INTRODUCTION:

Literature survey reveals that quinolones found to possess various wide spectrum of activities like anti-bacterial, anti-malarial, anti-tumor, anti-tubercular. Quinolones are also referred as fluoroquinolones, quinolone carboxylic acids and 4-quinolones.These drugs are the analogs of the earlier developed agent nalidixic acid. Nalidixic acid was discovered by Lesher¹ in early 1960s from a distillate during chloroquine synthesis Figure 1. Nalidixic acid and all quinolone agents are pure synthetic compounds and no structurally related compound has yet been identified as product of living organisms. More than thousand quinolone analogs have been synthesized so far.

With the introduction of oxolinic acid and cinoxacin the drugs showed improved activity against gram-negative bacteria. Another compound, viz. pipemidic acid, synthesized around the same time had limited activity against Pseudomonas aeruginosa². The broad-spectrum antibacterial activity has been found in the class of quinolone compounds via fluorination at C-6 position which gives a new class of compounds commonly known as fluoroquinolones².

In the late 1980, fluoroquinolone compounds have been successfully used for the treatment of urinary tract infections, including those caused by P. aeruginosa. Norfloxcin was the first quinolone antibacterial with the substitution of fluorine atom at the C-6 and piperazine group at the C-7 position, which led to further generations of fluoroquinolones which had a wider range of antibacterial activity.

Figure 1: Sequential landmarks in the development of fluoroquinolones.

General structural features of the quinolones:

The fluoroquinolones are synthetic derivatives of the basic structure of the first quinolone viz. nalidixic acid. Several consistent modifications in the development of the structure-activity relationships (SAR) have led to synthesized various new large numbers of compounds. Many of them also show potent activity against anaerobes and pathogens that are resistant to many other groups of antimicrobials³.

On the basis of structure, quinolones are broadly classified into two types: the true quinolone moiety (1) containing a bicyclic aromatic core containing carbon atom at the C-8 position while the naphthyridone ring (2) possesses nitrogen at the same position. The presence of the pyridone ring, a carboxylate group at the C-3 position and the ketonic functions at C-4 are essential for the antibacterial activity.

It is observed that a fluorine atom at C-6 position and a piperazine ring at C-7 position greatly enhance the spectrum of biological activity of these compounds. These compounds are much more active against aerobic gram-negative microorganisms but less active against gram-positive ones and hence are extremely useful for treatment of a wide variety of infections.

Historical development in the discovery of quinolone antibacterials shown in Figure 2.

Classification:

It has been recognized that there are several ways to categorize quinolones on their chemical structure, nature and their spectrum of activities. However, most convenient classification of quinolone compounds is on the basis of their historical development. Such classification is clearly arbitrary. The classification provided is based on their antibacterial spectrum and their physicochemical properties⁴. The fluoroquinolones are synthetic derivatives of nalidixic acid that display broad-spectrum anti-bacterial activity including anti-mycobacterial activity⁵. Modifications of fluoroquinolones structure for optimization of more potency against mycobacterial infections and development of the structure-activity relationships (SAR) in the fluoroquinolones have led to synthesize a large number of new quinolone compounds. Presently, more than 10,000 structurally related agents have been synthesized which exhibit improved activity against gram-positive pathogens compared to ciprofloxacin⁶.

Figure 2: Historical development in the discovery of quinolone antibacterials.

A. First generation quinolones:

The early experimentation on original nalidixic acid structure (1) (Figure 3) developed the first generations of quinolone derivatives. In that generation, all of these quinolones retain a nitrogen atom at the C-1 position while modifying the naphthyridone structure of nalidixic acid returning to the quinolone nucleus. Compounds belonging to this generation are oxolinic acid (2), cinoxacin (3), pipemidic acid (4), pirodimic acid (5), and flumequin (6). These compounds attain high concentrations in the urinary tract and hence are therapeutically useful for the treatment of urinary tract infections⁷. Cinnoxacin and nalidixic acid require more frequent dosing than the newer quinolones, since they achieve minimum serum level. Consequently they are more susceptible to the development of bacterial resistance.

B. Second generation quinolones:

The second generations of quinolones were synthesized so as to increase the efficacy of compounds against Gram-positive bacteria while maintaining the effect on Gram-negative microorganisms. In that generation include norfloxacin (7), pefloxacin (8), enoxacin (9), fleroxacin (10), lomefloxacin (11), ciprofloxacin (12), ofloxacin (13), rufloxacin (14), (Figure 4) which have contain fluoro substitution at the C-6 position and piperazine ring at the C-7 position⁷.

