Design and synthesis of N-(3-sulfamoylphenyl)amides as Trypanosoma brucei leucyl-tRNA synthetase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113319

The protozoan parasite Trypanosoma brucei (T. brucei) causes human African trypanosomiasis (HAT), which is a fatal and neglected disease in the tropic areas, and new treatments are urgently needed. Leucyl-tRNA synthetase (LeuRS) is an attractive target for the development of antimicrobial agents. In this work, starting from the hit compound thiourea ZCL539, we designed and synthesized a series of amides as effective T. brucei LeuRS (TbLeuRS) synthetic site inhibitors. The most potent compounds 74 and 91 showed IC₅₀ of 0.24 and 0.25 μM, which were about 700-fold more potent than the starting hit compound. The structure-activity relationship was also discussed. These compounds provided a new scaffold and lead compounds for further development of antitrypanosomal agents.

Full text of DOI:10.1016/j.ejmech.2021.113319

European Journal of Medicinal Chemistry 217 (2021) 113319
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and synthesis of N-(3-sulfamoylphenyl)amides as Trypanosoma
brucei leucyl-tRNA synthetase inhibitors
Zezhong Li, Weixiang Xin, Qing Wang, Mingyan Zhu, Huchen Zhou*
State Key Laboratory of Microbial Metabolism, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
The protozoan parasite Trypanosoma brucei (T. brucei) causes human African trypanosomiasis (HAT),
which is a fatal and neglected disease in the tropic areas, and new treatments are urgently needed.
Leucyl-tRNA synthetase (LeuRS) is an attractive target for the development of antimicrobial agents. In
this work, starting from the hit compound thiourea ZCL539, we designed and synthesized a series of
amides as effective T. brucei LeuRS (TbLeuRS) synthetic site inhibitors. The most potent compounds 74
Received 5 November 2020
Received in revised form
17 February 2021
Accepted 17 February 2021
Available online 8 March 2021
and 91 showed IC50 of 0.24 and 0.25 mM, which were about 700-fold more potent than the starting hit
compound. The structure-activity relationship was also discussed. These compounds provided a new
scaffold and lead compounds for further development of antitrypanosomal agents.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
Trypanosoma brucei (T. brucei)
N-(3-sulfamoylphenyl)amides
Leucyl-tRNA synthetase (LeuRS)
1. Introduction
site [15]. In this work, in an attempt to replace the thiourea and its
adjacent acetoxyl group with a single amide group we designed
Human African trypanosomiasis (HAT) is a fatal disease caused
by the protozoan Trypanosoma brucei [1e3], which is a neglected
disease and mostly affects the underdeveloped regions of Africa.
Transmitted by bite of tsetse fly, the parasite Trypanosoma brucei
(T. brucei) enters human bloodstream and lymphatic system, pro-
liferate and enter the central nervous system, and eventually causes
death if not treated. Drugs for the treatment of HAT are limited. For
a long time only five drugs were available [4,5], and these drugs
have serious problems such as low efficacy, toxic side effects,
complex administration, and risk of drug resistance [6,7]. Thus, new
drugs for the treatment of HAT are in urgent need.
Aminoacyl-tRNA synthetases (aaRSs) play critical roles in pro-
tein synthesis by catalyzing the attachment of cognate amino acids
to their corresponding tRNA [8]. Inhibition of aaRSs would cause
disruption of protein synthesis in microbial and lead to antimi-
crobial effect. In the past, aaRSs have been studied as antibacterial
and antifungal targets [9e11]. In recent years, it has also been re-
ported as antiparasitic targets [12,13].
and synthesized a new series of TbLeuRS inhibitors. Compound 74
(N-(3-(N-L-Leucylsulfamoyl) phenyl-4-methoxybenzamide) was
the most potent compound with IC50 value of 0.24 mM, which was
about 700-fold more potent than the starting hit compound. The
structure-activity relationship was also discussed. This work pro-
vided a new scaffold and lead compounds for the development of
new antitrypanosomal agents.
2. Chemistry
N-(sulfamoylphenyl)amides were synthesized following the
similar procedure we previously reported [15]. Preparation of N-(4-
leucylsulfamoylphenyl)amide derivatives was shown in Scheme 1
and Scheme 2. First, coupling of 4-aminobenzenesulfonamide (1)
with N-Boc-L-leucine in the presence of EDCI and DMAP gave
compound 2 with 36.2% yield. Removal of Boc by HCl gas gave
compound 3 with 63.5% yield (Scheme 1).
Preparation of compounds 15e22 is depicted in Scheme 2. After
protection with Cbz, compound 4 was reacted with N-Boc-L-
leucine in the presence of EDCI and DMAP to give compound 5 with
93.2% yield, which underwent hydrogenation to give amine 6 with
quantitative yield. Compounds 7e14 were prepared by reaction
with acyl chlorides prepared from their corresponding acids and
oxalyl chloride. Removal of Boc group with HCl gas gave the
products 15-22 with yields ranging from 32.0% to 81.0%.
Our group paid attention to leucyl-tRNA synthetase (LeuRS) and
has previously reported benzoxaborole-based inhibitors of Trypa-
nosoma brucei LeuRS (TbLeuRS) targeting the editing site [14] and
N-(4-sulfamoylphenyl)thiourea inhibitors targeting the synthetic
* Corresponding author.
E-mail address: hczhou@sjtu.edu.cn (H. Zhou).
Preparation of N-(3-leucylsulfamoylphenyl)amide derivatives
https://doi.org/10.1016/j.ejmech.2021.113319
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
50.0% yield. Sequential removal of Cbz and Boc groups gave com-
pound 82 with 61.0% yield. Acylation of 26 with various acyl chlo-
rides gave compounds 27e63. Eventually, removal of Boc group
gave the desired products 64-81 and 83-91 with yields ranging
from 17.0% to 55.0%.
3. Result and discussion
In our previous search for new T. brucei inhibitors, we identified
a
new scaffold ZCL539 (Fig. 1) with moderate activity
(IC50 ¼ 174 M). Considering that the solubility of thiourea linking
m
group in this hit compound is known to be relatively low and its
toxicity also present an issue, which would limit further develop-
ment of the scaffold, thus we made attempts to replace the
thiourea-acetoxy linking group with a simple amide group which
plays a critical role in clinically approved drugs and synthetic
bioactive molecules [16]. Besides, we introduced a leucyl group at
the terminal sulfonamide nitrogen with the aim to mimic the
endogenous substrate. This resulted in compound 15 which
Scheme 1. Synthesis of N-(4-sulfamoylphenyl)amide derivative 3. Condition and re-
agents: (a) N-Boc-L-leucine, EDCI, DMAP, DCM, 36.2%; (b) HCl (gas), EtOAc, 63.5%.
showed slightly improved activity (IC50 ¼ 96.0
Introduction of second leucyl group as in compound
(IC50 ¼ 106.8 M) showed slightly decreased activity, which indi-
mM) (Table 1).
a
3
m
cated that phenyl group was more favorable than leucyl at the right
terminus of the molecule. Thus, we kept the leucyl group on the left
terminal sulfonamide group, and explored a number of electron
withdrawing and donating substituents on the right terminal
phenyl group. It turned out that both electron withdrawing and
donating groups gave improved activity as exemplified by nitro
compound 19 and methyl compound 21 (IC50 ¼ 32.7 and 39.1
mM),
which showed roughly 3-fold increase of potency as compared to
compound 15. Bromo compound 16 also gave 3-fold increase of
potency. In the case of chloro compounds 17 and 18, the meta-
substitution was favorable, while in the case of nitro and methyl
compounds 19-22, the para-substitution was favorable. These ob-
servations indicated that both electron withdrawing and donating
groups were tolerated, which suggests that electronic effect was
not important in this case. Most derivatives in Table 1 showed
increased activity as compared to compound 15, however the
improvement was limited.
Scheme 2. Synthesis of N-(4-sulfamoylphenyl)amide derivatives. Condition and re-
agents: (a) CbzCl, NaHCO3, H2O/acetone, 84.3%; (b) N-Boc-L-leucine, EDCI, DMAP, DCM,
93.2%; (c) 10% Pd/C, H2, MeOH, quantitative yield; (d) RCOOH, oxalyl chloride, DMF,
DCM, then pyridine, THF; (e) HCl (gas), EtOAc, 32.0%e81.0%.
The substrate mimicking molecule Leu-AMS (Fig. 2a) is biolog-
ically stable and is often used in cocrystallization studies for LeuRS.
Its 3D geometry characteristics should provide useful insight for the
optimization of LeuRS inhibitors. Thus, compound 15 was super-
imposed with Leu-AMS atom-by-atom using Maestro 2015 for
comparison, and it was found that when the leucyl end of the two
molecules were overlapped, the other end of the molecule deviated
significantly from each other (Fig. 2b). While Leu-AMS has the two
extending branches spaced by a single oxygen atom in the sugar
ring, compound 15 with a 1,4-substitution pattern in its central
phenyl ring has the two branches space by two carbon atoms. So, it
implied that a 1,3-substitution pattern should make the geometry
of these LeuRS inhibitors more closely resembles Leu-AMS, which
might lead to higher affinity to the target, TbLeuRS. This hypothesis
was validated by the significant improvement of the inhibitory
activity as exemplified by comparing 1,4-substituted compound 15
(IC50 ¼ 96.0
m
M) and its 1,3-substituted analog 64 (IC50 ¼ 9.4
mM).
The superimposed structures in Fig. 2c also showed that compound
64 better resembles the geometry of Leu-AMS. Thus, a series of 1,3-
substitued compounds 65-86 was synthesized and evaluated as
shown in Table 2.
