Pan Assay Interference Compounds (PAINS) and ... - ACS Publications

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Pan Assay Interference Compounds (PAINS) and Other Promiscuous Compounds in Antifungal Research Miniperspective Martin Pouliot and Stephane Jeanmart* Syngenta Crop Protection Research, Schaffhauserstrasse, 4332 Stein, Switzerland

ABSTRACT: Every week, articles disclosing new antifungal leads reported as promising starting points for optimization projects are published. In many cases, the mechanism that accounts for their antifungal activity has not been fully elucidated. More significantly, the detrimental impact that could result from certain embedded chemical features has been underestimated or even overlooked. In the course of our research in the agrochemical area, we have concluded that in many cases such leads are actually nonoptimizable because they either contain what are now recognized as pan assay interference compounds (PAINS) or other promiscuous groups. This article is aimed at highlighting the pitfalls we have encountered and hopefully to steer other research groups away from them.

1. INTRODUCTION Fungicides are an important class of chemicals that are used widely for the treatment of fungal infections of humans, animals, and plants.1 Candidiasis is probably one of the most common human fungal diseases and is normally a harmless infection affecting the skin and/or the mucous membranes of the mouth, intestines, or vagina. However, for patients with disordered immune systems, either acquired or induced by chemotherapy, a Candida infection can be life-threatening.1a,b Unfortunately, there are a limited number of drugs available on the market to treat such fungal infections.1e Over the years, the over-reliance on the same medicines acting on a limited number of modes of action has induced a selection pressure among the originally susceptible pathogens. As a consequence, drug resistance has become increasingly common and has diminished the arsenal of effective antifungal drugs. 2 Furthermore, many of those drugs have additional significant limitations. For example, echinocandins need to be administrated intravenously because of their poor oral bioavailability, and amphotericin B has been known to induce adverse nephrotoxicity.1a In agriculture, extensive use of phytosanitary products has also induced a similar selection pressure toward resistant fungal pathogens. The shift of susceptibility of the plant pathogens is well documented in the literature.3 As a result, many fungicides are nowadays ineffective against fungal strains found in the field. This raises strong concerns, since the uncontrolled development of diseases can ravage crops and subsequently limit food availability and quality. The devastation of potato crops by potato blight which caused the 1840s Irish famine is a striking example of what can happen if a fungal plant disease spreads uncontrollably.4 © 2015 American Chemical Society

Taken together, the two examples mentioned above clearly show the strong need for new molecules that are able to control pathogens showing resistance to our current antifungal products. To bring new antifungals into development, researchers in the area of agrochemicals first need to identify new lead molecules. In order to achieve this, they often rely on screening techniques, with phenotypic screening of new compounds being the most preferred.5 New leads are commonly identified using in-house discovery programs, competitor monitoring, and public sources of information, such as the current literature or databases of active molecules (e.g., ChemBl, PubChem, ChemSpider, UniChem, etc.).5,6 Since the turn of the century there has been a significant increase in the number of publications with the word or concept “antifungal” (see Figure 1).7 The number of these papers has increased by more than 2-fold in less than 14 years. It could easily be perceived that this is a reflection of a similar increase in the number of hits or leads and subsequently numbers of antifungal projects in development in the pharmaceutical or agrochemical industry. Unfortunately, this has not been the case. The reality is that very few compounds are reaching development and this raises the question: Why? Many factors may come into play. To debate of all of them here would be more of a philosophical debate than a scientific endeavor. However, we believe that one key reason is the meager quality of some of these new inhibitors reported in the antifungal literature; many of which contain undesirable features. Received: March 4, 2015 Published: August 27, 2015 497

DOI: 10.1021/acs.jmedchem.5b00361 J. Med. Chem. 2016, 59, 497−503

Journal of Medicinal Chemistry

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Figure 1. Number of publications per year in SciFinder matching the concept antifungal.

