Optimization of Isothiazolo[4,3- b]pyridine-Based Inhibitors of Cyclin G Associated Kinase (GAK) with Broad-Spectrum Antiviral Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b00613

There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo[4,3-b]pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo[4,3-b]pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections.

Full text of DOI:10.1021/acs.jmedchem.8b00613

Subscriber access provided by - Access paid by the | UCSB Libraries
Article
Optimization of isothiazolo[4,3-b]pyridine-based inhibitors of cyclin
G associated kinase (GAK) with broad-spectrum antiviral activity
Szu-Yuan Pu, Randy Wouters, Stanford Schor, Jef Rozenski, Rina Barouch-Bentov, Laura I Prugar,
Cecilia M Obrien, Jennifer M Brannan, John M. Dye, Piet Herdewijn, Steven De Jonghe, and Shirit Einav
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00613 • Publication Date (Web): 28 Jun 2018
Downloaded from http://pubs.acs.org on July 1, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Optimization of isothiazolo[4,3ꢀb]pyridineꢀbased inhibitors of cyclin G associated kinase
(GAK) with broadꢀspectrum antiviral activity
#,†
§,†
#
§
#
§
SzuꢀYuan Pu, Randy Wouters, Stanford Schor, Jef Rozenski, Rina Barouch Bentov,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
◊
◊
◊
◊
Laura I. Prugar, Cecilia M. O’Brien, Jennifer M. Brannan, John M. Dye, Piet Herdewijn,
§
,*
#,*
Steven De Jonghe, Shirit Einav
#
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and
Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, California 94305, USA.
§
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49 – bus
1041, 3000 Leuven, Belgium.
◊
US Army Medical Research Institute of Infectious Diseases, Viral Immunology Branch, Fort
Detrick, Maryland 21702, USA.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
There is an urgent need for strategies to combat dengue and other emerging viral infections.
We reported that cyclin Gꢀassociated kinase (GAK), a cellular regulator of the clathrinꢀ
associated host adaptor proteins APꢀ1 and APꢀ2, regulates intracellular trafficking of multiple
unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the
discovery of potent, selective GAK inhibitors based on an isothiazolo[4,3ꢀb]pyridine scaffold,
albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery
of novel isothiazolo[4,3ꢀb]pyridines that maintain high GAK affinity and selectivity. These
compounds demonstrate improved in vitro activity against dengue virus, including in human
primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses.
Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral
action of these compounds. These findings demonstrate the potential utility of a GAKꢀ
targeted broadꢀspectrum approach for combating currently untreatable emerging viral
infections.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Emerging viral infections, such as those caused by dengue (DENV), Ebola (EBOV) and
chikungunya (CHIKV) viruses, represent major threats to global health. DENV is estimated to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
infect 390 million people annually in over 100 countries. The majority of individuals infected
with any of the four DENV serotypes remain asymptomatic or present with acute dengue
2
fever. A fraction (~5ꢀ20%) of dengue patients, particularly those secondarily infected with a
heterologous DENV serotype, will progress to severe dengue, manifested by bleeding, plasma
leakage, shock, organ failure, and death. The development of an effective vaccine for DENV
has been hampered by the need to generate simultaneous protection against the four distinct
DENV serotypes to avoid antibodyꢀdependent enhancement (ADE), with recent data
3
indicating an increase in dengue severity requiring hospitalization in vaccinated children.
4
–6
EBOV is the causative agent of a severe and often fatal hemorrhagic disease
.
The
unprecedented scope of the 2013ꢀ2016 Ebola virus disease (EVD) epidemic in western Africa
highlighted the need for effective medical countermeasures against this emerging infectious
7
disease. CHIKV is a reꢀemerging alphavirus that has been causing massive outbreaks in
8
various parts of Africa, Asia and more recently in Central and South America. There are
currently no vaccines available for the prevention of CHIKV infection. While an EBOV
9
vaccine has shown promise recently, it is not yet approved. Importantly, no effective antiviral
treatment is available against DENV, EBOV, CHIKV, and most other emerging viral
pathogens.
The majority of the currently approved antiviral drugs target viral enzymatic functions and
thus typically have a narrow spectrum of coverage and a low genetic barrier to resistance. An
attractive approach to overcome these limitations is to develop compounds that target host
10
factors broadly required for the effective replication of multiple viral pathogens. Such a
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hostꢀtargeted broadꢀspectrum approach is more scalable to address the large unmet clinical
need and is particularly attractive for the treatment of emerging viral infections lacking any
10
treatment.
Intracellular membrane trafficking is one of multiple cellular processes usurped by viruses.
Cyclin Gꢀassociated kinase (GAK) is a ubiquitously expressed host cell kinase that regulates
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
clathrinꢀmediated intracellular trafficking of cellular cargo proteins. GAK is a 160 kDa
serine/threonine kinase belonging to the numbꢀassociated kinase (NAK) family, which also
includes adaptorꢀassociated kinase 1 (AAK1), BMPꢀ2ꢀinducible kinase (BIKE/BMP2K) and
myristoylated and palmitoylated serine/threonine kinase 1 (MPSK1/STK16). Clathrinꢀ
mediated membrane trafficking is dependent on the action of oligomeric clathrin and adaptor
protein complexes (APs) that coordinate the specific recruitment and assembly of clathrin into
1
2–14
clathrinꢀcoated vesicles (CCVs) as well as its coupling to endocytic cargo.
The
heterotetrametic APꢀ1 and APꢀ2 complexes are major components of CCVs, responsible for
12,15
vesicle formation in the trans-Golgi network (TGN) and plasma membrane, respectively.
The µ subunit of these AP complexes recognizes and binds sorting signals in the form of
tyrosineꢀ or dileucineꢀbased motifs present on cargo molecules. The efficiency of cargo
protein binding to APs is regulated via phosphorylation of a threonine residue within their µ
subunit (Thr 144 in APꢀ1 and Thr 156 in APꢀ2), which induces a conformational
1
2,16,17
change.
cargo recruitment and vesicle assembly.
recruitment of clathrin and APꢀ2 to the plasma membrane, and GAK also controls the
Both GAK and AAK1 phosphorylate these Thr residues, thereby enhancing
18–21
Additionally, these kinases regulate the
12,14
uncoating of CCVs, enabling recycling of clathrin back to the cell surface.
We have previously demonstrated that depletion of GAK by siRNAs or ectopic expression of
phosphorylation mutants of APꢀ1 and APꢀ2 are dispensable for RNA replication of hepatitis C
virus (HCV), but significantly inhibit four temporally distinct steps of the HCV lifecycle,
ACS Paragon Plus Environment

