The optimization and characterization of functionalized sulfonamides derived from sulfaphenazole against Mycobacterium tuberculosis with reduced CYP 2C9 inhibition

Article abstract of DOI:10.1016/j.bmcl.2021.127924

In this study, a series of sulfonamide compounds was designed and synthesized through the systematic optimization of the antibacterial agent sulfaphenazole for the treatment of Mycobacterium tuberculosis (M. tuberculosis). Preliminary results indicate that the 4-aminobenzenesulfonamide moiety plays a key role in maintaining antimycobacterial activity. Compounds 10c, 10d, 10f and 10i through the optimization on phenyl ring at the R² site on the pyrazole displayed promising antimycobacterial activity paired with low cytotoxicity. In particular, compound 10d displayed good activity (MIC = 5.69 μg/mL) with low inhibition of CYP 2C9 (IC₅₀ > 10 μM), consequently low potential risk of drug-drug interaction. These promising results provide new insight into the combination regimen using sulfonamide as one component for the treatment of M. tuberculosis.

Full text of DOI:10.1016/j.bmcl.2021.127924

Bioorg. Med. Chem. Lett. 40 (2021) 127924
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The optimization and characterization of functionalized sulfonamides
derived from sulfaphenazole against Mycobacterium tuberculosis with
reduced CYP 2C9 inhibition
,
,
,*
Hui Chenb, Bin Wangc, Peng Lia, Hong Yanb, Gang Lia *, Haihong Huanga *, Yu Luc
a Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation & Chinese Academy of Medical Sciences Key Laboratory of Anti-DR TB Innovative
Drug Research, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, PR China
b Beijing Key Laboratory of Environmental and Viral Oncology, College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100124, PR China
c Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing
Chest Hospital, Capital Medical University, Beijing 101149, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, a series of sulfonamide compounds was designed and synthesized through the systematic opti-
mization of the antibacterial agent sulfaphenazole for the treatment of Mycobacterium tuberculosis
(M. tuberculosis). Preliminary results indicate that the 4-aminobenzenesulfonamide moiety plays a key role in
maintaining antimycobacterial activity. Compounds 10c, 10d, 10f and 10i through the optimization on phenyl
ring at the R2 site on the pyrazole displayed promising antimycobacterial activity paired with low cytotoxicity. In
Sulfonamide compounds
Optimization
Antimycobacterial activity
CYP 2C9
Cytotoxicity
particular, compound 10d displayed good activity (MIC = 5.69
10
into the combination regimen using sulfonamide as one component for the treatment of M. tuberculosis.
μ
g/mL) with low inhibition of CYP 2C9 (IC50
>
μ
M), consequently low potential risk of drug-drug interaction. These promising results provide new insight
Tuberculosis (TB) is a chronic disease that results from infection with
M. tuberculosis. TB is one of the top 10 causes of death worldwide, and
the leading cause of mortality stemming from a single infectious path-
ogen.1 In addition, the emergence of multidrug-resistant (MDR) TB and
extensively drug-resistant (XDR) TB have worsened the situation,
particularly in low-income countries.2,3 Renewed efforts have therefore
been focused on developing new antibiotics against TB.4 Meanwhile,
repurposing of efficacious antibiotics has emerged as an effective way to
discover new pharmacophores, resulting in the discovery of potentially
new drugs against M. tuberculosis.5
for bacterial viability. Furthermore, the combination of SMX and TMP
(SXT) has emerged as a potential treatment regimen to combat drug-
resistant TB.14 Notably, SXT has been successfully used to treat iso-
lated cases of drug-resistant tuberculosis,15 with further research
showing that several other related sulphonamide derivatives also dis-
played pronounced anti-TB effects.7,16,17 All these results encouraged us
to further evaluate sulfonamide derivatives against M. tuberculosis.
The initial hit compound, SPA, discovered through screening our in-
house library of clinically relevant sulfonamide compounds, displayed
good in vitro efficacy against M. tuberculosis H37Rv (Fig. 1). However,
SPA is also a selective, competitive inhibitor of CYP 2C9, which can
potentially lead to drug-drug interactions.18–20 In this work, a series of
sulfonamide compounds derived from SPA was designed and synthe-
sized to establish and explore structure–activity relationships (SARs),
with the aim to design-out the unwanted CYP 2C9 activity. Systematic
optimization led to compound 10d which displayed good anti-
mycobacterial activity and, importantly, a reduced CYP 2C9 inhibitory
profile.
