World Journal of Pharmaceutical Research Rafik et al. World Journal of Pharmaceutical Research SJIF Impact Factor 5.990 Volume 4, Issue 9, 334-360. Research Article ISSN 2277– 7105 ANTIBACTERIAL ACTIVITY OF NOVEL PRODRUGS OF AMOXICILLIN AND CEPHALEXIN Rafik Karaman*1,2, Samia Al-Kurd1, Reem Yaghmour1, Ahmad Amro1, Donia Karaman1 1Bioorganic Chemistry Department, Faculty of Pharmacy, Al-Quds University, P. O. Box 20002, Jerusalem. 2Department of Sciences, University of Basilicata, Via dell‘Ateneo Lucano 10, Potenza 85100, Italy. ABSTRACT Article Received on 03 July 2015, Two novel prodrugs of amoxicillin and cephalexin (amoxicillin ProD 1 Revised on 27 July 2015, and cephalexin ProD 1, respectively) were designed and synthesized to Accepted on 22 Aug 2015 DOI:10.20959/wjpr20159-4478 improve the stability and bitter sensation of their parent drugs. The in vitro susceptibility for both prodrugs was determined against Escherichia coli, staphylococcus epidermidis, staphylococcus aureus, *Correspondence For Author Klebsiella pneumonia, streptococcus group A and streptococcus group Dr. Rafik Karaman B, and was compared to that of their active parent drugs.The Bioorganic Chemistry antibacterial screening demonstrates that amoxicillin ProD 1 and Department, Faculty of cephalexin ProD 1 were found to be active and are considered among a Pharmacy, Al-Quds small number of prodrugs that have therapeutic activity themselves University, P. O. Box 20002, Jerusalem. before undergoing interconversion via enzymatic or chemical reaction to their corresponding active parent drugs. Both prodrugs exhibit their antibacterial activity against different types of bacterial strains due to the presence of β- lactam ring in their structures. In addition, it is expected that these novel prodrugs will be much more stable in aqueous media than their corresponding active parent drugs due to the fact that the chemically sensitive amine group contained in the active parent drug structures is replaced with an amide, more chemically stable group, in the corresponding prodrugs. KEYWORDS: Antibacterials, amoxicillin, cephalexin, prodrugs, bitter sensation, cleavage. www.wjpr.net Vol 4, Issue 09, 2015. 3 3 4 Rafik et al. World Journal of Pharmaceutical Research 1. INTRODUCTION 1.1 Historical Background Infectious diseases are as old as lifetime itself. In 1910, Ehrlich synthesized salvarsan for treatment of syphilis to become the first antimicrobial drug in the world. In 1929 Fleming observed that bacterial growth was inhibited in the presence of Penicillium notatum. This observation makes penicillin the first broad antibiotic used in 1940s and led to its broad use during World War II. In 1935 Domagk developed sulfonamides, followed by the discovery of quinolones (e.g. Ciprofloxacin) in 1962 and oxazolidinones in 1979. [1-3] Figure 1 shows the timeline discovery of antibiotics with natural and synthetic origins. PP hheennyyllpprrooppaannooiiddss AAmmiinnooggllyyccoossii cchhlloorraammpphheenniiccooll ddee 22nnddcceepphhaalloossppoorriinn 11994466 SSttrreeppttoommyycciinn 11996622 11994466 --11995500 CC hhlloorraammpphheenniiccooll DDaappttoommyycciinn 22000033 MMaaccrroolliiddee CC eerryytthhrroommyycciinn11996622 11993300 11994400 11995500 11996600 11997700 11998800 11999900 22000000 PPeenncciilllliinn 11992299--11994411 QQuuiinnoolloonneess1199 OOxxaazzoolliiddoonneess.. 6600--11996622 LLiinneezzoolliidd 11999977-- 22000000 TTeettrraaccyycclliinn GGllyyccooppeepp11ttii00dd aassee BBeettaa llaaccttaamm ee11994499 vvaannccoommyycciinn 11995566--11997766 ccaarrbbaappeenneemmss 11997766--11998855 11995566 Figure 1. Timeline discovery of antibiotics[3] 1.1 Discovery of Penicillin Sanderson and Robert independently noted that bacterial growth was prevented in the presence of fungi.[4] The same observation was made by Tyndall in 1876 upon surmising the antagonism of bacterial growth due to the low oxygen level, which presumably was consumed by fungi. The first in vitro work was done by Cornil and Babes. Both scientists assessed the microbial inhibition and antagonism and explained this observation as a substance produced by one microorganism that may serve as an antagonist for the growth of another.[4, 5] www.wjpr.net Vol 4, Issue 09, 2015. 