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
namely entry (APꢀ2), assembly (APꢀ2), release of cellꢀfree virus and direct cellꢀtoꢀcell spread
14–16,22
(APꢀ1).
Moreover, we discovered a requirement for GAK in early and late stages of the
lifecycle of other viruses, including DENV and EBOV, thereby validating GAK as a potential
15
cellular target for the development of broadꢀspectrum antiviral agents. Indeed, erlotinib
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(compound 1, Figure 1), an approved anticancer drug with potent, yet nonꢀselective, antiꢀ
GAK activity (dissociation constant (Kd) value of 3.1 nM) inhibits replication of multiple
15
viruses from six unrelated viral families in cell culture. Combinations of erlotinib and
sunitinib, an approved anticancer drug with potent antiꢀAAK1 activity, protected mice from
1
5
morbidity and mortality associated with DENV and EBOV infections. These data provide a
proofꢀofꢀconcept that smallꢀmolecule inhibitors of GAK and AAK1 are useful candidates for
broadꢀspectrum antiviral therapy. The safety and efficacy of erlotinib and/or sunitinib will be
evaluated in DENV infected patients in the near future and potentially in patients with EVD in
future outbreaks (ClinicalTrials.gov NCT02380625). Nevertheless, erlotinib is a potent, yet
nonꢀselective inhibitor whose side effects result not only from on target inhibition of EGFR
2
3–25
but also from inhibition of other cellular kinases and nonꢀkinase proteins.
A particularly
relevant side effect that often complicates combination regimens of erlotinib with sunitinib is
26
diarrhea, attributed in part to inhibition erlotinib’s cancer target, EGFR. This side effect may
27
limit erlotinib’s use in patients with EVD, who often present with diarrhea. To overcome
this challenge and avoid other potential toxicity, we have sought to develop novel, chemically
distinct and more selective inhibitors of GAK.
To the best of our knowledge, only two series of potent and selective GAK inhibitors have
been reported to date. The first, represented by 4ꢀanilinoquinoline analogue 2 (Figure 1),
displays low nM GAK inhibition, has over 50,000 fold greater selectivity to GAK than to
other members of the NAK subfamily of kinases, and shows selective GAK inhibition when
2
8
tested against a large panel of kinases. Nevertheless, this series has not been tested for its
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
antiviral activity. We have previously reported the discovery and synthesis of isothiazolo[4,3ꢀ
2
9
b]pyridines, a second, structurally distinct class of selective GAK inhibitors. The two most
prominent derivatives within this series (compounds 3 and 4, Figure 1) have a high GAK
29
affinity (Kd = 8 nM). We reported that these isothiazolo[4,3ꢀb]pyridines have antiviral
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
activity against both HCV and DENV, yet the measured 50% effective concentration (EC50
)
15,29
values were at a micromolar range.
Here, we describe our efforts to optimize the antiviral
activity of these isothiazolo[4,3ꢀb]pyridines, while maintaining their potency and selectivity
to GAK.
Figure 1. Known GAK inhibitors
Results and discussion
Synthesis of isothiazolo[4,3-b]pyridines
To synthesize isothiazolo[4,3ꢀb]pyridine derivatives, a known procedure was followed with
29,30
minor modifications (Scheme 1).
First, the nitro group of 3ꢀnitroꢀ5ꢀbromopyridineꢀ2ꢀ
carbonitrile 5 was reduced by treatment with iron under acidic conditions, generating the
desired aniline 6 as the major product. However, due to the acid hydrolysis of the cyano
group, a substantial amount (12%) of 3ꢀaminoꢀ5ꢀbromopicolinamide was also formed. These
two compounds were easily separated by silica gel flash chromatography. Thionation of 6
using phosphorus pentasulfide yielded thioamide 7 in moderate yield. Although this
procedure was successful on a small scale (< 100 mg), upon upscaling, the starting material 6
was not completely consumed, drastically reducing the reaction yield. Substituting
ACS Paragon Plus Environment

Page 7 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
phosphorus pentasulfide by Lawesson’s reagent allowed isolation of 3ꢀaminoꢀ5ꢀ
bromopyridineꢀ2ꢀcarbothioamide 7 in good yield at a larger (gram) scale. The isothiazole
moiety was formed by an oxidative ring closure using hydrogen peroxide in methanol,
generating 3ꢀaminoꢀ6ꢀbromoꢀisothiazolo[4,3ꢀb]pyridine 8. The subsequent Sandmeyer
reaction of the exocyclic amino group with sodium nitrite, hydrogen bromide and CuBr
yielded the key intermediate 3,6ꢀdibromoꢀisothiazolo[4,3ꢀb]pyridine 9 in moderate yield.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Workꢀup of this reaction was initially done by careful addition of solid K CO to the reaction
2
3
mixture. Since time consuming on a larger scale, this step was substituted with neutralization
with a 30% aqueous NaOH solution. Various nitrogenꢀnucleophiles were introduced at
position 3 of the isothiazolo[4,3ꢀb]pyridine scaffold, generating compounds 10aꢀt in yields
ranging from 25 to 97%. Subsequently, Suzuki coupling of compounds 10aꢀs using 4ꢀaminoꢀ
3ꢀmethoxyphenylboronic acid pinacol ester, potassium carbonate as a base and
tetrakis(triphenylphosphine)palladium(0) as a catalyst in a mixture of dioxane/water yielded
compound 11 and a series of isothiazolo[4,3ꢀb]pyridines 12aꢀs. Finally, acidic cleavage of the
Boc protecting group of compound 11 furnished compound 12t, giving rise to a small library
of isothiazolo[4,3ꢀb]pyridines 12aꢀt (Table 1), which was then evaluated biologically.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Reagents and conditions: a) Fe, CH
3
COOH, rt; b) Lawesson’s reagent, EtOH,
reflux; c) 30% aq. H , MeOH, 0 °C; d) NaNO
2
O
2
2
, HBr, CuBr, H
2
O, 0 °C to rt; e) R
3
H, EtOH
CO
or nꢀBuOH, reflux; f) 4ꢀaminoꢀ3ꢀmethoxyphenylboronic acid pinacol ester, K
Pd(PPh , dioxane/water, 100 °C; g) 37% HCl, dioxane, rt.
GAK binding affinity studies
2
3
,
3 4
)
All compounds were tested for GAK binding affinity using the KINOMEscan™ platform,
which quantitatively measures the ability of a compound to compete with an immobilized
3
1
activeꢀsiteꢀdirected ligand. Compound 4 was used as a positive control, as it has a potent
29
GAK affinity (Kd = 8 nM). Prior work revealed that position 3 of the isothiazolo[4,3ꢀ
b]pyridine scaffold can tolerate structural modifications without impairing GAK affinity.
2
9
30
30
Various amines , alkoxy and carboxamide groups have thus been inserted at this position.
Indeed, many of these molecules demonstrated high affinity to GAK (low µM range), with 3ꢀ
Nꢀmorpholinoꢀisothiazolo[4,3ꢀb]pyridines emerging as the most potent derivatives within this
series. In combination with the appropriate aryl substituent (e.g. 3,4,5ꢀtrimethoxyphenyl, 3ꢀ
methoxyꢀ4ꢀaminophenyl) at position 6, these compounds demonstrated Kd values in a low
ACS Paragon Plus Environment

