From hit to lead: Structure-based discovery of naphthalene-1-sulfonamide derivatives as potent and selective inhibitors of fatty acid binding protein 4

Article abstract of DOI:10.1016/j.ejmech.2018.05.007

Fatty acid binding protein 4 (FABP4) plays a critical role in metabolism and inflammatory processes and therefore is a potential therapeutic target for immunometabolic diseases such as diabetes and atherosclerosis. Herein, we reported the identification of naphthalene-1-sulfonamide derivatives as novel, potent and selective FABP4 inhibitors by applying a structure-based design strategy. The binding affinities of compounds 16dk, 16do and 16du to FABP4, at the molecular level, are equivalent to or even better than that of BMS309403. The X-ray crystallography complemented by the isothermal titration calorimetry studies revealed the binding mode of this series of inhibitors and the pivotal network of ordered water molecules in the binding pocket of FABP4. Moreover, compounds 16dk and 16do showed good metabolic stabilities in liver microsomes. Further extensive in vivo study demonstrated that 16dk and 16do exhibited a dramatic improvement in glucose and lipid metabolism, by decreasing fasting blood glucose and serum lipid levels, enhancing insulin sensitivity, and ameliorating hepatic steatosis in obese diabetic (db/db) mice.

Full text of DOI:10.1016/j.ejmech.2018.05.007

Accepted Manuscript
From hit to lead: Structure-based discovery of naphthalene-1-sulfonamide derivatives
as potent and selective inhibitors of fatty acid binding protein 4
Ding-Ding Gao, Hui-Xia Dou, Hai-Xia Su, Ming-Ming Zhang, Ting Wang, Qiu-Feng
Liu, Hai-Yan Cai, Hai-Peng Ding, Zhuo Yang, Wei-Liang Zhu, Ye-Chun Xu, He-Yao
Wang, Ying-Xia Li
PII:
S0223-5234(18)30411-2
10.1016/j.ejmech.2018.05.007
EJMECH 10417
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2018
Revised Date: 4 May 2018
Accepted Date: 6 May 2018
Please cite this article as: D.-D. Gao, H.-X. Dou, H.-X. Su, M.-M. Zhang, T. Wang, Q.-F. Liu, H.-Y.
Cai, H.-P. Ding, Z. Yang, W.-L. Zhu, Y.-C. Xu, H.-Y. Wang, Y.-X. Li, From hit to lead: Structure-based
discovery of naphthalene-1-sulfonamide derivatives as potent and selective inhibitors of fatty acid
binding protein 4, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.007.
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ACCEPTED MANUSCRIPT
From Hit to Lead: Structure-based Discovery of
Naphthalene-1-Sulfonamide Derivatives as Potent and Selective
Inhibitors of Fatty Acid Binding Protein 4
Ding-Ding Gao,§ Hui-Xia Dou,§ Hai-Xia Su,§ Ming-Ming Zhang,§ Ting Wang, Qiu-Feng Liu,
Hai-Yan Cai, Hai-Peng Ding, Zhuo Yang, Wei-Liang Zhu,* Ye-Chun Xu,* He-Yao Wang,* and
Ying-Xia Li *

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From
Hit
to
Lead:
Structure-based
Discovery
of
Naphthalene-1-Sulfonamide Derivatives as Potent and Selective
Inhibitors of Fatty Acid Binding Protein 4
Ding-Ding Gao,†,§ Hui-Xia Dou,‡,¶,§ Hai-Xia Su,♯,¶,§ Ming-Ming Zhang,†,§ Ting Wang,‡ Qiu-Feng Liu,♯
△
Hai-Yan Cai,♯, Hai-Peng Ding, † Zhuo Yang,♯ Wei-Liang Zhu,*, ♯ Ye-Chun Xu,*, ♯ He-Yao Wang,*, ‡
and Ying-Xia Li *, †
†School of Pharmacy, Fudan University, Shanghai 201203, China
‡State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
♯CAS Key Laboratory of Receptor Research；Drug Discovery and Design Center, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences (CAS), Shanghai 201203, China
¶University of Chinese Academy of Sciences, Beijing 100049, China
△Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of
Education, School of Medicine, Shanghai JiaoTong University, Shanghai 201203, China
*
Corresponding Authors
E-mail addresses: liyx417@fudan.edu.cn (Y. Li), hywang@simm.ac.cn (H. Wang), ycxu@simm.ac.cn (Y. Xu),
wlzhu@simm.ac.cn (W. Zhu).
§ These authors contributed equally to the work.
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Abstract
Fatty acid binding protein 4 (FABP4) plays a critical role in metabolism and inflammatory processes
and therefore is a potential therapeutic target for immunometabolic diseases such as diabetes and
atherosclerosis. Herein, we reported the identification of naphthalene-1-sulfonamide derivatives as
novel, potent and selective FABP4 inhibitors by applying a structure-based design strategy. The
binding affinities of compounds 16dk, 16do and 16du to FABP4, at the molecular level, are
equivalent to or even better than that of BMS309403. The X-ray crystallography complemented by the
isothermal titration calorimetry studies revealed the binding mode of this series of inhibitors and the
pivotal network of ordered water molecules in the binding pocket of FABP4. Moreover, compounds
16dk and 16do showed good metabolic stabilities in liver microsomes. Further extensive in vivo study
demonstrated that 16dk and 16do exhibited a dramatic improvement in glucose and lipid metabolism,
by decreasing fasting blood glucose and serum lipid levels, enhancing insulin sensitivity, and
ameliorating hepatic steatosis in obese diabetic (db/db) mice.
Keywords: FABP4 inhibitors, FABP3 sparing, Structure-based design strategy, X-ray crystallography.
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¢ Introduction
Lipids are critical modulators that regulate the metabolic, inflammatory and innate immune processes
in intracellular and extracellular signaling coordinately [1]. As intracellular lipid chaperones or fatty
acids shuttles, fatty acid binding proteins (FABPs) are a family of 14–15 kDa proteins that modulate
lipid fluxes, trafficking and signaling, and thus play important roles in lipid metabolism and
inflammation. At least ten members have been identified since the initial discovery of FABPs in 1972.
Each of them exhibits tissue-specific distribution and is expressed richly in tissues associated with
active lipid metabolism (LFABP or FABP1, liver; IFABP or FABP2, intestines; HFABP or FABP3,
heart; AFABP or FABP4, adipocyte; EFABP or FABP5, epidermis; IlFABP or FABP6, ileum; BFABP
or FABP7, brain; MFABP or FABP8, myelin; TFABP or FABP9, testis; LOC646486 or FABP12,
Human retinoblastoma cell lines) [2-6]. It is intriguing that these members possess only moderate
sequence homology ranging from 15% to 70%, but their overall 3-D structures are very similar
[2,7,8].
Fatty acid binding proteins 4 (also known as AFABP or aP2) is highly expressed in differentiated
adipocytes, macrophages and endothelial cells, and induced by insulin and/or insulin-like growth
factors-1 (IGF-1), fatty acids, as well as peroxisome-proliferator-activated receptor-γ (PPAR-γ)
agonists [9-12]. Epidemiological studies and animal knockout models showed that FABP4 is crucial to
in many aspects of metabolic syndrome. For example, FABP4-/- mice were protected from
obesity-induced insulin resistance, cardiovascular disease and hyperglycaemia [13,14]. Ablation of
FABP4 in apolipoprotein E (ApoE)-deficient mice showed protection from atherosclerosis [11].
Additionally, the FABP4-deficient mice show reduced lipolysis but increased lipogenesis [15]. FABP4
plays also an important role in carcinogenesis, such as ovarian, prostate, bladder, breast, renal cell
carcinoma and other types of cancer cells [16]. FABP3, another important member of the FABPs
family, is mainly expressed in cardiac as well as skeletal muscle tissues and has important roles in cell
proliferation, apoptosis and prevention of oxidative stress. Silencing of FABP3 in embryonic
carcinoma cells led to reduced proliferation and promoted apoptosis [17]. Also, specific deletion of
cardiac muscle FABP3 in zebrafish resulted in apoptosis-induced mitochondrial dysfunction and
impairment of cardiac development [18,19]. Accordingly, selective inhibition of FABP4 without
disruption of FABP3 is an important precondition to develop druggable FABP4 inhibitors for the
treatment of metabolic syndromes, such as obesity, insulin resistance, diabetes, and atherosclerosis.
To date, several classes of FABP4 inhibitors have been identified and exhibited good inhibitory
potency, for example, pyrazole derivatives (1), quinoline derivatives (2), indole derivatives (3),
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pyrimidine derivatives (4), thiophene derivatives (5), oxazole and imidazole derivatives (6, 7),
1,3,5-triisopropylbenzene derivatives (8) (Fig. 1) [20-28]. Among which, pyrazole compound 1 has
been utilized as a chemical tool for FABP4 inhibition both in vitro and in vivo [20]. In our previous
study, compound 9 bearing 2-((2-oxo-2-(phenylamino)ethyl)thio)acetic acid scaffold, with an IC50
value of 13.5 µM (Ki: 1.66 µM) against FABP4, was discovered by virtual screening [27]. Molecular
dynamics (MD) simulation and site-directed mutagenesis studies were carried out to identify the
binding pattern. Taking compound 9 as a hit to perform a structural optimization and SAR study led us
to design more potent inhibitors bearing naphthalene-1-sulfonamide scaffold. Furthermore, the X-ray
crystal structures combined with isothermal titration calorimetry (ITC) revealed the binding mode of
compounds 16d, 16dk, 16do, 16di with FABP4. We also performed a systematic study to validate the
potency and efficacy of 16dk and 16do both in vitro and in vivo, respectively.