Figure 3: First Generation of Quinolones.

Figure 4: Second Generation of Quinolones.

C. Third generation quinolones:

Third generation quinolones includes gatifloxacin (15), levofloxacin (16), pazofloxacin (17), grepafloxacin (18), sparfloxacin (19), and tosufloxacin (20) (Figure 5) compounds. Those having increased potency against staphylococcus aureus as well as an expanded spectrum, including anaerobic bacteria, Chlamydia and mycoplasma.

D. Fourth generation quinolones:

In that generation of quinolones includes clinafloxacin (21), trovafloxacin (22), and moxifloxacin (23) (Figure 6). These new quinolones have significant antimicrobial activity against anaerobes and pneumococcus while maintaining the gram-positive and gram-negative activity as well as activity against Pseudomonas species⁷.

Figure 5: Third Generation of Quinolones.

Figure 6: Fourth Generation of Quinolones.

Quinolone pharmacophore and functional domain:

Molecular structure of quinolone is structurally divided into certain domains depending upon their functions. The basic unit consists of the pyridone ring and a carboxyl group .In this unit the pyridone nitrogen (N-1) is the integral part, the groups attached though can be varied, the carboxyl at C-3 position is replaceable by a fused thiazolidone bioisoster, while very few C-4 variations have been reported (Figure 7).

The organic chemists have been successively trying to enhance the biological activity of quinolones against Gram-positive as well as Gram-negative bacteria by varying the substitutions at different positions like N-1, C-2, C-3, C-5, C-6, C-8 and primarily at C-7 with small groups like H, CH₃, F, cyclopropyl etc. to bulky moieties like benzoxazines.

It has been suggested that the quinolones have an auto-assembling domain, which forms a bioactive tetraplex with the DNA gyrase on the substrate DNA during the strand cleavages. Quinolone moiety can be divided in four major regions. The upper margin or the “northern front” of the quinolone consisting of pharmacophoric carboxyl group at C-3 and the ketone at C-4 is complementary to the DNA bases exposed and is suitable for hydrogen bonding with DNA. It is likely that certain substituents at C-5 may also contribute to this. It has been suggested that the substituents at C-2 must be small so as not to prevent the carboxyl group at C-3 from lining up in the plane of the quinolone ring as needed. The lower margin (southern front) of the molecule is considered to be a lipophilic and self-association region consisting of N-1 with its pendant substituents. The basic substituents at C-7 serve to orient the quinolone molecules so that carboxyl and protonated amino groups line up near each other in the adjoining quinolone molecules in the vertical and horizontal directions and hence are thought to contribute to their binding on the B subunit of the DNA gyrase (western front)⁸.

Figure 7: Structure activity relationship of quinolones.

Thus, the proposed model suggests three functional domains on the quinolone molecules: the DNA-binding domain, the drug self-association domain and the drug-enzyme interaction domain. Such functional domains for the quinolone derivatives provide a guideline for synthesizing improved quinolones with novel structures. For example, the substitution of groups with capabilities of generating amphoteric quinolones tends to yield more potent compounds.

Although the structure-activity relationships of fluoroquinolones have been extensively investigated, the optimum substituent at the C-7 position, which has a great impact on potency, spectrum, solubility, and pharmaco-kinetics, has not been precisely defined. The most extensively investigated substituents are piperazin-1-yl and its 4-substituted derivatives. For example, pefloxacin, the 4-methyl-norfloxacin, and other 4-substituted piperazin-1-yl prodrugs of norfloxacin were prepared to improve the bioavailability of the parent.

Mechanism of action

The fluoroquinolones have a rather unique mechanism of action for antimicrobial agents. They are known to interact with two related but distinct targets within the bacterial cell, viz DNA gyrase (or topoisomerase II) and topoisomerase IV⁹. Topoisomerase enzymes maintain cellular DNA in an appropriate state of supercoiling in both replicating and non-replicating regions of the bacterial chromosome. In the replication of DNA to occur the two strands of double-helical DNA must be separated. However, separation of the strands results in excessive positive supercoiling or over winding of the DNA in front of the separation point. This is avoided by the bacterial enzyme DNA gyrase, which is responsible for continually introducing negative supercoils into DNA in an ATP-dependent reaction. Both strands of the DNA are cut during this process thus allowing passage of a portion of the DNA through the break which is then resealed⁹.

