaldehyde (14.3 mg, 0.48
mmol) according to the general procedure. The crude material was
purified by column chromatography on silica gel (5% EtOAc/DCM).
White solid (29.3 mg, 56% yield). 1H NMR (400 MHz, CDCl3): δ 8.82
(s, 1H), 8.01 (s, 1H), 7.83 (s, 1H), 6.87 (s, 1H), 4.37 (s, 2H), 3.61 (s,
3H), 2.77 (s, 3H). LC−MS method 1: Rt = 0.74 min. MS-ESI (m/z):
calcd for C13H12Cl2N4S [M + H]+, 327.0; found, 327.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-6-fluoro-N-
methylbenzo[d]isothiazol-5-amine (59). Compound 59 was prepared
using 83 (56 mg, 0.19 mmol) and paraformaldehyde (17 mg, 0.57
mmol) according to the general procedure. The crude material was
purified by column chromatography on silica gel (40% EtOAc/DCM)
and then by reverse-phase column chromatography (5−60% MeCN/
0.1% HCOOH in water). Colorless oil (32 mg, 54% yield). 1H NMR
(500 MHz, CDCl3): δ 8.81 (d, J = 0.98 Hz, 1H), 7.69 (d, J = 7.83 Hz,
1H), 7.64 (d, J = 11.25 Hz, 1H), 6.91 (s, 1H), 4.35 (s, 2H), 3.65 (s, 3H),
2.79 (s, 3H). LC−MS method 1: Rt = 0.65 min. MS-ESI (m/z): calcd
for C13H12ClFN4S [M + H]+, 311.1; found, 311.1.
N-((5-Chlorothiazol-2-yl)methyl)isothiazolo[5,4-b]pyridin-5-
amine (57). Compound 57 was prepared using 81l (96.65 mg, 0.64
mmol) and 5-chlorothiazole-2-carbaldehyde (93.86 mg, 0.64 mmol)
according to the general procedure. The crude material was purified by
reverse-phase column chromatography (5−50% MeCN/0.1%
1
HCOOH in water). White solid (51.80 mg, 16% yield). H NMR
(500 MHz, CDCl3): δ 8.77 (s, 1H), 8.39 (d, J = 2.74 Hz, 1H), 7.57 (s,
1H), 7.39 (d, J = 2.74 Hz, 1H), 4.87 (br s, 1H), 4.69 (d, J = 6.04 Hz,
2H). LC−MS method 1: Rt = 0.88 min. MS-ESI (m/z): calcd for
C10H7ClN4S2 [M + H]+, 283.0; found, 283.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-6-
methylbenzo[d]isothiazol-5-amine (84). Compound 84 was prepared
using 81a (100 mg, 0.60 mmol) and 5-chloro-1-methyl-1H-imidazole-
2-carbaldehyde (97 mg, 0.67 mmol) according to the general
procedure. The crude material was purified by column chromatography
on silica gel (0−30% EtOAc/cyclohexane). White solid (50 mg, 29%
yield). 1H NMR (500 MHz, CDCl3): δ 8.76 (s, 1H), 7.67 (s, 1H), 7.26
(s, 1H), 6.97 (s, 1H), 4.43 (s, 3H), 3.65 (s, 3H), 2.38 (s, 3H). LC−MS
method 1: Rt = 0.56 min. MS-ESI (m/z): calcd for C13H13ClN4S [M +
H]+, 293.1; found, 293.0.
General Procedure for the Synthesis of Compounds 47, 48, 82,
and 83. A solution of the appropriate intermediate 81 (1 equiv) and 5-
chlorothiazole-2-carbaldehyde or 5-chloro-1-methyl-1H-imidazole-2-
carbaldehyde (1.5−2 equiv) in MeOH (0.1 M) was heated at 60 °C for
16 h. After cooling to 0 °C, NaBH4 (2 equiv) was added and the
reaction mixture was allowed to warm to rt and stirred for 2−3 h. The
mixture was quenched with a saturated aqueous NaHCO3 solution and
extracted with DCM. The organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness. The crude material
was purified by column chromatography on silica gel (5% EtOAc/
DCM).
6-Chloro-N-((5-chlorothiazol-2-yl)methyl)benzo[d]isothiazol-5-
amine (47). Compound 47 was prepared using 81b (18 mg, 0.10
mmol) and 5-chlorothiazole-2-carbaldehyde (21 mg, 0.15 mmol)
according to the general procedure. Pale yellow solid (16.9 mg, 55%
yield). 1H NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 7.93 (s, 1H), 7.57
(s, 1H), 7.19 (s, 1H), 5.32 (t, J = 5.37 Hz, 1H), 4.75 (d, J = 5.70 Hz,
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N,6-
dimethylbenzo[d]isothiazol-5-amine (60). Compound 60 was
prepared using 84 (27 mg, 0.09 mmol) and paraformaldehyde (8 mg,
0.27 mmol) according to the general procedure. The crude material was
purified by column chromatography on silica gel (0−50% EtOAc/
cyclohexane). White solid (12.2 mg, 43% yield). 1H NMR (500 MHz,
CDCl3): δ 8.81 (s, 1H), 7.90−7.65 (m, 2H), 6.90 (s, 1H), 4.28 (s, 2H),
3.53 (s, 3H), 2.64 (s, 3H), 2.51 (s, 3H). LC−MS method 1: Rt = 0.67
min. MS-ESI (m/z): calcd for C14H15ClN4S [M + H]+, 307.1; found,
307.0.
General Procedure for the Synthesis of Compounds 61−63. A
solution of the appropriate intermediate 87 (1 equiv) and 5-
chlorothiazole-2-carbaldehyde (1 equiv) in MeOH (0.1 M) was heated
5950
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
at 60 °C for 16 h. After cooling to 0 °C, NaBH4 (3 equiv) was added and
the reaction mixture was allowed to warm to rt and stirred for 2 h. The
mixture was quenched with a saturated aqueous NaHCO3 solution and
extracted with DCM. The organic layer was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness. The crude material
was purified by reverse-phase HPLC to give the target compounds.
