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Frontiers in Drug Discovery ·

Volume 6 - 2026 | https://doi.org/10.3389/fddsv.2026.1919483

Probing the activity of BM212, an antimycobacterial, and its derivatives against multiple life cycle stages of Plasmodium falciparum

  • Benedetta Salvucci1,2
  • Mariëtte van der Watt3
  • Chiara Tammaro1,4
  • Michela Guida1,5
  • Lucrezia Pantaleo1
  • Janette Reader6
  • Tayla Rabie6
  • Sizwe Tshabalala6
  • Mariska Naude6
  • Natanya Venter6
  • Tyrick Welcome6
  • Erica Erlank7
  • Lizette L. Koekemoer7
  • Mariangela Biava1
  • Lyn-Marié Birkholtz3,6,8*corresponding author
  • Giovanna Poce1*corresponding author
  1. 1Department of Drug Chemistry and Technologies, Sapienza University of Rome, Rome, Italy
  2. 2National Center Rare Diseases, Istituto Superiore di Sanità, Rome, Italy
  3. 3University of Pretoria Institute for Sustainable Malaria Control (UP ISMC), School of Health Systems and Public Health (SHSPH), University of Pretoria, Gezina, South Africa
  4. 4National Research Council (CNR) - Institute of Molecular Biology and Pathology (IBPM), Rome, Italy
  5. 5Department of Chemistry, Sapienza University of Rome, Rome, Italy
  6. 6Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Hatfield, South Africa
  7. 7Faculty of Health Sciences, Wits Research Institute for Malaria, University of the Witwatersrand, Centre for Emerging Zoonotic and Parasitic Diseases, National Institute for Communicable Diseases of the National Health Laboratory Services, Johannesburg, South Africa
  8. 8Department of Biochemistry, Africa Centre for Therapeutics Innovation, Stellenbosch University, Stellenbosch, South Africa
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Abstract

The emergence of resistance to frontline antimalarial drugs underscores the urgent need for new chemotypes capable of targeting multiple P. falciparum life-cycle stages, including transmission stages. Building on the antimycobacterial scaffold BM212, we generated and profiled a focused library of 1,3,5-trisubstituted pyrroles to define the structural features driving activity against asexual blood-stage (ABS) parasites and late-stage gametocytes (LGc) of P. falciparum. Clear structure -activity relationship (SAR) trends emerged: the C3 cyclic amine was the dominant driver of life-cycle stage preference, with methylpiperazine substituent conferring potent gametocytocidal activity (compound 7, IC50 0.093 μM), whereas the dimethyl-azasilinane group enabled potent dual-stage profiles (compound 1 IC50 values 0.48 against LGc and 1.08 against ABS). At N1 and C5, electron-deficient aryl substituents (4-F-phenyl, 2-CF3-phenyl) consistently enhanced potency and improved physicochemical properties, while aliphatic rings (e.g., cyclobutyl) at N1 optimized hydrophobic fit and solubility. Specific combinations of these motifs yielded balanced dual-stage activity without compromising lipophilicity or metabolic stability. Several optimized analogues retained activity against the multidrug-resistant P. falciparum Dd2 strain, with IC50 values ranging from 1.01 to 1.54 μM and exhibited potent transmission-blocking activity, with compound 9 achieving 81% transmission-reducing activity in the standard membrane feeding assay (SMFA). Overall, this work positions BM212 -derived pyrroles as a promising and versatile multistage antiplasmodial chemotype, while delineating the key structural determinants required to achieve potency, selectivity, and transmission-blocking efficacy.

