Dihydropyrazoles containing morpholine: design, synthesis and bioassay testing as potent antimicrobial agents

Abstract

A series of dihydropyrazole derivatives containing morpholine was designed and synthesized as antimicrobial agents. All of the synthesized compounds were characterized by ¹H-NMR and MS. Afterwards they were evaluated for in vitro antibacterial activity against four bacteria strains. Along with S. aureus TyrRS inhibition and cytotoxicity examination, some compounds proved to be of low toxicity and potent, especially against Gram-positive bacteria strains. Compound 4s exhibited the potential to be new a antibacterial drug with strong broad-spectrum antimicrobial activity and enzyme inhibitory activity. Docking simulation was performed to position compound 4s into the S. aureus TyrRS structure active site to investigate the probable binding mode. A 3D-QSAR model was also established to explain how structural alterations affect the activity and guide further study.

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1 Introduction

Antibiotics have long been researched and exploited for clinical use since the first emergence of antibiotics in the 1930s; to date numerous improved analogues are available.¹ However, after years of misuse, overuse and misdiagnosis, traditional clinical antibiotics tend to be feeble in the face of the remarkably increasing resistance of microbes.² By mutation in the binding site or directly bypassing the target functionality or being impermeable, bacteria have acquired the resistance to traditional drugs cunningly.³ Along with the resistance was the advent of higher rates of morbidity and mortality, providing a severe challenge to medicine research and development.^4,5 Therapeutic targets should be further studied and new antimicrobial drugs elaborated to ease the widening gap between supply and demand.

Although relevant theories have been developed and practical research has progressed, it still tends to be that the traditional antibacterial targets maintain their dominant position in drug design area, since the molecular screening of new genomic targets has turned out to be lackluster.⁶ Among all the validated targets, aminoacyl tRNA synthetases (aaRSs) are one of the classes with the most potential. The aaRSs are essential enzymes catalyzing the charging of tRNA, which is a vital process in the translation of mRNA into protein.⁷ The catalysis is performed with high fidelity, to ensure the correct amino acid is loaded to the tRNA and concomitantly making these enzymes highly conserved in catalytic domains. They are found in all kinds of organisms because of their indispensability and many vary in sequence between prokaryotes and eukaryotes.³ Taking all these factors (essential, conserved and difference between prokaryotes and eukaryotes) into consideration, it’s clear that the aaRSs family provides ideal and potential targets for antibacterial drugs. However, these enzymes remain underexploited since only Bactroban (also known as mupirocin) targeting isoleucin-tRNA synthetase (IleRS) has been approved as an antibiotic for clinical use. Research on the utilization of aaRSs as antibacterial targets has broad space and paves the way for the design of antimicrobial drugs. As part of the huge aaRSs family, tyrosyl-tRNA synthetase (TyrRS) holds importance for protein synthesis and has already received close attention. It functions differently between human being and microbes and this means that as antibacterial agents, drugs targeting TyrRS would have low toxicity to normal cells.⁸ Now the TyrRS family has been validated as a promising target against bacteria with rising resistance, and corresponding antagonists are being developed to meet the substantial clinical need.

As a kind of nitrogen-containing heterocycle, morpholine has received significant attention because of their broad spectrum of uses. It has been reported that many morpholines possess bioactivy such as anticancer,⁹ antimicrobial,¹⁰ acesodyne,¹¹ anti-inflammatory,¹² antiemetic¹³ and so on. The morpholine group is essential in many clinical medicines like gefitinib (Iressa), linezolid, pinaverium bromide and buparlisib (Fig. 1). Also, pyrazolines have various activities and have versatile use in medicinal chemistry. Among all the multitudinous pyrazoline derivatives, substituted dihydropyrazoles performed particularly well in manifold applications, including antitumor,¹⁴ anti-depressant,¹⁵ immunosuppression,¹⁶ antituberculotic,¹⁷ anti-inflammatory,¹⁸ antidiabetic,¹⁹ antibacteria,²⁰ antimalarial,²¹ antiamoebic.²² The significance dihydropyrazoles hold in pharmacy has led to increasing interest and has made them valuable in drug design. In our previous work, several series of pyrazole compounds were synthesized and bioassays have proved some possess potent bioactivity and low toxicity.²³ TryRS has also been exploited as an antibacterial target to examine the bioactivity of the synthesized compounds and based on these former research studies, a nitrogen atom-containing heterocyclic structurally resembling morpholine was found to be vital.^8,24 Thus it was of interest to implement a symbiotic approach to design novel candidates linking morpholine to dihydropyrazoles and investigate their bioactivity.

Fig. 1 Morpholine-containing drugs.

In this research, we have designed and synthesized a class of dihydropyrazoles bearing a morpholine ring as potential antibacterial drugs. The bioactivity assays suggest that these compounds possess potent antibacterial activity, especially against Gram-positive bacteria strains. The S. aureus TyrRS inhibition and cytotoxicity examination suggest these compounds are potent antagonists with low toxicity. Furthermore, docking simulations were performed using the X-ray crystallographic structure of the S. aureus TryRS (PDB code: 1JIJ) to explore the binding modes of these compounds at the active site. A 3D-QSAR model was constructed in order to explain how structural alterations impact the activity, paving the way for further study.

