Saccharin derivatives as inhibitors of interferon-mediated inflammation

Article abstract of DOI:10.1021/jm500409k

A series of novel, saccharin-based antagonists have been identified for the interferon signaling pathway. Through in vitro high-throughput screening with the Colorado Center for Drug Discovery (C2D2) Pilot Library, we identified hit compound 1, which was the basis for extensive structure-activity relationship studies. Our efforts produced a lead anti-inflammatory compound, tert-butyl N-(furan-2-ylmethyl)-N-{4-[(1,1,3-trioxo-2,3-dihydro-1λ⁶,2- benzothiazol-2-yl)methyl]benzoyl}carbamate CU-CPD103 (103), as a potent inhibitor using an established nitric oxide (NO) signaling assay. With further studies of its inhibitory mechanisms, we demonstrated that 103 carries out this inhibition through the JAK/STAT1 pathway, providing a drug-like small molecule inflammation suppressant for possible therapeutic uses.

Full text of DOI:10.1021/jm500409k

Subscriber access provided by University of South Dakota
Article
Saccharin Derivatives as Inhibitors of Interferon-Mediated Inflammation
Adam Csakai, Christina Smith, Emily Davis, Alexander Martinko, Sara Coulup, and Hang Yin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500409k • Publication Date (Web): 24 May 2014
Downloaded from http://pubs.acs.org on June 2, 2014
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Saccharin Derivatives as Inhibitors of
Interferon-Mediated Inflammation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Adam Csakai,† Christina Smith, † Emily Davis, Alexander Martinko, Sara Coulup, Hang Yin*
Department of Chemistry & Biochemistry and the BioFrontiers Institute, University of Colorado
Boulder, Boulder, Colorado 80309-0596
†These authors contributed equally to this work.
Abstract
A series of novel, saccharin-based antagonists have been identified for the interferon signaling
pathway. Through in vitro high-throughput screening with the Colorado Center for Drug
Discovery (C2D2) Pilot Library, we identified hit compound 1, which was the basis for extensive
structure activity relationship studies. Our efforts produced a lead anti-inflammatory compound,
tert‐butyl
N‐(furan‐2‐ylmethyl)‐N‐{4‐[(1,1,3‐trioxo‐2,3‐dihydro‐1λⁱ,2‐benzothiazol‐2‐yl)methyl]benzoyl}c
arbamate CU-CPD103 (103), as a potent inhibitor using an established nitric oxide (NO)
signaling assays. With further studies of its inhibitory mechanisms, we demonstrated that
103 carries out this inhibition through the JAK/STAT1 pathway, providing a drug-like small
molecule inflammation suppressant for possible therapeutic uses.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 30
1
2
3
4
Introduction
5
6
7
8
Interferons (IFN) are a linchpin of inflammatory signaling, assisting in host defense
against pathogens, antigen presentation, and immunomodulation. There are two main classes of
interferons: type I (IFN-α/β) and type II (IFN-γ).1 Interferons bind to their respective
transmembrane receptors, inducing dimerization and regulation of inflammatory gene expression
through the JAK/STAT signaling pathway.1 Janus kinases (JAKs) are tyrosine kinases that
interact with interferon receptors, resulting in recruitment and phosphorylation of signal
transduction and activator of transcription (STAT) proteins.2 The JAK/STAT association in turn
promotes transcription of pro-inflammatory genes including inducible nitric oxide synthase
(iNOS).3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Interferons coordinate the inflammation response in concert with other innate immune
pathways, particularly Toll-like receptor (TLR) signaling. TLRs are pattern recognition receptors
that respond to infectious markers and induce a pro-inflammatory response.4,5 These two
pathways synergistically interact in macrophages to elicit an immune response towards infective
threats. Macrophage priming with IFN-γ improves the inflammatory response to TLR ligands,
such as lipopolysaccharide (LPS) for TLR4. In turn, TLRs upregulate type I interferons, and NF-
κB assists in transcription of multiple interferon-inducible genes.6 Of particular interest, the
iNOS promoter has binding sites for both STAT1 and NF-κB.7 Transcription of iNOS may be
activated by multiple inflammatory factors, including LPS, type I, and type II interferons.8
STAT1 activation by IFN-β has an autocrine/paracrine mechanism preceding iNOS activation,
and serves as a necessary transcription factor for synthesis.9 It has been demonstrated that
knockdown of the interferon-α/β receptor 1 (IFNAR1) mitigates iNOS expression, even in the
2
ACS Paragon Plus Environment