the antifungal literature over the past 5 years, we estimate that those publications could cover up to 80% of the new molecules reported to have an antifungal effect. This review sets out to highlight a small sample of undesirable structural features that are repeatedly published in the literature. While the science described is more often than not excellent, key questions are often ignored: Why are the compounds active? The desired mode of action might have been confirmed in vitro, but is it truly responsible for the observed antifungal activity? Do some undesirable chemical features confer fungicidal activity owing to nonspecific binding? If the latter is true, what is the likelihood that such an unselective compound would ever reach the market when the huge hurdle of registration is taken into account? Scientists working in antifungal research understand that killing fungi is not the most difficult task to accomplish. Eradicating a pathogenic fungus from its host without having a negative impact on other living organisms or the environment, however, is a much more demanding endeavor. In this regard, pharmaceutical and agrochemical chemists share many of the same burdens of providing useful antifungal compounds that are safe to use while considering acute toxicity, chronic toxicity, and genotoxicity. An additional responsibility for agrochemical chemists is to ensure no adverse reproductive and developmental effect of the molecules. Bearing commercialization of a molecule as our ultimate goal, we believe that unselective inhibitors increase dramatically the risk of toxic side effects that would impair their registration as antifungals and, therefore, would suggest to us avoiding such molecules as strong leads or hits for new projects.

Frequent hitters, promiscuous compounds, and other false positive inhibitors have been well documented in the recent literature. Mostly published in relation to high-throughput screening (HTS) campaigns, rules describing unwanted molecules have been reported over the years, mainly by the pharmaceutical industry but also by academic groups. For example, GSK has reported filters that their medicinal chemists use to reject molecules containing undesired functional groups.8 Abbott reported their ALARM NMR tool and subsequent procedures to remove or flag potentially thiol reactive compounds.9 Eli-Lilly reported on the filters they use at the front end of their open-innovation platform in order to discriminate the molecules they are interested in testing from unwanted ones.10 In academia, the University of Dundee published a series of SMARTs filters to remove unwanted groups,11 and Sean Ekins et al. published a study toward the phenotypic screening of Mycobacterium tuberculosis and subsequent findings regarding unwanted groups.12 In the same year, Jonathan Baell from the Monash University published his report on pan assays interference compounds (PAINS), with a list of structural features of frequent hitters from six different and independent assays.13 Recently, 2aminothiazoles have been highlighted as promiscuous inhibitors,14 and the chemical mechanisms of assay interference of some thiol reactive promiscuous functions have been revealed.15 As explained earlier, unwanted compounds adversely affect not only enzyme assays but also phenotypic screens. They show biological activity for the wrong reasons but have nevertheless been followed up by various research groups. In vitro phenotypic screening of potential fungicides is particularly easy to set up, as it is technically close to an enzyme assay and does not require complex materials. It is therefore not surprising to see a long list of papers reporting molecules that are fungicidally active by virtue of some embedded undesirable feature. On the basis of our survey and analysis of

2. DISCUSSION One class of molecules often reported as antifungal are rhodanines and molecules containing related scaffolds. These molecules are attractive, since they are easily prepared in two chemical steps. An example from the recent patent literature 498



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Figure 2. PAINS and other promiscuous compounds from the antifungal literature.

potential lack of selectivity between the fungi and the other exposed living organisms. Another class of compounds frequently reported in the antifungal literature is known by synthetic chemists as Michael acceptors. One example of such a molecule reported to have antifungal activity against Aspergillus f umigatus, C. albicans, Botrytis cinerea, and Rhizoctonia solani and containing a conjugated ester as a Michael acceptor is the compound 2 (Figure 2).19 Our experience with this molecule revealed both a weak antifungal activity and reactivity toward thiol. We believe that the biological activity of compound 2 might arise from a nonspecific protein reactivity given its proven ability to form a thiol adduct. It is worth noting that the reactivity toward thiols

discloses (Z)-5-decylidenethiazolidine-2,4-dione (1) as a good antifungal against Candida albicans (Figure 2).16 As a potential carboxylic acid isostere, thiazolidine-2,4-diones may be sufficiently unreactive such that they can progress some way in development.17 Nevertheless, one should be aware of the thiol reactivity associated with this type of molecule, as highlighted by ALARM NMR and glutathione assays,18 and this is especially relevant when the compound contains an exocyclic alkene such as in 1. The fact that rhodanines are promiscuous compounds has been recently highlighted in the literature.13,18 The observed antifungal activity of 1 is certainly genuine; however, this could potentially be the result of in vivo promiscuous reactivity in which case the main issue lies in the 499



