Medicinal Chemistry I II nd Module Special Section (3) Major Antifungal Drugs

Transcription

1 Medicinal Chemistry I II nd Module Special Section (3) Major Antifungal Drugs

2 The Ideal Antifungal Agent Over the last 50 years, there has been a sustained effort to discover and develop an ideal treatment for fungal infections of human hosts. When comparing these advances in drug discovery, it is useful to consider what would be the ideal profile for an antifungal agent. Thus an ideal agent would be potently active against a fungal-specific target, i.e., one with no close mammalian homologs, which is essential and closely homologous in wide range of fungi: fungicidal rather than fungistatic due to its mechanism of action highly selective against all other mammalian targets, especially metabolising enzymes distributed systemically throughout the mammalian host, including into the central nervous system soluble enough for formulation by both oral and intravenous route, either as a stand-alone agent or by prodrug/formulation methodology. Medicinal Chemistry Page 2

3 Agents Affecting Ergosterol Ergosterol is the major product of sterol biosynthesis in fungi (and also in some trypanosomes), whereas mammalian systems synthesize cholesterol as the major membrane lipid. While both sterols play a similar role in membrane fluidity, this effect has been shown to be essential for aerobic growth of most fungi. Although cholesterol plays an equally important role in mammalian cells, most cholesterol is obtained through diet, hence inhibition of cholesterol biosynthesis as a side effect is not a major selectivity concern for antifungal agents. Most of the current antifungal agents interfere with ergosterol function in some way, either through inhibition of various steps in ergosterol biosynthesis (allylamines, azoles, morpholines) or by complexing directly with membrane ergosterol (polyenes). Medicinal Chemistry Page 3

4 Ergosterol Biosynthesis Like cholesterol, ergosterol is biosynthetically derived from acetyl-coenzyme A. The nine biosynthetic steps leading to the first sterol intermediate, lanosterol, are identical in both pathways; related enzymes in fungi and mammalian cells catalyze all but one of the remaining steps in either the cholesterol or ergosterol pathways. While there have been reports of compounds inhibiting the fungal-specific step of introduction of the 24-methyl group through sterol D24-methyltransferase, to date no drugs have arisen from this approach, hence in all cases selectivity for the fungal target is a key target to avoid toxicological consequences. The most successful targets have been squalene epoxidase (SE), lanosterol-14a-demethylase and D7-8 isomerase, which are discussed in the following sections. Medicinal Chemistry Page 4

5 Squalene Epoxidase Inhibitors SE is a microsomal enzyme catalyzing the conversion of squalene into 2,3-oxidosqualene, which is subsequently cyclized by squalene epoxide cyclase to form lanosterol. Whereas mammalian SE consists of an NADPH-cytochrome c reductase activity and a terminal oxidase, the fungal enzymes prefer NADH as the cofactor. The first SE inhibitor, naftifine, was discovered in the late 1970s, as a result of routine whole-cell screening. Based upon the structure of naftifine (1), the entire class of SE inhibitors are known as allylamines. Naftifine was found to have activity against a number of human pathogenic fungi, principally those of the dermatophyte class, e.g., Trichophyton rubrum. Naftifine was subsequently shown to be a potent blocker of ergosterol biosynthesis by fungal SE inhibition,1 with excellent selectivity over mammalian homologs. In Candida albicans, naftifine has been shown to have a noncompetitive mode of inhibition. Medicinal Chemistry Page 5

6 Squalene Epoxidase Inhibitors Naftifine s effect on ergosterol biosynthesis results in a fungicidal action against dermatophytes and other filamentous fungi, e.g., Aspergillus fumigatus, and against some yeasts, e.g., C. parapsilosis. In contrast, its action is fungistatic against most strains of C. albicans. The difference is believed to be due to two intrinsic physiological factors in the target fungi; in some cases growth inhibition is achievable at concentrations at which ergosterol biosynthesis is only partly inhibited, whereas in other fungi, total inhibition is necessary. In dermatophytes, only partial inhibition is necessary, which suggests that squalene accumulation may play an important role in causing cell death. This fungicidal action has a slow onset, implying that death is a secondary effect, unlike the polyenes. Ultrastructural studies of cells treated with naftifine show large numbers of lipid bodies, which may represent intracellular squalene accumulation. Naftifine was also found to be active in vivo in dermatophytosis models such as the guineapig trichophytososis model following topical application, though oral activity could only be demonstrated after administration of very high doses that were of no therapeutic relevance. Medicinal Chemistry Page 6

7 Squalene Epoxidase Inhibitors Initial SAR studies around naftifine demonstrated that its activity was specific since the basic nitrogen atom, the double bond, and the 1-substituted naphthalene ring were all important. In addition, the aromatic rings could not be interchanged. Changes in the distances between the function groups and the aromatic rings could not be altered. However, it was found that rigidification of the structure of naftifine was possible. Incorporation of the basic center into a piperidine ring provided a compound with antifungal activity both in vitro and in vivo (active piperidine analog (2)). Activity was restricted to the R-enantiomer and this compound was also found to exhibit significantly improved oral activity in the guinea-pig trichophytosis model. In subsequent SAR studies, the cinnamyl group of naftifine was replaced by long alkyl chains, with an increasing number of conjugated double bonds. Activity was maximized when two multiple bonds were present but was improved, especially in vivo, when the C4 C5 double bond was replaced by a triple bond. An E-configuration of the double and triple bonds was preferred (Terbinafine (3). While the nature of the terminal substituent on the triple bond had relatively little effect on potency, oral activity was optimized by incorporating a t-butyl substituent providing terbinafine. Detailed mechanistic studies using terbinafine demonstrated that its mechanism of action was identical to that of naftifine, though terbinafine was significantly more potent and had a superior pharmacokinetic profile. Medicinal Chemistry Page 7