Keywords

1 Introduction

Of the communicable infectious diseases, malaria remains a global health emergency, with progress in combating the disease stagnating, particularly due to the COVID-19 pandemic. According to the World Health Organization (WHO), an estimated 280 million malaria cases and 600,000 deaths occurred worldwide in 2024. The heaviest disease burden is still borne by the African Region, which accounts for 94% and 95% of estimated malaria cases and deaths worldwide (World Health Organization, 2025; Venkatesan, 2025). These figures indicate that global progress in reducing malaria morbidity and mortality has stalled since 2015, with incidence and mortality increasing by 9% and 12%, respectively, due at least in part to the emergence and spread of resistance to artemisinin and its partner drugs (Balikagala et al., 2022; Hawkes et al., 2015; Ikeda et al., 2018; Kayiba et al., 2021; Lu et al., 2017). Symptoms can manifest either as a non-specific febrile illness or a severe life-threatening disease with the most dangerous consequences in children (Kepha et al., 2016). P. falciparum has a complex life cycle, as echoed by the various developmental stages within different niches in the human host and mosquito vector (Burrows et al., 2017). Malaria transmission occurs when a female Anopheles mosquito injects parasites into the human peripheral blood system; sporozoites migrate to the liver to undergo exoerythrocytic replication, releasing merozoites that invade erythrocytes and sustain the development of asexual blood-stage parasites (ABS) (Vaughan and Kappe, 2017). A small portion of the schizonts produced during the ABS differentiate into sexual gametocytes, which differentiate through five morphological stages in P. falciparum parasites. Mature stage V gametocytes circulate in the peripheral blood and are the only forms capable of sustaining onward transmission (Day et al., 1998; Henry et al., 2019; Talman et al., 2004). Malaria elimination will therefore require a multipronged approach, including recently recommended vaccines such as RTS,S and R21/Matrix-M, together with strategies aimed at blocking parasite transmission (Birkholtz et al., 2022). Although drugs remain the mainstay of malaria control, most do not have transmission-blocking capabilities, largely due to the variant biology of these sexual life cycle stages of malaria parasites (Burrows et al., 2017; Birkholtz et al., 2022; van der Watt et al., 2022; Stampolaki et al., 2023). While immature gametocytes (stages I–IV) remain metabolically active and drug-sensitive (e.g., chloroquine, active on stage I-III or artemisinin derivatives active on stage I-IV gametocytes), mature stage V forms are metabolically quiescent, and most current drugs are therefore ineffective (Plouffe et al., 2016; Sinden et al., 2012; Reader et al., 2015). However, compounds that target both ABS parasites and mature gametocytes offer promise for reducing transmission rates and preventing the spread of resistance, once it has developed in the ABS stages (Reader et al., 2021). MMV1580843 (MMV’843) is a hit compound that emerged from a parallel screen of the Medicines for Malaria (MMV) Pandemic Response Box (PRB) (Reader et al., 2021). MMV’843 displayed stage-specific activity towards late-stage gametocytes (LGc; stage IV/V), with an IC50 of 0.11 µM, whilst being comparatively inactive against ABS parasites (IC50 > 5 µM) (Table 1). MMV’843 displays considerable structural similarities to an antitubercular compound, BM212 (Figure 1; pyrazole vs. pyrrole core). BM212 was originally identified during a phenotypic screen of a small library of azole, imidazole, pyrrole, and toluidine derivatives with activity against drug-sensitive M. tuberculosis (Mtb; Minimal Inhibitory Concentration, MIC = 5 µM) and multidrug-resistant Mtb (MIC = 8 µM) (Deidda et al., 1998). Mycobacterium membrane protein Large (MmpL3) has been proposed as the primary molecular target of BM212 based on whole-genome sequencing of resistant mutants generated by exposing M. tuberculosis, M. smegmatis, and M. bovis to high concentrations of the compound (La Rosa et al., 2012). MmpL3 is a conserved, essential inner membrane protein, involved in cell wall biosynthesis and belonging to the resistance, nodulation and cell division superfamily (Belardinelli et al., 2016). While several antimycobacterial agents have been found to exert non-specific activities (e.g., affecting membrane potential and the electrochemical gradient of the proton-motive force) (Belardinelli et al., 2016; Poce et al., 2016), BM212 inhibits trehalose monomycolate inner membrane flipping through direct binding with MmpL3 (Zhang et al., 2019). Based on the structural similarity between MMV’843 and BM212, we subsequently evaluated BM212 and a series of 1,3,5-trisubstituted pyrroles to evaluate the structure-activity relationships (SAR) of the BM212 scaffold as potential multistage active antiplasmodial compounds (Table 1). The study focused on ABS and transmissible stages of P. falciparum (gametocytes, gametes and oocysts), as they are responsible for malaria symptoms and parasite transmission, respectively. A focused medicinal chemistry campaign was conducted to explore SAR by modifying the substituents at N1, C5, and C3 while keeping the central core constant (Biava et al., 2005). Several compounds included in this study had been previously reported as BM212 analogues and evaluated for antitubercular activity (5, 25–31) (Poce et al., 2018; Li et al., 2018; Poce et al., 2013); however, these molecules were deliberately resynthesized and systematically re-evaluated within the present work to enable a coherent SAR analysis in the antiplasmodial context. A total of 33 compounds (Tables 2–6) were synthesized, strategically incorporating variations at the terminal aromatic and aliphatic regions to probe their impact on antimalarial activity. Compounds 1–3 represent BM212-derived analogues bearing modifications at the C3 cyclic amine. In particular, 1 incorporates the 4,4-dimethyl-1,4-azasilinane moiety, as in MMV’843, while 2 and 3 feature a morpholine and a 4,4-difluoropiperidine, respectively, with the aim of exploring the influence of these substitutions on biological activity and physicochemical properties. Subsequently, different substitutions were introduced at C5, including electron-withdrawing or moderately lipophilic groups such as 4- or 2-fluorophenyl (4, 5, 7, 9-11, 13-14, 16, 18, 23-26, 31, 33), 4-isopropylphenyl (21, 22, and 29), 4-trifluoromethylphenyl (19, 20 and 30) and heteroaromatic moieties such as 4-pyridyl (6, 8, 12, 15, 17, 27 and 28), to explore how different electronic properties influence biological activity. Diversification at the N1 position encompassed a broad range of substituents, from rigid aromatic groups (e.g., 4- or 2-fluorophenyl (4, 7, 8, 13, 15, 23-27, 30 and 32), 2-trifluoromethylphenyl (9, 14, 18)) to small aliphatic rings (e.g., cyclobutyl (5, 6, 11, 12, 16, 17, 19, 28, 31), cyclohexyl (21)) and nitrogen-containing heterocycles (e.g., three- or 4-methylpiperidinyl (10, 20, 22 and 29), enabling systematic evaluation of steric effects, and solubility. Matched-pair analyses, such as switching N1 substituents from aromatic to cyclic amines or aliphatic rings while maintaining a constant C5 group, provided insight into key pharmacophoric features. This modular optimization strategy was designed to aid the identification of structural motifs associated with ABS and gametocytocidal activity and to guide further optimization of potency and selectivity. All compounds were evaluated for their biological activity against ABS parasites of the drug-sensitive NF54 strain of P. falciparum (PfNF54 ABS) by detecting SYBR® Green I fluorescence as an indicator of parasite proliferation (Li et al., 2018), and compounds with IC50 < 1 µM were additionally profiled against the multidrug-resistant strain, PfDd2, to assess the potential for cross-resistance. Further, compounds were evaluated against late-stage gametocytes (stage IV/V) from PfNF54 (PfNF54 LGc), with viability detected with PrestoBlue® or Mitotracker® Deep Red and orthogonally evaluated for toxicity against a human hepatocellular carcinoma line, Hep G2.

TABLE 1. Antiplasmodial activity for the hit compounds MMV’843 and BM212.

CompoundMMV’843BM212
C34,4-Dimethyl-1,4-azasilin-1-ylN-methylpiperazin-1-yl
N14-Cl-phenyl4-Cl-phenyl
C54-Cl-phenyl4-Cl-phenyl
PfABS NF54/Dd2 IC50 (µΜ) [a]>5/ND>10/>10
PfNF54 LGc IC50 (µM) [a]0.110.18 ± 8.13
Pf gamete (% inhibition at 2 µM)58 (m), 64 (f)68 (m), 10 (f)
Oocyst formation (% inhibition) [b]80.262
Hep G2 EC50 (µΜ) [a]38.97.8
Solubility pH 6.5 (µΜ)9510
cLogD[c]4.224.19
tPSA[c]21.0611.4

[a]All cell growth inhibition data (IC50, EC50, % inhibition) are from ≥3 independent biological repeats each, each performed in technical triplicates, mean ± S.E. indicated.

[b]Transmission-reducing activity (TRA) after a 48 h drug treatment at 2 μM, of two independent biological repeats.

[c]cLogD and tPSA were calculated using StarDrop version 7.6.1.42965.