Sulfonamides have been intensively investigated since their discov-
ery as the first potent antibacterial agents, and notably, are still in use
today.6–10 Sulfonamides generally act as structural analogues of 4-ami-
nobenzoic acid and therefore inhibit dihydropteroate synthase in
bacteria.11–13 The 4-aminobenzenesulfonamides with antibacterial ac-
tivity are represented as sulfamethoxazole (SMX), sulfadiazine (SD),
sulfisoxazole (SIZ) and sulfaphenazole (SPA).
The antibacterial activity observed upon combining SMX with
trimethoprim (TMP) is a result of the sequential inhibition of the
biosynthesis of tetrahydrofolic acid within bacteria, which is essential
The synthesis of compounds 5a-i, 10a-k, 12a-c, 16a-f, 17 and 18a-g
is outlined in Schemes 1–4. Sulfonylation of commercially available 5-
* Corresponding authors.
E-mail addresses: ligang@imm.ac.cn (G. Li), joyce@imm.ac.cn (H. Huang), luyu4876@hotmail.com (Y. Lu).
https://doi.org/10.1016/j.bmcl.2021.127924
Received 22 December 2020; Received in revised form 8 February 2021; Accepted 25 February 2021
Available online 9 March 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127924
Fig. 1. Structure optimization through in-house screening.
amino-1-phenylpyrazole 1 with various sulfonyl chlorides afforded in-
termediates 2 and 3c-f, respectively. The desired products 5a-b were
obtained from 2 via cross-coupling reactions.21,22 Hydrogenation of 3c-f
led to the desired products 5c-f. Intermediate 4 was formed by the sul-
fonylation of 1 followed by simple hydrolysis. The condensation re-
actions of 4 with various alicyclic amines afforded the corresponding
products 5g-h in the presence of EDCI (N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide hydrochloride) and HOBt (1-hydroxybenzotriazole)
at room temperature. The condensation of 4 with Boc-piperazine fol-
lowed by deprotection of Boc group provided the target compound 5i
(Scheme 1).
Ethyl (E)-2-cyano-3-ethoxyacrylate 6 was treated with hydrazines
7a-k to form the pyrazole intermediates 8a-k. Decarboxylation of 8a-k
in the presence of phosphoric acid afforded the intermediates 9a-k,23
and subsequent sulfonylation with 4-acetamidobenzene sulfonyl chlo-
ride followed by the hydrolysis of acetyl group provided the target
compounds 10a-k. Intermediate 9e was subjected to Suzuki cross-
coupling conditions with the corresponding phenylboronic acids to
afford the desired biphenyl intermediates 11a-c. The subsequent sulfo-
nylation of 11a-c followed by hydrolysis of the acetyl group generated
the target compounds 12a-c (Scheme 2).
Sulfonylation of intermediates 13a-f with 4-acetamidobenzene sul-
fonyl chloride 14 afforded intermediates 15a-f, which following hy-
drolysis provided the desired compounds 16a-f. Methylation of 16a
(SPA) with methyl iodide generated the target compound 17 (Scheme
3).
Scheme 1. Synthesis of the target compounds 5a-i. Reagents and conditions:
(a) 4-Bromobenzenesulfonyl chloride, pyridine, reflux; (b) Arylsulfonyl chlo-
ride, pyridine, reflux; (c) Methyl 4-(chlorosulfonyl)benzoate, pyridine, reflux;
(d) 2 N NaOH aqueous solution, reflux; (e) Methylamine, Cu, microwave (MW),
110℃ for 5a; piperidine, Pd2(dba)3, rac-BINAP, K3PO4⋅H2O, DMF, reflux for 5b;
(f) H2, Pd/C, MeOH, rt; (g) Alicyclic amines, EDCI, HOBt, Et3N, DMF, rt for 5g,
5h and N-Boc 5i, then TFA, DCM, rt for 5i.
Various aryl amines were treated with 4-acetamidobenzene sulfonyl
chloride 14 followed by the hydrolysis of acetyl group to produce the
target compounds 18a-g (Scheme 4).
The synthesized library was screened for activity against
M. tuberculosis H37Rv and selected compounds were tested against an
XDR-TB isolated clinical strain. The minimum inhibitory concentration
2

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127924
Scheme 2. Synthesis of the target compounds 10a-k and 12a-c. Reagents and conditions: (a) EtOH, reflux; (b) H3PO4, 170 ℃; (c) 4-Acetamidobenzene sulfonyl
chloride, pyridine, reflux; (d) 2 N NaOH aqueous solution, reflux; (e) Phenylboronic acids, Na2CO3, PdCl2(dppf), dioxane/H2O, reflux.
