Chiral lactic hydrazone derivatives as potential bioactive antibacterial agents: Synthesis, spectroscopic, structural and molecular docking studies

Accepted Manuscript Chiral lactic hydrazone derivatives as potential bioactive antibacterial agents: Synthesis, spectroscopic, structural and molecular docking studies Nader Noshiranzadeh, Azam Heidari, Fakhri Haghi, Rahman Bikas, Tadeusz Lis PII:

S0022-2860(16)30922-X

DOI:

10.1016/j.molstruc.2016.09.006

Reference:

MOLSTR 22918

To appear in:

Journal of Molecular Structure

Received Date: 24 June 2016 Revised Date:

1 September 2016

Accepted Date: 1 September 2016

Please cite this article as: N. Noshiranzadeh, A. Heidari, F. Haghi, R. Bikas, T. Lis, Chiral lactic hydrazone derivatives as potential bioactive antibacterial agents: Synthesis, spectroscopic, structural and molecular docking studies, Journal of Molecular Structure (2016), doi: 10.1016/ j.molstruc.2016.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Chiral lactic hydrazone derivatives as potential bioactive antibacterial

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agents: Synthesis, spectroscopic, structural and molecular docking studies

Nader Noshiranzadeh,*a Azam Heidari,a Fakhri Haghi,b Rahman Bikas,a and Tadeusz

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Lisc

Department of Chemistry, University of Zanjan, 45195-313, Zanjan, I. R. Iran.

b

Department of Microbiology, School of Medicine, Zanjan University of Medical

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a

Sciences,45139-56111, Zanjan, I. R. Iran.

Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, Wroclaw 50-383, Poland

*

Correspondence should be addressed to:

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c

Nader Noshiranzadeh, Department of Chemistry, University of Zanjan, Zanjan, Iran.

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Fax: +98 241-2283203 Tel.: +98-243-3052583

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Email: [email protected] and [email protected]

ACCEPTED MANUSCRIPT Abstract A series of novel chiral lactic-hydrazone derivatives were synthesized by condensation of (S)lactic acid hydrazide with salicylaldehyde derivatives and characterized by elemental analysis and spectroscopic studies (FT-IR, 1H NMR and 13C NMR spectroscopy). The structure of one

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compound was determined by single crystal X-ray analysis. Antibacterial activity of the synthesized compounds was studied against Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli and Pseudomonas aeruginosa as bacterial cultures by broth

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microdilution method. All of the synthesized compounds showed good antibacterial activity with MIC range of 64 - 512 µg/mL. Compounds (S,E)-2-hydroxy-N-(2-hydroxy-5(5)

and

(S,E)-2-hydroxy-N-((3-hydroxy-5-

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nitrobenzylidene)propanehydrazide

(hydroxymethyl)-2-methylpyridin-4-yl)propanehydrazide

(7)

were

the

most

effective

antibacterial derivatives against S. aureus and E. coli respectively with a MIC value of 64 µg/mL. Bacterial biofilm formation assay showed that these compounds significantly

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inhibited biofilm formation of P. aeruginosa. Also, in silico molecular docking studies were performed to show lipoteichoic acid synthase (LtaS) inhibitory effect of lactic hydrazone derivatives. The association between electronic and structural effects of some substituents on

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the benzylidene moiety and the biological activity of these chiral compounds were studies. Structural studies show that compound with higher hydrogen bonding interactions show

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higher antibacterial activity. The results show chiral hydrazone derivatives based on lactic acid hydrazide could be used as potential lead compounds for developing novel antibacterial agents.

Keywords: Lactic hydrazone, Antibacterial activity, Spectroscopic studies, Molecular docking, X-ray structure, Structure effects on biological activity

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ACCEPTED MANUSCRIPT 1. Introduction In the twentieth century the discovery of the antimicrobial agents led to the significant reduction of deaths caused by infectious diseases. The appearance of antimicrobial resistance among bacterial pathogens has become a serious problem in healthcare settings worldwide.

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Intensive use of antimicrobial agents in human and veterinary medicine is associated with emerging of multi-drug resistant pathogens [1]. Furthermore, most serious bacterial infections are dependent to biofilm formation of pathogen which regulated by quorum sensing (QS)

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system [2]. Therefore, there is a need for new compounds with extended antimicrobial and ant virulence activity. One of the most efficient approaches in drug improvement is chemical

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modification of biologically active compounds from natural sources [3]. Lactic acid (2hydroxypropanoic acid, pKa 3.79), which is approved by the U.S. Food and Drug Administration, is produced by several microorganisms, e.g. lactic acid bacteria (Lactobacillales), in anoxic respiration and by fermentation processes. Lactic acid has various

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applications in food, pharmaceutical technology, cosmetics and other chemical industries because of its disinfectant and keratolytic properties [4]. It is reported that L isomeric form of lactic acid is more effective for pathogen inhibition in comparison with its D isomer [5].

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Transformation of lactic acid as natural chiral compound to other derivatives with potential

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biological behavior (like lactic acid hydrazide) is one of important ways to design new antibacterial agents.

Hydrazones obtained from the condensation of aromatic or aliphatic acid hydrazides (R−C(=O)−NH−NH2) with wide range of aldehydes and ketones are one of the important multipurpose class of compounds in various fields of science [6]. High stability, simple preparation and tendency towards crystallinity are desirable characteristics of hydrazones.