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
nM range. We therefore focused on the synthesis and biological evaluation of isothiazolo[4,3ꢀ
b]pyridine analogues bearing substituents at position 3 that closely resemble morpholine. The
aryl moiety at position 6 was fixed as a 4ꢀaminoꢀ3ꢀmethoxyphenyl residue, since this was
shown to be optimal for GAK affinity and antiviral activity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The isosteric replacement of the oxygen atom of the morpholino ring of compound 4 by a
carbon gave rise to the 3ꢀNꢀpiperidine analogue 12a. Compound 12a displayed reasonable
affinity to GAK (Kd = 0.97 µM), albeit 100 fold lower than that of compound 4 (Kd = 0.0089
µM). A number of substituents at position 4 of the piperidinyl moiety were introduced.
Whereas the 4,4ꢀdifluoropiperidine (compound 12b), 4ꢀaminopiperidine (compound 12t) and
4ꢀcarboxamidepiperidine (compound 12c) derivatives exhibit GAK Kd values exceeding 1
µM, the 4ꢀcyano analog (compound 12d) has a quite potent GAK affinity (Kd = 0.25 µM).
Remarkably, the 3ꢀcarboxamide congener (compound 12e) demonstrates potent affinity for
GAK (Kd = 0.16 µM), yet its 4ꢀsubstituted congener 12c is much less active. Replacing the
morpholino oxygen with a sulphon moiety yielded compound 12f, which is 20ꢀfold less active
as a GAK ligand relative to the morpholino containing derivative 4. We have previously
demonstrated that the presence of a piperazine ring in place of morpholine reduced GAK
29
affinity, suggesting that a basic nitrogen atom is not tolerated at this position. A lactam
containing analogue (compound 12h) was therefore prepared, with potent binding affinity to
GAK (Kd = 0.052 µM). Since the morpholine oxygen seems to play a crucial role in
mediating GAK affinity, it was kept fixed, whereas the morpholine nitrogen was changed to
an externally secondary amine group. The resulting compound 12g bound GAK with a Kd of
80 nM.
Expansion of the sixꢀmembered morpholino moiety towards a sevenꢀmembered
homomorpholino derivative yielded compound 12i. This 1,4ꢀoxazepine derivative is 20ꢀfold
less potent as a GAK ligand than the morpholine analogue (compound 4). Additionally, using
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a spirocyclic oxetane, a wellꢀknown isoster for morpholine, compound 12j was obtained, with
a GAK Kd value of 0.17 µM. Surprisingly, the bridged morpholine derivative 12k (with an 8ꢀ
oxaꢀ3ꢀazaꢀbicyclo[3.2.1]octane moiety) displayed potent GAK affinity (Kd = 61 nM). Since
this suggested a positive effect of aliphatic substituents, the SAR of the morpholine ring was
further explored by introducing methyl groups at various positions of the morpholine moiety.
Introducing a single methyl group at position 2 yielded compound 12l as a racemic mixture.
This compound demonstrated potent GAK affinity (Kd = 46 nM). The enantiopure
compounds 12m and 12n were thus prepared using optically pure building blocks. The two
enantiomers demonstrated very similar Kd values (26 nM and 19 nM, respectively). To avoid
the racemic mixtures and the high cost of enantiopure building blocks, a geminal dimethyl
group was introduced at position 2 of the morpholine ring, yielding compound 12o as a potent
GAK ligand (Kd = 35 nM). The stereochemistry of the 3ꢀmethyl substituted morpholine
analogues demonstrated a strong impact on GAK binding, with the Sꢀ3ꢀmethylmorpholine
derivative (compound 12p) showing a Kd value of 18 nM, and the corresponding Rꢀisomer
(compound 12q) demonstrating a 40ꢀfold reduction in GAK affinity. To expand the SAR at
position 2 of the morpholine moiety, two 2,6ꢀdimethylꢀsubstituted morpholine derivatives
were prepared. Both the cis (compound 12r) and trans (compound 12s) diastereoisomers
demonstrated potent GAK affinity (Kd values of 89 nM and 11 nM, respectively). Xꢀray
crystallography has previously demonstrated that compound 4 bound to the ATP binding site
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
of GAK according to a ‘type I’ binding mode. Given the close structural similarity between
the most potent congeners of the current series and compound 4, we predict that their mode of
binding to GAK is similar.
Anti-DENV activity of isothiazolo[4,3-b]pyridines
ACS Paragon Plus Environment

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
All the synthesized derivatives were tested for their activity against DENV, independently of
their affinity to GAK. Human hepatoma (Huh7) cells infected with DENV2 (New Guinea C
3
2,33
strain) harboring a luciferase reporter
hours. Antiviral activity (EC50 and EC90) was measured via luciferase assays. Cytotoxicity
CC50) was measured in the same cell culture wells via AlamarBlue assays (Table 1). In
were treated with the individual compounds for 48
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
general, isothiazolo[4,3ꢀb]pyridines demonstrating GAK binding displayed a doseꢀdependent
inhibition of DENV infection. The 3ꢀNꢀpiperidinyl isothiazolo[4,3ꢀb]pyridine analogues,
which displayed GAK Kd values in the range of 0.16 – 1.6 µM (compounds 12a, 12t, 12d and
12e), inhibited DENV with EC50 values ranging from 0.18 to 6.38 µM, albeit compound 12d
was more cytotoxic. As predicted, inactive GAK ligands with substitutions at the piperidinyl
ring (compounds 12b, 12c) demonstrated no antiviral activity. Nevertheless, the introduction
of other saturated heterocycles at position 3, such as a tetrahydroꢀ2Hꢀpyranꢀ4ꢀamine
(compound 12g) and a piperazinylꢀ2ꢀone (compound 12h), yielded analogues with potent
GAK affinity (Kds<100 nM), yet with limited antiviral activity (EC50> 20 µM and 4.03 µM,
respectively). Among the series of isothiazolo[4,3ꢀb]pyridines, which bear a closely related
analogue of morpholine as substituent at position 3, the homomorpholino (compound 12i) and
2ꢀoxaꢀ6ꢀazaspiro[3.3]heptane (compound 12j) analogues are the least active GAK binders.
Accordingly, these compounds demonstrated a moderate antiviral activity (EC50 values of 2
µM and 7.12 µM, respectively). The majority of the 3ꢀNꢀmorpholinylꢀisothiazolo[4,3ꢀ
b]pyridines (compounds 12kꢀp and 12rꢀs), which demonstrated potent affinity to GAK,
displayed potent antiviral activity, with EC values of ~1 µM. Notably, these new analogues
50
also displayed significantly lower EC values (at a low µM range) and reduced cytotoxicity
9
0
relative to the original lead compound 4. A comparison of the doseꢀresponse curves of
compound 12r, the most promising compound in this series, and the parental compound 4
reveals the improved antiviral activity and toxicity profiles of 12r (Figure 2). Several
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
derivatives within this series, such as the geminal 2,2ꢀdimethylmorpholino (compound 12o)
and the 3ꢀSꢀmethylmorpholino (compound 12p), demonstrated no antiviral activity beyond
toxicity. The 3ꢀRꢀmethylmorpholine analogue 12q, which had poor GAK affinity, also lacked
antiviral activity (Table 1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Compound 12r suppresses DENV infection more effectively than compound 4.
Cell viability (blue) and dose response of DENV infection (black) to compound 4 (A) and 12r
(B) measured by luciferase and alamarBlue assays, respectively, 48 hours after infection. Data
are plotted relative to vehicle control. Shown are representative experiments from at least 2
conducted, each with 6 biological replicates; shown are means ± SD.
Table 1. SAR at position 3 of isothiazolo[4,3ꢀb]pyridines
O
2
H N
N
S
N
R3
GAK
affinity
DENVꢀ2 antiviral
activity
Cytotox
3
Compound#
R
Kd
(µM)
EC50
(µM)
EC90c
CC50
(µM)
a
b
d
(µM)
4
0.0089
0.97
1.844
8.05
>10
17
12a
5.72
>10
>10
1
2b
4.3
>10
>10
ACS Paragon Plus Environment