2 Results and discussion

2.1 Chemistry

The synthesis of the 20 compounds followed the route depicted in Scheme 1. To a stirred solution of morpholine in DMSO, p-fluorobenzaldehyde was added and the reaction was heated for 4 h, poured into ice water to give compound 2. The chalcones 3a–3t were obtained by the condensation of compound 2 with various acetophenones, in an ice bath catalyzed by KOH. Under stirring, the chalcones were added into acetic acid along with phenylhydrazine. The reaction was then heated to reflux and furnished compounds 4a–4t. All of the target compounds 4a–4t are reported for the first time, and give satisfactory analytical and spectroscopic data. ¹H NMR and EI-MS spectra are consistent with the assigned structures.

Scheme 1 General synthesis of compounds (4a–4t). Reagents and conditions: (a) 1.0 equiv. p-fluorobenzaldehyde, DMSO, reflux 4 h; (b) 1.0 equiv. of the acetophenone, 3.0 equiv. KOH, CH₃CH₂OH, 0 °C, 2 h; (c) 1.22 equiv. phenylhydrazine, acetic acid, reflux, 6 h.

2.2 Biological activity

2.2.1 Antibacterial activity. Two Gram-negative bacterial strains: E. coli and P. aeruginosa and two Gram-positive bacterial strains: B. subtilis and S. aureus were exploited in the antimicrobial assay. The test followed the MIC method and used penicillin and kanamycin under identical conditions as control. The results are listed in Table 1, and as shown, the MIC (minimum inhibitory concentration) value indicated that some of the new compounds possess potent activity compared to the controls. On the whole, these compounds are stronger antagonists of Gram-positive bacterial strains than Gram-negative. Against S. aureus, compounds 4b, 4c, 4e, 4p, 4q and 4t (each with MIC of 3.13 μg mL⁻¹) are comparable to penicillin (MIC of 3.13 μg mL⁻¹). Compounds 4e, 4f, and 4t (MIC of 1.56 to 3.13 μg mL⁻¹) are comparable to penicillin (MIC of 3.13 μg mL⁻¹) and kanamycin (MIC of 1.56 μg mL⁻¹). Notably, compound 4s showed broad-spectrum antibacterial activity against all the four bacteria strains, with MIC of 0.78 to 3.13 μg mL⁻¹. These results suggest that this compound is more potent than penicillin and kanamycin overall. Though other compounds are less potent, many are close to the control. To enhance the antiseptic activity against S. aureus, electron-withdrawing groups tended to be preferable to electron-donating groups. This conclusion is especially true when it comes to the o-position substituents, because the order of electron-withdrawing potential is: –NO₂ > –F > –Cl > –H and the order of antibacterial potential is 4s > 4b > 4g > 4a. Also, 4s, 4b and 4g are more potent than 4m and 4p which have an electron-donating group at the o-position.

Table 1 Antibacterial activities (MIC μg mL⁻¹) of target compounds (4a–4t)

Compound	R	MIC (μg mL^−1)
		Gram-positive		Gram-negative
		B. subtilis	S. aureus	P. aeruginosa	E. coli
4a		12.50	12.50	12.50	25.00
4b		3.13	3.13	3.13	6.25
4c		6.25	3.13	6.25	12.50
4d		12.50	6.25	12.50	25.00
4e		3.13	3.13	3.13	6.25
4f		3.13	6.25	3.13	6.25
4g		6.25	12.50	12.50	12.50
4h		6.25	3.15	6.25	12.50
4i		12.50	6.25	12.50	12.50
4j		6.25	6.25	12.50	12.50
4k		6.25	6.25	12.50	6.25
4l		12.50	12.50	12.50	12.50
4m		12.50	6.25	12.50	25.00
4n		12.50	12.50	12.50	12.50
4o		6.25	6.25	3.13	6.25
4p		12.50	12.50	6.25	12.50
4q		12.50	6.25	12.50	12.50
4r		12.50	12.50	12.50	12.50
4s		1.56	0.78	3.13	1.56
4t		1.56	3.13	3.13	6.25
Penicillin	—	3.13	3.13	1.56	3.13
Kanamycin	—	1.56	1.56	3.13	1.56

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2.2.2 S. aureus TyrRS enzyme inhibition. The S. aureus TyrRS enzyme inhibitory activity of these compounds was investigated and the results are summarized in Table 2. As shown, to some extent the antibacterial potential is consistent with the enzyme inhibition of S. aureus TyrRS, with a few exceptions. In detail, compounds possessing potent activity against S. aureus are generally endowed with significant potential against the enzyme S. aureus TyrRS; compounds 4b, 4c, 4e, 4t and 4s inflicting more destruction to bacteria (with MIC of 0.78 to 3.13 μg mL⁻¹) possess relatively more potent inhibition to S. aureus TyrRS (with IC₅₀ of 1.63 to 24.1 μM). On the other hand, compounds 4a, 4n, 4g, 4i, 4j and 4l bearing lower antimicrobial properties (with MIC of 12.5 to 25 μg mL⁻¹) show poor enzyme inhibition activity (with IC₅₀ of 18.13 to 48.79 μM). From the data listed, a conclusion similar to the one above could be achieved as there is consistency between antibacterial activity and S. aureus TyrRS enzyme inhibition with little discrepancy: electron-withdrawing groups are preferable to electron-donating groups. In addition, taking electron-withdrawing groups into consideration, it can be concluded that compounds with substituents on the o-position and m-position, compounds 4b and 4c, are more potent than 4d (with IC₅₀ of 11.92, 4.53, 15.27 μM, respectively). This is also supported by compounds 4i, 4h, and 4g (with IC₅₀ of 32.22, 24.1, 30.73 μM, respectively).

