Introducing Broadened Antibacterial Activity to Rhodanine Derivatives Targeting Enoyl-Acyl Carrier Protein Reductase

Abstract

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 = 3.18 µM for Mtb InhA; IC₅₀ = 10 nM 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.

Introduction

Searching for novel drugs treating bacterial infections is of high priority because of emerging bacterial resistance^1,2) and clinical complication.^3,4) Recruiting known targets and interactions to seek for valid drug candidates is an available strategy.⁵⁾ Among present methods on controlling bacterial pathogens, blocking their biosynthesis of fatty acids has been convinced practicable.^6,7) In this pathway, the enoyl-acyl carrier protein reductase (InhA) is highly conserved and significant.⁸⁾ InhA has been validated as a potent target for tuberculosis treatment by the first-line drug isoniazid.⁹⁾ Since computer assistant drug design (CADD) was introduced,¹⁰⁾ various categories of InhA inhibitors have been discovered, including triclosan,¹¹⁾ pyrrolidine carboxamides,¹²⁾ isoniazid (INH)-nicotinamide adenine dinucleotide (NAD) analogues¹³⁾ and pyridomycin.¹⁴⁾ The in vitro inhibition of them on Mycobacterial tuberculosis (Mtb) was still not ideal. Moreover, during the aggravation of tuberculosis, serious complication including further bacterial injection often occurs along with the decline of immunity.¹⁵⁾ Therefore introducing broadened antibacterial activity to merely InhA inhibition seems essential.

In this work, we introduced broadened antibacterial activity to rhodanine derivatives targeting InhA. Maintaining the inhibition of Mtb InhA, feature of xacins was added to bring 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 rationalization of recruiting rhodanine moiety. Despite activities on various bacterial-relating targets including penicillin-binding proteins,¹⁶⁾ RNA polymerase¹⁷⁾ and oxyreductase enzymes,¹⁸⁾ it has been involved in enhancing inhibition of tightly related homologous Plasmodium falciparum FabI¹⁹⁾ and then InhA itself.²⁰⁾ Now we started from the check point of studied rhodanine derivatives with revealed potency and molecular interactions. The first task we completed was rationally bringing the cyclopropane feature of xacins. Molecular overlay was shown in Fig. 1A, with the linker regulated, potential candidates could mimic the cyclopropane feature of Moxifloxacin better. Meanwhile, they suggested similar conformation to previous reported InhA inhibitory compound with alanine (ALA) residue.²⁰⁾ Introducing benzene ring could not succeed in such an imitation as seen in Fig. 1B. Therefore we enriched the substitutes to obtain the series in this work.

Fig. 1. Molecular Overlay of (A) Linker-Regulated Candidates with Cyclopropane Feature and (B) Aryl Ring-Introduced Compounds

Experimental

Chemistry

All commercially available chemicals were directly used without further purification. ¹H-NMR and ¹³C-NMR spectra were scanned by Bruker AM 600 (Rhenistetten-Forchheim, Germany) spectrometer and resolved with MestreNova software. Mass spectra were obtained from an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS. Substituted benzaldehydes (1 mmol) was added to rhodanine (1 mmol) alcohol solution (10 mL). After urea (1.5 mmol) was added, the reaction continued in reflux for 5 h. After confirming the completion of the reaction by TLC the sediment was filtered, washed with ethanol and dried to obtain intermediates B. Then intermediates B (1 mmol) were initially dissolved in heated ethanol (8 mL), then the ethanol solution (2 mL) of (bromomethyl)cyclopropane (1 mmol) and sodium ethylate (1.5 mmol) was added dropwise. The mixture was heated to 55°C and kept reacting overnight. Target compounds Cy1–Cy14 were obtained through column chromatography and refined by recrystallization. Compounds Cx1–Cx6 for comparison were synthesized via the same method by using corresponding bromide to replace (bromomethyl)cyclopropane. Detailed information and NMR data of compounds Cy1–Cy14 and Cx1–Cx6 were presented in “Supplementary Materials.”

Determination of Minimum Inhibitory Concentration (MIC)

Mtb was grown in 7H9 medium with OADC for 24 h at 37°C under microaerobi conditions (5% O₂, 10% CO₂, and 85% N₂). Methicillin-sensitive Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27583 were grown in Brucella Broth supplemented with 10% heat-inactivated horse serum. Inoculums containing 1 × 10⁵ bacteria were seeded in 96 well plates with varying concentrations of test compounds and incubated at 37°C for 48 h. Absorbance was measured at 590 nm. Wells without test compounds and with media only served as control and blank, respectively. The percentages of growth inhibition were calculated, and the concentrations of compounds producing 80% inhibition were considered as MIC values.

