Design, Molecular Docking, Synthesis, and Antimicrobial Evaluation of new Dipeptide derivatives of Ceftizoxime sodium

 

Zahra N. Hachim1*, Shakir M. Alwan2, Mayada H. Al-Qaisi1

1Pharmaceutical Chemistry Department, College of Pharmacy, Al-Mustansyriah University.

2Department of Pharmacy, Al-Farabi University Collage.

*Corresponding Author E-mail: zahraanaji2020@gmail.com

 

ABSTRACT:

Newer cephalosporins that can be orally administered with significant oral bioavailability and resist β-lactamases are continuously and significantly requested. A method of using a potent third-generation cephalosporin, ceftizoxime, was suggested to synthesize new dipeptide derivatives. These derivatives were successfully synthesized by linking a dipeptide moiety, which includes L-Tryptophan-L-valine, L-Tryptophan-L-alanine, L-Tryptophan-L-histidine, and L-Tryptophan-L-phenylalanine as dipeptides to the aminothiazole moiety of ceftizoxime by an amide bond. Their chemical structures were confirmed by spectral analysis, including 1H-NMR,13CNMR, and FT-IR spectroscopy. Molecular docking for these new derivatives was performed on penicillin-binding proteins (PBPs) type 2a (3ZG0) of Methicillin-resistant Staphylococcus aureus, type 2X (5OJ0) of Streptococcus pneumonia and type 1b (5HLA) of E. coli, and has recorded higher affinity binding represented as PLP fitness on target enzymes. The binding scores were significant and may indicate better antimicrobial activities when compared with ceftizoxime. This improvement in affinity binding can be explained by the presence of indole and/or imidazole moieties in those derivatives. The indole and imidazole moieties are actual pharmacophores with various biological activities and may contribute to affinity binding, and the derivatives are considered molecular hybrids. Furthermore, a preliminary evaluation of the antibacterial activity of the synthesized derivatives was performed against two significant bacterial species (MRSA and E. coli), which showed better activity in compression to ceftizoxime. Moreover, the derivatives were tested on the protein oligopeptide (POT) family system and have recorded very interesting results for possible oral absorption when compared with Ceftizoxime, Val-acyclovir, and Val-Val-Acyclovir, as reference drugs. The Swiss ADME server was also used to analyze the pharmacokinetic characteristics and identify those likely to be absorbed orally.

 

KEYWORDS: Ceftizoxime, Dipeptides, Peptide transport system, MRSA, Molecular hybridization.

 

 


INTRODUCTION: 

Ceftizoxime (CZX) is a parenterally administered 3rd generation cephalosporin having an aminothiazole-methoxyimino moiety at the C-7 position of the cephem nucleus. It is administered intravenously or intramuscularly only due to poor absorption and bioavailability through the gastrointestinal tract (GI) because of their low lipophilicity due to the low PKa of the carboxyl group at the C-4 position of cephem nucleus1.

 

CZX exhibits a broad spectrum of activity against G-positive and G-negative bacteria isolated from patients with respiratory and urinary tract infections2,3. At concentrations close to its MIC, CZX is resistant to hydrolysis by various β-lactamases and non-β-lactamases producing bacteria and has approximately similar activity against both types3. It has been considered the most effective antibiotic against Klebsiella pneumonia and E. coli, suppressing 80% of the bacteria at a 0.025µg/ml concentration. Furthermore, it is more effective against Enterobacter cloacae, Proteus mirabilis, and indole-positive proteus than Cefoxitin, Cefoperazone, Cefotaxime, and Carbenicillin4. Based on the presence of a limited number of β-lactam antibiotics that are well absorbed orally5,6 and the great need to convert CZX or other cephalosporins of advanced generation (4th and 5th generations) to an orally absorbable form, several attempts were considered leading to limited success with moderate oral bioavailability, including the double ester approach on C3 carboxyl group of the dihydrothiazine ring, which was one of the solutions7.

 

An attempt was considered to link various dipeptides to CZX molecule via an amide bond to the aminothiazole moiety to fulfill the requirements of the peptides transport system. Peptide transporters have been identified in bacteria, fungi, plants, and mammalian cells. These transporters use the proton gradient across the brush border membrane to concentrate their substrates within the enterocyte before exiting through the basolateral surface into the bloodstream8,9. These transporters take up dipeptide and tripeptide nutrients andseveral peptide-like and peptide-containing drugs, which are rapidly taken up into intestinal epithelial cells by a specific apical peptide transporter encoded by the PEPT1 gene. This information provides the molecular basis for a rational drug design to increase the oral absorption of peptide-based drugs mediated by PEPT110. So, the new L-dipeptide-ceftizoxime derivatives are expected to overcome the very poor or devoid of oral bioavailability of ceftizoxime. Furthermore, these derivatives may enhance activity due to introducing another pharmacophore group to the acyl side chain of CZX, such as L-Tryptophan and L-Histidine, which contain indole11 and imidazole12 moieties, respectively. 