Table 2 S. aureus TyrRS enzyme inhibition (IC₅₀, μM) and cytotoxicity (CC₅₀, μM) data of all compounds

Compound	CC50^a (μM)	IC50^a (μM)
	Cytotoxicity	S. aureus TyrRS IC50
a Values are the average of three independent experiments run in triplicate. Variation was generally 5–10%.
4a	291.05	48.79
4b	204.71	11.92
4c	200.00	4.53
4d	182.48	15.27
4e	202.41	18.28
4f	231.07	22.52
4g	227.40	30.73
4h	225.22	24.10
4i	195.35	32.22
4j	207.90	30.99
4k	197.88	5.75
4l	176.24	42.08
4m	259.31	39.09
4n	223.62	18.13
4o	194.06	12.34
4p	230.10	21.67
4q	242.93	17.16
4r	220.07	20.38
4s	221.36	1.63
4t	252.19	3.72

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2.2.3 Cytotoxicity. To examine the safety of these compounds, all of the new compounds were evaluated for their toxicity against the human kidney epithelial cell 293T (median cytotoxic concentration (CC₅₀) data) using the MTT assay.²⁵ As shown in Table 2, the IC₅₀ value was employed to demonstrate the harm to 293T cells caused by these compounds and the results turned out to be favorable.

2.3 Docking

Docking is an effective and reliable approach to simulate the probable binding mode of ligands and proteins. In this study, we have performed the docking study by fitting the most potent compound 4s into the active center of the S. aureus TyrRS (PDB code: 1JIJ) using Discovery Studio 3.5. The results obtained are presented in Fig. 2 and 3. As shown, two amino acids, LYS84 and ARG88, are of significance in the binding of ligand with enzyme; in particular the LYS84 forms a cation–π interaction, an electrostatic interaction and two hydrogen bonds with 4s (angle O⋯H–N = 104.1°, distance = 2.28 Å; angle O⋯H–N = 92.5°, distance = 2.48 Å; however, in the 2D graph, these two hydrogen bonds are overlapped, which could be distinguished in the 3D graph), while a cation–π interaction was formed between ARG88 and 4s. The molecular docking results, along with the biological assay data, suggest that compound 4s is a potential inhibitor of S. aureus TyrRS.

Fig. 2 2D docking model of interactions between compound 4s with S. aureus TyrRS enzyme.

Fig. 3 3D docking model of interactions between compound 4s with S. aureus TyrRS enzyme: for clarity, only interacting residues are displayed.

2.4 3D-QSAR model

In consideration of follow-up research, a 3D-QASR model was built to study the systematic structure–activity relationship of these compounds. As intended, analysis and improvement suggestions can be gained by the 3D-QASR model, which plays a significant role in providing guidance in the quest for more powerful antagonists against S. aureus TyrRS. The process was carried out by the built-in QSAR software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd.), with all molecules converted to the active conformation and corresponding pIC₅₀ (μM) values. Concomitantly, these compounds were randomly partitioned into two groups: training set and test set. While the training set contains 80% of these compounds, the test set comprises the other four agents, as summarized in Table 3.

Table 3 Experimental and predicted inhibitory activity of compounds 4a–4t by a 3D-QSAR model based upon active conformation achieved by molecular docking

Compound^a	Actual pIC50	Predicted pIC50	Residual error
a The underlined compounds are the test set, and the rest are for training.
4a	4.31	4.34	−0.03
4b	4.92	4.89	−0.03
4c	5.34	5.32	0.02
4d	4.82	4.77	0.04
4e	4.74	4.95	−0.22
4f	4.65	4.66	−0.01
4g	4.51	4.74	−0.23
4h	4.62	4.56	0.06
4i	4.49	4.67	−0.18
4j̲	4.51	5.15	−0.64
4k	5.24	5.34	−0.10
4l	4.38	4.38	0.00
4m̲	4.41	4.78	−0.37
4n	4.74	4.47	0.26
4o̲	4.91	4.81	0.09
4p	4.66	4.71	−0.05
4q	4.77	4.79	−0.03
4r	4.69	4.50	0.19
4s	5.79	5.55	0.24
4t̲	5.43	5.13	0.29

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By default, each molecule was arranged to possess alignment conformation with the lowest CDOCKER_INTERACTION_ENENGY among all of the docked poses while the CHARMm force field and PLS regression were exerted in building the 3D-QSAR model. As shown in Fig. 4, a scatter plot with conventional R² of 0.816 was obtained, indicating a high degree of compatibility exists between the predicted pIC₅₀ and the actual pIC₅₀; hence this model possesses reliable predicting capability. Also, the information about critical regions (steric or electrostatic) affecting the binding affinity was gained: all of the compounds were aligned with the iso-surfaces of the 3D-QSAR model coefficients on electrostatic potential grids (Fig. 5(a)) and van der Waals grids (Fig. 5(b)). The electrostatic map presents information of favorable (in blue) or unfavorable (in red) electrostatic field regions in a contour plot, while the energy grids corresponding to the favorable (in green) or unfavorable (in yellow) steric effects are also marked out. Compounds are characterized as active if they bear strong van der Waals attraction in the green areas and polar groups in the blue electrostatic potential areas. A good compliance is observed between the model and actual situation for compounds under study. On the basis of this part of the study, optimized compounds possessing more potential against enzyme S. aureus TyrRS could be designed, with the activity easily and credibly predicted.

