Design, synthesis, and biological evaluation of a focused pyrrolo- and pyrido-quinazolinone natural products-templated library for anti-fungal and anthelmintic activities

Abstract

A focused pyrrolo- and pyrido-quinazoline natural products-templated library has been designed and synthesized using two novel synthetic approaches: an aza-Nazarov approach and a Dieckmann cyclization strategy. This privileged scaffold-based collection was screened against pathogenic fungi and parasitic helminths. Three compounds (23b, 25a, 13f) emerged as potent inhibitors of Candida auris adhesion, each causing more than 50 % inhibition at 10 μM, and two compounds (13k, 14h) displayed strong anthelmintic activity, demonstrating 7- to 28-fold enhanced activity against human hookworms relative to albendazole. These molecules represent promising leads for the design and synthesis of focused quinazolinone analogs to support SAR driven optimization against C. auris or helminth pathogens. Ongoing efforts aim to refine these bioactive leads by improving their pharmacokinetic and activity profiles through targeted structural modifications.

1. Introduction

Gastrointestinal nematodes (GINs) are among the most prevalent disease-causing agents of humans, livestock, and companion animals. Human GINs include the roundworm Ascaris lumbricoides, the whipworm Trichuris trichiura, and three hookworm species: Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum; they are among the most prevalent infections of Neglected Tropical Diseases (NTDs),¹ affecting people in the world’s poorest communities,^2–4 infecting approximately 2 billion individuals worldwide, mostly living in tropical countries.^5,6 These parasites have a dramatic impact on human health and the global economy and together perpetuate the poverty cycle via their harmful effect on human well-being, especially among hundreds of millions of children and pregnant women.^7,8 The cost and losses associated with parasitic nematodes of livestock are estimated to be tens of billions of dollars worldwide due to adverse effects on health and productivity. Morbidity associated with GINs infections includes growth stunting, anemia, and cognitive impairment. The adverse impact of GINs is challenging to quantify, but they are responsible for less than 2–3 million disability-adjusted life years (DALYs), with hookworms accounting for nearly half of those DALYs.^3,9

Children who received hookworm treatment showed increased school attendance and adult income.^4,10 GINs also have significant impacts on the health and productivity of farm animals.¹¹ GINs are generally prevalent in communities without clean water and proper sanitation in the Global South— sub-Saharan Africa, South America, and Southeast Asia. Although human GIN elimination was reported in the southern region of the USA following years of deworming campaigns, there are significant concerns about the potential persistence of GIN infections, especially in areas with poor sanitation and hygiene.¹² Alarmingly, 16.5 % of serum samples collected from the low-income central Texas community were positive for human soil-transmitted GINs,¹³ and 34 % of fecal samples collected from rural Alabama regions tested positive for hookworm N. americanus.¹⁴ Human GINs are controlled through regular annual or semi-annual administration of anthelmintic drugs. The benzimidazoles, albendazole and mebendazole, are currently the only two drugs used in mass drug administration targeting school-age children. Unfortunately, parasitic nematodes of livestock are resistant to benzimidazoles, and only a few classes of anthelmintic drugs are available today.¹⁵ Following the same track, drug resistance in human nematode parasites is underway, with multiple reports of reduced activity and warnings of a foreseen resistance catastrophe.¹⁶ New drugs against STNs are urgently needed to replace those diminishing due to drug resistance. Furthermore, the fungal pathogen Candida auris has recently emerged as a deadly drug-resistant superbug in healthcare settings worldwide.¹⁷ It was first cultured in 2009 from the external ear canal of a patient in Japan.¹⁸ Within a few short years, C. auris infections were reported at hospitals in the Indian subcontinent, South Africa, Kuwait, Brazil, Venezuela, UK, and USA.¹⁷ The simultaneous emergence of multidrug-resistant C. auris in multiple geographic locations is an epidemiological conundrum and a serious global public health concern (CDC, USA, and PHE, Public Health England, UK reports). The burden and threats posed by this superbug have a significant impact on human health. It has been described as the “perfect pathogen” for a serious pandemic. The CDC reported that the fungus can become fatal if it permeates one’s bloodstream. Each year in the United States, an estimated 2 million people become infected with drug-resistant C. auris, resulting in at least 23,000 deaths. There is an urgent need for safe, effective, and resistance-resilient therapeutic agents against Candida auris.

