Structure-Activity Relationship Studies to Identify Affinity Probes in Bis-aryl Sulfonamides That Prolong Immune Stimuli

Article abstract of DOI:10.1021/acs.jmedchem.9b00870

Agents that safely induce, enhance, or sustain multiple innate immune signaling pathways could be developed as potent vaccine adjuvants or coadjuvants. Using high-throughput screens with cell-based nuclear factor κB (NF-κB) and interferon stimulating response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound 1 that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure-activity relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group of compound 1 focused toward identification of affinity probes. The murine vaccination studies showed that compounds 1 and 33 when used as coadjuvants with monophosphoryl lipid A (MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can tolerate the introduction of aryl azide, biotin, and fluorescent rhodamine substituents to obtain several affinity and photoaffinity probes which will be utilized in concert for future target identification and mechanism of action studies.

Full text of DOI:10.1021/acs.jmedchem.9b00870

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Article
Structure-Activity Relationship Studies to Identify Affinity
Probes in Bis-aryl Sulfonamides that Prolong Immune Stimuli
Michael Chan, Fitzgerald S Lao, Paul J Chu, Jonathan Shpigelman, Shiyin Yao, Jason Nan, Fumi Sato-
Kaneko, Vicky Li, Tomoko Hayashi, Maripat Corr, Dennis A. Carson, Howard B. Cottam, and Nikunj M. Shukla
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00870 • Publication Date (Web): 11 Oct 2019
Downloaded from pubs.acs.org on October 12, 2019
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Structure-Activity Relationship Studies to Identify
Affinity Probes in Bis-aryl Sulfonamides that
Prolong Immune Stimuli
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a
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Michael Chan, Fitzgerald S Lao, Paul J. Chu, Jonathan Shpigelman, Shiyin Yao, Jason
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Nan, Fumi Sato-Kaneko, Vicky Li, Tomoko Hayashi, Maripat Corr, Dennis A. Carson,
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Howard B. Cottam, Nikunj M. Shukla*
a. Moores Cancer Center, University of California San Diego, La Jolla, CA 92093-0695, USA.
b. Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
KEYWORDS: Adjuvant, NF-κB, ISRE, LPS, Interferon, Toll-like receptor, Sulfonamide,
Affinity probe, Biotin, Fluorescent, Photoaffinity, Vaccine, Immunization
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Abstract: Agents that safely induce, enhance, or sustain multiple innate immune signaling
pathways could be developed as potent vaccine adjuvants or co-adjuvants. Using high-
throughput screens with cell-based nuclear factor kappa B (NF-κB) and interferon stimulating
response element (ISRE) reporter assays, we identified a bis-aryl sulfonamide bearing compound
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that demonstrated sustained NF-κB and ISRE activation after a primary stimulus with
lipopolysaccharide or interferon-α, respectively. Here, we present systematic structure-activity
relationship (SAR) studies on the two phenyl rings and amide nitrogen of the sulfonamide group
of compound 1 focused towards identification of affinity probes. The murine vaccination studies
showed that compounds 1 and 33 when used as co-adjuvants with monophosphoryl lipid A
(MPLA) showed significant enhancement in antigen ovalbumin-specific immunoglobulin
responses compared to MPLA alone. SAR studies pointed to the sites on the scaffold that can
tolerate the introduction of aryl azide, biotin and fluorescent rhodamine substituents to obtain
several affinity and photoaffinity probes which will be utilized in concert for future target
identification and mechanism of action studies.
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Introduction: Vaccines consisting of antigen and adjuvant rely primarily on adjuvants for
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enhancement of immune stimuli. These adjuvants include ligands for pattern recognition
receptors (PRRs) such as Toll-like receptors (TLR) -2, -4, -7, -8, and -9, nucleotide-binding
oligomerization domain-like receptors (NLRs), RIG-I-like receptors (RLRs) and cytokines such
as interferon-α (IFN-α), IFN-γ and IL-12.2-13 Some of these adjuvants have been approved for
human use by the U.S. Food and Drug Administration (FDA) including the TLR-4 agonist
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monophosphoryl Lipid A (MPLA), the TLR-9 agonist CpG 1018, and other adjuvants with
different mechanisms of action such as alum and squalene based adjuvants. Despite the
availability of approved adjuvants, the need for co-adjuvants is evident since single adjuvant
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vaccines often do not generate long lasting protective immunity. Alum has been used as an
effective single adjuvant for decades primarily due to its safety record and induction of increased
humoral immunity;17, 18 however it induces only weak cellular immunity and predominantly a T
helper (Th) type 2 associate response; whereas in some cases a T helper type 1 response would
be more effective for protection. In addition, it is not always sufficient for vaccinating
immunocompromised and elderly populations.17, 19 A co-adjuvant is a substance that may or may
not be an adjuvant by itself but can work with a known adjuvant to offer synergistic effects such
as enhanced antibody response. For example, IL-2 has been shown to be a co-adjuvant with
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alum-adsorbed hepatitis B vaccine. Similarly, combination adjuvants can be obtained using a
PRR or NLR ligand, an immunogenic protein, a delivery system or another adjuvant with a
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complementary mechanism of action. One such combination AS04 (Adjuvant System 04),
®
