In-silico design, synthesis, ADMET studies and biological evaluation of novel derivatives of Chlorogenic acid against Urease protein and H. Pylori bacterium

Background Plants have always played important role in treating human and animal diseases as a therapeutic agent for traditional medicine. Through extensive research throughout the world, potential of natural products have been identified to control the over activity of many enzymes. In-silico screening a library of chlorogenic acid derivatives highlighted some novel compounds which were found effective against urease enzyme and cancer causing H. Pylori bacterium. Selected top ligands possessing minimum binding energy and good docking score were synthesized in wet lab by suitable procedure and evaluated for urease enzyme inhibition and free radical scavenging property. Synthetic scheme includes three step reactions i. e protection of hydroxyl group of quinic acid part of chlorogenic acid with lactonisation process, anilide formation by reaction with substituted anilines followed by extraction with ethyl acetate under vacuum and deprotection of hydroxyl groups by treatment with hydrochloric acid. Results In-vitro results of the series concluded that compounds C4a, C4d and C4b (IC50 11.01 ± 0.013, 13.8 ± 0.041 and 15.86 ± 0.004 µM respectively in urease inhibition and 5.10 ± 0.018, 5.34 ± 0.007 and 6.01 ± 0.005 µM in antioxidant property against DPPH) were found to be significantly potent with excellent dock score − 10.091, − 10.603, − 9.833 and binding energy − 62.674, − 63.352, 56.267 kg/mol as compared to standard drugs thiourea and acetohydroxamic acid (− 3.459, − 3.049 and − 21.156 kJ/mol and − 17.454 kJ/mol) whereas compounds C4c, C4(e, h) exhibited moderate in vivo activity when compared to standard. Conclusions Selected candidates from the outcome of in vitro urease inhibitory were further examined for anti-H. Pylori activity by well diffusion method against H. pylori bacterium (DSM 4867). Compound C4a showed significant anti-H. Pylori activity with zone of inhibition 10.00 ± 0.00 mm and MIC value 500 μg/mL as compared to standard drug acetohydroxamic acid having zone of inhibition 9.00 ± 0.50 mm and MIC 1000 μg/mL. Molecular docking studies also showed that compounds show strong inhibition by forming strong hydrogen bonding interactions with residues of pocket site in target protein. Hence, the present investigation studies will provide a new vision for the discovery of potent agents against H. Pylori and urease associated diseases.