N-((5-Chlorothiazol-2-yl)methyl)benzo[d]isothiazol-6-amine
(61). Compound 61 was prepared using 87a (100 mg, 0.66 mmol) and
5-chlorothiazole-2-carbaldehyde (97 mg, 0.66 mmol) according to the
column chromatography on silica gel (0−30% EtOAc/cyclohexane).
White solid (12.7 mg, 47% yield). 1H NMR (400 MHz, DMSO-d6): δ
8.72 (s, 1H), 7.81 (s, 1H), 7.77 (s, 1H), 7.11 (s, 1H), 6.67 (t, J = 6.03
Hz, 1H), 4.70 (d, J = 5.92 Hz, 2H), 2.30 (s, 3H). LC−MS method 1: Rt
= 1.07 min. MS-ESI (m/z): calcd for C12H10ClN3S2 [M + H]+, 296.0;
found, 296.0.
N-((5-Chlorothiazol-2-yl)methyl)-5-fluorobenzo[d]isothiazol-6-
amine (67). Compound 67 was prepared using 90c (36 mg, 0.21
mmol) and 5-chlorothiazole-2-carbaldehyde (93 mg, 0.63 mmol)
according to the general procedure using DCE as a solvent and heating
the reaction mixture at 70 °C for 20 h. The crude material was purified
by column chromatography on silica gel (0−30% EtOAc/cyclohexane).
Yellow solid (33 mg, 54% yield). 1H NMR (400 MHz, DMSO-d6): δ
8.77 (s, 1H), 7.88 (d, J = 11.62 Hz, 1H), 7.78 (s, 1H), 7.33 (d, J = 7.45
Hz, 1H), 7.28−7.18 (m, 1H), 4.70 (d, J = 6.14 Hz, 2H). LC−MS
method 1: Rt = 1.05 min. MS-ESI (m/z): calcd for C11H7ClFN3S2 [M +
H]+, 300.0; found, 300.0.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-5-
methylbenzo[d]isothiazol-6-amine (68). Compound 68 was prepared
using 90b (15 mg, 0.09 mmol) and 5-chloro-1-methyl-1H-imidazole-2-
carbaldehyde (14.5 mg, 0.10 mmol) according to the general procedure
using DCM as a solvent and stirring the reaction mixture at rt for 36 h.
The crude material was purified by column chromatography on silica
gel (0−50% EtOAc/cyclohexane). White solid (10.3 mg, 39% yield).
1H NMR (400 MHz, DMSO-d6): δ 8.73 (d, J = 0.66 Hz, 1H), 7.80 (s,
1
general procedure. White solid (25 mg, 13% yield). H NMR (400
MHz, CDCl3): δ 8.68 (s, 1H), 7.84 (d, J = 8.77 Hz, 1H), 7.56 (s, 1H),
7.03 (d, J = 1.32 Hz, 1H), 6.82 (dd, J = 8.66, 2.08 Hz, 1H), 4.90 (t, J =
5.59 Hz, 1H), 4.69 (d, J = 5.92 Hz, 2H). LC−MS method 1: Rt = 1.03
min. MS-ESI (m/z): calcd for C11H8ClN3S2 [M + H]+, 282.0; found,
282.1.
N-((5-Chlorothiazol-2-yl)methyl)benzo[d]isothiazol-4-amine
(62). Compound 62 was prepared using 87b (80 mg, 0.53 mmol) and 5-
chlorothiazole-2-carbaldehyde (78 mg, 0.53 mmol) according to the
1
general procedure. White solid (30 mg, 20% yield). H NMR (400
MHz, CDCl3): δ 8.94 (s, 1H), 7.56 (s, 1H), 7.43−7.30 (m, 2H), 6.61−
6.48 (m, 1H), 5.32 (br s, 1H), 4.79 (d, J = 5.70 Hz, 2H). LC−MS
method 1: Rt = 1.08 min. MS-ESI (m/z): calcd for C11H8ClN3S2 [M +
H]+, 282.0; found, 282.1.
N-((5-Chlorothiazol-2-yl)methyl)benzo[d]isothiazol-7-amine
(63). Compound 63 was prepared using 87c (107 mg, 0.71 mmol) and
5-chlorothiazole-2-carbaldehyde (105 mg, 0.71 mmol) according to the
general procedure. Yellow solid (28.6 mg, 14% yield). 1H NMR (400
MHz, CDCl3): δ 8.90 (s, 1H), 7.59−7.53 (m, 2H), 7.33 (t, J = 7.78 Hz,
1H), 6.72 (d, J = 7.45 Hz, 1H), 4.80 (d, J = 5.70 Hz, 2H), 4.55 (br s,
1H). LC−MS method 1: Rt = 1.08 min. MS-ESI (m/z): calcd for
C11H8ClN3S2 [M + H]+, 282.0; found, 282.1.
General Procedure for the Synthesis of Compounds 64−68, 70,
and 71. A mixture of the appropriate intermediate 90 (1 equiv) and 5-
chloro-1-methyl-1H-imidazole-2-carbaldehyde or 5-chlorothiazole-2-
carbaldehyde (1−3 equiv) in DCM or DCE (0.1 M) and catalytic
AcOH was stirred at rt for 30 min. NaBH(OAc)3 (2−3 equiv) was
added and the reaction mixture was stirred at rt or heated at 70 °C for
8−48 h. Consumption of starting material was followed by LC−MS.
Upon completion, the mixture was treated with a saturated aqueous
NaHCO3 solution and extracted with DCM. The organic layer was
washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness. The crude material was purified as described for each
compound.
5-Chloro-N-((5-chlorothiazol-2-yl)methyl)benzo[d]isothiazol-6-
amine (64). Compound 64 was prepared using 90a (70 mg, 0.38
mmol) and 5-chlorothiazole-2-carbaldehyde (112 mg, 0.76 mmol)
according to the general procedure using DCE as a solvent and heating
the reaction mixture at 70 °C for 8 h. The crude material was purified by
column chromatography on silica gel (0−30% EtOAc/cyclohexane).
Yellow solid (58 mg, 48% yield). 1H NMR (400 MHz, DMSO-d6): δ
8.77 (d, J = 0.66 Hz, 1H), 8.20 (s, 1H), 7.78 (s, 1H), 7.35 (s, 1H), 7.06
(t, J = 6.14 Hz, 1H), 4.75 (d, J = 6.14 Hz, 2H). LC−MS method 1: Rt =
1.14 min. MS-ESI (m/z): calcd for C11H7Cl2N3S2 [M + H]+, 316.0;
found, 316.0.