Scheme 3. Synthesis of N-(3-sulfamoylphenyl)amide derivatives. Condition and Re-
agents: (a) CbzCl, NaHCO3, H2O/acetone, quantitative yield; (b) N-Boc-L-Leucine, EDCI,
DMAP, DCM, 50.0%; (c) Pd/C, H2, MeOH, quantitative yield; (d) RCOOH, SOCl2, then
pyridine, THF; (e) HCl (gas), EtOAc, 17.0%e61.0%.
As shown in Table 2, a variety of functional groups were intro-
duced to the phenyl ring to study the SAR of the derivatives. First,
halogen substitutions in compounds 65-67 gave inhibitory activity
comparable to unsubstituted compound 64. Next, we explored
substituents with different electronic effects and found that the
was shown in Scheme 3. First, 3-aminobenzenesulfonamide 23 was
protected by Cbz group followed by reaction with N-Boc-L-leucine
in the presence of EDCI and DMAP to afford compound 25 with
2

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
Fig. 1. Design of N-(sulfamoylphenyl)amide derivatives.
Table 1
Inhibitory activity of N-(4-sulfamoylphenyl)amide derivatives with various R groups
against TbLeuRS (Standard errors are within 0.05 M).
m
Compd
R
TbLeuRS
IC50 M)
(
m
3
106.8
15
96
16
17
31.2
115.5
18
19
42.8
32.7
20
21
22
>1000
39.1
59.7
electron-withdrawing nitro group gave comparable activity (68-70,
IC50 ¼ 8.6e14.7 M), methyl group (71-73, IC50 ¼ 9.5e14.3 M) also
failed to improve activity, while electron-donating methoxy group
(74-75, IC50 ¼ 0.24 and 2.4 M) gave nearly 40-fold increase of
m
m
Fig. 2. a. Chemical structures of Leu-AMS and compound 15 and 64. b. Overlay of Leu-
AMS (cyan) and compound 15 (grey) atom-by-atom using Maestro 2015. c. Overlay of
Leu-AMS and compound 64 (orange) atom-by-atom using Maestro 2015.
m
inhibitory activity as compared to compound 64. Comparison of
different substitution patterns in compounds 74-77 showed that
para-methoxy gave the best activity. At the same time, para-phe-
noxy compound 78 also showed better activity than its meta-analog
79. Replacement of the phenyl group by furan or thiophene gave
activity comparable to compound 64. Biphenyl substitution gave
improved activity, especially in compound 86 which also has a
benzyl and linear group such ethyl were well accommodated as
shown by compounds 88 and 91 (IC50 ¼ 0.50 and 0.25
mM), which
implied that sterically bulky substituents should be avoided. Thus,
we successfully replaced the linking group in the hit compound
ZCL539 from thiourea-acetoxy to a simple amide group and ob-
tained good TbLeuRS inhibitors with ~700-fold improvement of
inhibitory activity from the initial hit compound. The TbLeuRS in-
methoxy substitution (IC50 ¼ 1.8
mM). Those results indicated that
phenyl group could be replaced by electron-rich aromatic rings.
As the activity of phenoxy derivatives 74-78 were superior and
gave the most potent TbLeuRS inhibitor (compound 74) so far in
this study, it was worthwhile to modify the phenoxy group in order
to further improve the potency, for which we synthesized a number
of derivatives as shown in Table 3. First, the unsubstituted OH group
hibitors whose IC50 values were lower than 3
on the T. brucei 221 parasites, and among them compound 91
showed inhibition of parasite growth with an IC50 of 69.8 M, while
mM were also tested
m
others showed no obvious inhibition. The water solubility of these
compounds was tested and found to be low, thus the low in vitro
antiparasitic activity may be attributed to their low aqueous solu-
bility. Future optimization will be focused on their physicochemical
properties in order to achieve satisfactory antiparasitic activity.
in compound 87 also gave good activity (IC50 ¼ 0.30
mM). Next,
when the substituent was a sterically bulky group such as isopropyl
in compound 89, the activity was diminished by 10-fold as
compared to methoxy or OH groups, while planar group such as
3

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
Table 2
Table 2 (continued )
Inhibitory activity of N-(3-sulfamoylphenyl)amide with different R groups against
TbLeuRS (Standard errors are within 0.05 mM).
Compd
R
TbLeuRS
IC50
(mM)
85
86
3.6
1.8
Compd
R
TbLeuRS
IC50
(mM)
64
9.4
Table 3
65
66
6.4
4.9
Inhibirtory activity of N-(3-sulfamoylphenyl)amides with different ether sub-
stituents against TbLeuRS (Standard errors are within 0.05 M).
m
67
68
5.2
Compd
R
TbLeuRS
IC50 M)
14.7
(
m
87
88
H
0.30
0.50
69
70
8.6
89
3.9
11.5
90
91
0.80
0.25
71
72
73
14.0
9.5
4. Conclusions
14.3
Human African trypanosomiasis, a neglected and fatal tropical
disease, is in urgent need of new therapeutics, especially those in
new chemical classes. LeuRS, as a clinically validated antimicrobial
target, should serve as a promising antitrypanosomal target.
Herein, we reported the discovery of a new class of N-(3-
sulfamoylphenyl)amide TbLeuRS inhibitors. Compounds 74 and
74
0.24
75
76
2.4
0.66
91 showed IC50 of 0.24 and 0.25 mM, which were about 700-fold
more potent than the hit compound ZCL539. The structure-
activity relationship was also discussed. This study provided a
new scaffold for further development of antitrypanosomal agents.
77
1.48
5. Material and methods
78
79
1.3
8.6
5.1. Chemistry
All solvents and reagents were purchased from commercial
sources and used without further purification unless otherwise
noted. Reactions were not optimized for maximum yields. Column
chromatography was performed using Huanghai silica gel
80
81
82
3.7
6.2
4.0
(45e75
mm). NMR spectra were recorded on Bruker Avance III
400 MHz. Chemical shifts are expressed in parts per million (ppm)
relative to residual solvent as an internal reference (CDCl3:7.26;
MeOD: 3.31; DMSO‑d6: 2.50). High resolution mass spectra were
obtained on a Micromass GCT (electron ionization) or an Agilent
6530 Accurate Mass Q-TOF LC-MS (electrospray ionization). HPLC
analysis was performed on an Agilent 1200 with a flow rate of
1 mL minꢀ1 and two gradients: gradient A (10% v/v MeCN in H2O
83
84
3.6
5.8
(containing 0.1% v/v TFA) (t
¼
0.0 min) to 100% MeCN
(t ¼ 20.0 min)); gradient B (10% v/v MeCN in H2O (t ¼ 0.0 min) to
100% MeCN (t ¼ 20.0 min)), stopping at 20 or 25 min, using a DAD
detector. An Agilent Eclipse XDB-C18 column (4.6 mm ꢁ 150 mm,
4

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
5
m
m) was used. Purity was based on the integrated UV chro-
atomsphere. After 5 h, 30 mL DCM was added to quench the re-
action and the Pd/C was removed by filtration, the solution was
concentrated to gain 6 (3.2 g, 100% yield) as white solid. 1H NMR
matogram (254 nm). The purity of all biological assay compounds
was >95.0%.
(400 MHz, DMSO‑d6)
d
11.69 (s, 1H), 7.51 (d, J ¼ 8.4 Hz, 2H), 6.92 (d,
5.1.1. (S)-2-((Tert-Butoxycarbonyl)amino)-N-(4-((N0-tert-butoxy
carbonyl-L-leucylamino)sulfonyl)phenyl)-4-methylpentanamide (2)
J ¼ 8.0 Hz, 2H), 6.58 (d, J ¼ 8.4 Hz, 2H), 6.11 (s, 2H), 3.95 (br. s, 1H),
1.46e1.53 (m, 1H), 1.34 (s, 9H), 1.16e1.22 (m, 2H), 0.78e0.82 (m,
6H).
To
a
mixture of 4-aminobenzenesulfonamide
1 (172 mg,
1.00 mmol) and N-Boc-
L-lencine (694 mg, 3.00 mmol), EDCI (1.9 g,
10.00 mmol) and DMAP (366 mg, 3.00 mmol) were added at 0 ꢂC,
then the mixture was warmed to r.t. and stirred overnight. The
mixture was washed with 0.1 M HCl (20 mL ꢁ 3), 0.1 M NaOH
(20 mL ꢁ 3) and brine (20 mL), dried over anhydrous Na2SO4 and
concentrated. The residue was washed with DCM. Compound 2 was
obtained as a white solid (395 mg, 36.2% yield). 1H NMR (400 MHz,
5.1.6. General procedure 1 for preparation of 15e22
To a solution of organic acetic acids (1.2 eq) in DCM, oxalyl
chloride in DMF was added dropwise at 0 ꢂC. After 3 h, DCM was
removed by rotary evaporation. Acyl chloride dissolved in THF and
pyridine was added dropwise into a solution of 6 (1.0 eq) in THF at
0 ꢂC, then slowly warmed to r.t. and continued stirring for 5e6 h. 1
M HCl was added and extracted with ethyl acetate for three times.
The organic layer was combined, washed with brine for once, dried
over anhydrous Na2SO4 and concentrated, compounds 7e14 were
obtained. Then the intermediates 7e14 were dissolved in ethyl
acetate, treated with HCl gas for 0.5 h, the resulting precipitate was
filtered to obtain the products.