Introduction
Enzyme inhibition studies has been the interesting topic of research today since a large number of diseases are directly or indirectly associated with some enzyme. A hydrolytic enzyme, urease is found abundantly in nature in soil as well as in a large number of microbes like bacteria, algae, fungi. Urease is beneficial for plants as it provides nitrogen to them which is essential for their growth but its excess activity may harm environment by producing excess of ammonia. In human beings it serves the role of a pathogen and responsible for many serious disease like gastric cancer, peptic ulcer, crust formation in catheter, hepatic coma, hepatic encephalopathy, pyelonephritis, [1,2] ureter obstruction which can lead to kidney fibrosis [3], etc. Among various responsible factors for colonization of H. Pylori presence of urease within bacteria has been found to be most important virulence factor as it causes buffering of stomach acid, produce toxic effect on epithelium cells of stomach, cause disruption of junction of cells tight junctions and sheaths antigens [4][5][6][7]. Furthermore it spreads its virulence by catalyzing urea present in human and animal body into ammonia which causes elevation in pH of gastric media and thereby make comfortable environment for the various pathogenic bacteria containing ureases such as H. pylori to survive there and spread colonies [8][9][10][11][12][13][14][15][16][17].
These infections need attention and proper treatment which can be possible only by inhibiting the responsible enzyme; this compelled us to investigate on urease inhibitors. Drugs used clinically for the treatment of ailments caused by urease producing bacteria includes bismuth complex, phosphoramidates, imidazole derivatives, Antibiotics [18,19], proton-pump inhibitors [20,21], H 2 -blockers [22,23] and hydroxamic acids with double and triple therapy to eradicate the H. pylori infection [24,25]. But cost of therapy, antibiotic resistance, and associated side effects like teratogenic effects shown by hydroxamic acid and rapid disintegration of phosphoramidates at low pH [26][27][28] motivated researchers towards natural compounds.
A number of biologically active compounds from natural products have been studied against many diseases [29,30]. One of the most abundant polyphenol present in human being diet specially consumed in green coffee, fruits and vegetables; chlorogenic acid is a naturally occurring ester of quinic acid and caffeic acid [31]. The name chlorogenic acid was introduced in 1846 in response to explain the major component present in beans of green coffee (Payen 1846) [32]. Chlorogenic acid has antioxidant, antimicrobial, and anxiolytic activity antioxidant, antimicrobial, antibacterial, antiviral and anti-inflammatory capacities, which can contribute in the prevention of chronic and cardiovascular diseases, as described by several studies in-vivo and in-vitro [31,[33][34][35][36][37][38][39][40]. Recent reports indicate that this compound also exerts anti-proliferative effects against some types of cancer cell, such as those from liver, blood, or brain cancer [41][42][43]. Chlorogenic acids not only play a role as antioxidant of coffee, but also contribute to the aroma and taste profile [44,45]. In pharmaceutical industries it has been in continuous demand because of its beneficial effects [46].
According to literature studies a large number of medicinal plants having phenolic compound and polyphenols as active component have been reported for their anti-H. Pylori activity. Recent findings of many researchers stressed that phenolic compounds having strong antioxidant profile play important role in inhibiting H. pylori. Literature uncovers the antioxidant and H. Pylori urease inhibition profile of chlorogenic acid extracted from Oregano, Cranberry juices as well as extracts of other phytochemical extracts probably by inhibiting urease enzyme [47]. Konstantinopoulou et al. studied phenolic compound chlorogenic acid extracted from plant Anthemis altissima against H. Pylori bacterium and found that it was quite effective in inhibiting the growth of bacteria with MIC value 312.5-1250 μg/mL [48]. In an application for patent for cosmetic and dermatological composition for anti-rash also used chlorogenic acid extracted from aqueous green coffee extract as anti-urease agent [49]. Shang et al. reported that extract of L. japonica containing chlorogenic acid and luteolin glycoside as main component showed good therapeutic potential against H. Pylori [50]. Paun et al. reported urease inhibitory activities of extracts of Geranium spp., Helleborus spp. and Hyssopus spp. polyphenolic extracts having chlorogenic acid as active compound [51]. Furthermore the docking interactions with target protein help in optimization of lead molecules [52] which further help in designing the ligands as potential urease inhibitors. In this regards, targeting urease for treating pathogenic disorders caused by it may open a new line of treatment for infections caused by urease-producing bacteria. This research was conducted in search for newer pharmacophore with emphasis towards urease. ethylacetate and washing with water and brine solution. Anilides of chlorogenic acid was prepared in the second step by refluxing with substituted anilines (

NO 2
C4h F and completion of reaction was confirmed by single spot in TLC under UV lamp. Evaluation of structure of novel derivatives was confirmed by spectroscopic methods such as IR, 1 H NMR, 13 CNMR, elemental analysis. Derivatisation was confirmed by disappearance of peak of saturated acid (C=O) at 1716 cm −1 of chlorogenic acid and appearance of C=O amide peak at 1629-1683 cm −1 in compounds C4(a-h). Moreover peak of N-H amide at 3310-3334 cm −1 in compounds C4(a-h) also confirmed formation of anilides. 1 HNMR signals were interpreted by their value of chemical shift for particular protons of synthesized derivatives, coupling constant and multiplicities of signals. Singlet observed at δ 8.5-9.0 was due to NH of secondary amide in all derivatives and in 13 C NMR signal of carboxylic acid carbon at δ 180 ppm was shifted to 175 ppm due to formation of amide derivative confirmed formation of compound. Further and final confirmation process involves analyzing their mass spectrum for determination of molecular weight in which Q-ToF Micro instrument was used as ion source. Maximum number of the derivatives showed peak at M + (molecular ion peak), (M ++1 ), (M ++2 ) in positive chemical ionization and (M +1 ), (M +2 ), M + during negative chemical ionization mode. Elemental analysis of chlorogenic acid derivative was carried out in CHNS analyzer where C, H and N in percent were found within acceptable range.

