Synthesis, Identification, Theoretical Study, and Effect of the New Heterocyclic System from Ciprofloxacin Derivatives on the Activity of Some Liver Enzymes

: The target of this study was to synthesize several new Ciprofloxacin drug analogs by providing a nucleophilic substitution procedure that provides new functionality at the carboxylic group location. The analogs were synthesized, designed, and characterized by 1 HNMR, and FTIR. The synthetic path began from the reaction of ciprofloxacin drug with morpholine to give compound[B], ciprofloxacin derivative was linked with a variety of primary and secondary amines to give compounds[B1-B9]. The above-mentioned prepared compounds [B3 and B5] were applied to liver enzymes, and the increase in the activity of these enzymes was observed. In addition, a theoretical study was conducted to study the energies and properties of the prepared compounds.


Introduction:
Ciprofloxacin (CP, Fig. 1), is a broad-spectrum fluoroquinolone (FQ) 1 antibiotic with few adverse effects, it is widely used and has been found to inhibit cancer cell proliferation and induce apoptosis in a variety of cancer cell lines [2][3] . FQ is also employed in clinical trials as second-line medicine to treat tuberculosis because it has favorable pharmacokinetic profiles of good adsorption , and efficient penetration into host macrophages 4 . It is one of the most commonly given antibacterial medications, according to the World Health Organization 5 . Ciprofloxacin is approved for the treatment of a wide range of Gram-positive and Gram-negative bacterial illnesses 6,7 . Ciprofloxacin's COOH group was transformed into amide and ester derivatives, which have different antibacterial properties 8 . Ciprofloxacin was integrated into a new series of Schiff bases of 1,2,4-triazole via the Mannich process, and the antibacterial results were comparable to ciprofloxacin 9 . When compared to ciprofloxacin, NH-derivatives of ciprofloxacin demonstrated improved activity against Gramnegative bacteria 10 . Metal complexes are still a valuable resource for developing anticancer and antibacterial drugs in pharmaceutical chemistry, and they can be used to treat drug-resistant bacteria and a variety of viral illnesses [11][12][13] . As a result, fluoroquinolones are used to treat a variety of illnesses, including infections of the urinary system, respiratory tract, skin, gastrointestinal tract, and bones 14,15 . In this research ciprofloxacin has been incorporated into morphilene via Mannich reaction to give a new compound, then synthesis of several levofloxacin analogs by providing novel functionality at the carboxylic group position. For this reason, the carboxylic group at C-6 was reacted at the carbonyl carbon novel structural hybrids of ciprofloxacin with different amines to check the effectiveness of newly introduced remains on the activity of some liver enzymes. All melting points were recorded, uncorrected, using a Gallen Kamp capillary melting point equipment. Infrared (IR) spectra were registered using KBr disk on [Shimadzu model (FTIR-8400S)] spectrometer. 1 H NMR spectra in DMSO-d6 were acquired using an internal standard for the Varian Mercury-400 spectrometer, the reference used was tetramethylsilane (TMS). The purity of compounds was checked by TLC on aluminum-coated plates of 60 F245 (E. Merck), using) ethyl acetate and hexane, 2:1v:v) as the mobile phase and iodine was used as a visualizing agent. Fluka and Sigma-Aldrich provided all starting ingredients and solvents, which were used without further purification.

Synthesis of the Organic Compounds: Synthesis of Compound [B] 16 :
Stirred the blend of ciprofloxacin (0.01mol, 3.31g) and morpholine (0.01 mol,1.5 g) in 25 ml ethanol, and formaldehyde (37%CH2O) excess was added dropwise and heated to reflux for 4 hours at 70-75°C and allowed to cool to ambient temperature before freezing overnight to form crystals to make the title compound. The precipitate was filtered and recrystallized from [Ethanol with Water 2:8], the physical properties of synthesized compounds are shown in table.1.

Synthesis of Compounds [B1-B9] 17 :
Various ciprofloxacin derivatives were synthesized using aromatic or aliphatic amines (Diisopropylamine, 4-Chloroaniline, 2,5-Dimethylaniline, Dimethylamine, Dicyclohexylamine, 4-Chloro-2-nitroaniline, 4-Hydroxyaniline, Morphline, 4-Methylaniline). Ciprofloxacin (0.001 moles) was added to 20 mL of ethanol in a round-bottomed flask. After a few drops of sulphuric acid were added to the flask, the reaction was refluxed for 5 to 8 hours. Following the exhaustion of ciprofloxacin, 0.001 molar solutions of aromatic or aliphatic amines in ethanol were added one at a time. The reaction was refluxed for other 2 to 3 hours with constant stirring until complete. Evaporation was used to lower the reaction mixture's volume. The precipitates were filtered and recrystallized from the filtrate (DMF), the physical properties of synthesized compounds shown in table.1.

Materials and Methods of Biological Activity Section
Compounds (B3 and B5) have an effect on the activities of SGOT and SGPT. The following processes are used to colorimetrically determine the activity of SGOT or SGPT.
L-Alanine + oxoglutarate ALT Pyruvate + L-Glutamate L-Aspartate + oxoglutarate Oxalacetate+ L-Glutamate AST The pyruvate or oxaloacetate formed was measured in its derived from 2,4dinitrophenylhydrazine, which was absorbed at wave length 546 nm (SYRBIO kit).

