Structure-activity relationship studies in substituted sulfamoyl benzamidothiazoles that prolong NF-κB activation

Article abstract of DOI:10.1016/j.bmc.2021.116242

In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell-based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure–activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in the future.

Full text of DOI:10.1016/j.bmc.2021.116242

Journal Pre-proofs
Structure-Activity Relationship Studies in Substituted Sulfamoyl Benzamido-
thiazoles that Prolong NF-κB Activation
Nikunj Shukla, Michael Chan, Fitzgerald S. Lao, Paul J. Chu, Masiel
Belsuzarri, Shiyin Yao, Jason Nan, Fumi Sato-Kaneko, Tetsuya Saito,
Tomoko Hayashi, Maripat Corr, Dennis A. Carson, Howard B. Cottam
PII:
S0968-0896(21)00250-9
https://doi.org/10.1016/j.bmc.2021.116242
BMC 116242
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 May 2021
24 May 2021
25 May 2021
Please cite this article as: N. Shukla, M. Chan, F.S. Lao, P.J. Chu, M. Belsuzarri, S. Yao, J. Nan, F. Sato-Kaneko,
T. Saito, T. Hayashi, M. Corr, D.A. Carson, H.B. Cottam, Structure-Activity Relationship Studies in Substituted
Sulfamoyl Benzamidothiazoles that Prolong NF-κB Activation, Bioorganic & Medicinal Chemistry (2021), doi:
https://doi.org/10.1016/j.bmc.2021.116242
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Structure-Activity Relationship Studies in
Substituted Sulfamoyl Benzamidothiazoles that
Prolong NF-κB Activation
,
a
a
a
a
a
Nikunj Shukla,* Michael Chan, Fitzgerald S Lao, Paul J. Chu, Masiel Belsuzarri, Shiyin
a
a
a
a
a
b
Yao, Jason Nan, Fumi Sato-Kaneko, Tetsuya Saito, Tomoko Hayashi, Maripat Corr,
a
a
Dennis A. Carson, Howard B. Cottam
a. Moores Cancer Center, University of California San Diego, La Jolla, CA 92093-0809, USA.
b. Department of Medicine, University of California San Diego, La Jolla, CA 92093-0656, USA.
KEYWORDS: adjuvant, NF-κB, LPS, Toll-like receptor, vaccine, immunization, MPLA,
thiazoles, affinity probe.
1

Abstract: In the face of emerging infectious diseases, there remains an unmet need for vaccine
development where adjuvants that enhance immune responses to pathogenic antigens are highly
desired. Using high-throughput screens with a cell based nuclear factor κB (NF-κB) reporter
assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a
sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist,
lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR)
studies on compound 1 that indicated the sites on the scaffold that tolerated modification and
yielded more potent compounds compared to 1. The selected analogs enhanced release of
immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary
dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in
combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant,
monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific
antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a
photoaffinity probe which will aid the target identification and mechanism of action studies in
future.
2

Introduction: In the face of emerging infectious diseases where viruses continue to mutate to
escape host immunity or emergence of new viruses such as severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), there remains an unmet need for vaccine development.1-5
Vaccination is a cost-effective healthcare strategy to prevent infections and has been successful
in reducing the incidence of infectious diseases including diphtheria, pertussis, measles, polio,
rotavirus, rubella and tetanus. However, many vaccines require repeated injections and boosters
for protective immunity and yet do not confer extended protection. For vaccines against diseases
such as influenza, a broader immunoprotection would aide in the defense against viruses that
undergo antigenic drift and shift, which are the potential cause of epidemics and pandemics,
respectively.6-8 To address such needs, novel vaccine adjuvants are being investigated. Many
candidate adjuvants are components of bacterial or viral origin, or synthetic ligands for pattern
recognition receptors (PRRs) including Toll-like receptors (TLR) -2, -4, -7, -8, and -9,
nucleotide-binding oligomerization domain-like receptors (NLRs), and retinoic acid-inducible
gene I (RIG-I)-like receptors (RLRs).9-18 To date the U.S. Food and Drug Administration (FDA)
1
9
20
has approved a TLR-4 agonist monophosphoryl Lipid A (MPLA) , TLR-9 agonist CpG 1018,
alum and squalene based adjuvants for human use. Although these adjuvants were approved for
use in specific vaccines, the need for improving adjuvant activity led to our focus for the
2
1
discovery of co-adjuvants, that promote efficacy of a known adjuvant. For example, AS04
(Adjuvant System 04), consists of MPLA and alum, and is approved in a hepatitis B vaccine,
®
® 22-25
Fendrix , and a human papillomavirus vaccine, Cervarix .
Our efforts towards identifying co-adjuvants focused on compounds that may enhance the NF-
κB and/or interferon (IFN) stimulating response element (ISRE) activation induced by a TLR-4
agonist or IFN-α, respectively.26, 27 These compounds were clustered into multiple chemotypes
3

such as pyrimido[5,4-b]indoles that had distinct structural requirements for prolonging NF-κB
activation compared to a set of compounds that induced NF-κB activation as TLR-4 agonists.26,
2
8
Additional chemotypes included compounds that enhanced both NF-κB and ISRE signaling
such as 4H-chromene-3-carbonitriles which were found to be good adjuvant for cancer
immunotherapy and bis-aryl sulfonamides that were found to enhance adjuvanticity of TLR-4
2
9
agonists. All these compounds mentioned above were identified via cell-based phenotypic
assays, so we aim to first analyze the SAR studies to apply and utilize them to understand
mechanism of action and target receptor identification in the future. Here we report the structure-
activity relationship (SAR) studies in one such chemotype bearing a substituted sulfamoyl
benzamidothiazole structure that is specific towards enhancing the NF-κB activation. These SAR
studies are aimed at identifying more potent compounds as well as utilizing SAR understanding
towards design and synthesis of an affinity probe analog.
4

