Jasmine Choi, Department of Chemistry, Monash University

Abstract

Leishmaniasis are a complex of diseases with a significantly negative prognosis. High mortality rates are accentuated in the absence of medical intervention. Leishmania parasites have exhibited susceptibility to chelated heavy metals, specifically antimony and bismuth. Certain arrangements of ligands chelated to heavy metal centres are postulated to increase the permissibility of compounds through the parasitic membrane. Deliverance of the metal centre in a reduced and antimicrobial form may result in bactericidal effects while concurrently reducing the cytotoxic effect on human fibroblast tissues. A series of three simple substituted derived sulfonic acids are chelated to bismuth (III/V) via a variety of mechanisms, the effectiveness of which will be examined. The compound characterisation of 14 new chelated bismuth (III/V) substituted sulfonic acids were analysed via a range of analytical techniques with a biological assay of antimicrobial and cytotoxic effects. Compounds I–III displayed no toxic effects on either fibroblasts or promastigotes, whereas VI, X, XI and XIII appeared to show potent antimicrobial effects and displayed cytotoxic properties with the human fibroblast cell line. However, compound XIV displayed selective activities between promastigotes and fibroblast cells. Further analysis into the structure and function of these compounds may prove to be beneficial in drug development.

Keywords: leishmaniasis, bismuth, cytotoxicity, antimicrobial drug design, analytical chemistry, promastigotes

Introduction

Chemical synthesis of bismuth compounds

As a coordination metal, bismuth may be chelated by desired ligands with the correct application of experimental design. The sheer range and possibilities allow for different niches to be fulfilled by engineered chemical compounds, based on the biomedical and chemical properties of the chelated ligands (Ould-Ely, Thurston and Whitmi,2005: 1907–20).

Bismuth with an atomic weight of 208.98, displays several oxidations states with different ionic radii and stabilities. However, only two of these are of a biological interest (III and V). These are the only two biologically active oxidation states as bismuth is traditionally insoluble in aqueous media (Thomas, Bialek, & Hensel, 2011: 1). Bi (III) is a less powerful oxidant than Bi (V) and displays affinities to nitrogen and oxygen, allowing for hydrolysation in water, but preferentially coordinates to available thiolate groups (Yang and Sun, 2007: 2355). As Wang and Xu describes, Bi (III) chelated with citrate can be readily displaced by deprotonated thiolate functional groups of cysteine amino acids under the right chemical conditions. Replacing the attached citrate ligand forms a coordination complex through three sulfur interactions as Bi-S bonds. This indicates a promising outcome for synthesis and generation of sulfonic acid complexes as they are structurally similar to cysteine residues. The chelated sulfonic acid functional groups used are homo- and hetero-atom containing ligands; benzene substituted with dimethyl and dinitro functional groups paired with different combinations of bismuth metal centre oxidation states. Bi (V) complexes contain a higher-valent state relative to the III oxidation state, resulting in a strong but highly stable oxidant, especially in aqueous media (Yang and Sun, 2007: 2355).

Specific Bi (III) compounds were synthesised with a range of techniques, including sonication, solvent-free reactions/differential scanning calorimetry and solvent-mediated/reflux reactions. Alternatively, Bi (V) can be selectively chelated with the presence of oxidising agents (hydrogen peroxide, triethylamine) or mediated by a salt metathesis reaction. Both classes of reactions depend on molar ratios of reactants and reaction conditions for the select generation of the final compound and yields. Generally, experimental synthesis of product yields were relatively high as bismuth acts as a self-catalyst for chelation. The generated compounds were of a monodentate chelate mode to the metal centre. Analysis of a synthesised compound is vital for the identification and verification of a given chemical compound as there could potentially be a range of structural isomers. Analytical chemistry of the generated compounds includes melting point analysis, infrared spectroscopy, nuclear resonance spectroscopy and mass spectrometry, which assist in determining the identity, purity and product yield.

Leishmania parasites and Leishmaniasis

Leishmania parasites are subdivided between two groups, characterised by the differing developmental sites while within the female sand-fly vector, Old (Phlebotomus) and New World (Lutzomyia) (Herwaldt, 1999: 1191–93). There are two clinically relevant stages of the parasite: the intracellular amastigote and the extracellular promastigote (Pace, 2014: 12). Leishmaniasis manifests as a clinical spectrum of diseases of diverging severities, which are factored by the infecting protozoan strain, extent of medical interventions and host immunological responses (Mansueto et al., 2014: 563–74). This can range from a cutaneous skin disease, which results in lesions, to the disseminated visceral form of the disease, which is most often fatal. India and Sudan report the highest disease burden of visceral leishmaniasis. However, exposure to the sand-fly vectors during travels abroad can result in infections (Moore and Lockwood, 2011: 492–97). With the exception of Australia and Antarctica, leishmaniasis is endemic globally. Regarding mortality rates associated with human parasitic diseases, leishmaniasis reports are penultimate after malarial diseases (Pace, 2014: 10) with a reported mortality rate of 20,000–30,000 and 900,000 to 1,300,000 new cases annually (World Health Organization, 2016). The untreated disseminated or visceral form reports a fatality case rate of 95% (World Health Organization, 2016).

Leishmania treatment can vary from category to category due to the different clinical manifestations of the disease (González et al., 2008: 4-90). Since the 1940s, the frontline treatment includes multi-therapies of pentavalent antimonials (Sb; sodium stibogluconate and meglumine antimoniate), which are cost-effective as a long-term treatment option for cutaneous lesions (Vieira‐Gonçalves et al., 2015: 1–3) (Rocha, 2013: 1–7). However, Frézard Demicheli and Ribeiro (2009) commented on the limitations of pentavalent antimony treatments; common severe side effects and the requirement of a parenteral administration over three weeks with medical observations. Other leishmaniasis treatment options include liposomal Amphotericin B and Miltefosine, however, relatively high prices form access-to-treatment barriers (Chappuis et al., 2011: 455-456) and fluconazole which can be applied in the cutaneous form of the disease (Alrajhi et al., 2002: 891–95).

Emergence of clinically resistant parasitic strains, especially against pentavalent antimony drugs, have reached epidemic proportions in the North Bihar region of India. Leishmaniasis exhibits unique thiol metabolism, which is demonstrated to play a key function with the action of antimonial drugs. The primary low-molecular-mass thiol is trypanothione and, after interaction with biologically active trivalent antimony, is actively effluxed from the cell, resulting in disruptions to thiol homeostasis (Chakravarty and Sundar, 2010: 167–73). The emergence of antimony resistance implicates several different proteins and subsequently their associated mechanisms: multidrug resistance-associated protein A (MRPA), aquaglyceroporin, heat shock protein 70 and trypanothione reductase (Torres et al., 2013: 139), (Ashutosh, Sundar and Goyal, 2007: 144–49). Some other potential resistance mechanisms include diminished reduction of Sb (V) to Sb (III) (Ashutosh, Sundar and Goyal, 2007: 145–46) and coinfection with HIV, which increases carriage rates of drug-resistant parasites (Chakravarty and Sundar, 2010: 167–73). Subsequently, there is a demand for alternative, low-cost, safe, orally active treatments with reduced toxicity and side effects that do not exhibit cross-resistance with conventional antimonial drugs.

Antimicrobial activity of bismuth compounds in leishmaniasis

Bismuth is traditionally utilised as antimicrobial metallodrug compounds for its low cytotoxicity properties attributed to the insolubility within aqueous media (Yang et al., 2015: 389–90) (Ge and Sun, 2007: 267–74). The modern bismuth compounds are primarily used as treatment regimens for Helicobacter pylori eradication (Thomas, Bialek and Hensel, 2011: 1). The bioutility of bismuth-containing compounds includes colloidal bismuth subcitrate, bismuth subsalicylate and tripotassium dicitrato bismuthate in the treatment of gastric and small intestinal ulcers. These mechanisms of actions are proposed to be gastric mucus fortification, cytoprotective immune processes and mucosal bicarbonates. However, other antimicrobial bismuth preparations have contributed to the treatment of a range of microbial diseases including syphilis (Treponema pallidum), diarrhoea (enteric strains of Escherichia Coli) and colitis (Briand and Burford, 1999: 2602).