Scheme 3. Synthesis of the target compounds 16a-f and 17. Reagents and conditions: (a) 4-Acetamidobenzene sulfonyl chloride, pyridine, reflux; (b) 2 N NaOH
aqueous solution, reflux. (c) CH3I, Cs2CO3, MeCN, rt.
(MIC) was defined as the lowest concentration effecting a reduction in
fluorescence of ≥90% relative to the mean of replicate bacterium-only
controls.24 Tables 1–3 summarize the MIC data for all synthesized sul-
fonamide derivatives. Selected compounds were further tested activity
against one drug-resistant TB strain and mammalian cell cytotoxicity
using Vero cells measured at a concentration inhibiting 50% cell growth
(IC50) as compared to a no-treatment control in Table 5. Sulfaphenazole
(SPA) and isoniazid (INH) were used as the reference compounds.
Our initial MIC-based SAR studies against M. tuberculosis around the
hit compound SPA focused on exploring the 4-aminobenzenesulfona-
mide moiety on antitubercular activity. As shown in Table 1, the
replacement of the terminal amino group with the secondary amine in
5a as well as the bulky cyclic amine in 5b resulted in the loss of potency.
Compared to the parent SPA, an amino group at the meta-position dis-
played a markedly decreased potency against M. tuberculosis (5c), while
the introduction of a methyl group at the R4 site (17) also resulted in a
drop in potency. Furthermore, a phenyl group was not tolerated on the
4-aminobenzenesulfonamide moiety (5d). The introduction of elec-
tronically dissimilar substituents, via the installation of a methoxy or
fluoro substituent on the phenyl ring, led to inactive compounds (5e and
5f). To improve the potency and avoid CYP 2C9 inhibition by blocking
the NH2 group binding to CYP 2C9 iron, we attached benzamide frag-
ment with specific lipophilicity at the R1 site to produce compounds 5g-i
and to our delight, compound 5g showed moderate antimycobacterial
activity (MIC = 30.15
μg/mL). A bioisosteric replacement strategy to
replace the methylene (5g) with sulfur or nitrogen heteroatom (5h and
3

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127924
the parent SPA. Introducing a fluoro substituent onto the phenyl ring
afforded compound 10f with a MIC of 7.94 μg/mL, which showed better
potency than that of 10e with a bromo substituent at the same position.
Nitro substituted compound 10g displayed moderate activity with a MIC
of 10.31 μg/mL, while the introduction of a trifluoromethyl group (10h)
led to the loss of potency. We then turned our attention onto the position
of substitutions, with compound 10i, bearing a meta-fluoro group,
demonstrating equal activity to that of para-position 10f (MIC = 7.93 vs
7.94
μg/mL). In addition, compound 10j with meta-methyl group,
demonstrated moderate activity (MIC = 12.22 μg/mL) while the intro-
duction of the larger dimethyl group (10k) led to the loss of potency. The
addition of a simple phenyl group caused an obvious decrease in activity
(12a-c). Finally, we observed that introduction of a methylene bridge
between the pyrazole and phenyl ring (16b) was not tolerated.
Since the substitution at the R2 site has been investigated in detail,
our attention shifted to exploring substitution at the R3 site. As shown in
Table 2, a methyl substituent at the pyrazole ring (16c), provided a
moderately active derivative with a MIC of 15.38 μg/mL. The intro-
Scheme 4. Synthesis of the target compounds 18a-g. Reagents and conditions:
(a) 4-Acetamidobenzene sulfonyl chloride, pyridine, reflux; (b) 2 N NaOH
aqueous solution, reflux.
duction of larger substitutions, as exemplified by ethyl, tert-butyl and
phenyl groups, led to inactive compounds (16d-f).
We then turned our attention onto replacing the phenyl pyrazole
scaffold with various aromatic rings via a truncated scaffold hopping
strategy. As shown in Table 3, compound benzothiazole 18a demon-
5i) caused a loss of potency. In addition, the acetylation of the amino
group (15a) also led to a drop in potency. The above results clearly
indicate that the 4-aminobenzenesulfonamide moiety plays a key role in
maintaining antimycobacterial activity.
strated moderate activity (MIC = 14.26 μg/mL), while altering the po-
sition in which the phenyl ring is attached (18b) led to the loss of
potency. The replacement of benzothiazole (18a) with benzoxazole
(18c) resulted in a loss of potency. Introduction of phenyl groups
derived from 18a, such as 18d and 18e, led to an obvious decrease in
activity. Furthermore, addition of a phenyl ring between the pyrazole
and sulfonamide was not tolerated (18f). Replacing the pyrazole motif in
sulfaphenazole with thiophene (18g) caused a loss of antimycobacterial
activity. The above results indicated that the pyrazole core directly
connected with 4-aminobenzenesulfonamide moiety is favor of anti-TB
potency.
