Exploring novel thiazole-based minor groove binding agents as potential therapeutic agents against pathogenic Acanthamoeba castellanii

Abstract

Due to limited advances in diagnosis and targeted therapy, as well as poor understanding of pathophysiology, infections due to Acanthamoeba have remained a medical concern. With their ability to selectively bind to DNA sequences, minor groove binders have emerged as useful therapeutic agents against parasitic infections. Herein, 6 novel thiazole-based minor groove binders were synthesized. Purification of intermediate compounds was accomplished by utilising silica gel column chromatography, while thin-layer chromatography was utilised to monitor reactions. The purification of the final products was achieved using liquid chromatography. Confirmation of structures was achieved by NMR spectroscopy and mass spectrometry. All compounds were evaluated against pathogenic A. castellanii via in vitro assays. At micromolar concentrations, selected minor groove binder derivatives revealed potent effects against (i) A. castellanii trophozoites as observed using amoebicidal assays, (ii), against A. castellanii cysts as observed using excystation assays, and (iii) against A. castellanii-mediated host cell death utilising human cerebrovascular endothelial cells, but (iv) showed limited effects against host cells alone, using cytotoxicity assays. The binding interaction between minor groove binders and DNA was studied using isothermal titration calorimetry and molecular docking simulations to provide insights into their binding affinity and mode of interaction. The findings of our study underscore the therapeutic value of thiazole-based minor groove binders as potent agents against A. castellanii, demonstrating effective antiamoebic activity with a low propensity for human cell damage, thus supporting their further development as antiamoebic agents.

Acanthamoeba infections remain a medical challenge due to limited diagnosis, therapies, and unclear pathophysiology. DNA-targeting minor groove binders show promise as novel antiamoebic agents.

Introduction

Acanthamoeba is a free-living, opportunistic protozoan and a causative agent of skin lesions and deadly central nervous system infections, i.e., granulomatous amoebic encephalitis and blinding keratitis.^1–3 The genotypes of Acanthamoeba have been categorized into several genotypes, based on sequence differences in the 18S rRNA gene.⁴ Although there are around 23 genotypes,⁵ isolates belonging to the T4 genotype are frequently identified from the infection sites.^6,7

The Acanthamoeba life cycle consists of an active trophozoite form that reproduces asexually through mitosis, and a double-walled cyst form, which is dormant with minimal metabolic activity.^1–3,8 The physical structure of the cysts (multilayered walls) and minimal metabolic activity hinder and/or limit the efficacy of commercially available drugs.^9,10 This is expected as antimicrobial drugs often target metabolic activity, and hence the available antiamoebic compounds show limited activity against the cyst stage.^11,12 During the cyst stage, amoebae remain dormant but viable for decades while retaining their pathogenicity.¹³ Once the treatment is halted, amoebae may reemerge from the cyst stage resulting in infection recurrence; hence, it is important to identify drugs that can target both the cyst and trophozoite forms of Acanthamoeba.

Our recent studies showed that DNA minor groove binders have potential as anti-parasitic compounds.¹⁴ These compounds can impair DNA structure and function by binding to the minor groove of duplex DNA.^15,16 These substances have demonstrated therapeutic capabilities in the treatment of microbial and cancer illnesses due to their capacity to bind reversibly with minor grooves.^15,17–21 With the aim of creating both drugs and molecular probes for DNA polymorphism, numerous research projects have concentrated on targeting specific DNA sequences using synthetic ligands. Controlling gene expression, or the selective inhibition of transcription from particular regions by the targeted action of a ligand, has emerged as a viable research area. DNA minor groove binders exhibit diverse biological activities, including anticancer, antibacterial, antiparasitic, and antiviral effects, by disrupting essential DNA–protein interactions in target cells.^15,16 In this study, the design of the MGBs was guided by the three-dimensional NMR structure of the lead compound AIK18-51 bound to DNA (PDB: 2mne).^15,16 A series of novel analogues, incorporating modifications to the head, tail, and heterocyclic regions, were synthesized and their interactions with DNA were characterised using isothermal titration calorimetry (ITC) and molecular modelling. Molecular docking simulations were employed to predict their binding conformations and to identify the key interactions involved in stabilising the MGB–DNA complex. This structure-based drug design strategy facilitates the efficient discovery of new therapeutic candidates and enhances the success rate of drug development. Herein, we evaluated novel thiazole-based DNA minor groove binders for their effects against pathogenic A. castellanii.

Materials and methods

Chemistry

Chemicals and organic solvents were obtained from Sigma-Aldrich (Steinheim, Germany) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Proton (¹H) and carbon (¹³C) NMR spectra were recorded on a Bruker 500 MHz spectrometer, with chemical shift values (δ, in ppm) referenced to tetramethylsilane as the internal standard. Reaction progress was assessed by thin-layer chromatography (TLC) utilising silica gel plates (0.25 mm thickness, 60 F254, E. Merck). Intermediate compounds were isolated using silica gel column chromatography (particle size: 200–400 mesh), while the final purification was accomplished through preparative high-performance liquid chromatography (HPLC) on an Agilent 1260 Infinity system (Agilent Technologies, Palo Alto, CA, USA), equipped with a Zorbax C18 column (5 μm; 25 cm × 9.4 mm i.d.). The synthetic procedures used for both intermediate and final MGB compounds followed previously established methods.¹⁷ Structural verification of all synthesized products was performed using NMR and electrospray ionization mass spectrometry (ESI-MS) on a Micromass Quattro micro™ system (Waters Corp., Milford, MA, USA). Additional experimental details and full spectral data are available in the SI (S1).

Experimental procedure for the synthesis of methyl-5-isopropyl-2-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)thiazole-4-carboxylate (3)

To a stirred solution of methyl-2-amino-5-isopropyl thiazole-4-carboxylate (1) (1.0 g, 5.0 mmol) and TEA (2.07 mL, 15.0 mmol) in anhydrous DCM (8 mL), N-methyl-4-nitro-2-trichloroacetylpyrrole (2) (1.35 g, 5.0 mmol) in anhydrous DCM (10 mL) was added dropwise at 0 °C, and the reaction mixture was left stirring under N₂ for 1 h at rt. The solvent was removed under reduced pressure, a solution of aq. Na₂CO₃ (25 mL) was added, and the product was then filtered and washed with 40 : 60 MeOH : water and dried under reduced pressure to give pure compound 3.