3 3 5 Rafik et al. World Journal of Pharmaceutical Research In 1887 Garre observed that the staphylococcus pyogens growth was inhibited in the presence of Bacillus fluorescence. Another observation which was noted by Duschesne in 1897 is the antagonism between Penicillium and Escherichia bacteria. In 1941, Waksman named these observations as antibiosis.[4] The true story began in 1928 by Fleming who observed an accumulation of staphylococcus aureus culture plates on one edge of his laboratory board and a colony of mold growing on the other side of the plate where Staphylococcus aureus around this area disappeared. Fleming was interested in this observation and he sub-cultured the mold and studied it. The culture of the mold was in nutrient broth and was for a period of eight days at room temperature. Fleming noticed that there was complete inhibition of growth of many bacteria. This fluid was first called mold juice and later Fleming named the active substance ―penicillin‖.[6-8] During this time period, Clutterbuck, Lovell and Raistrich extracted the active compound from the mold. They found that a pure compound could be separated by ether and watery acidic medium extractions. Upon evaporating the ether they recognized that the activity of the compound was diminished which led them to conclude that the active ingredient (penicillin) is unstable compound in acidic aqueous medium.[7] 1.. β- lactam Antibiotics Structure Structurally, β-lactam antibiotic molecules contain β -lactam nucleus, 6-amino penicillanic acid (6-APA) or 7-amino cephalosporinic acid (7-ACA), which provide a key for synthesis and modification (Figure 2). H H N S 2 H H N 2 S N O N O O O O O HO HO 7-Aminocephalosporanic acid 6-Aminopenicillanic acid Figure 2. Chemical structures of 6-aminopenicillanic acid and 7-aminocephalosporanic acid. www.wjpr.net Vol 4, Issue 09, 2015. 3 3 6 Rafik et al. World Journal of Pharmaceutical Research Novel β-lactam agents can be synthesized by linking a unique side chain to 6-APA. Early work by Sheehan produced penicillin V by acylation of 6-APA. Thereafter, in 1960 methicillin was approved in the United States and became the first semisynthetic penicillin which is stable to enzymatic degradation, especially to penicillinase enzyme. In addition, in 1967 carbenicillin was produced as semisynthetic compound by adding a carboxyl group instead of the amino group of ampicillin. Abraham and Newton isolated a new family of β-lactam antibiotics from Cephalosporium acremonium called cephalosporin C which contains 7-ACA nucleus instead of 6-APA in penicillin.[5] Chemical modification on β-lactam antibiotics provided many semisynthetic compounds. For example, various salts or esters of penicillin such as procaine and bezathine were synthesized and used for intramuscular injection due to their poor solubility in water. The reactive β-lactam ring present in this class of antibiotics made these agents unstable and very labile. Therefore, a variety of modifications on the nucleus led to changes in their chemical properties such as an increase in their stability in acidic and basic media, a decrease in their degradation by enzymes and a broader spectrum of activity.[9] Penicillanic acid (Figure 2) represents the core structure of penicillin which upon conversion to its Na+ or K+ salts provides soluble compounds and upon substitution with benzathine gives insoluble agents. The most important modification in the structure of the core is on the R group (Figure 3) because the β-lactam ring‘s reactivity and stability depend on the side chain substitution. This is essential for the action of β-lactam antibiotics to act as anti- bacterial agents. The first semisynthetic modification was changing the side chain R in penicillin G with other side chains such as in phenoxyethyl, phenoxymethyl, where the β-lactam ring is less reactive to H+ due to the change of the electron distribution and a creation of more stable entities. In the basic chemical structure of cephalosporin which has a basic structure as penicillin, but it has six-member dihydrothiazine ring instead of the thiazolidine ring in penicillin both R 1 and R provide opportunity for essential side chains modifications which result in changes of 2 different properties of the agents (Figure 3). www.wjpr.net Vol 4, Issue 09, 2015. 3 3 7 Rafik et al. World Journal of Pharmaceutical Research 6-Amino group Thiazolidine ring O H H N S R N O -lactam ring O HO Pinicillins 6-Amino group Dihydrothiazine ring H H O N S R2 N R1 O HO O -lactam ring Cephalosporins Figure 3. Penicillin’s and cephalosporin’s core structures, where the R, R and R 1 2 groups are variable.