Biological activity
It has been recommended that phenolic moiety has been found to be important for antioxidant and urease inhibition property of synthesized compounds. The newly synthesized derivatives of chlorogenic acid were examined for their urease enzyme inhibition, free radical scavenging activity and for H. Pylori inhibition. As structure of urease from different sources has common structural features therefore urease inhibition studies were carried out on jack bean urease. Inhibition studies were carried out by indophenol technique described by Weatherburn [56] in which measurement of concentration of ammonia released during the reaction between reagents and compounds were done. Antioxidant property of derivatives was observed by their ability to donate hydrogen or electrons to DPPH [57][58][59]. According to our investigational data almost all the synthesized derivatives showed good potential in both activities than parent drug chlorogenic acid as well as standard drug having IC 50 value in range of 11.01-22.02 µM and 5.10-8.48 µM against urease inhibition with competitive mode of inhibition and free radical scavenging activity using thiourea and ascorbic acid as standard drugs respectively ( Table 2). According to experimental values derivative C4a, C4d, C4a and C4b bearing nitro and chloro at 2nd and 4th position and C4d bearing only nitro group at 3rd position) were found to possess excellent IC 50 value i.e. 11.01 ± 0.013, 13.8 ± 0.041 and 15.86 ± 0.004 µM respectively in urease inhibition (Fig. 1) and 5.10 ± 0.018, 5.34 ± 0.007 and 6.01 ± 0.005 µM in antioxidant property (Fig. 2) against DPPH. It was observed from the experimental data that substitution of electron withdrawing halogen specially chloro and nitro at anilide ring specifically 2nd and 4th position strengthen the potential of candidate towards enzyme inhibition while electron donating groups like methoxy and methyl groups at 3rd and 4 position (C4e, C4f and C4g) does not showed remarkable potential. Antioxidant activity was also highly influenced by addition of electron withdrawing groups in compound C4a and C4b (IC 50 5.10 ± 0.018 and 6.01 ± 0.005 µM) chloro and nitro group work synergistically and improves hydrogen releasing capacity of phenolic group of parent chlorogenic acid.
Derivatives C4a, C4b and C4d which were found best in urease inhibition and antioxidant activity as well as in terms of docking score were tested further against antibacterial efficiency against Helicobacter pylori DSM 4867 using AHA as a standard and DMSO as control. MIC 50 values, lowest concentration of compound inhibiting microbial growth after 24 h on agar plates in comparison with positive control [60][61][62][63], were calculated for compounds and the results revealed that C4a displayed its excellent potency with good zone of inhibition i.e. 10.00 ± 0.00 mm and MIC value 500 μg/mL as compared to standard drug acetohydroxamic acid having zone of inhibition 9.00 ± 0.50 mm and MIC 1000 μg/mL. In-vitro results concluded that compound C4a bearing nitro and chloro group at 2nd and 4th position indicated excellent antibacterial action against H. Pylori probably by interacting with the target protein efficiently and inhibited the growth of pathogenic bacteria.

Enzyme kinetics
Study of inhibitory effect of chlorogenic derivatives on jack bean urease was performed to check the inhibitory potential, kinetics studies and mechanism of inhibition in phosphate buffer and 1 mM EDTA at pH 8.2. Lineweaver-Burk plots (1/absorbance versus 1/urea) was constructed from kinetic data to determine the mechanism of enzyme inhibition by varying the concentration of substrate urea in the absence and presence of different concentrations of most potent compound C4a (Fig. 3). Inhibition constant (Ki) was determined as the intersection on x-axis of the plot of 1/V max and varying concentration of inhibitor obtained from Lineweaver-Burke plot and all experiments were performed in triplicate. Linear lines were formed which intersected at y-axis indicated competitive type of Inhibition as Km was different but V max was same at different concentrations of compound. Competitive inhibition infers that compound C4a will probably compete for binding at the site of substrate in enzyme active site pocket. Binding confirmations from molecular simulation studies also confirm the mode of inhibition as shown in Fig. 3.

