Structure-activity relationship study of the pyridine moiety of isothiazolo[4,3-b]pyridines as antiviral agents targeting cyclin G-associated kinase

Article abstract of DOI:10.1016/j.bmc.2019.115188

Previously, we reported the discovery of 3,6-disubstituted isothiazolo[4,3-b]pyridines as potent and selective cyclin G-associated kinase (GAK) inhibitors with promising antiviral activity. In this manuscript, the structure-activity relationship study was expanded to synthesis of isothiazolo[4,3-b]pyridines with modifications of the pyridine moiety. This effort led to the discovery of an isothiazolo[4,3-b]pyridine derivative with a 3,4-dimethoxyphenyl residue at position 5 that displayed low nanomolar GAK binding affinity and antiviral activity against dengue virus.

Full text of DOI:10.1016/j.bmc.2019.115188

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-activity relationship study of the pyridine moiety of isothiazolo[4,
3-b]pyridines as antiviral agents targeting cyclin G-associated kinase
Belén Martinez-Gualdaa, Szu-Yuan Pub, Mathy Froeyena, Piet Herdewijna, Shirit Einavb,
⁎
Steven De Jonghec,
a KU Leuven, Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Herestraat 49, 3000 Leuven, Belgium
b Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of
Medicine, Stanford, CA 94305, USA
c KU Leuven, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Herestraat
49, 3000 Leuven, Belgium
A R T I C L E I N F O
A B S T R A C T
Keywords:
Previously, we reported the discovery of 3,6-disubstituted isothiazolo[4,3-b]pyridines as potent and selective
cyclin G-associated kinase (GAK) inhibitors with promising antiviral activity. In this manuscript, the structure-
activity relationship study was expanded to synthesis of isothiazolo[4,3-b]pyridines with modifications of the
pyridine moiety. This effort led to the discovery of an isothiazolo[4,3-b]pyridine derivative with a 3,4-di-
methoxyphenyl residue at position 5 that displayed low nanomolar GAK binding affinity and antiviral activity
against dengue virus.
Cyclin G-associated kinase
Isothiazolo[4,3-b]pyridine
Dengue virus
Kinase inhibitor
Antiviral drugs
1. Introduction
hence, functions as a master regulator of viral infection. GAK is there-
fore a promising target for the discovery of novel antiviral agents. Such
Cyclin G-associated kinase (GAK), also known as auxilin 2, is a
cellular serine/threonine kinase that belongs to the numb-associated
kinases (NAK). This family also includes adaptor-associated kinase 1
(AAK1), BMP-2 inducible kinase (BIKE/BMP2K) and myristoylated and
palmitoylated serine/threonine kinase 1 (MPSK1, also known as serine/
threonine kinase 16 or STK16).1 GAK, ubiquitously expressed within
the cell in the Golgi apparatus, cytoplasm and nucleus, regulates the
clathrin-mediated intracellular trafficking of cellular cargo proteins.2
Interactions between clathrin- associated adaptor protein (AP) com-
plexes and transmembrane cargo play an important role in membrane
trafficking, as these AP complexes orchestrate the formation of vesicles
destined for transport in distinct intracellular pathways.3,4 Phosphor-
ylation of the µ subunits of AP1 and AP2 by GAK leads to a con-
formational change that enhances their binding to sorting motifs within
cargo proteins.5,6 GAK regulates the recruitment of clathrin and AP-2 to
the plasma membrane and of AP-1 to the trans-Golgi network
(TGN).3,7,8 GAK also controls the uncoating of clathrin coated vesicles
(CCVs) for recycling clathrin back to the cell surface.3,5
a host-targeted approach offers advantages, including a high barrier to
resistance and an opportunity to develop broad-spectrum antiviral
agents when focusing on host factors, such as GAK, that are essential for
the life cycle of multiple viruses11
.
Erlotinib (Fig. 1), an approved anticancer drug with potent GAK
affinity (dissociation constant value or KD = 3.1 nM), has shown anti-
viral activity against an array of unrelated viruses including hepatitis C
virus (HCV), dengue virus (DENV), and Zika virus (ZIKV) from the
Flaviviridae family, the filovirus Ebola (EBOV) and the alpha virus
Chikungunya (CHIKV).9 However, since erlotinib targets EGFR with a
comparable affinity to GAK and several other kinases at a lower affinity,
it is not an ideal chemical tool to study the role of GAK in viral infec-
tion. Several medicinal chemists were thus inspired to develop potent,
drug-like and selective GAK inhibitors. Exploring the chemistry of 4-
anilino-quinazolines and –quinolines12 led to the discovery of SGC-
GAK-1 (Fig. 1) that was endowed with very strong GAK affinity
(KD = 1.9 nM) and displayed an excellent kinase selectivity profile. This
compound was not investigated for its antiviral activity, yet it showed
cytotoxic activity against selected prostate cancer cell lines.13
Viruses hijack these intracellular membrane trafficking machineries,
as indicated by the important roles played by various AP complexes in
the life cycle of multiple unrelated viruses.7,9,10 It has been demon-
strated that GAK regulates early and late stages of the viral lifecycle and
Our group reported the discovery of 3,6-disubstituted isothiazolo
[4,3-b]pyridines as potent and selective GAK inhibitors. The original
series of compounds, as represented by compound 2 (Fig. 1), carried a
⁎ Corresponding author.
E-mail address: steven.dejonghe@kuleuven.be (S. De Jonghe).
https://doi.org/10.1016/j.bmc.2019.115188
Received 28 August 2019; Received in revised form 21 October 2019; Accepted 25 October 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:BelénMartinez-Gualda,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.115188

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Fig. 1. Previously studied GAK inhibitors.
morpholino residue at position 3 and was endowed with high affinity
for GAK (KD = 9 nM), but displayed only low µM antiviral activity
against HCV and DENV.14 Subsequent optimization revealed that a cis-
2,6-dimethylmorpholine residue at this position (compound 2, Fig. 1)
had a detrimental effect on the GAK affinity (KD = 89 nM), but a fa-
vourable effect on the activity against DENV, EBOV and CHIKV.15 The
incorporation of other amines, alkoxides, and carboxamides at position
3 yielded compounds that were much less active as GAK ligands and/or
showed weaker antiviral activity.16 To increase the structural variety at
this position, palladium-catalyzed cross couplings (such as Suzuki and
Sonogashira reactions) have been performed, leading to the discovery
pyridine ring of the isothiazolo[4,3-b]pyridine core, keeping the cis-2,6-
dimethylmorpholine substituent fixed. Several variations were in-
troduced. Besides additional Suzuki couplings, several linkers (amino,
alkenyl and alkynyl) were inserted between the phenyl ring at position
6 and the isothiazolo[4,3-b]pyridine scaffold. In addition, a number of
previously unknown 5-aryl-isothiazolo[4,3-b]pyridines were prepared.
All compounds were evaluated for their GAK affinity and the most
potent GAK ligands were also assessed for anti-DENV activity.
2. Chemistry
of
a congener (compound 3, Fig. 1) with high GAK affinity
For the synthesis of the 6-substituted isothiazolo[4,3-b]pyridines,
the known 6-bromo-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]
pyridine 4 was selected as the key intermediate (Scheme 1).15 Aryl
groups were conveniently introduced by Suzuki cross-couplings using
classical reaction circumstances: an appropriate arylboronic acid, Pd
(PPh3)4 as catalyst and potassium carbonate as base in a mixture of
dioxane/water. This procedure allowed to assemble a focused library of
6-aryl-isothiazolo[4,3-b]pyridines 5a-f in yields ranging from 78% to
91%. The insertion of an ethenyl linker between the central core
structure and the phenyl ring was performed by a palladium-catalyzed
cross-coupling between styrene and isothiazolo[4,3-b]pyridine 4 ap-
plying Heck reaction conditions, using Pd(OAc)2 as catalyst, N-pheny-
lurea as ligand and potassium carbonate as base (step b, Scheme 1).19
This allowed the isolation of the desired compound 5 g in high yield
(88%). Coupling of different anilines at position 6 of the isothiazolo
[4,3-b]pyridine scaffold was achieved via a Buchwald reaction using Pd
(KD = 41 nM) and a moderate antiviral activity against DENV.17 More
recently, the importance of the isothiazolo[4,3-b]pyridine core has been
evaluated by a scaffold hopping approach, yielding a series of novel
scaffolds that displayed a reduced GAK affinity relative to the original
scaffold.18
Although a broad structural variety has been introduced at position
3 of the isothiazolo[4,3-b]pyridine scaffold, and a wide range of het-
erocyclic core structures have been prepared, the substitution pattern
on the pyridine moiety of the isothiazolo[4,3-b]pyridine scaffold has
not been previously studied in detail. In our prior medicinal chemistry
optimization campaigns,14–18 an aryl group was always present at po-
sition 6 of the isothiazolo[4,3-b]pyridine scaffold, with a 3,4-di-
methoxyphenyl, 3,4,6-trimethoxyphenyl and 3-methoxy-4-amino-
phenyl being optimal for GAK affinity (Fig. 2). In the present work, we
describe our efforts to introduce structural modifications on the
Fig. 2. Medicinal chemistry strategy.
2

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Scheme 1. Reagents and conditions. (a) R6B(OH)2, Pd(PPh3)4, K2CO3, dioxane/water, 90 °C; (b) styrene, N-phenylurea, Pd(OAc)2, K2CO3, DMF, 120 °C, overnight; (c)
aniline or phenylacetylene, Pd(dba)2, SPhos, tBuOK, toluene, 90 °C; (d) (i) SOCl2, DCM, 40 °C, 3 h; (ii) amine, Et3N, rt, 3 h.
(dba)2 as catalyst, SPhos as ligand, potassium tert-butoxide as base and
toluene as solvent, yielding compounds 5 h-j in good yields ranging
from 73% to 85%.20 For the insertion of a phenylethynyl residue at
position 6 of isothiazolo[4,3-b]pyridine scaffold, classical Sonogashira
reaction conditions (using ethynylbenzene, Pd(PPh3)2Cl2 and copper(I)
iodide) were initially applied, but failed. However, when the same
Buchwald conditions, previously employed for the coupling of anilines,
were used for the coupling of phenylacetylene (step c, Scheme 1),
compound 5k was obtained in good yield (87%). Prior to the addition
of the catalyst, it was necessary to pass a continuous flow of argon for
5–10 min through the reaction mixture, as this led to increased reaction
yields in all these palladium-catalyzed cross-couplings.
easily introduced. Commercially available 6-chloro-2-cyano-3-ni-
tropyridine 8 was selected as starting material. Reduction of the nitro
group to the corresponding amino group led to a concomitant complete
hydrolysis of the cyano moiety affording 3-amino-6-chloropicolinic acid
as the sole product. As an alternative, we introduced the corresponding
aryl group via a Suzuki cross-coupling reaction in the first step of the
synthesis (Scheme 3). These reactions were carried out in toluene as
solvent (except for compound 9 h bearing a 3,4,5-trimethoxyphenyl
group) instead of the dioxane/water mixture that was commonly ap-
plied for the preparation of 6-substituted derivatives. Thus, several aryl
groups were introduced at position 6 of compound 8 generating deri-
vatives 9a-i in yields ranging from 76% to 96%. The nitro group of
compounds 9a-i was reduced by treatment with iron under acidic
conditions (acetic acid at 60 °C) affording 3-amino-6-aryl-picolinoni-
triles 10a-i. Inevitably, small amounts of the corresponding 3-amino-6-
phenylpicolinamides were formed, resulting from hydrolysis of the
cyano group. These mixtures were used as such for the next step.
Thionation of the nitrile functionality using phosphorus pentasulfide
yielded thioamides 11a-i. The isothiazole moiety was then constructed
by an oxidative ring closure using hydrogen peroxide in methanol. As
this reaction proceeded smoothly and cleanly, purification of the crude
residue was not necessary, and the corresponding 5-aryl-3-amino-iso-
thiazolo[4,3-b]pyridines 12a-i were isolated in excellent yields. The
conversion of the amino group to a bromine was achieved via a Sand-
meyer reaction. Applying the classical reaction conditions (NaNO2,
HBr, CuBr, water) was successful for compounds 13a-d and 13 h.
However, for the analogues (compounds 12e-g), in which the 5-phenyl
moiety carried one or two electron-donating substituents (methyl or
methoxy groups), none to very low yields the most were obtained. This
result was attributed to multiple brominations at different positions of
the 5-phenyl ring. To minimise these aromatic bromination side reac-
tions, milder conditions (tBuONO, CuBr2 and dry acetonitrile) were
used.23 This slightly improved the yields of compounds 13e-g
(34–51%), but not of compound 13i. Finally, cis-2,6-dimethylmorpho-
line was introduced at position 3 of the isothiazolo[4,3-b]pyridine
scaffold, affording the desired compounds 14a-h in yields ranging from
55% to 91%.
The carboxylic acid moiety of compound 5f was converted to sev-
eral amides.21 The acid chloride was generated in situ by reaction with
thionyl chloride, followed by reaction with a number of amines gen-
erating target compounds 6a-f in yields varying from 68% to 85%. Si-
milarly, the amino group of compound 2 was also used to prepare a
number of amides (Scheme 2). The formylamino congener 7a was
prepared using MnO2 as catalyst and formamide as solvent.22 The re-
action of compound 2 with acetic anhydride, cyclopropanecarbonyl
chloride and benzoyl chloride provided compounds 7b, 7c and 7d,
respectively.
For the synthesis of 5-substituted isothiazolo[4,3-b]pyridines, an
alternative synthetic pathway was followed. Initial attempts were fo-
cused on the synthesis of an intermediate (i.e. 3-bromo-5-chloro-iso-
thiazolo[4,3-b]pyridine) that could serve as key synthon from which
structural variety at two different positions of the scaffold could be
To circumvent the Sandmeyer reaction, a 3-N-morpholinyl moiety
was directly synthesized from the corresponding 3-amino congeners.
Reaction of compounds 12f, 12 g and 12i with 2-bromoethyl ether and
potassium carbonate as a base generated the desired compounds 15f,
15 g and 15i in decent yields (71–77%).
Scheme 2. Reagents and conditions. (a) formamide, MnO2, 150 °C, 5 h; (b) acetic
anhydride or RCOCl, Et3N, DCM, rt, overnight.
3

