Antimicrobial and anti-tubercular activities of isolates and semi-synthetic derivatives of lichen Ramalina leiodea ( Nyl . ) Nyl

EXPERIMENTAL Antimicrobial activities The determinations of both the antibacterial and antifungal activities for measuring the antimicrobial properties were realized according to the standard well plate method.1 For the antibacterial activities, all the selected test strains (Salmonella enterica ATCC 35664, Pseudomonas aeruginosa ATCC 15442, Bacillus subtilis ATCC 23857 and Staphylococcus aureus ATCC 25923) were initially activated and grown in nutrient agar. Whereas, for antifungal studies, the test strain used, Candida albicans, was grown on potato dextrose agar medium. All compounds were re-dissolved in DMSO to obtain a final concentration of 1 mg mL-1, which were used as stock solutions. The compounds were used for activity studies and the concentration of each sample was 1 μg mL-1 along with standard and control. The media, Petri dishes were autoclaved at 121 °C for 15 min. After sterilization, the agar plates were prepared by pouring 25 mL of agar medium followed by incubation at room temperature for 30 min for solidification under a sterile environment. These plates were inoculated with 60 μL of test inoculums using sterile cotton swabs. Wells 8 mm in diameter were made with a sterile cork borer and into each well exactly 100 μL of sample were loaded. The control and standard were placed in separate wells. The plates were initially incubated for 20–30 min at 4 °C to allow the compounds to diffuse into the agar, and then subsequently incubated for 24 h at 37 °C for the bacteria and 48 h at 28 °C for the fungi. The zone diameters were measured in mm using a calibrated scale. The experiments were conducted in triplicate with aliquots to minimize the deviations and the average values are reported. The compounds having better anti-microbial activities were selected for the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) studies against Salmonella enterica, P. aeruginosa, B. subtilis, S. aureus and C. albicans according to a reported method.2 The concentrations of test samples were serially diluted from 1 to 0.001 μg mL-1 and one tube without drug served as the control. All the tubes were inoculated with 1 mL of respective cultures having an OD of 0.2 (≈McFarland standard) and the tubes were incubated at 37 °C for 12–16 h. The turbidity of each tube was measured with respect to the control tube. The MIC values are defined as the lowest concentration of a compound at which


INTRODUCTION
Ramalina leiodea (Nyl.)Nyl. is a fruticose lichen belonging to Ramalinaceae that is found in regions of eastern India.Generally, Ramalina genus has about 246 species distributed around the world, of which only 118 species have been investigated for their chemical and biological properties.A diversity of secondary metabolites has been isolated from the Ramalina genus, including dibenzofuran derivatives, depsides, depsidones and orcinol derivatives. 1,2Moreover, biological screening of this genus resulted in the identification of antioxidant, 3 anti-inflammatory, 4 anticancer, 5 antimicrobial 3 and anti-tubercular 6 activities.
As a part of ongoing research of anti-infective agents from lichens, this study presents the outcomes of chemical and biological investigation of R. leiodea.

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TATIPAMULA and VEDULA Moreover, previously some novel benzohydrazide derivatives were synthesized that were found to be very active against the Mycobacterium tuberculosis H37Rv strain. 7Now, the acquired synthetic knowledge was applied towards naturally occurring secondary metabolite and some semi-synthetic analogues, i.e., benzohydrazides, were derived and screened along with the isolated metabolites for their antimicrobial and anti-tubercular (anti-TB) activities.

General
Commercially available chemicals were used for the extraction, isolation and purification protocols.TLC was performed on silica gel 60 F254 plates (Merck, Germany); elemental analysis was realized on a Carlo Erba 1108 analyzer; nuclear magnetic resonance (NMR) spectra were recorded in CDCl 3 , DMSO-d 6 or acetone-d 6 using an Avance (400 MHz) instrument for both 1 H-NMR and 13 C-NMR; the FT-IR spectra were recorded on a Bruker Alpha-T FT-IR spectrometer (Bruker Optik, Switzerland) and the EI-MS spectra on a JEOL-M-JMS-D-300 instrument at 70eV (Jeol, Tokyo, Japan).The melting points (MP) were recorded on a Boitus melting point apparatus and the UV-Vis spectra on an Electron 420 series spectrophotometer, Elico India Ltd.