Page 3 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
presence of LPS.10 IFN-γ activates STAT1 through interaction with its cognate receptor IFNGR,
upregulating iNOS.11 Thus, activation of the TLR and interferon interrelated pathways can be
regulated at their convergence in JAK/STAT signaling. Nonetheless, it has been a significant
challenge to regulate individual pathways with high specificity and selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Generally, inflammatory signaling is beneficial, and may protect the host against
infection. However, in autoimmune pathologies such as systemic lupus erythematosus and
multiple sclerosis, an overabundance of interferon signaling can have deleterious effects.12–14
Indeed, it has been noted that among centenarian women, polymorphisms that result in decreased
IFN-γ may contribute to longevity.15 Additionally, a small molecule modulator of IFN signaling
may provide a useful tool in the study and treatment of autoimmune diseases. Here, we report a
small molecule that is able to inhibit the interferon-induced JAK/STAT1 signaling pathway,
without compromising the effectiveness of other components of the innate immune system, such
as TLRs.
Results and Discussion
C2D2 Compound Library Screening
We started our search for anti-inflammatory agents by looking for inhibitors of LPS-
induced TLR4 activation. The Colorado Center for Drug Discovery (C2D2) compound library.16
The C2D2 pilot library consists of 2,200 drug-like compounds that represent a variety of diverse
and commercially available scaffolds. The initial screen was performed using LPS-activated
RAW 264.7 cells in a 96-well plate format to monitor nitric oxide (NO) production. This assay
uses a Sandmeyer reaction to convert 2,3-diaminonapthalene to fluorescent napthalenetriazole in
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 30
1
2
3
4
5
6
7
8
9
the presence of NO. As NO is produced in the TLR inflammatory response, this readout provides
information on the extent of TLR signaling. Initial screening yielded 31 hits, representing 5
scaffolds (Figure S1, supplementary information). We selected the scaffold with an isothiazolone
1,1-dioxide core for further development as it produced the most numerous and potent hits.
Compounds 1 and 2 (Figure 1), represent two of the more potent hits.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
S
Ar
R
N
H
N
N
H3C
O
O
1
2
R
O
Figure 1. Generic scaffold of hit molecules selected from the screening of the Colorado Center
for Drug Discovery (C2D2) Pilot Library. Both variable functionalities (Ar = substituted benzene
ring, R= various amide moieties) shown in these representative structures have been
subsequently optimized.
Synthesis and Structure Activity Relationship (SAR)
We have developed an efficient synthetic route for the generic scaffold in Figure 1. As an
example, the synthesis of the most potent hit molecule, 1 (Scheme 1), began with the oxidative
dimerization of commercially available 3 to give dithiodipropionic acid 4.17 Conversion to the
dithiodipropionyl chloride was completed with thionyl chloride, and subsequent treatment with
anhydrous ammonia gave dithiodipropionamide 5 as a mixture of diastereomers.17 As previously
described by Lewis and co-workers, oxidative cyclization was performed with sulfuryl chloride
to give an inseparable mixture of 6 and 7.17 This mixture of isothiazolinones was then
deprotonated with sodium hydride, and alkylated with benzyl chloride 9. The alkylation yields
4
ACS Paragon Plus Environment

Page 5 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
two synthetically useful intermediates 12 and 13, which are easily separable by column
chromatography. Chloro-intermediate 12 was oxidized with meta-chloroperoxybenzoic acid (m-
CPBA) to give the isothiazolone 1,1-dioxide core, 14.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Isothiazolinone Synthesis
O
O
H3C
H3C
OH
OH
NH2
NH2
Y
S
H3C
CO2H
SH
i
ii
iii
S
S
S
S
NH
H3C
O
H3C
H3C
O
O
3
4
5
6, Y = Cl
7, Y = H
Cl
Cl
R'
R =
N
N
iv
O
Cl
R
9
10, R' = H
O
O
v
11, R' = Boc
8
Y
S
N
vi
H3C
6/7
(≈ 1:1.54)
O
N
O
12, Y = Cl
13, Y = H
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 30
1
2
3
4
5
6
7
8
9
H3C
H3C
O
O
O
O
Ar =
Cl
Ar
S
S
N
N
viii
vii
H3C
H3C
12
1
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
O
O
14
15
O
N
O
N
O
S
S
N
N
x
ix
13
H3C
H3C
O
O
N
N
O
O
16
17
i. I2, KI, NaOH, H2O, (4 = 99%), ii. 1) SOCl2 2) NH3, CH2Cl2 (5 = 66%), iii. SO2Cl2, EtOAc (6 &
7 = 51%), iv. piperidine or furfurylamine, DIPEA, CH2Cl2 (9 = 95%, 10 = 99%), v. Boc2O,
DMAP, THF (11 = 81%), vi. 1) NaH, DMF 2) 9 (12 = 70%, 13 = 67%), vii. m-CPBA, CH2Cl2
(14 = 49%), viii. ArB(OH)2, Pd(dppf)Cl2•CH2Cl2, K2CO3, 1,4-dioxane (1 = 47%, 15 = 32%), ix.
4-bromopyridine•HCl, Pd(OAc)2, KOAc, DMA (16 = 54%), x. m-CPBA, CH2Cl2 (17 = 84%)
Chloro-intermediate 14 was coupled to 3,4-dimethylphenylboronic acid, using previously
reported conditions to give the parent library hit, 1.18 Identical conditions were used to synthesize
a related β-naphthalene analog, 15. These conditions failed, however, when 4-pyridinylboronic
acid was used. Making use of intermediate 13 with Heck conditions and 4-bromopyridine•HCl
successfully gave 16. The coupling product 16 was fully oxidized at both sulfur and the pyridyl
nitrogen to give 17. The synthesis of compounds 15 and 17 provide analogs that exhibit both
higher and lower calculated logP values, 4.70 and 1.29 respectively, as compared to 1 (4.46).
6
ACS Paragon Plus Environment