Fig. 4 Plot of experimental vs. predicted S. aureus TyrRS enzyme inhibitory activities of training set and test set.

Fig. 5 (a) 3D QSAR model coefficients on electrostatic potential grids. Blue represents positive coefficients; red represents negative coefficients. (b) 3D QSAR model coefficients on van der Waals grids. Green represents positive coefficients; yellow represents negative coefficients.

3 Conclusion

In this study, a suite of new S. aureus TyrRS inhibitors has been designed and synthesized; their potential has been evaluated in the following bioassays, which suggest these compounds possess moderate to potent antibacterial activity and S. aureus TyrRS inhibitory activity. The cytotoxicity test employing human kidney epithelial cell 293T also indicates high safety. Among all these compounds, compound 4s showed the most potent inhibition activity against four bacteria strains (with MIC of 1.56, 0.78, 3.13, 1.56 μg mL⁻¹) and S. aureus TyrRS enzyme (with IC₅₀ of 1.63 μM). The probable binding mode proposed by the docking simulation may be a good explanation of the impressive performance of 4s, in which 4s binds well with S. aureus TyrRS via two hydrogen bonds and two cation–π interactions. In addition, a reliable 3D-QSAR model was obtained to analyze the structure–activity relationship, and to guide further study seeking for more potent agents against S. aureus TyrRS.

4 Experiments

4.1 Materials and measurements

All chemicals and reagents used in the current study were analytical grade. All the ¹H NMR spectra were recorded on a Bruker DPX 400 model Spectrometer in DMSO-d₆ and chemical shifts (δ) are reported as parts per million (ppm). EI-MS spectra were recorded on a Mariner System 5304 Mass spectrometer. Melting points were determined on a XT4 MP apparatus (Taike Corp, Beijing, China). Thin layer chromatography (TLC) was performed on silica gel plates (Silica Gel 60 GF254) and visualized in UV light (254 nm). Column chromatography was performed using silica gel (200–300 mesh) eluting with ethyl acetate and petroleum ether.

4.2 General procedure for the preparation of compound 2

Under stirring, morphine (50 mmol) was added to DMSO (20 mL); afterwards an equivalent amount of p-fluorobenzaldehyde was added into the solution. The reaction was heated to reflux for 4 h, and then poured into a large amount of ice water to give a yellow precipitate. The crude product was then washed with cold ethanol and water three times to give compound 2.

4.3 General procedure for the preparation of compounds 3a–3t

The compound 2 was then added and dissolved in 20 mL ethanol, along with an equivalent amount of substituted acetophenone. The solution was then moved to an ice bath and stirred. After 5 min, 3 equiv. KOH was added slowly into the reaction, which was then poured into saturated salt water to give crude product in 2 h. The product was then washed with cold ethanol and water three times to give compounds 3a–3t.

4.4 General procedure for the preparation of compounds 4a–4t

To a stirred solution of compound 3a–3t in 20 mL acetic acid, 1.2 equiv. phenylhydrazine was added dropwise; the reaction was then heated to reflux for 6 h and then ended by pouring into ice water, furnishing the target compounds 4a–4t, which were subsequently purified by column chromatography.

4.4.1 4-(4-(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4a). Yellow crystals, yield 67.1%, m.p. 184–186 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.79–7.70 (m, 2H, ArH), 7.47–7.34 (m, 3H, ArH), 7.15 (dt, J = 8.6, 3.5 Hz, 4H, ArH), 7.03 (d, J = 7.8 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 6.71 (t, J = 7.2 Hz, 1H, ArH), 5.39 (dd, J = 12.1, 6.3 Hz, 1H, CH), 3.87 (dd, J = 17.4, 12.2 Hz, 1H, CH₂), 3.72–3.68 (m, 4H, CH₂), 3.10 (d, J = 6.3 Hz, 1H, CH₂), 3.08–3.03 (m, 4H, CH₂). MS (EI): 383.1 (M⁺).

4.4.2 4-(4-(5-(2-Fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4b). Yellow crystals, yield 55.7%, m.p. 141–143 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.96–7.87 (m, 1H, ArH), 7.41 (td, J = 7.2, 3.5 Hz, 1H, ArH), 7.31–7.23 (m, 2H, ArH), 7.16 (t, J = 8.0 Hz, 4H, ArH), 7.03 (d, J = 7.8 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 6.73 (t, J = 7.3 Hz, 1H, ArH), 5.40 (dd, J = 12.2, 6.3 Hz, 1H, CH), 3.94 (dd, J = 16.4, 12.4 Hz, 1H, CH₂), 3.73–3.64 (m, 4H, CH₂), 3.13 (dd, J = 6.3, 2.1 Hz, 1H, CH₂), 3.11–3.04 (m, 4H, CH₂). MS (EI): 401.2 (M⁺).