consisting of MPLA and alum, has been FDA approved in a hepatitis B vaccine Fendrix and
human papillomavirus vaccine Cervarix®.21-24 Alternative combinations involving approved
adjuvants, TLR agonists, NOD agonists, and delivery systems are being explored.25-28
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Our approach towards identifying novel co-adjuvants focused on small molecules that may not
lead to immune activation by themselves but may enhance the primary immune activation such
as nuclear factor kappa B (NF-κB) or IFN stimulating response element (ISRE) activation
induced by a TLR-4 agonist (LPS or MPLA). The rationale behind the approach is as follows:
Upon vaccine administration, local antigen presenting cells (APCs) at the site of injection, such
as dendritic cells and Langerhans cells, are activated by the TLR-4 agonist. These APCs engulf
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antigen and travel to local draining lymph nodes where the antigen is presented to T cells. The
activation levels of APCs induced by a TLR-4 agonist peaks at 2-6 hours and then decays due to
negative feedback mechanisms.30-37 Because it takes approximately 12-24 hours for an APC to
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travel to the lymph node after vaccination, APCs are arriving during the decay phase of the
activation. This rationale is well supported from a report that showed that the absence of
interleukin-1 receptor associated kinase M (IRAK-M, a negative regulator of TLR signaling)39
increases NF-κB activation, improves migration of dendritic cells (DCs) to lymph nodes thereby
increasing the lifespan of the activated DCs and secretion of Th1-skewed cytokines and
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chemokines. Thus, we hypothesize that prolonging or sustaining the activation of APCs
induced by the TLR-4 agonist for 12-24 hours will lead to optimal presentation of antigen to the
T cells which would enhance the initial immune response and potentially allow for a longer
lasting response. Our hypothesis is supported by reports that enhanced responses to vaccinations
were observed in mice with genetic disruption of either IRAK-M, an inhibitor of the NF-κB
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pathway, or of UBP43, a negative regulator of type 1 IFN signaling. Thus, to address this
issue, we sought HTS methods directed towards identification of co-adjuvants that prolonged
activation of an immune response induced by a primary stimulus.41, 42
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These cell based HTS campaigns tested protraction of a TLR-4 agonist lipopolysaccharide (LPS)
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stimulus through the NF-κB pathway or of IFN-α signaling via the interferon stimulating
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response element (ISRE) pathway. Compounds that prolonged LPS induced NF-κB signaling
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included a distinct set of pyrimido[5,4-b]indoles that were also found to be effective co-
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adjuvants with MPLA, an FDA approved adjuvant, in murine vaccination studies. In parallel,
compounds that prolonged IFN-α induced ISRE signaling in vitro were also evaluated as co-
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adjuvants in vivo which led to identification of a potent bis-aryl sulfonamide compound 1 (Fig.
) bearing 4-chloro-2,5-dimethoxy and 4-ethoxy substituted phenyl groups connected by a
1
sulfonamide functional group. Compound 1 did not possess any NF-κB or ISRE activity when
tested alone but it enhanced their activation when tested in presence of LPS or IFN-α
respectively, compared to the stimulus alone. The further drug development of such hits
identified through cell-based phenotypic assays and involved in cell signaling pathways is
hampered without the knowledge of the target receptor or the compound’s mechanism of
action.43
Figure 1. Structure of compound 1 and sites of modification on the scaffold. SAR studies on the
bis-aryl sulfonamide compound 1 were approached by modifying one site at a time.
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This necessitated SAR studies focused towards identification of affinity probes which involved
evaluation of structural variations for compound 1 that were unexplored in the HTS with an aim
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 or large substituents such as biotin
and fluorescence moieties to generate affinity and fluorescent probes, respectively.44-50
Exploration of several different functional groups and substituents will allow us to systematically
identify the position and size of the affinity probe as well as the reactive handle to be used for
introducing these probes. These chemical probes would then be useful tools for future
mechanistic and functional receptor studies. In addition, the chemical handle would allow one to
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covalently conjugate the small molecule to peptides or protein antigens to make self-adjuvanting
vaccine constructs which are widely explored in vaccine development.51-56
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Results and Discussion: Approximately 3400 differently substituted bis-aryl sulfonamide
compounds were screened in the original HTS libraries and a scatter plot showing activation data
for these compounds in both cell-based NF-κB and ISRE assays is shown in Supporting
Information Fig. S1. These results provided preliminary SAR analysis indicating the
substituents on the two aryl rings necessary for activity and pointed to compound 1 as an
advanced lead. Hence, we approached further SAR studies on compound 1 by first identifying
three areas (sites A, B and C) of potential modification as shown in Fig. 1. To standardize the
reaction, we began with synthesis of compound 1 by reaction of 4-ethoxysulfonyl chloride (3a)
and 4-chloro-2,5-dimethoxy aniline (2a) in the presence of an organic base (Scheme 1).