DMSO‑d6)
d 12.10 (s, 1H), 10.39 (s, 1H), 7.88e7.78 (m, 4H), 7.11 (d,
J ¼ 7.6 Hz, 1H), 6.99 (d, J ¼ 7.7 Hz, 1H), 1.63 (s, 1H), 1.51 (dd, J ¼ 18.6,
9.8 Hz, 2H), 1.37 (s, 9H), 1.30 (s, 9H), 1.23 (s, 3H), 0.88 (dd, J ¼ 6.3,
3.9 Hz, 6H), 0.79 (dd, J ¼ 9.8, 6.7 Hz, 6H). 13C NMR (151 MHz,
DMSO‑d6)
d 173.16, 172.72, 155.99, 155.81, 129.23, 118.98, 78.69,
78.56, 54.15, 53.48, 28.11, 24.81, 24.66, 23.37, 23.31, 22.00, 21.64.
Mp: 212e213 ꢂC. HRMS: [MþNa]þ calcd [C28H46N4O8SþNa]þ
621.2934, found 621.2937. HPLC: gradient B, 98.0% purity, 16.3 min.
5.1.7. N-(4-(N-L-Leucylsulfamoyl)phenyl)benzamide (15)
Compound 15 (25.8 mg, 32.0% yield) was prepared from 7
(73 mg, 0.19 mmol) and benzoic acid (25 mg, 0.21 mmol) following
5.1.2. (S)-2-Amino-N-(4-((L-leucylamino)sulfonyl)phenyl)-4-
methyl-pentanamide (3)
general procedure 1. 1H NMR (400 MHz, DMSO‑d6)
d 10.71 (s, 1H),
Compound 2 (100 mg, 0.17 mmol) was dissolved in ethyl acetate,
the solution was treated with HCl gas, and 3 was obtained as a
white precipitate (50 mg, 63.5% yield). 1H NMR (400 MHz,
8.22 (s, 3H), 8.04 (d, J ¼ 8.8 Hz, 2H), 8.00 (d, J ¼ 8.0 Hz, 2H), 7.95 (d,
J ¼ 8.9 Hz, 2H), 7.64 (t, J ¼ 7.3 Hz, 1H), 7.57 (t, J ¼ 7.4 Hz, 2H), 3.80 (s,
1H), 1.66e1.49 (m, 3H), 0.86 (dd, J ¼ 6.3, 1.3 Hz, 6H). 13C NMR
DMSO‑d6)
d
12.82 (s, 1H), 11.56 (s, 1H), 8.50 (s, 3H), 8.29 (s, 3H), 7.91
(101 MHz, DMSO‑d6) d 169.50, 166.61, 144.53, 134.72, 132.57, 129.39,
(q, J ¼ 9.0 Hz, 4H), 4.10 (s, 1H), 3.79 (s, 1H), 1.68 (d, J ¼ 3.8 Hz, 3H),
128.96, 128.38, 126.98, 120þ.26, 51.86, 23.87, 23.16, 21.90. Mp:
257e259 ꢂC. HRMS: [MþH] calcd [C19H23N3O4SþH]þ 390.1488,
found 390.1486. HPLC: gradient A, 98.9% purity, 7.2 min.
1.60e1.46 (m, 3H), 0.92 (t, J ¼ 5.4 Hz, 6H), 0.82 (d, J ¼ 6.1 Hz, 6H). 13
C
NMR (151 MHz, DMSO‑d6)
d 169.21, 129.56, 119.64, 52.22, 24.25,
23.88, 23.09, 22.67, 22.04. Mp: 72e73 ꢂC. HRMS: [MþH]þ calcd
[C18H30N4O4SþH]þ 399.2066, found 399.2056. HPLC: gradient A,
97.0% purity, 5.5 min.
5.1.8. N-(4-(N-L-Leucylsulfamoyl)phenyl)-4-bromobenzamide (16)
Compound 16 (88.3 mg, 61.9% yield) was prepared from 8
(108 mg, 0.28 mmol) and 4-bromobenzoic acid (67.8 mg,
0.33 mmol) following general procedure 1. 1H NMR (400 MHz,
5.1.3. Benzyl 4-sulfamoylphenylcarbamate (4)
CbzCl (10.0 mL, 116.28 mmol) was added dropwise to a solution
of 1 (10.0 g, 58.14 mmol) and NaHCO3 (7.4 g, 88.10 mmol) in H2O/
acetone (40/150 mL) at 0 ꢂC, then the mixture was slowly warmed
to r.t. After the mixture was stirred overnight, acetone was removed
by rotary evaporation and dried. The white residue was triturated
with H2O and filtered. The dried precipitate was washed with cold
DCM and ether, 4 was obtained as white solid (15.0 g, 84.3% yield).
DMSO‑d6) d 12.97 (s, 1H), 10.94 (s, 1H), 8.42 (s, 3H), 8.08 (d,
J ¼ 8.4 Hz, 2H), 8.00 (d, J ¼ 8.0 Hz, 2H), 7.94 (d, J ¼ 8.4 Hz, 2H), 7.75
(d, J ¼ 8.0 Hz, 2H), 3.87 (s, 1H), 1.62e1.54 (m, 3H), 0.84 (d, J ¼ 4.4 Hz,
6H). 13C NMR (101 MHz, DMSO‑d6)
d 168.82, 165.08, 143.89, 133.16,
130.05, 128.77, 125.81, 119.86, 51.20, 23.31, 22.53, 21.54. Mp:
261e262 ꢂC. HRMS: [MþH]þ calcd [C19H22BrN3O4SþH]þ 468.0592,
found 468.0596. HPLC: gradient A, 98.6% purity, 8.2 min.
1H NMR (300 MHz, DMSO‑d6)
d
7.75 (d, J ¼ 8.8 Hz, 2H), 7.63 (d,
J ¼ 8.8 Hz, 2H), 7.33e7.46 (m, 5H), 5.18 (s, 2H).
5.1.9. N-(4-(N-L-Leucylsulfamoyl)phenyl)-4-chlorobenzamide (17)
Compound 17 (100.6 mg, 81.0% yield) was prepared from 9
(103.8 mg, 0.27 mmol) and 4-chlorobenzoic acid (50.5 mg,
0.32 mmol) following general procedure 1. 1H NMR (400 MHz,
5.1.4. Benzyl(4-(N-(N-tert-butoxycarbonyl-L-leucyl)sulfamoyl)
phenyl) carbamate (5)
EDCI (9.3 g, 48.51 mmol) and DMAP (5.9 g, 48.51 mmol) were
added to a solution of 4 (5.0 g, 16.17 mmol) and N-Boc-L-leucine
(7.5 g, 32.34 mmol) in DCM at 0 ꢂC, the mixture was slowly warmed
to r.t. and stirred overnight. The mixture was washed with 1 M HCl
for three times and brine once, dried over anhydrous Na2SO4 and
concentrated to give the residue, the residue was then purified by
column chromatography to afford 5 (7.9 g, 93.2% yield). 1H NMR
DMSO‑d6) d 12.84 (s, 1H), 10.83 (s, 1H), 8.28 (s, 3H), 8.05 (d,
J ¼ 8.8 Hz, 2H), 8.04 (d, J ¼ 8.4 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz, 2H), 7.62
(d, J ¼ 8.4 Hz, 2H), 3.84 (s, 1H), 1.64e1.52 (m, 3H), 0.84 (dd, J ¼ 6.0,
2.4 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 168.77, 164.97, 143.95,
136.86, 132.81, 129.85, 128.87, 128.48, 119.87, 51.25, 23.29, 22.57,
21.40. Mp: 253e255 ꢂC. HRMS: [MþH]þ calcd [C19H22ClN3O4SþH]þ
424.1098, found 424.1095. HPLC: gradient A, 97.7% purity, 8.1 min.
(400 MHz, DMSO‑d6)
d
12.07 (s, 1H), 10.28 (s, 1H), 7.81 (d, J ¼ 8.8 Hz,
2H), 7.65 (d, J ¼ 8.8 Hz, 2H), 7.35e7.45 (m, 5H), 6.99 (d, J ¼ 8.0 Hz,
1H), 5.19 (s, 2H), 3.95 (br. s, 1H), 1.50 (br. s, 1H), 1.23e1.38 (m, 10H),
1.14 (s, 1H), 0.80 (dd, J ¼ 9.2, 6.8 Hz, 6H).
5.1.10. N-(4-(N-L-Leucylsulfamoyl)phenyl)-3-chlorobenzamide (18)
Compound 18 (41.2 mg, 33.2% yield) was prepared from 10
(103.8
(50.5 mg,0.32 mmol) following general procedure 1. 1H NMR
(400 MHz, DMSO‑d6)
mg,
0.27
mmol)
and
3-chlorobenzoic
acid
5.1.5. N-(N-Tert-butoxycarbonyl-L-leucyl)-4-
aminobenzenesulfonamide (6)
To a solution of 5 (4.2 g, 8.00 mmol) in methanol (100 mL), 10%
Pd/C (420 mg) was added. The reaction was stirred at r.t under H2
d
10.89 (s, 1H), 8.36 (s, 3H), 8.08 (d, J ¼ 2.0 Hz,
2H), 8.05 (s,1H), 7.97 (t, J ¼ 9.0 Hz, 3H), 7.71 (ddd, J ¼ 8.0, 2.0, 0.9 Hz,
1H), 7.60 (t, J ¼ 7.9 Hz, 1H), 3.86 (s, 1H), 1.68e1.49 (m, 3H), 0.85 (dd,
J ¼ 6.3, 1.9 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 168.83, 164.58,
5

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
143.75, 136.09, 133.17, 131.77, 130.37, 128.80, 127.58, 126.70,þ119.89,
51.21, 23.31, 22.54, 21.48. Mp: 241e242 ꢂC. HRMS: [MþH] calcd
[C19H22ClN3O4SþH]þ 424.1098, found 424.1097. HPLC: gradient A,
95.6% purity, 8.1 min.