Fig. 1. Structures of representative FABP4 inhibitors.
¢ Results and discussion
Chemistry
Methods for the synthesis of compounds 12 and 14a-14g were outlined in Scheme 1. Commercially
available 10a-10d were transformed to corresponding amino acid methyl ester hydrochlorides 11a-11d
in good yields with the thionyl chloride/CH3OH system. Then the desired compounds (12, 14a-14g)
were obtained through a two-step consecutive reaction. Treatment of the intermediates 11a-11d with
3,5-dichlorobenzoyl chloride or substitutive benzenesulfonyl chloride in dichloromethane at room
temperature resulted in the formation of corresponding product precursors. Subsequent sodium
hydroxide hydrolysis of the esters gave compounds 12 and 14a-14g.
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Reagents and conditions: (a) SOCl2, CH3OH, 0 °C to rt ; (b) (i) 11a-11d ,CH2Cl2, pyridine, 0° C to rt. (ii) NaOH, H2O,
CH3OH, rt.
Scheme 1. Synthesis of Compounds 12, 14a-14g
Compounds 16a-16d and 16da-16dv were synthesized as described in Scheme 2. The reaction of
commercially available naphthalene derivatives with chlorosulfonic acid in chloroform led to the
formation of the corresponding naphthalene-1-sulfonyl chlorides (15a-15d), which were further
reacted with 11c or amino derivatives using the same method as the synthesis of compound 12.
a Reagents and conditions: (a) ClSO2OH, CHCl3, 0 °C to rt; (b) (i) 11c or amino derivatives, CH2Cl2, pyridine, 0 °C to
rt. (ii) NaOH, H2O, CH3OH, rt.
Scheme 2. Synthesis of compounds 16a-16d, 16da-16dv
Hit optimization and identification of compounds 16dk and 16do.
From our reported binding mode of compound 9 (hit) with FABP4 protein, the aromatic ring is located
in a large hydrophobic region made of Y19, M20, V23, V25, A33, F57, and A75. Also, it interacts
with the electron-rich phenyl ring of F16 via π-π stacking and the carboxylic acid group makes polar
interactions with R126 and Y128 (Fig. S4) [27]. In an effort to obtain more potent FABP4 inhibitors,
the structure optimization was first focused on the linker between the carboxylic acid and the aromatic
ring of hit compound 9, and the results were shown in Table 1. Reversing the amide and replacing the
sulfur atom with a carbon atom obtained the compound 12 of which the Ki value is 4.32 µM, worse
than that of compound 9 (Ki: 1.66 µM). Interestingly, the activity is slightly improved when the amide
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(12) was changed into a sulfonamide (14a, Ki: 2.55 µM). Subsequently, the length of the aliphatic
chain of 14a was explored and compound 14c (Ki: 1.19 µM) with four carbon atoms between the
sulfonamide and the carboxylic acid group was more potent than compounds 14a, 14b and 14d (Table
1).
Table 1. Inhibitory activities of designed compounds
Compd.
Structure
Ki (µM) a
1.66±0.19
9
4.32±0.36
12
2.55±0.32
6.51±0.31
14a
14b
1.19±0.01
4.84±0.36
0.35±0.04
14c
14d
1
a Each compound was tested in triplicate and the data are presented as the mean ± SD.
Molecular docking (Schrodinger, Maestro suite) of compound 14c with FABP4 (PDB code: 2HNX)
showed that the aromatic ring was located in a large hydrophobic region and the carboxylate makes
H-bonding interactions with R126 and Y128 (Fig. 2A). Additionally, one oxygen atom of the
sulfonamide group forms additional two H-bonds with R78 and Q95. We envisioned that the
introduction of more hydrophobic groups on the phenyl ring or using bulkier hydrophobic fragment
may improve the potency. To validate our hypothesis, a series of sulfonamide compounds were
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designed and synthesized, and their inhibitions to FABP4 were tested. As summarized in Table 2,
introducing more methyl groups on the phenyl ring was favorable, as the Ki value of 14e, 14f and 14g,
with three, four and five methyl groups, respectively, is gradually decreased. Replacement of the
phenyl ring with a naphthyl ring further improved the hydrophobic interaction. It’s interesting that the
substitution at the C-4 position of the naphthyl ring has a remarkable effect on target inhibition.
Compared to compound 16a (Ki: 2.16 µM) with a fluorine substitution, the inhibitory activities
increased when a chlorine (16b, Ki: 1.85 µM), a bromine (16c, Ki: 1.16 µM), or a methoxy group (16d,
Ki: 0.59 µM) was introduced at this position.
Fig. 2. (A) The predicted binding mode of compound 14c with FABP4 resulted from a molecular docking (PDB code:
2HNX). (B)The X-ray crystal structure of FABP4 in complex with compound 16d (PDB code: 5Y12). (C) An overlay
of the predicted binding mode of compound 14c (green) with the complex structure of FABP4-16d (yellow). The
dashed lines in black represent hydrogen bonds.
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Table 2. Inhibitory Activities of Designed Compounds
Compd.
R
Ki (µM) a
2.69±0.07
1.95±0.17
0.91±0.11
2.16±0.00
1.85±0.18
1.16±0.07
0.59±0.03
0.35±0.04
14e
14f
14g
16a
16b
16c
16d
1
a Each compound was tested in triplicate and the data are presented as the mean ± SD.
To further elucidate the binding modes of this series of inhibitors, the high-resolution crystal structures
of FABP4 in complex with compound 16d were solved (PDB code 5Y12). As depicted in Fig. 2B, the
naphthyl ring of compound 16d occupies the large hydrophobic pocket and forms edge-to-face
π-stacking interactions with the phenyl ring of F16. The carboxyl group is engaged in direct H-bonds
with R126 and Y128, together with two water-mediated H-bonds with R106 and Y128. In particular,
the sulfonamide group participates in many H-bonding interactions with neighboring residues. In
detail, one oxygen atom of the sulfonamide group forms H-bonds with R78 as well as Q95, and the
second oxygen establishes a few water-bridged H-bonds with E72, A75 and R106. Besides, through a
water molecule, the NH group make H-bond interactions with residues Y19, R78 and Q95.
Guided by the crystal structures of FABP4 in complex with compound 16d, we further focused on
modifying the aliphatic chain moiety and the electronic nature of the carboxyl group. In the first step,
all or partial of the aliphatic chain moiety was replaced with a benzene ring to reduce the flexibility
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(16da-16dh) so as to increase the compounds’ binding affinities against FABP4 (Table 3). The
carboxyl or carboxymethyl placed at the meta-position of the phenyl ring was better than at the
para-position (16da vs 16dd, 16db vs 16de). Changing the para-carboxylphenyl group (16da) into a
para-carboxylbenzyl one (16dc) improved the binding affinity from 1.28 µM to 0.76 µM, while
replacing the meta-carboxylphenyl group (16dd) with a meta-carboxylbenzyl one (16df) or its
derivative (16dg) led to decreased binding affinity. In comparison with 16de, the addition of a
methylene between the phenyl ring and the carboxyl group (16dh) slightly increased the binding
affinity which indicated that the length of methylene and activities did not correlate very well. Next,
on the basis of the most potent compound 16dd, structural variations were conducted by mainly
introducing electron-donating or -withdrawing groups on the phenyl ring to change the electronic
property of the carboxyl group (Table 3). Interestingly, only the small electron-withdrawing fluorine
atom at the C-4 (16dk, Ki: 0.21 µM) or C-6 (16do, Ki: 0.20 µM) position led to improvement of the
activities due to the increased salt bridge interaction between the carboxyl group and R126. The larger
electron-withdrawing group (6-Cl, 16dp; 6-OCH3, 16dr) or electron-donating group (4-CH3, 16dl;
6-CH3, 16dq) at these positions resulted in decreased binding affinities, suggesting that steric clashes
of these substitutions with residues might occur. Moreover, the attachment of two fluorine substituents
at both the C-4 and C-6 positions (16du) failed to increase the binding affinity compared to
compounds 16dk and 16do. When the fluorine or chlorine was attached to the C-5 position (16dm and
16dn), their binding affinity were 2-3-fold less potent compared with 16dd. Replacing the benzene
ring with an electron-poor aromatic system such as a pyridine ring (16ds and 16dt) also reduced the
binding affinity dramatically. Intriguingly, neither the small electron-withdrawing fluorine atom (16di
and 16dv) nor the electron-donating group (16dj) at the C-2 position resulted in more potent
compounds. All of these results indicated that the pyridine ring or a big substitution at the phenyl
moiety was not tolerable. The replacement of a fluorine atom at the C-4 or C-6 position of the phenyl
resulted in compounds (16dk and 16do) with higher potency than 1, but it is detrimental when the
fluorine was placed at the C-2 or C-5 position.