However, the reaction not only provided the desired compound 1, but also formed the bis-
sulfonamide side-product in high yields. This undesired side-product was formed in situ by
further reaction of compound 1 with another equivalent of 4-ethoxysulfonyl chloride (3a). We
were able to isolate this bis-sulfonamide side-product but observed that it was somewhat
unstable. Limited hydrolysis by lithium hydroxide facilitated the complete conversion of this bis-
sulfonamide side-product to compound 1 without further hydrolysis of the mono-sulfonamide
bond thereby improving reaction yields for compound 1 (Scheme 1). This reaction strategy was
utilized for synthesis of several site A and site B modified compounds for SAR analysis.
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Scheme 1. Syntheses of compound 1 and its site A and B modified analogs.
O
NH2
O
HN S
O
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R1
+ Cl S
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2a-g
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1, 4-9, 11-17, 19, 22-27, 31
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O O
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Reagents and conditions: a) Et N, CH Cl ; b) LiOH. MeOH, THF, H O.
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SAR studies were initiated by modifying the substituents at site A (Fig. 1). These compounds
were synthesized according to Scheme 1 using different anilines (2a-g). We probed the removal
of one aryl substituent at a time to obtain compounds 4, 5, and 6 lacking the 2-methoxy, 3-
methoxy and 4-chloro substituent, respectively. Replacement of 4-chloro by a 4-bromo
substituent gave compound 7 and migration of the 2-methoxy substituent to the 3-position gave
compound 8. These compounds were evaluated for sustained activation of both NF-κB and ISRE
pathways using LPS and IFN-α as primary stimuli, respectively. The SAR studies pointed to the
importance of the methoxy substituents at the 2 and 5 positions of the aryl ring, because either
removal of any one of the substituents as in compound 4 and 5 or its displacement to another
position on the ring as in 8 led to complete loss of activity. Removal of the 4-chloro as in
compound 6 or its replacement with a spatially larger bromo substituent as in compound 7
retained activity (Table 1). Thus, to further explore position 4 on the phenyl ring, we synthesized
analogs with 4-nitro (9) substitution and its 4-amino (10) derivative. However, both these
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analogs were inactive suggesting that only hydrophobic substituents at this site are tolerated
(Table 1).
Table 1. Structure and bioactivity data for site A modified compounds.
1
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Com-
pound
Reagent
Site A
Substitution
NF-κBb
%
ISREb
%
MTTb
a
2
%
Activa SEM Activa SEM
Viabi
lity
SEM
tion
tion
1
4
2a
2b
O
O
200
-
200
-
73.5
83.7
1.0
Cl
O
126
2.4
99
1.1
1.8
Cl
5
6
7
2c
2d
2e
O
141
116
176
7.8
6.9
109
96
3.1
2.3
6.6
78.3
73.1
68.8
1.5
1.1
2.0
Cl
O
O
O
O
10.2
235
Br
8
9
2f
2g
--c
O
179
133
101
7.7
2.0
7.1
218
110
102
6.0
1.9
2.1
71.4
79.2
92.2
2.5
3.4
2.2
Cl
O
O
O
O
O2N
10
O
H2N
aCompounds 1, 4-9 were obtained by reaction of reagent 2 with 4-ethoxybenzenesulfonyl chloride (3a) as
b
shown in Scheme 1. The % activation values in NF-ĸB and ISRE induction assays were two point
normalized between compound 1 as 200% and LPS (10 ng/mL) for NF-κB or IFN-α (100 U/mL) for ISRE
as 100%. The mean SEAP response in NF-ĸB assay for compound 1 + LPS and LPS alone was 3.44 ± 0.08
and 0.56 ± 0.06 µg/mL, respectively. The mean emission ratio in ISRE assay for compound 1 + IFN-α and
IFN-α alone was 1.88 ± 0.04 and 0.69 ± 0.05 µg/mL, respectively. The % viability values for compounds
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in MTT assay were normalized to DMSO as 100%. The mean OD value at 405 nm for DMSO was 1.24 ±
c
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.03. All raw values used for normalization are represented as mean ± SEM. Compound 10 was derived
from compound 9 as shown in Scheme 2.