Cytotoxicity

293T (human embryonic kidney cell line) was used for cytotoxicity evaluation via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method according to reference.²¹⁾

Mtb InhA Inhibition Assay

Mtb InhA was expressed in BL21(DE3) Escherichia coli strain. After incubation overnight in Kanamycin-containing plates, continuous incubation in 2 mL 2× Yt medium at 37°C 200 r/min was conducted for 14 h. Then 1 mL fluid was added into 100 mL 2× Yt medium and incubated at 37°C 240 r/min till OD₆₀₀ was 0.6. The expression was induced by 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 4 h and controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bacteria were harvested by 5000 r/min centrifugation and suspended in purification buffer (20 mM Tris–HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0). Ultrasonication was performed and purification was conducted through Nit-NTA Superflow column with 10–500 mM imidazole eluent. After examining the absorption under 280 nm, we collected the protein and checked by SDS-PAGE. According to method in reference,²⁰⁾ Mtb InhA inhibition was measured by monitoring the initial velocities (120 s) of the decrease in the absorbance of reduced nicotinamide adenine dinucleotide (NADH) at 340 nm, 25°C. The standard reaction mixture of 1.0 mL included buffer (30 mM PIPES, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 6.8), 200 µM trans-2-decenoyl-N-acetyl cysteamine, 100 µM NADH and 1 µg recombinant Mtb InhA. The data of positive control was close to literature value. All assays were performed in triplicates.

In Silico Assay

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) study was conducted by Calculate Molecular Properties in Small Molecules module of Discovery Studio 3.5 (Accelrys, Inc., San Diego, CA, U.S.A.). The ADMET properties map and data were provided by the ADMET Descriptors tool. Molecular docking was carried out by Discovery Studio 3.5 (Accelrys, Inc.) CDOCKER protocol as described in literature.²¹⁾ In this work, the crystal structures of Mtb InhA (PDB code: 4R9S) and S. aureus DNA Gyrase B (PDB code: 4URO) were used instead.

Results and Discussion

The general synthesis method and the structures of cyclopropylmethyl rhodanine derivatives Cy1–Cy14 (Cy was from ‘Cyclo’) were organized in Chart 1. They were all synthesized and tested for the first time. Rhodanine was added to different substituted benzaldehyde with the participation of urea to yield intermediate B. Subsequent reaction with (bromomethyl)cyclopropane and sodium ethylate in ethanol led to the target compounds. Compounds Cx1–Cx6 (Cx was from ‘X-factor’) for comparison were synthesized via the same method by using corresponding bromide. Refined compounds were obtained through recrystallisation. All of them gave satisfactory analytical and spectroscopic data.

Chart 1. General Synthesis of Cy1–Cy14, Cx1–Cx6 and Their Structures

Before bioassay, ADMET properties as a preliminary evaluation for the basic druggability were shown in Figure S1. Since the design of linker and substitutes was strict, all compounds indicated practicable pharmacokinetics potential such as log P and PSA (polar surface area) values.

With methods according to references,^20,22) the Mtb InhA inhibitory effect, growth blocking of Mtb and S. aureus DNA Gyrase B inhibition were evaluated. The designing concept of this series included two major concerns. One was maintaining the inhibition of Mtb InhA, the other was introducing DNA Gyrase B inhibition to block the growth of Mtb and impede secondary infection. A general glance of all synthesized compounds indicated a majority of them achieved both.