Page 13 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
2t
1.6
7.2
6.38
>10
>10
>10
>10
>10
12c
12d
0.25
0.16
0.21
0.08
0.18
5.28
5.305
>20
0.56
>10
>10
>20
2.09
>10
>10
>20
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12e
12f
12g
1
2h
12i
2j
2k
2l
12m
2n
2o
2p
12q
2r
2s
0.052
0.19
4.03
2
>10
4.15
>10
>10
2.74
2.9
>10
>10
>10
>10
21.5
>25
20.68
4.14
6.32
>10
>25
>10
1
0.17
7.12
2.735
0.76
1.1
1
0.061
0.046
0.026
0.019
0.035
0.018
0.8
1
1
2.09
0.8416
0.47
>10
6.5
1
3.92
1.34
>10
1.76
2.15
1
1
0.089
0.011
0.82
0.70
1
a
Kd = dissociation constant. Values represent the average of two independent experiments.
EC50 = halfꢀmaximal effective concentration.
b
c
EC90 = 90% effective concentration.
d
CC = halfꢀmaximal cytotoxic concentration.
50
Compound 12r inhibits DENV infection in human primary monocyte-derived dendritic
cells.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To further determine the therapeutic potential of compound 12r as an antiꢀDENV compound,
we studied its antiviral effect in human primary monocyteꢀderived dendritic cells (MDDCs);
34
an established ex vivo model system for DENV. We measured a doseꢀdependent inhibition
of DENV infection with minimal cytotoxicity following a 3ꢀday compound treatment with an
EC50 of 3.537 ꢁM and CC50 > 20 ꢁM by plaque assays and alamarBlue assays, respectively
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
5
(
Figure 3). Dendritic cells represent the primary target of DENV in humans. Moreover,
35
primary cells model human physiology and disease better than immortalized cell lines. Our
finding that 12r treatment exhibits antiviral efficacy in MDDCs may therefore more
accurately reflect the dependence of DENV on GAK during human infection and support the
biological relevance of this approach.
Figure 3. Ex vivo antiviral activity of 12r in human primary dendritic cells. Cell viability
(blue) and dose response of DENV infection (black) to 12r measured by plague assays and
alamarBlue assays, respectively, 72 hours after infection of primary human monocyteꢀderived
dendritic cells (MDDCs). Data are plotted relative to vehicle control. Shown is a
representative experiment with cells from a single donor, out of 2 independent experiments
conducted with cells derived from 2 donors, each with 5 biological replicates; shown are
means ± SD.
ACS Paragon Plus Environment

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Broad-spectrum antiviral activity of 3-N-morpholinyl-isothiazolo[4,3-b]pyridine analogues
To explore the spectrum of coverage of the new 3ꢀNꢀmorpholinylꢀisothiazolo[4,3ꢀb]pyridine
analogues beyond DENV, we studied their antiviral effects against two unrelated viruses:
15
EBOV (Filoviridae family), which was previously shown to hijack GAK , and CHIKV
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Togaviridae family). Huh7 cells were infected with authentic EBOV and treated for 48 hours
with individual isothiazolo[4,3ꢀb]pyridine derivatives. Antiviral activity was measured via an
immunofluorescence assay and cytotoxicity via a Cell TiterGlo assay (Table 2). Whereas
treatment with compound 4 showed no antiꢀEBOV activity beyond cytotoxicity, treatment
with 12r and 3ꢀNꢀmorpholinylꢀisothiazolo[4,3ꢀb]pyridine analogues (compounds 12l, 12m
and 12n) resulted in a doseꢀdependent inhibition of EBOV infection, with EC50 values of ~2
ꢁM and CC50 > 10 ꢁM (Figure 4A, Table 2). Additionally, treatment of Vero cells with 12r
commencing 48 hours before infection resulted in a doseꢀdependent decrease in CHIKV
infection measured by plaque assay 48 hours following infection (Figure 4B). These data
expand the possible future indications of these compounds as antiviral agents beyond
Flaviviridae infections to other emerging RNA viral infections.
Figure 4: Compound 12r suppresses EBOV and CHIKV infections. Cell viability (blue)
and dose response of EBOV (A) or CHIKV (B) infection (black) to compound 12r measured
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
by immunofluorescence (A) or plaque (B) assays in Huh7 (A) or Vero (B) cells 48 hours after
infection. Data are plotted relative to vehicle control. Shown are representative experiments
from at least 2 conducted, each with 6 biological replicates; shown are means ± SD.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
GAK
EBOV antiviral activity
Cytotox
Compound#
affinity a
b
c
d
Kd (µM)
EC50 (µM)
11.86
EC90 (µM)
21.91
CC50 (µM)
11
4
12r
12l
0.0089
0.089
0.046
0.026
0.019
2.436
7.732
>10
9.398
>10
2.001
7.865
12m
2.728
8.062
12n
4.345
>10
>10
a
Kd = dissociation constant. Values represent the average of two independent experiments.
b
EC50 = halfꢀmaximal effective concentration.
c
EC90 = 90% effective concentration.
d
CC = halfꢀmaximal cytotoxic concentration.
50
Table 2. Activity against EBOV
The antiviral effect of compound 12r correlates with functional inhibition of GAK
To confirm that the observed antiviral activity is correlated with functional inhibition of GAK
activity, we measured levels of the phosphorylated form of the µ subunit of the APꢀ2
complex, AP2M1, upon treatment with compound 12r. Since AP2M1 phosphorylation is
2
7
transient (due to phosphatase PP2A activity) , to allow capturing of the phosphorylated state,
Huh7 cells were incubated for 30 minutes in the presence of the PP2A inhibitor calyculin A
prior to lysis. Treatment with compound 12r reduced AP2M1 phosphorylation (Figure 5),
indicating modulation of AP2M1 phosphorylation via GAK inhibition.
ACS Paragon Plus Environment