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Next, we focused on site B as shown in Fig. 1. The compounds were synthesized as discussed
earlier (Scheme 1) using different aryl sulfonyl chlorides (3a-p) and 4-chloro-2,5-dimethoxy
aniline (2a). Some of the aryl sulfonyl chlorides were commercially available, while the others
were synthesized as shown in Supporting Information Scheme S1. We first probed the
homologous series of 4-O-alkylated compounds starting with 4-hydroxy analog 11, 4-methoxy
analog 12, 4-propoxy analog 13 and 4-butoxy analog 14 compared to 4-ethoxy analog compound
1
. Bioactivity evaluation of these compounds showed that only the smaller homolog as in 4-
methoxy compound 12 was tolerated while the hydrophilic interaction with hydroxy group of 11
without any hydrophobic alkyl group was not tolerated. The higher 4-alkoxy chains showed
gradual loss of activity (Table 2). While the 4-propoxy substituted compound was weakly active,
the 4-propargyloxy compound 15, designed to use the alkyne as a handle for click chemistry
reactions, was found to be inactive. Removal of the ether oxygen to obtain 4-propyl substituted
compound 16 also led to loss of activity suggesting a crucial role of hydrogen bond interaction
by the ether oxygen. Other functional groups that could be involved in such hydrogen bond
interactions led to the syntheses of 4-nitro analog 17 and its amine bearing derivative 18
(Scheme 2) obtained by reduction of the nitro group. Also, the 4-nitrile analog 19, N-Boc
methylamine derivative 20 obtained by in situ N-Boc protection during the reduction of the
nitrile group and its free methylamine derivative 21 (Scheme 2) were synthesized. All these
compounds were also evaluated but found to be either weakly active or completely inactive. A
prior report indicated that analogs bearing a 4-O-phenyl substitution exhibited ubiquitin ligase
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57
inhibition activity, so we synthesized the 4-O-phenyl analog 22, but this compound was
inactive. Encouraged by the activity of 4-methoxy substituted analog 12, we synthesized 3-
methoxy and 2-methoxy substituted compounds 23 and 24, respectively. However, none of these
molecules was active. In order to find an additional handle for modification, bromine was
introduced to obtain a 3-bromo-4-methoxy substituted compound 25, which was also found to be
inactive. Learning from the requirement of a hydrogen bonding functional group at site B for
activity, we probed the addition of another oxo-containing group to obtain the 4-methylester
analog 26 and an amide analog 27. Ester hydrolysis of compound 26 yielded the 4-carboxyl
derivative 28 (Scheme 2). While the methyl ester bearing analog 26 was active, the hydrolyzed
carboxylic acid analog 28 and the amide linked compound 27 lost activity (Table 2).
Hypothesizing that the lack of hydrophobic alkyl group interaction could be a cause for the loss
of activity, compound 28 was further derivatized to obtain the ethyl ester analog 29, and the N-
methylamide analog 30 (Scheme 2). While analog 29 retained partial activity, compound 30 was
completely inactive suggesting that only hydrogen bond accepting substituents were tolerated
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
(
Table 2). An additional analog (compound 31, Scheme 1) was synthesized by inversing the
sulfonamide bond obtained by reaction of 2-ethoxyaniline and 4-chloro-2,5-
dimethoxybenzenesulfonyl chloride, but the inactivity of this analog suggested that the
positioning of the sulfonamide functional group was also critical for activity.
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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
Scheme 2. Syntheses of site A and B modified derivatives.
O
O
HN S
O
HN S
O
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
a
a
b
O
O
O
O
O
O
O N
2
H N
2
9
10
O
O
HN S
NO2
HN S
NH2
O
O
O
O
O
O
Cl
Cl
17
18
O
O
X
HN S
CN
HN S
O
O
O
O
O
O
Cl
Cl
c
19
20, X = -NHBoc
1, X = -NH2
2
O
O
O
O
Y
HN S
HN S
d
O
O
O
O
O
O
O
f
Cl
Cl
26
28, Y = -OH
29, Y = -OCH CH3
30, Y = -NHCH3
e
2
Reagents and conditions: a) Pd/C, H (50psi), EtOAc b) (Boc) O, Pd/C,
2
2
H (50 psi), MeOH c) 4N HCl/dioxane d) LiOH, MeOH, THF, H O
2
2
e) TMSCl, EtOH f) EtNH , HATU, Et N, DMF.
2
3
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Table 2. Structure and bioactivity data for site B modified compounds.
NF-κBb
%
ISREb
%
MTTb
%
Com-
pound
Reagent
Site B
Substitution
a
3
Activa
tion
SEM
Activa
tion
SEM
Viabi
lity
SEM
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
11
3b
3c
OH
O
98
3.1
6.8
104
219
2.3
6.5
85.2
69.6
1.0
1.3
1
1
2
3
226
3d
3e
149
101
8.1
2.3
125
106
5.6
1.3
71.3
95.3
0.7
1.5
O
1
4
O
O
1
1
1
5
6
7
3f
3g
3h
--c
3i
97
102
101
101
97
2.6
2.3
7.2
0.6
2.2
3.4
99
1.6
5.1
0.6
3.1
1.5
3.4
80.9
85.7
0.9
4.3
3.4
2.5
3.7
1.4
103
103
103
106
101
NO2
NH2
97.4
18
96.0
1
2
2
2
9
0
1
2
CN
102.9
109.2
NHBoc
--c
100
NH2
OPh
--c
3j
103
98
3.5
4.4
111
95
5.4
4.8
97.0
91.3
2.5
1.6
2
2
3
4
3k
3l
116
96
7.3
5.3
3.3
104
101
101
0.9
1.2
1.6
79.6
92.1
85.0
1.4
2.7
3.6
O
O
Br
O
2
2
5
6
3m
87
3n
3o
--c
--c
--c
CO2Me
CONH2
CO2H
191
103
89
6.4
2.6
202
94
6.5
3.1
1.2
9.8
2.2
74.4
91.0
98.2
70.8
77.0
0.7
1.5
3.0
0.8
2.2
27
2
2
3
8
9
0
2.3
104
169
105
CO2Et
189
103
10.1
2.4
CONHCH3
aCompounds 11-17, 19, 22-27 were obtained by reaction of reagent 3 with 4-chloro-2,5-dimethoxyaniline
b
(2a) as shown in Scheme 1. The % activation values in NF-ĸB and ISRE induction assays were two point
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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
normalized between compound 1 as 200% and LPS (10 ng/mL) for NF-κB or IFN-α (100 U/mL) for ISRE
as 100%. The mean SEAP response in NF-ĸB assay for compound 1 + LPS and LPS alone was 3.44 ± 0.08
and 0.56 ± 0.06 µg/mL, respectively. The mean emission ratio in ISRE assay for compound 1 + IFN-α and
IFN-α alone was 1.88 ± 0.04 and 0.69 ± 0.05 µg/mL, respectively. The % viability values for compounds
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
in MTT assay were normalized to DMSO as 100%. The mean OD value at 405 nm for DMSO was 1.24 ±
c
0
.03. All raw values used for normalization are represented as mean ± SEM. The compounds were
synthesized as shown in Scheme 2.