Intriguingly, through our screening against parasitic nematodes, we have identified multiple antifungal compounds with broad activity against nematodes, among which are many azoles.^19,20 Interestingly, cross-reactivity between anthelmintic drugs and fungi has been frequently reported,^21,22; for example, mebendazole was found to inhibit the growth of Cryptococcus neoformans and Candida gattii and to enhance the antifungal activity of amphotericin B.²² On the other hand, many fungicidal drugs were found to have potent and broad-spectrum anthelmintic activity, among which are sulconazole, econazole, miconazole, and clotrimazole.^19,20,23,24 Similarly, de novo-synthesized chemical derivatives were found to carry similar dual activity against parasitic nematodes and fungal pathogens. For example, benzofuran derivatives were found to carry fungicidal and anthelmintic activity against Candida albicans and parasitic worms, respectively.²¹ The similarity in drug susceptibility between nematode parasites and fungal pathogens may result from convergent evolution of structurally and functionally similar, essential targets crucial for parasitism in vertebrate hosts. We envisioned this convergence could be tapped to develop dual-active leads for both parasitic nematodes and pathogenic Fungi via the quinazolinone natural product scaffolds.

2. Results and Discussion

2.1. Synthesis of a focused NP-based library

Among quinazolinone natural products (Fig. 1), Luotonin A, an alkaloid extracted from the Peganum genus, a perennial herb popular in Chinese traditional medicine, has been used to treat fungal infections and to expel gastrointestinal worms.^25–27 Luotonin A of P. nigellastrum, with a medicinally proven quinazolinone pharmacophore, is an attractive choice for synthesizing the modified derivatives for screening.^28–30 Thus, we have established the chemistry necessary for synthesizing and modifying quinazolinone natural products, particularly luotonin A and vascionone,^31,32 with an aim to screen these quinazolinone synthetic derivatives for potential antifungal and anthelmintic activities. Such a natural product-based medicinal chemistry approach requires the development of viable methodologies to access variously substituted NP analogues. We achieved this via two approaches, a novel aza-Nazarov approach and a Dieckmann condensation approach to access the fused quinazolinones, and the synthetic chemistry has been communicated earlier^31,32 and summarized below (Scheme 1). Accordingly, we have developed a new one-pot aza-Nazarov-based synthetic route to access pentacyclic C-ring substituted luotonins along with vasicinones. Our aza-Nazarov studies required identifying suitable conditions to cyclize the enones 11 into tricyclic enols 12. After exhaustive research, we found that triflic acid (TfOH) in different stoichiometries and solvent combinations effected the cyclizations in a regioselective manner. Thus, treatment of enones 11 in a relatively poor proton environment (lower amounts of TfOH in solvents) led to the formation of angular tricyclic enols 12a’-l’ that, upon one-pot Friedlander quinoline synthesis, formed C-aryl angular luotonins 13a-l.

Fig. 1.

Representative structures of bioactive quinazolinone natural products.

Scheme 1.

Synthesis Methodologies a) One-pot aza-Nazarov-Friedlander Sequence b) Dieckmann-Friedlander Sequence.

Alternatively, when quinazolinonyl enones 11 were treated in the presence of a proton-rich environment (large equivalents of TfOH), linear tricyclic enols 12a-o were obtained, which were then subjected to Friedlander quinoline synthesis to yield a series of C-aryl luotonins 14a-n.¹¹

