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Naringenin derivatives as glucosamine-6-phosphate synthase inhibitors: synthesis, antioxidants, antimicrobial, preservative efficacy, molecular docking and in silico ADMET analysis

Naringenin derivatives as glucosamine-6-phosphate synthase inhibitors: synthesis, antioxidants,... Background: Preservatives have to be added in food, pharmaceuticals and cosmetics products to maintain their shelf life. However, the existing chemical based preservatives have been associated with severe side effects that compel the researchers to find better safe preservatives based on natural products. G-6-P synthase is an important enzyme for bacterial and fungal cell wall synthesis and offers as a potential target to find better G-6-P synthase inhibi- tors based antimicrobial compounds. Naringenin, a flavanone, has been reported for a wide range of pharmacological activities including antimicrobial activity, which makes it a potential candidate to be explored as novel G-6-P synthase inhibitor. Results: The synthesis of naringenin derivatives with potent G-6-P synthase inhibitor having remarkable antioxi- dant, antimicrobial and preservative efficacy was performed. Among the synthesized compounds, the compound 1 possessed good antioxidant activity (IC value, 6.864 ± 0.020 µM) as compared to standard ascorbic acid (IC value, 50 50 8.110 ± 0.069 µM). The antimicrobial activity of synthesized compounds revealed compound 1 as the most potent compound (pMIC 1.79, 1.79, 1.49, 1.49, 1.49 and 1.49 μM/mL for P. mirabilis, P. aeruginosa, S. aureus, E. coli, C. albicans and A. niger respectively) as compared to standard drugs taken. The compound 2 showed comparable activity against P. mirabilis (pMIC 1.14 μM/mL), C. albicans (pMIC 1.14 μM/mL) while the compound 3 also showed comparable activity against C. albicans (pMIC 1.16 μM/mL) as well A. niger (pMIC 1.46 μM/mL), likewise the compound 4 showed compa- rable activity against P. mirabilis (pMIC 1.18 μM/mL) as compared to the standard drugs streptomycin (pMIC 1.06, 1.36, 1.06 and 1.96 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli respectively), ciprofloxacin (pMIC 1.12, 1.42, 1.12 and 1.42 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli respectively), ampicillin (pMIC 1.14, 0.84, 0.84 and 1.74 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli respectively) and fluconazole (pMIC 1.08 and 1.38 μM/mL for C. albicans and A. niger respectively). The molecular docking with the target G-6-P synthase pdb id 1moq resulted with an better dock score for compound 1 (− 7.42) as compared to standard antimicrobial drugs, ciprofloxacin *Correspondence: dranuragkhatkarmdurtk@gmail.com; anuragpharmacy@gmail.com Laboratory for Preservation Technology and Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, India Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lather et al. BMC Chemistry (2020) 14:41 Page 2 of 15 (− 5.185), ampicillin (− 5.065) and fluconazole (− 5.129) that supported the wet lab results. The preservative efficacy test for compound 1 in White Lotion USP showed the log CFU/mL value within the prescribed limit and results were comparable to standard sodium benzoate, ethyl paraben and propyl paraben as per USP standard protocol. Conclusions: The synthesized naringenin derivatives exhibited significant G-6-P synthase inhibitory potential with good selectivity towards the selected target G-6-P synthase. Compound 1, bearing nitro group showed good antioxi- dant, antimicrobial and preservative efficacy compared with the standard drugs taken. The mechanistic insight about the compounds within the active site was completed by molecular docking that supported the results for novel synthesized G-6-P synthase inhibitors. Keywords: G-6-P synthase, Naringenin derivatives, DPPH, Preservative efficacy Introduction Naringenin is a naturally occurring bioflavonoid pre - The use of packaged foods containing various additive’s sent in various fruits, vegetables and honey which is used viz. artificial sweeteners, colorants, stabilizers, preserva - as a dietary supplement due to its low toxicity [19–21]. tives etc. has greatly increased in recent years. As per Naringenin has been reported for its diverse pharma- recent data available it is estimated that 75% of the con- cological profile including its antibacterial property as temporary diet is packaged food and on an average every shown in Fig. 2 [22–41]. person consumes 3.6 to 4.5 kg of food additives per year Further, naringenin could be utilized as a potential [1]. candidate for evaluation of its G-6-P synthase inhibitory Among other additives the preservative such as response. Hence, it was planned to synthesize and inves- sodium benzoate, ethyl paraben, propyl paraben, butyl- tigate the naringenin derivatives for their antioxidant, ated hydroxytoluene (BHT), butylated hydroxyanisole antimicrobial, preservative efficacy and in silico evalua - (BHA), etc. plays a vital role to maintain the shelf life tion for G-6-P synthase inhibition. of various food, pharmaceuticals and cosmetic prod- ucts [2–4]. However, the existing chemical preservatives Results and discussion have been associated with serious side effects viz. estro - Chemistry genic effect, breast cancer, malignant melanoma, contact Naringenin derivatives were synthesized according to eczema, endocrine disruption, etc. [5–12]. Hence, there Kriza et  al. 2011 with slight modifications as shown in is an urgent need for the discovery of novel and safer pre- Scheme 1 [42]. The chemical structures of all the synthe - 1 13 servatives for use in food, pharmaceuticals and cosmetic sized compounds were confirmed by FTIR, H NMR, C products. NMR, mass spectroscopy and elemental analysis which G-6-P synthase is a complex enzyme involved in the were in agreement with the structures. formation of UDP-N-acetyl glucosamine and catalyzes For the synthesis of naringenin derivatives substituted the initial step in hexosamine biosynthesis. One of these aniline (0.01 mol) was taken in a round bottom flask and catalyzed products, N-acetyl glucosamine, is an impor- concentrated hydrochloric acid was added drop wise with tant part of the peptidoglycan layer of bacterial and fun- continuous stirring. Equimolar concentration of narin- gal cell wall. Hence, G-6-P synthase may act as potential genin (0.01  mol) was dissolved in ethanol (50  mL) and target for discovery of novel antimicrobial compounds was refluxed for 80–100 h at 80 °C on heating mantle. All which could be evaluated for their preservative efficacy to the compounds in series were synthesized according to find better and safe preservatives [13, 14]. the standard procedure outlined in Scheme  1. Comple- The complex 3-D crystal structure of G-6-P syn - tion of reaction was confirmed by TLC under UV lamp thase can be utilized for molecular docking to and FTIR spectra. explore the structural requirements for the pharma- Formation of compound 1, 2, 3 and 4 was confirmed by cophore complex. Flavonoids such as luteolin, cat- peaks of IR, NMR, mass spectroscopy. In positive chemi- echin, (4S)-2-Methyl-2-phenylpentane-1,4-diol, cal ionization most of the naringenin derivatives showed 7-Methoxy-2,3-dihydro-2-phenyl-4 quinolone, 3-(tert- (M++1), M+ (molecular ion peak), (M++2) and in neg- Butoxycarbonyl)-6-(3 benzoylprop-2-yl)phenol and ative chemical ionization mode showed (M+1), (M+2), (3R,4S)-4-(methylamino)-1-phenylpent-1-en-3-ol also M+. The elemental analysis established the synthesis of have been explored for G-6-P synthase inhibition [15– naringenin derivatives where the percentage of C, H and 18]. Some flavonoids along with their G-6-P synthase N in the synthesized compounds was observed within inhibitory dock score have been shown in Fig. 1. defined limits. The reaction mixture was concentrated, La ther et al. BMC Chemistry (2020) 14:41 Page 3 of 15 Fig. 1 G-6-P synthase inhibitory profile of flavonoids and their derivatives cited in the recent literature Antimicrobial activity after that precipitates formed were filtered off and dried. Minimum inhibitory concentration Crude products were recrystallized by alcohol which The antimicrobial activity of synthesized compounds yielded the final compounds 1–4. revealed compound 1 as the most potent compound (pMIC 1.79, 1.79, 1.49, 1.49, 1.49 and 1.49  μM/mL for Antioxidant activity P. mirabilis, P. aeruginosa, S. aureus, E. coli, C. albi- DPPH radical