Sulfonamide-Derived Four-Component Molecular Hybrids as Novel DNA-Targeting Membrane Active Potentiators against Clinical Escherichia coli

Article abstract of DOI:10.1021/acs.molpharmaceut.8b01021

Novel sulfonamide-based four-component molecular hybrids as potential DNA-targeting antimicrobial agents were developed from marketed acetanilide through convenient procedures. Biological assays indicated that a few of the target compounds showed significant inhibitory efficiencies toward the tested bacteria and fungi. Noticeably, metronidazole hybrid 6a exhibited a lower minimum inhibitory concentration (MIC) value of 0.019 mM against clinical drug-resistant Escherichia coli (E. coli), which showed to be 84-fold more active than clinical norfloxacin and had no obvious toxicity toward human breast cancer MCF-7 cells. Synergistic combinations of compound 6a with clinical antibacterial or antifungal drugs could improve the antimicrobial efficiency. Further molecular modeling indicated that the active molecule 6a could bind with THR-199, HIS-64, and GLN-92 residues of human carbonic anhydrase isozyme II through hydrogen bonds and was also able to insert into base-pairs of the DNA hexamer duplex by forming hydrogen bonds. The preliminary exploration of the antibacterial mechanism suggested that compound 6a was capable of disturbing the E. coli membrane effectively and intercalating into clinical resistant E. coli bacterial DNA through noncovalent bonds to form a supramolecular complex, thus exerting its powerful antimicrobial activity. This might suggest a great possibility for hybrid 6a to be a DNA-targeting membrane active potentiator against clinical drug-resistant E. coli.

Full text of DOI:10.1021/acs.molpharmaceut.8b01021

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Article
Sulfonamide-derived Four-component Molecular Hybrids as Novel DNA-
targeting Membrane Active Potentiators against Clinical Escherichia coli
Yuan-Yuan Hu, Rammohan R. Yadav Bheemanaboina, Narsaiah Battini, and Cheng-He Zhou
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01021 • Publication Date (Web): 14 Jan 2019
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Sulfonamide-derived Four-component Molecular
Hybrids as Novel DNA-targeting Membrane Active
Potentiators against Clinical Escherichia coli
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Yuan-Yuan Hu, Rammohan R. Yadav Bheemanaboina, Narsaiah Battini, Cheng-He Zhou
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of
Chongqing Municipality, School of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, China
TOC Graphics
O
O2N
HO
HN
CH3
O
S
6a
N
Im =
H2N
N
N
Im
N
O
H3C
MIC = 0.019 mM against drug-resistant E. coli strains.
Intercalation into DNA to block DNA replication. Efficient membrane permeability.
Low cytotoxicity to mammalian cells. Stronger antibacterial ability in drug combination use.
6a
Compound
6a
Compound
Culture
Isolation
6a
Drug-resistant
Escherichia
coli
6a
Intercalating into
DNA
Disturbing
E. coli
membrane
E. coli
DNA
membrane
E. coli
E. coli
ABSTRACT
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Novel sulfonamide-based four-component molecular hybrids as potentially DNA-targeting
antimicrobial agents were developed from marketed acetanilide through convenient procedures.
Biological assays indicated that a few of the target compounds showed significant inhibitory
efficiencies toward the tested bacteria and fungi. Noticeably, metronidazole hybrid 6a exhibited
lower minimum inhibitory concentrations (MIC) value of 0.019 mM against clinical drug-
resistant Escherichia coli (E. coli), which showed 84-fold more active than clinical norfloxacin
and no obvious toxicity toward human breast cancer MCF-7 cells. Synergistic combinations of
compound 6a with clinical antibacterial or antifungal drugs could improve the antimicrobial
efficiency. Further molecular modeling indicated that the active molecule 6a could bind with
THR-199, HIS-64 and GLN-92 residues of human carbonic anhydrase isozyme II through
hydrogen bonds and was also able to insert into base-pairs of DNA hexamer duplex by forming
hydrogen bonds. The preliminary exploration of antibacterial mechanism suggested that
compound 6a was capable of disturbing E. coli membrane effectively, and intercalating into
clinical resistant E. coli bacterial DNA through non-covalent bonds to form supramolecular
complex, thus exerting its powerful antimicrobial activity. This might suggest a great possibility
for hybrid 6a to be a DNA-targeting membrane active potentiator against clinical drug-resistant
E. coli.
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KEYWORDS: sulfonamide, azole, Escherichia coli, DNA, antibacterial
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INTRODUCTION
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Antibiotics have been playing crucial roles in treating deadly microbial infections. In particular,
the success of antibacterial sulfonamides immensely promoted the development of antimicrobial
agents.1 However, the frequently clinical use of various types of antimicrobial drugs caused the
emergence and spread of resistant strains such as fluoroquinolone-resistant Escherichia coli (E.
coli), vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus
aureus (MRSA).2,3 More seriously, E. coli has shown resistance to a variety of clinical
antibacterials such as trimethoprim-sulfamethoxazoles, aminoglycosides and carbapenems and
so on.4,5 Consequently, the discovery of antimicrobial agents with new structural hybrids against
resistant bacterial strains has been an urgent task worldwide.6,7
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Sulfonamides with characteristic p-aminobenzene sulfonamide skeleton are the first class of
artificial synthetic chemotherapeutic agents which were extensively used for the cure and
prevention of bacterial infections in early 1935.8 Antimicrobial mechanism revealed that this
type of drugs could competitively bind enzymes with p-aminobenzoic acid (PABA) in the
biosynthesis of bacterial dihydrofolate and affected the synthesis of nucleic acid precursors,
thereby inhibiting bacterial growth and reproduction.9 Continuous structural modification of the
core framework of p-aminophenyl sulfonamide has been receiving special interest in the
development of novel sulfonamides, and a large number of sulfonamides especially aromatic
heterocycle derivatives like pyrimidine, pyridazine, pyridine, isoxazole, and thiazole ones have
been marketed and extensively used in clinic, such as sulfadiazine, sulfachlorpyridazine,
sulfathiazole and sulfisoxazole. All the sulfonamide drugs provide a great encouragement and
guidance for the rational design of broad-spectrum, high-activity, and low-toxicity new
sulfonamide derivatives, and put forward idea to develop sulfonamide-based multicomponent
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molecular hybrids which might have a large promise as potentially novel DNA-targeting
antimicrobial agents.10-12
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Ethanol as an important component of disinfectant is extensively employed for anti-infective
medicinal application. It is generally considered to denature the protein, break bacterial cell wall
and destroy enzyme system. Meanwhile, it is prevalent that hydroxyethyl fragment is universally
employed to construct new active molecules. Especially, lots of hydroxyethyl derivatives such as
antibacterial metronidazole and antifungal fluconazole were clinical drugs.13,14 Therefore, the
exploration of potentially hydroxyethyl-based antimicrobial agents has provided an important
step forward for current therapies.
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Heterocyclic azoles are helpful structural fragments for biological activity and extensively
used in drug design and development because they could easily interact with the active sites of
organisms through non-covalent interactions like coordination bond, hydrogen bond, π-π
stacking, etc. to improve the physicochemical and pharmacokinetic properties, thus improving
their bioavailability and target selectivity.15−17 In particular, five-membered azoles such as
imidazole,18−20 triazole21−23 and tetrazole24,25 played an important role in the field of medicine,
and some azole compounds have been successfully used in clinic like antibacterial secnidazole
and ornidazole as well as antifungal fluconazole and voriconazole.26−28 This further promotes
increasingly effort towards fused azoles including benzimidazole,29−32 benzotriazole and
carbazole,33−35 and some fused azoles like carbazomycins and murrayafoline have been
marketed.36−38
Piperazine as a significant heterocyclic building block in medicinal chemistry can effectively
regulate physicochemical properties such as lipid-water partition coefficient (logP), binding
affinity and acid-base equilibrium constant.39,40 In the meantime, piperazine provides an easy
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route to access highly functionalized segments of clinical drugs like quinolones, using
straightforward, base-mediated or reductive N-alkylation reactions.41,42
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On the basis of the above considerations, we would like to combine the core antibacterial p-
aminobenzene sulfonamide skeleton, hydroxyethyl, azole and piperazine four effective structural
fragments into one molecule to produce a novel kind of potential four-component antimicrobial
hybrids (Figure 1). Meanwhile, this type of new sulfonamide hybrids may be helpful for
overcoming drug resistance, broadening antimicrobial spectrum, and opening a new avenue to
develop effective multi-targeting antimicrobial agents. All these target molecules would be tested
for antimicrobial activities, and bacterial membrane permeabilization, cytotoxicity, resistance of
bacteria, bactericidal potential and drug combination of the highly active compound were
investigated. To further evaluate the preliminary antibacterial mechanism of this highly active
molecule, molecular docking with DNA hexamer duplex and human carbonic anhydrase isozyme
II as well as interaction with DNA were also evaluated.
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R1
N
R2
N
Hydroxyethyl group is an important fragment in
exerting antimicrobial activity, and widely present in
N
N
N
N
a
lot of clinical drugs, such as antibacterial
N
N
R3
N
metronidazole, antifungal fluconazole and so on.
R4
Various azoles
were inserted to
investigate the
effect on
p-Aminobenzene sulfonamide
is a core structural fragment
HO
S
N
O
S
in
antibacterial
such
drug
as
Z
N
R5
H2N
N
N
Im
bioactivities.
sulfonamides
O
sulfadiazine, sulfathiazole etc.
which have been widely used
for the prevention and cure of
bacterial infection.