Results and Discussion:
Preliminary SAR studies based on HTS data of the screening library compounds: The
library used in the HTS included 248 compounds that belonged to the substituted sulfamoyl
benzamidothiazole chemotype. These 248 compounds provided a preliminary SAR
understanding which is important from the perspective of further designing novel and potent
compounds as it guides drug design. The detailed scaffold classification for these 248
compounds and the potency in enhancing NF-κB signaling activity in the presence of LPS as part
of HTS is presented in Supporting Information Fig. S1.
The subset of 21 compounds that were identified to be active in enhancing NF-κB activity in the
presence of LPS after 12 hours in HTS were then subjected to confirmatory kinetic screening for
NF-κB enhancement activity at 5h and 12h in the presence of LPS. A scatter plot showing the
activation data for these compounds in the cell-based NF-κB FRET assay for 12h (Y axis) and 5h
(X-axis) incubation is shown in Supporting Information Fig. S2. Unlike previously identified
chemotypes that enhanced NF-κB activity which could be clearly classified into 4 groups based
2
6
on the 4 quadrants of the kinetic plot; here this series of compounds did not statistically affect
the activity at 5h but significantly enhanced the NF-κB activity by LPS at 12h. Of all these
compounds, compound 1 was found to be the most potent (Supporting Information Fig. S2).
The structure of compound 1 bearing a 2,5-dimethylphenyl substituent on the 4 position of the
thiazole ring and benzoyl-4-(sulfonylpiperidine) substituent connected at the 2-amino position of
the thiazole ring is shown in Fig. 1. Compound 1 was then subjected to further SAR studies with
an aim to not only identify more potent compounds but also to understand the positions on the
scaffold that could tolerate modification. We systematically divided the structure into 6 different
sites of modification (A, B, C, D, E and F) as shown in Fig. 1. Site A was assigned the 4-
5

substituent of the thiazole ring, site B was the thiazole ring itself, site C was the central amide
bond in the structure, site D was the benzene ring bearing the 1,4-carboxyl and the sulfamoyl
groups, site E was the sulfonamide functional group and site F was the amine substituent of the
sulfonamide group. SAR studies on compound 1 were approached by modifying one site at a
time.
Sites
of
A
B
C
D
E
F
modification
N
O
S N
O
NH
O
S
Compound 1
Figure 1. Structure and sites of modification of compound 1. The structure of compound 1 was
reduced to six constituent components (A-F) based on the substituents and the synthetic strategy
as shown in alternate blue and black colors.
Syntheses of analogs of compound 1 and SAR studies: We began with the synthesis of
compound 1 to understand the chemistry that may be utilized in the syntheses of analogs. The
synthesis of compound 1 required the two advanced synthons (compounds 4 and 8a) as shown in
Scheme 1. Compound 4 was obtained by formation of the sulfonamide bond by reaction of
compounds 2 and 3. Compound 8a was obtained by first activation of the alpha carbon by
bromination of the 2,5-dimethyl acetophenone (6a) to obtain compound 7a, followed by fusing
the activated ketone with thiourea. These two synthons were coupled using the HATU reagent
via amide bond to yield compound 1. A similar strategy was used for the synthesis of several
other SAR analogs where the advanced synthon 4 was used for syntheses of site A and site B
6

modified compounds while synthon 8a was utilized for syntheses of site D, E and F modified
compounds.
Site A modified analogs: With the preliminary SAR analysis in mind, we began the further SAR
studies with site A modified analogs by first eliminating one methyl unit at a time of the 2,5-
dimethylphenyl substituent to obtain 2-methyl and 3-methyl substituted compounds 12b and 12c,
respectively (Scheme 1). We evaluated these compounds for NF-κB activation to evaluate the
NF-κB inducing effects at longer incubation times (20h). NF-κB activation was measured by
THP1-Blue™ NF-κB Cells, that secreted alkaline phosphatase (SEAP) in response to NF-κB.
Both of the des-methyl compounds were found to be inactive suggesting a bis-substituted phenyl
may be necessary for activity (Table 1). Thus, for the next two compounds, we exchanged the
methyl group at each position with a bromo substituent, aiming to verify the functional group
compatibility and to use it as a reactive intermediate for further aromatic nucleophilic
substitution type reactions. 2-Bromo-5-methyl substituted compound 12d and 5-bromo-2-methyl
substituted compound 12e were then synthesized as shown in Scheme 1. Since the corresponding
acetophenone reagents (6d and 6e) for making these two compounds were not commercially
available, we had to synthesize them from carboxylic acid (5d and 5e) precursors by a Weinreb
amide synthesis, followed by Grignard reaction with methyl magnesium bromide. Both these
bromo substituted analogs were active suggesting these could be utilized for further modification
(Table 1). Next, we spaced the 2,5-dimethyl substituent away from the thiazole ring with a
methylene unit to obtain compound 12f. However, the compound was inactive suggesting a bi-
aryl conformation may be necessary for activity (Table 1). In the next set of compounds, we
probed if the phenyl ring could be replaced with nonpolar aliphatic substituents of small and
bulky sizes. Thus, 4-thiazole substituted methyl (12g), ethyl (12h), propyl (12i), iso-pentyl (12j)
7