scavenging activity cans and A. niger respectively) as compared to standard All the synthesized compounds were evaluated for drugs taken. The compound 2 showed comparable activ - antioxidant profile by using DPPH radical scaveng - ity against P. mirabilis (pMIC 1.14  μM/mL), C. albicans ing assay method (Table  1). The compound 1 was (pMIC 1.14 μM/mL) while the compound 3 also showed observed as the most potent antioxidant compound (IC comparable activity against C. albicans (pMIC 1.16  μM/ 6.864 ± 0.020  µM) as compared to standard L-ascorbic mL) as well A. niger (pMIC 1.46  μM/mL), likewise the acid (IC 8.110 ± 0.069  µM). However, compounds compound 4 showed comparable activity against P. mira- 3 and 4 showed moderate antioxidant activity (IC bilis (pMIC 1.18  μM/mL) as compared to the standard 7.170 ± 0.028  µM and 7.801 ± 0.077  µM, respectively) drugs streptomycin (pMIC 1.06, 1.36, 1.06 and 1.96 μM/ as compared to standard. The electron withdrawing mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli, strongly deactivating nitro group in compound 1 may be respectively), ciprofloxacin (pMIC 1.12, 1.42, 1.12 and responsible for better antioxidant activity. The presence 1.42 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and of weakly deactivating electron withdrawing chloro and E. coli, respectively), ampicillin (pMIC 1.14, 0.84, 0.84 fluoro groups present in compound 3 and 4 have moder - and 1.74 μM/mL for P. mirabilis, P. aeruginosa, S. aureus ate antioxidant activity. I C value of synthesized narin- and E. coli, respectively) and fluconazole (pMIC 1.08 and genin derivatives has been shown in Fig. 3. 1.38 μM/mL for C. albicans and A. niger, respectively). In Lather et al. BMC Chemistry (2020) 14:41 Page 4 of 15 Fig. 2 Pharmacological potential of Naringenin general, the results of MIC studies (Table 2) revealed that and degree of microbial log reduction has been repre- the synthesized compounds have better anti bacterial sented in Fig. 5. and anti fungal potential as compared to standard drugs streptomycin, ciprofloxacin, ampicillin and fluconazole. Structure activity relationship (SAR) studies The graphically representation of the pMIC values of test Design strategy of naringenin derivative for G-6-P inhi- and standard compounds have been shown in Fig. 4. bition and antioxidant activity has been represented in Fig.  6. The structure activity relationship of the synthe - Preservative efficacy study sized naringenin derivatives with their antioxidant activ- The most active antimicrobial compound 1 was selected ity results were summarized as: for the evaluation of its preservative efficacy. The results of preservative efficacy testing performed in triplicate (1) Substitution of naringenin with aliphatic amines and were reported as mean values in Table 3. produced biological activity but aromatic substitu- Compound 1 showed the values of log CFU/mL reduc- tion showed greater activity than aliphatic i.e. com- tion within the prescribed limit and the results were pound 2 showed the lowest activity as compared to comparable to that of the standard preservatives sodium other. benzoate, propyl paraben and methyl paraben. The pre - servative efficacy of compound 1 in White lotion USP La ther et al. BMC Chemistry (2020) 14:41 Page 5 of 15 Cl NOH O OH (4) HO Ethanol, conc.HCL Refluxed at 80 C Cl for70-75 h NH NO OH Ethanol, conc.HCL F o Refluxed at 80 C HO O NH NOH 2 for80-85 h NO NOH Ethanol, conc.HCL OH O Refluxed at 80 C O OH for90-95 h O OH HO NH HO (1) OH OH (3) Ethanol, conc.HCL Refluxed at 80 C NH 2 for100-105 h OH OH NOH O OH HO (2) Scheme 1 Synthetic route for the synthesis of naringenin derivatives tivity i.e. compound 1 was more active than com- Table 1 Antioxidant IC values of synthesized compounds pound 3 and 4. S. no. Compound(s) IC (µM) 50 (3) Replacement of para position with nitro group produced the highest activity i.e. compound 1 was 1. Compound 1 6.864 ± 0.020 most active in the series. 2. Compound 2 26.210 ± 0.151 (4) Exchange at para position produced more activity 3. Compound 3 7.170 ± 0.028 as compared to ortho position substitution. 4. Compound 4 7.801 ± 0.077 5. Naringenin 13.765 ± 0.408 6. Standard (l -ascorbic acid) 8.110 ± 0.069 Molecular docking study Values are expressed as mean ± SEM, n = 3 Molecular docking studies were carried out to identify the binding affinities and interaction between the inhibi - tors and pdb id 1moq of G-6-P synthase protein by using (2) Substitution with aromatic amine at para position Glide software (Schrodinger Inc. U.S.A. Maestro version increased the activity with increase in electronega- 11). Dock score and binding of compound 1, 2, 3 and 4 Lather et al. BMC Chemistry (2020) 14:41 Page 6 of 15 Compound 1 Compound 2Compound 3 Compound 4Naringenin Standard (L- Ascorbic acid) Fig. 3 IC value of different synthesized compunds with respect to standard l -ascarbic acid Table 2 pMIC values (μM/mL) of synthesized naringenin derivatives against different standard microbial strains Compound(s) PMIC values in μM/mL P. mirabilis P. aeruginosa S. aureus E. coli C. albicans A. niger Compound 1 1.79 1.79 1.49 1.49 1.49 1.49 Compound 2 1.14 1.14 0.83 1.14 1.14 0.83 Compound 3 0.86 1.16 0.86 0.86 1.16 1.46 Compound 4 1.18 0.88 0.88 1.18 0.88 1.18 Naringenin < 0.73 < 0.73 < 0.73 < 0.73 < 0.73 < 0.73 Streptomycin 1.06 1.36 1.06 1.96 – – Ciprofloxacin 1.12 1.42 1.12 1.42 – – Ampicillin 1.14 0.84 0.84 1.74 – – Fluconazole – – – – 1.08 1.38 1.8 1.6 P. mirabilis 1.4 P. aeruginosa 1.2 1 S. aureus 0.8 E. coli 0.6 C. albicans 0.4 A. niger 0.2 Fig. 4 Antimicrobial activity (pMIC in µM/mL) of synthesized naringenin derivatives against different microorganisms pMIC values in uM/mL IC Vale 50 La ther et al. BMC Chemistry (2020) 14:41 Page 7 of 15 Table 3 Log CFU/mL values of the selected compound 1 Compound(s) E. coli P. aeruginosa S. aureus C. albicans A. niger CFU/mL after days 14 days 28 days 14 days 28 days 14 days 28 days 14 days 28 days 14 days 28 days Compound 1 3.190 ± 0.008 3.496 ± 0.12 3.306 ± 0.16 3.406 ± 0.016 3.486 ± 0.012 3.486 ± 0.012 3.200 ± 0.081 3.313 ± 0.016 3.306 ± 0.016 3.463 ± 0.020 Sodium Benzoate 3.213 ± 0.012 3.323 ± 0.24 3.282 ± 016 3.210 ± 0.037 3.863 ± 0.044 3.166 ± 0.047 3.076 ± 0.088 2.800 ± 0.081 3.166 ± 0.012 3.320 ± 0.014 Propyl Paraben 3.280 ± 0.57 3.246 ± 0.36 3.310 ± 0.016 3.306 ± 0.016 3.883 ± 0.023 3.516 ± 0.012 3.940 ± 0.028 3.530 ± 0.016 3.113 ± 0.065 3.403 ± 0.012 Ethyl Paraben 3.336 ± 0.020 3.090 ± 0.148 3.246 ± 0.36 3.340 ± 0.014 3.166 ± 0.047 3.210 ± 0.008 3.520 ± 0.014 3.200 ± 0.018 3.043 ± 0.041 3.300 ± 0.081 5 6 # Initial microbial count in inoculums 1 × 10 –1 × 10 Lather et al. BMC Chemistry (2020) 14:41 Page 8 of 15 4.5 3.5 2.5 Compound 1 Sodium Benzoate 1.5 Propyl Paraben Ethyl Paraben 0.5 14 28 14 28 14 28 14 28 14 28 days days days days days days days days days days E.coli P.aeruginosaS.aureus C.albicansA.niger Fig. 5 Preservative efficacy of compound 1 in White lotion USP and degree of microbial log reduction NO NO NH 2 NOH 4-nitrobenzenamine OH OH O OH NOH HO OH OH (1) NH O OH HO 1,3-dihydroxy-isopropylamine (2) OH HO O OH O Naringenin NOH NH 2-fluorobenzenamine O OH HO Cl NOH (3) Cl O OH NH HO 2-chlorobenzenamine (4) Fig. 6 Design strategy of Naringenin derivatives for G-6-P synthase inhibition and antioxidant activity Log reduction count La ther et al. BMC Chemistry (2020) 14:41 Page 9 of 15 with G-6-P synthase have been shown in Table  4 and Table 4 G-6-P synthase inhibition showed by  synthesized naringenin derivatives Fig.  7. After, docking results of compound 1 with G-6-P synthase protein suggested the formation of the hydro- S. no. Compound(s) Structure of G-6-P synthase Dock gen bond between NO and Thr 402. Additionally, the inhibitors score molecule has been stabilized by residues such as Ser 347, 1. Compound 1 − 7.42 Thr 352, Ser 303, Gln 348, Ala 602, Asn 600 and Asp 354. The binding orientation of compound 2 within the cata - lytic site of G-6-P synthase exhibited backbone hydrogen bonding with Glu 488. The molecule is stabilized by resi - dues such as Asp 354, Lys 603, Glu 488, Lys 487 and Ala 400. The compound 3 showed interaction with Arg 599. The molecule was enclosed by residues such as Val 399, Thr 302, Lys 487 and Leu 484. In compound 4 hydrogen bonding was shown by Thr 606 and ligand was entrapped by the residue sequence of Val 399, Lys 487, Cys 300 and Ser 328. Docking results of G-6-P synthase showed that 2. Compound 