Collected as half-white color solid; yield (70%); m.p: 130–132 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.67 (d, J = 1.4 Hz, 1H), 7.29 (d, J = 1.8 Hz, 1H), 4.14 (sept, J = 6.9 Hz, 1H, (CH₃)₂CH–), 4.08 (s, 3H, –OCH₃), 3.88 (s, 3H, –NCH₃), 1.38 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, CDCl₃) δ 161.8, 157.8, 154.4, 153.3, 135.7, 132.3, 128.5, 123.6, 109.9, 52.4, 38.5, 27.8, 24.8. ESI-MS: m/z calcd for C₁₄H₁₆N₄O₅S, 352.08 found 353.02 [M + H]⁺.

Experimental procedure for the synthesis of compounds 5a and 5b

To a stirred solution of compound 3 (0.88 g, 2.5 mmol) in THF (6 mL) and H₂O (4 mL), LiOH (0.18 g, 7.5 mmol) was added, and the reaction mixture was left stirring for 6 h. The organic solvent was evaporated under reduced pressure, and the aqueous layer was acidified by using 1 N HCl solution. The desired product was precipitated, filtered, and washed with distilled water; it was then dried under vacuum to obtain the desired acid intermediate as a white color solid, which was directly dissolved in DMF (5 mL), and HBTU (1.13 g, 3 mmol), TEA (1.0 mL, 7.5 mmol) and compound 4a or 4b (3 mmol) at 0 °C were added sequentially and the reaction mixture was left stirring under N₂ for 15 h at rt. After 15 h, the reaction mixture was diluted with sat. aq. Na₂CO₃ (25 mL), filtered the precipitated compound, washed with H₂O (30 mL), and dried under reduced pressure to give compounds 5a and 5b.

N-(3-(Dimethylamino)propyl)-5-isopropyl-2-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)thiazole-4-carboxamide (5a)

Collected as a pale-yellow color solid; m.p: 221–223 °C; yield: 60%; m.p. ¹H NMR (500 MHz, CDCl₃) δ 7.69 (d, J = 1.5 Hz, 1H), 7.63 (d, J = 1.8 Hz, 1H), 7.54 (t, J = 5.8 Hz, 1H), 4.40 (sept, J = 6.9 Hz, 1H, (CH₃)₂CH–), 4.09 (s, 3H, –NCH₃), 3.43 (dd, J = 12.9, 6.7 Hz, 2H, –NCH₂CH₂CH₂N–), 2.40 (t, J = 7.1 Hz, 2H, –NCH₂CH₂CH₂N–), 2.25 (s, 6H, –NCH₃), 1.79 (p, J = 6.9 Hz, 2H, –NCH₂CH₂CH₂N–), 1.33 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C NMR (126 MHz, CDCl₃) δ 162.8, 157.9, 152.8, 148.9, 136.1, 135.5, 128.4, 124.2, 109.7, 57.5, 45.6, 38.6, 37.7, 27.5, 27.2, 25.1. ESI-MS: m/z calcd for C₁₈H₂₆N₆O₄S, 422.17 found 423.08 [M + H]⁺.

5-Isopropyl-2-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)-N-(3-morpholinopropyl)thiazole-4-carboxamide (5b)

Collected as a pale-yellow color solid; m.p: 207–209 °C; yield: 55%; m.p. ¹H NMR (500 MHz, DMSO-d₆) δ 8.29 (d, J = 1.2 Hz, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.82 (t, J = 5.9 Hz, 1H), 4.19 (sept, J = 6.9 Hz, 1H, (CH₃)₂CH–), 3.98 (s, 3H, –NCH₃), 3.60–3.54 (m, 4H, –NCH₂CH₂O), 3.29 (dd, J = 13.2, 6.7 Hz, 2H, –NCH₂CH₂CH₂N–), 2.38–2.29 (m, 6H), 1.66 (p, J = 7.0 Hz, 2H, –NCH₂CH₂CH₂N–), 1.26 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C NMR (126 MHz, DMSO-d₆) δ 162.4, 158.6, 153.8, 145.9, 136.9, 134.6, 130.2, 124.4, 110.7, 66.6, 56.5, 53.79, 38.4, 37.5, 26.6, 26.5, 25.2. ESI-MS: m/z calcd for C₂₀H₂₈N₆O₅S, 464.18 found 465.12 [M + H]⁺.

Experimental procedure for the synthesis of final products 7–12

Nitro derivatives 5a/5b (0.43 mmol) in DMF : MeOH : THF (0.5 : 1.0 : 4.0 mL) with 10% Pd/C (90 mg) were flushed with H₂ at 0 °C under N₂, then stirred under H₂ at room temperature. After 4 h, the reaction mixture was then filtered through a short path celite pad to remove the Pd-catalyst and the collected filtrate was evaporated under reduced pressure to afford the amine compound. Without further purification, the obtained amine compound was then diluted with DMF (1 mL) and added dropwise to a stirred solution of corresponding acids 6a–6c (0.36 mmol), HBTU (163 mg, 0.43 mmol) and TEA (0.3 mL, 2.16 mmol) in DMF (2 mL) and stirring was continued at room temperature, under N₂. After 15 h, the reaction mixtures were diluted with EtOAc (20 mL) and H₂O (40 mL). The organic layer was then separated and washed with sat. aq. NaHCO₃ solution (10 mL) and H₂O (10 mL) followed by sat. aq. NaCl solution (10 mL) and finally dried over NaSO₄, filtered, and evaporated under reduced pressure to afford the crude compounds. The crude products obtained were purified through reverse-phase HPLC employing a gradient elution protocol. The products were collected and dried using a freeze-drying method to obtain the desired products 7–12 in moderate to good yields.