 

MATERIALS AND METHODS:

Melting points were determined using Stuart, UK's electrical melting point apparatus. The infrared spectra were performed by FT-IR spectroscopy of Bruker tensor 27, Germany. The 1HNMR analysis was performed using a Bruker Avance NEO spectrometer Switzerland. Ethyl chloroformate (ECF), L-valine, L-alanine, L-histidine, L-phenylalanine, hydrochloric acid (HCl), and ceftizoxime sodium were purchased from Sigma Aldrich/USA. Boc-Tryptophan was purchased from Shanghai World Yang Chemical/China. Trimethylamine (TEA) and trifluoroacetic acid (TFA) were obtained from Thomas Baker/India.

 

General procedure for the synthesis of BOC-L-dipeptides (1a-d):

The synthesis of dipeptides was performed starting with BOC-L-Tryptophan with certain L- amino acids using a modified mixed anhydride method13 and as illustrated in scheme 1 and as follows:

 

A cold solution of ethyl-chloroformate (ECF, 10mmol, 0.94ml) in dry chloroform (10ml) was prepared at (-2 to -4°C). A slightly yellowish solution was obtained by dissolving Boc-L-Tryptophan (10mmol, 3.04g) in dry chloroform (40ml) containing triethylamine (TEA, 10mmol, 1.39ml), which was cooled to (-2 to -4°C), added to the ECF solution, and stirred for 2 hrs. A cold solution at 0–5°C was prepared by dissolving L-amino acids [10mmol (1.17g of L-valine, 0.89g of L-alanine, 1.55 of L-histidine, and 1.65g of L-phenylalanine) separately in distilled water (10ml) containing TEA (20 mmol, 2.78ml) was added all at once with vigorous stirring for 2hrs and then stirred overnight at room temperature. A yellowish-orange solution resulted in the chloroform layer. Acidification of the mixture with a few drops of hydrochloric acid (1N) to adjust the pH to 3-4. The two layers were separated using a separator funnel, and the aqueous layer was removed. The organic layer was washed with distilled water (3 x 30ml), and the aqueous layer was discarded. The chloroform layer was dried using anhydrous calcium chloride and then evaporated under reduced pressure in a rotary evaporator to obtain yellowish residues. Residues were triturated with diethyl or petroleum ether to produce a crystalline powder, which was dried in an oven at 35oC.  Compounds 1a-d are soluble in chloroform, acetone, tetrahydrofuran, 1,4-dioxane, and dichloromethane. At the same time, they are insoluble in water, methanol, and toluene. Compound 1a was collected as creamy beige powder, Yielding; 77% m.p. 168-170 oC. FT-IR spectra of compound 1a recorded the following characteristic absorption bands (ʋ, cm-1); 3419; N-H stretch. Vib. of secondary amide, 3402; N-H stretch. Vib. of the indole ring, 3200; O-H stretch. Vib of carboxylic acid, 1740; C=O stretch. vib. of Boc group, 1705; C=O stretching vib of carboxylic acid, 1630; C=C stretch. vib of the aromatic ring. Compound 1a recorded the following characteristic chemical shifts (δ, ppm) in 1HNMR analysis; 10.9 (s,1H, indole NH), 6.9-7.8 (m, 4H, aromatic CH), 7.7(s, 1H, 2° amide NH), 1.34 (s, 9H, BOC CH3), 0.8 (d, 6H, valine CH3) and recorded the following characteristic chemical shifts (δ, ppm) in 13 CNMR analysis;110-118 (m,4C. Ar carbon), 18.3 (m, 2C, valine CH3), 172 (s,1C, 2°Amide carbonyl), 28.2 (m, 3C, Boc CH3), 174 (s, 1C, COOH),110(s, 1C, indole C), 124(d,1C ,indole CH).

 

Compound 1b was collected as an off-white powder, Yielding 75% m.p. 143-144 oC. Compound 1b recorded the following FT- IR spectra (ʋ, cm-1); 3419, NH stretch. vib. of secondary amide; 3400, NH stretch. vib. of indole ring; 3180, O-H stretch. vib of carboxylic acid; 1750, C=O stretch. vib. of Boc carboxyl group; 1705, C=O stretch. vib of carboxylic acid; 1640, C=C stretch. vib. of the aromatic ring. Compound 1b recorded the following characteristic chemical shifts (δ, ppm) in 1HNMR analysis; 10.9 (s,1H, indole NH), 6.8-7.8 (m, 4H, aromatic CH), 7.1(s, 1H, 2° amide NH), 1.33 (s, 9H, BOC CH3), 1.2 (d, 3H, alanine CH3).And recorded the following characteristic chemical shifts (δ, ppm) in 13 CNMR analysis; 110-123 (m,4C. Ar carbon), 17 (m, 2C, alanine CH3), 174 (s,1C , 2°Amide carbonyl), 28.5 (m, 3C, Boc CH3), 174.5 (s, 1C, COOH) , 110(s, 1C, indole C), 124(d,1C ,indole CH).

