Antimony and bismuth as antimicrobial agents

ARTICLE IN PRESS

Antimony and bismuth as antimicrobial agents Rebekah N. Duffin, Melissa V. Werrett, Philip C. Andrews∗ School of Chemistry, Monash University, Melbourne, VIC, Australia ∗ Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Synthetic strategies for antimony and bismuth complexes 3. Bismuth and antimony as anti-leishmanials 3.1 Bismuth(III) indole-carboxylates 3.2 Bismuth and antimony α-hydroxy acetate complexes 3.3 Triphenyl antimony (V) and bismuth(V) acetate complexes 3.4 Selectivity of bismuth(V) and antimony (V) 4. Bismuth as an antibacterial 4.1 Bismuth(III) indole-carboxylate complexes 4.2 Bismuth(III) thiolates 4.3 Bismuth(III) complexes derived from phosphinic acids 5. Conclusions and future perspectives Acknowledgments References

2 3 6 6 9 15 22 24 25 30 38 43 44 45

Abstract There is an emerging interest in the application of metal complexes in the treatment of microbial infections and colonization. Their unique modes of action and the difficulty that microbes face in evolving mechanisms for resistance toward metal complexes, makes them attractive. Metal complexes offer a way to incorporate a diverse range of ligands and therefore easily tune physico-chemical properties, which in turn can affect the overall behavior of the complex in a biological system. For more than a decade, we have synthesized and characterized a range of bismuth and antimony complexes in the +III and +V oxidation states and evaluated their efficacy toward the treatment of Leishmania or bacteria, including but not limited to: Helicobacter pylori, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Escherichia coli. In many cases these novel complexes outperform the current medicinal compounds employed. There is still work to be done to understand the relationship between the chemical structure of metal complexes and their corresponding antimicrobial activity. We have compiled recent results in the area of metal complexes against microbes, to uncover some of the trends related to antimicrobial activity.

Advances in Inorganic Chemistry ISSN 0898-8838 https://doi.org/10.1016/bs.adioch.2019.10.001

#

2019 Elsevier Inc. All rights reserved.

1

ARTICLE IN PRESS 2

Rebekah N. Duffin et al.

1. Introduction A range of metal ions are essential for human function (e.g., iron, calcium, magnesium) and a shortage of these metal ions can lead to disease.1 Conversely, an excess of essential metal ions (or others, including heavy metals such as lead and mercury) can lead to toxicity in humans.1 Metals and metal-based compounds play an important role in medicine. Historically, metals have been used to treat a range of ailments and disease since ancient times. In more recent years, metal-compounds have been heavily researched and investigated as active pharmaceuticals for the treatment of disease. Antimony and bismuth are found in group 15 of the periodic table and although surrounded by toxic heavy metals (e.g., Tl, Sn, Pb, As) are of great importance in medicinal chemistry. Both metals have a long history of use in medicine. Bismuth has been used since the 18th century, commonly in the subnitrate or subcitrate form and is mainly associated with the treatment of gastrointestinal disorders.2 Some of the earliest reports of medically relevant antimony can be traced back to the ancient Egyptians who used antimony salts and ores for the treatment of fevers and skin irritation.3,4 Antimony-based drugs have been prescribed against cutaneous and mucocutaneous leishmaniasis since the beginning of the 20th century. Leishmaniasis is a group of diseases that is caused by the parasite Leishmania. It is classified by the World Health Organization (WHO) as a severely neglected tropical parasitic disease which affects 350 million people across 90 tropical and subtropical countries.5,6 Even after 80 years the frontline treatments include pentavalent antimonial complexes, sodium stibogluconate (Pentostam™) and meglumine antimonate (Glucantime™), despite toxic side effects and evidence of growing resistance of the parasite against antimony in some part of India.7 Organic alternatives, such as liposomal amphotericin B, miltefosine and pentadiame,8–13 are also employed but are expensive, possess narrow therapeutic windows, and exhibit teratogenicity/toxicity.8,14–18 Research has also investigated the potential of antimony compounds as antivirals (against the hepatitis C virus) and as anti-cancer agents.19,20 Bismuth, in the form of bismuth subsalicylate, was first marketed in the United States (as Pepto-Bismol™) in the early 1900s and along with colloidal bismuth subcitrate (CBS, De-Nol®),21 is still used widely today for the treatment of stomach ulcers caused by the Gram negative bacterium Helicobacter pylori.22 Furthermore, bismuth compounds have found applications in

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

3

imaging (as computed tomography contrast agents),23 as anti-cancer agents,24,25 in the treatment of microbial infections,26,27 and in retarding biofilm growth on surfaces.28,29 Bismuth has also been approved by the FDA (Food and Drug Administration) for use as the radiopaque material for catheters (bismuth subcarbonate), dental fillers (Bi2O3), and orthopedic tools (Bi2O3).30–32 Antimony conversely has a reputation as a highly toxic metalloid, though this toxicity is highly dependent on oxidation state and stability of the antimony complexes.33,34 Sb(III) was found to be more toxic than its pentavalent analog, with an LD50 ranging from 172 to 4000 mg kg
1 in mice and rats.35 For Sb(V), when a 10-fold increase of the daily recommended concentration for sodium stibogluconate was administered to humans, no serious toxicity was observed.36 However, side effects of the pentavalent complexes do range from mild to severe, with three reported cases of death.37 For antimony and bismuth, the activity and toxicity of the compound is highly dependent on several factors, including composition, solubility, reactivity, oxidation state, stability, and lability of any ligands.38–41 Our ongoing interest in this field involves investigations into the development and application of both antimony and bismuth complexes as anti-parasitic and antimicrobial agents. With the emergence of increasing numbers of multidrug-resistant bacteria, and the ever-shortening times in which resistance develops toward new natural and synthetic drugs, research into metal-based antimicrobials has become once again of great importance. Moreover, the need for new, safe, affordable drugs for the treatment of neglected tropical diseases (such as Leishmaniasis) has also motivated our research into metal-based anti-parasitic compounds. Herein, we present our recent work into the biological and medical applications of both bismuth and antimony against a series of microbes including Gram negative and Gram positive bacteria and the Leishmania parasite. The potential biological applications of each will be discussed along with a comparison to other related literature. We have focused on our work from the past 3 years within the context of others working in the field of Bi and Sb antimicrobial complexes.

2. Synthetic strategies for antimony and bismuth complexes Many of the relevant metal-organic and inorganic bismuth complexes described above contain the “sub” prefix, including subsalicylate and subcitrate. This highlights an important point that many bismuth compounds

ARTICLE IN PRESS 4

Rebekah N. Duffin et al.

are hydrolytically sensitive, resulting in the formation and/or decomposition to oxide containing materials.42 Even those bismuth formulations still used as pharmaceuticals today can suffer from a lack of stability, difficulties in synthetic reproducibility, insolubility and limited characterization.43 Therefore, the focus on developing robust synthetic methods and carrying out accurate characterization is a priority. Some of these synthetic approaches are based on solvent-mediated methodologies, such as reflux, microwave and salt metathesis reactions, using a range of bismuth precursors to control reactivity and substitution of ligands around the metal center. Other approaches employ solvent-free methodologies for the reproducible and high yielding formation of bismuth-organic complexes.44,45 Characterization methods include but are not limited to; solution (1H, 13C, 31P) and solid-state (13C, 31P) NMR, mass spectrometry, infrared spectroscopy, ICP (for Bi content), TGA/DSC, single crystal X-ray crystallography and powderX-ray diffraction for the identification of complexes, structure and stability. Much of the early work on Bi and Sb was forming complexes in the +III oxidation state. Metal containing starting materials for these reactions include tris-aryl bismuth or antimony compounds [MAr3], and salts such as Bi(NO3)3.5H2O, SbCl3/BiCl3, BiPh2Cl and BiPhCl2. These are reacted with acids, or their salts (often Na or Ag), to form the target organometallic or metal-organic complexes.46–49 Formation of complexes in the +V oxidation state are often done in one-pot and involve the use of an oxidizing agent, such as hydrogen peroxide or tertiary-butyl peroxide.50,51 Other strategies involve starting from a pre-formed +V complex, such as BiPh5/SbPh5, Ph3BiCl2/Ph3SbCl2, and carrying out reactions with the desired organic acids or their salts.52–54 The methods commonly employed to synthesize the Bi and Sb complexes described herein are summarized in Scheme 1 and have been used with a range of acids (¼LH, where H is the acidic proton) including carboxylic acids, sulfonic acids, phosphinic acids and thiols, or their sodium or silver salts. General procedure 1 (GP1) is one of the cleanest reaction routes, involving triphenyl antimony or triphenyl bismuth. As a result of the relative pKas of benzene and the acids commonly employed, [BiPh3]/[SbPh3] undergo protolysis resulting in benzene elimination.55 Another relatively clean reaction route for bismuth(III) complexes is to use bismuth tert-butoxide [Bi(OtBu)3] (GP2), which behaves as a stronger base than [BiPh3]. These reactions can often lead to single products, but due to the hydrolytic instability of [Bi(OtBu)3], reactions are normally conducted under inert atmosphere conditions and reactants combined at low temperature

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

5

Scheme 1 General reaction conditions employed in the synthesis of antimony and bismuth +III and +V complexes.

(
78 °C).56 One significant benefit from GP1 to GP2 is that they avoid possible salt contamination, which can often complicate salt metathesis reactions such as those outlined in GP3–GP5. Despite the potential for salt contamination these reactions though do offer a way to control the substitution of the required ligand around the metal center. To eliminate possible contamination of the product by unwanted salts, an oxidative addition reaction can be employed (GP6). This leads to the formation of the desired [MAr3L2] complex in high yield, producing only water and tert-butanol as by-products.50,57,58 As well as the diverse variety of starting materials listed above, a range of reaction conditions exist to achieve the synthesis and isolation of bismuth and antimony complexes in the +III and +V oxidation states. The most diverse set of reaction conditions are available when using GP1, which involves tris-aryl bismuth or antimony precursors, and is a solvothermal synthesis, referring to reactions carried out in organic solvents, generally at high temperatures. Some systems offer the possibility to conduct these reactions

ARTICLE IN PRESS 6

Rebekah N. Duffin et al.

without heat, especially when strong acids are employed. Synthesis in a microwave reactor can also be employed as a way to reduce reaction times, and is often considered more efficient and environmentally benign. Solventfree synthesis is another technique often employed in the synthesis of Bi(III) complexes and is considered a “green chemistry” technique. Many of the synthetic details for both metals have been summarized in recent publications by the groups of Yang,47 Sun,40 Gasser and Andrews,41 and Andleeb.46

3. Bismuth and antimony as anti-leishmanials Research into antimony reveals an emphasis on its use as an antiparasitic, notably for Leishmania infections.59 A more detailed history of bismuth and antimony against Leishmaniasis is given in the recent review Metal Compounds against Neglected Tropical Diseases by Ong et al.41 Despite the target population for new anti-leishmanial drugs being extremely large, its occurrence in lower socioeconomic communities, who are generally unable to afford expensive medications, means it is of little interest to major global pharmaceutical companies, severely hindering progress in the field.60–62 The lifecycle of the Leishmania parasite consists of two stages: the promastigote and the amastigote. The initial stage is the promastigote, which is the motile form that resides in the female sandfly vector (species Phlebotomus and Lutzomyia). Upon infection into a mammalian host, the promastigote differentiates into the clinically relevant amastigote form.59 It is important in the study of new drugs that they are tested against both forms of the parasite since compounds can act differently toward the two forms.63 Metal(V) complexes often exhibit their activity through reductive pathways to the metal(III) complex.64 The pentavalent complex is reduced in vivo to the more bioactive trivalent form on reaction with redox active proteins, such as glutathione and trypanothione.65,66 This inhibits the primary function of these proteins, which is oxidative stress protection, leading to increased production of reactive oxygen species (ROS) and eventual cell death.67

3.1 Bismuth(III) indole-carboxylates Our studies on the potential application of bismuth(III) indole-carboxylates were able to provide a baseline activity against both human fibroblasts and promastigotes of the L. major species of parasite. The complexes were also tested for their anti-bacterial activity against H. pylori, which will be discussed in Section 4.1.68 The complexes were synthesized using indole

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

7

Fig. 1 Bismuth indole carboxylate complexes: [Bi(O2CR)3] (B1–B5) and [BiPh(O2CR)2] (B6 and B7).

acetic acid, (IAA-H), indole propanoic acid, (IPA-H), indole butanoic acid, (IBA-H), 1-methylindole-3-carboxylic acid, (MICA-H) and indole glyoxylic acid (IGA-H). B1–B5 shown in Fig. 1 were synthesized from [Bi(OtBu)3] (following GP2, Scheme 1) to isolate the tris-substituted complexes [Bi(O2CR)3]. The bis-substituted complexes, [BiPh(O2CR)2] B6 and B7 were isolated from reactions with [BiPh3], following GP6 (Scheme 1). Stability of the bismuth(III) indole-carboxylate compounds was assessed thoroughly. Given that bismuth carboxylates are known to undergo hydrolysis to oxido clusters, an assessment of stability in air, moisture and in an acidic medium was examined.69 The compounds were found to be stable to degradation in both air and moisture through a study in d6-DMSO over a period of 2 months. Stability in an acidic medium was also investigated to identify whether the bismuth(III) indole-carboxylates would be stable for enough time in the highly acidic environment of the stomach to have any potential absorption into the bloodstream. The complexes were found to gradually release the free carboxylic acids in the acidic environment, generating HCl soluble BiOCl as a by-product. The complexes and their corresponding indole-carboxylic acids were then assessed for their anti-leishmanial activity and their mammalian cytotoxicity. As theorized, all the indole-carboxylic acids showed neither mammalian cytotoxicity nor anti-leishmanial activity. The tris-carboxylato complexes B1–B5 showed little to no activity against the L. major promastigotes, with minimal activity also toward human fibroblasts.

ARTICLE IN PRESS 8

Rebekah N. Duffin et al.