5-Chloro-N-((5-chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo-
[d]isothiazol-6-amine (65). Compound 65 was prepared using 90a
(120 mg, 0.65 mmol) and 5-chloro-1-methyl-1H-imidazole-2-carbal-
dehyde (188 mg, 1.3 mmol) according to the general procedure using
DCE as a solvent and heating the reaction mixture at 70 °C for 20 h.
The crude material was purified by column chromatography on silica
gel (20−50% EtOAc/cyclohexane). Yellow solid (90 mg, 45% yield).
1H NMR (400 MHz, DMSO-d6): δ 8.76 (s, 1H), 8.17 (s, 1H), 7.49 (s,
1H), 6.94 (s, 1H), 6.63 (t, J = 5.26 Hz, 1H), 4.51 (d, J = 5.26 Hz, 2H),
3.61 (s, 3H). LC−MS method 1: Rt = 0.66 min. MS-ESI (m/z): calcd
for C12H10Cl2N4S [M + H]+, 313.0; found, 313.0.
N-((5-Chlorothiazol-2-yl)methyl)-5-methylbenzo[d]isothiazol-6-
amine (66). Compound 66 was prepared using 90b (15 mg, 0.09
mmol) and 5-chlorothiazole-2-carbaldehyde (26.5 mg, 0.18 mmol)
according to the general procedure using DCM as a solvent and stirring
the reaction mixture at rt for 36 h. The crude material was purified by
1H), 7.32 (br s, 1H), 7.23 (s, 1H), 6.31 (br s, 1H), 4.60 (s, 2H), 3.69 (s,
3H), 2.29 (s, 3H). LC−MS method 1: Rt = 0.57 min. MS-ESI (m/z):
calcd for C13H13ClN4S [M + H]+, 293.1; found, 293.1.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-5-fluorobenzo-
[d]isothiazol-6-amine (70). Compound 70 was prepared using 90c
(36 mg, 0.21 mmol) and 5-chloro-1-methyl-1H-imidazole-2-carbalde-
hyde (91 mg, 0.63 mmol) according to the general procedure using
DCE as a solvent and heating the reaction mixture at 70 °C for 20 h.
The crude material was purified by column chromatography on silica
gel (20−60% EtOAc/cyclohexane). Yellow solid (32 mg, 52% yield).
1H NMR (400 MHz, DMSO-d6): δ 8.75 (s, 1H), 7.83 (d, J = 11.84 Hz,
1H), 7.47 (d, J = 7.67 Hz, 1H), 6.93 (s, 1H), 6.82 (td, J = 5.26, 2.19 Hz,
1H), 4.46 (d, J = 5.48 Hz, 2H), 3.60 (s, 3H). LC−MS method 1: Rt =
0.55 min. MS-ESI (m/z): calcd for C12H10ClFN4S [M + H]+, 297.0;
found, 297.1.
N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)benzo[d]-
isothiazol-6-amine (71). Compound 71 was prepared using 87a (64
mg, 0.43 mmol) and 5-chloro-1-methyl-1H-imidazole-2-carbaldehyde
(62 mg, 0.43 mmol) according to the general procedure using DCM as
a solvent and stirring the reaction mixture at rt for 24 h. The crude
material was purified by column chromatography on silica gel (15%
MeOH/DCM). White solid (35 mg, 30% yield). 1H NMR (400 MHz,
CDCl3): δ 8.68 (s, 1H), 7.83 (d, J = 8.55 Hz, 1H), 7.11 (d, J = 1.10 Hz,
1H), 6.96 (s, 1H), 6.84 (dd, J = 8.77, 1.97 Hz, 1H), 4.88 (br s, 1H), 4.42
(d, J = 4.60 Hz, 2H), 3.62 (s, 3H). LC−MS method 1: Rt = 0.48 min.
MS-ESI (m/z): calcd for C12H11ClN4S [M + H]+, 279.0; found, 279.0.
Synthesis of N-((5-Chloro-1-methyl-1H-imidazol-2-yl)methyl)-N-
methylbenzo[d]isothiazol-6-amine (69). NaH (60% w/w in mineral
oil, 4 mg, 0.2 mmol) was added to a solution of 71 (25 mg, 0.09 mmol)
in dry DMF (1 mL) and cooled to 0 °C. The reaction mixture was
stirred at rt for 15 min, MeI (6 μL, 0.1 mmol) was added, and stirring
was continued for 3 h. The mixture was treated with a saturated aqueous
NH4Cl solution and extracted with EtOAc. The combined extracts were
washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness. The crude material was purified by column chromatography
1
on silica gel (5% MeOH/DCM). White solid (3 mg, 11% yield). H
NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 7.89 (d, J = 8.77 Hz, 1H),
7.25 (d, J = 1.32 Hz, 1H), 7.10 (dd, J = 8.99, 1.97 Hz, 1H), 6.93 (s, 1H),
4.63 (s, 2H), 3.51 (s, 3H), 3.05 (s, 3H). LC−MS method 1: Rt = 0.56
min. MS-ESI (m/z): calcd for C13H13ClN4S [M + H]+, 293.1; found,
293.0.
Synthesis of 5-Chloro-N-((5-chloro-1-methyl-1H-imidazol-2-yl)-
methyl)-N-methylbenzo[d]isothiazol-6-amine (72). A mixture of the
compound 65 (60 mg, 0.19 mmol) and paraformaldehyde (29 mg, 0.95
mmol) in DCE (3 mL) and catalytic AcOH was stirred at rt for 30 min.
5951
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
NaBH(OAc)3 (121 mg, 0.57 mmol) was added and the reaction
mixture was heated at 40 °C for 48 h. The mixture was treated with
water and extracted with DCM. The organic layer was washed with
brine, dried over Na2SO4, filtered, and evaporated to dryness. The
crude material was purified by column chromatography on silica gel
of the graph of the natural log of percentage parent remaining versus
incubation time. Clearance was estimated from the rate of depletion k
(min−1), the volume of the incubation V (mL), and the amount of
microsomal proteins in the incubation M (mg) scaled for the protein in
the incubation relative to that in the liver.