and tert-butyl (12k) analogs were synthesized. None of these molecules had any NF-κB activity
suggesting that a bis-substituted phenyl group is necessary for activity (Table 1).
Scheme 1. Syntheses of compound 1 and site A modified compounds.
O
S
O
S
R
N
O
S
i
MeOOC
Cl
HN
HOOC
N
NH
O
N
v
O
O
S
O
2
3
4
1, 12b-q
R =
R
OH
ii
R
iii
R
iv
R
N
Br
NH2
O
O
O
S
5d-e
6a-f, p-q
7a-f, p
8a-q
1
12b
Br
12c
12f
N
Br
N
R
N
S
vi
vii
viii
NH
Boc
NH
Boc
NH
Boc
S
S
Br
9
10
11l-o
Br
2d
1
12e
N
S
O
S
iii, iv
4, v
NH
O
H3C
12g
N
O
N
n
O
12h
12i
12m
12p
12j
12k
n
n
NH2
S
12r-s
13r-s
14r-s
n = 1, r, n = 2, s
X
Br
1
2l
12n
ix
4, v
N
S
O
NH
O
S
N
S
S
O
N
N
NH2
NH2
X = -C CH, 12t
X = -CH2CH3, 12u
x
12o
12q
8
e
15
Reagents and conditions: i. (a) Et3N, CH2Cl2, (b) LiOH, MeOH/THF/H2O; ii. (a) N,O-dimethylhydroxylamine hydrochloride, HATU, Et3N, DMF, (b)
MeMgBr, THF; iii. Br2, CHCl3; iv. thiourea, EtOH, 100 oC; v. HATU, Et3N, DMF, 45 oC; vi. N,N-diisopropylamine, n-BuLi, THF; vii. (a) RB(OH)2,
Pd(PPh3)4, Na2CO3, DMF/H2O; viii. 4N HCl/dioxane; ix. (a) TMS acetylene, CuI, Pd(PPh3)4, DMF (b) TBAF, THF; x. Pd/C, H2 (50psi), MeOH.
Thus, we then varied the substitution pattern of the two methyl groups on the phenyl to identify
if any pattern correlated with activity. Molecules bearing all the different bis-methyl substituted
configurations at ortho (o), meta (m), and para (p) positions were synthesized. This included o,m
(2,3-dimethyl, 12l), o,p (2,4-dimethyl, 12m), m,p (3,4-dimethyl, 12n), m,m (3,5-dimethyl, 12o),
and o,o (2,6-dimethyl, 12p) analogs as shown in Scheme 1. In order to evaluate an alternate
route to synthesize these analogs from the perspective of improving synthetic yields and
8

accessing aryl compounds for which the ketone synthon may not be available, we began with
3
0
tert-butyl (5-bromothiazol-2-yl)carbamate (9), performed a halogen dance reaction to migrate
the bromo to position 4 on the thiazole ring and then used it as a common precursor for different
Suzuki coupling reactions, followed by N-Boc removal using HCl/dioxane and a HATU assisted
coupling reaction with 4 to obtain the differently substituted bis-methyl analogs 12l-12o. Since
our attempts to perform the Suzuki coupling reaction with 2,6-dimethylphenylboronic acid to
obtain compound 12p failed, likely due to steric hindrance, we used the ketone precursor 6p to
obtain the bis ortho analog 12p. Of all these compounds, only the ortho and meta substitution
bearing compounds 12l and 12p retained activity while the others were inactive (Table 1).
Notably, compound 1 was o,m (2,5-dimethyl) substituted suggesting that substitution closer to
the thiazole ring is tolerated while expansion on the ring at the para position results in loss of
activity. Based on this observation, we elected to expand on the o,m position on the same side of
the phenyl ring to obtain a α-naphthyl analog 12q, which was found to be equally potent, thus
justifying our hypothesis (Table 1). Encouraged by the acceptance of steric bulk around the
thiazole ring, we elected to lock the conformation of the phenyl and the thiazole ring by forming
a 5 or 6 membered ring connecting the ortho position of phenyl ring to position 5 of the thiazole
to obtain compound 12r and 12s, respectively. However, both these compounds were weakly
active suggesting the possibility of a different conformation for optimal activity (Table 1). We
then utilized our existing SAR findings to derivatize the bromo to expand at the ortho and meta
positions on the phenyl ring. Our attempts to displace the bromo substituent with an ethynyl unit
at the ortho position of 12d failed, but we were able to obtain the corresponding ethynyl
substituted compound 12t using a Sonogashira coupling reaction. This analog was further
reduced using catalytic hydrogenation to obtain the chain expanded 5-ethyl-2-methyl analog 12u.
9

While the ethynyl analog 12t was inactive, releasing the rigidity of the carbon-carbon bond as in
2u regained the NF-κB activity (Table 1).
1
Site B modified analogs: All the compounds explored in the HTS contained a thiazole ring and
most of them were modified at position 4 on the thiazole with very few compounds representing
5
-substituted thiazole analogs (Supporting Information Fig. S1). Thus, we were interested to
explore alternate heterocyclic replacement of thiazole as well as compounds modified at the 5-
position on the thiazole ring. We synthesized imidazole analog 18a, different bioisosteric
pyridine analogs including 2-amino-6-aryl (18b), 2-amino-5-aryl (18c) and 3-amino-6-aryl (18d)
substituted pyridine analogs. In addition, flipping the thiazole moiety yielded 5-phenyl
substituted compound 18e and addition of another nitrogen atom at position 5 yielded thiadiazole
analog 18f (Scheme 2). However, none of these molecules that replaced the thiazole
functionality was active, suggesting that thiazole may be involved in key interactions with the
receptor component necessary for activity (Table 1). Since thiazole was indispensable, we
decided to probe further by substituting at position 5 on the ring. Bromination of compound 8a
using N-bromosuccinimide allowed for the introduction of 5-bromo on the thiazole. The 5-bromo
was displaced using different reagents to obtain substituted amine analogs 18g and 18h, Suzuki
reaction derived phenyl analog 18i and furan analog 18j. Furthermore, ethanethiol and ethoxide
displaced the 5-bromo to obtain corresponding analogs 18k and 18l, respectively. All of these
compounds were completely inactive except for the ethoxy analog 18l, which showed partial
activity suggesting that linear chains may be tolerated (Table 1). Thus, we first synthesized a 5-
methyl analog 18m using the chemistry shown in Scheme 2 as above. The 5-bromo-2-
aminothaizole was amide-linked to obtain the bromo analog (18n) of compound 1, which was
derivatized to obtain a series on carboalkyl chains including 2-carbon, 4-carbon, and 5-carbon
1
0