8 Squalene Epoxidase Inhibitors Further SAR studies around terbinafine have shown that the naphthalene ring of terbinafine can be replaced by lipophilic heterocycles. Variation of the position of substitution and further substituents on a benzo[b]thiophenyl ring system led to the 3-chloro-derivative (SDZ (4)), which is the most potent SE inhibitor reported. SDZ was also found to be highly effective in in vivo animal infection models. While optimization of potency led to the pharmaceutically unique enyne fragment, further SAR explorations on the allyl side chain led to the discovery of the homopropargylamines and benzylamines. Para substitution on the benzylamine was found to be essential for high antifungal activity and the t-butyl derivative, butenafine (5), was subsequently developed for topical treatment of dermatophytoses. Medicinal Chemistry Page 8

9 Pharmacokinetic profiles Squalene Epoxidase Inhibitors There have been limited reports on the pharmacokinetics of naftifine. Following topical application in a 1% cream formulation, naftifine penetrates the epidermis in sufficient concentrations of inhibit fungal growth but only 3.8% of the dose was recovered in the urine. Clearance of naftifine was rapid following oral administration, leading to 15 different metabolites. Some 14.3% of the dose was recovered as metabolites, with naphthoic acid predominating. None of the metabolites exhibited significant antifungal activity. As observed in animal models, and in contrast to naftifine, terbinafine achieves clinically effective concentrations following oral administration. Approximately 70 80% of an orally administered dose is absorbed from the gastrointestinal tract and bioavailability is little affected by food. Following administration of the efficacious dose of 250 mg, a peak plasma concentration of 0.9 mg L1 was achieved within 2 h in healthy volunteers. On multiple dosing to patients, peak and trough concentrations of 3.62 and 1.44 mg L1 were measured at steady state, which is reached after days.12 Terbinafine is a highly lipophilic base and consequently has a very high volume of distribution (13.5Lkg1 at steady state), a result of strong, nonspecific binding to plasma proteins. Terbinafine is distributed to all tissues with particularly high concentrations in the liver, pancreas, and sebum (45 mg kg1). Terbinafine is extensively metabolized in humans: the major routes of metabolism are N-demethylation, N- oxidation, and oxidation of the t-butyl group. Plasma elimination occurs with a half-life of h in healthy volunteers. However, administration of radiolabeled drug identified an additional elimination phase with a half-life of h. The second phase is believed to reflect the slow redistribution of terbinafine from adipose tissue. Total plasma clearance in healthy volunteers was 76Lh1. Approximately 80% of an oral dose was excreted in urine within 72 h. Medicinal Chemistry Page 9

10 Clinical profiles Squalene Epoxidase Inhibitors Clinical trials with naftifine (Exoril) employed the 1% cream formulation against mycologically confirmed dermatophytosis, e.g. tinea pedis (athlete s foot), for periods of weeks. The predominant infecting organisms were Trichophyton rubrum and T. mentagrophytes, though activity against cutaneous candidiases has also been observed. Twicedaily application gave the best results, though a once-daily regimen was also effective. Mycological cure rates were high (84%), with similar activities to the first-generation azole, clotrimazole, being observed. Despite its fungicidal mode of action in vitro, modest cure rates (19%) have been observed. There have been few reports of systemic adverse events following topical application of naftifine. In clinical trials, a few patients developed mild local irritation in the early stages of treatment. Clinical trials with terbinafine (Lamisil) have shown it to be effective in the treatment of dermatophyte infections following either oral (250 mg day1) or topical (1% cream) administration. Mycological cures have been observed in approximately 90% on patients with tinea pedis, with associated clinical cures in 80% of cases. Oral terbinafine also shows impressive success in the treatment of finger-and toenail infections following oral administration for 3 12 months. Terbinafine is less effective against skin infections caused by Candida spp. when given orally, though it is efficacious following topical treatment. There have been few reports of the activity of terbinafine against systemic candidoses. Terbinafine is well tolerated following oral administration. The predominant side effects are gastrointestinal disturbances and skin reactions, though more unusual reports include tongue discoloration. The potential for drug drug interaction with terbinafine are low due to its lack of cytochrome P-450 inhibition, though drugs that inhibit or enhance P-450 activity can alter the metabolism of terbinafine. Medicinal Chemistry Page 10

11 Squalene Epoxidase Inhibitors Drug resistance In contrast to the azoles, there are relatively few reports of primary resistance to allylamines, although resistant mutants can be induced through ultraviolet irridation or chemical mutagenesis. In the one primary resistant example, a patient who failed oral terbinafine therapy was found to have a resistant strain of Trichophyton rubrum, which was sensitive to azoles but cross-resistant to other SE inhibitors. The remaining examples of terbinafine resistance have been observed in organisms where the multidrug efflux transporter CDR2 has been induced Medicinal Chemistry Page 11