Extraction
The extraction procedure is based on modified procedures of Jug et al. 2017. 8The lichen specimens were collected gently and shade dried.The dried lichen material (150 g) was suspended in ethanol-water (1:1) for a week and evaporated under reduced pressure to obtain the hydroalcoholic extract of R. leiodea (RL-HA, 40.11 g).The RL-HA was re-extracted with acetone solvent, dried over anhydrous sodium sulfate and concentrated to obtain the dry acetone extract of R. leiodea (RL-Ac, 14.50 g).The RL-Ac was subjected to column chromatography using Sephadex LH-20 resulting in four bioactive fractions.Fraction I (612 mg) obtained from 10 % ethyl acetate in hexane, on further purification using column chromatography (hexane in dichloromethane, 9:1) to obtain 1 (236 mg) as yellow needles.Fraction II (1.50 g), obtained from 30 % ethyl acetate in hexane, was subjected to column chromatography (ethyl acetate in dicholormethane, 3:7) to obtain 2 (153 mg) as a greenish solid and 3 (911 mg) as white crystals.Fraction III (4.6 g), obtained in 40 % ethyl acetate in hexane, was recrystallized using acetone and hexane, 9.5:0.5, yielding 4 (4.05 g) as pale-yellow crystals.Fraction IV (20 mg), obtained in 50 % ethyl acetate in hexane, was subjected to column chromatography (ethyl acetate in hexane, 1:1) to obtain 5 (11 mg) as a white solid.
Procedure for the synthesis of 2,6-dihydroxy-4-methylbenzohydrazide (4a) 4 (2.0 g) was dissolved in ethanol (50 mL) and hydrazine hydrate (3.0 eq., 1.06 mL) was added to the solution.The resultant suspension was refluxed for 6 h and the progress of the reaction was monitored by TLC.After completion of the reaction, ethanol and excess hydra-________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2019 SCS.
POTENT ANTI-TUBERCULAR DERIVATIVES FROM R. leiodea 557 zine were removed under vacuum to afford the crude solid product (2.5 g) that was washed and recrystallized from acetone to give 4a (1.91 g, 95.5 mass %) as a pale yellowish solid.
General procedure for the synthesis of benzohydrazide derivatives (4b-e) 4a (1.0 mmol) was dissolved in ethanol and different aldehydes (4b-e, 1.1 mmol) was added to the solution and the reaction mixture was heated to reflux for 2-3 h.TLC confirmed the completion of the reaction, the solvent was evaporated under vacuum to obtain a solid that was stirred in n-hexane:acetone (1:1) and filtered.The obtained solid was dried under vacuum to afford the solid products 4b-e.

RESULTS AND DISCUSSION
The chemical examination of acetone extract of R. leiodea revealed the presence of five known metabolites (1-5), except 1, all were reported for the first time from this species.By elemental and spectral analysis, 1-5 (Fig. 1) were identified as dibenzofuran derivatives, i.e., usnic acid (1); a monocyclic aromatic ester, ethyl everninate (2); a depside derivative, scrobiculin (3); an orcinol derivative, methyl 2,6-dihydroxy-4-methylbenzoate (4) and a depside derivative, 4-[(2-hydroxy-4-methoxy-6-propylbenzoyl)oxy]-2-methoxy-6-propylbenzoic acid (5).These metabolites showed moderate to potent antibacterial activity against gram-positive (Staphylococcus aureus and Bacillus subtilis) and gram-negative (Pseudomonas aeruginosa and Salmonella typhi) strains and antifungal activity against Candida albicans and mild anti-tubercular activity against the Mycobacterium tuberculosis H37Rv strain.It is well known that many today drugs that are commercially available and in the late stage of clinical trials are of natural origin.Additionally, structural modification of natural metabolites is a tool to obtain bioactive molecules with smaller adverse effects than their natural counterparts.Based on the aforementioned observations, an attempt was made to develop new structurally diverse molecular scaffolds from natural metabolites.This paper focuses on the derivatization of 2,6-dihydroxybenzoate into novel benzohydrazides that possess potent anti-tubercular activity.In addition, the potential anti-tubercular activities of ben-558 TATIPAMULA and VEDULA zohydrazide derivatives coupled with functionalities that are present on these natural metabolites make them well suited for semi-synthetic derivatization for the development of more efficacious compounds.Hitherto, there have been no synthetic studies that established or reported on benzohydrazide substituted with 2,6-dihydroxy derivatives.Thus, five analogs were synthesized and evaluated for their antimicrobial and anti-tubercular activities.
As shown in Scheme 1, compounds 4a-e were prepared in good yield from 4. Initially, the terminal ester group in compound 4 was cleaved by hydrazine monohydrate to yield 4a, which was further coupled with different aldehydes to yield the corresponding benzohydrazides (4b-e).The structures of the 4a-e were characterized by elemental analysis, and IR, ESI-MS, 1 H-and 13 C-NMR spectral data.The presence of a carbonyl carbon was confirmed for the benzohydrazides 4a-e by the presence of a carbon signal at δ C 166-174 ppm in 13 C-NMR spectra and an intense FT-IR absorption in the range from 1620-1700 cm -1 .Similarly, the amine proton (-NH) of the benzohydrazides was confirmed by a proton signal at δ H 7.41-8.84ppm, while the phenolic group was confirmed by a proton signal at δ H 9.3-10.2ppm in the 1 H-NMR spectra and an intense FT-IR absorption at around 3200 cm -1 .The complete NMR and positive ESI-MS analyses confirmed the structures for 4a-e, which are shown in Scheme 1.