alkynyls 18o, 18p, and 18q, and their corresponding alkyl derivatives 18r, 18s, and 18t,
respectively, obtained by catalytic hydrogenation of the alkynes. As hypothesized, these
compounds were potent in inducing NF-κB activity and we saw a chain length dependent activity
trend as shown in Fig. 2 (Table 1). The smaller alkyl chain analogs 18r and 18s retained activity
while the higher homologs showed dramatic loss in activity. In contrast, the ethynyl 18o had
weak activity while higher chain homologs 18p and 18q were very potent surpassing the activity
of compound 1 when tested alone (Fig. 2). These data, especially the potency of 5-alkyne
substituted thiazole compounds suggested the presence of a hydrophobic pocket with the
possible involvement of π-π interaction in the target receptor. Another possible hypothesis for the
observed difference in potency could be the different orientation of the substituent attached to an
alkyne or an ethyl group as observed with the enhanced potency of ponatinib where the ethynyl
3
1
group functions as linker to span the bulky residues on the target protein. Overall, site B
modification suggested the necessity to preserve the thiazole ring, but showed that NF-κB
activity can be enhanced with compounds bearing 5-alkyne substitution.
1
1

Scheme 2. Syntheses of site B modified compounds.
i
ii
iii
O
N
N
N
O
S
NH
NH2
NH
O
N
O
NH
16
NH
17
NH
O
6a
18a
H
N
X
O
N
S
N
iv
iii
N
H
O
OH
X
NH2
N
B
OH
Br
NH2
X
X
N
N
H
N
N
2
0b-d
O
H
H
19
21b-d
18b-d
b
c
d
v
S
iii
S
O
S
NH2
NH
O
N
N
N
O
2
2
18e
vi
iii
OH
S
S
O
S
NH2
NH
O
N
O
N
N
N
N
O
23
24
18f
vii
viii
iii
N
N
S
N
N
O
NH2
NH2
NH2
NH
S
N
S
S
S
Br
Y
Y
O
O
18g-l
8
a
25
26g-l
N
N
g
i
l
h
k
Y =
ix
iii
N
N
O
S
NH2
NH
O
S
O
N
O
S
S
O
O
j
27
28
18m
N
N
S
O
N
O
S
iii
x
NH2
NH
O
NH
S
N
N
S
S
Br
Br
O
R
O
O
1
8o-t
xi
2
5
18n
R = X
R = X
X = -H, 18o
X = -CH2CH3, 18p
X = -CH2CH2CH3, 18q X = -CH2CH2CH3, 18t
X = -H, 18r
X = -CH2CH3, 18s
Reagents and conditions: i. (a) Br2, CHCl3, (b) 1-acetylguanidine, DMF; ii. 3M HCl, MeOH, 55 oC; iii. 4, HATU, Et3N, DMF, 45 oC; iv. Pd(PPh3)4, Na2CO3,
DMF/H2O; v. (a) 9, Pd(PPh3)4, Na2CO3, DMF/H2O, (b) 4N HCl/dioxane; vi. thiosemicarbazide, POCl3, 105 oC; vii. N-bromosuccinimide, CH2Cl2; viii. For
compound 26g: Pd(dppf)Cl2, Et2NH, dioxane, for compound 26h: piperidine, NaH, DMF, for compounds 26i-j: Y-B(OH)2, Pd(PPh3)4, Na2CO3, DMF/H2O; for
compound 26k: NaSEt, DMF, for compound 26l: NaOEt, EtOH; ix. (a) Br2, CHCl3, (b) thiourea, EtOH, 100 oC; x. For compound 18o: (a) TMS acetylene,
Pd(PPh3)4, CuI, Et2NH, (b) TBAF, THF, For compounds 18p-q: RH, Pd(PPh3)4, CuI, Et2NH; xi. Pd/C, H2 (50 psi), MeOH.
1
2

Figure 2. Chain length dependent NF-κB activity profile in 5-thiazolyl site B modified analogs.
Differential activity profiles were observed for alkane v/s alkyne bearing analogs at the 5-
thiazolyl position of compound 1. Chain length (n) dependent reduction in activity was observed
for 5-thiazolyl modified alkanes (green squares) but an inverse activity relationship was observed
with corresponding alkynes (blue circles). The % activation values in NF-κB induction assays
were two-point normalizedor DMSO (0.5%) as 100% (grey dotted line) and compound 1 as
2
00% (grey dotted line). The mean SEAP response in NF-ĸB assay for compound 1 and DMSO
was 0.4 ± 0.02 and 0.033 ± 0.001 µg/mL, respectively. The relative reduction in the activity for
terminal alkane compounds was significantly greater than the terminal alkynes for the same
chain length suggesting involvement of π-π interactions. Alkyne derivatives are shown in blue
circles and alkane derivative activity is shown in green squares. Data are presented as mean ±
SD. **p<0.01 and *p<0.05 for alkyne bearing compounds compared to alkane bearing
compounds for the same chain length using one-way ANOVA followed by Kruskal-Wallis test.
1
3