[1] The main entity contained in both structures, penicillins and cephalosporins, and essential for the antibacterial activity is the β-lactam ring. This entity interacts with active sites in the bacteria and produces the desired antibacterial effect. This happens when C—N bond in the β-lactam opens and binds to a carbon atom in the bacteria‘s site of action by a covalent bond, resulting in an acylation of an important group needed for cell wall synthesis.[1] 1.4 Mechanism of Action Cell wall in bacteria is an important structure in both Gram positive and Gram negative bacteria because of stress bearing and shape maintaining function.[10] It is a complicated structure which is composed of multiple types of polymers, peptidoglycans, teichoice acid and lipopolysaccharides. However, the most important among these is peptidoglycan because it is essential for cells living under normal growth conditions. Transpeptidase enzyme interacts with the peptide linkage contained in the pentapeptide chain of the uncrossed linked peptidoglycan (terminal D-alanine). This interaction results in D– alanine release and an acyl enzyme intermediate formation. Penicillin behaves like terminal D-alanine in the pentapeptide chain. The CO-N bond in β- lactam structure crosses bond to the peptide bond during trans-peptidation. Thus new www.wjpr.net Vol 4, Issue 09, 2015. 3 3 8 Rafik et al. World Journal of Pharmaceutical Research transition state is formed and peptide bond is cleaved; when the enzyme cleaves the β-lactam ring forms a stable pencicilloyl-enzyme complex resulting in an inhibition of the transpeptidase enzyme.[11, 12] 1.5 Amoxicillin In 1972 amoxicillin was synthesized in the UK. It has the same activity as ampicillin, but with higher bioavailability.[13] Later a combination of amoxicillin with clavulanic acid was developed to introduce better oral bioavailability and broad spectrum activity against a variety of pathogens that produce β-lactamase enzyme.[14] As amoxicillin acts on cell wall of bacteria; it has bactericidal action against both gram positive and gram negative. Amoxicillin is used for many indications; treatment middle ear infection [15] laryngitis, bronchitis, pneumonia,[16] and typhoid fever.[15] Amoxicillin is the most commonly prescribed antibiotic for children, it is well absorbed after oral administration, used for treatment in a variety of infections not only for broad spectrum also for outstanding advantage compared to other penicillins with higher bioavailability of 70-90%, and reaches C within 1-2 hours. [15] Amoxicillin is widely distributed in the body max and the apparent volume of distribution is 0.26 - 0.31mL/kg, it has half-life 1-1.5 hours. [17] It is excreted by the renal route and approximately 10-25% of the drug is bio-transformed into penicillanic acid. [15] 1.6 Cephalosporin Cephalosporins are related to penicillin β-lactam antibiotics; they act on cell wall of bacteria; interfering and lysing bacterial cell wall. This action is achieved by drug‘s crossing and binding to penicillin binding protein in the bacteria‘s cell wall (site of action).[18] Cephalosporin has no activity against enterococcus due to low affinity on penicillin binding protein. However, it has different activities against Pseudomonas aeruginosa and Enterobacteriacea, because of differences in binding on the active site located on the bacteria‘s cell wall.[19] Structure activity relationship and differences in side chain substitution at C7 position of the main core of cephalosporins can lead to various pharmacokinetics properties, spectra of activity, and β-lactamase stability. www.wjpr.net Vol 4, Issue 09, 2015. 3 3 9 Rafik et al. World Journal of Pharmaceutical Research It was reported that alteration of the substituent on C7 by the addition of methoxy group (cephamycin) or replacing the sulfur in dihydrothiazine ring with oxygen (moxalactam) leads to an increase in stability against enzymatic hydrolysis by β-lactamase.[18] Conventional oral suspensions and solutions of antibiotics dispensed as powders need to be reconstituted with water at the time of use. The reconstitution process of penicillins and cephalosporins allows acceptable but short life of the antibacterial agent with storage in refrigerator.