Only the bis-carboxylato complexes exhibited any sort of biological activity, with excellent anti-promastigote activity observed. However, both complexes also proved cytotoxic to the human fibroblasts at low concentrations. A selectivity index was then calculated: that being the IC50 of the complexes against mammalian cells, divided by the IC50 of the complexes against the microbe. The FDA defines any compound with less than a twofold difference between IC50(mammalian):IC50(microbe) as having a narrow therapeutic index.70 As both B6 and B7 exhibit over a sevenfold difference, they make ideal candidates for future testing (Table 1). It has been noted in our previous work with bismuth(III) carboxylates that a similar structure activity was observed. Heteroleptic mono-phenyl Bi(III) benzoates, [Bi{(CH3CONH)2C6H3CO2}3(H2O)3], [PhBi(o-NO2 C6H4CO2)2], [PhBi(o-MeOC6H4CO2)2(bipy)], [PhBi(m-MeOC6H4CO2)2 (H2O)2], [PhBi(C9H12N2O3CO2)2(H2O)], were found to inhibit both the growth of L. major promastigotes and human fibroblasts at all ranges of concentrations tested (1.95–500 μg/mL), whereas the tris-substituted analogs showed no activity against either Leishmania or mammalian cells.71 In terms of comparative literature, little has been done in recent years on bismuth(III) as an anti-leishmanial. From group 15, antimony complexes remain the most selective. A study conducted by Keogan et al. on Sb(III) hydroxamates observed moderate to excellent activity against two separate strains of Leishmania, L. chagasi and L. amazonensis.72 Despite using antimony in the more toxic +III oxidation state, the complexes showed a good degree of selectivity (Table 2). Through analysis of both homo- and heteroleptic complexes with O, N and S donors, an idea about structure-activity relationships can be elucidated. Observations from our most recent work, along with some of our previous research, are that the nature and degree of substitution at the bismuth center can have a significant effect on the biological activity.68,71,73–76 The presence of the various aryl groups in heteroleptic complexes appears to contribute to the degree of mammalian toxicity observed, though this has to Table 1 IC50 values of complexes B6 and B7 against both human fibroblasts and L. major promastigotes, selectivity indices are given by IC50(mammalian)/IC50(parasite). IC50: IC50: L. major Selectivity Compound Fibroblasts (μM) promastigotes (μM) index

B6 [BiPh(MICA)2]

1.43

0.15

9.53

B7 [BiPh(IGA)2]

1.38

0.18

7.66

ARTICLE IN PRESS 9

Antimony and bismuth complexes as antimicrobial agents

Table 2 IC50 values of the Sb(III) hydroxamate/complexes for RAW macrophage and L. amazonensis and L. chagasis promastigotes.72 IC50 macrophage IC50 L. amazonensis IC50 L. chagasis (μM) (μM) (μM) Compound

[Sb(C7H6NO2)Cl]

11.8

1.2

0.8

[Sb(C6H5N2O2)Cl]

209.5

6.3

4.9

[Sb(C7H7N2O2) (C7H9N2O2)]Cl2

>500

>14.2

>14.6

[Sb(C7H7NO3)Cl]

75.8

3.3

3.9

[Sb(C14H19N2O3) (C14H18N2O3)]

13.9

0.8

0.6

be considered in light of other chemical and physical factors associated with the bismuth complexes. Detailed biochemical, mechanistic and metallomic studies need to be undertaken if we are to understand the structure-activity relationship of these complexes and their future complex design.

3.2 Bismuth and antimony α-hydroxy acetate complexes Our recent studies into both antimony and bismuth as anti-leishmanials follows on from our previous studies into α-hydroxy carboxylato bismuth(III) complexes and tris-aryl bismuth(V) carboxylato complexes. The α-hydroxy carboxylato complexes were found to have little to no activity against the human fibroblast controls, but this non-toxicity extended to the promastigote form of the Leishmania parasite. Remarkably, however, the two glycolate complexes, [Bi(Gly)(Gly-H).H2O] A and [Bi(Gly)(NO3) (H2O)] B, (GlyH ¼ glycolic acid) exhibited a good degree of activity toward the clinically relevant amastigote form (Fig. 2). The nitrate substituted complex in particular presented with a very low % infection value of 1.8 + 0.9, though at a concentration of 50 μM.77 Despite being delivered as Bi(III), this was five times higher than we had previously observed for a series pentavalent antimony complexes.78 As such, we turned to the possible efficacy of Bi(V) complexes. The current Sb(V) drugs based on gluconate and meglumine are too hydrophilic to be absorbed effectively through the stomach. For an oral administration route to be effective the compounds need to be more lipophilic. In this respect, tris-aryl Bi(V) complexes may offer some potential. It is theorized that once taken up by the parasite there could be a mechanistic

ARTICLE IN PRESS 10

Rebekah N. Duffin et al.

Fig. 2 Bismuth glycolate complexes, graphs of the anti-amastigote activity at concentrations of 50, 10 and 1 μM are shown against a positive control, DMSO and amphotericin B.

Fig. 3 α-Hydroxy acids used in the study.

relationship to the current Sb(V) compounds. These tris-aryl bismuth carboxylates studied had a range of activities and selectivity ranging from poor to excellent and were synthesized by a one-pot oxidative addition reaction, which gave clean products in high yield.50,57 Due to the activity observed with the Bi(III) α-hydroxy carboxylates, the Sb(V) and Bi(V) analogs were next explored. Eight complexes were synthesized and characterized, and of these, six were assessed for their anti-leishmanial activity. Of the complexes synthesized, four of these were novel, with the remaining four having been previously characterized. The α-hydroxy acids selected for this study were glycolic acid (GlyH2), R and S enantiomers of mandelic acid (R/S-ManH2) and benzilic acid (BenH2) (Fig. 3). A summary of these complexes and their structures is given in Fig. 4.51,79,80

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

11

Fig. 4 General structures of the Sb(V) and Bi(V) triphenyl α-hydroxy carboxylates: B8 [BiPh3(GlyH)2], B9r [BiPh3(R-ManH)2], B9s [BiPh3(S-ManH)2], B10 [BiPh3(BenH)2], S1 [SbPh3(Gly)], S2r [SbPh3(R-ManH)2], S2s [SbPh3(S-ManH)2], S2r0 [SbPh3(R-Man)], S2s0 [SbPh3(S-Man)], and S3 [SbPh3(BenH)2].

Both solution and solid-state analysis of the complexes was able to be conducted, with four of them undergoing analysis via single crystal X-ray diffraction to elucidate the solid-state structures. The complexes of the bis-substituted antimony mandelates, S2r [SbPh3(R-ManH)2] and S2s [SbPh3(S-ManH)2], were found to exist in two different forms depending on the polarity and water miscibility of the solvent system used. When synthesized in toluene, the complexes would form bis-substituted complexes of the general formula [SbPh3(O2(OH)CR)2]. However, if this bis-substituted complex was dissolved in DMSO, cyclometallation would occur in which one ligand is cleaved off and the remaining deprotonates at the α-hydroxyl, resulting in a di-anionic five membered chelate of the general formula, [SbPh3(O2C(O)CR)], S2s0 /r0 . The proton lost from the α-hydroxy chelate is taken by the cleaved mandelate to form an equivalent of mandelic acid, as outlined in Scheme 2. As this cyclometallation occurs in DMSO, it can be assumed that this form, [SbPh3(Man)], is the bioactive form. This occurs as all stock solutions for biological analysis are made in DMSO.

Scheme 2 Cyclometallation of complex 2Ss/r to 2ss0 /r0 by proton exchange in water miscible solvents.

All bismuth structures, B8 [BiPh3(GlyH)2], B9r [BiPh3(R-ManH)2], B9s [BiPh3(S-ManH)2], B10 [BiPh3(BenzH)2] and the remaining two antimony complexes, S1 [SbPh3(Gly)] and S3, [SbPh3(BenzH)2], were found to be stable in DMSO for long periods of time, eluding to a high degree of hydrolytic stability.

ARTICLE IN PRESS 12

Rebekah N. Duffin et al.

Consistent results from repeated elemental analysis also proved a high degree of solid-state stability, with no absorption of water or hydrolysis present in any of the crystalline complexes. Similar to the bismuth(III) complexes, stability in potential biological media must be assessed. Previously, we observed that complexes of the general formula [BiPh3(O2CR)2] would undergo rapid decomposition in mammalian cell culture media, Dulbecco’s Modified Eagle Medium (DMEM). Though the exact process behind this decomposition has yet to be elucidated, it was observed that glucose may be a contributing factor due to its high concentration in the culture media.81 Our study with an example complex, [Bi(o-Tol)3(asp)2] (where asp ¼ aspirin), showed that decomposition occurred in the presence of pure glucose, though at a slower rate: taking over a week rather than 1 h in culture media. Further insights into this decomposition process are ongoing. The bismuth benzoate complexes were found to undergo rapid one-phase exponential decay, with half-lives of approximately 2 h.50,57 When the same method was employed on the α-hydroxy carboxylate complexes, [BiPh3(ManH)2] and [BiPh3(GlyH)2], they were found to be surprisingly stable, as evidenced by their long calculated half-lives. Decomposition did occur over time, but the decay was found to be linear rather than exponential (R2 ¼ 0.9990 and 0.9986, respectively) (Fig. 5) and half-lives were determined to exceed 50 h (the limit of the experiment). These half-lives are significantly longer than our previous Bi(V) carboxylates, indicating their higher stability.50,57 Insights into this increased stability in media are ongoing. Unsurprisingly, the antimony complexes proved to be stable in culture media.

Integration

Complex B8

Complex B9r

1

1

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

00

5

10

15

20

25

30

35

40

45 50

0 0

5

10

15

20

25

30

35

40

45

50

Time (Hours)

Fig. 5 Decay curves of complex B8 [BiPh3(GlyH)2] (left) and complex B9r [BiPh3(R-ManH)2] (right) in DMEM culture media over a period of 50 h. Adapted from Duffin, R.N.; Blair, V.L.; Kedzierski, L.; Andrews, P.C. Comparative Stability, Toxicity and Anti-leishmanial Activity of Triphenyl Antimony(V) and Bismuth(V) α-Hydroxy Carboxylato Complexes. Dalton Trans. 2018, 47, 971–980.

ARTICLE IN PRESS 13

Antimony and bismuth complexes as antimicrobial agents

All complexes were then assessed for their activity against both human fibroblasts and L. major promastigotes. The complexes exhibited excellent anti-promastigote activity with values ranging from 3.58 to 20.7 μM. In contrast to our previous experiments with bismuth, the Bi(V) α-hydroxy carboxylates were found to be highly toxic to human fibroblasts. All bismuth complexes exhibited a higher degree of mammalian cytotoxicity than their antimony counterparts. An IC50 value range of 5.83–7.01 μM was calculated (Fig. 6; Table 3). The toxicity of the Bi(V) triphenyl α-hydroxy carboxylate complexes was potentially higher than that calculated from the cell viability assay, since each bismuth complex formed a slightly opaque solution when injected into the culture media. Though solubility proved an issue for the bismuth complexes, their apparent activity was still able to be qualitatively compared to that of the analogous antimony complexes. Each antimony complex S1, [SbPh3(Gly)], S2r0 /s0 , [SbPh3(Man)] and S3, [SbPh3(BenzH)2] were found to be selectively toxic, exhibiting little to no effect on the human fibroblasts while actively eradicating the parasite. This selective activity and increased stability has been observed in our previously synthesized tris-aryl antimony carboxylates.78 As all antimony complexes were found to be superior to their bismuth analogs, they were taken forward for further testing against the clinically relevant amastigote form of the parasite (Fig. 7). A good degree of activity was % Viability vs Concentration complexes S1-S3, B8-B10 against Human Fibroblasts

% Viability vs Concentration complexes S1-S3, B8-B10 against L. major 150

100 % Viability

% Viability

100

50

50

0 0

50 Concentration mM

100

0

Sb Sb Bi Bi

0

50 Concentration mM

100

Fig. 6 Comparison of %Cell viability of complexes S1–S3, B8–B10, against Human fibroblasts (left) and L. major promastigotes (right). Dose response curves were generated over a range of concentrations (48 nm–100 μM) in the appropriate culture media from 10 mM DMSO stock solutions. All readings were compared spectroscopically to non-treated control and the percent growth inhibition calculated. Adapted from Duffin, R.N.; Blair, V.L.; Kedzierski, L.; Andrews, P.C. Comparative Stability, Toxicity and Anti-leishmanial Activity of Triphenyl Antimony(V) and Bismuth(V) α-Hydroxy Carboxylato Complexes. Dalton Trans. 2018, 47, 971–980.

ARTICLE IN PRESS 14

Rebekah N. Duffin et al.

Table 3 IC50 values for both fibroblasts and L. major promastigotes for Sb(V) and Bi(V) triphenyl α-hydroxy carboxylates. IC50: Fibroblasts IC50: L. major Complex (μM) (μM) Selectivity index

B8 [BiPh3(GlyH)2]

5.83

6.33

0a

S1 [SbPh3(Gly)]

100

12.5

8.00b

B9r [BiPh3(R-ManH)2]

6.15

5.39

1.14

S2r [SbPh3(R-ManH)2]

>100

20.7

4.80b

B9s [BiPh3(S-ManH)2]

6.25

3.58

1.75

S2s [SbPh3(S-ManH)2]

72.8

14.5

5.16

B10 [BiPh3(BenH)2]

7.01

5.07

1.38

S3 [SbPh3(BenH)2]

100

20.0

5.00b

a

Negative values assumed to be 0. Values would be greater than calculated due to 100 μM activity. Selectivity index ¼ (IC50: fibroblasts/IC50: parasite). b

Fig. 7 Infected macrophages after 48 h treatment with complexes S1, S2r, S2s and S3. Number of infected macrophage was determined microscopically, in duplicate of fixed specimens. Amphotericin B (AmpB) was used as a positive control at 10 μM concentration. A DMSO control was also employed at a 1% concentration. Error bas indicate SEM, one-way ANOVA. Dunnett’s multiple comparison test was used to determine the statistical significance between all test compounds and a positive control lacking treatment (+ve). Adapted from Duffin, R.N.; Blair, V.L.; Kedzierski, L.; Andrews, P.C. Comparative Stability, Toxicity and Anti-leishmanial Activity of Triphenyl Antimony(V) and Bismuth(V) α-Hydroxy Carboxylato Complexes. Dalton Trans. 2018, 47, 971–980.

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

15

observed for the complexes against the amastigote form. The R/S mandelates were found to exhibit the greatest degree of activity suggesting that the cyclometallation of the acid may contribute to the increased activity. This also alludes to the general understanding whereby promastigote activity is not always an accurate indication of activity in the amastigote. The Sb(V) R-mandelate complex, S2r0 , [SbPh3(R-Man)] presented with the lowest anti-promastigote activity of 20.7 μM, but exhibited a greater degree of activity than both the benzylic and glycolic complexes. The S-mandelate, S2s0 , [SbPh3(S-Man)], proved to be the best candidate overall, suggesting that not only cyclometallation, but chirality may also be important in conferring activity. Potentially the elongated carbon chain could have an effect on stability, as previous complexes have used direct carboxylates as opposed to two carbon chain backbones.57,71 Synthesis of Sb(V) mandelate complexes with chemical variation on the aryl group (directly coordinated to the Sb center) could be used to further probe the determinant for antileishmanial activity. Our previous studies have explored variations in aryl groups on Sb(V) and Bi(V) complexes which led to changes in both activity and selectivity.50,78 Studies on tris o-, m-, p-tolyl antimony(V) benzoates showed that both the activity and selectivity seemed to be contributed to the substitution of the aryl group. [Sb(Ar)3L2] complexes incorporating 2-methoxybenzoate exhibited a greater degree of selectivity in the order of m-tol > p-tol > o-tol > Ph.78 By exploring differentiation in both the aryl group and the carboxylate moiety, further insight into the structure-activity relationship of these group 15 pentavalent complexes could be established.