1
(20−50% EtOAc/cyclohexane). White solid (10 mg, 16% yield). H
Clint = k × V/M
NMR (400 MHz, CDCl3): δ 8.75 (s, 1H), 8.08 (s, 1H), 7.68 (s, 1H),
6.89 (s, 1H), 4.41 (s, 2H), 3.57 (s, 3H), 2.81 (s, 3H). LC−MS method
1: Rt = 0.75 min. MS-ESI (m/z): calcd for C13H12Cl2N4S [M + H]+,
327.0; found, 327.1.
CLi values were expressed as μL/min/mg liver microsomes.
Plasma Protein Binding. Equipment and Chemical Reagents.
Unless otherwise stated, all analytical reagents were of at least standard
analytical laboratory reagent grade.
Plasma from different species, according to sponsor requirements,
was obtained in-house or purchased from external suppliers (Sera
Laboratories International Ltd. or B&K Universal Ltd.).
Rapid equilibrium dialysis device (Thermo Scientific RED Device)
or 96 well dialyzer apparatus (HTDialysis LLC).
Solubility Assay. Equipment and Chemical Reagents. Control
compounds: ibuprofen for high solubility and progesterone for low
solubility.
DMSO evaporation device: Genevac HT-4X.
Shaking device: DVX 2500 Multi Tube Vortex VWR.
Filtration device: MultiScreen Resist vacuum manifold Millipore
Phenomenex 96 well glass fiber filter 0.45 μm plate Agilent HPLC
system with DAD, UV detection provided at 230 and 254 nm.
Sample plate: NUNC 400.
Experimental Design. 10 μL of a 10 mM compound stock solution
in DMSO was dispensed in a well in duplicate and dried under vacuum.
200 μL of phosphate-buffered saline buffer with pH = 7.4 was added in
each well, sonicated for 5 min, and then mixed with heavy shaking at rt
for 2 h. The final concentration was 500 μM. Then, the solutions were
filtered, 160 μL of filtrate was transferred in a well, and 40 μL of MeOH
was added. The sample was analyzed by HPLC/UV.
RSP Microlab STARlet (Hamilton).
Refrigerated centrifuge, GS 6KR (Beckman).
Microtiter stirrer with thermostatic Cupola mod. 715 CT+ (Asal).
Experimental Design. The dialysis buffer was prepared as follows:
Na2HPO4 8.69 g, KH2PO4 1.90 g, and NaCl 4.11 g were dissolved in
1 L of Milli-Q water and the pH was adjusted to pH 7.4 with H3PO4.
An appropriate amount of test item was dissolved in DMSO (or an
appropriate solvent) to give a 10 mM solution. Further dilutions, to
obtain 50 μM WS, were prepared using 50% ACN in Milli-Q water. 50
μM WS used to spike the plasma to obtain a final concentration of 0.5
μM.
In Vitro Clearance in Liver Microsomes. Equipment and Chemical
Reagents. All chemicals and solvents were of reagent or LC grade.
Water was purified in house (Milli-Q, Millipore) and used in the
preparation of solutions.
350 μL of test item-free isotonic phosphate buffer was dispensed in
the buffer compartment and 200 μL of the spiked matrix was loaded in
the matrix compartment of the RED Device.
50 mM phosphate buffer at pH 7.4: 11.2 mL of potassium phosphate
monobasic and 38.8 mL of potassium phosphate dibasic diluted to 1 L
with Milli-Q water.
Alternatively, 150 μL of test item-free buffer was dispensed in the
buffer compartment and 150 μL of the spiked matrix was loaded in the
matrix compartment of a 96-well dialyzer apparatus (HTDialysis LLC).
At the end of the equilibration period (device is sealed and shaken at
500 rpm for 5 h at 37 °C), 50 μL of the dialyzed matrix was added to 50
μL of the corresponding test item-free dialyzed buffer, and vice versa for
buffer, such that the volume of buffer to matrix to be the same. Samples
were extracted by protein precipitation with 300 μL of ACN containing
rolipram (for positive ionization mode) or diclofenac (for negative
ionization mode) as the internal standard and centrifuged for 10 min at
2800 rpm. Supernatants collected (100 μL), diluted with 18% ACN in
Milli-Q water (200 μL), and injected into an HPLC MS/MS or UPLC
MS/MS system.
Regenerating system: reduced nicotinamide adenine dinucleotide
phosphate (NADPH) regenerating system solution prepared by
dissolving 1.7 mg of nicotinamide adenine dinucleotide phosphate,
7.8 mg of glucose 6-phosphate (G6P), and 6 units of G6P
dehydrogenase in 1 mL of 2% sodium bicarbonate solution (prepared
by dissolving 20 g of NaHCO3 in 1 L of Milli-Q water).
Liver microsomes were supplied by an accredited supplier.
Experimental Design. Liver microsomes were rapidly thawed in a
water bath at 37 °C and kept on ice until use. Liver microsomes were
diluted with 50 mM potassium phosphate buffer pH 7.4 to a protein
concentration of 0.56 mg/mL.
Plasma protein binding was determined with the following formulas:
5 μL of the test compound and positive control working solutions
(WS) (to give a final concentration of 0.5 μM) were added to 445 μL of
microsomes and 50 μL of the regenerating system.
Automated assay steps were performed using RSP Freedom EVO
(Tecan) and Hamilton Systems:
Afu = buffer/plasma
where afu = the apparent fraction unbound; buffer = the analyte/
internal standard ratio determined in the buffer compartment; and
plasma = the analyte/internal standard ratio determined in the plasma
compartment.
150 μL of quenching solution was taken in each well of 96 deep
well 1 mL plates.
Fucr = (1/D)/([(1/Afu − 1) + 1/D])
where fucr = the fraction unbound corrected; D = the matrix dilution
factor (D = 1 for plasma); and % binding = (1 − fucr) × 100; %
unbound = 100 − % bound.
Caco-2 Cell Permeability. Equipment and Chemical Reagents.
Digoxin at a final target concentration of 10 μM.
Atenolol (low permeable reference) at a final concentration of 3 μM.