[20] The highly strained β–lactam ring that presents in both penicillin and cephalosporin structures is unstable in solution; hydrolysis occurs and as a result the antibacterial agent loses its activity. The degradation process is an irreversible chemical change in the organic molecular structure of the antibacterial agent.[21] The degradation of penicillin can occur in different conditions; acidic or alkaline, in the presence of weak nucleophile as water and β-lactamase enzyme. Therefore, methods to increase the stability of penicillins and cephalosporins are crucially needed. The palatability of the active ingredient of a drug is a significant obstacle in developing a patient friendly dosage form. Organoleptic properties such as taste are an important factor when selecting a certain drug from the generic products available in the market that have the same active ingredient. The problem of the bitter taste of drugs in pediatrics and geriatrics formulations still creates a challenge to pharmacists. Thus, different strategies should be developed in order to overcome this serious problem.[22-27] In the past few years we have been engaging in studying intramolecularity and concluded that there is a need to research the mode and action by which intramolecular processes proceed in order to utilize them in the design of novel prodrugs. Unraveling the mechanism of intramolecular processes such as enzyme models would open the door widely for a precise design of chemical devices to be exploited as promoities to be covalently attached to commonly used drugs for providing prodrugs with better bioavailability and less adverse effects than their corresponding active parent drugs. Among the intramolecular processes (enzyme models) that we have calculated using quantum mechanics and molecular mechanics methods are: (1) proton transfer between two oxygens and proton transfer between nitrogen and oxygen in Kirby‘s enzyme model; (2) acid- www.wjpr.net Vol 4, Issue 09, 2015. 3 4 0 Rafik et al. World Journal of Pharmaceutical Research catalyzed hydrolysis in Kirby‘s N-alkylmaleamic acids; (3) proton transfer between two oxygen atoms in Menger‘s rigid hydroxy-acids; (4) acid-catalyzed lactonization of hydroxy- acids as investigated by Cohen and (5) cyclization in dicarboxylic semi-esters as researched by Bruice and Pandit. Prodrugs in which the above mentioned enzyme models were utilized as linkers which covalently are attached to drugs having poor bioavailability or/and bitter sensation were designed and synthesized. The controlled (programmed) intraconversion rates of the novel designed prodrugs to release their active parent drugs are solely determined on the structural features of the linker and there is no need to an involvement of metabolic enzymes.[28-64] For example, unraveling the mechanism for the intramolecular proton transfer in Kirby‘s acetals.[65-73] revealed a design and synthesis of novel prodrugs of aza-nucleosides for the treatment of myelodysplastic syndromes,[74] and statins to lower cholesterol concentration in the systemic blood circulation.[75] In these cases, the prodrug linker was covalently linked to the hydroxyl group in the active drug such that the prodrug has the capability to undergo a chemical cleavage upon reaching a physiological environment such as stomach, intestine, and/or blood circulation, with rates that are determined only by the structural features of the pharmacologically inactive linker (Kirby‘s acetal). Kirby‘s N-alkylmaleamic acids enzyme model.[65-73] was also studied as linkers in the design of tranexamic acid prodrugs for treating bleeding conditions [76] acyclovir prodrugs for the treatment of Herpes Simplex,[77] and atovaquone prodrugs as antimalarial agents.[78-80] The intramolecular proton transfer in Menger‘s Kemp acid enzyme model.[81-85] was also explored and used for the design of dopamine prodrugs for Parkinson‘s disease cases. [86] In addition, dimethyl fumarate prodrugs for treating psoriasis cases were also designed and developed..[87] The novel prodrugs approach was also applied for masking the bitter sensation of the pain killer paracetamol, the anti-hypertensive agent atenolol, the decongestant phenylephrine, the anti-inflammatory agents, diclofenac and mefenamic acid and the bitter antibacterials cefuroxime, amoxicillin and cephalexin.