Identification of benzofused five-membered sultams, potent dual NOD1/NOD2 antagonists in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2020.112575

Nucleotide-binding oligomerization domain-containing proteins 1 and 2 play important roles in immune system activation. Recently, a shift has occurred due to the emerging knowledge that preventing nucleotide-binding oligomerization domains (NODs) signalin

Full text of DOI:10.1016/j.ejmech.2020.112575

Journal Pre-proof
Identification of benzofused five-membered sultams, potent dual NOD1/NOD2
antagonists in vitro and in vivo
Yao Ma, Xueyuan Li, Yameng Pi, Jingjia Ye, Xiduan Wei, Jingshu Yang, Guangxu Si,
Jingyuan Tian, Yi Dong, Gang Liu,
PII:
S0223-5234(20)30547-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112575
EJMECH 112575
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 February 2020
Revised Date: 28 March 2020
Accepted Date: 11 June 2020
Please cite this article as: Y. Ma, X. Li, Y. Pi, J. Ye, X. Wei, J. Yang, G. Si, J. Tian, Y. Dong, G. Liu,
Identification of benzofused five-membered sultams, potent dual NOD1/NOD2 antagonists in vitro and in
vivo, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112575.
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Identification of benzofused five-membered sultams, potent dual
NOD1/NOD2 antagonists in vitro and in vivo
a,1
b,1
b,1
b
b
b
Yao Ma , Xueyuan Li , Yameng Pi , Jingjia Ye , Xiduan Wei , Jingshu Yang ,
b
b
a,
b,
Guangxu Si , Jingyuan Tian , Yi Dong *, Gang Liu *
a
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, 2A Nanwei Rd, Xicheng Dist, Beijing 100050, P.R. China
b
School of Pharmaceutical Sciences, Tsinghua University, Haidian Dist，Beijing 100084, P.R.
China
Abstract
Nucleotide-binding oligomerization domain-containing proteins 1 and 2 play
important roles in immune system activation. Recently, a shift has occurred due to the
emerging knowledge that preventing nucleotide-binding oligomerization domains
(NODs) signaling could facilitate the treatment of some cancers, which warrants the
search for dual antagonists of NOD1 and NOD2. Herein, we undertook the synthesis
and identification of a new class of derivatives of dual NOD1/NOD2 antagonists with
novel benzofused five-membered sultams. Compound 14k was finally demonstrated
to be the most potent molecule that inhibits both NOD1- and NOD2-stimulated
NF-κB and MAPK signaling in vitro and in vivo.
Keywords: Five-membered sultams, Dual NOD1/NOD2 antagonist, NF-κB signaling,
MAPK signaling
1
These authors contributed equally.
E-mail address: dongyi@imm.ac.cn (Y. Dong), gangliu27@tsinghua.edu.cn (G. Liu)

1
. Introduction
Nucleotide-binding oligomerization domain-like receptor (NLR) family proteins
play a crucial role in mammalian innate immunity, serving as intracellular pattern
recognition receptors (PRRs) involved in the detection of cytoplasmic
pathogen-associated molecular patterns (PAMPs) and danger-associated molecular
patterns (DAMPs) [1,2]. Nucleotide-binding oligomerization domain-containing
proteins 1 and 2 (NOD1 and NOD2, respectively), which belong to the nodosome
subgroup of NLRs, play an important role in host defense because they are the only
proteins that recognize bacterial peptidoglycan components, leading to activation of
NF-κB and MAPKs, resulting in an inflammatory response [3−5].
The NOD1 and NOD2 pathways have been linked to a range of inflammatory
disorders [6], including inflammatory bowel disease (IBD) [7,8], Blau syndrome
[
9,10], rheumatoid arthritis [11,12], sarcoidosis [13], insulin resistance [14] and
asthma [15−17]. Mutations and overactivation of the NOD1 and NOD2 genes have
both been shown to cause immune dysregulation, which is also associated with a
number of human diseases, including inflammatory disorders and cancer [5]. Recently,
a shift has occurred due to the emerging knowledge that preventing NOD signaling
could facilitate the treatment of some cancers [18,19]. For example, Zitvogel’s group
has identified the NOD2 receptor as a ‘gut immune checkpoint’ based on evidence
that NOD2 weakens the antitumor effect of cyclophosphamide by preventing the
translocation of two gut commensal species, Enterococcus hirae and Barnesiella
intestinihominis, into the spleen [20]. Ferri and coworkers demonstrated NOD1 to be

a putative therapeutic target in colon cancer metastasis because NOD1 augments
colon cancer metastasis via the p38 mitogen activated protein kinase pathway [21].
All of the above evidence warrants the urgent development of NOD1 and NOD2
antagonists.
Recently,
several
representative
molecules
based
on
indoline,
tetrahydroisoquinoline, benzimidazole and purine scaffolds were reported to have
potent NOD inhibitory activity [5]. For example, the purine-2-6-diones (1) [22] and
benzimidazole (2) [23,24] were identified as active and selective NOD 1 inhibitors,
while another benzimidazole (3) [25,26] developed at GSK was reported to
selectively inhibit NOD2-mediated NF-κB signaling. Tetrahydroisoquinoline (4) [23]
and indoline (5) [23,27] derivatives were found to inhibit both NOD 1 and NOD2
signaling pathways with similar inhibitory activity for NOD 1 and NOD2. We also
reported that an antagonist (7, DY-16-43) of NOD2 signaling, conjugated muramyl
dipeptide (MDP) and paclitaxel (PTX), produced sensitization to paclitaxel therapy
and prevented tumor metastasis [28]. Another conjugate of an MDP derivative and
docetaxel (DTX) (8) was also characterized as an antagonist of NOD1 signaling and
was superior to DTX in breast cancer treatment [29]. Subsequently, we demonstrated
that 1,4-benzodiazepine-2,5-dione (BZD) derivatives, as dual NOD1/2 antagonists (6),
could enhance the effect of PTX in the treatment of Lewis lung carcinoma (LLC) [30].
The above evidence suggests that both NOD1 and NOD2 are indispensable for tumor
survival and invasion. Effective inhibitors used for cancer treatment should
antagonize both NOD1 and NOD2 signaling.