2 − 4.29 the synthetic compounds have comparable docking score as compared to the standard drugs taken. All the ligands showed variable degrees of hydrogen bond interaction, hydrophobic interactions, electrostatic interactions, ionic interactions and π–π stacking with the various amino acid residues in the binding pockets of G-6-P synthase. ADME study The evaluation of different ADME parameters has been 3. Compound 3 − 3.30 represented in Table  5. It was observed that all the syn- thesized compounds fulfilled the standard Rule of Five [43]. All the synthesized compounds qualified the condi - tions for various descriptors like LogP, HBA, HBD and MW. All these parameters were in suitable range for drug-like characteristics. In addition, according to Veber et  al., 2002 for better bioavailability rotatable bonds should be ≤ 10 as the rotatable bonds in ligand impart elasticity [44]. The values of QPlogBB should be > 1.0 CNS active compounds and value < 1.0 CNS inactive 4. Compound 4 − 4.02 compounds. QPPCaco cell permeability should be in a range from 4–70 [45–47]. In the present study, all the synthesized compounds exhibited a suitable drug-like profile. Conclusion In conclusion, the above mentioned wet and dry labo- ratory studies highlight the underlying mechanism of G-6-P synthase inhibition. The rational development 5. Naringenin − 6.36 of inhibitors and specificity of naringenin derivatives to be discovered as the novel preservatives. Moreover, the synthesized compounds were also found as wonderful antioxidants towards DPPH with remarkable potential as compared to the reference compounds. Lather et al. BMC Chemistry (2020) 14:41 Page 10 of 15 Table 4 (continued) Experimental Materials and methods S. no. Compound(s) Structure of G-6-P synthase Dock All the chemicals required for experiments were of inhibitors score analytical grade and were purchased from Loba Che- 6. Standard Streptomycin − 5.795 mie (Mumbai, India), SRL (Mumbai, India), and Sigma Ciprofloxacin − 5.185 Aldrich (Germany). Nutrient agar, nutrient broth, sab- ouraud dextrose agar and sabouraud dextrose broth Ampicillin − 5.065 required for antimicrobial and preservative efficacy were Fluconazole − 5.129 obtained from Hi-media Laboratories. Streptomycin, Compound 1 Compound 2 Compound 3 Compound 4 Fig. 7 Binding of compounds 1, 2, 3 and 4 with G-6-P synthase La ther et al. BMC Chemistry (2020) 14:41 Page 11 of 15 Table 5 ADMET profile of various newly synthesized naringenin derivatives Compound(s) Mol. Wt. No. of rotatable DonorHB AcceptHB QPlogPo/w QPlogBB QPPMDCK QPPCaco bond Compound 1 392.10 5 5 4 2.084 0.081 0.053 1.877 Compound 2 345.12 3 3 3 2.490 0.138 11.251 2.773 Compound 3 365.11 4 2 4 4.29 2.445 0.282 10.982 Compound 4 381.08 3 4 2 1.278 3.355 0.162 20.169 ciprofloxacin, ampicillin and fluconazole were obtained 8.04 (s, 1H), 7.29 (d, J = 9.3  Hz, 2H), 7.28 (d, J = 7.5  Hz, as gift sample from Belco Pharma, Bahadurgarh, India. 2H), 6.80 (d, J = 7.3  Hz, 2H), 6.28 (s, 1H), 6.27 (s, 1H), Microbial strains S. aureus MTCC 3160, P. aeruginosa 5.28 (t, J = 9.0  Hz, 1H), 3.15 (d, J = 7.3  Hz, 1H), 3.00 (d, MTCC 1934, E. coli MTCC 45, C. albicans MTCC 183 J = 7.3 Hz, 1H); C NMR (400 MHz, C DCL ) δ = 166.11, and A. niger MTCC 282 strains wer e purchased from 165.34, 163.98, 161.90, 153.71, 152.59, 146.42, 133.96, MTCC, Chandigarh, India. Chemical reactions were 131.14, 126.43, 125.72, 124.08, 123.64, 117.42, 103.05, monitored by TLC on silica gel plates in iodine and UV 97.89, 95.36, 77.13, 38.79, 27.19, 22.70; MS ES + (ToF): chambers. Sonar melting point apparatus in open capil- m/z 392.10 [M +2]; CHNS: Calc (C H N O ): C, 64.28; 12 16 2 2 lary tube was used for the recording of melting points. H, 4.11; N, 7.14; O, 24.47; Found C, 64.25; H, 4.14; N, 1 13 H NMR and C NMR spectra were confirmed in 7.17; O, 24.44. DMSO and deuterated CDCl on Bruker Avance II 400 NMR spectrometer at a frequency of 400  MHz down- 4‑(1,3‑dihydroxypropan‑2‑ylimino)‑2‑(4‑hydroxyphenyl) field to tetramethyl silane standard. FTIR spectra were chroman‑5,7‑diol recorded on Perkin Elmer FTIR spectrophotometer with R TLC mobile phase: Chloroform: Acetone (8:5) = 0.66; the help of KBr pellets technique. Waters Micromass Yield = 50%; M.P . = 173–175  °C; M.Wt. = 345.32; IR (KBr −1 Q-ToF Micro instrument was used for Mass spectrum pellets) cm : 1074 (–C–O–C–), 1251 (–C–C–), 1513 (– recording. C=C–), 1631 (–C=N–), 2831 (–C–H–), 3295 (–OH–); H NMR (400  MHz, DMSO-d ) δ = 11.60 (s, 1H), 11.10 General procedure for the synthesis of naringenin (s, 1H), 8.04 (s, 1H), 7.28 (d, J = 7.2  Hz, 2H), 6.80 (d, derivatives J = 7.3  Hz, 2H), 6.28 (s, 1H), 6.24 (s, 1H), 5.25–5.24 (m, Substituted aniline (0.01  mol) was taken in a round bot- 1H), 3.95 (d, J = 8.1 Hz, 2H), 3.64 (q, J = 9.0 Hz, 2H), 3.50 tom flask, concentrated hydrochloric acid was added (q, J = 9.6 Hz, 2H), 3.47–3.45 (m, 1H), 3.13 (d, J = 8.6  Hz, drop wise with continuous stirring. Equimolar concen- 1H), 2.86 (d, J = 8.6  Hz, 1H); C NMR (400  MHz, tration of naringenin (0.01  mol) was dissolved in etha- CDCL ) δ = 164.81, 162.47, 161.87, 161.58, 158.28, nol (50  mL) and was re fluxed for 80-100  h on heating 130.84, 128.42, 115.98, 107.15, 103.33, 96.95, 78.20, 72.30, mantle. All the compounds in the series were synthe- 63.75, 37.92, 27.60, 22.32, 14.16; MS ES + (ToF): m/z sized according to the standard procedures as outlined 345.12 [M +2]; CHNS: Calc (C H NO ): C, 62.60; H, 18 19 6 in Scheme  1. Completion of reaction was monitored by 5.55; N, 4.06; O, 27.80; Found C, 62.63; H, 5.52; N, 4.09; TLC. Reaction mixture was concentrated and the pre- O, 27.82. cipitates formed were filtered off and dried. The crude product was recrystallized using alcohol which yielded 4‑(2‑fluorophenylimino)‑2‑(4‑hydroxyphenyl) the final compounds 1-4. chroman‑5,7‑diol R TLC mobile phase: Chloroform: Acetone (8:5) = 0.64; Spectral data Yield = 23%; M.P . = 165-167  °C; M.Wt. = 365.35; IR (KBr −1 2‑(4‑hydroxyphenyl)‑4‑(4‑nitrophenylimino) chroman‑5, pellets) cm : 753 (–F–), 1082 (–C–O–C), 1241 (–C– 7‑diol C–), 1612 (–C=C–), 1632 (–C=N–), 2833 (–C–H–), R TLC mobile phase: Chloroform: Acetone (8:5) = 0.63; 3350 (–OH–); H NMR (400 MHz, DMSO-d ) δ = 11.78 Yield = 55%; M.P . = 190–192  °C; M.Wt. = 317.29; IR (s, 1H), 11.10 (s, 1H), 8.04 (s, 1H), 7.47 (d, J = 8.8  Hz, −1 (KBr pellets) cm : 1081 (–C–O–C), 1156 (–C–C–), 1H), 7.31 (dt, J = 15.7, 8.4  Hz, 2H), 7.28–7.26 (m, 3H), 1305 (–NO ), 1599 (–C=C–), 1632 (–C=N–), 2921 6.80 (d, J = 7.4 Hz, 2H), 6.31 (s, 1H), 6.28 (s, 1H), 5.33 (t, (–C–H–), 3479 (–OH–); H NMR (400 MHz, DMSO-d ) J = 8.5 Hz, 1H), 3.04 (d, J = 7.7 Hz, 1H), 2.92 (d, J = 8.5 Hz, δ = 11.94 (s, 1H), 11.10 (s, 1H), 8.17 (d, J = 8.5  Hz, 2H), 1H); C NMR (400  MHz, C DCL ) δ = 165.92, 165.91, 3 Lather et al. BMC Chemistry (2020) 14:41 Page 12 of 15 strength nutrient broth I.P. (bacteria) or sabouraud dex- 165.24, 163.73, 161.86, 132.64, 132.61, 126.96, 126.94, trose broth I.P. (fungi) [51, 52]. The slants of E. coli, P. 126.50, 126.48, 125.25, 114.89, 114.86, 102.91, 97.83, aeruginosa, P. mirabilis and S. aureus were incubated 95.53, 72.64, 39.18, 20.46; MS ES+ (ToF): m/z 365.11 at the 30-35  °C for 24  h. The slants of C. albicans were [M +2]; CHNS: Calc (C H FNO ): C, 69.04; H, 4.41; F, 21 16 4 incubated at 20–25 °C for 48 h whereas; the slants of A. 5.20; N, 3.83; O, 17.52; Found C, 69.01; H, 4.44; F, 5.23; N, niger were incubated at 20–25  °C for 5  days. After the 3.84; O, 17.55. incubation period sterilized 0.9% NaCl solution was used to harvest the bacterial and fungal cultures from agar 4‑(2‑chlorophenylimino)‑2‑(4‑hydroxyphenyl) slant through proper shaking and then the suspensions chroman‑5,7‑diol of microorganisms were diluted with the sterile 0.9% R TLC mobile phase: Chloroform: Acetone (8:5) = 0.66; NaCl solution to CFU count was adjusted by adjust- Yield = 60%; M.P . = 155-157  °C; M.Wt. = 381.81; IR (KBr −1 ing the density of microorganism suspension to that of pellets) cm : 754 (–Cl–Str), 1062 (–C–O–), 1155 (–C– 0.5 McFarland standards by adding distilled water. The C–), 1602 (–C=C–) 1633 (–C=N–), 2834 (–C–H–), number of CFU was determined by dilution pour-plate 3284 (–OH–); H NMR (400 MHz, DMSO-d ) δ = 11.78 method [53]. A serial dilution of 50  µg/mL, 25  µg/mL, (s, 1H), 11.10 (s, 1H), 8.04 (s, 1H), 7.55 (d, J = 6.9 Hz, 1H), 12.5  µg/mL, 6.25  µg/mL, 3.12  µg/mL and 1.62  µg/mL 7.39 (t, J = 8.0  Hz, 1H), 7.28 (d, J = 8.0  Hz, 2H), 7.26 (d, was used for determination of MIC. The samples tubes J = 8.3 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 7.5 Hz, were incubated at 37  °C for 24  h (bacteria), at 25  °C for 2H), 6.19 (s, 1H), 6.17 (s, 1H), 5.34 (t, J = 8.9 Hz, 1H), 3.04 7 days (A. niger), and at 37 °C for 48 h (C. albicans) and (d, J = 8.7  Hz, 1H), 2.94 (d, J = 9.1  Hz, 1H); C NMR the results were recorded in pMIC. (400 MHz, CDCL ) δ = 165.10, 163.08, 161.26, 159.81, 143.28, 139.86, 129.24, 128.98, 128.45, 128.28, 127.73, Preservative effectiveness 127.42, 126.85, 124.29, 107.38, 102.08, 95.02, 76.72, 38.77, White lotion USP was utilized as the medium for the 17.39, 14.71; MS ES+ (ToF): m/z 381.08 [M +2]; CHNS: testing of preservative effectiveness. Calc (C H ClNO ): C, 66.06; H, 4.22; Cl, 9.29; N, 3.67; 21 16 4 Ingredients: Zinc sulfate 40 gm, sulfurated potash 40 O, 16.76; Found C, C, 66.09; H, 4.20; Cl, 9.26; N, 3.69; O, gm and purified water q.s. to 1000 mL. 16.72. Firstly, zinc sulphate and sulfurated potash were dis- solved in 450  mL of water separately and filtered. Then, Antioxidant activity sulfurated potash solution was added to zinc sulfate with DPPH radical scavenging assay stirring. At last, the required amount of water was added Antioxidant activity of the synthesized compounds was and mixed thoroughly and sterilized. For preservative determined by DPPH (2, 2-diphenyl-1-pycrilhydrazil efficacy testing, the White lotion USP was prepared using hydrate) radical scavenging method. Briefly, 0.1 mM solu - the equimolar amount of compounds 1-4 as novel pre- tion of DPPH in methyl alcohol was prepared and 1  mL servatives by replacing sodium benzoate, methyl paraben of this solution was added to 3 mL of sample or standard and propyl paraben from the formula [54]. with a concentration of 12.5, 25, 50, 75 and 100  μg/mL. Discolorations were measured at 517 nm after incubation Challenge microorganism for 30 min at 30 °C in the dark. Lower absorbance of the Staphylococcus aureus MTCC 3160, P. aeruginosa MTCC reaction mixture indicates higher free radical scavenging 1934, E. coli MTCC 45, C. albicans MTCC 183 and A. activity. The IC values of given samples were calculated niger MTCC 282 were used as common contaminants in by using formula: the study as prescribed in USP for preservative efficacy IC = (A − A ) × 100/A 50 c s c testing in the pharmaceutical preparations. Here, A was the absorbance of the control and A was c s Preparation of ioculums the absorbance of the sample [48, 49]. The slants of E. coli, P. aeruginosa and S. aureus were incubated at the 30–35 °C for 24 h. The slants of C. albi- Antimicrobial activity cans were incubated at 20–25  °C for 48  h whereas; the Minimum inhibitory concentration (MIC) slants of A. niger were incubated at 20–25  °C for 5  days The antimicrobial activity of the synthesized compounds [55]. were performed against S. aureus MTCC 3160, P. aerugi- nosa MTCC 1934, E. coli MTCC 45, P. mirabilis MTCC Test procedure 3310, C. albicans MTCC 183 and A. niger MTCC 282 White lotions USP was added in final containers and by using the tube dilution method [50]. Dilutions of were used in challenge test. The preparation was test and standard compounds were prepared in double La ther et al. BMC Chemistry (2020) 14:41 Page 13 of 15 Ligand Preparation inoculated with 0.5–1% volume of microbial inocu- 5 6 The three-dimensional structural library was prepared lum having a concentration of 1 × 10 –1 × 10   CFU/ using the Chemdraw software and proceeded for energy mL [56]. Inoculated samples were mixed thoroughly minimization using the LigPrep tool for the correction of to ensure homogeneous microorganism distribution coordinates, ionization, stereochemistry and tautomeric and incubated. The CFU/mL of the product was deter - structure to gain the appropriate conformation through mined at an interval of 0 days, 7 days, 14 days, 21 days, the addition or removal of hydrogen bonds. The partial and 28 days in agar plates. Log CFU/mL of white lotion charges were computed according to the OPLS-2005 USP was calculated as not less than 2.0 log reductions force field (32 stereo isomers, tautomers and ionization) from initial count at 14  days of incubation and no at biological pH and used for molecular docking studies. increase in CFU from 14  days count at 28  days in case of bacteria and no increase from the initial calculated count at 14 and 28 days [57]. Abbreviations ADMET: Absorption, distribution, metabolism, excretion & toxicity; G-6-P Synthase: Glucosamine-6-phosphate synthase; CYP P450: Cytochromes P450; In silico molecular docking studies OATP1B1: Solute carrier organic anion transporter family member 1B1; DHEAS: The Schrodinger, Inc. (New York, USA) software Maestro Dehydroepiandrosterone; PPAR: Peroxisome proliferator-activated receptors; 11 was used for the computational calculations and dock- DPPH: 2,2-Diphenyl-1-picrylhydrazyl; UDP-N-acetyl glucosamine: Uridine diphosphate N-acetylglucosamine; FTIR: Fourier-transform infrared spec- ing studies. Laboratory for Enzyme Inhibition Studies, 1 13 troscopy; H NMR: Proton nuclear magnetic resonance; C NMR: Carbon 13 Department of Pharmaceutical Sciences, M.D. University, nuclear magnetic resonance; UV: Ultra violet; TLC: Thin layer chromatography; Rohtak, INDIA was used for the computational work. The IC : Inhibitory concentration; MIC: Minimum inhibitory concentrations; CFU: Colony forming unit; HBA: Hydrogen bond acceptor; HBD: Hydrogen bond receptor-grid files were generated by grid-receptor gen - donor; MW: Molecular weight; MTCC : Microbial type culture collection; DMSO: eration program Glide [58]. Grid-based ligand docking Dimethyl sulfoxide; BOD: Biological oxygen demand; USP: United States utilized the hierarchical sequence of filters to produce pos - Pharmacopoeia; PDB ID: Protein Data Bank Identification; OPLS: Optimized potential for liquid simulations; Q.S.: Quantity sufficient. sible conformations of the ligand in the active-site region of the protein receptor. At this stage, crude score values Acknowledgements and geometric filters were prepared out unlikely binding The authors are highly thankful to the Head, Department of Pharmaceutical Sciences, M.D. University, Rohtak for providing essential facilities to accomplish modes. The next filter phase involves a grid-based force this research study. The authors are also thankful to Dr. Vinod Devaraji Applica- field evaluation and refinement of docking experiments tion Scientist Schrödinger LLC for his support to carry out the computational including torsional and rigid-body movements of the work. ligand [59]. The remained docking evaluations were sub - Authors’ contributions jected to a Monte Carlo procedure to minimize the energy The authors AL, SS and AK have designed, synthesized and carried out score. A conjugate gradient minimization protocol was the work in equal contribution. All authors read and approved the final manuscript. used in all calculations [60]. The energy differences were calculated using the Funding equation: No funding received for this research work from outside sources. E = E − E − E complex ligand protein Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Protein preparation Competing interests The X-ray protein structure co-ordinates of pdb id 1moq The authors declare that they have no competing interests. were downloaded from Protein Data Bank from www. Author details rcbs.org [61] and were prepared with the help of the Department of Pharmaceutical Sciences, Maharshi Dayanand University, Schrödinger protein preparation wizard ‘Prepwiz’ [62, Rohtak, Haryana, India. Department of Pharmaceutical Sciences, G.J.U.S.&T., Hisar, India. Laboratory for Preservation Technology and Enzyme Inhibition 63]. PDB id 1moq (resolution 1.57 A°) was selected on Studies, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical the basis of the lowest resolution and availability. All the Sciences, Maharshi Dayanand University, Rohtak, Haryana, India. waters molecules except metals co-ordinated and pre- Received: 30 July 2019 Accepted: 11 June 2020 sent between the ligand and protein were removed. The energy-restrained structure of the protein G-6-P syn- thase was constructed with the help of OPLS-2005 force field. References 1. 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Naringenin derivatives as glucosamine-6-phosphate synthase inhibitors: synthesis, antioxidants, antimicrobial, preservative efficacy, molecular docking and in silico ADMET analysis