Keeping the 4-aminobenzenesulfonamide moiety as the side chain,
we then evaluated the effects of various substituents at the R2 site. As
shown in Table 2, the replacement of benzene in SPA with cyclohexane
(10a) and cyclopentane (10b) led to an obvious decrease in activity. We
then introduced various substituents onto the phenyl ring to more
deeply explore the SAR. Compounds bearing methyl and methoxy
groups on the phenyl ring (10c and 10d) showed good anti-
mycobacterial activities (MIC = 7.70 and 7.92 μg/mL), and similar to
Table 1
SAR of sulfonamide compounds at R1 and R4 sites.
Compds.
R1
R4
H
MICa
(
μg/mL)
Compds.
R1
R4
H
MICa
(μg/mL)
5a
>32
5e
>32
5b
H
H
>32
>32
5f
H
H
>32
15a
5g
30.15
5c
17
H
>32
>32
5h
5i
H
H
>32
>32
CH3
5d
H
>32
SPA
H
5.51
a
MIC against M. tuberculosis H37Rv.
4

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127924
Table 2
SAR of sulfonamide compounds at R2 and R3 sites.
Compds.
R2
R3
H
MICa
(μg/mL)
Compds.
R2
R3
H
MICa
(μg/mL)
10a
>32
10k
>32
10b
H
>32
12a
H
>32
10c
H
7.70
12b
H
31.04
10d
10e
H
H
7.92
12c
16b
H
H
>32
>32
14.54
10f
H
H
7.94
16c
16d
CH3
15.38
10g
10.31
CH2CH3
>32
10h
H
>32
16e
C(CH3)3
>32
10i
10j
H
H
7.93
16f
>32
12.22
SPA
H
5.51
a
MIC against M. tuberculosis H37Rv.
Table 3
SAR of sulfonamide compounds at R5 site.
Compds.
R5
MICa
(μg/mL)
Compds.
R5
MICa
(μg/mL)
18a
14.26
18e
>32
18b
18c
>32
>32
18f
>32
>32
18g
SPA
18d
>32
5.51
a
MIC against M. tuberculosis H37Rv.
5

H. Chen et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127924
Table 4
Table 5
CYP 2C9 inhibition of the selected compounds.
In vitro activity of selected compounds against drug-resistant TB strain and Vero
cells.
Compds.
Compds.
CYP 2C9
Inhibition (%)a
MIC (
μ
g/mL)
Vero IC50 (μg/mL)
IC50 (μM)
H37Rv
14862a
5g
0.2
52.3
9.1
–
10c
10d
10f
12b
16c
18a
SPA
–
11.5
–
10c
10d
10f
7.33
5.69
6.88
6.87
0.02
11.93
10.62
10.88
12.59
>10
>64
>64
>64
>64
–
45.7
2.2
–
SPA
45.0
4.8
–
INH
–
a
Resistance to isoniazid (INH), streptomycin (SM), rifampicin (RFP), etham-
butol (EMB), paza-aminosalicylate (PAS), prothionamide (1321) and capreo-
mycin (CPM).
48.3
0.63
a
Test concentration: 0.5 µM.
The selected sulfonamide compounds with anti-TB potency and the
reference compound SPA were further evaluated for inhibition of CYP
2C9 (Table 4). The replacement of the free amino group with a benza-
mide fragment (5g) led to a distinct loss of inhibition of CYP 2C9 as
expected through negating the amine-metal binding event between the
active compound and CYP 2C9. The compounds bearing relatively small
substituents at R2 and R3 sites remained similar inhibitory activity
against CYP 2C9 (10c, 10f, 16c) compared to SPA. To our delight, the
involvement of methoxy (10d) or methylphenyl (12b) groups at R2 site
resulted in an obvious decrease of the inhibitory effects toward CYP 2C9
with <10% inhibition at the tested concentration of 0.5 µM. The intro-
duction of benzothiazole at R5 site (18a) also led to reduced inhibition of
CYP 2C9. In particular, compound 10d with good anti-TB activity
showed a 18-fold reduction of CYP 2C9 inhibition relative to SPA (IC50
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by the CAMS Innovation Fund for Medical
Sciences (CAMS-2016-I2M-1-010).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127924.