Structure activity relationship
Studying results from antioxidant nature and urease inhibition activities of newly synthesized morin derivatives, structure activity relationship can be derived (Fig. 4).
1. Incorporation of aromatic ring bearing chloro and nitro group ortho and para position improved urease inhibition and hydrogen donating capacity of compound (compound C4a, C4b and C4d bearing nitro at 3rd position). Nitro and chloro group probably worked synergistically and make the ligands suitable for proper fitting into the active site of protein thereby increased urease inhibition property, also predicted by molecular docking studies (Fig. 5).

Presence of Nitro group (C4a, C4b and C4d)
improved biological profile of compound. 3. Addition of aromatic ring bearing electron withdrawing groups (C4e, g) did not much improve the urease efficiency of compound might be by stabilizing the molecule. 4. Incorporation of aromatic ring having bromo and fluoro substituents only improved the antioxidant nature of parent compound (C4c, C4h).

Molecular docking study
Molecular docking is an informative tool for investigating affinity between ligand and protein target. With the aim to insight the binding interaction of the compounds with jack bean urease protein [64][65][66], Schrodinger's maestro package of molecular docking software was used for in-silico studies of newly designed library of chlorogenic acid proposed derivatives. Plant urease jack bean having PDB ID 3LA4 having good resolution about 2.0 Å, crystallographic complex was selected for docking the ligands by induced fit docking (IFD) method. Docking parameters revealed that designed ligands possess excellent docking score and binding energy as compared to standard drugs thiourea and AHA (Table 3). Exhaustive ligand protein interaction studies showed that newly synthesized ligands bind within active site of enzyme firmly by hydrogen bond formation, pi-pi stacking and hydrophobic interaction (

Substitution of methyl and floro at ortho position did not show much improvement in both activities to large extant.
Nitro substitution at para position increase the urease inhibitory activity much better than bromo substitution

Ligands I nteraction Diagram Protein-ligand Interaction Description
binding capability for protein as compared to parent and standard compounds.

Admet studies
In drug discovering the ADME profile of drug like molecules is very important and for this purpose Schrodinger's maestro molecular modeling package Qikprop module was utilized. The Absorption, distribution, metabolism and excretion descriptors of the designed molecules are given in Table 5. Blood brain barrier partition coefficient (QPlogBB), estimated IC 50 value for HERG K + channels obstruction (log HERG), estimation of human serum albumin binding (QPlogKhsa), permeation through skin estimation (QPlogKp), apparent Caco-2 cell permeability estimation in nm/sec (QPP-Caco) and apparent MDCK cell permeability estimation in nm/sec (QPPMDCK), partition coefficient in octanol and water Log P, solubility in aqueous media log S, Lipinski's rule of five and Percent Human Oral Absorption (%HOA). Results revealed that ADME parameters of each ligand within the bounds of satisfactory range without violating Lipinski's rules [67][68][69][70].

Materials
Chlorogenic acid was purchased from CDH and Jack bean urease from Sigma Aldrich. Analytical grade reagents and solvents were utilized as a part of study and obtained locally. Progression of reaction was observed via. Thin layer chromatography and recrystallization of products was done in order to purify the compounds which were again checked for purity by thin layer chromatography (TLC) performed on precoated plates (0.25 mm) purchased from Merk and visulisation was done under UV lamp. Measurement of melting point was done on Sonar melting point apparatus in open capillary tubes and was uncorrected. The spectral data, IR and 1 H NMR, 13 CNMR were measured by standard procedures. Brucker 12060280, Germany Software: OPUS 7.2.1394 spectrophotometer in cm −1 was used for recording IR spectra of derivatives and elemental analysis was done on Perkin-Elmer 2400 C, H, N analyzer. The 1 HNMR and 13 CNMR spectra were recorded in DMSO-d 6 on a Bruker DRX-400 FTNMR instrument. Coupling constants (J) were reported in Hertz (Hz) and chemical shifts were depicted as δ (parts per million). Waters Micromass Q-ToF Micro instrument was used for Mass spectra recording.
General procedure for synthesis of compounds (C3ah) Amide derivatives of chlorogenic acid was prepared by refluxing a solution of compound 2 (0.1 g, 0.46 mmol) in dichloromethane (5 mL) with substituted aniline (0.7 mmol) at 80 °C for 2-3 h (scheme 1). Reaction progress was continuously monitored with the help of TLC. After the completion of the reaction as indicated by single spot on precoated plate, the solvent was evaporated and diluted with ethyl acetate (25 mL). Mixture was washed with water (250 mL) followed by brine solution (30 mL). The crude product was obtained which was purified by dichloromethane to afford the pure product (C3a-h).
General procedure for synthesis of compounds (C4ah) For deprotecting the hydroxyl groups of synthesized amide derivatives, synthesized compounds C (3a-h) (5.0 g) was dissolved in water (10 mL), then added aq. 1 M HCl (40 mL) at room temperature. The reaction mixture was stirred at room temperature for 1-2 h. Solvent was removed under reduced pressure. The residue HCl was removed by adding EtOEt (20 × 3 mL) under vacuum [53-