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Table 3. Inhibitory activities of designed compounds
Compd.
16da
16db
16dc
16dd
16de
16df
R
Ki (µM) a
1.28±0.03
1.67±0.02
0.76±0.02
0.40±0.02
0.84±0.02
2.12±0.07
>6.14
Compd.
16dm
16dn
16do
16dp
16dq
16dr
16ds
16dt
16du
16dv
1
R
Ki (µM) a
1.15±0.09
0.82±0.01
0.20±0.01
0.93±0.01
2.76±0.09
>6.14
>6.14
16dg
16dh
16di
0.58±0.01
7.75±0.09
>6.14
>6.14
0.23±0.01
7.38±0.15
0.35±0.04
16dj
0.21±0.01
>6.14
16dk
16dl
a Each compound was tested in triplicate and the data are presented as the mean ± SD.
Structural and thermodynamic characterization of 16dk, 16do and 16di binding to FABP4
To understand why incorporation of a fluorine atom at the C-4 or C-6 position of the phenyl ring was
favorable while it was detrimental with the 2-fluorinated benzene, the complex structures of FABP4
with 16dk, 16do and 16di (PDB code: 16dk, 5Y0F; 16do, 5Y0G; 16di, 5Y0X) were determined. As
depicted in Fig. 3, the naphthyl and sulfonamide group of these three inhibitors formed the similar
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interactions with FABP4 as those found with compound 16d. Furthermore, compounds 16dk and 16do
which possess a fluorine atom at the C-4 position or the C-6 position of the phenyl ring have the same
binding mode within FABP4. However, the orientation of the phenyl ring of compound 16di shifted
due to the introduction of a fluorine at the C-2 position in this compound (Fig. 3A). We speculated that
the 2-F substituent had clashes with the oxygen atom of the sulfonamide group, which led to the
changed orientation of the phenyl. To test this, we added a fluorine atom to the C-2 position of
compound 16dk in silico, and the resulting distance between the added fluorine and one oxygen atom
of the sulfonamide group was 2.3 Å (Fig. S3). Given the high electronegativity of both oxygen and
fluorine and the sum of the Van der Waals radius of two atoms (2.99 Å), the orientation of the phenyl
had to be altered to avoid the repulsive force as well as clashes between two atoms. Such an
orientation change of the phenyl ring caused the side-chain movement of R126 together with F16, as
cation-π interactions are formed between these two residues (Fig. 3B). The side-chain movement of
R126 weakened its H-bonding interactions with the carboxyl oxygen of 16di. Notably, a network of
the ordered water molecules surrounding compound 16do was disrupted in the complex of FABP4
bound with 16di (Fig. 3C and 3D). In comparison with the network in the complex structures of
FABP4-16dk and FABP4-16do, three water molecules around the carboxyl group disappeared in the
complex of FABP4-16di, resulting that the H-bonding interactions between the carboxyl oxygen of
16di and R106 was thus broken. This together with the weakened H-bond between R126 and the
carboxyl oxygen may explain why the binding affinity of 16di to FABP4 was reduced compared to
that of 16dk or 16do.
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Fig. 3. X-ray structures of FABP4 in complex with compounds 16dk (green) (PDB code: 5Y0F), 16do (yellow) (PDB
code: 5Y0G) and 16di (blue) (PDB code: 5Y0X). (A) An overlay of complex structures of FABP4-16dk, FABP4-16do
and FABP4-16di. Residues are shown in stick. Inhibitors are shown in ball and stick. The light blue shadow marks the
change of the phenyl ring and the adjacent residues with a shifted side-chain. (B) The superimposed structures of the
FABP4-16do and FABP4-16di complexes. (C) A critical water network in the complex structure of FABP4-16do. The
water molecules are shown in red sphere. (D) The disrupted water network in the complex structure of FABP4-16di.
To gain more insight into how the changes of the phenyl ring orientations was well as the surrounding
water network affected the binding free energy [31,32], thermodynamic properties of 16di and 16do
binding to FABP4 in solution was investigated by ITC measurements. The averaged dissociation
constant (Kd), binding free energy (∆G), enthalpy (∆H), and entropy term (−T∆S) resulted from three
independent ITC measurements on each compound were listed in Table 4. Consistent with the
different binding patterns revealed by the complex structures, compound 16di and 16do displayed
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significantly different binding thermodynamics. The multiple H-bonding as well as hydrophobic
interactions of 16do with the protein is in accord with the huge enthalpy (∆H: -59.64 kJ/mol) resulted
from the ITC measurements, while it is much less for 16di (∆H: -25.87 kJ/mol) due to, as mentioned
above, the less interactions formed between 16di, in particular the carboxyl group, and FABP4.
Although the binding of both compounds with FABP4 were mainly driven by the enthalpic term, the
entropy contribution to their binding were just opposite for two compounds. Binding of compound
16do to FABP4 exhibited a substantial entropic penalty (-T∆S: 25.72 kJ/mol), whereas in the case of
16di the complex formation benefited from the entropic effect (-T∆S: -4.38 kJ/mol). Accordingly, the
binding free energies for two compounds are close to each other, but the enthalpic and entropic
contributions to the binding in two cases are distinctive. In comparison with 16do, the lower binding
enthalpy and positive contribution of the entropy for 16di binding to FABP4 could be ascribed to the
disruption of the H-bonds and the water-molecule-network resulted from the orientation change of the
phenyl ring, and the releasing of three ordered water molecules from the network.
Table 4. Thermodynamic parameters of 16di and 16do binding to FABP4
Compd.
16di
Kd (M)a
∆G (kJ/mol)a
-30.25 ± 0.13
-33.92 ± 0.13
∆H (kJ/mol)a
-25.87 ± 0.15
-59.64 ± 0.53
- T∆S (kJ/mol)a
-4.38 ± 0.02
(6.03 ± 0.31) × 10-6
(1.40 ± 0.07) × 10-6
25.72 ± 0.66
16do
a Each compound was tested in triplicate and the data are presented as the mean ± SD.
The selectivity of representative compounds against FABP3 and other fatty acid targets
As FABP3 has important roles in cell proliferation, apoptosis and prevention of oxidative stress in
cardiac and red skeletal muscle tissues [17], the selectivity between FABP3 and FABP4 is a crucial
issue for design of FABP4 inhibitors. Therefore, the inhibition of four compounds, which have good
binding affinity to FABP4, to FABP3 were examined and listed in Table 5. Remarkably, our
compounds showed a good selectivity of FABP4 over FABP3. It is even better than the selectivity of 1
and 2 to FABP3. We then superimposed the crystal structure of 16do bounded FABP4 with the
structure of FABP3 in complex with the palmitic acid to exploit the difference in the ligand binding
sites. As shown in Fig. 4, the sub-pocket for the phenyl ring of 16do includes residues V115 and C117
in FABP4 but the corresponding residues in FABP3 are L115 and L117, with a larger side-chain. It
seems that compound 16do would have clashes with these two amino acids in FABP3. Therefore, the
inappropriate complementarity between the compound and the ligand binding pocket of FABP3
accounts for the weaker FABP3 activity of 16do. Moreover, the selectivity of compounds 16dk and
16do on other fatty acid targets was evaluated to exclude the off-target effects. The results show that
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none of them exhibit activity towards GPR40 (Free fatty acid receptor 1), GPR120 (Free fatty acid
receptor 4), DGAT1 (Acyl coenzyme A: diacylglycerol acyltransferase 1) and PPARγ (Peroxisome
proliferator activated receptor γ) (see Table S3).
Table 5. Selectivity of representative compounds against FABP3
FABP4 Ki (µM)a
FABP4 IC50 (µM)a
FABP3 IC50 (µM)a
FABP3/FABP4
Compd.
0.40±0.02
0.21±0.01
0.20±0.01
0.23±0.01
0.35±0.04
0.087±0.002
3.22±0.17
1.69±0.12
1.65±0.12
1.84±0.10
2.82±0.35
0.71±0.06
>100
>100
>31
>59
>60
>54
14.5
6.5
16dd
16dk
16do
16du
1
>100
>100
40.83±3.27
4.6±0.41
2
a Each compound was tested in triplicate and the data are presented as the mean ± SD.
Fig. 4. (A) The binding mode of Compound 16do in the pocket of FABP4 (PDB code: 5Y0G). (B) The superimposed
structures of the FABP4-16do complex and FABP3 (PDB code 4TKB). 16do and the structure of FABP3 were shown.
The ligand binding pockets are shown with their inter-molecular surface.
Effects of 16dk and 16do on adipocytes.