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ACCEPTED MANUSCRIPT There is continuous interest in the chemistry of hydrazone compounds due to their wide variety of biological relevance since they can show anticonvulsant [7], analgesic [8], antiinflammatory [9], antidepressant [10], antiplatelet [11], antimalarial [12], antileshmania [13], antimicrobial [14,15], antimycobacterial [16,17], anticancer [18,19], antihepatitis C [20], and

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insecticidal [21] activities. Hydrazone derivatives are versatile compounds for preparing numerous metal complexes and have been widely employed as ligands in coordination chemistry [22]. Due to facile keto–enol tautomerization and the availability of several

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potential donor sites, hydrazone derivatives can interact with wide range of metal ions in different oxidation states [23]. It is obvious that nucleophilic and electrophilic characteristic

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groups of N-acylhydrazones, –C(=O)–NH–N=CH–, are responsible for improvement of main interactions between binding sites and active sites of biological receptors [24]. Also, they have several hydrogen bonding donor and acceptor groups which increase their interactions with targeting proteins or DNA, resulting strong inhibition of cell proliferation [25].

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Therefore, hydrazones have found applications in the treatment of several diseases such as tuberculosis [26], for the treatment of Fe overload disease [27], and inhibition of DNA synthesis and cell growth [28].

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It is a fact that enantiomers of chiral compounds have different biological activities [29].

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Interaction between a chiral compound and its chiral binding site is responsible for biological effects. When chiral molecule fits a receptor site, this fitting interaction leads to an active response. In contrast, there is no active response due to interactions between the inactive enantiomer and its receptor through the same way [30]. Although several hydrazone derivatives have been widely used as bioactive compounds, there are few reports on the chiral hydrazones. This can be attributed to the fact that most of the

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ACCEPTED MANUSCRIPT industrially available hydrazone derivatives are not chiral, and chiral hydrazones derivatives are scarce and expensive. The aim of present study was the synthesis of a novel, cheap and chiral hydrazone derivative as a new hydrazone based bioactive compound for antibacterial assay. (S)-Lactic acid hydrazide is a simple chiral hydrazide which can be easily obtained

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from lactic acid or lactic esters as a cheap and readily available bioactive compound. In addition, we are interested in studying the effects of structural parameters (like hydrogen bonding interactions and the effects of various substituents on the phenyl ring) in the

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antibacterial activity of hydrazone based compounds. In this study we report synthesis, spectroscopic and structural studies of some novel N-acylated chiral lactic hydrazone

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derivatives as potential materials for antimicrobial screening against various pathogenic bacteria and biofilm formation against P. aeruginosa. Also, newly synthesized compounds were screened by molecular docking studies for LtaS as an extra cellular target for

2. Experimental

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development of antibiotics against drug resistant Gram positive pathogens.

2.1. Materials and Methods

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All chemical materials for preparing the compounds were purchased from Sigma-Aldrich and

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used as received. Solvents of the highest grade commercially available (Merck) were used without further purification. Melting points were determined in open capillary tubes in a Thomas–Hoover melting point apparatus and were uncorrected. Thin-layer chromatography (TLC) was performed on silica gel plates with visualization by UV. 1H and 13C NMR spectra of compounds in DMSO-d6 solution were measured on a Bruker 250 and 62.9 MHz spectrometer, respectively, and chemical shifts are indicated in ppm relative to

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ACCEPTED MANUSCRIPT tetramethylsilane (TMS). The IR spectra were recorded on a Nicolet iS10 FT-IR spectrophotometer as KBr pellets in the range of 400-4000 cm-1. The resolution of FT-IR instrument and the scan's numbers are 4 and 10, respectively.

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2.2. Synthesis of (S)-2-hydroxypropanehydrazide ((S)-lactic acid hydrazide)

Equimolar amounts of (S)-(-)-ethyl lactate and hydrazine hydrate were mixed in the absence of any solvent and the mixture was refluxed for 3 hours. Then, the reaction mixture was

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transferred to a beaker and the obtained oily liquid was kept in a reduced pressure at room temperature for two days to remove the produced water and ethanol. The final oily product

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was used in future syntheses. Yield: 98%. Anal. Calc. for C3H8N2O2 (MW = 104.11) C, 34.61; H, 7.75; N, 26.91%. Found: C, 34.56; H, 7.72; N, 26.98%. FT-IR (KBr, cm-1): 3203 (br, vs); 3026 (s); 2993 (s); 2981 (s); 2942 (m); 2890 (s); 2817(s); 2664 (m); 1682 (vs); 1660 (vs); 1549 (s); 1515 (s); 1464 (m); 1457 (m); 1442 (m); 1415 (m); 1374 (s); 1329 (s); 1283

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(m); 1272 (m); 1248 (s); 1131 (vs); 1091 (m); 1043 (s); 950 (s); 886 (s); 733 (w); 661 (br, m); 581 (m); 550 (m), 452 (s). 1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.15 (3H, d, J= 6.75 Hz); 3.96 (1H, q, J = 6.75 Hz); 4.36 (2H, s, NH2); 5.37 (1H, s, OH); 8.85 ppm (1H,

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ppm.

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s, NH).13C NMR (62.90 MHz, DMSO-d6); δ: 21.39 (-CH3), 67.04 (-CHOH), 173.82 (C=O)

2.3. General procedure of the synthesis of lactic-hydrazone compounds (1-6) General procedure: The lactic-hydrazone compounds (1-6) were prepared in a similar manner by refluxing an equivalent molar ratio of (S)-lactic acid hydrazide and aromatic 2hydroxybenzaldehyd derivatives (compounds 1-6) or pyridoxal (compound 7) in 20 mL methanol (Scheme 1). The mixture was refluxed for 3-5 hours and completion of the

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ACCEPTED MANUSCRIPT reactions was checked by TLC on silica gel plates. When the reaction completed, the solution was evaporated on a steam bath to 5 mL and cooled to room temperature. The obtained solids were separated and filtered off, washed with 5 mL of cooled methanol and then dried in air.