3.3 Triphenyl antimony (V) and bismuth(V) acetate complexes To gauge whether differentiation in the carbon backbone chain of the carboxylate ligand can contribute to both selectivity and stability, we focused our studies on the synthesis, characterization and biological application of triphenyl antimony(V) and bismuth(V) acetate complexes, which share a similar two carbon backbone to the α-hydroxy acids.82 The acetic acids used in the synthesis of these complexes are shown in Fig. 8. The synthesis of the complexes utilized the same oxidative addition reaction, in which the [MPh3] precursor is oxidized by a peroxide (GP6, Scheme 1) in the presence of the acetic acid (Fig. 8), to form the final Bi(V) or Sb(V) complexes.58 Using eight different acetic acids, eight Sb(V) complexes, S4 [SbPh3(oTA)2], S5 [SbPh3(mTA)2], S6 [SbPh3(pTA)2], S7 [SbPh3(2MA)2], S8 [SbPh3(3MA)2], S9 [SbPh3(4MA)2], S10 [SbPh3(AOA)2], S100 [SbPh3(AOA)OH], S11

ARTICLE IN PRESS 16

Rebekah N. Duffin et al.

Fig. 8 Acetic acid derivatives utilized in the formation of Bi and Sb acetate complexes.

Fig. 9 Structures of the complexes S4 [SbPh3(oTA)2], S5 [SbPh3(mTA)2], S6 [SbPh3(pTA)2], S7 [SbPh3(2MA)2], S8 [SbPh3(3MA)2], S9 [SbPh3(4MA)2], S10 [SbPh3(AOA)2], S100 [SbPh3(AOA)OH], S11 [SbPh3(POA)2], B11 [BiPh3(oTA)2], B12 [BiPh3(mTA)2], B13 [BiPh3(pTA)2], B14 [BiPh3(2MA)2], B15 [BiPh3(3MA)2], B16 [BiPh3(4MA)2], B17 [BiPh3(AOA)2] and B18 [BiPh3(POA)2].

[SbPh3(POA)2] and eight Bi(V) complexes B11 [BiPh3(oTA)2], B12 [BiPh3 (mTA)2], B13 [BiPh3(pTA)2], B14 [BiPh3(2MA)2], B15 [BiPh3(3MA)2], B16 [BiPh3(4MA)2], B17 [BiPh3(AOA)2] and B18 [BiPh3(POA)2], were isolated, as outlined in Fig. 9. Of these 17 complexes, 13 were found to be novel. Solid-state structures of 15 of the complexes were determined by single-crystal X-ray diffraction and were found to exhibit analogous coordination geometry to the

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

17

previously synthesized triphenyl mandelates. 14 of the bismuth and antimony acetate complexes were screened for their anti-leishmanial activity. We had previously examined complexes S5 and S11 for their antileishmanial activity.78 Complexes S7 and S9 had underwent previous anti-leishmanial testing against the L. tropica strain of Leishmania, (IC50: 0.58 and 0.67 μg/mL, respectively)83 therefore, due to the physiological difference between species, testing on the L. major variant was conducted. A study by Escobar et al. found that L. major is one of the more resilient strains to the commercially available drugs on the market and therefore a more difficult vector to combat.11 To determine their stability all complexes were monitored by 1H NMR spectroscopy in d6-DMSO for 24–48 h and subjected to repeated elemental and melting point analysis. All complexes were found to be hydrolytically stable except for the oxido bridged complex S10. When dissolved into DMSO, S10 underwent hydrolysis in polar protic solvents to form a mono-hydroxido complex, S100 (Scheme 3).

Scheme 3 Hydrolysis of complex S10 to two equivalents of complex S100 .

Complex S100 was isolated as a pure solid via extraction using a 50:50 H2O:DMSO mixture. Similar to the cyclometallates previously isolated, re-arrangement occurs in DMSO and therefore the biologically active form of the complex is the mono-hydroxido. This was confirmed by 1H NMR spectrum of the complex in d6-DMSO.82 After the initial stability studies in d6-DMSO, the stability of the complex in culture media was determined. Example complexes of the p-tolylacetate, B13 [BiPh3(pTA)2] and S6 [SbPh3(pTA)2] were placed into the culture media in an NMR tube and monitored by 1H NMR spectroscopy over a period of 24 h. Similar to the α-hydroxy acetates, the bismuth complex was found to once again exhibit a linear decay in the culture media (R2 ¼ 0.9923) (Fig. 10). Though this decay happened at a more rapid rate that the α-hydroxy complexes, a half-life of 5.2 h was calculated, along with the zero-order rate constant of 59.5 μM/h (based on an initial concentration of 500 μM). Unsurprisingly, the antimony complexes exhibited no change over a 12-h period. Though the bismuth complexes were known to be unstable

ARTICLE IN PRESS 18

Rebekah N. Duffin et al.

Fig. 10 Decomposition curve of [BiPh3(pTA)2], B13 in DMEM culture media at 25 °C vs time. Half-life ¼ 5.2 h, rate constant ¼ 59.5 μM/h. Reprinted from Duffin, R.N.; Blair, V.L.; Kedzierski, L.; Andrews, P.C. Comparative Stability, Cytotoxicity and Anti-leishmanial Activity of Analogous Organometallic Sb(V) and Bi(V) Acetato Complexes: Sb Confirms Potential While Bi Fails the Test. J. Inorg. Biochem. 2018, 189, 151–162, with permission from Elsevier.

in culture media, an analysis of their activity against both L. major and human fibroblasts was conducted regardless. With a half-life of 5.2 h, the complexes are well within a therapeutic time-frame. Several of our previous complexes have shown to be active with substantially quicker decay rates.50 All antimony complexes tested exhibited sufficient solubility in the culture media. For bismuth complexes B17 [BiPh3(AOA)2] and B18 [BiPh3(POA)2], a small degree of complex precipitation was observed in the culture medium, impacting the integrity of the results obtained from the assays. Once again, the bismuth complexes were observed to be non-selectively toxic to both the promastigotes and the mammalian control cells, while the antimony complexes provided a good level of selectivity. The bismuth complexes did prove to be substantially more potent to the promastigote form of the parasite, with an IC50 range of 2.06–4.63 μM (compared to 6.18–19.1 μM for the antimony complexes). However, with a cytotoxic concentration range of 11.4–19.8 μM for B11–B18, compared to the lack of mammalian activity of the antimony complexes (only S100 , [SbPh3(AOA)OH] proved to exhibit any activity, IC50: 73.8), disqualified the bismuth complexes as potential lead compounds and therefore no further

ARTICLE IN PRESS 19

Antimony and bismuth complexes as antimicrobial agents

% Viability vs Concentration complexes S4-S11, B11-B18 against L. Major promastigotes

% Viability vs Concentration complexes S4-S11, B11-B18 against Human Fibroblasts

100

% Viability

% Viability

100

50

0 0

20

40 60 Concentration (mM)

80

Sb Sb Bi Bi

100

50

0

0

20

40 60 Concentration (mM)

100

80

Fig. 11 Comparison of %Cell viability of complexes B11–B18 and S4, S6–S100 , against Human fibroblasts (left) and L. major promastigotes (right). Dose response curves were generated over a range of concentrations (48 nm–100 μM) in the appropriate culture media from 10 mM DMSO stock solutions. All readings were compared spectroscopically to non-treated control and the percent growth inhibition calculated. Reprinted from Duffin, R.N.; Blair, V.L.; Kedzierski, L.; Andrews, P.C. Comparative Stability, Cytotoxicity and Anti-leishmanial Activity of Analogous Organometallic Sb(V) and Bi(V) Acetato Complexes: Sb Confirms Potential While Bi Fails the Test. J. Inorg. Biochem. 2018, 189, 151–162, with permission from Elsevier.

Table 4 IC50 values for both fibroblasts and L. major promastigotes for complexes B11–B18 and S4, S6–S100 . Complex B11 B12 B13 B14 B15 B16 B17 B18

IC50: Fibroblasts (μM)

11.8

12.1

16.4

19.8

18.7

12.9

12.4

11.4

IC50: L. major (μM)

4.63

2.09

2.59

2.06

3.19

3.02

2.77

3.73

Selectivity index

2.54

5.79

6.33

9.61

5.86

4.27

4.77

3.06

Complex

S4

S6

S7

S8

S9

S100

IC50: Fibroblasts (μM)

>100

>100

>100

>100

>100

73.8

IC50: L. major (μM)

19.1

Selectivity index

>5.23

6.18 a

>16.2

11.8 a

>8.47

14.4 a

>6.94

12.1 a

>8.26

6.62 a

11.1

Values would be greater than calculated due to 100 μM activity. Selectivity index ¼ (IC50: fibroblasts/IC50: parasite).

a

biological analysis was conducted. IC50 values and selectivity are given below (Fig. 11; Table 4). Though the selectivity indices of the bismuth complexes were found to be in a therapeutic range, their instability and solubility issues meant there

ARTICLE IN PRESS 20

Rebekah N. Duffin et al.

was little value in evaluating then in the amastigote assay. All antimony complexes, except for S100 , are likely to have indices greater than what was calculated as no activity was observed at the highest fixed concentration of 100 μM. By repeating the assay at a higher concentration gradient, the actual value could be accurately determined. Other tris-aryl Sb(V) complexes described in the literature have been observed to exhibit a greater degree of activity, with tris-aryl Sb(V) cinnamates reported by Mustaq et al. presenting with an IC50 range of 0.028–7.49 μM.84 However, no cytotoxic studies on these compounds have been reported or the activity against the amastigotes determined, therefore the possibility of these complexes presenting as potential lead compounds is uncertain. A vital aspect of any biological assessment is the selectivity, without selectivity it is unknown what adverse effects the compounds could potentially have on the human population. Another two separate studies on trisaryl Sb(V) carboxylates yielded the same indeterminate prospects, with excellent anti-promastigote activity observed but no exploration into anti-amastigote activity pursued (Tables 5 and 6).85,86 Other metals/metal complexes have been explored as anti-leishmanials in recent years, with gold, zinc and ruthenium providing a potential alternative to the current group 15 metallodrugs.87–89 Gold complexes synthesized by Zhang et al. and Chaves et al. have exhibited excellent anti-leishmanial activity against both forms of the parasite (IC50 amastigote: 1.41–4.25 μM,90 0.19–11.1 μM91). Unfortunately, the selectivity’s of these Table 5 IC50 of the Sb(V) tris-aryl carboxylate complexes synthesized by Saleem et al. against L. tropica promastigotes.85 IC50: Macrophage IC50: Leishmania (μg/mL) (μg/mL) Complex

[SbPh3((C6H4Br)CO2)2]

4.19

–

[SbPh3{(C6H5)2CHCO2}2]

4.25

–

[SbPh3{C6H4(NO2)CO2}2]

1.76

–

[SbPh3(C12H17NO4S2)2]

2.22

–

[Sb(p-tol)3{(C6H4Br)CO2}2]

1.49

–

[Sb(p-tol){(C6H5)2CHCO2}2]

0.68

–

[Sb(p-tol)3{C6H4(NO2)CO2}2]

0.24

–

[SbPh3(C12H17NO4S2)2]

0.17

–

ARTICLE IN PRESS 21

Antimony and bismuth complexes as antimicrobial agents

Table 6 IC50 of the Sb(V) tris-aryl carboxylate complexes synthesized by Mushtaq et al. against mammalian macrophages and L. tropica promastigotes.86 IC50: Macrophage IC50: Leishmania (μg/mL) (μg/mL) Complex

[SbPh3(C6H4NH2CO2)2]

0.100

20.45

[SbPh3(C6H4(CH3)CO2)2]

0.480

25.94

[SbPh3{C6H4(C6H4NH2)CO2}2]

0.077

11.23

[SbPh3{(C6H3Cl2)CO2}2]

0.260

20.86

[SbPh3(C3H5O2)2]

0.230

19.10

[SbPh3(C6H5NO2)2]

0.400

35.93

[SbPh3{C6H4(OCH3)CH2CO2}2]

0.580

21.95

[SbPh3{C6H4(OCH3)CH2CO2}2]

0.670

18.24

[SbPh3{C6H4(Cl)CH2CO2}2]

0.380

20.93

NHC gold(I) complexes were limited, with a significant range of activity observed against the murine macrophage controls (IC50: 0.23–7.08 μM,90 0.66–33.4 μM91).90,91 Due to a high degree of selectivity, the remaining antimony complexes, S4, S6–S9, were assessed for their anti-amastigote activity. Similar to the α-hydroxy carboxylates a varying degree of activity was observed for the complexes. A % infection range of 7.75–40.5% was observed, indicating the complexes exhibited moderate to mild activity at 10 μM. Complex S4 [SbPh3(oTA)2], proved to be the least effective anti-leishmanial, with complex S9 [SbPh3(4MA)2], proving to be the best candidate. From the amastigote invasion assay it was found that a trend existed for not only the functional group on the phenyl ring of the ligand, but its position on the ring. The following trend of the activity of the complexes was concluded to be: o-Tol > p-Tol > o-OMe > m-OMe > p-OMe. Trends in activity related to structure have been observed for other complexes of a similar structure. The previously mentioned tris-aryl Sb(V) carboxylate complexes (Table 4) synthesized by Saleem et al. were also found to exhibit some structure-activity relationships, with the position of the methyl group of the tolyl leading to a difference in anti-leishmanial activity85 (Fig. 12).83 A similar structural trend was observed by Iftikhar et al., with positioning of the methyl group at the para position on the phenyl ring having a strong influence on the biological activity observed.

ARTICLE IN PRESS 22

Rebekah N. Duffin et al.