12 Squalene Epoxidase Inhibitors Synthesis of terbinafine and butenafine The preparation this agent begins with the alkylation of the common intermediate (10-1) with propargyl bromide. The product (10-4) is then coupled with 1-bromo-2- tertiary-butylacetylene by means of a copper salt catalyzed reaction in the presence of a mild base such as a tertiary amine. Construction of the allylamine function takes advantage of a reaction characteristic of propargyl amines Thus reaction of intermediate (10-5) with lithium di-iso-propylaluminum hydride proceeds to give a selective reduction of the double bond closest to the amino group; the observed selectivity can be rationalized by assuming the intermediacy of a complex of the hydride reagent with basic nitrogen. There is thus obtained the antifungal agent terbinafine (10-6) [11]. Anifungal activity is retained when the complex side chain in that compound is replaced by a substituted aromatic ring. Thus alkylation of the ubiquitous naphthylamine (10-1) with 4-tert-butylbenzyl bromide (10-7) affords the antifungal agent butenafine (10-8) Medicinal Chemistry Page 12

13 Squalene Epoxidase Inhibitors Medicinal Chemistry Page 13

14 Enzymology Lanosterol-14 -Demethylase Inhibitors Demethylation of the 14a-methyl of lanosterol is catalyzed by a specific cytochrome P450 (CYP51 in Candida albicans) via a multistep mechanism based on sequential oxidation of the methyl group to the aldehyde oxidation state, followed by an oxidative rearrangement and elimination to leave a double bond. The vast majority of known lanosterol demethylase inhibitors and all of the drugs make use of a mechanism involving coordination to the P450 heme ferric cation core of the enzyme through a metal-chelating group which is part of a structure capable of mimicking the sterol backbone. The azole drugs are selective for CYP51 inhibition over the human CYP3A4, the major metabolizing enzyme Medicinal Chemistry Page 14

15 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry The first inhibitors of lanosterol demethylase were discovered by the Janssen group as part of an effort to synthesize and evaluate the biological activity of a series of imidazole derivatives. The initial leads were 1-phenethylimidazoles bearing a hydroxyl or amino group attached to the benzylic position. Further synthesis around the initial lead demonstrated that a wide range of analogs retained biological activity, including a series of ethers and cyclic acetals derived from intermediate ketones. Among the analogs prepared was the bis-(2,4- dichlorophenyl) ether derivative, miconazole, which became the first drug of the azole class; subsequent analogs in the same series have led to three further drugs from the Janssen group alone. The initial studies found that the substitution on the parent primary amine gave moderate activity against dermatophytes but poor activity against yeasts (and bacteria). The ketals were found to have a similar biological profile but in both series activity improved when substituents were introduced on to the aromatic ring. Reduction of several of the ketones and formation of the corresponding benzylethers provided a series with very potent activity against dermatophytes combined with modest activity against C. albicans and Aspergillus fumigatus. Medicinal Chemistry Page 15

16 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry Whereas substitution on the first aryl ring produced a moderate potency increase in the amine series, over a 100-fold increase in anti-candida activity was achieved with 2,4-dichloro aryl examples. Unexpectedly, similar substitution patterns were optimal at both positions in miconazole, while substitution at the branch point with a methyl group was not tolerated (Table 3). Two other marketed drugs, econazole (6) (also Janssen) and tioconazole (12) (discovered by Pfizer), are members of the same chemical series. Medicinal Chemistry Page 16

17 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry Follow-on studies around miconazole established that the ether oxygen was not essential for antifungal activity since it was possible to replace the benzylether side chain with alkyl groups. While most of the analogs were highly active against dermatophytes, and some had excellent activity against yeasts, none were active against bacteria. The SAR relationships for aryl substitution were the same as in the miconazole series, while alkyl chains of more than four carbons provided compounds with activity both in vitro and in guinea-pig dermatophyte models. In parallel with the research that led to miconazole and econazole, scientists at Bayer discovered that the ethyl linker between the imidazole and aryl rings was not essential for biological activity. Instead, trityl substitution on the imidazole ring was found to be sufficient, leading to the 2-chloroderivative, clotrimazole (7). Medicinal Chemistry Page 17

18 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry Although the first generation of antifungal azoles were potent in vitro against a wide range of fungi of clinical interest, and were successful in the topical treatment of human infections, their main drawback was the low systemic levels following oral administration. Continuing work at Janssen found that introduction of an additional substituent on the ketal ring of their initial leads was tolerated, leading to ketoconazole. The in vitro activity of ketoconazole (8) appeared to be inferior to that of miconazole under standard conditions (Sabouraud s broth) but, in contrast, the activity of ketoconazole was improved by the addition of serum to the antifungal assay while that of miconazole was dramatically reduced. This improvement was mirrored in in vivo experiments in candidosis models, where ketoconazole displayed a dose response in both prophylactic (starting on day of treatment) and curative treatment (starting on day 3 following infection) regimens. Medicinal Chemistry Page 18

19 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry While each of the early azole derivatives makes use of an imidazole ring to coordinate with the heme, the Janssen group found that the triazole analog of ketoconazole (terconazole (9)) had a similar in vitro profile (Table 4). Surprisingly, in view of later developments, terconazole had an inferior profile compared to ketoconazole in vaginal candidosis models following oral administration. Medicinal Chemistry Page 19

20 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry Further elaboration on the terminal piperazine substituent of terconazole led to a series of triazolones with broadspectrum activity both in vitro and in vivo. N- alkylation on the triazolone ring was found to be essential for oral activity, and in this series, the triazole derivatives were found to be superior to the corresponding imidazoles. Extension of the alkyl substituent on the triazolone with straight chains had little effect on the oral activity of the series but introduction of a branching carbon resulted in a dramatic increase in systemic activity with the final marketed drug from this series, itraconazole. The 2,4-difluorophenyl analog of itraconazole (10), saperconazole, was taken into development but was found to carcinogenic in long-term toxicology studies. Medicinal Chemistry Page 20