Unlike the unstable and unisolable angular enols 12a’-l’ the linear tricyclic enols 12a-o upon NaBH₄ reduction afforded the C-aryl vasicinones 20b-o that can be regarded as vasicinone/nigellastrine analogues. The C-ring modification renders these quinazolinone analogues TOPO-I inactive through perturbation of the flat nature required for intercalation, thus reducing their cytotoxicity to the host.²⁷ Additionally, we have also developed the Dieckmann-Friedlander condensation approach for accessing B-ring substituted luotonins and rutaecarpines, along with modified versions of the same. Therein, we have cyclized the diesters 15a and 15b into the tricyclic ketones 18 and 19, respectively. These ketones, upon Friedlander condensation with o-amino phenyl systems, provided the B-ring substituted luotonins 21a-e and homoluotonins 24a-e. Fischer-indolization of these same ketones 18 and 19 with substituted hydrazines provided the nor-rutaecarpines 23a-c and rutaecarpines 22a-d, respectively. A few modified rutaecarpines were also obtained via bromination of the ketones 18 and 19 and treatment with bisnucleophiles such as thiourea to obtain modified rutaecarpines 22e-f (Fig. 2). The ketones 18 and 19 also enabled the synthesis of isaindigotones 25a-b and homoisaindigotones 26a-e through Wittig olefinations. The reductive amination of ketone 19 enabled access to homo-vasnetine 27, and L-selectride reduction gave homo-vasicinone. Thus, we have succesfully assembled a 70-member quinazolinone NPs-templated library (Fig. 2; please see the SI for the synthesis of compounds and characterization data) for lead identification by tapping the diversity built into the two in-house approaches, attesting to the potential of our synthetic methodologies to access larger librararies for lead optimization in the future. With this quinazolinone NPs-templated library of compounds in hand, we set out to identify potent validated lead for further optimization into powerful dual active NCEs to treat fungal and parasitic infections, representing a significant improvement over how they are currently treated. The structures of the templated library of various quinazolinone natural products and their analogues subjected to biological assays are listed in Fig. 2, and details of the screening studies are described in the following sections.

Fig. 2.

Structures of the compounds amassed via the aza-Nazarov and Dieckmann cyclizations.

2.2. Anthelmintic activity against the human hookworm Ancylostoma ceylanicum and Trichuris muris whipworms

Because the adult stages of GIN parasites are responsible for the infection burdens and disease manifestation, we decided to screen the synthetic collection of quinazolinone derivatives directly against the adult parasites of two evolutionarily distant GIN parasites: A. ceylanicum hookworm and T. muris whipworms, using automated motility readout (Fig. 3). Fifty-eight quinazolinone derivatives were tested against the adult stages of A. ceylanicum hookworms at 50 μM (Fig. 4). Because these helminth parasites are ensheathed in highly protective cuticles or teguments, higher screening concentrations (30–100 μM) are typically used when screening against helminth parasites.^{19,20,24,33–40} Of the 58 compounds, we identified 13 that significantly reduced the average motility of six adult hookworms by ≥50 % after 48 h of incubation at 50 μM. Interestingly, the angular luotonin 13k, and the C-aryl luotonin 14h were the most active against hookworms. These two compounds were tested against A. ceylanicum hookworms at doses of 0, 12.5, 25, 50, 100, and 200 μM, with four adults per concentration to generate their 50 % motility inhibition concentration (IC₅₀) values. The angular luotonin 13k was more effective than the C-aryl luotonin 14h, with an IC₅₀ of 12.93 μM compared to 54.2 μM. For comparison, the IC₅₀ of albendazole against the adult stages of GIN parasites is >376 μM against hookworms and > 750 μM against whipworms.^37,41,42 Albendazole is one of the two benzimidazole compounds recommended by the WHO for the MDA to control human GINs, including hookworms. Therefore, 13k and 14h demonstrated 7- to 28-fold enhanced activity against hookworms relative to albendazole.

Fig. 3.

Adult parasites screening. (A): Adult GIN parasites of Trichuris muris whipworms, Scale bar 1 mm. (B): Motility readout screenshot from a 48-well plate with a single adult worm using Wormassay-6 software and the Worminator imaging setup. Red depicts pixel changes from frame to frame (movement) and blue depicts no change in pixel (dead or very low movement). (C). Example of calculated motility readout, compounds considered active if the average motility inhibition ≤50 % relative to the DMSO control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Anthelmintic activity (%) of quinazolinone synthetic derivatives against the Human A. ceylanicum hookworms. IVM: Ivermectin, positive control.

Because we aimed to identify derivatives with broad activity against parasitic nematodes, we tested hookworm-active compounds against T. muris whipworms. Unfortunately, at 50 μM, most hookworm-active compounds demonstrated no significant activity against whipworms (Fig. 5). Interestingly, the hookworm active 24c demonstrated moderate activity against whipworms (42 % motility inhibition relative to DMSO control, P = 0.0025). Further optimization of 24c could significantly enhance its broad anthelmintic activity.