Fig. 12 Infected macrophages after 48 h treatment with S4, S6, S7, S8 and S9. Number of infected macrophage was determined microscopically, in duplicate of fixed specimens. Amphotericin B (AmpB) was used as a positive control at 10 μM concentration. A DMSO control was also employed at a 1% concentration. Error bars indicate SEM, one-way ANOVA. Dunnett’s multiple comparison test was used to determine the statistical significance between all test compounds and a positive control lacking treatment (+ve). Reprinted from Duffin, R.N.; Blair, V.L.; Kedzierski, L.; Andrews, P.C. Comparative Stability, Cytotoxicity and Anti-leishmanial Activity of Analogous Organometallic Sb(V) and Bi(V) Acetato Complexes: Sb Confirms Potential While Bi Fails the Test. J. Inorg. Biochem. 2018, 189, 151–162, with permission from Elsevier.

3.4 Selectivity of bismuth(V) and antimony (V) From our observations over the past 3 years it seems that tris-aryl carboxylato bismuth(V) complexes tend to be reactive and unstable in a biological medium, while the analogous antimony complexes are more stable and can exhibit selective activity against the parasite alone. This contrast in stability and selectivity is most likely attributed to the reductive potential of bismuth vs antimony in the +V oxidation state (Eo ¼ +V/+III ¼ 0.59 and 2.03 V, respectively). The current front-line Sb(V) drugs are very specific, acting on the amastigote form of the parasite alone. Studies into the activity of both the pentavalent antimonials and the organically derived miltefosine (MILT) and amphotericin B (AmpB), showed that the organic drugs were active against both stages of the parasite at similar concentrations. Pentostam™, however, lacked any significant activity against the promastigote stage (Table 7).92

ARTICLE IN PRESS 23

Antimony and bismuth complexes as antimicrobial agents

Table 7 Different activity of amphotericin B (AmpB), miltefosine (MILT) and sodium stibogluconate (Pentostam) against different growth phases of L. donavani.92 AmpB MILT Pentostam

Log-phase promastigotes

+

+




Established axenic amastigotes

NA

NA

NA

Ex vivo axenic amastigotes

+

+




Intracellular amastigotes

+

+

+

+, active; NA, not applicable;
, inactive.

In contrast, our current tris-aryl Sb(V) complexes show significant activity against both stages of the parasite, alluding to more than just a redox active pathway as the potential mechanism of action. Mammalian cells and parasites alike are rich in sulfur containing proteins. Trypanothione (TPH) is a parasite specific protein, with the mammalian equivalent being glutathione (GSH). Both of these proteins play key roles in reductive pathways, aiming to maintain cellular pH and reducing potential oxidative stress within the cell. These proteins have also been observed to play a key role in the interaction of metallodrugs within the cell and allude to the potential mechanisms of action.93,94 It has been documented that Glucantime™ (meglumine antimonate) is reduced in vivo by both parasitic trypanothione and mammalian glutathione, with an increase in reduction observed in the TPH as opposed to the GSH.95 The GSH reaction tends to be mediated by body temperature and a low pH environment, such as that found in the phagolysosome of mammalian macrophages. As trypanothione exists as a dithiol, this was theorized to have contributed to the increased reactivity of the Sb(V) drug with the TPH.96 In a study conducted by Sun et al. it was observed that reduction of the Sb(V) precursor by the action of glutathione led to the formation of SbO3
, oxidized GSH (GSSG) and the glutathione complex of antimony Sb(GS)3.97 It was also suggested by Frezard et al. that Sb(V) organometallic complexes, for example, S4–S11, tend to exhibit a slower rate of reduction in the complexed state, suggesting that the Sb(V) may have to undergo dissociation prior to reduction.98 Though a pH of 5 was found to induce a higher rate of reduction, it is still probable that the antimony complexes are able to traverse the acidic conditions of the macrophage before entering the parasite cytosol.98 Observations into the binding mode of reduced Sb(III) with TPH has been identified by protein crystallography, disclosing the interaction of the Sb(III) along with the mechanism of action in the trivalent state. Sb(III) has a high binding

ARTICLE IN PRESS 24

Rebekah N. Duffin et al.

affinity for the sulfur rich proteins, especially TPH, the metal center interacts directly with the two cysteine moieties, one threonine and one histidine of the active site. This interaction of the metal in the active site pocket blocks the ability for the protein to undergo further reduction and hydride transfer.99 Compared with antimony, bismuth is a much more thiophilic metal and it has been observed that Bi(III) carboxylates will rapidly exchange their ligands for thiol containing moieties, therefore it can be assumed that the more reactive +V state will readily reduce in the presence of redox active thiol proteins.100 This high degree of redox chemistry could be what is leading to the non-selective activity of these complexes. Bi(V) is undergoing rapid and non-selective reduction to Bi(III), consequently oxidizing the sulfur rich proteins, and potentially other redox active molecules of both mammalian cells and parasites, rendering them inactive and causing a cascade to eventual apoptosis. This unstable redox activity of metals is a common occurrence in biological systems both intracellularly and extracellularly. Two of the major contributing metals are chromium +VI and manganese +IV. The reduction of these metals has been theorized to undergo a similar mechanism to that of Bi(V), mediated by the interaction of the highly oxidizing metals with cellular thiols such as GSH and cysteine.101,102 Though both manganese and chromium exhibit large reduction potentials (1.22 V Mn(IV)/Mn(III) and 1.36 V Cr(VI)/Cr(III)) which contribute to generalized oxidative stress of the cell, the reduction potential of bismuth is larger still at 2.03 V for Bi(V)/Bi(III).103 This infers a high level of reactivity of the bismuth in biological systems and may be one of the major contributing factors in the non-selective toxicity observed.40

4. Bismuth as an antibacterial Despite being a heavy metal, the use of bismuth in medicine spans more than two centuries, and today is generally considered to be of low toxicity in humans and environmentally safe.21 Antimicrobial resistance (AMR) is a global threat requiring urgent action which has led to a resurgence in the interest and research into metal-based antimicrobial agents.104 Bismuth has most commonly been used in the treatment of H. pylori and continues to be used today. Bismuth therapy is highly effective in those areas with multiresistant strains of H. pylori and high rates of antibiotic resistance.105 It should be noted that bismuth is one of the few antimicrobials to which resistance has not yet developed.26 Furthermore, studies have shown the synergistic effect

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

25

that bismuth can exhibit against H. pylori when combined with antibiotics (e.g., metronidazole).29,106 Despite significant efforts in the design and testing of new bismuth compounds for applications as antimicrobials, there is still much to be explored regarding specific structure-activity relationship. It is known that the specific ligand systems surrounding bismuth as well as the overall oxidation state of the metal can have a significant influence on stability (e.g., pH dependent ligand exchange and cluster formation), targeted activity and toxicity. The mechanism of action of Bi(III) complexes on bacteria is still not well known. However, investigations are beginning to uncover some mechanisms by which bismuth is acting on the bacteria, particularly through studying the effects of typical bismuth drugs on H. pylori. For example, bismuth is known to inactivate enzymes involved in respiration, such as F1-ATPase in H. pylori.107 Bismuth can interfere with a range of proteins, in particular Zn(II) and Fe(III) regulating proteins, cause cytoplasmic degradation, lead to the formation of Bi-glycoproteins, and act on metallo-enzymes such as urease and alcohol dehydrogenase.66 Against Escherichia coli (E. coli), bismuth can act to reduce intracellular ATP levels and collapses the membrane potential.108 A recent breakthrough from the group of Prof. Hongzhe Sun shows that colloidal bismuth subcitrate (CBS), and related Bi(III) compounds inhibit metallo-β-lactamases (MBLs).109 To inhibit MBLs is crucial to revitalize the efficacy of the existing class of beta-lactam antibiotics, for which resistance has become a major issue. Most of the susceptibility and mechanistic studies on Bi complexes have been carried out against H. pylori but more recently, our group and others have started to investigate the potential of Bi complexes to act against a larger suite of both Gram positive and Gram negative bacteria, including drug resistant strains. The following sections will address bismuth complexes and their activity against both H. pylori and other bacteria of interest. We will aim to break down the recent results to further understand what is required for achieving both potent antimicrobial activity as well as limited mammalian cell toxicity, providing an acceptable selectivity index.

4.1 Bismuth(III) indole-carboxylate complexes As outlined in Section 3.1, a series of bismuth(III) complexes derived from indole-carboxylic acids was synthesized: five homoleptic [Bi(IAA)3], [Bi(IPA)3], [Bi(IBA)3], [Bi(MICA)3] and [Bi(IGA)3] (B1–B5) and two

ARTICLE IN PRESS 26

Rebekah N. Duffin et al.

heteroleptic [BiPh(MICA)3] and [BiPh(IGA)3] (B6 and B7), as defined in Fig. 1.68 These complexes were assessed for their activity against H. pylori: a Gram-negative bacterium effecting half the world population and that is formally recognized as a bacterial carcinogen.110 The three particular stains of interest: 251, B128 and 26,695, contain virulence factors which have been demonstrated to be predictors of severe clinical outcomes.111 The free parent indole-carboxylic acids did not show any antibacterial activity at the highest concentrations tested (100 μg/mL). The bismuth derivatives were tested against all three strains of H. pylori at a concentration range of 1.56–100 μg/mL. Unlike the activity toward L. major promastigotes, which was only evident for the bis-substituted indole carboxylate complexes (B6 and B7) all of the synthesized bismuth complexes, including both the bis- (B6 and B7) and tris-analogs (B1–B5), displayed MIC values of 6.25 μg/mL (7.66–9.85 μM) against H. pylori. This suggests that the antibacterial activity is insensitive to the degree of substitution at the Bi(III) center or the composition of the indole-carboxylate ligands. Furthermore, the MIC values were consistent against all three strains of H. pylori assessed (251, B128 and 26,695). The MIC value of 6.25 μg/mL for the indole carboxylate complexes against H. pylori is consistent with previous studies on a series of bismuth(III) carboxylate complexes derived from a wide range of non-steroidal anti-inflammatory drugs (NSAIDs), for example, sulindac and tolfenamic acid.74 These results are also comparable to commercially available bismuth based anti-bacterial agents such as bismuth subsalicylate, ranitidine bismuth citrate and colloidal bismuth subcitrate, which give MIC values of 12.5, 8 and 12.5 μg/mL respectively, against H. pylori. Furthermore, the IC50 values of B6 [BiPh(MICA)2] and B7 [BiPh(IGA)2] toward human fibroblasts are well below the MIC values, at 0.90 μg/mL (1.43 μM) for B6 and 0.91 μg/mL (1.38 μM) for B7. This lack of selectivity is undesirable and indicates these heteroleptic complexes are unsuitable for use in the treatment of H. pylori. Conversely, the IC50 values for the homoleptic complexes (B1–B5) are >100 μM: well above the MIC of 6.25 μg/mL and are therefore more viable as antibacterial agents than their heteroleptic counterparts. Conversely, the results from the study of Bi(III) indolecarboxylates, do not compare well with other oxygen-bound bismuth(III) complexes, in particular those complexes containing hydroxamates,56 arenesulfonates75 or amino arenesulfonates.76 These complexes display significantly lower MIC values against H. pylori, as summarized in Table 8.

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

27

Table 8 Comparative activities of bismuth hydroxamates, bismuth arenesulfonates and bismuth amino arenesulfonates.

Those compounds with MIC values in bold/gray display potent activity in the nanomolar (nM) range.

Complexes B19–B25 (Fig. 13) were synthesized from a series of hydroxamic acids including: benzohydroxamic acid (H2-BHA), salicylhydroxamic acid (H3-SHA), acetohydroxamic acid (H2-AHA), N-methylfurohydroxamic acid (H-MFHA) and N-benzoyl-N-phenylhydroxamic acid (H-BPHA), and

ARTICLE IN PRESS 28

Rebekah N. Duffin et al.

Fig. 13 Bismuth(III) hydroxamate complexes displaying potent antibacterial activity against H. pylori.

display exceptional antibacterial activity against H. pylori, in the high nanomolar or low micromolar range. The complexes are a mixture of tris-hydroxamato complexes containing only monoanionc ligands (B20, B22, B24 and B25) or mixed anion complexes (B19, B21 and B23). For each of the hydroxamic acids, coordination to bismuth has significantly improved the antibacterial activity toward H. pylori, with the free hydroxamic acids displaying negligible activity. Toxicity tests against human skin fibroblast cells proved that all of the bismuth hydroxamate complexes display exceptional selectivity and were non-toxic up to 100 μM, with the exception of B22, which proved lethal at this concentration (but was nontoxic at 5 μM, therefore providing an acceptable selectivity index). Similar antibacterial activity was observed for the tris-sulfonate bismuth(III) complexes, B26–B29, which were synthesized by a solvent free method (GP1, Scheme 1) reacting [BiPh3] with S-(+)-camphorsulfonic acid (CamSO3H), benzenesulfonic acid (PhSO3H), toluenesulfonic acid monohydrate (TolSO3H) or mesitylenesulfonic acid dihydrate (MesSO3H). The free sulfonic acids are inactive against H. pylori but upon coordination to bismuth, nanomolar activity against H. pylori is observed (Table 8).75 An interesting trend with the arylsulfonate complexes is that the bismuth(III) trissulfonate complexes B26–B29 ([Bi(O3SR)3]) showed a significant increase in bactericidal activity against H. pylori compared with their analogous heteroleptic complexes containing two phenyl groups.112 The MIC values for all mono-sulfonate complexes ([Ph2Bi(O3SPh)]) were all 6.25 μg/mL, compared to the tris complexes which were all below 1.21 μg/mL. This is also notable because [BiPh3] is essentially inactive (>64 μg/mL), similar to the free sulfonic acids. Therefore, it appears that the addition of a single sulfonate group magnifies the bactericidal properties of the bismuth complexes dramatically, and furthermore, the addition of

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

29

three sulfonate groups provides activity that is an order of magnitude better (complexes B26–B29, Table 8). The tris(sulfonato) bismuth(III) complexes gave MIC values as low as 0.06 μM against H. pylori and the tris(amino-arene sulfonato) bismuth(III) complexes B30–B33 give comparable MIC values (Table 8).76 Again, the free amino-arenesulfonic acids: ortho-aminobenzenesulfonic acid (2ABSO3H), ortho-aminonaphthalenesulfonic acid (2AN-SO3H), 5-Amino1-naphthalenesulfonic acid was (5AN-SO3H) and 4-amino-3-hydroxy naphthalenesulfonic acid (4-A-3HN-SO3H) and are inactive toward H. pylori. One significant difference for the amino-arene sulfonate derivatives is that they appear to be less active against the 26,695 strain of H. pylori when compared to the hydroxamate and tris-sulfonate bismuth(III) complexes. Overall, the activity of the complexes does differ depending on the particular stain of H. pylori. Based on the current state of literature, this result is still not fully understood. Generally, the better performance (as demonstrated by the significantly lower MIC values obtained) of the bismuth sulfonates, vs the indole-carbolyate derivatives, has been explained by the chelating nature, greater covalency and reduced lability of carboxylates in comparison with the sulfonates.75,76 However, reports from 2014, investigating a series of bismuth(III) complexes derived from α-amino acids shows that the carboxylate chelate binding nature can still produce complexes with nanomolar activity toward H. pylori.113 Table 8 shows a select few of these complexes, derived from the reaction of L-cysteine (Cys), D,L-serine (Ser) or L-glutamic acid (Glu) with [Bi(OtBu)3] (B34–B36, respectively). The MIC values range significantly depending on the solvent used to dissolve the complex for the biological assays. This has been reported as a function of the hydrolysis of the complexes in an aqueous environment, leading to a reduced activity. Generally, when the complexes were tested in DMSO, they were more potent than when they were delivered in water. Expanding from the O-coordinating nature of carboxylates, sulfonates and amino acids, the thiophilic nature of bismuth has led research groups to investigate the synthesis, structural chemistry and antibacterial potential of a range of bismuth thiolates. In this field, bismuth(III) β-thioxoketonates have been explored as antibiotic agents against H. pylori.73 Within the context of the bismuth(III) complexes discussed herein, the thioxoketonates chelate to the bismuth center through O and S, as depicted in Fig. 14.