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Scheme 3. Reagents and conditions. (a) R3B(OH)2, Pd(PPh3)4, K2CO3, toluene or dioxane/H2O, 95 °C; (b) Fe, CH3COOH, 60 °C; (c) P2S5, EtOH, 75 °C; (d) 35% aq·H2O2,
MeOH, rt; (e) NaNO2, HBr, CuBr, H2O, 0 °C to rt; (f) tBuONO, CuBr2, dry CH3CN, −10 °C to rt; (g) 2,6-dimethylmorpholine, EtOH, reflux, overnight; (h) 2-bromoethyl
ether, K2CO3, DMF, 100 °C, 4–5 h.
3. GAK binding affinity
3.33 µM, respectively. This indicated that the methoxy group at posi-
tion 3 is more important for GAK binding than the one at position 4.
Therefore, keeping the 3-methoxy group fixed, a number of derivatives
were prepared with alternative functional groups at position 4 of the
aromatic ring. The presence of a 4-carboxylic acid resulted in com-
pound 5f that showed very potent GAK inhibition (IC50 = 0.042 µM).
The carboxylic acid was used as a chemical handle to make a small set
of amides, including a simple carboxamide analogue (compound 6a),
cycloaliphatic amides (compounds 6b-d) and aromatic amides (6e-f).
Based on the X-ray crystallographic data that we previously reported,14
this region (i.e. the 6-phenyl moiety) is oriented towards the solvent
and therefore tolerates structural variety. This is in agreement with the
experimentally determined IC50 values, as several of these compounds,
and particularly the carboxamide congener 6a (IC50 = 0.022 µM), show
good GAK affinity. Because of the good affinity of these amides, a
number of reverse amides (compounds 7a-d) with aliphatic and aro-
matic side chains were prepared, displaying GAK IC50 values in the
range of 0.1 to 0.4 µM. To further explore the SAR, the effect of various
linkers between the isothiazolo[4,3-b]pyridine scaffold and the 6-
phenyl moiety on GAK affinity were investigated. An alkenyl (com-
pound 5 g) or alkynyl (compound 5 k) spacer gave compounds that
were completely devoid of GAK affinity. Albeit weak, an anilino sub-
stituent at position 6 (compound 5 h) gave rise to GAK affinity with an
IC50 of 1.63 µM. In an effort to restore GAK affinity of the 6-anilino
congener, the 3,4-dimethoxy- (compound 5i) and 3,4,5-trimethoxy-
anilino (compound 5j) congeners were prepared. Consistent with the 6-
phenyl series, the 3,4,5-trimethoxyanilino derivative 5j showed the
most potent GAK affinity, with an IC50 of 0.458 µM.
The binding affinity of compounds 5a-k, 6a-f, 7a-d, 14a-h, 15f,
15 g and 15i for GAK was evaluated via a commercially available
LanthaScreen® Europium kinase binding assay.24 In this assay format,
an Alexa Fluor 647-labeled conjugate or tracer competes with the GAK
ligand for the ATP binding site. Signal detection is dependent on the
time-resolved fluorescence resonance energy transfer (TR-FRET) signal
resulting from the antibody and tracer binding. When the ATP site is
occupied by the tracer, there is a high TR-FRET signal. When the tracer
is displaced by the GAK inhibitor, a reduction in TR-FRET signal is
observed. The GAK binding affinity of reference compound 2 was
measured via a LanthaScreen assay with an IC50 value of 0.0394 µM,
which is in agreement with the KD of 0.089 µM previously measured via
the DiscoverX screening assay.15 To drive the subsequent structure-
activity relationship (SAR) study, the GAK binding affinity of all com-
pounds was evaluated in the Lanthascreen assay (Tables 1 and 2).
Prior SAR studies of 3-N-morpholino-isothiazolo[4,3-b]pyridines
demonstrated that electron-donating substituents (e.g. methoxy,
amino) at the 6-phenyl moiety are optimal for GAK binding. Yet,
combination of a cis-2,6-dimethylmorpholinyl substituent at position 3,
which is known to confer potent antiviral activity,15 with structural
variation of the 6-phenyl moiety has not been previously explored.
Based on the beneficial effect of methoxy groups on GAK binding,14 the
6-(3,4-dimethoxy)- and 6-(3,4,5-trimethoxyphenyl) derivatives were
synthesized (Table 1). The presence of a 3,4-dimethoxyphenyl residue
(compound 5b) caused a 3-fold drop in GAK affinity (IC50 = 0.136 µM)
relative to compound 2. Conversely, the 3,4,5-trimethoxyphenyl con-
gener (compound 5c) showed
a
slightly improved GAK affinity
To expand the SAR, a series of regioisomeric 5-aryl-isothiazolo[4,3-
b]pyridines (compounds 14a-i) was synthesized (Table 2). Because of
synthetic difficulties (vide supra), a number of derivatives (compounds
15f, 15 g and 15i) carrying a morpholine instead of a cis-2,6-di-
methylmorpholino residue were synthesized. An unsubstituted phenyl
(compound 14a), halogen substituted phenyls (compounds 14c-d) and
(IC50 = 0.071 µM). Removal of the methoxy groups furnished com-
pound 5a which shows an IC50 of 0.661 µM, which is about 10-fold less
potent than the trimethoxyphenyl congener 5c. Replacement of one of
the methoxy groups by an endocyclic pyridine nitrogen gave rise to
compounds 5d and 5e that display IC50 values of 0.545 µM and >
4

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Table 1
Table 2
GAK binding affinity of 6-substituted isothiazolo[4,3-b]pyridines.
GAK binding affinity of 5-aryl-isothiazolo[4,3-b]pyridines.
R6
N
S
N
N
O
Cmpd
R
R5
GAK IC50 (µM)
Cmpd
R6
GAK IC50 (µM)
0.0394
14a
14b
CH3
CH3
0.364
O
2
H2
N
> 0.37
5a
5b
0.661
0.136
14c
14d
CH3
CH3
0.11
O
0.262
O
5c
0.071
14e
14f
CH3
CH3
0.214
0.099
5d
5e
0.545
> 3.33
14g
14h
CH3
CH3
0.024
0.422
5f
0.042
15f
H
H
0.136
0.124
5g
5h
> 3.33
1.63
15g
15i
H
0.126
5i
5j
1.29
0.458
the 6-phenyl ring is known to be advantageous for GAK affinity,14 the
phenyl at position 5 was also substituted with one, two or three
methoxy groups affording compounds 14f-h, 15f-g and 15i. This series
displayed GAK IC50 values in the 0.1–0.5 µM range, with compound
14 g (carrying a 3,4-dimethoxyphenyl at position 5 and a cis-2,6-di-
methylmorpholinyl group at position 3) having the most potent GAK
affinity (IC50 = 0.024 µM). In contrast, the corresponding 3-N-mor-
pholinyl derivative (compound 15 g) is 5-fold less active as a GAK li-
gand (IC50 = 0.124 µM).
5k
6a
> 10
0.022
6b
6c
0.118
0.083
6d
6e
6f
0.101
> 0.37
0.073
4. Antiviral activity against DENV
The compounds with the strongest GAK binding affinity
(IC50 < 0.1 µM) were assessed for antiviral activity in human hepa-
toma (Huh7) cells infected with DENV2 expressing a luciferase reporter
(Table 3). Their effect on DENV2 infection was measured at 48 h post-
infection via luciferase assays, and the half-maximal effective con-
centration (EC50 value) was calculated. In parallel, the cytotoxicity of
the compounds in the infected cells (expressed as the half-maximal
cytotoxic concentration or CC50 value) was measured via an Alamar-
Blue assay. Compound 2 was included as a reference compound, as it
was previously shown to have a promising anti-DENV2 activity, with an
EC50 of 0.82 µM and no apparent cytotoxicity.9 The 6-(3,4,5-tri-
methoxyphenyl) derivative 5c reduces the anti-DENV activity by 3 folds
relative to compound 2. This is in line with its slightly reduced GAK
7a
7b
0.113
0.132
7c
7d
0.173
0.37
affinity. Although compound 5f is
a very potent GAK ligand
(IC50 = 0.042 µM), it lacks antiviral activity. This might be due to im-
paired cellular permeability resulting from the presence of a polar
carboxylic acid group. The corresponding amides (compounds 6a and
6c) and particularly compound 6f (EC50 = 5.83 µM) show an improved
antiviral activity when compared to the carboxylic acid derivative 5f.
These findings suggest that this site of the molecule tolerates some
structural variation with respect to GAK affinity, and that this position
can therefore be used to tune physicochemical properties, such as
p-tolyl (compound 14e) at position 5 of the isothiazolo[4,3-b]pyridine
scaffold yielded compounds that displayed GAK IC50 values in the
0.1–0.3 µM range. Only the 4-trifluoromethylphenyl analogue (com-
pound 14b) was less active as GAK ligand. As methoxy substitution of
5

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Table 3
Antiviral activity against DENV2.
Cmpd
2
R
R5
H
R6
DENV EC50
(µM)a
DENV CC50
(µM)b
CH3
CH3
0.82
> 25
5c
H
2.79
> 10
5f
CH3
CH3
H
H
> 10
10.05
> 10
> 10
Fig. 3. Autodock Vina docking of compound 14 g (cyan carbons) superimposed
on compound 1 (magenta carbons). Amino acids having van der Waals inter-
actions are coloured as green surface.
6a
6c
6f
CH3
CH3
H
H
8.85
5.83
> 10
> 10
During this docking process, a rigid enzyme conformation and a flexible
ligand by variable torsion angles was applied. Taking into account in-
duced fit effects, the observed interactions may in fact be more fa-
vourable. For instance, the Lys69.B side chain is within interaction
distance with the oxygen atom of the morpholino ring, possibly forming
a hydrogen bond, that is currently not detected.
14f
CH3
CH3
H
H
> 10
1.049
> 10
> 10
14g
Erlotinib
–
–
–
1.22
> 10
6. Conclusion
a
EC50 = half-maximal effective concentration.
CC50 = half-maximal cytotoxic concentration.
Isothiazolo[4,3-b]pyridines are well known as potent and selective
GAK inhibitors with antiviral activity. Prior research has focused on
structural modifications of the scaffold and the substituent at position 3.
In this manuscript, the SAR of the pyridine moiety of the isothiazolo
[4,3-b]pyridine core structure was studied in detail. The insertion of
various linkers between the 6-phenyl ring and the central scaffold
generated compounds with decreased GAK affinity. However, position
4 of the 6-phenyl moiety tolerates some structural variety with respect
to GAK affinity, and structural variation at this site can be used to
modulate the antiviral activity. Furthermore, a novel class of 5-sub-
stituted isothiazolo[4,3-b]pyridines was synthesized, yielding selected
congeners with potent GAK affinity and anti-DENV activity.
b
aqueous solubility and cellular permeability. Within the 5-aryl-iso-
thiazolo[4,3-b]pyridines, two compounds were investigated for anti-
viral activity. Whereas compound 14f did not show antiviral activity,
the 5-(3,4-dimethoxyphenyl) derivative 14g was a potent GAK inhibitor
(IC50 = 0.024 µM) demonstrating potent antiviral activity (EC50 and
CC50 values of 1.049 µM and > 10 µM, respectively). Despite the fact
that compound 14g is less active as GAK ligand when compared to
erlotinib (KD = 3.1 nM), both compounds are equally active as anti-
DENV agents. As erlotinib is known to affect multiple other kinases
besides GAK, compound 14g might be a better lead for the discovery of
GAK inhibitors as antiviral drugs.
7. Experimental section
5. Molecular docking of compound 14g into GAK
7.1. Chemistry
To rationalize the potent GAK affinity of compound 14g, molecular
docking was initiated starting from the co-crystal X-ray structure of
GAK and compound 1 (PDB 4Y8D),14 with a docking protocol as de-
scribed before,17 using the software packages Autodocktools and Au-
todock vina.25,26 Compound 14g has a slightly different substituent at
position 3 of the isothiazolo[4,3-b]pyridine scaffold from the original
crystallized GAK inhibitor 1. More specifically, compound 14g bears a
cis-2,6-dimethylmorpholino ring of which the intrinsic torsion angles
are not flexible during docking. However, since the choice of the ring
conformation might be important for the final docking orientation and
since morpholine can exist in different conformations in crystallized
structures,27 we used different conformations of the morpholino ring of
compound 14g for docking. This analysis revealed that a chair con-
formation with both methyl groups in an equatorial orientation was
optimal. The nitrogen has more a sp2 character and lays in the same
plane as the aromatic ring system. Compound 14g docked in a fa-
vourable way in the ATP binding site. Primarily hydrophobic interac-
tions were observed between GAK and compound 14g, similarly to
compound 1 (Fig. 3). One hydrogen bond with Thr123.B was observed.
For all reactions, analytical grade solvents were used. Argon was
used to carry out reactions under an inert atmosphere. Melting points
were recorded with a Stuart SMP20 melting point apparatus. 1H and 13
C
NMR spectra were recorded on a Bruker Avance 300 MHz instrument
(1H NMR, 300 MHz; 13C NMR, 75 MHz), 500 MHz instrument (1H NMR,
500 MHz; 13C NMR, 125 MHz) or a 600 MHz instrument (1H NMR,
600 MHz; 13C NMR, 150 MHz, 19F NMR, 471 MHz), using tetra-
methylsilane as internal standard for 1H NMR spectra and DMSO‑d6
(39.5 ppm) or CDCl3 (77.2 ppm) for 13C NMR spectra. Abbreviations
used are s = singlet, d = doublet, t = triplet, q = quartet, m = multi-
plet, b = broad. Coupling constants are expressed in Hz. High resolu-
tion mass spectra were acquired on a quadrupole orthogonal accelera-
tion time-of-flight mass spectrometer (Synapt G2 HDMS, Waters,
Milford, MA). Samples were infused at 3 mL/min and spectra were
obtained in positive or negative ionization mode with a resolution of
15,000 (FWHM) using leucine enkephalin as lock mass. Precoated
aluminum sheets (Fluka silica gel/TLC-cards, 254 nm) were used for
TLC. Column chromatography was performed on silica gel
0.060–0.200 mm, 60 (Acros Organics). Purity of final compounds was
6