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with a cysteine residue.32 Taken together, these effects suggest that the naphthoquinone moiety and its heteroaromatic analogues might inhibit enzymatic activity via nonspecific interactions or by interfering with the redox chemistry of the cell via the depletion of its essential level of glutathione.33,34 Dahlin et al. also earlier suggested that under their experimental conditions, the naphthoquinone moiety seemed an important structural factor of both thiol reactivity and redox activity.15 Another type of promiscuous behavior arises from oxidative metabolism of nitrogen-containing compounds that results in the formation of N-oxides and nitroso intermediates that could react unselectively with proteins. For example, compound 9 depicted in Figure 2 was reported to show antifungal activity on B. cinerea, Pyricularia oryzae, Gibberella zeae, and Sclerotinia sclerotiorum at 50 ppm.35 Inspection of the chemical features of molecule 9 also reveals an activated thioether group. Were this compound to be progressed, the potential for promiscuous reactivity should cause one to do so with utmost caution, especially as again there is the potential for oxidative activation to even more reactive species in vivo. Further, this compound has a hydrazide-type amine group, and these are known to undergo metabolic oxidation to reactive nitrogen species.36 There is no hard evidence that this feature could be accountable for the antifungal activity, but one should be alerted of the potential toxicity risk that it represents. Molecules 10 and 11 have been reported as plant (F. oxysporum) and human (C. albicans, Cryptococcus neoformans, and Aspergillus f lavus) antifungal leads, respectively (Figure 2).37,38 Both of these compounds contain acylhydrazone-like groups. Acylhydrazones are known to be electrophilic, particularly in acidic media such as that of some cellular compartments where the imine carbon could even be reactive toward water.39 Confounding this reactivity is that a potentially toxic hydrazide, discussed above, is released after such hydrolysis. Each of compounds 10 and 11 have further potential liabilities. For example, there is a thiocarbonyl in 10 that could plausibly be oxidized to its sulfene analogue unraveling a site of nucleophilic attack.40 Furthermore, the molecule 10 also contains an acyclic aminal which could readily generate an electrophilic iminium species in vivo. Compound 11 contains an electron poor imine carbon adjacent to a thioether that is likely to be electrophilic. Moreover, very similar analogues to compound 10 have been reported to be effective Fe(III) chelators.41 In the same way, it was described that compounds closely related to 11 could chelate a metal cation via both thiazol-4-one and hydrazine nitrogens, and indeed the observed insecticidal activity was found to be dependent on the metal cation being complexed.42 Metal chelation has been associated with different toxicity issues including developmental toxicity,43 genotoxicity,44 and cytotoxicity.45 While independently it might be possible to overcome these issues, the associated risks might increase the likelihood of observing in vivo adverse effects that could adversely affect the commercialization of the compound. Another moiety that could be treated with caution is the αketocarbonyl functionality, exemplified in molecule 12 (Figure 2). Molecule 12 was claimed to have antifungal activity against different Aspargillus species by the company F2G Limited.46 It has been mentioned in papers compiling promiscuous groups that α-ketocarbonyl functionality can be electrophilic.10,47 We are aware that this is certainly not always the case, as the intrinsic electrophilicity depends on both the steric environment and the geometry of the ketocarbonyl functional group.