Metoprolol (high permeable reference) or propranolol at a final
concentration of 3 μM.
Palmitoyl carnitine at a final concentration of 0.3 mM.
Elacridar HCl (GF120918) at a final concentration of 10 μM.
DMSO.
800 μL aliquots of NADPH regenerating system were
prewarmed at 37 °C for 5 min.
5 μL of 50 μM test items and control were added to 445 μL of
the 0.56 mg/mL microsome solution and the incubation mixture
was prewarmed in a 96-deep-well 2 mL plate (incubation plate)
at 37 °C for 5 min.
Incubation reactions were initiated by adding 50 μL of the
prewarmed NADPH regenerating system to the incubation mixtures.
50 μL aliquots were taken from incubation mixtures at: 0, 3, 6, 9, 15,
and 30 min.
Samples were centrifuged at 3000 rpm for 10 min and further diluted
in order to optimize the analytical conditions prior to the LC−MS/MS
analysis.
Lucifer yellow at a final concentration 100 μM.
HBSS with 12.5 mM HEPES.
Peak areas for test and control items were integrated using Integrator
software from Agilent or MultiQuant Software from AB Sciex and
exported to XLFit or Morphit (The Edge) designed to calculate in vitro
metabolic stability parameters. The observed rate constant (kobs) for
parent degradation was calculated by determining the slope of the line
Caco-2 cells used originated from American Type Culture Collection
and cultured in house.
Experimental Design. Test item stock solutions and reference
compound solutions were diluted with DMSO to generate a 200×
solution.
5952
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
200× solutions were then diluted 1:200 with transport buffer to have
a 3 or 10 μM final concentration (0.5% DMSO).
On the day of the experiment, the culture medium present in the
feeder tray was replaced with prewarmed transport buffer and the apical
wells were refilled with transport buffer. Incubation of the cells was
carried out at 37 °C for 30 min.
Blood, colon, and ileum were collected at 6, 24, and 48 h postdose (n =
six animals per time point).
Group 2 (M19−M36): 18 male C57BL6 mice each received a single
oral administration of test item at a nominal dose level of 3 mg/kg.
Blood, colon, and ileum were collected at 6, 24, and 48 h postdose (n =
six animals per time point).
During preincubation, Trans Epithelial Electrical Resistance
(TEER) measurements were taken from selected wells (at least 20%)
using a Millicell ERS meter (Electrical Resistance System, from
Millipore).
After preincubation, the transport of the test items was measured in
two directions, [A > B] and [B > A], in the absence or presence of
palmitoyl carnitine at 0.3 mM or 10 μM GF120918, where applicable.
At the end of the incubation, aliquots from receiver, donor, and
dosing solutions were transferred into the MS plate. Samples were
extracted by protein precipitation with ACN, centrifuged for 10 min at
3000 rpm, and then injected into an LC−MS/MS system.
The apparent permeability (Papp) was determined in [A > B] and [B
> A] directions in the absence or in the presence of palmitoyl carnitine
or GF120918.
Group 3 (M37−M54): 18 male C57BL6 mice each received a single
oral administration of test item at a nominal dose level of 100 mg/kg.
Blood, colon, and ileum were collected at 6, 24, and 48 h postdose (n =
six animals per time point).
Experimental Procedure. 50 μL blood samples were collected from
tail vein into tubes containing anticoagulant (e.g., K3EDTA, lithium
heparin), stored on ice, and centrifuged to prepare plasma.
Plasma, colon, ileum, and feces samples were stored at approximately
−20 °C, pending analysis.
Plasma and tissue samples were assayed using a method based on
protein precipitation with ACN, followed by LC−MS/MS analysis.
Results for test substances in plasma were subjected to non-
compartmental PK analysis using Phoenix WinNonlin 6.3.
This experiment was carried out in accordance with the PHS Policy,
the National Research Council Guide for the Care and Use of
Laboratory Animals (8th edition), and National Laboratory Animal
Management Regulations (2017), and according to the company
policies, SOPs, and guidelines on the care and use of laboratory animals.
All the studies were approved by the Institutional Animal Care and Use.
Assessment of Compound 64 on the Gastromotility Model. Male
CD-1 mice (6 weeks old; Charles River, Calco, Como, Italy) were
housed in groups on a 12 h light−dark cycle (light on at 0600 h) and
handled 3 days before the test, to minimize the stress due to
manipulation on the day of the experiment; access to food and water
was allowed ad libitum. On the night before the experiment, the mice
were housed in single cages provided with grid and removable tray on
the bottom, for the habituation to the experimental environment. On
the day of the test, mice were treated with test compound (1−10−100
mg/kg) or vehicle by oral administration and returned to their cage and
allowed access to their standard food and top water ad libitum. Twelve
mice for each group of treatment were included, and treatments were
randomly distributed on the rack of cages. The group size of 12 was
selected as the minimal number necessary to measure a statistical
difference (at least p < 0.05) fixing the statistical power of 0.8.
1, 2, 3, and 6 h after treatment, feces for each mouse were collected,
counted, and weighted.
The following parameters were calculated:
1. Papp (apparent permeability) values calculated for directions [A
> B] and [B > A], according to the following equation
i
j
j
y
z
z
idQ y
1
C0
1
A
i
j
y
z
z
z
j
j
j
z
z
z
P
=
× j z × j
app
j
j
z
z
j
dt
k
{
k
{
k
{
where dQ/dt = the permeability rate (dQ/dt is the amount of test/
control items within the incubation period); C0 = the initial
concentration of test/control items in the donor compartment; and
A = the surface area of the filter, corresponding to the surface area of the
cell monolayer. The Papp value (nm/s) reported as average Papp (nm/s)
standard deviation from three monolayers for [A > B] and for [B > A]
directions, where applicable.
2. Monolayer efflux ratios were derived using the mean Papp [A > B]
and [B > A] direction according to the following equation
i
j
y
z
B − AP (nm/s)
app
j
j
z
z
Efflux ratio =
j
z
j
j
z
z
A − BP (nm/s)
app
k
{
PK Studies. Animals. All experiments involving animals were carried
out in accordance with the European Directive 2010/63/UE governing
animal welfare and protection, which is acknowledged by the Italian
Legislative Decree no. 26/2014 and according to the company policy
on the care and use of laboratories animals. All the studies were revised
by the Animal Welfare Body and approved by Italian Ministry of Health
(authorization no. 181/2018-PR, authorization no. 67/2014-PR).