ARTICLE IN PRESS 30

Rebekah N. Duffin et al.

Fig. 14 Bismuth(III) β-thioxoketonates (B37–B44) and their corresponding MIC values against H. pylori (strain B128).73

Against the three strains of H. pylori tested (B128, 26,695 and 251), the β-thioxoketonates give MIC values in the range of 3.125–6.25 μg/mL and do not provide nanomolar activity. Differentiating the substituents on the thioxoketonate has an impact on the MIC value, as demonstrated by the series B37–B41; however, no clear trend is evident. Therefore, the β-thioxoketonates are not comparable to the sulfonate classes discussed above (Table 8). Their activity is closer to that of the indole-carboxylate complexes (B1–B7), which all displayed MIC values of 6.25 μg/mL against H. pylori.

4.2 Bismuth(III) thiolates The allure of investigating bismuth-sulfur complexes is due to the thiophilic nature of bismuth as well as the high lability of BidS bonds114 alongside the hydrolytic and thermodynamic stability of bismuth thiolates.23,26 It is reported that the thiophilic nature of bismuth leads to biological targets, such as proteins and peptides rich in the S-based amino acids cysteine and methionine.21,66 While many heteroleptic thiolato complexes of bismuth have been reported previously,115,116 only a small number have been assessed for their antimicrobial and/or antibiofilm activity.106,117–122 For example, much of the work related to bismuth thiols (BTs) has been published and patented by Domenico and co-workers (Microbion Biosciences, USA). They have used a range of simple thiols, including: ethanedithiol, 2,3-dimercaptopropanol and 3,4-dimercaptotoluene to produce antimicrobial bismuth thiol agents, with varying Bi:S ratios. The most simple and

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

31

potent antimicrobial to come from this work is bismuth ethanedithiol (BisEDT).122 BisEDT is effective against methicillin-resistant Staphylococcus epidermidis strains at 0.9–1.8 μM Bi(III) and against Staphylococcus aureus and S. epidermidis at 2.4 at 0.1 μM Bi(III), respectively.107 More recent work has demonstrated the efficacy of bismuth-thiols against a series of ESKAPE pathogens. The term “ESKAPE” includes six pathogens with growing multidrug resistance: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. The WHO have listed these ESKAPE pathogens in a list of 12 bacteria for which new antibiotics are urgently required.123 Interestingly, BiEDT has also been reported as toxic toward human epithelial cells (16HBE14o or A549) with LD50 values of 5 and 13 μM, respectively.124 Work was therefore carried out using non-toxic BisEDT levels (no toxicity observed at 2 μM) in combination with a known antibiotic, tobramycin. These combinations worked to reduce the MIC of tobramycin against strains of Burkholderia cenocepacia and Burkholderia multivorans tested (these are opportunistic human pathogens, particularly for cystic fibrosis patients).125 Similar action has recently been published by Sun et al. regarding the synergistic effect of colloidal bismuth subsalicylate (CBS) with a carbapenem (meropenem) against carbapenem resistant bacterial strains.109 Furthermore, in tests against Methicillin-resistant Staphylococcus aureus (MRSA), bismuth-2,3dimercaptopropanol (BisBAL) outperformed silver sulfadiazine at relatively lower molar concentrations (2011). These studies highlight the relevance of bismuth thiols and their great potential as antibiotics and for the treatment of bacterial biofilms.29 The medicinal significance of heterocyclic compounds led to the expansion upon simple bismuth thiols to the investigation of bismuth-heterocyclic derivatives. Heterocyclic compounds are an important class in the drug development and pharmaceutical fields. The diverse options for functionalization mean these molecules can be easily diversified. Furthermore, their structures often allow for coordination to metals and therefore we investigated the synthesis and biological activity of a series of bismuth(III) thiolato complexes derived from a variety of sulfur containing heterocycles. The Bi(III) thiolate complexes display powerful antibacterial properties against, among others the multidrug-resistant bacterial strains, MRSA and vancomycin-resistant Enterococcus (VRE).119–121 In 2015 a series of seven heteroleptic bismuth(III) thiolate complexes [BiPh(R-PTOT)2] derived from 5-substituted phenylthiazole oxadiazo -lethiones [R-PTOT(H)] (where R ¼ CH3, OCH3, SCH3, F, Cl, Br, CF3)

ARTICLE IN PRESS 32

Rebekah N. Duffin et al.

Fig. 15 Mono-phenyl-bisthiolatobismuth(III) complexes, [BiPh(R-PTOT)2] (B45–B51) synthesized from a series of 5-substituted phenylthiazole oxadiazolethiones [R-PTOT(H)].119

were synthesized and evaluated for their activity against bacteria. The oxadiazole moiety and its derivatives have both a proven and potentially wide range of applications in biology and medicine including as anti-HIV, anticancer, anti-inflammatory, fungicidal and as analgesic agents.126–128 This led the motivation to explore the closely related oxadiazolethiones (or thiols) which have also shown promise as anticancer agents. The structure of the bismuth(III) complexes [BiPh(R-PTOT)2], are depicted in Fig. 15. The synthetic protocol producing the highest yields of the Bi(III) complexes, [BiPh(R-PTOT)2] (51–75%) was achieved using GP4, Scheme 1, employing the sodium salt of the oxadiazolethiones ([Na(R-PTOT)]). The same product was isolated by reacting the oxadiazolethiones [R-PTOT(H)] with [BiPh3] at reflux or under microwave conditions (GP1, Scheme 1); however, yields were overall lower (50–53%). The free thiols and the bismuth(III) thiolato complexes (B45–B51) were assessed for their antibacterial activity against Staphylococcus aureus (S. aureus), MRSA, VRE and Enterococcus faecalis (E. faecalis). Despite many studies reporting the antimicrobial activity of oxadiazoles and derivatives,129,130 the oxadiazolethiones [R-PTOT(H)] in this study, were inactive against all bacteria tested (based on a disc diffusion assay).119 Upon coordination to the bismuth center, the activity of the free oxadiazolethiones was increased, and the seven bismuth(III) thiolato complexes B45–B51 [BiPh (R-PTOT)2] gave MIC values between 1.1 and 2.1 μM against S. aureus, MRSA, VRE and E. faecalis.119 E. coli was less susceptible to [BiPh (R-PTOT)2] complexes with MIC values between 41 and 120 μM. This reduced activity against E. coli is consistent with reports from Kotani et al. on cyclic organobismuth(III) compounds and it is often thought that the double membrane of Gram negative bacteria can translate to lower permeability and therefore lower activity.131 Cytotoxicity studies on the seven [BiPh(R-PTOT)2] complexes, revealed that at 10 times the MIC value, the complexes were non-toxic toward COS-7 cells (MTT assay).

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

33

Another pharmaceutically relevant class of heterocycles are the heterocyclic thioamides which are used clinically today and have demonstrated fungicidal and antimicrobial activity.132–134 A series of homo- and heteroleptic bismuth thiolates were derived from the biologically relevant and structurally similar heterocyclic thiols: 1-methyl-1H-tetrazole-5-thiol (1-MMTZ(H)); 4-methyl-4H-1,2,4-triazole-3-thiol (4-MTT(H)); 1-methyl-1H-imidazole2-thiol (2-MMI(H)); 5-methyl-1,3,4-thiadiazole-2-thiol (5-MMTD(H)); 1,3,4-thiadiazole-2-dithiol (2,5-DMTD(H)2); and 4-(4-bromophenyl)thiazole-2-thiol (4-BrMTD(H)), as shown in Fig. 16.121 All six N-heterocyclic thiols and their sodium salts were successfully reacted with [BiPh3] or BiPhCl2 (as described in GP1 or GP4, Scheme 1) to give monophenylbismuth(III) thiolate complexes [BiPh(SR)2], where SR is the deprotonated N-heterocyclic thiol. Furthermore, to explore structure-activity relationship, the corresponding tris-thiolate bismuth(III) complexes [Bi(SR)3], were synthesized by reacting the N-heterocyclic thiols with [Bi(OtBu)3] (GP2, Scheme 1). The [BiPh(SR)2] complexes, B52–B57 and the tris analogs [Bi(SR)3], B58–B63 are listed in Table 9. All complexes and their corresponding thiols (thiones) were assessed for their in vitro antibacterial activity against S. aureus, MRSA, VRE, E. coli and E. faecalis. The antibacterial assays revealed that the thiols (thiones) are relatively poor antimicrobial agents and displayed no bactericidal activity. The observed antibacterial activities of the various bismuth(III) thiolate complexes demonstrates some marked differences based on ligand type and

Fig. 16 Six N-heterocyclic compounds used in the synthesis of Bi(V) and Bi(III) thiolate complexes: 1-methyl-1H-tetrazole-5-thiol (1-MMTZ(H)); 4-methyl-4H-1,2,4-triazole-3-thiol (4-MTT(H)); 1-methyl-1H-imidazole-2-thiol (2-MMI(H)); 5-methyl-1,3,4-thiadiazole-2-thiol (5-MMTD(H)); 1,3,4-thiadiazole-2-dithiol (2,5-DMTD(H)2); and 4-(4-bromophenyl)thiazole2-thiol (4-BrMTD(H)). The compounds are presented in their thiol for but can tautomerize to the thione.

ARTICLE IN PRESS 34

Rebekah N. Duffin et al.

Table 9 Antibacterial activities of heteroleptic ([BiPh(SR)2]) and homoleptic ([Bi(SR)3]) bismuth(III) thiolate complexes (B52–B63) against MRSA, VRE and E. coli. Bacteria (MIC μM) MRSA

VRE

E. coli

B52 [BiPh(4-MTT)2]

9.72

19.44

19.44

B53 [BiPh(2-MMI)2]

190.51

190.51

190.51

B54 [BiPh(1-MMTZ)2]

9.68

4.84

19.36

B55 [BiPh(5-MMTD)2]∞

180.23

180.23

36.46

B56 [BiPh{2,5-DMTD(H)}2]∞

171.07

171.07

171.07

B57 [BiPh(4-BrMTD)2]

6.06

6.06

48.52

B58 [Bi(4-MTT)3]

180.13

180.13

180.13

B59 [Bi(2-MMI)3]

182.33

182.33

182.33

B60 [Bi(1-MMTZ)3]

180.37

180.37

180.37

B61 [Bi(5-MMTD)3]

66.23

66.23

165.56

B62 [Bi{2,5-DMTD(H)}3]

60.98

60.98

152.44

B63 [Bi(4-BrMTD)3]

90.78

39.14

39.14

Compound

[BiPh(SR)2]

[Bi(SR)3]

MIC values presented in μM.

substitution pattern, as summarized in Table 9. Comparisons between the homo- and heteroleptic bismuth thiolates, derived from N-heterocylic thiones revealed some interesting trends between these two forms: [Bi(SR)3] and [BiPh(SR)2], respectively. Generally, the heteroleptic complexes ([BiPh(SR)2]) were more active against a range of bacteria, with MIC values as low 2.5 μg/mL (4.84 μM) while the homoleptic analogs ([Bi(SR)3]) were overall less effective with the lowest MIC reported at 40 μg/mL (39.14 μM).121 Interestingly, the MIC values for the heteroleptic containing the anionic ligands 1-methyl-1H-imidazole-2-thiol (2-MMI), 5-methyl-1,3,4-thiadiazole-2-thiolate (5-MMTD) and 1,3,4-thiadia-zole2-dithiolate (2,5-DMTD) were 100 μg/mL (190.51, 180.23 and 71.07 μM, respectively). Table 9 also highlights another trend in that the Gram negative bacteria are generally less susceptible to the synthesized Bi(III) complexes, except B55 [BiPh(5-MMTD)2]∞ which appears to oppose this trend (Table 9).121

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

35

The study from 2014 revealed that the activity of bismuth thiolates, derived from N-heterocylic thiones appeared to be sensitive to the type of ligand and its substituents as well as the homo- or heterolepticity of the complexes. In light of this, a recent study (2016) has investigated, from a structure-activity perspective, whether the presence of a Ph group is crucial in improving antibacterial activity or if more generally, it is the presence of two or three different ligands on the bismuth atom which is the key to achieving low MIC values against bacteria.118 Luqman et al. report the synthesis, characterization and antibacterial activities for a series of five mixed bismuth(III) thiolate complexes. These complexes are shown in Fig. 17 and include: [BiPh-(5-MMTD)2{4-MMT(H)}] (B64), [Bi(1-MMTZ)2{(PYM)(PYM(H))2}] (B65), [Bi(MBT)2(5-MMTD)] (B66), [Bi(4-BrMTD)3{2-MMI(H)}] (B67) and [Bi(1-MMTZ)2{1-MMTZ (H)}(2-MMI){2-MMI(H)2}] (B68) were synthesized from imidazole-, thiazole-, thiadiazole-, triazole-, tetrazole- and pyrimidine-based heterocyclic thiones.118 Each complex was synthesized by reacting different ratios of the respective ligands with [BiPh3] in toluene (except for B68, which can be synthesized from [Bi(4-(MeO)Ph)3]) either at reflux or in a microwave reactor (as outlined in GP1, Scheme 1). This study provided further insights into how the lepticity of the complex as well as the ligands surrounding the bismuth center, influence antimicrobial activity. Complex B64 [BiPh-(5-MMTD)2{4-MMT(H)}], contains one phenyl, two anionic thiadiazole thiolato ligands and one neutral triazole thione ligand. As outlined in Table 10, it shows good activity

Fig. 17 Heteroleptic bismuth(III) complexes, B64–B68, synthesized from a series of heterocyclic thiones.118

ARTICLE IN PRESS 36

Rebekah N. Duffin et al.