5.1.16. Benzyl(3-(N-(N-tert-butoxycarbonyl-L-leucyl)sulfamoyl)
phenyl) carbamate (25)
To a solution of 24 (2.0 g, 6.53 mmol) and N-Boc-L-leucine (3.0 g,
12.99 mmol), EDCI (5.0 g, 26.08 mmol), DMAP (1.6 g,12.99 mmol) in
50 mL DCM, the mixture was stirred at r.t. overnight. Then reaction
finished, wash with 1 M HCl and organic layer dried by anhydrous
Na2SO4, evaporated to remove solvent, the residue was purified by
column chromatography to afford 25 (1.65 g, 50.0% yield). 1H NMR
5.1.11. N-(4-(N-L-Leucylsulfamoyl)phenyl)-4-nitrobenzamide (19)
Compound 19 (65.8 mg, 55.8% yield) was prepared from 11
(96 mg, 0.25 mmol) and 4-nitrobenzoic acid (50 mg, 0.30 mmol)
following general procedure 1. 1H NMR (400 MHz, DMSO‑d6)
d 11.20
(400 MHz, DMSO‑d6) d 12.30 (s,1H),10.18 (s,1H), 8.18 (s,1H), 7.66 (s,
2H), 7.35e7.50 (m, 7H), 7.00 (d, J ¼ 6.0 Hz, 1H), 5.18 (s, 2H), 3,96 (br.
(s, 1H), 8.39 (s, 3H), 8.37 (s, 2H), 8.28 (d, J ¼ 8.8 Hz, 2H), 8.10 (d,
J ¼ 8.9 Hz, 2H), 7.98 (d, J ¼ 8.8 Hz, 2H), 3.88 (s, 1H), 1.67e1.53 (m,
3H), 0.85 (dd, J ¼ 6.1, 1.9 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
s, 1H), 1.49 (br. s, 1H), 1.32 (s, 9H), 1.11 (s, 2H), 0.80 (s, 6H).
5.1.17. N-(N-Tert-butoxycarbonyl-L-leucyl)-3-aminobenzene-
sulfonamide (26)
To a solution of compound 25 (1.5 g, 2.89 mmol) in 30 mL
methanol and 10% Pd/C (150 mg) was added. The reaction was
stirred at r.t under H2 atomsphere, after 5 h, added 30 mL DCM and
filtered, concentrated to afford compound 26 (1.1 g, 100% yield). 1H
d
168.85, 164.44, 149.31, 143.58, 139.73, 133.14, 129.49, 128.84,
123.44, 120.02, 51.21, 23.31, 22.54, 21.49; Mp: 259e260 ꢂC. HRMS:
[MþH]þ calcd [C19H22N4O6SþH]þ 435.1338, found 435.1334. HPLC:
gradient A, 97.1% purity, 7.7 min.
5.1.12. N-(4-(N-L-Leucylsulfamoyl)phenyl)-3-nitrobenzamide (20)
Compound 20 (84.9 mg, 65.6% yield) was prepared from 12
(105 mg, 0.27 mmol) and 3-nitrobenzoic acid (55 mg, 0.33 mmol).
NMR (400 MHz, DMSO‑d6)
d 11.96 (br. s, 1H), 7.09e7.21 (m, 2H),
6.95e6.99 (m, 2H), 6.79 (d, J ¼ 8.0 Hz, 1H), 5.63 (s, 2H), 3.96e4.02
(m, 1H), 1.48e1.53 (m, 1H), 1.34 (s, 8H), 1.90 (s, 2H), 0.81 (t,
J ¼ 7.7 Hz, 6H).
1H NMR (400 MHz, DMSO‑d6)
d 12.96 (s, 1H), 11.20 (s, 1H), 8.83 (s,
1H), 8.51 (d, J ¼ 7.7 Hz, 1H), 8.47 (d, J ¼ 8.3 Hz, 1H), 8.40 (s, 3H), 8.10
(d, J ¼ 8.7 Hz, 2H), 7.98 (d, J ¼ 8.7 Hz, 2H), 7.86 (t, J ¼ 8.0 Hz, 1H),
3.87 (s, 1H), 1.67e1.54 (m, 3H), 0.86 (d, J ¼ 5.1 Hz, 6H). 13C NMR
5.1.18. General procedure 2 for preparation of 64-81, 83-91
To a solution of organic acids (1.1 eq) in SOCl2, the reaction was
stirred at reflux, then finished, SOCl2 was removed and 5 mL THF
was added and dropwise to compound 26 (1.0 eq). Mixture was
stirred at r.t overnight. Then solvent was removed, H2O and ethyl
acetate was added, organic layer was dried by anhydrous Na2SO4
and concentrated. Next, to a solution of compounds gained above in
ethyl acetate, HCl gas was added for 0.5 h, filtered the solid to afford
products.
(101 MHz, DMSO‑d6) d 168.87, 163.92, 147.66, 143.57, 135.53, 134.37,
130.16,128.83,126.48,122.66þ,120.07, 51.23, 23.32, 22.54, 21.49. Mp:
269e271 ꢂC. HRMS: [MþH] calcd [C19H22N4O6SþH]þ 435.1338,
found 435.1335. HPLC: gradient A, 95.6% purity, 7.7 min.
5.1.13. N-(4-(N-L-Leucylsulfamoyl)phenyl)-4-methylbenzamide
(21)
Compound 21 (73.7 mg, 63.9% yield) was prepared from 13
(100 mg, 0.26 mmol) and 4-methylbenzoic acid (42.7 mg,
0.32 mmol) following general procedure 1. 1H NMR (400 MHz,
5.1.19. N-(3-(N-L-Leucylsulfamoyl)phenyl)benzamide (64)
Compound 64 (21 mg, 25.9% yield) was prepared from 26
(73 mg, 0.19 mmol) and benzoic acid (25 mg, 0.21 mmol) following
DMSO‑d6)
d
10.68 (s, 1H), 8.33 (s, 3H), 8.06 (d, J ¼ 8.8 Hz, 2H), 7.94
(dd, J ¼ 8.6, 1.8 Hz, 4H), 7.36 (d, J ¼ 8.1 Hz, 2H), 3.84 (s, 1H), 2.40 (s,
general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
d 13.10 (s, 1H),
3H), 1.65e1.52 (m, 3H), 0.85 (dd, J ¼ 6.3, 1.6 Hz, 6H). 13C NMR
10.77 (s, 1H), 8.60 (s, 1H), 8.40 (s, 3H), 8.12 (d, J ¼ 8.1 Hz, 1H),
8.07e8.01 (m, 2H), 7.68 (d, J ¼ 7.9 Hz, 1H), 7.62 (td, J ¼ 7.5, 4.1 Hz,
2H), 7.56 (t, J ¼ 7.4 Hz, 2H), 3.86 (s, 1H), 1.66e1.50 (m, 3H), 0.85 (d,
(101 MHz, DMSO‑d6) d 168.84, 165.84, 144.18, 142.11, 132.52, 131.23,
128.88, 128.74, 127.95, 119.72, 51.21, 23.33, 22.54, 21.57, 20.97. Mp:
271e273 ꢂC. HRMS: [MþH]þ calcd [C20H25N3O4SþH]þ 404.1644,
found 404.1641. HPLC: gradient A, 97.1% purity, 7.8 min.
J ¼ 5.5 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 169.58, 166.30,
140.31, 134.66, 132.42, 129.89, 128.91, 128.34, 125.59, 122.98,þ119.32,
51.77, 23.88, 23.04, 22.19. Mp: 252e253 ꢂC. HRMS: [MþH] calcd
[C19H23N3O4SþH]þ 390.1488, found 390.1483. HPLC: gradient A,
99.6% purity, 7.0 min.
5.1.14. N-(4-(N-L-Leucylsulfamoyl)phenyl)-3-methylbenzamide
(22)
Compound 22 (43.6 mg, 33.8% yield) was prepared from 14
(111 mg, 0.293 mmol) and 3-methylbenzoic acid (47.8 mg,
0.35 mmol) following general procedure 1. 1H NMR (400 MHz,
5.1.20. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-fluorobenzamide (65)
Compound 65 (28 mg, 33.2% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-fluorobenzoic acid (29 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
DMSO‑d6)
d
10.74 (s,1H), 8.38 (s, 3H), 8.07 (d, J ¼ 8.8 Hz, 2H), 7.95 (d,
J ¼ 8.8 Hz, 2H), 7.82 (dd, J ¼ 9.8, 4.8 Hz, 2H), 7.44 (d, J ¼ 4.7 Hz, 2H),
3.86 (s, 1H), 2.41 (s, 3H), 1.65e1.53 (m, 3H), 0.85 (d, J ¼ 5.6 Hz, 6H).
d
10.77 (s, 1H), 8.58 (s, 1H), 8.36 (s, 3H), 8.16e8.09 (m, 3H), 7.69 (d,
13C NMR (101 MHz, DMSO‑d6)
d 168.81, 166.14, 144.12, 137.72,
J ¼ 7.9 Hz, 1H), 7.62 (t, J ¼ 7.9 Hz, 1H), 7.40 (t, J ¼ 8.8 Hz, 2H), 3.86 (s,
1H), 1.67e1.51 (m, 3H), 0.90e0.79 (m, 6H). 13C NMR (101 MHz,
134.11, 132.55, 128.77, 128.30, 128.26, 125.04, 119.72, 51.19, 23.32,
22.54, 20.84. Mp: 252e253 ꢂC. HRMS: [MþH]þ calcd
[C20H25N3O4SþH]þ 404.1644, found 404.1644. HPLC: gradient A,
95.7% purity, 7.7 min.