Site C modified analogs: Next, site C modified compounds required us to make new synthons
as the amide linking synthetic strategy utilized so far could not be used since we wanted to
evaluate the effect of changing this amide linkage. Thus, an amine linked analog 34a and an
inverse amide linked compound 34b were synthesized as shown in Scheme 3. The alkylation of
the amide nitrogen yielded N-methyl analog 34c and N-propargyl analog 34d. The importance of
the hydrogen bond forming capabilities and positioning of the amide link was evident as all these
site C modified analogs were inactive except the inverse amide linked compound 34b that
retained only weak activity (Table 1).
Scheme 3. Syntheses of site C modified compounds.
O
S
O
S
O
S
i
ii
iv
ii
NC
Cl
NC
N
N
O
O
H2N
O
2
9
a
30
31
v
N
O
S
NH
34a
N
O
O
S
O
iii
NCS
N
S
Br
6
32
36
33
37
O
O
O
i
O2N
S
Cl
O2N
S
N
H2N
S
N
O
O
O
3
5
a
viii
O
N
O
S
N
O
S
HN
vi
vii
O
O
N
34b
N
O
O
S
OH
S
6
38
39
ix
R
N
N
O
S
N
O
S
NH
N
N
S
S
O
O
O
O
3
4c-d
1
R = -CH3, 34c
=
-CH2-C CH, 34d
Reagents and conditions: i. K2CO3, DMF; ii. Pd/C, H2 (50 psi), MeOH; iii. (a) Br2, CHCl3, (b) NaSCN, EtOH; iv. HBr/AcOH, 130 oC; v. Pd(OAc)2, dioxane; vi. (a) Br2,
CHCl3, (b) ethylthioxamate, EtOH, 120 oC; vii. LiOH, MeOH, THF, H2O; viii. HATU, Et3N, DMF; ix. for compound 34c: iodomethane, NaH, DMF, for compound 34d:
propargyl bromide, NaH, DMF.
Site D modified analogs: Site D modification included bio-isosteric replacement of the benzene
ring with pyridine to obtain compounds 42a and 42b and thiophene to obtain compound 42c
(Scheme 4). However, these bioisosteres of the benzene ring led to loss of activity, so we
1
4

prepared the next set of substituted benzene analogs. These included addition of a small group on
the position next to sulfonamide (position 3) such as 3-chloro (42d), 3-bromo (42e), 3-nitro
(42f), and 3-methoxy (42g). Also, the 3-nitro group on compound 42f was reduced to obtain the
3
-amino analog 42k. While the 3-chloro and 3-bromo substituted compounds retained NF-κB
activity, compounds containing the hydrogen bond forming substituents including 3-nitro, 3-
methoxy and 3-amino showed a decline in activity. This drop suggested the need for
hydrophobic components on the benzene ring. We queried the importance of hydrophobicity of
the substituent on the benzene ring and whether the position on the ring matters as well. So, we
synthesized the next set of compounds with substituents on the position next to the carboxamide
(position 2) including 2-chloro (42h), 2-bromo (42i) and 2-methoxy (42j). The NF-κB activity
significantly dropped for these analogs suggesting the presence of a small hydrophobic pocket on
the receptor at position 3 of the chemotype.
1
5

Scheme 4. Syntheses of site D modified compounds.
Site E modified analogs: Next, site E modification involved changing the sulfonamide bond.
This included elimination of hydrophilic interaction capable atoms like the sulfonamide nitrogen
and sulfone oxygens to obtain thioether analog 45a, which was oxidized to yield sulfone analog
4
5b. Replacement of sulfonamide with carboxamide at position 4 and 3 provided compounds 45c
and 45d, respectively, as shown in Scheme 5. Surprisingly, none of these modifications at site E
was tolerated (Table 1). Overall, the SAR studies suggested that the sulfamoyl benzamide
thiazole component consisting of sites B, C, D and E were critical for activity and may be the
core pharmacophore. Thus, for curiosity we swapped the carboxamide bond with the
sulfonamide bond to obtain compound 51 (Scheme 6) which was also found to be inactive
(Table 1).
1
6

Scheme 5. Syntheses of site E modified compounds.
Br
HO
i
ii
HO
Z
N
Z
NH
O
O
Z
O
S
43, Z = SH
44, Z = S
Z = S, 45a
Z = SO2, 45b
iii
HO
O
OH
O
iv
ii
HN
HO
O
N
O
N
N
NH
O
S
O
46
47
45c
O
O
O
OH
N
iv
ii
N
HO
O
HN
HO
O
N
NH
O
S
48
49
45d
Reagents and conditions: i. K2CO3, DMF, 45 oC; ii. 8a, Et3N, HATU, DMF, 45 oC; iii. 3-chloroperoxybenzoic acid; iv. HATU, Et3N, DMF.
Scheme 6. Syntheses of substituent swap analog.
O
O
O
i
OH
ii
N
MeOOC
S
Cl
+
N
N
N
NH
S
NH
S
O
NH2
S
S
S
O
O
O
O
2
8a
50
51
Reagents and conditions: i. (a) Et3N, CH2Cl2, (b) LiOH, MeOH, THF, H2O; ii. piperidine, HATU, Et3N, DMF.
Site F modified analogs: Last in the series, site F modified compounds were obtained by
coupling different carboxylic acid analogs with compound 8a to obtain several sulfonamide
analogs. This included increasing bis-alkyl chain length on sulfonamide analogs 54a (N-ethyl, N-
propyl), 54b (N,N-dipropyl), and 54c (N,N-dibutyl) (Scheme 7). Up to a propyl chain length
substituent was tolerated while dibutyl substituted compound 54c lost activity (Table 1). Thus,
bulky steric groups may not be tolerated. Replacing one alkyl chain with a phenyl group afforded
5
5
4d while extending the carbocyclic ring by one atom yielded N-cyclohexyl sulfonamide analog
4e. Both these molecules were inactive indicating that the aryl substitution or mono-alkyl
1
7