N-(3-(Dimethylamino)propyl)-5-isopropyl-2-(4-(5-isopropyl-2-(nicotinamido)thiazole-4-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)thiazole-4-carboxamide (7)

Collected as a yellow color solid; m.p: 220–222 °C; yield: 49%; ¹H NMR (500 MHz, CDCl₃) δ 10.98 (bs, 1H), 9.66 (s, 1H), 9.28 (bs, 1H), 8.80 (d, J = 7.9 Hz, 1H), 8.69 (d, J = 4.8 Hz, 1H), 8.13 (s, 1H), 7.71–7.60 (m, 1H), 7.56 (s, 1H), 7.09 (s, 1H), 4.39 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 4.12 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 3.93 (s, 3H, –NCH₃), 3.53–3.44 (m, 2H, –NCH₂CH₂CH₂N–), 3.26–3.17 (m, 2H, –NCH₂CH₂CH₂N–), 2.87 (s, 3H, –NCH₃), 2.87 (s, 3H, –NCH₃), 2.16–2.07 (m, 2H, –NCH₂CH₂CH₂N–), 1.36 (d, J = 6.8 Hz, 6H, (CH₃)₂CH–), 1.23 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, CDCl₃) δ 163.9, 162.5, 162.2, 159.6, 158.6, 154.3, 153.1, 150.3, 148.6, 135.6, 134.4, 130.5, 125.6, 122.3, 122.2, 120.2, 117.5, 115.2, 106.1, 56.0, 43.2, 37.4, 35.7, 27.4, 27.1, 25.1, 24.9, 24.8. ESI-MS: m/z calcd for C₃₁H₃₉N₉O₄S₂, 665.26 found 666.45 [M + H]⁺.

HPLC purification method for compound 7

Column: 250 × 9.4 mm, 5 μm; solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; UV: 220 nm; injection volume: 50 μl. Stop time: 32 minutes. Collection time: start at 12.68 min and end at 15.2 min. Briefly, the chromatographic method began at 0 min with a flow rate of 5 mL min⁻¹, using a mobile phase composed of 70% water (containing 0.1% TFA) and 30% acetonitrile (also containing 0.1% TFA). Over the course of 25 min, the composition was gradually adjusted to 55% water and 45% acetonitrile, while maintaining the same flow rate. At 25.2 min, the mobile phase was rapidly shifted to 100% acetonitrile with 0.1% TFA and held at this composition until 28 min. Subsequently, at 28.2 min, the original mobile phase composition of 70% water and 30% acetonitrile was reintroduced and maintained until the end of the run at 32 min. The flow rate remained constant at 5 mL min⁻¹ throughout the entire procedure.

5-Isopropyl-2-(4-(5-isopropyl-2-(nicotinamido)thiazole-4-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-N-(3-morpholinopropyl)thiazole-4-carboxamide (8)

Collected as a pale-yellow color solid; m.p: 205–207 °C; yield: 40%. ¹H NMR (500 MHz, CDCl₃) δ 11.65 (bs, 1H), 10.86 (bs, 1H), 9.59 (s, 1H), 9.27 (bs, 1H), 8.78–8.67 (m, 2H), 8.18 (s, 1H), 7.62–7.51 (m, 2H), 7.12 (s, 1H), 4.39 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 4.15 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 4.04–3.83 (m, 7H), 3.54–3.45 (m, 4H), 3.26–3.17 (m, 2H, –NCH₂CH₂CH₂N–), 3.02–2.81 (m, 2H, –NCH₂CH₂O), 2.22–2.10 (m, 2H, –NCH₂CH₂CH₂N–), 1.35 (d, J = 6.8 Hz, 6H, (CH₃)₂CH–), 1.22 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, CDCl₃) δ 164.2, 160.9, 159.4, 158.6, 154.2, 153.0, 150.1, 148.6, 146.9, 144.5, 141.3, 135.4, 134.6, 130.1, 125.4, 122.3, 122.1, 119.8, 105.8, 63.8, 55.4, 52.2, 37.3, 35.7, 27.3, 27.1, 25.2, 24.9, 23.9. ESI-MS: m/z calcd for C₃₃H₄₁N₉O₅S₂, 707.27 found 708.32 [M + H]⁺.

HPLC purification method for compound 8

Column: 250 × 9.4 mm, 5 μm; solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; UV: 220 nm; injection volume: 50 μl. Stop time: 32 min. Collection time: start at 13.45 min and end at 15.25 min.

2-Benzamido-N-(5-((4-((3-(dimethylamino)propyl)carbamoyl)-5-isopropylthiazol-2-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)-5-isopropylthiazole-4-carboxamide (9)

Collected as a half white color solid; m.p: 162–164 °C; yield: 45%. ¹H NMR (500 MHz, CDCl₃) δ 11.10 (bs, 2H), 8.93 (s, 1H), 8.25–8.15 (m, 2H), 8.14 (bs, 1H), 7.65–7.57 (m, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.23 (s, 1H), 4.34 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 4.13 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 3.98 (s, 3H, –NCH₃), 3.52–3.38 (m, 2H, –NCH₂CH₂CH₂N–), 3.19–3.08 (m, 2H, –NCH₂CH₂CH₂N–), 2.82 (s, 6H, –NCH₃CH₃), 2.15–2.00 (m, 2H, –NCH₂CH₂CH₂N–), 1.35 (d, J = 6.8 Hz, 6H, (CH₃)₂CH–), 1.19 (d, J = 6.8 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, CDCl₃) δ 165.4, 163.5, 159.8, 158.9, 154.9, 154.4, 149.7, 148.8, 134.8, 133.4, 133.2, 131.8, 129.0, 128.2, 123.5, 122.1, 119.9, 107.2, 56.0, 43.3, 37.5, 35.7, 27.3, 27.1, 24.9, 24.9, 24.7. ESI-MS: m/z calcd for C₃₂H₄₀N₈O₄S₂, 664.26 found 665.42 [M + H]⁺.