21 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry While itraconazole has potent antifungal activity in its own right, one of the main metabolites, involving hydroxylation of the alkyl side chain on the triazolone ring, is also highly potent. Scientists at Schering incorporated this finding in their closely related series featuring a tetrahydrofuran ring in place of the dioxolane ring of saperconazole. The pentyl side chain of their original preclinical candidate, SCH , was substituted with a hydroxyl group to give posaconazole (SCH-56592: 11) Medicinal Chemistry Page 21

22 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry Following on from their discovery of tioconazole, researchers at Pfizer adopted an alternative approach to obtaining systemic activity, by aiming for low clearance compounds by reducing lipophilicity. After experimenting with a number of series, they opted for the tertiary alcohol series because this was the most polar series known to have antifungal activity. After extensive effort in this series, they concluded that the imidazole substituent was a metabolic flaw and replaced it with alternative heterocycles, including the 1,2,4-triazole. The triazole UK (13) was found to give improved in vivo activity, despite a sixfold reduction in in vitro potency. Based on the hypothesis that the triazole was more metabolically stable than the imidazole but retained affinity for the target enzyme, they replaced the hexyl chain with another triazole unit. The initial lead, UK (13), was 100 times more potent in a mouse candidosis model than ketoconazole but was found to have modest activity under the standard in vitro assay conditions. Subsequently, it became clear that complex media, especially those containing peptones, antagonized the activity of the more polar azoles, and the assay conditions were modified to a simple medium. Medicinal Chemistry Page 22

23 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry Although UK had outstanding activity in a wide range of systemic and superficial infection models, it was found to be hepatotoxic in mice and dogs, and teratogenic in rats. An intensive follow-up provided over 100 bis-triazole analogs, many with very good in vivo activity. The preferred compound, fluconazole (14), has a 2,4-difluorophenyl substituent in place of the dichloroaryl ring of UK This modification retained the activity of the initial lead in the infection models but was devoid of teratogenicity or hepatoxicity. Medicinal Chemistry Page 23

24 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry During its development, it became clear that fluconazole had superior efficacy to the existing azoles (miconazole, ketoconazole) for the treatment of infections due to Candida albicans and Cryptococcus neoformans. However, it was poorly effective against Aspergillus infections compared to itraconazole. Pfizer scientists found that introducing a methyl group adjacent to one of the triazole rings of fluconazole increased potency against A. fumigatus, while retaining potency against the other fungal pathogens. Replacement of the triazole ring with sixmembered heterocycles provided compounds with broad-spectrum in vitro activity, and, surprisingly, a fungicidal mechanism of action against Aspergillus spp. The optimal compound in the series, voriconazole (15), features a 5-fluoro-4- pyrimidinyl substituent. Voriconazole is a single enantiomer whose opposite enantiomer is at least 500-fold less active. The difference in activity of fluconazole and voriconazole against CYP51 in Aspergillus has been rationalized as a difference in binding mode. Medicinal Chemistry Page 24

25 Lanosterol-14 -Demethylase Inhibitors Medicinal chemistry The beneficial impact of the -methyl group was also discovered by Sumitomo scientists, who found that one of the triazoles of fluconazole could be replaced by a methyl sulfone substituent (genaconazole: 16; Table 5). The racemate, genaconazole, was co-developed with Schering but was subsequently found to be toxic in long-term safety studies. Resolution of genaconazole demonstrated that the activity resided on one enantiomer, as for voriconazole, but the enantiomer proved to have a similar toxicology profile to the racemate. Interestingly, there is a 60-fold difference in the in vitro potency of genaconazole against Candida albicans and Aspergillus fumigatus, an effect consistent with the proposed difference in binding modes. Medicinal Chemistry Page 25

26 Lanosterol-14 -Demethylase Inhibitors Pharmacokinetic profiles The first generation of the azoles, which were characterized by poor systemic activity, suffered from a combination of incomplete absorption, high volumes of distribution, and high clearance. For example, the oral bioavailability of miconazole in humans is 25 30%,; its volume of distribution is 21 Lkg1; and less than 1% of the administered dose was excreted unchanged. Consequently, this generation of drug found greater usage by topical or intravaginal application. Thus econazole, administered topically as its nitrate salt, provided high drug levels in the dermal layers but very low systemic levels. Across a number of pharmacokinetic studies, 7% or less of the drug was absorbed. Even when absorbed, econazole undergoes a series of metabolic transformations, resulting in the identification of over 20 urinary metabolites in monkey. Ketoconazole is better absorbed than the early agents, especially under acidic conditions where the drug is fully dissolved. Coadministration with antacids such as cimetidine reduced blood levels. Although ketoconazole is highly protein-bound (99%) in humans, it is widely distributed, including by penetration into the cerebrospinal fluid. Like the other early azoles, ketoconazole is extensively metabolized, including by oxidation of the imidazole ring and degradation of the piperazine ring. While some of this metabolism occurs on first pass during absorption, ketoconazole is a potent inhibitor of hepatic CYP 3A4, resulting in inhibition of its own metabolism. As a consequence, the half-life of ketoconazole is both dose- and patient-dependent. Medicinal Chemistry Page 26