Synthesis

of

(S,E)-2-hydroxy-N-(2-hydroxy-3-

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2.3.1.

methoxybenzylidene)propanehydrazide (1)

Yield: 91%. M.p. 122-125 °C. Anal. Calc. for C11H14N2O4 (MW = 238.24) C, 55.46; H, 5.92;

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N, 11.76%. Found: C, 55.42; H, 5.95; N, 11.72%. FT-IR (KBr, cm-1): 3497 (m); 3381 (m); 3212 (m); 3194 (w); 3079 (w); 1732 (w); 1686 (s); 1631 (m); 1612 (m); 1574 (m); 1469 (m);

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1424 (w); 1371 (w); 1336 (w); 1328 (w); 1313 (m); 1270 (s); 1251 (s); 1236 (s); 1224 (s); 1178 (w); 1134 (m); 1110 (w); 1079 (s); 1047 (m); 985 (m); 969 (m); 941 (m); 883 (s); 838 (m); 774 (m); 744 (m); 728 (m); 700 (m); 569 (w); 526 (w). 1H NMR (250.13 MHz, DMSOd6, 25 °C, TMS); δ: 1.28 (3H, d, J = 6 Hz); 3.75 (3H, s, -OCH3); 4.14 (1H, q, J = 6 Hz); 5.70

(1H, s, OH).

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(OH, d, J = 4.25 Hz); 6.76-7.01 (3H, m); 8.57 (1H, s, CH=N); 11.10 (1H, s, NH); 11.49 ppm C NMR (62.90 MHz, DMSO-d6); δ: 21.25 (-CH3), 56.22 (-OCH3), 67.46 (-

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CHOH), 114.29, 119.07, 119.41, 121.67, 147.67, 148.31, 149.19, 171.35 (C=O) ppm.

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2.3.2. Synthesis of (S,E)-2-hydroxy-N-(2-hydroxybenzylidene)propanehydrazide (2) Yield: 88%. M.p. 179-180 °C. Anal. Calc. for C10H12N2O3 (MW = 208.21) C, 57.68; H, 5.81; N, 13.45%. Found: C, 57.72; H, 5.83; N, 13.40%. FT-IR (KBr, cm-1): 3286 (br, m); 1673 (s); 1617 (s); 1604 (m); 1569 (w); 1538 (s); 1488 (m); 1463 (w); 1403 (w); 1363 (m); 1324 (w); 1317 (w); 1276 (br, m); 1258 (br, m); 1239 (m); 1222 (m); 1203 (br,m); 1159 (w); 1129 (m); 1122 (m); 1046 (w); 1034 (m); 985 (w); 974 (m); 961 (s); 895 (w); 877 (w); 868 (m); 786

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ACCEPTED MANUSCRIPT (m); 766 (s); 713 (br, m); 660 (w); 634 (br, w); 588 (w); 558 (w). 1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.27 (3H, d, J = 6.75 Hz); 4.15 (1H, q, J = 6.75 Hz); 5.68 (OH, d, J = 5.25 Hz); 6.83-7.40 (4H, m); 8.57 (1H, s, CH=N); 11.32 (1H, s, NH); 11.51 ppm (1H,

119.71, 130.22, 131.67, 149.12, 157.91, 171.27 (C=O) ppm.

2.3.3.

Synthesis

of

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s, OH). 13C NMR (62.90 MHz, DMSO-d6); δ: 21.32 (-CH3), 67.46 (-CHOH), 116.83, 118.93,

(S,E)-2-hydroxy-N-(2-hydroxy-5-

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bromobenzylidene)propanehydrazide (3)

Yield: 94%. M.p. 222-224 °C. Anal. Calc. for C10H11BrN2O3 (MW = 287.11) C, 41.83; H,

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3.86; N, 9.76%. Found: C, 41.88; H, 3.84; N, 9.81%. FT-IR (KBr, cm-1): 3467 (m); 3288 (s); 3256 (br, s); 3234 (br, m); 3218 (m); 2878 (w); 1689 (vs); 1666 (vs); 1625 (w); 1610 (s); 1544 (s); 1480 (vs); 1448 (w); 1384 (w); 1366 (m); 1350 (s); 1319 (w); 1265 (vs); 1239 (w); 1226 (w); 1207 (w); 1181 (m); 1129 (vs); 1080 (w); 974 (m); 963 (w); 944 (m); 875 (m); 819

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(s); 781 (m); 728 (m); 704 (s); 691 (s); 633 (m); 595 (m); 560 (m).1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.26 (3H, d, J = 6.5 Hz); 4.12 (1H, q, J = 6.5 Hz); 5.68 (OH, d, J = 4.75 Hz); 6.84 (1H, d, J = 8.75 Hz); 7.37 (1H, d, J = 8.75 Hz); 7.64 (1H, s); 8.52 (1H, s,

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CH=N); 11.31 (1H, s, NH); 11.58 ppm (1H, s, OH).

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C NMR (62.90 MHz, DMSO-d6); δ:

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21.26 (-CH3), 67.42 (-CHOH), 110.78, 119.06, 121.56, 131.17, 133.89, 146.51, 156.77, 171.45 (C=O) ppm.

