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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-CHar), δ = 147858 ((NO2)2-CHar), 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  
appendix_3-1_bsa.jpg
7.772 7.692 7.558
DNSA  
appendix_3-2_dnsa.jpg
8.524 8.222 8.653
DMSA  
appendix_3-3_dmsa.jpg
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

Appendix 5

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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 .