Page 7 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
However, 15 only showed a small improvement in potency, and 17 showed greatly reduced
potency as compared to 1 (Table 1).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. SAR Summary and Toxicity of 4-Aryl-3-methylisothiazolone 1,1-dioxide Analogs
O
O
R1
S
N
ID #
IC50
Key:
H3C
R1
O
LC50
H3C
H3C
N
1
O
16.5 ± 0.55 µM
>100 µM
15
9.61 ± 2.56 µM
47.9 ± 5.79 µM
O
17
79.4 ± 13.6 µM
> 100 µM
N
Summary of IC50 values and toxicity for structure activity relationship studies. IC50 values were
obtained using RAW 264.7 cells treated with 20 ng/mL LPS and varying concentrations of
compound. A cell viability assay was used to determine cytotoxicity at each tested concentration.
LC50 is the concentration at which cytotoxicity results in 50% cell viability. The purity of tested
compounds was evaluated via 1H NMR (>95% sample purity).
The 2-benzyl-4-methyl-5-phenylisothiazol-3-one 1,1-dioxide core of 1, 15, and 17 was
sensitive to a variety of mild reaction conditions. We commonly observed complex mixtures in
efforts to synthesize other analogs. The few successfully synthesized analogs exhibited only
modest activity, so we thought it best to make more drastic structural changes. In an effort to
simplify synthesis and increase stability, we took inspiration from saccharin 18, (Scheme 2).
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 30
1
2
3
4
5
6
Scheme 2. Saccharin Derived Analogs
7
8
9
O
O
R'
S
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R =
N
O
N
O
N
S
xi
NH
O
19
CU-CPD103 (103), R' = Boc
20, R' = H
xii
O
R
O
18
xi. 1) NaH, DMF 2) 9 (19 = 65%) or 11 (103 = 62%), xii. TFA, CH2Cl2 (20 = 87%)
Saccharin inspired analogs, 19 and CU-CPD103 (103), were easily synthesized from
commercially available saccharin and previously synthesized benzylchlorides 9 and 11. 103 is
easily deprotected with TFA to give 20. A significant improvement in activity was observed with
intermediate 103, so we sought related analogs 26-43 (Scheme 3; detailed syntheses can be
found in the supporting information). Compounds 19, 20, 103, and 26-43 contain the same
piperidine or furfuryl amide moieties (Figure 1) that were present in our initial library screen so
we could have a consistent basis for comparison. The lithium aluminum hydride reduction of
saccharin 18, previously described by Porter and coworkers,19 provided 2,3-dihydro-1,1-dioxo-
1,2-benzisothiazole 21. Alkylation of this sultam with 9 and 11 provided analogs 26 and 27.
Commercially available 1-isoindolinone 22, phthalimide potassium salt 23, 1,2-benzisothiazol-
3(2H)-one 24, and 3-hydroxybenzisoxazole 25, were alkylated with 9 and 11 to give analogs 29,
30, 32, 33, 35, 36, 38, and 39 (Scheme 3). Treatment of analogs 35 and 36 with m-CPBA at 0 °C
gave analogs 38 and 39 respectively, as racemic mixtures. All Boc protected analogs were
treated with trifluoroacetic acid in methylene chloride to give deprotected analogs 28, 31, 34, 37,
40 and 43.
8
ACS Paragon Plus Environment

Page 9 of 30
Journal of Medicinal Chemistry
1
2
3
4
Scheme 3. Fused Ring Analogs
5
6
7
8
9
X
N
Z
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
X
N H
Z
R
O
21, X = SO2 , Z = CH2
22, X = CH2, Z = C(O)
23, X = Z = C(O) •K salt
24, X = S, Z = C(O)
25, X = O, Z = C(O)
R'
R =
N
R =
N
O
R' = Boc
R' = H
xiv
xiv
xvi
xv
21
27
28
21
22
23
24
26
29
32
35
xvi
xvii
xix
22
23
24
30
33
31
34
37
xviii
xviii
xx
xx
xxi
36
xxii
xxii
xxiii
xxv
39
42
40
43
38
41
xxiv
xxiv
25
25
xiv. 1) NaH, DMF 2) 9 (26 = 84%) or 11 (27 = 81%), xv. TFA, CH2Cl2 (28 = 92%), xvi. 1) NaH,
DMF 2) 9 (29 = 57%) or 11 (30 = 10%), xvii. TFA, CH2Cl2 (31 = 71%), xviii. 18-crown-6, DMF,
9 (32 = 84%) or 11 (33 = 81%), xix. TFA, CH2Cl2 (34 = 81%), xx. 1) NaH, DMF 2) 9 (35 =
48%) or 11 (36 = 27%), xxi. TFA, CH2Cl2 (37 = 97%), xxii. m-CPBA, CH2Cl2, 35 (38 = 85%) or
36 (39 = 79%), xxiii. TFA, CH2Cl2 (40 = 92%), xxiv. 1) NaH, DMF 2) 9 (41 = 55%) or 11 (42 =
14%), xxv. TFA, CH2Cl2 (43 = 87%)
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 30
1
2
3
4
5
6
7
8
The removal of the saccharin series 3-position carbonyl gave us the sultam series 26, 27,
and 28 (Table 2). As we had seen with the parent saccharin series, the Boc protected amide 27
was the most potent of the series. 27, however, was unable to match the potency 103. For this
reason, we believe the 3-position carbonyl to be somewhat important to either binding or cell
permeability. Replacement of the saccharin series 1-position SO2 moiety with a methylene unit
gave us the isoindolinone series 29, 30, and 31. Unlike others, the analog bearing a Boc protected
amide 30 shows the lowest potency. All isoindolinone analogs, however, are poor inhibitors.
This led us to believe that the 1-position SO2 moiety makes a significant interaction, perhaps as a
hydrogen bond acceptor. As previously discussed, the 3-position carbonyl of 103 appears less
significant, so the carbonyl of the isoindolinone 30 may be making a weaker hydrogen bond
interaction as a poor bioisostere of the SO2 moiety. Otherwise, the isoindolinone series would
likely have even lower potency.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To further investigate the role of the SO2 moiety, the 3-position carbonyl of 103 was
maintained, and the SO2 moiety replaced with an additional carbonyl, as shown with the
phthalimide analogs 32, 33, and 34. The activity of 103 is nearly equal with 33. As with the
parent saccharin analogs, 32 and 34 show little or no activity. This suggests that a carbonyl is a
suitable bioisostere to the SO2 moiety, so long as the 3-position carbonyl is intact, unlike 30.
Furthermore, a loss of one, or both, oxygen atoms from the SO2 moiety of 103 results in a
complete loss of activity, as seen with 36 and 39. As seen before, the deprotected species 37 and
40 show poor activity. As expected, the piperidine amide analog, 38 shows a further decrease in
10
ACS Paragon Plus Environment