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Antiviral effect of compound 12r correlates with functional inhibition of GAK.
The effect of 12r on AP2M1 phosphorylation by Western analysis in lysates derived from
Huh7 cells. Shown are a representative membrane (from two independent experiments)
blotted with antiꢀphosphoꢀAP2M1 (pꢀAP2M1) and antiꢀAP2M1 antibodies and quantitative
data of pꢀAP2M1/total AP2M1 protein ratio normalized to DMSO controls.
GAK is a molecular target underlying the antiviral effect of compound 12r
Next, we conducted gainꢀofꢀfunction assays, to confirm that inhibition of GAK is a
mechanism underlying the antiꢀDENV effect of compound 12r. A doxycyclineꢀinducible cell
line was established to overexpress GAK using the FlpꢀIn™ recombination system in TꢀREx
36
2
93 cells. Following doxycyclineꢀmediated induction of GAK expression, these cells were
infected with a luciferase reporter DENV and treated with compound 12r for 72 hours prior to
luciferase and viability assays. Doxycyclineꢀinduced GAK expression either partially or
completely reversed the antiviral effect of compound 12r relative to uninduced cells (Figure
6
). These results validate GAK as a key mediator of the antiꢀDENV effect of compound 12r.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. GAK is a molecular target underlying the antiviral effect of compound 12r.
A. Expression of GAK in TꢀREx 293 cells induced with doxycycline and uninduced control
cells. B. DENV infection normalized to cellular viability in the induced cells (+DOX, grey) or
uninduced control cells (ꢀDOX, black) 72 hours following infection with a luciferase reporter
DENV and 12r treatment, via luciferase and alamarBlue assays, respectively. Shown is a
representative experiment out of two conducted, each with 5 replicates. **P<0.01 relative to
induced or uninduced cells treated with DMSO by oneꢀway ANOVA with Dunnett’s multiple
comparisons test. Means ± SD are shown.
Kinase selectivity profile of compound 12r
Due to its excellent GAK affinity, good antiviral activity, and relatively low cytotoxicity,
compound 12r was selected as a lead compound for further analysis. While our data provide
evidence that GAK is an important mediator of the observed antiviral effect of compound 12r,
additional cellular kinases may mediate its antiviral activity. This is particularly relevant as Xꢀ
ray crystallography has previously demonstrated targeting of the ATP binding site of GAK by
29
compound 3, thereby classifying it as a classical type I kinase inhibitor. Since the ATP
binding site is highly conserved across the kinome, achieving high selectivity of kinase
3
1
inhibitors is quite challenging. To determine whether the improved antiviral activity of
ACS Paragon Plus Environment

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compound 12r resulted from reduced selectivity, we screened it against a diverse panel of 468
kinases (74 mutant and 394 wildꢀtype kinases) using an in vitro ATPꢀsite competition binding
assay (DiscoveRX, KinomeScan). This assay measures binding and not functional activity, is
conducted in the absence of ATP and is quite heterogeneous, yet it is commonly used for
selectivity assessment. Overall, the selectivity profile of compounds 3 and 12r is comparable,
as illustrated by their kinome trees (Figure 7), with selectivity indices S(35) of 0.033 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.057, respectively. Beyond GAK, at a concentration of 10 µM, compound 12r targets only 8
other kinases with greater than 90% inhibition (<10% control): ABL1, AURKC, EPHA7,
FLT3, KIT, MAP2K5, MAPK14 and PDGRFB. ABL1 (Abelson ProtoꢀOncogene 1), a
member of the Abl family of nonꢀreceptor tyrosine kinases, is implicated in the lifecycle of
37
38
both DENV and EBOV , and thus represents another target of 12r that may be contributing
to the observed antiviral effect. We previously reported that siRNAꢀmediated silencing of KIT
1
5
(
KIT ProtoꢀOncogene Receptor Tyrosine Kinase) reduced DENV infection. Nevertheless,
KIT’s role in DENV infection remained questionable since its suppression substantially
39,15
reduced cellular viability (likely due to its role in cell survival and proliferation).
Moreover, the antiꢀDENV activity of the tested derivatives does not correlate with the binding
affinity to KIT. Compound 4 exhibits potent KIT affinity (Kd = 0.0045 µM), yet only
moderate antiviral activity, whereas compound 12r is 300ꢀfold less potent as a KIT inhibitor
(
Kd = 1.3 µM), but displays an improved antiviral activity. These data suggest that KIT is
unlikely to be an important molecular target underlying the antiviral effect of these
compounds.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Kinase selectivity of compound 12r. Kinome tree comparison of 3 (left) and 12r
(right). Red circles indicate kinases inhibited by more than 90% at a concentration of 10 µM
of the indicated compounds.
ADME profiling
Compounds 4 and 12h exhibit a large (200 fold) or moderate (75 fold) difference between
their GAK affinity and antiꢀDENV cellular activity, respectively (Table 1). In contrast, the
antiviral activity of the structurally related isothiazolo[4,3ꢀb]pyridine 12r is only 10 fold
lower than its GAK affinity (Table 1). We thus tested our hypothesis that the aqueous
solubility and/or cellular permeability of these compounds account for these observed
differences. We measured the solubility of compounds 4, 12h and 12r, as well as of three
reference compounds (propranolol, ketoconazole and tamoxifene) in phosphate buffered
saline at a pH of 7.4 (Table 3). Compound 12r exhibited very poor aqueous solubility yet
potent antiviral activity, compound 4 demonstrated low solubility and intermediate antiviral
activity, whereas compound 12h demonstrated high solubility but reduced antiviral activity
relative to compounds 4 and 12r (Table 3). The membrane permeability properties of these
compounds was then measured via MDR1ꢀMDCK permeability assays. The permeability
measured in the apical to basolateral (AꢀtoꢀB) direction was higher (compound 4) or slightly
lower (compounds 12h, 12r) than that of metoprolol (a highly permeable compound) and
ACS Paragon Plus Environment