DMSO‑d6)
d 169.58, 165.20, 140.22, 131.09, 129.95, 125.58, 123.06,
119.35, 116.02, 115.80, 51.87, 23.88, 23.08, 22.01. Mp: 262e264 ꢂC.
HRMS: [MþH]þ calcd [C19H22FN3O4SþH]þ 408.1393, found
408.1388. HPLC: gradient A, 97.3% purity, 7.3 min.
5.1.15. Benzyl (3-sulfamoylphenyl)carbamate (24)
To
a
solution of 3-aminobenzenesulfonamide 23 (5.0 g,
5.1.21. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-chlorobenzamide (66)
Compound 66 (30.5 mg, 35.1% yield) was prepared from 26
(73 mg, 0.19 mmol) and benzoic acid (32.5 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
29.07 mmol) and NaHCO3 (3.7 g, 43.61 mmol) in 65 mL H2O/
acetone, CbzCl (5 mL) was added, the mixture was stirred overnight
at r.t. under N2 atomsphere. After the reaction, the mixture was
evaporated and solid was washed by H2O, DCM and ether to afford
d
10.82 (s, 1H), 8.58 (s, 1H), 8.35 (s, 3H), 8.11 (d, J ¼ 8.0 Hz, 1H), 8.07
24 (10.0 g, 100% yield). 1H NMR (400 MHz, DMSO‑d6)
d
10.09 (s, 1H),
(d, J ¼ 8.6 Hz, 2H), 7.69 (d, J ¼ 8.0 Hz, 1H), 7.63 (t, J ¼ 8.6 Hz, 3H),
8.07 (s, 1H), 7.58e7.60 (m, 1H), 7.34e7.49 (m, 9H), 5.18 (s, 2H).
3.86 (s,1H),1.68e1.47 (m, 3H), 0.85 (dd, J ¼ 6.0,1.6 Hz, 6H). 13C NMR
6

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
(101 MHz, DMSO‑d6)
d
165.23, 140.10, 137.30, 133.39, 130.27, 129.98,
1.64e1.48 (m, 3H), 0.85 (dd, J ¼ 6.1, 2.2 Hz, 6H). 13C NMR (101 MHz,
129.03, 123.72, 123.17, 119.37, 51.86, 23.87, 23.10, 21.99. Mp:
270e271 ꢂC. HRMS: [MþH]þ calcd [C19H22ClN4O6SþH]þ 424.1098,
found 424.1101. HPLC: gradient A, 99.3% purity, 9.3 min.
DMSO‑d6) d 169.53, 166.14, 142.55, 140.40, 131.81, 129.93, 129.46,
128.32, 122.89, 119.26, 51.81, 23.87, 23.09, 22.00, 21.52. Mp:
259e260 ꢂC. HRMS: [MþH]þ calcd [C20H25N3O4SþH]þ 404.1644,
found 404.1640. HPLC: gradient A, 99.1% purity, 7.8 min.
5.1.22. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-bromobenzamide
(67)
Compound 67 (27 mg, 28.2% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-bromobenzoic acid (41 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
5.1.27. N-(3-(N-L-Leucylsulfamoyl)phenyl)-3-methylbenzamide
(72)
Compound 72 (29.3 mg, 35.2% yield) was prepared from 26
(73 mg, 0.19 mmol) and 3-methylbenzoic acid (28 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
d
10.82 (s, 1H), 8.58 (s, 1H), 8.35 (s, 3H), 8.11 (d, J ¼ 7.9 Hz, 1H), 7.99
(d, J ¼ 8.5 Hz, 2H), 7.78 (d, J ¼ 8.5 Hz, 2H), 7.69 (d, J ¼ 8.0 Hz, 1H),
7.63 (t, J ¼ 8.0 Hz,1H), 3.86 (s, 1H), 1.68e1.50 (m, 3H), 0.92e0.79 (m,
d
10.65 (s, 1H), 8.58 (s, 1H), 8.08 (d, J ¼ 8.1 Hz, 1H), 7.83 (s, 1H), 7.80
(t, J ¼ 4.3 Hz, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.62 (t, J ¼ 7.9 Hz, 1H), 7.44
(d, J ¼ 5.0 Hz, 2H), 3.83 (s, 1H), 2.42 (s, 3H), 1.64e1.51 (m, 3H),
6H). 13C NMR (101 MHz, DMSO‑d6)
d 165.36, 140.09, 133.77, 131.97,
130.42, 129.97, 126.27, 125.59, 123.17, 119.40, 51.89, 23.88, 23.09,
22.01. Mp: 277e278 ꢂC. HRMS: [MþH]þ calcd [C19H22BrN3O4SþH]þ
468.0593, found 468.0597. HPLC: gradient A, 98.9% purity, 8.3 min.
0.88e0.82 (m, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 166.43, 140.34,
138.27, 134.70, 133.02, 128.85, 128.71, 125.46, 122.96, 119.24þ, 51.83,
23.87, 23.09, 21.99, 21.43. Mp: 266e267 ꢂC. HRMS: [MþH] calcd
[C20H25N3O4SþH]þ 404.1644, found 404.1637. HPLC: gradient A,
98.3% purity, 7.7 min.
5.1.23. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-nitrobenzamide (68)
Compound 68 (25.7 mg, 35.7% yield) was prepared from 26
(59 mg, 0.15 mmol) and 4-nitrobenzoic acid (28 mg, 0.17 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
d
11.12
5.1.28. N-(3-(N-L-Leucylsulfamoyl)phenyl)-2-methylbenzamide
(73)
Compound 73 (25.2 mg, 30.2% yield) was prepared from 26
(73 mg, 0.19 mmol) and 2-methylbenzoic acid (28 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
(s, 1H), 8.60 (t, J ¼ 1.8 Hz, 1H), 8.39 (dd, J ¼ 6.9, 2.0 Hz, 5H),
8.30e8.25 (m, 2H), 8.14 (d, J ¼ 8.8 Hz,1H), 7.75e7.70 (m,1H), 7.65 (t,
J ¼ 8.0 Hz, 1H), 3.87 (s, 1H), 1.68e1.51 (m, 3H), 0.85 (dd, J ¼ 6.1,
1.6 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 169.60, 164.71, 149.83,
140.30, 139.84, 130.00, 124.06, 123.52, 119.51, 51.82, 23.88, 23.09,
22.02. Mp: 271e273 ꢂC. HRMS: [MþH]þ calcd [C19H22N4O6SþH]þ
435.1338, found 435.1336. HPLC: gradient A, 99.9% purity, 8.7 min.
d
10.73 (s, 1H), 8.59 (s, 1H), 8.36 (s, 3H), 7.94 (d, J ¼ 8.3 Hz, 1H), 7.68
(d, J ¼ 7.9 Hz, 1H), 7.61 (t, J ¼ 7.9 Hz, 1H), 7.50 (d, J ¼ 7.6 Hz, 1H), 7.42
(t, J ¼ 7.5 Hz, 1H), 7.33 (d, J ¼ 7.6 Hz, 2H), 3.84 (s, 1H), 2.40 (s, 3H),
1.67e1.54 (m, 3H), 0.85 (d, J ¼ 6.0 Hz, 6H). 13C NMR (101 MHz,
5.1.24. N-(3-(N-L-Leucylsulfamoyl)phenyl)-3-nitrobenzamide (69)
Compound 69 (23.2 mg, 32.2% yield) was prepared from 26
(59 mg, 0.15 mmol) and 3-nitrobenzoic acid (28 mg, 0.17 mmol)
DMSO‑d6) d 169.57, 168.68, 140.32, 139.97, 136.96, 135.88, 131.12,
130.46, 129.98, 127.79, 126.17, 123.08, 118.49, 99.99, 51.90, 23.88,
23.08, 22.04, 19.81. Mp: 248e250 ꢂC. HRMS: [MþH]þ calcd
[C20H25N3O4SþH]þ 404.1644, found 404.1638. HPLC: gradient A,
99.2% purity, 8.6 min.
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
d 11.17
(s,1H), 8.86 (t, J ¼ 1.9 Hz,1H), 8.59 (t, J ¼ 1.8 Hz,1H), 8.52 (dd, J ¼ 7.9,
1.3 Hz, 1H), 8.47 (dd, J ¼ 2.3, 0.9 Hz, 1H), 8.45e8.37 (m, 3H),
8.21e8.15 (m, 1H), 7.86 (t, J ¼ 8.0 Hz, 1H), 7.76e7.70 (m, 1H), 7.66 (t,
J ¼ 8.0 Hz, 1H), 3.90 (s, 1H), 1.68e1.56 (m, 3H), 0.85 (dd, J ¼ 6.1,
5.1.29. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-methoxybenzamide
(74)
Compound 74 (30.6 mg, 37.2% yield) was prepared from 26
(70 mg, 0.18 mmol) and 4-methoxybenzoic acid (30 mg,
0.20 mmol) following general procedure 2. 1H NMR (400 MHz,
1.8 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 169.56, 164.18, 148.23,
139.80, 136.04, 134.84, 130.74, 130.10, 126.98, 125.82, 123.47, 123.11,
119.59, 51.78, 23.88, 23.07, 22.07. Mp: 268e269 ꢂC. HRMS: [MþH]þ
calcd [C19H22N4O6SþH]þ 435.1338, found 435.1328. HPLC: gradient
A, 99.5% purity, 8.6 min.