N
R6
N
Z
R7
R8
N
Piperazine ring is a benefical flexible
linker in regulating lipid-water partition
coefficient and flexibility of molecules.
Z = CH, N
R9
Figure 1. Design of novel four-component sulfonamide hybrids.
MATERIALS AND METHODS
Chemicals and Measurements. Unless specifically stated, all materials were commercially
available and did not require further purification for use. Thin layer chromatography (TLC) was
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characterized by UV light (254 nm). The masses were measured by a microbalance. Silica gel
(500-600 mesh size) was used for column chromatography. Determination of melting point (mp)
was employed by a X-6 melting point apparatus. Bruker AVANCE III 600 MHz spectrometer
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was used to record H NMR and C NMR (150 MHz) spectra of target compounds. ESI-TOF
method was used to analyze high resolution mass spectra (HRMS) by IonSpec FT-ICR mass
spectrometer. The following abbreviations were representative for the signal of NMR spectra: s =
singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet. Hertz unit (Hz) was used to
express the coupling constants (J). The following abbreviations were used to designate structural
fragments: Im = imidazole, Bim = benzimidazole, Ph = phenyl, Cb = carbazole, Pip = piperazine.
Synthesis. Sulfonamide-based four-component hybrids were conveniently synthesized from
commercially available acetanilide 1. The sulfonylation of acetanilide 1 was carried out with
chlorosulfonic acid at 60 °C for 2 h without using any additional solvent to afford acetamido
benzenesulfonyl chloride 2 in a high yield of 90.2%, which was further reacted with piperazine
in dichloromethane at 0 °C for 1 h to generate intermediate 3 with the yield of 43.4%. The key
intermediate 4 with moderate yield of 64.4% was obtained by the reaction of compound 3 with
epichlorohydrin in acetonitrile employing potassium carbonate at 80 °C for 18 h. The
intermediate 4 was used as scaffold to provide a library of compounds using imidazoles, triazoles
and tetrazoles on the basis of the synthetic route outlined in Scheme 1, thioether-bridged azoles
in Scheme 2, benzimidazoles, benzotriazole and carbazoles in Scheme 3. Sulfonamide
intermediates 5a−d, 7a−b, 9, 11, 13a−c and 15a−e with the yields ranging from 18.8% to 35.2%
were synthesized in acetonitrile using triethylamine (TEA) as base at 80 °C for 24 h. Finally, all
the sulfonamide intermediates were deprotected with hydrochloric acid (40%) in ethanol at 78 °C
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for 1 h to afford their deacetylated products 6a−d, 8a−b, 10, 12, 14a−c and 16a−e with the yields
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ranging from 56.4% to 77.3%.
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All the sulfonamide hybrids with new structures were confirmed by H NMR, C NMR and
HRMS. The spectral data were described in the experimental protocols.
Experimental procedures for the synthesis of intermediate (2). Compound 2 was obtained
according to the reported procedures.12
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Experimental procedures for the synthesis of intermediate (3). Compound 3 was obtained
according to the reported procedures.43
N-(4-((4-(Oxiran-2-ylmethyl)piperazin-1-yl)sulfonyl)phenyl)acetamide (4).
A
mixture
solution of compound 3 (1.50 g, 5.30 mmol), acetonitrile (25 mL) and potassium carbonate (1.46
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g, 10.60 mmol) was stirred at 80 C for 1 h, then epichlorohydrin (1.00 mL, 10.60 mmol) was
added and allowed to stir another 18 h. After disappearance of the starting material by TLC,
potassium carbonate was separated by suction filtration, then the solvent was removed and the
crude product was subjected to column chromatography (eluent, petroleum
ether/dichloromethane (10/1, V/V)) to afford the desired compound 3 as light yellow liquid.
1
Yield: 64.4%; H NMR (DMSO-d6) δ: 10.43 (s, 1H, NHCOCH3), 7.84 (d, J = 7.9 Hz, 2H, Ph-
2,6-H), 7.67 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 2.93 (dd, J = 6.2, 3.0 Hz, 1H, epoxypropyl-CH2),
2.87 (s, 4H, Pip-N-(CH2)2), 2.65 (dd, J = 8.6, 4.1 Hz, 2H, epoxypropyl-CH2), 2.56−2.52 (m, 2H,
Pip-N-CH2), 2.49−2.45 (m, 2H, Pip-N-CH2), 2.42 (dd, J = 4.9, 2.6 Hz, 1H, epoxypropyl-CH),
2.16 (dd, J = 13.4, 6.7 Hz, 1H, cyclopropyl-CH2), 2.10 (s, 3H, COCH3) ppm; 13C NMR (DMSO-
d6) δ: 169.6, 144.06, 129.3, 119.1, 60.0, 52.4, 50.1, 46.3, 44.4, 24.6 ppm.
N-(4-((4-(2-Hydroxy-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (5a). A mixture solution of 2-methyl-5-nitro-1H-imidazole (0.23
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g, 1.80 mmol), acetonitrile (10 mL) and TEA (1.40 mL, 10.00 mmol) was stirred at 80 oC for 1 h,
after compound 4 (0.41 g, 1.20 mmol) was added and the system was allowed to stir additional
24 h. After disappearance of the starting material, the solvent was removed and the crude product
was subjected to column chromatography (eluent, dichloromethane/methanol (300/1, V/V)) to
produce the desired compound 5a as yellow liquid. Yield: 26.8%; 1H NMR (DMSO-d6) δ: 10.46
(s, 1H, NHCOCH3), 8.18 (s, 1H, Im-4-H), 7.84 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.67 (d, J = 8.6
Hz, 2H, Ph-3,5-H), 5.09 (s, 1H, OH), 4.02 (d, J = 12.0 Hz, 1H, CHOH), 3.92−3.84 (m, 2H, Im-
N-CH2CHOH), 3.08 (dd, J = 6.9, 3.6 Hz, 1H, Pip-N-CH2CHOH), 2.87 (s, 4H, Pip-N-(CH2)2),
2.47 (s, 4H, Pip-N-(CH2)2), 2.37 (s, 1H, Pip-N-CH2CHOH), 2.29 (s, 3H, Im-CH3), 2.10 (s, 3H,
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COCH3) ppm; C NMR (DMSO-d6) δ: 169.7, 146.1, 145.6, 144.0, 129.2, 128.8, 123.2, 119.2,
67.3, 61.2, 52.7, 51.2, 46.3, 24.6, 13.3 ppm.
N-(4-((4-(2-Hydroxy-3-(4-nitro-1H-imidazol-1-yl)propyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (5b). Sulfonamide intermediate 5b was generated by the same
experimental procedure for the synthesis of molecule 5a. Compound 4 (0.41 g, 1.20 mmol), 4-
nitro-1H-imidazole (0.20 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used as starting
o
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materials. The pure product 5b was obtained as white solid. Yield: 33.1%; mp: 180−182 C; H
NMR (DMSO-d6) δ: 10.38 (s, 1H, NHCOCH3), 8.26 (s, 1H, Im-2-H), 7.83 (d, J = 8.7 Hz, 2H,
Ph-2,6-H), 7.72 (s, 1H, Im-5-H), 7.68 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 5.12 (d, J = 4.0 Hz, 1H,
OH), 4.11 (dd, J = 17.2, 6.6 Hz, 1H, CHOH), 3.91−3.87 (m, 2H, Im-N-CH2CHOH), 2.89 (d, J =
8.3 Hz, 4H, Pip-N-(CH2)2), 2.52 (s, 2H, Pip-N-CH2), 2.46 (d, J = 4.6 Hz, 2H, Pip-N-CH2), 2.28
(dd, J = 12.7, 5.0 Hz, 1H, Pip-N-CH2CHOH), 2.22 (dd, J = 12.7, 5.9 Hz, 1H, Pip-N-CH2CHOH),
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2.10 (s, 3H, COCH3) ppm; C NMR (DMSO-d6) δ: 147.1, 144.0, 138.3, 129.3, 128.9, 122.7,
67.1, 61.0, 52.7, 52.2, 46.3, 24.6 ppm.
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N-(4-((4-(2-Hydroxy-3-(2-phenyl-1H-imidazol-1-yl)propyl)piperazin-1-
5
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yl)sulfonyl)phenyl)acetamide (5c). Sulfonamide intermediate 5c was generated by the similar
experimental procedure for the synthesis of molecule 5a. Compound 4 (0.41 g, 1.20 mmol), 2-
phenyl-1H-imidazole (0.26 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used as starting
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materials. The pure product 5c was obtained as white solid. Yield: 35.2%; mp: > 250 C; H
NMR (DMSO-d6) δ: 10.52 (s, 1H, NHCOCH3), 7.91 (d, J = 8.8 Hz, 2H, Im-2-Ph-2,6-H), 7.68 (d,
J = 8.8 Hz, 2H, Ph-2,6-H), 7.54−7.51 (m, 2H, Ph-3,5-H), 7.28 (d, J = 1.0 Hz, 1H, Im-5-H), 7.25
(t, J = 7.4 Hz, 1H, Im-2-Ph-4-H), 7.16 (t, J = 7.6 Hz, 2H, Im-2-Ph-3,5-H), 6.94 (d, J = 1.0 Hz,
1H, Im-4-H), 5.13 (s, 1H, OH), 4.12 (q, J = 6.7 Hz, 1H, CHOH), 3.77 (d, J = 9.7 Hz, 2H, Im-N-
CH2CHOH), 2.72 (s, 4H, Pip-N-(CH2)2), 2.39 (d, J = 21.0 Hz, 4H, Pip-N-(CH2)2), 2.22−2.19 (m,
2H, Pip-N-CH2CHOH), 2.14 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6) δ: 169.6, 147.2, 144.1,
131.6, 129.3, 128.5, 127.9, 122.7, 119.2, 68.3, 61.4, 52.7, 50.8, 46.6, 24.6 ppm.