It has been reported that genetic ablation or pharmacological inhibition of FABP4 can inhibit lipolysis
and increase lipogenesis in adipocytes [15,33]. To investigate the effects of 16dk and 16do on
adipocytes, lipolysis assay, triglyceride assay and oil red staining were carried out one by one. Firstly,
the cytotoxicity of 16dk and 16do were tested using MTT assay. As illustrated in Fig. S1, they had no
significant cytotoxicity up to 80 µM in 3T3-L1 pre-adipocytes while 1 at a concentration of 80 µM
caused the death of ~50% cells. As shown in Fig. 5A, both compounds reduced glycerol levels in
mature adipocytes supernatants at 25 µM and 50 µM, exhibiting an inhibition of forskolin-stimulated
lipolysis in a dose-dependent manner. They also significantly increased intracellular triglyceride
content (Fig. 5B) and lipid droplets (Fig. 5C and 5D) during adipocytes differentiation. The above
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results suggested that both 16dk and 16do could inhibit lipolysis and enhance lipid accumulation.
These results are in agreement with the inhibitory activity data of 16dk and 16do on FABP4 and
consistent with the phenotypes of targeted FABP4 deletion in adipocytes, which further conform that
16dk and 16do are FABP4 inhibitors.
Fig. 5. Compounds 16dk and 16do inhibit lipolysis and increase lipid accumulation. (A) Effect of compounds 16dk
and 16do on the release of free glycerol from forskolin-stimulated mature 3T3-L1 adipocyte. Fully differentiated
3T3-L1 were incubated with either DMSO (control) or compounds 16dk and 16do (25 µM and 50 µM) for 24 hours,
then the supernatant was collected and its glycerol level was measured by an assay kit. (B, C, D) Effect of compounds
16dk and 16do on the lipid accumulation during differentiation. DMSO (control), 16dk and 16do (50 µM) were added
with differentiation induction media in the whole differentiation period. At day 6, intracellular triglyceride was
measured and oil red staining followed by quantification of the extracted oil red dye were performed (Scale bar =20
µm.). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. Values are presented as mean ± S.E.M from three
independent experiments (n = 3).
Stabilities of 16dk and 16do tested with in vitro microsomes
The stabilities of compounds 16dk and 16do in microsome were evaluated using liver microsome
preparations from mouse, rat, and human. It shows that both compounds were very stable in
microsomes (Table 6). The percentage of the parent remaining was more than 90% in rat and mouse
microsomes after 60 min. More than 70% of parent compounds were maintained after metabolism in
human liver microsome. Furthermore, both compounds metabolized slowly with T1/2 values ranging
from 4.66 to 24.6 h and showed low CLint in all species. All of these data indicate that compounds
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16dk and 16do have good metabolic stabilities in liver microsomes.
Table 6. Microsome stability of 16dk and 16do.
Species
Human
Rat
T1/2 (h)
4.66
11.1
Clint (mL/min/gprot)
% parent @ 60 min a
Compd.
16dk
16dk
16dk
16do
7.51
3.15
1.4
72.3
93.4
96.9
73.6
93.9
94.8
Mouse
Human
Rat
24.6
4.99
12.1
14.3
7.02
2.90
2.4
16do
Mouse
16do
a Mean percentage remaining of parent compounds (0.1 µM) 60 min after incubation with the indicated liver
microsomes.
In vivo efficacy of 16dk and 16do
The leptin receptor-deficient db/db mice, with severe obesity and insulin resistance, and their lean
littermates C57/BL6 were used to investigate the effect of 16dk and 16do on glucose, lipid
metabolism and insulin sensitivity. As shown in Fig. S2, oral administration of 16dk and 16do did not
show significant body weight loss and observable abnormalities of main organs such as liver, kidney
and pancreas, and relative lipid content of abdominal adipose tissue at a dose of 100 mg/kg for a
period of 5-week treatment. In contrast, the weekly fasting blood glucose levels of 16dk and 16do
treated-groups were decreased 20-35% (Fig. 6A). Glucose area under the curve (AUC) during glucose
tolerance tests (OGTT) in all groups after 2- and 4-week compounds treatment reduced significantly
compared with the vehicle group, revealing a significant improvement in glucose metabolism (Fig.
6B-E). Insulin tolerance tests (ITT, Fig. 6F) along with the western blot (Fig. 8B) resulted in
significantly increased insulin sensitivity in the db/db mice treated with 16dk and 16do. Additionally,
compounds 16dk and 16do could reduce the serum levels of ALT and AST (Fig. 7A and 7B), two liver
enzymes indicating liver damage, indicating an improvement of the compounds on liver function.
Furthermore, 16dk and 16do treatment also significantly decreased serum levels of triglyceride (TG)
and non-esterified fatty acid (NEFA) (Fig. 7C and 7E), suggesting the favorable effect of the
compounds on lipid metabolism. However, the level of total cholesterol (TCH) was almost unchanged
(Fig. 7D). Compared with the lean mice, the vehicle-treated db/db mice exhibited severe hepatic
steatosis (hematoxylin & eosin staining, HE staining) and inflammatory infiltration (F4/80 staining
and F4/80 gene expression). Compounds 16dk and 16do treatment could significantly reduce the
hepatic lipid accumulation (Fig. 8A) and inflammatory infiltration (Fig. 8C) of db/db mice. The
staining and relative expression of F4/80 of the adipose tissue in compounds-treated groups also
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revealed decreased inflammation infiltration, and the irregular cell morphology seemed to be
improved somewhat after treatment with 16dk and 16do (Fig. 8A and 8D). Meanwhile, hepatic
proteins of glucose-stimulated mice were collected and western blot was performed. The serine 473
phosphorylation level of Akt (protein kinase B, PKB), as the key signaling node in hepatic insulin
action was significantly elevated, suggesting the hepatic insulin resistance was ameliorated (Fig. 8B).
These results indicated that compounds 16dk and 16do could effectively improve glucose and lipid
metabolism disorder in obese diabetic db/db mice.
Fig. 6. Effects of compounds 16dk and 16do on fasting glucose level, glucose tolerance and insulin sensitivity in db/db
mice. db/db and C57 mice were treated with or without compounds 16dk and 16do (100 mg/kg) for 5 weeks. (A)
Fasting blood glucose levels were measured regularly. (B-E) After 2- and 4-week treatment, the mice were subjected to
oral glucose tolerance test (OGTT). Glucose concentrations of indicated time points and the area under curve (AUC) of
OGTT were shown. (F) After 4-week treatment, mice were subjected to insulin tolerance test (ITT), the glucose curve
was recorded. *p<0.05, **p<0.01, ***p<0.001 vs. vehicle group. Values are expressed as mean ± S.E.M from eight
independent experiments (n = 8 animals/group).
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Fig. 7. Biochemical estimations of ALT, AST, TG, TCH, and NEFA in the serum. (A–E) ALT, AST, TG, TCH, and
NEFA concentrations in the serum of the vehicle, compound-treated and lean groups. All indexes were measured using
assay kits. *p<0.05, **p<0.01, ***p<0.001 vs. vehicle group. Values are given as mean ± S.E.M from eight
independent experiments (n = 8 aninmals/group).
Fig. 8. Compounds 16dk and 16do improve liver steatosis, insulin resistance, and attenuate inflammatory infiltration of
liver and adipose tissue in db/db mice. (A) HE staining of liver and adipose tissue. (B) Representative blots of Akt and
p-Akt and histogram of the statistical results of Western blot analysis data in the liver. (C-D) Immunostaining and
relative expression for F4/80 of liver (C) and adipose tissue (D). Scale bar =100µm. *p<0.05, **p<0.01, ***p<0.001
versus Vehicle group. Values are given as mean ± S.E.M from eight independent experiments (n = 8 aninmals /group).
Plasma protein binding
As animal efficacy of a compound is determined by its inhibitory activity and exposure in tissues to its
unbound, or “free” fraction, the plasma protein binding experiment of compound 16dk was conducted.
As shown in the Table 7, compound 16dk show a high plasma protein binding (98.53%) in the mouse
plasma at 1µM concentration. This provide a possible reason that high plasma protein binding
contribute to the high dose required in our in vivo study.
Table 7. The plasma protein binding rate of compound 16dk
Compd.
Species
Mouse
Concentration
% Bound a
98.53%
1 µM
16dk
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Mouse
1 µM
89.11%
Propranolol
a % Bound, Fraction bound; Mean percentage of three independent experiments.
Conclusion
In summary, we reported the identification of naphthalene-1-sulfonamide derivatives as novel, potent
and selective FABP4 inhibitors. Systematic SAR explorations resulted in the discovery of compounds
16dk and 16do as potential FABP4 inhibitors for further development. In particular, crystal structures
of 16dk, 16do and 16di in complex with FABP4 together with the ITC study revealed the binding
mode of these compounds and the importance of the water-molecule network in the binding pocket of
FABP4. Furthermore, both 16dk and 16do showed good metabolic stabilities in liver microsomes and
a dramatic improvement in glucose and lipid metabolism in db/db mice. All these data indicated that
16dk and 16do would be promising lead compounds for further development.