 

Compound 1c was obtained as yellow powder; Yield: 75%, m.p. 151-153oC. Compound 1c recorded the following FT- IR spectra (ʋ, cm-1); 3383, N-H stretch. vib. of indole ring; 3307, C=O stretch. vib. of imidazole ring; 1730, C=O stretch. vib. of histidine and Boc carboxyl groups; 1688, C=O stretch. vib. of secondary amide; 1620, C=C stretch. vib. of aromatic ring. Compound 1c recorded the following characteristic chemical shifts δ (ppm) in 1HNMR analysis; 10.8 (s,1H, indole NH), 6.8-7.8 (m, 4H, aromatic CH), 7.5(s, 1H, 2° amide NH), 1.34 (s, 9H, BOC CH3), 8.4 (s, 1H, imidazole NH). and recorded the following characteristic chemical shifts (δ, ppm) in 13 CNMR analysis; 110-121 (m,4C. Ar carbon), 172 (s,1C , 2°Amide carbonyl), 28.2 (m, 3C, Boc CH3), 174 (s, 1C, COOH) , 110 (s, 1C, indole C), 124(d,1C ,indole CH), 124.5(s, 1C, imidazole C), 117.4(d,1C , imidazole CH)

 

Compound 1d was obtained as creamy beige; Yield:  98%, m.p. 146-148oC. Compound 1d recorded the following FT-IR spectra (ʋ, cm-1); 3309, N-H stretch. vib. of indole ring; 1718, C=O stretch. vib. of phenylalanine and Boc carboxyl groups; 1701, C=O stretch. vib of Boc group; 1686, C=O stretch. vib. of secondary amide; 1655, C=C stretch. vib. of aromatic ring. Compound 1d recorded the following characteristic chemical shifts (δ, ppm) in 1HNMR analysis; 10.8 (s,1H, indole NH), 6.8-7.9 (m, 9H, aromatic CH), 8.1 (s, 1H, 2° amide NH), 1.35 (s, 9H, BOC CH3). and recorded the following characteristic chemical shifts (δ, ppm) in 13 CNMR analysis;110-129 (m,9C. Ar carbon), 172 (s,1C , 2°Amide carbonyl), 28.6 (m, 3C, Boc CH3), 174 (s, 1C, COOH) ) , 110 (s, 1C, indole C), 124(d,1C ,indole CH).

 

General procedure for the synthesis of BOC-L-Dipeptide-Ceftizoxime intermediates (2a-d):

The synthesis of BOC-L-Dipeptides-Ceftizoxime intermediates (2a-d) was performed starting with BOC-L-Dipeptides (1a-d), as previously described, which were reacted with ceftizoxime using the modified mixed anhydride method 13, as illustrated in scheme 1 and as previously described in detail.

 

Ethyl chloroformate (ECF, 10 mmol, 0.94 ml) was reacted with Boc-L-Dipeptides 1a-d (10 mmol). Ceftizoxime (10 mmol, 3.83 g) in tetrahydrofuran (20 ml) containing TEA (20 mmol, 2.78 ml) was used as the amine-containing compound. The reaction mixture was a yellowish-orange suspension containing a white precipitate of (TEA. HCl). The solvent mixture was evaporated using reduced pressure in a rotary evaporator to obtain yellowish residues. Distilled water/chloroform (30/30ml) was added and a yellowish orange two-layered solution had been obtained. Acidification of the mixture with a few drops of hydrochloric acid (1N) to adjust the pH to 3-4 and separation of the two layers by using a separator funnel. The aqueous layer was removed, and the organic layer was washed with distilled water (5 x 30ml) to remove TEA. HCl. The chloroform layer was dried using anhydrous calcium chloride and then evaporated under reduced pressure in a rotary evaporator to obtain yellowish residues. Residues were triturated with diethyl ether or petroleum ether to produce a crystalline powder, which was dried in an oven at 35°C. Compounds 2a-d appeared to be soluble in acetone, tetrahydrofuran, methanol and 1,4-dioxane, while they are insoluble in water, chloroform, toluene, and dichloromethane. 

 

Compound 2a recorded the following characteristic absorption bands (ʋ, cm-1); 3420; N-H stretch. vib. of secondary amide, 3402; N-H stretch. vib. of indole ring, 3188; O-H stretch. vib of carboxylic acid, 1764; C=O stretch. vib. of Ceftizoxime carboxyl group,1740; C=O stretch. vib. of Boc group, 1670; C=O stretch. vib. of secondary amide, 1630; C=C stretch. vib. of an aromatic ring, 702; C-S-C stretch. vib. of ceftizoxime. compound 2a recorded the following characteristic chemical shifts δ (ppm) in 1HNMR analysis; 10.8 (s,1H, indole NH) , 6.5-7.7 (m, 4H, aromatic CH) , 8.18 ( s, 1H, 2° amide NH ), 1.33 (s, 9H, BOC CH3), 0.8 (d, 6H, valine CH3), 3.5(s, 3H, O-CH3), 5 (d, 1H, S-CH-N),8.3(s,1H, N-C=CH-S) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 110-127 (m,4C. Ar carbon), 18.6 (m, 2C, valine CH3), 171 (s,2C , 2°Amide carbonyl), 30.3 (m, 3C, Boc CH3), 172 (s, 1C, COO), 110(s, 1C, indole C), 123.7 (d,1C ,indole CH), 163(s, 1c, β-lactam carbonyl), 20(t, 1C , β-lactam CH2), 70.2(q, 1C, methoxy CH3).

