In Silico Evaluation of Biopharmaceutical Properties of Chloramphenicol Derivatives and their Iron Complexes

Evaluation of Biopharmaceutical Properties of Chloramphenicol Derivatives and their Complexes. Abstract Context and The use of chloramphenicol (CAM) has been reduced due to the side effects associated with its use (Bone marrow depression, neurotoxicity) and the increase in resistance to CAM that some microbes develop. To overcome these difficulties, two CAM derivatives, L1 and L2, and their respective iron complexes were synthesized to evaluate in silico their biopharmaceutical properties. The substrate (CAM), as well as the basic reagents (AAP and AASC) were purified from commercial pharmaceuticals. The CAM derivatives (L1 and L2) and also their iron complexes (C1, C2, and C3) were synthesized and showed maximum absorbance at 335 nm for CAM, 325 nm for L1, 395 nm for L2, at 330 nm for C1, at 325 nm for C2, and at 335 nm for C3. The in silico simulations performed with the above-mentioned tools showed that all the ligands (CAM, L1, and L2) present good similarities with the drugs, a good bioavailability because they were compliant with the Lipinski rule. The complexes, although bioavailable, did not conform to Lipinski's rule. CAM showed efficacy in enzymatic inhibition. However, L1 and L2 ligands perform better in ion channel modulation, kinase, and protease inhibition. This suggests that the ligands have better therapeutic performance and may well address several clinical needs. The C3 complex was the compound that showed better bioavailability and high bioactivity thus it was the most bioactive. L1, L2, and C3 could therefore be potential and promising candidates for CAM substitution. Permeability ; hERG : human Ether-a-go-go-Related Gene); GPCR : G protein-coupled receptor; NRL : Nuclear receptor ligand ; ICM : Ion channel modulation; KI : Kinase inhibition ; PI : Protease inhibition ; EI : Enzyme activity inhibition ; MLCT : Metal to Ligand Charge Transfer ; AAP : Acetaminophen ; AASC : Acetylsalicylic acid ; CAM : Chloramphenicol ; C1 : Ferric complex of CAM-O-AAP (L1) ; C2 : CAM-O-AASC iron complex (L2) ; C3 : CAM iron complex ; FeCAM : CAM iron complex; FeCAM-O-AAP : CAM-O-AAP iron complex (L1) ; FeCAM-O-AASC : CAM-O-AASC iron complex (L2) ; L1 : 2-(4-Acetylaminophenoxy)-2-chloro-N-[1,3-dihydroxy-1-(4-nitrophenyl) propan-2-yl] "CAM-O-AAP; : 2-(2-Acetoxybenzoyloxy)-2-chloro-N-[1,3-dihydroxy-1-(4-nitrophenyl) propan-2-yl] the L2 ligand (from 335 nm for CAM to 395 nm for L2). These observations would thus be evidence for the formation of L1 and L2 compounds. In addition, the UV-Vis spectra of the C1, C2, and C3 complexes compared to the spectra of their respective ligands (L1, L2, and CAM) showed different types of effects, in particular the hyperchromatic effect in the case of the C1 and C3 complexes justified by the increase of the absorption maximum and the hypsochromatic effect in the case of C2 (from 395 nm for L2 to 315 nm for C2). These observations could well indicate the formation of C1, C2, and C3 complexes.


Introduction
The CAM, a bacteriostatic antibiotic of the phenicol family, has an effective bacteriostatic action against grampositive and gram-negative bacteria and was the first broad-spectrum antibiotic to be used medically against typhoid infections. Although effective, its use is nowadays reduced because of its side effects and the increase of resistance developed by microbes and bacteria [1,2]. The CAM was first isolated in 1947 from Streptomyces venezuelae, and