Given the potential value of NOD1/2 dual antagonists, we have developed and
optimized a cell-based screening assay platform in which either NOD1 or NOD2
activation stimulates a NF-κB-responsive SEAP reporter gene as we previously
described [30]. Continuously working engaged in the design, synthesis and evaluation
of NOD1 and NOD2 antagonists, we found a new class of derivatives of dual
NOD1/NOD2 based on a novel benzofused five-membered sultam skeleton from our
compound library, which are structurally different from previously identified
antagonists. Among these compounds, compound 14k was demonstrated to be the
most potent molecule that inhibited both NOD1- and NOD2- stimulated NF-κB and
MAPK signaling in vitro and in vivo. The results obtained also lead to a better
understanding of the structural requirements for NOD1 and NOD2 inhibition
O
R1
R
O
N
HN
R1
R2
N
N
N
R2
N
O
N
O
Me
S
N
O
O
1
(purine scaffold)
a, R1 = CH2CH=CHCH3; R = S(CH2)3Ph
b, R1 = (CH2)4CH3; R2 = S(CH2)2Ph
c, R1 = CH2Ph; R2 = CH(CH3)2
2
NH2
HN
1
1
1
N
2 (benzimidazole scaffold)
a, R = Me; 2b, R = Cl
3 (benzimidazole scaffold)
2
3a, R1 = H, R2 = H, IC50 (NOD2) = 3.3
3b, R1 = H, R2 = Me, IC50 (NOD2) = 0.4
3c, R1 = Cl, R2 = H, IC50 (NOD2) = 1.3
M
M
1
1
1
a, IC50 (NOD1/NOD2) = 1.54 M/12.7
b, IC50 (NOD1/NOD2) = 0.94 M/>20
c, IC50 (NOD1/NOD2) = 1.22 M/18.1
M
M
M
2a, IC50 (NOD1/NOD2) = 0.56 M/>20
2b, IC50 (NOD1/NOD2) = 0.09 M/19.9
M
M
M
O
O
S
N
H
R3
O
N
N
OH
O
Me
O
R2
N
O
N
Me
H
R
H
Me
O
4
(tetrahydroisoquinoline scaffold)
O
NH
O
Me
R
O
Me
4
a, R = CO2Et; 4b, R = SMe; 4c, R = OCF3
O
Me
5
(indoline scaffold)
O
OH
4
4
4
a, IC50 (NOD1/NOD2) = 0.15 M/0.40
b, IC50 (NOD1/NOD2) = 0.24 M/0.26
c, IC50 (NOD1/NOD2) = 0.15 M/0.40
M
M
M
5
a, R = OMe; 5b, R = Cl; 5c, R = NO2
O
O
X
O
5
5
5
a, IC50 (NOD1/NOD2) = 0.51 M/0.39
b, IC50 (NOD1/NOD2) = 1.01 M/0.60
c, IC50 (NOD1/NOD2) = 0.26 M/0.20
M
M
M
O
NH
Me2N
O
O
O
O
Me
O
O
NH
O
S
O
H
O
R1
N
N
O
N
N
CONH2
BnO
R1
H
H
N
H
N
O
CONH2
HN nPr
H
O
R3
R2
conjugated compound
6
O
7
8
, R1 = 4-Cl, R2 = Ph, R3 = Ac, X = CH2
, R1 = 2-Cl, 4-F, R2 = Ph, R3 = O Bu, X = C(O)CH2
IC50(NOD1)= 2.36
IC50(NOD2)= 4.16
M
M
t
benzofused Five-membered Sultam
Scheme 1. Representative NOD1/2 antagonists

2
. Results and discussion
2
.1. Chemistry
To evaluate the antagonistic activity of NOD1/2, a series of benzofused
five-membered sultams were designed and synthesized. As depicted in Scheme 2,
acetylation of commercially available benzenesulfonamides (9a-9j) produced N-Ac
protected analogues, which underwent Rh-catalyzed C–H alkenylation/annulation
with acrylates to generate five-membered sultams (10a-10l) in good to excellent
yields [31]. Removal of acetyl by treatment with K CO resulted in amino-free sultam
2
3
11, which underwent alkylation, alkenylation and acylation to yield the target
t
compounds 12, 13 and 14. The -butyl ester sultam analogue (14w) was transformed
into the corresponding acid (15) by treatment with trifluoroacetate, followed by
amidation to produce the target amide compound 16.
Scheme 2. General procedure for the synthesis of benzofused five-membered sultam

derivatives. Reaction conditions: (a) ZnCl , Ac O, rt.; (b) [RhCp*Cl ] , Cu(OAc) ,
2
2
2 2
2
2
3
acrylates, toluene, 100°C. (c) K CO , R OH, 60°C. (d) K CO , R X, DMF, rt. (e)
2
3
2
3
3
R OH, PPh , DEAD, THF, rt. (f) N-methylmorpholine, ethyl propiolate, DCM, rt. (g)
3
MeSO Cl, Et N, DCM, rt. (h) RC(O)Cl, Et N, DCM, rt. (i) TFA, DCM, rt. (j) (COCl) ,
2
3
3
2
5
6
DMF(cat.), DCM, rt. (k) R R NH, Et N, DCM, rt.
3
In presence of Cs CO in DMF at 120°C, compound 10a transformed into the
2
3
alkene
intermediate
17,
which
was
subsequently
treated
with
4
-(trifluoromethyl)benzoyl chloride to afford the target compound 18. When
compound 10a was treated with LiAlH , deacetylated alcohol intermediate 19 was
4
obtained, followed by acylation and butylation to produce the target compound 21.
Scheme 3. Synthesis of compounds 18 and 21. Reaction conditions: (a) Cs CO , DMF,
2
3
1
20°C. (b) 4-CF -Ph-C(O)Cl, Et N, DCM, rt. (c) LiAlH , THF, Ar, 0°C-rt. (d)
3 3 4
4
-CF -Ph-COOH, HOSu, DIC, DMF, rt. e) 1-iodobutane, K CO , acetone, rt.
3
2
3
Acid intermediate 15 underwent amidation with (E)-N'-hydroxypentanimidamide
to produce the intermediate 22, followed by dehydration to yield the target
,2,4-oxadiazole compound 23. Additionally, the hydrazide intermediate 24 was
1
prepared from acid intermediate 15, followed by acylation with butyric anhydride to
generate the intermediate 25, which subsequently underwent dehydration to yield the

target 1,3,4-oxadiazole compound 26.
Scheme 4. Synthesis of compounds 23 and 26. Reaction conditions: (a) (COCl)2,
DMF (cat.), DCM, rt. (b) (E)-N'-hydroxypentanimidamide, Et N, DCM, rt. (c) toluene,
3
reflux. (d) hydrazine hydrate, Et N, DCM, rt. (e) butyric anhydride, Et N, THF, rt. (f)
3
3
I , PPh , Et N, DCM, rt.
2
3
3
Sultam 11a was treated with DppoNH in the presence of NaH to produce
2
sulfonyl
hydrazide
27,
which
further
underwent
acylation
with
4
-(trifluoromethyl)benzoyl chloride to yield the target compound 28. 2-Methyl
benzamide underwent Rh-catalyzed C–H alkenylation/annulation with butyl acrylate
to yield a five-membered lactam (29), followed by catalytic hydrogenation and
acylation to produce the target lactam compound 31.
Scheme 5. Synthesis of compounds 28 and 31. Reaction conditions: (a) NaH,
DppoNH , THF, 0°C-rt. (b) 4-CF -Ph-C(O)Cl, Et N, DCM, rt. (c) [RhCp*Cl ] ,
2
3
3
2 2