Structures of the pyrazole and pyrrole cores. The MMV PRB compound **MMV1580843** (**MMV’843**) displays a pyrazole core and azasilinane moiety at C3. **BM212** is structurally related but has a pyrrole core and methylpiperazine moiety at C3.
Figure 1. Structures of the pyrazole and pyrrole cores. The MMV PRB compound MMV1580843 (MMV’843) displays a pyrazole core and azasilinane moiety at C3. BM212 is structurally related but has a pyrrole core and methylpiperazine moiety at C3.

2 Materials and methods

2.1 Chemistry

The synthesis of the pyrrole library was based on a modular and efficient strategy, previously optimized in our research group, to provide rapid access to structurally diverse derivatives in good overall yields while minimizing the number of synthetic steps. This approach enabled the preparation of a sufficiently diverse compound set to support a comprehensive SAR investigation of this new class of antiplasmodial agents (Poce et al., 2013). Briefly, the 1,4-diketones 36a-g were synthesized through a Stetter reaction between methyl vinyl ketone (35) and the appropriate benzaldehydes (34a-g). Subsequent cyclization of 36a-g with the corresponding amines (37a-i) afforded the desired 1,5-disubstituted pyrroles 38a-r (Scheme 1). Compounds 1–9, 11–19, 21, 23–28, and 30–33 were then obtained by Mannich reaction of the appropriate 1,5-disubstituted pyrroles (38a-f, 38h, 38j, 38L-n, and 38p-r) with formaldehyde and the corresponding amines (Scheme 1). The 1,5-disubstituted pyrroles 39a-b and 41 were prepared by deprotection of the corresponding N-Boc-protected intermediates (38g, 38o, and 38i) using trifluoroacetic acid (TFA), followed by reductive methylation of intermediates 40a-b and 42 with formaldehyde and sodium triacetoxyborohydride (NaBH(CH3COO)3). Subsequent Mannich reaction of 40a-b and 42 with formaldehyde and the appropriate amines afforded compounds 10, 20, and 29 (Scheme 2). The synthesis of compound 22 is outlined in Scheme 3. Owing to experimental limitations, it was not possible to employ the same synthetic sequence used for compounds 10, 20, and 29 (Schemes 1 and 2). Therefore, the synthesis was accomplished through an alternative route. First, a Mannich reaction of the N-Boc-protected 1,5-disubstituted pyrrole 38k with morpholine afforded intermediate 43. Subsequent removal of the N-Boc protecting group with TFA yielded intermediate 44, which was finally subjected to reductive methylation using formaldehyde and NaBH(CH3COO)3 to furnish the target compound 22.

General procedure to prepare final compounds **1–9**, **11**–**19**, **21**, **23**–**28**, and **30**–**33**.
Figure 2. General procedure to prepare final compounds 1–9, 11–19, 21, 23–28, and 30–33.
**(A)** General procedure to prepare final compounds **10** and **29**; **(B)** general procedure to prepare final compound **20**.
Figure 3. (A) General procedure to prepare final compounds 10 and 29; (B) general procedure to prepare final compound 20.
General procedure for the preparation of the final compound **22**.
Figure 4. General procedure for the preparation of the final compound 22.

2.2 ADME

2.2.1 Aqueous solubility

The solubility of the compounds was measured using an adapted miniaturised shake-flask method as described (Zhou et al., 2007). Briefly, 4 μL of a 10 mM stock in dimethylsulfoxide (DMSO) was added to a 96-well plate and then evaporated using a GeneVac system. Phosphate buffer (pH 6.5) was then added to the wells and the plate was incubated for 24 h at 25 °C with shaking. Samples were subsequently centrifuged (3500g for 15 min) then transferred to a clean analysis plate. Analysis was then performed by HPLC- UV (Agilent 1200 rapid resolution HPLC coupled to a Diode Array Detector), and solubility of each sample determined from the corresponding calibration curve (concentration range: 10–220 μM).

2.2.2 Physicochemical predictions

ADME predictions were made using models derived from Medicines for Malaria Venture’s (MMV) proprietary data using the StarDrop Auto-Modeller module (https://optibrium.com/products/stardrop/modules/auto-modeller/).

2.3 Biology

Biology Ethics statement for in vitro biology. The in vitro work described holds ethics approval from the University of Pretoria (Health Sciences (506/2018) and Natural Sciences (180000094) Research Ethics Committee and the University of the Witwatersrand Human Research Ethics Committee (M130569) and Animal Ethics Committee (20190701−7O).

2.3.1 Asexual blood stage

P. falciparum asexual parasites were cultured from drug-sensitive NF54 (PfNF54) and multidrug-resistant strains PfDd2. Parasites were maintained in human erythrocytes suspended at 5% hematocrit in RPMI-1640 culture medium (23.81 mM sodium bicarbonate, 0.024 mg/mL gentamycin, 25 mM HEPES pH 7.5, 0.2% D-glucose, 0.2 mM hypoxanthine and Albumax II) under hypoxic conditions (5% CO2, 5% O2 and 90% N2) with moderate shaking (60 rpm) at 37 °C. Cultures were synchronized to >95% in the ring stage with 5% (w/v) D-sorbitol treatment (Smilkste et al., 2004).

2.3.2 Gametocytes

Gametocytogenesis was induced on highly synchronized (>95%) asexual ring stage parasites from PfNF54 at 0.5% parasitemia and 6% hematocrit and thereafter maintained in glucose-deprived media as previously described (Reader et al., 2015; Reader et al., 2022). After 3 days, the hematocrit was decreased to 4%. Parasites were maintained in complete culture medium supplemented with 50 mM N-acetyl-glucosamine for 5–10 days post-induction to inhibit proliferation of residual asexual blood stages. Gametocyte progression and morphology were also monitored microscopically using Giemsa -stained thin smears.

2.3.3 SYBR green I assay

Activity against ABS parasites of P. falciparum was evaluated using the SYBR Green I-based fluorescence assay as previously described (Reader et al., 2021; Leshabane et al., 2021). Cellular proliferation was measured on synchronized in vitro PfNF54 or PfDd2 ring-stage parasites (1% parasitemia, 2% hematocrit), following drug pressure for 96 h. Chloroquine was used as the positive drug control for complete inhibition of parasite proliferation. Half-maximal inhibitory concentration (IC50) values were calculated through nonlinear regression and statistical analysis performed using GraphPad Prism 8.3.0. All IC50s are reported as the means from three independent biological replicates, each performed in technical triplicates, with standard error indicated.