Extraction, purification, and identification of chemicals used
For CAM, open the capsules and collect the powder. Weigh an amount equivalent to 20 g of chloramphenicol (80 capsules) and dissolve them in 100 ml of ethyl acetate in a beaker. Heat while stirring the mixture until boiling to ensure total dissolution of the solutes; Filter while hot, then let the filtrate cool until crystals form; Filter and dry under vacuum, then collect the residue greenish-white (Yield: 80%). For its identification, add to 1 g of CAM, a mixture of 5 drops of phenol, and 0.20 g of potassium hydroxide. Bring to a boil over a small flame and shake; a red-brown coloration develops. A red-orange color is produced and ammonia is released when the CAM is heated with a 50% sodium hydroxide solution [9]. For PAA, weigh 20 g of PAA equivalent to 40 paracetamol tablets and dissolve in 100 ml of acetone in a beaker. Heat while stirring the mixture until boiling to ensure total dissolution of the solutes. Filter while hot and collect the filtrate. Evaporate the solvent by heating, then collect the white crystals (Yield: 70%). For its identification, dissolve 0.10 g of PAA in 10 ml of water and add 0.5 ml of ferric chloride 25g/l; it appears an intense blue coloration (European Pharmacopoeia, 2017). For AASC, weigh 25 g of AASC equivalent to about 50 aspirin tablets and dissolve in 60 ml of isopropanol in a beaker; Heat while stirring the mixture until boiling to ensure total dissolution of solutes; Filter while hot, and collect the filtrate; Allow the filtrate to cool, and add about 180 ml of ice-cold distilled water; Collect the precipitates by vacuum filtration (Blank, yield: 79%).
Finally, TLC evaluated the purity. To identify it, heat 0.5 mg of AASC in a test tube; the molten product gives off a strong acetic acid smell. Further heating turns the product from yellow to brown to black. Place a small amount of the AASC on a white ceramic plate or watch glass placed on a white background and add 1 drop of potassium hydroxide in ethanol. After 1 minute, add 1 drop of ferric chloride 25 g/l; a purple coloration appears [10].

Synthesis and identification of ligands and complexes
The ligand syntheses were done as described by Omar et al. [11] and the complex syntheses as described by Lawal and Obaleyo [12]. The formation of the products proceeds from the formation of the ether function (R-O-R) between the carbocation of CAM and the hydroxyl function of PAA and/or AASC. The ether function can form the oxonium ion in an acidic medium. However, the C-O bond can be broken, in the presence of hydrogen halide [13]. This is the basis for the identification of the ether function.

Synthesis of the ligand CAM-O-AAP (L1):
In a 100 ml flask equipped with a refrigerator and a magnetic bar is introduced a mixture of acetone (70 ml), CAM (10 g, 0.03 mol), APP (3.596 g, 0.0238 mol), and sodium carbonate (2.524 g, 0.0238 mol). The reaction mixture is heated at reflux. After 5 hours, it is hot filtered, then the filtrate is cooled and left for 48 hours for crystallization. The residue is filtered and the crystals obtained are washed with ethanol (Yellowish-white, yield: 91.17%).

Synthesis of the ligand CAM-O-AASC (L2):
In a flask (100 ml) equipped with a refrigerator and a magnetic bar is introduced a mixture of acetone (70 ml), CAM (10 g, 0.03 mol), AASC (4.2867 g, 0.0238 mol), and sodium carbonate (2.524 g, 0.0238 mol). The reaction mixture is heated at reflux. After 5 hours, it is filtered while hot. After cooling, two phases were observed, the non-ketone phase was washed with acetone, dried with Na2SO4, collected (Brown liquid, yield: 81%) and finally the purity of the product was evaluated by TLC.

Purification and identification of reagents
The substrate (CAM) and reagents were purified to separate them from the excipients. The pharmaceuticals were treated with several solvents to remove compounds such as starch, cellulose, talc, and others. The active substances were obtained in the form of white crystals with yields varying between 70 and 80% (CAM: 80%, AAP: 70%, and AASC: 79%). The identification tests of CAM, PAA, and AASC were positive. Indeed, the mixture of CAM, phenol and KOH, when boiled, produced a red-brown coloration. In addition, upon heating, in the presence of sodium hydroxide, a red-orange coloration was produced, and there was a release of ammonia. The identification of PAA was confirmed by the appearance of an intense blue coloration in the presence of ferric chloride [10]. The presence of AASC was confirmed by the release of acetic acid upon heating and obtaining a yellow color after intense heating. Furthermore, the addition of potassium hydroxide (in ethanol) in the presence of ferric chloride produced a purple color [10].

Synthesis and characterization of ligands and complexes
The ligands were synthesized from CAM as substrate with PAA for the synthesis of ligand L1 (Yellowish-white solid) on the one hand and with AASC for the synthesis of ligand L2 (Highly viscous brown liquid) on the other hand. All ligands were characterized by the presence of the R-O-R bond formed between the substrate and the reagent. All ligands were synthesized, purified and obtained with good yields, i.e. 91.12% for L1 and 81% for L2.
Like the ligands, all the complexes were also synthesized, by reaction between the L1 ligands with iron to give the C1 complex (Greyish solid) and between the L2 ligand and iron to give the C2 complex (Red-brown solid) on the one hand as well as between CAM and iron giving the C3 complex (Yellow-red solid).