AgSbF , Cu(OAc) , dioxane, 120°C. (d) Pd/C, H , rt.
6
2
2
2
.2. Structure-activity relationship (SAR) studies
As we previously described [30], screening for dual NOD1/2 antagonists was
conducted using HEK-Blue hNOD1-secreted alkaline phosphatase (SEAP) reporter
and HEK-Blue hNOD2 SEAP reporter cells activated by lauroyl-γ-D-Glu-mDAP
(C12-iE-DAP) and MDP, respectively. Hit compounds were then profiled in
dose-response assays and counter screened to eliminate cytotoxic compounds by the
sulforhodamine B (SRB) assay.
n
A variety of 7-methyl benzofused five-membered sultams inserted via a butyl
ester chain at the C-3 position with different N-2 substituents were evaluated for their
NOD1/2 antagonistic activity. The results clearly showed that the antagonistic activity
of these sultam analogues was significantly dependent on the structure of the N-2
substituent (Table 1). In this SAR exploration, a series of N-2 substituents including
hydrogen (11a), alkyl (12a-12c), allyl (12d), benzyl (12e), alkenyl (12h), acetyl (10a),
mesyl (13), methoxycarbonyl (14a), and vinylcarbonyl (14b-14d) groups lead to low
NOD1/2 inhibition rates in a single dose, while the derivatives with N-benzoyl groups
containing Cl (14h), CN (14i) and CF (14j) exhibited high NOD1/2 antagonistic
3
activity with an IC of 1-5 ꢀM. These results suggested that N-benzoyl is an essential
5
0
group for NOD1/2 antagonistic activity and shows unique SAR dependence. When
methyl-substituted benzoyl (14e) or furanoyl (14f) was attached to the nitrogen atom,
no NOD1/2 antagonistic activity was observed, implying that the electron-rich
aromatic membered was likely not a suitable structural unit for this activity.

1
a
Table 1. SAR of N-2 Substituent (R ) Exploration
1
b
Compd.
1a
2a
2b
2c
R
NOD1/2 inhibition %
IC (NOD1/2) ꢀM
50
1
H
-1.05±3.20/-16.81±5.22
-25.85±1.85/4.63±5.27
ND/ ND
ND/ ND
1
Me
1
-3.53±1.41/-1.00±3.49
-1.50±0.60/9.07±1.49
ND/ ND
ND/ ND
1
1
2d
2e
2h
-6.10±3.26/5.52±6.62
-18.12±4.34/41.38±1.44
6.38±4.59/20.57±1.26
ND/ ND
ND/ND
ND/ ND
1
1
*
CO Et
2
10a
-13.86±4.51/6.59±2.55
ND/ ND
1
3
-29.24±4.45/5.29±0.97
39.26±4.75/-0.67±0.98
2.70±12.71/-28.20±23.55
ND/ ND
ND/ ND
ND/ ND
14a
14b
1
4c
34.71±0.86/34.99±3.42
7.18±11.59/-25.07±32.49
ND/ ND
ND/ ND
14d
14e
16.65±7.39/24.27±5.73
ND/ ND

1
4f
-5.44±1.21/1.83±6.79
23.70±1.24/55.55±2.29
79.32±0.41/87.04±0.80
66.87±2.68/65.62±1.32
76.90±2.47/83.99±1.01
ND/ ND
14g
ND/ ND
14h
2.14±0.22/1.74±0.05
3.83±0.43/5.45±0.19
1.31±0.20/1.18±0.17
1
4i
1
4j
a
All compounds were tested in C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue
hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not
b
determined. The compounds were tested at 10 ꢀM.
Next, we performed a detailed SAR investigation on the structural skeleton of
the benzofused five-membered sultam and mainly focused on analyzing the effect of
the diversity of substituents on the benzene ring with regard to the NOD1/2
antagonistic activity (Table 2). The results showed that some representative
electron-donating (Me, OMe) or electron-withdrawing groups (F, Cl, ester) at
different positions had little influence on the NOD1/2 antagonistic activity. However,
a larger conjugated structure with an alkenyl substitution obviously lead to the loss of
activity (14u), which implied that the benzene ring is an important and key structural
unit for maintaining NOD1/2 antagonistic activity.
2
a
Table 2. SAR of R Substituent Exploration

b
Compd.
4j
Ar
NOD1/2 inhibition %
76.90±2.47/83.99±1.01
IC (NOD1/2) ꢀM
50
1
1.31±0.20/1.18±0.17
2.46±0.09/1.85±0.05
8.37±5.08/9.83±4.6
9.03±3.6/6.70±1.07
5.82±0.42/4.05±0.32
1
4m
75.64±0.33/85.73±0.50
68.24±2.77/58.18±1.39
62.61±2.60/66.59±3.21
73.93±3.79/76.28±0.64
1
4n
1
4o
1
4p
1
4q
89.00±0.47/92.43±0.15
88.10±1.70/76.98±1.25
1.63±0.03/1.39±0.05
7.14±1.75/7.034±3.36
1
4r
4s
1
74.32±1.70/65.75±0.64
69.52±0.24/66.73±2.71
3.15±0.17/3.96±0.28
2.93±0.15/4.29±0.25
1
4t
1
4u
31.93±11.96/31.44±6.09
ND/ ND
a
All compounds were tested on C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue

hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not
b
determined. The compounds were tested at 10 ꢀM.
3
Subsequently, the C-3 substituent (R ) was further considered, and the results are
summarized in Table 3. Ester-terminated analogues exhibited high NOD1/2
antagonistic activity, including different lengths of aliphatic chain substitution (14j,
1
4v), and sterically demanding substitution (14w), but the corresponding acid (15)
and amide (16a-16d) analogues were likely not good NOD1/2 antagonists due to their
low NOD1/2 inhibition. Removal of the carbonyl group from the ester structure
resulted in the loss of activity (21). These results suggested that the ester group at the
C-3 position plays an important role in inhibiting NOD1/2. In addition, we targeted
the methylene (CH ) attached to the C-3 position and evaluated its antagonistic effect
2
on NOD1/2 in particular. When a C=C double bond was inserted at the C-3 position
(
18), low NOD1/2 inhibition was observed, implying that this CH group, similar to
2
the ester group attached to it, is necessary for NOD1/2 antagonistic activity.
3
a
Table 3. SAR of R Substituent Exploration
3
b
Compd.
4j
4v
4w
R
NOD1/2 inhibition %
IC (NOD1/2) ꢀM
50
1
76.90±2.47/83.99±1.01 1.31±0.20/1.18±0.17
74.35±0.74/76.36±0.79 2.98±0.30/3.55±0.41
54.72±0.98/66.48±1.32 8.12±0.59/4.65±0.24
1
1