HPLC purification method for compound 9

Column: 250 × 9.4 mm, 5 μm; solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; UV: 220 nm; injection volume: 50 μl. Stop time: 32 min. Collection time: start at 8.9 min and end at 11.8 min.

2-Benzamido-5-isopropyl-N-(5-((5-isopropyl-4-((3-morpholinopropyl)carbamoyl)thiazol-2-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)thiazole-4-carboxamide (10)

Collected as a white color solid; m.p: 156–158 °C; yield: 39%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.68 (s, 1H), 12.06 (s, 1H), 9.80 (s, 1H), 8.09 (d, J = 7.5 Hz, 2H), 8.05 (t, J = 6.0 Hz, 1H), 7.67 (dd, J = 11.7, 4.3 Hz, 1H), 7.58 (t, J = 7.7 Hz, 2H), 7.53 (s, 1H), 7.40 (d, J = 1.7 Hz, 1H), 4.28–4.16 (m, 2H, (CH₃)₂CH–), 4.04–3.94 (m, 2H, –NCH₂CH₂O), 3.91 (s, 3H, –NCH₃), 3.70–3.60 (m, 2H, –NCH₂CH₂O), 3.37–3.30 (m, 4H), 3.18–3.08 (m, 2H, –NCH₂CH₂CH₂N–), 3.15–3.03 (m, 2H, –NCH₂CH₂O), 1.96–1.85 (m, 2H, –NCH₂CH₂CH₂N–), 1.33 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–), 1.28 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, DMSO-d₆) δ 165.4, 162.6, 159.5, 158.9, 154.2, 153.8, 146.2, 145.5, 136.4, 135.9, 132.8, 131.8, 128.7, 128.2, 121.9, 121.5, 120.4, 107.1, 63.4, 54.2, 51.2, 39.5, 36.7, 35.8, 26.5, 26.3, 24.8, 24.7, 23.8. ESI-MS: m/z calcd for C₃₄H₄₂N₈O₅S₂, 706.27 found 707.38 [M + H]⁺.

HPLC purification method for compound 10

Column: 250 × 9.4 mm, 5 μm; solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; UV: 220 nm; injection volume: 50 μl. Stop time: 32 min. Collection time: start at 9.36 min and end at 12.45 min.

N-(3-(Dimethylamino)propyl)-5-isopropyl-2-(4-(5-isopropyl-2-(thiazole-4-carboxamido)thiazole-4-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)thiazole-4-carboxamide (11)

Collected as a pale-brown color solid; m.p: 210–212 °C; yield: 48%. ¹H NMR (500 MHz, CDCl₃) δ 11.78 (s, 1H), 10.45 (s, 1H), 9.15 (s, 1H), 8.87 (d, J = 2.0 Hz, 1H), 8.39 (d, J = 2.0 Hz, 1H), 7.91 (t, J = 5.8 Hz, 1H), 7.63 (d, J = 1.3 Hz, 1H), 7.20 (d, J = 1.4 Hz, 1H), 4.42 (sept, J = 6.8 Hz, 1H, (CH₃)₂CH–), 4.25 (sept, J = 6.9 Hz, 1H, (CH₃)₂CH–), 4.01 (s, 3H, –NCH₃), 3.56–3.48 (m, 2H, –NCH₂CH₂CH₂N–), 3.25–3.15 (m, 2H, –NCH₂CH₂CH₂N–), 2.87 (s, 6H, –NCH₃CH₃), 2.19–2.08 (m, 2H, –NCH₂CH₂CH₂N–), 1.39 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–), 1.32 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, CDCl₃) δ 163.5, 159.8, 158.7, 158.5, 154.7, 153.8, 152.6, 150.0, 148.8, 148.6, 135.6, 134.1, 126.0, 122.4, 122.3, 120.5, 106.2, 55.9, 43.3, 37.5, 35.8, 27.4, 27.2, 25.1, 24.9, 24.8. ESI-MS: m/z calcd for C₂₉H₃₇N₉O₄S₂, 671.21 found 672.22 [M + H]⁺.

HPLC purification method for compound 11

Column: 250 × 9.4 mm, 5 μm; solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; UV: 220 nm; injection volume: 50 μl. Stop time: 32 min. Collection time: start at 6.88 min and end at 11.36 min.

5-Isopropyl-2-(4-(5-isopropyl-2-(thiazole-4-carboxamido)thiazole-4-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)-N-(3-morpholinopropyl)thiazole-4-carboxamide (12)

Collected as a half white color solid; m.p: 202–204 °C; yield: 37%. ¹H NMR (500 MHz, CDCl₃) δ 12.55 (bs, 1H), 10.81 (s, 1H), 10.45 (bs, 1H), 9.12 (bs, 1H), 8.88 (d, J = 2.0 Hz, 1H), 8.39 (d, J = 2.0 Hz, 1H), 7.83 (t, J = 5.7 Hz, 1H), 7.61 (d, J = 1.1 Hz, 1H), 4.42 (sept, J = 6.9 Hz, 1H, (CH₃)₂CH–), 4.28 (sept, J = 6.9 Hz, 1H, (CH₃)₂CH–), 4.10–3.85 (m, 7H), 3.52–3.42 (m, 4H), 3.20–3.11 (m, 2H, –NCH₂CH₂CH₂N–), 2.90–2.80 (m, 2H, –NCH₂CH₂O), 2.19–2.09 (m, 2H, –NCH₂CH₂CH₂N–), 1.39 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–), 1.32 (d, J = 6.9 Hz, 6H, (CH₃)₂CH–). ¹³C {¹H} NMR (126 MHz, CDCl₃) δ 163.2, 159.9, 158.7, 158.4, 154.6, 153.7, 152.6, 150.1, 148.9, 148.6, 135.69, 134.3, 126.0, 122.3, 122.3, 120.7, 106.1, 63.9, 55.8, 52.3, 37.5, 36.2, 27.4, 27.2, 25.1, 24.9, 23.8. ESI-MS: m/z calcd for C₃₁H₃₉N₉O₅S₃, 713.22 found 714.31 [M + H]⁺.