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
verified to be > 95% by HPLC analysis. HPLC conditions to assess
purity were as follows: Shimadzu HPLC equipped with a LC-20AT
pump, DGU-20A5 degasser, and a SPD-20A UV–VIS detector; Symmetry
C18 column (5 µm, 4.6 mm × 150 mm); gradient elution of H2O/
CH3CN from 95/5 or 70/30 to 5/95 over 25 min; flow rate 1 mL/min;
wavelength, UV 254 nm. Preparative HPLC purifications were per-
formed using a Phenomenex Gemini 110A column (C18, 10 µM,
21.2 mm × 250 mm).
2 × CH3), 2.92 (dd, J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.91 (s, 3H,
OCH3), 3.93–3.97 (m, 8H, 2 × OCH, 2 × OCH3), 4.52 (dd, J = 12.7,
1.9 Hz, 2H, 2 × NCH), 6.85 (s, 2H, arom H), 7.87 (d, J = 2.1 Hz, 1H,
arom H), 8.62 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C NMR (126 MHz,
CDCl3) δ: 18.8 (CH3), 55.4 (CH2), 56.2 (OCH3), 61.0 (OCH3), 71.2 (CH),
104.6 (CH), 125.3 (CH), 133.3 (C), 134.1 (C), 135.8 (C), 138.5 (C),
144.1 (CH), 153.7 (C), 156.2 (C), 172.9 (C) ppm. HR-MS m/z [M+H]+
calcd for C21H25N3O4S: 416.1638, found 416.1629.
7.1.1. Synthesis of 6-aryl-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-
b]pyridines
7.1.1.4. 6-(2-Methoxy-4-pyridyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5d). This compound was obtained using 2-
methoxypyridine-4-boronic acid. The crude residue was purified by
silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 7:3) as mobile phase, affording the title compound
as an amorphous yellow solid in 87% yield (47.2 mg, 0.13 mmol). Mp
160.5–162.1 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
2 × CH3), 2.93 (dd, J = 12.6, 10.7 Hz, 2H, 2 × NCH), 3.91–3.97 (m,
2H, 2 × OCH), 4.01 (s, 3H, OCH3), 4.52 (dd, J = 12.7, 1.9 Hz, 2H,
2 × NCH), 7.01–7.02 (m, 1H, arom H), 7.15 (dd, J = 5.3, 1.5 Hz, 1H,
arom H), 7.94 (d, J = 2.1 Hz, 1H, arom H), 8.27–8.29 (m, 1H, arom H),
8.59 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ:
18.8 (CH3), 53.6 (CH), 55.4 (CH2), 71.2 (OCH3), 108.9 (CH), 115.2
(CH), 126.4 (CH), 133.0 (C), 135.0 (C), 142.9 (CH), 147.7 (CH), 147.8
(C), 155.7 (C), 164.9 (C), 173.2 (C) ppm. HR-MS m/z [M+H]+ calcd
for C18H20N4O2S: 357.1380, found 357.1374.
General procedure. A solution of 6-bromo-3-(cis-2,6-dimethylmor-
pholino) isothiazolo[4,3-b]pyridine (1 equiv) in a mixture of dioxane/
water (ratio 4:1) was degassed with argon and subsequently, were
added the corresponding boronic acid (1.2 equiv), Pd(PPh3)4 (0.02
equiv) and K2CO3 (2 equiv). The mixture was degassed a second time,
filled with argon and stirred at 90 °C overnight. After completion of the
reaction as monitored by TLC, the volatiles were evaporated to dryness
and the crude residue was diluted with EtOAc (20 mL) and washed with
water (2 × 20 mL). The organic layer was dried over anhydrous Na2SO4
and concentrated in vacuo. The resulting residue was purified by silica
gel flash chromatography, yielding the corresponding 6-substituted-3-
(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]pyridines.
The
fol-
lowing compounds were made according to this general procedure.
7.1.1.1. 3-(cis-2,6-Dimethylmorpholino)-6-(phenyl)isothiazolo[4,3-b]
pyridine (5a). This compound was obtained using phenylboronic acid.
The crude residue was purified by silica gel flash chromatography using
a mixture of hexane and ethyl acetate (in a ratio of 4:1) as mobile
phase, affording the title compound as an amorphous yellow solid in
89% yield (88.3 mg, 0.27 mmol). Mp 141.5–142.3 °C. 1H NMR
(500 MHz, CDCl3) δ: 1.31 (d, J = 6.2, Hz, 6H, 2 × CH3), 2.91 (dd,
J = 12.6, 10.7 Hz, 2H, 2 × NCH), 3.91–3.99 (m, 2H, 2 × OCH),
4.50–4.54 (m, 2H, 2 × NCH), 7.41–7.45 (m, 1H, arom H), 7.48–7.52
(m, 2H, arom H), 7.62–7.68 (m, 2H, arom H), 7.90 (d, J = 2.1 Hz, 1H,
arom H), 8.63 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C NMR (126 MHz,
CDCl3) δ: 18.8 (CH3), 55.4 (CH2), 71.2 (CH), 125.6 (CH), 127.4 (CH),
128.4 (CH), 129.1 (CH), 134.2 (C), 135.8 (C), 137.6 (C), 144.3 (CH),
156.3 (C), 172.9 (C) ppm. HR-MS m/z [M+H]+ calcd for C18H19N3OS:
326.1322, found 326.1319.
7.1.1.5. 6-(2-Methoxy-5-pyridyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5e). This compound was obtained using 2-
methoxy-5-pyridineboronic acid. The crude residue was purified by
silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 7:3) as mobile phase, affording the title compound
as an amorphous yellow solid in 88% yield (47.7 mg, 0.13 mmol). Mp
160.2–162.1 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
2 × CH3), 2.92 (dd, J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.91–3.97 (m,
2H, 2 × OCH), 4.00 (s, 3H, OCH3), 4.51 (dd, J = 12.7, 1.9 Hz, 2H,
2 × NCH), 6.88 (dd, J = 8.6, 0.6 Hz, 1H, arom H), 7.82–7.87 (m, 2H,
arom H), 8.47 (dd, J = 2.5, 0.6 Hz, 1H, arom H), 8.57 (d, J = 2.1 Hz,
1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 53.7
(OCH3), 55.4 (CH2), 71.2 (CH), 111.4 (CH), 125.0 (CH), 126.5 (C),
132.6 (C), 134.1 (C), 137.4 (CH), 143.4 (CH), 145.4 (CH), 156.0 (C),
164.2 (C), 173.0 (C) ppm. HR-MS m/z [M+H]+ calcd for C18H20N4O2S:
357.1380, found 357.1374.
7.1.1.2. 6-(3,4-Dimethoxyphenyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5b). This compound was obtained using 3,4-
dimethoxyphenylboronic acid. The crude residue was purified by silica
gel flash chromatography using a mixture of hexane and ethyl acetate
(in a ratio of 7:3) as mobile phase, affording the title compound as an
amorphous yellow solid in 90% yield (52.7 mg, 0.13 mmol). Mp
156.2–157.9 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz,
6H, 2 × CH3), 2.91 (dd, J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.92–3.96
(m, 5H, OCH3, 2 × OCH), 3.96 (s, 3H, OCH3), 4.51 (dd, J = 12.7,
1.9 Hz, 2H, 2 × NCH), 7.00 (d, J = 8.3 Hz, 1H, arom H), 7.16 (d,
J = 2.1 Hz, 1H, arom H), 7.24 (dd, J = 8.3, 2.1 Hz, 1H, arom H), 7.85
7.1.1.6. 6-(3-Methoxy-4-carboxyphenyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5f). This compound was obtained using 3-
methoxy-4-carboxyphenylboronic acid. After completion of the
reaction, a solution of HCl (1 M) was added until reach pH acid. The
crude residue was purified by silica gel flash chromatography using a
mixture of dichloromethane and methanol (in a ratio of 20:1) as mobile
phase, affording the title compound as an amorphous yellow solid in
78% yield (47.4 mg, 0.12 mmol). Mp 251.3–253.1 °C. 1H NMR
(600 MHz, DMSO) δ: 1.20 (d, J = 6.2 Hz, 6H, 2 × CH3), 2.91 (dd,
J = 12.1, 11.0 Hz, 2H, 2 × NCH), 3.83–3.89 (m, 2H, 2 × OCH), 3.94 (s,
3H, OCH3), 4.52 (d, J = 12.0 Hz, 2H, 2 × NCH), 7.45 (dd, J = 8.0,
1.4 Hz, 1H, arom H), 7.52 (d, J = 1.2 Hz, 1H, arom H), 7.75 (d,
J = 7.9 Hz, 1H, arom H), 8.15 (d, J = 2.0 Hz, 1H, arom H), 8.81 (d,
J = 2.0 Hz, 1H, arom H) ppm. 13C NMR (151 MHz, DMSO) δ: 18.6
(CH3), 54.8 (CH2), 56.1 (OCH3), 70.6 (CH), 111.5 (CH), 118.9 (CH),
121.1 (C), 125.7 (CH), 131.5 (CH), 133.8 (C), 134.0 (C), 141.4 (C),
143.8 (CH), 155.4 (C), 158.7 (C), 167.1 (C), 172.5 (C) ppm. HR-MS m/z
[M + H]+ calcd for C20H21N3O4S: 400.1325, found 400.1324.
(d, J = 2.1 Hz, 1H, arom H), 8.63 (d, J = 2.1 Hz, 1H, arom H) ppm. 13
C
NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 55.4 (CH2), 56.0 (OCH3), 56.0
(OCH3), 71.2 (CH), 110.3 (CH), 111.6 (CH), 119.8 (CH), 124.8 (CH),
130.3 (C), 133.9 (C), 135.5 (C), 144.3 (CH), 149.5 (C), 149.5 (C), 156.4
(C), 172.9 (C) ppm. HR-MS m/z [M + H]+ calcd for C20H23N3O3S:
386.1532, found 386.1527.
7.1.1.3. 6-(3,4,5-Trimethoxyphenyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5c). This compound was obtained using
3,4,5-trimethoxyphenylboronic acid. The crude residue was purified
by silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 7:3) as mobile phase, affording the title compound
as an amorphous yellow solid in 91% yield (57.7 mg, 0.14 mmol). Mp
154.1–155.5 °C. 1H NMR (500 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
7.1.1.7. 6-(trans-Styryl)-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]
pyridine (5g). A solution of 6-bromo-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (1 equiv) in DMF was degassed with argon.
Styrene (1.5 equiv), N-phenylurea (0.02 equiv), Pd(OAc)2 (0.01 equiv)
7

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
and K2CO3 (2 equiv) were added. The resulting mixture was degassed a
second time, filled with argon and stirred at 120 °C overnight. After
completion of the reaction as monitored by TLC, the volatiles were
evaporated to dryness and the crude residue was diluted with EtOAc
(20 mL) and washed with a 0.5 M HCl solution (2 × 10 mL) and water
(20 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The resulting residue was purified by silica gel
flash chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 4:1) as mobile phase, affording the title compound as an
amorphous yellow solid in 88% yield (47 mg, 0.13 mmol). Mp
177.4–178.5 °C. 1H NMR (600 MHz, CDCl3) δ: 1.31 (d, J = 6.3 Hz,
6H, 2 × CH3), 2.89 (dd, J = 12.2, 10.9 Hz, 2H, 2 × NCH), 3.90–3.97
(m, 2H, 2 × OCH), 4.48 (dd, J = 12.7, 1.6 Hz, 2H, NCH), 7.12 (d,
J = 16.4 Hz, 1H, double bond CH), 7.28 (d, J = 16.4 Hz, 1H, double
bond CH), 7.32 (t, J = 7.3 Hz, 1H, arom H), 7.40 (t, J = 7.7 Hz, 2H,
arom H), 7.56 (d, J = 7.5 Hz, 2H, arom H), 7.74 (d, J = 1.8 Hz, 1H,
arom H), 8.61 (d, J = 1.9 Hz, 1H, arom H) ppm. 13C NMR (151 MHz,
CDCl3) δ: 18.8 (CH3), 55.4 (CH2), 71.2 (CH), 124.9 (CH), 125.3 (CH),
126.8 (CH), 128.4 (CH), 128.8 (CH), 131.7 (CH), 132.2 (C), 134.0 (C),
136.5 (C), 143.4 (CH), 156.4 (C), 172.7 (C) ppm. HR-MS m/z [M+H]+
calcd for C20H21N3OS: 352.1478, found 352.1473.
J = 8.2 Hz, 1H, arom H), 7.06 (d, J = 2.4 Hz, 1H, arom H), 8.05 (d,
J = 2.5 Hz, 1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.9
(CH3), 55.5 (CH2), 56.1 (OCH3), 56.4 (OCH3), 71.4 (CH), 105.1, (CH),
106.5 (CH), 112.2 (CH), 113.9 (CH), 129.7 (C), 133.9 (C), 139.8 (CH),
140.9 (C), 146.1 (C), 149.9 (C), 157.5 (C), 172.1 (C) ppm. HR-MS m/z
[M+H]+ calcd for C20H24N4O3S: 401,1642, found 401,1638.
7.1.2.3. 6-(3,4,5-Trimethoxyanilino)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5j). This compound was obtained using
3,4,5-trimethoxyaniline. The crude residue was purified by silica gel
flash chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 3:2) as mobile phase, affording the title compound as an
amorphous yellow solid in 73% yield (47.8 mg, 0.11 mmol). Mp
252.2–253.5 °C. 1H NMR (300 MHz, CDCl3) δ: 1.29 (d, J = 6.3 Hz,
6H, 2 × CH3), 2.86 (dd, J = 12.5, 10.7 Hz, 2H, 2x NCH), 3.83 (s, 6H,
2 × OCH3), 3.83 (s, 3H, OCH3), 3.86–3.98 (m, 2H, 2 × OCH), 4.41 (dd,
J = 12.8, 2.0 Hz, 2H, 2 × NCH), 5.88 (bs, 1H, NH), 6.42 (s, 2H, arom
H), 7.18 (d, J = 2.5 Hz, 1H, arom H), 8.08 (d, J = 2.5 Hz, 1H, arom H)
ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 55.3 (CH2), 56.2
(OCH3), 61.0 (OCH3), 71.2 (CH), 98.2 (CH), 106.4 (CH), 129.8 (C),
134.3 (C), 136.8 (C), 139.7 (C), 139.8 (CH), 154.0 (C), 157.2 (C), 172.1
(C) ppm. HR-MS m/z [M+H]+ calcd for C21H26N4O4S: 431.1747,
found 431.1745.
7.1.2. Synthesis of 6-anilino-3-(cis-2,6-dimethylmorpholino)isothiazolo
[4,3-b]pyridines
General procedure. Pd(dba)2 (0.03 equiv) and SPhos (0.06 equiv)
were added to a degassed solution of toluene (15 mL). The resulting
mixture was stirred under argon for 5–10 min. Subsequently, 6-bromo-
3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]pyridine (1 equiv),
the appropriate aniline or ethynylbenzene (1.5 equiv) and t-BuOK (1.5
equiv) were added and the mixture was degassed a second time. The
reaction was filled with argon and stirred at 90 °C for 4–5 h. After
completion of the reaction as monitored by TLC, the volatiles were
evaporated to dryness. The crude residue was diluted with EtOAc
(20 mL) and washed twice with a 0.5 M HCl solution (2 × 10 mL) and
brine (20 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The resulting residue was purified by silica gel
flash chromatography, yielding the corresponding 6-substituted-3-(cis-
2,6-dimethylmorpholino)isothiazolo[4,3-b]pyridines. The following
compounds were made according to this procedure.
7.1.2.4. 3-(cis-2,6-Dimethylmorpholino)-6-(phenylethynyl)isothiazolo
[4,3-b]pyridine (5k). This compound was obtained using ethynylbenzene.
The crude residue was purified by silica gel flash chromatography using a
mixture of hexane and ethyl acetate (in a ratio of 4:1) as mobile phase,
affording the title compound as an amorphous yellow solid in 87% yield
(46.3 mg, 0.13 mmol). Mp 162.4–163.8 °C. 1H NMR (500 MHz, CDCl3) δ:
1.31 (d, J = 6.1 Hz, 6H, 2 × CH3), 2.90 (t, J = 11.5 Hz, 2H, 2 × NCH),
3.90–3.98 (m, 2H, 2 × OCH), 4.47 (d, J = 12.5 Hz, 2H, 2 × NCH),
7.35–7.41 (m, 3H, arom H), 7.56–7.61 (m, 2H, arom H), 7.89 (s, 1H,
arom H), 8.39 (s, 1H, arom H) ppm. 13C NMR (126 MHz, CDCl3) δ: 18.8
(CH3), 55.4 (CH2), 71.2 (CH), 86.6 (C), 93.4 (C), 119.4 (C), 122.5 (C),
128.5 (CH), 128.9 (CH), 130.9 (CH), 131.8 (CH), 133.6 (C), 145.5 (CH),
155.0 (C), 173.0 (C) ppm. HR-MS m/z [M+H]+ calcd for C20H19N3OS:
350.1322, found 350.1315.
7.1.3. Synthesis of 6-(benzamide-4-yl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridines
7.1.2.1. 3-(cis-2,6-Dimethylmorpholino)-6-(phenylamino)isothiazolo[4,3-
b]pyridine (5h). This compound was obtained using aniline. The crude
residue was purified by silica gel flash chromatography using a mixture
of hexane and ethyl acetate (in a ratio of 7:3) as mobile phase, affording
the title compound as an amorphous yellow solid in 85% yield (44 mg,
0.13 mmol). Mp 186.1–187.5 °C. 1H NMR (600 MHz, CDCl3) δ: 1.29 (d,
J = 6.3 Hz, 6H, 2 × CH3), 2.83–2.89 (m, 2H, 2 × NCH), 3.87–3.95 (m,
2H, 2 × OCH), 4.41 (d, J = 12.3 Hz, 2H, 2 × NCH), 6.04 (bs, 1H, NH),
7.04 (t, J = 7.3 Hz, 1H, arom H), 7.17 (d, J = 7.7 Hz, 2H, arom H), 7.26
(s, 1H, arom H), 7.32 (t, J = 7.8 Hz, 2H, arom H), 8.10 (d, J = 2.4 Hz,
1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 55.3
(CH2), 71.2 (CH), 106.7 (CH), 119.6 (CH), 122.9 (CH), 129.5 (CH),
129.9 (C), 139.2 (C), 140.1 (CH), 140.8 (C), 157.2 (C), 172.0 (C) ppm.
HR-MS m/z [M+H]+ calcd for C18H20N4OS: 341.1431, found
341.1431.
General procedure. To a solution of compound 5f (6-(3-methoxy-4-
carboxyphenyl)-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]pyr-
idine) (1 equiv) in dry dichloromethane (15 mL) was added thionyl
chloride (SOCl2) (2 equiv). The mixture was stirred at 40 °C for 3 h.
Then, triethylamine (2 equiv) and the appropriate amine (2 equiv) were
added and the resulting mixture was stirred at room temperature
overnight. After completion of the reaction as monitored by TLC, the
residue was diluted with dichloromethane (20 mL) and washed with
0.5 M HCl solution (2 × 10 mL) and water (20 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated in vacuo. The re-
sulting residue was purified by silica gel flash chromatography, yielding
the title compound. The following compounds were made according to
this procedure.
7.1.3.1. 6-(2-Methoxybenzamide-4-yl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (6a). This compound was obtained using
aqueous ammonia (25%) (2 mL). After completion of the reaction as
monitored by TLC, water (15 mL) was added, followed by extraction
with dichloromethane (2 × 20 mL). The organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuo. The resulting residue was
7.1.2.2. 6-(3,4-Dimethoxyphenylamino)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (5i). This compound was obtained using 3,4-
dimethoxyaniline. The crude residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio
of 3:2) as mobile phase, affording the title compound as an amorphous
yellow solid in 79% yield (48 mg, 0.12 mmol). Mp 207.1–208.9 °C. 1H
NMR (600 MHz, CDCl3) δ: 1.29 (d, J = 6.3 Hz, 6H, 2 × CH3). 2.86 (dd,
J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.86 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 3.90–3.94 (m, 2H, 2 × OCH), 4.40 (dd, J = 12.6, 1.6 Hz, 2H,
2 × NCH), 5.78 (bs, 1H, NH), 6.75–6.79 (m, 2H, arom H), 6.85 (d,
purified by silica gel flash chromatography, using
a mixture of
dichloromethane and methanol (20:0.5) as mobile phase, yielding the
title compound as an amorphous yellow solid in 67% yield (33.4 mg,
0.08 mmol). Mp 270.6–272.2 °C. 1H NMR (300 MHz, DMSO) δ: 1.20 (d,
J = 6.2 Hz, 6H, 2 × CH3), 2.90 (dd, J = 12.2, 11.0 Hz, 2H, 2 × NCH),
8