Animals were housed in groups on a 12 h light/dark schedule with
food and water ad libitum and fed with Mouse House and iso-BLOX
until the day of experiment.
The number and weight of feces were expressed in terms of the total
number and dry weight per mouse (drying at 105 °C, 24 h). After
recording the weight, the wet feces for each mouse were dried at 105 °C
for 24 h to calculate the percentage of water of feces as follows
fecal water content (%)
= (feces weight before dried − feces weight after dried)
/feces weight before dried × 100
Compound 61 PK Profile after Intravenous (IV) and Oral (PO)
Administration in Male CD-1 Mouse: Experimental Design. The
study was conducted on two groups of animals:
Group 1 (M1−M3): 3 male CD-1 mice each received a single IV
administration of test item at a nominal dose level of 1 mg/kg. Serial
blood samples were collected from each animal at up to 8 time points of
50 μL each over 24 h postdose. After the completion of the last time
point, mice were killed with CO2.
Statistical analysis was performed by one-way ANOVA followed by
Dunnett’s test.
Mice were also monitored for the general health status at the same
time points of feces collection, in particular coat conditions, posture,
locomotion, and general behavior. After the completion of the
experiment, mice were killed with CO2.
Group 2 (M4−M6): 3 male CD-1 mice each received a single oral
administration of test item at a nominal dose level of 10 mg/kg. Serial
blood samples were collected from each animal at up to 8 time points of
50 μL each over 24 h postdose. After the completion of the last time
point, mice were killed with CO2.
Group 1 (IV): 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose.
Group 2 (PO): 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose.
Compound 64 PK Profile after Oral (PO) Administration in Male
C57BL6 Mouse: Experimental Design. The study was conducted on
three groups of animals:
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00065.
Molecular formula string (CSV)
LC-MS traces and 1H NMR spectra of compounds 25, 61,
and 64; compound 64 safety scan panel; and compound
64 cytotoxicity data (cell viability ATPlite assay (PDF)
Group 1 (M1−M18): 18 male C57BL6 mice each received a single
oral administration of test item at a nominal dose level of 1 mg/kg.
5953
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
electron-withdrawing group; FLIPR, fluorometric imaging
plate reader; FMP, FLIPR membrane potential; LE, ligand
efficiency; LLE, lipophilic ligand efficiency; Met ID, metabolite
identification; TRPA, transient receptor potential cation
channel subfamily A; TRPC, transient receptor potential cation
channel subfamily C; TRPM, transient receptor potential cation
channel subfamily M; TRPP, transient receptor potential cation
channel subfamily P; TRPV, transient receptor potential cation
channel subfamily V
AUTHOR INFORMATION
Corresponding Author
Alessio Barilli − Aptuit, an Evotec Company, Verona 37135,
Italy; orcid.org/0000-0001-5923-2269;
Email: Alessio.barilli@evotec.com
■
Authors
Laura Aldegheri − Aptuit, an Evotec Company, Verona 37135,
Italy
Federica Bianchi − Aptuit, an Evotec Company, Verona 37135,
Italy
REFERENCES
■
Laurent Brault − Aptuit, an Evotec Company, Verona 37135,
Italy
Daniela Brodbeck − Aptuit, an Evotec Company, Verona
37135, Italy
Laura Castelletti − Aptuit, an Evotec Company, Verona 37135,
Italy
Aldo Feriani − Aptuit, an Evotec Company, Verona 37135,
Italy
Iain Lingard − Aptuit, an Evotec Company, Verona 37135, Italy
Richard Myers − Takeda Pharmaceuticals, San Diego,
California 92121, United States
Selena Nola − Aptuit, an Evotec Company, Verona 37135, Italy
Laura Piccoli − Aptuit, an Evotec Company, Verona 37135,
Italy
Daniela Pompilio − Aptuit, an Evotec Company, Verona
37135, Italy
Luca F. Raveglia − Aptuit, an Evotec Company, Verona 37135,
Italy
Cristian Salvagno − Aptuit, an Evotec Company, Verona
37135, Italy
Sabrina Tassini − Aptuit, an Evotec Company, Verona 37135,
Italy
Caterina Virginio − Aptuit, an Evotec Company, Verona
37135, Italy
Mark Sabat − Takeda Pharmaceuticals, San Diego, California
92121, United States
(1) Gees, M.; Colsoul, B.; Nilius, B. The role of transient receptor
potential cation channels in Ca2+ signaling. Cold Spring Harbor Perspect.
Biol. 2010, 2, a003962.
(2) Nilius, B.; Owsianik, G. The transient receptor potential family of
ion channels. Genome Biol. 2011, 12, 218−229.
(3) Wu, L.-J.; Sweet, T.-B.; Clapham, D. E. International union of
basic and clinical pharmacology. LXXVI. Current progress in the
mammalian TRP ion channel family. Pharmacol. Rev. 2010, 62, 381−
404.
(4) Gees, M.; Owsianik, G.; Nilius, B.; Voets, T. TRP channels.
Compr. Physiol. 2012, 2, 563−608.
(5) Owsianik, G.; D’hoedt, D.; Voets, T.; Nilius, B. Structure-function
relationship of the TRP channel superfamily. Rev. Physiol., Biochem.
Pharmacol. 2006, 156, 61−90.
(6) Hasan, R.; Zhang, X. Ca2+ regulation of TRP ion channels. Int. J.
Mol. Sci. 2018, 19, 1256.
(7) Pérez, C. A.; Huang, L.; Rong, M.; Kozak, J. A.; Preuss, A. K.;
Zhang, H.; Max, M.; Margolskee, R. F. A transient receptor potential
channel expressed in taste receptor cells. Nat. Neurosci. 2002, 5, 1169−
1176.
(8) Zhang, Y.; Hoon, M. A.; Chandrashekar, J.; Mueller, K. L.; Cook,
B.; Wu, D.; Zuker, C. S.; Ryba, N. J. P. Coding of sweet, bitter, and
umami tastes: different receptor cells sharing similar signalling
pathways. Cell 2003, 112, 293−301.