1
5
5.49±3.90/10.27±2.74
-8.79±4.30/-9.14±2.08
14.67±4.32/17.69±2.93
ND/ ND
ND/ ND
ND/ ND
16a
16b
16c
-2.01±11.43/-21.15±27.28
ND/ ND
1
6d
14.20±2.59/30.30±5.05
27.00±1.10/54.40±1.20
34.91±4.37/45.91±1.71
ND/ ND
ND/ ND
ND/ ND
1
8
1
2
a
All compounds were tested on C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue
hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not
b
determined. The compounds were tested at 10 ꢀM.
To examine the necessity of the sultam skeleton and its N-carbonyl group,
several representative compounds were evaluated (Table 4). Removal of the carbonyl
group (12f) or addition of CH (12g) or NH (28) between the carbonyl and
2
sulfonamide groups caused a loss of NOD1/2 antagonistic activity, suggesting that the
N-carbonyl is an essential functional group for inhibition of NOD1/2. Interestingly,
the corresponding lactam analogue (31) was also a good NOD1/2 antagonist with an
IC for NOD1/2 similar to that of the sultam analogue (14j).
5
0
a
Table 4. SAR of X and Y Substituent Exploration

b
Compd.
X
Y
NOD1/2 inhibition %
IC (NOD1/2) ꢀM
50
1
1
4j
2f
76.90±2.47/83.99±1.01 1.31±0.20/1.18±0.17
21.69±5.72/42.54±3.41
24.41±3.12/7.20±4.52
28.87±16.83/59.99±3.78
ND/ ND
ND/ ND
ND/ ND
1
2g
2
8
1
3
72.02±1.11/82.73±0.74 2.47±0.19/2.36±0.29
a
All compounds were tested on C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue
hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not
b
determined. The compounds were tested at 10 ꢀM.
The NOD1/2 antagonistic effect for the different substituent positions of CF was
3
examined. As shown in Table 5, meta-CF (14k) or ortho-CF (14l) substituted
3
3
analogues showed a similar antagonistic activity, which suggested that different
substituent positions of CF had little effect on the antagonistic activity. The isostere
3
of ester group was further designed and the corresponding analogues were prepared
and evaluated. As shown by compounds 23 and 26, replacement of the ester group by
1
,2,4-oxadiazole or 1,3,4-oxadiazole led to NOD1/2 antagonistic activity similar to

that of ester analogue 14k, which suggested that the ester group at the C-3 position or
its isostere is a key structural unit necessary for NOD1/2 antagonistic activity.
a
Table 5. SAR of Z and CF Substituent Exploration
3
b
Compd.
4j
Z
o-, m- or p-CF3 NOD1/2 inhibition %
IC (NOD1/2) ꢀM
50
1
76.90±2.47/83.99±1.01 1.31±0.20/1.18±0.17
87.73±0.33/89.65±0.29 1.05±0.05/0.93±0.03
80.32±0.96/90.38±1.17 1.10±0.06/0.48±0.02
73.07±1.64/78.43±0.60 0.23±0.02/5.74±5.24
85.03±2.24/91.3±0.28 1.74±0.16/4.65±0.24
1
4k
1
4l
2
3
6
2
a
All compounds were tested on C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue
hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not
b
determined. The compounds were tested at 10 ꢀM.
In summary, 45 compounds were synthesized and tested on C12-iE-DAP (50
ng/mL)-stimulated HEK-Blue hNOD1 cells and MDP (100 ng/mL)-stimulated
HEK-Blue hNOD2 cells to evaluate their NOD1/2 antagonistic activity, and the SAR
is summarized in Fig. 1. For the A structural fragment, a series of groups including H,
F, Cl, Me, OMe and ester groups at different positions were well tolerated with regard

to NOD1/2 antagonistic activity, but an alkenyl substituent resulted in a low level of
NOD1/2 inhibition. For the B structural fragment, a C=C double bond at the C-3
position caused a loss of activity, implying that the ɑ, β-unsaturated side chain at the
C-3 position is not beneficial for NOD1/2 antagonistic activity. Compared to the
ester-terminated analogue and its isostere, the amide-terminated compounds did not
exhibit obvious NOD1/2 antagonistic activity, and a free carboxyl group appeared to
result in a complete loss of NOD1/2 antagonistic activity because it could prevent the
compound from interacting with NOD1/2. For the C structural fragment, the
corresponding lactam analogue showed similar inhibitory activity to the sultam,
implying that this sultam or lactam structure unit is necessary for NOD1/2
antagonistic activity. For the D structural fragment, only the N-benzoyl group
substituted by Cl, CN or CF resulted in potent NOD1/2 antagonistic activity.
3
Additionally, removal of the carbonyl or addition of CH or NH resulted in a complete
2
loss of NOD1/2 antagonistic activity, which suggests that N-benzoyl is a key
structural unit for inhibition of NOD1/2. Based on the SAR described above,
compound 14k was selected as a model compound to evaluate the cytotoxicity and
anti-inflammatory effects in animals.