[88-95]The role of the linker in the antibacterial prodrugs is to block the amine or/and hydroxyl groups which are believed to be responsible for the drug bitter sensation. The difference between the designed antibacterials prodrugs and their active parent drugs is that the free amine moiety in the active drug is replaced with an amide group. Replacing the amine with an amide eliminate the capability of the antibacterial www.wjpr.net Vol 4, Issue 09, 2015. 3 4 1 Rafik et al. World Journal of Pharmaceutical Research to form hydrogen bonds with the bitter taste receptor, thus masking the bitter sensation of the parent antibacterial drug. Based on DFT calculations and experimental values obtained from intramolecular acid catalyzed hydrolysis in nine N-alkylmaleamic acids, we have designed and synthesized two prodrugs of amoxicillin (amoxicillin ProD 1) and cephalexin (cephalexin ProD 1) by reacting the antibacterial agent with a maleic anhydride linker (Figure 4) aiming to: (1) improve the stability and aqueous solubility of the antibacterial agent (2) provide antibacterial agents lacking bitter sensation. The two synthesized prodrugs were designed such that the amine group in the active parent drugs is replaced with the more stable amide group. In this manuscript we report the antibacterial spectrum of two novel prodrugs, amoxicillin ProD 1 and cephalexin ProD 1. O O NH2 H H OH N S HN O O HO O N HN H S O O OH HO N O O O OH Maleic anhydride O Amoxicillin Amoxicillin ProD 1 O O NH2 H H N S OH O HN O O N H H O N S O O OH O N O O OH Maleic anhydride Cephalexin Cephalexin ProD 1 Figure 4. Synthesis of amoxicillin ProD1 and cephalexin ProD 1. 1.7 Antimicrobial Activity of Amoxicillin and Cephalexin 1.7.1 Amoxicillin Activity Penicillins have been divided into classes based on their spectrum of activity; the first agent that was used clinically to treat infections is the natural penicillin (penicillin G), but after the www.wjpr.net Vol 4, Issue 09, 2015. 3 4 2 Rafik et al. World Journal of Pharmaceutical Research emergence of penicillinase in staphylococci penicillins became inefficient for these organisms. Therefore, development of penicillinase resistant-penicillins was initiated; this led to the development of three categories of penicillins: the aminopenicillins, carboxypencillins and ureidopencillin.[96] Aminopenicillins was the first class of penicillin antibiotic that has activity to both gram positive and gram negative bacteria; ampicillin compared to natural penicillin has more activity against enterococci, but somewhat less activity against pyogens, streptococcus pneumonia, and Neisseria species. On the other hand, it has some activity against E.coli, proteus Mirabella, salmonella, shigella, listeria, which are gram negative bacteria.[96] Amoxicillin has shown to be effective against a variety of infections, which are caused by gram positive and gram negative bacteria in humans and in animals. [97] Amoxicillin has a higher activity against gram positive than gram negative microorganisms.[98] In addition, it has greater efficacy relative to penicillinV and other antimicrobial such as ampicillin [15] and cefuroxime.[99] Different study reports showed that amoxicillin was effective at MIC in the range of 0.06 μg/mL- 4 μg/mL against variety of microorganism, except staphylococcus .epi 64 μg/mL and staphylococcus aureus MIC up to 256 μg/mL.[100] In one study, amoxicillin and ampicillin showed that the kill rates for amoxicillin was higher than ampicillin for E.coli, and the rate of killing was the same for both agents for Staphylococcus Aureus, but amoxicillin showed longer bacteriostatic phase which was not observed with ampicillin.[15] In another study an investigation on the antibacterial activity of amoxicillin and ampicillin against 30 isolates of each of proteus mirabilis, Klebsiella, E.coli, Enterobacter and idol positive proteus was carried out, and the results obtained are as follows: 89% of the E.coli strains were inhibited by both drugs at 10 μg or less per mL, whereas only 5μg or less were sufficient in the case of Proteus. mirabilas. On the other hand, high response of resistance to amoxicillin and ampicillin was seen among strains of Klebsiella, enterobacter and idol positive species.[101] In addition, other studies have demonstrated that amoxicillin was quite active against group A hemolytic streptococci, penicillin G susceptible staphylococcus aureus and pneumococci, www.wjpr.net Vol 4, Issue 09, 2015. 3 4 3