Fig. 5.

Anthelmintic activity (%) of hookworm active compounds against the T. muris whipworms. LEV: Levamisole, positive control.

2.3. Antifungal activity of quinazolinone synthetic derivatives against Candida auris B11221 strain

Planktonic growth and plastic adhesion assays were performed on C. auris B11221 in the presence of the quinazolinone synthetic derivatives, each at the highest concentration based on its solubility (Figs. 6 and 7). When analyzing C. auris planktonic growth in the presence of the compounds, only eight (13f, 14j, 22b, 22d, 24b, 24d, 25b, and 26e) exhibited over 20 % of growth inhibition, among which the isaindigotone 25b and the rutaecarpine 22d inhibited growth by <35 %. Noticeably, most compounds had a minimal impact on C. auris growth (Fig. 6 and SI Table 5).

Fig. 6.

Growth Inhibition (%) of C. auris B11221 in presence of quinazolinone synthetic derivatives. CAS: Caspofungin, positive control.

Fig. 7.

Inhibition (%) of C. auris B11221 surface adhesion in presence of quinazolinone synthetic derivatives.

Fungal surface adhesion is the initial step in biofilm formation, laying the foundation for the multilayer structure. This process allows the microbe to attach to surrounding surfaces, extending its survival in the environment while increasing the chance for fungal species transmission. Candida auris is a drug-resistant human pathogen known for causing severe infections and outbreaks, particularly in healthcare settings, where it can persist on surfaces. The B11221 strain from clade III is an isolate associated with severe disease and exhibits a significantly higher level of adhesion compared to other C. auris isolates. Therefore, inhibiting adhesion is a crucial aspect to evaluate for this pathogen. When we evaluated C. auris adhesion in the presence of the quinazolinones, several compounds reduced attachment to the polystyrene surface (Fig. 7 and SI Table 5). Among them, the angular luotonin 13f, the nor-rutaecarpine 23b, and the isaindigotone 25a showed high activity, each achieving more than 50 % inhibition of C. auris adhesion at just 10 μM, with no statistically significant differences between them (p < 0.05) (Fig. 7A). Additionally, the B-ring substituted luotonin 21d, homoluotonin 24a, and rutaecarpines 22c and 22d inhibited over 50 % of yeast adhesion to the surface at 50 μM (Fig. 7B). Within each group, the addition of halogens like bromide, chloride, or fluoride enhanced the activity, with the para position being particularly important. The tricyclic enol compounds (20b-o) were the least soluble and exhibited the lowest activity in inhibiting both growth and adhesion of C. auris, even at 500 μM (Fig. 7C). These results highlight that growth inhibition and adhesion inhibition assess different aspects of antifungal activity. While most compounds had little effect on C. auris planktonic growth, several displayed potent anti-adhesion effects, underscoring that quinazolinones may act primarily by interfering with biofilm initiation rather than by directly inhibiting fungal proliferation.

2.4. SAR Analysis

The molecules evaluated in this study share a quinazolinone core, yet the library displays substantial structural diversity, spanning tricyclic vasicinones, isaindigotone derivatives, pentacyclic luotonins, and rutaecarpines with varied substitution patterns. This structural heterogeneity makes it challenging to derive a unified SAR, and because the primary objective of this phase of research was lead identification, detailed SAR mapping will be pursued during subsequent lead-optimization studies.

Anthelmintic Activity:

Screening against helminths revealed several SAR trends: 1. Tricyclic vasicinone analogues were generally inactive, whereas the pentacyclic natural-product-derived scaffolds showed more promising activity. 2. Within the pentacyclic series, angular luotonins were more active than their linear counterparts. Halogen-substituted angular luotonins exhibited enhanced potency: 13c (p-Cl) and 13g (p-F) both outperformed the unsubstituted analogue 13a. Another notable finding was the strong influence of methyl substitution position on hookworm motility inhibition: Ortho-methyl (o-CH₃) substitution conferred markedly improved activity relative to para-methyl (p-CH₃). Analogue 13k (o-CH₃) emerged as the most potent hookworm motility inhibitor in the series. Homoluotonin A analogues also displayed promising activity and tolerated various quinoline-ring substitutions, though they remained slightly less potent than 13k—reinforcing 13k as a strong lead candidate for hookworm-focused development. Also, species-specific differences were observed: 13k, while effective against hookworm, showed limited activity against whipworm. Analogue 24c demonstrated the highest whipworm potency, making it a promising scaffold for future work aimed at broad-spectrum anthelmintic activity.

