Introducing broadened antibacterial activity to rhodanine derivatives targeting enoyl-acyl carrier protein reductase

Article abstract of DOI:10.1248/cpb.c18-00663

Broadened antibacterial activity was introduced to rhodanine derivatives targeting Mycobacterial tuberculosis enoyl-acyl carrier protein reductase (Mtb InhA) by recruiting feature of xacins to bring DNA Gyrase B inhibitory capability. This is significant for preventing further bacterial injections in the tuberculosis treatment. The most potent compound Cy14 suggested comparable bioactivity (IC₅₀=3.18μM for Mtb InhA; IC₅₀=10nM for DNA Gyrase B) with positive controls. Structure–activity relationship discussion and molecular docking model revealed the significance of rhodanine moiety and derived methoxyl on meta-position, pointing out orientations for future modification.

Full text of DOI:10.1248/cpb.c18-00663

Vol. 67, No. 2
Chem. Pharm. Bull. 67, 125–129 (2019)
125
Regular Article
Introducing Broadened Antibacterial Activity to Rhodanine Derivatives
Targeting Enoyl-Acyl Carrier Protein Reductase
,a
Zhi-Gang Sun,a,b,# Yun-Jie Xu,a,# Jian-Fei Xu,a Qi-Xing Liu,a Yu-Shun Yang,* and
,a
Hai-Liang Zhu*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University; Nanjing 210023, China: and
b Central Laboratory, Linyi Central Hospital; Linyi 276400, Shandong, China.
Received August 21, 2018; accepted November 8, 2018
Broadened antibacterial activity was introduced to rhodanine derivatives targeting Mycobacterial tuber-
culosis enoyl-acyl carrier protein reductase (Mtb InhA) by recruiting feature of xacins to bring DNA Gyrase
B inhibitory capability. This is significant for preventing further bacterial injections in the tuberculosis
treatment. The most potent compound Cy14 suggested comparable bioactivity (IC50 =3.18 µM for Mtb InhA;
IC50 =10nM for DNA Gyrase B) with positive controls. Structure–activity relationship discussion and mo-
lecular docking model revealed the significance of rhodanine moiety and derived methoxyl on meta-position,
pointing out orientations for future modification.
Key words bacterial infection; enoyl-acyl carrier protein reductase; DNA Gyrase B; computer assistant drug
design; rhodanine; molecular docking
self.20) Now we started from the check point of studied rhoda-
Introduction
Searching for novel drugs treating bacterial infections is nine derivatives with revealed potency and molecular interac-
of high priority because of emerging bacterial resistance1,2) tions.ꢀTheꢀfirstꢀtaskꢀweꢀcompletedꢀwasꢀrationallyꢀbringingꢀtheꢀ
and clinical complication.3,4) Recruiting known targets and cyclopropane feature of xacins. Molecular overlay was shown
interactions to seek for valid drug candidates is an available in Fig. 1A, with the linker regulated, potential candidates
strategy.5) Among present methods on controlling bacte- couldꢀmimicꢀtheꢀcyclopropaneꢀfeatureꢀofꢀMoxifloxacinꢀbetter.ꢀ
rial pathogens, blocking their biosynthesis of fatty acids has Meanwhile, they suggested similar conformation to previous
been convinced practicable.6,7) In this pathway, the enoyl-acyl reported InhA inhibitory compound with alanine (ALA) resi-
carrier protein reductase (InhA) is highly conserved and sig- due.20)ꢀIntroducingꢀbenzeneꢀringꢀcouldꢀnotꢀsucceedꢀinꢀsuchꢀanꢀ
nificant.8) InhA has been validated as a potent target for tuber- imitation as seen in Fig. 1B. Therefore we enriched the substi-
culosisꢀtreatmentꢀbyꢀtheꢀfirst-lineꢀdrugꢀisoniazid.9) Since com- tutes to obtain the series in this work.