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
much higher than that of the low permeable control, atenolol. In the basolateral to apical (Bꢀ
toꢀA) direction, the permeability of compound 4 was comparable to that of metoprolol,
whereas the permeability of compound 12r was lower than metoprolol’s. Notably, compound
12h exhibited permeability in the BꢀA direction that was higher than that of quinidine’s, a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound that is subjected to efflux (Table 3). Accordingly, the calculated efflux ratios (Bꢀ
toꢀA/AꢀtoꢀB) (Table 3) suggest that compound 12h is likely subjected to efflux, which may
explain in part its diminished antiꢀDENV activity relative to compounds 4 and 12r. In
contrast, compound 12r has reduced efflux compared to compounds 4 and 12h, suggesting
that it may achieve a higher intracellular concentration, contributing to its enhanced antiviral
activity.
Taken together, these data suggest that differences in the efflux ratio of these compounds, but
not their aqueous solubility, may in part account for the measured differences between their
GAK affinity and antiviral activity. Additionally, GAK binding rather than enzymatic activity
was measured. It is therefore possible that the degree of the functional inhibition of GAK
activity by these compounds is different from their GAK affinity, particularly in a cellular
environment where high ATP concentrations and other cellular proteins are present.
Table 3: Solubility (in PBS) and Permeability values of compounds 4, 12h and 12r
6
Solubility
µM)
Papp x 10
(cm/s)
c
Compound
Efflux ratio
(
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
AꢀB
BꢀA
AꢀB
BꢀA
AꢀB
BꢀA
45.45 ± 0.07
4
3.44 ± 0.03
51.30 ± 0.02
0.52 ± 0.23
b
a
b
a
b
0.68
1.43
0.58
30.80 ± 0.08
30.32 ± 0.01
43.48 ± 0.02
31.33 ± 0.03
18.28 ± 0.26
1
2h
12r
d
Propanolol
Ketoconazole
Tamoxifene
117 ± 0.15
32.00 ± 0.03
1.32 ± 0.09
ND
d
ND
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d
ND
a
b
a
b
a
b
AꢀB
BꢀA
AꢀB
BꢀA
AꢀB
BꢀA
39.29 ± 0.04
30.66 ± 0.00
1.21 ± 0.00
1.25 ± 0.00
13.03 ± 0.01
41.99 ± 0.01
d
Metoprolol
Atenolol
ND
0.78
1.03
3.22
d
ND
d
Quinidine
ND
a
AꢀB: Apical to Basolateral
BꢀA: Basolateral to Apical
Efflux ratio: BꢀA/AꢀB
b
c
d
ND: not determined
Conclusions
Based on our earlier discovery of isothiazolo[4,3ꢀb]pyridine (compound 4) as a potent and
selective GAK inhibitor with moderate antiviral activity, we embarked on an optimization
campaign aimed at improving the antiviral activity while maintaining potent and selective
GAK inhibition. Synthesis of novel isothiazolo[4,3ꢀb]pyridines with structural variation at
position 3 of the scaffold led to the discovery of 3ꢀ(cisꢀ2,6ꢀdimethylmorpholino)ꢀ6ꢀ(4ꢀaminoꢀ
3
ꢀmethoxypheny)isothiazolo[4,3ꢀb]pyridine 12r. Compound 12r maintains potent activity and
high selectivity to GAK and displays significantly improved antiviral activity against DENV
with reduced cellular toxicity relative to the parental compound 4. Our gainꢀofꢀfunction
assays provide evidence that GAK is an important molecular target underlying the antiviral
effect of compound 12r. Compound 12r treatment exhibits antiviral efficacy in human
primary dendritic cells with minimal toxicity to host cells, further supporting the biological
relevance of this approach. Lastly, our finding that compound 12r also demonstrates antiviral
activity against EBOV and CHIKV establishes the broadꢀspectrum potential of this and
related GAK inhibitors.
ACS Paragon Plus Environment

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Experimental section
Chemistry
For all reactions, analytical grade solvents were used. All moistureꢀsensitive reactions were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
carried out in ovenꢀdried glassware (125 °C). H and C NMR spectra were recorded on a
1
13
Bruker Avance 300 MHz instrument ( H NMR, 300 MHz; C NMR, 75 MHz), 500 MHz
1
13
1
instrument ( H NMR, 500 MHz; C NMR, 125 MHz) or a 600 MHz instrument ( H NMR,
13
1
600 MHz; C NMR, 150 MHz), using tetramethylsilane as internal standard for H NMR
1
3
6 3
spectra and DMSOꢀd (39.5 ppm) or CDCl (77.2 ppm) for C NMR spectra. Abbreviations
used are s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. Coupling
constants are expressed in Hz. High resolution mass spectra were acquired on a quadrupole
orthogonal acceleration timeꢀofꢀflight mass spectrometer (Synapt G2 HDMS, Waters,
Milford, MA). Samples were infused at 3uL/min and spectra were obtained in positive or
negative ionization mode with a resolution of 15000 (FWHM) using leucine enkephalin as
lock mass. Precoated aluminum sheets (Fluka silica gel/TLCꢀcards, 254 nm) were used for
TLC. Column chromatography was performed on silica gel 0.060 – 0.200 mm, 60 Å (Acros
Organics). Purity of final compounds was verified to be >95% by HPLC analysis. HPLC
conditions to asses purity were as follows: Shimadzu HPLC equipped with a LCꢀ20AT pump,
DGUꢀ20A5 degasser, and a SPDꢀ20A UVꢀVIS detector; Symmetry C18 column (5 ꢁm, 4.6
2 3
mm × 150 mm); gradient elution of H O/CH CN from 95/5 or 70/30 to 5/95 over 25 minutes;
flow rate 1 mL/min; wavelength, UV 254 nm. Preparative HPLC purifications were
performed using a Phenomenex Gemini 110A column (C18, 10 µM, 21.2 mm x 250 mm).
3ꢀAminoꢀ5ꢀbromopyridineꢀ2ꢀcarbonitrile (6)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To a stirred solution of iron powder (4.900 g, 87.7 mmol) in acetic acid (50 mL) at room
temperature, was added 5ꢀbromoꢀ3ꢀnitropyridineꢀ2ꢀcarbonitrile 5 (10.00 g, 43.9 mmol) in one
portion. The resulting reaction mixture was stirred for 3 hours. After completion, ethyl acetate
was added and the reaction mixture was filtered through a paper filter. The filtered cake was
washed thoroughly with ethyl acetate and the filtrate was evaporated under reduced pressure.
The crude residue was purified by silica gel flash column chromatography (using
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
dichloromethane as mobile phase) to yield the title compound (4.845 g, 56 %). H NMR (300
MHz, DMSOꢀd
6
): δ = 8.08 – 7.66 (m, 1H), 7.55 – 7.26 (m, 1H), 6.58 (bs, 2H) ppm. HRMS
BrN : 197.96618, found 197.9656.
+
m/z [M+H] calcd for C
H
6 4
3
3ꢀAminoꢀ5ꢀbromoꢀ2ꢀpyridinecarbothioamide (7)
To a solution of 3ꢀaminoꢀ5ꢀbromopyridineꢀ2ꢀcarbonitrile 6 (5.395 g, 25.00 mmol) in absolute
ethanol (50 mL) was added Lawesson’s reagent (20.22 g, 50.0 mmol). The mixture was
refluxed for 24 hours. When the starting material was completely consumed according to TLC
analysis, the solvent was evaporated under reduced pressure. The residue was redissolved in
water and extracted three times with dichloromethane. The combined organic layers were
evaporated under reduced pressure. The crude residue was purified by silica gel flash column
chromatography (using a mixture of heptane/acetone in a ratio of 60:40 as mobile phase)
1
yielding the title compound (4.513 g, 78 %). H NMR (300 MHz, DMSO) δ 9.64 (bs, 1H),
9.60 (bs, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.81 (s, 1H), 7.50 (d, J = 2.0 Hz, 1H) ppm.
3
ꢀAminoꢀ6ꢀbromoisothiazolo[4,3ꢀb]pyridine (8)
To a solution of 3ꢀaminoꢀ5ꢀbromopyridineꢀ2ꢀcarbothioamide 7 (2.0 g, 8.62 mmol) in
methanol (30 mL) at 0 °C was added dropwise a 30% aqueous H solution (2.22 mL, 21.54
2
O
2
mmol). The mixture was stirred overnight at room temperature. After disappearance of the
ACS Paragon Plus Environment