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
3.87 (dd, J = 12.3, 4.3 Hz, 2H, 2 × OCH), 4.03 (s, 3H, OCH3), 4.51 (d,
J = 12.3 Hz, 2H, 2 × NCH), 7.48 (dd, J = 8.1, 1.5 Hz, 1H, arom H),
7.53 (s, 1H, arom H), 7.62 (bs, 1H, NH), 7.70 (bs, 1H, NH), 7.91 (d,
J = 8.0 Hz, 1H, arom H), 8.15 (d, J = 2.1 Hz, 1H, arom H), 8.81 (d,
J = 2.1 Hz, 1H, arom H) ppm. 13C NMR (75 MHz, DMSO) δ: 18.7 (CH3),
55.0 (CH2), 56.3 (OCH3), 70.7 (CH), 111.1 (CH), 119.4 (CH), 122.7 (C),
125.6 (CH), 131.6 (CH), 133.8 (C), 134.1 (C), 140.9 (C), 143.9 (CH),
155.5 (C), 157.9 (C), 166.0 (C), 172.5 (C) ppm. HR-MS m/z [M+H]+
calcd for C20H22N4O3S: 399.1485, found 399.1483.
calcd for C24H28N4O4S: 469.1904, found 469.1898.
7.1.3.5. 3-(cis-2,6-Dimethylmorpholino)-6-(N-phenyl-2-
methoxybenzamide-4-yl)isothiazolo[4,3-b]pyridine (6e). This compound
was obtained using aniline. The crude residue was purified by silica gel
flash chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 3:2) as mobile phase, affording the title compound as an
amorphous yellow solid in 76% yield (45 mg, 0.09 mmol). Mp
251.1–252.5 °C. 1H NMR (600 MHz, CDCl3) δ: 1.33 (d, J = 6.3, 6H,
2 × CH3), 2.94 (dd, J = 12.6, 10.7 Hz, 2H, 2 × NCH), 3.92–3.98 (m,
2H, 2 × OCH), 4.14 (s, 3H, OCH3), 4.53 (dd, J = 12.6, 1.7 Hz, 2H,
2 × NCH), 7.13–7.16 (m, 1H, arom H), 7.27 (d, J = 1.5 Hz, 1H, arom
H), 7.36–7.40 (m, 2H, arom H), 7.42 (dd, J = 8.1, 1.6 Hz, 1H, arom H),
7.69–7.71 (dd, J = 8.5, 1.0 Hz, 2H, arom H), 7.95 (d, J = 2.1 Hz, 1H,
arom H), 8.40 (d, J = 8.1 Hz, 1H, arom H), 8.64 (d, J = 2.1 Hz, 1H,
arom H), 9.79 (s, 1H, NH) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8
(CH3), 55.4 (CH2), 56.4 (OCH3), 71.2 (CH), 110.5 (CH), 120.4 (CH),
120.6 (CH), 121.6 (C), 124.3 (CH), 126.1 (CH), 129.0 (CH), 133.4 (CH),
134.5 (C), 138.3 (C), 142.6 (C), 143.5 (CH), 155.8 (C), 157.6 (C), 162.7
(C), 173.1 (C) ppm. HR-MS m/z [M+H]+ calcd for C26H26N4O3S
475.1798, found 475.1799.
7.1.3.2. 6-(N-Cyclopropyl-2-methoxybenzamide-4-yl)-3-(cis-2,6-
dimethylmorpholino) isothiazolo[4,3-b]pyridine (6b). This compound
was obtained using cyclopropylamine. The crude residue was purified
by silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 3:7) as mobile phase, affording the title compound
as an amorphous yellow solid in 70% yield (38 mg, 0.09 mmol). Mp
195.1–196.8 °C. 1H NMR (600 MHz, CDCl3) δ: 0.61–0.64 (m, 2H, CH2),
0.86–0.91 (m, 2H, CH2), 1.33 (d, J = 6.3 Hz, 6H, 2 × CH3), 2.91–2.99
(m, 3H, CH, 2 × NCH), 3.92–3.99 (m, 2H, 2 × OCH), 4.03 (s, 3H,
OCH3), 4.53 (dd, J = 12.7, 1.8 Hz, 2H, 2 × NCH), 7.20 (d, J = 1.6 Hz,
1H, arom H), 7.38 (dd, J = 8.1, 1.6 Hz, 1H, arom H), 7.91 (d,
J = 2.8 Hz, 1H, NH), 7.93 (d, J = 2.1 Hz, 1H, arom H), 8.35 (d,
J = 8.1 Hz, 1H, arom H), 8.63 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C
NMR (151 MHz, CDCl3) δ: 6.9 (CH2), 18.8 (CH3), 22.9 (CH), 55.4 (CH2),
56.1 (OCH3), 71.2 (CH), 110.3 (CH), 120.3 (CH), 121.3 (C), 126.1 (CH),
133.1 (CH), 134.5 (C), 134.8 (C), 142.1 (C), 143.6 (CH), 155.9 (C),
157.8 (C), 166.1 (C), 173.1 (C) ppm. HR-MS m/z [M+H]+ calcd for
7.1.3.6. 6-(N-Benzyl-2-methoxybenzamide-4-yl)-3-(cis-2,6-
dimethylmorpholino) isothiazolo[4,3-b]pyridine (6f). This compound was
obtained using benzylamine. The crude residue was purified by silica
gel flash chromatography using a mixture of hexane and ethyl acetate
(in a ratio of 1:1) as mobile phase, affording the title compound as an
amorphous yellow solid in 85% yield (51.2 mg, 0.11 mmol). Mp
172.2–173.4 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.2 Hz,
6H, 2 × CH3), 2.93 (dd, J = 12.2, 11.0 Hz, 2H, 2 × NCH), 3.91–3.98
(m, 2H, 2 × OCH), 4.00 (s, 3H, OCH3), 4.53 (d, J = 11.6 Hz, 2H,
2 × NCH), 4.72 (d, J = 5.6 Hz, 2H, CH2), 7.21 (d, J = 0.8 Hz, 1H, arom
H), 7.27–7.31 (m, 1H, arom H), 7.33–7.42 (m, 5H, arom H), 7.93 (d,
J = 1.9 Hz, 1H, arom H), 8.20 (m, 1H, NH), 8.38 (d, J = 8.1 Hz, 1H,
arom H), 8.63 (d, J = 1.9 Hz, 1H, arom H) ppm.13C NMR (151 MHz,
CDCl3) δ: 18.8 (CH3), 43.8 (CH2), 55.4 (CH2), 56.1 (OCH3), 71.2 (CH),
110.3 (CH), 120.3 (CH), 121.3 (C), 126.1 (CH), 127.3 (CH), 127.5 (CH),
128.7 (CH), 133.3 (CH), 134.5 (C), 134.7 (C), 138.7 (C), 142.2 (C),
143.6 (CH), 155.9 (C), 157.9 (C), 164.8 (C), 173.1 (C) ppm. HR-MS m/z
[M+H]+ calcd for C27H28N4O3S: 489.1955, found 489.1946.
C
23H26N4O3S: 439.1798, found 439.1798.
7.1.3.3. (4-(3-(cis-2,6-Dimethylmorpholino)isothiazolo[4,3-b]pyridin-6-
yl)-2-methoxyphenyl)(pyrrolidin-1-yl)methanone (6c). This compound
was obtained using pyrrolidine. The crude residue was purified by
silica gel flash chromatography using a mixture of dichloromethane and
methanol (in a ratio of 10:0.1) as mobile phase, affording the title
compound as an amorphous yellow solid in 65% yield (36.6 mg,
0.08 mmol). Mp 173.1–174.9 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32
(d, J = 6.3 Hz, 6H, 2 × CH3), 1.86–1.92 (m, 2H, CH2), 1.94–2.01 (m,
2H, CH2), 2.93 (dd, J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.30 (t,
J = 6.7 Hz, 2H, CH2), 3.68 (t, J = 7.0 Hz, 2H, CH2), 3.92 (s, 3H,
OCH3), 3.93–3.98 (m, 2H, 2 × OCH), 4.53 (dd, J = 12.7, 1.8 Hz, 2H,
2 × NCH), 7.16 (d, J = 1.4 Hz, 1H, arom H), 7.27 (dd, J = 7.7, 1.5 Hz,
1H, arom H), 7.40 (d, J = 7.7 Hz, 1H, arom H), 7.90 (d, J = 2.1 Hz, 1H,
arom H), 8.61 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C NMR (151 MHz,
CDCl3) δ: 18.8 (CH3), 24.6 (CH2), 25.8 (CH2), 45.6 (CH2), 47.7 (CH2),
55.4 (CH2), 55.7 (OCH3), 71.2 (CH), 110.2 (CH), 119.9 (CH), 125.8
(CH), 127.5 (C), 128.6 (CH), 134.3 (C), 135.3 (C), 139.9 (C), 143.9
(CH), 155.8 (C), 156.0 (C), 167.2 (C), 173.0 (C) ppm. HR-MS m/z [M
+H]+ calcd for C24H28N4O3S: 453.1955, found 453.1945.
7.1.3.7. 6-(3-Methoxy-4-formamidophenyl)-3-(cis-2,6-
dimethylmorpholino)isothiazolo[4,3-b]pyridine (7a). To a solution of 6-
(4-amino-3-methoxyphenyl)-3-(cis-2,6-dimethylmorpholino)isothiazolo
[4,3-b]pyridine (1 equiv) in formamide (10 mL) was added MnO2 (5%
mol) in one portion. The reaction mixture was stirred at 150 °C for 5 h.
After completion of the reaction as monitored by TLC, the volatiles
were evaporated to dryness and the residue was purified by silica gel
flash chromatography, using a mixture of hexane and acetyl acetate,
yielding the title compound as an amorphous yellow solid in 73% yield
(39.2 mg, 0.10 mmol). Mp 234.3–235.7 °C. Doubling of signals for the
protons of the amine and the formyl group in the 1H NMR spectrum at
25 °C was observed, due to restricted rotation around the CeN amide
bond. Based on the integration of the signals in 1H NMR spectrum, the
ratio of the isomers was determined to be 95:5 (cis:trans).28 When
running the 1H NMR spectrum at 50 °C, coalescence of both signals is
observed (see Supporting Information). 1H NMR (600 MHz, DMSO) δ:
1.19 (d, J = 6.2 Hz, 6H, 2 × CH3), 2.89 (t, J = 11.5 Hz, 2H, 2 × NCH),
3.83–3.89 (m, 2H, 2 × OCH), 3.98 (s, 3H, OCH3), 4.51 (d, J = 12.0 Hz,
1H, 2 × NCH), 7.42 (dd, J = 8.3, 1.3 Hz, 1H, arom H), 7.50 (d,
J = 1.4 Hz, 1H, arom H), 8.06 (d, J = 1.8 Hz, 1H, arom H), 8.31 (d,
J = 8.3 Hz, 1H, arom H), 8.35 (d, J = 1.3 Hz, 1H, HCO), 8.80 (d,
J = 1.8 Hz, 1H, arom H), 9.80 (s, 1H, NH) ppm. 13C NMR (151 MHz,
DMSO) δ: 18.6 (CH3), 54.8 (CH2), 56.2 (OCH3), 70.6 (CH), 110.0 (CH),
119.5 (CH), 120.5 (CH), 124.3 (CH), 127.5 (C), 132.3 (C), 133.3 (C),
134.5 (C), 144.0 (CH), 149.0 (C), 155.7 (C), 160.3 (HCO), 172.3 (C)
7.1.3.4. (4-(3-cis-2,6-Dimethylmorpholino)isothiazolo[4,3-b]pyridin-6-
yl)-2-methoxyphenyl)(morpholino)methanone (6d). This compound was
obtained using morpholine. The crude residue was purified by silica gel
flash chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 3:7) as mobile phase, affording the title compound as an
amorphous yellow solid in 68% yield (39 mg, 0.08 mmol). Mp
134.2–135.9 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz,
6H, 2 × CH3), 2.90–2.97 (m, 2H, 2 × NCH), 3.27–3.40 (m, 2H, CH2),
3.58–3.71 (m, 2H, CH2), 3.76–3.89 (m, 4H, 2 × CH2), 3.93 (s, 3H,
OCH3), 3.93–3.98 (m, 2H, 2 × OCH), 4.53 (d, J = 11.3 Hz, 2H,
2 × NCH), 7.15 (d, J = 1.2 Hz, 1H, arom H), 7.29 (dd, J = 7.7,
1.4 Hz, 1H, arom H), 7.39 (d, J = 7.7 Hz, 1H, arom H), 7.90 (d,
J = 1.7 Hz, 1H, arom H), 8.60 (d, J = 1.9 Hz, 1H, arom H) ppm. 13C
NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 42.2 (CH2), 47.4 (CH2), 55.4
(CH2), 55.7 (OCH3), 66.8 (CH2), 67.0 (CH2), 71.2 (CH), 110.0 (CH),
120.2 (CH), 125.3 (C), 125.9 (CH), 128.9 (CH), 135.1 (C), 140.3 (C),
143.7 (CH), 155.8 (C), 167.3 (C), 173.1 (C) ppm. HR-MS m/z [M+H]+
9