substitution on sulfonamide were not tolerated (Table 1). The preliminary SAR suggested that
all compounds bearing sulfonamide linked to piperazine were inactive (Fig. 1), but this set of
compounds only included ethyl carbamate linked to 4-nitrogen of the piperazine ring, while the
piperazine or its N-amide and N-alkyl analogs were not explored. Thus, en route towards the
syntheses of substituted piperazine analogs, we made N-Boc protected piperazine compound 54f.
The N-Boc was removed to obtain compound 54i, followed by acetylation to obtain 54j or
alkylation to obtain N-propyl analog 54k (Scheme 7). All the piperazine bearing molecules were
found to be inactive, prompting us to focus on the substituted piperidine analogs (Table 1). Thus,
we synthesized 4-ethoxy and 4-propyl substituted piperidine analogs 54g and 54h, respectively,
as shown in Scheme 7. Both these 4-substituted piperidine analogs were found to be very potent
(Table 1).
Scheme 7. Syntheses of sites F modified compounds.
O
S
O
S
O
O
i
ii
Cl + H R
R
N
O
S
NH
X O
HO
R
O
O
S
O
X = -H (52) or -CH (2)
53a-f, j, k
O
3
5
4a-f, j, k
R
=
N
N
N
N
N
N
N Boc
N
O
N
H
54a
54b
54c
54d
54e
54f
54g
54h
iii
N
O
S
N
S
O
S
NH
O
N
Boc
NH
N
Y
N
N
S
O
O
O
54f
Y = -H, 54i
Y = -COCH , 54j
iv
v
3
Y = -CH -CH -CH , 54k
2
2
3
Reagents and conditions: i. For X = H: Et N, CH Cl , For X = -CH : (a) Et N, CH Cl , (b) LiOH, MeOH, THF, H O; ii. 8a, HATU, Et N,
3
2
2
3
3
2
2
2
3
o
DMF, 45 C; iii. 4N HCl/dioxane; iv. CH COCl, Et N, DMF; v. C H I, K CO , DMF.
3
3
3
7
2
3
1
8

Hybrid compounds: All the SAR studies so far led us to identify three compounds, namely 12d,
8q, and 54h each from site A, B, and F modified, respectively; that were most active in NF-κB
1
inducing assays (Table 1, Supporting Information Fig. S3). These compounds bear structural
features that made them more potent than compound 1 and we wondered if synergistic activities
could be obtained by combining these chemical features to obtain hybrid compounds. Combining
the structural feature of 2-bromo substitution on the 4-phenylthiazole as in 12d (site A modified
compound) with the 4-propylpiperidine feature as in 54h (site F modified compound) yielded
compound 59 as shown in Scheme 8. Similarly, combining the 5-pentynyl substitution as in 18q
(Site B modified compound) and 4-propylpiperidine feature as in 54h provided compound 61.
Similarly, combining features of compound 12d and 18q provided Site A and B modified hybrid
compound 60. Inclusion of all the structural features of the above-mentioned compounds led us
to compound 61 (Scheme 8). Bioactivity analysis of these hybrid compounds showed that while
compounds 55 and 60 retained NF-κB activity, they did not show any synergistic increase in
activity. In contrast, compounds 57 and 61, both of which had the 4-propylpiperidine substituent
as Site F, led to significant loss of activity. Ideally, combining the chemical attributes such as
substituents necessary for activity in potent compounds leads to enhancement of activity,
however, in this case, combining the structural features of potent compounds to obtain hybrid
compounds did not yield any synergistic effects. This could possibly be due to overall
enhancement of the hydrophobicity or the size of these hybrid compounds.
1
9

Scheme 8. Syntheses of hybrid analogs.
8d
N
O
S
NH
O
N
Br
S
O
O
O
S
55
i
N
HO
O
53h
25
N
O
S
N
O
S
iii
NH
O
NH
O
N
N
S
S
Br
O
O
56
57
iv
ii
v
N
N
N
N
O
NH2
NH2
NH2
NH
R
S
N
Br
Br
Br
Br
S
S
S
S
I
O
O
8d
58
59
R = -H, 60
R = -CH2CH2CH3, 61
Reagents and conditions: i. (a) HATU, Et3N, DMF, 45 oC; ii. pentyne, Pd(PPh3)4, CuI, DMF; iii 53h, HATU, Et3N, DMF; iv. N-iodosuccinimide, CH2Cl2; v. for compound
0: 4, HATU, Et N, DMF, 45 oC and for compound 61: 53h, HATU, Et N, DMF, 45 oC.
6
3
3
Cellular toxicity assessment: All these compounds were evaluated for toxicity using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. MTT data analysis
showed that most of the compounds did not exhibit any significant toxicity with %viability
>
80% of vehicle control values. Some of the compounds enhanced cell growth as seen by
viability over 100% (Table 1). The few exceptions included a site B modified imidazole group
18a), a substituent swap analog (51) and a site F modified 4-propylpiperazine bearing
%
(
compound (54k) that showed lower than 80% viability despite the compound being inactive in
NF-κB assays.
Dose response NF-ĸB activity profile: The bioactivity data (Table 1) and the SAR studies
pointed us to the most potent compounds on sites A, B and F as 12d, 18q and 54h, respectively
(Supporting Information Fig. S3). These compounds selected based on the NF-κB SEAP assay
were further characterized by dose responses in NF-κB induction in combination with LPS as
these compounds prolonged NF-κB activation in the HTS (Fig. 3). The dose response curves
more finely differentiated the potencies in terms of EC values in the presence of LPS (10
5
0
2
0