Moving forward, we wanted to probe the expansion at site C on the nitrogen of the sulfonamide
function of compound 1. These compounds were synthesized by derivatization of compound 1 as
shown in Scheme 3. The first extensive series of compounds were the N-alkylated derivatives
including N-methyl (33), N-propyl (34), N-butyl (35), N-pentyl (36), N-hexyl (37), N-heptyl (38),
and N-dodecyl (39). A clear correlation of bioactivity with the alkyl chain length was observed
with potency gradually decreasing with increased alkyl chain length and compounds bearing
alkyl chain lengths greater than N-pentyl were completely inactive (Table 3). We also probed the
effect of steric bulk around the core structure by synthesizing N-isopropyl (40) and N-isobutyl
(41) derivatives. Steric bulk closer to the core structure, as in compound 40, eliminated the NF-
κB activity while retaining ISRE activity. In contrast, spacing the isopropyl group away by one
methylene unit as in compound 41 regained the activity in both the NF-κB and ISRE assays.
Encouraged by these results, we desired to synthesize alkyne bearing compounds with an
additional aim to utilize the functional group as a biorthogonal reactive site. A homologous
series of alkyne bearing molecules including N-propargyl (42), N-butynyl (43), and N-pentynyl
(44) were synthesized (Scheme 3). Activity data showed that while N-alkyl derivatization with
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increasing alkyl chain length led to dramatic loss of activity, the corresponding N-alkynyl
derivatives retained activity almost equivalent to that of compound 1 (Fig. 2, Table 3) for the
corresponding alkyl chain length. As shown in Fig. 2, the retention of activity for the N-alkynyl
compounds compared to loss in activity for the analogous N-alkyl derivatives for the same
carbon unit chain length suggested the possible involvement of π-π interactions in near proximity
with the target receptor(s). We were therefore interested in evaluation of a triethyleneglycol
linked alkyne derivative (45) which would allow us to conveniently place the reactive functional
group distant from the core. However, the 12-atom chain length equivalent to N-dodecyl
compound 39 was too long to retain 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
Figure 2. Differential activity profile for terminal alkane v/s terminal alkyne bearing derivatives
of compound 1. Chain length (n) dependent reduction in activity was observed for site C
modified terminal alkane (closed symbols) and terminal alkyne (open symbols) bearing
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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
compounds. The % activation values in NF-ĸB and ISRE induction assays were two point
normalized between compound 1 as 200% (grey dotted line) and LPS (10ng/mL) for NF-κB or
IFN-α (100 U/mL) for ISRE as 100% (grey dotted line). 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. NF-κB activity is shown in blue squares
and ISRE activity is shown in red circles. Structure of the compounds is shown to the right with
variable chain length ‘n’ varying from 1 to 3. Data are presented as mean ± SEM. **p<0.01 and
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
*
**p<0.001 for alkyne bearing compounds compared to alkane bearing compounds for the same
chain length using two-way ANOVA followed by Bonferroni post hoc analysis.
These results for the alkyne bearing compounds led us to propose making compounds where
substituents can form enhanced π-π interactions. Thus N-benzyl (46) and N-phenethyl (47)
derivatives were synthesized and were also found to be potent analogs (Table 3). Since the N-
isopropyl analog 40 was inactive, we desired to determine if steric bulk was the only reason for
its inactivity and if that could be mitigated by some hydrogen bonding functional group such as
acetyl. Thus, the N-acetyl derivative (48) was synthesized and the bioactivity assays showed that
the compound was active. However, before proceeding with syntheses of additional acylated
analogs, we wanted to evaluate its stability in stock solutions since during the assay this
compound could behave as a prodrug by undergoing deacetylation to release active compound 1.
While the stock of compound 48 in DMSO was stable, incubation of compound with assay
media showed release of compound 1 (data not shown), suggesting that the bioactivity could be
due to a prodrug effect and not true interaction with the receptor. Thus, syntheses of additional
acylated analogs were not pursued.