lactam skeleton
similar activity
carbonyl group is
H, F, Cl, OMe, Me
at different position
necessary for activity
Me
O
A
B
D
O
7
similar activity
1
O
6
5
S
2N
C
removal of carbonyl,
addition of CH or NH
2
4
3
removal of carbonyl
replacement by amide
CF
3
O
nBu
loss of activity
O
1
4k
Cl, CN, o-CF , p-CF3
3
loss of activity
replacement by isostere
similar activity
similar activity
Fig. 1. SAR exploration for benzofused five-membered sultam compounds
2
.3. Biological profiling
HEK-Blue hNOD1 and HEK-Blue hNOD2 cells were preincubated with
different concentrations of compound 14k for 3 h and then stimulated with
C12-iE-DAP (50 ng/mL) and MDP (100 ng/mL) for an additional 20 h. SEAP was
quantified as described in the Experimental Section. As shown in Fig. 2A, compound
1
4k exhibited promising potent inhibitory activity against NOD1 and NOD2 with IC50
values of 1.05 and 0.93 ꢀM, respectively, and no cytotoxicity (Fig. 2B).
Fig. 2. (A) Compound 14k inhibits C12-iE-DAP and MDP-induced NF-κB activation.
HEK-Blue hNOD1 and HEK-Blue hNOD2 cells were preincubated with different
concentrations of compound 14k for 3 h and then stimulated with C12-iE-DAP (50
ng/mL) and MDP (100 ng/mL) for an additional 20 h. SEAP was quantified as

described in the Experimental Section. Data are presented as the mean ± standard
deviation (SD) (n = 3). (B) Compound 14k had little or no effect on cell growth.
Following SEAP quantification, cells were fixed with 80% TCA at 4°C for 1 h and
stained with SRB in 1% acetic acid for 10 minutes. Then, the plates were washed five
times and air-dried at room temperature (rt). Bound stain was subsequently
solubilized with 10 mM Tris base (150 ꢀL), and the absorbance was measured with a
spectrophotometer at 515 nm. The inhibition rate of cell growth was calculated by the
following formula: [(C-T)/C] × 100 (C, OD of control group; T, OD of the compound
1
4k-treated group). Data are presented as the mean ± SD (n=3).
Fig. 3. Suppression of IL-6 expression in THP-1 cells by 14k. THP-1 cells were
treated with or without compound 14k (1 ꢀM or 10 ꢀM) for 1 h and then stimulated
with (left) C12-iE-DAP (500 ng/mL) or (right) MDP (20 ꢀg/mL) for 90 minutes. The
mRNA level of IL-6 was determined by qRT-PCR. Data are presented as the mean ±
standard deviation (SD) (n =3): (∗) p < 0.05, (∗∗) p < 0.01, (∗∗∗) p < 0.001 vs the
C12-iE-DAP- or MDP-treated group.
The activity of compound 14k on endogenous NOD1 and NOD2 receptors was
determined by inhibition of either C12-iE-DAP- or MDP-stimulated IL-6
transcription, respectively, using THP-1 human myeloid leukemia mononuclear cells.
These cells have been demonstrated to express native functional NOD1 and NOD2
and respond to NOD activation with increased NF-κB activity and secretion of

inflammatory cytokines [32]. As shown in Fig. 3, pretreatment of THP-1 cells with
compound 14k consistently and dose-dependently reduced the transcription of IL-6
stimulated by C12-iE-DAP and MDP, respectively, which suggested that compound
1
4k inhibits both the NOD1 and NOD2 signaling pathways in human immune cells.
Fig. 4. Compound 14k suppresses inflammation via NOD1 and NOD2 activation.
Serum-starved THP1 cells were pretreated with or without compound 14k (1 ꢀM or
1
0 ꢀM) for 1 h prior to (A) C12-iE-DAP (800 ng/mL) or (B) MDP (10 ꢀg/mL)
treatment for 30 minutes. The levels of IκBα, total and phosphorylated RIP2, JNK,
p38, and ERK1/2 were determined by Western blotting. β-Actin was used as a loading
control.
The activation of NOD1 and NOD2 signaling, which leads to the release of
proinflammatory chemokines, is mediated by the phosphorylation of a cascade of
effector proteins, including RIP2, extracellular signal-regulated kinase (ERK), p38,
c-Jun N-terminal kinase (JNK), and IκBα [33]. To determine whether compound 14k

inhibited both the NOD1 and NOD2 pathways, the effects on total IκBα and
phosphorylated p38, p-JNK and p-ERK levels were tested in THP-1 cells stimulated
with C12-iE-DAP or MDP, respectively. Pre-treatment of cells with compound 14k at
1
and 10 µM prevented C12-iE-DAP-induced increases in p-RIP2, p-p38, p-JNK, and
p-ERK and C12-iE-DAP-induced reduction in total IκBα in THP-1 cells. As expected,
compound 14k inhibited similar effects induced by MDP (Fig. 4B). These findings
demonstrate that compound 14k inhibits both the NF-κB and MAPK signaling
pathways following activation of NOD1 and NOD2.
Fig. 5. Compound 14k inhibited MDP-induced inflammatory responses in vivo.
Intravenous injection of compound 14k (50 mg/kg) produced moderate inhibition of
serum KC in mice following an intraperitoneal challenge with MDP (4 mg/kg). Data
are presented as the mean ± SEM (n =10): (∗∗) p < 0.01 vs MDP-treated group.
To demonstrate the utility of compound 14k in vivo, we employed a model in
which the systemic inflammatory response to an intraperitoneal injection of MDP or
Tri-DAP was measured in mice after administration of 50 mg/kg of compound 14k. A

single intravenous dose of 50 mg/kg of compound 14k produced moderate inhibition
of serum KC (the rodent orthologue of IL-8) in mice following intraperitoneal
challenge with MDP (4 mg/kg) (Fig. 5). However, intraperitoneal injection of
Tri-DAP (up to 20 mg/kg) produced only weak induction of serum KC, and a
reduction of this level by compound 14k was barley observed (data not shown). Since
compound 14k only had moderate inhibition effect on cytokine production in
response to stimulation with MDP in vivo, which is less effective in mice than
compound (6) that we previously reported. It may currently not good enough to be an
ideal drug candidate used for adjuvant cancer chemotherapy. Nonetheless, our
preliminary studies have indicated that these sultam derivatives had potential value
for continued development. Meanwhile, given the limited structure number of the
currently reported NOD1/2 antagonists, the discovery of benzofused five-membered
sultam dual NOD1/NOD2 antagonist increased the scaffold diversity of NOD1/2
antagonists, and potentially provided a novel lead compound for further optimization
to get more ideal NOD1/2 antagonists. Further work focused on improvement of
potency and pharmacological profile of 14k will be performed.
3
. Conclusions
We report the synthesis and identification of a new class of derivatives of dual
NOD1/NOD2 antagonists based on a novel benzofused five-membered sultam
skeleton, which is structurally different from previously identified antagonists.
Compound 14k was demonstrated to be the most potent molecule that suppressed
both NOD1- and NOD2- stimulated NF-κB and MAPK signaling in vitro and in vivo.