= 11.5 vs 0.63 μM). We therefore predict that compound 10d will lead to
low potential risk of drug-drug interactions.
Representative sulfonamide compounds 10c, 10d, 10f, SPA and the
reference compound INH were further tested for activity against XDR-TB
as well as for cytotoxicity against Vero cells. As shown in Table 5, these
compounds displayed equivalent potency against an XDR-TB isolated
clinical strain compared to SPA. Additionally, compounds 10c, 10d, 10f
and SPA displayed very low cytotoxicity against Vero cells (IC50 > 64
References
1 World Health Organization, Global Tuberculosis Report; Geneva, 2020.
2 Dheda K, Gumbo T, Maartens G, et al. Lancet Resp Med. 2017;5:291–360.
3 Singh R, Dwivedi SP, Gaharwar US, et al. J Appl Microbiol. 2020;128:1547–1567.
4 Lambert N, Abdalla AE, Duan X, et al. J Drug Target. 2017;25:296–306.
5 Bahuguna A, Rawat DS. Med Res Rev. 2020;40:263–292.
6 Bentley R. J Ind Microbiol Biot. 2009;36:775–786.
μ
g/mL). The results indicated that sulfonamide compounds with good
´
ˇ
´
´
7 Kratký M, Vinsova J, Volkova M, et al. Eur J Med Chem. 2012;50:433–440.
8 Naaz F, Srivastava R, Singh A, et al. Bioorg Med Chem. 2018;26:3414–3428.
9 Wilkinson BL, Bornaghi LF, Wright AD, et al. Bioorg Med Chem Lett. 2007;17:
1355–1357.
anti-TB activity have the potential as the component in a combination
regimen for the treatment of TB including MDR or XDR-TB.
In summary, we report the design, synthesis and SAR of a series of
sulfonamide compounds that have the potential to act as anti-TB agents.
All synthesized compounds were evaluated for their in vitro activities
against M. tuberculosis H37Rv and the established SAR indicated that the
4-aminobenzenesulfonamide moiety was most favorable for anti-
mycobacterial activity. We observed that large structural modification
of the sulfaphenazole core caused a significant loss of potency. More
nuanced optimization, through substituted phenyl derivatives at the R2
site on the pyrazole fragment resulted in the identification of compounds
10c, 10d, 10f and 10i with promising antimycobacterial activity.
Moreover, compound 10d containing a 4-methoxy phenyl fragment,
demonstrated good in vitro activity against both drug-susceptible and
drug-resistant tuberculosis, low cytotoxicity as well as low inhibition of
CYP 2C9. These promising results laid the foundation for applying sul-
fonamide as the component in the combination regimen for the treat-
ment of M. tuberculosis.
10 Sui YF, Li D, Wang J, et al. Bioorg Med Chem Lett. 2020;30, 126982.
11 Brown GM. J Biol Chem. 1962;237:536–540.
˝
12 Sweberg G, Castensson S, Skold O. J Bacteriol. 1979;137:129–136.
13 Bock L, Miller GH, Schaper KJ, et al. J Med Chem. 1974;17:23–28.
14 Forgacs P, Wengenack NL, Hall L, et al. Antimicrob Agents Ch. 2009;53:4789–4793.
15 Palomino JC, Martin A. Future Microbiol. 2016;11:539–547.
16 Krasavin M, Lukin A, Vedekhina T, et al. Eur J Med Chem. 2019;166:125–135.
17 Santos JL, Yamasaki PR, Chin CM, et al. Bioorg Med Chem. 2009;17:3795–3799.
18 Ha-Duong NT, Dijols S, Marques-Soares C, et al. J Med Chem. 2001;44:3622–3631.
19 Mancy A, Dijols S, Poli S, et al. Biochemistry. 1996;35:16205–16212.
20 Ha-Duong NT, Marques-Soares C, Dijols S, et al. Arch Biochem Biophys. 2001;394:
189–200.
21 Jiao J, Zhang XR, Chang NH, et al. J Org Chem. 2011;76:1180–1183.
22 Yempalla KR, Munagala G, Singh S, et al. ACS Med Chem Lett. 2015;6:1041–1046.
23 Silva TB, Bernardino AMR, Ferreira MLG, et al. Bioorg Med Chem. 2016;24:
4492–4498.
24 Li P, Wang B, Zhang X, et al. Eur J Med Chem. 2018;160:157–170.
6