Evidence on the quinolones targeting DNA topoisomerase IV has only been recently established¹⁰. This enzyme is also a bacterial II DNA topoisomerase but unlike the gyrase it cannot supercoil to DNA. Topoisomerase IV carries out the ATP-dependent relaxation of DNA and is a more potent decatenase than DNA gyrase. Thus, it helps separate the daughter DNA molecules after DNA replication. It is well accepted that fluoroquinolone inhibition of these two enzymes are important in their mechanism of action. Generally, fluoroquinolone apparently bind to a complex of the gyrase and the DNA, which stabilizes the complex leading to the lethal effect of these drugs. It leads to induction of DNA strand breaks and freezing of the replication fork or both¹¹. Complexes of DNA, quinolone and active topoisomerase IV appear to form physical barriers to DNA replication. However, both of these complexes appear to be insufficient by themselves breaking the double-strand of DNA which is thought to be necessary for quinolone produced bacterial lethality¹¹.

A topoisomerase that controls the supercoiling of the nucleic acid and is a product of the gyrA and gyrB genes exerts its influence on the antibacterial activity by providing additional affinity for the bacterial enzymes, enhancing cell penetration or altering the pharmacokinetics. The inhibitory activities of many of these enzyme are driven by ATP hydrolysis^12,13while subunit A of the gyrase enzyme is thought to be the direct target of these drugs (Figure 8).

Figure 8: Proposed quinolone-DNA cooperative binding model for DNA gyrase inhibition.

Therapeutic activities of quinolones:

A) Antibacterial activity:

Nalidixic acid was the first quinolone introduced in clinical practice in the early 1960s and it is still in use today for the treatment of urinary tract infections caused by gram-negative pathogens. Numerous analogues of this prototype have been synthesized and used as antibacterial agents. In the 1980s quinolones containing fluorine atoms were developed which were significantly more potent, had broader antibacterial spectra, and were easily absorbed upon administration. The major antibacterial effects of fluoroquinolones are considered to be mediated via binding to DNA gyrase and topoisomerase IV. The antibacterial activity of fluoroquinolones depends not only on the bicyclic hetero-aromatic pharmacophore but also on the nature of the peripheral substituents and their spatial relationship. It has been concluded that DNA gyrase is a primary target of fluoroquinolones in gram-negative bacteria such as Escherichia coli, Neisseria gonorrhoeae, and Klebsiella pneumonia whereas topoisomerase IV is the primary target in gram–positive bacteria such as Staphylococcus aureus, and Streptococcus pneumoniae^{14, 15}.

B) Antimalarial activity:

Quinolones were collected as byproducts during the synthesis of chloroquine, the lead antimalarial drug; it was therefore a logical step to check whether they exert any anti-parasitic activity. As expected, quinolones indeed exhibit effective antimalarial activity against chloroquine-sensitive as well as chloroquine-resistant P. falciparam¹⁶.Fluoroquinolones showed excellent inhibitory activity not only against erythrocytic stages of P. falciparum but also against blood stages and hepatic stages of Plasmodium yoelii and P.falciparam. Consequently some of the quinolone derivatives like Grepafloxacin, trovafloxacin, and ciprofloxacin exhibit IC₅₀ values even less than 10 µg/ml. Antimalarial potency of ciprofloxacin in vitro against P. falciparam is similar to travofloxacin, while norfloxacin is comparatively ineffective. It has been anticipated that fluoroquinolones may show increased efficacy on combination therapy.

In general, a limited use of quinolones in antimalarial drug therapy is possibly due to their low intraerthyrocytic concentration attainable, while tolerance of other antibiotics like tetracycline and azithralone seems to be more acceptable.

C) Antimycobacterial activity:

In this context some of the newer fluoroquinolones have demonstrated activity in vitro and in vivo against resistant infections¹⁷. Tuberculosis caused by M.tuberculosis, M.leprae, M.avium and atypical mycobacteria such as M. fortuitum respectively, level of susceptibility being dependant upon the mycobacterial species under study involved¹⁸. Use of fluoroquinolones for the clinical control of MDR-TB is advocated for several reasons. For example, there are no reports of cross-resistance or antagonism for fluoroquinolones with other classes of antimycobacterial drugs. Secondly, fluoroquinolones can be administered orally with good absorption and favorable pharmacokinetics including efficient penetration into the tissues and host macrophages. Finally, incidence and severity of adverse effects are generally low for most fluoroquinolones¹⁹.