N-(4-((4-(2-Hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (5d). Sulfonamide intermediate 5d was generated by the similar
experimental procedure for the synthesis of molecule 5a. Compound 4 (0.41 g, 1.20 mmol),
triazole (0.12 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used as starting materials.
The pure product 5d was obtained as yellow liquid. Yield: 30.6%; 1H NMR (DMSO-d6) δ: 10.37
(s, 1H, NHCOCH3), 8.36 (s, 1H, triazole 5-H), 7.89 (s, 1H, triazole 3-H), 7.83 (d, J = 8.7 Hz, 2H,
Ph-2,6-H), 7.67 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 4.95 (d, J = 5.0 Hz, 1H, OH), 4.17 (dd, J = 13.8,
3.6 Hz, 1H, triazole N-CH2CHOH), 4.03 (dd, J = 13.8, 7.5 Hz, 1H, triazole N-CH2CHOH),
3.92−3.87 (m, 1H, CHOH), 2.85 (s, 4H, Pip-N-(CH2)2), 2.49 (s, 4H, Pip-N-(CH2)2), 2.28 (dq, J =
13.0, 6.5 Hz, 2H, Pip-N-CH2CHOH), 2.10 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6) δ: 169.6,
151.5, 145.1, 144.0, 129.3, 128.8, 119.2, 66.9, 61.3, 53.6, 52.8, 46.4, 24.6 ppm.
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yl)propan-2-ol (6a). A mixture solution of compound 5a (0.10 g, 0.20 mmol), ethanol (10 mL)
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and hydrochloric acid (5 mL, 40%) was stirred at 78 C for 1 h. After disappearance of the
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starting material, the reaction solvent was evaporated and neutralized with saturated sodium
bicarbonate solution to produce the desired compound 6a as yellow liquid. Yield: 61.5%; mp: >
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250 C; H NMR (DMSO-d6) δ: 8.19 (s, 1H, Im-4-H), 7.35 (d, J = 8.6 Hz, 2H, Ph-2,6-H), 6.66
(d, J = 8.6 Hz, 2H, Ph-3,5-H), 6.08 (s, 2H, Ph-NH2), 5.09 (d, J = 5.0 Hz, 1H, OH), 4.03 (dd, J =
14.0, 2.3 Hz, 1H, Im-N-CH2CHOH), 3.87 (d, J = 5.4 Hz, 1H, CHOH), 3.79 (dd, J = 14.0, 8.4 Hz,
1H, Im-N-CH2CHOH), 2.81 (s, 4H, Pip-N-(CH2)2), 2.52 (s, 2H, Pip-N-CH2), 2.47 (s, 2H, Pip-N-
CH2), 2.30 (s, 5H, Pip-N-CH2CHOH, COCH3) ppm; 13C NMR (DMSO-d6) δ: 153.7, 146.1,
145.6, 130.1, 123.2, 119.9, 113.2, 67.4, 61.4, 52.8, 51.3, 46.3, 13.3 ppm; HR-ESIMS m/z calcd
for C17H24N6O5S [M + H]+, 425.1607; found, 425.1606.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(4-nitro-1H-imidazol-1-yl)propan-2-ol
(6b). Sulfonamide derivative 6b was produced by the similar experimental procedure for the
synthesis of molecule 6a. Compound 5b (0.10 g, 0.22 mmol) and hydrochloric acid (5 mL, 40%)
were used as starting materials. The pure product 6b was acquired as yellow solid. Yield: 68.9%;
mp: 120−122 oC; 1H NMR (DMSO-d6) δ: 8.27 (d, J = 0.7 Hz, 1H, Im-2-H), 7.73 (d, J = 0.9 Hz,
1H, Im-5-H), 7.35 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 6.66 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.08 (s, 2H,
Ph-NH2), 5.13 (d, J = 4.3 Hz, 1H, OH), 4.12 (q, J = 6.5 Hz, 1H, CHOH), 3.88 (q, J = 7.6 Hz, 2H,
Im-N-CH2CHOH), 2.81 (s, 4H, Pip-N-(CH2)2), 2.51 (s, 2H, Pip-N-CH2), 2.45 (s, 2H, Pip-N-
CH2), 2.27 (dd, J = 12.7, 5.0 Hz, 1H, Pip-N-CH2CHOH), 2.22 (dd, J = 12.7, 6.1 Hz, 1H, Pip-N-
CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 153.7, 147.1, 138.4, 130.1, 122.7, 119.9, 113.2, 67.0,
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61.2, 52.8, 52.2, 46.3 ppm; HR-ESIMS m/z calcd for C16H22N6O5S [M + H]+, 411.1451; found,
411.1450.
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(2-phenyl-1H-imidazol-1-yl)propan-2-ol
(6c). Sulfonamide derivative 6c was produced by the same experimental procedure for the
synthesis of molecule 6a. Compound 5c (0.10 g, 0.21 mmol), and hydrochloric acid solution (5
mL, 40%) were used starting materials. The pure product 6c was acquired as yellow solid. Yield:
66.6%; mp: 76−78 oC; 1H NMR (DMSO-d6) δ: 7.56 (d, J = 7.3 Hz, 2H, Ph-2,6-H), 7.38−7.34 (m,
4H, Im-2-Ph-2,3,5,6-H), 7.20 (t, J = 7.7 Hz, 2H, Ph-3,5-H), 7.03 (s, 1H, Im-2-Ph-4-H), 6.73 (d, J
= 8.7 Hz, 2H, Im-4,5-H), 6.19 (s, 2H, Ph-NH2), 5.17 (s, 1H, OH), 4.16 (q, J = 6.5 Hz, 1H,
CHOH), 3.78 (d, J = 9.3 Hz, 2H, Im-N-CH2CHOH), 2.69 (s, 4H, Pip-N-(CH2)2), 2.41 (d, J =
28.6 Hz, 4H, Pip-N-(CH2)2), 2.24 (d, J = 4.4 Hz, 2H, Pip-N-CH2CHOH) ppm; 13C NMR
(DMSO-d6) δ: 153.8, 147.0, 130.7, 130.2, 129.4, 129.0, 128.7, 127.1, 123.0, 119.5, 113.2, 68.2,
61.5, 52.8, 51.0, 46.4 ppm; HR-ESIMS m/z calcd for C22H27N5O3S [M + H]+, 442.1913; found,
442.1915.
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol
(6d).
Sulfonamide derivative 6d was produced by the similar process depicted for compound 6a.
Compound 5d (0.10 g, 0.24 mmol) and hydrochloric acid solution (5 mL, 40%) were used
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starting materials. The pure product 6d was acquired as yellow liquid. Yield: 69.2%; H NMR
(DMSO-d6) δ: 8.37 (s, 1H, triazole 5-H), 7.90 (s, 1H, triazole 3-H), 7.35 (d, J = 8.7 Hz, 2H, Ph-
2,6-H), 6.66 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.08 (s, 2H, Ph-NH2), 4.98 (s, 1H, OH), 4.18 (dd, J =
13.8, 3.6 Hz, 1H, triazole N-CH2CHOH), 4.03 (dd, J = 13.8, 7.5 Hz, 1H, triazole N-CH2CHOH),
3.91−3.88 (m, 1H, CHOH), 2.79 (s, 4H, Pip-N-(CH2)2), 2.48 (s, 4H, Pip-N-(CH2)2), 2.31−2.25
(m, 2H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 153.7, 151.5, 145.1, 130.1, 113.2,
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66.9, 61.4, 53.7, 52.8, 46.3 ppm; HR-ESIMS m/z calcd for C15H22N6O3S [M + H]+, 367.1552;
found, 367.1551.
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N-(4-((4-(2-Hydroxy-3-(1H-tetrazol-1-yl)propyl)piperazin-1-yl)sulfonyl)phenyl)acetamide
(7a). Sulfonamide intermediate 7a was generated by the similar process like compound 5a.
Compound 4 (0.41 g, 1.20 mmol), tetrazole (0.13 g, 1.80 mmol) and TEA (1.40 mL, 10.00
mmol) were used starting materials. The pure product 7a was acquired as yellow liquid. Yield:
30.5%; 1H NMR (DMSO-d6) δ: 10.46 (s, 1H, NHCOCH3), 9.24 (s, 1H, tetrazole 5-H), 7.84 (d, J
= 8.7 Hz, 2H, Ph-2,6-H), 7.67 (d, J = 8.8 Hz, 2H, Ph-3,5-H), 4.48 (dd, J = 14.0, 3.3 Hz, 1H,
tetrazole N-CH2CHOH), 4.31 (dd, J = 14.0, 7.8 Hz, 1H, tetrazole N-CH2CHOH), 3.97−3.92 (m,
1H, CHOH), 2.86 (s, 4H, Pip-N-(CH2)2), 2.50 (s, 4H, Pip-N-(CH2)2), 2.30 (d, J = 6.2 Hz, 2H,
Pip-N-CH2CHOH), 2.10 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6) δ: 169.6, 145.0, 144.8,
144.0, 129.3, 119.2, 66.7, 61.0, 52.7, 52.3, 46.4, 24.6 ppm.