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
ppm. HR-MS m/z [M+H]+ calcd for C20H22N4O3S: 399.1485, found
7.20 (d, J = 1.9 Hz, 1H, arom H), 7.35 (dd, J = 8.3, 1.9 Hz, 1H, arom H),
7.50–7.55 (m, 2H, arom H), 7.56–7.60 (m, 1H, arom H), 7.90 (d,
J = 2.1 Hz, 1H, arom H), 7.91–7.95 (m, 2H, arom H), 8.62 (bs,
J = 8.2 Hz, 1H, NH), 8.66–8.69 (m, 2H, arom H) ppm. 13C NMR
(151 MHz, CDCl3) δ: 18.8 (CH3), 55.4 (CH2), 56.0 (OCH3), 71.2 (CH),
108.8 (CH), 120.2 (CH), 120.3 (CH), 125.0 (CH), 127.1 (CH), 128.2 (C),
128.8 (CH), 131.9 (CH), 133.2 (C), 134.1 (C), 135.1 (C), 135.4 (C), 144.2
(CH), 148.6 (C), 156.3 (C), 165.3 (C), 172.9 (C) ppm. HR-MS m/z [M
+H]+ calcd for C26H26N4O3S: 475.1798, found 475.1794.
399.1485.
7.1.4. Synthesis of 6-(4-aminyl-3-methoxyphenyl)-3-(cis-2,6-dimethyl-
morpholino) isothiazolo[4,3-b]pyridine
General procedure. To a solution of the precursor (6-(4-amino-3-
methoxyphenyl)-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]pyr-
idine) (1 equiv) in dry dichloromethane (15 mL), was added trimethy-
lamine (2 equiv). The reaction mixture was stirred under argon for
10 min and cooled to 0 °C. Then, the appropriate acid chloride (1.5
equiv) or acetic anhydride (2 equiv) was added and the resulting
mixture was stirred at room temperature overnight. After completion of
the reaction as monitored by TLC, dichloromethane was added (10 mL)
and the reaction mixture was washed with 0.5 M HCl solution
(2 × 10 mL) and water (20 mL). The organic layer was dried over an-
hydrous Na2SO4 and concentrated in vacuo. The resulting residue was
purified by silica gel flash chromatography, yielding the title com-
pound. The following compounds were made according to this proce-
dure.
7.1.4.4. Synthesis of 6-aryl-3-nitropyridine-2-carbonitriles (9a-i). General
procedure. A solution of 6-chloro-2-cyano-3-nitropyridine (1 equiv) in
toluene or dioxane/water (see compound description) was degassed
with argon and subsequently, the corresponding aryl boronic acid (1.2
equiv), Pd(PPh3)4 (0.02 equiv) and K2CO3 (2 equiv) were added. The
mixture was degassed a second time, filled with argon and stirred at
95 °C overnight. After completion of the reaction as monitored by TLC,
the volatiles were evaporated to dryness and the crude residue was
diluted with EtOAc (20 mL) and washed with water (2 × 20 mL). The
organic layer was dried over anhydrous Na2SO4 and concentrated in
vacuo. The resulting residue was purified by silica gel flash
chromatography, yielding the corresponding 6-aryl-3-nitropyridine-2-
carbonitrile. The following compounds were made according to this
procedure.
7.1.4.1. 6-(3-Methoxy-4-acetamidophenyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (7b). This compound was obtained using acetic
anhydride. The crude residue was purified by silica gel flash
chromatography using a mixture of dichloromethane and acetone (in a
ratio of 95:5) as mobile phase, affording the title compound as an
amorphous yellow solid in 73% yield (32.5 mg, 0.08 mmol). Mp
255.1–256–4 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
2 × CH3), 2.24 (s, 3H, CH3), 2.92 (dd, J = 12.6, 10.7 Hz, 2H, 2 × NCH),
3.92–3.98 (m, 5H, 2 × OCH, OCH3), 4.52 (dd, J = 12.8, 1.9 Hz, 2H,
2 × NCH), 7.14 (d, J = 1.9 Hz, 1H, arom H), 7.28 (dd, J = 8.3, 1.9 Hz,
1H, arom H), 7.82 (s, 1H, NH), 7.86 (d, J = 2.1 Hz, 1H, arom H), 8.49 (d,
J = 8.4 Hz, 1H, arom H), 8.63 (d, J = 2.1 Hz, 1H, arom H) ppm. 13C
NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 25.0 (CH3), 55.4 (CH2), 55.8
(OCH3), 71.2 (CH), 108.7 (CH), 120.1 (CH), 120.2 (CH), 125.0 (CH),
128.1 (C), 132.9 (C), 134.0 (C), 135.4 (C), 144.1 (CH), 148.1 (C), 156.3
(C), 168.2 (C), 172.9 (C) ppm. HR-MS m/z [M+H]+ calcd for
7.1.4.5. 3-Nitro-6-phenylpyridine-2-carbonitrile (9a). This compound
was obtained using phenyl boronic acid and toluene as solvent. The
crude residue was purified by silica gel flash chromatography using a
mixture of hexane and ethyl acetate (in a ratio of 8:2) as mobile phase,
affording the title compound as an amorphous light yellow solid in 81%
yield (397.6 mg, 1.8 mmol). 1H NMR (300 MHz, CDCl3) δ: 8.65 (d,
J = 8.9 Hz, 1H, arom H), 8.11–8.19 (m, 3H, arom H), 7.53–7.62 (m,
3H, arom H) ppm. HR-MS m/z [M+H]+ calcd for C12H7N3O2:
226.0611, found 226.0611.
7.1.4.6. 6-(4-(Trifluoromethyl)phenyl)-3-nitropyridine-2-carbonitrile
(9b). This compound was obtained using 4-(trifluoromethyl)phenyl
boronic acid and toluene as solvent. The crude residue was purified by
C21H24N4O3S: 413.1642, found 413.1638.
7.1.4.2. 6-(3-Methoxy-4-cyclopropylamidophenyl)-3-(cis-2,6-dimethyl-
morpholino) isothiazolo[4,3-b]pyridine (7c). This compound was
obtained using cyclopropanecarbonyl chloride. The crude residue was
purified by silica gel flash chromatography using a mixture of hexane
and ethyl acetate (in a ratio of 6:4) as mobile phase, affording the title
compound as an amorphous yellow solid in 79% yield (37 mg,
0.08 mmol). Mp 256.4–257.8 °C. 1H NMR (500 MHz, CDCl3) δ:
0.85–0.91 (m, 2H, CH2), 1.10–1.14 (m, 2H, CH2), 1.32 (d, J = 6.3 Hz,
6H, 2 × CH3), 1.57–1.64 (m, 1H, CH), 2.92 (dd, J = 12.5, 10.7 Hz, 2H,
2 × NCH), 3.94 (m, 2H, 2 × OCH), 3.98 (s, 3H, OCH3), 4.51 (dd,
J = 12.6, 1.7 Hz, 2H, 2 × NCH), 7.15 (d, J = 1.9 Hz, 1H, arom H),
7.26–7.28 (m, 1H, arom H), 7.86 (d, J = 2.1 Hz, 1H, arom H), 8.05 (bs,
1H, NH), 8.48 (d, J = 8.2 Hz, 1H, arom H), 8.64 (d, J = 2.1 Hz, 1H,
arom H) ppm. 13C NMR (126 MHz, CDCl3) δ: 8.1 (CH2), 18.8 (CH3),
55.4 (CH2), 55.8 (CH3), 71.2 (CH), 108.8 (CH), 120.0 (CH), 120.2 (CH),
124.9 (CH), 128.4 (C), 132.6 (C), 134.0 (C), 135.5 (C), 144.2 (CH),
148.0 (C), 156.3 (C), 171.8 (C), 172.9 (C) ppm. HR-MS m/z [M+H]+
calcd for C23H26N4O3S: 439.1798, found 439.1794.
silica gel flash chromatography using a mixture of hexane and
dichloromethane (in a ratio of 4:6) as mobile phase, affording the
title compound as an amorphous light yellow solid in 85% yield
(543.3 mg, 1.85 mmol). 1H NMR (600 MHz, CDCl3) δ: 7.84 (dd,
J = 8.7, 0.5 Hz, 2H, arom H), 8.22 (d, J = 8.8 Hz, 1H, arom H), 8.27
(dd, J = 8.8, 0.7 Hz, 2H, arom H), 8.72 (d, J = 8.8 Hz, 1H, arom H)
ppm. HR-MS m/z [M+Na]+ calcd for C13H6F3N3O2: 316.0305, found
316.0312.
7.1.4.7. 6-(4-Fluorophenyl)-3-nitropyridine-2-carbonitrile
(9c). This
compound was obtained using 4-fluorophenylboronic acid and
toluene as solvent. The crude residue was purified by silica gel flash
chromatography using a mixture of hexane and dichloromethane (in a
ratio of 4:6) as mobile phase, affording the title compound as an
amorphous light yellow solid in 94% yield (498.3 mg, 2.05 mmol). 1H
NMR (300 MHz, CDCl3) δ: 7.21–7.29 (m, 2H, arom H), 8.11 (d,
J = 8.9 Hz, 1H, arom H), 8.14–8.20 (m, 2H, arom H), 8.65 (d,
J = 8.9 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
C
12H6FN3O2: 244.0517, found 244.0521.
7.1.4.3. 6-(3-Methoxy-4-phenylamidophenyl)-3-(cis-2,6-dimethylmopholino)
isothiazolo[4,3-b]pyridine (7d). This compound was obtained using
benzoyl chloride. The crude residue was purified by silica gel flash
chromatography using a mixture of dichloromethane and acetone (in a
ratio of 95:5) as mobile phase, affording the title compound as an
amorphous yellow solid in 81% yield (41.1 mg, 0.09 mmol). Mp
195.4–196.3 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
2 × CH3), 2.93 (dd, J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.93–3.98 (m, 2H,
2 × OCH), 4.02 (s, 3H, OCH3), 4.53 (dd, J = 12.7, 1.8 Hz, 2H, 2 × NCH),
7.1.4.8. 6-(3-Chlorophenyl)-3-nitropyridine-2-carbonitrile
(9d). This
compound was obtained using 3-chlorophenylboronic acid and
toluene as solvent. The crude residue was purified by silica gel flash
chromatography using a mixture of hexane and dichloromethane (in a
ratio of 4:6) as mobile phase, affording the title compound as an
amorphous white solid in 98% yield (545.2 mg, 2.1 mmol). 1H NMR
(300 MHz, CDCl3) δ: 7.47–7.60 (m, 2H, arom H), 8.01 (dt, J = 7.3,
1.6 Hz, 1H, arom H), 8.12–8.17 (m, 2H, arom H), 8.67 (d, J = 8.8 Hz,
10

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
1H, arom H) ppm. HR-MS m/z [M+Na]+ calcd for C12H6ClN3O2:
to dryness and the crude was diluted with EtOAc (50 mL) and filtered
through a paper filter. The filter cake was washed repeated times with
ethyl acetate. The organic phase was washed with aqueous solution 1 N
NaOH (3 × 30 mL), dried over anhydrous Na2SO4 and evaporated to
dryness. This mixture of two compounds (3-amino-6-aryl-pyridine-2-
carbonitrile as the major and 3-amino-6-aryl-pyridine-2-carboxamide
as the minor compound) was used as such in the next reaction.
282.0041found 282.0044.
7.1.4.9. 6-(4-Methylphenyl)-3-nitropyridine-2-carbonitrile
(9e). This
compound was obtained using 4-methylphenylboronic acid and
toluene as solvent. The crude residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio
of 7:3) as mobile phase, affording the title compound as an amorphous
light yellow solid in 93% yield (485 mg, 2.03 mmol). 1H NMR
(300 MHz, CDCl3) δ: 2.46 (s, 3H, CH3), 7.36 (d, J = 8.1 Hz, 2H, arom
H), 8.04 (d, J = 8.3 Hz, 2H, arom H), 8.10 (d, J = 8.9 Hz, 1H, arom H),
8.60 (d, J = 8.9 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
7.1.4.15. Synthesis of 3-amino-6-aryl-pyridine-2-carbothioamides (11a-
i). General procedure. To a solution of the crude mixture of 3-amino-6-
aryl-pyridine-2-carbonitrile and 3-amino-6-aryl-pyridine-2-carboxamide
in absolute ethanol (40 mL), was added phosphorus pentasulfide (1.2
equiv). The corresponding solution was then heated overnight at 75 °C
overnight. After the completion of the reaction as monitored by TLC, the
solvent was evaporated to dryness and the residue was washed with an
aqueous 1 N NaOH (2 × 30 mL) solution, dried over anhydrous Na2SO4
and evaporated to dryness. The residue was purified by silica gel flash
chromatography yielding the 3-amino-6-aryl-pyridine-2-carbothioamide.
The following compounds were made according to this procedure.
7.1.4.15.1. 3-Amino-6-phenylpyridine-2-carbothioamide (11a). This
C
13H9N3O2: 240.0767, found 240.0775.
7.1.4.10. 6-(3-Methoxyphenyl)-3-nitropyridine-2-carbonitrile (9f). This
compound was obtained using 3-methoxyphenylboronic acid and
toluene as solvent. The crude residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio
of 7:3) as mobile phase, affording the title compound as an amorphous
yellow solid in 94% yield (523 mg, 2.05 mmol). 1H NMR (300 MHz,
CDCl3) δ: 3.92 (s, 3H, OCH3), 7.09–7.15 (m, 1H, arom H), 7.47 (t,
J = 8.0 Hz, 1H, arom H), 7.63–7.68 (m, 1H, arom H), 7.68–7.72 (m,
1H, arom H), 8.13 (d, J = 8.9 Hz, 1H, arom H), 8.63 (d, J = 8.9 Hz, 1H,
arom H) ppm. HR-MS m/z [M+H]+ calcd for C13H9N3O3: 256.0717,
found 256.0715.
compound was obtained from
6-phenylpyridine-2-carbonitrile
a
crude mixture of 3-amino-
3-amino-6-phenylpyridine-2-
and
carboxamide. The residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 8:2) as mobile phase, affording the title compound as an
amorphous yellow solid in 71% yield (166.8 mg, 0.73 mmol). 1H NMR
(300 MHz, DMSO) δ: 7.35 (m, 2H, arom H), 7.43 (m, 2H, arom H), 7.84
(bs, 2H, NH2), 7.90 (d, J = 8.7 Hz, 1H, arom H), 8.08 (d, J = 7.4 Hz, 2H,
arom H), 9.66 (bs, 1H, NH), 9.74 (bs, 1H, NH) ppm. HR-MS m/z [M
+H]+ calcd for C12H11N3S: 230.0746, found 230.0743.
7.1.4.11. 6-(3,4-Dimethoxyphenyl)-3-nitropyridine-2-carbonitrile
(9g). This compound was obtained using 3,4-dimethoxyphenylboronic
acid and toluene as solvent. The crude residue was purified by silica gel
flash chromatography using a mixture of hexane and acetone (in a ratio
of 7:3) as mobile phase, affording the title compound as an amorphous
orange solid in 78% yield (485.1 mg, 1.7 mmol). 1H NMR (300 MHz,
DMSO) δ: 3.87 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.17 (d, J = 8.6 Hz,
1H, arom H), 7.77 (d, J = 2.0 Hz, 1H, arom H), 7.87 (dd, J = 8.5,
2.1 Hz, 1H, arom H), 8.54 (d, J = 9.0 Hz, 1H, arom H), 8.75 (d,
J = 9.0 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
7.1.4.15.2. 3-Amino-6-(4-trifluoromethylphenyl)pyridine-2-
carbothioamide (11b). This compound was obtained from a crude
mixture of 3-amino-6-(4-trifluoromethylphenyl)pyridine-2-carbonitrile
and 3-amino-6-(4-trifluoromethylphenyl)pyridine-2-carboxamide. The
residue was purified by silica gel flash chromatography using a mixture
of hexane and ethyl acetate (in a ratio of 7:3) as mobile phase, affording
the title compound as an amorphous yellow solid in 81% yield (183 mg,
0.62 mmol). 1H NMR (300 MHz, DMSO) δ: 7.37 (d, J = 8.8 Hz, 1H,
arom H), 7.75 (d, J = 8.4 Hz, 2H, arom H), 7.99 (d, J = 8.8 Hz, 1H,
arom H), 7.93 (bs, 2H, NH2), 8.32 (d, J = 8.2 Hz, 2H, arom H), 9.74 (s,
1H, NH), 9.78 (s, 1H, NH) ppm. HR-MS m/z [M+H]+ calcd for
C
14H11N3O4: 286.0822, found 286.0822.
7.1.4.12. 6-(3,4,5-Trimethoxyphenyl)-3-nitropyridine-2-carbonitrile
(9h). This compound was obtained using 3,4,5-trimethoxyphenylboronic
acid and dioxane/water (in ratio 4:1) as solvent. The crude residue was
purified by silica gel flash chromatography using a mixture of hexane and
acetone (in a ratio of 7:3) as mobile phase, affording the title compound as
an amorphous orange solid in 96% yield (659.8 mg, 2.1 mmol). 1H NMR
(300 MHz, CDCl3) δ: 3.94 (s, 3H, OCH3), 4.00 (s, 6H, 2 × OCH3), 7.37 (s,
2H, arom H), 8.09 (d, J = 8.9 Hz, 1H, arom H), 8.61 (d, J = 8.9 Hz, 1H,
arom H) ppm. HR-MS m/z [M+H]+ calcd for C15H13N3O5: 316.0928,
found 316.0931.
C
13H10F3N3S: 298.0620, found 298.0614.
7.1.4.15.3. 3-Amino-6-(4-fluorophenyl)pyridine-2-carbothioamide
(11c). This compound was obtained from a crude mixture of 3-amino-
6-(4-fluorophenyl)pyridine-2-carbonitrile
and
3-amino-6-(4-
fluorophenyl)pyridine-2-carboxamide. The residue was purified by
flash chromatography using a mixture of hexane and ethyl acetate (in
a ratio of 8:2) as mobile phase, affording the title compound as an
amorphous yellow solid in 79% yield (160.4 mg, 0.65 mmol). 1H NMR
(300 MHz, DMSO) δ: 7.23 (t, J = 8.8 Hz, 2H, arom H), 7.34 (d,
J = 8.8 Hz, 1H, arom H), 7.83 (s, 2H, NH2), 7.88 (d, J = 8.8 Hz, 1H,
arom H), 8.15 (dd, J = 8.7, 5.6 Hz, 2H, arom H), 9.75 (bs, 1H, NH),
9.66 (bs, 1H, NH) ppm. HR-MS m/z [M+H]+ calcd for C12H10FN3S:
248.0652, found 248.0651.
7.1.4.13. 6-(3,5-Dimethoxyphenyl)-3-nitropyridine-2-carbonitrile
(9i). This compound was obtained using 3,5-dimethoxyphenylboronic
acid and toluene as solvent. The crude residue was purified by silica gel
flash chromatography using a mixture of hexane and acetone (in a ratio
of 7:3) as mobile phase, affording the title compound as an amorphous
orange solid in 81% yield (503.5 mg, 2.1 mmol). 1H NMR (300 MHz,
CDCl3) δ: 3.91 (s, 6H, 2 × OCH3), 6.66 (t, J = 2.2 Hz, 1H, arom H),
7.25 (d, J = 2.2 Hz, 2H, arom H), 8.10 (d, J = 8.9 Hz, 1H, arom H),
8.62 (d, J = 8.9 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
C14H11N3O4: 286.0822, found 286.0818.
7.1.4.15.4. 3-Amino-6-(3-chlorophenyl)pyridine-2-carbothioamide
(11d). This compound was obtained from a crude mixture of 3-amino-6-
(3-chlorophenyl)pyridine-2-carbonitrile and 3-amino-6-(3-chlorophenyl)
pyridine-2-carboxamide. The residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio
of 8:2) as mobile phase, affording the title compound as an amorphous
yellow solid in 71% yield (173 mg, 0.62 mmol). 1H NMR (300 MHz,
DMSO) δ: 7.31–7.48 (m, 3H, arom H), 7.86 (bs, 2H, NH2), 7.94 (d,
J = 8.8 Hz, 1H, arom H), 8.04 (d, J = 7.6 Hz, 1H, arom H), 8.19 (t,
J = 1.6 Hz 1H, arom H), 9.68 (bs, 1H, NH), 9.79 (bs, 1H, NH) ppm. HR-
MS m/z [M+H]+ calcd for C12H10ClN3S: 264.0357, found 264.0353.
7.1.4.15.5. 3-Amino-6-(4-methylphenyl)pyridine-2-carbothioamide
7.1.4.14. Synthesis of 3-amino-6-aryl-pyridine-2-carbonitriles (10a-
i). General procedure. To a stirred suspension of iron powder (3
equiv) in acetic acid (40 mL) at 60 °C, the corresponding 6-aryl-3-
nitropyridine-2-carbonitrile (1 equiv) was added. The reaction mixture
was stirred at 60 °C until disappearance of the starting material as
monitored by TLC (3–6 h). After cooling, the volatiles were evaporated
11