2.3.4. Synthesis of (S,E)-2-hydroxy-N-(2-hydroxy-5-iodobenzylidene)propanehydrazide (4)

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ACCEPTED MANUSCRIPT Yield: 90%. M.p. 231- 234 °C. Anal. Calc. for C10H11IN2O3 (MW = 334.11) C, 35.95; H, 3.32; N, 8.38%. Found: C, 35.91; H, 3.34; N, 8.35%. FT-IR (KBr, cm-1): 3464 (w); 3287 (m); 3262 (m); 3242 (br, m); 3060 (w); 2985 (w); 2932 (w); 2876 (w); 1688 (vs); 1667 (vs); 1623 (m); 1540 (s); 1477 (s); 1448 (w); 1384 (w); 1364 (m); 1349 (m); 1319 (w); 1266 (vs); 1241

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(m); 1225 (m); 1207 (w); 1181 (m); 1128 (vs); 1084 (w); 1073 (w); 1040 (w); 974 (m); 966 (w); 944 (m); 899 (w); 875 (m); 817 (s); 780 (w); 729 (w); 697 (br, s); 622 (w); 594 (w); 556 (w). 1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.27 (3H, d, J = 6.25 Hz); 4.12 (1H,

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q, J = 6.25 Hz); 5.68 (OH, d, J = 5.00 Hz); 6.72 (1H, d, J = 8.5 Hz); 7.51 (1H, d, J = 8.5 Hz); 7.78 (1H, s); 8.50 (1H, s, CH=N); 11.30 (1H, s, NH); 11.57 ppm (1H, s, OH).

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C NMR

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(62.90 MHz, DMSO-d6); δ: 21.25 (-CH3), 67.40 (-CHOH), 81.57 (C−I), 119.46, 122.11, 137.06, 139.64, 146.44, 157.31, 171.42 (C=O) ppm.

2.3.5. Synthesis of (S,E)-2-hydroxy-N-(2-hydroxy-5-nitrobenzylidene)propanehydrazide

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(5)

Yield: 89%. M.p. 224-226 °C. Anal. Calc. for C10H11N3O5 (MW = 253.21) C, 47.43; H, 4.38; N, 16.59%. Found: C, 47.46; H, 4.40; N, 16.55%. FT-IR (KBr, cm-1): 3223 (br, m); 3204 (br,

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m); 3040 (br, w); 2991 (br, w); 2918 (br, w); 1698 (m); 1661 (vs); 1630 (s); 1613 (s); 1574

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(w); 1550 (m); 1525 (s); 1429 (w); 1376 (m); 1344 (vs); 1282 (s); 1244 (m); 1213 (w); 1193 (w); 1128 (m); 1119 (m); 1110 (m); 1093 (s); 1080 (m); 1050 (w); 1032 (w); 973 (w); 964 (w); 932 (w); 885 (w); 847 (m); 836 (m); 785 (w); 752 (s); 733 (w); 706 (br, m); 639 (m); 592 (w); 558 (w); 544 (w); 534 (w).1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.28 (3H, d, J = 5.75 Hz); 4.15 (1H, q, J = 6.25 Hz), 5.73 (1H, s, OH); 7.03 (1H, d, J = 8.50 Hz); 8.08 (1H, d, J = 8.00 Hz); 8.40 (1H, s); 8.64 (1H, s, CH=N); 11.69 (1H, s, NH); 12.20 ppm

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ACCEPTED MANUSCRIPT (1H, s, OH).

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C NMR (62.90 MHz, DMSO-d6); δ: 21.24 (-CH3), 67.44 (-CHOH), 117.52,

120.14, 124.57, 126.84, 140.26, 145.31, 163.01, 171.69 (C=O) ppm.

2.3.6. Synthesis of (S,E)-N-(2,5-dihydroxybenzylidene)-2-hydroxypropanehydrazide (6)

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Yield: 87%. M.p. 235-237 °C. Anal. Calc. for C10H12N2O4 (MW = 224.21) C, 53.57; H, 5.39; N, 12.49%. Found: C, 53.54; H, 5.42; N, 12.53%. FT-IR (KBr, cm-1): 3257 (br, m); 3091 (br, w); 3085 (br, w); 1671 (br, m); 1667 (br, m); 1660 (br, m); 1551 (br, w); 1504 (br, w); 1466

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(w); 1455 (w); 1371 (w); 1368 (w); 1320 (br, w); 1311 (br, w); 1282 (w); 1235 (m ); 1221 (m); 1161 (m); 1122 (m); 1094 (m); 1083 (w); 1034 (w); 972 (s); 962 (s); 889 (w); 863 (m);

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794 (m); 743 (w); 646 (w); 544 (w); 507 (w). 1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.25 (3H, d, J = 6.75 Hz); 4.10 (1H, q, J = 6.75 Hz); 5.62 (OH, d, J = 4.75 Hz); 6.26 (1H, s); 6.30 (1H, dd, J = 8.50 Hz, 4J = 2.00 Hz); 7.15 (1H, d, J = 8.25 Hz); 8.42 (1H, s, CH=N); 9.90 (1H, s, OH); 11.28 (1H, s, NH); 11.48 ppm (1H, s, OH). 13C NMR (62.90 MHz,

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DMSO-d6); δ: 21.36 (-CH3), 67.42 (-CHOH), 103.06, 108.01, 110.85, 131.94, 149.89, 159.92, 160.98, 170.83 (C=O) ppm.