Page 11 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
potency. However, 35 inexplicably shows comparable potency as compared to 103, and is a great
improvement over the related analog 26. When the unoxidized sulfur of 36 is replaced with a
smaller oxygen atom 42, the activity is once again comparable to that of 103. Again, the
deprotected analog 43 shows significantly lower activity, though 41 shows some modest activity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We further investigated the role of the SO2 moiety by synthesizing the fully reduced and
partially reduced variants, 35-40. If the SO2 moiety does play the role of hydrogen bond
acceptor, then these reduced analogs would be less efficacious. As expected, we observed either
a loss in activity, or no change in activity from analogs 36-40. The fully reduced analog 35,
however, inexplicably shows activity comparable to that of 103. We also synthesized 41-43 as
smaller, more basic bioisosteres of 35-37. This modification resulted with a drop in activity for
41 as compared to 35, and a great improvement with 42 as compared to 36. In fact, the potency
of 42 is comparable to that of 103, 27, and 33. This suggests that an oxygen hydrogen bond
acceptor is important to activity. The steric constrains of this position seem flexible, given two of
the more active analogs in this series, 103 and 42, are the largest and smallest of the series,
respectively. The less active of this series are of varying size, but are consistently less basic than
the more active analogs.
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 30
1
2
3
4
Table 2. SAR Summary and Toxicity of Saccharin Inspired Analogs
5
6
7
8
9
Het
ID #
IC50
LC50
R2
Key:
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R2
H
N
Boc
N
O
O
N
Het
O
O
19
50.6 ± 8.48 µM
69.2 ± 1.01 µM
103
2.61 ± 0.40 µM
> 100 µM
20
24.3 ± 1.87 µM
> 100 µM
S
N
O
O
O
26
44.3 ± 3.72 µM
> 100 µM
27
10.6 ± 2.44 µM
> 100 µM
28
68.4 ± 24.25 µM
> 100 µM
S
N
29
21.9 ± 2.41 µM
> 100 µM
30
51.8 ± 6.71 µM
95.2 ± 10.9
31
49.7 ± 24.3 µM
> 100 µM
N
O
O
32
>100 µM
> 100 µM
33
3.45 ± 1.93 µM
> 100 µM
34
56.0 ± 5.85 µM
> 100 µM
N
O
S
35
5.38 ± 1.60 µM
70.3 ± 8.89 µM
36
68.0 ± 30.5 µM
> 100 µM
37
19.8 ± 1.72 µM
> 100 µM
N
O
O
38
49.0 ± 4.68 µM
> 100 µM
39
68.0 ± 7.83 µM
87.3 ± 6.78 µM
40
23.3 ± 0.68 µM
> 100 µM
S
N
O
O
41
16.8 ± 5.93 µM
66.2 ± 4.18 µM
42
6.69 ± 1.70 µM
> 100 µM
43
49.3 ± 7.02 µM
> 100 µM
N
O
Summary of IC50 values and toxicity for structure activity relationship studies. IC50 values were
obtained using RAW 264.7 cells treated with 20 ng/mL LPS and varying concentrations of
compound. A cell viability assay was used to determine cytotoxicity at each tested concentration.
LC50 is the concentration at which cytotoxicity results in 50% cell viability. The purity of tested
compounds was evaluated via 1H NMR (>95% sample purity).
12
ACS Paragon Plus Environment