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
(11e). This compound was obtained from a crude mixture of 3-amino-6-
(4-methylphenyl)pyridine-2-carbonitrile and 3-amino-6-(4-methylphenyl)
pyridine-2-carboxamide. The residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio of
8:2) as mobile phase, affording the title compound as an amorphous
yellow solid in 83% yield (242.3 mg, 1.0 mmol). 1H NMR (300 MHz,
DMSO) δ: 2.34 (s, 3H, CH3), 7.23 (d, J = 8.0 Hz, 2H, arom H), 7.32 (d,
J = 8.8 Hz, 1H, arom H), 7.80 (bs, 2H, NH2), 7.86 (d, J = 8.8 Hz, 1H,
arom H), 7.97 (d, J = 8.2 Hz, 2H, arom H), 9.63 (bs, 1H, NH), 9.72 (bs,
1H, NH) ppm. HR-MS m/z [M+H]+ calcd for C13H13N3S: 244.0903,
found 244.0916.
starting material as monitored by TLC, the solvent was evaporated to
dryness affording the desired 3-amino-5-aryl-isothiazolo[4,3-b]pyr-
idine, that was used as such in the next reaction without any further
purification. The following compounds were made according to this
procedure.
7.1.5.1. 3-Amino-5-(phenyl)isothiazolo[4,3-b]pyridine
(12a). This
compound was prepared from 3-amino-6-phenylpyridine-2-
carbothioamide affording the title compound as an amorphous
yellow solid in 95% yield (89.4 mg, 0.39 mmol). 1H NMR (600 MHz,
DMSO) δ: 7.41–7.46 (m, 1H, arom H), 7.48–7.53 (m, 2H, arom H),
7.81 (d, J = 9.2, 1H, arom H), 7.96 (d, J = 9.3 Hz, 1H, arom H),
8.20–8.24 (m, 2H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
7.1.4.15.6. 3-Amino-6-(3-methoxyphenyl)pyridine-2-carbothioamide
(11f). This compound was obtained from a crude mixture of 3-amino-6-(3-
methoxyphenyl)pyridine-2-carbonitrile and 3-amino-6-(4-methoxyphenyl)
pyridine-2-carboxamide. The residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio
of 8:2) as mobile phase, affording the title compound as an amorphous
yellow solid in 72% yield (261.4 mg, 1.0 mmol). 1H NMR (300 MHz,
DMSO) δ: 3.83 (s, 3H, OCH3), 6.91 (dd, J = 8.1, 2.5 Hz, 1H, arom H),
7.30–7.38 (m, 2H, arom H), 7.57–7.66 (m, 2H, arom H), 7.89 (d,
J = 8.8 Hz, 1H, arom H), 9.66 (bs, 1H, NH), 9.72 (bs, 1H, NH) ppm. HR-
MS m/z [M+H]+ calcd for C13H13N3OS: 260.0852, found 260.0848.
7.1.4.15.7. 3-Amino-6-(3,4-dimethylphenyl)pyridine-2-
C
12H9N3S: 228.0590, found 228.0583.
7.1.5.2. 3-Amino-5-(4-trifluoromethylphenyl)isothiazolo[4,3-b]pyridine
(12b). This compound was prepared from 3-amino-6-(4-fluorophenyl)
pyridine-2-carbothioamide affording the title compound as an
amorphous yellow solid in 97% yield (120.3 mg, 0.41 mmol). 1H
NMR (300 MHz, DMSO) δ: 7.79–7.89 (m, 3H, arom H), 8.01 (d,
J = 9.4 Hz, 1H, arom H), 8.10 (bs, 2H, NH2), 8.44 (d, J = 8.1 Hz, 2H,
arom H) ppm. HR-MS m/z [M+H]+ calcd for C13H8F3N3S: 296.0464,
found 296.0471.
carbothioamide (11g). This compound was obtained from a crude
mixture of 3-amino-6-(3,4-dimethylphenyl)pyridine-2-carbonitrile and
3-amino-6-(3,4-dimethylphenyl)pyridine-2-carboxamide. The residue
was purified by silica gel flash chromatography using a mixture of
hexane and ethyl acetate (in a ratio of 7:3) as mobile phase, affording
the title compound as an amorphous yellow solid in 73% yield
(165.4 mg, 0.57 mmol). 1H NMR (300 MHz, DMSO) δ: 3.79 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 6.99 (d, J = 8.4 Hz, 1H, arom H), 7.32 (d,
J = 8.8 Hz, 1H, arom H), 7.55–7.60 (m, 1H, arom H), 7.62 (d,
J = 1.9 Hz, 1H, arom H), 7.76 (bs, 2H, NH2), 7.86 (d, J = 8.8 Hz, 1H,
arom H), 9.62 (bs, 1H, NH), 9.71 (bs, 1H, NH) ppm. HR-MS m/z [M
+H]+ calcd for C14H15N3O2S: 290.0958, found 290.0941.
7.1.5.3. 3-Amino-5-(4-fluorophenyl)isothiazolo[4,3-b]pyridine
(12c). This compound was prepared from 3-amino-6-(4-fluorophenyl)
pyridine-2-carbothioamide affording the title compound as an
amorphous yellow solid in 98% yield (106.9 mg, 0.44 mmol). 1H
NMR (600 MHz, DMSO) δ: 7.30–7.36 (m, 2H, arom H), 7.79 (d,
J = 9.3 Hz, 1H, arom H), 7.90 (bs, 2H, NH2), 7.91 (d, J = 9.4 Hz, 1H,
arom H), 8.25–8.30 (m, 2H, arom H) ppm. HR-MS m/z [M+H]+ calcd
for C12H8FN3S: 246.0496, found 246.0506.
7.1.5.4. 3-Amino-5-(3-chlorophenyl)isothiazolo[4,3-b]pyridine
(12d). This compound was prepared from 3-amino-6-(3-chlorophenyl)
pyridine-2-carbothioamide affording the title compound as an
amorphous yellow solid in 97% yield (192.9 mg, 0.74 mmol). 1H
NMR (300 MHz, DMSO) δ: 7.45–7.57 (m, 2H, arom H), 7.79 (d,
J = 9.4 Hz, 1H, arom H), 7.97 (d, J = 9.4 Hz, 1H, arom H), 8.07 (bs,
2H, NH2), 8.15 (d, J = 7.4 Hz, 1H, arom H), 8.36 (s, 1H, arom H) ppm.
HR-MS m/z [M+H]+ calcd for C12H8ClN3S: 262.0200, found
262.0196.
7.1.4.15.8. 3-Amino-6-(3,4,5-trimethoxyphenyl)pyridine-2-
carbothioamide (11h). This compound was obtained from a crude
mixture of 3-amino-6-(3,4,5-trimethoxyphenyl)pyridine-2-carbonitrile
and 3-amino-6-(3,4,5-trimethoxyphenyl)pyridine-carboxamide. The
residue was purified by silica gel flash chromatography using a
mixture of hexane and ethyl acetate (in a ratio of 6:4) as mobile
phase, affording the title compound as an amorphous yellow solid in
76% yield (170.1 mg, 0.53 mmol). 1H NMR (600 MHz, CDCl3) δ: 3.90
(s, 3H, OCH3), 3.95 (s, 6H, 2 × OCH3), 6.94 (bs, 2H, NH2), 7.05 (s, 2H,
arom H), 7.15–7.18 (m, 1H, arom H), 7.30 (bs, 1H, NH), 7.62–7.64 (m,
1H, arom H), 9.64 (bs, 1H, NH) ppm. HR-MS m/z [M+H]+ calcd for
C15H17N3O3S: 320.1063, found 320.1072.
7.1.5.5. 3-Amino-5-(4-methylphenyl)isothiazolo[4,3-b]pyridine
(12e). This compound was prepared from 3-amino-6-(4-methylphenyl)
pyridine-2-carbothioamide affording the title compound as an
amorphous yellow solid in 98% yield (194.0 mg, 0.8 mmol). 1H NMR
(300 MHz, DMSO) δ: 2.37 (s, 3H, CH3), 7.31 (d, J = 8.1 Hz, 2H, arom
H), 7.78 (d, J = 9.4 Hz, 1H, arom H), 7.93 (d, J = 9.3 Hz, 1H, arom H),
7.96 (bs, 2H, NH2), 8.13 (d, J = 8.1 Hz, 2H, arom H) ppm. HR-MS m/z
[M+H]+ calcd for C13H11N3S: 242.0746, found 242.0750.
7.1.4.15.9. 3-Amino-6-(3,5-dimethylphenyl)pyridine-2-
carbothioamide (11i). This compound was obtained from a crude
mixture of 3-amino-6-(3,5-dimethylphenyl)pyridine-2-carbonitrile and
3-amino-6-(3,5-dimethylphenyl)pyridine-2-carboxamide. The residue
was purified by silica gel flash chromatography using a mixture of
hexane and ethyl acetate (in a ratio of 7:3) as mobile phase, affording
the title compound as an amorphous yellow solid in 76% yield
(258.3 mg, 1.17 mmol). 1H NMR (300 MHz, DMSO) δ: 3.81 (s, 6H,
2 × OCH3), 6.48 (t, J = 2.1 Hz, 1H, arom H), 7.18 (d, J = 2.2 Hz, 2H,
arom H), 7.32 (d, J = 8.8 Hz, 1H, arom H), 7.81 (bs, 2H, NH2), 7.88 (d,
J = 8.8 Hz, 1H, arom H), 9.65 (bs, 1H, NH), 9.70 (bs, 1H, NH) ppm.
HR-MS m/z [M+H]+ calcd for C14H15N3O2S: 290.0958, found
290.0963.
7.1.5.6. 3-Amino-5-(3-methoxyphenyl)isothiazolo[4,3-b]pyridine
(12f). This compound was prepared from 3-amino-6-(3-methoxyphenyl)
pyridine-2-carbothioamide affording the title compound as an
amorphous yellow solid in 91% yield (117.4 mg, 0.46 mmol). 1H NMR
(600 MHz, DMSO) δ: 3.86 (s, OCH3), 7.02 (dd, J = 8.1, 2.2 Hz, 1H, arom
H), 7.41 (t, J = 7.9 Hz, 1H, arom H), 7.78 (d, J = 7.7 Hz, 1H, arom H),
7.81–7.85 (m, 2H, arom H), 8.01 (d, J = 9.0 Hz, 2H, arom H) ppm. HR-
MS m/z [M+H]+ calcd for C13H11N3OS: 258.0696, found 258.0688.
7.1.5. Synthesis of 3-amino-5-(aryl)isothiazolo[4,3-b]pyridines (12a-i)
General procedure. To a solution of a 3-amino-6-(aryl)pyridine-2-
carbothioamides (1 equiv) in methanol (50 mL) was added dropwise a
35% H2O2 (2.5 equiv) solution in water at 0 °C. The reaction mixture
was stirred overnight at room temperature. After disappearance of the
7.1.5.7. 3-Amino-5-(3,4-dimethoxyphenyl)isothiazolo[4,3-b]pyridine
(12g). This compound was prepared from 3-amino-6-(3,4-
dimethylphenyl)pyridine-2-carbothioamide affording the title compound
as an amorphous yellow solid in 92% yield (79.3 mg, 0.28 mmol). 1H
NMR (600 MHz, DMSO) δ: 3.83 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.05
12

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
(d, J = 8.5 Hz, 1H, arom H), 7.72 (dd, J = 8.4, 2.1 Hz, 1H, arom H), 7.76
(d, J = 9.4 Hz, 1H, arom H), 7.91 (d, J = 2.0 Hz, 1H, arom H), 7.94 (d,
J = 9.4 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
1H, arom H), 8.20 (d, J = 9.3 Hz, 1H, arom H), 8.30 (d, J = 8.4 Hz, 2H,
arom H) ppm. HR-MS m/z [M+H]+ calcd for C13H6BrF3N2S: 358,9460,
found 358,9464.
C
14H13N3O2S: 288.0801, found 288.0798.
7.1.6.3. 3-Bromo-5-(4-fluorophenyl)isothiazolo[4,3-b]pyridine
(13c). This compound was prepared from 3-amino-5-(4-fluorophenyl)
isothiazolo[4,3-b]pyridine and the residue was purified by flash
chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 9:1) as mobile phase, affording the title compound as an
amorphous white solid in 60% yield (105 mg, 0.34 mmol). 1H NMR
(300 MHz, CDCl3) δ: 7.21 (m, 2H, arom H), 7.86 (d, J = 9.4 Hz, 1H,
arom H), 8.14 (d, J = 9.4 Hz, 1H, arom H), 8.17–8.23 (m, 2H, arom H)
ppm. HR-MS m/z [M+H]+ calcd for C12H6BrFN2S: 308,9492, found
308,9503.
7.1.5.8. 3-Amino-5-(3,4,5-trimethoxyphenyl)isothiazolo[4,3-b]pyridine
(12h). This compound was prepared from 3-amino-6-(3,4,5-
trimethylphenyl)pyridine-2-carbothioamide
affording
the
title
compound as an amorphous orange solid in 94% yield (112.1 mg,
0.35 mmol). 1H NMR (300 MHz, DMSO) δ: 3.73 (s, 3H, OCH3), 3.92 (s,
6H, 2 × OCH3), 7.54 (s, 2H, arom H), 7.78 (d, J = 9.4 Hz, 1H, arom H),
8.02 (d, J = 9.4 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
C
15H15N3O3S: 318.0907, found 318.0904.
7.1.5.9. 3-Amino-5-(3,5-dimethoxyphenyl)isothiazolo[4,3-b]pyridine
(12i). This
compound
was
prepared
from
3-amino-6-(3,5-
7.1.6.4. 3-Bromo-5-(3-chlorophenyl)isothiazolo[4,3-b]pyridine
dimethylphenyl)pyridine-2-carbothioamide affording the title compound
as an amorphous yellow solid in 93% yield (92.2 mg, 0.35 mmol). 1H
NMR (300 MHz, DMSO) δ: 3.85 (s, 6H, 2 × OCH3), 6.57 (bs, 1H, arom H),
7.40 (s, 2H, arom H), 7.77 (d, J = 9.4 Hz, 1H, arom H), 7.95 (d,
J = 9.4 Hz, 1H, arom H), 8.05 (bs, 2H, NH2) ppm. HR-MS m/z [M
+H]+ calcd for C14H13N3O2S: 288.0801, found 288.0803.
(13d). This compound was prepared from 3-amino-5-(3-chlorophenyl)
isothiazolo[4,3-b]pyridine and the residue was purified by flash
chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 8:2) as mobile phase, affording the title compound as an
amorphous white solid in 51% yield (25.4 mg, 0.08 mmol). 1H NMR
(300 MHz, CDCl3) δ: 7.41–7.45 (m, 2H, arom H), 7.87 (d, J = 9.4 Hz,
1H, arom H), 8.02–8.09 (m, 1H, arom H), 8.17 (d, J = 9.4 Hz, 1H, arom
H), 8.21 (s, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
7.1.6. Synthesis of 5-aryl-3-bromoisothiazolo[4,3-b]pyridines (13a-h)
General procedure A. A solution of 5-aryl-3-aminoisothiazolo[4,3-b]
pyridine (1 equiv) in HBr (30 mL) was stirred for 10 min at room
temperature, and then CuBr was added (2 equiv). The resulting mixture
was cooled at 0 °C. A solution of sodium nitrite (3 equiv) in H2O
(15 mL) was added dropwise over a period of 30 min. The reaction
mixture was stirred for 2 h at 0 °C and overnight at room temperature.
The mixture was cooled at 0 °C and carefully neutralized with a 2 N
NaOH solution. The mixture was extracted with ethyl acetate
(3 × 30 mL) and the combined organic layers were dried over anhy-
drous Na2SO4 and evaporated to dryness. The crude residue was pur-
ified by silica gel flash chromatography (using a mixture of hexane/
ethyl acetate as mobile phase), yielding the corresponding 5-aryl-3-
bromoisothiazolo[4,3-b]pyridine.
C
12H6BrClN2S: 324.9197, found 324.9191.
The following compounds were made according to the general
procedure B:
7.1.6.5. 3-Bromo-5-(4-methylphenyl)isothiazolo[4,3-b]pyridine
(13e). This compound was prepared from 3-amino-5-(4-methylphenyl)
isothiazolo[4,3-b]pyridine and the residue was purified by silica gel
flash chromatography using a mixture of hexane and ethyl acetate (in a
ratio of 9:1) as mobile phase, affording the title compound as an
amorphous white solid in 51% yield (25.5 mg, 0.08 mmol). 1H NMR
(300 MHz, CDCl3) δ: 2.44 (s, 3H, CH3), 7.34 (d, J = 8.1 Hz, 2H, arom
H), 7.89 (d, J = 9.4 Hz, 1H, arom H), 8.07–8.15 (m, 3H, arom H) ppm.
HR-MS m/z [M+H]+ calcd for C13H9BrN2S: 304.9743, found
304.9748.
General procedure B. To a stirred solution of CuBr2 (1.2 equiv) and 5-
aryl-3-aminoisothiazolo[4,3-b]pyridine (1 equiv) in dry acetonitrile
(15 mL) under argon atmosphere at −10 °C, was slowly added tert-
butylnitrite (1.3 equiv). The resulting mixture was stirred at 0 °C for 2 h
and additional 3 h at room temperature. After disappearance of the
starting material as monitored by TLC, the volatiles were evaporated
and the crude residue was purified by silica gel flash column chroma-
tography (using a mixture of hexane/ethyl acetate as eluent) yielding
the corresponding 5-aryl-3-bromoisothiazolo[4,3-b]pyridine.
The following compounds were made according to the general
procedure A:
7.1.6.6. 3-Bromo-5-(3-methoxyphenyl)isothiazolo[4,3-b]pyridine
(13f). This compound was prepared from 3-amino-5-(3-methoxyphenyl)
isothiazolo[4,3-b]pyridine and the residue was purified by silica gel flash
chromatography using a mixture of hexane and ethyl acetate (in a ratio
of 9:1) as mobile phase, affording the title compound as an amorphous
light yellow solid in 34% yield (62 mg, 0.19 mmol). 1H NMR (300 MHz,
CDCl3) δ: 3.93 (s, 3H, OCH3), 7.06 (dd, J = 8.0, 2.3 Hz, 1H, arom H),
7.44 (t, J = 8.0 Hz, 1H, arom H), 7.72 (d, J = 7.8 Hz, 1H, arom H),
7.77–7.81 (m, 1H, arom H), 7.89 (d, J = 9.4 Hz, 1H, arom H), 8.14 (d,
J = 9.4 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
7.1.6.1. 3-Bromo-5-(phenyl)isothiazolo[4,3-b]pyridine
(13a). This
C13H9BrN2S: 320.9692, found 320.9693.
compound was prepared from 3-amino-5-phenylisothiazolo[4,3-b]
pyridine and the residue was purified by flash chromatography using
a mixture of hexane and ethyl acetate (in a ratio of 9:1) as mobile
phase, affording the title compound as an amorphous white solid in
61% yield (39 mg, 0.13 mmol). 1H NMR (300 MHz, CDCl3) δ: 7.48–7.58
(m, 3H, arom H), 7.90 (d, J = 9.3 Hz, 1H, arom H), 8.11–8.22 (m, 3H,
arom H) ppm. HR-MS m/z [M+H]+ calcd for C12H7BrN2S: 290.9587,
found 290.9590.
7.1.6.7. 3-Bromo-5-(3,4-dimethoxyphenyl)isothiazolo[4,3-b]pyridine
(13g). This compound was prepared from 3-amino-5-(3,4-
dimethoxyphenyl)isothiazolo[4,3-b]pyridine and the residue was
purified by silica gel flash chromatography using a mixture of hexane
and ethyl acetate (in a ratio of 8:2) as mobile phase, affording the title
compound as an amorphous light yellow solid in 56% yield (58.2 mg,
0.17 mmol). 1H NMR (300 MHz, CDCl3) δ: 3.97 (s, 3H, OCH3), 4.04 (s,
3H, OCH3), 6.98 (d, J = 8.4 Hz, 1H, arom H), 7.68 (dd, J = 8.4, 2.1 Hz,
1H, arom H), 7.89 (dd, J = 6.6, 6.0 Hz, 2H, arom H), 8.09 (d,
J = 9.4 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
7.1.6.2. 3-Bromo-5-(4-trifluoromethylphenyl)isothiazolo[4,3-b]pyridine
(13b). This
compound
was
prepared
from
3-amino-5-(4-
trifluoromethylphenyl)isothiazolo[4,3-b]pyridine and the residue was
purified by flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 9:1) as mobile phase, affording the title compound as
an amorphous white solid in 57% yield (48.3 mg, 0.13 mmol). 1H NMR
(300 MHz, CDCl3) δ: 7.79 (d, J = 8.4 Hz, 2H, arom H), 7.91 (d, J = 9.3 Hz,
C14H11BrN2O2S: 350.9798, found 350.9796.
7.1.6.8. 3-Bromo-5-(3,4,5-trimethoxyphenyl)isothiazolo[4,3-b]pyridine
(13h). This compound was prepared from 3-amino-5-(3,4,5-
trimethoxyphenyl)isothiazolo[4,3-b]pyridine and the residue was
13