might vary with the pH and could potentially be catalyzed by a protein. The analogues of the natural product kakuol, molecules 3a and 3b (Figure 2),20 are two other examples of antifungal molecules containing a Michael acceptor functionality; they were both reported to control the growth of plant pathogens such as Phytophthora infestans and Pythium ultimum. In addition to their intrinsic potential to react with thiols, compounds 3a and 3b incorporate a 1,3-benzodioxole moiety which raises, in our opinion, further concerns. Indeed, 1,3-benzodioxole itself has been reported to behave as a quasi-irreversible cytochrome P450 (CYP) inactivator.21 It is well-known in the literature that inhibition of CYP51 is an excellent mode of action for the control of fungi.22 However, if the observed antifungal activity of compounds 3a and 3b is due to a nonselective quasiirreversible inhibition of a broad scope of CYP enzymes, this could result in toxic side effects. While some reactive functional groups can be identified rather easily, others are much less obvious and might pass the scrutiny of a watchful chemist unnoticed. One such case might be when a reactive and undesirable functionality is only revealed in the conditions used for screening. During the course of our research, we investigated the antifungal activity of lactone 4 (Figure 2), a prototypal compound of a class of published and patented antifungal lactones active against C. albicans, A. f umigatus, and Fusarium oxysporum.23 We observed in our testing platform that at physiological pH lactone 4 is protonated and in equilibrium with its open chain ester 5, revealing through this retro-Michael reaction a reactive olefin. After 24 h of incubation, cell culture extracts showed complete degradation of both compounds 4 and 5, and LC/MS analysis could only reveal a hydroxylated metabolite.24 While we did not attempt to identify a covalently bound protein adduct resulting from addition to the Michael acceptor 5, our observations would indicate that such a scenario is plausible. Other types of electrophiles delivering fungicidal activity against various C. albicans isolates are depicted in the compound 6 (Figure 2).25 Medicinal chemists would recognize two activated thioether linkages in this molecule, one adjacent to a pyrimidin-2-yl and another adjacent to an oxadiazol-2-yl. In both cases the respective heterocycles are strongly electronwithdrawing and an experienced medicinal chemist would probably agree that both of those moieties could show nonspecific reactivity with biological nucleophiles. In fact, we have actually observed thiol reactivity for this compound in our test systems, which we suggest could plausibly arise through nucleophilic attack of either (or both) of these activated thioether groups. Furthermore, thioether groups are known to have a propensity to undergo metabolic oxidation in fungi,26 which in this case would give rise to even more reactive compounds. Quinazolinedione 7 and naphthoquinone 8 depicted in Figure 2 are among a series of quinone derivatives that have been reported in the recent literature to deliver antifungal activity against diverse Candida and Aspergillus species.27,28 Generation of hydrogen peroxide via redox cycling of quinone has been described in the literature.29 Recently, quinazolinediones have been specifically reported to act as redox modulators.30 Moreover, the thiol reactivity of electrophilic quinones has also been highlighted by a fluorescence-based high throughput assay.31 In yet another example, 2,3-bis(2hydroxyethylsulfanyl)naphthalene-1,4-dione was shown to inhibit the tumor marker S100B by forming a covalent bond 500



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chemical design. Is the activity genuine? All the compounds described in Figure 2 have shown some level of antifungal activity in our screens. Are they a useful starting point for optimizing the antifungal activity? We believe the answer is “no”. None of the above-mentioned compounds have shown useful activity once promoted to our in-house higher tier tests.54 We think that this is likely to be the result of those compounds interfering with nontarget organisms in the same unselective manner as they do with the fungi in phenotypic screening. We realize that the task of discovering an antifungal is challenging and the funding of research limited, and this is why we believe that all researchers should be aware of certain pitfalls before embarking in extensive (and expensive) research programs. While we are happy to see the number of papers dealing with antifungal research increasing over the years, we would prefer even more to see an increasing number of successful cures being discovered. Ultimately, this should be our only goal.

For example, rapamycin which contains a masked tricarbonyl moiety is a commercial drug.48 It is interesting to note that before being surpassed by its immunosuppressant activities, rapamycin was initially developed for its antifungal properties.48b The huge success of rapamycin notwithstanding, we believe that a α-ketocarbonyl group is a functionality that one should incorporate in molecular design understanding the possible pitfalls described above. The molecule 13 has been reported to possess antifungal activity against plant pathogenic pathogens like Fusarium solani and Alternaria brassicicolla by researchers in Japan (Figure 2).49 This compound is a gallate, effectively containing two catechol moieties. Catechols are well documented PAINS moieties, being able to redox-cycle to reactive quionoids and to chelate metals.13 In fact, it has been reported that gallic acid itself induces apoptosis in promyelocytic leukemia HL-60RG cells by the participation of reactive oxygen species (ROS),50 and ROS have been reported to kill fungi.51 This could represent the antifungal mechanism of 13. Indeed, its antifungal activity could be due to gallic acid, which would be produced should the ester bond in 13 undergo esterase-mediated hydrolysis intracellularly. If this is the case, the SAR of molecule 13 and its analogues reported in the paper could thus be a consequence of the different physicochemical properties of the various esters that might vary in esterase reactivity and fungal cell wall permeability.49 While ROS could effectively control a fungal infection, generated nonspecifically and in high concentration in mammalian cells, this might not be a desirable feature with regard to putative toxicity.33 The last example we disclose is illustrated in phenazine 14 (Figure 2) which was reported as an antifungal agent in the literature.52 This type of moiety is an archetypal example of a very flat molecule that may generate some fungicidal activity as a result of their intercalation into the DNA. It has been published in the literature that phenazine 1-carboxamides similar to 14 can interact with DNA and act as potential antitumor agents.53 Should one investigate further molecules with such type of mode of action with the aim of treating an invasive fungal infection? Our experience would guide us away from such moiety because of the challenge associated with delivering such an antifungal molecule without detrimental side effects or toxicity. The molecules presented above are only meant to represent a small sample of the many antifungal compounds published. Our learning from several years of research and surveys of the antifungal literature area is similar to what was observed in other areas,36 and we believe that even though PAINS were identified based on target-based HTS, they also have great relevance to phenotypic screening hits. Although not discussed in this paper, membrane-perturbing amphiphilic compounds are another issue that can be encountered upon phenotypic screening. Experience has taught us that when a class of molecules presents a potentially reactive functionality, a group recognized by many medicinal chemists as promiscuous or belonging to the long list of PAINS, it is extremely difficult to progress a compound to the market. This has led us to conclude that precious resources would be better placed in projects arising from leads unblemished by promiscuous functionality.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +41 62 866 02 05. Notes