27 Lanosterol-14 -Demethylase Inhibitors Pharmacokinetic profiles As a consequence of its high lipophilicity and low aqueous solubility, gastric acidity is also required for itraconazole absorption. It is best absorbed when administered with food, though there is considerable interpatient variability. The oral bioavailability of itraconazole from a 100mg solution dose was 55%. Like ketoconazole, bioavailability and half-life are dosedependent, indicating saturable metabolism. Once absorbed, itraconazole is highly plasma protein-bound (99.8%) and widely distributed (10.7Lkg1). Although itraconazole is widely metabolized, it does produce an active metabolite by hydroxylation of the triazolone side chain. Following multiple-dose administration of itraconazole for 14 days, concentrations of itraconazole and the hydroxyl metabolite were 1.9 and 3.2mgmL1, respectively, at steady state. In contrast to all previous antifungal azoles, fluconazole is a polar, water-soluble, metabolically stable molecule. Fluconazole is very well absorbed, reaching peak plasma concentrations in 1 2 h and virtually complete bioavailability following oral administration. Neither food nor gastric acidity had any effect. Its volume of distribution is low ( L kg1), approximating to body water, with very low plasma protein binding (12%). Fluconazole also crosses membranes easily such that cerebrospinal fluid-to-plasma ratios of are achieved. There is very little evidence for metabolism of fluconazole, which is mainly excreted through the renal elimination route (60 90% of dose). However, there is extensive tubular reabsorption, resulting in a long plasma elimination half-life of h, enabling once-daily dosing for systemic candidoses and a single-dose therapy for vaginal candidiasis. Medicinal Chemistry Page 27

28 Lanosterol-14 -Demethylase Inhibitors Pharmacokinetic profiles Like fluconazole, voriconazole is polar, with moderate aqueous solubility (0.5mgmL1), resulting in rapid absorption (maximum concentration achieved in less than 2 h) and oral bioavailability (96%). Moderate food effects have been observed. Distribution is wide with a steady-state volume of 4.6Lkg1 and moderate binding to plasma proteins (58%). Unlike fluconazole, little (2%) of orally administered voriconazole is renally eliminated. Instead, voriconazole is metabolized by a range of cytochrome P450s (CYPs), the major route being N-oxidation of the pyrimidine ring by CYP2C19 to give an inactive metabolite. The elimination half-life of voriconazole is approximately 6 h. Medicinal Chemistry Page 28

29 Clinical profiles Lanosterol-14 -Demethylase Inhibitors Econazole can be used to treat dermatophyte infections using a twice-daily regimen for 2 6 weeks, resulting in high cure rates (B90%). In vaginal candidosis, a 3-day treatment regimen using a 15mg suppository once daily was nearly as effective as a longer treatment (15 days) at a lower dose (50 mg). Both treatments are well tolerated. The first orally available azole, ketoconazole, is effective in patients with dermatophyte or yeast skin infections, and in systemic infections due to Candida and Histoplasma spp. For most conditions a 200 mg daily dose is recommended, the exception being vaginal candidosis, where twice-daily dosing for 5 days is required. Administration at higher doses is limited by the ketoconazole s side-effect profile, including elevated liver enzymes and gynecomastia in males. Due to its wide spectrum of activity, itraconazole is used to treat a wide range of infections. High cure rates were achieved in fingernail and toenail onchomycosis (200 mg day1 for 3 months), dermatophytosis (100 mg day1 for 2 4 weeks), and vaginal candidiasis (400 mg single dose). Unlike ketoconazole, there are few side effects and liver toxicity is rare. Medicinal Chemistry Page 29

30 Clinical profiles Lanosterol-14 -Demethylase Inhibitors Fluconazole is effective against oropharyngeal and esophageal candidiasis when used orally once daily either as a treatment or prophylactically in patients with acquired immunodeficiency syndrome (AIDS) or undergoing cancer therapy. It is also effective in patients with cryptococcal meningitis, especially as a maintenance therapy. In general a loading dose of twice the daily dose is recommended on the first day of treatment, for example, doses of 200 mg then 100 mg are used for oropharyngeal candidiasis. Higher doses are equally well tolerated and are used for esophagal infections and cryptococcal meningitis. Vaginal candidiasis can be treated using a single 50 mg dose. Fluconazole is available for both oral and intravenous administration, particularly for seriously ill patients, with few side effects. Owing to its inferior pharmacokinetic profile, voriconazole is dosed twice daily; intravenous doses of 6mg kg1 are used on the first day, followed by 200 mg orally or continued intravenous dosing at 4mg kg1. Voriconazole is recommended for the treatment of adults with invasive aspergillosis and can be used for rare infections caused by Fusarium spp. and Scedosporium apiospermum, where treatment with other agents has failed. In Europe, it can be used to treat fluconazole-resistant serious Candida infections. Its primary use is in immunocompromised patients with progressive, life-threatening infections. Although generally well tolerated, adverse effects such as visual disturbances and skin rash have been observed. Medicinal Chemistry Page 30