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
starting material, the mixture was cooled to 0 °C. The precipitate was filtered off and washed
1
with cold methanol to yield the title compound (1.0 g, 50 %). H NMR (300 MHz, DMSO) δ
+
8.29 (d, J = 2.0 Hz, 1H), 8.07 (s, 2H), 8.03 (d, J = 2.0 Hz, 1H) ppm. HRMS m/z [M+H]
calcd for C BrN S: 229.9383, found 229.9380.
6
H
6
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3,6ꢀDibromoisothiazolo[4,3ꢀb]pyridine (9)
A solution of 3ꢀaminoꢀ6ꢀbromoisothiazolo[4,3ꢀb]pyridine 8 (1.1 g, 4.78 mmol) in HBr (50
mL) was stirred for 10 min at room temperature, and then CuBr was added in one portion
(1.37 g, 9.56 mmol, 2.0 equiv). The resulting mixture was cooled to 0 °C. A solution of
2
sodium nitrite (0.99 g, 14.34 mmol, 3.0 equiv) in H O (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 to 0 °C and carefully neutralized with a 30% aqueous
NaOH solution. The formed precipitate was filtered over Celite and thoroughly washed with
dichloromethane and methanol. The filtrate was evaporated in vacuo and the crude residue
was purified by silica gel flash chromatography (using a mixture of cyclohexane and
1
ethylacetate in a ratio of 95:5 as mobile phase), yielding the title compound (1.1 g, 78%). H
NMR (300 MHz, DMSOꢀd ): δ 8.92 (d, J = 2.04 Hz, 1H), 8.70 (d, J = 2.01 Hz, 1H) ppm.
6
+
HRMS m/z [M+H] calcd for C
6
H
2
Br
2
N
2
S: 292.8379, found 292.8373.
Synthesis of 6ꢀbromoꢀ3ꢀsubstitutedꢀisothiazolo[4,3ꢀb]pyridines (10aꢀt)
General procedure
To a solution of 3,6ꢀdibromoisothiazolo[4,3ꢀb]pyridine 9 in ethanol or as specified was added
an appropriate nitrogen nucleophile (3 equivalents or specified otherwise). The reaction was
stirred at reflux overnight or as specified. When TLC analysis indicated complete
disappearance of the starting material, the solvent was evaporated in vacuo and the crude
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
residue was purified either by silica gel flash chromatography or extraction with
dichloromethane yielding the 3ꢀsubstitutedꢀ6ꢀbromoisothiazolo[4,3ꢀb]pyridine derivatives.
The following compounds were made according to this procedure:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6ꢀBromoꢀ3ꢀ(piperidinꢀ1ꢀyl)isothiazolo[4,3ꢀb]pyridine (10a)
This compound was prepared from compound 9 (0.68 mmol, 200 mg) and piperidine (2.04
mmol, 174 mg). The product was purified by silica gel flash column chromatography (using a
mixture of dichloromethane/ethyl acetate in a ratio of 90:10 as mobile phase) yielding the title
1
compound (180 mg, 60%). H NMR (300 MHz, DMSOꢀd
6
): δ = 8.32 (d, J = 2.0 Hz, 1H), 8.07
+
(
d, J = 2.0 Hz, 1H), 4.00 – 3.70 (m, 4H), 1.74 – 1.62 (m, 6H) ppm. HRMS m/z [M+H] calcd
for C H BrN S: 298.0009, found 298.0014.
11
12
3
6ꢀBromoꢀ3ꢀ(4,4ꢀdifluoropiperidinꢀ1ꢀyl)isothiazolo[4,3ꢀb]pyridine (10b)
This compound was prepared from compound 9 (0.34 mmol, 100 mg), 4,4ꢀdifluoropiperidine
hydrochloride (1.02 mmol, 161 mg) and DIPEA (1.70 mmol, 296 µL). The product was
purified by silica gel flash column chromatography (using a mixture of heptane/ethyl acetate
1
in a ratio of 60:40 as mobile phase) yielding the title compound (110 mg, 97%). H NMR
(300 MHz, DMSOꢀd
6
): δ = 8.40 (d, J = 1.4 Hz, 1H), 8.16 (d, J = 1.4 Hz, 1H), 4.20 – 3.98 (m,
+
4
H), 2.32 – 2.16 (m, 4H) ppm. HRMS m/z [M+H] calcd for C11
H12BrN
3
S: 333.9820, found
333.9823.
1ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)piperidineꢀ4ꢀcarboxamide (10c)
This compound was prepared from compound 9 (0.68 mmol, 200 mg) and piperidineꢀ4ꢀ
carboxamide (1.36 mmol, 174 mg, 2 eq). The crude product was resuspended in water and
washed three times with dichloromethane. The combined organic layers evaporated in vacuo
ACS Paragon Plus Environment