The authors declare the following competing financial interest(s): The authors are employees of Syngenta Crop Protection Münchwilen AG. Biographies Martin Pouliot is a team leader chemist in the Fungicide Research Group at Syngenta. He received his Ph.D. in Organic Chemistry from the Université Laval, Canada, under the supervision of Prof. John Boukouvalas, where he worked on the synthesis of natural products. He then carried out postdoctoral research on radical chemistry in the group of Prof. Philippe Renaud at the University of Bern, Switzerland. His research interests at Syngenta are focused on the design and synthesis of small molecule agrochemicals for the antifungal indication. Stephane Jeanmart obtained his B.S. and M.S. in Chemistry from the University of Namur, Belgium. He received his Ph.D. degree in Organic Chemistry from the same university. After 18 months of a postdoctoral position at the University of York, U.K., he joined Syngenta as a team leader working toward the discovery of new herbicides. In 2009, he moved within the same company to perform research in the fungicide indication at the Stein Research Center in Switzerland. He is currently the Head of Fungicide Lead Generation at Syngenta.

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ACKNOWLEDGMENTS The authors thank Dr. Guillaume Berthon for useful discussions on promiscuous compounds, Dr. Chris Godfrey and Dr. Andy Edmunds for their help in preparing this manuscript, and all our colleagues from the fungicide indication for their help in preparing some of the compounds described in this article. The referees are also thanked for their precious advice and comments to shape the final version of this manuscript.

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ABBREVIATIONS USED CYP, cytochrome P450; HTS, high throughput screening; PAINS, pan assay interference compounds; ROS, reactive oxygen species

3. CONCLUDING REMARKS Every day new papers claiming antifungal activity are published with PAINS or promiscuous groups embedded in their 501



