Quinolones: Synthesis and Antibacterial Activity

After the concept of selective toxicity in chemotherapy was introduced at the beginning of the 20th century, (Ehrlich, 1913), classes of substances with antibacterial properties, produced by microorganisms or created through synthesis were obtained. After the discovery of penicillin, the first antibiotic introduced in clinical use in man in 1940s, a large number of different types of antibiotics were produced. Antibiotics such as beta-lactams, macrolides, aminoglycozides and tetracyclines were discovered and introduced during an extremely short period. These were obtained either by isolation from fungi or by chemically modification of the naturally isolated substrates. These dominated the antimicrobial industry, while synthetically obtained substances only played a minor role. (Chu & Fernandes, 1991)


Introduction
After the concept of selective toxicity in chemotherapy was introduced at the beginning of the 20 th century, (Ehrlich, 1913), classes of substances with antibacterial properties, produced by microorganisms or created through synthesis were obtained.After the discovery of penicillin, the first antibiotic introduced in clinical use in man in 1940s, a large number of different types of antibiotics were produced.Antibiotics such as beta-lactams, macrolides, aminoglycozides and tetracyclines were discovered and introduced during an extremely short period.These were obtained either by isolation from fungi or by chemically modification of the naturally isolated substrates.These dominated the antimicrobial industry, while synthetically obtained substances only played a minor role.(Chu & Fernandes, 1991) In 1962, G. Y. Lesher and his collaborators introduced the first quinolone derivative, nalidixic acid (1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxilyc acid), (1, Lesher et al. 1962) which had moderate activity against gram-negative organisms and was used for treating urinary tract infections.In the following years, a large gamma of derivatives from common elements were synthesized, which could be grouped by: cinoline (cinoxacin), pyrido-pyrimidine (pipemidic acid; piromidic acid), naphthyridine (nalidixic acid) and quinolones (oxolinic acid, miloxacin , tioxacin, etc.).These derivatives, with differentiated structures, have 2 common pharmacological properties, which allowed them to be classified as first generation biologically active derivatives with quinolone structure.The two common characteristics for first generation quinolones are: a narrow antibacterial spectrum, designed especially for enterobacteriaceae; -a pharmacokinetic which allows for rapid elimination and reduced tissue absorption, only allowing them to be used as urinary antiseptics.The success of first generation quinolones spurred the research in this area, which led to the obtainment through synthesis, after 1980, of a new series of compounds with stronger antibacterial properties and a broader spectrum of antibacterial activity which included gram positive and gram negative organisms, and which where defined by their ability to be applied on all localized infections.Koga and his collaborators introduced Norfloxacin into clinical use in 1980, the first quinolone with a fluorine atom substituted at the C-6 position and a piperazine C-7.Norfloxacin (Koga et al. 1980) was the first quinolone with increased antimicrobial activity, acting on a large spectrum of gram positive and gram negative microorganisms, including Pseudomonas aeruginosa.Until now a large number of antibacterial substances belonging to the above mentioned class have been used in medicine.Quinolones are used when treating infections of the urinary tract, the respiratory tract, intestinal infections, ear/nose/throat infections, STD's, soft tissue and skin infections, meningitis caused by gram negative and Staphilococci bacteria, liver and bile infections, septicemia and endocarditis, prophylaxis and surgical infections and on patients with immune deficiencies.
The mechanism of action of quinolone antibacterial agents involves the inhibition of DNA gyrase (a bacterial topoisomerase II) resulting in a rapid bactericidal effect.
The antibacterial activity of quinolones (measured in terms of MIC), however, is the result of the combination of bacterial cell penetration and DNA gyrase inhibitory activity.The antibacterial activity of quinolones depends not only on the bicyclic heteroaromatic system combining the 1,4-dihydro-4-pyridine-3-carboxylic acid moiety and an aromatic ring, but also on the nature of the peripheral substituents and their spatial relationships.These substituents exert their influence on bacterial activity by providing additional affinity for bacterial enzimes, enhancing cell penetration or altering the pharmacokinetics.
The research for an ideal quinolone continues worldwide.Such a quinolone must be biologically active on a large spectrum of gram positive and gram negative bacteria, aerobes and anaerobes and mycobacteria, must have as few side effects as possible, excellent solubility in water and oral bioavailability.
In figure 5, the most common synthesized chemical variations obtained during the research for new quinolones with antibacterial activity, are visible.