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
purified by silica gel flash chromatography using a mixture of hexane
and ethyl acetate (in a ratio of 7:3) as mobile phase, affording the title
compound as an amorphous light yellow solid in 35% yield (19.7 mg,
0.05 mmol). 1H NMR (300 MHz, CDCl3) δ: 3.93 (s, 3H, OCH3), 4.01 (s,
6H, 2 × OCH3), 7.42 (s, 2H, arom H), 7.87 (d, J = 9.4 Hz, 1H, arom H),
8.14 (d, J = 9.3 Hz, 1H, arom H) ppm. HR-MS m/z [M+H]+ calcd for
149.6 (C), 155.3 (C), 163.4 (d, J = 248.9 Hz, C), 172.50 (C) ppm. 19F
NMR (471 MHz, CDCl3) δ: −111.93 ppm. HR-MS m/z [M+H]+ calcd
for C18H18FN3OS: 344.1227, found 344.1235.
7.1.7.4. 5-(3-Chlorophenyl)-3-(cis-2,6-dimethylmorpholino)isothiazolo
[4,3-b]pyridine (14d). This compound was prepared from 3-bromo-5-
(3-chlorophenyl)isothiazolo[4,3-b]pyridine and the residue was
purified by silica gel flash chromatography using a mixture of hexane
and ethyl acetate (in a ratio of 4:1) as mobile phase, affording the title
compound as an amorphous yellow solid in 78% yield (34.5 mg,
0.09 mmol). Mp 160.3–161.9 °C. 1H NMR (600 MHz, CDCl3) δ: 1.33
(d, J = 6.3 Hz, 6H, 2 × CH3), 2.95–3.01 (m, 2H, 2 × NCH), 3.93–4.00
(m, 2H, 2 × OCH), 4.65 (d, J = 12.5 Hz, 2H, 2 × NCH), 7.36–7.43 (m,
2H, arom H), 7.67 (d, J = 9.4 Hz, 1H, arom H), 7.82–7.85 (m, 2H, arom
H), 8.00–8.02 (m, 1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ:
18.8 (CH3), 55.6 (CH2), 71.3 (CH), 120.7 (CH), 124.6 (CH), 126.9 (CH),
128.8 (CH), 129.7 (CH), 130.0 (CH), 134.8 (C), 134.9 (C), 140.8 (C),
148.8 (C), 155.4 (C), 172.7 (C) ppm. HR-MS m/z [M+H]+ calcd for
C
15H13BrN2O3S: 380.9903, found 380.9888.
7.1.7. Synthesis of 5-aryl-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-
b]pyridines (14a-h)
General procedure. To a solution of a 5-substituted-3-bromoisothia-
zolo[4,3-b]pyridine (1 equiv) in absolute ethanol (20 mL) was added
cis-2,6-dimethylmorpholine (3 equiv) and the mixture was stirred at
reflux overnight. After disappearance of the starting material as mon-
itored by TLC, the volatiles were evaporated to dryness and the residue
was purified by silica gel flash column chromatography yielding the 5-
substituted-3-(cis-2,6-dimethylmorpholino)isothiazolo[4,3-b]pyridine.
The following compounds were made according to this procedure:
C
18H18ClN3OS: 360.0932, found 360.0932.
7.1.7.1. 3-(cis-2,6-Dimethylmorpholino)-5-phenylisothiazolo[4,3-b]
pyridine (14a). This compound was prepared from 3-bromo-5-
phenylisothiazolo[4,3-b]pyridine. The residue was purified by silica
gel flash chromatography using a mixture of hexane and ethyl acetate
(in a ratio of 4:1) as mobile phase, affording the title compound as an
amorphous yellow solid in 91% yield (40.5 mg, 0.12 mmol). Mp
154.1–155.8 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz,
6H, 2 × CH3), 2.96 (dd, J = 12.5, 10.8 Hz, 2H, 2 × NCH), 3.92–4.00
(m, 2H, 2 × OCH), 4.66 (d, J = 11.6 Hz, 2H, 2 × NCH), 7.40–7.44 (m,
1H, arom H), 7.47–7.50 (m, 2H, arom H), 7.72 (d, J = 9.4 Hz, 1H, arom
H), 7.83 (d, J = 9.4 Hz, 1H, arom H), 7.97–8.01 (m, 2H, arom H) ppm.
13C NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 55.6 (CH2), 71.2 (CH), 121.2
(CH), 126.7 (CH), 128.8 (CH), 128.9 (CH), 129.5 (CH), 134.8 (C),
139.1, (C), 150.6 (C), 155.5 (C), 172.5 (C) ppm. HR-MS m/z [M+H]+
calcd for C18H19N3OS: 326.1322, found 326.1321.
7.1.7.5. 3-(cis-2,6-Dimethylmorpholino)-5-(4-methylphenyl)isothiazolo
[4,3-b]pyridine (14e). This compound was prepared from 3-bromo-5-
(4-methylphenyl)isothiazolo[4,3-b]pyridine. The residue was purified
by silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 4:1) as mobile phase, affording the title compound
as an amorphous orange solid in 88% yield (39.1 mg, 0.12 mmol). Mp
198.1–199.8 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
2 × CH3), 2.42 (s, 3H, CH3), 2.95 (dd, J = 12.6, 10.7 Hz, 2H,
2 × NCH), 3.93–4.00 (m, 2H, 2 × OCH), 4.66 (d, J = 11.2 Hz, 2H,
2 × NCH), 7.30 (d, J = 7.9 Hz, 2H, arom H), 7.70 (d, J = 9.4 Hz, 1H,
arom H), 7.82 (d, J = 9.4 Hz, 1H, arom H), 7.89 (d, J = 8.2 Hz, 2H,
arom H) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 21.3 (CH3),
55.6 (CH2), 71.2 (CH), 121.1 (CH), 126.6 (CH), 129.4 (CH), 129.5 (CH),
134.7 (C), 136.4 (C), 139.0 (C), 150.7 (C), 155.5 (C), 172.3 (C) ppm.
HR-MS m/z [M+H]+ calcd for C19H21N3OS: 340.1478, found
340.1473.
7.1.7.2. 5-(4-Trifluoromethylphenyl)-3-(cis-2,6-dimethylmorpholino)
isothiazolo[4,3-b]pyridine (14b). This compound was prepared from 3-
bromo-5-(4-trifluoromethylphenyl)isothiazolo[4,3-b]pyridine.
The
7.1.7.6. 3-(cis-2,6-Dimethylmorpholino)-5-(3-methoxyphenyl)isothiazolo
[4,3-b]pyridine (14f). This compound was prepared from 3-bromo-5-
(3-methoxyphenyl)isothiazolo[4,3-b]pyridine and the residue was
purified by silica gel flash chromatography using a mixture of hexane
and ethyl acetate (in a ratio of 7:3) as mobile phase, affording the title
compound as an amorphous yellow solid in 91% yield (40 mg,
0.11 mmol). Mp 146.2–147.9 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32
(d, J = 6.3 Hz, 6H, 2 × CH3), 2.97 (dd, J = 12.5, 10.8 Hz, 2H,
2 × NCH), 3.89 (s, 3H, OCH3), 3.93–4.00 (m, 2H, 2 × OCH), 4.67 (d,
J = 11.6 Hz, 2H, 2 × NCH), 6.98 (ddd, J = 8.2, 2.6, 0.7 Hz, 1H, arom
H), 7.40 (t, J = 7.9 Hz, 1H, arom H), 7.55–7.57 (m, 1H, arom H),
7.58–7.61 (m, 1H, arom H), 7.71 (d, J = 9.4 Hz, 1H, arom H), 7.83 (d,
J = 9.4 Hz, 1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8
(CH3), 55.2 (OCH3), 55.5 (CH2), 71.2 (CH), 111.9 (CH), 114.8 (CH),
119.2 (CH), 121.2 (CH), 129.5 (CH), 129.8 (CH), 134.7 (C), 140.5 (C),
150.3 (C), 155.5 (C), 160.0 (C), 172.5 (C) ppm. HR-MS m/z [M+H]+
calcd for C19H21N3O2S: 356.1427, found 356.1427.
residue was purified by silica gel flash chromatography using a
mixture of hexane and ethyl acetate (in a ratio of 7:3) as mobile
phase, affording the title compound as an amorphous yellow solid in
85% yield (37 mg, 0.09 mmol). Mp 238.2–239.7 °C. 1H NMR (600 MHz,
CDCl3) δ: 1.33 (d, J = 6.3 Hz, 6H, 2 × CH3), 2.99 (dd, J = 12.6,
10.7 Hz, 2H, 2 × NCH), 3.93–3.99 (m, 2H, 2 × OCH), 4.64 (d,
J = 11.8 Hz, 2H, 2 × NCH), 7.72 (d, J = 9.4 Hz, 1H, arom H), 7.74
(d, J = 8.2 Hz, 2H, arom H), 7.87 (d, J = 9.4 Hz, 1H, arom H), 8.09 (d,
J = 8.1 Hz, 2H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ: 18.9
(CH3), 55.6 (CH2), 71.2 (CH), 121.0 (CH), 124.2 (q, J = 272.0 Hz, C),
125.7 (bq, J = 3.2 Hz, CH), 126.9 (CH), 129.8 (CH), 130.6 (q,
J = 32.4 Hz, C), 135.0 (C), 142.5 (C), 148.9 (C), 155.3 (C), 173.0 (C)
ppm. 19F NMR (471 MHz, CDCl3) δ: −62.23 ppm. HR-MS m/z [M+H]+
calcd for C19H18F3N3OS: 394.1195, found 394.1200.
7.1.7.3. 5-(4-Fluorophenyl)-3-(cis-2,6-dimethylmorpholino)isothiazolo
[4,3-b]pyridine (14c). This compound was prepared from 3-bromo-5-
(4-fluorophenyl)isothiazolo[4,3-b]pyridine. The residue was purified
by silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 7:3) as mobile phase, affording the title compound
as an amorphous yellow solid in 91% yield (40 mg, 0.12 mmol). Mp
165.5–166.9 °C. 1H NMR (600 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 6H,
2 × CH3), 2.95 (dd, J = 12.5, 10.7 Hz, 2H, 2 × NCH), 3.92–3.99 (m,
2H, 2 × OCH), 4.62 (dd, J = 12.5, 1.3 Hz, 1H, 2 × NCH), 7.13–7.20
(m, 2H, arom H), 7.66 (d, J = 9.4 Hz, 1H, arom H), 7.83 (d, J = 9.4 Hz,
1H, arom H), 7.94–7.98 (m, 2H, arom H) ppm. 13C NMR (151 MHz,
CDCl3) δ: 18.8 (CH3), 55.6 (CH2), 71.2 (C), 115.70 (d, J = 21.6 Hz, CH),
120.9 (CH), 128.43 (d, J = 8.2 Hz, CH), 129.7 (C), 134.7 (C), 135.3 (C),
7.1.7.7. 3-(cis-2,6-Dimethylmorpholino)-5-(3,4-dimethoxyphenyl)
isothiazolo[4,3-b]pyridine (14g). This compound was prepared from 3-
bromo-5-(3,4-dimethoxyphenyl)isothiazolo[4,3-b]pyridine. The residue
was purified by silica gel flash chromatography using a mixture of
hexane and ethyl acetate (in a ratio of 7:3) as mobile phase, affording
the title compound as an amorphous yellow solid in 55% yield (24 mg,
0.06 mmol). Mp 200.4–201.7 °C. 1H NMR (600 MHz, CDCl3) δ: 1.31 (d,
J = 6.3 Hz, 6H, 2 × CH3), 2.95 (dd, J = 12.3, 10.8 Hz, 2H, 2 × NCH),
3.93–4.00 (m, 8H, 2 × OCH3, 2 × OCH), 4.64 (d, J = 11.4 Hz, 2H,
2 × NCH), 6.96 (d, J = 8.3, 1H, arom H), 7.52 (dd, J = 8.4, 2.0 Hz, 1H,
arom H), 7.68 (d, J = 2.0 Hz, 1H, arom H), 7.69–7.72 (m, 1H, arom H),
14