puter assistant drug design (CADD) was introduced,10) various
categories of InhA inhibitors have been discovered, including
Experimental
Chemistry All commercially available chemicals were
triclosan,11) pyrrolidine carboxamides,12)ꢀ isoniazidꢀ (INH)-
nicotinamide adenine dinucleotide (NAD) analogues13) and directlyꢀ usedꢀ withoutꢀ furtherꢀ purification.ꢀ 1H-NMR and
pyridomycin.14) The in vitro inhibition of them on Mycobacte- 13C-NMR spectra were scanned by Bruker AM 600 (Rhe-
rial tuberculosis (Mtb) was still not ideal. Moreover, during nistetten-Forchheim, Germany) spectrometer and resolved
the aggravation of tuberculosis, serious complication including with MestreNova software. Mass spectra were obtained
further bacterial injection often occurs along with the decline from an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS.
of immunity.15) Therefore introducing broadened antibacterial Substitutedꢀ benzaldehydesꢀ (1ꢀmmol)ꢀ wasꢀ addedꢀ toꢀ rhodanineꢀ
activity to merely InhA inhibition seems essential.
(1mmol) alcohol solution (10mL). After urea (1.5mmol) was
In this work, we introduced broadened antibacterial activ- added,ꢀtheꢀreactionꢀcontinuedꢀinꢀrefluxꢀforꢀ5ꢀh.ꢀAfterꢀconfirm-
ity to rhodanine derivatives targeting InhA. Maintaining the ing the completion of the reaction by TLC the sediment was
inhibition of Mtb InhA, feature of xacins was added to bring filtered,ꢀ washedꢀ withꢀ ethanolꢀ andꢀ driedꢀ toꢀ obtainꢀ intermedi-
DNA Gyrase B inhibitory activity. The blocking effect on the
growth of other Gram-positive (Staphylococcus aureus) and
Gram-negative (Pseudomonas aeruginosa) pathogenic bacteria
was also evaluated.
Preliminary design of our series came from the rational-
izationꢀ ofꢀ recruitingꢀ rhodanineꢀ moiety.ꢀ Despiteꢀ activitiesꢀ onꢀ
various bacterial-relating targets including penicillin-binding
proteins,16) RNA polymerase17)ꢀ andꢀ oxyreductaseꢀ enzymes,18)
it has been involved in enhancing inhibition of tightly related
homologous Plasmodium falciparum FabI19) and then InhA it-
Fig. 1. Molecular Overlay of (A) Linker-Regulated Candidates with Cy-
clopropane Feature and (B) Aryl Ring-Introduced Compounds
# These authors contributed equally to this work.
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀys_yang@nju.edu.cn;ꢀzhuhl@nju.edu.cn
© 2019 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 67, No. 2 (2019)
Chart 1. General Synthesis of Cy1–Cy14, Cx1–Cx6 and Their Structures
ates B. Then intermediates B (1mmol) were initially dissolved (IPTG) for 4h and controlled by sodium dodecyl sulfate-
in heated ethanol (8mL), then the ethanol solution (2mL) of polyacrylamide gel electrophoresis (SDS-PAGE). The bacteria
(bromomethyl)cyclopropane (1mmol) and sodium ethylate were harvested by 5000r/min centrifugation and suspended in
(1.5mmol) was added dropwise. The mixture was heated to purificationꢀ bufferꢀ (20ꢀmMꢀ Tris–HCl,ꢀ 500ꢀmMꢀ NaCl,ꢀ 20ꢀmMꢀ
55°C and kept reacting overnight. Target compounds Cy1– imidazole,ꢀ pHꢀ 8.0).ꢀ Ultrasonicationꢀ wasꢀ performedꢀ andꢀ puri-
Cy14 were obtained through column chromatography and ficationꢀ wasꢀ conductedꢀ throughꢀ Nit-NTAꢀ Superflowꢀ columnꢀ
refinedꢀ byꢀ recrystallization.ꢀ Compoundsꢀ Cx1–Cx6 for com- withꢀ 10–500ꢀmMꢀ imidazoleꢀ eluent.ꢀ Afterꢀ examiningꢀ theꢀ ab-
parisonꢀ wereꢀ synthesizedꢀ via the same method by using cor- sorption under 280nm, we collected the protein and checked
responding bromide to replace (bromomethyl)cyclopropane. by SDS-PAGE. According to method in reference,20) Mtb InhA
Detailed information and NMR data of compounds Cy1–Cy14 inhibition was measured by monitoring the initial velocities
and Cx1–Cx6 were presented in “Supplementary Materials.”