Antifungal Activity:

The quinazolinone natural-product library overall showed limited activity against C. auris growth, yet several analogues displayed noteworthy inhibition of fungal adhesion, an important virulence factor. Three natural-product–derived analogues stood out: 13f (luotonin A derivative), 23b (rutaecarpine derivative), 25a (isaindigotone derivative); noteworthy is that all three contain halogen substitutions, suggesting that halogenation may enhance antifungal adhesion-blocking activity.

3. Conclusions

We have successfully designed and synthesized a focused library of 70 pyrrolo/pyrido-fused quinazolinone natural-product–inspired compounds, to establish this fused quinazolinone scaffold as a promising privileged substructure for developing antifungal and anthelmintic agents. Screening studies identified angular luotonin 13k and C-aryl luotonin 14h as the most potent anti-nematode compounds, while angular luotonin 13f, nor-rutaecarpine 23b, and isaindigotone 25a as the most effective inhibitors of Candida auris adhesion, an early and critical step in biofilm formation. The mechanisms underlying these activities are unknown and will be investigated through the design and synthesis of expanded libraries to enable more detailed SAR analyses of quinazolinone natural product leads. As this study represents an initial in vitro screening and lead-identification effort, comprehensive assessments of drug-like properties and safety profiling will be addressed in future lead-optimization efforts and subsequent in vivo studies, and will be reported in due course.

4. Experimental Details

4.1. Synthesis details of the quinazolinone NP-templated library

General Info:

All the ¹H and ¹³C NMRs were recorded on a Bruker AVANCE III HD 400 MHz High-Performance Digital NMR spectrometer ¹H (400 MHz), ¹³C (101 MHz), with complete proton decoupling for 13C. Chemical shifts were analyzed on MestReNova software. Chemical shifts are reported in parts per million with the solvent resonance as the internal standard (CDCl₃, ¹H: 7.26 ppm, ¹³C: 77.16 ppm; DMSO, ¹H: 2.50 ppm, ¹³C: 39.52 ppm). Coupling constants are reported in Hertz (Hz). Abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, dq = doublet of quartet, dtd = doublet of triplet of doublet. Infrared spectra were collected on a Bruker ALPHA Compact FT-IR Spectrometer (ATR, solid/liquid).

General Procedure for the one-pot synthesis of angular luotonins (13a-l):

To a pressure vial containing the corresponding quinazolinonyl enone (11a-l, 1 mmol, 1 equiv) in dry DCE (15 mL) was added methane sulfonic acid (MsOH, 2 equiv) dropwise, and the resulting contents were heated at 80 °C for ~45 min - 1 h, followed by the addition of amino benzaldehyde (1.1 equiv) in one portion. The reaction mixture then continued to reflux at 80 °C until the disappearance of the starting material (per TLC). After the complete consumption of the reactants, the contents were cooled to room temperature and diluted with dichloromethane (~30 mL). The contents were then transferred to a separatory funnel, followed by the addition of saturated NaHCO₃ (~15 mL) and extracted with DCM (2X50mL). The combined organic layer was dried over Na₂SO₄ and reduced under vacuum to a final volume of ~2 mL. At this stage, the desired compound precipitated out, which was further cooled down to enhance the precipitation. The precipitate was collected by filtration, washed with cold diethyl ether, and dried to afford pure angular luotonins (13a-l).

General procedure for the synthesis of compounds (14a-n).

To a reaction vial containing the tricyclic enol (12a-n, 0.25 mmol) in ethanol (4 mL) was added 2-aminobenzaldehyde (0.312 mmol, 1.25 equiv), and the contents were stirred at 75 °C (the product starts to precipitate while the reaction is in progress) until the disappearance of the starting material. The reaction mixture was then reduced to ~0.5 mL under vacuum and cooled in an ice bath to maximize the precipitation. The contents were then filtered, washed with 3 to 4 drops of ice-cold ethanol, and dried to yield the product as a solid. It was re-suspended in water, followed by the addition of 10 % NaHCO₃ to remove any acidic impurities, and was filtered, washed with water, and dried to yield substituted luotonin (14a-n).