Page 13 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
From this series of molecules, there are five analogs that have IC50 values of less than or
roughly equal to 10 μM. Four of these five analogs have the same Boc-protected furfurylamide
(103, 27, 33, and 42). All four of these analogs lose significant activity when deprotected. This
implies that there is an important interaction(s) being made with the Boc group, and/or that an
amide N-H causes a deleterious interaction. To better understand this observation, analogs 50,
52, 54, 55, 56, 57, and 58 were synthesized from the corresponding carboxylic acid 45 (Scheme
4).20
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 30
1
2
3
4
Scheme 4. Amide Modifications
5
6
O
O
O
O
O
O
S
S
S
7
8
9
N
N
N
xxvi
xxvii
xxiii
18
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
O
Ot-Bu
OH
Cl
O
O
O
44
45
46
R
O
O
S
O
R'
N
amines, or xxiv
47, or 48
NR'2
N
O
N
OCH3
O
O
49, R' = H
53, R' = H
57
58
xxv
xxvii
R
50, R' = Boc
54, R' = Boc
O
CH3
N
O
O
N
55
NR'CH3
O
O
CH3
O
H3CO(O)CHN
AcHN
51, R' = H
xxvi
56
NHBoc
52, R' = Boc
47
48
xxvi. 1) NaH, DMF 2) 4-bromomethylbenzoic acid tert-butyl ester (44 = 62%), xxvii. TFA,
CH2Cl2 (45 = 98%), xxiii. SOCl2 (>99%), xxiv. Procedure A: 46 in CH2Cl2, then ammonia in
THF (49 = 92%), or methylamine in THF (51 = 79%), or 2-furan-2-yl-ethylamine and DIPEA
(53 = 85%), or N-methylfurfurylamine and DIPEA (55 = 86%). Procedure B: 1) tert-
butylcarbamate, LiHMDS, THF 2) 46 in CH2Cl2. Procedure C: 1) NaH in DMF, then 47 or 48 2)
46 in CH2Cl2 (57 = 41%, 58 = 17%), xxv. Boc2O, DMAP, CH2Cl2 (50 = 73%), xxvi. Boc2O,
DMAP, CH2Cl2 (52 = 81%), xxvii. Boc2O, DMAP, CH2Cl2 (54 = 33%)
Interestingly, intermediate ester 44 showed modest activity (Table 3), while intermediate
carboxylic acid 45 shows no activity. This may imply that the previously successful analogs
14
ACS Paragon Plus Environment

Page 15 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
bearing Boc groups might be benefiting from hydrophobic interactions with the tertiary butyl
moiety. Boc protected amides 52 and 56, however, have shown very poor activity. Initial efforts
to synthesize 56 produced the double-Boc protected amide 50, which also has poor activity,
suggesting that the furan substituent was necessary. We also investigated a very minor change
by synthesizing the extended linker analog 54. Surprisingly, a significant loss in activity was
observed.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To test if the presence of an acidic amide N-H could be causing a negative effect on
activity, we synthesized 55. However, we observed significantly lower potency, perhaps
suggesting that the rotational constraints of a tertiary amide are not conducive to activity. Given
this observation, we speculated that perhaps a carbonyl component was still required for activity.
However, the N-methylcarbamate and N-acyl analogs, 57 and 58, were significantly less potent
than even 20. We concluded that the furfurylamide component was essential, and that Boc
protection was the optimal substituent. Based on these results, 103 was selected as our lead
compound.
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 30
1
2
3
4
Table 3. SAR Summary and Toxicity of Saccharin Derived Analogs
5
6
O
O
7
S
8
N
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
ID #
IC50
LC50
ID #
IC50
LC50
Key:
Key:
R3
R3
R3
O
44
10.6 ± 2.44 µM
> 100 µM
52
42.0 ± 3.20 µM
> 100 µM
N(Boc)CH3
Ot-Bu
103
45
74.7 ± 9.92 µM
> 100 µM
53
76.6 ± 6.49 µM
> 100 µM
2.61 ± 0.40 µM
OH
N
H
O
> 100 µM
Boc
N
49
50.6 ± 10.3 µM
> 100 µM
54
48.6 ± 7.15 µM
> 100 µM
R3 =
O
NH2
N
O
Boc
CH3
56
64.7 ± 8.30 µM
> 100 µM
55
79.6 ± 3.69 µM
> 100 µM
NHBoc
N
O
O
N
N(Boc)2
50
33.4 ± 3.31 µM
> 100 µM
57
76.0 ± 8.20 µM
> 100 µM
OCH3
O
Ac
51
37.7 ± 1.21 µM
> 100 µM
58
42.6 ± 5.19 µM
> 100 µM
NHCH3
N
O
Summary of IC50 values and toxicity for structure activity relationship studies. IC50 values were
obtained using RAW 264.7 cells treated with 20 ng/mL LPS and varying concentrations of
compound. A cell viability assay was used to determine cytotoxicity at each tested concentration.
LC50 is the concentration at which cytotoxicity results in 50% cell viability. The purity of tested
compounds was evaluated via 1H NMR (>95% sample purity).
16
ACS Paragon Plus Environment