Efficacy of bismuth compounds in the medical treatment of H. pylori are clinically significant (Schlesinger et al., 2014: 4223–24) and exceed that of regular proton pump inhibitor therapies (Yang and Sun, 2007: 2355–56). Arenesulfonates-complexed bismuth preparations showed efficacy against H. pylori in vitro with low toxicity to human-derived cells (Andrews et al., 2012a: 7583–85). Andrews et al. (2011a) also synthesised a suite of bismuth (III) thiosaccharinate and saccharinate compounds that showed anti-leishmanial properties unapparent in the precursor molecules of thiosaccharin, saccharin and BiPh3. The same H. pylori bactericidial activity was viewed with bismuth (III) sulfonate (Andrews et al., 2012b: 11798–806) and bismuth (III) β-thioketonate compounds (Andrews et al., 2014: 1278–91).

Thiol functional groups are substantial within enzyme active site structures (Leung-Toung, Li, Tam and Kaarimian, 2002: 979–1000). A potential mechanism for the observed antimicrobial effect manifests in a chelated bismuth metal centre entropically displacing particular thiol groups via reduction to decrease protozoan cell viability (Thomas, Bialek and Hensel, 2011: 2).

The indication of anti-leishmanial promastigotes activity using libraries with different ligands of bismuth metallodrugs appear to have therapeutical potential (Andrews et al., 2011b; Andrews et al., 2014; Luqman et al., 2014; Ong et al., 2014; Loh et al., 2015; Ong et al., 2015). Further studies on metallodrug candidates with high efficacy and low cytotoxicity with good oral bioavailability would be ideal to minimise the requirement for frequent parenteral treatments.

Results and Discussion

Generation of compounds

A chemical library of three commercially available sulfonic acids (Figure 1) was used to generate a series of 14 Bi(III/V) compounds (Table 1).

Table 1: Synthesis of bismuth (III/V) sulfonic acid compounds I-XIV with respective precursors, mode of synthesis, final appearance and yield.
Compound	Bismuth Precursor	Sulfonic Acid	Mode of Synthesis	Solvent	Appearance	Yield (%)
I	Bi(NO3)3.5H2O	BSA	Sonication	H2O	White powder + crystals	0.91
II	Bi(NO3)3.5H2O	DNSA	Sonication	H2O	Yellow powder + crystals	1.02
III	Bi2O3	DMSA	Sonication	H2O	Light brown powder	23.81
IV	Bi2O3	DNSA	SM	Toluene	Light brown powder	25.92
V	Bi(Ph)3	BSA	OA	H2O	Light brown powder	38.99
VI	BiPh3Cl2	BSA	SMet	THF	White powder	48.24
VII	BiPh3Cl2	BSA	TEA	Acetone	White crystals	11.88
VIII	BiPh3Cl2	BSA	SF	Solvent free	Light brown powder	38.73
IX	BiPh3Cl2	BSA	SM	H2O	Light brown powder	20.90
X	BiPh3Cl2	DNSA	TEA	Acetone	White crystals	20.72
XI	BiPh3Cl2	DMSA	SM	Diethyl ether	White powder + crystals	98.52
XII	BiPh3Cl2	DNSA	SF	Solvent free	Brown powder	40.51
XIII	BiPh3Cl2	DMSA	TEA	Acetone	White crystals	18.22
XIV	BiPh3	BSA	TEA	Acetone	White crystals	16.07

Table 1: Synthesis of bismuth (III/V) sulfonic acid compounds I-XIV with respective precursors, mode of synthesis, final appearance and yield.
Solvent free (SF); Solvent mediated (SM); Triethylamine (TEA); Salt Metathesis (SMet); Oxidative Addition (OA); Tetrahydrofuran (THF)

Bi (III) complexes could be generated from five different precursors: 1. Bi₂O₃; 2. Bi(NO₃)₃.5H₂O; 3. Bi(Ph)₃; 4. BiCl₃; 5. Bi₂Mo₃O₁₂. Notably, Bi(NO₃)₃.5H₂O experimentally reacts stronger with sulfonic acids than Bi₂O₃.

An ultrasound bath is used to sonicate reactants in a variety of solvents; the mechanism of action is suggested to be due to turbulence and pressure generated by imploding cavities in the liquid medium (Suslick, 1989: 80–86). Reflux and solvent-mediated synthesis generates products through solvent selectivity (toluene and diethyl ether) with rotary evaporation after reaction completion. Solvent-free or Differential Scanning Calorimetry (DSC), a thermoanalytical technique, can be used in Bi (III) compound synthesis due to the speed, simplicity and is readily available. DSC was used to augment the reaction between a triphenylbismuth (III) dichloride precursor and a chosen sulfonic acid equivalent (BSA-VIII, DNSA-XII). The DSC plot of the reaction between DNSA and BiPh₃Cl₂ is shown in Figure 2 (compound XII). The trace displays an exothermic reaction at 100–125°C, which indicates a protolysis of the BiPh₃Cl₂ and chloride dissociation. The reaction was then conducted at 115°C for optimal reaction progress.

Figure 2: DSC scan of the solvent-free reaction generation of compound XII ((BiPh₃(O₃SC₆H₃(NO₂)₂)₂) during a substitution reaction between three equivalents of DNSA with one equivalent of BiPh₃Cl₂

Bi (V) complexes could be generated with an oxidative addition reaction, where the oxidative state of Bi (III) in the form of Bi(Ph)₃ is mixed with a sulfonic acid derivative in the presence of 30% hydrogen peroxide in a 1:2:1 stoichiometric ratio (Scheme 1). Acting as an oxidising agent, hydrogen peroxide oxidises the Bi (III) to Bi (V). Subsequent collision of the metal centre with the sulfonic acid functional group then allows for chelation.

Scheme 1: General synthesis of tris-aryl bismuth(V) bis-sulfonic complexes with a hydrogen peroxide catalyst

BiPh3+ 2 RSO3H H₂O2 → -H₂O BiPh3(RSO3)2

Triethylamine (TEA) reactions utilise basic properties to scavenge excess protons (Scheme 2), through salt precipitation as triethylamine hydrochloride and catalysing the formation of the product. When acetone is added to the reaction mixture, formations of bismuth complexed crystals throughout the solution can be observed with each sulfonic acid entrance in the library – DMSA (XIII), DNSA (X) and BSA (VII, XIV). The crystals generated by synthesis of compound XIV are exhibited in Appendix 4.

Scheme 2: General synthesis of tris-aryl bismuth(V) bis-sulfonic complexes using a TEA catalyst

BiPh3Cl2+2C6H5SO3HTriethyl amine ⟶ -HCl BiPh3(C6H5SO3)2

Comparatively, Scheme 3 utilises a salt metathesis scheme for silver oxide. This then generates a deprotonated form of the sulfonic acid. Subsequent complexion of the reactive mixture with a Bi (V) metal precursor then allows for substitution.

Scheme 3: General synthesis of tris-aryl bismuth(V) bis-sulfonic complexes using a metal mediated approach

Ag2O+2 C6H5SO3HTHF → 90˚C 2 Ag+(C6H5SO3)-+ H₂O

BiPh3Cl2+2Ag+(C6H5SO3)-THF → 90˚C BiPh3(C6H5SO3)2+2AgCl

There were different efficiencies seen throughout the different modes of synthesis. Bi (V) complexes yields were varied across all modes of synthesis with DSC generally producing higher yields; however, TEA reactions producing the highest quality crystal formations with the highest purity (sharp, small melting point range) and a modest yield (Figure 3). It is suggested that the reaction is incomplete and leads to a heteroleptic compound.