¢ Experimental section
Chemistry methods
All reagents and solvents were purchased from commercial suppliers such as Adamas-beta®, Alfa
Aesar, Acros, Bide pharmatech, etc and used without further purification unless otherwise indicated.
Melting points were measured on a WRS-1B digital melting point apparatus. Flash chromatography
was performed on silica gel (200–300 mesh) and visualized under UV light monitor at 254 nm.
Nuclear magnetic resonance (NMR) spectroscopy were recorded with a 400 MHz Varian or a Bruker
600 MHz NMR spectrometer at ambient temperature. Chemical shifts (δ) were expressed in parts per
million (ppm) downfield from tetramethylsilan, and coupling constants (J) values were described as
hertz. MS was measured on Agilent 6120 quadrupole LC/MS. High resolution mass spectrometry
(HRMS) determinations for all new compounds were carried out on AB SCIWX TRIPLETOF 5600+.
The purity of all the tested compounds was analyzed using an Agilent 1200 HPLC system and were
conformed to be ≥95% (Table S1).
General procedure for the synthesis of intermediates 11a-11d
To a solution of amino substituted fatty acid derivatives 10a-10d (10 mmol) in 20 mL methanol was
added thionyl chloride (20 mmol) dropwise under ice bath. After stirring at room temperature for
about 2 h, the solution was evaporated to give the desired products 11a-11d.
Methyl 4-aminobutanoate hydrochloride (11a)
White solid; yield 96.0 %; 1H NMR (400 MHz, D2O) δ 3.46 (s, 3H), 2.79 (t, J = 7.6 Hz, 2H), 2.28 (t, J
= 7.3 Hz, 2H), 1.77-1.67 (m, 2H). ESI-MS: calcd for [M+H-HCl]+ m/z : 118.1, found: 118.1.
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Methyl 3-aminopropanoate hydrochloride (11b)
White solid; yield 98.0 %; 1H NMR (400 MHz, D2O) δ 3.70 (s, 3H), 3.24 (t, J = 6.5 Hz, 2H), 2.78 (t, J
= 6.5 Hz, 2H). ESI-MS: calcd for [M+H-HCl]+ m/z : 104.1, found: 104.1.
Methyl 5-aminopentanoate hydrochloride (11c)
White solid; yield 95.0 %; 1H NMR (400 MHz, D2O) δ 3.64 (s, 3H), 2.95 (t, J = 6.1 Hz, 2H), 2.43 –
2.37 (m, 2H), 1.67 – 1.59 (m, 4H). ESI-MS: calcd for [M+H-HCl]+ m/z : 132.1, found: 132.1.
Methyl 6-aminohexanoate hydrochloride (11d)
White solid; yield 96.0 %; 1H NMR (400 MHz, D2O) δ 3.44 (s, 3H), 2.74 (t, J = 7.6 Hz, 2H), 2.17 (t, J
= 7.4 Hz, 2H), 1.48 – 1.34 (m, 4H), 1.21 – 1.09 (m, 2H). ESI-MS: calcd for [M+H-HCl]+ m/z : 146.1,
found: 146.1.
4-(3, 5-Dichlorobenzamido)butanoic acid (12)
To a solution of compound 11a (154 mg, 1.0 mmol) in anhydrous CH2Cl2 (10 mL) was added 3,
5-dichlorobenzoyl chloride (209 mg, 1.0 mmol) and pyridine (241µL 3.0 mmol) under ice bath. After
stirring at room temperature for about 8 h, the solvent was removed in vacuum. The resulting residue
was dissolved in NaOH (1 M) water solution (3 mL) and CH3OH (3 mL) and stirred for another 2 h.
Then the solvent was removed under vacuum, and the residue was acidified to pH = 2 or below with
HCl (1 M) water solution. The solution was extracted with ethyl acetate twice and the combined
organics were dried over anhydrous sodium sulfate and concentrated in vacuum. The resulting residue
was purified by silica gel chromatography to give the desired compound as a white solid (234 mg,
1
yield 85.0 %), mp 145-147 °C. H NMR (400 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.69 (t, J = 5.3 Hz,
1H), 7.83 (d, J = 1.9 Hz, 2H), 7.78 (t, J = 1.9 Hz, 1H), 3.27 – 3.20 (m, 2H), 2.26 (t, J = 7.3 Hz, 2H),
1.77 – 1.67 (m, 2H). ESI-MS: calcd for [M - H]- m/z : 274.0, found: 274.1.
4-((3, 5-Dichlorophenyl)sulfonamido)butanoic acid (14a)
The title compound was prepared from 13a and 11a following general procedure for the synthesis of
compound 12. White solid, yield 35.4 %, mp 169-170 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.12 (s,
1H), 7.99 (t, J = 1.9 Hz, 1H), 7.97 (s, 1H), 7.77 (d, J = 1.9 Hz, 2H), 2.81 (t, J = 6.9 Hz, 2H), 2.22 (t, J
= 7.3 Hz, 2H), 1.64 – 1.54 (m, 2H). ESI-MS: calcd for [M - H]- m/z : 310.0, found: 310.0.
3-((3, 5-Dichlorophenyl)sulfonamido)propanoic acid (14b)
The title compound was prepared from 13a and 11b following general procedure for the synthesis of
compound 12. White solid, yield 39.1 %, mp 138-140 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s,
1H), 8.00 (s, 1H), 7.96 (t, J = 1.9 Hz, 1H), 7.76 (d, J = 1.9 Hz, 2H), 2.97 (t, J = 6.7 Hz, 2H), 2.36 (t, J
= 6.7 Hz, 2H). ESI-MS: calcd for [M - H]- m/z : 296.0, found: 296.0.
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5-((3, 5-Dichlorophenyl)sulfonamido)pentanoic acid (14c)
The title compound was prepared from 13a and 11c following general procedure for the synthesis of
compound 12. White solid, yield 45.3 %, mp 131-132 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.07 (s,
1H), 7.98 (t, J = 1.9 Hz, 1H), 7.90 (t, J = 5.4 Hz, 1H), 7.77 (d, J = 1.9 Hz, 2H), 2.78 (dd, J = 11.6, 6.2
Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H), 1.54 – 1.32 (m, 4H). ESI-MS: calcd for [M - H]- m/z : 324.0, found:
324.0.
6-((3, 5-Dichlorophenyl)sulfonamido)hexanoic acid (14d)
The title compound was prepared from 13a and 11d following general procedure for the synthesis of
compound 12. White solid, yield 43.8 %, mp 130-131 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.03 (s,
1H), 7.98 (t, J = 1.8 Hz, 1H), 7.87 (t, J = 5.7 Hz, 1H), 7.77 (d, J = 1.8 Hz, 2H), 2.77 (q, J = 6.7 Hz,
2H), 2.15 (t, J = 7.3 Hz, 2H), 1.47 – 1.32 (m, 4H), 1.27 – 1.18 (m, 2H). ESI-MS: calcd for [M - H]-
m/z : 338.0, found: 338.0.
5-((2, 4, 6-Trimethylphenyl)sulfonamido)pentanoic acid (14e)
The title compound was prepared from 13b and 11c following general procedure for the synthesis of
compound 12. White solid, yield 76.5 %, mp 104-105 °C. 1HNMR (400MHz, CD3OD) δ 7.04 (s, 2H),
2.86 (t, 2H, J = 6.8 Hz), 2.63 (s, 6H), 2.32 (s, 3H), 2.20 (t, 2H, J = 6.8Hz), 1.57 – 1.43 (m, 4H). 13C
NMR (100MHz, CD3OD) δ 175.8, 142.0, 138.8, 134.2, 131.5, 127.7, 123.4, 41.4, 32.8, 28.6, 23.3,
21.7, 21.6, 19.5. ESI-MS: calcd for [M - H]- m/z : 298.1, found: 298.1.
5-((2, 3, 5, 6-Tetramethylphenyl)sulfonamido)pentanoic acid (14f)
The title compound was prepared from 13c and 11c following general procedure for the synthesis of
compound 12. White solid, yield 84.5 %, mp 118-121 °C. 1HNMR (400MHz, CD3OD) δ 7.21 (s, 1H),
2.87 (t, 2H, J = 6.8 Hz), 2.56 (s, 6H), 2.30 (s, 6H), 2.19 (t, 2H, J = 6.8), 1.60 – 1.40 (m, 4H). 13C NMR
(100MHz, CD3OD) δ 175.8, 142.0, 138.2, 135.8, 135.0, 134.7, 125.4, 41.4, 32.8, 28.6, 23.3, 21.6, 20.3,
19.6, 16.7. ESI-MS: calcd for [M - H]- m/z : 312.1, found: 312.1.
5-((2, 3, 4, 5, 6-Pentamethylphenyl)sulfonamido)pentanoic acid (14g)
The title compound was prepared from 13d and 11c following general procedure for the synthesis of
compound 12. White solid, yield 79.3 %, mp 124-125 °C. 1HNMR (400MHz, CD3OD) δ 2.85 (t, 2H, J
= 6.8), 2.58 (s, 6H), 2.31 (s, 3H), 2.27 (s, 6H), 2.17 (t, 2H, J = 7.2Hz), 1.60 – 1.40 (m, 4H). 13C NMR
(100MHz, CD3OD) δ 175.8, 139.0, 136.3, 135.0, 134.4, 133.9, 127.4, 41.5, 32.8, 28.6, 23.3, 21.6, 20.3,
17.8, 16.4, 15.7. ESI-HRMS [M - H]- calcd for C16H24NO4S: 326.1432, found: 326.1432.