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(13) Baell, J. B.; Holloway, G. A. New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for their Exclusion in Bioassays. J. Med. Chem. 2010, 53, 2719−2740. (14) Devine, S. M.; Mulcair, M. D.; Debono, C. O.; Leung, E. W. W.; Nissink, J. W. M.; Lim, S. S.; Chandrashekaran, I. R.; Vazirani, M.; Mohanty, B.; Simpson, J. S.; Baell, J. B.; Scammells, P. J.; Norton, R. S.; Scanlon, M. J. Promiscuous 2-Aminothiazoles (PrATs): A Frequent Hitting Scaffold. J. Med. Chem. 2015, 58, 1205−1214. (15) Dahlin, J.; Nissink, W.; Strasser, J. M.; Francis, F.; Higgins, L. A.; Zhou, H.; Zhang, Z.; Walters, M. A. PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed During a Sulfhydryl-Scavenging HTS. J. Med. Chem. 2015, 58, 2091−2113. (16) Srebnik, M.; Polacheck, I.; Steinberg, D.; Jabbour, A.; Sionov, E. Novel Anti-Biofilm Agents. WO2010/058402 A1, 2010. (17) Baell, J. B.; Ferrins, L.; Falk, H.; Nikolakopoulos, G. PAINS: Relevance to Tool Compound Discovery and Fragment-Based Screening. Aust. J. Chem. 2013, 66, 1483−1494. (18) Tomosic, T.; Masic, L. P. Rhodanine as a Scaffold in Drug Discovery: a Critical Review of its Biological Activities and Mechanisms of Target Modulation. Expert Opin. Drug Discovery 2012, 7, 549−560. (19) (a) Lu, A.; Wang, J.; Liu, T.; Han, J.; Li, Y.; Su, M.; Chen, J.; Zhang, H.; Wang, L.; Wang, Q. Small Changes Results in Large Differences: Discovery of (−)-Incrustoporin Derivatives as Novel Antiviral and Antifungal Agents. J. Agric. Food Chem. 2014, 62, 8799− 8807. (b) Pour, M.; Spulak, M.; Balsanek, V.; Kunes, J.; Buchta, V.; Waisser, K. 3-Phenyl-5-Methyl-2H,5H-Furan-2-ones: Tuning Antifungal Activity by Varying Substituents on the Phenyl Ring. Bioorg. Med. Chem. Lett. 2000, 10, 1893−1895. (20) Musso, L.; Dallavalle, S.; Merlini, L.; Farina, G. Synthesis and Antifungal Activity of 2-Hydroxy-4,5-methylenedioxyaryl Ketones as Analogues of Kakuol. Chem. Biodiversity 2010, 7, 887−897. (21) Orr, S. T. M.; Ripp, S. L.; Ballard, T. E.; Henderson, J. L.; Scott, D. O.; Obach, R. S.; Sun, H.; Kalgutkar, A. S. Mechanism-Based Inactivation (MBI) of Cytochrome P450 Enzymes: Structure-Activity Relationships and Discovery Strategies to Mitigate Drug-Drug Interaction Risks. J. Med. Chem. 2012, 55, 4896−4933. (22) Choi, J. Y.; Podust, L. M.; Roush, W. R. Drug Strategies Targeting CYP51 in Neglected Tropical Diseases. Chem. Rev. 2014, 114, 11242−11271. (23) (a) Bardiot, D.; Cammue, B.; Chalatin, P.; Marchand, A.; Thevissen, K. Preparation of Morpholinylacetamide Derivatives as Antifungal Agents. WO2011072345 A1, 2011. (b) Bardiot, D.; Thevissen, K.; De Brucker, K.; Peeters, A.; Cos, P.; Taborda, C. P.; McNaughton, M.; Maes, L.; Chaltin, P.; Cammue, B. P. A.; Marchand, A. 2-(2-Oxo-morpholin-3-yl)-acetamide Derivatives as Broad-Spectrum Antifungal Agents. J. Med. Chem. 2015, 58, 1502−1512. (24) We putatively assigned this metabolite as the product of water addition to the reactive olefin. (25) Kaplancikli, Z. A. Synthesis of Some Oxadiazole Derivatives as New Anticandidal Agents. Molecules 2011, 16, 7662−7671. (26) Roberts, T. R.; Huston, D. H. Organophosphorus Insecticides. In Metabolic Pathways of Agrochemicals, Part 2: Insecticides and Fungicides; Roberts, T. R., Huston, D. H., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 1999; pp 182−522. (27) Ryu, C.-K.; Kim, Y. H.; Im, H. A.; Kim, J. Y.; Yoon, J. H.; Kim, A. Synthesis and Antifungal Activity of 6,7-bis(Arylthio)-quinazoline5,8-diones and Furo[2,3-f ]quinazolin-5-ols. Bioorg. Med. Chem. Lett. 2012, 22, 500−503. (28) (a) Ibis, C.; Tuyun, A. F.; Bahar, H.; Ayla, S. S.; Stasevych, M. V.; Musyanovych, R. Y.; Komarovska-Porokhnyavets, O.; Novikov, V. Nucleophilic Substitution Reactions of 1,4-Naphthoquinone and Biologic Properties of Novel S-, S,S-, N-, and N,S-Substituted 1,4Naphthoquinone Derivatives. Med. Chem. Res. 2014, 23, 2140−2149. (b) For another related example see the following: Ryu, C.-K.; Shim, J.-Y.; Chae, M. J.; Choi, I. H.; Han, J.-Y.; Jung, O.-J.; Lee, J. Y.; Jeong, S. H. Synthesis and Antifungal Activity of 2/3-Arylthio- and

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