4.4.3 4-(4-(5-(3-Fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4c). Yellow crystals, yield 78.3%, m.p. 140–141 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.64 (d, J = 8.1 Hz, 2H, ArH), 7.24 (d, J = 8.0 Hz, 2H, ArH), 7.17–7.12 (m, 4H, ArH), 7.03–6.99 (m, 2H, ArH), 6.89 (d, J = 8.8 Hz, 2H, ArH), 6.70 (t, J = 7.2 Hz, 1H, ArH), 5.35 (dd, J = 12.0, 6.3 Hz, 1H, CH), 3.83 (dd, J = 17.4, 12.1 Hz, 1H, CH₂), 3.71 (dd, J = 11.3, 7.0 Hz, 4H, CH₂), 3.09–2.98 (m, 5H, CH₂). MS (EI): 401.2 (M⁺).

4.4.4 4-(4-(5-(4-Fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4d). Yellow crystals, yield 51.9%, m.p. 138–140 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.75 (dd, J = 7.5, 1.9 Hz, 1H, ArH), 7.52 (d, J = 7.1 Hz, 1H, ArH), 7.44–7.33 (m, 2H, ArH), 7.19–7.12 (m, 4H, ArH), 7.02 (d, J = 7.9 Hz, 2H, ArH), 6.89 (d, J = 7.9 Hz, 2H, ArH), 6.73 (t, J = 7.3 Hz, 1H, ArH), 5.39 (dd, J = 11.7, 5.9 Hz, 1H, CH), 4.04–3.93 (m, 1H, CH₂), 3.73–3.66 (m, 4H, CH₂), 3.18 (dd, J = 17.5, 6.2 Hz, 1H, CH₂), 3.05 (d, J = 3.4 Hz, 4H, CH₂). MS (EI): 401.2 (M⁺).

4.4.5 4-(4-(5-(2,4-Difluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4e). Yellow crystals, yield 49.6%, m.p. 139–141 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.94–7.88 (m, 1H, ArH), 7.46–7.38 (m, 1H, ArH), 7.31–7.23 (m, 2H, ArH), 7.16 (d, J = 8.5 Hz, 3H, ArH), 7.03 (d, J = 7.8 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 6.73 (t, J = 7.2 Hz, 1H, ArH), 5.40 (dd, J = 12.2, 6.3 Hz, 1H, CH), 3.94 (dd, J = 17.2, 11.9 Hz, 1H, CH₂), 3.75–3.66 (m, 4H, CH₂), 3.18–3.00 (m, 5H, CH₂). MS (EI): 419.1 (M⁺).

4.4.6 4-(4-(5-(2-Fluoro-4-methylphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4f). Yellow crystals, yield 66.2%, m.p. 154–156 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.48 (dd, J = 8.9, 4.7 Hz, 2H, ArH), 7.34 (t, J = 8.1 Hz, 1H, ArH), 7.19–7.11 (m, 4H, ArH), 7.02 (d, J = 7.9 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 6.72 (t, J = 7.3 Hz, 1H, ArH), 5.40 (dd, J = 12.1, 6.3 Hz, 1H, CH), 3.83 (dd, J = 17.5, 12.2 Hz, 1H, CH₂), 3.74–3.66 (m, 4H, CH₂), 3.20–2.99 (m, 5H, CH₂), 2.27 (s, 3H, CH₃). MS (EI): 415.1 (M⁺).

4.4.7 4-(4-(5-(2-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4g). Yellow crystals, yield 70.9%, m.p. 135–136 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.75 (dd, J = 7.3, 2.1 Hz, 1H, ArH), 7.52 (dd, J = 7.4, 1.8 Hz, 1H, ArH), 7.43–7.33 (m, 2H, ArH), 7.15 (t, J = 7.3 Hz, 4H, ArH), 7.02 (d, J = 7.9 Hz, 2H, ArH), 6.89 (d, J = 8.7 Hz, 2H, ArH), 6.73 (t, J = 7.3 Hz, 1H), 5.39 (dd, J = 12.1, 6.3 Hz, 1H, CH), 3.97 (dd, J = 17.4, 12.1 Hz, 1H, CH₂), 3.72–3.65 (m, 4H, CH₂), 3.18 (dd, J = 17.4, 6.3 Hz, 1H, CH₂), 3.09–3.01 (m, 4H, CH₂). MS (EI): 418.1 (M+).

4.4.8 4-(4-(5-(3-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4h). Yellow crystals, yield 80.6%, m.p. 146–148 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, J = 7.4 Hz, 1H, ArH), 7.72 (d, J = 7.9 Hz, 1H, ArH), 7.55 (d, J = 7.9 Hz, 1H, ArH), 7.49–7.34 (m, 3H, ArH), 7.15 (dd, J = 13.5, 8.1 Hz, 3H, ArH), 7.04 (d, J = 7.8 Hz, 1H, ArH), 6.91 (t, J = 9.5 Hz, 2H, ArH), 6.73 (t, J = 7.2 Hz, 1H, ArH), 5.44 (dd, J = 12.2, 6.1 Hz, 1H, CH), 3.85 (dd, J = 17.4, 12.3 Hz, 1H, CH₂), 3.71 (dd, J = 11.0, 6.9 Hz, 4H, CH₂), 3.18–3.02 (m, 5H, CH₂). MS (EI): 418.1 (M⁺).