2.3.4 Gametocyte viability assays

In vitro activity against the sexual stages of P. falciparum was evaluated using the PrestoBlue® fluorescence assay as previously described (Reader et al., 2021). Activity was measured on PfNF54 stage IV/V gametocytes (2% gametocytemia, 5% hematocrit) previously seeded with compounds (in non-lethal DMSO) for 48 h drug pressure, after which PrestoBlue® reagent was added and incubated for 2 h. Fluorescence was detected in the supernatant at 612 nm. Dihydroartemisinin (DHA) was used as a positive drug control. Alternatively, P. falciparum late-stage gametocyte sensitivity was measured using a flow cytometric readout of parasitemia post-compound treatment. Late (stage IV/V) gametocyte cultures were mixed with 25 µL 1xSYBR™ Green I and 200 nM Mitotracker™ Deep Red in PBS. The samples were incubated for 30 min at 37 °C, washed and diluted to 200 µL in PBS. Stained cells were analyzed on the BD Accuri™ C6 Plus flow cytometer; recording a minimum of 100 000 events per sample. All data is reported for three independent biological repeats, each performed in technical triplicates, S.E. indicated.

2.3.5 Male gamete exflagellation inhibition assay (EIA)

The EIA was performed using video microscopy by capturing the movement of exflagellation centres over time as previously described (Reader et al., 2021; Coetzee et al., 2020). Mature PfNF54 stage V gametocytes (>95%) were treated with 2 µM of drug for 48 h before the activation of exflagellation, without removing drug pressure. Movement was semi-automatically quantified from 15 videos of 8–10 s each between 15 and 22.5 min after incubation. Each video was analyzed using ICY-bio image analysis software.

2.3.6 Female gamete inhibition assay

Female gametes were activated as described previously (Arendse et al., 2022) and monoclonal anti-Pfs25 antibody conjugated to FITC was used to detect female gametes. Image acquisition was performed using a Zeiss Axio Lab. A1 epifluorescence microscope with a 100/1.4 numerical aperture (NA) oil immersion. Thirty images were captured per sample and analyzed manually.

2.3.7 Standard membrane feeding assay (SMFA)

SMFA was performed using glass feeders as are outlined (Reader et al., 2021; Coetzee et al., 2020). Mature stage V gametocytes (>95%) were treated with 2 µM of each compound for 48 h prior to feeding. Anopheles coluzzii females were allowed to feed on gametocyte cultures (1.5%–2.5% gametocytaemia, 50% haematocrit in A+ male serum with fresh erythrocytes) in the dark for 40 min. All unfed or partially fed mosquitoes were removed, and remaining females housed for 8–10 days. Mosquitoes were dissected to remove midguts and oocysts counted under bright-field illumination at ×20–×40 magnification. The percentage of block in transmission (transmission-blocking activity; TBA) and the percentage of reduction in the number of oocysts (transmission-reducing activity; TRA) were calculated after normalizing the control untreated sample. The experiment was performed for two independent repeats, with a minimum of 10 mosquitoes dissected per treatment.

2.3.8 In vitro activity against Hep G2 cells

Human hepatocellular liver carcinoma cells (Hep G2) were cultivated in vitro in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin/streptomycin (Verlinden et al., 2016). Cells were detached with trypsin treatment at 80% confluency and were seeded in 96-well plates to adhere for 24 h under 5% CO2, humid (95%) conditions. Cells were subsequently exposed to drug pressure for 48 h and cytotoxicity was then determined using the lactate dehydrogenase (LDH) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release through the colorimetric detection at 450 and 570 nm, respectively (Reader et al., 2021; Leshabane et al., 2021; Sinha et al., 2019). All data are reported for three independent biological repeats, each performed in technical triplicates, S.E. indicated.

2.3.9 Data analysis and silico predictions

All biological data are from a minimum of three independent biological repeats (each performed with technical triplicates) unless otherwise indicated. Assay reproducibility was evaluated by Z′-factors, all at >0.8. Half-maximal inhibitory concentrations (IC50) were calculated by nonlinear regression using a four-parameter logistic dose-response model. Statistical evaluation was performed with paired, two-tailed t-test (95% CI) and analysed using GraphPad Prism 8.3.0. The physicochemical properties of the compounds were predicted using StarDrop version: 7.4.0.35635 (Optibrium Ltd.). The source data is provided in the Supplementary Material S1.

3 Results

3.1 Initial activity profiles exploring changes at C3

BM212 displayed >50-fold increased activity against LGc in vitro (IC50 = 0.18 µM) over ABS parasites (IC50 > 10 µM against both PfNF54 and PfDd2) (Table 2). This preferential gametocytocidal activity was accompanied by approximately 40-fold selectivity over mammalian Hep G2 cells (EC50 = 7.8 µM) (Table 2). Compounds 1–3 were designed as BM212 analogues bearing modifications at the C3 cyclic amine while retaining the 4-chlorophenyl substituents at both the N1 and C5 positions (Table 2). Replacement of the dimethyl azasilinane moiety with morpholine (2) or 4,4-difluoropiperidine (3) resulted in a marked reduction in activity against LGc (IC50 > 10 and >4 μM, respectively). In contrast, both analogues retained measurable activity against ABS parasites, with IC50 values of 4.37 and 1.41 µM, respectively, against PfNF54. These modifications were therefore associated with a marked shift in the biological profile, from preferential gametocytocidal activity to greater activity against ABS parasites. By comparison, the dimethyl azasilinane analogue 1 retained potent gametocytocidal activity (LGc IC50 = 0.48 µM) while displaying improved activity against ABS parasites (PfNF54 IC50 = 1.08 µM), suggesting that dual-stage activity can be achieved within the BM212 pyrrole scaffold, in contrast to the structurally related pyrazole MMV'843, which predominantly displays gametocytocidal activity (Reader et al., 2021). Moreover, compound 1 showed comparable activity against the multidrug-resistant PfDd2 strain (IC50 = 1.01 µM), suggesting that its activity is retained in this resistant parasite line. Overall, these findings suggest that the nature of the C3 cyclic amine strongly influences the stage-selective activity of this scaffold, with the dimethyl azasilinane moiety providing the most balanced activity profile among the analogues examined.