The results obtained also lead to a better understanding of the structural requirements
for NOD1 and NOD2 inhibition and enriched the scaffold diversity of NOD1/2
antagonists. Further improvement of the potency and druggability of these compounds
will be elaborated.
4
. Experimental
4
.1 General
Toluene, DCM, n-BuOH, t-BuOH, DMF and other commercial reagents were
purchased from domestic corporations and used without further purification.
Analytical thin-layer chromatography (TLC) plates, preparative TLC plates, and silica
gel for column chromatography were purchased from Qingdao Haiyang Chemical and
Special Silica Gel Co, Ltd. High-resolution LCMS was conducted using Agilent
LC/MSD TOF with an Agilent ZORBAX SB-C18 (rapid resolution, 3.5 ꢀm, 2.1 mm
×
30 mm) column at a flow rate of 0.40 mL/min. The solvent was MeOH/water
(75:25 (v/v)) containing 5 mmol/L ammonium formate. The ion source was
electrospray ionization (ESI). The automatic liquid chromatography−mass
spectrometry (LCMS) analysis was performed on a Waters SQ Advantage mass
spectrometer equipped with an ultraperformance LC (UPLC) system and an eluent
splitter (5% eluent was split into the MS system).
Proton nuclear magnetic resonance was performed on a Bruker Advance 300NMR
1
or 400 NMR spectrometer depending on its necessary. The chemical shifts of the H
NMR spectra are reported in units of parts per million (ppm) downfield from SiMe4
(δ 0.0) and relative to the signals of chloroform-d (δ = 7.26 ppm, singlet).

Multiplicities are described as follows: s (singlet), brs (broad singlet), d (doublet), t
triplet), q (quartet), dd (doublet of doublets), and m (multiplets). The number of
(
1
3
protons (n) for a given resonance is indicated by nH. The chemical shifts of the C
NMR spectra are reported in units of ppm downfield from SiMe4 (δ 0.0) and relative
to the signals of chloroform-d (δ = 77.16 ppm, triplet).
The compounds 10a-10l, 11a, 12a-12e, 12g-12h, 13, 14a, 14j were synthesized
according to our previous report [31].
General synthetic procedure of compounds 12f and 12g. K CO (2.0 equivalent,
2
3
3
2
7.6mg) and R X (1.5 equivalent) were added to a solution of compound 11a (0.1
mmol, 30 mg) in 5 mL of DMF, and the reaction mixture was stirred at room
temperature and monitored by LCMS until all of the starting material was consumed
completely. Solvent was removed in vacuo, and the residue was diluted with 10 mL of
ethyl acetate, washed with 10 mL water, and extracted with ethyl acetate two times.
Then the organic phase was combined, washed with brine, and dried over anhydrous
Na SO . The organic layer was concentrated and purified with column
2
4
chromatography on silica gel to afford compounds 12f and 12g.
Butyl
3
3
7
7
1
-(7-methyl-1,1-dioxido-2-(4-(trifluoromethyl)benzyl)-2,3-dihydrobenzo[d]isothiazol-
1
-yl)-2-oxopropanoate (12f). (36.8 mg, 81% yield). H NMR (400 MHz, CDCl ) δ
3
.59 (q, J = 8.4 Hz, 4H), 7.49 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.16 (d, J =
.7 Hz, 1H), 4.82 (t, J = 5.7 Hz, 1H), 4.64 (s, 2H), 4.02 – 3.87 (m, 2H), 2.83 (dd, J =
6.4, 6.1 Hz, 1H), 2.74 (dd, J = 16.4, 5.7 Hz, 1H), 2.69 (s, 3H), 1.51 – 1.42 (m, 2H),

13
1
.24 (dq, J = 14.5, 7.2 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, CDCl )
3
δ 170.4, 140.4 137.4, 134.6, 133.2, 133.1, 131.2, 130.2, 128.8, 125.8 (q, J = 3.7 Hz),
1
6
4
24.2 (d, J = 272.0 Hz), 121.4, 65.3, 57.5, 46.8, 40.0, 30.5, 19.1, 17.1, 13.7. mp:
+
8−70 °C. HRMS (ESI): m/z (M+H ) calcd for C H O NF S, 456.1451, found:
22
25
4
3
56.1451.
Butyl
3
-(7-methyl-1,1-dioxido-2-(2-oxo-2-(4-(trifluoromethyl)phenyl)ethyl)-2,3-dihydrobenz
1
o[d]isothiazol-3-yl)-2-oxopropanoate (12g). (36.2 mg, 75% yield). H NMR (400
MHz, CDCl ) δ 8.05 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 8.1 Hz, 2H), 7.52 (t, J = 7.6 Hz,
3
1
5
2
7
1
3
H), 7.31 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 5.15 (dd, J = 8.8, 3.4 Hz, 1H),
.01 (q, J = 18.7 Hz, 2H), 4.06 (t, J = 6.7 Hz, 2H), 2.98 (dd, J = 17.2, 8.9 Hz, 1H),
.86 (dd, J = 17.2, 3.6 Hz, 1H), 2.66 (s, 3H), 1.60 – 1.51 (m, 2H), 1.31 (dt, J = 15.0,
1
3
.6 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, CDCl ) δ 193.9, 171.4,
3
38.2, 137.6, 135.1 (q, J = 32.7 Hz), 134.3, 133.8, 133.2, 131.0, 128.5, 126.0 (q, J =
.6 Hz), 123.6 (d, J = 272.9 Hz), 121.3, 65.3, 58.5, 51.4, 41.5, 30.6, 19.2, 17.1, 13.8.
+
mp: 88−90 °C. HRMS (ESI): m/z (M+H ) calcd for C H O NF S, 484.1400; found:
2
3
25
5
3
4
84.1400.
General synthetic procedure of compound 14b-14w. Et N (3.0 equivalent, 41 mg)
3
4
and R C(O)Cl (1.5 equivalent) was added to a solution of compound 11a (0.1 mmol,
3
0 mg) in 1 mL of DCM, and the reaction mixture was stirred at room temperature
and monitored by LCMS until all of the starting material was consumed completely.
Solvent was removed in vacuo, and the residue was diluted with 10 mL of ethyl