HPLC purification method for compound 12

Column: 250 × 9.4 mm, 5 μm; solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; UV: 220 nm; injection volume: 50 μl. Stop time: 32 min. Collection time: start at 7.1 min and end at 10.02 min.

In vitro cultivation of Acanthamoeba spp.

Acanthamoeba castellanii (genotype T4) was procured from American Type Culture Collection (ATCC 50492). The amoebae were cultivated in 10 mL of peptone-yeast extract-glucose (PYG) growth media, consisting of 0.75% yeast extract, 0.75% proteose peptone, and 1.5% glucose, within tissue culture flasks, following previously established protocols.²² Cultures were placed at 30 °C until the cells reached full confluency. The adherent trophozoites indicative of the active, replicative stage were detached by laying the flasks on ice and gently tapping. These harvested trophozoites were subsequently used for experimental assays.²²

In vitro cultivation of human cerebral microvascular endothelial cells

Human cerebral microvascular endothelial cells (HCMECs) (Addexbio) were cultivated in Dulbecco's modified Eagle medium and Nutrient Mixture F12 media containing l-glutamine, sodium pyruvate and glucose in T-75 tissue culture flasks as detailed beforehand.²³ The flasks containing human cells were incubated at 37 °C in a 5% CO₂ incubator with over 95% humidity. The cells were observed regularly under an inverted microscope. Once confluency was reached, the cell monolayers were trypsinized using trypsin-EDTA and cells were collected by centrifugation at 2700 × g for 5 min and the pellet was resuspended in media for subsequent use in assays.

Amoebicidal assay

Antiamoebic properties of thiazole-based DNA minor groove binders were determined using amoebicidal assays.¹⁴ In brief, 3 × 10⁵ amoebae were incubated with different concentrations of the tested compounds and the final volume was adjusted to 500 μL in 24-well plates. The plates were placed in a 30 °C incubator for 24 h. For negative controls, amoebae were incubated alone, while the amoebae incubated with chlorhexidine were used as the positive control for 100% amoebae death. Following incubation, 1% trypan blue was added, and viable amoebae were enumerated with a haemocytometer. Live cells expel the dye, and they appear as unstained or light blue during haemocytometer counting, while dead amoebae appear dark blue stained. The results are representative of at least three independent experiments, displayed as the mean values ± standard error, and the statistical difference was calculated using the Student's t-test, paired, two-tailed distribution. The significance level was set at a p-value threshold of less than 0.05.

Inhibition of cysts

A. castellanii trophozoites were converted into the cyst form by placing active trophozoites on bacteriological (non-nutrient) agar plates as before.²³ In brief, 10⁷ amoebae were deposited on a non-nutrient agar plate and incubated at 30 °C for two weeks. Next, 1 mL PBS was inoculated onto the plate and amoebae cysts were scraped using a cell scraper. Finally, cysts were collected by centrifugation at 3000 × g for 10 min. After that, 3 × 10⁵ cysts were incubated with and without 100 μM of the tested compounds in 1 mL of PYG media in 24-well plates. The plates were kept at 30 °C for various intervals of time with regular observation under an inverted microscope. For negative controls, amoebae were incubated without any treatment, whereas amoebae exposed to chlorhexidine served as the positive control. Once amoebae were observed in their active trophozoite form in control wells, experiments were halted, and amoebae trophozoites were enumerated in all wells using a haemocytometer.²³

Cytotoxicity assay

To test the toxicity of thiazole-based DNA minor groove binders on HCMEC in vitro, cytotoxicity assays were performed. HCMECs were cultivated in 96-well plates and were incubated with thiazole-based DNA minor groove binders. Plates were then incubated at 37 °C for 24 h in a 5% CO₂ incubator with 95% humidity. Following this, the supernatant was collected, and compound cytotoxicity was established using a cytotoxicity kit that detects/measures the extent of lactate dehydrogenase (LDH) released. HCMEC incubated with media alone was utilised as a negative control, and HCMEC treated with 1% Triton X-100-treated was the positive control.²³

Cytopathogenicity assay

To determine whether thiazole-based DNA minor groove binders inhibit amoeba-mediated host cell death, cytopathogenicity experiments were accomplished.²³ In brief, 3 × 10⁵ trophozoites were incubated with thiazole-based DNA minor groove binder compounds at 30 °C as explained in the methods section; however, incubation was limited to 2 h. Next, amoebae were centrifuged for 10 min at 3000 × rpm and then resuspended in 200 μL of RPMI-1640 and placed on HCMECs grown in 96-well plates. After this incubation, supernatants were collected, and the LDH release and percent cytotoxicity were determined as follows: % HCMEC toxicity = sample value − negative control value/positive control value − negative control value × 100.

Thermodynamic analysis by isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC) was employed to establish the thermodynamic parameters governing the interaction between the minor groove binders (MGBs) and the DNA sequence d(GCGAGTACTCGC)₂. The experiments were conducted at 25 °C using a MicroCal PEAQ-ITC instrument. The sample cell was filled with 10 μM of the DNA, and the MGB compound (40 μL) was injected through 19 individual 2 μL injections, with 180-second intervals to ensure thorough mixing. Stirring was maintained at 500 rpm, with the power reference set to 10 kcal s⁻¹. ITC experimentations were carried out in MES buffer at pH 7 and 25 °C. Heat changes from the binding interactions were corrected for dilution effects, and the adjusted data were analyzed using a one-site binding model in the MicroCal PEAQ-ITC software to calculate binding enthalpy as well as additional thermodynamic parameters.