Table 10 Antibacterial activities of bismuth(III) thiolate complexes (B64–B69) against MRSA, VRE and E. coli.

MIC values presented in μM. Those compounds with MIC values in bold/gray display potent activity in the nanomolar (nM) range.

against MRSA and VRE (7.49 and 3.74 μM) but is less active against E. coli, however, still with some reasonable activity (15 μM). To assess the role of the Ph group as well as lepticity, comparisons can be made to those bismuth complexes containing just one of the two thioligands contained in B64. The bis- and tris-thiolato complexes, [BiPh (5-MMTD)2] (B55) and [Bi(5-MMTD)3] (B61) both contain the anionic ligand, 5-methyl-1,3,4,-thiadiazole-2-thiolate (5-MMTD). Notably the homoleptic complex, [Bi(5-MMTD)3] is not effective against MRSA, VRE or E. coli with (MIC values shown in Table 10). More interestingly, the heteroleptic complex, [BiPh(5-MMTD)2] (B55) is even less effective than the homoleptic analog against the Gram positive bacteria but more effective against E. coli. Looking at the activity of complexes containing 4-methyl-4H-1,2,4-triazole-3-thione (MMT, the other ligand contained in B64) the activity of the [Bi(SR)3] analog (B58) is poor against all strains shown in Table 10 and the heteroleptic analogs B52, B69 ([BiPh(SR)2]) are

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

37

more effective. Based on the results presented in Table 10 the introduction of the thione ligand 4-MMT(H) improves the antibacterial activity of the complex. Complex, [BiPh(4-MMT)2{4-MMT(H)}2] (B69), which also contains the triazole in its neutral form, shows remarkable activity against VRE and MRSA, with nanomolar activity against VRE. When trying to compare the structure-activity relationship, it is clear that heterolepticity is important for antibacterial activity but is not the only determining factor for antibacterial effectiveness. The specific ligand, coordination number of the bismuth and the binding mode of the ligand are all factors that appear to be affecting the antibacterial activities of the complexes. When each of these five mixed thiolatobismuth(III) complexes were assessed for their toxicity against cultured COS-7 cells (via the MTT assay) it was revealed that they exhibited negligible toxicity at concentrations of 20 μg/mL. The one exception being, [BiPh(4-MTT)2] (B52), which reduced cell viability by 25% at 20 μg/mL. This increase in toxicity is not clearly understood at this stage. The studies above show that the antibacterial activity is sensitive to subtle changes in the ligand, therefore a subsequent study was carried out to look at the effect of the ligand itself. Fig. 18 shows the thiols studied and their corresponding homoleptic and heteroleptic complexes (B57, B63, B70 and B71).120 Complexes B70, B71 and B57 generally showed higher antimicrobial activities against the Gram-positive bacteria (MRSA and VRE) than against the Gram negative bacterium, E. coli. This observation supports the other studies on five-membered heterocyclic Bi(III) thiolates,121 as well as those of Kotani131 on cyclic organobismuth-(III) compounds, and Domenico’s studies on Bi(III) thiol chelators.135 The effect on toxicity of changing the ligand from 4-BrMTD to MBT is quite dramatic, as shown in Table 11. The tris-thiolato complex [Bi(MBT)3] (B70) shows excellent

Fig. 18 Bismuth(III) complexes, B70 [Bi(MBT)3], B71 [BiPh(MBT)2], B63 [Bi(4-BrMTD)3] and B57 [BiPh(4-BrMTD)2].

ARTICLE IN PRESS 38

Rebekah N. Duffin et al.

Table 11 Antibacterial activities of bismuth(III) thiolate complexes (B57, B63, B70 and B71) against MRSA, VRE and E. coli. Bacteria (MIC μM) Compound

MRSA

VRE

E. coli

MBT(H)

Inactive

Inactive

Inactive

4Br-MTD(H)

Inactive

Inactive

Inactive

B70 [Bi(MBT)3]

1.49

59.64

149.10

B71 [BiPh(MBT)2]

1.52

1.27

127.26

B63 [Bi(4-BrMTD)3]

90.78

39.14

39.14

B57 [BiPh(4-BrMTD)2]

6.06

6.06

48.52

MIC values presented in μM.

activity against MRSA with an MIC value of 1.49 μM. This is the lowest MIC values observed for any tris-thiolato complex that we have studied. Interestingly, the bromo analog [Bi(4-BrMTD)3] (B63) gave a significantly higher MIC of 90.78 μM against MRSA. B70 [Bi(MBT)3] was less active than B63 [Bi(4-BrMTD)3] against E. coli. Thus, complex B63 appears to display some of the toxicity traits normally associated with mono-phenyl bis-thiolato complexes. The hetroleptic complex [BiPh(MBT)2] (B71) displayed excellent activity toward the Gram positive having MIC values of 1.27 μM (VRE), and 1.52 μM (MRSA). For MRSA, this level of inhibition is comparable to that of vancomycin.136 These results are superior to those obtained using [BiPh(4-BrMTD)2] (B57); however, MICs against E. coli appear to be the one exception to this apparent rule. Intriguingly, the activity of the bismuth thiolate complexes B57, B63, B70 and B71 against fibroblasts shows that there is a generalized toxicity. The IC50 values range between 1.56 and 6.25 μM, whereas only B71 [BiPh(MBT)2] gave a lower level of activity (improved selectivity) against VRE at 1.27 μM. This aligns with some of the toxicity observed for Domenico’s bismuth thiol, BiEDT (as discussed above).124

4.3 Bismuth(III) complexes derived from phosphinic acids Moving away from the area of drug development, metals and metal compounds have found applications as antimicrobial additives for use within materials, surfaces and medical devices to help prevent the spread of high-risk multidrug-resistant bacteria, especially within hospitals and healthcare facilities. Silver and its compounds are often employed as antimicrobial additives

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

39

as they display broad spectrum antimicrobial activity at low concentrations. The move toward bismuth was inspired by the ongoing uncertainty surrounding silver due to environmental toxicity, accumulation and acquired bacterial resistance.137 As the opinion around silver and its extensive use remains divided, consequently, there is a crucial need to find safe, new alternatives to silver-based antimicrobial additives. This is where the work in bismuth phosphinates was applied. Phosphinic acids, like sulfonic acids, have low pKa values, generate ligands which complex through their O atoms, and would be expected to generate similarly labile complexes. Despite a number of reports on the synthesis and characterization of organo-phosphinato bismuth compounds in the literature,138,139 prior to 2018, no reports on their antimicrobial efficacy had been published. We have recently reported the synthesis and characterization of two series of phosphinato bismuth(III) complexes, one series homoleptic, [Bi(OP(¼O) R1R2)3] and the other, heteroleptic, [BiPh(OP(¼O)R1R2)2].140 The two series were synthesized and investigated to further probe and understand structure-activity relationships of Bi(III) complexes. Five phosphinic acids, R1R2P(¼O)OH: (i) diphenyl-, (R1 ¼ R2 ¼ Ph); (ii) bis(4-methoxyphenyl)-, (R1 ¼ R2 ¼ p-OMePh); (iii) bis(3-nitrophenyl)-, (R1 ¼ R2 ¼ m-NO2Ph); (iv) phenyl-, (R1 ¼ Ph, R2 ¼ H); (v) dimethyl-, (R1 ¼ R2 ¼ Me), were employed in the synthesis of 10 complexes (B72–B81) in total. The synthesis of B72– B76 is adapted from GP6, Scheme 1, where the heteroleptic complexes are isolated from the reaction of two equivalents of phosphinc acid with triphenyl bismuth. The tris-substituted phosphinato bismuth(III) complexes (B77–B81) were isolated from the reaction of bismuth(III) tertiary-butoxide with the phosphinic acids in a 1:3 M ratio, as depicted in GP2, Scheme 1. The complex codes and structures are depicted in Fig. 19. All complexes were isolated as sparingly soluble white/cream powders. Due to the very low solubility of the complexes a qualitative solid-state antibacterial assay was employed as a first analysis. Sterile pipette tips were used to transfer a small quantity (<1 mg) of the solid complex onto agar plates (gently pierced) spread with overnight bacterial cultures (E. coli, S. aureus, MRSA and VRE) and incubated at 37 °C overnight. It was immediately evident, observing the agar plates the next day, that all bacteria tested were susceptible to the heteroleptic complexes tested [BiPh(OP(¼O) R1R2)2] (B72–B76) whereas [Bi(OP(¼O)R1R2)3] (B77–B81) produced no zones of inhibition and therefore it was concluded that the bacteria tested were not susceptible to these tris-phosphinato bismuth(III) complexes.

ARTICLE IN PRESS 40

Rebekah N. Duffin et al.

Fig. 19 Structures and codes for two series of phosphinato bismuth(III) complexes: one series heteroleptic [BiPh(OP(¼O)R1R2)2] and the other homoleptic [Bi(OP(¼O)R1R2)3]

This is in line with observations from the bismuth thiolate work described herein.121 Again, it appears vital that for activity against bacteria, the complexes need a phenyl group directly connected to the bismuth center, or for the complexes to be simply heteroleptic. It must also be noted that the antibacterial activity cannot solely be related to solubility as the homoleptic complexes (superior antibacterial activity) are more water soluble than the heteroleptic analogs. Considering the low solubility of the active bis-phosphinato bismuth(III) complexes, it was proposed that they could be used to produce composites for use as antibacterial materials and surfaces. As a proof-of-concept, the bismuth complexes were incorporated into microfibrillated (nano-) cellulose generating a bismuth-cellulose composite as paper sheets. The loading of the bismuth complex was easily adjusted and a range of sheets were made with loadings of complex from 0 to 20 wt% (with respect to nanocellulose). Hole-punches were taken from the nanocelluose composite sheets and placed onto agar (spread with bacterial colonies) for a zone of inhibition test to be conducted. Fig. 20 shows a representative example of how the lower

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

41

Fig. 20 Zone of inhibition tests against VRE using the bismuth paper with calculated loadings (wt%) of complex B72: 0% (0, blank), 0.05% (A), 0.34% (B), 0.64% (C) and 4.06% (D). Ag: silver sulfadiazine at 0.43 wt%. Reprinted from Werrett, M.V.; Herdman, M.E.; Brammananth, R.; Garusinghe, U.; Batchelor, W.U.; Crellin, P.K.; Coppel, R.L.; Andrews, P.C. Bismuth Phosphinates in Bi-Nanocellulose Composites and Their Efficacy Towards Multi-Drug Resistant Bacteria. Chem. Eur. J. 2018, 24, 12938–12949.

limit for antibacterial activity was determined, using complex B72 [BiPh(OP (¼ O)Ph2)2] against VRE. The bismuth-loaded discs produced clear zones of inhibition, against the Gram -positive bacteria MRSA, VRE and S. aureus at loadings as low as 0.34 wt% complex (0.005–0.007 mg of bismuth complex per disc). For the Gram-negative bacterium (E. coli) 4.06 wt% complex loading (0.051 mg bismuth complex per disk) was required for a zone of inhibition to be observed. This suggests that the Bi-cellulose composite is less active toward Gram negative bacteria, as we have observed before. Fig. 20 also shows the small zone of inhibition present around the Ag-cellulose composite which was produced using silver sulfadiazine as a comparison. It is important to note that the zone of inhibition test is not always directly related to an MIC value; however, for a surface application this assay suggests that VRE is more susceptible to bismuth complex B72, [BiPh(OP(¼O)Ph2)2], over silver sulfadiazine, at similar loadings. This was also observed for MRSA and S. aureus, however, not for E. coli, where the silver composite outperformed the bismuth. Wu et al. report the antibacterial activity of silver nanoparticle containing bacterial cellulose nanofibers (AgNP-BC). Samples loaded with 1.01 wt% Ag produced small zones of inhibition against E. coli, S. aureus and P. aeruginosa. The zone sizes reported were all below 8 mm (6.5 mm cellulose sample size + 1.5 mm growth inhibition ring).141 Based on the zones of inhibition, the bismuth loaded cellulose samples are equal if not more active than the Ag-NP samples.

ARTICLE IN PRESS 42

Rebekah N. Duffin et al.

Fig. 21 Zone of inhibition tests against S. aureus, MRSA and E. coli (left to right) using [Bi(OP(¼O)R1R2)3] samples. “6” paper loaded with 3.87 wt% of complex [Bi(OP(¼O) Ph2)3] B77; “9”: paper loaded with 0.42 wt% of complex [Bi(OP(¼O)((Ph)(H))2)3] B80. “10”: blank. Each sample in duplicate on the agar plate. Reprinted from Werrett, M.V.; Herdman, M.E.; Brammananth, R.; Garusinghe, U.; Batchelor, W.U.; Crellin, P.K.; Coppel, R.L.; Andrews, P.C. Bismuth Phosphinates in Bi-Nanocellulose Composites and Their Efficacy Towards Multi-Drug Resistant Bacteria. Chem. Eur. J. 2018, 24, 12938–12949.

Complexes B77 [Bi(OP(¼O)Ph2)3] and B80 [Bi(OP(¼O)(Ph)(H))3] (homoleptic analogs of B72 and B75) were incorporated into cellulose in the same manner as the corresponding bis-phosphinato bismuth complex. The loading of the complexes B77 and B80 was determined to be 3.87 and 0.42 wt%, respectively. Preliminary results showed that although paper containing B77 gave small haloes against MRSA and S. aureus, no clear zones could be observed (Fig. 21). Comparatively, cellulose loaded with B72 at 4.06 wt% gave clear zones of 16 mm against MRSA and 25 mm against S. aureus. Likewise, the paper loaded with B80 (0.42 wt%) elicited no effect whereas the same loading of the analogous bis-phosphinato complex B75 (0.43 wt%) in cellulose, produced zones of 9.67 and 11.67 mm against MRSA and S. aureus, respectively. These results suggest that activity of tris complexes B77 and B80 are not equivalent to their bis-phosphinato analogs (B72 and B75). The difference in activity observed between the homo- and heteroleptic complexes is not simply a diffusion or solubility issue, as the less active homoleptic complexes ([Bi(OP(¼O)R1R2)3]) demonstrate increased hydrophilicity.140 It is still not clear why a stark difference in activity is observed here, or even for the bismuth thiolato complexes discussed above. However, as a next step into this understanding, bacterial and human cell uptake experiments are under way to probe whether it is simply that the heteroleptic complexes are able to be up taken (to a larger extent than the homoleptic analogs) and impart activity or if there is a deeper mechanistic difference at play.