(120s) of the decrease in the absorbance of reduced nicotin-
Determination of Minimum Inhibitory Concentration amide adenine dinucleotide (NADH) at 340nm, 25°C. The
(MIC) Mtb was grown in 7H9 medium with OADC for 24h standard reaction mixture of 1.0mL included buffer (30mM
at 37°C under microaerobi conditions (5% O2, 10% CO2, and PIPES, 150mM NaCl, 1mM ethylenediaminetetraacetic acid
85% N2). Methicillin-sensitive Staphylococcus aureus ATCC (EDTA), pH 6.8), 200µM trans-2-decenoyl-N-acetyl cyste-
25923, methicillin-resistant Staphylococcus aureus ATCC amine, 100µM NADH and 1µg recombinant Mtb InhA. The
29213 and Pseudomonas aeruginosa ATCC 27583 were grown data of positive control was close to literature value. All as-
in Brucella Broth supplemented with 10% heat-inactivated says were performed in triplicates.
horse serum. Inoculums containing 1×105 bacteria were
In Silico Assay The absorption, distribution, metabolism,
seeded in 96 well plates with varying concentrations of test excretion, and toxicity (ADMET) study was conducted by
compounds and incubated at 37°C for 48h. Absorbance was Calculate Molecular Properties in Small Molecules module of
measured at 590nm. Wells without test compounds and with Discovery Studio 3.5 (Accelrys, Inc., San Diego, CA, U.S.A.).
media only served as control and blank, respectively. The The ADMET properties map and data were provided by the
percentages of growth inhibition were calculated, and the ADMET Descriptors tool. Molecular docking was carried out
concentrations of compounds producing 80% inhibition were by Discovery Studio 3.5 (Accelrys, Inc.) CDOCKER protocol
considered as MIC values.
as described in literature.21) In this work, the crystal structures
Cytotoxicity 293T (human embryonic kidney cell line) of Mtb InhA (PDB code: 4R9S) and S. aureus DNA Gyrase B
was used for cytotoxicity evaluation via 3-(4,5-dimethylthia- (PDB code: 4URO) were used instead.
zol-2-yl)-2,5-diphenyltetrazoliumꢀ bromideꢀ (MTT)ꢀ methodꢀ ac-
cording to reference.21)
Results and Discussion
Mtb InhA Inhibition Assay Mtb InhA was expressed in
The general synthesis method and the structures of cy-
BL21(DE3) Escherichia coli strain. After incubation overnight clopropylmethyl rhodanine derivatives Cy1–Cy14 (Cy was
in Kanamycin-containing plates, continuous incubation in fromꢀ ‘Cyclo’)ꢀwereꢀ organizedꢀinꢀChartꢀ 1.ꢀTheyꢀwereꢀallꢀsyn-
2mL 2× Yt medium at 37°C 200r/min was conducted for 14h. thesizedꢀ andꢀ testedꢀ forꢀ theꢀ firstꢀ time.ꢀ Rhodanineꢀ wasꢀ addedꢀ
Thenꢀ 1ꢀmLꢀ fluidꢀ wasꢀ addedꢀ intoꢀ 100ꢀmLꢀ 2× Yt medium and toꢀ differentꢀ substitutedꢀ benzaldehydeꢀ withꢀ theꢀ participationꢀ
incubated at 37°C 240r/min till OD600 was 0.6. The expression of urea to yield intermediate B. Subsequent reaction with
was induced by 1mM isopropyl β-D-thiogalactopyranoside (bromomethyl)cyclopropane and sodium ethylate in ethanol

Vol. 67, No. 2 (2019)
Chem. Pharm. Bull.