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Abstract

Background: Preservatives have to be added in food, pharmaceuticals and cosmetics products to maintain their shelf life. However, the existing chemical based preservatives have been associated with severe side effects that compel the researchers to find better safe preservatives based on natural products. G-6-P synthase is an important enzyme for bacterial and fungal cell wall synthesis and offers as a potential target to find better G-6-P synthase inhibi- tors based antimicrobial compounds. Naringenin, a flavanone, has been reported for a wide range of pharmacological activities including antimicrobial activity, which makes it a potential candidate to be explored as novel G-6-P synthase inhibitor. Results: The synthesis of naringenin derivatives with potent G-6-P synthase inhibitor having remarkable antioxi- dant, antimicrobial and preservative efficacy was performed. Among the synthesized compounds, the compound 1 possessed good antioxidant activity (IC value, 6.864 ± 0.020 µM) as compared to standard ascorbic acid (IC value, 50 50 8.110 ± 0.069 µM). The antimicrobial activity of synthesized compounds revealed compound 1 as the most potent compound (pMIC 1.79, 1.79, 1.49, 1.49, 1.49 and 1.49 μM/mL for P. mirabilis, P. aeruginosa, S. aureus, E. coli, C. albicans and A. niger respectively) as compared to standard drugs taken. The compound 2 showed comparable activity against P. mirabilis (pMIC 1.14 μM/mL), C. albicans (pMIC 1.14 μM/mL) while the compound 3 also showed comparable activity against C. albicans (pMIC 1.16 μM/mL) as well A. niger (pMIC 1.46 μM/mL), likewise the compound 4 showed compa- rable activity against P. mirabilis (pMIC 1.18 μM/mL) as compared to the standard drugs streptomycin (pMIC 1.06, 1.36, 1.06 and 1.96 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli respectively), ciprofloxacin (pMIC 1.12, 1.42, 1.12 and 1.42 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli respectively), ampicillin (pMIC 1.14, 0.84, 0.84 and 1.74 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli respectively) and fluconazole (pMIC 1.08 and 1.38 μM/mL for C. albicans and A. niger respectively). The molecular docking with the target G-6-P synthase pdb id 1moq resulted with an better dock score for compound 1 (− 7.42) as compared to standard antimicrobial drugs, ciprofloxacin *Correspondence: dranuragkhatkarmdurtk@gmail.com; anuragpharmacy@gmail.com Laboratory for Preservation Technology and Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, India Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lather et al. BMC Chemistry (2020) 14:41 Page 2 of 15 (− 5.185), ampicillin (− 5.065) and fluconazole (− 5.129) that supported the wet lab results. The preservative efficacy test for compound 1 in White Lotion USP showed the log CFU/mL value within the prescribed limit and results were comparable to standard sodium benzoate, ethyl paraben and propyl paraben as per USP standard protocol. Conclusions: The synthesized naringenin derivatives exhibited significant G-6-P synthase inhibitory potential with good selectivity towards the selected target G-6-P synthase. Compound 1, bearing nitro group showed good antioxi- dant, antimicrobial and preservative efficacy compared with the standard drugs taken. The mechanistic insight about the compounds within the active site was completed by molecular docking that supported the results for novel synthesized G-6-P synthase inhibitors. Keywords: G-6-P synthase, Naringenin derivatives, DPPH, Preservative efficacy Introduction Naringenin is a naturally occurring bioflavonoid pre - The use of packaged foods containing various additive’s sent in various fruits, vegetables and honey which is used viz. artificial sweeteners, colorants, stabilizers, preserva - as a dietary supplement due to its low toxicity [19–21]. tives etc. has greatly increased in recent years. As per Naringenin has been reported for its diverse pharma- recent data available it is estimated that 75% of the con- cological profile including its antibacterial property as temporary diet is packaged food and on an average every shown in Fig. 2 [22–41]. person consumes 3.6 to 4.5 kg of food additives per year Further, naringenin could be utilized as a potential [1]. candidate for evaluation of its G-6-P synthase inhibitory Among other additives the preservative such as response. Hence, it was planned to synthesize and inves- sodium benzoate, ethyl paraben, propyl paraben, butyl- tigate the naringenin derivatives for their antioxidant, ated hydroxytoluene (BHT), butylated hydroxyanisole antimicrobial, preservative efficacy and in silico evalua - (BHA), etc. plays a vital role to maintain the shelf life tion for G-6-P synthase inhibition. of various food, pharmaceuticals and cosmetic prod- ucts [2–4]. However, the existing chemical preservatives Results and discussion have been associated with serious side effects viz. estro - Chemistry genic effect, breast cancer, malignant melanoma, contact Naringenin derivatives were synthesized according to eczema, endocrine disruption, etc. [5–12]. Hence, there Kriza et  al. 2011 with slight modifications as shown in is an urgent need for the discovery of novel and safer pre- Scheme 1 [42]. The chemical structures of all the synthe - 1 13 servatives for use in food, pharmaceuticals and cosmetic sized compounds were confirmed by FTIR, H NMR, C products. NMR, mass spectroscopy and elemental analysis which G-6-P synthase is a complex enzyme involved in the were in agreement with the structures. formation of UDP-N-acetyl glucosamine and catalyzes For the synthesis of naringenin derivatives substituted the initial step in hexosamine biosynthesis. One of these aniline (0.01 mol) was taken in a round bottom flask and catalyzed products, N-acetyl glucosamine, is an impor- concentrated hydrochloric acid was added drop wise with tant part of the peptidoglycan layer of bacterial and fun- continuous stirring. Equimolar concentration of narin- gal cell wall. Hence, G-6-P synthase may act as potential genin (0.01  mol) was dissolved in ethanol (50  mL) and target for discovery of novel antimicrobial compounds was refluxed for 80–100 h at 80 °C on heating mantle. All which could be evaluated for their preservative efficacy to the compounds in series were synthesized according to find better and safe preservatives [13, 14]. the standard procedure outlined in Scheme  1. Comple- The complex 3-D crystal structure of G-6-P syn - tion of reaction was confirmed by TLC under UV lamp thase can be utilized for molecular docking to and FTIR spectra. explore the structural requirements for the pharma- Formation of compound 1, 2, 3 and 4 was confirmed by cophore complex. Flavonoids such as luteolin, cat- peaks of IR, NMR, mass spectroscopy. In positive chemi- echin, (4S)-2-Methyl-2-phenylpentane-1,4-diol, cal ionization most of the naringenin derivatives showed 7-Methoxy-2,3-dihydro-2-phenyl-4 quinolone, 3-(tert- (M++1), M+ (molecular ion peak), (M++2) and in neg- Butoxycarbonyl)-6-(3 benzoylprop-2-yl)phenol and ative chemical ionization mode showed (M+1), (M+2), (3R,4S)-4-(methylamino)-1-phenylpent-1-en-3-ol also M+. The elemental analysis established the synthesis of have been explored for G-6-P synthase inhibition [15– naringenin derivatives where the percentage of C, H and 18]. Some flavonoids along with their G-6-P synthase N in the synthesized compounds was observed within inhibitory dock score have been shown in Fig. 1. defined limits. The reaction mixture was concentrated, La ther et al. BMC Chemistry (2020) 14:41 Page 3 of 15 Fig. 1 G-6-P synthase inhibitory profile of flavonoids and their derivatives cited in the recent literature Antimicrobial activity after that precipitates formed were filtered off and dried. Minimum inhibitory concentration Crude products were recrystallized by alcohol which The antimicrobial activity of synthesized compounds yielded the final compounds 1–4. revealed compound 1 as the most potent compound (pMIC 1.79, 1.79, 1.49, 1.49, 1.49 and 1.49  μM/mL for Antioxidant activity P. mirabilis, P. aeruginosa, S. aureus, E. coli, C. albi- DPPH radical scavenging activity cans and A. niger respectively) as compared to standard All the synthesized compounds were evaluated for drugs taken. The compound 2 showed comparable activ - antioxidant profile by using DPPH