ng/mL). Compound 12d showed a rapid fall in activity at 2.5 µM (EC = 4.5 µM) compared to
5
0
compound 1 with EC of 2.3 µM. Compounds 18q (EC = 1.7 µM) and 54h (EC = 0.9 µM)
5
0
50
50
were more potent than compound 1.
6
1
12d
18q
54h
4
2
0.3
1
Compound Concentration (M)
LPS (10 ng/mL)
LPS
+
Figure 3. Dose response curves for selected active analogs with LPS. Compounds 1, 12d, 18q,
and 54h were evaluated for enhancement of NF-κB signaling at graded concentrations. Site A
modified compound 12d was less potent than compound 1 while sites B and F modified
compounds 18q and 54h were more potent. The activity of LPS alone is shown as a grey bar.
Data are presented as mean ± SEM for experiments performed in triplicates in two independent
experiments. The NF-κB activation was measured as SEAP induced in the culture supernatant.
Cytokine and chemokine induction: Activation of NF-κB is usually characterized by
production of cytokines and chemokines including IL-6, IL-8, IL-12 and TNF-α. Because NF-κB
2
1

controls multiple intracellular functions, including cytokine and chemokine expression, we were
interested to see if the potent compounds enhanced production of a representative cytokine.
THP-1 cells (wild type) were treated with compounds with and without LPS and NF-κB
downstream chemokine IL-8 was measured by ELISA. The selected compounds (5 µM
concentration) significantly enhanced the IL-8 production when co-incubated with LPS (1
ng/mL) compared to LPS alone. When cells were treated with compound alone, the treated cells
released significantly higher levels of IL-8 in comparison to the vehicle control (Fig. 4).
*
*
*
*
1
*
*
*
0.1
0.01
Veh 1 12d 18q 54h Veh 1 12d 18q 54h
LPS (1 ng/mL)
+
2
2

Figure 4. IL-8 release by selected active analogs with and without LPS in THP-1 cells.
Compounds 1, 12d, 18q and 54h (5 µM) not only induced IL-8 but significantly enhanced its
induction in the presence of LPS (1 ng/mL, shaded boxes) and alone as well (unshaded boxes).
Compounds 1 and 54h also induced significantly higher cytokines compared to that of 12d and
1
8q. **p<0.001 and *p<0.01 compared to Veh or Veh+LPS by one-way ANOVA analysis and
Bonferroni’s post hoc testing.
Since the aim of this project is to verify the adjuvanticity of compounds in an animal model such
as mice, we chose to first verify if these compounds induced cytokine secretion by primary
murine derived bone marrow dendritic cells (BMDCs) similar to that observed in the human
THP-1 cell line. Murine BMDCs treated with compound alone did not release detectable
amounts of IL-12, but when BMDCs were treated with compounds in the presence of LPS, a
significant enhancement in IL-12 secretion in the culture supernatant was observed compared to
IL-12 secretion induced by LPS alone (Supporting Information Fig. S4). The cytokine
induction profile for these compounds in murine BMDCs was similar to IL-8 induction in human
THP-1 cells illustrating their activity in both human and mice cells as well as primary immune
cells.
In vivo adjuvanticity: Encouraged by the potency of compounds in both human and murine
cells, we were interested to verify the potency as co-adjuvants with MPLA (FDA approved TLR-
4
agonistic adjuvant), for in vivo evaluation. Immunization experiments in mice (5 mice/group)
were performed to evaluate the co-adjuvanticity of these selected lead compounds (50
nmol/mice) with low dose of MPLA (10 ng/mice) using ovalbumin (OVA, 20 µg/mice) as a
model antigen. Examination of OVA-specific IgG antibodies showed that co-immunization of
MPLA with compounds 18q and 54h induced statistically significant increases in antigen-
2
3

specific antibody titers when compared to mice immunized with MPLA alone (Fig. 5, Left).
There was also no demonstrable systemic toxicity, as indicated by behavior change or weight
loss. These results showed that selected potent compounds that prolonged NF-κB stimulation
enhanced the adjuvanticity of MPLA. Although adjuvanticity is a complex biological response
involving different immune cell types, tissue distribution, antigen presentation and antibody
maturation, we wondered if there was any correlation between NF-κB inducing activity and total
IgG responses to immunization for these 4 potent compounds. We plotted the mean anti-OVA
IgG titers obtained post immunization against potency (EC s from Fig. 3) of these compounds in
5
0
the THP-1 NF-κB SEAP assay (Fig. 5, Right). It is notable that there was good correlation
2
(
P<0.0007, R = 0.9986; Pearson two-tailed correlation) between the mean antibody titers and
compound potency in the THP-1 reporter cells. The results suggested that dose response potency
measurements (EC calculation) in vitro may be a useful tool to predict adjuvantic potency in
5
0
vivo for these series of compounds.
2
4