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N-(4-((4-(2-Hydroxy-3-(5-methyl-1H-tetrazol-1-yl)propyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (7b). Sulfonamide intermediate 7b was generated by the same
process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 5-methyl-1H-tetrazole (0.15 g,
1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The pure product 7b
was acquired as white solid. Yield: 29.5%; mp: 95−97 oC; 1H NMR (DMSO-d6) δ: 10.38 (s, 1H,
NHCOCH3), 7.83 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.67 (d, J = 8.8 Hz, 2H, Ph-3,5-H), 5.10 (s, 1H,
OH), 4.37 (dd, J = 14.3, 3.4 Hz, 1H, tetrazole N-CH2CHOH), 4.16 (dd, J = 14.3, 7.9 Hz, 1H,
tetrazole N-CH2CHOH), 3.92 (d, J = 3.6 Hz, 1H, CHOH), 2.86 (s, 4H, Pip-N-(CH2)2), 2.51 (s,
4H, Pip-N-(CH2)2), 2.45 (s, 3H, tetrazole CH3), 2.37 (dd, J = 12.9, 6.6 Hz, 1H, Pip-N-
CH2CHOH), 2.32 (dd, J = 12.8, 6.0 Hz, 1H, Pip-N-CH2CHOH), 2.09 (s, 3H, COCH3) ppm; 13C
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NMR (DMSO-d6) δ: 169.6, 153.4, 144.0, 129.3, 128.8, 119.2, 67.3, 61.1, 52.8, 51.2, 46.4, 24.6,
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(1H-tetrazol-1-yl)propan-2-ol
(8a).
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Sulfonamide derivative 8a was produced by the similar process depicted for compound 6a.
Compound 7a (0.10 g, 0.24 mmol) and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 8a was acquired as white solid. Yield: 67.6%; mp: 94−96
oC; 1H NMR (DMSO-d6) δ: 9.24 (s, 1H, tetrazole 5-H), 7.35 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 6.66
(d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.08 (s, 2H, Ph-NH2), 5.15 (d, J = 5.1 Hz, 1H, OH), 4.49 (dd, J =
14.0, 3.2 Hz, 1H, tetrazole N-CH2CHOH), 4.31 (dd, J = 14.0, 7.8 Hz, 1H, tetrazole N-
CH2CHOH), 3.95 (dd, J = 9.4, 6.5 Hz, 1H, CHOH), 2.80 (s, 4H, Pip-N-(CH2)2), 2.50 (s, 4H, Pip-
N-(CH2)2), 2.30 (d, J = 6.2 Hz, 2H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 153.7,
144.9, 130.1, 119.9, 113.2, 66.7, 61.1, 52.8, 52.3, 46.3 ppm; HR-ESIMS m/z calcd for
C14H21N7O3S [M + H]+, 368.1505; found, 368.1505.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(5-methyl-1H-tetrazol-1-yl)propan-2-ol
(8b). Sulfonamide derivative 8b was produced by the similar process depicted for compound 6a.
Compound 7b (0.10 g, 0.24 mmol) and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 8b was acquired as white solid. Yield: 70.2%; mp: 100−102
oC; 1H NMR (DMSO-d6) δ: 7.35 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 6.66 (d, J = 8.7 Hz, 2H, Ph-3,5-
H), 6.08 (s, 2H, Ph-NH2), 5.12 (s, 1H, OH), 4.38 (dd, J = 14.3, 3.2 Hz, 1H, tetrazole N-
CH2CHOH), 4.17 (dd, J = 14.2, 7.8 Hz, 1H, tetrazole N-CH2CHOH), 3.94 (s, 1H, CHOH), 2.80
(s, 4H, Pip-N-(CH2)2), 2.51 (s, 4H, Pip-N-(CH2)2), 2.46 (s, 3H, tetrazole CH3), 2.37 (d, J = 15.9
Hz, 2H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 153.7, 153.4, 130.1, 119.8, 113.2,
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67.3, 61.2, 52.8, 51.2, 46.3, 9.1 ppm; HR-ESIMS m/z calcd for C15H23N7O3S [M + H]+,
382.1661; found, 382.1659.
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yl)sulfonyl)phenyl)acetamide (9). Sulfonamide intermediate 9 was generated by the similar
process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 1H-1,2,4-triazole-3-thiol (0.18 g,
1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The pure product 9
was acquired as yellow liquid. Yield: 28.4%; 1H NMR (DMSO-d6) δ: 10.38 (s, 1H, NHCOCH3),
8.31 (s, 1H, triazole 5-H), 7.83 (d, J = 8.6 Hz, 2H, Ph-2,6-H), 7.66 (d, J = 8.7 Hz, 2H, Ph-3,5-H),
3.80 (dt, J = 11.5, 5.9 Hz, 1H, CHOH), 3.23 (dd, J = 13.2, 4.5 Hz, 1H, triazole N-CH2CHOH),
2.99 (dd, J = 13.2, 7.0 Hz, 1H, triazole N-CH2CHOH), 2.84 (s, 4H, Pip-N-(CH2)2), 2.46 (s, 4H,
Pip-N-(CH2)2), 2.38 (dd, J = 12.8, 5.5 Hz, 1H, Pip-N-CH2CHOH), 2.33 (dd, J = 12.7, 6.4 Hz,
1H, Pip-N-CH2CHOH), 2.10 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6) δ: 169.6, 147.0, 144.0,
129.3, 128.9, 119.2, 67.6, 62.8, 52.7, 46.4, 37.5, 24.6 ppm.
1-((1H-1,2,4-Triazol-3-yl)thio)-3-(4-((4-aminophenyl)sulfonyl)piperazin-1-yl)propan-2-ol
(10). Sulfonamide derivative 10 was produced by the similar process depicted for compound 6a.
Compound 9 (0.10 g, 0.23 mmol) and hydrochloric acid solution (5 mL, 40%) were used starting
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materials. The pure product 10 was acquired as white solid. Yield: 69.9%; mp: 96−98 C; H
NMR (DMSO-d6) δ: 8.31 (s, 1H, triazole 5-H), 7.34 (d, J = 8.5 Hz, 2H, Ph-2,6-H), 6.65 (d, J =
8.6 Hz, 2H, Ph-3,5-H), 6.06 (s, 2H, Ph-NH2), 4.94 (s, 1H, OH), 3.82−3.78 (m, 1H, CHOH), 3.23
(dd, J = 13.2, 4.5 Hz, 1H, triazole N-CH2CHOH), 3.00 (dd, J = 13.2, 7.0 Hz, 1H, triazole N-
CH2CHOH), 2.78 (s, 4H, Pip-N-(CH2)2), 2.46 (d, J = 15.3 Hz, 4H, Pip-N-(CH2)2), 2.39−2.31 (m,
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2H, Pip-N-CH2CHOH) ppm; C NMR (DMSO-d6) δ: 153.7, 147.0, 130.1, 120.0, 113.2, 67.6,
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62.9, 52.8, 46.3, 37.6 ppm; HR-ESIMS m/z calcd for C15H22N6O3S2 [M + H]+, 399.1273; found,
399.1271.
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yl)sulfonyl)phenyl)acetamide (11). Sulfonamide intermediate 11 was prepared according to the
same process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 1-methyl-1H-imidazole-2-
thiol (0.21 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The
pure product 11 was acquired as light yellow liquid. Yield: 27.6%; 1H NMR (DMSO-d6) δ: 10.42
(s, 1H, NHCOCH3), 7.84 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.66 (d, J = 8.8 Hz, 2H, Ph-3,5-H), 7.17
(s, 1H, Im-5-H), 6.86 (d, J = 1.0 Hz, 1H, Im-4-H), 3.78−3.74 (m, 1H, CHOH), 3.52 (s, 3H, Im-
CH3), 3.10 (dd, J = 13.3, 4.5 Hz, 1H, Im-N-CH2CHOH), 2.92 (dd, J = 13.3, 6.7 Hz, 1H, Im-N-
CH2CHOH), 2.82 (s, 4H, Pip-N-(CH2)2), 2.47 (s, 4H, Pip-N-(CH2)2), 2.37 (dd, J = 12.8, 5.8 Hz,
1H, Pip-N-CH2CHOH), 2.32 (dd, J = 12.7, 6.4 Hz, 1H, Pip-N-CH2CHOH), 2.10 (s, 3H, COCH3)
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ppm; C NMR (DMSO-d6) δ: 169.6, 144.0, 141.5, 129.3, 128.6, 123.4, 119.2, 67.9, 62.5, 52.7,
46.3, 39.4, 33.3, 24.6 ppm.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-((1-methyl-1H-imidazol-2-yl)thio)propan-
2-ol (12). Sulfonamide derivative 12 was produced by the similar process depicted for compound
6a. Compound 11 (0.10 g, 0.22 mmol) and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 12 was acquired as white solid. Yield: 58.7%; mp: 178−180
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oC; H NMR (DMSO-d6) δ: 7.34 (d, J = 8.6 Hz, 2H, Ph-2,6-H), 7.17 (s, 1H, Im-5-H), 6.87 (s,
1H, Im-4-H), 6.66 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.09 (s, 2H, Ph-NH2), 3.80−3.75 (m, 1H,
CHOH), 3.53 (s, 3H, Im-CH3), 3.11 (dd, J = 13.3, 4.5 Hz, 1H, Im-N-CH2CHOH), 2.94−2.91 (m,
1H, Im-N-CH2CHOH), 2.77 (s, 4H, Pip-N-(CH2)2), 2.48 (s, 4H, Pip-N-(CH2)2), 2.40 (dd, J =
12.8, 5.6 Hz, 1H, Pip-N-CH2CHOH), 2.35 (dd, J = 12.7, 6.5 Hz, 1H, Pip-N-CH2CHOH) ppm;
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13C NMR (DMSO-d6) δ: 153.7, 141.5, 130.1, 128.5, 123.4, 119.8, 113.2, 67.8, 62.5, 52.7, 46.2,
39.4, 33.3 ppm; HR-ESIMS m/z calcd for C17H25N5O3S2 [M + H]+, 412.1477; found, 412.1478.