Page 17 of 30
Journal of Medicinal Chemistry
1
2
3
4
Anti-Inflammatory Mechanism Studies of 103
5
6
7
8
Our initial screen identified compounds that could inhibit the LPS-induced inflammatory
response. To determine if 103 specifically targets TLRs, NO signaling was assessed with three
different TLR ligands. LPS (TLR4), Poly I:C (TLR3), and Pam2CSK4 (TLR2/6) were chosen to
encompass the most variety in signaling, including differences in TLR localization and adaptor
proteins (Figure S3, supplementary information). As Figure 2 shows, 103 inhibits NO signaling
regardless of ligand treatment. This suggests that 103 does not bind specifically to an individual
TLR, but rather inhibits a common downstream factor of these TLRs. The dose-response curves
for all three ligands show comparable inhibition, with minor deviations due to TLR expression
levels and the effectiveness of the ligand to induce inflammation. Primary macrophage cells
demonstrated the same behavior as RAW 264.7 cells, with 103 inhibiting LPS signaling with an
IC50 value of 9.61 ± 1.45 µM (Figure S2, supplementary information). It is important to note that
103 shows no cytotoxicity at concentrations up to 100 μM (Figure S4, supplementary
information).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. 103 inhibits NO signaling mediated by different TLRs with comparable IC50s. RAW
264.7 cells were treated with LPS (TLR4 ligand), Poly I:C (TLR3 ligand), or Pam2CSK4
(TLR2/6 ligand). The IC50 values are 2.61 ± 0.40 µM, 10.9 ± 0.74 µM, and 1.69 ± 0.43 µM,
respectively. Treatment with 103 decreased NO production with all TLR ligands in a dose-
dependent fashion. These results demonstrate that 103 does not specifically inhibit a particular
TLR, but rather, a common downstream effector. Data was normalized [(raw data-untreated
cells)/(TLR agonist + solvent control-untreated cells)] such that TLR agonist + solvent is 100%
activation, and untreated cells are 0% activation. Data points shown are the average of nine
replicates, with error bars represented as the standard deviation.
18
ACS Paragon Plus Environment

Page 19 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
To further confirm that iNOS was being down regulated by treatment with 103,
quantitative real time polymerase chain reaction (RT-PCR) and western blot experiments were
performed. RT-PCR data was obtained using RAW 264.7 cells treated with LPS and varying
concentrations of 103. Figure 3 demonstrates that treatment with 103 decreases iNOS mRNA in
a dose-dependent fashion. Western blots were performed with a pan NOS antibody, and again
iNOS is seen to decrease in a dose-dependent fashion (Figure 4), indicating that 103 suppresses
iNOS at mRNA, protein, and cell signaling levels. While LPS was used as the inflammation-
inducing ligand in the NO production assay to maintain consistency, its effects are indirect.
Therefore, a secondary assay that monitors IFN-γ induced mRNA changes was carried out to
confirm the validity of the NO assay as the primary readout for understanding SAR and
compound optimization (Figure S7, supplementary information). These results demonstrated the
same trend of inhibition for 103, 36, and 49 (49 is commercially available from Enamine Ltd.).
Compound 103 is able to inhibit greater than 70% of IFN-γ induced mRNA,
whereas 36 and 49 show a weaker potency, with maximum inhibition of 50%. Importantly, these
results corroborate the IC50 values determined in the previously described NO production assay.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. 103 treatment decreases iNOS mRNA in a dose-dependent fashion. RAW 264.7 cells
were incubated with 20 ng/mL LPS and varying concentrations of 103 for 20 hours. Data is
shown with ligand-induced cells normalized to a fold change of 1. Treatment with 103 decreases
iNOS mRNA up to 67%. Data shown is the average quantification of three biological replicates,
each in technical duplicate, with error bars represented as the standard deviation. ** p ≤0.01,
**** p ≤0.0001.
20
ACS Paragon Plus Environment

Page 21 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. A) 103 treatment reduces iNOS protein expression in a dose-dependent fashion. The
iNOS protein is induced by LPS treatment, and decreases with compound treatment, suggesting
that compound reduces the inflammation that results in iNOS expression. The image shown is a
representative image, with brightness and contrast adjusted for clarity. B) Quantification of
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 30
1
2
3
4
5
6
7
8
9
iNOS western blot. Data was normalized to GAPDH as a loading control. Data shown is the
average quantification of three biological replicates, with error bars represented as the standard
deviation. *** p ≤ 0.001.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Regardless of their ligand or localization, all TLRs activate NF-κB (Figure S3,
supplementary information). In order to test the inhibitory effects of 103 on NF-κB activity, a
secreted embryonic alkaline phosphatase (SEAP) assay was performed in HEK 293T cells.
When tested at concentrations up to 100 μM, the compound did not down regulate NF-κB
activation through TLR3 or TLR4 (Figure S5, supplementary information). To determine if any
modulation occurs through other NF-κB pathways, TNF-α was used to activate NF-κB signaling.
As seen in the Figure S4 in the supplementary information, NF-κB signaling through tumor
necrosis factor receptor (TNFR) is also unaffected. This data suggests that 103 does not directly
modulate the TLR signaling pathway at any point, as NF-κB is essential to all TLR signaling.
We next sought to confirm this result through observation of NF-κB-induced cytokines,
particularly TNF-α. A commercially available enzyme-linked immunosorbent assay (ELISA)
was used to measure TNF-α in RAW 264.7 cells. Figure S6 in the supplementary information
shows that there was no change in TNF-α cytokine levels with compound treatment. These
results confirm in two cell types that there is no modulation of NF-κB by 103, regardless of
ligand or signaling pathway. However, previous results demonstrated that TLR-induced NO
activation is inhibited by 103. The iNOS promotor has binding sites for both NF-κB and STAT1.
Since NF-κB activation is not being affected with 103 treatment, the inhibition of iNOS was
therefore likely to occur within the JAK/STAT1 pathway.
22
ACS Paragon Plus Environment