Protocol of in-silico study
In-silico studies of the designed derivatives of chlorogenic acid were done by Induced fit docking protocol of Schrodinger's Maestro molecular modeling software. Urease from plant source Jack bean having resolution 2.05 Å (PDB code: 3LA4) was retrieved from protein databank online (http://www.rcsb.org) from the Research Collaboratory for Structural bioinformatics (RCSB) and has been used as for the purpose of docking the newly designed ligands in X-ray crystallized structure form for finding the active lead compound.

Protein preparation
With the help of protein preparation wizard named prepwiz, protein was prepared for docking studies. In this process assigning bond orders, partial charges and hydrogens were added, side chains and loops having missing atoms were fabricated, all waters (with exception of those which were coordinated to metals) and unnecessary atoms were expelled and alternate conformations were performed.

Generation of grid
After preparation of protein area for docking interaction with ligands was selected on protein by forming a grid over it. Grid generation was done by grid generation module of Schrodinger Maestro programming according to the conventions.

Ligand preparation
Proposed structure of ligand were drawn by Chemdraw Ultra 8.0 (ChemOffice Package) software which were finally saved as mol. file format and finally prepared by LigPrep module. Addition or removal of hydrogen bond were done for ideal confirmation of ligands, partial charges were computed according to the OPLS-2005 force field with 32 stereo isomers, tautomers, and other options like ionization at the physiological pH 7 ± 2 were set as default options. Ligands were selected for simulation studies after their preparation based upon their minimum potential energy. Geometry of ligand was minimized by application of force field algorithm according to protocol of software.

Molecular docking
Ligand-receptor interaction study using maestro package was done according to the given protocol. While performing Grid-based Ligand Docking ligands were allocated at various positions of grid within range of 1 Å and were rotated in all three angles and glide score (GScore Gridbased Ligand Docking with Energetics) scoring function was obtained as the output for each ligand given in Table 2. Derivatives possessing higher score in terms of glide score and those possessing lower binding energy were selected for further synthesis process [65][66][67][68][69][70].

Urease inhibitory assay
Urease enzyme inhibition investigation studies for all synthesized compounds were done by the method developed by Weatherburn (1967) named Indophenol against Jack Bean Urease [56]. Briefly, incubation of solution of Jack bean protein 25 μL was done at temperature of 30 °C for a period of 15 min with 55 μL of buffers solution having 100 mM urea and 5 μL of test solution in 96-well plates. 45 μL phenol solution composing phenol 1% w/v and solution of sodium nitroprusside 0.005% w/v and alkali reagent 70 μL composed of sodium hydroxide 0.5% w/v and active sodium hypochloride solution 0.1% were mixed to every well. The measurement of increase in absorption at 625 nm was done after 50 min by help of a microplate reader (Molecular Device, USA). Readings were recorded in triplicate set in a final volume of 200 μL using thiourea as standard. Each assay was performed at pH 8.2 (0.01 M K 2 HPO 4 .3H 2 O, 0.01 M LiCl 2 and 1 mM EDTA). Calculation of Inhibition in percentage of synthesized derivatives was done by the formula given as: where, A control is control absorbance; A sample is test absorbance.