 

Compound 2b recorded the following FT-IR spectra; 3300, NH stretch. vib. of secondary amide and indole ring; 3063, O-H stretch. vib. of carboxylic acid; 1741, C=O stretch. vib. of Boc and Ceftizoxime carboxyl groups; 1680; C=O stretch. vib. of secondary amide,1606, C=C stretch. vib. of an aromatic ring, 700; C-S-C stretch. vib. of ceftizoxime. compound 2b recorded the following characteristic chemical shifts δ (ppm) in 1HNMR analysis; 10.9 (s,1H, indole NH) , 6.8-7.9 (m, 4H, aromatic CH) , 8.3 ( s, 1H, 2° amide NH ), 1.33 (s, 9H, BOC CH3), 1.2 (d, 3H, alanine CH3), 3.3(s, 3H, O-CH3), 5 (d, 1H, S-CH-N),8.2(s,1H, N-C=CH-S) and  recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 118-127 (m,4C Ar carbons), 18.6 (m, 1C, alanine CH3), 171 (s,2C , 2°Amide carbonyl), 29.4 (m, 3C, Boc CH3), 163 (s, 1C, COO) ,111(s, 1C, indole C), 124.1 (d,1C ,indole CH), 164(s, 1c, β-lactam carbonyl), 28.6 (t, 1C, β-lactam CH2), 66.7 (q, 1C, methoxy CH3).

 

Compound 2c recorded the following FT-IR spectra; 3381, N-H stretch. vib of indole ring; 3309, C=O stretch. vib. of imidazole ring; 1731, 3052; O-H stretch. vib. of Ceftizoxime carboxylic acid, C=O stretch. vib. of histidine and Boc carboxyl groups; 1687, C=O stretch. vib. of secondary amide; 1517, C=C stretch. vib. of an aromatic ring, 699; C-S-C stretch. vib. of Ceftizoxime compound 2c recorded the following characteristic chemical shifts δ (ppm) in 1HNMR analysis; 10.9 (s,1H, indole NH) , 6.8-7.9 (m, 4H, aromatic CH) , 8.2 ( s, 1H, 2° amide NH ), 1.33 (s, 9H, BOC CH3), 8.3 (s, 1H, imidazole NH), 3.4(s, 3H, O-CH3), 5 (d, 1H, S-CH-N),8.4(s,1H, N-C=CH-S) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 118-121 (m,4C Ar carbon), 171 (s, 2C , 2°Amide carbonyl), 29.4 (m, 3C, Boc CH3), 164 (s, 1C, COO) ,111.7(s, 1C, indole C), 124 (d,1C ,indole CH), 164(s, 1c, β-lactam carbonyl), 28.6 (t, 1C , β-lactam CH2), 66.7 (q, 1C, methoxy CH3),127.9(s, 1C, Imidazole C), 118 (d, 1C, imidazole CH).

 

Compound 2d recorded the following FT-IR spectra; 3321, N-H stretch. vib. of indole ring; 1718, 3058; O-H stretch. vib of Ceftizoxime carboxylic acid, C=O stretch. vib of phenylalanine and Boc carboxyl groups; 1701, C=O stretch. vib. of Boc group; 1686, C=O stretch. vib. of secondary amide; 1655, C=C stretch. vib. of an aromatic ring, 699; C-S-C stretch. vib. of ceftizoxime.  compound 2d recorded the following characteristic chemical shifts δ (ppm) in 1HNMR analysis; 10.9 (s,1H, indole NH) , 6.7-7.8 (m, 9H, aromatic CH) , 8.25 ( s, 1H, 2° amide NH ), 1.34 (s, 9H, BOC CH3), 3.4(s, 3H, O-CH3), 5 (d, 1H, S-CH-N), 8.4 (s,1H, N-C=CH-S) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 120-131 (m,9 C, Ar. carbon), 171 (s,2C , 2°Amide carbonyl), 29.4 (m, 3C, Boc CH3), 166 (s, 1C, COO) ,112(s, 1C, indole C), 124 (d,1C ,indole CH), 164(s, 1c, β-lactam carbonyl), 27 (t, 1C , β-lactam CH2), 64 (q, 1C, methoxy CH3).

 

General procedure for synthesis of the L-Dipeptides-Ceftizoxime derivatives (1-4)

Removal of the BOC protecting group was achieved using Trifluoroacetic acid 14.

Compounds 2a–d [2 mmol (1.337g of 2a, 1.28g of 2b, 1.413g of 2c, and 1.433g of 2d)] were separately used and dissolved in dichloromethane, DCM (15 ml), which was then chilled to 0°C in an ice bath. Trifluoroacetic acid, TFA (15ml) was added with continuous stirring for 1 hr in the presence of anisole (3 ml). The solvents were removed under reduced pressure using a rotary evaporator. Then ethanol (30 ml) was used to dissolve the residue and the PH was adjusted to 7 using a 5% ethanolic solution of NaOH. The solvent was evaporated under reduced pressure using a rotary evaporator and the residue was washed with chloroform to remove unreacted compounds, which was then filtered, washed with acetone, and recrystallized from ethyl acetate: petroleum ether (9:1). The precipitate was collected and dried in an oven at 35°C. Compounds 1-4 were collected as colored powders.