DMSO‑d6)
d
10.54 (s, 1H), 8.58 (s, 1H), 8.29 (s, 3H), 8.09 (d, J ¼ 7.9 Hz,
1H), 8.03 (d, J ¼ 8.8 Hz, 2H), 7.66 (d, J ¼ 7.9 Hz,1H), 7.60 (t, J ¼ 7.9 Hz,
1H), 7.09 (d, J ¼ 8.9 Hz, 2H), 3.86 (s, 3H), 3.84e3.81 (m, 1H),
1.67e1.44 (m, 3H), 0.89e0.80 (m, 6H). 13C NMR (101 MHz,
5.1.25. N-(3-(N-L-Leucylsulfamoyl)phenyl)-2-nitrobenzamide (70)
Compound 70 (27.3 mg, 37.8% yield) was prepared from 26
(59 mg, 0.15 mmol) and 2-nitrobenzoic acid (28 mg, 0.17 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
DMSO‑d6)
d 169.59, 165.66, 162.69, 140.49, 130.25, 129.86, 126.72,
125.44, 122.74, 119.24, 114.18þ, 55.97, 51.94, 23.88, 23.11, 21.93. Mp:
266e268 ꢂC. HRMS: [MþH] calcd [C20H25N3O5SþH]þ 420.1593,
found 420.1587. HPLC: gradient A, 99.3% purity, 7.2 min.
d
11.20 (s, 1H), 8.47 (s, 1H), 8.40 (s, 3H), 8.18 (d, J ¼ 8.2 Hz, 1H),
7.95e7.87 (m, 2H), 7.85e7.77 (m, 2H), 7.72 (d, J ¼ 8.0 Hz, 1H), 7.64 (t,
J ¼ 8.0 Hz, 1H), 3.86 (s, 1H), 1.69e1.53 (m, 3H), 0.92e0.81 (m, 6H).
13C NMR (101 MHz, DMSO‑d6)
d
169.60, 165.04, 146.87, 139.88,
5.1.30. N-(3-(N-L-Leucylsulfamoyl)phenyl)-3-methoxybenzamide
(75)
Compound 75 (23.2 mg, 28.2% yield) was prepared from 26
(70 mg, 0.18 mmol) and 3-methoxybenzoic acid (30 mg,
0.20 mmol) following general procedure 2. 1H NMR (400 MHz,
134.69, 132.52, 131.78, 130.27, 129.81, 124.85, 123.50, 118.61, 51.84,
23.88, 23.07, 22.03. Mp: 244e246 ꢂC. HRMS: [MþH]þ calcd
[C19H22N4O6SþH]þ 435.1338, found 435.1338. HPLC: gradient A,
99.8% purity, 7.8 min.
DMSO‑d6)
d
10.73 (s, 1H), 8.59 (t, J ¼ 1.8 Hz, 1H), 8.39 (s, 3H), 8.13 (d,
5.1.26. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-methylbenzamide
(71)
J ¼ 8.6 Hz, 1H), 7.71e7.66 (m, 1H), 7.62 (dd, J ¼ 11.3, 4.4 Hz, 2H),
7.59e7.57 (m, 1H), 7.47 (t, J ¼ 7.9 Hz, 1H), 7.21e7.17 (m, 1H), 3.87 (s,
1H), 3.86 (s, 3H), 1.67e1.45 (m, 3H), 0.87e0.82 (m, 6H). 13C NMR
Compound 71 (30.9 mg, 36.8% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-methylbenzoic acid (28 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
(101 MHz, DMSO‑d6) d 169.56,166.03,159.69,140.24, 136.08,130.01,
125.64, 123.02, 120.51, 119.40, 118.20, 113.52, 55.90, 51.85, 23.88,
23.08, 22.04. Mp: 257e258 ꢂC. HRMS: [MþH]þ calcd
[C20H25N3O5SþH]þ 420.1593, found 420.1588. HPLC: gradient A,
98.9% purity, 7.3 min.
d
12.99 (s, 1H), 10.61 (s, 1H), 8.59 (s, 1H), 8.26 (s, 3H), 8.09 (d,
J ¼ 8.2 Hz,1H), 7.93 (d, J ¼ 8.2 Hz, 2H), 7.67 (d, J ¼ 8.0 Hz,1H), 7.62 (t,
J ¼ 7.9 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 2H), 3.83 (s, 1H), 2.40 (s, 3H),
7

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
5.1.31. N-(3-(N-L-Leucylsulfamoyl)phenyl)-3,4-
dimethoxybenzamide (76)
Compound 76 (19.8 mg, 21.5% yield) was prepared from 26
(73 mg, 0.19 mmol) and 3,4-dimethoxybenzoic acid (38 mg,
0.21 mmol) following general procedure 2. 1H NMR (400 MHz,
(d, J ¼ 2.7 Hz,1H), 6.73 (dd, J ¼ 3.5,1.7 Hz,1H), 3.81 (s,1H),1.67e1.51
(m, 3H), 0.85 (dd, J ¼ 6.1, 2.3 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 156.92, 147.44, 146.69, 139.70, 129.93, 125.33, 123.03, 119.25,
115.89, 112.72, 52.01, 23.88, 23.10, 21.91. Mp: 237e239 ꢂC. HRMS:
[MþH]þ calcd [C17H21N3O5SþH]þ 380.1280, found 380.1275. HPLC:
gradient A, 96.3% purity, 6.1 min.
DMSO‑d6)
d
10.57 (s, 1H), 8.54 (s, 1H), 8.34 (s, 3H), 8.13 (d, J ¼ 6.6 Hz,
1H), 7.69 (d, J ¼ 8.5 Hz, 1H), 7.63e7.59 (m, 2H), 7.09 (d, J ¼ 8.1 Hz,
1H), 4.01 (s, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 1.54 (s, 3H), 0.84 (s, 6H).
5.1.36. N-(3-(N-L-Leucylsulfamoyl)phenyl)thiophene-2-
carboxamide (81)
13C NMR (101 MHz, DMSO‑d6)
d 169.57, 165.66, 152.42, 148.79,
140.47, 129.83, 126.66, 125.65, 122.67, 121.90, 119.40, 111.67, 111.45,
56.20, 51.85, 23.89, 23.07, 22.07. Mp: 261e262 ꢂC. HRMS: [MþH]þ
calcd [C21H27N3O6SþH]þ 450.1699, found 450.1698. HPLC: gradient
A, 98.1% purity, 6.9 min.
Compound 81 (23 mg, 28.0% yield) was prepared from 26
(73 mg, 0.19 mmol) and thiophene-2-carboxylic acid (27 mg,
0.21 mmol) following general procedure 2. 1H NMR (400 MHz,
DMSO‑d6)
d
10.82 (s,1H), 8.53 (s,1H), 8.36 (s, 3H), 8.24 (d, J ¼ 3.6 Hz,
1H), 8.14 (d, J ¼ 8.1 Hz,1H), 7.90 (d, J ¼ 5.0 Hz,1H), 7.68 (d, J ¼ 7.9 Hz,
5.1.32. N-(3-(N-L-Leucylsulfamoyl)phenyl)-3,4,5-
trimethoxybenzamide (77)
1H), 7.62 (t, J ¼ 7.9 Hz, 1H), 7.28e7.23 (m, 1H), 3.87 (s, 1H), 1.67e1.51
(m, 3H), 0.89e0.80 (m, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 169.57,
Compound 77 (25.3 mg, 32.7% yield) was prepared from 26
(59 mg, 0.15 mmol) and 3,4,5-trimethoxybenzoic acid (36 mg,
0.17 mmol) following general procedure 2. 1H NMR (400 MHz,
160.74, 139.92, 132.98, 130.42, 130.00, 128.70, 125.46, 122.91,þ119.24,
51.86, 23.88, 23.07, 22.01. Mp: 257e258 ꢂC. HRMS: [MþH] calcd
[C17H21N3O4S2þH]þ 396.1052, found 396.1053. HPLC: gradient A,
98.7% purity, 6.8 min.
DMSO‑d6)
d
10.51 (s, 1H), 8.47 (s, 1H), 8.08 (s, 4H), 7.65 (d, J ¼ 7.4 Hz,
1H), 7.61 (d, J ¼ 8.0 Hz, 1H), 7.32 (s, 2H), 3.87 (s, 6H), 3.73 (s, 3H),
3.65e3.58 (m, 1H), 1.65e1.43 (m, 3H), 0.84 (d, J ¼ 6.0 Hz, 6H). 13
C
5.1.37. Benzyl (3-(N-L-leucylsulfamoyl)phenyl)carbamate (82)
Compound 82 (50 mg, 61.0% yield) was prepared from 25
(100 mg, 0.18 mmol) in the presence of HCl gas. 1H NMR (400 MHz,
NMR (151 MHz, DMSO‑d6)
d 165.62, 153.15, 141.09, 140.13, 129.95,
129.72, 122.96, 119.57, 105.91, 60.63, 56.63, 52.04, 23.88, 23.16,
21.83. Mp: 242e244 ꢂC. HRMS: [MþH]þ calcd [C22H29N3O7SþH]þ
480.1804, found 480.1803. HPLC: gradient A, 99.5% purity, 7.2 min.
DMSO‑d6)
d
10.25 (s, 1H), 8.34 (s, 3H), 8.25 (s, 1H), 7.70 (d, J ¼ 7.7 Hz,
1H), 7.59e7.52 (m, 2H), 7.47e7.32 (m, 5H), 5.19 (s, 2H), 3.83 (s, 1H),
1.68e1.46 (m, 3H), 0.83 (d, J ¼ 6.1 Hz, 6H). 13C NMR (101 MHz,
5.1.33. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-phenoxybenzamide
(78)
Compound 78 (18.7 mg, 23.5% yield) was prepared from 26
(59 mg, 0.15 mmol) and 4-phenoxybenzoic acid (36 mg, 0.17 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
DMSO‑d6) d 153.80, 140.28, 136.84, 130.08, 128.96, 128.62, 128.60,
121.95, 117.06, 66.52, 51.87, 23.85, 23.06, 22.00. Mp: 252e253 ꢂC.