N-(4-((4-(3-(1H-Benzo[d]imidazol-1-yl)-2-hydroxypropyl)piperazin-1-
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yl)sulfonyl)phenyl)acetamide (13a). Sulfonamide intermediate 13a was prepared according to
the similar process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), benzimidazole (0.21 g,
1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The pure product 13a
was acquired as yellow liquid. Yield: 27.3%; 1H NMR (DMSO-d6) δ: 10.42 (s, 1H, NHCOCH3),
8.07 (s, 1H, Bim-2-H), 7.85 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.67 (d, J = 8.7 Hz, 2H, Ph-3,5-H),
7.56 (d, J = 7.7 Hz, 1H, Bim-7-H), 7.49 (d, J = 7.6 Hz, 1H, Bim-4-H), 7.17−7.12 (m, 2H, Bim-
5,6-H), 5.02 (d, J = 4.6 Hz, 1H, OH), 4.24 (dd, J = 14.3, 3.7 Hz, 1H, Bim-N-CH2CHOH), 4.09
(dd, J = 14.3, 7.0 Hz, 1H, Bim-N-CH2CHOH), 3.93 (d, J = 5.1 Hz, 1H, CHOH), 2.83 (s, 4H, Pip-
N-(CH2)2), 2.45 (dd, J = 30.3, 3.2 Hz, 4H, Pip-N-(CH2)2), 2.27 (d, J = 6.2 Hz, 2H, Pip-N-
CH2CHOH), 2.10 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6) δ: 169.6, 145.1, 144.0, 143.7,
134.9, 129.3, 122.3, 121.6, 119.6, 119.2, 111.1, 66.9, 61.4, 55.38, 52.8, 46.3, 24.6 ppm.
N-(4-((4-(2-Hydroxy-3-(5-methyl-1H-benzo[d]imidazol-1-yl)propyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (13b). Sulfonamide intermediate 13b was prepared according to
the same process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 5-methyl-1H-
benzoimidazole (0.24 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting
materials. The pure product 13b was acquired as yellow liquid. Yield: 26.5%; 1H NMR (DMSO-
d6) δ: 10.44 (s, 1H, NHCOCH3), 8.00 (d, J = 12.0 Hz, 1H, Bim-2-H), 7.86 (dd, J = 8.7, 1.6 Hz,
2H, Ph-2,6-H), 7.68 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 7.44−7.25 (m, 2H, Bim-6,7-H), 6.97 (dd, J =
15.3, 8.4 Hz, 1H, Bim-4-H), 5.03 (d, J = 9.4 Hz, 1H, OH), 4.21−4.17 (m, 1H, Bim-N-
CH2CHOH), 4.08−4.04 (m, 1H, Bim-N-CH2CHOH), 3.92 (s, 1H, CHOH), 2.85 (s, 4H, Pip-N-
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Hz, 2H, Pip-N-CH2CHOH), 2.11 (s, 3H, COCH3) ppm; C NMR (DMSO-d6) δ: 169.6, 144.9,
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24.6, 21.8 ppm.
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N-(4-((4-(3-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-hydroxypropyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (13c). Sulfonamide intermediate 13c was prepared according to
the similar process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), benzotriazole (0.21 g,
1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The pure product 13c
was acquired as yellow liquid. Yield: 31.1%; 1H NMR (DMSO-d6) δ:10.47 (s, 1H, NHCOCH3),
7.87 (d, J = 8.5 Hz, 3H, Ph-2,6-H, benzotriazole 4-H), 7.78 (d, J = 8.4 Hz, 1H, benzotriazole 7-
H), 7.64 (d, J = 8.8 Hz, 2H, Ph-3,5-H), 7.44 (t, J = 7.6 Hz, 1H, benzotriazole 6-H), 7.29 (t, J =
7.6 Hz, 1H, benzotriazole 5-H), 5.10 (d, J = 4.9 Hz, 1H, OH), 4.70 (dd, J = 14.2, 4.5 Hz, 1H,
benzotriazole N-CH2CHOH), 4.58 (dd, J = 14.2, 6.8 Hz, 1H, benzotriazole N-CH2CHOH),
4.11−4.07 (m, 1H, CHOH), 2.71 (s, 4H, Pip-N-(CH2)2), 2.43 (s, 4H, Pip-N-(CH2)2), 2.40 (d, J =
6.9 Hz, 1H, Pip-N-CH2CHOH), 2.33 (dd, J = 12.8, 5.6 Hz, 1H, Pip-N-CH2CHOH), 2.12 (s, 3H,
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COCH3) ppm; C NMR (DMSO-d6) δ: 169.6, 145.5, 144.0, 134.3, 129.3, 127.1, 123.9, 119.2,
111.7, 67.4, 61.7, 52.7, 49.1, 46.2, 24.6 ppm.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(1H-benzo[d]imidazol-1-yl)propan-2-ol
(14a). Sulfonamide derivative 14a was generated by the similar process depicted for compound
6a. Compound 13a (0.10 g, 0.22 mmol), and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 14a was acquired as yellow solid. Yield: 71.1%; mp:
120−122 oC; 1H NMR (DMSO-d6) δ: 8.07 (s, 1H, Bim-2-H), 7.58 (d, J = 7.6 Hz, 1H, Bim-7-H),
7.50 (d, J = 7.7 Hz, 1H, Bim-4-H), 7.35 (d, J = 8.6 Hz, 2H, Ph-2,6-H), 7.19−7.13 (m, 2H, Ph-
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3,5-H), 6.68 (d, J = 8.6 Hz, 2H, Bim-5,6-H), 6.08 (s, 2H, Ph-NH2), 5.00 (d, J = 4.5 Hz, 1H, OH),
4.24 (dd, J = 14.3, 3.2 Hz, 1H, Bim-N-CH2CHOH), 4.09 (dd, J = 14.3, 7.0 Hz, 1H, Bim-N-
CH2CHOH), 3.93 (s, 1H, CHOH), 2.79 (s, 4H, Pip-N-(CH2)2), 2.45 (d, J = 30.7 Hz, 4H, Pip-N-
(CH2)2), 2.27 (d, J = 6.1 Hz, 2H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 153.7, 145.1,
143.7, 134.9, 130.1, 122.4, 121.6, 119.8, 113.2, 111.1, 67.0, 61.4, 52.9, 49.1, 46.3 ppm; HR-
ESIMS m/z calcd for C20H25N5O3S [M + H]+, 416.1756; found, 416.1754.
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(5-methyl-1H-benzo[d]imidazol-1-
yl)propan-2-ol (14b). Sulfonamide derivative 14b was generated by the similar process depicted
for compound 6a. Compound 13b (0.10 g, 0.21 mmol) and hydrochloric acid solution (5 mL,
40%) were used starting materials. The pure product 14b was obtained as white solid. Yield:
70.9%; mp: 140−142 oC; 1H NMR (DMSO-d6) δ: 8.05 (d, J = 19.6 Hz, 1H, Bim-2-H), 7.48−7.38
(m, 2H, Bim-6,7-H), 7.36 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.00 (dd, J = 20.0, 8.2 Hz, 1H, Bim-4-
H), 6.68 (dd, J = 8.6, 1.5 Hz, 2H, Ph-3,5-H), 6.11 (s, 2H, Ph-NH2), 5.13 (s, 1H, OH), 4.24−4.19
(m, 1H, Bim-N-CH2CHOH), 4.08 (dd, J = 14.4, 6.9 Hz, 1H, Bim-N-CH2CHOH), 3.96 (s, 1H,
CHOH), 3.07 (q, J = 7.3 Hz, 1H, Pip-N-CH2CHOH), 2.84 (s, 4H, Pip-N-(CH2)2), 2.58 (s, 4H,
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Pip-N-(CH2)2), 2.38 (d, J = 15.2 Hz, 3H, Bim-CH3), 2.35 (s, 1H, Pip-N-CH2CHOH) ppm; C
NMR (DMSO-d6) δ: 153.8, 144.8, 144.5, 130.1, 124.0, 123.4, 119.1, 113.3, 110.9, 66.7, 61.0,
52.7, 49.1, 46.0, 21.8 ppm; HR-ESIMS m/z calcd for C21H27N5O3S [M + H]+, 430.1913; found,
430.1912.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(1H-benzo[d][1,2,3]triazol-1-yl)propan-2-
ol (14c). Sulfonamide derivative 14c was generated by the similar process depicted for
compound 6a. Compound 13c (0.10 g, 0.22 mmol) and hydrochloric acid solution (5 mL, 40%)
were used starting materials. The pure product 14c was acquired as brown solid. Yield: 56.4%;
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mp: 248−250 C; H NMR (DMSO-d6) δ: 7.97−7.78 (m, 2H, benzotriazole 4,7-H), 7.47 (s, 2H,
benzotriazole 5,6-H), 7.34 (s, 2H, Ph-2,6-H), 6.68 (d, J = 8.6 Hz, 2H, Ph-2,6-H), 6.13 (s, 2H, Ph-
NH2), 5.17−5.05 (m, 1H, OH), 4.73 (dd, J = 14.2, 7.6 Hz, 1H, benzotriazole N-CH2CHOH), 4.60
(dd, J = 14.2, 8.0 Hz, 1H, benzotriazole N-CH2CHOH), 4.03 (d, J = 71.1 Hz, 1H, CHOH), 3.51
(d, J = 81.0 Hz, 2H, Pip-N-CH2CHOH), 2.67 (s, 4H, Pip-N-(CH2)2), 2.43 (s, 4H, Pip-N-(CH2)2)
ppm; 13C NMR (DMSO-d6) δ: 153.7, 145.5, 134.3, 130.1, 127.3, 124.0, 119.2, 113.2, 111.8,
71.4, 63.9, 55.4, 52.7, 46.1 ppm; HR-ESIMS m/z calcd for C19H24N6O3S [M + H]+, 417.1709;
found, 417.1708.