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
7.81 (d, J = 9.4 Hz, 1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ:
18.8 (CH3), 55.5 (CH2), 55.6 (OCH3), 56.0 (OCH3), 71.2 (CH), 109.4
(CH), 111.0 (CH), 119.4 (CH), 120.8 (CH), 129.5 (CH), 131.9 (C), 149.2
(C), 150.1 (C), 150.2 (C), 155.5 (C), 172.1 (C) ppm. HR-MS m/z [M
+H]+ calcd for C20H23N3O3S: 386.1533, found 386.1526.
7.1.8.3. 5-(3,5-Dimethoxyphenyl)-3-(N-morpholino)isothiazolo[4,3-b]
pyridine (15i). This compound was prepared from 3-amino-5-(3,5-
dimethoxyphenyl)isothiazolo[4,3-b]pyridine. The residue was purified
by silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 3:2) as mobile phase, affording the title compound
as an amorphous yellow solid in 71% yield (17.5 mg, 0.05 mmol). Mp
149.2–150.8 °C. 1H NMR (600 MHz, CDCl3) δ: 3.87 (s, 6H, 2 × OCH3),
3.96–3.99 (m, 4H, 2 × OCH2), 4.01–4.04 (m, 4H, 2 × NCH2), 6.54 (t,
J = 2.3 Hz, 1H, arom H), 7.14 (d, J = 2.3 Hz, 2H, arom H), 7.67 (d,
J = 9.4 Hz, 1H, arom H), 7.84 (d, J = 9.4 Hz, 1H, arom H) ppm. 13C
NMR (151 MHz, CDCl3) δ: 50.5 (CH2), 55.4 (OCH3), 66.2 (CH2), 100.7
(CH), 105.2 (CH), 121.5 (CH), 129.5 (CH), 134.9 (C), 141.3 (C), 150.7
(C), 155.5 (C), 161.1 (C), 173.0 (C) ppm. HR-MS m/z [M+H]+ calcd
for C18H19N3O3S: 358.1220, found 358.1221.
7.1.7.8. 3-(cis-2,6-Dimethylmorpholino)-5-(3,4,5-trimethoxyphenyl)
isothiazolo[4,3-b]pyridine (14h). This compound was prepared from 3-
bromo-5-(3,4,5-trimethoxyphenyl)isothiazolo[4,3-b]pyridine.
The
residue was purified by silica gel flash chromatography using a
mixture of hexane and ethyl acetate (in a ratio of 3:2) as mobile
phase, affording the title compound as an amorphous yellow solid in
80% yield (34.8 mg, 0.08 mmol). Mp 196.3–198.1 °C. 1H NMR
(600 MHz, CDCl3) δ: 1.31 (d, J = 6.3 Hz, 6H, 2 × CH3), 2.94–3.00
(m, 2H, 2 × NCH), 3.92 (s, J = 2.5 Hz, 3H, OCH3), 3.95–3.99 (m, 9H,
2 × OCH, 2 × OCH3), 4.64–4.68 (m, 2H, 2 × NCH), 7.27 (s, 2H, arom
H), 7.69 (d, J = 9.4 Hz, 1H, arom H), 7.84 (d, J = 9.4 Hz, 1H, arom H)
ppm. 13C NMR (151 MHz, CDCl3) δ: 18.8 (CH3), 55.5 (CH2), 56.1
(OCH3), 61.0 (OCH3), 71.2 (CH), 104.0 (CH), 121.0 (CH), 129.6 (CH),
134.7 (C), 139.2 (C), 150.2 (C), 153.5 (C), 155.4 (C), 172.3 (C) ppm.
HR-MS m/z [M+H]+ calcd for C21H25N3O4S: 416.16384, found
416.1636.
7.2. GAK LanthaScreen™ Eu binding assay
The compounds were subjected to a LanthaScreen™ binding assay in
which 10 titrations of dissolved test compound in DMSO are transferred
to a 384-well plate. Sequential addition of the kinase buffer (50 mM
HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl and 1 mM EGTA), the 2X
kinase antibody (Eu Anti GST) mixture and the 4X Tracer 222 solution
was performed. After shaking for 30 s and a one hour incubation period
at room temperature, the plate was read on a fluorescence plate reader.
When the bound tracer in the active site was displaced by the test
compound, fluorescence was not observed. The collected data were
then compared to a 0% displacement control with pure DMSO and a
100% displacement control with staurosporine, a known inhibitor of
GAK and plotted against the logarithmic concentration parameter. The
IC50 was subsequently extracted.
7.1.8. Synthesis of 5-aryl-3-(N-morpholino)isothiazolo[4,3-b]pyridines
General procedure. To a solution of 3-amino-5-arylisothiazolo[4,3-b]
pyridine (1 equiv) in DMF (15 mL), was added K2CO3 (2 equiv) and 2-
bromoethyl ether (0.7 equiv). The reaction mixture was stirred at
100 °C for 4–5 h. After disappearance of the starting material as mon-
itored by TLC, the volatiles were evaporated to dryness and the residue
was purified by silica gel flash chromatography, yielding the corre-
sponding 5-aryl-3-(N-morpholino)isothiazolo[4,3-b]pyridines. The fol-
lowing compounds were made according to this procedure:
7.3. Antiviral assays
Virus construct. DENV2 (New Guinea C strain)29,30 Renilla reporter
plasmid used for the antiviral assays was a gift from Pei-Yong Shi (The
University of Texas Medical Branch).
7.1.8.1. 5-(3-Methoxyphenyl)-3-(N-morpholino)isothiazolo[4,3-b]
pyridine (15f). This compound was prepared from 3-amino-5-(3-
methoxyphenyl)isothiazolo[4,3-b]pyridine. The residue was purified
by silica gel flash chromatography using a mixture of hexane and
ethyl acetate (in a ratio of 7:3) as mobile phase, affording the title
compound as an amorphous yellow solid in 77% yield (24.5 mg,
0.08 mmol). Mp 169.1–170.6 °C. 1H NMR (600 MHz, CDCl3) δ: 3.89
(s, 3H, OCH3), 3.97–4.00 (m, 4H, 2 × OCH2), 4.02–4.04 (m, 4H,
2 × NCH2), 6.98 (ddd, J = 8.2, 2.5, 0.9 Hz, 1H, arom H), 7.40 (t,
J = 7.9 Hz, 1H, arom H), 7.55–7.58 (m, 2H, arom H), 7.71 (d,
J = 9.4 Hz, 1H, arom H), 7.85 (d, J = 9.4 Hz, 1H, arom H) ppm. 13C
NMR (151 MHz, CDCl3) δ: 50.5 (CH2) 55.3 (OCH3), 66.2 (CH2), 112.6
(CH), 114.3 (CH), 119.4 (CH), 121.4 (CH), 129.6 (CH), 129.8 (CH),
135.0 (C), 140.6 (C), 150.8 (C), 155.5 (C), 160.0 (C), 173.0 (C) ppm.
HR-MS m/z [M+H]+ calcd for C17H17N3O2S: 328.1114, found
328.1111.
Cells. Huh7 (Apath LLC) cells were grown in DMEM (Mediatech)
supplemented with 10% FBS (Omega Scientific), nonessential aminoa-
cids, 1% L-glutamine, and 1% penicillin-streptomycin (Thermo-Fisher
Scientific) and maintained in a humidified incubator with 5% CO2 at
37 °C.
Virus Production. DENV2 RNA was transcribed in vitro using
mMessage/mMachine (Ambion) kits. DENV was produced by electro-
porating RNA into BHK-21 cells, harvesting supernatants on day 10 and
titering via standard plaque assays on BHK-21 cells. Virus titers were
determined via standard plaque assay on Vero76 cells.
Infection assays. Huh7 cells were infected with DENV in replicates
(n = 3–10) for 4 h at MOI of 0.05. Overall infection was measured at
48 h by standard luciferase assays.
Viability assays. Viability was assessed using AlamarBlue® reagent
(Invitrogen) according to manufacturer's protocol. Fluorescence was
detected at 560 nm on InfiniteM1000 plate reader.
7.1.8.2. 5-(3,4-Dimethoxyphenyl)-3-(N-morpholino)isothiazolo[4,3-b]
pyridine (15g). This compound was prepared from 3-amino-5-(3,4-
dimethoxyphenyl)isothiazolo[4,3-b]pyridine. The residue was purified
by silica gel flash chromatography using a mixture of hexane and ethyl
acetate (in a ratio of 3:2) as mobile phase, affording the title compound
as an amorphous yellow solid in 73% yield (18.1 mg, 0.05 mmol). Mp
151.2–152.9 °C. 1H NMR (600 MHz, CDCl3) δ: 3.95 (s, 3H, OCH3),
3.97–4.00 (m, 7H, OCH3, 2 × OCH2), 4.01–4.04 (m, 4H, 2 × NCH2),
6.98 (d, J = 8.3 Hz, 1H, arom H), 7.55 (dd, J = 8.3, 2.1 Hz, 1H, arom
H), 7.62 (d, J = 2.1 Hz, 1H, arom H), 7.70 (d, J = 9.4 Hz, 1H, arom H),
7.84 (d, J = 9.4 Hz, 1H, arom H) ppm. 13C NMR (151 MHz, CDCl3) δ:
50.6 (CH2), 55.8 (OCH3), 56.0 (OCH3), 66.2 (CH2), 109.8 (CH), 111.1
(CH), 119.7 (CH), 121.1 (CH), 129.6 (CH), 132.1 (C), 135.0 (C), 149.2
(C), 150.2 (C), 150.8 (C), 155.4 (C), 172.6 (C) ppm. HR-MS m/z [M
+H]+ calcd for C18H19N3O3S: 358.1220, found 358.1220.
Declaration of Competing Interest
None.
Acknowledgments
This work was supported by award number W81XWH-16-1-0691
from the Department of Defense (DoD), Congressionally Directed
Medical Research Programs (CDMRP) to S.E, P.H; Grant 12393481 from
the Defense Threat Reduction Agency (DTRA), Fundamental Research
to Counter Weapons of Mass Destruction to S.E. and P.H.
15

B. Martinez-Gualda, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxxx
Appendix A. Supplementary material
Brannan JM, et al. J Med Chem. 2018;61:6178–6192.
16. Li J, Kovackova S, Pu SY, et al. Isothiazolo[4,3-b]pyridines as inhibitors of cyclin G
associated kinase: synthesis, structure–activity relationship studies and antiviral ac-
tivity. Med Chem Comm. 2015;6:1666–1672.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.115188.
17. Wouters R, Pu SY, Froeyen M, et al. Cyclin G-associated kinase (GAK) affinity and
antiviral activity studies of a series of 3-C-substituted isothiazolo[4,3-b]pyridines.
Eur J Med Chem. 2019;163:256–265.
References
18. Wouters R, Tian J, Herdewijn P, De Jonghe S. A scaffold-hopping strategy toward the
identification of inhibitors of cyclin G associated kinase. ChemMedChem.
2018;13:1–19.
1. Sorrell FJ, Szklarz M, Abdul Azeez KR, Elkins JM, Knapp S. Family-wide structural
analysis of human Numb-associated protein kinases. Structure. 2016;24:401–411.
2. Edeling MA, Smith C, Owen D. Life of a clathrin coat: insights from clathrin and AP
structures. Nat Rev Mol Cell Biol. 2006;7:32–44.
19. Cui X, Zhou Y, Wang N, Liu L, Guo QX. N-Phenylurea as an inexpensive and efficient
ligand for Pd-catalyzed Heck and room-temperature Suzuki reactions. Tetrahedron
Lett. 2007;48:163–167.
20. Hanthorn JJ, Valgimigli L, Pratt DA. Preparation of highly reactive pyridine- and
pyrimidine-containing diarylamine antioxidants. J Org Chem. 2012;77:6908–6916.
21. Zhongzhong Y, Aiping L, Mingzhi H, et al. Design, synthesis, DFT study and anti-
fungal activity of the derivatives of pyrazolecarboxamide containing thiazole or
oxazole ring. Eur J Med Chem. 2018;149:170–181.
3. Zhang CX, Engqvist-Goldstein AEY, Carreno S, Owen DJ, Smythe E, Drubin DG.
Multiple roles for cyclin G-associated kinase in clathrin-mediated sorting events.
Traffic. 2005;6:1103–1113.
4. Korolchuk VI, Banting G. CK2 and GAK/auxilin2 are major protein kinases in cla-
thrin-coated vesicles. Traffic. 2002;3:428–439.
22. Subhash LY, Denvert SD, Bhalchandra MB. MnO2 catalyzed formylation of amines
and transamidation of amides under solvent-free conditions. RSC Adv.
2015;5:80441–80449.
5. Ghosh P, Kornfeld S. AP-1 binding to sorting signals and release from clathrin-coated
vesicles is regulated by phosphorylation. J Cell Biol. 2003;160:699–708.
6. Umeda A, Meyerholz A, Ungewickell E. Identification of the universal cofactor
(auxilin 2) in clathrin coat dissociation. Eur J Cell Biol. 2000;79:336–342.
7. Neveu G, Barouch-Bentov R, Ziv-Av A, Gerber D, Jacob Y, Einav S. Identification and
targeting of an interaction between a tyrosine motif within hepatitis C virus core
protein and AP2M1 essential for viral assembly. PLoS Pathog. 2012;8:e1002845.
8. McMahon HT, Boucrot E. Molecular mechanism and physiological functions of cla-
thrin-mediated endocytosis. Nat Rev Mol Cell Biol. 2011;12:517–533.
9. Bekerman E, Neveu G, Shulla A, et al. Anticancer kinase inhibitors impair in-
tracellular viral trafficking and exert broad-spectrum antiviral effects. J Clin Invest.
2017;127:1338–1352.
23. Striela R, Urbelis G, Sudzius J, Stoncius S, Sadzeviciene R, Labanauskas L. Synthesis
of bromocyclopropylpyridines via the Sandmeyer reaction. Tetrahedron Lett.
2017;58:1681–1683.
24. Lebakken CS, Riddle SM, Singh U, Frazee WJ, Eliason HC, et al. Development and
applications of a broad-coverage, TR-FRET-based kinase binding assay platform. J
Biomol Screening. 2009;14:924–935.
25. Morris GM, Huey R, Lindstrom W, et al. Autodock4 and AutoDockTools4: automated
docking with selective receptor flexibility. J Comput Chem. 2009;16:2785–2791.
26. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with
a new scoring function, efficient optimization and multithreading. J Comput Chem.
2010;31:455–461.
10. Neveu G, Ziv-Av A, Barouch-Bentov R, Berkerman E, Mulholland J, Einav S. AP-2-
associated protein kinase 1 and cyclin G-associated kinase regulate hepatitis C virus
entry and are potential drug targets. J Virol. 2015;8:4387–4404.
27. Bandaru SSM, Kapdi AR, Schulzkea C. Crystal structure of 4-(pyrazin-2-yl)morpho-
line. Acta Crystallogr E Crystallogr Commun. 2018;74:137–140.
11. Bekerman E, Einav S. Infectious disease. Combating emerging viral threats. Science.
2015;348:282–283.
28. Quintanilla-Licea R, Colunga-Valladares JF, Caballero-Quintero A, et al. NMR
Detection of isomers arising from restricted rotation of the C-N amide bond of N-
formyl-o-toluidine and N,N’-bis-formyl-o-tolidine. Molecules. 2002;7:662–673.
29. Xie X, Gayen S, Kang C, Yuan Z, Shi P-Y. Membrane topology and function of dengue
virus NS2A protein. J Virol. 2013;87:4609–4622.
12. Asquith CRM, Laitinen T, Bennett JM, et al. Identification and optimization of 4-
anilinoquinolines as inhibitors of cyclin G associated kinase. ChemMedChem.
2018;13:48–66.
13. Asquith CRM, Berger BT, Wan J, et al. SGC-GAK-1: a chemical probe for cyclin G
associated kinase (GAK). J Med Chem. 2019;62:2830–2836.
30. Zou G, Xu HY, Qing M, Wang QY, Shi PY. Development and characterization of a
stable luciferase dengue virus for high-throughput screening. Antivir Res.
2011;91:11–19.
14. Kovackova S, Chang L, Bekerman E, et al. Selective inhibitors of cyclin G associated
kinase (GAK) as anti-hepatitis C agents. J Med Chem. 2015;58:3393–3410.
15. Pu SY, Wouters R, Schor S, Rozenski J, Barouch-Bentov R, Prugar LI, O’Brien CM,
16