Trabajo colectivo durante la construcción de los distritos de riego

During the synthesis of the antimalarial agent, chloroquine, chemists at Sterling-Winthorp laboratories isolated compound 1 as a by-product (Figure 3.1). Due to the antibacterial properties exhibited by compound 1 a series of derivatives were synthesised in an attempt to find a novel antibacterial agent.¹⁰⁹ In 1963, one of these derivatives, nalidixic acid, was introduced into the clinical settings for the treatment of UTIs (Table 3.1).^109-110 Since the discovery of nalidixic acid numerous quinolone derivatives have been developed, many of which have made it to the clinical setting.

Figure 3.1: By-product isolated during chloroquine production, compound 1.¹¹⁰ Quinolones are classified into generations based on their spectrum of activity against bacteria (Table 3.1). The first-generation quinolones include the naphythyridone derivative, nalidixic acid, and the dioxolane bearing compounds, cinoxacin and oxolinic acid. These compounds were active against Enterobacteria with cinoxacin and oxolinic acid exhibiting increased activity in comparison to nalidixic acid.¹⁰⁹ The introduction of a piperazine ring at C-7, as in pipemidic acid, gave rise to activity against P. aeruginosa.¹⁰⁹ In the 1980’s, the combination of the C-7 piperazine and a fluorine atom at the C-6 position resulted in the fluoroquinolone, norfloxacin (Table 3.1). In comparison to the first-generation quinolones, norfloxacin demonstrated improved anti-Gram-negative bacteria and some activity against Gram-positive bacteria.¹⁰⁹ Replacement of the N-1 ethyl group with a cyclopropyl group produced one of the most well-known fluoroquinolones, ciprofloxacin (Table 3.1).

Ciprofloxacin exhibited enhanced activity against both negative and Gram-positive bacteria and was the first quinolone to be used in the treatment of infections other than UTIs.¹⁰⁹

96 Table 3.1: Quinolone classification^109,111

Generation Quinolone Structure Activity Clinical application

1^st

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a Withdrawn due to adverse side effects.

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In the third-generation quinolones the introduction of a C-8 moiety has led to improved activity against Gram-positive bacteria.^109,111b The fourth-generation quinolones exhibit increased potency and a broader spectrum of activity, including anaerobic bacteria.^109,111b Currently under development are two quinolone compounds JNJ-Q2 and nemonoxacin (Table 3.1). These compounds exhibit activity against MDR S. pneuminiae as well as quinolone-resistant MRSA.111b,112-113

The quinolones are attractive antibacterial agents not only because of their broad spectrum activity but also because of their molecular target. Quinolone antibacterials work by interfering with bacterial DNA replication.^109,114 DNA consists of two polynucleotide strands paired together through H-bonds which results in the formation of a double helix structure (Figure 3.2).¹⁴ During DNA replication, the two strands are unwound and pulled apart, forming a replication bubble, allowing each strand to act as a template for the synthesis of two new strands (Figure 3.2).¹⁴ DNA replication proceeds in both directions thus the replication bubble expands laterally, which can induce supercoiling ahead of the replication fork.¹⁴

Figure 3.2: DNA replication.

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In bacteria, an enzyme known as DNA gyrase (a type II topoisomerase) is responsible for relieving the strain caused by supercoiling during DNA replication.^109,115 As shown in Figure 3.3, DNA gyrase is a tetramer consisting of two GyrA units and two GyrB units. When double stranded DNA (the G-segment) binds to the DNA gyrase a conformational change occurs resulting in the dimerization of the GyrA units, (2) Figure 3.3. The binding of ATP (indicated by *) initiates the dimerization of the ATPase which leads to the capture of a second segment of double stranded DNA, the T-segment (3). A conformational cascade occurs, leading to the cleavage of the G-segment and passage of the T-segment through the DNA gate (4). The G-segment is then resealed and the T-segment released (5), relieving the supercoiling. The GyrA units dimerise and ATP is hydrolysed regenerating the initial enzyme state.