4.4.9 4-(4-(5-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4i). Yellow crystals, yield 47.8%, m.p. 129–130 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.74 (d, J = 8.6 Hz, 2H, ArH), 7.48 (d, J = 8.6 Hz, 2H, ArH), 7.20 (s, 2H, ArH), 7.14 (dd, J = 20.0, 12.4 Hz, 4H, ArH), 7.01 (d, J = 7.9 Hz, 2H, ArH), 6.72 (t, J = 7.2 Hz, 1H, ArH), 5.45 (dd, J = 12.0, 6.0 Hz, 1H, CH), 3.93–3.84 (m, 1H, CH₂), 3.80 (d, J = 17.8 Hz, 4H, CH₂), 3.17 (s, 4H, CH₂), 3.07 (dd, J = 17.5, 6.3 Hz, 1H, CH₂). MS (EI): 418.1 (M⁺).

4.4.10 4-(4-(5-(3,4-Dichlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4j). Yellow crystals, yield 50.7%, m.p. 153–154 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J = 1.7 Hz, 1H, ArH), 7.69 (dt, J = 18.0, 5.1 Hz, 2H, ArH), 7.14 (dd, J = 14.4, 8.4 Hz, 4H, ArH), 7.04 (d, J = 7.9 Hz, 2H, ArH), 6.89 (d, J = 8.7 Hz, 2H, ArH), 6.73 (t, J = 7.2 Hz, 1H, ArH), 5.44 (dd, J = 12.2, 6.2 Hz, 1H, CH), 3.84 (dd, J = 17.6, 12.3 Hz, 1H, CH₂), 3.73–3.65 (m, 4H, CH₂), 3.15–3.07 (m, 1H, CH₂), 3.07–3.00 (m, 4H, CH₂). MS (EI): 451.0 (M⁺).

4.4.11 4-(4-(1-Phenyl-5-(3,4,5-trichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4k). Yellow crystals, yield 46.4%, m.p. 166–167 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.72 (dd, J = 26.5, 8.7 Hz, 2H, ArH), 7.17 (t, J = 8.1 Hz, 4H, ArH), 7.04 (d, J = 7.8 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 6.76 (t, J = 7.3 Hz, 1H, ArH), 5.46 (dd, J = 12.2, 6.3 Hz, 1H, CH), 4.01 (dd, J = 17.5, 12.2 Hz, 1H, CH₂), 3.75–3.66 (m, 4H, CH₂), 3.21 (dd, J = 17.4, 6.3 Hz, 1H, CH₂), 3.11–3.02 (m, 4H, CH₂). MS (EI): 485.0 (M⁺).

4.4.12 4-(4-(5-(3-Iodophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4l). Yellow crystals, yield 41.1%, m.p. 167–169 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.79 (d, J = 8.4 Hz, 2H, ArH), 7.53 (d, J = 8.4 Hz, 2H, ArH), 7.15 (t, J = 8.5 Hz, 4H, ArH), 7.02 (d, J = 7.9 Hz, 2H, ArH), 6.90 (d, J = 8.6 Hz, 2H, ArH), 6.72 (t, J = 7.3 Hz, 1H, ArH), 5.41 (dd, J = 12.2, 6.3 Hz, 1H, CH), 3.84 (dd, J = 17.4, 12.3 Hz, 1H, CH₂), 3.76–3.65 (m, 4H, CH₂), 3.19–2.92 (m, 5H, CH₂). MS (EI): 508.9 (M⁺).

4.4.13 4-(4-(1-Phenyl-5-(m-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4m). Yellow crystals, yield 73.0%, m.p. 138–141 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.60 (s, 1H, ArH), 7.53 (d, J = 7.4 Hz, 1H, ArH), 7.32 (t, J = 7.6 Hz, 1H, ArH), 7.16 (dd, J = 18.2, 9.4 Hz, 5H, ArH), 7.02 (d, J = 7.8 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H, ArH), 6.71 (t, J = 7.2 Hz, 1H, ArH), 5.39 (dd, J = 12.1, 5.9 Hz, 1H, CH), 3.85 (dd, J = 17.2, 12.0 Hz, 1H, CH₂), 3.70 (d, J = 4.6 Hz, 4H, CH₂), 3.07 (dd, J = 11.2, 6.3 Hz, 5H, CH₂), 2.36 (s, 3H, CH₃). MS (EI): 397.1 (M⁺).

4.4.14 4-(4-(1-Phenyl-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4n). Yellow crystals, yield 65.9%, m.p. 134–135 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.63 (d, J = 8.1 Hz, 2H, ArH), 7.23 (d, J = 8.0 Hz, 2H, ArH), 7.13 (dt, J = 7.2, 3.5 Hz, 4H, ArH), 7.00 (d, J = 7.9 Hz, 2H, ArH), 6.88 (d, J = 8.7 Hz, 2H, ArH), 6.69 (t, J = 7.2 Hz, 1H, ArH), 5.34 (dd, J = 12.0, 6.3 Hz, 1H, CH), 3.83 (dd, J = 17.4, 12.1 Hz, 1H, CH₂), 3.72–3.66 (m, 4H, CH₂), 3.09–2.99 (m, 5H, CH₂), 2.33 (s, 3H, CH₃). MS (EI): 397.1 (M⁺).