Figure 3: Experimental bismuth compounds with absolute percentage yield (%) as measured reactants and generated species weight displayed in groups according to the chemical synthesis approach utilised

Chemical analysis

Compound	MP (°C)	FT-IR	1H-NMR	13C-NMR	Mass Spectrometry	Indication of Reaction
I	>300	o	-	✓	✓	Positive
II	>300	✓	✓	✓	✓	Positive
III	259	o	✓	✓	✓	Positive
IV	210	o	-	✓	✓	Positive
V	82	✓	✓	-	-	Positive
VI	179	-	✓	-	-	Inconclusive
VII	149	✗	o	-	-	Negative
VIII	133	-	✗	✓	-	Positive
IX	159	✓	✓	✓	-	Positive
X	159	-	✓	-	-	Positive
XI	>300	✓	✓	✓	✓	Positive
XII	127–129	-	-	-	-	Inconclusive
XIII	140		✓	✓	-	Positive
XIV	85	✓	✓	✓	-	Positive

Table 2: Completion of different chemical analysis modules for compounds I–XIV and confirmation of a successful reaction
Melting Point (MP), Fourier Transformation Infrared Radiation Spectroscopy (FT-IR), ¹³C-Nuclear Magnetic Resonance (¹³C-NMR),¹H-Nuclear Magnetic Resonance Spectroscopy (¹H-NMR), Electrospray Ionisation Mass Spectrometry (Mass Spectrometry), (-) chemical test was not performed, (✓) chemical test is indicative of a successful reaction, (✗) chemical test indicative of an unsuccessful reaction, (o) inconclusive and ambiguous test results

Melting Point (MP), Fourier Transformation Infrared Radiation Spectroscopy (FT-IR), 13C-Nuclear Magnetic Resonance (¹³C-NMR),1H-Nuclear Magnetic Resonance Spectroscopy (¹H-NMR), Electrospray Ionisation Mass Spectrometry (Mass Spectrometry), (-) chemical test was not performed, (✓) chemical test is indicative of a successful reaction, (✗) chemical test indicative of an unsuccessful reaction, (○) inconclusive and ambiguous test results

Table 2 describes the different conformational analysis of compounds I-XIV, through the following chemical analysis: Melting Point (MP), Fourier Transformation – Infrared Radiation Spectroscopy (FT-IR), 13C-Nuclear Magnetic Resonance (¹³C-NMR), 1H-Nuclear Magnetic Resonance Spectroscopy (¹H-NMR), Electrospray Ionisation Mass Spectrometry (Mass Spectrometry). Appendix 3 displays the sulfonic acid vibrational frequencies for comparison for the generated ¹H-NMR spectrum analysis of each bismuth sulfonic acid complex when dissolved in dimethyl sulfoxide (DMSO). When contrasted with the spectra of precursor bismuth compounds, the results display low-frequency shifts for their complexed sulfonic acid resonances compared with their parent free acids, indicating complexion to their respective bismuth precursors. Further ¹³C-NMR analysis measured expected carbon environments within a characteristic chemical shift environment, generating a proportional number of quaternary carbon environments, which indicates the formation of expected products. Full details are provided in Appendix 2.

Melting point comparison with respective precursor bismuth compounds and the generated products are indicative of the occurrence of a chemical reaction. As observed in Figure 4, there were significant shifts between compounds III and VI and the common precursor Bi2O3. Compound XI displayed high melting point, indicating a high compound stability. A different melting point was generated in each reaction class, except the TEA reaction (Scheme 3) which exhibited little deviation from precursor melting points.

Figure 4: The mode of bismuth compound chemical synthesis when contrasted with observed melting points with precursor bismuth references for successful reaction synthesis indications

Biological analysis

Compounds I, II, III, VI, X, XI, XIII and XIV were performed against L. major promastigotes and human fibroblasts. All tested bismuth compounds were dissolved in DMSO; however, compounds I, II, III, XI and XIII remained cloudy and with sediment remaining following sonication, which indicates partial insolubility in DMSO. The DMSO control showed no apparent effect on any of the promastigotes or human fibroblast cells. Amphotericin B was the reference anti-leishmanial reagent, fixed at concentrations equivalent to that of the compound concentrations (48 nM to 100 μM). Full experimental assay methodologies are detailed in Appendix 2.

Figure 5: Anti-leishmanial activity of complexes I, II, III, VI, X, XI, XIII and XIV after 48 hours of exposure towards L. major promastigotes at 2-fold serial dilutions of compound concentration between 100 to 0.048 μM

For each individual compound entry, percentage viabilities were calculated with complex concentrations and we analysed the data to obtain the IC50 for each complex based on their activity against promastigotes and fibroblasts. A selectivity index was calculated from the ratio n of the fibroblasts to the IC50 of the promastigotes. The resulting IC50 towards fibroblasts, IC50 towards promastigotes and selectivity index values are presented in Table 3. Figure 5 displayed the observed antimicrobial activity present from compounds VI, X, XI, XIII and XIV with a relatively poor antimicrobial performance from the sonicated compounds (I, II and III) at the given biological concentrations. For anti-promastigote activity, compound X ((BiPh3(O3SC6H3(NO2)2)2) and compound XI ((BiPh3(O3SC6H3(CH3)2)2) displayed similar IC50 values: 1.09 μM and 1.02 μM respectively, followed by generated (BiPh3(O3SC6H5)2 compounds by compound XII synthesised by TEA (2.13 μM), and lastly by VI synthesised by a salt metastasis reaction at 3.08 μM.

Figure 6: Cytotoxicity activity of complexes I, II, III, VI, X, XI, XIII and XIV after 48 hours of exposure towards human fibroblasts at serial compound concentration dilutions between 100 to 0.048 μM

For fibroblast cell assay (Figure 6), the same trends from the promastigote assays were also observed, with the sonicated samples (I, II and III) deriving little cytotoxic properties. The highest IC50 concentration towards fibroblasts with a similar trend was observed in the promastigote assays (Figure 5). In this assay, compounds that exhibited cytotoxic properties at the lowest concentrations were compound XI (5.74 μM) and compound X (6.01 μM). Compounds VI (9.03 μM) and XII (9.69 μM) also produced a similar yet relatively higher IC50 value. Compound XIV exhibited little to no cytotoxicity with an IC50 value greater than the measured 100 μM. However, it still displayed an antimicrobial effect, as observed with an IC50 value of 43.35.

From IC50 comparison across different sulfonic acid ligands, it can be observed that while compound X and XI were more toxic to L. major promastigotes, they were also cytotoxic. This was likewise exhibited by compounds VI and XIII but with a greater therapeutic window. The sonicated series were relatively identical in relation to both parasites and fibroblasts activities and did not display selectivity. Compound XIV yielded the greatest potential for further study as it exhibited little to no cytotoxic properties and yet partially inhibited promastigote viability.

Compound	IC50(µM)	IC50 (µM)	Therapeutic Index
	Fibroblasts	Promastigotes
I	>100	>100	1
II	>100	>100	1
III	>100	>100	1
IV	-	-	-
V	-	-	-
VI	9.03	3.08	2.93
VII	-	-	-
VIII	-	-	-
IX	-	-	-
X	6.01	1.09	5.51
XI	5.74	1.02	5.63
XII	-	-	-
XIII	9.69	2.13	4.55
XIV	>100	43.35	2.306

Table 3: Biological assay screens of compounds I–XIV, with IC50 concentrations against fibroblasts, promastigotes and the derived therapeutic index.
(-) Biological assays were not performed

Conclusion

The formation of a set of homoleptic and heteroleptic bismuth (III/V) sulfonic acid complexes through the reaction of bismuth precursors with a set of three sulfonic acids was performed with six reaction schemes. Eight of the respective 14 compounds were screened by biological assays. Out of the screened assays, the most promising results were of compounds VI, X, XI, XIII and XIV, which demonstrated some antimicrobial activity against promastigotes. These were attributed to the chelated sulfonic acids. Compounds I-III, which chelated bismuth metal in a +3 oxidation state via sonication, displayed little effect on either fibroblasts or promastigotes. Suggested project extensions include crystallography of generated crystalline bismuth structures and diffraction data for structural resolution; performance of biological assays in water as a solvent; for assessment of a bioavailable drug delivery and the effect of water on the potency of the metallodrugs as antimicrobials. A further analysis, based on the selectivity index, of the most promising compounds (VI, X, XI, XIII and XIV), could potentially be assayed against L. major amastigotes, the most pertinent form during disease states. The biological assays generated from compound XIV is a potential candidate for multi-therapy treatment as it exhibited little cytotoxicity with some anti-leishmanial activity. There are research opportunities in studying the effects of different ligands chelating bismuth metal centres in the discovery of further potent antimicrobials.