radical scaveng - ity against P. mirabilis (pMIC 1.14  μM/mL), C. albicans ing assay method (Table  1). The compound 1 was (pMIC 1.14 μM/mL) while the compound 3 also showed observed as the most potent antioxidant compound (IC comparable activity against C. albicans (pMIC 1.16  μM/ 6.864 ± 0.020  µM) as compared to standard L-ascorbic mL) as well A. niger (pMIC 1.46  μM/mL), likewise the acid (IC 8.110 ± 0.069  µM). However, compounds compound 4 showed comparable activity against P. mira- 3 and 4 showed moderate antioxidant activity (IC bilis (pMIC 1.18  μM/mL) as compared to the standard 7.170 ± 0.028  µM and 7.801 ± 0.077  µM, respectively) drugs streptomycin (pMIC 1.06, 1.36, 1.06 and 1.96 μM/ as compared to standard. The electron withdrawing mL for P. mirabilis, P. aeruginosa, S. aureus and E. coli, strongly deactivating nitro group in compound 1 may be respectively), ciprofloxacin (pMIC 1.12, 1.42, 1.12 and responsible for better antioxidant activity. The presence 1.42 μM/mL for P. mirabilis, P. aeruginosa, S. aureus and of weakly deactivating electron withdrawing chloro and E. coli, respectively), ampicillin (pMIC 1.14, 0.84, 0.84 fluoro groups present in compound 3 and 4 have moder - and 1.74 μM/mL for P. mirabilis, P. aeruginosa, S. aureus ate antioxidant activity. I C value of synthesized narin- and E. coli, respectively) and fluconazole (pMIC 1.08 and genin derivatives has been shown in Fig. 3. 1.38 μM/mL for C. albicans and A. niger, respectively). In Lather et al. BMC Chemistry (2020) 14:41 Page 4 of 15 Fig. 2 Pharmacological potential of Naringenin general, the results of MIC studies (Table 2) revealed that and degree of microbial log reduction has been repre- the synthesized compounds have better anti bacterial sented in Fig. 5. and anti fungal potential as compared to standard drugs streptomycin, ciprofloxacin, ampicillin and fluconazole. Structure activity relationship (SAR) studies The graphically representation of the pMIC values of test Design strategy of naringenin derivative for G-6-P inhi- and standard compounds have been shown in Fig. 4. bition and antioxidant activity has been represented in Fig.  6. The structure activity relationship of the synthe - Preservative efficacy study sized naringenin derivatives with their antioxidant activ- The most active antimicrobial compound 1 was selected ity results were summarized as: for the evaluation of its preservative efficacy. The results of preservative efficacy testing performed in triplicate (1) Substitution of naringenin with aliphatic amines and were reported as mean values in Table 3. produced biological activity but aromatic substitu- Compound 1 showed the values of log CFU/mL reduc- tion showed greater activity than aliphatic i.e. com- tion within the prescribed limit and the results were pound 2 showed the lowest activity as compared to comparable to that of the standard preservatives sodium other. benzoate, propyl paraben and methyl paraben. The pre - servative efficacy of compound 1 in White lotion USP La ther et al. BMC Chemistry (2020) 14:41 Page 5 of 15 Cl NOH O OH (4) HO Ethanol, conc.HCL Refluxed at 80 C Cl for70-75 h NH NO OH Ethanol, conc.HCL F o Refluxed at 80 C HO O NH NOH 2 for80-85 h NO NOH Ethanol, conc.HCL OH O Refluxed at 80 C O OH for90-95 h O OH HO NH HO (1) OH OH (3) Ethanol, conc.HCL Refluxed at 80 C NH 2 for100-105 h OH OH NOH O OH HO (2) Scheme 1 Synthetic route for the synthesis of naringenin derivatives tivity i.e. compound 1 was more active than com- Table 1 Antioxidant IC values of synthesized compounds pound 3 and 4. S. no. Compound(s) IC (µM) 50 (3) Replacement of para position with nitro group produced the highest activity i.e. compound 1 was 1. Compound 1 6.864 ± 0.020 most active in the series. 2. Compound 2 26.210 ± 0.151 (4) Exchange at para position produced more activity 3. Compound 3 7.170 ± 0.028 as compared to ortho position substitution. 4. Compound 4 7.801 ± 0.077 5. Naringenin 13.765 ± 0.408 6. Standard (l -ascorbic acid) 8.110 ± 0.069 Molecular docking study Values are expressed as mean ± SEM, n = 3 Molecular docking studies were carried out to identify the binding affinities and interaction between the inhibi - tors and pdb id 1moq of G-6-P synthase protein by using (2) Substitution with aromatic amine at para position Glide software (Schrodinger Inc. U.S.A. Maestro version increased the activity with increase in electronega- 11). Dock score and binding of compound 1, 2, 3 and 4 Lather et al. BMC Chemistry (2020) 14:41 Page 6 of 15 Compound 1 Compound 2Compound 3 Compound 4Naringenin Standard (L- Ascorbic acid) Fig. 3 IC value of different synthesized compunds with respect to standard l -ascarbic acid Table 2 pMIC values (μM/mL) of synthesized naringenin derivatives against different standard microbial strains Compound(s) PMIC values in μM/mL P. mirabilis P. aeruginosa S. aureus E. coli C. albicans A. niger Compound 1 1.79 1.79 1.49 1.49 1.49 1.49 Compound 2 1.14 1.14 0.83 1.14 1.14 0.83 Compound 3 0.86 1.16 0.86 0.86 1.16 1.46 Compound 4 1.18 0.88 0.88 1.18 0.88 1.18 Naringenin < 0.73 < 0.73 < 0.73 < 0.73 < 0.73 < 0.73 Streptomycin 1.06 1.36 1.06 1.96 – – Ciprofloxacin 1.12 1.42 1.12 1.42 – – Ampicillin 1.14 0.84 0.84 1.74 – – Fluconazole – – – – 1.08 1.38 1.8 1.6 P. mirabilis 1.4 P. aeruginosa 1.2 1 S. aureus 0.8 E. coli 0.6 C. albicans 0.4 A. niger 0.2 Fig. 4 Antimicrobial activity (pMIC in µM/mL) of synthesized naringenin derivatives against different microorganisms pMIC values in uM/mL IC Vale 50 La ther et al. BMC Chemistry (2020) 14:41 Page 7 of 15 Table 3 Log CFU/mL values of the selected compound 1 Compound(s) E. coli P. aeruginosa S. aureus C. albicans A. niger CFU/mL after days 14 days 28 days 14 days 28 days 14 days 28 days 14 days 28 days 14 days 28 days Compound 1 3.190 ± 0.008 3.496 ± 0.12 3.306 ± 0.16 3.406 ± 0.016 3.486 ± 0.012 3.486 ± 0.012 3.200 ± 0.081 3.313 ± 0.016 3.306 ± 0.016 3.463 ± 0.020 Sodium Benzoate 3.213 ± 0.012 3.323 ± 0.24 3.282 ± 016 3.210 ± 0.037 3.863 ± 0.044 3.166 ± 0.047 3.076 ± 0.088 2.800 ± 0.081 3.166 ± 0.012 3.320 ± 0.014 Propyl Paraben 3.280 ± 0.57 3.246 ± 0.36 3.310 ± 0.016 3.306 ± 0.016 3.883 ± 0.023 3.516 ± 0.012 3.940 ± 0.028 3.530 ± 0.016 3.113 ± 0.065 3.403 ± 0.012 Ethyl Paraben 3.336 ± 0.020 3.090 ± 0.148 3.246 ± 0.36 3.340 ± 0.014 3.166 ± 0.047 3.210 ± 0.008 3.520 ± 0.014 3.200 ± 0.018 3.043 ± 0.041 3.300 ± 0.081 5 6 # Initial microbial count in inoculums 1 × 10 –1 × 10 Lather et al. BMC Chemistry (2020) 14:41 Page 8 of 15 4.5 3.5 2.5 Compound 1 Sodium Benzoate 1.5 Propyl Paraben Ethyl Paraben 0.5 14 28 14 28 14 28 14 28 14 28 days days days days days days days days days days E.coli P.aeruginosaS.aureus C.albicansA.niger Fig. 5 Preservative efficacy of compound 1 in White lotion USP and degree of microbial log reduction NO NO NH 2 NOH 4-nitrobenzenamine OH OH O OH NOH HO OH OH (1) NH O OH HO 1,3-dihydroxy-isopropylamine (2) OH HO O OH O Naringenin NOH NH 2-fluorobenzenamine O OH HO Cl NOH (3) Cl O OH NH HO 2-chlorobenzenamine (4) Fig. 6 Design strategy of Naringenin derivatives for G-6-P synthase inhibition and antioxidant activity Log reduction count La ther et al. BMC Chemistry (2020) 14:41 Page 9 of 15 with G-6-P synthase have been shown in Table  4 and Table 4 G-6-P synthase inhibition showed by  synthesized naringenin derivatives Fig.  7. After, docking results of compound 1 with G-6-P synthase protein suggested the formation of the hydro- S. no. Compound(s) Structure of G-6-P synthase Dock gen bond between NO and Thr 402. Additionally, the inhibitors score molecule has been stabilized by residues such as Ser 347, 1. Compound 1 − 7.42 Thr 352, Ser 303, Gln 348, Ala 602, Asn 600 and Asp 354. The binding orientation of compound 2 within the cata - lytic site of G-6-P synthase exhibited backbone hydrogen bonding with Glu 488. The molecule is stabilized by resi - dues such as Asp 354, Lys 603, Glu 488, Lys 487 and Ala 400. The compound 3 showed interaction with Arg 599. The molecule was enclosed by residues such as Val 399, Thr 302, Lys 487 and Leu 484. In compound 4 hydrogen bonding was shown by Thr 606 and ligand was entrapped by the residue sequence of Val 399, Lys 487, Cys 300 and Ser 328. Docking results of G-6-P synthase showed that 2. Compound 2 − 4.29 the synthetic compounds have comparable docking score as compared to the standard drugs taken. All the ligands showed variable degrees of hydrogen bond interaction, hydrophobic interactions, electrostatic