acetate, washed with 10 mL water, and extracted with ethyl acetate two times. Then
the organic phase was combined, washed with brine, and dried over anhydrous
Na SO . The organic layer was concentrated and purified with column
2
4
chromatography on silica gel to afford compounds 14b-14w.
Butyl
3
-(2-acryloyl-7-methyl-1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-3-yl)-2-oxopropan
1
oate (14b). (30.2 mg, 86% yield). H NMR (400 MHz, CDCl ) δ 7.56 (t, J = 7.7 Hz,
3
1
H), 7.35 (dd, J = 12.3, 7.6 Hz, 2H), 7.08 (dd, J = 16.6, 10.4 Hz, 1H), 6.61 (dd, J =
6.6, 1.4 Hz, 1H), 5.97 (dd, J = 10.4, 1.4 Hz, 1H), 5.76 (dd, J = 7.7, 3.3 Hz, 1H), 4.07
1
(t, J = 6.7 Hz, 2H), 3.16 (dd, J = 16.0, 3.4 Hz, 1H), 2.95 (dd, J = 16.0, 7.7 Hz, 1H),
2
3
1
.65 (s, 3H), 1.57 – 1.49 (m, 2H), 1.27 (dq, J = 14.2, 7.2 Hz, 2H), 0.88 (t, J = 7.4 Hz,
1
3
H). C NMR (100 MHz, CDCl ) δ 169.8, 162.9, 135.4, 135.0, 134.3, 132.2, 132.1,
3
+
31.6, 127.9, 122.1, 65.1, 55.0, 39.3, 30.5, 19.1, 17.0, 13.8. HRMS (ESI): m/z (M+H )
calcd for C H O NS, 352.1213, found: 352.1213.
1
7
22
5
Butyl
(E)-3-(2-(3-(4-chlorophenyl)acryloyl)-7-methyl-1,1-dioxido-2,3-dihydrobenzo[d]isoth
1
iazol-3-yl)-2-oxopropanoate (14c). (34.6 mg, 75% yield). H NMR (400 MHz, CDCl )
3
δ 7.83 (d, J = 15.4 Hz, 1H), 7.56 (dd, J = 8.1, 6.5 Hz, 3H), 7.59 – 7.52 (m, 3H), 7.40 –
7
.31 (m, 5H), 4.08 (td, J = 6.6, 0.9 Hz, 2H), 3.20 (dd, J = 16.0, 3.4 Hz, 1H), 2.99 (dd,
J = 16.0, 7.6 Hz, 1H), 2.67 (s, 3H), 1.57 – 1.49 (m, 2H), 1.30 – 1.24 (m, 2H), 0.86 (t,
1
3
J = 7.4 Hz, 3H). C NMR (100 MHz, CDCl ) δ 169.8, 163.0, 144.7, 136.9, 135.5,
3
1
35.0, 134.3, 132.8, 132.3, 131.6, 129.9, 129.3, 122.1, 118.1, 65.1, 55.1, 39.3, 30.5,

+
1
9.1, 17.0, 13.8. mp: 144−146 °C. HRMS (ESI): m/z (M+H ) calcd for
C H O NClS, 462.1136, found: 462.1138.
23
25
5
Butyl
(E)-3-(2-(3-(3-chloro-5-fluorophenyl)acryloyl)-7-methyl-1,1-dioxido-2,3-dihydrobenz
1
o[d]isothiazol-3-yl)-2-oxopropanoate (14d). (34.5 mg, 72% yield). H NMR (400
MHz, CDCl ) δ 8.22 (d, J = 15.4 Hz, 1H), 7.77 (dd, J = 8.8, 6.0 Hz, 1H), 7.57 (t, J =
3
7
.7 Hz, 1H), 7.40 – 7.28 (m, 3H), 7.18 (dd, J = 8.4, 2.6 Hz, 1H), 7.03 (td, J = 8.2, 2.5
Hz, 1H), 5.81 (dd, J = 7.6, 3.3 Hz, 1H), 4.08 (t, J = 6.7 Hz, 2H), 3.21 (dd, J = 16.0,
.4 Hz, 1H), 3.00 (dd, J = 16.0, 7.6 Hz, 1H), 2.66 (s, 3H), 1.57 – 1.49 (m, 2H), 1.27
3
1
3
(
dq, J = 14.9, 7.4 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, CDCl ) δ
3
1
1
2
1
4
69.8, 162.6, 163.7 (d, J = 253.1 Hz), 140.7, 136.6 (d, J = 10.4 Hz), 135.5, 135.0,
34.3, 132.2, 131.6, 129.7 (d, J = 9.2 Hz), 129.0 (d, J = 3.7 Hz), 122.1, 119.7 (d, J =
.1 Hz), 117.6 (d, J = 24.8 Hz), 115.0 (d, J = 21.7 Hz), 65.1, 55.2, 39.3, 30.6, 19.1,
+
7.0, 13.7. mp: 92−94 °C. HRMS (ESI): m/z (M+H ) calcd for C H O NClFS,
2
3
24
5
80.1042, found: 480.1050.
Butyl
3
-(2-benzoyl-7-methyl-1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-3-yl)-2-oxopropano
1
ate (14e). (37.3 mg, 93% yield). H NMR (400 MHz, CDCl ) δ 7.90 (d, J = 7.5 Hz,
3
2
H), 7.65 – 7.46 (m, 4H), 7.39 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.5 Hz, 1H), 6.14 (t, J
5.7 Hz, 1H), 4.09 (t, J = 6.7 Hz, 2H), 3.14 – 2.99 (m, 2H), 2.55 (s, 3H), 1.58 – 1.44
=
1
3
(
m, 2H), 1.26 (dq, J = 14.9, 7.2 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H). C NMR (100 MHz,
CDCl ) δ 169.6, 168.5, 135.4, 134.9, 134.6, 134.1, 132.8, 131.6, 128.7, 128.5, 122.1,
3

+
6
5.2, 54.9, 40.1, 30.5, 19.1, 16.9, 13.7. mp: 59−61 °C. HRMS (ESI): m/z (M+H )
calcd for C H O NS, 402.1370, found: 402.1373.
2
1
24
5
Butyl
3
-(2-(furan-2-carbonyl)-7-methyl-1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-3-yl)-2-
1
oxopropanoate (14f). (34.8 mg, 89% yield). H NMR (400 MHz, CDCl ) δ 7.72 (s,
3
1
H), 7.66 (s, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.43 – 7.31 (m, 2H), 6.61 (s, 1H), 6.18 (t, J
4.7 Hz, 1H), 4.07 (t, J = 6.0 Hz, 2H), 3.08 – 2.94 (m, 2H), 2.64 (s, 3H), 1.55 – 1.46
=
1
3
(
m, 2H), 1.30 – 1.19 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, CDCl ) δ
3
1
6
69.6, 156.2, 147.3, 146.0, 135.4, 134.9, 134.1, 132.8, 131.7, 120.0, 120.3, 112.4,
5.2, 54.8, 40.2, 30.5, 19.1, 17.0, 13.8. mp: 180−182 °C.
Butyl
2
-(7-methyl-2-(4-methylbenzoyl)-1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-3-yl)acet
1
ate (14g). (39.4 mg, 95% yield). H NMR (400 MHz, CDCl ) δ 7.82 (d, J = 8.0 Hz,
3
2
H), 7.56 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.7 Hz, 3H), 6.15
(t, J = 5.6 Hz, 1H), 4.08 (t, J = 6.6 Hz, 2H), 3.11 – 2.99 (m, 2H), 2.55 (s, 3H), 2.43 (s,
13
3
H), 1.57 – 1.47 (m, 2H), 1.26 (dq, J = 15.0, 7.3 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H). C
NMR (100 MHz, CDCl ) δ 169.6, 168.5, 143.6, 135.4, 134.8, 134.0, 131.8, 131.5,
3
1
29.2, 128.8, 122.0, 65.2, 54.8, 40.1, 30.5, 21.9, 19.1, 16.9, 13.7. mp: 89−90 °C.
+
HRMS (ESI): m/z (M+H ) calcd for C22 H26 O5 N S, 416.1526, found: 416.1528.
Butyl
2
-(2-(4-chlorobenzoyl)-7-methyl-1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-3-yl)acet
1
ate (14h). (39.6 mg, 91% yield). H NMR (400 MHz, CDCl ) δ 7.85 (d, J = 8.3 Hz,
3