General Procedure for the Synthesis of Intermediates 15a-15d
Substituted naphthalene (5.0 mmol) was dissolved in 15 mL of chloroform, chlorosulfonic acid (658
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µL, 10.0 mmol) was added under ice bath and stirred for about 4 h at 0° C. The mixture was poured
slowly into 50 mL of ice water and extrated with ethyl acetate for three times. The combined organic
layer was was dried over anhydrous sodium sulfate and concentrated in vacuum to give 15a-15d,
which were used directly for the next step.
5-(4-Fluoronaphthalene-1-sulfonamido)pentanoic acid (16a)
The title compound was prepared from 15a and 11c following general procedure for the synthesis of
compound 12. White solid, yield 85.2 %, mp 114-115 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s,
1H), 8.69 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.14 (dd, J = 8.2, 5.5 Hz, 1H), 8.01 (t, J = 5.7
Hz, 1H), 7.90 – 7.73 (m, 2H), 7.49 (dd, J = 10.1, 8.3 Hz, 1H), 2.76 (q, J = 6.4 Hz, 2H), 2.04 (t, J = 7.0
Hz, 2H), 1.47 – 1.21 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 174.65, 159.66, 132.56, 130.08,
129.75, 129.47, 128.11, 125.50, 124.05, 121.39, 109.14, 42.47, 33.45, 29.02, 21.94. ESI-HRMS [M -
H]- calcd for C15H15FNO4S: 324.0711, found: 324.0713.
5-(4-Chloronaphthalene-1-sulfonamido)pentanoic acid (16b)
The title compound was prepared from 15b and 11c following general procedure for the synthesis of
compound 12. White solid, yield 80.6 %, mp 158-163 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.97 (s,
1H), 8.78 – 8.68 (m, 1H), 8.42 – 8.31 (m, 1H), 8.16 – 8.03 (m, 2H), 7.91 – 7.80 (m, 3H), 2.78 (q, J =
6.2 Hz, 2H), 2.05 (t, J = 7.0 Hz, 2H), 1.43 – 1.26 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 174.66,
136.50, 132.20, 129.45, 129.28, 129.22, 129.19, 129.11, 128.24, 127.97, 125.96, 42.54, 33.46, 29.08,
21.94. ESI-HRMS [M - H]- calcd for C15H15ClNO4S: 340.0416, found: 340.0414.
5-((4-Bromonaphthalene)-1-sulfonamido)pentanoic acid (16c)
The title compound was prepared from 15c and 11c following general procedure for the synthesis of
compound 12. White solid, yield 68.6 %, mp 177-179 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.97 (s,
1H), 8.78 – 8.68 (m, 1H), 8.36 – 8.30 (m, 1H), 8.14 – 8.03 (m, 2H), 8.00 (d, J = 7.9 Hz, 1H), 7.90 –
7.76 (m, 2H), 2.78 (q, J = 6.2 Hz, 2H), 2.05 (t, J = 7.0 Hz, 2H), 1.43 – 1.26 (m, 4H). 13C NMR (100
MHz, DMSO-d6) δ 174.70, 136.49, 135.87, 130.95, 129.27, 129.16, 129.00, 128.93, 125.95, 125.67,
125.20, 42.54, 33.50, 29.08, 21.96. ESI-HRMS [M - H]- calcd for C15H15BrNO4S: 383.9911, found:
383.9911.
5-(4-Methoxynaphthalene-1-sulfonamido)pentanoic acid (16d)
The title compound was prepared from 15d and 11c following general procedure for the synthesis of
compound 12. White solid, yield 56.9 %, mp 149-153 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s,
1H), 8.59 (d, J = 8.5 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.84 – 7.74 (m, 1H),
7.74 – 7.68 (m, 1H), 7.67 – 7.60 (m, 1H), 7.09 (d, J = 8.4 Hz, 1H), 4.06 (s, 3H), 2.69 (q, J = 6.2 Hz,
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2H), 2.04 (t, J = 7.0 Hz, 2H), 1.43 – 1.23 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 174.67, 158.97,
130.99, 129.23, 128.59, 127.56, 126.67, 125.75, 125.19, 122.74, 103.22, 56.68, 42.40, 33.48, 28.99,
21.99. ESI-HRMS [M - H]- calcd for C16H18NO5S: 336.0911, found: 336.0909.
4-((4-Methoxynaphthalene)-1-sulfonamido)benzoic acid (16da)
The title compound was prepared from 15d and methyl 4-aminobenzoate following general procedure
1
for the synthesis of compound 12. White solid, yield 59.9 %, mp 226-229 °C. H NMR (400 MHz,
DMSO-d6) δ 12.68 (s, 1H), 11.11 (s, 1H), 8.66 (d, J = 8.6 Hz, 1H), 8.32 – 8.22 (m, 2H), 7.84 – 7.67 (m,
3H), 7.64 (t, J = 7.6 Hz, 1H), 7.16 – 7.06 (m, 3H), 4.03 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
167.15, 159.77, 142.44, 133.06, 131.11, 129.16, 128.98, 126.94, 125.63, 125.56, 125.34, 124.53,
123.00, 117.45, 103.30, 56.80. ESI-MS: calcd for [M - H]- m/z : 356.1, found: 356.1.
2-(4-((4-Methoxynaphthalene)-1-sulfonamido)phenyl)acetic acid (16db)
The title compound was prepared from 15d and methyl 2-(4-aminophenyl)acetate following general
procedure for the synthesis of compound 12. White solid, yield 74.1 %, mp 216-218 °C. 1H NMR (400
MHz, DMSO-d6) δ 12.25 (s, 1H), 10.52 (s, 1H), 8.66 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H),
8.18 (d, J = 8.4 Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.08 – 6.98 (m, 3H), 6.94 (d,
J = 8.4 Hz, 2H), 4.02 (s, 3H), 3.37 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 172.42, 158.83, 136.08,
131.86, 129.85, 128.49, 128.29, 126.17, 125.64, 124.98, 124.16, 122.27, 118.57, 102.65, 56.09.
ESI-HRMS [M - H]- calcd for C19H16NO5S: 370.0755, found: 370.0757.
4-(((4-Methoxynaphthalene)-1-sulfonamido)methyl)benzoic acid (16dc)
The title compound was prepared from 15d and methyl 4-(aminomethyl)benzoate following general
procedure for the synthesis of compound 12. White solid, yield 63.8 %, mp 227-228 °C.1H NMR (400
MHz, DMSO-d6) δ 12.87 (s, 1H), 8.60 (d, J = 8.5 Hz, 1H), 8.46 (t, J = 6.2 Hz, 1H), 8.25 (d, J = 8.4 Hz,
1H), 8.06 (d, J = 8.3 Hz, 1H), 7.76 – 7.67 (m, 3H), 7.64 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 8.0 Hz, 2H),
7.02 (d, J = 8.4 Hz, 1H), 4.03 (d, J = 8.4 Hz, 5H). 13C NMR (150 MHz, DMSO-d6) δ 166.88, 158.48,
142.65, 130.60, 129.23, 128.78, 128.53, 128.02, 127.21, 126.86, 126.03, 125.10, 124.53, 122.11,
102.55, 56.06, 45.37. ESI-HRMS [M - H]- calcd for C19H16NO5S: 370.0755, found: 370.0753.
3-((4-Methoxynaphthalene)-1-sulfonamido)benzoic acid (16dd)
The title compound was prepared from 15d and methyl 3-aminobenzoate following general procedure
1
for the synthesis of compound 12. White solid, yield 63.3 %, mp 236-240 °C. H NMR (400 MHz,
DMSO-d6) δ 13.00 (s, 1H), 10.82 (s, 1H), 8.67 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.21 (d, J
= 8.4 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.67 – 7.60 (m, 2H), 7.50 – 7.44 (m, 1H), 7.32 – 7.24 (m, 2H),
7.07 (d, J = 8.4 Hz, 1H), 4.02 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 177.83, 164.21, 141.45, 137.26,
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135.23, 133.86, 133.68, 131.56, 130.99, 130.36, 129.53, 127.66, 123.93, 108.03, 61.47. ESI-MS: calcd
for [M - H]- m/z : 356.1, found: 356.1.