Antimicrobial activity
The antimicrobial activity 9 of the isolated metabolites 1-5 and their derivatives 4a-e were evaluated against S. aureus, B. subtilis, P. aeruginosa, S. enterica and C. albicans.Among the isolated metabolites, 3 and 5 having the same skeleton (i.e., depside) depicted better antimicrobial activity against all the tested strains, when compared to the standard drug (streptomycin), which suggested that the presence of depside nucleus plays a vital role in conferring antimicrobial activity.Furthermore, compound 1 that contains a benzofuran moiety showed potent activity only against the tested bacterial strains whereas moderate activity against the fungal strains (Table I).However, compounds 2, 3 and 4a-e showed only moderate activity against all the tested microbial strains (Table I).Moreover, to explore the importance of the isolates 1-5, their minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations ________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2019 SCS.
POTENT ANTI-TUBERCULAR DERIVATIVES FROM R. leiodea 559 (MBC) against selected microbial strains were evaluated, along with streptomycin as the standard using 100 µg/mL each.The MIC data suggested that all the selected strains were susceptible to 1, 3 and 5 and were more potent than streptomycin.However, most potent activity was found against P. aeruginosa (nosocomial infections), with an MIC value for 1 and 5 of 0.007 µg mL -1 , whereas the value for streptomycin was 0.03 µg mL -1 ; against S. aureus (causes disease due to direct infection/production of toxins) with an MIC value of 0.007 µg mL -1 for 3, while the value for streptomycin was 0.03 µg mL -1 ; 5 showed potent activity against S. typhi (typhoid fever) with an MIC of 0.007 µg mL -1 , whereas, it was 0.01 µg mL -1 for streptomycin (Table II).The calculated MBC values against P. aeruginosa were found to be 0.03 µg mL -1 for 1, 3 and 5; against S. aureus, it was found to be 0.01 µg mL -1 for 3 and against S. enterica, it was found to be 0.01 µg mL -1 for 5 (Table II).

Anti-tubercular activity
The anti-TB activity 7 of the isolated compounds 1-5 and benzohydrazide derivatives 4a-e was investigated against the M. tuberculosis H37Rv strain since the benzohydrazides are also known for their anti-TB properties. 7The results are summarized in Table III.They confirmed that the benzohydrazides 4a-e showed better inhibitory profiles against the M. tuberculosis H37Rv strain than the isolated compounds 1-5 (Table III).Among the semi-synthetic derivatives, compounds 4b and 4d depicted a better MIC value of about 1.6 µg mL -1 , which suggested that the presence of benzyl moiety played a major role in conferring anti-TB activity.In addition, 4c exhibited an equivalent MIC to that of streptomycin with an MIC of 6.25 µg mL -1 (Table III).The interesting mechanism of action attributed to 4a-e derivatives, especially inhibition of synthesis of mycolic acids, arises due to their structural similarity with the first line anti-TB drug, isoniazid. 10

Structure-activity relationship of benzohydrazides as anti-tubercular agents
The in vitro anti-TB activity assisted in the identification of the potency of benzohydrazides against M. tuberculosis, thereby in elucidating the structure--activity relationship.The syntheses of phenyl containing analogues (4b and d) were found to increase the anti-TB activity.Additionally, the replacement of electron donating groups such as methoxy (4b) with a phenyl moiety led to better anti-TB activity than substitution with an electron withdrawing group such as chlorine (4e).In addition, the electron releasing (such as alkyl, 4c) derivatives improved the anti-TB activity.
________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2019 SCS.To conclude, the present study involved the isolation, semi-synthesis and biological profile of metabolites from the lichen R. leiodea.Among all the compounds, 3 and 5 revealed good antibacterial as well as antifungal activity.Additionally, compound 1 showed significant antibacterial activity against the tested strains.Moreover, the semi-synthetic analogs 4a-e exhibited better anti-TB activity with moderate antimicrobial activity.Hence, this research suggested a solid foundation for further lead optimization of this class of benzohydrazides by a systematic refinement, including the synthesis of compounds to improve their overall biological properties.

SUPPLEMENTARY MATERIAL
The structural elucidation and spectral data of all the isolated metabolites and semisynthetic derivatives were provided as Supplementary Material, available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request.

TABLE I .
Diameters of the zones of inhibition for all the isolates and benzohydrazide derivatives of R. leiodea against bacterial and fungal stains; loading concentration: 100 µL per well a n = 3; the diameter of the inhibition zone includes the diameter of the well

TABLE II .
MIC values for all the isolates and benzohydrazide derivatives of R. leiodea against bacterial and fungal stains ________________________________________________________________________________________________________________________ Available on line at www.shd.org.rs/JSCS/(CC) 2019 SCS.

TABLE III .
In vitro anti-tubercular activity of all the isolates and benzohydrazide derivatives of Ramalina leiodea against the M. tuberculosis H37Rv strain; n = 3; MIC: minimum inhibitory