2
H), 7.57 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.32
d, J = 7.5 Hz, 1H), 6.12 (t, J = 5.7 Hz, 1H), 4.07 (t, J = 6.7 Hz, 2H), 3.05 (d, J = 5.7
Hz, 2H), 2.55 (s, 3H), 1.56 – 1.45 (m, 2H), 1.25 (dq, J = 14.6, 7.4 Hz, 3H), 0.86 (t, J =
(
1
3
7
1
6
4
.3 Hz, 3H). C NMR (100 MHz, CDCl ) δ 169.5, 167.5, 139.3, 135.2, 134.9, 134.2,
3
32.9, 132.6, 131.6, 130.2, 128.9, 122.1, 65.3, 54.9, 40.0, 30.5, 19.1, 16.9, 13.7. mp:
+
2−64 °C. HRMS (ESI): m/z (M+H ) calcd for C H O NClS, 436.0980, found:
2
1
23
5
36.0977.
Butyl
2
-(2-(4-cyanobenzoyl)-7-methyl-1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-3-yl)aceta
1
te (14i). (37.9 mg, 89% yield). H NMR (400 MHz, CDCl ) δ 7.96 (d, J = 8.1 Hz, 2H),
3
7
7
2
3
1
7
4
.79 (d, J = 8.0 Hz, 2H), 7.59 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.34 (d, J =
.5 Hz, 1H), 6.08 (t, J = 5.6 Hz, 1H), 4.07 (t, J = 6.7 Hz, 2H), 3.07 (d, J = 5.7 Hz, 2H),
.54 (s, 3H), 1.56 – 1.44 (m, 2H), 1.24 (dq, J = 14.8, 7.4 Hz, 2H), 0.86 (t, J = 7.4 Hz,
1
3
H). C NMR (100 MHz, CDCl ) δ 169.3, 166.9, 138.3, 135.0, 135.0, 134.4, 132.3,
3
32.3, 131.8, 129.2, 122.1, 118.0, 116.1, 65.3, 55.0, 39.8, 30.5, 19.1, 16.9, 13.7. mp:
+
3−75 °C. HRMS (ESI): m/z (M+H ) calcd for C H O N S, 427.1322, found:
2
2
23
5
2
27.1329.
Butyl
2
-(7-methyl-1,1-dioxido-2-(3-(trifluoromethyl)benzoyl)-2,3-dihydrobenzo[d]isothiazol
1
-
3-yl)acetate (14k). (43.1 mg, 92% yield). H NMR (400 MHz, CDCl ) δ 8.17 – 8.07
3
(m, 2H), 7.86 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H),
7
.40 (d, J = 7.7 Hz, 1H), 7.33 (d, J = 7.5 Hz, 1H), 6.12 (t, J = 5.6 Hz, 1H), 4.09 (t, J =

6
1
1
.7 Hz, 2H), 3.08 (d, J = 5.7 Hz, 2H), 2.55 (s, 3H), 1.57 – 1.47 (m, 2H), 1.26 (dq, J =
1
3
4.9, 7.3 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, CDCl ) δ 169.5,
3
67.3, 135.3, 135.2, 135.0, 134.3, 132.5, 131.9, 131.7, 131.2 (q, J = 33.0 Hz), 129.3
(dd, J = 7.2, 3.5 Hz), 129.1, 125.9 (q, J = 3.8 Hz), 123.7 (d, J = 272.6 Hz), 122.1, 65.4,
+
5
4
5.0, 40.0, 30.5, 19.1, 16.9, 13.7. HRMS (ESI): m/z (M+H ) calcd for C H O NF S,
2
2
23
5
3
70.1244, found: 470.1245.
Butyl
2
-(7-methyl-1,1-dioxido-2-(2-(trifluoromethyl)benzoyl)-2,3-dihydrobenzo[d]isothiazol
1
-
(
(
3-yl)acetate (14l). (42.2 mg, 90% yield). H NMR (400 MHz, CDCl ) δ 7.82 – 7.73
3
m, 2H), 7.72 – 7.64 (m, 2H), 7.58 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.33
d, J = 7.5 Hz, 1H), 5.99 – 5.90 (m, 1H), 4.11 (t, J = 6.7 Hz, 2H), 3.23 (d, J = 14.3 Hz,
1
1
1
H), 3.07 (dd, J = 15.0, 6.7 Hz, 1H), 2.53 (s, 3H), 1.59 – 1.50 (m, 2H), 1.27 (dd, J =
1
3
4.7, 7.2 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). C NMR (100 MHz, CDCl ) δ 169.6,
3
65.3, 135.04 (d, J = 5.3 Hz), 134.3, 132.5, 132.0, 131.7, 131.4, 131.3, 129.2, 127.0
(q, J = 3.9 Hz), 122.3, 122.2 (d, J = 9.3 Hz), 65.3, 55.2, 39.4, 30.6, 19.1, 17.0, 13.8.
+
mp: 84−85 °C. HRMS (ESI): m/z (M+H ) calcd for C H O NF S, 470.1244, found:
2
2
23
5
3
4
70.1239.
General synthetic procedure of compound 14m-14u. Et N (3.0 equivalent, 41 mg)
3
and 4-(Trifluoromethylbenzoylchloride (1.5 equivalent) was added to a solution of
compound 11 (0.1 mmol) in 1 mL of DCM, and the reaction mixture was stirred at
room temperature and monitored by LCMS until all of the starting material was
consumed completely. Solvent was removed in vacuo, and the residue was diluted