Acknowledgements

I would like to thank the Andrews Research Group for support and guidance. A special mention goes to Dimuthu Chanaka for guiding me through the very confusing induction into research and Dr Lukasz Kedzierski for his work in the biological examination of synthesised compounds on human primary fibroblasts and L. major promastigotes. Thank you to Sasha and Rahad for all their help with putting this together. This would not have been possible without you all.

Appendices

Appendix 1: Abbreviations

AmpB	Amphotericin B
BSA	Benzenesulfonic acid
CL	Cutaneous Leishmaniasis
DMSO	Dimethyl sulfoxide
DSC	Differential Scanning Calorimeter
DMSA	2,5-dimethylbenzenesulfonic acid
DNSA	2,4-dinitrobenzenesulfonic acid
ESI-MS	Electrospray ionisation mass spectrometry
FT-IR	Fourier Transform Infrared Spectrometer
L. major	Leishmania major
M	Medium (Peak)
MP	Melting Point
NMR	Nuclear Magnetic Resonance
NSAID	Non-steroidal anti-inflammatory drug
OA	Oxidatative addition
RBF	Round Bottom Flask
s	Strong (Peak)
SF	Solvent free
sh	Shoulder (Peak)
SM	Solvent mediated
SMet	Salt Metathesis
THF	Tetrahydrofuran
TEA	Triethylamine
VL	Visceral Leishmaniasis
w	Weak (Peak)

Appendix 2: Biological Cellular Assays

Cell viability assay

Anti-leishmanial properties were screened with Celltiter Blue Cell Viability Assay (Promega, Madison, WI, USA). Compounds were dissolved in DMSO at 10 mmol L−1 working stock and sero-diluted within an appropriate culture media. The assay was set up according to the manufacturer’s instructions with duplicates in 96-well plates. Concentrations of 106 mL−1 and 105 mL−1 of promastigotes and primary human fibroblasts, respectively were used. All plates were assessed microscopically. Cell viability was assessed by fluorescence measurements at 550 nm (excitation) and 590 nm (emission). Celltiter Blue dye was added to samples at the T=0 of the assay and values derived from a background/blank (negative control with no cells) were subtracted from all readings. A mean was calculated from the duplicate readings. Percentage growth inhibition was calculated using assay controls (Lackovic et al., 2010: 1712–19).

Cell culture

L. major virulent clone V121 was derived from the L. major isolate LRC-L137 and maintained at 26°C in M199 medium supplemented with 10% (v/v) heat inactivated foetal bovine serum (FBS). The human primary fibroblast cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% HI-FBS at 37°C in 5% CO2 (Lackovic et al., 2010: 1714).
Data analysis
Statistical analysis IC50 values were calculated by Graphpad Prism through the non-linear regression (curve fit), dose-response inhibition, log (inhibitor) vs. normalised response, variable slope standard formats.

General procedure

Reactions were conducted in stoichiometric ratios and at 25°C with the exception of solvent-mediated reactions. Tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), diethyl ether, acetone were purchased from Merck. Melting points were collected with a digital Stuart Scientific melting point apparatus SMP10. IR spectra were collected with an Agilent Technologies Cary 630 FT-IR spectrometer. NMR readings were collected with a Bruker Avance III 400 and 600 Spectrometers and processed with Bruker Topspin 2.1 program. MS readings were collected with a Micromass Platform QMS spectrometer with an electrospray source and a cone voltage of 35 eV.

Synthesis of (Bi(O3SC6H5)3), Compound I

Sonication. Benzene sulfonic acid (0.527 g, 3.0 mmol) was dissolved in 17mL of distilled water in a 100 mL RBF and Bi(NO₃)₃.5H₂O (0.495 g, 1.0 mmol) added after dissolution of the benzene sulfonic acid. This solution was sonicated for four hours at room temperature, after which a white precipitate was formed in a colourless solution. Following centrifugation at 1,140 RPM for 10 minutes, the solution was then separated from the filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 0.91% (0.00621g, 9.132 x 10-3 mmol); MP; >300°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 3.349 (5H, s, H₂O), δ = 2.511 (1H, s, DMSO); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 128.898 (CHar), δ = 128.113 (CHar), δ = 125.950 (CHar), δ = 40.431 (C-C), δ = 40.222 (C-C), δ = 40.013 (C-C), δ = 39.804 (C-C), δ = 39.595 (C-C); IR[cm^-1]: 3408 (w), 2110 (w), 1351 (s), 1307 (sh), 1092 (s), 1026 (s), 1016 (sh), 959 (sh), 803 (m), 761(w), 729 (w).

Synthesis of (Bi(O3SC6H3(NO2)2)3), Compound II

Sonication. 2,4-dinitrobenzenesulfonic acid (0.7597 g, 3.0mmol) and Bi(NO₃)₃.5H₂O (0.495 g, 1.0 mmol) was crushed with a mortar and pestle and dissolved into 10 mL of distilled water in a 100 mL RBF. This solution was then sonicated at for five hours at room temperature, after which a clay brown precipitate was formed in a yellow, clear solution. The solution was then separated from the filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 1.02% (0.00962g, 0.0137 mmol); MP; >300°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.552 (3H, d, CHc), δ = 8.420 (3H, dd, CHa), δ = 8. 400 (3H, d, CHc), δ = 3.622 (77H, s, H₂O), δ = 2.509 (3H, s, DMSO); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 147.888 ((NO₂)₂-CH_ar), δ = 147858 ((NO₂)₂-CH_ar), δ = 131.219 (CHar), δ = 126.157 (CHar), δ = 118.815 (CHar), δ = 39.517 (C-C); IR[cm^-1]: 3334 (w), 3101 (sh), 2121 (w), 1607 (m), 1534 (s), 1356 (s), 1236 (s), 1181 (sh), 1117 (s), 1022 (s), 1016 (sh), 905 (s), 836 (s), 666(sh), 741 (s).

Synthesis of (Bi(O3SC6H3(CH3)2)3), Compound III

Sonication. 2, 5-dimethylbenzenesulfonic acid (1.129 g, 3.0mmol) was dissolved into 12mL of distilled water in a 100mL RBF and Bi2O3 (0.476 g, 1.0mmol) added after dissolution. This solution was sonicated at for five hours at room temperature, after which a white precipitate was formed in a clear solution. The solution was then separated from the filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 23.81% (0.0938g, 0.0162 mmol); MP; 259°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 7.5457 (6H, s, CHa), δ = 7.044 (18H, s, CHb), δ = 3.402 (44H, s, H₂O), δ = 2.500 (7H, s, DMSO), δ = 2.241 (3H, s, CHc); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 133.585 (CH3-CHar), δ = 132.315 (CH3-CHar), δ = 130.591 (CH3-CHar), δ = 129.086 (CH3-CHar), δ = 127.096 (CHar), δ = 39.769 (C-C); IR[cm^-1]: 3397 (m), 3218 (w), 1488 (m), 1177 (s), 1141 (sh), 1089 (s), 1017 (s), 925 (sh), 907 (sh), 706(m), 663 (s).