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
yielding the title compound (160 mg, 69%). The compound was used as such for further
reaction.
1ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)piperidineꢀ4ꢀcarbonitrile (10d)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
This compound was prepared from compound 9 (0.68 mmol, 200 mg) and piperidineꢀ4ꢀ
carbonitrile (1.36 mmol, 150 mg, 2 eq). The crude product was resuspended in water and
washed with dichloromethane three times. The combined organic phases were evaporated in
1
vacuo to yield the title compound (190 mg, 86%). H NMR (300 MHz, DMSOꢀd
6
): δ = 8.37
(d, J = 2.0 Hz, 1H), 8.13 (d, J = 2.0 Hz, 1H), 4.29 – 4.02 (m, 2H), 3.86 – 3.58 (m, 2H), 3.29 –
+
3
.08 (m, 1H), 2.15 – 2.00 (m, 2H), 2.00 – 1.85 (m, 2H) ppm. HRMS m/z [M+H] calcd for
C H BrN S: 322.9961, found 322.9958.
12
11
4
1ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)piperidineꢀ3ꢀcarboxamide (10e)
This compound was prepared from compound 9 (0.68 mmol, 200 mg) and piperidineꢀ3ꢀ
carboxamide (1.36 mmol, 174 mg, 2 eq). The crude product was resuspended in water and
washed three times with dichloromethane. The combined organic layers were evaporated in
1
vacuo to yield the title compound (150 mg, 65%). H NMR (300 MHz, DMSOꢀd ): δ = 8.36
6
(d, J = 2.0 Hz, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.44 (bs, 1H), 6.99 (bs, 1H), 4.51 (dd, J = 60.1,
11.6 Hz, 2H), 3.51 – 3.12 (m, 2H), 2.00 – 1.79 (m, 2H), 1.77 – 1.58 (m, 2H) ppm. HRMS m/z
+
[M+H] calcd for C12
H13BrN
4
OS: 341.0067, found 341.0064.
4
ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)thiomorpholine 1,1ꢀdioxide (10f)
This compound was prepared from compound 9 (1.02 mmol, 300 mg) and thiomorpholineꢀ
,1ꢀdioxide (3.06 mmol, 414 mg). The product was purified by silica gel flash column
1
chromatography (using a mixture of dichloromethane/acetone in a ratio 95:5 as mobile phase)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
generating the title compound (90 mg, 25%). H NMR (300 MHz, DMSOꢀd ): δ = 8.44 (d, J =
6
+
2.1 Hz, 1H), 8.21 (d, J = 2.1 Hz, 1H), 4.42 (m, 4H), 3.42 (m, 4H) ppm. HRMS m/z [M+H]
3 2 2
calcd for C10H10BrN O S : 347.9471, found 347.9480.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
ꢀBromoꢀNꢀ(tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl)isothiazolo[4,3ꢀb]pyridinꢀ3ꢀamine (10g)
This compound was prepared from compound 9 (0.68 mmol, 200 mg), 4ꢀaminotetrahydroꢀ
Hꢀpyran hydrochloride (3.4 mmol, 468 mg) and DIPEA (3.4 mmol, 595 µL). The product
2
was purified by silica gel flash column chromatography (using a mixture of
dichloromethane/acetone in a ratio 95:5 as mobile phase) yielding the title compound (90 mg,
1
4
1
2%). H NMR (300 MHz, DMSOꢀd
6
): δ = 8.81 (d, J = 8.07 Hz, 1H), 8.30 (d, J = 1.98 Hz,
H), 8.05 (d, J = 1.86 Hz, 1H), 3.92 (dd, J = 1.86 Hz, J = 10.08 Hz, 2H), 3.58 (m, 1H), 3.42
(
td, J = 2.1 Hz, J = 11.78 Hz, 2H), 1.96 (dd, J = 12.7 Hz, J = 2.2 Hz, 2H), 1.68 (m, 2H) ppm.
+
HRMS m/z [M+H] calcd for C11
H
12BrN
3
OS: 313.9958, found 313.9954.
4ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)piperazinꢀ2ꢀone (10h)
This compound was prepared from compound 9 (0.68 mmol, 200 mg) and 2ꢀpiperazinone
(
2.06 mmol, 206 mg) and stirred for 7 days. The crude product was purified by silica gel flash
column chromatography (eluting with a mixture of dichloromethane/MeOH in a ratio of 95:5
1
as mobile phase) yielding the title compound (150 mg, 70%). H NMR (300 MHz, DMSOꢀ
d
6
): δ = 8.40 – 8.33 (m, 1H), 8.22 – 8.04 (m, 1H), 4.42 – 4.29 (m, 2H), 4.24 – 3.94 (m, 2H),
3.65 – 3.49 (m, 2H) ppm.
4
ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)ꢀ1,4ꢀoxazepane (10i)
This compound was prepared from compound 9 (0.34 mmol, 100 mg) and 1,4ꢀoxazepane
(
1.02 mmol, 103 mg). The product was purified by silica gel flash column chromatography
ACS Paragon Plus Environment

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
using a mixture of dichloromethane/acetone in a ratio 95:5 as mobile phase) affording the
1
title compound (100 mg, 96%). H NMR (300 MHz, DMSOꢀd ): δ = 8.27 (s, 1H), 8.04 (s,
6
1
2
3
H), 4.18 – 4.12 (m, 2H), 4.069 – 4.0 (m, 2H), 3.87 (d, J = 3.9 Hz, 2H), 3.76 – 3.68 (m, 2H),
+
.08 – 1.91 (m, 2H) ppm. HRMS m/z [M+H] calcd for C11
13.9959.
3
H12BrN OS: 313.9957, found
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)ꢀ2ꢀoxaꢀ6ꢀazaspiro[3.3]heptane (10j)
This compound was prepared from compound 9 (0.34 mmol, 100 mg) and 2ꢀoxaꢀ6ꢀ
azaspiro[3.3]heptane (1.02 mmol, 206 mg). The crude product was purified by silica gel flash
column chromatography (using a mixture of dichloromethane/ethyl acetate in a ratio of 90:10
1
as mobile phase) yielding the title compound (90 mg, 85%). H NMR (300 MHz, CDCl ): δ =
3
8
.29 – 8.16 (m, 1H), 7.98 – 7.81 (m, 1H), 4.86 (s, 4H), 4.61 – 4.47 (m, 4H) ppm. HRMS m/z
+
[
M+H] calcd for C11
H10BrN
3
OS: 311.9801, found 311.9815.
3ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)ꢀ8ꢀoxaꢀ3ꢀazabicyclo[3.2.1]octane (10k)
This compound was prepared from compound 9 (0.68 mmol, 200 mg) and 8ꢀoxaꢀ3ꢀ
azabicyclo[3.2.1]octane (1.36 mmol, 154 mg, 2 eq). The crude product was purified by silica
gel flash column chromatography (using a mixture of heptane/acetone in a ratio 8:2 as mobile
1
phase) yielding the title compound (155 mg, 70%). H NMR (300 MHz, DMSOꢀd
6
): δ = 8.35
(d, J = 2.1 Hz, 1H), 8.13 (d, 1H), 4.51 (s, 2H), 4.20 (d, J = 12.4 Hz, 2H), 3.43 (dd, J = 12.4,
+
2.1 Hz, 2H), 2.05 – 1.78 (m, 4H) ppm. HRMS m/z [M+H] calcd for C H BrN OS:
12
12
3
3
25.9957, found 325.9964.
4
ꢀ(6ꢀBromoisothiazolo[4,3ꢀb]pyridinꢀ3ꢀyl)ꢀ2ꢀmethylmorpholine (10l)
ACS Paragon Plus Environment