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Since the hydrophobic alkyl and alkynyl groups were well tolerated at site C, we examined if
incorporating a hydrophilic group that could serve as a handle for further chemical modification
would be acceptable for activity. A pair of compounds bearing a precursor to a reactive handle
such as carboxylic esters were synthesized by alkylation of compound 1 to obtain the N-ethyl
glycinate (49) and N-ethyl butanoate (50) analogs (Scheme 3). Our attempts to make a stable
propionate analog failed after several attempts likely due to retro Michael type reaction, and
despite isolating a few milligrams of the tert-butyl propionate ester derivative, activity studies
were not pursued due to stability concerns. Both the ethyl ester substituted compounds 49 and 50
retained dual NF-κB and ISRE activities (Table 3). To avoid additional substitution closer to the
core sulfonamide pharmacophore, we chose a propylene spacer for further analogs. A terminal
hydroxy bearing analog as in N-propan-3-ol (51) and the N-Boc protected aminopropane analog
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
(52) were then synthesized. The ethyl ester of compound 50 was de-esterified using lithium
hydroxide to obtain its carboxylic acid analog 53, which was converted to the ethyl amide analog
5
5
4 (Scheme 4). Similarly, a free amine bearing molecule was obtained by N-Boc deprotection of
2 to obtain compound 55. Biological evaluation showed that the terminal hydroxy analog 51
retained activity in both the assays while the N-Boc protected compound 52 showed reduction in
activity, which was recovered when the N-Boc group was removed as in compound 55. Both the
free carboxylic acid and ethyl amide derivatives retained activity, which was more skewed
towards the NF-κB pathway (Table 3).
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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
Scheme 3. Syntheses of sulfonamide N-substituted derivatives of compound 1.
O
O
R
HN S
O
N S
O
a or b
O
O
O
O
+
R-X
O
O
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
Cl
Cl
1
32a-t
33-52
o
Reagents and conditions: a) K CO , DMF, 45 C. For compound 48: b) Et N, CH Cl .
2
3
3
2
2
Scheme 4. Syntheses of chemically reactive handle bearing analogs of compound 1.
O
O
O
O
OH
NH
O
S
O
S
O
S
N
O
a
N
O
b
N
O
O
O
O
O
O
O
O
O
O
Cl
Cl
Cl
50
53
54
Boc
NH
NH3+Cl-
O
S
O
N
O
N
S
O
c
O
O
O
O
O
O
Cl
Cl
52
55
Reagents and conditions: a) LiOH, MeOH, THF, H O b) EtNH , HATU, Et N, DMF; c) 4N HCl/dioxane.
2
2
3
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Table 3. Structure and bioactivity data for site C modified compounds.
NF-κBb
ISREb
MTTb
Com-
pound
Reagent
Site C Substitution
Variable -R Group
a
32
%
%
%
Viabi
lity
Activa
tion
SEM
Activa
tion
SEM
SEM
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
CH3
3
3
3
4
32a
32b
235
227
9.0
215
199
7.3
2.7
74.5
71.6
1.3
2.4
14.1
CH3
CH3
3
3
5
6
32c
32d
192
135
11.0
6.8
156
105
4.2
3.5
77.7
72.9
3.2
2.4
CH3
37
32e
81
4.0
99
2.1
95.2
3.3
CH3
CH3
3
8
32f
32g
32h
108
97
5.8
3.1
3.5
95
1.2
2.6
7.0
90.4
93.0
1.2
2.7
6.1
39
CH3
100
169
CH3
CH3
4
4
0
1
76
103.2
32i
32j
CH3
175
232
9.8
193
213
9.6
4.3
76.7
72.0
2.0
2.2
H3C
4
2
13.8
CH
4
4
3
4
32k
32l
206
182
3.3
5.8
201
184
6.2
7.3
70.1
70.7
1.9
2.1
CH
CH
4
5
32m
O
107
2.9
130
4.4
73.7
1.6
O
O
CH
4
4
6
7
32n
32o
181
131
3.9
6.7
159
141
5.9
7.1
71.7
44.0
2.5
1.3
Ph
Ph
O
4
4
8
9
32p
32q
32r
184
202
199
10.3
10.6
4.7
244
184
199
4.9
7.5
2.5
72.0
81.4
80.1
1.7
2.5
2.2
CH3
CH3
O
O
O
O
50
51
32s
32t
--c
OH
NHBoc
219
134
230
14.0
7.7
178
140
151
8.5
3.2
5.0
68.5
75.6
73.4
1.8
1.4
1.8
5
5
2
3
O
OH
7.9
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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
O
5
5
4
5
--c
--c
NH
NH2
206
189
4.4
137
182
7.3
6.4
78.2
71.0
1.8
0.7
10.6
a
b
Compounds 33-52 were obtained by reaction of reagent 32 with compound 1 as shown in Scheme 3. The
%
activation values in NF-ĸB and ISRE induction assays were two point normalized between compound 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
as 200% and LPS (10 ng/mL) for NF-κB or IFN-α (100 U/mL) for ISRE as 100%. The mean SEAP response
in NF-ĸB assay for compound 1 + LPS and LPS alone was 3.44 ± 0.08 and 0.56 ± 0.06 µg/mL, respectively.