A Compound [(Stock Solution , 0.01 M) (B3 and B5)]
By dissolving the compounds (B3 and B5) in distilled water, a stock solution (0.01 M) was created, and the following concentrations [(10-2, 10-3, 10-4, 10-5 M)] were created by diluting with distilled water. The activity of the enzymes SGOT and SGPT were determined in human serum using the same procedures as these enzymes, but with 100 µl of buffer replaced with 100 l of substances (B3 and B5). The activation % was estimated by comparing efficiency under identical conditions with and without chemicals (B3 and B5), using the equation: % Activation =100× the activity in the presence of activator/the activity in the absence of activator -100. The activation constant (Ka) was calculated according to the following equation: is an activator concentration

A Constant Concentration of Compounds (B3 and B5) (10 -2 M)
To investigate the kind of activation, a constant concentration of chemicals (B3 and B5) (

Synthesis:
The synthetic approach ,detailed in Scheme 1 ,was used to produce derivatives (B-B9) in modest yields. FTIR and 1 H NMR spectrum data was used to determine the structures of the produced compounds.

Comp.
No. The objective of this study is to look into the effects of SGOT and SGPT enzyme compounds (B3 and B5). The biochemical assays demonstrated that these substances stimulated the activity of the SGOT and SGPT enzymes. The impact of various chemical concentrations (B3 and B5) on the activity of SGOT and SGPT enzymes in human serum is shown in Table. 4. This study aims to look into the effects of SGOT and SGPT enzyme compounds (B3 and B5).

Compounds structure 1 HNMR data of (δ-H) in ppm
The biochemical experiments demonstrated that these substances had an activating effect on the activity of the SGOT and SGPT enzymes. In compound(B3) and compound(B5), the SGOT and SGPT enzyme activity were (11 and 14 U/L) correspondingly. Figs 2 and 3 show the association between chemical concentrations (B3 and B5) and enzyme activity (1 and 2). According to these findings, the proportion of enzyme activation increased with each rise in compound concentration.  Competitive, non-competitive and uncompetitive activators can easily be distinguished by using the Lineweaver-Burk plot's double reciprocal plot. The concentration of enzymes was kept constant during both groups of rate determinations. The speed of the inactivated enzyme was calculated in the first experiment, and a constant amount of activator was inserted in each enzyme test in the second experiment. Various substances are capable of reducing or eliminating the catalytic activity of a particular enzyme 20 . Table. 5 with Figs. 4 and 5 display the kind of enzyme activation using Line weaver-Burk plot for compounds (B3 and B5) on SGOT and SGPT activity. The Km values and Vmax were determined with (10 -2 M) of compounds (B3 and B5) and without it. Vmax without compounds (B3 and B5) was greater than Vmax in the presence of compounds (B3 and B5). A liquate 10 -2 M of compounds (B3 and B5) were non-competitive activation for enzymes activity. Non-competitive activation altered the enzyme's Vmax but not its Km. The Ka values of enzymes for compounds investigated at various concentrations were calculated using the Lineweaver-Burk equation.  Amino acid metabolism, as well as the urea and tricarboxylic acid cycles, rely heavily on enzymes. We hypothesized that compounds (B3 and B5) include (-NH, =C, and =O) groups, which activate the active sides of amino acids in SGOT and SGPT enzymes by boosting their alliance for substrate reaction.

Theoretical Study
The optimize geometry and HOMO and LUMO energies for the compounds (B3 and B5) were calculated by using the semi-empirical (PM3) method. Figs 6 and 7 respectively show the shapes and contain the energy values for all bonding molecular orbitals [including the highest occupied molecular orbital HOMO, plus the lowest unoccupied molecular orbital LUMO] for (B3 and B5) compounds. They also demonstrated the symmetry assignment for each molecular orbital. Both HOMO and LUMO are the main orbitals in chemical stability. According to the semi-empirical, the reactive ability of the above compounds is related to the molecular orbitals (MO) that are the HOMO and the LUMO 21 Table. 6, shows (EHOMO) which represents the capacity to donate an electron, 22 . While (ELUMO) indicates the capacity to obtain electron. The energy variation between HOMO and LOMO explains the ability of compounds (B3 and B5) to donate electrons and thus stimulate the ends of amino acids present in the enzyme, and this is consistent with the practical results that showed that these compounds (B3 and A5) are enzymes activator.  positively charged organisms are more likely to attack a molecule with high electrostatic potential 23 .

Conclusion:
Various methodology involving structural modifications have been attempted for the synthesis of new ciprofloxacin derivatives featuring amide functional groups at carbon C-3 of the fluoroquinolone scaffold. As detailed above, ten carboxamide analogues have been synthesized and characterized. B3 and B5 compounds were studied evaluated for their activity on some liver enzymes (GOT and GPT). The ciprofloxacin derivatives were found to have activating effects on the GOT and GPT enzymes in biochemical experiments. Also, we worked on a theoretical study in order to compare the findings to those of the experiment results. Our theoretical results are in a good agreement with the experimental ones.