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2.3.7. Synthesis of (S,E)-2-hydroxy-N-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-

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4-yl)propanehydrazide (7)

Compound 7 was synthesized by the reaction of pyridoxal with equimolar amount of (S)lactic acid hydrazide according the same procedure used for compounds 1-6. Yield: 92%. M.p. 193-195 °C. Anal. Calc. for C11H15N3O4 (MW = 253.25) C, 52.17; H, 5.97; N, 16.59%. Found: C, 52.24; H, 6.00; N, 16.52%. FT-IR (KBr, cm-1): 3353 (s); 3299 (s); 3229 (s); 3032 (m); 2934 (w); 2730 (s); 1964 (m); 1723 (vs); 1683 (w); 1621 (m); 1531 (s); 1476 (m); 1383

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ACCEPTED MANUSCRIPT (m); 1368 (m); 1345 (m); 1317 (m); 1302 (w); 1257 (s); 1211 (m); 1193 (m); 1157 (w); 1110 (s); 1066 (w); 1041 (s); 1023 (s); 988 (w); 976 (w); 933 (w); 921 (m); 892 (w); 882 (m); 859 (w); 802 (m); 785 (m); 752 (w); 720 (w); 695 (w); 658 (m); 637 (m); 565 (w); 534 (w). 1H NMR (250.13 MHz, DMSO-d6, 25 °C, TMS); δ: 1.29 (3H, d, J = 4.75 Hz); 2.59 (3H, s,

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CH3); 4.26 (1H, q, J = 4.75 Hz);4.70 (2H, s, CH2OH);5.09 (OH, s); 5.73(OH, d, J = 5.00 Hz); 8.14 (1H, s, CH=N); 8.94 (1H, s, CH=N, Ar-H); 12.46 (1H, s, NH); 13.15 ppm (1H, s, OH).

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C NMR (62.90 MHz, DMSO-d6); δ: 14.65 (-CH3), 21.09 (-CH3), 48.97, 58.21 (-

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CH2OH), 67.33 (-CHOH), 127.34, 129.06, 137.51, 143.96, 153.41, 161.05, 172.40 (C=O)

2.4. X-ray crystallography

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ppm.

A summary of the crystal data and refinement details for compound 1 are given in Table 1. Single crystal data collection for 1 was performed on a Kuma KM4 four-circle diffractometer

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with CCD detector, equipped with an Oxford Cryosystems open-flow nitrogen cryostat, using ω-scan and a graphite-monochromated Mo Kα (λ= 0.71073 Å) radiation at 100 K. Data collection, cell refinement, data reduction and analysis, and absorption correction were

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carried out with the Xcalibur R software, CrysAlisPro [31]. The structure was solved by direct

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methods with SHELXS-97 [32], and refined with full-matrix least-squares techniques on F2 with SHELXL-2013 [33]. The C-bonded hydrogen atoms were calculated in idealized geometry riding on their parent atoms. The N− and O− bonded hydrogen atoms were located from the difference map and refined isotropically. The molecular structure plots were prepared using Diamond [34].

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ACCEPTED MANUSCRIPT 2.5. Antibacterial activity assay Antibacterial activity of the synthesized compounds was determined against Gram-positive (S. aureus PTCC 1112, S. pneumonia PTCC 1240) and Gram-negative bacteria (E. coli ATCC 25922, P. aeruginosa PAO1). S. aureus PTCC 1112 and S. pneumonia PTCC 1240

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were kindly obtained from Persian Type Culture Collection of Iranian research organization for science and technology (Tehran, Iran) and were grown on blood agar. Because of E. coli ATCC 25922 is a standard strain for antimicrobial activity assay, it was used as quality

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control.

Minimum inhibitory concentration (MIC) of synthesized compounds was determined by the

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standard broth microdilution method according to the Clinical Laboratory and Standards Institute (CLSI) guidelines [35] in Mueller Hinton broth (MHB) using ~5 × 105 CFU/mL of inoculums concentration. Stock solution of tested compounds was prepared in dimethyl sulfoxide (DMSO) with the final concentration of 1 mg/mL. Two fold serial dilutions of

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compounds (512, 256, 128, 64, 32, 16, 8, 4, 2, 1 µg/mL) were prepared. Diluted compounds were inoculated with 0.1 ml of overnight culture of tested bacterial isolates containing 5×106 CFU/mL and incubated at 37 ºC for 24 h. Furthermore, the bacterial control without

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synthesized compounds and compounds control without bacterial strains were used as quality

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control for elimination of technical errors. Minimal inhibitory concentration (MIC) was calculated as the lowest concentration that inhibited visible growth of the organism. Gentamicin, as an extended spectrum antibiotic was used to compare the antibacterial activity of synthesized compounds. All assays were performed in triplicate.

2.6. Biofilm forming Capacity

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ACCEPTED MANUSCRIPT According to MIC results, compounds 5 and 7 were selected to investigate the biofilm formation in P. aeruginosa POA1. Biofilm-forming capacity was determined by using a microtitre plate assay, as described previousely [36]. 100µL of P. aeruginosa PAO1 cultures (0.1 mL) were incubated for 24 h at 37 °C in LB medium with and without sub-MIC

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concentrations (1/4 and 1/16 MIC) of compounds 5 and 7. Planktonic cells were removed and biofilms were washed three times with sterile PBS and fixed with 150 µL of 99% (v/v) methanol. The wells were stained with 0.5 % (w/v) crystal violet for 15 min at room

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temperature. Crystal violet was dissolved using 33% (v/v) glacial acetic acid for 20 min and the absorbance was measured at 570 nm. Biofilm-forming capacity was calculated as A570 of

are the mean of three measurements.