(9) Clapp, T. R.; Medler, K. F.; Damak, S.; Margolskee, R. F.;
Kinnamon, S. C. Mouse taste cells with G protein-coupled taste
receptors lack voltage-gated calcium channels and SNAP-25. BMC Biol.
2006, 4, 7.
(10) Kaske, S.; Krasteva, G.; König, P.; Kummer, W.; Hofmann, T.;
Gudermann, T.; Chubanov, V. TRPM5, a taste-signaling transient
receptor potential ion-channel, is a ubiquitous signalling component in
chemosensory cells. BMC Neurosci. 2007, 8, 49.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00065
Author Contributions
(11) Wyatt, A.; Wartenberg, P.; Candlish, M.; Krasteva-Christ, G.;
Flockerzi, V.; Boehm, U. Genetic strategies to analyze primary TRP
channel-expressing cells in mice. Cell Calcium 2017, 67, 91−104.
(12) Brixel, L. R.; Monteilh-Zoller, M. K.; Ingenbrandt, C. S.; Fleig, A.;
Penner, R.; Enklaar, T.; Zabel, B. U.; Prawitt, D. TRPM5 regulates
This manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
glucose-stimulated insulin secretion. Pflugers Arch. 2010, 460, 69−76.
̈
The authors declare no competing financial interest.
(13) Colsoul, B.; Schraenen, A.; Lemaire, K.; Quintens, R.; Van
Lommel, L.; Segal, A.; Owsianik, G.; Talavera, K.; Voets, T.;
Margolskee, R. F.; Kokrashvili, Z.; Gilon, P.; Nilius, B.; Schuit, F. C.;
Vennekens, R. Loss of high-frequency glucose-induced Ca2+ oscillations
in pancreatic islets correlates with impaired glucose tolerance in
Trpm5-/- mice. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5208−5213.
(14) Philippaert, K.; Pironet, A.; Mesuere, M.; Sones, W.; Vermeiren,
L.; Kerselaers, S.; Pinto, S.; Segal, A.; Antoine, N.; Gysemans, C.;
Laureys, J.; Lemaire, K.; Gilon, P.; Cuypers, E.; Tytgat, J.; Mathieu, C.;
Schuit, F.; Rorsman, P.; Talavera, K.; Voets, T.; Vennekens, R. Steviol
glycosides enhance pancreatic beta-cell function and taste sensation by
potentiation of TRPM5 channel activity. Nat. Commun. 2017, 8, 14733.
(15) Steele, S. P.; Melchor, S. J.; Petri, W. A., Jr. Tuft cells: new players
in colitis. Trends Mol. Med. 2016, 22, 921−924.
⊥Retired.
ACKNOWLEDGMENTS
■
We thank Derek Cole and Jill Wykosky for the helpful
discussions, guidance, and support on the project; Gyorgy
Snell and Hui Wang for their input on target structure; Luana
Griffin, Robyn Fabrey, Brian Russell, and Deepika Balakrishna
for additional rodent studies; Takafumi Takai for safety
evaluation; Sree Gopal, Mark Hixon, Jangir Selimkhanov,
Donavon McConn, Elisa Gironda, and Monica Castagna for in
vitro ADME, PK, and PD discussions; and Chiara De Santi for
NMR spectroscopy support.
(16) Zierler, S.; Hampe, S.; Nadolni, W. TRPM channels as potential
therapeutic targets against pro-inflammatory diseases. Cell Calcium
2017, 67, 105−115.
ABBREVIATIONS
CHO, Chinese hamster ovary cells; CRC, concentration−
response curve; EDG, electron-donating groups; EWG,
■
(17) Prawitt, D.; Monteilh-Zoller, M. K.; Brixel, L.; Spangenberg, C.;
Zabel, B.; Fleig, A.; Penner, R. TRPM5 is a transient Ca2+-activated
5954
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
cation channel responding to rapid changes in [Ca2+]i. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 15166−15171.
(18) Philippaert, K.; Kerselaers, S.; Voets, T.; Vennekens, R. A
thallium-based screening procedure to identify molecules that
modulate the activity of Ca2+-activated monovalent cation-selective
channels. SLAS Discovery 2018, 23, 341−352.
(19) Guinamard, R.; Simard, C.; Del Negro, C. Flufenamic acid as an
ion channel modulator. Pharmacol. Ther. 2013, 138, 272−284.
(20) Ullrich, N. D.; Voets, T.; Prenen, J.; Vennekens, R.; Talavera, K.;
Droogmans, G.; Nilius, B. Comparison of functional properties of the
Ca2+-activated cation channels TRPM4 and TRPM5 from mice. Cell
Calcium 2005, 37, 267−278.
(21) Gees, M.; Alpizar, Y. A.; Luyten, T.; Parys, J. B.; Nilius, B.;
Bultynck, G.; Voets, T.; Talavera, K. Differential effects of bitter
compounds on the taste transduction channels TRPM5 and IP3
receptor type 3. Chem. Senses 2014, 39, 295−311.
(22) Sung, D.-J.; Kim, J.-G.; Won, K. J.; Kim, B.; Shin, H. C.; Park, J.-
Y.; Bae, Y. M. Blockade of K+ and Ca2+ channels by azole antifungal
agents in neonatal rat ventricular myocytes. Biol. Pharm. Bull. 2012, 35,
1469−1475.
(23) Luo, J.; Zhu, Y.; Zhu, M. X.; Hu, H. Cell-based calcium assay for
medium to high throughput screening of TRP channel functions using
FlexStation 3. J. Visualized Exp. 2011, 54, No. e3149.
(24) Miller, M.; Wu, M.; Xu, J.; Weaver, D.; Li, M.; Zhu, M. X. High-
throughput screening of TRPC channel ligands using cell-based assays.
TRP Channels; CRC Press/Taylor & Francis: Boca Raton, FL, 2011;
Chapter 1.
(25) Zicha, S.; Radresa, O.; Laplante, P.; Morton, M.; Jones, K.; Main,
M.; Trivedi, S.; Julien, R. P.; Griffin, A.; Labrecque, J.; Ahmad, S.;
Brown, W. Novel methodology to identify TRPV1 antagonists
independent of Capsaicin activation. J. Biomol. Screening 2012, 18,
544−555.