TABLE 2. Initial C3 exploration around BM212 scaffold.[a].

Cp IDC3N1C5PfNF54 LGc IC50 (µM) [CI][a]PfABS IC50 (µM) NF54 [CI] [a]PfABS IC50 (µM) Dd2 [CI] [a]Hep G2 EC50 (µM) [CI] [a]/(SI)[b]Solubility pH 6.5 (µM)cLogD[c]
BM212N-methylpiperazin-1-yl4-Cl-phenyl4-Cl-phenyl0.18 ± 8.13 [-34.80–35.1]>10>107.8/(1x)104.19
14,4-Dimethyl-1,4-azasilin-1-yl4-Cl-phenyl4-Cl-phenyl0.48 ± 0.063 [0.21–0.75]1.08 ± 0.046 [0.88–1.27]1.01 ± 0.16 [0.32–1.70]34.9 ± 10.8 [-11.57–81.37]/(32x)<54.38
2Morpholin-1-yl4-Cl-phenyl4-Cl-phenyl>104.37 ± 0.72 [1.27–7.47]ND[d]19.5 ± 6.13 [-6.88–45.88]/(4x)<52.45
34,4-Difluoro-piperidin-1-yl4-Cl-phenyl4-Cl-phenyl>41.41 ± 0.63 [-1.30–4.12]ND[d]>20/(>14x)165.00

[a]All cell growth inhibition data are from ≥3 independent biological repeats, each performed in technical triplicates, mean ± S.E., indicated. 95% confidence intervals (CI) are indicated in each instance in square brackets.

[b]S.I., selectivity index as ratio between EC50 determined against Hep G2 cells and the corresponding IC50 values for PfNF54 ABS, parasites.

[c]cLogD was calculated using StarDrop version 7.6.1.42965.

[d]ND, not determined.

3.2 Activity of the dimethyl azasilinane at C3

Since compound 1 displayed dual-stage activity (Table 2), further optimization focused on modifications at the N1 and C5 positions while retaining the C3 dimethyl azasilinane moiety as a favorable structural feature. Replacing the parent 4-chlorophenyl substituents with 4-fluorophenyl groups at both N1 and C5 largely preserved biological activity, although with a modest reduction in potency against LGc (Table 3), suggesting that a para-substituted electron-withdrawing (EWG) aromatic group at both positions is beneficial for activity. LGc activity was maintained when the N1 phenyl group was replaced with the smaller cyclobutyl substituent, affording compound 5, which displayed comparable activity against ABS and LGc (PfNF54 ABS IC50 = 1.69 µM; LGc IC50 = 1.15 µM; ABS/LGc fold difference = 1.5), together with improved aqueous solubility. In contrast, replacement of the C5 fluorophenyl group with a pyridyl ring (6) resulted in a complete loss of gametocytocidal activity despite a marked improvement in solubility, suggesting that increased polarity at this position is detrimental to biological activity. Although BM212 exhibited poor aqueous solubility, modifications at the N1 position generally improved both solubility and lipophilicity (cLogD <3). Among this series, compound 5 emerged as the most balanced analogue, combining dual-stage activity with improved physicochemical properties. Guided by these findings, additional analogues were designed by introducing a broader range of substituents at N1 to explore steric effects and optimize solubility and membrane permeability, while diversification of the C5 position with electron-withdrawing or moderately lipophilic groups was undertaken to further investigate the influence of electronic properties on biological activity.

TABLE 3. Antiplasmodial activity of the dimethyl azasilinane scaffold.[a].

Cp IDC3N1C5PfNF54 LGc IC50 (µM) [CI][a]PfABS IC50 (µM) NF54 [CI][a]Hep G2 EC50 (µM)[CI] [a]/(SI)[b]Solubility pH 6.5 (µM)cLogD[c]
44,4-Dimethyl-1,4-azasilin-1-yl4-F-phenyl4-F-phenyl4.40 (n = 1)0.61 ± 0.20 [-0.25–1.47]112 ± 50.2 [-104–328]/(184x)493.74
54,4-Dimethyl-1,4-azasilin-1-ylCyclobut-1-yl4-F-phenyl1.15 (n = 1)1.69 ± 0.061 [1.43–1.95]34.1 ± 12.9 [-24.40–86.60]/(20x)872.80
64,4-Dimethyl-1,4-azasilin-1-ylCyclobut-1-ylPyridin-1-yl>104.79 ± 0.31 [3.46–6.12]217 ± 10.8 [170.5–263.5]/(45x)2401.54

[a]All cell growth inhibition data are from ≥3 independent biological repeats, each performed in technical triplicates, mean ± S.E., indicated. 95% confidence intervals (CI) are indicated in each instance in square brackets.

[b]S.I., selectivity index as ratio between EC50 determined against Hep G2 cells and the corresponding IC50 values for PfNF54 ABS, parasites.

[c]cLogD was calculated using StarDrop version 7.6.1.42965.

3.3 Activity of the methylpiperazines at C3

Most modifications at the N1 and C5 positions were well tolerated within the methylpiperazine series, affording compounds that largely retained their potency against late-stage gametocytes while showing approximately fourfold improved activity against ABS parasites compared with BM212 (PfNF54 IC50 = 2.7–5.4 µM) (Table 4). Maintaining EWG at N1 and C5, either as fluorophenyl (7 and 9) or pyridyl (8) groups, preserved potent gametocytocidal activity, with the difluorinated analogue 7 displaying a twofold improvement over BM212 (LGc IC50 = 0.093 µM). Notably, this trend differed from that observed in the azasilinane series, where introduction of a pyridyl substituent resulted in complete loss of activity, suggesting that the methylpiperazine scaffold tolerates increased polarity at C5. These modifications also led to marked improvements in aqueous solubility, reaching 185, 195, and 180 µM for compounds 7, 8, and 9, respectively, together with reduced lipophilicity (cLogD <4; Table 4). However, a clear limitation of this series emerged. Replacement of the N1 chlorophenyl group with cyclobutyl was well tolerated in the presence of a C5 fluorophenyl substituent (11; LGc IC50 = 0.26 µM), whereas the corresponding pyridyl analogue (12) showed a >30-fold reduction in both ABS and LGc activity. These findings suggest that simultaneously reducing the lipophilicity at both N1 and C5 is detrimental to biological activity. Importantly, all compounds retained activity against the multidrug-resistant PfDd2 strain, displaying less than threefold differences in potency relative to PfNF54, and showed no detectable cytotoxicity against Hep G2 cells (EC50 > 25 µM) (Table 4). Overall, the methylpiperazine series combined potent and selective gametocytocidal activity with improved aqueous solubility and favorable selectivity indices (SI > 200–678), highlighting this scaffold as a promising starting point for the development of transmission-blocking antimalarial agents.