The mean emission ratio in ISRE assay for compound 1 + IFN-α and IFN-α alone was 1.88 ± 0.04 and 0.69
±
0.05 µg/mL, respectively. The % viability values for compounds in MTT assay were normalized to
DMSO as 100%. The mean OD value at 405 nm for DMSO was 1.24 ± 0.03. All raw values used for
c
normalization are represented as mean ± SEM. The compounds were derived from compounds 50 and 52
as shown in Scheme 4.
All these compounds were evaluated for toxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assays. All the active compounds showed viability between
6
9% and 81%. Some of the inactive compounds were completely non-toxic. Compound 47 with
the N-phenethyl substitution was an exception showing somewhat higher toxicity (% viability =
4%) suggesting that an aryl group connected by an ethylene unit near the core sulfonamide
4
structure may lead to toxicity (Tables 1-3).
The bioactivity data from both the assays for all the compounds were plotted to verify the
correlation between the chemical structure and bioactivity. Most of the compounds were active
in both NF-κB and ISRE bioassays and showed a good correlation (Pearson two-tailed,
2
R =0.6812, P<0.0001, Fig. 3). The SAR trends however varied depending on the site of
modification. Site A modifications involving removal of the methoxy substituent (compounds 4
and 5) led to significant loss of activity (Fig. 3). On the other hand, nonpolar modifications at
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position 4 of site A (compounds 7 and 8) showed slightly skewed ISRE activity compared to
compound 1, while hydrogen bond forming substituents at this position led to loss of activity
(compounds 9 and 10). Most of the site B modified compounds were inactive suggesting
restricted SAR tolerance due to limited spatial availability in the target receptor. Only short alkyl
groups connected via ether-linkage as in compounds 1, 12 and 13 or carboxyl (ester)-linkage as
in compounds 26 and 29 retained activity. A good correlation was seen, however, between the
two assays for these compounds. In contrast, most of the site C modified compounds were active
in both the bioassays suggesting that only a part of the substituent may be involved in receptor
interaction and the rest of the group subtends out of the target receptor(s). A notable variation
was observed in sterically hindered bulky groups close to the core structure as in compound 40
which led to a loss of NF-κB activity, while still retaining ISRE activity. On the other hand,
another subset of compounds bearing a reactive handle such as carboxylic acid analog 53 and its
amidated derivative 54, showed reduction in ISRE activity while retaining the NF-κB 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
(Fig. 3). This suggested that a negative charge on the compound may be a deterrent for ISRE
activity.
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Bioactivity analysis for synthesized bis-aryl sulfonamide analogs. Scatter plot showing
the ISRE activity on the Y-axis for cells treated with each compound and NF-κB activity on X-
axis for cells treated with compound and LPS. Data are shown as a two-point normalization
between the active hit compound 1 as 200% (purple star) and vehicle (0.1% DMSO) as 100%.
Site A, B, and C modified analogs are designated as blue, red, and green squares, respectively.
Pearson two-tailed correlation was significant (P<0.0001) for the two activities for all these
compounds.
Continuing with the focus on compounds that retain dual NF-κB and ISRE activity similar to
original hit compound 1, we selected site B modified compound 12, site C modified compound
3
3 and an aliphatic amine bearing compound 55 for dose response experiments and EC50
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determination as these compounds are nearly equipotent in both the assays when evaluated at 5
µM concentration. As shown in Fig. 4, both compounds 12 and 33 showed relatively higher NF-
κB activity at 5 µM concentration, but the activity of compound 12 decreased faster at lower
concentrations which led to EC value of 1.85 µM. Compound 1 and 33 were almost equipotent
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
5
0
with EC values of 0.60 µM and 0.69 µM, respectively. Compound 55 was relatively weaker
5
0
with EC of 3.32 µM. The potency trends for these compounds remained the same in ISRE
50
activity with compounds 1, 12 and 33 exhibiting EC of 0.66 µM, 1.4 µM, and 0.84 µM,
5
0
respectively, and compound 55 with EC = 3.04 µM. Even though the activity of compound 55
5
0
was slightly attenuated, the amine handle can be utilized for derivatization to obtain affinity
probes.
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0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Dose response curves for selected active analogs. Compounds 1, 12, 33, and 55 were
evaluated for enhancement of NF-κB and ISRE signaling at graded concentrations. Compound
3
5
3 was equipotent as compound 1 while chemically reactive handle (amino) bearing compound
5 retained activity even though slightly attenuated. The activity of stimulus alone is shown as
grey bar. Data are presented as mean ± SEM. The NF-κB activity is measured as amount of
SEAP induced, while ISRE activity is measured as emission ratio for the FRET based assay.