Molecular docking

The initial 3D structure of the DNA duplex d-(CGACTAGTCG)₂ complexed with compound 11 was derived from the solution-refined NMR structure (PDB code: 2MNE). The compound 11 structure was generated by modifying the ligand “AIK18-51” within 2MNE using Discovery Studio Visualizer (2021). Molecular docking with the DNA duplex d(CGAGTACTCG)₂ was performed using the HADDOCK web server (version 2.4, https://wenmr.science.uu.nl/haddock2.4/). The docked structure with the highest HADDOCK score, indicating the most favourable interaction, was selected. Ligand–target interactions were subsequently evaluated using Discovery Studio Visualizer (2021).

Results

Chemical synthesis

The synthetic procedure for the thiazole-based DNA minor groove binders investigated herein was as previously explained.^17,20 Briefly, direct coupling of compounds 1 and 2 under anhydrous conditions in the presence of triethylamine produced amide compound 3 in a good yield (70%). The methyl ester group in compound 3 was hydrolysed using LiOH to obtain an acid intermediate which was then coupled with dimethylaminopropylamine (4a) and 3-morpholinopropylamine (4b) using HBTU to afford the nitro compounds 5a (90%) and 5b (80%). The obtained nitro compounds 5a & 5b were further reduced by catalytic hydrogenation with 10% Pd/C to give amine intermediates and then directly coupled with acids 6a–6c using HBTU under basic conditions to obtain the final products 7 (49%), 8 (40%), 9 (45%), 10 (39%), 11 (48%) and 12 (37%) in moderate to good yields (Scheme 1). The final yields were determined following HPLC purification and subsequent lyophilization.

Scheme 1. Synthesis of final products 7–12. Selected DNA minor groove binder derivatives reduced A. castellanii viability.

Amoebicidal assays were accomplished to comprehend the impact of thiazole-based DNA minor groove binders on the viability of A. castellanii. The results demonstrated that the selected thiazole-based DNA minor groove binders showed amoebicidal properties against A. castellanii. The number of amoebae incubated alone was considered as 100%, and the relative change was verified consequently. When compared to the solvent control, all tested derivatives displayed considerable activity against A. castellanii at 100 μM. In particular, compounds 9, 10 and 12 showed substantial amoebicidal activity, and resulted in significant killing of amoebae trophozoites (P < 0.05) (Fig. 1). Overall, all thiazole-based DNA minor groove binders tested reduced the amoebae viability (Fig. 1).

Fig. 1. Thiazole-based minor groove binders (7–12) significantly reduced A. castellanii viability. The amoebicidal effects of various drugs were determined using trypan blue staining. Briefly, 3 × 10⁵ of A. castellanii were treated with various derivatives at a concentration of 100 μM for 24 h at 30 °C. The data are presented as the mean ± standard error; P-values were calculated using a two-sample t-test with a two-tailed distribution, where (*) indicates P ≤ 0.05. CHX (chlorhexidine) was used as an antiamoebic drug.

Minor groove binder derivatives reduced A. castellanii excystation

Excystation is the process through which a latent cyst transforms into the trophozoite stage under favourable conditions resulting in infection recurrence. Hence, one way to block infection recurrence is to inhibit excystation. To achieve this, excystation assays were performed using thiazole-based DNA minor groove binders. Cysts incubated with PYG alone were considered as 100%, and the relative change was determined accordingly. When mature A. castellanii cysts were challenged, all thiazole-based DNA minor groove binders investigated herein significantly reduced the trophozoite reemergence (P < 0.05) (Fig. 2). Among the minor groove binder derivatives tested, the most active compounds were 9, 10, and 11 (Fig. 2).

Fig. 2. Inhibition of cyst assays was performed in the presence of thiazole-based minor groove binders (7–12) against A. castellanii cysts as described in the Materials and methods section. The data are presented as the mean ± standard error; P-values were calculated using a two-sample t-test with a two-tailed distribution, where (*) indicates P ≤ 0.05. CHX (chlorhexidine) was used as an antiamoebic drug.

Minor groove binder derivatives demonstrated limited cytotoxicity versus human cells

To ascertain the properties of thiazole-based DNA minor groove binders tested on human cell cytotoxicity, lactate dehydrogenase assays were accomplished using human cerebral microvascular endothelial cells. Cytotoxicity of HCMECs incubated with the detergent was considered as 100%, and the relative change was established accordingly. All thiazole-based DNA minor groove binders tested showed significantly reduced HCMEC toxicity (P < 0.05) (Fig. 3). Among the minor groove binder derivatives tested, compounds 7 and 12 showed the least toxicity against HCMECs (Fig. 3).

Fig. 3. Cytotoxicity assays were performed to determine the toxicity of thiazole-based minor groove binders (7–12) against human cerebrovascular endothelial cells as described in the Materials and methods section. All the employed compounds displayed limited cytotoxicity. The data are presented as the mean ± standard error; P-values were calculated using a two-sample t-test with a two-tailed distribution, where (*) indicates P ≤ 0.05. +ve indicates 100% cell death, i.e., cells treated with Triton as described in the Materials and Methods section. −ve indicates 0% cell death, i.e., cells incubated alone.

Pre-treatment of A. castellanii with selected minor groove derivatives significantly inhibited parasite-mediated host cell death

To ascertain whether thiazole-based DNA minor groove binders affect amoeba-mediated host cell death, cytopathogenicity experiments were accomplished. Cytotoxicity of HCMECs incubated with amoebae alone was considered as 100%, and the relative change was elucidated accordingly. The results showed that pre-treatment with thiazole-based DNA minor groove binders showed selective effects (Fig. 4). Among the minor groove binder derivatives tested, compounds 9 and 10 reduced A. castellanii-mediated HCMEC toxicity significantly (P < 0.05) (Fig. 4).

Fig. 4. Thiazole-based minor groove binders (7–12) inhibited the amoeba-mediated cytopathogenic effect against human cells. After treating 3 × 10⁵ amoebae with 100 μM compounds for 2 hours, the compounds showed varying degrees of reduction in the amoeba-mediated host cell death. The data are presented as the mean ± standard error; P-values were calculated using a two-sample t-test with a two-tailed distribution, where (*) indicates P ≤ 0.05. CHX (chlorhexidine) was used as an antiamoebic drug.