2-(3-((4-Methoxynaphthalene)-1-sulfonamido)phenyl)acetic acid (16de)
The title compound was prepared from 15d and methyl 2-(3-aminophenyl)acetate following general
procedure for the synthesis of compound 12. White solid, yield 59.0 %, mp 170-174 °C. 1H NMR (400
MHz, acetone-d6) δ 9.26 (s, 1H), 8.75 (d, J = 8.6 Hz, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 8.4 Hz,
1H), 7.69 (t, J = 7.7 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.16 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.99 (d, J
= 8.3 Hz, 2H), 6.90 (d, J = 7.4 Hz, 1H), 4.06 (s, 3H), 3.47 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ
172.12, 158.86, 137.63, 135.73, 132.01, 128.70, 128.51, 128.29, 126.15, 125.58, 125.00, 124.16,
124.15, 122.25, 119.33, 116.54, 102.61, 56.08, 40.50. ESI-HRMS [M - H]- calcd for C19H16NO5S:
370.0755, found: 370.0751.
3-(((4-Methoxynaphthalene)-1-sulfonamido)methyl)benzoic acid (16df)
The title compound was prepared from 15d and methyl 3-(aminomethyl)benzoate following general
procedure for the synthesis of compound 12. White solid, yield 51.2 %, mp 200-205 °C. 1H NMR (400
MHz, DMSO-d6) δ 8.54 (d, J = 8.6 Hz, 1H), 8.41 (t, J = 6.3 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.05 (d,
J = 8.3 Hz, 1H), 7.70-7.62 (m, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6
Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 4.01 (s, 3H), 3.99 (d, J = 6.1 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 167.04, 158.59, 138.13, 131.79, 130.73, 130.35, 128.59, 128.30, 128.12, 128.05, 127.78,
126.96, 126.11, 125.19, 124.58, 122.22, 102.64, 56.14, 45.43. ESI-HRMS [M - H]- calcd for
C19H16NO5S: 370.0755, found: 370.0756.
3-(1-((4-Methoxynaphthalene)-1-sulfonamido)ethyl)benzoic acid (16dg)
The title compound was prepared from 15d and methyl 3-(1-aminoethyl)benzoate following general
procedure for the synthesis of compound 12. White solid, yield 59.7 %, mp 218-221 °C. 1H NMR (400
MHz, DMSO-d6) δ 8.48 (d, J = 8.6 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.5 Hz, 1H), 7.95 (d,
J = 8.3 Hz, 1H), 7.66 – 7.58 (m, 1H), 7.57 – 7.44 (m, 2H), 7.18 (d, J = 7.8 Hz, 1H), 7.04 – 6.96 (m,
1H), 6.89 (d, J = 8.5 Hz, 1H), 4.31 – 4.21 (m, 1H), 3.97 (s, 3H), 1.11 (d, J = 6.8 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 167.02, 158.50, 143.24, 130.91, 130.12, 130.05, 128.51, 127.91, 127.65,
127.37, 127.00, 126.76, 125.92, 124.98, 124.52, 122.08, 102.45, 56.03, 52.43, 23.24. ESI-HRMS [M -
H]- calcd for C20H18NO5S: 384.0911, found: 384.0911.
3-(3-((4-Methoxynaphthalene)-1-sulfonamido)phenyl)propanoic acid (16dh)
The title compound was prepared from 15d and methyl 3-(3-aminophenyl)propanoate following
1
general procedure for the synthesis of compound 12. White solid, yield 60.9 %, mp 161-162 °C. H
24

ACCEPTED MANUSCRIPT
NMR (400 MHz, DMSO-d6) δ 10.27 (s, 1H), 8.44 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.98
(d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 6.86 – 6.74 (m, 2H), 6.67 (s, 1H),
6.60 – 6.50 (m, 2H), 3.80 (s, 3H), 2.41 (t, J = 7.5 Hz, 2H), 2.14 (t, J = 7.5 Hz, 2H). 13C NMR (150
MHz, DMSO-d6) δ 173.34, 158.85, 141.65, 137.63, 132.03, 128.74, 128.52, 128.27, 126.14, 125.60,
124.97, 124.18, 123.05, 122.23, 118.35, 116.20, 102.61, 56.08, 34.71, 29.97. ESI-HRMS [M - H]-
calcd for C20H18NO5S: 384.0911, found: 384.0913.
2-Fluoro-3-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16di)
The title compound was prepared from 15d and methyl 3-amino-2-fluorobenzoate following general
procedure for the synthesis of compound 12. White solid, yield 53.3 %, mp 235-236 °C. 1H NMR (400
MHz, DMSO-d6) δ 10.53 (s, 1H), 8.64 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 8.4
Hz, 1H), 7.74 – 7.66 (m, 1H), 7.66-7.59 (m, 1H), 7.53-7.46 (m, 1H), 7.45-7.38 (m, 1H), 7.12 (t, J =
8.0 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 4.00 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.51, 159.12,
155.24, 152.65, 131.61, 128.80, 128.61, 128.39, 127.85, 126.40, 125.97, 125.79, 125.13, 124.44,
124.11, 122.35, 102.73, 56.25. ESI-HRMS [M - H]- calcd for C18H13FNO5S: 374.0504, found:
374.0500.
3-((4-Methoxynaphthalene)-1-sulfonamido)-2-methylbenzoic acid (16dj)
The title compound was prepared from 15d and methyl 3-amino-2-methylbenzoate following general
procedure for the synthesis of compound 12. White solid, yield 65.8 %, mp 183-187 °C. 1H NMR (400
MHz, DMSO-d6) δ 12.90 (s, 1H), 9.87 (s, 1H), 8.64 (d, J = 8.5 Hz, 1H), 8.27 (d, J = 8.3 Hz, 1H), 7.92
(d, J = 8.3 Hz, 1H), 7.69 (t, J = 7.3 Hz, 1H), 7.66 – 7.60 (m, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.07 (t, J =
7.8 Hz, 1H), 7.02 – 6.96 (m, 2H), 4.01 (s, 3H). 2.07 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 168.80,
158.86, 135.91, 134.77, 133.07, 131.30, 129.43, 128.72, 128.25, 127.57, 126.89, 126.35, 125.81,
125.16, 124.66, 122.35, 102.81, 56.24, 14.97. ESI-HRMS [M - H]- calcd for C19H16NO5S: 370.0755,
found: 370.0754.
2-Fluoro-5-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16dk)
The title compound was prepared from 15d and methyl 5-amino-2-fluorobenzoate following general
procedure for the synthesis of compound 12. White solid, yield 50.8 %, mp 220-222 °C. 1H NMR (400
MHz, DMSO-d6) δ 10.71 (s, 1H), 8.61 (d, J = 8.6 Hz, 1H), 8.23 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 8.4
Hz, 1H), 7.74 (t, J = 7.3 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.47 (dd, J = 6.2, 2.7 Hz, 1H), 7.24 – 7.16
(m, 1H), 7.13 – 7.06 (m, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.99 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 164.36, 159.17, 158.58, 156.06, 133.77, 132.17, 128.61, 128.51, 126.45, 125.09, 124.11, 122.48,
122.09, 119.48, 117.86, 117.62, 102.78, 56.27. ESI-HRMS [M - H]- calcd for C18H13FNO5S: 374.0504,
25

ACCEPTED MANUSCRIPT
found: 374.0505.
5-((4-Methoxynaphthalene)-1-sulfonamido)-2-methylbenzoic acid (16dl)
The title compound was prepared from 15d and methyl 5-amino-2-methylbenzoate following general
procedure for the synthesis of compound 12. White solid, yield 35.1 %, mp 214-217 °C. 1H NMR (400
MHz, DMSO-d6) δ 12.83 (s, 1H), 10.58 (s, 1H), 8.64 (d, J = 8.7 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H),
8.13 (d, J = 8.3 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.60 (t, J = 7.9 Hz, 1H), 7.46 (d, J = 1.8 Hz, 1H),
7.10-7.02 (m, 3H), 4.00 (s, 3H), 2.29 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 168.06, 159.04,
135.45, 133.87, 132.15, 131.08, 128.56, 126.39, 125.40, 125.10, 124.25, 122.51, 121.84, 122.35,
120.64, 102.77, 56.27, 20.44. ESI-HRMS [M - H]- calcd for C19H16NO5S: 370.0755, found: 370.0754.
3-Fluoro-5-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16dm)
The title compound was prepared from 15d and methyl 3-amino-5-fluorobenzoate following general
procedure for the synthesis of compound 12. White solid, yield 54.8 %, mp 247-249 °C. 1H NMR (400
MHz, DMSO-d6) δ 11.14 (s, 1H), 8.61 (d, J = 8.6 Hz, 1H), 8.27-8.21 (m, 2H), 7.75 (t, J = 7.8 Hz, 1H),
7.62 (t, J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.17 (d, J = 8.7 Hz, 1H), 7.10-7.02 (m, 2H), 4.01 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 165.57, 163.14, 160.71, 159.40, 133.70, 132.46, 128.77, 128.43,
126.53, 125.15, 124.74, 123.94, 122.56, 114.77, 110.08, 108.57, 102.84，56.32. ESI-HRMS [M - H]-
calcd for C18H13FNO5S: 374.0504, found: 374.0505.