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N-(4-((4-(3-(9H-Carbazol-9-yl)-2-hydroxypropyl)piperazin-1-yl)sulfonyl)phenyl)acetamide
(15a). Sulfonamide intermediate 15a was produced according to the similar process like
compound 5a. Compound 4 (0.41 g, 1.20 mmol), 9H-carbazole (0.30 g, 1.80 mmol) and TEA
(1.40 mL, 10.00 mmol) were used starting materials. The pure product 15a was acquired as light
yellow liquid. Yield: 29.6%; 1H NMR (DMSO-d6) δ: 10.44 (s, 1H, NHCOCH3), 8.01 (d, J = 7.7
Hz, 2H, Cb-4,5-H), 7.88 (d, J = 8.7 Hz, 2H, Cb-1,8-H), 7.64 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.52
(d, J = 8.2 Hz, 2H, Ph-3,5-H), 7.33 (t, J = 7.7 Hz, 2H, Cb-2,7-H), 7.12 (t, J = 7.4 Hz, 2H, Cb-3,6-
H), 4.96 (d, J = 4.9 Hz, 1H, OH), 4.36 (dd, J = 14.8, 4.8 Hz, 1H, Cb-N-CH2CHOH), 4.24 (dd, J
= 14.8, 6.5 Hz, 1H, Cb-N-CH2CHOH), 4.00 (dd, J = 11.4, 5.9 Hz, 1H, CHOH), 2.69 (s, 4H, Pip-
N-(CH2)2), 2.40 (dd, J = 12.5, 6.5 Hz, 4H, Pip-N-(CH2)2), 2.32 (dd, J = 12.7, 5.7 Hz, 2H, Pip-N-
CH2CHOH), 2.12 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6) δ: 169.6, 144.0, 141.1, 129.3,
125.8, 122.5, 120.4, 119.2, 119.0, 110.2, 67.2, 62.1, 52.8, 49.1, 46.2, 24.6 ppm.
N-(4-((4-(3-(2-Bromo-9H-carbazol-9-yl)-2-hydroxypropyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (15b). Sulfonamide intermediate 15b was produced according to
the same process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 2-bromo-9H-carbazole
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(0.44 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The pure
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product 15b was acquired as white solid. Yield: 25.6%; mp: > 250 C; H NMR (DMSO-d6) δ:
10.42 (s, 1H, NHCOCH3), 8.05 (d, J = 7.7 Hz, 1H, Cb-4-H), 7.98 (d, J = 8.2 Hz, 1H, Cb-5-H),
7.87 (d, J = 8.8 Hz, 2H, Ph-2,6-H), 7.76 (s, 1H, Cb-1-H), 7.66 (d, J = 8.8 Hz, 2H, Ph-3,5-H),
7.56 (d, J = 8.3 Hz, 1H, Cb-8-H), 7.38 (t, J = 7.5 Hz, 1H, Cb-7-H), 7.24 (d, J = 8.3 Hz, 1H, Cb-
3-H), 7.16 (t, J = 7.4 Hz, 1H, Cb-6-H), 5.02 (d, J = 5.0 Hz, 1H, OH), 4.36 (dd, J = 14.9, 4.2 Hz,
1H, Cb-N-CH2CHOH), 4.23 (dd, J = 14.9, 6.2 Hz, 1H, Cb-N-CH2CHOH), 3.99−3.94 (m, 1H,
CHOH), 2.80 (s, 4H, Pip-N-(CH2)2), 2.45 (d, J = 11.0 Hz, 4H, Pip-N-(CH2)2), 2.37 (d, J = 7.5
Hz, 1H, Pip-N-CH2CHOH), 2.27 (dd, J = 12.6, 5.0 Hz, 1H, Pip-N-CH2CHOH), 2.12 (s, 3H,
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COCH3) ppm; C NMR (DMSO-d6) δ: 169.6, 144.1, 142.1, 141.4, 129.3, 128.7, 126.3, 122.0,
121.7, 120.6, 119.6, 119.2, 118.7, 113.2, 110.7, 67.4, 61.6, 52.9, 48.1, 46.3, 24.6 ppm.
N-(4-((4-(3-(3,6-Dibromo-9H-carbazol-9-yl)-2-hydroxypropyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (15c). Sulfonamide intermediate 15c was produced according to
the similar process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 3,6-dibromo-9H-
carbazole (0.59 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials.
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The pure product 15c was acquired as white solid. Yield: 18.8%; mp: 175−177 C; H NMR
(DMSO-d6) δ: 10.45 (s, 1H, NHCOCH3), 8.34 (d, J = 1.0 Hz, 2H, Cb-4,5-H), 7.88 (d, J = 8.7 Hz,
2H, Cb-1,8-H), 7.66 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 7.53 (d, J = 8.8 Hz, 2H, Ph-3,5-H), 7.49 (dd,
J = 8.7, 1.5 Hz, 2H, Cb-2,7-H), 4.95 (d, J = 3.7 Hz, 1H, OH), 4.34 (dd, J = 14.9, 4.0 Hz, 1H, Cb-
N-CH2CHOH), 4.24 (dd, J = 14.9, 6.7 Hz, 1H, Cb-N-CH2CHOH), 3.96 (s, 1H, CHOH), 2.75 (s,
4H, Pip-N-(CH2)2), 2.43 (s, 4H, Pip-N-(CH2)2), 2.37 (d, J = 6.4 Hz, 1H, Pip-N-CH2CHOH), 2.30
(dd, J = 12.6, 5.6 Hz, 1H, Pip-N-CH2CHOH), 2.11 (s, 3H, COCH3) ppm; 13C NMR (DMSO-d6)
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δ: 169.6, 144.0, 140.2, 129.3, 128.9, 123.5, 123.3, 119.2, 112.7, 111.6, 67.2, 61.7, 52.8, 48.3,
46.3, 24.6 ppm.
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yl)sulfonyl)phenyl)acetamide(15d). Sulfonamide intermediate 15d was produced according to
the same process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 3-iodo-9H-carbazole
(0.53 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials. The pure
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product 15d was acquired as white solid. Yield: 23.7%; mp: 186−188 C; H NMR (DMSO-d6)
δ: 10.43 (s, 1H, NHCOCH3), 8.42 (s, 1H, Cb-5-H), 8.06 (d, J = 7.7 Hz, 1H, Cb-4-H), 7.88 (d, J =
8.7 Hz, 2H, Ph-2,6-H), 7.65 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 7.60 (d, J = 8.7 Hz, 1H, Cb-8-H),
7.53 (d, J = 8.3 Hz, 1H, Cb-2-H), 7.41 (d, J = 8.6 Hz, 1H, Cb-1-H), 7.36 (t, J = 7.6 Hz, 1H, Cb-
7-H ), 7.14 (t, J = 7.4 Hz, 1H, Cb-6-H ), 4.94 (d, J = 4.7 Hz, 1H, OH), 4.34 (dd, J = 14.8, 4.3 Hz,
1H, Cb-N-CH2CHOH), 4.22 (dd, J = 14.8, 6.6 Hz, 1H, Cb-N-CH2CHOH), 3.98 (dd, J = 10.4, 5.1
Hz, 1H, CHOH), 2.73 (s, 4H, Pip-N-(CH2)2), 2.39 (d, J = 13.0 Hz, 4H, Pip-N-(CH2)2), 2.37 (s,
1H, Pip-N-CH2CHOH), 2.31 (dd, J = 12.6, 5.5 Hz, 1H, Pip-N-CH2CHOH), 2.12 (s, 3H, COCH3)
ppm; 13C NMR (DMSO-d6) δ: 169.6, 144.0, 141.1, 140.3, 133.6, 129.3, 128.9, 126.5, 125.2,
121.3, 120.8, 119.5, 119.2, 112.9, 110.5, 81.8, 67.2, 61.9, 52.8, 48.2, 46.2, 24.6 ppm.
N-(4-((4-(3-(3,6-Di-tert-butyl-9H-carbazol-9-yl)-2-hydroxypropyl)piperazin-1-
yl)sulfonyl)phenyl)acetamide (15e). Sulfonamide intermediate 15e was produced according to
the similar process like compound 5a. Compound 4 (0.41 g, 1.20 mmol), 3,6-di-tert-butyl-9H-
carbazole (0.50 g, 1.80 mmol) and TEA (1.40 mL, 10.00 mmol) were used starting materials.