With data in Table 1 compared, preliminary structure–activity relationship (SAR) studies on Mtb InhA and DNA Gyrase B were provided. For enhancing Mtb InhA inhibition, several hints could be organized. Initially, para-position basically required electron-donating groups, with the corresponding order –NHAc (Cy9, IC₅₀ = 6.85 µM) > –N(Me)₂ (Cy8, IC₅₀ = 7.73 µM) > –OMe (Cy6, IC₅₀ = 18.2 µM) > –NO₂ (Cy7, IC₅₀ = 22.3 µM) > –Br (Cy4, IC₅₀ = 24.8 µM) > –Me (Cy5, IC₅₀ = 27.1 µM) > –H (Cy1, IC₅₀ = 28.5 µM) > –F (Cy2, IC₅₀ = 29.8 µM) > –Cl (Cy3, IC₅₀ = 31.4 µM). With electron-donating group methoxyl picked, substitute position indicated meta-(Cy12, IC₅₀ = 14.5 µM) > para-(Cy6, IC₅₀ = 18.2 µM) > ortho-(Cy11, IC₅₀ = 35.2 µM). When multi-positions were picked, effect brought by meta- and para- substitutes could be overlying, according to the fact that trimethoxyl (Cy14, IC₅₀ = 3.18 µM) and dimethoxyl (Cy13, IC₅₀ = 11.7 µM) both exceeded single methoxyl at any position. Comparisons Cx1–Cx6 were introduced based on the original (Phenyl) and optimized (3,4,5-trimethoxyphenyl) situations of Cy series. N-substituent factors including –CH₂CH₂CH₂COOH, –CH₂COOH and –CH₂CN were involved to bring more SAR discussion. Agreeing with the above SAR, trimethoxyl on benzene ring could improve the activity during the variation of N-substituent factors. Moreover, for N-substitutes, it seemed that shorter aliphatic chain was preferred (Cx1 > Cx3; Cx2 > Cx4) and electron-withdrawing was not the good choice (Cy1 > Cx3 > Cx5; Cy14 > Cx4 > Cx6). It was attractive that the top hits of our series showed comparable Mtb InhA inhibition effect with the top one with aliphatic chain^17–20). For bringing DNA Gyrase B inhibition, hints seemed a bit different. The high electronic density requirement still existed but was more ambiguous with the tendency –NHAc (Cy9, IC₅₀ = 15 nM) > –NO₂ (Cy7, IC₅₀ = 20 nM) > –N(Me)₂ (Cy8, IC₅₀ = 23 nM) > –Me (Cy5, IC₅₀ = 32 nM) > –OMe (Cy6, IC₅₀ = 35 nM) > –H (Cy1, IC₅₀ = 47 nM) > –F (Cy2, IC₅₀ = 76 nM) > –Cl (Cy3, IC₅₀ = 89 nM) > –Br (Cy4, IC₅₀ = 110 nM). Then fixed electron-donating group methoxyl inferred choice on positions as meta- (Cy12, IC₅₀ = 21 nM) > ortho-(Cy11, IC₅₀ = 28 nM) > para- (Cy6, IC₅₀ = 35 nM). Viewing multi-substituent situations, the result was similar with trimethoxyl (Cy14, IC₅₀ = 10 nM) and dimethoxyl (Cy13, IC₅₀ = 18 nM) both performed better potency. The comparisons (Cx1–Cx6) and reference compounds (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.

Table 1. The Mtb InhA Inhibitory, Mtb Growth Blocking and DNA Gyrase B Inhibitory Activities of Cy1–Cy14

Compounds	IC50 (µM) Mtb InhA	MIC (µM) Mtb	IC50 (nM) DNA Gyrase B
Cy1	28.5 ± 1.82	15.3 ± 1.36	47 ± 4.5
Cy2	29.8 ± 2.04	17.6 ± 1.58	76 ± 6.8
Cy3	31.4 ± 2.51	18.2 ± 2.02	89 ± 10
Cy4	24.8 ± 2.01	20.1 ± 1.77	110 ± 13
Cy5	27.1 ± 1.98	12.5 ± 0.97	32 ± 2.5
Cy6	18.2 ± 0.91	8.24 ± 1.13	35 ± 3.1
Cy7	22.3 ± 1.76	9.86 ± 1.08	20 ± 2.1
Cy8	7.73 ± 0.89	1.75 ± 0.31	23 ± 2.1
Cy9	6.85 ± 0.91	0.52 ± 0.09	15 ± 1.1
Cy10	40.1 ± 2.86	21.8 ± 1.93	43 ± 3.8
Cy11	35.2 ± 1.72	18.1 ± 1.84	28 ± 3.2
Cy12	14.5 ± 1.19	4.18 ± 0.57	21 ± 1.8
Cy13	11.7 ± 1.06	1.68 ± 0.21	18 ± 2.3
Cy14	3.18 ± 0.57	0.35 ± 0.04	10 ± 1.0
Cx1	83.6 ± 7.15	68.2 ± 5.79	>1000
Cx2	73.9 ± 6.88	65.1 ± 5.92	>1000
Cx3	58.4 ± 5.11	45.1 ± 4.03	830 ± 60
Cx4	50.7 ± 4.71	40.4 ± 3.81	750 ± 50
Cx5	71.2 ± 6.84	63.9 ± 5.72	>1000
Cx6	67.1 ± 6.22	59.5 ± 5.37	>1000
Ref20–1	120 ± 10.2	85.5 ± 6.84	720 ± 50
Ref20–17	19.1 ± 1.32	1.99 ± 0.14	>1000
Triclosan	6.30 ± 1.20	8.84 ± 1.12	>1000
Moxifloxacin	>200	1.73 ± 0.21	43 ± 3.5
Epalrestat	>200	>200	>1000