Isothermal titration calorimetry studies

Isothermal titration calorimetry (ITC) studies are utilised to understand ligand–DNA interactions as they provide direct measurements of the thermodynamic parameters involved in binding. ITC enables the measurement of binding affinity, stoichiometry, changes in enthalpy and entropy, as well as Gibbs free energy of interactions, without the need for labelling or altering the ligand or DNA.^16,17,20 Herein, ITC was used to investigate the interaction of the bioactive compounds (# 9, 10, 11 and 12) with a short DNA duplex sequence, d(CGCAGTACTGCG)₂. This sequence was commonly used to investigate the binding of MGBs with DNA.^19,20 The MGB ligands were initially titrated into the buffer as a control experiment, generating endothermic peaks. The heats of ligand dilution consistently decreased with each injection into the sample cell, suggesting ligand aggregation in the buffered solution (Fig. 5). Subsequently, MGB solutions were titrated into the DNA solution of d(GCGAGTACTCGC)₂, and the resulting binding enthalpograms from the titration are shown in Fig. 6. The ITC data were fitted using a one-site binding model and the generated thermodynamic parameters are summarized in Table 1.

Fig. 5. ITC control titration of A) compound 9, B) compound 10, C) compound 11 and D) compound 12 in MES buffer at 25 °C and pH 7.

Fig. 6. ITC titration of A) compound 9, B) compound 10, C) compound 11 and D) compound 12 with d(GCGAGTACTCGC)₂ in MES buffer at 25 °C and pH 7. The top panel signifies the raw data for the serial injection of ligands into the sample cells and the bottom panel shows integrated heat data after correction of heat of dilution. The line represents the fit obtained from a single-site binding model.

Table 1. ITC derived thermodynamic parameters for the binding of compounds 9, 10, 11 and 12 with d(GCGAGTACTCGC)₂.

Ligand	DNA	T [°C]	N (ligand/DNA)	ΔH [kcal mol−1]	TΔS [kcal mol−1]	ΔG [kcal mol−1]	K [M−1]
Compound 9	AGTACT	25	2	−22.5 ± 1.0	−16.0	−6.5	6.09 ± 0.2 × 104
Compound 10	AGTACT	25	2	−12.2 ± 1.3	−5.3	−6.9	1.07 ± 0.2 × 105
Compound 11	AGTACT	25	2	−66.5 ± 1.5	−55.7	−10.8	8.84 ± 0.2 × 107
Compound 12	AGTACT	25	2	−14.8 ± 0.3	−5.43	−9.4	6.89 ± 0.2 × 106

The titration of all bioactive compounds resulted in an overall binding driven by favorable enthalpy (ΔH = −22.5 ± 5.12 kcal mol⁻¹ for 9, −12.2 ± 1.34 kcal mol⁻¹ for 10, −66.5 ± 1.57 kcal mol⁻¹ for 11 and −14.8 ± 0.364 kcal mol⁻¹ for 12) and the unfavorable negative entropy (TΔS = −16.0 kcal mol⁻¹ for 9, −5.31 kcal mol⁻¹ for 10, −55.7 kcal mol⁻¹ for 11, −5.43 kcal mol⁻¹ for 12; Table 1). These results indicate that hydrogen bonding and van der Waals forces were the main contributors to these interactions (evidenced by the negative ΔH). Moreover, the observed negative unfavorable entropy (−TΔS) suggests that the complexation process led to rigidification or distortion of the DNA structure upon binding. The negative binding free energy (ΔG) indicates the spontaneity of these interactions. The titration of the investigated MGBs to the DNA dodecamer d(GCGAGTACTCGC)₂ showed that the binding stoichiometry is 2 : 1 [ligand : DNA] (Table 1). These findings are consistent with the modelling studies and previous ITC results for similar MGB compounds.^20,21

The ITC titration results showed that compound 11 exhibited the strongest binding affinity with DNA (K = 8.84 ± 0.2 × 10⁷ M⁻¹), driven by favorable enthalpy and opposed by entropic penalty, while compound 9 showed the weakest binding affinity with d(GCGAGTACTCGC)₂. These findings illustrate the role of the terminal thiazole ring in recognizing G-DNA bases.

Molecular docking

Molecular docking analysis was conducted using the HADDOCK web server²⁴ to investigate the binding interactions of the active compound 11 identified by ITC as having the highest binding affinity and the DNA sequence d(CGAGTACTCG)₂. This specific sequence was selected based on prior research, indicating that structurally related MGBs exhibit strong binding affinity to this sequence.¹⁹ The NMR structure (PDB code: 2MNE) of the DNA sequence d-(CGACTAGTCG)₂ complexed with AIK18-51 (Alniss et al., 2014),¹⁵ an analogue of the studied compounds, was utilized to generate the initial structures for the modelling experiment. The HADDOCK results are summarized in Table 2. The scoring function of HADDOCK consists of various energies including electrostatic, van der Waals, restraint violation and desolvation energies. These energies are integrated into a formula to achieve the final HADDOCK score, which represents the best fit confirmation in the binding site. The docking findings indicate that compound 11 displayed strong negative scores (−103.3), demonstrating the stability of its binding to the AGTACT binding site. Additionally, the docking results revealed that compound 11 binds to 5′-AGTACT-3′ as antiparallel dimers, side by side in the minor groove of the DNA (Fig. 7A).

Table 2. The HADDOCK docking results of compound 11 with d-(CGAGTACTCG)₂.

	Compound 11
HADDOCK score	−103.3 ± 1.9
RMSD (Å)	0.5 ± 0.4
van der Waals energy (kcal mol−1)	−62.7 ± 3.7
Electrostatic energy (kcal mol−1)	−182.8 ± 12.3
Desolvation energy	−4.2 ± 0.7
Buried surface area (Å2)	1405.9 ± 21.5

Fig. 7. A) 3D structural representation of the best docked pose for compound 11 with d(CGAGTACTCG)₂. B) 2D drawing showing the non-covalent interactions of compound 11 with d(CGAGTACTCG)₂, generated by BIOVIA Discovery Studio Visualizer (2021).