127
led to the target compounds. Compounds Cx1–Cx6 (Cx was IC50 =18.2 µM)>ortho-(Cy11, IC50 =35.2 µM). When multi-
fromꢀ ‘X-factor’)ꢀ forꢀ comparisonꢀ wereꢀ synthesizedꢀ via the positions were picked, effect brought by meta- and para-
sameꢀ methodꢀ byꢀ usingꢀ correspondingꢀ bromide.ꢀ Refinedꢀ com- substitutes could be overlying, according to the fact that
pounds were obtained through recrystallisation. All of them trimethoxyl (Cy14, IC50 =3.18 µM) and dimethoxyl (Cy13,
gave satisfactory analytical and spectroscopic data.
IC50 =11.7 µM) both exceeded single methoxyl at any posi-
Before bioassay, ADMET properties as a preliminary tion. Comparisons Cx1–Cx6 were introduced based on
evaluation for the basic druggability were shown in Figure S1. theꢀ originalꢀ (Phenyl)ꢀ andꢀ optimizedꢀ (3,4,5-trimethoxyphe-
Since the design of linker and substitutes was strict, all com- nyl) situations of Cy series. N-substituent factors includ-
pounds indicated practicable pharmacokinetics potential such ing –CH2CH2CH2COOH, –CH2COOH and –CH2CN were
as logP and PSA (polar surface area) values.
involved to bring more SAR discussion. Agreeing with the
With methods according to references,20,22) the Mtb InhA aboveꢀ SAR,ꢀ trimethoxylꢀ onꢀ benzeneꢀ ringꢀ couldꢀ improveꢀ
inhibitory effect, growth blocking of Mtb and S. aureus DNA the activity during the variation of N-substituent factors.
Gyrase B inhibition were evaluated. The designing concept of Moreover, for N-substitutes, it seemed that shorter aliphatic
this series included two major concerns. One was maintaining chain was preferred (Cx1>Cx3; Cx2>Cx4) and electron-
the inhibition of Mtb InhA, the other was introducing DNA withdrawing was not the good choice (Cy1 >Cx3>Cx5;
Gyrase B inhibition to block the growth of Mtb and impede Cy14 >Cx4>Cx6). It was attractive that the top hits of our
secondaryꢀinfection.ꢀAꢀgeneralꢀglanceꢀofꢀallꢀsynthesizedꢀcom- series showed comparable Mtb InhA inhibition effect with the
pounds indicated a majority of them achieved both.
top one with aliphatic chain17–20). For bringing DNA Gyrase
With data in Table 1 compared, preliminary structure– B inhibition, hints seemed a bit different. The high elec-
activity relationship (SAR) studies on Mtb InhA and DNA tronic density requirement still existed but was more ambigu-
Gyrase B were provided. For enhancing Mtb InhA inhibi- ous with the tendency –NHAc (Cy9, IC50 =15nM)>–NO2
tion,ꢀseveralꢀhintsꢀcouldꢀbeꢀorganized.ꢀInitially,ꢀ para-position (Cy7, IC50 =20nM)>–N(Me)2 (Cy8, IC50 =23nM)>–Me
basically required electron-donating groups, with the cor- (Cy5, IC50 =32nM)>–OMe (Cy6, IC50 =35nM)>–H (Cy1,
responding order –NHAc (Cy9, IC50 =6.85µM)>–N(Me)2 IC50 =47nM)>–F
(Cy2,
IC50 =76nM)>–Cl
(Cy3,
(Cy8, IC50 =7.73 µM)>–OMe (Cy6, IC50 =18.2 µM)> IC50 =89nM)>–Br (Cy4, IC50 =ꢀ110ꢀnM).ꢀThenꢀfixedꢀelectron-
–NO2 (Cy7, IC50 =22.3µM)>–Br (Cy4, IC50 =24.8µM)> donating group methoxyl inferred choice on positions as meta-
–Me (Cy5, IC50 =27.1 µM)>–H (Cy1, IC50 =28.5µM)>–F (Cy12, IC50 =21nM)>ortho-(Cy11, IC50 =28nM)>para-
(Cy2, IC50 =29.8 µM)>–Cl (Cy3, IC50 =31.4 µM). With (Cy6, IC50 =35nM). Viewing multi-substituent situations,
electron-donating group methoxyl picked, substitute posi- the result was similar with trimethoxyl (Cy14, IC50 =10nM)
tion indicated meta-(Cy12, IC50 =14.5 µM)>para-(Cy6, and dimethoxyl (Cy13, IC50 =18nM) both performed better
potency. The comparisons (Cx1–Cx6) and reference com-
Table 1. The Mtb InhA Inhibitory, Mtb Growth Blocking and DNA Gy-
rase B Inhibitory Activities of Cy1–Cy14
pounds (Ref20–1; Ref20–17) did not indicate potency against
DNA Gyrase B, inferring the importance of introducing the
cyclopropane feature. Moreover, the Mtb InhA inhibitory, Mtb
growth blocking and DNA Gyrase B inhibitory activities of
Cy1–Cy14 presented basically consistency according to Fig. 2.