2.7. Molecular docking

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the treated PAO1 strain (ODs)/ A570 of the untreated PAO1 strain (ODc). Reported values

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Molecular docking studies of lactic hydrazide derivatives were carried out by Auto Dock Tool (ADT, version 4.2.6) [36b] in order to understand the various interactions between ligand and protein in detail. For docking studies, high resolution crystal structure of LtaS

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(PDB ID: 2W5T) was downloaded from the protein data bank website (PDB ID: 2W5T) and

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molecular docking studies were analyzed on same software. The 3D crystal structure of LtaS was prepared for docking by removing the co-crystallized ligands and waters. Also, polar hydrogen was added and Kollman and Gastegier charges were calculated. The ligands were prepared for docking by energy minimized using the Molecular mechanics MM+ and the semi-empirical AM1 methods with HyperChem Professional Release 8.0 program. The active site of the LtaS was defined to include residues of the active site within the grid size of 60 Å

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ACCEPTED MANUSCRIPT × 60 Å × 60 Å. The most stable docked conformation of test compounds was identified based on binding energy and also by analyzing the hydrogen bonds and hydrophobic interactions at the active site. The graphical representations of docked pose were performed using UCSF

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Chimera 1.10.2 program.

3. Results and discussion 3.1. Synthesis and characterization

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A series of novel chiral lactic-hydrazone derivatives were synthesized by condensation of (S)lactic acid hydrazide and salicylaldehyde derivatives. Also, pyridoxal was used as aldehyde

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derivatives for syntheses of lactic-hydrazone compounds. Hydrazones 1–7 were obtained following the same synthetic strategy which is shown in Scheme 1. All the newly synthesized hydrazones gave satisfactory elemental analyses for the proposed structure. The proposed structures were also confirmed on the basis of the spectral (IR, 1H NMR and 13C NMR) data

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and single crystal X-ray analysis (for compound 1).

3.1.1. Crystal structure of compound 1

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In order to define the structure of prepared lactic-hydrazones conclusively a single-crystal X-

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ray diffraction study was made for the crystal of compound 1. Compound 1 crystallizes in monoclinic system and chiral P21 space group. The asymmetric unit contains four independent molecules of compound 1. These four molecules (Fig. 1a) have similar conformation (Fig. 1b) and dimensions (Table 2). In compound 1, the bond lengths and bonding angles are in the normal range reported for hydrazone compounds [37]. The molecule adopts an E configuration with respect to the C=N bond and S configuration is seen

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ACCEPTED MANUSCRIPT around the C2 atom. The structure is stabilized by several C–H···O, C–H···N and C–H···π interactions (Fig. 2a). There is an intramolecular O–H···N and two intermolecular O–H···O and N–H···O hydrogen bonding interactions in the crystal of compound 1 which connects

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each molecule to others and form 1D polymeric chains (Fig. 2b).

3.1.2. Spectroscopic studies

The FT-IR spectra of compounds are shown in Figs. S1-S8 in the electronic supporrting

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information (ESI) file. The FT-IR spectra of the synthesized lactic-hydrazones (1–7) exhibit a broad band around 3100–3300 cm−1 due to –NH-vibrations. Also, in FT-IR spectra of these

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compounds very strong band appear around (1660-1723) cm−1 due to the amidic C=Ovibration [38]. In addition a broad band is centered at 3400-3490 cm-1 in 1–6 is due to the O– H groups of the phenolic and alcoholic moieties, probably involved in intramolecular and intermolecular hydrogen bonding interactions. The infrared spectra of 1–7 display FT-IR

frequency [39]. 1

H and

13

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absorption band around 1610-1650 cm-1 which can be assigned to the C=N stretching

C NMR spectral data of 1–7 in DMSO-d6 (Figs. S9-S23in ESI file) confirmed the

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proposed structure for them (Scheme 1). The chemical shifts for these compounds are

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comparable and very close to each other. In the 1H NMR spectrum of 1–7 a doublet peak (at about δ 1.27 ppm) and a quartet peak (at about δ 4.15 ppm) appear which are due to the –CH3 and –CH– groups, respectively. The signals at δ 11.10-12.64 and 11.48-13.15 ppm in the spectra of 1–7 are assigned to the amidic NH and phenolic OH groups, respectively. These signals rapidly loss when D2O is added to the solution (see Fig. S14). Also, the signal at about δ 5.68 ppm in the 1H NMR spectra of 1–7 is lost upon addition of D2O to the solution.

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ACCEPTED MANUSCRIPT Hence, this signal is assigned to the aliphatic –OH group of lactic moiety. The singlet resonances between δ 8.14-8.63 are assigned to the azomethine (–CH=N–) in the spectra of 1–7. In all of these compounds the number and intensity of aromatic hydrogen atoms (which appear between δ 6.25-8.50 ppm) are in agreement with the proposed structures. In the

13

C

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NMR spectrum of these compounds the peaks of –CH3 and –CHOH– groups are seen at about δ 21.25 and 67.43 ppm, respectively. The peaks around δ 171.4 and 147.0 ppm can be attributed to the C=O and C=N moieties, respectively. In compound 4 the signal of aromatic

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carbon atom comprising iodine substituent is observed at δ 81.57 ppm which is in the normal

3.2. Antibacterial activity

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range reported for aromatic C–I groups.

The antibacterial activity of these new compounds were evaluated on four

species of

microorganisms (S. aureus, S. pneumoniae, E. coli, and P. aeuroginosa) using the broth

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microdilution method. The screened compounds were dissolved individually in DMSO (dimethyl sulfoxide) in order to make up a solution of 1 mg/mL. All synthesized compounds showed antibacterial activity with MIC range of 64-512 µg/mL. According to Table 3, and

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comparing the MIC of lactic hydrazone derivatives, it is seen that the compounds with

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electron withdrawing groups like -I, -Br or -NO2 (compounds 3, 4 and 5) generally showed higher antibacterial activity in comparison with the compounds containing electron donating OCH3 or -OH groups (compounds 1 and 6). In general, compounds 5 and 7 showed higher antibacterial activities than the others. The high antibacterial activity of compound 5 is possibly attributed to the presence of the highly electronegative -NO2 substitute in this compound. Among the synthesized compounds, 5 and 7 were found to be the most active

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ACCEPTED MANUSCRIPT against S. aureus and E. Coli, respectively with MIC value of 64 µg/mL. The MIC of other compounds for bacterial pathogens was determined with range of 128-512 µg/mL. However, these compounds had lower activity than Gentamycin as a reference antibiotic. The high level antibacterial activity of compound 5 against S. aureus may be related to partial

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negative surface area and several hydrogen bonding donor/acceptor sites of this compound. According to Aslan et al. Report [40], high polarity of the molecule leads to higher binding ability, H-bond formation and intermolecular interactions. Also, strong nucleophilic nature of

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molecule induces higher antibacterial effect.