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It was important to examine the adjuvanticity of the most potent compounds to verify if
prolongation of immune stimulus by this chemotype leads to enhancement of in vivo antibody
responses and if prolonged activation of the innate immune system could lead to systemic
inflammation that may be harmful to the host.58, 59 In addition, it was also important to verify that
the modifications on the scaffold that yielded potent compounds in vitro would retain the
adjuvanticity in vivo as well. All the compounds administered to mice had low toxicity in the
MTT assays. Since these vaccine co-adjuvants are designed to be administered locally (mostly
intramuscularly) and show negligible toxicity (based on MTT data), we did not anticipate an
excessive systemic inflammatory response. In the past, we utilized LPS as a widely recognized
activator of the innate immune system and well characterized TLR-4 ligand to screen over
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
60,000 compounds for their ability to enhance APC activation.41, 42 However, to test these
compounds for potency as co-adjuvants, we switched to the FDA approved TLR-4 adjuvant,
MPLA for in vivo evaluation. Immunization experiments in mice (8 mice/group) were performed
to evaluate the co-adjuvanticity of the lead compounds 1, 12 or 33 using ovalbumin (OVA) as a
model antigen and MPLA as an adjuvant. Amine handle bearing compound 55 was not selected
for immunization since it was designed for further derivatization as an intermediate to make
probes as discussed below. Examination of OVA-specific IgG antibodies showed that co-
immunization of MPLA with compounds 1 and 33 induced statistically significant increases in
antigen-specific antibody titers when compared to mice immunized with MPLA alone (Fig. 5),
without demonstrable systemic toxicity, as indicated by behavior change or weight loss. These
results verified our approach that selected bis-aryl sulfonamide compounds that prolong immune
stimulation could enhance the adjuvanticity of MPLA and that modified compounds that retained
potency in vitro were equally potent in vivo as well.
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**
1
1
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*
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8
9
0
10
1
0
10
1
0
OVA+
MPLA MPLA+ MPLA+ MPLA+
12 33
OVA+
OVA+
OVA+
1
Figure 5. Co-adjuvanticity of potent bis-aryl sulfonamide compounds with MPLA. Mice (n=8
per group) were immunized on day 0 and day 21 with antigen (ovalbumin, 20 µg/animal), MPLA
(
10 µg/animal) and compound 1, 12 or 33 (100 nmol/animal). The immunized mice were bled on
day 21 and OVA-specific IgG titers were measured using ELISA. Note, that the potent
compounds 1 and 33 showed significant enhancement of antibody titers when co-adjuvanted
with MPLA compared to MPLA alone. **p<0.01 and *p<0.05 compared to MPLA group using
one-way ANOVA followed by Dunnett’s post hoc testing.
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Now that we had confirmed the in vitro and in vivo potency of selected active compounds, we
were further interested to utilize the SAR studies for designing affinity probes. The activity data
guided us to utilize site C for the introduction of an identifiable tag by derivatizing compound
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
5
5. Although compound 55 was less potent than compound 1, the changes in the hydrophobic
interaction after amine derivatization may improve the potency. Compound 55 was derivatized to
obtain fluorescein labeled compound 56, rhodamine labeled compound 57 and biotin labeled
compound 58 (Scheme 5). In primary screens, the biotin labeled compound 58 was equipotent to
compound 1 and thus could serve as the affinity probe (Fig. 6, Table 4). The rhodamine analog
5
7 showed reduced activity compared to compound 1 in both the NF-κB and ISRE assays likely
due to the presence of a fixed charge on the molecule similar to the amine bearing compound 55.
In contrast, the fluorescein analog 56 was completely inactive in both the assays (Fig. 6, Table
4
).
Scheme 5. Syntheses of fluorescent and biotin labeled derivatives of compound 1.
+
-
NH Cl
HN R
3
O
O
N S
O
N S
O
a or b
O
O
O
O
O
O
Cl
Cl
55
56-58
O
O
S
O
O
HO
O
R =
N
H
O
H
O
56
57
58
HN
N
O
N+
O
N
HO
O
OH
H
H
Reagents and conditions: For compound 56: a) 5(6)-SFX SE, Et N, DMF;
3
For compound 57: b) NHS-Rhodamine, Et N, DMF;
3
For compound 58: b) biotin, HATU, Et N, DMF.
3
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0
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Having validated specific site C modifications that tolerated the introduction of a trackable tag,
we were interested to find a position where a photoreactive group such as aryl azide could be
introduced to make photoaffinity probes. This prompted us to derivatize compounds 10 and 25,
even though these were inactive but surmising that a change in the hydrogen bonding properties
may have an opposite effect. The aromatic amine on position 4 at site A of compound 10 was
converted to aryl azide using diazotization reaction to obtain compound 59 (Scheme 6). In
parallel, the 3-bromo substitution at site B of compound 25 was reacted with sodium azide using
copper catalyzed reaction. However, the major product of this reaction was aromatic amine
analog 60, which was further converted to azide using the earlier described diazotization
chemistry to obtain compound 61 (Scheme 6). The photoreactive aryl azide bearing compounds
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
5
9 and 61 and the aromatic amine analog 60 were then evaluated in the primary screens. While
compound 61 was inactive just like its precursor bromo analog 25, the reversal of hydrogen
bonding capacity in compound 60 led to resurgence of activity in both the assays possibly due to
hydrophilic interaction with the aromatic amine (Fig. 6, Table 4). In contrast, the reversal of
hydrogen bonding capacity of compound 10 led us to a potent aryl azide bearing analog 59
which was then utilized for making photoaffinity probes (Fig. 6, Table 4).
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