ADME prediction:
The latter consists of the prediction of ADME properties. These were predicted using the PreADMET, and PkCSM tools (Table 3).  values of skin permeability, suggesting that it would not be important for the compounds to be administered by the transdermal route. Most drugs in plasma would exist in equilibrium between either an unbound or protein-bound state in serum. The effectiveness of a given drug may be affected by the degree to which it binds to proteins in the blood. Indeed, the more bound that molecule is, the lower the efficiency to cross the cell membrane and diffuse [15].

Parameters
clearance but also potential contraindications. Drug clearance appears to be the combination of hepatic clearance (metabolism in the liver and biliary clearance) and renal clearance (excretion via the kidneys). All compounds were not likely substrates for OCT2, and showed negative values of total clearance except for CAM and L1, which have positive values and thus possess good extrusion.

Toxicity prediction:
Similar to the prediction of ADME properties, the toxicity prediction was performed using the PreADMT and PkCSM tools (Table 4). Both tools revealed in general that the products studied are all mutagenic. However, for their hERG inhibition, CAM presented a low risk, L1 presented a high risk, and an equivocal result is obtained for L2. On the other hand, all complexes (C1, C2, and C3) showed a medium risk. All studied compounds were negative in TA100_10RLI, TA100_NA and TA1535_10RLI toxicity tests and appeared to be hepatotoxic except for CAM and its C3 complex;

Parameters/Toxicity
they are not likely to be associated with skin sensitization. Tetrahymena pyriformis is a protozoan bacterium whose toxicity is often used as the limit or endpoint of toxicity. For a given compound, if the plGC 50 value (the negative logarithm of the concentration required for 50% growth inhibition) is greater than -0.5 log μg/L, that compound would be toxic [15]. All of the compounds tested, given that they have positive plGC 50 values and greater than -0.5 log μg/L, exhibited T. Pyriformis. In addition, minnow toxicity is expressed by the lethal concentration (LC50) values, which represent the concentration of a molecule required to cause the death of 50% of minnows. A compound is considered to have high acute toxicity if it has an LC 50 value less than -0.3 [15]. All of the compounds tested had valued above -0.3, so they are non-toxic to minnow toxicity, except for C3, which had a value of -0.324.

Bioactivity scores:
Prediction of the bioactivity score was performed using the Molinspiration tool (Table 5).
Bioactivity is considered considerable when the bioactivity score is greater than 0.00; moderate when the bioactivity score is between -0.5 and 0.00 and inactive when the bioactivity score is less than -0.5 [16].

GPCR Ligand
Ion channel modulation

Conclusion
In this work, two derivatives of chloramphenicol and their iron complexes were synthesized to see the possibilities of improving the efficacy and pharmaceutical properties of chloramphenicol on the one hand and reducing its toxicity, consequently its side effects, on the other hand. The synthesized compounds were collected with a good yield and characterized by analytical (chemical), spectral and theoretical methods for the determination of the probable structures of the complexes. And these structures allowed a theoretical study (in silico prediction) of the biopharmaceutical properties of the compounds. In general, CAM, L1, and L2 show good similarities with drugs, good bioavailability and bioactivity since they were by Lipinski's five rule. The complexes, on the other hand, did not comply with Lipinski's rule. On the other hand, the ADME properties showed that C3 was presented a good efficiency of absorption and distribution in the organism about the other synthesized compounds and CAM as a reference compound. Concerning toxicity, all the compounds seemed to have toxicity almost identical to that of CAM. All the compounds studied have overall moderate bioactivity while L1 and L2 showed better bioactivity scores than the complexes. CAM showed only efficacy in enzyme inhibition. However, L1 and L2 ligands have better performance especially in kinase (score of -0.26 and -0.37 respectively for L1 and L2), protease (score of -0.10 for L1, and -0.11 for L2) inhibition. This suggests the ligands have better therapeutic performance and could well address several clinical needs. The synthesized compounds (L1 and L2) appeared to be potential candidates to substitute chloramphenicol, hence it could be interesting to continue the studies by preparing and characterizing (by advanced spectral methods) other derivatives, by adding other pharmacophoric moieties on chloramphenicol, and synthesizing their complexes; and finally to evaluate their biopharmaceutical properties by in silico predictions and by in vitro and in vivo studies on mice.

Credit authorship contribution statement
KME designed the study, KMM, LKE & KKO carried out the study, the analysis, the writing of the manuscript, the editing, the revision, the methodology with the help of MKM.

Declaration of competing ınterest
The authors declare that they have no potential competing interests