 

Compound 1 (C29H31N8O7S2.Na) was obtained as orange colored powder; Yield: 74%, m.p. 210-212 oC. Compound 1 recorded the following characteristic FT-IR spectra (ʋ, cm-1): 3385, N-H stretch. vib. of primary amine; 3310, N-H stretch. vib. of indole ring; 1731, C=O stretch. vib. of C3 carboxylic of Ceftizoxime; 1687, C=O stretch. vib. of secondary amide. Compound 1 recorded the following characteristic chemical shifts (δ, ppm); 10.9 (s,1H, indole NH), 6.8-7.5 (m, 4H, aromatic CH), 8.15 (s, 1H, 2° amide NH), 0.7 (d, 6H, valine CH3), 3.4 (s, 3H, O-CH3), 4.9 (d, 1H, S-CH-N), 8.5 (s,1H, N-C=CH-S), 8.1 (s. 2H 1° amine) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 110-127 (m,4C. Ar carbon), 18.6 (m, 2C, valine CH3), 171 (s,2C , 2°Amide carbonyl), 172 (s, 1C, COO) ,110(s, 1C, indole C), 123.7 (d,1C ,indole CH), 163(s, 1c, β-lactam carbonyl), 20(t, 1C , β-lactam CH2), 70.2(q, 1C, methoxy CH3).

 

Compound 2 (C27H27N8O7S2.Na)was collected as orange-colored powder; Yield: 74%, m.p.163-164 oC. Compound 2 recorded the following FT-IR absorption bands; 3385, N-H stretch. vib. of primary amine; 3309, N-H stretch. vib. of indole ring; 1731, C=O stretch. of C3 of carboxyl group of Ceftizoxime; 1687, C=O stretch. vib. of secondary amide.

 

Compound 2 recorded the following chemical shifts (δ, ppm); 10.9 (s,1H, indole NH), 6.8-7.5 (m, 4H, aromatic CH), 8.15 (s, 1H, 2° amide NH), 1.2 (d, 3H, alanine CH3), 3.4 (s, 3H, O-CH3), 5.1 (d, 1H, S-CH-N), 8.4 (s,1H, N-C=CH-S), 8.2 (s. 2H 1° NH2) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 118-127 (m,4C. Ar carbon), 18.6 (m, 1C, alanine CH3), 171 (s,2C , 2°Amide carbonyl), 163 (s, 1C, COO) ,111(s, 1C, indole C), 124.1 (d,1C ,indole CH), 164(s, 1c, β-lactam carbonyl), 28.6 (t, 1C , β-lactam CH2), 66.7 (q, 1C, methoxy CH3).

 

Compound 3 (C30H29N10O7S2. Na) was obtained as light brown powder; Yield: 88%, m.p. 167-169 oC. Compound 3 recorded the following FT-IR absorption bands (ʋ, cm-1): 3367, N-H stretch. vib. of 1°- NH); 3311, N-H stretch. vib. of indole ring; 3279, N-H stretch. vib. of imidazole ring; 1729, C=O stretch. vib. of C3 carboxyl of Ceftizoxime; 1669, C=O stretch. vib. of secondary amide. Compound 3 recorded the following chemical shifts (δ, ppm); 11.1 (s,1H, indole NH), 6.8-7.5 (m, 4H, aromatic CH), 8.25 (s, 1H, 2° amide NH), 8.3 (s, 1H, imidazole NH), 3.4 (s, 3H, O-CH3), 5.1 (d, 1H, S-CH-N), 8.4 (s,1H, N-C=CH-S), 8.2 (s. 2H 1° NH2) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 118-121 (m,4C. Ar carbon), 171 (s,2C , 2°Amide carbonyl), 164 (s, 1C, COO) ,111.7(s, 1C, indole C), 124 (d,1C ,indole CH), 164(s, 1c, β-lactam carbonyl), 28.6 (t, 1C , β-lactam CH2), 66.7 (q, 1C, methoxy CH3),127.9(s, 1C, Imidazole C), 118(d, 1C, imidazole CH).

 

Compound 4 (C33H31N8O7S2.Na) was collected as yellowish-orange powder; Yield 72%, m.p. 166-169 oC. Compound 4 recorded the following FT-IR absorption bands: 3385, N-H stretch. vib. of primary amine; 3307, N-H stretch. vib. of indole ring; 1730, C=O stretch. vib. of C3-carboxyl of Ceftizoxime; 1687, C=O stretch. vib. of secondary amide.  Compound 4 recorded the following chemical shifts (δ, ppm); 10.9 (s,1H, indole NH), 6.8-7.5 (m, 9H, aromatic CH), 8.2 (s, 1H, 2° amide NH), 3.4(s, 3H, O-CH3), 5.0 (d, 1H, S-CH-N),8.4 (s,1H, N-C=CH-S), 8.2 (s. 2H, 1°- NH2) and recorded the following characteristic chemical shifts δ (ppm) in 13CNMR analysis ; 120-131 (m,9 C, Ar. carbon), 171 (s,2C , 2°Amide carbonyl), 166 (s, 1C, COO) ,112(s, 1C, indole C), 124 (d,1C ,indole CH), 164(s, 1c, β-lactam carbonyl), 27 (t, 1C , β-lactam CH2), 64 (q, 1C, methoxy CH3).