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(DMSO-d6) δ: 10.40 (s, 1H, NHCOCH3), 8.10 (s, 2H, Cb-4,5-H), 7.86 (d, J = 8.6 Hz, 2H, Ph-
2,6-H), 7.68 (d, J = 8.6 Hz, 2H, Ph-3,5-H), 7.38 (q, J = 8.9 Hz, 4H, Cb-1,2,7,8-H), 4.88 (d, J =
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4.8 Hz, 1H, OH), 4.28 (dd, J = 14.8, 4.0 Hz, 1H, Cb-N-CH2CHOH), 4.15 (dd, J = 14.8, 6.9 Hz,
1H, Cb-N-CH2CHOH), 3.97 (dd, J = 9.9, 4.7 Hz, 1H, CHOH), 2.84 (s, 4H, Pip-N-(CH2)2), 2.45
(s, 4H, Pip-N-(CH2)2), 2.38 (dd, J = 12.5, 6.3 Hz, 1H, Pip-N-CH2CHOH), 2.32 (dd, J = 12.6, 5.9
Hz, 1H, Pip-N-CH2CHOH), 2.09 (s, 3H, COCH3), 1.39 (s, 18H, Cb-3,6-C(CH3)3) ppm; 13C
NMR (DMSO-d6) δ: 169.5, 144.0, 141.3, 139.6, 129.3, 123.3, 122.5, 119.1, 116.4, 109.6, 67.7,
61.9, 52.9, 48.1, 46.4, 34.8, 32.4, 24.6 ppm.
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(9H-carbazol-9-yl)propan-2-ol
(16a).
Sulfonamide derivative 16a was generated by the similar process depicted for compound 6a.
Compound 15a (0.10 g, 0.20 mmol) and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 16a was acquired as yellow solid. Yield: 73.5%; mp: > 250
oC; 1H NMR (DMSO-d6) δ: 8.14−8.03 (m, 2H, Cb-4,5-H), 7.63−7.52 (m, 2H, Cb-1,8-H), 7.35 (s,
4H, Ph-2,3,5,6-H), 7.20−7.12 (m, 2H, Cb-2,7-H), 6.70 (d, J = 6.7 Hz, 2H, Cb-3,6-H), 6.15 (s,
2H, Ph-NH2), 4.98 (s, 1H, OH), 4.37 (d, J = 11.1 Hz, 1H, Cb-N-CH2CHOH), 4.26 (d, J = 6.8 Hz,
1H, CHOH), 4.06 (d, J = 62.1 Hz, 1H, Cb-N-CH2CHOH), 3.17 (s, 2H, Pip-N-CH2CHOH), 2.67
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(s, 4H, Pip-N-(CH2)2), 2.39 (s, 4H, Pip-N-(CH2)2) ppm; C NMR (DMSO-d6) δ: 141.1, 130.1,
125.8, 122.6, 120.4, 119.1, 113.3, 110.3, 67.3, 62.1, 52.9, 49.1, 46.2 ppm; HR-ESIMS m/z calcd
for C25H28N4O3S [M + H]+, 465.1960; found, 465.1960.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(2-bromo-9H-carbazol-9-yl)propan-2-ol
(16b). Sulfonamide derivative 16b was generated by the same process depicted for compound
6a. Compound 15b (0.10 g, 0.17 mmol) and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 16b was acquired as brown solid. Yield: 72.2%; mp:
188−190 oC; 1H NMR (DMSO-d6) δ: 8.15 (d, J = 7.4 Hz, 1H, Cb-4-H), 8.09 (d, J = 8.0 Hz, 1H,
Cb-5-H), 7.95 (s, 1H, Cb-1-H), 7.71 (d, J = 7.6 Hz, 1H, Cb-8-H), 7.47 (t, J = 7.3 Hz, 1H, Cb-7-
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H), 7.37 (d, J = 8.6 Hz, 2H, Ph-2,6-H), 7.33 (d, J = 8.0 Hz, 1H, Cb-3-H), 7.23 (t, J = 7.3 Hz, 1H,
Cb-6-H), 6.68 (d, J = 8.6 Hz, 2H, Ph-3,5-H), 6.21 (s, 2H, Ph-NH2), 5.94 (s, 1H, OH), 4.45 (s,
1H, CHOH), 4.38 (d, J = 4.9 Hz, 2H, Cb-N-CH2CHOH), 3.60 (d, J = 12.1 Hz, 4H, Pip-N-
(CH2)2), 3.33−3.13 (m, 4H, Pip-N-(CH2)2), 2.73 (t, J = 11.1 Hz, 1H, Pip-N-CH2CHOH),
2.67−2.59 (m, 1H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 154.2, 142.1, 141.3, 130.2,
126.6, 122.2, 121.9, 120.8, 120.0, 119.0, 113.3, 110.6, 65.0, 59.2, 52.2, 50.9, 47.5 ppm; HR-
ESIMS m/z calcd for C25H27BrN4O3S [M + H]+, 543.1065; found, 543.1064.
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(3,6-dibromo-9H-carbazol-9-yl)propan-2-
ol (16c). Sulfonamide derivative 16c was generated by the similar process depicted for
compound 6a. Compound 15c (0.10 g, 0.15 mmol) and hydrochloric acid solution (5 mL, 40%)
were used starting materials. The pure product 16c was acquired as white solid. Yield: 57.9%;
o
1
mp: 180−182 C; H NMR (DMSO-d6) δ: 8.47 (s, 2H, Cb-4,5-H), 7.68 (s, 2H, Cb-1,8-H), 7.60
(s, 2H, Cb-2,7-H), 7.36 (d, J = 7.8 Hz, 2H, Ph-2,6-H), 6.68 (d, J = 6.9 Hz, 2H, Ph-3,5-H), 6.19
(s, 2H, Ph-NH2), 5.92 (s, 1H, OH), 4.39 (s, 3H, CHOH, Cb-N-CH2CHOH), 3.58 (s, 4H, Pip-N-
(CH2)2), 3.21 (s, 4H, Pip-N-CH2), 2.71 (s, 2H, Pip-N-CH2), 2.62 (s, 1H, Pip-N-CH2CHOH), 2.39
(s, 1H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ: 140.2, 139.3, 130.2, 129.2, 123.7,
113.7, 113.3, 112.8, 111.4, 64.8, 59.0, 52.3, 49.1, 43.2 ppm; HR-ESIMS m/z calcd for
C25H26Br2N4O3S [M + H]+, 621.0171; found, 621.0170.
1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(3-iodo-9H-carbazol-9-yl)propan-2-ol
(16d). Sulfonamide derivative 16d was generated by the same process depicted for compound
6a. Compound 15d (0.10 g, 0.16 mmol) and hydrochloric acid solution (5 mL, 40%) were used
starting materials. The pure product 16d was acquired as yellow solid. Yield: 58.1%; mp:
o
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196−198 C; H NMR (DMSO-d6) δ: 8.54 (s, 1H, Cb-5-H), 8.19 (d, J = 7.5 Hz, 1H, Cb-4-H),
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7.69 (d, J = 7.8 Hz, 2H, Cb-2,8-H), 7.56 (d, J = 8.1 Hz, 1H, Cb-1-H), 7.49−7.44 (m, 1H, Cb-7-
H), 7.36 (d, J = 8.4 Hz, 2H, Ph-2,6-H), 7.22 (t, J = 7.4 Hz, 1H, Cb-6-H), 6.68 (d, J = 8.4 Hz, 2H,
Ph-3,5-H), 6.20 (s, 2H, Ph-NH2), 5.95 (s, 1H, OH), 4.43 (s, 1H, CHOH), 4.38 (s, 2H, Cb-N-
CH2CHOH), 3.63−3.47 (m, 4H, Pip-N-(CH2)2), 3.31−3.09 (m, 4H, Pip-N-(CH2)2), 2.74−2.67 (m,
1H, Pip-N-CH2CHOH), 2.64−2.58 (m, 1H, Pip-N-CH2CHOH) ppm; 13C NMR (DMSO-d6) δ:
154.2, 141.1, 140.3, 133.9, 130.2, 129.0, 126.8, 125.4, 121.5, 121.1, 119.9, 113.3, 113.0, 110.5,
82.3, 64.9, 52.2, 50.7, 49.1, 47.5 ppm; HR-ESIMS m/z calcd for C25H27IN4O3S [M + H]+,
591.0927; found, 591.0926.
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1-(4-((4-Aminophenyl)sulfonyl)piperazin-1-yl)-3-(3,6-di-tert-butyl-9H-carbazol-9-
yl)propan-2-ol (16e). Sulfonamide derivative 16e was generated by the similar process depicted
for compound 6a. Compound 15e (0.10 g, 0.16 mmol) and hydrochloric acid solution (5 mL,
40%) were used starting materials. The pure product 16e was acquired as white solid. Yield:
o
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77.3%; mp: 204−206 C; H NMR (DMSO-d6) δ: 8.12 (s, 2H, Cb-4,5-H), 7.40 (s, 4H, Cb-
1,2,7,8-H), 7.36 (d, J = 8.7 Hz, 2H, Ph-2,6-H), 6.68 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.09 (s, 2H,
Ph-NH2), 4.86 (s, 1H, OH), 4.29 (dd, J = 14.7, 3.8 Hz, 1H, Cb-N-CH2CHOH), 4.16 (s, 1H, Cb-
N-CH2CHOH), 3.99 (s, 1H, CHOH), 2.81 (s, 4H, Pip-N-(CH2)2), 2.46 (s, 4H, Pip-N-(CH2)2),
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2.39−2.32 (m, 2H, Pip-N-CH2CHOH), 1.40 (s, 18H, Cb-3,6-C(CH3)3) ppm; C NMR (DMSO-
d6) δ: 141.4, 139.6, 130.1, 123.4, 122.5, 116.5, 113.2, 109.6, 67.6, 61.9, 52.9, 49.1, 46.3, 34.8,
32.4 ppm; HR-ESIMS m/z calcd for C33H44N4O3S [M + H]+, 577.3212; found, 577.3211.