General procedure for the synthesis of compounds (20b-o).

To a reaction vial containing the corresponding tricyclic enol compound (12a-n, 0.25 mmol) in methanol (5 mL) was added NaBH₄ (0.5 mmol, 2 equiv) at 0 °C and the contents were stirred for 15–20 min at rt. After completion of the reaction, as evidenced by TLC, the reaction mixture was reduced to ~1 mL under vacuum, to which water (~0.5 mL) was added and stirred for 5 min. The resulting precipitate was filtered, washed with water, and dried to yield compounds 20b-o. The resulting alcohols were pure enough for analytical purposes. Recrystallization was performed, when necessary, in DCM: MeOH.

General Procedure for Preparation of luotonins (21b-d, 24b-d).

To a suspension of dione 18 or 19 (1.0 eq) and TsOH·H₂O (0.2 eq) in EtOH (10 mL) was added 2’-aminophenyl ketone (1.1–1.2 eq) in a pressure tube. The reaction mixture was heated at 100–110 °C for 5 h and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to obtain the luotonins.

General Procedure for Preparation of Rutaecarpines (22b-c, 23b-c).

To a solution of dione 18 or 19 (150 mg, 1.0 eq) in H₃PO₄ (2 mL) was added substituted phenylhydrazine hydrochloride (2.0 eq). The reaction mixture was heated at 180 °C for 1 h and then poured into ice. It was neutralized with solid NaHCO₃ to a pH of 5–6. The mixture was extracted with ethyl acetate (3 × 15 mL). The organic layers were combined and dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexane/ethyl acetate/DCM = 3:1:1) to obtain the Rutaecarpines.

General procedure of Wittig reactions for the synthesis of Isaindigotone and homo-Isaindigotone analogues (25a-b, 26a-e).

To a solution of phosphonium salt (1.50 mmol, 1.5 eq) in 10 mL of DCM was added t-BuOK (1.70 mmol, 1.70 eq). The color of the reaction mixture changed from yellow to orange-red quickly. After stirring at RT for 30–60 min, a solution of dione 18 or 19 (1.00 mmol, 1.0 eq) in 10 mL of DCM was added and stirred at RT overnight. The reaction mixture was diluted with a saturated aqueous solution of NH₄Cl (20 mL) and then separated. The aqueous layer was extracted with ethyl acetate (3 × 10 mL). The combined organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel using hexane/ethyl acetate as the eluent to obtain the product.

4.2. Anthelmintic activity against parasitic nematodes

Ethics statement.

All animal experiments were conducted according to the recommendations of the National Institute of Health (USA) Guide for Care and Use of Laboratory Animals and the Animal Welfare Act. The animal protocols used in this study (PROTO202000044 and PROTO202000071) were approved by the Institutional Animal Care and Use Committee (IACUC) of UMASS Chan Medical School.

Parasite maintenance in laboratory animals: Following standard protocols, A. ceylanicum hookworm parasites were maintained in golden Syrian hamsters,⁴³ and T. muris whipworm parasites were maintained in STAT6−/− mice.⁴⁴

Human hookworm parasites.

Quinazolinone synthetic compounds were initially tested against the parasitic stages of adult hookworm parasites. A. ceylanicum hookworm adults were harvested 17–22 days post-inoculation from the small intestines of infected hamsters. Harvested worms were washed and incubated in prewarmed hookworm culture medium (RPMI1640 of pH 7.2 supplemented with 10 % fetal bovine serum, 100 U penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin). Worms were manually picked into each well of the 48-well assay plates, six wells/test drug, containing 297 μL of hookworm medium. 3 μL of 5 mM in 100 % DMSO tested compounds were added to each well, giving a final concentration of 1 % DMSO and 50 μM test compound. Each compound was dispensed into screening wells, giving a final concentration in assay plates of 50 μM of test compounds and 1 % DMSO with duplicate wells/drug. Ivermectin was used as positive control at 50 μM. Assay plates were incubated for 48 h at 37 °C and 5 % CO₂. Worm motility was measured using an in-house assembled worminator and worm assay software⁴⁵ for 3 min after 24 and 48 h. Drug activity was determined using the percentage of motility inhibition compared to the DMSO control. Drugs were considered active if the 24- and/or 48-h average motility of six wells (1 worm/well) was reduced by at least 50 % compared to the negative control.