 

Molecular Docking:

Cambridge Crystallographic Data Centre (CCDC) software, namely Genetic Optimization for Ligand Docking (GOLD) version 2020.3.0 was used to conduct the docking investigation15,16 of the newly synthesized L-dipeptide -CZX derivatives at the target proteins including penicillin-binding proteins (PBP2X from Streptococcus pneumonia, PBP2b from E-Coli and PBP2a from methicillin-resistant staphylococcus aureus) and one of the POT family transporters extracted from Staphylococcus homines which were obtained from the Protein Data Bank. The visualizer program Hermes CCDC (version 2020.3.0) calculates the bond length and displays ligands, receptors, hydrogen, and hydrophobic bonding interactions. Chemdraw Professional (version-16.0) was used to draw the molecular structures of our ligands. The results are illustrated in Tables 1-4.

 

Preliminary antimicrobial evaluation:

The preliminary antimicrobial evaluation was performed using the well diffusion method 17,18. Two bacterial species were utilized as in vitro test subjects for the antibacterial activity of four of the synthesized compounds: E. coli as a form of G-negative bacteria, and the G-positive bacteria, Methicillin-resistant S. aureus (MRSA). Ceftizoxime sodium was the positive control for antibacterial activity.

 

The agar well diffusion method, which requires the utilization of cultures for all microbial species, was used to screen the synthetic compounds' antibacterial activities. Utilizing the number 0.5 McFarland turbidity standard for bacteria, the surface of previously prepared Mueller Hinton (MHA) agar Petri was inoculated with bacteria at a concentration of 1.5108 CFU/ml by the carpet method.

 

These were then set aside to dry. Upon solidification, a sterile cork borer (6 mm in diameter) was used to make wells in agar plates containing bacterial suspension. The MHA plate's wells were then filled with 100 μl of synthesized derivative dilutions and the reference drug (Ceftizoxime sodium) in the concentrations of (500, 250, 125, 62.5, 30, and 10) μg/ml for each well. The negative control utilized was DSMO. For 24 hrs, the plates were kept at 37°C. By measuring the distance from the inhibitory zone, the antibacterial activity could be determined. The antimicrobial screening was conducted using the diameter of the inhibition zone that formed across the well.

 

Analytical Statistics:

All of the study's data were statistically analyzed by using the Microsoft Office Excel 2010 and IBM-SPSS version 26(Statistical Package for the Social Sciences), based in one way ANOVA, LSD and Independent t test at p. value < 0.05.

 

SWISS ADME19:

The Swiss ADME server was used to predict the synthesized derivatives' pharmacokinetic properties. One of the Swiss ADME investigations included Lipinski's rule of five, which relates to the molecular parameters of drugs that must be present to be passively orally absorbed.

 

RESULTS AND DISCUSSION:

Amides are highly significant moieties in chemistry and biochemistry, as they form important linkages in peptides, proteins, pharmaceuticals, natural products, and Peptidomimetics20. In general, amide bonds have relatively higher enzymatic stability than ester bonds. They are stable for several hours or even several days in the absence of specific enzymes. Amide bond creation should ideally be fast, quantitative, and performed under mild conditions that do not affect adjacent stereogenic centers, do not cause secondary reactions or produce easily removable byproducts21. It is ideal to form a covalent bond with carboxylic acid and an amine through dehydrative condensation22. One of these well-known processes includes activating the carboxyl group to the mixed carboxylic-carbonic acid anhydride, which is typically carried out at low temperatures in anhydrous organic solvents. The resultant anhydride is reacted with the appropriate nucleophile (amine). The anhydride is produced as a result of an alkyl chloroformate (ethyl chloroformate) in the presence of triethylamine (TEA) 23, as illustrated in scheme 1. Furthermore, the synthesis of target derivatives required the cleavage of tert-butyl carbamates, which occurs under anhydrous acidic conditions with the generation of carbon dioxide and tert-butyl cations.

 

Scavengers, such as anisole are used to prevent alkylation (t-butylation) of nucleophilic L-Tryptophan during the deprotection process.

 

Scheme 1: General chemical synthesis of intermediates and target compounds.

Molecular Docking of L-Dipeptides-Ceftizoxime on 5OJ0 fromStr. Pneumonia:

Ceftizoxime showed a PLP fitness on penicillin-binding protein 2X(5OJ0) 24 from Str. Pneumonia of 60.8, while all the other new derivatives have recorded higher values, with the highest affinity binding for L-Trp-L-His-CZX and L-Trp-L-Phe-CZX approaching 96.4 and 89.3, respectively (Table 1). This may indicate much better activities of the new L-dipeptide- CZX derivatives on Str. Pneumonia. These are expected results since imidazole 11, and indole 12 moieties may contribute to the affinity binding to receptors and antimicrobial activities.

 

The amino acids, Asn337, Gln554, Tyr568, and His594 of the PBP2X (5OJ0) have been demonstrated to interact with CZX by three different types of each of the hydrogen and hydrophobic bonding. Amino acids, including one or two that interacted with CZX, have also been shown to interact with the L-Dipeptide-CZX derivatives via three to four hydrogen bonds. The hydrophobic bonding ranges from 4 to 9 (Table 1). L-Trp-L-Val-CZX and L-Trp-L-Ala-CZX derivatives had the greatest 7 and 9 bonds, respectively. The introduction of L-valine and L-alanine moieties may contribute to the hydrophobicity of these molecules and cause the level of elevation of such bonding.