31 Drug resistance Lanosterol-14 -Demethylase Inhibitors Until the late 1980s, resistance to the azole derivatives was rare as they were generally used topically. Since the introduction of the systemically active azoles as the mainstay of antifungal chemotherapy, resistance has become much more widely reported in the literature. A number of different resistance mechanisms have also emerged, ranging from decreased cell wall permeability or increased efflux to increased expression or mutation of the target enzyme, and alteration of the target pathway. In a related fashion to the way human cells use the adenosine triphosphate-driven pump P-glycoprotein to remove xenobiotics from cancer cells, analogous genes, e.g., CDR, are expressed by fungal cells to efflux azoles. In Candida albicans another type of protein (MDR) from the major facilitator family or transporters uses membrane potential to efflux azoles, particularly fluconazole. Changes in the affinity of the target CYP51 is another important mechanism of resistance that has been described in C. albicans and Cryptococcus neoformans. However, it is important to distinguish between acquired resistance and insensitivity of the specific fungal CYP51, e.g., the Aspergillus fumigatus isozyme is much less sensitive to inhibition by fluconazole than the Candida albicans CYP51. One clear example was the discovery of four separate mutated CYP51 gene products from resistant C. albicans isolates. These genes were expressed in yeast and were shown to have different affinity for different azoles compared to the wild-type enzyme. Overexpression of CYP51 is less common but in one case threefold increased levels were observed in resistant isolates. Another less common change is an alteration in the sterol biosynthetic pathway. In the presence of azoles, the D-5,6-desaturase continues to synthesize 14-methyl sterols from 14 -methylfecosterol, resulting in the formation of a toxic membrane sterol. Mutation of the desaturase then results in the nontoxic methylfecosterol rather than ergosterol, hence the mutant strains are also resistant to polyenes. Medicinal Chemistry Page 31

32 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF ITRACONAZOLE (SPORANOX) The discovery pathway to itraconazole (1) (Heeres and Backx, 1980, 1981; Heeres et al., 1988) can be traced back through the earlier Janssen antifungal drugs from miconazole in a series of publications (Heeres et al., 1979, 1983, 1984). The ethyl spacer between the azole and phenyl rings was quickly found to be optimal, as was 2,4-dichlorosubstitution on the aryl ring. The strategy of elaboration of the remaining side-chain from ethers (miconazole, econazole) to aryloxymethyldioxolanes (ketoconazole) was also continued in the synthesis of itraconazole, albeit with the requisite azole installed at the first stage. Consequently, the route to itraconazole is relatively lengthy and linear. Compared to ketoconazole, itraconazole is much less active in in vitro tests, possibly due to its low solubility in the test medium. However, it is still active at low concentrations (0.1 1 mg/ml) against dermatophytes (skin infections) and against the three main fungal pathogens (Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus) at 0.1 mg/ml. An entirely different picture is seen in animal infection models where itraconazole is much more potent than ketoconazole. Due to its wide spectrum of activity, itraconazole is used to treat a wide range of infections in man. Clinical trials demonstrated that high cure rates could be achieved in fingernail and toenail onchomycosis (200 mg/day for 3 months), dermatophytosis (100 mg/day for 2 4 weeks) and vaginal candidiasis (400 mg single dose).compared to ketoconazole, itraconazole is much less active in in vitro tests, possibly due to its low solubility in the test medium. However, it is still active at low concentrations (0.1 1 mg/ml) against dermatophytes (skin infections) and against the three main fungal pathogens (Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus) at 0.1 mg/ml. An entirely different picture is seen in animal infection models where itraconazole is much more potent than ketoconazole. Due to its wide spectrum of activity, itraconazole is used to treat a wide range of infections in man. Clinical trials demonstrated that high cure rates could be achieved in fingernail and toenail onchomycosis (200 mg/day for 3 months), dermatophytosis (100 mg/day for 2 4 weeks) and vaginal candidiasis (400 mg single dose). Medicinal Chemistry Page 32

33 Lanosterol-14 -Demethylase Inhibitors Medicinal Chemistry Page 33

34 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF ITRACONAZOLE (SPORANOX) The synthesis of itrazonazole (Scheme 5.2) started by ketalization of 2,4-dichloroacetophenone with glycerine under mildly acidic condition in refluxing benzene (Heeres et al., 1979). The crude product was then brominated at 40 C to give 10 in 91% yield for the two steps. The primary alcohol was protected as the benzoate by treatment with benzoyl chloride in pyridine, enabling isolation of a crystalline solid in 50% yield. Treatment of the bromide with the sodium salt of 1,2,4-triazole (generated in situ from triazole and sodium hydride) in DMSO at 130 C gave a mixture of the regioisomeric triazole derivatives, which were saponified using a mixture of aqueous sodium hydroxide and dioxane (Heeres et al., 1983). The isomers were separated by chromatography, with the major product (approximately 10 : 1) being the desired 1- substituted triazole isomer. Medicinal Chemistry Page 34

35 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF ITRACONAZOLE (SPORANOX) Following activation of the alcohol through mesylation with mesyl chloride in pyridine (87%), a second nucleophilic displacement with the sodium salt of N-acetyl-4-hydroxyphenylpiperazine at 80 C, again generated in situ with sodium hydride in DMSO, provided the triazole analog of ketoconazole in 64% yield (Heeres et al., 1984). Deacetylation was achieved with sodium hydroxide in n-butanol under refluxing conditions (70%) before attachment of the final phenyl ring by nucleophilic aromatic substitution using 4- chloronitrobenzene under mildly basic conditions in DMSO at 120 C. Medicinal Chemistry Page 35

36 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF ITRACONAZOLE (SPORANOX) Catalytic hydrogenation over platinum on carbon in ethylene glycol monomethyl ether at 50 C gave the corresponding poorly soluble aniline derivative, which required the crude reaction mixture to be heated to avoid filtration of the product along with the catalyst. Carbamoylation of the aniline derivative with phenyl chloroformate in a mixture of chloroform and pyridine gave an activated carbamate, which was treated with hydrazine to yield the semicarbazide in 86% yield over two steps. Condensation of the semicarbazide with formamidine acetate in DMF at 130 C for 3 h gave the cyclized triazolone (62% yield). Subsequent alkylation with 2-bromobutane was achieved using powdered potassium hydroxide in DMSO to give itraconazole (1) as a crystalline solid from toluene. Medicinal Chemistry Page 36