HRMS: [MþH]þ calcd [C20H25N3O5SþH]þ 420.1593, found
420.1587. HPLC: gradient A, 98.0% purity, 8.2 min.
d
10.62 (s, 1H), 8.57 (s, 1H), 8.24 (s, 2H), 8.11e8.03 (m, 3H), 7.67 (d,
5.1.38. N-(3-(N-L-Leucylsulfamoyl)phenyl)-(1,10-biphenyl)-3-
carboxamide (83)
Compound 83 (52 mg, 55.0% yield) was prepared from 26
(73 mg, 0.19 mmol) and (1,10-biphenyl)-3-carboxylic acid (41 mg,
0.21 mmol) following general procedure 2. 1H NMR (400 MHz,
J ¼ 8.0 Hz, 1H), 7.61 (t, J ¼ 8.0 Hz, 1H), 7.48 (t, J ¼ 8.0 Hz, 2H), 7.25 (t,
J ¼ 7.4 Hz, 1H), 7.12 (dd, J ¼ 8.2, 5.0 Hz, 4H), 3.82 (s, 1H), 1.67e1.49
(m, 3H), 0.85 (d, J ¼ 4.8 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d
169.64, 165.51, 160.60, 155.89, 140.34, 130.78, 130.62, 129.88,
129.22, 125.43, 124.97, 122.90, 120.13, 119.29, 117.85, 51.93, 23.88,
23.10, 21.99. Mp: 268e269 ꢂC. HRMS: [MþH]þ calcd
[C25H27N3O5SþH]þ 482.1750, found 482.1752. HPLC: gradient A,
99.1% purity, 9.2 min.
DMSO‑d6)
d
10.81 (s, 1H), 8.59 (s, 1H), 8.29 (s, 4H), 8.14 (d, J ¼ 8.1 Hz,
1H), 8.00 (d, J ¼ 7.9 Hz, 1H), 7.92 (d, J ¼ 7.9 Hz, 1H), 7.83e7.77 (m,
2H), 7.72e7.67 (m, 1H), 7.67e7.59 (m, 2H), 7.53 (t, J ¼ 7.6 Hz, 2H),
7.44 (t, J ¼ 7.3 Hz, 1H), 3.85 (s, 1H), 1.68e1.46 (m, 3H), 0.86 (d,
J ¼ 5.7 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 169.60, 166.23,
5.1.34. N-(3-(N-L-Leucylsulfamoyl)phenyl)-3-phenoxybenzamide
(79)
Compound 79 (15.1 mg, 19.5% yield) was prepared from 26
(59 mg, 0.15 mmol) and 3-phenoxybenzoic acid (36 mg, 0.17 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
140.83,140.28,139.90,135.38,130.63,129.84,129.52,128.38,127.46,
126.46, 125.63, 123.06, 119.þ40, 51.84, 23.89, 23.10, 22.03. Mp:
258e259 ꢂC. HRMS: [MþH] calcd [C25H27N3O4SþH]þ 466.1801,
found 466.1810. HPLC: gradient A, 99.8% purity, 10.8 min.
d
10.69 (s, 1H), 8.55 (s, 1H), 8.24 (s, 2H), 8.06 (d, J ¼ 8.0 Hz, 1H), 7.81
5.1.39. N-(3-(N-L-Leucylsulfamoyl)phenyl)-(1,10-biphenyl)-4-
carboxamide (84)
Compound 84 (50 mg, 53.0% yield) was prepared from 26
(73 mg, 0.19 mmol) and (1,10-biphenyl)-4-carboxylic acid (41 mg,
0.21 mmol) following general procedure 2. 1H NMR (400 MHz,
(d, J ¼ 8.2 Hz, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.65e7.63 (m, 1H),
7.62e7.59 (m,1H), 7.57 (d, J ¼ 7.9 Hz,1H), 7.45 (t, J ¼ 7.8 Hz, 2H), 7.26
(dd, J ¼ 8.2, 2.4 Hz, 1H), 7.21 (t, J ¼ 7.3 Hz, 1H), 7.09 (d, J ¼ 8.4 Hz,
2H), 3.81 (s, 1H), 1.68e1.45 (m, 3H), 0.84 (dd, J ¼ 5.9, 3.9 Hz, 6H). 13
C
NMR (101 MHz, DMSO‑d6)
d
165.46, 157.32, 156.69, 140.05, 136.58,
DMSO‑d6)
d
10.73 (s, 1H), 8.62 (s, 1H), 8.25 (s, 3H), 8.12 (t, J ¼ 7.2 Hz,
130.77, 130.70, 129.92, 124.41, 123.20, 123.16, 122.44, 119.41, 119.37,
118.12, 52.00, 23.87, 23.12, 21.88. Mp: 273e275 ꢂC. HRMS: [MþH]þ
calcd [C25H27N3O5SþH]þ 482.1750, found 482.1751. HPLC: gradient
A, 99.8% purity, 9.1 min.
3H), 7.87 (d, J ¼ 8.5 Hz, 2H), 7.81e7.76 (m, 2H), 7.69 (d, J ¼ 8.0 Hz,
1H), 7.63 (t, J ¼ 7.9 Hz, 1H), 7.53 (t, J ¼ 7.5 Hz, 2H), 7.45 (t, J ¼ 7.3 Hz,
1H), 3.83 (s, 1H), 1.67e1.52 (m, 3H), 0.86 (d, J ¼ 5.4 Hz, 6H). 13C NMR
(101 MHz, DMSO‑d6) d 169.56, 165.95, 143.94, 140.35, 139.47, 133.41,
129.97, 129.57, 129.03, 128.72, 127.42, 127.13, 125.60, 123.03,þ119.32,
51.82, 23.88, 23.09, 22.04. Mp: 279e280 ꢂC. HRMS: [MþH] calcd
[C25H27N3O4SþH]þ 466.1801, found 466.1799. HPLC: gradient A,
99.3% purity, 10.8 min.
5.1.35. N-(3-(N-L-Leucylsulfamoyl)phenyl)furan-2-carboxamide
(80)
Compound 80 (18.9 mg, 23.9% yield) was prepared from 26
(73 mg, 0.19 mmol) and furan-2-carboxylic acid (24 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
5.1.40. N-(3-(N-L-Leucylsulfamoyl)phenyl)-30-methoxy-(1,10-
biphenyl)-3-carboxamide (85)
d
10.64 (s, 1H), 8.52 (s, 1H), 8.25 (s, 3H), 8.07 (d, J ¼ 7.9 Hz, 1H), 7.98
(d, J ¼ 1.0 Hz, 1H), 7.67 (d, J ¼ 8.0 Hz, 1H), 7.60 (t, J ¼ 7.9 Hz, 1H), 7.47
Compound 85 (28 mg, 25.2% yield) was prepared from 26
8

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
(80 mg, 0.21 mmol) and 30-methoxy-(1,10-biphenyl)-3-carboxylic
acid (52 mg, 0.23 mmol) following general procedure 2. 1H NMR
NMR (101 MHz, DMSO‑d6) d 169.58, 165.67, 160.99, 140.53, 130.34,
126.31, 122.68, 119.21, 115.43, 69.99, 60.22, 23.88, 23.07, 22.13,
21.23, 14.55. Mp: 267e268 ꢂC. HRMS: [MþH]þ calcd
[C22H29N3O5SþH]þ 448.1906, found 448.1900. HPLC: gradient A,
98.2% purity, 8.4 min.
(400 MHz, DMSO‑d6)
d 13.13 (s, 1H), 10.95 (s, 1H), 8.63 (s, 1H), 8.46
(s, 3H), 8.33 (s, 1H), 8.20 (d, J ¼ 8.1 Hz, 1H), 8.02 (d, J ¼ 7.8 Hz, 1H),
7.92 (d, J ¼ 7.7 Hz, 1H), 7.71 (d, J ¼ 7.9 Hz, 1H), 7.64 (t, J ¼ 7.9 Hz, 2H),
7.43 (t, J ¼ 8.1 Hz, 1H), 7.39e7.34 (m, 2H), 7.02e6.97 (m, 1H), 3.91 (s,
1H), 3.86 (s, 3H),1.69e1.52 (m, 3H), 0.86 (d, J ¼ 5.9 Hz, 6H). 13C NMR
5.1.45. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-butoxybenzamide
(90)
(101 MHz, DMSO‑d6) d 169.57, 166.23, 160.32, 141.43, 140.71, 140.32,
135.33, 130.64, 129.93, 129.61, 127.65, 126.56, 125.71, 123.03, 119.76,
119.45, 113.96, 113.02, 55.77, 51.84, 23.90, 23.07, 22.13. Mp:
232e235 ꢂC. HRMS: [MþH]þ calcd [C26H29N3O5SþH]þ 496.1906,
found 496.1916. HPLC: gradient A, 99.6% purity, 9.1 min.