Page 23 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
As no direct antagonism was observed through TLRs and NF-κB, additional tests were
carried out to identify the potential anti-inflammatory mechanism of 103. The interferon I (IFN-
α/β) and interferon II (IFN-γ) pathways cause upregulation of iNOS, which results in production
of NO. As such, we speculated that observed NO inhibition might occur through inhibition of the
JAK/STAT signaling pathway. To test this hypothesis, IFN-γ was used as a ligand to activate
iNOS in RAW 264.7 cells. Inhibition of NO occurred in a dose-dependent fashion with treatment
of 103. The IC50 value with IFN-γ is 7.88 ± 1.25 μM, which corroborates the IC50 value of LPS
(Figure 5). This indicated that the JAK/STAT1 pathway is involved in the inhibitory function of
103. Additionally, as TLR activation results in production of type I interferons, synonymous
inhibition with IFN-γ proposes a shared target between these two pathways. Thus, it is likely that
the molecular target of 103 lies in the interferon-induced STAT1 pathway.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 30
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. 103 inhibits IFN-γ signaling in a dose-dependent fashion. RAW 264.7 cells were
treated with 5 ng/mL IFN-γ to activate JAK/STAT1 signaling. Treatment with 103 decreased
NO production in a dose-dependent fashion. These results suggest that 103 inhibits NO through
the JAK/STAT1 pathway. Data was normalized [(raw data-untreated cells)/(TLR agonist +
solvent control-untreated cells)] such that TLR ligand + solvent is 100% activation, and
untreated cells are 0% activation. Data points shown are the average of nine replicates, with error
bars represented as the standard deviation.
Additionally, a commercial JAK/STAT RT-PCR array was used to determine if
JAK/STAT pathway signaling is by and large modulated with 103 treatment. A summary of the
24
ACS Paragon Plus Environment

Page 25 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
results is available in Table S1. The modified genes, including NOS2, Cebpb, and Gtp1 suggest
that 103 modulates STAT1 signaling.3,21,22 Additional validation is required to target the specific
site of action for 103. Taken together, our results provide consistent evidence that 103 functions
through the interferon-induced JAK/STAT1 signaling pathway, suppressing iNOS.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
In summary, we report the identification of a group of novel IFN inhibitors based on a
saccharine core. Extensive SAR studies have shown a narrow tolerance for change at the lone
amido position. A Boc protected furfurylamide has proven to be the most consistently successful
substituent. Our results demonstrate that 103 is a potent inhibitor of iNOS on both an mRNA
and protein expression level. However, this occurs without NF-κB modulation, indicating a
TLR-independent mechanism. Further biochemical studies imply potential inhibition in the
STAT1 pathway, as this is shared between type I and II interferons, and associated genes were
observed to change via PCR array. 103 may provide therapeutic insight for inflammatory
diseases such as systemic lupus erythematosus and multiple sclerosis.
Associated Content
Supporting Information Available: general chemistry information, synthetic methods,
compound characterization, and cell-based assay data. This material is available free of charge
via the Internet at http://pubs.acs.org.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 30
1
2
3
4
Author Information
5
6
Corresponding Author:
7
8
9
*Hang Yin: phone, +1-303-492-6786; e-mail, hubert.yin@colorado.edu
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notes:
The authors declare no competing interests.
Acknowledgements
We thank the National Institutes of Health (Grants GM101279 and GM103843) for
financial support. We thank Noah Kastelowitz for critical reading of the manuscript and
assistance with statistical calculations.
Abbreviations Used
Nitric oxide (NO), Interferon (IFN), Janus kinase (JAK), signal transduction and activator of
transcription (STAT), inducible nitric oxide synthase (iNOS), Toll-like receptor (TLR),
lipopolysaccharide (LPS), Nuclear Factor Kappa B (NF-κB), interferon-α/β receptor 1
(IFNAR1), interferon-γ receptor (INFGR), secreted embryonic alkaline phosphatase (SEAP),
tumor necrosis factor-alpha (TNF- α), tumor necrosis factor receptor (TNFR), real time
polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA)
References
(1)
Schroder, K.; Hertzog, P.; Ravasi, T.; Hume, D. Interferon-Γ: An Overview of Signals,
Mechanisms and Functions. J. Leukoc. … 2004, 75, 163–189.
26
ACS Paragon Plus Environment