Structural features
Quinolone derivatives are an important class of antibacterial agents with wide action.Basic structure of these compounds (Figure 6) is a bicyclic structure, which contains a ring of type A 4-piridinona combined with aromatic or heteroaromatic ring B. The ring type A 4piridinona is a ring with absolute necessity: an unsaturation in position 2-3, a free acid function in position 3 and a substituent at nitrogen in position 1.

Position 1
The studies on quinolones indicated that in order for the compound to have antibacterial activity, the N-1 position requires a substituent.Many quinolones contain in N-position : ethyl (norfloxacin, pefloxacin, lomefloxacin) , fluoroethyl (fleroxacin), vinyl, clloroethyl, trifluoroethyl, aminoethyl,, cyclopropyl (ciprofloxacin), t-butyl, bicyclopentyl,pfluorophenyl,2,4-difluorophenyl (Scott 1997) Position 2 Quinolones contain at C-2 hydrogen (R 2 =H).The replacement at hydrogen has generally proven to be disadvantageous.However, some compounds containing a suitable C-1, C-2 ringhave recently been shown to possess biological activity.(Figure7 -Segawa 1992) (Figure 8 Position 3 The C-3 carboxylic acid moiety is most commonly encountered.(Chu &Fernandes1991) .In the late of 1980s, a modification was described that eliminated the need for C-3 carboxylic acid.A fused izothiazolone ring was identified as serving as a carboxylic acid mimic,The compound A-62824 (Figure 9) have been found with biological activity.

Position 4
The C-4 oxo group of the quinolones nucleus is generally considered to be essential for antibacterial activity.

Position 5
The choice of the C-5 substituent appears to be dictated by the the steric regulations and the nature of the N-1 and C-8 substituent (Chu &Fernandes1991).(R 5 = methyl, halogen, amino when X = CF).

Position 6
The nature of the C-6 substituent have a great impact on the DNA-gyrase inhibitory activity and cell penetration.(Domagala et al. 1986).The R 6 can be H, Cl, F, NO 2 , NH 2 , CN, CH 3 SCH 3 , COCH 3 ) (Koga et al. 1980) Position 7 The choice of the C-7 substituent is a key issue which continues to guide the design of new antibacterial quinolones.The R 7 can be substituted/unsubstituted piperazines, aminopyrrolidines, aminoalkylpyrrolidines, (Figure 5) (Chu & Fernandes 1991) (Scott 1997).

Method requiers the reaction of isatoic anhydride with sodium ethyl formyl acetate
Another synthesis method requiers the reaction of isatoic anhydride with sodio ethyl formyl acetate (Figure 13).2,4,5-trihalobenzoic acid (XX) is reacted with an appropiate amine, and then is treated with the compound :R 2 R 3 CO (R 2 = R 3 = Cl, CCl 3 O or R 2 = C 1-10 alkyl and R 3 = Cl) to produce benzoxazindione (XXII).The benzoxazindione (XXII) is then condensed with compound (HOCH=CHCO 2 Et) to provide key compound (V).CO V VI VII Fig. 13.Method requiers the reaction of isatoic anhydride with sodium ethyl formyl acetate
Treatment with a base induces cyclization to produce the quinolone (V).

Intramolecular nucleophilic displacement cyclization route to quinolones (b)
A synthesis method similar to that described above is shown in Figure 15.This method involves intramolecular cyclization of the compound (VIII).(Egawa et al. 1987).

Antibacterial activity of the new compunds
The new compounds were evaluated for "in vitro" activity by determining minimum inhibitory concentration against of bacteria Escherichia.Coli, Staphylococcus.Aureus and Pseudomonas .aeruginosa,by agar dilution method (Buiuc 1998) (NCCLS 2003).(  Table 2. "In vitro" antibacterial activity of the new quinolones.

Fig. 3 .Fig. 4 .
Fig. 2. Second-generation quinolones.Research in the field of derivatives with a quinolone structure have lead to new compounds obtained recently, which have been classified as third and fourth generation systemic quinolones, largely effective against Staphilococcus aureus.Their large antibacterial spectrum includes anaerobes, Chlamydia and Mycoplasma.(Brighty & Gootz, 2000)