TABLE 4. Antiplasmodial activity of the methylpiperazine scaffold.[a].

Cp IDC3N1C5PfNF54 LGc IC50 (µM) [CI][a]PfABS IC50 (µM) NF54 [CI] [a]PfABS IC50 (µM) Dd2 [CI] [a]Hep G2 EC50 (µM) [CI] [a]/(SI)[b]Solubility pH 6.5 (µM)cLogD[c]
7N-methylpiperazin-1-yl4-F-phenyl4-F-phenyl0.093 ± 0.010 [0.050–0.14]2.707.97 ± 0.92 [4.01–11.93]20/(7.4x)1853.43
8N-methylpiperazin-1-yl4-F-phenylPyridin-1-yl0.27 ± 0.020 [0.18–0.35]3.66 ± 0.19 [2.84–4.48]ND[d]>260/(>70x)1952.66
9N-methylpiperazin-1-yl2-Trifluoromethyl-phenyl4-F-phenyl0.19 ± 0.061 [-0.24–0.28]4.71 ± 0.92 [0.75–8.67]ND[d]129 ± 60.8 [-132.6–390.6]/(27x)1804.37
10N-methylpiperazin-1-yl1-Methyl-piperidin-1-yl4-F-phenyl>104.95 ± 1.14 [0.045–9.86]ND[d]163 ± 26.5 [48.98–277.0] [/(33x)2711.62
11N-methylpiperazin-1-ylCyclobut-1-yl4-F-phenyl0.26 ± 0.0096 [0.22–0.30]5.12 ± 1.03 [0.69–9.55]ND[d]84.1 ± 3.35 [69.69–98.51]/(16x)1652.31
12N-methylpiperazin-1-ylCyclobut-1-ylPyridin-1-yl>10>10ND[d]>300/(>10x)3261.32

[a]All cell growth inhibition data are from ≥3 independent biological repeats, each performed in technical triplicates, mean ± S.E., indicated. 95% confidence intervals (CI) are indicated in each instance in square brackets.

[b]S.I., selectivity index as ratio between EC50 determined against Hep G2 cells and the corresponding IC50 values for PfNF54 ABS, parasites.

[c]cLogD was calculated using StarDrop version 7.6.1.42965.

[d]ND, not determined.

3.4 Activity of the diflouropiperidines at C3

Substitution at the C3 position with a 4,4-difluoropiperidine moiety (13–17) facilitated a fine-tuning of the physicochemical profile, yielding a significant improvement in aqueous solubility (up to 163 µM for 17) and a reduction in cLogD (Table 5). However, although ABS activity was maintained with EWG fluorophenyls (13, PfABS IC50 1.4 µM), none of the substitutions resulted in any improvements over the activity for the diflouropiperidine (3) initially evaluated (Tables 2, 5). Interestingly, maintenance of the flourophenyl groups (reference compound 3) combined with the introduction of EWG substituents at both the C5 and N1 positions shifted the compounds from a potential dual-acting profile toward selective ABS activity.

TABLE 5. Antiplasmodial activity of the diflouropiperidine scaffold.[a].

Cp IDC3N1C5PfABS IC50 (µM) NF54 [CI] [a]PfABS IC50 (µM) Dd2 [CI] [a]Solubility pH 6.5 (µM)cLogD[b]
134,4-Difluoro-piperidin-1-yl4-F-phenyl4-F-phenyl1.41 ± 0.67 [-1.47–4.29]1.44 ± 0.41 [-0.32–3.20]454.44
144,4-Difluoro-piperidin-1-yl2-triFluoromethyl-phenyl4-F-phenyl2.84 ± 0.41 [1.08–4.60]ND[c]345.00
154,4-Difluoro-piperidin-1-yl4-F-phenylPyridin-1-yl6.83 ± 1.16 [1.83–11.82]ND[c]1013.64
164,4-Difluoro-piperidin-1-ylCyclobut-1-yl4-F-phenyl7.57 ± 1.57 [0.81–14.33]ND[c]733.47
174,4-Difluoro-piperidin-1-ylCyclobut-1-ylPyridin-1-yl>10ND[c]1632.32

[a]All cell growth inhibition data are from ≥3 independent biological repeats, each performed in technical triplicates, mean ± S.E., indicated. 95% confidence intervals (CI) are indicated in each instance in square brackets.

[b]cLogD was calculated using StarDrop version 7.6.1.42965.

[c]ND, not determined.

3.5 Activity of the morpholino scaffold at C3

The introduction of a morpholine moiety at C3 was explored as a bioisosteric replacement for the piperazine and azasilinane rings. From a medicinal chemistry perspective, morpholine is a privileged heterocycle commonly used to reduce basicity relative to piperazine, improve metabolic stability, and introduce an additional hydrogen-bond acceptor through its ether oxygen. Although the initial morpholine analogue (2) showed limited activity against both ABS parasites and LGc, subsequent modifications at the N1 and C5 positions led to modest improvements in ABS activity, with compounds 18–20 displaying IC50 values below 2.5 µM (Table 6). More importantly, incorporation of the morpholine ring consistently improved the physicochemical profile of the series by reducing lipophilicity and increasing aqueous solubility. Potent gametocytocidal activity was restored by introducing 4- or 2-fluorophenyl substituents (25, 26, and 32), all of which retained sub-micromolar activity against late-stage gametocytes. Interestingly, exchanging the N1 and C5 substituents (33) resulted in a marked change in the biological profile relative to compound 32, indicating that the position of these substituents strongly influences biological activity. Consistent with the trends observed in the azasilinane and methylpiperazine series, the combination of a 4-fluorophenyl group at C5 and a cyclobutyl substituent at N1 (31) was associated with improved activity, suggesting that small saturated substituents at N1 are generally well tolerated. In contrast, introduction of either a para-trifluoromethyl (19, 20, and 30) or para-isopropyl group (21, 22, and 29) at C5 markedly reduced gametocytocidal activity while retaining moderate activity against ABS parasites, indicating that both the steric and electronic properties of the C5 substituent influence biological activity. Furthermore, the difluorophenyl analogue 26 exhibited substantially improved aqueous solubility (180 µM) compared with the parent morpholine analogue 2 (<5 µM). Finally, all compounds retained activity against the multidrug-resistant PfDd2 strain and showed no detectable cytotoxicity toward Hep G2 cells, confirming a favorable selectivity profile. Overall, incorporation of the morpholine scaffold significantly improved the physicochemical properties of the series, particularly aqueous solubility, while biological activity proved to be highly dependent on the substitution pattern at the N1 and C5 positions, defining a relatively narrow but informative SAR.