Synthesis of (Bi(O3SC6H3(NO2)2)3), Compound IV

Solvent-mediated synthesis. 2,4-dinitrobenzenesulfonic acid (1.519 g, 3. 0mmol) and Bi2O3 (.476 g, 1.0 mmol) was crushed with a motor and pestle, dissolved into 10 mL of toluene in a RBF. This solution was then vacuum sealed and heated with a condenser for 40 minutes at 110˚C, and further stirred for three hours, after which a brown precipitate was formed in a clear solution. The solution was then separated from the filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 25.92% (0.2462 g, 0.0425 mmol); MP; 210°C; ¹H-NMR (400MHz, DMSO, 25°C):δ = 8.072 (2H, s,CHa), δ = 7.801 (2H, t, CHb), δ = 7.604 (2H, t, CHc), δ = 7.735 (6H, d, o-Ph), δ = 7.398 (5H, t, m-Ph), δ = 7.313 (5H, t, p-Ph), δ = 3.402 (37H, s, H₂O), δ = 2.500 (5H, s, DMSO); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 147.421 (NO2-CHar), δ = 144.625 (NO2-CHar), δ = 130.724 (CHar), δ = 125.674 (CHar), δ = 118.336 (CHar), δ = 39.595 (C-C); IR[cm^-1]: 3388 (w), 3110 (m), 2112 (w), 1603 (w), 1525 (s), 1357 (s), 1229 (s), 1169 (s), 1121 (s), 1073 (s) 1022 (s), 896 (m), 836 (m), 741(s).

Synthesis of (BiPh3(O3SC6H5)2), Compound V

Oxidising reagent-mediated synthesis. Benzene sulfonic acid (0.7597 g, 2.0 mmol) was dissolved into 10 mL of distilled water in a RBF and Bi(Ph)3 (0.445 g, 1.0 mmol) added after dissolution with sonication. 10 mL of 30% H₂O2 was then added and the solution stirred for 50 minutes at room temperature, after which a brown crystalline product was formed in a clear, yellow solution. The solution was then separated from the filtrate without suction via paper filtration and the product was desiccated for two days. Yield (precipitate): 38.99% (0.3627 g); MP; 82°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 3.349 (5H, s, H₂O), δ = 2.511 (1H, s, DMSO); IR[cm^-1]: 3404 (w), 2110 (w), 1562 (m), 1473 (m), 1426 (m), 1156 (m), 1110 (m), 983 (m), 724(s), 694s (s).

Synthesis of (BiPh3(O3SC6H5)2), Compound VI

Metal translocation-mediated synthesis. Benzene sulfonic acid (0.3515 g, 2.0 mmol) was dissolved into 6 mL of THF in a RBF and sonicated with silver oxide (0.231g, 1.0 mmol) until dissolution occurs. This mixture was then refluxed at 90˚C and the filtrate removed. The subsequent solution was then combined with BiPh3Cl2 (.256 g, 1.0mmol) and topped up with 10mL of additional THF. This solution was then refluxed at for two hours at 66°C with constant addition of THF, after which a purple precipitate was formed in a clear solution. The solution was then separated from the filtrate via paper filtration, washed with acetone and the product was desiccated for two days. Yield (precipitate): 48.24% (0.3637 g, 0.535 mmol); MP; 179°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.091 (4H, d, o-Ph), δ = 7.887 (4H, t, m-Ph), δ = 7.704 (2H, t, p-Ph), δ = 7.604 (7H, m, CHa), δ = 7.336 (5H, m, CHb), δ = 7.313 (5H, m, CHc), δ = 3.402 (31H, s, H₂O), δ = 2.500 (4H, s, DMSO).

Synthesis of (BiPh3(O3SC6H5)2), Compound VII

Triethylamine-mediated synthesis. Benzene sulfonic acid (0.352 g, 2.0 mmol) and BiPh3Cl2 (0.511 g, 1.0 mmol) was dissolved into 12 mL of distilled water in a RBF with 5mL of acetone and 0.28 mL of triethylamine. This solution was stirred overnight at room temperature, after which clear needle-like crystals formed in a clear solution. The solution was then separated from the filtrate via paper filtration, further crystal growth developed after a few days in solution. Yield (precipitate): 11.88% (0.00621 g); MP: 149°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 7.742 (5H, d, o-Ph), δ = 7.723 (5H, t, CHa), δ = 7.415 (3H, t, m-Ph), δ = 7.398 (3H, t, CHb), δ = 7.336 (2H, t p-Ph), δ = 7.315 (1H, t, CHc), δ = 3.402 (31H, s, H₂O), δ = 2.500 (4H, s, DMSO).

Synthesis of (BiPh3(O3SC6H5)2), Compound VIII

Solvent-free synthesis. Benzene sulfonic acid (0.7597 g, 3.0mmol) was ground with BiPh3Cl2 (0.495 g, 1.0mmol) and placed in a pear-shaped vessel and underwent a solvent-free synthesis at 105.0°C for 30 minutes. Upon completion was quenched with 10 mL of distilled water. The milky white solution was then separated from the gum like-brown filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 38.73% (0.316 g); MP; 133°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.373 (10H, d, o-Ph), δ = 7.780 (10H, t, m-Ph), δ = 7.614 (5H, t, p-Ph), δ = 3.350 (159H, s, H₂O), δ = 2.500 (16H, s, DMSO); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 138.39 (CHar), δ = 137.51 (CHar), δ = 134.21 (CHar), δ = 132.20 (CHar), δ = 131.73 (CHar), δ = 128.79 (CHar), δ = 39.595 (C-C).

Synthesis of (BiPh3(O3SC6H5)2), Compound IX

Solvent-mediated synthesis. Benzene sulfonic acid (0.7597 g, 3.0 mmol) was dissolved into 10mL of distilled water in a RBF and BiPh3Cl2 (.495 g, 1.0 mmol) added after dissolution. This solution was refluxed for five hours at 100˚C, with additional topping up of water, after which a clay brown precipitate was formed in a colourless, clear solution. The solution was then separated from the filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 20.90% (0.1476 g); MP; 159°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.379 (10H, d, o-Ph), δ = 7.776 (5H, t p-Ph), δ = 7.775 (5H, t, CHc), δ = 7.629 (2H, t, m-Ph), δ =7.628(2H, t, CHb), δ = 7.608 (1H, t, CHa), δ = 3.392 (26H, s, H₂O), δ = 2.500 (2H, s, DMSO); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 137.31 (CHar), δ = 134.20 (CHar), δ = 132.20 (CHar), δ = 131.63 (CHar), δ = 39.595 (C-C); IR[cm^-1]: 3052 (w), 2925 (w), 1559(m), 1496(m), 1436 (m), 1328 (w), 1164 (m), 985 (s), 732(s), 680 (s).

Synthesis of (BiPh3(O3SC6H3(NO2)2)2), Compound X

Triethylamine-mediated synthesis. 2,4-dinitrobenzenesulfonic acid (0.496 g, 2.0 mmol) was dissolved into 10 mL of distilled water in a RBF with 5mL of acetone, 0.28 mL of triethylamine and BiPh3Cl2 (0.511 g, 1.0 mmol). This solution was stirred overnight at room temperature, after which clear needle-like crystals formed in a clear solution. The solution was then separated from the filtrate via paper filtration, further crystal growth developed after a few days within solution. Yield (precipitate): 20.72% (0.3627 g); MP: 159°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.379 (10H, d, o-Ph), δ = 7.776 (5H, t p-Ph), δ = 7.775 (5H, t, CHc), δ = 7.629 (2H, t, m-Ph), δ =7.628(2H, t, CHb), δ = 7.608 (1H, t, CHa), δ = 3.392 (26H, s, H₂O), δ = 2.500 (2H, s, DMSO).