4.4.15 4-(4-(1-Phenyl-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4o). Yellow crystals, yield 76.3%, m.p. 151–153 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.73 (d, J = 7.9 Hz, 2H, ArH), 7.47 (d, J = 8.4 Hz, 2H, ArH), 7.14 (dd, J = 13.3, 6.0 Hz, 4H, ArH), 7.02 (d, J = 7.9 Hz, 2H, ArH), 6.88 (d, J = 7.4 Hz, 2H, ArH), 6.71 (t, J = 7.2 Hz, 1H, ArH), 5.39 (s, 1H, CH), 3.82 (t, J = 14.8 Hz, 1H, CH₂), 3.69 (s, 4H, CH₂), 3.04 (s, 5H, CH₂). MS (EI): 451.2 (M⁺).

4.4.16 4-(4-(5-(2-Methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4p). Yellow crystals, yield 68.4%, m.p. 183–185 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.89 (d, J = 7.7 Hz, 1H, ArH), 7.36 (t, J = 7.1 Hz, 1H, ArH), 7.14 (t, J = 7.7 Hz, 4H, ArH), 7.08 (d, J = 8.3 Hz, 1H, ArH), 7.01 (dd, J = 11.4, 8.0 Hz, 3H, ArH), 6.90 (d, J = 8.5 Hz, 2H, ArH), 6.70 (t, J = 7.2 Hz, 1H, ArH), 5.30 (dd, J = 11.9, 6.4 Hz, 1H, CH), 3.91 (dd, J = 10.3, 7.5 Hz, 1H, CH₂), 3.80 (s, 3H, CH₃), 3.73–3.67 (m, 4H, CH₂), 3.20–3.02 (m, 5H, CH₂). MS (EI): 413.1 (M⁺).

4.4.17 4-(4-(5-(3-Methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4q). Yellow crystals, yield 56.5%, m.p. 179–180 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (s, 1H), 7.71 (d, J = 7.9 Hz, 1H, ArH), 7.54 (d, J = 7.9 Hz, 1H, ArH), 7.40 (ddd, J = 11.6, 10.0, 5.0 Hz, 3H, ArH), 7.16–7.11 (m, 3H, ArH), 7.03 (d, J = 7.8 Hz, 1H, ArH), 6.90 (t, J = 9.5 Hz, 2H, ArH), 6.72 (t, J = 7.2 Hz, 1H, ArH), 5.43 (dd, J = 12.2, 6.1 Hz, 1H, CH), 3.90 (dd, J = 10.3, 7.5 Hz, 1H, CH₂), 3.78 (s, 3H, CH₃), 3.72–3.66 (m, 4H, CH₂), 3.18–3.01 (m, 5H, CH₂). MS (EI): 413.1 (M⁺).

4.4.18 4-(4-(5-(4-Methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4r). Yellow crystals, yield 56.5%, m.p. 177–179 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.57 (d, J = 8.9 Hz, 2H, ArH), 7.37–7.29 (m, 3H, ArH), 7.09 (dd, J = 8.7, 3.1 Hz, 4H, ArH), 7.03 (s, 2H, ArH), 6.98 (dd, J = 8.0, 3.8 Hz, 1H, ArH), 6.89 (s, 1H, ArH), 5.54 (dd, J = 12.0, 4.9 Hz, 1H, CH), 3.89 (dd, J = 17.6, 12.0 Hz, 1H, CH₂), 3.81 (s, 3H, CH₃), 3.71–3.65 (m, 4H, CH₂), 3.15 (dd, J = 17.6, 5.0 Hz, 1H, CH₂), 3.07–3.01 (m, 4H, CH₂). MS (EI): 413.1 (M⁺).

4.4.19 4-4-(4-(5-(2-Nitrophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4s). Red crystals, yield 39.2%, m.p. 179–181 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.09 (s, 1H, ArH), 7.72 (d, J = 7.9 Hz, 2H, ArH), 7.23 (t, J = 7.8 Hz, 1H, ArH), 7.19–7.11 (m, 4H, ArH), 7.03 (d, J = 7.9 Hz, 2H, ArH), 6.89 (d, J = 8.7 Hz, 2H, ArH), 6.73 (t, J = 7.3 Hz, 1H, ArH), 5.42 (dd, J = 12.2, 6.1 Hz, 1H, CH), 3.84 (dd, J = 17.5, 12.3 Hz, 1H, CH₂), 3.73–3.68 (m, 4H, CH₂), 3.12–3.03 (m, 5H, CH₂). MS (EI): 428.1 (M⁺).