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

43

5. Conclusions and future perspectives Generally, deprotonation of organic acids (studied throughout) and subsequent complexation of the resulting ligand(s) to bismuth or antimony significantly changes their biological activity. However, this is also dependent on the lepticity of the complex and the oxidation state, and therefore the physico-chemical properties of the final complex formed. The studies presented herein also highlight that the binding mode (denticity) of the ligands can influence the activity of the overall complex. Oxidation state has been found to play a crucial role in determining the reactivity and selectivity of anti-leishmanial complexes. The Sb(V) complexes discussed in Section 3 were sufficiently stable and selective in their activity. The analogous Bi(V) complexes generally lacked both stability and selectivity. Bi(III) was observed to be a more favorable oxidation state, with many Bi(III) compounds exhibiting activity against a variety of microbes, including Leishmania.68,71,73,76,77,121,140 Therefore, the focus should be placed onto Bi(III) as opposed to Bi(V) for future drug screening. Tris-aryl Sb(V) carboxylates have proven to be better candidates for antileishmanial compounds, with our studies concluding a high level of activity and stability, consistent with reports from other research groups.51,78,82–86 The use of other donor systems as opposed to the conventional carboxylate ligand should be explored to provide insight into whether different ligand systems will play an essential role in the structure-activity relationship. The use of transition metals as antileishmanials has provided a good reference point for future metallodrug research, with gold in particular exhibiting a good degree of activity.90,91 Further explorations into other transition and main group metals may provide details on the different mechanisms of action the metallodrugs exert on the parasite, potentially opening up new areas of research into the treatment of the tropical disease. The antibacterial activity of the complexes discussed herein has shown to be highly dependent of the donor atoms of the bound ligands. Generally, the better performance (lower MIC values) of the bismuth sulfonates, vs the bismuth carboxylate derivatives, has been explained by the chelating nature, greater covalency and reduced lability of carboxylates in comparison with the sulfonates. However, this observed trend is challenged by the nanomolar activity (against H. pylori) of a series of bismuth(III) complexes derived from

ARTICLE IN PRESS 44

Rebekah N. Duffin et al.

α-amino acids. [Bi2(Cys)3] and [Bi2(Glu)3] had MIC values as low as 60 nM which is comparable to some of the bismuth sulfonate complexes discussed. Bismuth thiolate complexes, derived from heterocyclic thiones, were tested against a range of Gram positive and Gram negative bacteria including MRSA, VRE and E. coli (not including H. pylori). Bismuth is thiophilic and the BidS bond114 is highly labile. The general trends observed within the bismuth(III) thiolates explored was their general improved activity toward the Gram positive bacteria. Furthermore, the mixed ligand systems (heteroleptic complexes [BiPh(SR)2] or [Bi(SR)x(SR0 )y]) generally had lower MIC values against the bacteria tested when compared to their heteroleptic analogs: [Bi(SR)3]. This trend was also observed for the bismuth phosphinate complexes: [BiPh(OP(¼O)R1R2)2] were active whereas [Bi(OP(¼O) R1R2)3] displayed no antibacterial activity. Again, these trends are challenged and for the tris-thiolato complex [Bi(MBT)3] excellent activity was observed against MRSA with an MIC value of 1.49 μM: comparable to the heteroleptic analog [BiPh(MBT)2] (1.52 μM). Finally, significant differences in antimicrobial activity were observed when subtle changes were made to the thiols. Our studies on the bismuth(III) phosphinate complexes impregnated within cellulose highlight a real opportunity for Bi based complexes to be replacing the less desirable silver additives in a range of materials relevant to healthcare (e.g., surfaces, medical devices). In this field, work is also expanding into bismuth doped-NP and other low solubility bismuth complexes. There is still much work to be done to completely understand not only the structure-activity relationship but how the complexes are acting on and displaying efficacy toward bacteria. Furthermore, this understanding must expand to mammalian cells to ensure complexes can be designed to display acceptable selectivity indices. In light of this, our group is undertaking work to look at the difference in cellular uptake of the various Bi and Sb complexes described here, as a step toward understanding the differences in activity.

Acknowledgments The authors would like to thank the Australian Research Council (DP 170103624) and The National Health and Medical Research Council (APP1139844) for financial support and Monash University.

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

45

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

Lippard, S. Bioinorganic Chemistry. University Science Books: California, 1994. Mjos, K. D.; Orvig. C. Chem. Rev. 2014, 114, 4540–4563. Martin, G. J. Chem. Educ. 1979, 56, A352. McCallum, R. I. Antimony in Medical History: An Account of the Medical Uses of Antimony and its Compounds since Early Times to the Present. Pentland Press, 1999. WHO. Control of the Leishmaniases: Report of a WHO Expert Committee. World Health Organization: Geneva, 1989. WHO. Investing to Overcome the Global Impact of Neglected Tropical Diseases: Third WHO Report on Neglected Tropical Diseases 2015. World Health Organization: Geneva, 2015. Ponte-Sucre, A.; Gamarro, F.; Dujardin, J.-C.; Barrett, M. P.; Lo´pez-V
elez, R.; Garcı´a-Herna´ndez, R.; Pountain, A. W.; Mwenechanya, R.; Papadopoulou, B. PLoS Negl. Trop. Dis. 2017, 11, e0006052. Baker, N.; de Koning, H. P.; M€aser, P.; Horn, D. Trends Parasitol. 2013, 29, 110–118. Purkait, B.; Kumar, A.; Nandi, N.; Sardar, A. H.; Das, S.; Kumar, S.; Pandey, K.; Ravidas, V.; Kumar, M.; De, T. Antimicrob. Agents Chemother. 2011, 56, 1031–1041. Sundar, S.; Jha, T. K.; Thakur, C. P.; Bhattacharya, S. K.; Rai, M. Trans. R. Soc. Trop. Med. Hyg. 2006, 100, S26–S33. Escobar, P.; Matu, S.; Marques, C.; Croft, S. L. Acta Trop. 2002, 81, 151–157. Chulay, J.; Fleckenstein, L.; Smith, D. Trans. R. Soc. Trop. Med. Hyg. 1988, 82, 69–72. Pathak, M. K.; Yi, T. J. Immunol. 2001, 167, 3391–3397. Stauch, A.; Duerr, H.-P.; Dujardin, J.-C.; Vanaerschot, M.; Sundar, S.; Eichner, M. T. PLoS Negl. Trop. Dis. 2012, 6, e1973. Griensven, J.; van Balasegaram, M.; Meheus, F.; Alvar, J.; Lynen, L.; Boelaert, M. Lancet Infect. Dis. 2010, 10, 184–194. Rees, P.; Kager, P.; Keating, M.; Hockmeyer, W. Lancet 1980, 316, 226–229. Croft, S. L.; Engel, J. Trans. R. Soc. Trop. Med. Hyg. 2006, 100, S4–S8. Sundar, S.; Kumar, K.; Chakravarty, J.; Agrawal, D.; Agrawal, S.; Chhabra, A.; Singh, V. Trans. R. Soc. Trop. Med. Hyg. 2006, 100, 698–700. Yeh, C.-T.; Hwang, D.-R.; Lai, H.-Y.; Hsu, J. T. I. Biochem. Biophys. Res. Commun. 2003, 310, 537–541. Tiekink, E. R. Crit. Rev. Oncol. Hematol. 2002, 42, 217–224. Sadler, P. J.; Li, H.; Sun, H. Coord. Chem. Rev. 1999, 185-186, 689–709. Skoglund, M.; Watters, K. Gastroenterology 1988, 94, A430. Salvador, J. A.; Figueiredo, S. A.; Pinto, R. M.; Silvestre, S. M. Future Med. Chem. 2012, 4, 1495–1523. Yu, S. B.; Watson, A. D. Chem. Rev. 1999, 99, 2353–2377. Halwani, M.; Hebert, S.; Suntres, Z. E.; Lafrenie, R. M.; Azghani, A. O.; Omri, A. Int. J. Pharm. 2009, 373, 141–146. Keogan, D. M.; Griffith, D. M. Molecules 2014, 19, 15258–15297. Kowalik, M.; Masternak, J.; Barszcz, B. Curr. Med. Chem. 2019, 26, 729–759. Hernandez-Delgadillo, R.; Velasco-Arias, D.; Jose Martinez-Sanmiguel, J.; Diaz, D.; Zumeta-Dube, I.; Arevalo-Nino, K.; Cabral-Romero, C. C. Int. J. Nanomedicine 2013, 8, 1645–1652. Folsom, J. P.; Baker, B.; Stewart, P. S. J. Appl. Microbiol. 2011, 111, 989–996. FDA. Premarket Notification, Special 510(k): 8F FlowGate2™ Balloon Guide Catheter (K153729). Food and Drug Administration (FDA), 2016. FDA. Original 510(k) Premarket Notification: DSM Biomedical DPR Cable (K143716). Food and Drug Administration (FDA), 2015. FDA Original 510(k) Premarket Notification: MTA Fillapex (K140247). Food and Drug Administration (FDA), 2014.

ARTICLE IN PRESS 46

Rebekah N. Duffin et al.

33. Sundar, S.; Chakravarty, J. Int. J. Environ. Res. Public Health 2010, 7, 4267–4277. 34. Seiler, H.; Sigel, A.; Sigel, H. Handbook on Metals in Clinical and Analytical Chemistry. CRC Press, 1994. 35. Gebel, T. In Umweltmedizinisches Handbuch; Wichmann, H. E., Schlipk€ oter, H. W., F€ ulgraff, G., Eds.; 1999 p 17. 36. Yan, S.; Jin, L.; Sun, H. Metallotherapeutic Drugs and Metal-Based Diagnostic Agents: The Use of Metals in Medicine. 2005; pp. 441–461. 37. Oliveira, L. F.; Schubach, A. O.; Martins, M. M.; Passos, S. L.; Oliveira, R. V.; Marzochi, M. C.; Andrade, C. A. Acta Trop. 2011, 118, 87–96. 38. Levason, W.; Reid, G. In: Comprehensive Coordination Chemistry II; McCleverty, J. A., Meyer, T. J., Eds.; Oxford: Pergamon, 2003 pp 465–544. 39. Sun, H.; Zhang, L.; Szeto, K.-Y. Met. Ions Biol. Syst. 2004, 41, 333–378. 40. Sun, H. Biological Chemistry of Arsenic, Antimony and Bismuth. Wiley Online Library, 2011. 41. Ong, Y. C.; Roy, S.; Andrews, P. C.; Gasser, G. Chem. Rev. 2018, 119, 730–796. 42. Mehring, M. Coord. Chem. Rev. 2007, 251, 974–1006. 43. Briand, G. G.; Burford, N. Chem. Rev. 1999, 99, 2601–2657. 44. Albat, M.; Inge, A. K.; Stock, N. Z. Kristallogr. Cryst. Mater. 2017, 232, 245–253. 45. Andrews, P. C.; Deacon, G. B.; Junk, P. C.; Spiccia, N. F. Green Chem. 2007, 9, 1319–1327. 46. Andleeb, S.; Imtiaz-ud-Din J. Organomet. Chem. 2019, 898, 120871. 47. Yang, Y.; Ouyang, R.; Xu, L.; Guo, N.; Li, W.; Feng, K.; Ouyang, L.; Yang, Z.; Zhou, S.; Miao, Y. J. Coord. Chem. 2015, 68, 379–397. 48. Ozturk, I. I.; Urgut, O. S.; Banti, C. N.; Kourkoumelis, N.; Owczarzak, A. M.; Kubicki, M.; Hadjikakou, S. K. Polyhedron 2014, 70, 172–179. 49. Ozturk, I. I.; Banti, C. N.; Kourkoumelis, N.; Manos, M. J.; Tasiopoulos, A. J.; Owczarzak, A. M.; Kubicki, M.; Hadjikakou, S. K. Polyhedron 2014, 67, 89–103. 50. Ong, Y. C.; Blair, V. L.; Kedzierski, L.; Tuck, K. L.; Andrews, P. C. Dalton Trans. 2015, 44, 18215–18226. 51. Duffin, R. N.; Blair, V. L.; Kedzierski, L.; Andrews, P. C. Dalton Trans. 2018, 47, 971–980. 52. Hoppe, S.; Whitmire, K. H. Organometallics 1998, 17, 1347–1354. 53. Rocha, M. N.; Nogueira, P. M.; Demicheli, C.; de Oliveira, L. G.; da Silva, M. M.; Fr
ezard, F.; Melo, M. N.; Soares, R. P. Bioinorg. Chem. Appl. 2013, 2013, 1–7. 54. de Oliveira, L. G.; Silva, M. M.; Paula, F.; Pereira-Maia, E. C.; Donnici, C. L.; de Simone, C. A.; Fr
ezard, F.; Ju´nior, E. N.; Demicheli, C. Molecules 2011, 16, 10314–10323. 55. Andrews, P. C.; Deacon, G. B.; Junk, P. C.; Kumar, I.; Silberstein, M. Dalton Trans. 2006, 40, 4852–4858. 56. Pathak, A.; Blair, V. L.; Ferrero, R. L.; Junk, P. C.; Tabor, R. F.; Andrews, P. C. Dalton Trans. 2015, 44, 16903–16913. 57. Ong, Y. C.; Blair, V. L.; Kedzierski, L.; Andrews, P. C. Dalton Trans. 2014, 43, 12904–12916. 58. Moiseev, D. V.; Malysheva, Y. B.; Shavyrin, A. S.; Kurskii, Y. A.; Gushchin, A. V. J. Organomet. Chem. 2005, 690, 3652–3663. 59. Von Stebut, E. Exp. Dermatol. 2007, 16(4), 340–346. 60. Boelaert, M.; Meheus, F.; Sanchez, A.; Singh, S.; Vanlerberghe, V.; Picado, A.; Meessen, B.; Sundar, S. Trop. Med. Int. Health 2009, 14, 639–644. 61. Gossage, S. M.; Rogers, M. E.; Bates, P. A. Int. J. Parasitol. 2003, 33, 1027–1034. 62. Alvar, J.; V
elez, I. D.; Bern, C.; Herrero, M.; Desjeux, P.; Cano, J.; Jannin, J.; den Boer, M.; Team, W. L. C. PLoS One 2012, 7 e35671. 63. Killick-Kendrick, R. Ann. Parasitol. Hum. Comp. 1989, 65, 37–42. 64. Fr
ezard, F.; Demicheli, C.; Ribeiro, R. R. Molecules 2009, 14, 2317–2336.