 

Molecular Docking of CZX and L-dipeptide-CZX derivativeson PBP2a:

The molecular docking of the CZX on PBP2a(3ZG0)25from MRSA recorded 48.9 PLP fitness, while all the new derivatives recorded much higher PLP fitness ranging between 73.8 and 82.3 (Table 2). As expected, L-Trp-L-His-CZX andL-Trp- L-Phe-CZXrecorded the highest affinity binding of 82.3 and 78.6, respectively, due to additional functional moieties of the amino acids linked to ceftizoxime. CZX interacted with seven amino acids of this target, while the L-dipeptide-CZX derivatives interacted with 4 to 7 amino acids from the same target (Table 2).

 


 

Table 1: Molecular docking of the CZX and L-Dipeptide-CZX derivatives on PBP 2X from Str.Pneumonia.

Compound

PLP

fitness

PLP

Part. rer.

H bonding

Hydrophobic bonding

Amino acids of the target enzyme involved in the interaction

CZX

60.8

0.1817

3

3

Asn337, Tyr568, Gln552, His594

1

89.3

0.4699

4

7

Asn337, Trp374, Gln452, Met527, Ser548, Thr550, Gln552, Phe570, His594

2

82.5

0.4160

3

9

Ser548, His594, Asn377, Gly549, Thr550, Phe450, Ile371, Gln552.

3

96.4

0.3393

4

5

Gln552, Asn377, Thr550, His594, Tyr595, Ile598

4

87.5

0.8465

4

4

Gln552, Lys340, Asn397, Trp374, Ile371


Table 2: Molecular docking of the CZX and L-dipeptide-CZX derivatives on PBP 2a from MRSA

Compound

PLP-fitness

PLP

Part. rer.

H bonding

hydrophobic bonds

Amino acids of the target enzyme involved in the interaction

CZX

48.9

0.4554

3

6

Tyr272, Ala276, Val277, His293, Glu239, Lys148, Asp295,

1

73.8

0.5380

5

14

Val277, Val256, Met372,   Ser240, H2O2035, H2O2036

2

75.6

0.4743

0.8411

4

4

Lys148, Glu150, Ser240, Val277, Glu239

3

82.3

0.5405

5

14

Lys148, Ser240, Val277, His293, Met372, H2O2035, H2O2036.

4

78.6

0.7910

6

9

Lys148, Glu239, Asp274, Val277, His293, Asp295, Ala276.

H2O822, H2O956

 

Table 3: Molecular docking of the CZX and L-dipeptide-CZX derivatives on PBP 1b from E-Coli

Compound

PLP.Fitness

PLP

Part. rer

Hydrogen

Bonds

Hydrophobic bonds

Amino acids of target enzyme involved in the interaction

CZX

57.066

0.4868

5

5

Glu233, Asn275, Tyr310

Lys355, Ala357, Ser358.

1

71.7

0.4470

5

6

Tyr315, Gln318, Arg325, Ser358

2

67.5

0.7468

4

4

Lys274, Tyr315, Ser358

3

76.58

0.2903

3

3

Tyr315, Gln318, Ser358

4

75.7

0.6160

4

4

Gln271, Asn275, Tyr315,Ala357, Ser358.

 


Molecular docking at 5HLA:

The molecular docking of the CZX on PBP1b (5HLA) 26from E-Coli demonstrated PLP fitness of 57.066; however, all other novel derivatives recorded higher values, with the maximum affinity binding for L-Trp-L-His-CZX and L-Trp-L-Phe-CZX derivatives approaching 76.58 and 75.7, respectively (Table 3). The amino acids, Glu233, Asn275, Tyr310, Lys355, Ala357, and Ser358 of the PBP1b (5HLA) have demonstrated interaction with CZX and with five different types of hydrophobic and hydrogen bonding. At the same time, L-Dipeptides-CZX derivatives interacted with 3-5 of the amino acids of the same target enzyme (Table 3).

Molecular Docking on POT family transporter type 6GZ9:

All the new derivatives have recorded higher PLP fitness on the POT family transporter, type 6GZ927, extracted from staphylococcus homines, compared with CZX, which has very poor absorption. However, Val-Acyclovir and Val-Val-Acyclovir have been tested on this protein transporter and recorded less PLP fitness (Table 4). This test reflects the possibility of absorption via an intestinal tract protein transporter. All the new derivatives have recorded the highest values, which may refer to the possibility of absorption through the intestinal tract.


 

Table 4: Molecular docking of L- dipeptide -CZX derivatives on POT family transporter extracted from staphylococcus homines (6GZ9).

Compound

PLP

fitness

PLP

Part. rer.

H bonding

hydrophobic bonding

Amino acids of target enzyme involved in the interaction

L-Val-Acyclovir

60.9

0.7574

2

5

Tyr41, Tyr79, Tyr163, Ala170,  Asn347,  Pro348

L-Val-L-Val-Acyclovir

72.3

0.6725

2

9

Tyr163, Asn167,

Gln310, Ser422.