TABLE 6. Antiplasmodial activity of the morpholine scaffold. [a].

Cp. IDC3N1C5PfNF54 LGc IC50 (µM) [CI][a]PfABS IC50 (µM) NF54 [CI] [a]PfABS IC50 (µM) Dd2 [CI] [a]Hep G2 EC50 (µM) [CI] [a]/(SI)[b]Solubility pH 6.5 (µM)cLogD[c]
18Morpholin-1-yl2-Trifluoromethyl-phenyl4-F-phenyl0%1.02 ± 0.071 [0.71–1.33]1.54 ± 0.35 [0.034–3.05]49.3 ± 12.9 [-6.20–104.8]/(48x)464.50
19Morpholin-1-ylCyclobut-1-yl4-triFluoromethyl-phenyl>102.06 ± 0.16 [1.37–2.75]ND[d]>50/(>24x)963.55
20Morpholin-1-yl1-Methyl-piperidin-3-yl4-triFluoromethyl-phenyl0%2.45 ± 0.82 [-1.08–5.98]ND[d]ND[d]2282.97
21Morpholin-1-ylCyclohex-1-yl4-Isopropyl-phenyl15.82%3.36 ± 0.11 [2.88–3.83]ND[d]ND[d]674.02
22Morpholin-1-yl1-Methyl-piperidin-3-yl4-Isopropyl-phenyl0%3.80 ± 0.062 [3.53–4.07]ND[d]ND[d]1632.79
23Morpholin-1-yl4-F-phenyl4-F-phenyl0%>10ND[d]ND[d]523.86
24Morpholin-1-yl2-F-phenyl4-F-phenyl0%>10ND[d]ND[d]474.01
25Morpholin-1-yl4-F-phenyl2-F-phenyl0.29 ± 0.015 [0.23–0.35]>10ND[d]25.2 ± 8.21 [-10.12–60.52]/(>2x)1733.88
26Morpholin-1-yl2-F-phenyl2-F-phenyl0.53 ± 0.013 [0.49–0.57]>10ND[d]34.7 ± 4.35 [15.98–53.42]/(>3x)1803.56
27Morpholin-1-yl4-F-phenylPyridin-1-yl0%>10ND[d]ND[d]1552.93
28Morpholin-1-ylCyclobut-1-ylPyridin-1-yl32.14%>10ND[d]ND[d]3311.76
29Morpholin-1-yl1-Methyl-piperidin-1-yl4-isoPropyl-phenyl0%3.90 ± 0.32 [2.52–5.28]ND[d]ND[d]2022.36
30Morpholin-1-yl4-F-Phenyl4-triFluoromehtyl-phenyl>104.16 (n = 1)ND[d]100%<51.82
31Morpholin-1-ylCyclobut-1-yl4-F-phenyl64.80%4.93 ± 2.00 [-3.68–13.54]ND[d]ND[d]1172.99
32Morpholin-1-yl4-F-phenylPhen-1-yl0.42 ± 0.30 [-0.87–1.71]5.30 ± 1.06 [0.74–9.86]ND[d]64.9 ± 22.1 [(-30.19–160.0]/(12x)703.83
33Morpholin-1-ylPhen-1-yl4-F-phenyl0%9.00 ± 0.98 [4.78–13.22]ND[d]ND[d]703.83

[a]All cell growth inhibition data are from ≥3 independent biological repeats, each performed in technical triplicates, mean ± S.E., indicated. 95% confidence intervals (CI) are indicated in each instance in square brackets.

[b]S.I., selectivity index as ratio between EC50 determined against Hep G2 cells and the corresponding IC50 values for PfNF54 ABS, parasites.[c]cLogD was calculated using StarDrop version 7.6.1.42965.

[d]ND, not determined.

3.6 Transmission-blocking activity of BM212 and analogs

Since BM212 showed preferential activity against LGc, we next investigated whether this activity translated into transmission-blocking efficacy by assessing its effects on gamete and oocyst formation. BM212 inhibited male gamete exflagellation by 68%, resulting in a 62% TRA as measured using the SMFA (Table 1). Based on these findings, two optimized analogues representing the methylpiperazine (9) and morpholine (26) series were selected for further transmission-blocking evaluation because they exhibited sub-micromolar activity against LGc. Both compounds inhibited male gamete formation (Table 7); however, they showed a markedly stronger effect on female gamete formation, a profile that differs from that reported for many antimalarial candidates (Delves et al., 2013). This observation may suggest a mechanism of action distinct from that of compounds that primarily interfere with cell division during male gametogenesis (van der Watt et al., 2022; Delves et al., 2013), although the molecular target(s) remain unknown. Consistent with their activity against gamete formation, both compounds also reduced parasite transmission to the mosquito vector. In particular, compound 9, which inhibited >50% of male gamete formation and completely blocked female gamete formation, achieved an 81% transmission-reducing activity.

TABLE 7. Additional in vitro life cycle stage profiling.

Cp IDPfNF54 gamete (% inhibition at 2 µM)Oocyst formation (% inhibition)
MaleFemaleTRA[a]TRB[b]
BM21267.9 ± 7.69106238
954.0 ± 6.441008148
2642.9 ± 10.4100ND[c]ND[c]

[a]Transmission-reducing activity (TRA) after a 48 h drug treatment at 2 μM, of two independent biological repeats.

[b]Transmission-blocking activity (TBA) after a 48 h drug treatment at 2 μM, of two independent biological repeats.

[c]ND, not determined.