Balasubramanian and coworkers have evaluated antimicrobial activity of some of the new generation of quinolone such as moxifloxacin, ofloxacin, sparfloxacin along with ciprofloxacin against M.tubercuosis H₃₇R_V in mice, wherein sparfloxacin was found to be five fold more potent than the other compounds²⁰.

Shindikar et al have synthesized novel fluoroquinolones with the substituents at 4^th position of piperazine being [-2(pyiridine-4-caronyl) hydrazone] and -2[(pyrazine-2-carbonyl) amino] ethyl and have evaluated their activity against M.tubercuosis H₃₇R_V in mice. These compounds exhibit activity comparable to that of sparfloxacin²¹.

D) Antitumor activity:

The major drawback of quinolones as therapeutic agents is their cytotoxicity while pursuing their use as antibacterials which, however, can be a desirable trait when developing them as antiproliferative agents. Since quinolones are found to accumulate in the urinary tract, their use as adjuvant therapeutic agents for treating tumors of the bladder and urinary tract is a logical step since the urinary concentration of these quinolones after oral administration is found to be high and they are found to be excreted unchanged.

The research groups of Chu et al. and Yamashita et al. independently demonstrated that antitumor quinolones have a closely related structure consisting of two halogens at C-6 and C-8 group and a cyclopropyl group at N-1 of the quinolone skeleton. Several fluoroquinolones are capable of being used as antitumor agents, and Clement et al. have developed a new quinolone compound that shows distinctive activity against murine and human tumors experimentally²².

Zehavi-Willner and Shalit reported that ciprofloxacin at a concentration of 100 mg/L significantly inhibited the proliferation of human bladder carcinoma cells (BS-5867)²³.

Earlier work has confirmed the inhibitory potential of ciprofloxacin in case of bladder tumour cells and has established the induction of cell cycle arrest at the S/G2-M checkpoint. It also shows a down regulation of antiapoptotic protein Bcl-2 resulting in alteration of Bax: Bcl-2 ratio in the hormone resistant prostate cancer PC-3 cell line and thus favoring apoptosis²⁴.

Additional target for the quinolone action is thought to be the telomerase enzyme, which is expressed in almost all malignant cells and not in human somatic cells. It is an enzyme that is thought to be responsible for unlimited cell proliferation in vivo and in vitro and its expression increases with increase in proportion to the grade of tumor malignancy. Ofloxacin and levofloxacin decreased telomerase activity within 24 hrs of culture incubation at 50 µg/ml concentrations²⁵.

Mukherjee and coworkers have observed that enoxacin (9) induced significant growth inhibition of MCF-7 cells with cell cycle inhibition in the G2/M phase, leading to inhibition of cell growth. The growth inhibition was dose dependent, time dependent and irreversible in nature, with alteration of cellular morphology in-vitro culture condition. Increase in population doubling time and decrease in saturation density were also observed in enoxacin treated cells. Thus, human breast cancer MCF-7cells are found to be highly responsive to fluoroquinolones antibiotic enoxacin treatment²⁶.

E) Anti-inflammatory activity:

In addition to the bactericidal activities, fluoroquinolones also show anti-inflammatory activity by modulating the production and secretion of cytokines. Ciprofloxacin inhibits the synthesis of tumor necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 in humanmonocytes²⁷.

Moxifloxacin inhibits IL-1α, IL-1β, IL-8 and TNF-α from LPS-stimulated but not pansorbin-stimulated monocytes, whereas it does not affect the production of IL-4, IL-6 or IL-12²⁸.

Grepafloxacin inhibits the protein synthesis of IL-1α, TNF-α and IL-8 and the expression of their mRNAs in LPS-stimulated monocytes, but it stimulates the synthesis of IL-2²⁹.

CONCLUSION:

From the above discussion and extensive literature survey it is concluded that recently developed quinolones have wider potential applications and a broader spectrum of activity as compared to earlier quinolones compounds. From the quinolones pharamcophore and function domain we can synthesize novel quinolones structure and improve its therapeutic activites. Newer quinolones exhibit promising the different therapeutic activities and hence should be studied further for mechanistic details.

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Received on 11.05.2010 Modified on 29.05.2010

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C) Antimycobacterial activity:

Zehavi-Willner and Shalit reported that ciprofloxacin at a concentration of 100 mg/L significantly inhibited the proliferation of human bladder carcinoma cells (BS-5867)23.

Zehavi-Willner and Shalit reported that ciprofloxacin at a concentration of 100 mg/L significantly inhibited the proliferation of human bladder carcinoma cells (BS-5867)²³.