Synthesis of (BiPh3(O3SC6H3(CH3)2)2), Compound XI

Solvent-mediated synthesis. 2,5-dimethylbenzenesulfonic acid (0.376 g, 2.0 mmol) was dissolved into 10mL of diethyl ether in a RBF and BiPh3Cl2 (0.511 g, 1.0 mmol) added after dissolution. This solution was then vacuum sealed and refluxed at 30 minutes at 60˚C, and further refluxed at 80˚C for one hour, after which large amounts of white crystalline structures in a white powder was formed in a clear solution. The solution was then separated from the filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 98.52% (0.798g); MP: >300°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.728 (6H, d, o-Ph), δ = 8.083 (7H, t, m-Ph), δ = 7.558 (5H, s, CHa), δ = 7.451 (3H, t, p-Ph), δ =7.010(12H, d, CHc), δ = 3.392 (16H, s, H₂O), δ = 2.500 (4H, t, DMSO), δ =2.398 (4H, s, CHb); 13C- NMR (100MHz, DMSO, 25°C): δ = 146.185 (CH3- CHar), δ = 36.355 (CHar), δ = 133.436 (CH3- CHar), δ = 133.111 (CHar), δ = 130.473 (CHar), δ = 128.420 (CHar), ), δ = 127.103 (CHar)δ = 39.734 (C-C), δ = 20.563 (CH3- CHar), δ = 19.643 (CH3- CHar); IR[cm^-1]: 3049 (w), 2922 (m), 2117 (w), 1464 (m), 1438 (m), 1186 (sh), 1030 (m), 1086 (s), 1011 (s), 983 (s), 832 (s), 730(s), 707 (s), 678 (s).

Synthesis of (BiPh3(O3SC6H3(NO2)2)2), Compound XII

Solvent-free synthesis. 2,4-dinitrobenzenesulfonic acid (0.496 g, 2.0 mmol) was ground with BiPh3Cl2 (0.511 g, 1.0mmol) and placed in a pear-shaped vessel and underwent a solvent-free synthesis at 115.3°C for 30 minutes, upon completion, was quenched with 10 mL of distilled water. The milky white solution was then separated from the gum like-brown filtrate via paper filtration and the product was desiccated for two days. Yield (precipitate): 40.51% (0.316 g, 9.132 x 10-3 mmol); MP; 127-129°C;

Synthesis of (BiPh3(O3SC6H3(CH3)2)2), Compound XIII

Triethylamine-mediated synthesis. 2,5-dimethylbenzenesulfonic acid (0.376 g, 2.0 mmol) was dissolved into 10mL of distilled water in a RBF with 5mL of acetone, 0.28 mL of triethylamine and BiPh3Cl2 (0.511g, 1.0 mmol). This solution was stirred overnight at room temperature, after which clear needle-like crystals formed in a clear solution. The solution was then separated from the filtrate via paper filtration, further crystal growth developed after a few days in solution. Yield (precipitate): 18.22% (0.1476 g); MP: 140°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 8.360 (6H, d, o-Ph), δ = 7.611 (5H, t p-Ph), δ = 7.611 (5H, t, p-Ph), δ = 7.536 (1H, t, CHa), δ =7.370(3H, t, CHc),, δ = 3.392 (26H, s, H₂O), δ = 2.500 (2H, s, DMSO) δ = 2.241 (3H, s, CHb); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 137.290 (CH3-CHar), δ = 134.036 (CH3-CHar), δ = 131.720 (CH3-CHar), δ = 130.279 (CH3-CHar), δ = 127.472 (CH3-CHar), δ = 39.999 (C-C), δ = 39.019 (C-C); IR[cm^-1]: 3049 (w), 1562 (s), 1469(m), 1438 (m), 1352 (m), 1188 (m), 1024 (m), 985 (m), 830 (w), 683 (s).

Synthesis of (BiPh3(O3SC6H5)2), Compound XIV

Triethylamine-mediated synthesis. Benzene sulfonic acid (0.475g, 3.0 mmol) was dissolved into 10mL of distilled water in a RBF with 5mL of acetone, 41.58 mL of triethylamine and BiPh3 (0.440 g, 1.0 mmol). This solution was stirred overnight at room temperature, after which clear needle-like crystals formed in a clear solution. The solution was then separated from the filtrate via paper filtration, further crystal growth developed after a few days in solution. Yield (precipitate): 16.07% (0.12114 g, 0.0178 mmol); MP: 85°C; ¹H-NMR (400MHz, DMSO, 25°C): δ = 7.774 (2H, s, CHb), δ = 7.753 (5H, d, CHa), δ = 7.744 (5H, d, o-Ph), δ = 7.726 (2H, t, CHc), δ =7.402(2H, t, m-Ph), δ = 7.318 (1H, t, p-Ph), δ = 3.392 (26H, s, H₂O), δ = 2.500 (2H, s, DMSO); ¹³C-NMR (100MHz, DMSO, 25°C): δ = 134.20 (CHar), δ = 132.20 (CHar), δ = 131.63 (CHar), δ = 41.23 (C-C); IR[cm^-1]: 3052 (w), 1559 (m), 1469 (m), 1436 (m), 1328 (w), 1164 (m), 985 (s), 732 (s).

Appendix 3 Significant ¹H-NMR peaks of sulfonic acid ligands (BSA, DMSA, DNSA)

	Sulfonic Acid	CHa	CHb	CHc
BSA		7.772	7.692	7.558
DNSA		8.524	8.222	8.653
DMSA		7.760	2.415	7.366

Appendix 4 Biological Assays for compounds I, II, III, VI, X, XI, XIII and XIV and the given experimental controls