4 Discussion

In this study, a medicinal chemistry campaign based on the antimycobacterial scaffold BM212 led to the identification of pyrrole derivatives displaying distinct and tunable antiplasmodial profiles across multiple P. falciparum life cycle stages. A comprehensive SAR analysis suggested that both the nature of the cyclic amine at C3 and the electronic properties of the N1/C5 substituents influence not only potency but also the apparent life cycle preferential activity between ABS parasites and LGc. Notably, modulation of the C3 cyclic amine was associated with marked changes in the biological profile of the scaffold; more basic and conformationally constrained amines, such as the dimethyl azasilinane and N-methylpiperazine moieties, tended to be associated with dual-stage activity. This trend was exemplified by compounds 1 and 7, which combined potent gametocytocidal activity (LGc IC50 down to 0.093 µM) with improved ABS inhibition and favorable physicochemical properties. By contrast, the morpholine and difluoropiperidine series generally displayed greater stage preference, with morpholine-containing analogues showing a preference for gametocytocidal activity, as observed for compounds 25, 26, and 32. Across the series, fluorophenyl substituents at N1 and C5 were consistently associated with improved potency, broader life cycle activity, and enhanced aqueous solubility (up to 180–195 µM), while maintaining low cytotoxicity. Conversely, bulkier substituents at the C5 position, such as para-trifluoromethylphenyl or isopropylphenyl groups, were generally associated with reduced gametocytocidal activity. Given the interesting life cycle fingerprint of this scaffold, and the fact that Plasmodium does not have an MmpL3 homolog (as primary target of BM212 in mycobacteria), it is important for future work to identify the molecular target(s) or understand the mode of action of this scaffold. Typical methodologies available within the malaria community (Cowell and Winzeler, 2018; Cowell et al., 2018; Godinez-Macias et al., 2025) including label-free methods such as enhanced evolution techniques to select for resistant parasites that could be present in the target gene would be applicable to this series. Alternatively, proteomics techniques, including label-free (e.g., thermal protein profiling) or using labelled versions of lead compounds to perform chemical pulldown proteomics could directly show protein target engagement in the parasites. Interestingly, other well-known MmpL3 inhibitors like ethambutol and the ethylenediamine SQ109 have previously also shown activity against P. falciparum parasites, with a similar preference towards LGc compared to ABS parasites (Reader et al., 2021; Watson et al., 2024; Stampolaki et al., 2023). The involvement of MmpL3 or resistance nodulation-division superfamily proteins (to which MmpL3 belongs) has been excluded for these compounds, rather pointing to ionophore activity (Reader et al., 2021). A similar phenotype for the BM212 derivatives is unlikely. Importantly, in light of the resistance pressure that current antimalarials are under, including the de novo development of artemisinin kelch-13 resistance mutations in African parasite populations, it would be important to determine the minimum inoculum of resistance for this series to evaluate their potential propensity to resistance (Rosenthal et al., 2024; Deni et al., 2023; Duffey et al., 2021). However, the sensitivity towards PfDd2 parasites by this series implies that mutations in the chloroquine resistance transporter (PfCRT), antifolate resistance mutations or copy number variation in multidrug resistance transporter (MDR) does not confer resistance to the compounds evaluated here. These parameters are critical to evaluate the useful candidates from the series.

With this initial investigation, we provide a new scaffold that could progress in a formal hit-to-lead campaign, where the effects of the changes to active compounds have to be investigated in light of enhanced metabolic stability in microsomes and hepatocytes, leading to pharmacokinetic characterization and in vivo efficacy studies to assess the potential early lead antimalarial candidates. Collectively, these findings identify the BM212 pyrrole scaffold as a promising starting point for the development of multistage antiplasmodial agents and provide SAR trends that may guide the future optimization of transmission-blocking antimalarial compounds.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fddsv.2026.1919483/full#supplementary-material

Supplementary material Supplementary material at the source

Supplementary material Supplementary material at the source

Footnotes

Edited by: Elliasu Yakubu Salifu, Tshwane University of Technology, South Africa

Reviewed by: Hari Madhav, International Centre for Genetic Engineering and Biotechnology, India

Ganesh Sharma, Bhupal Noble’s Institute of Pharmaceutical Sciences, India

Statements

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author GP declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This project was in part supported by the Medicines for Malaria Venture (RD-19-001), Bill and Melinda Gates Foundation Grand Challenges Africa Program (GCA/DD2/Round10/021/001) and the Department of Science and Innovation South African Research Chairs Initiative Grants managed by the National Research Foundation (UID: 84627) to LM-B. This work was supported in part by Research Grant RM120172A80094C4 from the Sapienza University of Rome research grants.

Data availability

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics approval

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

Author contributions

BS: Conceptualization, Writing – original draft, Writing – review and editing, Data curation, Investigation, Methodology. MW: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review and editing. CT: Data curation, Investigation, Writing – original draft, Writing – review and editing. MG: Investigation, Writing – review and editing. LP: Investigation, Writing – review and editing. JR: Writing – review and editing, Data curation. TR: Data curation, Investigation, Writing – original draft. ST: Data curation, Investigation, Writing – review and editing. MN: Data curation, Investigation, Writing – review and editing. NV: Data curation, Investigation, Writing – review and editing. TW: Investigation, Writing – review and editing. EE: Data curation, Investigation, Writing – review and editing. LK: Conceptualization, Data curation, Writing – review and editing. MB: Data curation, Writing – review and editing. L-MB: Conceptualization, Data curation, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing. GP: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing.

Acknowledgements

We thank Shante da Rocha and Marche Mare from the University of Pretoria and Nelius NV from WITS University for technical assistance.

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Citation

Salvucci, B., et al. (2026). Probing the activity of BM212, an antimycobacterial, and its derivatives against multiple life cycle stages of Plasmodium falciparum. Frontiers in Drug Discovery, Volume 6. https://doi.org/
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6 - 2026

Copyright

Copyright © 2026 Salvucci, van der Watt, Tammaro, Guida, Pantaleo, Reader, Rabie, Tshabalala, Naude, Venter, Welcome, Erlank, Koekemoer, Biava, Birkholtz and Poce.

License: https://creativecommons.org/licenses/by/4.0/

*Correspondence: Lyn-Marié Birkholtz; Giovanna Poce

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