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NHCOCH3
NHCOCH3
i
O
NH
ii
H3COCHN
H3COCHN
S N
O
3
7
8
9
SO2Cl
R3
2
1
iii
v
O
R2
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O
N
iv
N
X
S N
4
O
N
N
O
R1
H3COCHN
S N
5a-d
HO
R3
N
O
R4
N
R2
vi
N
N
O
N
HO
N
X
H3COCHN
S N
O
7a-b
O
N
N
R1
H2N
S N
HO
6a-d
vi
N
O
R4
N
X = CH, R = CH3, R = H, R3 = NO2
1
2
5, 6:
a,
b,
c,
d,
N
O
N
N
X = CH, R = H, R2 = NO2, R3 = H
1
7, 8:
H2N
S N
O
4
X = CH, R = H, R2 = Ph, R3 = H
1
a,
b,
R = H
HO
8a-b
4
X = N, R1 = H, R2 = H , R3 = H
R = CH3
Scheme 1. Synthesis of five-membered azole sulfonamide hybrids. Reagents and conditions: (i)
o
o
chlorosulfonic acid, 0 C, 2 h; (ii) piperazine, dichloromethane, 0 C, 1 h; (iii) potassium
o
carbonate, epichlorohydrin, acetonitrile, 80 C, 18 h; (iv) imidazoles or triazole, triethylamine,
acetonitrile, 80 oC, 24 h; (v) tetrazoles, triethylamine, acetonitrile, 80 oC, 24 h; (vi) hydrochloric
acid, ethanol, 78 oC, 1 h.
N
O
N
O
N
S
viii
N
H3C
H3COCHN
H3COCHN
H2N
S N
O
H3COCHN
S N
O
O
HO
4
11
vii
ix
ix
N
N
N
O
N
S
NH
NH
O
N
S
S N
O
N
H3C
H2N
S N
O
HO
9
HO
12
N
N
O
N
S
S N
O
HO
10
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Scheme 2. Synthesis of thioether-bridged sulfonamide hybrids. Reagents and conditions: (vii)
5
6
o
1H-1,2,4-triazole-3-thiol, triethylamine, acetonitrile, 80 C, 24 h; (viii) 1-methyl-1H-imidazole-
7
8
9
2-thiol, triethylamine, acetonitrile, 80 oC, 24 h; (ix) hydrochloric acid, ethanol, 78 oC, 1 h.
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O
O
N
N
R6
R7
xi
H3COCHN
H3COCHN
H3COCHN
S N
O
S N
O
O
HO
N
4
xii
x
N
R5
15a-e
X
R8
N
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N
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N
R6
R7
S N
O
H2N
S N
O
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HO
X = CH, R5 = H
X = CH, R5 = CH3
X = N, R5 = H
xii
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13, 14:
16a-e
R8
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R = H, R = H,
R8 = H
R8 = H
R8 = Br
R8 = H
8
6
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15, 16: a,
H2N
S N
O
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7
b,
c,
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e,
R = Br, R = H,
HO
N
6
7
R = H, R = Br,
R5
X
6
7
R = H, R = I,
N
14a-c
6
7
R = H, R = C(CH3)3, R = C(CH3)3
Scheme 3. Synthesis of fused-azole sulfonamide hybrids. Reagents and conditions: (x)
o
benzimidazoles or benzotriazole, triethylamine, acetonitrile, 80 C, 24 h; (xi) carbazoles,
triethylamine, acetonitrile, 80 oC, 24 h; (xii) hydrochloric acid, ethanol, 78 oC, 1 h.
Antibacterial assay. The prepared sulfonamide hybrids 4−16 were tested for their antibacterial
activities against five Gram-positive bacteria (drug-resistant Enterococcus faecalis and
Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus N315 (MRSA),
Staphylococcus aureus ATCC 29213 and Staphylococcus aureus ATCC 25923) and six Gram-
negative bacteria (drug-resistant Pseudomonas aeruginosa, Escherichia coli, Acinetobacter
baumanii, and Klebsiella pneumonia, Escherichia coli ATCC 25922, Pseudomonas aeruginosa
ATCC 27853). Detailed protocols were performed according to the literature report.44 The
minimum inhibitory concentrations (MICs) for compounds 4−16 were summarized in Table 1.
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Antifungal assay. The synthesized sulfonamide hybrids 4−16 were also tested for their
antifungal activities against five fungi (drug-resistant Candida albicans, Candida tropical and
Aspergillus fumigates, Candida parapsilosis ATCC 22019 and Candida albicans ATCC 90023).
Detailed protocols were performed according to the literature report.44 The MICs for compounds
4−16 were summarized in Table 2.
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Bacterial membrane permeabilization assay. A 5 h grown culture (mid log phase) of E. coli
was harvested (5 min, 3500 rpm), washed, and resuspended in glucose (5 mM) and HEPES
buffer (5 mM) in 1:1 ratio (pH = 7.2). The tested compound 6a (10 μL, 12 × MIC) was added to
a cuvette containing the bacterial suspension (2 mL) and propidium iodide (PI) (10 μM).
Fluorescence was monitored at 535 nm (excitation wavelength) and 617 nm (emission
wavelength). PI, as a fluorescence dye for membrane permeability, was monitored by increasing
fluorescence for 2 h.
Cytotoxicity assay. A standard MTT assay was applied to evaluate the cytotoxicity of
compound 6a against human breast cancer MCF-7 cells. Cells were seeded in a 96-well plate at a
density of about 5 × 104 and cultured in a DMEM culture medium (10% serum and 1%
penicillin/streptomycin) in an incubator for 24 h (37 °C, 5% CO2). Samples of different
concentrations were then added into different wells and incubated with cells for another 48 h.
Afterwards, each well was added the MTT stock solution (25 μL, 5 mg/mL in PBS). After
incubation for another 4 h, the DMEM medium was removed and DMSO (150 μL) was added.
The optical density per well at 570 nm was measured by a microplate reader (Bio-Rad 680). The
absorbance values were normalized to wells in which cells were not treated with compound 6a.
Data were presented as the average values with standard deviations.
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Resistance study. The most active metronidazole derivative 6a was further investigated for the
bacterial resistance. The detailed protocols were performed according to the literature.4
Bactericidal kinetic assay. The rate of bactericidal activity, namely, the rate at which the
compounds killed bacteria was evaluated by performing time-kill kinetics. Briefly, E. coli strains
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o
grew for 6 hours in a suitable medium at 37 C and were diluted in the appropriate medium.
Metronidazole derivative 6a and norfloxacin were added to the bacterial solution (E. coli of
approximately 5.0  104 CFU/mL) at concentrations of 4  MIC in a 96-well plate and incubated
o
at 37 C. At different time intervals, aliquots from the solution (30 µL) were taken out and 10-
fold diluted in 0.9% saline. And then the dilutions (30 µL) were plated on respective agar plates
o
and incubated at 37 C for 24 h. The results after counting bacterial colonies represented in
logarithmic scale: log (CFU/mL) vs time (in hour).
Drug combination assay. The drug combination studies between the most active metronidazole
derivative 6a and standard drugs norfloxacin and fluconazole were investigated by 2-fold
dilution checkerboard assay method with concentration values from 1/64 to 4-fold the MIC value
of each molecule. The drug combination effect is usually expressed using fractional inhibitory
concentration (FIC) index. The FIC value may be calculated as FIC = MIC of molecule A in
mixture/MIC of molecule A alone + MIC of molecule B in mixture/MIC of molecule B alone.
Using this method, FIC  0.5 represents synergism, FIC > 0.5 and  1.0 represents additivism,
FIC > 1 and  2 represents an indifferent effect, and FIC > 2 represents antagonism.45
Isolating genomic DNA from drug-resistant E. coli strain. The genomic DNA of E. coli was
extracted according to the literature.44
RESULTS AND DISCUSSION
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Antibacterial activity. The antibacterial screening in vitro demonstrated that few of the target
compounds displayed good efficacy as shown in Table 1. Remarkably, metronidazole hybrid 6a
was the most active molecule against clinical drug-resistant Escherichia coli, which could be
further investigated as a potential antibacterial agent.
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In the series of imidazole sulfonamides, hybrids 5a−b and 6a−c exhibited moderate
antibacterial activities against Gram-negative P. aeruginosa, A. baumanii and E. coli as well as
Gram-positive E. faecalis and S. aureus, of which compounds 5a (MIC = 0.069 mM) and 5b
(MIC = 0.071 mM) showed better activity against E. coli. Nitroimidazole derivative 6b displayed
weak inhibition against E. faecalis (MIC = 0.156 mM), interestingly it improved the potency
against E. coli (MIC = 0.039 mM). Typically, metronidazole sulfonamide 6a displayed broad
antibacterial spectrum, which had MIC values ranging from 0.038 to 0.075 mM toward P.
aeruginosa, E. faecalis and A. baumanii, and 42, 11, 11-fold greater in comparison to reference
drug norfloxacin (MIC = 1.605 or 0.803 mM), respectively. It was noticeable that derivative 6a
highlighted superior activity in the inhibition of E. coli strains (MIC = 0.019 mM), which was
84-fold more potent than norfloxacin (MIC = 1.605 mM). However, triazole compounds 5d
(MIC = 0.078 mM) and 6d (MIC = 0.087 mM) were primarily susceptive to E. coli. While
tetrazole sulfonamides 7a-b and 8a-b were sensitive toward some tested bacterial strains. These
results dramatically manifested that the imidazole hybrids were more favorable for the
antibacterial activity. Especially, derivative 6a could have great potential to become an E. coli
inhibitor, therefore we selected this target compound for further studies. Nevertheless, the
triazole and tetrazole sulfonamides were almost not conducive to the inhibiting ability of bacteria
other than E. coli.
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