2-Hydroxy-N-((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)propanehdrazide

(7)

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with highest MIC value against E. coli contains two bioactive sites which are the lactic moiety [CH3-CHOH-C(=O)-] and pyridoxal (one form of Vitamin B6). Vitamin B6, as a component which involves in many enzymatic reactions in living organisms, is of interest as a starting material for preparing bioactive compounds. Because of the presence of multiple

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functional groups, and H-bond acceptance and donation [41], this compound can be considered as a safe pyridoxal based Schiff base candidate for other biological aspects.

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3.3. Effect on biofilm formation

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The effect of compounds 5 and 7 on biofilm formation is shown in Fig. 3. Both compounds showed a significant decrease (p<0.001) in biofilm formation when PAO1 strain was grown with 1/4 and 1/16 MIC of components in comparison with control. The inhibitory effect was concentration dependent.

3.4. Molecular docking

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ACCEPTED MANUSCRIPT Molecular docking was performed with a new potential antibiotic target of lipoteichoic acid synthase (LtaS) enzyme in S. aureus for prediction of binding ability of each synthesized compound at the active site pocket of the LtaS. It is an enzyme responsible for production of LTA in S. aureus which is required for staphylococcal growth, indicating that this enzyme is

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an extra cellular target for development of new antibiotics against Gram positive pathogens [42]. In order to understand the interaction of lactic hydrazone derivatives with LtaS, it was necessary to study the binding site of the lactic hydrazone derivatives in LtaS. The

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synthesized compounds were docked to LtaS to find out the preferred binding sites on the protein. The results of molecular docking analysis are given in Table 4. These results showed

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that docking energy range of all compounds is between -6.9 and -7.5 kcal/mol and number of H-bonds range is between 1 and 7. The main amino acid residue which is involved in H-bond interactions with the potent compounds is Thr 300. According to results, compound 5 in the most stable docked pose showed lowest interaction energy of -7.5 kcal/mol and was involved

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in the four hydrogen bonding interactions with Asp 349, His 416, Trp 354, Thr 300 in the active site of LtaS. The amino acid Asp 349 had formed hydrogen bonding with oxygen of hydroxyl group in lactic moiety (1.87 Å). The amino acids Trp 354 (2.11 Å), Thr 300 (1.81

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Å), and His 416 (2.95 Å) had formed hydrogen bonds with oxygen atoms of nitro substituent

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in phenyl ring (Fig. 4). On the basis of activity data and docking results, it was found the compound 5 had potential to inhibit LtaS enzyme. Furthermore, an in silico study of synthesized compounds was performed in order to prediction of absorption, distribution, metabolism and excretion (ADME) properties (Table 5). For this purpose, we computed drug likeness properties such as logarithm of partition coefficient (miLogP), topological polar surface area (TPSA), molecular weight (MW),

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ACCEPTED MANUSCRIPT number of hydrogen bond acceptors and donors (n-O/N, n-OH/NH), number of rotatable bonds (n-ROTB), molecular volume (MV), and Lipinski’s rule of five [43] using Molinspiration online software [44]. It was illustrated that all compounds obeyed the Lipinski’s rule of five. According to Lipinski’s rule of five, a candidate molecule -could be

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developed as an orally active if: the logP (octanol–water partition coefficient) ≤ 5, the molecular weight ≤ 500, the number of hydrogen bond acceptors ≤ 10 and the number of hydrogen bond donors ≤ 5 [45]. Our study showed compounds 1-7 followed these criteria and

4. Conclusion

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candidates and lead compounds for further research.

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they have good drug likeness score with no violations and, maybe developed as oral drug

A series of novel chiral lactic-hydrazone derivatives have been synthesized by condensation of (S)-lactic acid hydrazide and aromatic-aldehyde derivatives and characterized by elemental 13

C NMR spectroscopy and single crystal X-ray analysis.

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analysis, FT-IR, 1H NMR,

Antimicrobial activities of the synthesized compounds were investigated against Staphylococcus aureus PTCC 1112, Streptococcus pneumonia PTCC 1240, Escherichia coli

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ATCC 25922, and Pseudomonas aeruginosa PAO1 by broth microdilution method. All of

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synthesized compounds showed antibacterial activity with MIC range of 64-512 µg/mL. Also both of compounds 5 and 7 revealed a significant decrease in biofilm formation against PAO1. The docking result revealed that compound 5 can form hydrogen bond interaction with active residues of Asp 349, His 416, Trp 354, and Thr 300. The findings of molecular docking studies enhanced our understanding about interactions between these derivatives and LtaS active sites in detail and thereby help to further structural modification.

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ACCEPTED MANUSCRIPT Supporting Information CCDC 1445770 contains the supplementary crystallographic data for compound 1. This data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:

including the FT-IR, 1H NMR and

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(+44) 1223-336-033; or e-mail: [email protected]. Supplementary data to this article 13

C NMR spectra of the reported materials can be found

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online at DOI: Acknowledgments

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The authors are grateful to the University of Zanjan and Zanjan University of Medical Sciences for financial support of this study.