(38) Bingham, M. J.; Huggett, M.; Kiyoi, Y.; Napier, S. E.; Nimz, O.
Preparation of 9-azabicyclo[3.3.1]nonane derivatives for therapeutic
use as dopamine and serotonin reuptake inhibitors. WO 2007039563
A1, 2007.
(39) Jin, B.; Dong, Q.; Hung, G. Preparation of substituted pyrazoles
and imidazoles as hemoglobin modifier compounds and uses thereof.
WO 2017218960 A1, 2017.
(40) Fink, D. M.; Strupczewski, J. T. Preparation of 6-fluorobenzi-
sothazoles via a regioselective nucleophilic aromatic substitution
reaction. Tetrahedron Lett. 1993, 34, 6525−6528.
(41) Bollinger, S. R.; Engers, D. W.; Panarese, J. D.; West, M.; Engers,
J. L.; Loch, M. T.; Rodriguez, A. L.; Blobaum, A. L.; Jones, C. K.; Gray,
A. T.; Conn, P. J.; Lindsley, C. W.; Niswender, C. M.; Hopkins, C. R.
Discovery, structure−activity relationship, and biological character-
ization of a novel series of 6-((1H-Pyrazolo[4,3-b]pyridin3-yl)amino)-
benzo[d]isothiazole-3-carboxamides as positive allosteric Modulators
of the metabotropic glutamate receptor 4 (mGlu4). J. Med. Chem. 2019,
62, 342−358.
(42) Messaoudi, S.; Brion, J.-D.; Alami, M. Copper-catalyzed direct
amination of halo(hetero)arenes with sodium azide as the amino
source. Mini-Rev. Org. Chem. 2011, 8, 448−454.
(43) Markiewicz, J. T.; Wiest, O.; Helquist, P. Synthesis of primary
aryl amines through a copper-assisted aromatic substitution reaction
with sodium azide. J. Org. Chem. 2010, 75, 4887−4890.
(44) Holzer, P.; Masuya, K.; Furet, P.; Kallen, J.; Valat-Stachyra, T.;
Ferretti, S.; Berghausen, J.; Bouisset-Leonard, M.; Buschmann, N.;
̀
Pissot-Soldermann, C.; Rynn, C.; Ruetz, S.; Stutz, S.; Chene, P.; Jeay, S.;
Gessier, F. Discovery of a dihydroisoquinolinone derivative (NVP-
CGM097): a highly potent and selective MDM2 inhibitor undergoing
phase 1 clinical trials in p53wt tumors. J. Med. Chem. 2015, 58, 6348−
6358.
(45) Mathes, C.; Friis, S.; Finley, M.; Liu, Y. QPatch: the missing link
between HTS and ion channel drug discovery. Comb. Chem. High
Throughput Screening 2009, 12, 78−95.
(26) Reubish, D. S.; Emerling, D. E.; Defalco, J.; Steiger, D.; Victoria,
C. L.; Vincent, F. Functional assessment of temperature-gated ion-
channel activity using a real-time PCR machine. Biotechniques 2009, 47,
iii−ix.
(27) Castillo, B.; Pörzgen, P.; Penner, R.; Horgen, F. D.; Fleig, A.
Development and optimization of a high-throughput bioassay for
TRPM7 ion channel inhibitors. J. Biomol. Screening 2010, 15, 498−507.
(28) Bryant, R. W.; Lee, S. P.; Palmer, R. K.; Yang, Q.; Buber, M. N. T.
High throughput screening assay for the TRPM5 ion channel. WO
2007056160 A2, 2007.
(46) Obergrussberger, A.; Stölzle-Feixet, S.; Becker, N.; Bruggemann,
̈
A.; Möller, C. Novel screening techniques for ion channel targeting
drugs. Channels 2015, 9, 367−375.
(47) Bers, D. M.; Patton, C. W.; Nuccitelli, R. A practical guide to the
preparation of Ca2+ buffers. Methods Cell Biol. 2010, 99, 1−26.
(29) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful
metric for lead selection. Drug Discovery Today 2004, 9, 430−431.
(30) Abad-Zapatero, C. Ligand efficiency indices for effective drug
discovery. Expert Opin. Drug Discovery 2007, 2, 469−488.
́
̋
(31) Tarcsay, A.; Nyíri, K.; Keseru, G. M. Impact of lipophilic
efficiency on compound quality. J. Med. Chem. 2012, 55, 1252−1260.
(32) Zhou, M.; Ping, J.; Chen, J.; Xiu, A.; Zhao, Y.; Zhan, Y.; Chen, P.;
Zhang, J. Laxative effects of Salecan on normal and two models of
experimental constipated mice. BMC Gastroenterol. 2013, 13, 52.
(33) Kirk, K. L. Facile synthesis of 2-substituted imidazoles. J. Org.
Chem. 1978, 43, 4381−4383.
(34) Ramirez, S. M.; Layman, J. M.; Bissel, P.; Long, T. E. Ring-
opening polymerization of imidazole epoxides for the synthesis of
imidazole-substituted poly(ethylene oxides). Macromolecules 2009, 42,
8010−8012.
(35) Kwon, S.; Han, Y. T.; Jung, J.-W. Studies on the synthesis of
indothiazinone and its derivatives via direct 3-acylation of indole. Synth.
Commun. 2015, 45, 1662−1668.
(36) Kouznetsov, V. V.; Vargas Méndez, L. Y.; Sortino, M.; Vásquez,
Y.; Gupta, M. P.; Freile, M.; Enriz, R. D.; Zacchino, S. A. Antifungal and
cytotoxic activities of some N-substituted aniline derivatives bearing a
hetaryl fragment. Bioorg. Med. Chem. 2008, 16, 794−809.
(37) Fache, F.; Cros, F.; Piva, O.; Lefebvre, F. Easy access to 2-alkyl
and 2-arylthiazolines using a modified heteropolyacid catalysis:
application to the synthesis of bacillamide A. Synth. Commun. 2012,
42, 2098−2109.
5955
https://doi.org/10.1021/acs.jmedchem.1c00065
J. Med. Chem. 2021, 64, 5931−5955