3-Chloro-5-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16dn)
The title compound was prepared from 15d and methyl 3-amino-5-chlorobenzoate following general
procedure for the synthesis of compound 12. White solid, yield 43.6 %, mp 223-226 °C. 1H NMR (400
MHz, DMSO-d6) δ 11.13 (s, 1H), 8.61 (d, J = 8.6 Hz, 1H), 8.27 – 8.20 (m, 2H), 7.80 – 7.73 (m, 1H),
7.66-7.60 (m, 1H), 7.56 (t, J = 1.7 Hz, 1H), 7.40 (t, J = 1.7 Hz, 1H), 7.25 (t, J = 2.0 Hz, 1H), 7.10 (d, J
= 8.5 Hz, 1H), 4.02 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.45, 159.43, 139.59, 133.62, 133.55,
132.47, 128.81, 128.43, 126.57, 125.16, 124.75, 124.06, 123.17, 122.68, 121.03, 117.19, 102.88,
56.37. ESI-HRMS [M - H]- calcd for C18H13ClNO5S: 390.0208, found: 390.0212.
4-Fluoro-3-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16do)
The title compound was prepared from 15d and methyl 3-amino-4-fluorobenzoate following general
procedure for the synthesis of compound 12. White solid, yield 62.5 %, mp 228-230 °C. 1H NMR (400
MHz, DMSO-d6) δ 13.11 (s, 1H), 10.57 (s, 1H), 8.65 (d, J = 8.6 Hz, 1H), 8.25 (d, J = 8.3 Hz, 1H),
8.02 (d, J = 8.4 Hz, 1H), 7.86-7.80 (m, 1H), 7.74-7.68 (m, 1H), 7.67 – 7.59 (m, 2H), 7.20 – 7.11 (m,
1H), 7.02 (d, J = 8.5 Hz, 1H), 4.01 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 165.72, 159.05, 158.36,
156.67, 131.42, 128.52, 128.24, 127.84, 127.22, 126.27, 125.77, 125.02, 124.76, 124.30, 122.22,
26

ACCEPTED MANUSCRIPT
116.17, 102.58, 56.15. ESI-HRMS [M - H]- calcd for C18H13FNO5S: 374.0504, found: 374.0504.
4-Chloro-3-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16dp)
The title compound was prepared from 15d and methyl 3-amino-4-chlorobenzoate following general
procedure for the synthesis of compound 12. White solid, yield 53.3 %, mp 235-239 °C. 1H NMR (400
MHz, DMSO-d6) δ 13.23 (s, 1H), 10.35 (s, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H),
7.98 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.71-7.65 (m, 1H), 7.65-7.59 (m, 2H), 7.40 (d, J = 8.3 Hz, 1H),
7.01 (d, J = 8.5 Hz, 1H), 4.01 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.93, 159.13, 134.03,
133.09, 131.40, 130.14, 130.14, 128.74, 128.23, 127.61, 127.23, 126.36, 126.36, 125.15, 124.80,
122.34, 102.70, 56.26. ESI-HRMS [M - H]- calcd for C18H13ClNO5S: 390.0208, found: 390.0213.
4-Methyl -3-((4-methoxynaphthalene)-1-sulfonamido) benzoic acid (16dq)
The title compound was prepared from 15d and methyl 3-amino-4-methylbenzoate following general
procedure for the synthesis of compound 12. White solid, yield 51.0 %, mp 233-236 °C. 1H NMR (400
MHz, DMSO-d6) δ 12.81 (s, 1H), 9.91 (s, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.94
(d, J = 8.4 Hz, 1H), 7.69 (t, J = 7.4 Hz, 1H), 7.66 – 7.59 (m, 2H), 7.55 (d, J = 7.8 Hz, 1H), 7.12 (d, J =
7.9 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 4.02 (s, 3H), 1.90 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
166.65, 158.92, 145.78, 138.70, 135.20, 131.40, 130.82, 129.00, 128.66, 128.27, 126.69, 126.64,
126.37, 125.15, 124.62, 122.46, 102.76, 56.24, 17.78. ESI-HRMS [M - H]- calcd for C19H16NO5S:
370.0755, found: 370.0759.
4-Methoxy-3-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16dr)
The title compound was prepared from 15d and methyl 3-amino-4-methoxybenzoate following
1
general procedure for the synthesis of compound 12. White solid, yield 53.7 %, mp 229-231 °C. H
NMR (400 MHz, DMSO-d6) δ 12.70 (s, 1H), 9.78 (s, 1H), 8.69 (d, J = 8.5 Hz, 1H), 8.24 (d, J = 8.4 Hz,
1H), 7.91 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.72 – 7.66 (m, 1H), 7.65 – 7.59 (m, 2H), 6.96
(d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 3.99 (s, 3H), 3.18 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 166.61, 158.80, 155.83, 131.09, 128.91, 128.23, 127.95, 126.65, 126.19, 125.89, 125.37,
125.07, 125.05, 122.72, 122.06, 111.15, 102.46, 56.22, 55.29. ESI-HRMS [M - H]- calcd for
C19H16NO6S: 386.0704, found: 386.0708.
2-((4-Methoxynaphthalene)-1-sulfonamido)isonicotinic acid (16ds)
The title compound was prepared from 15d and methyl 2-aminoisonicotinate following general
procedure for the synthesis of compound 12. White solid, yield 33.7 %, mp 294-297 °C.1H NMR (400
MHz, DMSO-d6) δ 8.67 (d, J = 8.6 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H), 8.11 (d,
J = 4.7 Hz, 1H), 7.71 (t, J = 7.4 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.52 (s, 1H), 7.19 (d, J = 4.5 Hz,
27

ACCEPTED MANUSCRIPT
1H), 7.10 (d, J = 8.5 Hz, 1H), 4.02 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.45, 158.87, 153.01,
140.98, 131.76, 128.68, 128.34, 126.96, 126.23, 125.08, 124.59, 122.41, 115.57, 112.89, 111.71,
102.74, 56.27. ESI-HRMS [M - H]- calcd for C17H13N2O5S: 357.0551, found: 357.0554.
5-((4-Methoxynaphthalene)-1-sulfonamido)nicotinic acid (16dt)
The title compound was prepared from 15d and methyl 5-aminonicotinate following general
procedure for the synthesis of compound 12. White solid, yield 45.5 %, mp 252-255 °C. 1H NMR (400
MHz, DMSO-d6) δ 13.49 (s, 1H), 11.13 (s, 1H), 8.64-8.58 (m, 2H), 8.41 (d, J = 2.5 Hz, 1H), 8.27-8.20
(m, 2H), 7.87 (t, J = 2.0 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.08 (d, J = 8.5 Hz,
1H), 4.02 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 165.63, 159.43, 144.73, 143.31, 134.59, 132.46,
128.82, 128.40, 126.72, 126.58, 125.63, 125.15, 124.70, 123.96, 122.67, 102.91, 56.35. ESI-HRMS
[M - H]- calcd for C17H13N2O5S: 357.0551, found: 357.0551.
2, 4-Difluoro-5-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16du)
The title compound was prepared from 15d and methyl 5-amino-2, 4-difluorobenzoate following
1
general procedure for the synthesis of compound 12. White solid, yield 60.8 %, mp 211-214 °C. H
NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.96
(d, J = 8.4 Hz, 1H), 7.72-7.60 (m, 3H), 7.24 (t, J = 10.3 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 4.00 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ 163.57, 159.17, 157.81, 157.70, 131.60, 129.55, 128.65,
128.37, 126.39, 125.71, 125.12, 124.41, 122.35, 121.11, 115.68, 106.05, 102.71, 56.27. ESI-HRMS
[M - H]- calcd for C18H12F2NO5S: 392.0410, found: 392.0411.
2, 6-Difluoro-3-((4-methoxynaphthalene)-1-sulfonamido)benzoic acid (16dv)
The title compound was prepared from 15d and methyl 3-amino-2, 6-difluorobenzoate following
1
general procedure for the synthesis of compound 12. White solid, yield 53.9%, mp 211-214 °C. H
NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.60 (d, J = 8.5 Hz, 1H), 8.26 (d, J = 7.9 Hz, 1H), 8.00
(d, J = 8.4 Hz, 1H), 7.74-7.68 (m, 1H), 7.67-7.60 (m, 1H), 7.31 – 7.19 (m, 1H), 7.07 (t, J = 8.7 Hz,
1H), 7.01 (d, J = 8.5 Hz, 1H), 4.01 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 161.56, 159.16, 155.01,
154.08, 131.58, 128.78, 128.62, 128.39, 126.41, 125.87, 125.15, 124.39, 122.38, 121.43, 112.58,
112.08, 102.75, 56.27. ESI-HRMS [M - H]- calcd for C18H12F2NO5S: 392.0410, found: 392.0408.
In vitro FABPs inhibition assay
The inhibitory activities of the compounds against FABPs were measured in a fluorescence-based
assay using the probe 8-anilino-1-naphthalene-sulfonic acid (1, 8-ANS) [29,30]. Briefly, 10 µmol/L of
1, 8-ANS in 0.01M phosphate buffered solution (PBS, pH 7.4) was mixed with FABPs protein to a
final concentration of 10 µmol/L, then various concentrations of compounds were added and
28