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

47

65. Haldar, A. K.; Sen, P.; Roy, S. Mol. Biol. Int. 2011, 2011, 1–23. 66. Ge, R.; Sun, H. Acc. Chem. Res. 2007, 40, 267–274. 67. Mookerjee Basu, J.; Mookerjee, A.; Sen, P.; Bhaumik, S.; Sen, P.; Banerjee, S.; Naskar, K.; Choudhuri, S. K.; Saha, B.; Raha, S.; Roy, S. Antimicrob. Agents Chemother. 2006, 50, 1788–1797. 68. Pathak, A.; Blair, V. L.; Ferrero, R. L.; Kedzierski, L.; Andrews, P. C. J.Inorg. Biochem. 2017, 177, 266–275. 69. Lambert, J.; Midolo, P. Aliment. Pharmacol. Ther. 1997, 11, 27–33. 70. Abughazaleh, R. D.; Tracy, T. S. Wiley Encyclopedia of Clinical Trials. Wiley, 2007. 71. Andrews, P. C.; Frank, R.; Junk, P. C.; Kedzierski, L.; Kumar, I.; MacLellan, J. G. J. Inorg. Biochem. 2011, 105, 454–461. 72. Keogan, D.; Oliveira, S.; Sangenito, L.; Branquinha, M.; Jagoo, R.; Twamley, B.; Santos, A.; Griffith, D. Dalton Trans. 2018, 47, 7245–7255. 73. Andrews, P. C.; Blair, V. L.; Ferrero, R. L.; Junk, P. C.; Kedzierski, L.; Peiris, R. M. Dalton Trans. 2014, 43, 1279–1291. 74. Andrews, P. C.; Ferrero, R. L.; Junk, P. C.; Kumar, I.; Luu, Q.; Nguyen, K.; Taylor, J. W. Dalton Trans. 2010, 39, 2861–2868. 75. Andrews, P. C.; Busse, M.; Deacon, G. B.; Ferrero, R. L.; Junk, P. C.; MacLellan, J. G.; Vom, A. Dalton Trans. 2012, 41, 11798–11806. 76. Busse, M.; Trinh, I.; Junk, P. C.; Ferrero, R. L.; Andrews, P. C. Chem. A Eur. J. 2013, 19, 5264–5275. 77. Loh, A.; Ong, Y. C.; Blair, V. L.; Kedzierski, L.; Andrews, P. C. J. Biol. Inorg. Chem. 2015, 20, 1193–1203. 78. Ali, M. I.; Rauf, M. K.; Badshah, A.; Kumar, I.; Forsyth, C. M.; Junk, P. C.; Kedzierski, L.; Andrews, P. C. Dalton Trans. 2013, 42, 16733–16741. 79. Barucki, H.; Coles, S. J.; Costello, J. F.; Gelbrich, T.; Hursthouse, M. B. Dalton Trans. 2000, 14, 2319–2325. 80. Barucki, H.; Coles, S. J.; Costello, J. F.; Hursthouse, M. B. J.Organomet. Chem. 2001, 622, 265–273. 81. Arora, M. Mater. Methods 2013, 3, 24. 82. Duffin, R. N.; Blair, V. L.; Kedzierski, L.; Andrews, P. C. J. Inorg. Biochem. 2018, 189, 151–162. 83. Iftikhar, T.; Rauf, M. K.; Sarwar, S.; Badshah, A.; Waseem, D.; Tahir, M. N.; Khan, A.; Khan, K. M.; Khan, G. M. J. Organomet. Chem. 2017, 851, 89–96. 84. Sarwar, S.; Iftikhar, T.; Rauf, M. K.; Badshah, A.; Waseem, D.; Tahir, M. N.; Khan, K. M.; Khan, G. M. Inorg. Chim. Acta 2018, 476, 12–19. 85. Saleem, L.; Altaf, A. A.; Badshah, A.; Rauf, M. K.; Waseem, A.; Danish, M.; Azam, S. S.; Arshad, M. N.; Asiri, A. M.; Ahmad, S. Inorg. Chim. Acta 2018, 474, 148–155. 86. Mushtaq, R.; Rauf, M. K.; Bolte, M.; Nadhman, A.; Badshah, A.; Tahir, M. N.; Yasinzai, M.; Khan, K. M. Appl. Organomet. Chem. 2017, 31 e3606. 87. Rice, D. R.; Vacchina, P.; Norris-Mullins, B.; Morales, M. A.; Smith, B. D. Antimicrob. Agents Chemother. 2016, 60, 2932–2940. 88. Costa, M. S.; Gonc¸alves, Y. G.; Nunes, D. C.; Napolitano, D. R.; Maia, P. I.; Rodrigues, R. S.; Rodrigues, V. M.; Von Poelhsitz, G.; Yoneyama, K. A. J. Inorg. Biochem. 2017, 175, 225–231. 89. do Nascimento, N. R. F.; de Aguiar, F. L. N.; Santos, C. F.; Costa, A. M. L.; de Jesus Hardoim, D.; da Silva Calabrese, K.; Almeida-Souza, F.; de Sousa, E. H. S.; de Franc¸a Lopes, L. G.; Teixeira, M. J. I. Acta Trop. 2019, 192, 61–65. 90. Chaves, J. D. S.; Tunes, L. G.; Franco, C. H.; de Franco, J.; Francisco, T. M.; Correa, C. C.; Murta, S. M.; Monte-Neto, R. L.; Silva, H.; Fontes, A. P. S.; de Almeida, M. V. Eur. J. Med. Chem. 2017, 127, 727–739.

ARTICLE IN PRESS 48

Rebekah N. Duffin et al.

91. Zhang, C.; Delmas, S. B.; Alvarez, A. F.; Valentin, A.; Hemmert, C.; Gornitzka, H. Eur. J. Med. Chem. 2018, 143, 1635–1643. 92. Vermeersch, M.; da Luz, R. I.; Tot
e, K.; Timmermans, J.-P.; Cos, P.; Maes, L. Antimicrob. Agents Chemother. 2009, 53, 3855–3859. 93. Walker, J.; Vasquez, J.-J.; Gomez, M. A.; Drummelsmith, J.; Burchmore, R.; Girard, I.; Ouellette, M. Mol. Biochem. Parasitol. 2006, 147, 64–73. 94. Prowse, A. B.; McQuade, L. R.; Bryant, K. J.; Van Dyk, D. D.; Tuch, B. E.; Gray, P. P. Proteomics 2005, 5, 978–989. 95. Fr
ezard, F.; Demicheli, C.; Ferreira, C. S.; Costa, M. A. Antimicrob. Agents Chemother. 2001, 45, 913–916. 96. Mukhopadhyay, R.; Dey, S.; Xu, N.; Gage, D.; Lightbody, J.; Ouellette, M.; Rosen, B. P. Proc. Natl. Acad. Sci. 1996, 93, 10383–10387. 97. Sun, H.; Yan, S. C.; Cheng, W. S. Eur. J. Biochem. 2000, 267, 5450–5457. 98. dos Santos Ferreira, C.; Martins, P. S.; Demicheli, C.; Brochu, C.; Ouellette, M.; Fr
ezard, F. Biometals 2003, 16, 441–446. 99. Baiocco, P.; Colotti, G.; Franceschini, S.; Ilari, A. J. Med. Chem. 2009, 52, 2603–2612. 100. Phillips, H. A.; Eelman, M. D.; Burford, N. J. Inorg. Biochem. 2007, 101, 736–739. 101. Zhitkovich, A. Chem. Res. Toxicol. 2005, 18, 3–11. 102. Eriksson, H.; M€agiste, K.; Plantin, L.-O.; Fonnum, F.; Hedstr€ om, K.-G.; Theodorsson-Norheim, E.; Kristensson, K.; Sta˚lberg, E.; Heilbronn, E. Arch. Toxicol. 1987, 61, 46–52. 103. Haynes, W. M. CRC Handbook of Chemistry and Physics. CRC press, 2014. 104. Chen, L. M.; Alali, W. Front. Microbiol. 2018, 9, 2412. 105. O’Morain, N. R.; Dore, M. P.; O’Connor, A. J. P.; Gisbert, J. P.; O’Morain, C. A. Helicobacter 2018, 23, e12519. 106. Domenico, P.; Baldassarri, L.; Schoch, P. E.; Kaehler, K.; Sasatsu, M.; Cunha, B. A. Antimicrob. Agents Chemother. 2001, 45, 1417–1421. 107. Beil, W.; Birkholz, C.; Wagner, S.; Sewing, K. F. Pharmacology 1995, 50, 333–337. 108. Sox, T. E.; Olson, C. A. Antimicrob. Agents Chemother. 1989, 33, 2075–2082. 109. Wang, R.; Lai, T. P.; Gao, P.; Zhang, H.; Ho, P. L.; Woo, P. C.; Ma, G.; Kao, R. Y.; Li, H.; Sun, H. Nat. Commun. 2018, 9, 439. 110. Kusters, J. G.; van Vliet, A. H. M.; Kuipers, E. J. Clin. Microbiol. Rev. 2006, 19, 449–490. 111. Shiota, S.; Suzuki, R.; Yamaoka, Y. J. Dig. Dis. 2013, 14, 341–349. 112. Andrews, P. C.; Busse, M.; Deacon, G. B.; Ferrero, R. L.; Junk, P. C.; Huynh, K. K.; Kumar, I.; MacLellan, J. G. Dalton Trans. 2010, 39, 9633–9641. 113. Busse, M.; Border, E.; Junk, P. C.; Ferrero, R. L.; Andrews, P. C. Dalton Trans. 2014, 43, 17980–17990. 114. Yang, N.; Sun, H. Coord. Chem. Rev. 2007, 251, 2354–2366. 115. Stavila, V.; Whitmire, K. H.; Rusakova, I. Chem. Mater. 2009, 21, 5456–5465. 116. Farrugia, L. J.; Lawlor, F. J.; Norman, N. C. J. Chem. Soc. Dalton Trans. 1995, 13, 1163–1171. 117. Chauhan, H. P. S.; Singh, U. P. Appl. Organomet. Chem. 2006, 20, 404–410. 118. Luqman, A.; Blair, V. L.; Brammananth, R.; Crellin, P. K.; Coppel, R. L.; Andrews, P. C. Eur. J. Inorg. Chem. 2016, 2016, 2738–2749. 119. Luqman, A.; Blair, V. L.; Brammananth, R.; Crellin, P. K.; Coppel, R. L.; Andrews, P. C. Eur. J. Inorg. Chem. 2015, 2015, 4935–4945. 120. Luqman, A.; Blair, V. L.; Brammananth, R.; Crellin, P. K.; Coppel, R. L.; Kedzierski, L.; Andrews, P. C. Eur. J. Inorg. Chem. 2015, 2015, 725–733. 121. Luqman, A.; Blair, V. L.; Brammananth, R.; Crellin, P. K.; Coppel, R. L.; Andrews, P. C. Chem. A Eur. J. 2014, 20, 14362–14377. 122. Domenico, P. Microbiol. Exp. 2015, 2, 9–10.

ARTICLE IN PRESS Antimony and bismuth complexes as antimicrobial agents

49

123. Mulani, M. S.; Kamble, E. E.; Kumkar, S. N.; Tawre, M. S.; Pardesi, K. R. Front. Microbiol. 2019, 10, 539. 124. Wu, C. L.; Domenico, P.; Hassett, D. J.; Beveridge, T. J.; Hauser, A. R.; Kazzaz, J. A. Am. J. Respir. Cell Mol. Biol. 2002, 26, 731–738. 125. Veloira, W. G.; Domenico, P.; LiPuma, J. J.; Davis, J. M.; Gurzenda, E.; Kazzaz, J. A. J. Antimicrob. Chemother. 2003, 52, 915–919. 126. Savarino, A. Expert Opin. Investig. Drugs 2006, 15, 1507–1522. 127. Bostrom, J.; Hogner, A.; Llinas, A.; Wellner, E.; Plowright, A. T. J. Med. Chem. 2012, 55, 1817–1830. 128. Rubina, B.; Dharam, P. P.; Garima, K.; Ravi, K.; Manni, D. Indian J. Pharm. Edu. Res. 2019, 53, S1–S16. 129. Bala, S.; Kamboj, S.; Kajal, A.; Saini, V.; Prasad, D. N. Biomed. Res. Int. 2014, 2014, 1–18, article id 172791. 130. Nagalakshmi, G. Indian J. Pharm. Sci. 2008, 70, 49–55. 131. Kotani, T.; Nagai, D.; Asahi, K.; Suzuki, H.; Yamao, F.; Kataoka, N.; Yagura, T. Antimicrob. Agents Chemother. 2005, 49, 2729–2734. 132. Nakamura, H.; Noh, J. Y.; Itoh, K.; Fukata, S.; Miyauchi, A.; Hamada, N. J. Clin. Endocrinol. Metab. 2007, 92, 2157–2162. 133. Matysiak, J.; Skrzypek, A.; Niewiadomy, A. Heteroat. Chem. 2010, 21, 533–540. 134. Banala, S.; Sussmuth, R. D. Chembiochem 2010, 11, 1335–1337. 135. Domenico, P.; Salo, R. J.; Novick, S. G.; Schoch, P. E.; van Horn, K.; Cunha, B. A. Antimicrob. Agents Chemother. 1997, 41, 1697–1703. 136. Prakash, V.; Lewis, J. S.; Jorgensen, J. H. Antimicrob. Agents Chemother. 2008, 52, 4528. 137. Gunawan, C.; Marquis, C. P.; Amal, R.; Sotiriou, G. A.; Rice, S. A.; Harry, E. J. ACS Nano 2017, 11, 3438–3445. 138. Nema, M.; Jonsson, M.; Varga, R. A.; Silvestru, A.; Silvestru, C. Rev. Roum. Chim. 2007, 52, 51–58. 139. Metre, R. K.; Kundu, S.; Narayanan, R. S.; Chandrasekhar, V. Phosphorus Sulfur Silicon Relat. Elem. 2015, 190, 2134–2141. 140. Andrews, P. C.; Werrett, M.; Herdman, M.; Brammananth, R.; Garusinghe, U.; Batchelor, W.; Crellin, P.; Coppel, R. Chem. A Eur. J. 2018, 49, 12938–12949. 141. Wu, J.; Zheng, Y.; Song, W.; Luan, J.; Wen, X.; Wu, Z.; Chen, X.; Wang, Q.; Guo, S. Carbohydr. Polym. 2014, 102, 762–771.