CZX

53

0.2965

2

0

Asn426, H20604

1

99.3

0.1475

2

9

 Ile351, Asn426, Trp445,

Tyr163, Val166

2

97.9

0.9946

6

2

Tyr41, Ala170, Lys137, Asn167

H2O604, H2O605

3

101.8

0.5030

5

10

Val166, Asn426, Ile351, Gln310, Gln344, Ser314, Tyr41,

H2O602, H2O605

4

102.9

0.6701

3

4

Gln310, Asn426, Asn167,

Val160, Leu446

 


Preliminary antimicrobial evaluation:

As described previously, the synthesized derivatives were subjected to antimicrobial evaluation by well-diffusion method 26, 27. Interestingly, all the L-dipeptides-Ceftizoxime derivatives have better activity on E. Coli at higher concentrations, while they have slightly less activity at lower concentrations when compared to Ceftizoxime (Table 5). Moreover, these derivatives recorded significant increases in activities on MRSA at high concentrations and very close activities at lower concentrations, except for compound 1 (L-Trp-L-Val-CZX), which has recorded similar activity to ceftizoxime. The dipeptides containing aromatic moieties, such as indole 11and imidazole 12, have contributed to the activities of these derivatives, as these moieties are actual pharmacophores. Therefore, these derivatives may be considered molecular hybrids due to the presence of the basic cephem nucleus and those pharmacophore moieties. Applying molecular hybridization approach 13-20 on cephalosporins may provide better activities, stability against β-lactamases, and, hopefully, better pharmacokinetic properties. These L-dipeptides-CZX derivatives may also provide better penetration into soft tissues or tissues that have never been penetrated21-25. The results are summarized in Table 5.


 

Table 5: The antimicrobial evaluation of the L-dipeptide-CZX derivatives on E. Coli and MRSA.

Compound

Description

Microbe

Concentration  (µg/ml)

Zone of Inhibition (mm diameter)

500

250

125

60

30

10

CZX Na+

Ceftizoxime sodium

E. Coli

30

25

25

25

25

25

1

(L-Trp-L-Val)-Ceftizoxime

40

30

25

25

20

15

2

(L-Trp-L-Ala-) Ceftizoxime

36

32

30

25

25

16

3

(L-Trp-L-His)-Ceftizoxime

40

36

30

25

25

20

4

(L-Trp-L-Phe)-Ceftizoxime

36

34

30

25

20

20

CZX Na+

Ceftizoxime sodium

MRSA

20

20

20

20

20

20

1

(L-Trp-L-Val-)-Ceftizoxime

20

20

20

20

18

18

2

(L-Trp-L-Ala)-Ceftizoxime

30

25

25

25

20

20

3

(L-Trp-L-His)-Ceftizoxime

30

30

30

25

25

20

4

(L-Trp-L-Phe)-Ceftizoxime

35

30

25

25

18

15

 


Characterization of the L-dipeptides-Ceftizoxime derivatives by Swiss ADME Server:

The Swiss ADME server analyzed the synthesized derivatives and recorded the ADME properties 17,26-30. One of the Swiss ADME investigations included Lipinski's rule of five, which relates to the features of drugs that must be possessed to be passively orally absorbed31-36. Since the Lipinski rule of five cannot be applied to these novel L-dipeptide-CZX derivatives due to their large molecular weights, as shown in Table 6, passive oral absorption is not expected37-43.

 

Table 6: Violation of Lipinski rule.

Compound

Violation of Lipinski Rule

No. of H- donor

No. of H- acceptor

CZX

0 violation

2

7

1

2 violations: MW>500, N or O>10

5

10

2

2 violations: MW>500, N or O>10

5

10

3

3 violations: MW>500, N or O>10, NH or OH>5

6

11

4

2 violations: MW>500, N or O>10

5

10

 

However, these derivatives may serve as substrates for p-gp and refer to their capacity to be absorbed via transporter systems, particularly peptide transport systems.

L-dipeptide-CZX derivatives1-3 solely inhibits CYP3A4, while 4inhibits CYP2C9 and CYP3A4 subtypes44-45.   

 

CONCLUSION:

The designated target derivatives were synthesized smoothly and successfully with an acceptable yield. These derivatives had higher docking scores than ceftizoxime upon docking at different types of PBPs, which may refer to better antibacterial activity. A preliminary evaluation of the antibacterial activity of the synthesized derivatives against two significant bacterial species (MRSA and E. coli) 46-48showed higher antibacterial activities compared with CZX at higher concentrations and similar activity at lower concentrations.

 

The ADME investigation showed that none of the synthesized derivatives met or complied with the Lipinski rule, making these derivatives incapable of being passively absorbed. However, molecular docking data at the POT enzyme demonstrated that these derivatives are possible substrates for peptide transporters in the GIT since higher docking scores than ceftizoxime were recorded.

 

CONFLICT OF INTEREST

The authors have no conflicts of interest regarding this investigation.

 

ABBREVIATION:

1HNMR           Proton nuclear magnetic resonance

ADME            Absorption, Distribution, Metabolism and Excretion

Ala                  Alanine

BOC               Tert-butoxycarbonyl

CYP                Cytochrome p450 enzyme

CZX                Ceftizoxime

FT-IR              Fourier transform infrared

GIT                 Gastrointestinal tract

His                  Histidine

MIC                Minimum inhibitory concentration

MW                Molecular weight

Phe                  Phenylalanine

POT                Proton dependent oligopeptide transporter

Trp                  Tryptophan

Val                  Valine 

 

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Received on 16.09.2022           Modified on 23.03.2023

Accepted on 17.07.2023          © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):249-258.

DOI: 10.52711/0974-360X.2024.00039