To judge whether introducing DNA Gyrase B inhibition
brought broad-spectrum antibacterial potency, we used Gram-
positive MSSA (methicillin-sensitive Staphylococcus aureus
ATCC 25923), MRSA (methicillin-resistant Staphylococcus
aureus ATCC 29213) and Gram-negative (Pseudomonas aeru-
ginosaꢀATCCꢀ27583)ꢀpathogenicꢀbacteria.ꢀTopꢀfiveꢀcompoundsꢀ
(Cy14, Cy9, Cy8, Cy13 and Cy12) were selected as repre-
IC50 (µM)
Mtb InhA
MIC (µM)
IC50 (nM)
DNA Gyrase B
Compounds
Mtb
Cy1
Cy2
Cy3
Cy4
Cy5
Cy6
Cy7
Cy8
Cy9
Cy10
Cy11
Cy12
Cy13
Cy14
Cx1
Cx2
Cx3
Cx4
Cx5
Cx6
28.5 1.82
29.8 2.04
31.4 2.51
24.8 2.01
27.1 1.98
18.2 0.91
22.3 1.76
7.73 0.89
6.85 0.91
40.1 2.86
35.2 1.72
14.5 1.19
11.7 1.06
3.18 0.57
83.6 7.15
73.9 6.88
58.4 5.11
50.7 4.71
71.2 6.84
67.1 6.22
15.3 1.36
17.6 1.58
18.2 2.02
20.1 1.77
12.5 0.97
8.24 1.13
9.86 1.08
1.75 0.31
0.52 0.09
21.8 1.93
18.1 1.84
4.18 0.57
1.68 0.21
0.35 0.04
68.2 5.79
65.1 5.92
45.1 4.03
40.4 3.81
63.9 5.72
59.5 5.37
47 4.5
76 6.8
89 10
110 13
32 2.5
35 3.1
20 2.1
23 2.1
15 1.1
43 3.8
28 3.2
21 1.8
18 2.3
10 1.0
>1000
>1000
830 60
750 50
>1000
>1000
Ref20–1
120 10.2
19.1 1.32
85.5 6.84
1.99 0.14
720 50
Ref20–17
>1000
Triclosan
6.30 1.20
>200
>200
8.84 1.12
1.73 0.21
>200
>1000
43 3.5
>1000
Moxifloxacin
Epalrestat
Fig. 2. The Mtb InhA Inhibitory, Mtb Growth Blocking and DNA Gy-
rase B Inhibitory Activities Presented Basically Consistency

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Chem. Pharm. Bull.
Vol. 67, No. 2 (2019)
Table 2. Broad-Spectrum Antibacterial and Cytotoxic Performances of Representatives
Compounds
MIC (µM) MSSA
MIC (µM) MRSA
MIC (µM) P. aeruginosa
IC50 (µM) 293T
Cy14
0.17 0.02
1.27 0.11
2.11 0.20
0.56 0.05
0.81 0.06
0.16 0.02
0.18 0.02
2.05 0.18
3.12 0.25
0.37 0.04
0.86 0.10
0.11 0.01
1.42 0.15
2.18 0.20
3.04 0.19
2.56 0.22
7.25 0.83
3.60 0.32
>200
175 15.8
188 16.5
>200
>200
>200
Cy9
Cy8
Cy13
Cy12
Moxifloxacin
Fig. 3. Docking Models of Representative Cy14 into (A) Mtb InhA and (B) DNA Gyrase B
Significantꢀinteractionsꢀandꢀresiduesꢀwereꢀexhibited.