37 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF FLUCONAZOLE (DIFLUCAN) In contrast to the evolutionary discovery process that lead to itraconazole, the discovery of fluconazole (Narayanaswami and Richardson, 1983; Richardson, 1982) appears more revolutionary. The Pfizer team decided to target compounds with in vivo activity from the outset, deliberately focusing on compounds with a good ADME profile at the expense of in vitro potency (Richardson et al., 1988). Having reviewed the profiles of each of the known series of antifungal imidazole derivatives, they concentrated their efforts on the tertiary alcohol series, as examples often gave activity in animal infection models. However, when pharmacokinetic studies indicated that the imidazole ring was still susceptible to metabolism, a number of alternative heterocyclic replacements were investigated. Only the 1-linked 1,2,4- triazole offered any encouragement in vivo, despite being less active than the corresponding imidazole derivative in vitro. In continuation of the focus on pharmacokinetic properties, the hexyl side-chain of the lead compound was replaced by a second 1,2,4-triazole, on the assumption that the resulting derivatives would have low lipophilicity. It was hoped that this would be consistent with high blood levels and, due to reduced protein binding, higher free drug levels. This hypothesis turned out to be true and the series of bis-triazoles were found to be highly active in animal infection models. Fluconazole, the 2,4-difluorophenyl analog, emerged as the only compound to combine good aqueous solubility, a long half-life, and an excellent safety profile. Medicinal Chemistry Page 37

38 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF FLUCONAZOLE (DIFLUCAN) Fluconazole is effective against oropharyngeal and esophageal candidiasis when used orally once daily either as a treatment or prophylactically in patients with AIDS or undergoing cancer therapy. It is also effective in patients with cryptococcal meningitis, especially as a maintenance therapy. Vaginal candidiasis can be treated using a single 50-mg dose. Fluconazole is available for both oral and i.v. administration, particularly for seriously ill patients, with few side-effects. Fluconazole (Goa and Barradell, 1995) is very well absorbed, reaching peak plasma concentrations in 1 2 h with virtually complete bioavailability following oral administration. Neither food nor gastric acidity has any effect. Its volume of distribution is low ( L/kg), approximating to body water, with very low plasma protein binding (12%). Fluconazole also crosses membranes easily such that CSF : plasma ratios of are achieved. There is very little evidence for metabolism of fluconazole, which is excreted mainly through the renal elimination route (60 90% of dose). However, there is extensive tubular reabsorption, resulting in a long plasma elimination half-life of h, enabling once-daily dosing for systemic candidoses and a single-dose therapy for vaginal candidiasis. Medicinal Chemistry Page 38

39 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF FLUCONAZOLE (DIFLUCAN) In keeping with the simplicity of its structure, fluconazole can be synthesized in two steps from commercially available starting materials (Scheme 5.3). Metallation of 1-bromo-2,4-difluorobenzene with butyllithium in ether gave the corresponding aryllithium, which was trapped with 1,3-dichloroacetone. The crude alcohol intermediate was reacted with 1,2,4-triazole in DMF in the presence of potassium carbonate at 70 C to give a mixture of regioisomeric triazoles from which the desired bis-1-linked triazole could be isolated by chromatography in 26% overall yield. Medicinal Chemistry Page 39

40 Lanosterol-14 -Demethylase Inhibitors SYNTHESIS OF FLUCONAZOLE (DIFLUCAN) An alternative larger scale chromatography-free four-step route has also been described in the patent literature (Narayanaswami and Richardson, 1983; Richardson, 1982). Aluminum-chloride-catalysed chloroacetylation of 1,3- difluorobenzene using chloroacetyl chloride without solvent at 50 C afforded 10- chloro-2,4-difluoroacetophenone in 73% yield. Displacement of the chloride with 1,2,4-triazole using triethylamine as base in refluxing ethyl acetate gave a modest yield (40%) of the desired regioisomeric triazolylketone as the hydrochloride salt. Corey epoxidation using the sulfoxonium ylide [generated in situ from trimethylsulfoxonium iodide under phase transfer conditions (cetrimide/toluene/aqueous sodium hydroxide)] at 60 C provided the epoxide, which was isolated as its mesylate salt in 56% yield. The epoxide ring was opened with a second equivalent of 1,2,4-triazole using potassium carbonate as base to give a mixture of triazole isomers. The minor, undesired isomer is even more water soluble than fluconazole and consequently, can be removed by a simple water wash of the crude product solution in chloroform. Fluconazole (2) was isolated as a crystalline free base in 44% yield from isopropanol. Medicinal Chemistry Page 40