The stability of this complex is primarily attributed to hydrogen bonding interactions between the NH groups of the MGB amide and DNA bases within the minor groove, along with additional hydrogen bonds formed between the thiazole rings of compound 11 and guanine bases of the DNA (Fig. 7B). The HADDOCK results for compound 11 with d(CGAGTACTCG)₂ exhibit a similar binding pattern to those obtained for MGB analogues. Notably, the terminal thiazole head group demonstrated its ability to selectively form a hydrogen bond with guanine at position 2 within the recognition site, AG₂TACT. Overall, these results indicate that the binding of MGBs with DNA is driven by non-covalent reversible interactions.

Discussion

Despite advances in antimicrobial chemotherapy, infections due to pathogenic A. castellanii have remained a threat to public health. Furthermore, Acanthamoeba can serve as a reservoir for various microbes, facilitating their spread in the environment and to vulnerable hosts.²⁵ Recent advances in chemotherapy include a mixture of diamidines, biguanides, and antifungals that are mostly repurposed drugs,²⁶ but successful prognosis is often complicated due to awareness, delayed diagnosis, and efficacy of available compounds. Recently, the DNA minor groove has been recognised as a valuable target, and several compounds have been shown to exact their effects by binding to the DNA minor groove. The term ‘minor groove binders’ refers to a large class of substances that can bind to the minor groove of DNA with high affinity.²⁷ Polyamides, in addition to being antibacterial and antifungal drugs, are used in antiparasitic therapy. As antiparasitic drugs, recent studies revealed a number of amidine-related dicationic flexible triaryl bis-guanidines.²⁸ More recently, selected minor groove binders have been shown to exhibit antiamoebic effects,^17,20 highlighting this as a potential avenue for further research to identify minor groove binder derivatives against A. castellanii.

In this study, we synthesized 6 novel thiazole-based minor groove binders: 7, 8, 9, 10, 11 and 12 and tested their properties against A. castellanii of the T4 genotype. Among all minor groove binder derivatives tested, minor groove binder derivative 10 exhibited potent antiamoebic effects using amoebicidal excystation assays as well as reduced amoeba-mediated host cell death. Interestingly, we observed that the selected minor groove binder derivatives exhibited potent effects against the trophozoite form of A. castellanii. Previous work has shown that minor groove binders modulate gene expression by interfering with gene expression at the transcription level and inhibiting cell proliferation,²⁹ which may explain our findings. Of note, minor groove binder derivatives can be designed to target specific DNA sequences, allowing precision in therapeutic and research applications; hence, it is a promising area for further research. Notably, cysts incubated with the growth medium (PYG) in excystation assays would result in increased gene expression associated with metabolism and reproduction; hence, all minor groove binder derivatives tested showed more prominent effects in excystation assays, as observed in our study. Minor groove binder derivatives showed limited toxic effects against human cells. This is in contrast to earlier studies, which showed that minor groove binders produce toxic effects against cancer cell lines.²⁹ A likely explanation is that primary human cells used in our study were fully confluent, and once complete monolayers are formed, metabolic activity is reduced resulting in limited available targets.

Our previous studies have elucidated key structural features of the ligand that govern its sequence selectivity and preferred orientation within the minor groove of DNA, relative to the 3′ and 5′ ends.¹⁴ These investigations demonstrated that the MGBs can bind to the 5′-AGTACT-3′ sequence as antiparallel dimers. The findings also highlighted several important structure–activity relationships (SARs): (i) the position of the basic tail group determines the ligand's preferred orientation within the minor groove along the 5′–3′ axis; (ii) the thiazole ring selectively recognizes guanine bases through hydrogen bonding with the exocyclic amino group of guanine. (iii) Amide linkages are essential for establishing hydrogen bonds with DNA and for maintaining the molecular curvature required to conform to the groove's helical geometry; and (iv) the spatial alignment of these amide linkages with the 5′ and 3′ ends is critical for effective hydrogen bonding.

The differences in activity observed against A. castellanii among the tested MGB derivatives are likely due to their varying capacities to bind specific DNA sequences with different affinities, as supported by ITC data. MGB derivatives containing thiazole rings tend to favor GC-rich sequences, whereas those featuring N-methylpyrrole units show a preference for AT-rich regions.^14–21 Moreover, the DNA-binding affinities of individual minor group binding derivatives can differ substantially; even minor structural variations may significantly influence binding strength and, consequently, biological activity.

The MGB derivatives designed herein incorporate structural features that facilitate DNA interactions. Molecular docking suggests that MGB derivatives bind reversibly to the minor groove through non-covalent hydrogen bonds involving the amide NH groups or the nitrogen atoms of thiazole rings and the DNA bases lining the groove. This reversible binding mode helps avoid permanent DNA damage, thereby minimizing cytotoxicity. Additionally, the crescent-shaped conformation of the MGB derivatives aligns well with the curvature of the DNA minor groove, further enhancing their binding efficacy. While more investigations are needed to ascertain mechanistic mechanisms behind the efficacy of minor groove binder derivatives, our findings showed clearly that minor groove binder derivatives can target pathogenic A. castellanii with limited host cell death.

Author contributions

NAK and HYA conceived the study amid discussions with RS, YAM and HMA. SS, MD, HMA, AR, YAM, and BSA designed the study, provided resources and carried out all experiments under the supervision of RS, HYA and NAK. HYA developed the synthetic procedures for the reported MGB compounds and performed the molecular modeling studies. RS, HYA and NAK prepared the first draft of the manuscript. All authors corrected the manuscript and approved the final manuscript.

Conflicts of interest

Hasan Y. Alniss has a pending patent related to the MGBs reported in this work. The other authors declare no conflicts of interest.

Data availability

Supplementary information is available: Spectral characterization (NMR and mass spectrometry) and HPLC purity analysis of the reported MGB compounds. See DOI: https://doi.org/10.1039/D5MD00475F.

The data supporting this article have been included as part of the SI.