Figure 5. Co-adjuvanticity of potent analogs with MPLA. Left: mice (n=5 per group) were
immunized on day 0 and day 21 with antigen (OVA, 20 µg/mice), MPLA (10 ng/mice) and
compound 1, 12d, 18q or 54h (50 nmol/mice). The immunized mice were bled on day 28 and
OVA-specific IgG titers were measured using ELISA. Left: Box and whisker plot showing
statistically significant enhancement of antibody titers for compounds 18q and 54h when co-
adjuvanted with MPLA compared to MPLA alone. **p<0.05 compared to MPLA group using
Bartlett’s one-way ANOVA followed by Dunnett’s post hoc testing. Right: Pearson two-tailed
2
correlation ((P<0.0007, R = 0.9986) of mean (positive standard deviation) of OVA-specific IgG
titers and EC values obtained for NF-κB SEAP assay in THP-1 cells with LPS.
5
0
Affinity probe: The further drug development of HTS hits like compound 1 which are identified
through cell-based phenotypic assays and are involved in cell signaling pathways can likely
proceed with additional knowledge of the target receptor or the compound’s mechanism of
2
5

action.32 Thus, another aspect of SAR studies is to identify positions on the scaffold that can
tolerate the introduction of small functional groups such as aryl azide or diazirine to make
photoreactive probes.29, 33-37 Such chemical probes would then be useful tools for future
mechanistic and functional receptor studies. Based on the data here and our experience with
design and synthesis of photoreactive probes, we decided to introduce azide functionality at site
A by displacing bromo to obtain 3-azido and 2-azido analogs 63 and 66, respectively as shown in
Scheme 9. While 2-azido compound 66 was inactive, the 3-azido compound 63 retained activity,
thus allowing us to utilize site A for introducing a photoreactive aryl azide functional group.
Scheme 9. Syntheses of aryl azide and alkyne bearing analogs.
N3
Br
N3
N
O
S
i
ii
N
N
NH
O
NH2
NH2
N
S
S
S
O
8e
62
63
i
iii
ii
N
O
S
N
NH
O
NH2
N
N3
S
Br
O
N3
O
N3
S
O
6d
64
65
66
O
O
S
R
N
R
iv
v
HOOC
S
Cl + HN
HOOC
N
O
S
R
N
R'
NH
O
O
R'
O
R'
S
O
52
67a-e
68a-e
R
NH
N
N
N
N
R'
=
68a
68b
68c
68d
o
Reagents and conditions: i. CuI, 1,2-Dimethylethylenediamine, NaN , DMF, microwave 100 C; ii. 4, HATU, Et N, DMF;
3
3
o
iii. (a) Br , CHCl , (b) thiourea, EtOH, 100 C; iv. Et N, CH Cl ; v. 8a, HATU, Et N, DMF.
2
3
3
2
2
3
Next, in order to introduce a reactive tag such as an alkyne for pull down approaches, we decided
to probe site F. A series of propargyl bearing compounds such as N-propargyl 68a, N-ethyl,N-
2
6

propargyl 68b and N-propyl,N-propargyl 68c were synthesized as shown in Scheme 9. While the
fully reduced compounds synthesized earlier including analogs 54a and 54b (Scheme 7) were
active (Table 1), the corresponding alkyne bearing compounds (68a and 68b) lost their activity.
We next utilized the position 4 of the piperidine ring to introduce the ethyne group to obtain
compound 68d. Bioactivity data showed that 68d was modestly active which allowed us to
proceed towards making the photoaffinity probe by combining the active aryl azide attribute of
compound 63 and alkyne attribute of compound 68d. This was done by HATU coupling of
compounds 62 and 67d to obtain photoaffinity probe 69 as shown in Scheme 10. As a test of
potency of the photoaffinity probe 69, we compared it against compound 1 for NF-kB induction
in presence of LPS. A perfect overlapping dose-response curve (Fig. 6) confirmed the activity of
the photoaffinity probe which will be utilized in future for target identification studies.
Scheme 10. Synthesis of photoaffinity probe.
N3
N3
O
i
N
+
N
O
S
HOOC
S N
O
NH2
NH
O
N
S
S
O
62
67d
69
Reagents and conditions: i. HATU, Et N, DMF.
3
2
7

Figure 6. Potency of analogs towards design of photoaffinity probe. A) Potency of photoreactive
aromatic azide and alkyne bearing analogs compared to compound 1. Azide bearing compound
6
3 and alkyne bearing compound 68d were identified to be active in inducing NF-κB induction
in the presence of LPS. B) Comparison of dose-dependent NF-κB enhancing activity in presence
of LPS of compound 1 and photoaffinity probe 69 showing identical potency.
Conclusion: Compounds bearing sulfamoyl benzamidothiazole functionalities were identified
through HTS that enhance NF-κB signaling from a primary TLR-4 stimulus. Systematic SAR
studies involving six different sites of modifications on the scaffold led to identification of more
potent compounds than the original hit compound 1. These compounds not only enhanced the
NF-κB response but also augmented the induction of immunostimulatory IL-8 in THP-1 cells
and IL-12 in mouse BMDCs. Overall, these compounds were found to be safe in both in vitro
and in vivo assays and did not induce systemic inflammatory responses. Vaccine co-adjuvanticity
experiments in mice showed that these compounds enhanced antigen-specific antibody responses
with the adjuvant MPLA compared to MPLA alone. These studies indicate that future evaluation
of these potent compounds in infectious disease and vaccine models is warranted. Additionally,
2
8

the SAR studies presented here also helped synthesize a photoaffinity probe which will further
help in target identification and mechanisms of action studies.
2
9