Human fibroblast viabilities after 48 hours with serial dilution series of compounds I, II, III, VI, X, XI, XIII and XIV and given experimental controls
Conc (μM)		0.048		0.097		0.19		0.39	0.78		1.56		3.125		6.25		12.5		25		50		100		positive	blank
I		388527		406254		423908		419997	420042		416544		423330		432114		423563		442585		415033		412745		419049	23887
		388179		417676		431179		432010	440432		440949		423678		425477		449183		428389		426956		413198		361506	21943
mean		388353		411965		427543.5		426003.5	430237		428746.5		423504		428795.5		436373		435487		420994.5		412971.5		390277.5	22915
minus blank		365438		389050		404628.5		403088.5	407322		405831.5		400589		405880.5		413458		412572		398079.5		390056.5		367362.5	0
percentage		99.5		105.9		110.1		109.7	110.9		110.5		109.0		110.5		112.5		112.3		108.4		106.2		100.0	0.0
II		383925		418352		423135		435571	450184		429801		424068		435260		426651		425997		412208		362113
		389581		412571		443101		436229	453216		455464		431283		457199		451680		430251		404736		365488
mean		386753		415461.5		433118		435900	451700		442632.5		427675.5		446229.5		439165.5		428124		408472		363800.5
minus blank		363838		392546.5		410203		412985	428785		419717.5		404760.5		423314.5		416250.5		405209		385557		340885.5
percentage		99.0		106.9		111.7		112.4	116.7		114.3		110.2		115.2		113.3		110.3		105.0		92.8
III		384973		415289		425962		426620	427368		431195		430912		418626		425295		413364		423407		410118
		360048		365862		381574		381979	379143		383936		375028		375651		379695		370559		383128		381130
mean		372510.5		390575.5		403768		404299.5	403255.5		407565.5		402970		397138.5		402495		391961.5		403267.5		395624
minus blank		349595.5		367660.5		380853		381384.5	380340.5		384650.5		380055		374223.5		379580		369046.5		380352.5		372709
percentage		95.2		100.1		103.7		103.8	103.5		104.7		103.5		101.9		103.3		100.5		103.5		101.5
VI		380900		387328		382552		397365	393050		394904		379974		308162		61406		26099		25529		27664
		390111		411747		412561		420245	411636		416012		411162		327140		73882		26284		26412		27526
mean		385505.5		399537.5		397556.5		408805	402343		405458		395568		317651		67644		26191.5		25970.5		27595
minus blank		362590.5		376622.5		374641.5		385890	379428		382543		372653		294736		44729		3276.5		3055.5		4680
percentage		98.7		102.5		102.0		105.0	103.3		104.1		101.4		80.2		12.2		0.9		0.8		1.3
X		401792		408997		420961		414596	421169		409635		342763		194308		146847		29105		25918		27374
		397458		417383		424615		432133	407532		426023		358851		194415		150084		28045		25970		29800
mean		399625		413190		422788		423364.5	414350.5		417829		350807		194361.5		148465.5		28575		25944		28587
minus blank		376710		390275		399873		400449.5	391435.5		394914		327892		171446.5		125550.5		5660		3029		5672
percentage		102.5		106.2		108.8		109.0	106.6		107.5		89.3		46.7		34.2		1.5		0.8		1.5
XI		393102		421215		421187		426054	420937		425450		341493		174507		132372		26183		26748		28854
		398760		415087		427817		422679	421515		434076		334084		187460		139686		26846		26930		30707
mean		395931		418151		424502		424366.5	421226		429763		337788.5		180983.5		136029		26514.5		26839		29780.5
minus blank		373016		395236		401587		401451.5	398311		406848		314873.5		158068.5		113114		3599.5		3924		6865.5
percentage		101.5		107.6		109.3		109.3	108.4		110.7		85.7		43.0		30.8		1.0		1.1		1.9
XIII		381738		392764		395900		419985	421871		429310		420469		259279		174370		94413		30987		26541
		369197		376882		375944		380492	369679		380353		378466		245468		163591		71324		27311		25106
mean		375467.5		384823		385922		400238.5	395775		404831.5		399467.5		252373.5		168980.5		82868.5		29149		25823.5
minus blank		352552.5		361908		363007		377323.5	372860		381916.5		376552.5		229458.5		146065.5		59953.5		6234		2908.5
percentage		96.0		98.5		98.8		102.7	101.5		104.0		102.5		62.5		39.8		16.3		1.7		0.8
XIV		352868		372193		378974		387883	379825		386426		389959		390272		398713		405081		365305		360660
		362006		402516		406265		408204	407401		410989		409894		417987		413964		430016		413652		362229
mean		357437		387354.5		392619.5		398043.5	393613		398707.5		399926.5		404129.5		406338.5		417548.5		389478.5		361444.5
minus blank		334522		364439.5		369704.5		375128.5	370698		375792.5		377011.5		381214.5		383423.5		394633.5		366563.5		338529.5
percentage		91.1		99.2		100.6		102.1	100.9		102.3		102.6		103.8		104.4		107.4		99.8		92.2
Bi(NO3)3		380609		395447		416289		415879	419915		416008		429921		433504		432693		422221		411024		399177
		381863		407703		449368		422791	425255		426838		472423		425821		454576		433387		419294		391535
mean		381236		401575		432828.5		419335	422585		421423		451172		429662.5		443634.5		427804		415159		395356
minus blank		358321		378660		409913.5		396420	399670		398508		428257		406747.5		420719.5		404889		392244		372441
percentage		97.5		103.1		111.6		107.9	108.8		108.5		116.6		110.7		114.5		110.2		106.8		101.4
Ph2BiCl2		381184		413955		421689		424971	437037		423384		412123		279074		147233		95313		25404		24416
		384026		408432		425541		425755	435609		437970		421324		270933		150324		107430		25032		24307
mean		382605		411193.5		423615		425363	436323		430677		416723.5		275003.5		148778.5		101371.5		25218		24361.5
minus blank		359690		388278.5		400700		402448	413408		407762		393808.5		252088.5		125863.5		78456.5		2303		1446.5
percentage		97.9		105.7		109.1		109.6	112.5		111.0		107.2		68.6		34.3		21.4		0.6		0.4
2,5-dimethyl benzene sulfonic acid		377273		404977		415850		417923	417026		418964		423454		421746		414793		422159		411221		384833
		357372		360775		374223		366166	364001		366973		368394		369556		370550		369220		362187		352542
mean		367322.5		382876		395036.5		392044.5	390513.5		392968.5		395924		395651		392671.5		395689.5		386704		368687.5
minus blank		344407.5		359961		372121.5		369129.5	367598.5		370053.5		373009		372736		369756.5		372774.5		363789		345772.5
percentage		93.8		98.0		101.3		100.5	100.1		100.7		101.5		101.5		100.7		101.5		99.0		94.1
2,4-dinitor benze sulfonic acid		397233		384425		396712		387737	378035		403220		401448		393453		403997		398603		387028		403401
		387243		415551		370718		419704	418496		429731		421469		424073		469795		418795		412474		410157
mean		392238		399988		383715		403720.5	398265.5		416475.5		411458.5		408763		436896		408699		399751		406779
minus blank		369323		377073		360800		380805.5	375350.5		393560.5		388543.5		385848		413981		385784		376836		383864
percentage		100.5		102.6		98.2		103.7	102.2		107.1		105.8		105.0		112.7		105.0		102.6		104.5
benzene sulfonic acid		392851		401895		427508		407517	421497		402953		434993		416219		424341		426403		413021		405464
		393086		411009		431114		422821	417786		460309		423708		426872		438331		435355		416960		397982
mean		392968.5		406452		429311		415169	419641.5		431631		429350.5		421545.5		431336		430879		414990.5		401723
minus blank		370053.5		383537		406396		392254	396726.5		408716		406435.5		398630.5		408421		407964		392075.5		378808
percentage		100.7		104.4		110.6		106.8	108.0		111.3		110.6		108.5		111.2		111.1		106.7		103.1
DMSO		393848		413200		424389		425367	461049		433307		409350		409620		444694		425799		418002		402932
		394696		402614		436992		416540	486148		467668		416887		427942		444903		433701		412553		413508
mean		394272		407907		430690.5		420953.5	473598.5		450487.5		413118.5		418781		444798.5		429750		415277.5		408220
minus blank		371357		384992		407775.5		398038.5	450683.5		427572.5		390203.5		395866		421883.5		406835		392362.5		385305
percentage		101.1		104.8		111.0		108.4	122.7		116.4		106.2		107.8		114.8		110.7		106.8		104.9
L. major promastigote viabilities after 48 hours with serial dilution series of compounds I, II, III, VI, X, XI, XIII and XIV and given experimental controls
Conc (μM)	0.048		0.097		0.19		0.39		0.78	1.56		3.125		6.25		12.5		25		50		100		positive			blank
I	107824		106579		109442		103630		105436	103488		106414		107226		107351		106230		103068		107950		104777			17755