41 Lanosterol-14 -Demethylase Inhibitors Medicinal Chemistry Page 41

42 Other Ergosterol Biosynthesis Inhibitors Other than the SE inhibitors (allylamines) and lanosterol demethylase inhibitors (azoles), the only other pharmaceutically marketed ergosterol biosynthesis inhibitor is amorolfine (23). Amorolfine is a member of the morpholine class of antifungal agent, which includes fenpropiomorph (24), a marketed agrofungicide. The mechanism of action of amorolfine is thought to be through inhibition of D7 D8 isomerase (IC mmoll1), though amorolfine also inhibits D14-reductase (IC mmol L1), which may result in a synergistic effect. Amorolfine possesses a broad antifungal spectrum, including dermatophytes and yeasts; however, it is inactive against Aspergillus spp. In most cases, the fungicidal concentration is very close to its minimum inhibitory concentration (MIC). Like the SE inhibitors, this fungicidal effect, particularly, against dermatophytes, may be due to squalene accumulation. Alternatively, electron microscope studies reveal an accumulation of chitin in the cell membrane, which may be the direct effect. Amorolfine is formulated as a 5% lacquer for the treatment of nail infections. Levels of amorolfine exceed the MICs of most fungi causing nail infections within 24 h of dosing. Penetration into the deepest nail slices at well over the MIC levels are observed within 4 weeks of treatment. Medicinal Chemistry Page 42

43 Fungal Cell Wall Synthesis Inhibitors Since the fungal cell wall, an essential structure for maintaining cell integrity, is not found in mammalian cells, it offers the opportunity for specific antifungal targets. Although the exact structure of the fungal cell wall is not fully understood, it consists of a complex mixture of proteins and polysaccharides, including glucan, mannans, and chitin. Each of these carbohydrate-based polymers plays an important role in the cell wall via surface binding or structural processes. However, few of the potential targets have been isolated as pure enzymes. Instead, the primary method for identifying new targets has been whole-cell screening, followed by extensive biochemistry to identify the specific target. In addition, due to the polysaccharide nature of many of the enzyme substrates, few of the targets are amenable to small-molecule inhibitors and, consequently, inhibitors are usually natural products or semisynthetic analogs based on the natural products. Medicinal Chemistry Page 43

44 Glucan Synthase Inhibitors The first class of glucan synthase inhibitors to be discovered were the echinocandins, which were isolated from fermentation broths of Aspergillus cultures in the early 1970s. The echinocandins are a family of closely related lipopeptides, of which echinocandin B is the major component. Structurally, the echinocandins are composed of a complex hexapeptide whose N-terminus is acylated by a long chain carboxylic acid (27; Table 6). For echinocandin B, the cyclic peptide is made up of 4,5-dihydroxyornithine, two threonines, 3-hydroxyproline, 3-hydroxy- 4-methylproline and 3,4- dihydroxyhomotryosine. Medicinal Chemistry Page 44

45 Other Ergosterol Biosynthesis Inhibitors Other than the SE inhibitors (allylamines) and lanosterol demethylase inhibitors (azoles), the only other pharmaceutically marketed ergosterol biosynthesis inhibitor is amorolfine (23). Amorolfine is a member of the morpholine class of antifungal agent, which includes fenpropiomorph (24), a marketed agrofungicide. The mechanism of action of amorolfine is thought to be through inhibition of D7 D8 isomerase (IC mmoll1), though amorolfine also inhibits D14-reductase (IC mmol L1), which may result in a synergistic effect. Amorolfine possesses a broad antifungal spectrum, including dermatophytes and yeasts; however, it is inactive against Aspergillus spp. In most cases, the fungicidal concentration is very close to its minimum inhibitory concentration (MIC). Like the SE inhibitors, this fungicidal effect, particularly, against dermatophytes, may be due to squalene accumulation. Alternatively, electron microscope studies reveal an accumulation of chitin in the cell membrane, which may be the direct effect. Amorolfine is formulated as a 5% lacquer for the treatment of nail infections. Levels of amorolfine exceed the MICs of most fungi causing nail infections within 24 h of dosing. Penetration into the deepest nail slices at well over the MIC levels are observed within 4 weeks of treatment. Medicinal Chemistry Page 45

46 Fungal Cell Wall Synthesis Inhibitors Medicinal Chemistry Page 46

47 Fungal Cell Wall Synthesis Inhibitors Following the discovery of the echinocandins, several related classes of natural product were discovered, including the mulundocandins (featuring a serine residue in place of one threonine), the pneumocandins (where the same threonine is replaced by a 3-hydroxyglutamine), and the sporiofungins (both threonines are substituted by serines). The family of lipopeptides are characterized by excellent in vitro activity against Candida albicans (0.1 1 mgml1). Several compounds were also protective against candidiasis in animal models following intraperitonal dosing. Mechanistic studies suggested that the echinocandins and related lipopeptides inhibited cell wall biosynthesis. The compounds were found to inhibit the formation of (1,3)-b-glucan, which is present as the major strucutural component of fungal cell walls. Microfibrils of the glucan are extruded into the interstitial space of the cell, where they are crosslinked by enzymes such as glycosyl transferases to construct the growing cell wall. Interference with the process leads to a weakened wall, cell content leakage, and cell death. The point of intervention was found to be (1,3)-b-glucan synthase, which is a membrane-bound, multisubunit enzyme, made up of an insoluble catalytic subunit and a soluble regulatory subunit. The active complex binds and polymerizes uridine diphosphate glucose to form glucan. The lipopeptides act via a noncompetitive mechanism but their exact mechanism of action is unknown. However, it has been shown that simultaneous disruption of the genes (FKS1 and FKS2) coding for glucan synthase is lethal and that inhibition with the lipopeptides results in a fungicidal mechanism of action. Since glucan synthesis does not occur in humans, the lipopeptides were thought likely to have the potential for a selective mechanism of action, with low mammalian toxicity. Although this was found to be generally true, unfortunately, several of the early natural products were also found to have a hemolytic action. Medicinal Chemistry Page 47