Fig. 2. The Mtb InhA Inhibitory, Mtb Growth Blocking and DNA Gyrase B Inhibitory Activities Presented Basically Consistency

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 aeruginosa ATCC 27583) pathogenic bacteria. Top five compounds (Cy14, Cy9, Cy8, Cy13 and Cy12) were selected as representatives. Cytotoxicity was also assessed using 293T human embryonic kidney cell line. Seen in Table 2, all selected compounds suggested considerable broad-spectrum antibacterial capability and low toxicity.

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	0.18 ± 0.02	1.42 ± 0.15	>200
Cy9	1.27 ± 0.11	2.05 ± 0.18	2.18 ± 0.20	175 ± 15.8
Cy8	2.11 ± 0.20	3.12 ± 0.25	3.04 ± 0.19	188 ± 16.5
Cy13	0.56 ± 0.05	0.37 ± 0.04	2.56 ± 0.22	>200
Cy12	0.81 ± 0.06	0.86 ± 0.10	7.25 ± 0.83	>200
Moxifloxacin	0.16 ± 0.02	0.11 ± 0.01	3.60 ± 0.32	>200

Molecular docking simulation was conducted before synthesis to give preliminary screening. Now it was also used to visualize possible binding information. The active site of Mtb InhA (PDB code: 4R9S) and DNA Gyrase B (PDB code: 4URO) were defined as binding sites. The 2D maps of the most potent compound Cy14 within both sites were depicted in Fig. 3. The three possible hydrogen bonds (O^…H-O, 2.27 Å, 141.868° with SER20; S^…H-N, 2.36 Å, 146.443° with LYS165; O^…H-O, 1.94 Å, 144.035° with THR196) fixed Cy14 tightly into Mtb InhA. Meanwhile, possible interactions including hydrogen bond (S^…H-N, 2.25 Å, 145.242° with ARG144) and π-cation (4.41 Å) might cause better import of DNA Gyrase B inhibitory activity. From the maps we could know the significance of rhodanine moiety and derived methoxyl on meta-position. Besides, 3D patterns and receptor surface models of Cy14 into Mtb InhA and DNA Gyrase B in Fig. 4 indicated deep occupation into the active pocket. Therefore future modification could be hinted as involving substitutes to offer deeper impaction and adding moiety on rhodanine to supply interactions with outer residues.

Fig. 3. Docking Models of Representative Cy14 into (A) Mtb InhA and (B) DNA Gyrase B

Significant interactions and residues were exhibited.

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

In conclusion, we have introduced broadened antibacterial activity to InhA inhibitors containing rhodanine moiety by bringing feature of xacins. Through rational design and verification of biological activity, the synthesized series indicated that a majority of them achieved both maintaining Mtb InhA inhibition and introducing DNA Gyrase B inhibition. The most potent compound Cy14 suggested comparable bioactivity (IC₅₀ = 3.18 µM for Mtb InhA; IC₅₀ = 10 nM 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 nonnegligible interactions were visualized. According to the binding situations and SAR discussion, future modification with substitutes and stretching moiety were recommended. Information in this work provided potential orientations for treating tuberculosis and preventing serious complication at the same time, therefore hinted bi-target possibility in further relevant researches.

Acknowledgments

This work is supported by the Project of Medical and Health Science Technology Development Program in Shandong Province of China (No. 2016WS0242), the Linyi Science and Technology Innovation and Development Project(Medicine) (No. 201818033), the National Undergraduate Innovation Program and the National Science Foundation of China (No. J1210026). We thank Miss Huairuo Xu from International Department American Division, Nanjing Jinling High School, Nanjing, China for the synthesis of compounds Cx1–Cx6 of this paper.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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