	107297		109510		106164		106290		104921	105260		106662		103422		106368		104483		103603		104492		98175			17623
mean	107560.5		108044.5		107803		104960		105178.5	104374		106538		105324		106859.5		105356.5		103335.5		106221		101476			17689
minus blank	89871.5		90355.5		90114		87271		87489.5	86685		88849		87635		89170.5		87667.5		85646.5		88532		83787			0
percentage	107.3		107.8		107.6		104.2		104.4	103.5		106.0		104.6		106.4		104.6		102.2		105.7		100.0			0.0
II	105840		109503		106353		109797		105475	105737		106134		105770		106044		103397		104672		104694
	107294		106668		107663		105928		108235	107213		105004		105957		103946		102898		103604		109442
mean	106567		108085.5		107008		107862.5		106855	106475		105569		105863.5		104995		103147.5		104138		107068
minus blank	88878		90396.5		89319		90173.5		89166	88786		87880		88174.5		87306		85458.5		86449		89379
percentage	106.1		107.9		106.6		107.6		106.4	106.0		104.9		105.2		104.2		102.0		103.2		106.7
III	105511		108348		105286		108105		103498	108174		107271		105660		105222		101529		104723		110212
	101954		100585		106314		106507		101389	105173		105748		100708		103292		105554		102096		106235
mean	103732.5		104466.5		105800		107306		102443.5	106673.5		106509.5		103184		104257		103541.5		103409.5		108223.5
minus blank	86043.5		86777.5		88111		89617		84754.5	88984.5		88820.5		85495		86568		85852.5		85720.5		90534.5
percentage	102.7		103.6		105.2		107.0		101.2	106.2		106.0		102.0		103.3		102.5		102.3		108.1
VI	111213		102981		102834		108453		105860	99845		57356		21857		18922		18851		18681		19543
	102405		109020		107670		104395		103609	92576		59788		20831		18314		18739		18362		19858
mean	106809		106000.5		105252		106424		104734.5	96210.5		58572		21344		18618		18795		18521.5		19700.5
minus blank	89120		88311.5		87563		88735		87045.5	78521.5		40883		3655		929		1106		832.5		2011.5
percentage	106.4		105.4		104.5		105.9		103.9	93.7		48.8		4.4		1.1		1.3		1.0		2.4
X	110499		105025		107297		100384		77037	40706		28835		23629		19115		17771		18260		19437
	109028		108562		106201		103081		68525	38875		28074		23518		19376		18592		18561		20771
mean	109763.5		106793.5		106749		101732.5		72781	39790.5		28454.5		23573.5		19245.5		18181.5		18410.5		20104
minus blank	92074.5		89104.5		89060		84043.5		55092	22101.5		10765.5		5884.5		1556.5		492.5		721.5		2415
percentage	109.9		106.3		106.3		100.3		65.8	26.4		12.8		7.0		1.9		0.6		0.9		2.9
XI	109911		106966		109475		101346		66949	35034		27818		23343		18905		18221		18479		17219
	107534		103038		109806		100603		73630	35848		27061		23552		18689		18537		18220		17695
mean	108722.5		105002		109640.5		100974.5		70289.5	35441		27439.5		23447.5		18797		18379		18349.5		17457
minus blank	91033.5		87313		91951.5		83285.5		52600.5	17752		9750.5		5758.5		1108		690		660.5		-232
percentage	108.6		104.2		109.7		99.4		62.8	21.2		11.6		6.9		1.3		0.8		0.8		-0.3
XIII	107607		104590		105956		106734		109560	44912		47937		27731		22612		20605		19109		19293
	99492		101381		101655		108536		108730	93643		37630		29458		22585		20476		18454		18609
mean	103549.5		102985.5		103805.5		107635		109145	69277.5		42783.5		28594.5		22598.5		20540.5		18781.5		18951
minus blank	85860.5		85296.5		86116.5		89946		91456	51588.5		25094.5		10905.5		4909.5		2851.5		1092.5		1262
percentage	102.5		101.8		102.8		107.4		109.2	61.6		30.0		13.0		5.9		3.4		1.3		1.5
XIV	95978		97016		98329		99551		97920	105585		106595		101312		84197		65532		55355		54972
	100249		105071		106666		105403		105317	108395		109537		106058		91970		68826		58238		53907
mean	98113.5		101043.5		102497.5		102477		101618.5	106990		108066		103685		88083.5		67179		56796.5		54439.5
minus blank	80424.5		83354.5		84808.5		84788		83929.5	89301		90377		85996		70394.5		49490		39107.5		36750.5
percentage	96.0		99.5		101.2		101.2		100.2	106.6		107.9		102.6		84.0		59.1		46.7		43.9
Bi(NO3)3	103167		100332		104104		100406		102371	101757		104311		104303		103379		106843		102652		98129
	104709		102853		102390		103049		103352	101852		103321		100575		102973		105504		101240		103095
mean	103938		101592.5		103247		101727.5		102861.5	101804.5		103816		102439		103176		106173.5		101946		100612
minus blank	86249		83903.5		85558		84038.5		85172.5	84115.5		86127		84750		85487		88484.5		84257		82923
percentage	102.9		100.1		102.1		100.3		101.7	100.4		102.8		101.1		102.0		105.6		100.6		99.0
Ph2BiCl2	106581		107204		107005		109460		108237	75970		36104		27652		22548		19079		18441		19498
	108322		105058		108440		109995		111389	79340		35446		27766		22147		19026		17942		20517
mean	107451.5		106131		107722.5		109727.5		109813	77655		35775		27709		22347.5		19052.5		18191.5		20007.5
minus blank	89762.5		88442		90033.5		92038.5		92124	59966		18086		10020		4658.5		1363.5		502.5		2318.5
percentage	107.1		105.6		107.5		109.8		110.0	71.6		21.6		12.0		5.6		1.6		0.6		2.8
2,5-dimethyl benzene sulfonic acid	105559		103677		107359		103755		109136	106039		105412		103691		102016		103312		102672		99364
	101779		101334		102321		103219		104817	107169		108629		101822		104487		104655		103070		101445
mean	103669		102505.5		104840		103487		106976.5	106604		107020.5		102756.5		103251.5		103983.5		102871		100404.5
minus blank	85980		84816.5		87151		85798		89287.5	88915		89331.5		85067.5		85562.5		86294.5		85182		82715.5
percentage	102.6		101.2		104.0		102.4		106.6	106.1		106.6		101.5		102.1		103.0		101.7		98.7
2,4-dinitor benze sulfonic acid	96052		96645		97000		97975		96994	100419		102077		97855		100790		103705		99002		103123
	94782		97855		102945		97827		102298	102901		103436		101500		103600		104229		103579		100203
mean	95417		97250		99972.5		97901		99646	101660		102756.5		99677.5		102195		103967		101290.5		101663
minus blank	77728		79561		82283.5		80212		81957	83971		85067.5		81988.5		84506		86278		83601.5		83974
percentage	92.8		95.0		98.2		95.7		97.8	100.2		101.5		97.9		100.9		103.0		99.8		100.2
benzene sulfonic acid	96964		101063		104396		99840		103357	99539		102341		105866		101546		107606		103357		105369
	99827		99727		107078		102110		104264	100272		103380		105582		106332		108241		105681		104636
mean	98395.5		100395		105737		100975		103810.5	99905.5		102860.5		105724		103939		107923.5		104519		105002.5
minus blank	80706.5		82706		88048		83286		86121.5	82216.5		85171.5		88035		86250		90234.5		86830		87313.5
percentage	96.3		98.7		105.1		99.4		102.8	98.1		101.7		105.1		102.9		107.7		103.6		104.2
DMSO	94858		101883		106359		102850		105679	101552		105082		104723		106591		108787		105167		106454
	93180		96405		102630		99116		106592	103472		100825		108953		103168		107414		102413		108658
mean	94019		99144		104494.5		100983		106135.5	102512		102953.5		106838		104879.5		108100.5		103790		107556
minus blank	76330		81455		86805.5		83294		88446.5	84823		85264.5		89149		87190.5		90411.5		86101		89867
percentage	91.1		97.2		103.6		99.4		105.6	101.2		101.8		106.4		104.1		107.9		102.8		107.3
AmpB					95203		97816		98043	86559		81512		54034		40024		33729		28518		26705
					89821		91699		84651	85480		81695		57815		40722		32150		27133		26264
mean					92512		94757.5		91347	86019.5		81603.5		55924.5		40373		32939.5		27825.5		26484.5
minus blank					74823		77068.5		73658	68330.5		63914.5		38235.5		22684		15250.5		10136.5		8795.5
percentage					89.3		92.0		87.9	81.6		76.3		45.6		27.1		18.2		12.1		10.5
NOTE: AmpB concentration is in ug/ml and starts from 4ug/ml

Appendix 5: Crystal growth of compound XIV following 48 hours of evaporation within an acetone solution

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To cite this paper please use the following details: Choi, J. (2017), 'Synthesis and Biological Activity of Bismuth (III/V) Sulfonic Acids', Reinvention: an International Journal of Undergraduate Research, Volume 10, Issue 1, http://www.warwick.ac.uk/reinventionjournal/issues/volume10issue1/acids Date accessed [insert date]. If you cite this article or use it in any teaching or other related activities please let us know by e-mailing us at .