B inhibitory activity. From the maps we could know the sig-
nificanceꢀofꢀrhodanineꢀmoietyꢀandꢀderivedꢀmethoxylꢀonꢀmeta-
position. Besides, 3D patterns and receptor surface models
of Cy14 into Mtb InhA and DNA Gyrase B in Fig. 4 indi-
cated deep occupation into the active pocket. Therefore future
modificationꢀ couldꢀ beꢀ hintedꢀ asꢀ involvingꢀ substitutesꢀ toꢀ offerꢀ
deeper impaction and adding moiety on rhodanine to supply
interactions with outer residues.
In conclusion, we have introduced broadened antibacterial
activity to InhA inhibitors containing rhodanine moiety
by bringing feature of xacins. Through rational design and
verificationꢀ ofꢀ biologicalꢀ activity,ꢀ theꢀ synthesizedꢀ seriesꢀ in-
dicated that a majority of them achieved both maintaining
Mtb InhA inhibition and introducing DNA Gyrase B inhibi-
tion. The most potent compound Cy14 suggested comparable
bioactivity (IC50 =3.18 µM for Mtb InhA; IC50 =10nM for
DNA Gyrase B) with positive controls. The growth blocking
performance on Mtb (MIC=0.35 µM) was also favorable. It
exhibited broadened antibacterial effect and low cytotoxicity.
Via molecular docking simulation, the binding patterns with
Fig. 4. 3D Model (A) and Receptor Surface Model (B) of Cy14 into
Mtb InhA; 3D Model (C) and Receptor Surface Model (D) of Cy14 into
DNA Gyrase B
sentatives. Cytotoxicity was also assessed using 293T human nonnegligibleꢀ interactionsꢀ wereꢀ visualized.ꢀ Accordingꢀ toꢀ theꢀ
embryonic kidney cell line. Seen in Table 2, all selected com- bindingꢀ situationsꢀ andꢀ SARꢀ discussion,ꢀ futureꢀ modificationꢀ
pounds suggested considerable broad-spectrum antibacterial with substitutes and stretching moiety were recommended.
capability and low toxicity.
Information in this work provided potential orientations for
Molecular docking simulation was conducted before syn- treating tuberculosis and preventing serious complication at
thesis to give preliminary screening. Now it was also used the same time, therefore hinted bi-target possibility in further
toꢀ visualizeꢀ possibleꢀ bindingꢀ information.ꢀ Theꢀ activeꢀ siteꢀ ofꢀ relevant researches.
Mtb InhA (PDB code: 4R9S) and DNA Gyrase B (PDB code:
4URO)ꢀ wereꢀ definedꢀ asꢀ bindingꢀ sites.ꢀ Theꢀ 2Dꢀ mapsꢀ ofꢀ theꢀ
Acknowledgments This work is supported by the Project
most potent compound Cy14 within both sites were depicted of Medical and Health Science Technology Development Pro-
in Fig. 3. The three possible hydrogen bonds (O…H-O, 2.27Å, gram in Shandong Province of China (No. 2016WS0242), the
141.868° with SER20; S…H-N, 2.36Å, 146.443° with LYS165; Linyi Science and Technology Innovation and Development
O…H-O,ꢀ 1.94ꢀÅ,ꢀ 144.035°ꢀ withꢀ THR196)ꢀ fixedꢀ Cy14 tightly Project(Medicine) (No. 201818033), the National Undergradu-
into Mtb InhA. Meanwhile, possible interactions including ate Innovation Program and the National Science Foundation
hydrogen bond (S…H-N, 2.25Å, 145.242° with ARG144) and of China (No. J1210026). We thank Miss Huairuo Xu from
π-cation (4.41Å) might cause better import of DNA Gyrase International Department American Division, Nanjing Jinling

Vol. 67, No. 2 (2019)
Chem. Pharm. Bull.
129
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Conflict of Interest Theꢀ authorsꢀ declareꢀ noꢀ conflictꢀ ofꢀ
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