Figure 3.3: Topoisomerase II mechanism.^114-115

Quinolones can bind to the gyrase-DNA complexes resulting in a quinolone-stabilised cleavage complex.109,111b,114

The stabilisation of the enzyme-DNA complex blocks replication fork movement and therefore inhibits DNA synthesis.^111b,114 However, this process is reversible meaning that other events must be involved in the bactericidal activity of quinolones.^111b,114 Cell death is believed to occur as a result of

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the release of DNA breaks.^111b,114 The exact molecular mechanisms involved in the release of DNA breaks are not fully understood.^111b,114

Bacteria also have a second type II topoisomerase, known as topoisomerase IV. Like DNA gyrase, toposiomeras IV is a tetramer and is involved in the separation of linked DNA molecules.¹⁰⁹ Topoisomerase IV is said to be the main target in positive bacteria whereas DNA gyrase is believed to be the main target in Gram-negative bacteria.¹⁰⁹ A most favourable feature of the quinolone antibacterial agents is their selectivity for bacterial topoisomerase over mammalian topoisomerase. A number of quinolones, including ciprofloxacin, have demonstrated that much greater inhibitory concentrations are required to inhibit mammalian topoisomerase II reactions in comparison to those required to inhibit the bacterial enzyme reactions.¹¹⁶ 3.1.1 Aim

The quinolones are broad spectrum antibacterial agents that have found use in a variety of infections including UTIs, sexually transmitted diseases, bone, skin and soft-tissue infections.¹¹⁷ Fluoroquinolones are also used in the treatment of tuberculosis and some quinolone derivatives have been shown to exhibit anticancer and anti-HIV activity.¹¹⁷ Modifications to the basic quinolone structure such as the introduction of the C-7 piperazine, a fluorine atom at C-6 and the N-1 cyclopropyl group have resulted in the generation of compounds with increased potency and an expanded spectrum of activity.¹⁰⁹ However, as can be seen from Table 3.1 (section 3.1) the C-3 carboxylic acid moiety, believed to be particularly important for the activity of the quinolones, has remained throughout the quinolone generations.

In this study we wanted to synthesise a basic quinolone molecule with an alternative C-3 moiety, a bioisostere. A bioisostere is a group that can be used in place of another group while maintaining the desired biological activity.¹¹ Bioisosteres are often used to investigate the structure-activity relationship of a drug or to replace substituents that are important for target interaction but responsible for toxic side effects.¹¹ Replacement of a functional group with a bioisostere has also been shown to improve activity. For example, replacement of the amide moiety of the dopamine

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antagonist, sultopride, with a pyrrole ring resulted in increased activity and selectivity (Figure 3.4).

NH O

NEt

EtO2S

OMe

EtO2S

OMe NH

NEt

(a) (b)

Figure 3.4: Replacement of the amide functionality of sultopride (a) by a non-classical isostere pyrrole to give DU 122290 (b), resulted in increased activity and

selectivity.¹¹

Tetrazoles are popular bioisosteres for carboxylic acids (Figure 3.5). Similar to a carboxylic acid, tetrazoles contain an acidic proton and are planar in structure.¹¹ In comparison to the carboxylate anion, tetrazoles are also 10 times more lipophilic.¹¹ Furthermore, many modern day drugs contain the tetrazole moiety, for example, Cefazolin (Figure 3.6), a broad spectrum first generation cephalosporin antibiotic.¹¹⁸ Therefore, in an effort to improve activity the 1H-tetrazole was chosen as the bioisostere for the quinolone C-3 carboxylic acid.

Figure 3.5: Similarities in structure and acidity of the carboxylic acid, the (1H)-tetrazole and the hydroxamic acid.

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Figure 3.6: The tetrazole-containing antibiotic, Cefazolin.

In addition to the C-3 tetrazole, a second functional group was chosen to replace the carboxylic acid moiety, a hydroxamic acid. Hydroxamic acids, as well as being acidic, also possess a similar structure to that of a carboxylic acid (Figure 3.5).

Furthermore, a wide spectrum of biological properties have been associated with hydroxamic acids including, anticancer, antifungal and antibacterial activity.¹¹⁹

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3.2 Results and Discussion

The structure of each of the compounds synthesised in this section was elucidated using LC/TOF-MS, IR, ¹H and ¹³C NMR spectoscopies. Starting materials were obtained commercially and used without further purification.

The retrosynthetic analysis for the C-3 (1H)-tetrazole and its carboxylic acid analogue can be seen in Scheme 3.1.

Scheme 3.1: Retrosynthetic analysis for the C-3 (1H)-tetrazole quinolone and its