Dpph assay for antioxidant activity
Antioxidant nature of the synthesized derivatives was measured by the help of DPPH method. According to this method synthesized compounds were allowed to react for 0.5 h at 37 °C with stable free radical, 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH). The DPPH was taken in a concentration of 300 µM. Decrease in value of absorption at 515 nm was measured after the period of incubation using a microplate reader using ascorbic acid as a standard. Molecule capable of donating proton to DPPH and cause its reduction acts as an antioxidant. Following reduction deep violet coloured DPPH solution changes to yellow depending upon the nature of antioxidant compound, which brings a measurable decrease in value of absorption at 517 nm [57][58][59]. The proton or electron donating capacity of the derivative was estimated from the fading of deep purple colored methanolic solution of 1, 1-diphenylpicrylhydrazyl (DPPH). Incubation of mixture was done at room temperature for 30 min and measurements were done at 517 nm against blank. Calculation of percentage inhibition was done by following formula: where, A control is control absorbance; A sample is test absorbance.

Anti-H. pylori activity
Since H. Pylori is not acidophilic in nature therefore it requires urease, which produced ammonia by hydrolyzing urea present in stomach, for its survival in acidic gastric medium. Because of virulence nature of urease researchers decided to target its inhibition for anti-H. Pylori drugs. Novel synthesized derivatives were investigated against H. pylori bacterium (DSM 4867, AHA as standard) for their antibacterial activity by using Well diffusion technique. The stock solution of compounds in DMSO (1000 μg/mL) were prepared. Cell suspension was prepared from culture grown on BHI broth. The cell suspensions of all the cultures were adjusted to 1-2 × 10 5 cells/mL. Helicobacter pylori (100 µL) was inoculated by spread plate technique on agar plates (90 mm). Agar wells (5 mm) were made on H. pylori inoculated media and impregnated with 2500 µg of each sample and standard which were incubated @ 35 °C for 24-48 h with 5% CO 2 and observed for zone of inhibition around the well. MIC was determined against Helicobacter pylori by micro broth dilution technique as per NCCLS method [60][61][62][63].

Kinetic studies
Kinetic studies of the derivatives were done to calculate the rate constant Vmax and Michaelis constant Km by plotting Lineweaver bruke plot of 1/[S] (substrate concentration) against 1/[V] velocity. To examine the mode of inhibition compound C4a was solubilized in DMSO and further dilutions were prepared accordingly. Plot was prepared by using varying concentration of substrate (urea) in with varying concentration of inhibitor C4a. Inhibitory constant values (Ki) were determined as the interaction on the X-axis of the plots of the slope against different concentration of derivatives C4a. Mode of inhibition was found to be competitive as value of Km was different but V max was same. All experiments were performed in triplicate and kinetic study for interaction of enzyme with inhibitor was computed with Graphpad prism 7.0 software.

Statistical analysis
Output of statistical analysis has been depicted in mean ± SEM whereas statistical examination of data collected experimentally was done by one-way analysis of variance (ANOVA). Considerable difference revealed by ANOVA p < 0.05 was regarded significant. Evaluation of statistical data was done by Graph Pad Prism 7.0 Version for Windows (San Diego, CA, USA).

Conclusion
In conclusion, selected derivatives of chlorogenic acid were synthesized after in silico screening of library of designed derivatives against jack bean urease enzyme. Computational studies helped in selection of potential candidate also shed light into the underlying mechanism of inhibition. Synthetic procedure includes three step reactions which involve lactone formation of chlorogenic acid followed by reaction with substituted anilines. Novel synthesized derivatives were evaluated for their urease inhibitory, DPPH-free radical scavenging and Anti-H. Pylori activity by in vitro method. Among the series all molecules were found effective and specific in their action with good IC 50 values and MIC against H. Pylori. Designed candidate C4a was found top most in all parameters and hence can be the potential lead compound in future for treatment of pathologies caused by urease as well as against H. Pylori infection.