Whipworm parasites.

Next, we screened against T. muris whipworm parasites. T. muris adult whipworms were harvested from the cecum and large intestines of infected STAT6 −/− mice. Single worms were manually picked into each well of a 48-well plate, six wells/test drug, a final concentration of 50 μM and 1 % DMSO. Levamisole at 50 μM was used as positive control. Assay plates were incubated at 37 °C, and 5 % CO_2. T. muris whipworm motility was measured using an in-house assembled worminator and worm assay software⁴⁵ for 3 min after 24 and 48 h. Drug activity was determined using the percent of relative motility compared to the DMSO control. Drugs were considered active if the 24- and/or 48-h average motility of six wells (1 worm/well) was reduced by at least 50 % compared to the negative control.

Dose-response against human hookworms:

The top two hookworm active compounds, 13k and 14h, were tested against the adult stages of hookworms using a dose-response of 0, 12.5, 25, 50, 100, and 200 μM. Each dose was tested against four adult worms. Drug activity was determined using a worminator motility readout. GraphPad Prism generated the IC₅₀ values utilizing the 48-h average motility of 4 individual worms, normalized to the DMSO control.

4.3. Anti-fungal Activity

Preparation of stock solutions of quinazolinone synthetic derivatives.

Stock solutions of compounds were prepared at 100 or 10 mM in DMSO (depending on its solubility), and dilutions were made in each assay to have the desired concentration.

Inhibition of C. auris planktonic growth.

C. auris strain B11221 was obtained from the CDC. Recovery procedures were taken from instructions provided by the CDC. Later, C. auris B11221 was streaked on agar plates containing yeast extract, peptone, and dextrose (YPD) and grown at 30 °C prior to the assay. Single colonies were sub-cultured in YPD liquid medium at 30 °C overnight in a rotator at 180 rpm. Harvested cells were washed with PBS and resuspended in Synthetic Complete (SC) medium containing 2 % glucose. Optical density was measured at 600 nm (OD₆₀₀). For the growth curve assay, each well of a flat-bottom 96-well polystyrene plate was loaded with 200 μL of an inoculum of 0.05 OD/mL. Quinazolinone synthetic derivative solutions (2 μL) were added to each well to a final DMSO concentration of 1 %. Fluconazole was used as a control (256 μg/mL). Each condition was repeated in triplicate. The plate was incubated at 30 °C for 40 h, and absorbance measurements at 600 nm were done every 15 min in a BioTek Synergy H1 plate reader with shaking. After incubation, OD₆₀₀ was measured to monitor growth, and the percentage growth inhibition was calculated.

Inhibition of C. auris adhesion.

C. auris B11221 was grown in the same conditions as explained above. Then, cells were added to fresh SC + 2 % glucose medium to reach a final concentration of 0.5 OD/mL. Cell suspension (200 μL) was aliquoted into each well of a flat-bottom 96-well polystyrene plate. Compound solutions (2 μL) were added to each well to a final DMSO solvent concentration of 1 %. DMSO (1 %) alone was used as a control. Each condition was repeated in triplicate. Plates were covered and incubated at 37 °C for 4 h. After incubation, unattached cells were discarded, and 40 μL of 0.5 % Crystal Violet solution (CV) was added to each well for 45 min at room temperature to stain the attached C. auris cells on the polystyrene plates. The excess dye was discarded, and plates were washed 6 times with diH₂O and gently tapped onto a paper towel to remove residual diH₂O. To dissolve the CV in the attached cells, 200 μL of 75 % methanol was added to each well, and the plates were incubated at room temperature for 30 min. Absorbance of adhesion-retained crystal violet dye was measured at 590 nm and used as a measurement of adhesion to the plastic surface. Then 590 nm absorbance of each compound was divided by that of the DMSO control, and then subtracted by 100 % as the inhibition of adhesion³² . Please refer to SI for the data analysis.

Supplementary Material

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmc.2025.118517.

Data availability

No data was used for the research described in the article.