Page 27 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(2)
(3)
Schindler, C.; Levy, D. E.; Decker, T. JAK-STAT Signaling: From Interferons to
Cytokines. J. Biol. Chem. 2007, 282, 20059–20063.
Kota, R. S.; Rutledge, J. C.; Gohil, K.; Kumar, A.; Enelow, R. I.; Ramana, C. V.
Regulation of Gene Expression in RAW 264.7 Macrophage Cell Line by Interferon-
Gamma. Biochem. Biophys. Res. Commun. 2006, 342, 1137–1146.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(4)
(5)
Takeda, K.; Akira, S. TLR Signaling Pathways. Semin. Immunol. 2004, 16, 3–9.
Buchanan, M. M.; Hutchinson, M.; Watkins, L. R.; Yin, H. Toll-like Receptor 4 in CNS
Pathologies. J. Neurochem. 2010, 114, 13–27.
(6)
(7)
Schroder, K.; Sweet, M. J.; Hume, D. a. Signal Integration between IFNgamma and TLR
Signalling Pathways in Macrophages. Immunobiology 2006, 211, 511–524.
Lowenstein, C.; Alley, E.; Raval, P.; Snowman, A.; Snyder, S.; Russell, S.; Murphy, W.
Macrophage Nitric Oxide Synthase Gene: Two Upstream Regions Mediate Induction by
Interferon Gamma and Lipopolysaccharide. Proc. Natl. Acad. Sci. U. S. A. 1993, 90,
9730–9734.
(8)
(9)
Gessani, S.; Belardelli, F.; Pecorelli, A.; Puddu, P.; Baglioni, C. Bacterial
Lipopolysaccharide and Gamma Interferon Induce Transcription of Beta Interferon
mRNA and Interferon Secretion in Murine Macrophages. J. Virol. 1989, 63, 2785–2789.
Jacobs, A.; Ignarro, L. J. Lipopolysaccharide-Induced Expression of Interferon-Beta
Mediates the Timing of Inducible Nitric-Oxide Synthase Induction in RAW 264.7
Macrophages. J. Biol. Chem. 2001, 276, 47950–47957.
(10) Vadiveloo, P. K.; Vairo, G.; Hertzog, P.; Kola, I.; Hamilton, J. a. Role of Type I
Interferons during Macrophage Activation by Lipopolysaccharide. Cytokine 2000, 12,
1639–1646.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 30
1
2
3
4
5
6
(11) Aktan, F. iNOS-Mediated Nitric Oxide Production and Its Regulation. Life Sci. 2004, 75,
639–653.
7
8
9
(12) Santiago-Raber, M.-L.; Baccala, R.; Haraldsson, K. M.; Choubey, D.; Stewart, T. a; Kono,
D. H.; Theofilopoulos, A. N. Type-I Interferon Receptor Deficiency Reduces Lupus-like
Disease in NZB Mice. J. Exp. Med. 2003, 197, 777–788.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(13) Panitch, H. S.; Hirsch, R. L.; Schindler, J.; Johnson, K. P. Treatment of Multiple Sclerosis
with Gamma Interferon: Exacerbations Associated with Activation of the Immune
System. Neurology 1987, 37, 1097–1102.
(14) Baccala, R.; Hoebe, K.; Kono, D. H.; Beutler, B.; Theofilopoulos, A. N. TLR-Dependent
and TLR-Independent Pathways of Type I Interferon Induction in Systemic
Autoimmunity. Nat. Med. 2007, 13, 543–551.
(15) Lio, D.; Scola, L.; Crivello, A.; Bonafè, M.; Franceschi, C.; Olivieri, F.; Colonna-
Romano, G.; Candore, G.; Caruso, C. Allele Frequencies of +874T-->A Single Nucleotide
Polymorphism at the First Intron of Interferon-Gamma Gene in a Group of Italian
Centenarians. Exp. Gerontol. 2002, 37, 315–319.
(16) www.c2d2.org.
(17) Lewis, S.; Miller, G.; Hausman, M.; Szamborski, E. Isothioazoles I: 4-Isothiazolin-3-
Ones. A General Synthesis from 3, 3’-Dithiodipropionamides. J. Heterocycl. Chem. 1971,
8, 571–580.
(18) Combs, A. P.; Yue, E. W.; Bower, M.; Ala, P. J.; Wayland, B.; Douty, B.; Takvorian, A.;
Polam, P.; Wasserman, Z.; Zhu, W.; Crawley, M. L.; Pruitt, J.; Sparks, R.; Glass, B.;
Modi, D.; McLaughlin, E.; Bostrom, L.; Li, M.; Galya, L.; Blom, K.; Hillman, M.;
Gonneville, L.; Reid, B. G.; Wei, M.; Becker-Pasha, M.; Klabe, R.; Huber, R.; Li, Y.;
28
ACS Paragon Plus Environment

Page 29 of 30
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Hollis, G.; Burn, T. C.; Wynn, R.; Liu, P.; Metcalf, B. Structure-Based Design and
Discovery of Protein Tyrosine Phosphatase Inhibitors Incorporating Novel
Isothiazolidinone Heterocyclic Phosphotyrosine Mimetics. J. Med. Chem. 2005, 48, 6544–
6548.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(19) Porter, N. A.; Carter, R. L.; Mero, C. L.; Roepel, M. G.; Curran, D. P. Penultimate Group
Effects in Free Radical Telomerizations of Acrylamides. Tetrahedron 1996, 52, 4181–
4198.
(20) Vidal, J. A. G.; Seglar, J. F. D.; Chavez, M. T. Derivatives of Benzo[d]isothiazoles as
Histones Deacetylase Inhibitors. US20090312377 A1, 2009.
(21) Samson, S. I.; Richard, O.; Tavian, M.; Ranson, T.; Vosshenrich, C. a J.; Colucci, F.;
Buer, J.; Grosveld, F.; Godin, I.; Di Santo, J. P. GATA-3 Promotes Maturation, IFN-
Gamma Production, and Liver-Specific Homing of NK Cells. Immunity 2003, 19, 701–
711.
(22) Elser, B.; Lohoff, M.; Kock, S.; Giaisi, M.; Kirchhoff, S.; Krammer, P. H.; Li-Weber, M.
IFN-Gamma Represses IL-4 Expression via IRF-1 and IRF-2. Immunity 2002, 17, 703–
712.
29
ACS Paragon Plus Environment