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[42] A. Gründling, O. Schneewind, Proc. Natl. Acad. Sci. USA. 104 (2007) 8478–8483. [43] C.A. Lipinski, L. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug. Delivery. Rev. 46 (2001) 326. Molinspiration

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[45] P. Ertl, B. Rohde, P. Selzer, J. Med. Chem. 43 (2000) 3714-3717.

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Republic,

Available

from:

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Scheme 1. The synthetic pathway of lactic-hydrazone derivatives

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Fig. 1. a) Four independent moolecules of compound 1 b) an overlay of four independent

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molecules in the crystal of compound 1

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Fig. 2. a) C−H···O, C−H···N and C−H···π interactions; b) O−H···O, N−H···O and O−H···N

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hydrogen bonding interaction in the crystal of compound 1 shown as pink dashed lines

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Fig. 3. Effect of sub-MIC concentrations of compounds 5 and 7 on biofilm formations. The

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results are presented as means ±SD, P< 0.001.

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a)

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c)

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Fig. 4. a) Ribbion view of docked pose of compound 5 (yellow colour stick model) with LtaS at the active site. b) Hydrogen bonding interactions between the docked pose of compound 5 (green colour stick model) and amino acids in the active site of LtaS. c) Surface view of compound 5 (green colour stick model) with LtaS.

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Table 1. Crystallographic data of compound 1 C11H14N2O4 Net formula Mr/g mol−1 238.24 T/K 100 Radiation MoKα Diffractometer Kuma KM4 Crystal system Monoclinic Crystal shape, color Block, yellow space group P21 a/Å 5.9119(10) b/Å 20.926(5) c/Å 18.940(4) β/° 94.36(2) V/Å3 2336.3(8) Z 8 calc. density/g cm−3 1.355 0.10 µ/mm−1 F(000) 1008 θ range 2.9–36.9 h,k,l −9→9, −32→32, −31→31 Rint 0.031 R(Fobs) 0.045 Rw(F2) 0.119 S 1.03 Absorption correction Analytical hydrogen refinement Mixed measured reflections 35969 independent reflections 17545 reflections with I > 2σ(I) 15731 Parameters 665 Restraints 1 Max electron density/e Å−3 0.55 −3 Min electron density/e Å −0.24

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ACCEPTED MANUSCRIPT Table 2. Selected bond lengths (Å) and bond angles (°) in 1 A 1.2313(17) 1.4226(19) 1.347(2) 1.3806(17) 1.285(2) 124.33(14) 119.86(11) 115.54(12)

B 1.2351(17) 1.4206(19) 1.342(2) 1.3794(18) 1.290(2) 125.24(13) 121.75(12) 114.51(12)

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D 1.2343(17) 1.4203(19) 1.3467(19) 1.3799(17) 1.2895(19) 124.73(13) 121.17(11) 114.90(12)

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1 1.2335(16) 1.4180(17) 1.3450(17) 1.3809(16) 1.2926(17) 124.91(12) 121.36(11) 113.87(11)

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Molecule O1—C1 O2—C2 N1—C1 N1—N2 N2—C4 O1−C1−N1 C1−N1−N2 N1−N2−C4

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1 2 3 4 5 6 7 DMSO Gentamycin

Bacteria strains E. Coli P.aeruginosa S.pneumonia S.aureus 128 128 256 256 256 256 256 512 128 256 128 128 128 256 128 256 64 128 128 128 128 256 256 256 256 64 128 128 16 2 16 16

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Compound

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Table 4. Docking energy, hydrogen bonds and interacting residues at the active site of LtaS. Interacting residues of LtaS His 476 Ser 256, Ser 480, Gly 478 Asp 349, Thr 300 (2), Arg 356 Tyr 417, His 476, Thr 300 (2), Trp 354 Asp 349, His 416, Trp 354, Thr 300 Thr 300 (2), His 347, Arg 356 (2) Lys 299, Tyr 477, His 476, Thr 300 (2)

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Hydrogen bond(s) 1 3 4 5 4 5 5

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Compound Docking energy (kcal/mol) -6.9 1 -6.9 2 -7.3 3 -7.1 4 -7.5 5 -7.0 6 -6.9 7

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Table 5. Drug likeness properties for compounds 1-7

91.15 81.92 81.92 81.92 127.74 102.15 115.04

MW

n- ON

≤ 500 238.24 208.22 287.11 334.11 253.21 224.22 253.26

n-OHNH donor

n-RoTB

MV

acceptors

Lipinski's violations

≤ 10 6 5 5 5 8 6 7

≤5 3 3 3 3 3 4 4

4 3 3 3 4 3 4

213.66 188.11 206.00 212.10 211.450 196.13 225.34

≤1 0 0 0 0 0 0 0

EP

TE D

M AN U

SC

≤5 0.66 1.06 1.84 2.12 0.99 0.56 0.12

TPSA

AC C

Rule 1 2 3 4 5 6 7

miLogP

RI PT

Compound

35

ACCEPTED MANUSCRIPT

Highlights New Chiral lactic hydrazone derivatives were synthesized and characterized by spectroscopic methods and single crystal X-ray analysis.

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Chiral lactic hydrazone derivatives as potential bioactive antibacterial agents showed antibacterial activity with MIC range of 64 - 512 µg/mL.

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Lipoteichoic acid synthase (LtaS) inhibitory effect of lactic hydrazone derivatives were shown by molecular docking studies.

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Molecular properties prediction and drug likeness score were calculated for the synthesized compounds.

AC C

EP

TE D

M AN U

SC

RI PT

•