Compound 90 (28 mg, 29.5% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-butoxybenzoic acid (41 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
d
10.52 (s, 1H), 8.57 (s, 1H), 8.31 (s, 3H), 8.08 (d, J ¼ 8.0 Hz, 1H), 8.00
(d, J ¼ 8.9 Hz, 2H), 7.64 (d, J ¼ 8.0 Hz,1H), 7.59 (t, J ¼ 7.9 Hz,1H), 7.06
(d, J ¼ 8.9 Hz, 2H), 4.06 (t, J ¼ 6.5 Hz, 2H), 3.84 (s, 1H), 1.76e1.67 (m,
2H), 1.63e1.51 (m, 3H), 1.43 (dt, J ¼ 14.6, 7.4 Hz, 2H), 0.93 (t,
J ¼ 7.4 Hz, 3H), 0.83 (dd, J ¼ 6.0, 1.7 Hz, 6H). 13C NMR (101 MHz,
5.1.41. N-(3-(N-L-Leucylsulfamoyl)phenyl)-40-methoxy-(1,10-
biphenyl)-3-carboxamide (86)
Compound 86 (32 mg, 28.8% yield) was prepared from 26
(80 mg, 0.21 mmol) and 40-methoxy-(1,10-biphenyl)-3-carboxylic
acid (52 mg, 0.23 mmol) following general procedure 2. 1H NMR
DMSO‑d6)
d 169.51, 165.66, 162.14, 140.54, 139.71, 130.30, 129.83,
126.50, 125.50, 122.69, 119.24, 114.56, 67.96, 51.83, 31.09,þ23.88,
23.06, 22.09, 19.16, 14.15. Mp: 253e255 ꢂC. HRMS: [MþH] calcd
[C23H31N3O5SþH]þ 462.2063, found 462.2062. HPLC: gradient A,
99.7% purity, 9.3 min.
(400 MHz, DMSO‑d6) d 10.84 (s,1H), 8.61 (s,1H), 8.37 (s, 3H), 8.25 (s,
1H), 8.16 (d, J ¼ 8.1 Hz,1H), 7.94 (d, J ¼ 7.9 Hz,1H), 7.87 (d, J ¼ 7.9 Hz,
1H), 7.78e7.73 (m, 2H), 7.72e7.68 (m, 1H), 7.62 (dt, J ¼ 11.1, 7.9 Hz,
2H), 7.12e7.06 (m, 2H), 3.88 (s, 1H), 3.83 (s, 3H), 1.68e1.54 (m, 3H),
0.86 (d, J ¼ 5.9 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d
166.33,
5.1.46. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-benzyloxybenzamide
(91)
Compound 91 (37 mg, 36.6% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-(benzyloxy)benzoic acid (48 mg,
0.21 mmol) following general procedure 2. 1H NMR (400 MHz,
159.73,140.49,140.30,135.34,132.20,130.07,129.95,129.60,128.56,
126.72, 125.87, 123.03, 119.41, 114.94, 55.72, 51.89, 23.89, 23.10,
22.03. Mp: 257e258 ꢂC. HRMS: [MþH]þ calcd [C26H29N3O5SþH]þ
496.1906, found 496.1896. HPLC: gradient A, 99.3% purity, 9.1 min.
DMSO‑d6)
d
10.56 (s, 1H), 8.56 (s, 1H), 8.36 (s, 3H), 8.09 (d, J ¼ 7.7 Hz,
5.1.42. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-hydroxybenzamide
(87)
Compound 87 (15.2 mg, 18.1% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-hydroxybenzoic acid (29 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
1H), 8.02 (d, J ¼ 8.7 Hz, 2H), 7.64 (d, J ¼ 8.0 Hz,1H), 7.58 (t, J ¼ 7.9 Hz,
1H), 7.47 (d, J ¼ 7.3 Hz, 2H), 7.40 (t, J ¼ 7.3 Hz, 2H), 7.34 (t, J ¼ 7.1 Hz,
1H), 7.15 (d, J ¼ 8.7 Hz, 2H), 5.21 (s, 2H), 3.84 (s, 1H), 1.63e1.50 (m,
3H), 0.83 (d, J ¼ 5.5 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 169.55,
165.64, 161.72, 140.50, 137.09, 130.27, 129.85, 128.96, 128.46, 128.25,
126.92, 122.74, 119.23, 115.01þ, 69.92, 51.87, 23.88, 23.08, 22.02. Mp:
243e246 ꢂC. HRMS: [MþH] calcd [C26H29N3O5SþH]þ 496.1906,
found 496.1906. HPLC: gradient A, 98.6% purity, 9.2 min.
d
12.86 (s, 1H), 10.40 (s, 1H), 10.22 (s, 1H), 8.56 (s, 1H), 8.22 (s, 3H),
8.05 (d, J ¼ 7.9 Hz,1H), 7.91 (d, J ¼ 8.7 Hz, 2H), 7.64 (d, J ¼ 7.9 Hz,1H),
7.58 (t, J ¼ 7.9 Hz, 1H), 6.90 (d, J ¼ 8.7 Hz, 2H), 3.81 (s, 1H), 1.66e1.49
(m, 3H), 0.85 (dd, J ¼ 6.1, 1.6 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6)
d
165.85, 140.59, 130.35, 125.34, 125.11, 122.56, 119.17, 115.48, 70.26,
5.2. TbLeuRS assay [17]
23.88, 23.10, 21.98. Mp: 216e217 ꢂC. HRMS: [MþH]þ calcd
[C19H23N3O5SþH]þ 406.1437, found 406.1429. HPLC: gradient A,
99.9% purity, 6.2 min.
T. brucei LeuRS was cloned and expressed using pET21a vector in
E. coli strain BL21(DE3)-RIPL. The concentration of LeuRS with the
CPM value of 1000 was defined as 1 activity unit (1 U). Brewer’s
yeast tRNA was purchased from Roche.
5.1.43. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-ethoxybenzamide
(88)
LeuRS inhibition IC50 measurement was performed in 70
mL
Compound 88 (18.8 mg, 21.0% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-ethoxybenzoic acid (35 mg, 0.21 mmol)
following general procedure 2. 1H NMR (400 MHz, DMSO‑d6)
reaction mixtures containing 50 mM HEPES-KOH (pH 7.8), 5 mM
MgCl2 and 45 mM KCl, 1 mM DTT, 0.02%(w/v) BSA, 0.4 mg/mL
brewer’s yeast tRNA (Roche), 1 U T. brucei LeuRS, 3 m
Ci/mL [14C]
d
10.55 (s, 1H), 8.56 (s, 1H), 8.37 (s, 3H), 8.09 (d, J ¼ 7.5 Hz, 1H), 8.01
leucine (318 mCi/mmol, PerkinElmer), ddH2O, and compounds at
different concentrations. The mixture was pre-incubated at 37 ꢂC
without ATP for 20 min, then added 4 mM ATP at 37 ꢂC for 15 min.
(d, J ¼ 8.7 Hz, 2H), 7.64 (d, J ¼ 7.9 Hz, 1H), 7.58 (t, J ¼ 7.9 Hz,1H), 7.04
(d, J ¼ 8.9 Hz, 2H), 4.11 (q, J ¼ 7.0 Hz, 2H), 3.84 (s, 1H), 1.65e1.45 (m,
3H), 1.35 (t, J ¼ 7.0 Hz, 3H), 0.83 (d, J ¼ 5.2 Hz, 6H). 13C NMR
Then three 20
mL aliquots were spotted on 3 mm filter paper
(101 MHz, DMSO‑d6)
d
165.67, 161.97, 130.29, 129.85, 126.53, 125.49,
(Whatman), washed 3 times in 5% trichloroacetic acid and 3 times
in alcohol. Filter papers were then dried in oven under 85 ꢂC for
20 min and the precipitated [14C] leucine tRNALeu were quantified
by liquid scintillation counting using a Beckman Coulter LS 6500
liquid scintillation counter. Data in duplicate was averaged to
generate an IC50 value using GraphPad 5 for each test compound
122.70, 119.23, 114.54, 63.92,þ51.85, 23.88, 23.07, 22.05, 15.00. Mp:
271e273 ꢂC. HRMS: [MþH] calcd [C21H27N3O5SþH]þ 434.1750,
found 434.1744. HPLC: gradient A, 99.9% purity, 7.8 min.
5.1.44. N-(3-(N-L-Leucylsulfamoyl)phenyl)-4-
isopropoxybenzamide (89)
and standard errors are within 0.05 mM.
Compound 89 (15.6 mg, 17.0% yield) was prepared from 26
(73 mg, 0.19 mmol) and 4-isopropoxybenzoic acid (38 mg,
0.21 mmol) following general procedure 2. 1H NMR (400 MHz,
5.3. In vitro T. brucei assay
DMSO‑d6)
d
10.50 (s, 1H), 8.55 (s, 1H), 8.28 (s, 3H), 8.07 (d, J ¼ 7.7 Hz,
All in vitro anti-parasite assays were conducted with the
bloodstream-form Trypanosoma brucei brucei 221 strain. Parasites
were cultured in T-25 vented cap flasks and kept in humidified
incubators at 37 ꢂC and 5% CO2. The parasite culture media was
1H), 7.98 (d, J ¼ 8.7 Hz, 2H), 7.63 (d, J ¼ 7.8 Hz,1H), 7.57 (t, J ¼ 7.9 Hz,
1H), 7.04 (d, J ¼ 8.7 Hz, 2H), 4.75 (dt, J ¼ 12.1, 6.1 Hz,1H), 3.81 (s,1H),
1.63e1.51 (m, 3H),1.29 (d, J ¼ 6.0 Hz, 6H), 0.83 (d, J ¼ 5.8 Hz, 6H). 13
C
9

Z. Li, W. Xin, Q. Wang et al.
European Journal of Medicinal Chemistry 217 (2021) 113319
complete HMI-11 medium [18]. To ensure log growth phase, try-
References
panosomes were sub-cultured at appropriate dilutions every 2e3
days. Log phase cultures were diluted 1:10 in HMI-11 and 10 mL was
counted using hemocytometer to determine parasite concentra-
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2-fold working concentration for assay. Compounds to be tested
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The authors declare that they have no known competing
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Acknowledgments
We thank National Key Research and Development Program of
China (2017YFA0505200), National Science Foundation of China
(81573264),and E-Institutes of Shanghai Universities (EISU)
Chemical Biology Division for financial support of this work.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113319.
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