4.4.20 4-4-(4-(5-(3-Nitrophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)morpholine (4t). Red crystals, yield 48.9%, m.p. 176–178 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.26 (d, J = 9.0 Hz, 2H, ArH), 7.96 (d, J = 8.9 Hz, 2H, ArH), 7.18 (ddd, J = 37.2, 19.1, 8.2 Hz, 8H, ArH), 6.79 (t, J = 7.2 Hz, 1H, ArH), 5.61 (dd, J = 12.5, 6.1 Hz, 1H, CH), 3.94 (dd, J = 17.5, 12.5 Hz, 1H, CH₂), 3.80 (s, 4H, CH₂), 3.16 (dd, J = 17.6, 5.8 Hz, 5H, CH₂). MS (EI): 428.1 (M⁺).

4.5 Antibacterial activity²⁶

Two Gram-negative bacterial strains: E. coli ATCC 25922 and P. aeruginosa ATCC 27853 and two Gram-positive bacterial strains: B. subtilis ATCC 530 and S. aureus ATCC 25923 were employed in the antibacterial activities test, using the method recommended by National Committee for Clinical Laboratory Standards (NCCLS).

By the two-fold serial dilution method, the in vitro activities of the compounds were tested in nutrient broth (NB) for bacteria. Seeded broth (broth containing microbial spores) was prepared in NB from 24 h old bacterial cultures on nutrient agar (Hi-media) at 37 °C. The bacterial suspension was adjusted with sterile saline to a concentration of 1 × 10⁴ to 10⁵ CFU mL⁻¹. The tested compounds and reference drugs were prepared by two-fold serial dilution to obtain the required concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 μg mL⁻¹. The tubes were incubated in BOD incubators at 37 °C for bacteria. The MICs were recorded by visual observations after 24 h (for bacteria) of incubation. Kanamycin B and penicillin were used as standards for bacterial. The observed MICs are presented in Table 1.

4.6 Preparation of the TyrRS and enzyme assay

S. aureus TyrRS enzyme was over-expressed in E. coli bacteria and purified to near homogeneity (∼98% as judged by SDS-PAGE) using standard purification procedures. TyrRS activity was measured by aminoacylation using modifications to previously described methods. The assays were performed at 37 °C in a mixture containing (final concentrations) 100 mM Tris–Cl pH 7.9, 50 mM KCl, 16 mM MgCl₂, 5 mM ATP, 3 mM DTT, 4 mg mL⁻¹ E. coli MRE600 tRNA (Roche) and 10 μM L-tyrosine (0.3 μM L-[ring-3,5-3H] tyrosine (PerkinElmer, specific activity: 1.48–2.22 TBq mmol⁻¹), 10 μM carrier). TyrRS (0.2 nM) was pre-incubated with a range of inhibitor concentrations for 10 min at room temperature followed by the addition of pre-warmed mixture at 37 °C. After specific intervals, the reaction was terminated by adding aliquots of the reaction mix into ice-cold 7% trichloroacetic acid and harvesting onto 0.45 mm hydrophilic Durapore filters (Millipore Multiscreen 96 well plates) and counted by liquid scintillation. The rate of reaction in the experiments was linear with respect to protein and time with less than 50% total tRNA acylation. IC₅₀ values correspond to the concentration at which half of the enzyme activity is inhibited by the compound. The results are presented in Table 2.

4.7 Cytotoxicity test

Cells were incubated in a 96 well plate at a density of 10⁵ cells per well with various concentrations of compounds for 48 h. For the cytotoxicity assay, 20 μL of MTT (5 mg mL⁻¹) was added per well 4 h before the end of the incubation. After removing the supernatant, 200 μL DMSO was added to dissolve the formazan crystals. The absorbance at λ 570 nm was read on an ELISA reader (Tecan, Austria).

4.8 Experimental protocol for docking study

Molecular docking of compound 4s into the three dimensional X-ray structure of S. aureus TyrRS (PDB code: 1JIJ) was carried out using the Discovery Studio (version 3.5) as implemented through the graphical user interface DS-CDOCKER protocol. The three-dimensional structures of the aforementioned compounds were constructed using Chem. 3D ultra 12.0 software [Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2010)], then they were energetically minimized by using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal structures of protein complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound water and ligands were eliminated from the protein. The molecular docking was performed by inserting compound 4s into the binding pocket of S. aureus TyrRS based on the binding mode. Types of interactions of the docked protein with ligand-based pharmacophore model were analyzed after the end of molecular docking.

4.9 3D-QSAR

A ligand-based 3D-QSAR approach was performed by QSAR software of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd.). The training sets were composed of inhibitors with the corresponding pIC₅₀ values which were converted from the obtained IC₅₀ (μM), and test sets comprising compounds of data sets as listed in Table 3. All the definitions of the descriptors can be seen in the “Help” of DS 3.5 software and they were calculated by QSAR protocol of DS 3.5. The alignment conformation of each molecule was the one with lowest interaction energy in the docked results of CDOCKER. The predictive ability of 3D-QSAR modeling can be evaluated based on the cross-validated correlation coefficient, which qualifies the predictive ability of the models. Scrambled test (Y scrambling) was performed to investigate the risk of chance correlations. The inhibitory potencies of compounds were randomly reordered 30 times and subjected to a leave-one-out validation test, respectively. The models were also validated by test sets, in which the compounds are not included in the training sets.

Acknowledgements

The work was financed by a grant (no. J1103512) from National Natural Science Foundation of China.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra15201h

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