interactions, ionic interactions and π–π stacking with the various amino acid residues in the binding pockets of G-6-P synthase. ADME study The evaluation of different ADME parameters has been 3. Compound 3 − 3.30 represented in Table  5. It was observed that all the syn- thesized compounds fulfilled the standard Rule of Five [43]. All the synthesized compounds qualified the condi - tions for various descriptors like LogP, HBA, HBD and MW. All these parameters were in suitable range for drug-like characteristics. In addition, according to Veber et  al., 2002 for better bioavailability rotatable bonds should be ≤ 10 as the rotatable bonds in ligand impart elasticity [44]. The values of QPlogBB should be > 1.0 CNS active compounds and value < 1.0 CNS inactive 4. Compound 4 − 4.02 compounds. QPPCaco cell permeability should be in a range from 4–70 [45–47]. In the present study, all the synthesized compounds exhibited a suitable drug-like profile. Conclusion In conclusion, the above mentioned wet and dry labo- ratory studies highlight the underlying mechanism of G-6-P synthase inhibition. The rational development 5. Naringenin − 6.36 of inhibitors and specificity of naringenin derivatives to be discovered as the novel preservatives. Moreover, the synthesized compounds were also found as wonderful antioxidants towards DPPH with remarkable potential as compared to the reference compounds. Lather et al. BMC Chemistry (2020) 14:41 Page 10 of 15 Table 4 (continued) Experimental Materials and methods S. no. Compound(s) Structure of G-6-P synthase Dock All the chemicals required for experiments were of inhibitors score analytical grade and were purchased from Loba Che- 6. Standard Streptomycin − 5.795 mie (Mumbai, India), SRL (Mumbai, India), and Sigma Ciprofloxacin − 5.185 Aldrich (Germany). Nutrient agar, nutrient broth, sab- ouraud dextrose agar and sabouraud dextrose broth Ampicillin − 5.065 required for antimicrobial and preservative efficacy were Fluconazole − 5.129 obtained from Hi-media Laboratories. Streptomycin, Compound 1 Compound 2 Compound 3 Compound 4 Fig. 7 Binding of compounds 1, 2, 3 and 4 with G-6-P synthase La ther et al. BMC Chemistry (2020) 14:41 Page 11 of 15 Table 5 ADMET profile of various newly synthesized naringenin derivatives Compound(s) Mol. Wt. No. of rotatable DonorHB AcceptHB QPlogPo/w QPlogBB QPPMDCK QPPCaco bond Compound 1 392.10 5 5 4 2.084 0.081 0.053 1.877 Compound 2 345.12 3 3 3 2.490 0.138 11.251 2.773 Compound 3 365.11 4 2 4 4.29 2.445 0.282 10.982 Compound 4 381.08 3 4 2 1.278 3.355 0.162 20.169 ciprofloxacin, ampicillin and fluconazole were obtained 8.04 (s, 1H), 7.29 (d, J = 9.3  Hz, 2H), 7.28 (d, J = 7.5  Hz, as gift sample from Belco Pharma, Bahadurgarh, India. 2H), 6.80 (d, J = 7.3  Hz, 2H), 6.28 (s, 1H), 6.27 (s, 1H), Microbial strains S. aureus MTCC 3160, P. aeruginosa 5.28 (t, J = 9.0  Hz, 1H), 3.15 (d, J = 7.3  Hz, 1H), 3.00 (d, MTCC 1934, E. coli MTCC 45, C. albicans MTCC 183 J = 7.3 Hz, 1H); C NMR (400 MHz, C DCL ) δ = 166.11, and A. niger MTCC 282 strains wer e purchased from 165.34, 163.98, 161.90, 153.71, 152.59, 146.42, 133.96, MTCC, Chandigarh, India. Chemical reactions were 131.14, 126.43, 125.72, 124.08, 123.64, 117.42, 103.05, monitored by TLC on silica gel plates in iodine and UV 97.89, 95.36, 77.13, 38.79, 27.19, 22.70; MS ES + (ToF): chambers. Sonar melting point apparatus in open capil- m/z 392.10 [M +2]; CHNS: Calc (C H N O ): C, 64.28; 12 16 2 2 lary tube was used for the recording of melting points. H, 4.11; N, 7.14; O, 24.47; Found C, 64.25; H, 4.14; N, 1 13 H NMR and C NMR spectra were confirmed in 7.17; O, 24.44. DMSO and deuterated CDCl on Bruker Avance II 400 NMR spectrometer at a frequency of 400  MHz down- 4‑(1,3‑dihydroxypropan‑2‑ylimino)‑2‑(4‑hydroxyphenyl) field to tetramethyl silane standard. FTIR spectra were chroman‑5,7‑diol recorded on Perkin Elmer FTIR spectrophotometer with R TLC mobile phase: Chloroform: Acetone (8:5) = 0.66; the help of KBr pellets technique. Waters Micromass Yield = 50%; M.P . = 173–175  °C; M.Wt. = 345.32; IR (KBr −1 Q-ToF Micro instrument was used for Mass spectrum pellets) cm : 1074 (–C–O–C–), 1251 (–C–C–), 1513 (– recording. C=C–), 1631 (–C=N–), 2831 (–C–H–), 3295 (–OH–); H NMR (400  MHz, DMSO-d ) δ = 11.60 (s, 1H), 11.10 General procedure for the synthesis of naringenin (s, 1H), 8.04 (s, 1H), 7.28 (d, J = 7.2  Hz, 2H), 6.80 (d, derivatives J = 7.3  Hz, 2H), 6.28 (s, 1H), 6.24 (s, 1H), 5.25–5.24 (m, Substituted aniline (0.01  mol) was taken in a round bot- 1H), 3.95 (d, J = 8.1 Hz, 2H), 3.64 (q, J = 9.0 Hz, 2H), 3.50 tom flask, concentrated hydrochloric acid was added (q, J = 9.6 Hz, 2H), 3.47–3.45 (m, 1H), 3.13 (d, J = 8.6  Hz, drop wise with continuous stirring. Equimolar concen- 1H), 2.86 (d, J = 8.6  Hz, 1H); C NMR (400  MHz, tration of naringenin (0.01  mol) was dissolved in etha- CDCL ) δ = 164.81, 162.47, 161.87, 161.58, 158.28, nol (50  mL) and was re fluxed for 80-100  h on heating 130.84, 128.42, 115.98, 107.15, 103.33, 96.95, 78.20, 72.30, mantle. All the compounds in the series were synthe- 63.75, 37.92, 27.60, 22.32, 14.16; MS ES + (ToF): m/z sized according to the standard procedures as outlined 345.12 [M +2]; CHNS: Calc (C H NO ): C, 62.60; H, 18 19 6 in Scheme  1. Completion of reaction was monitored by 5.55; N, 4.06; O, 27.80; Found C, 62.63; H, 5.52; N, 4.09; TLC. Reaction mixture was concentrated and the pre- O, 27.82. cipitates formed were filtered off and dried. The crude product was recrystallized using alcohol which yielded 4‑(2‑fluorophenylimino)‑2‑(4‑hydroxyphenyl) the final compounds 1-4. chroman‑5,7‑diol R TLC mobile phase: Chloroform: Acetone (8:5) = 0.64; Spectral data Yield = 23%; M.P . = 165-167  °C; M.Wt. = 365.35; IR (KBr −1 2‑(4‑hydroxyphenyl)‑4‑(4‑nitrophenylimino) chroman‑5, pellets) cm : 753 (–F–), 1082 (–C–O–C), 1241 (–C– 7‑diol C–), 1612 (–C=C–), 1632 (–C=N–), 2833 (–C–H–), R TLC mobile phase: Chloroform: Acetone (8:5) = 0.63; 3350 (–OH–); H NMR (400 MHz, DMSO-d ) δ = 11.78 Yield = 55%; M.P . = 190–192  °C; M.Wt. = 317.29; IR (s, 1H), 11.10 (s, 1H), 8.04 (s, 1H), 7.47 (d, J = 8.8  Hz, −1 (KBr pellets) cm : 1081 (–C–O–C), 1156 (–C–C–), 1H), 7.31 (dt, J = 15.7, 8.4  Hz, 2H), 7.28–7.26 (m, 3H), 1305 (–NO ), 1599 (–C=C–), 1632 (–C=N–), 2921 6.80 (d, J = 7.4 Hz, 2H), 6.31 (s, 1H), 6.28 (s, 1H), 5.33 (t, (–C–H–), 3479 (–OH–); H NMR (400 MHz, DMSO-d ) J = 8.5 Hz, 1H), 3.04 (d, J = 7.7 Hz, 1H), 2.92 (d, J = 8.5 Hz, δ = 11.94 (s, 1H), 11.10 (s, 1H), 8.17 (d, J = 8.5  Hz, 2H), 1H); C NMR (400  MHz, C DCL ) δ = 165.92, 165.91, 3 Lather et al. BMC Chemistry (2020) 14:41 Page 12 of 15 strength nutrient broth I.P. (bacteria) or sabouraud dex- 165.24, 163.73, 161.86, 132.64, 132.61, 126.96, 126.94, trose broth I.P. (fungi) [51, 52]. The slants of E. coli, P. 126.50, 126.48, 125.25, 114.89, 114.86, 102.91, 97.83, aeruginosa, P. mirabilis and S. aureus were incubated 95.53, 72.64, 39.18, 20.46; MS ES+ (ToF): m/z 365.11 at the 30-35  °C for 24  h. The slants of C. albicans were [M +2]; CHNS: Calc (C H FNO ): C, 69.04; H, 4.41; F, 21 16 4 incubated at 20–25 °C for 48 h whereas; the slants of A. 5.20; N, 3.83; O, 17.52; Found C, 69.01; H, 4.44; F, 5.23; N, niger were incubated at 20–25  °C for 5  days. After the 3.84; O, 17.55. incubation period sterilized 0.9% NaCl solution was used to harvest the bacterial and fungal cultures from agar 4‑(2‑chlorophenylimino)‑2‑(4‑hydroxyphenyl) slant through proper shaking and then the suspensions chroman‑5,7‑diol of microorganisms were diluted with the sterile 0.9% R TLC mobile phase: Chloroform: Acetone (8:5) = 0.66; NaCl solution to CFU count was adjusted by adjust- Yield = 60%; M.P . = 155-157  °C; M.Wt. = 381.81; IR (KBr −1 ing the density of microorganism suspension to that of pellets) cm : 754 (–Cl–Str), 1062 (–C–O–), 1155 (–C– 0.5 McFarland standards by adding distilled water. The C–), 1602 (–C=C–) 1633 (–C=N–), 2834 (–C–H–), number of CFU was determined by dilution pour-plate 3284 (–OH–); H NMR (400 MHz, DMSO-d ) δ = 11.78 method [53]. A serial dilution of 50  µg/mL, 25  µg/mL, (s, 1H), 11.10 (s, 1H), 8.04 (s, 1H), 7.55 (d, J = 6.9 Hz, 1H), 12.5  µg/mL, 6.25  µg/mL, 3.12  µg/mL and 1.62  µg/mL 7.39 (t, J = 8.0  Hz, 1H), 7.28 (d, J = 8.0  Hz, 2H), 7.26 (d, was used for determination of MIC. The samples tubes J = 8.3 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 7.5 Hz, were incubated at 37  °C for 24  h (bacteria), at 25  °C for 2H), 6.19 (s, 1H), 6.17 (s, 1H), 5.34 (t, J = 8.9 Hz, 1H), 3.04 7 days (A. niger), and at 37 °C for 48 h (C. albicans) and (d, J = 8.7  Hz, 1H), 2.94 (d, J = 9.1  Hz, 1H); C NMR the results were recorded in pMIC. (400 MHz, CDCL ) δ = 165.10, 163.08, 161.26, 159.81, 143.28, 139.86, 129.24, 128.98, 128.45, 128.28, 127.73, Preservative effectiveness 127.42, 126.85, 124.29, 107.38, 102.08, 95.02, 76.72, 38.77, White lotion USP was utilized as the medium for the 17.39, 14.71; MS ES+ (ToF): m/z 381.08 [M +2]; CHNS: testing of preservative effectiveness. Calc (C H ClNO ): C, 66.06; H, 4.22; Cl, 9.29; N, 3.67; 21 16 4 Ingredients: Zinc sulfate 40 gm, sulfurated potash 40 O, 16.76; Found C, C, 66.09; H, 4.20; Cl, 9.26; N, 3.69; O, gm and purified water q.s. to 1000 mL. 16.72. Firstly, zinc sulphate and sulfurated potash were dis- solved in 450  mL of water separately and filtered. Then, Antioxidant activity sulfurated potash solution was added to zinc sulfate with DPPH radical scavenging assay stirring. At last, the required amount of water was added Antioxidant activity of the synthesized compounds was and mixed thoroughly and sterilized. For preservative determined by DPPH (2, 2-diphenyl-1-pycrilhydrazil efficacy testing, the White lotion USP was prepared using hydrate) radical scavenging method. Briefly, 0.1 mM solu - the equimolar amount of compounds 1-4 as novel pre- tion of DPPH in methyl alcohol was prepared and 1  mL servatives by replacing sodium benzoate, methyl paraben of this solution was added to 3 mL of sample or standard and propyl paraben from the formula [54]. with a concentration of 12.5, 25, 50, 75 and 100  μg/mL. Discolorations were measured at 517 nm after incubation Challenge microorganism for 30 min at 30 °C in the dark. Lower absorbance of the Staphylococcus aureus MTCC 3160, P. aeruginosa MTCC reaction mixture indicates higher free radical scavenging 1934, E. coli MTCC 45, C. albicans MTCC 183 and A. activity. The IC values of given samples were calculated niger MTCC 282 were used as common contaminants in by using formula: the study as prescribed in USP for preservative efficacy IC = (A − A ) × 100/A 50 c s c testing in the pharmaceutical preparations. Here, A was the absorbance of the control and A was c s Preparation of ioculums the absorbance of the sample [48, 49]. The slants of E. coli, P. aeruginosa and S. aureus were incubated at the 30–35 °C for 24 h. The slants of C. albi- Antimicrobial activity cans were incubated at 20–25  °C for 48  h whereas; the Minimum inhibitory concentration (MIC) slants of A. niger were incubated at 20–25  °C for 5  days The antimicrobial activity of the synthesized compounds [55]. were performed against S. aureus MTCC 3160, P. aerugi- nosa MTCC 1934, E. coli MTCC 45, P. mirabilis MTCC Test procedure 3310, C. albicans MTCC 183 and A. niger MTCC 282 White lotions USP was added in final containers and by using the tube dilution method [50]. Dilutions of were used in challenge test. The preparation was test and standard compounds were prepared in double La ther et al. BMC Chemistry (2020) 14:41 Page 13 of 15 Ligand Preparation inoculated with 0.5–1% volume of microbial inocu- 5 6 The three-dimensional structural library was prepared lum having a concentration of 1 × 10 –1 × 10   CFU/ using the Chemdraw software and proceeded for energy mL [56]. Inoculated samples were mixed thoroughly minimization using the LigPrep tool for the correction of to ensure homogeneous microorganism distribution coordinates, ionization, stereochemistry and tautomeric and incubated. The CFU/mL of the product was deter - structure to gain the appropriate conformation through mined at an interval of 0 days, 7 days, 14 days, 21 days, the addition or removal of hydrogen bonds. The partial and 28 days in agar plates. Log CFU/mL of white lotion charges were computed according to the OPLS-2005 USP was calculated as not less than 2.0 log reductions force field (32 stereo isomers, tautomers and ionization) from initial count at 14  days of incubation and no at biological pH and used for molecular docking studies. increase in CFU from 14  days count at 28  days in case of bacteria and no increase from the initial calculated count at 14 and 28 days [57]. Abbreviations ADMET: Absorption, distribution, metabolism, excretion & toxicity; G-6-P Synthase: Glucosamine-6-phosphate synthase; CYP P450: Cytochromes P450; In silico molecular docking studies OATP1B1: Solute carrier organic anion transporter family member 1B1; DHEAS: The Schrodinger, Inc. (New York, USA) software Maestro Dehydroepiandrosterone; PPAR: Peroxisome proliferator-activated receptors; 11 was used for the computational calculations and dock- DPPH: 2,2-Diphenyl-1-picrylhydrazyl; UDP-N-acetyl glucosamine: Uridine diphosphate N-acetylglucosamine; FTIR: Fourier-transform infrared spec- ing studies. Laboratory for Enzyme Inhibition Studies, 1 13 troscopy; H NMR: Proton nuclear magnetic resonance; C NMR: Carbon 13 Department of Pharmaceutical Sciences, M.D. University, nuclear magnetic resonance; UV: Ultra violet; TLC: Thin layer chromatography; Rohtak, INDIA was used for the computational work. The IC : Inhibitory concentration; MIC: Minimum inhibitory concentrations; CFU: Colony forming unit; HBA: Hydrogen bond acceptor; HBD: Hydrogen bond receptor-grid files were generated by grid-receptor gen - donor; MW: Molecular weight; MTCC : Microbial type culture collection; DMSO: eration program Glide [58]. Grid-based ligand docking Dimethyl sulfoxide; BOD: Biological oxygen demand; USP: United States utilized the hierarchical sequence of filters to produce pos - Pharmacopoeia; PDB ID: Protein Data Bank Identification; OPLS: Optimized potential for liquid simulations; Q.S.: Quantity sufficient. sible conformations of the ligand in the active-site region of the protein receptor. At this stage, crude score values Acknowledgements and geometric filters were prepared out unlikely binding The authors are highly thankful to the Head, Department of Pharmaceutical Sciences, M.D. University, Rohtak for providing essential facilities to accomplish modes. The next filter phase involves a grid-based force this research study. The authors are also thankful to Dr. Vinod Devaraji Applica- field evaluation and refinement of docking experiments tion Scientist Schrödinger LLC for his support to carry out the computational including torsional and rigid-body movements of the work. ligand [59]. The remained docking evaluations were sub - Authors’ contributions jected to a Monte Carlo procedure to minimize the energy The authors AL, SS and AK have designed, synthesized and carried out score. A conjugate gradient minimization protocol was the work in equal contribution. All authors read and approved the final manuscript. used in all calculations [60]. The energy differences were calculated using the Funding equation: No funding received for this research work from outside sources. E = E − E − E complex ligand protein Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Protein preparation Competing interests The X-ray protein structure co-ordinates of pdb id 1moq The authors declare that they have no competing interests. were downloaded from Protein Data Bank from www. Author details rcbs.org [61] and were prepared with the help of the Department of Pharmaceutical Sciences, Maharshi Dayanand University, Schrödinger protein preparation wizard ‘Prepwiz’ [62, Rohtak, Haryana, India. Department of Pharmaceutical Sciences, G.J.U.S.&T., Hisar, India. Laboratory for Preservation Technology and Enzyme Inhibition 63]. PDB id 1moq (resolution 1.57 A°) was selected on Studies, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical the basis of the lowest resolution and availability. All the Sciences, Maharshi Dayanand University, Rohtak, Haryana, India. waters molecules except metals co-ordinated and pre- Received: 30 July 2019 Accepted: 11 June 2020 sent between the ligand and protein were removed. The energy-restrained structure of the protein G-6-P syn- thase was constructed with the help of OPLS-2005 force field. References 1. 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Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, Sanschagrin PC, Mainz DT (2006) Extra precision glide: docking and scor- ing incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49:6177–6196 Ready to submit your research ? Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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