Oxadiazon Derivatives Elicit Potent Intracellular Growth Inhibition against Toxoplasma gondii by Disrupting Heme Biosynthesis

Abstract

Infections of Toxoplasma gondii can cause severe and sometimes fatal diseases in immunocompromised individuals. The de novo heme biosynthesis pathway is required for intracellular growth and pathogenesis, making it an appealing therapeutic target. We synthesized a small library of derivatives of the herbicide oxadiazon, a known inhibitor of the penultimate reaction within the heme biosynthesis pathway in plants, catalyzed by protoporphyrinogen oxidase (PPO). Seven of the 18 analogs exhibit potent intracellular growth inhibition of wild-type T. gondii (IC₅₀ = 1 to 2.4 μM). An assay of the compounds against Toxoplasma PPO knockout and complementation strains confirmed the mode of action to be due to the potent inhibition of PPO. The most potent compounds have no detectable cytotoxicity against human foreskin fibroblast cells up to 100 μM. This study suggests that oxadiazon derivatives may represent a new molecular scaffold for the effective treatment of T. gondii infections.

Graphical Abstract

Toxoplasma gondii is an obligate intracellular human protozoan pathogen belonging to the Apicomplexa phylum.¹ This parasite infects nearly all warm-blooded animals,² and it is estimated that one-third of the human population is actively infected, making it one of the most widespread parasitic organisms known to man.³ As an opportunistic pathogen, Toxoplasma infections can cause severe, even fatal, complications in immunocompromised patients, such as HIV carriers and those undergoing chemotherapy.^4,5 In immunocompetent individuals, acute T. gondii infections are generally asymptomatic and are therefore usually overlooked and left untreated. Thus the parasites can penetrate the host’s blood–brain barrier to hibernate within the brain and transition to chronic infections,^4,5 which can occasionally be reactivated to acute infections when the host’s immunity is compromised.^4,5 In addition, recent evidence has shown a significant correlation between changes in behavior and mental capabilities associated with infection.^6–9 T. gondii infections are responsible for several serious congenital disorders, especially when pregnant women are initially exposed during their first trimester.^1–3,10,11 The current chemical therapy mainly uses pyrimethamine and sulfadiazine; however, their strong side effects limit their use in certain populations.¹² The 16-membered ring macrolide spiramycin can be given to female toxoplasmosis patients in the first trimester of pregnancy, but it cannot cross the placenta to treat fetal toxoplasmosis.^13–15 In addition, there are no available drugs for treating chronic toxoplasmosis. Therefore, the development of novel antibiotics is urgently needed for clinical toxoplasmosis management.

Recently, a few publications have revealed that Toxoplasma parasites encode an active plant-like heme biosynthesis pathway and require de novo heme production for intracellular survival.^16–18 In addition, heme-deficient parasites do not cause death in murine models, suggesting that the disruption of heme biosynthesis significantly reduces the acute virulence of Toxoplasma in vivo.¹⁶ The efficient clearance of the heme-deficient parasites during acute infection by the host’s innate immunity potentially blocks their access to the brain for cyst formation. However, it remains unknown if the parasites rely extensively on their heme production to maintain chronic infections. These previous findings underscore the pathway’s potential as an untapped druggable target for controlling Toxoplasma acute infections.

By phylogenetic analysis, Toxoplasma protoporphyrinogen oxidase (PPO) is closely grouped with plant orthologs rather than mammalian counterparts.¹⁶ A series of commercially available herbicides are used for weed control by inhibiting plant PPO and demonstrate high specificity and low toxicity.^19,20 Hence, we hypothesized that the synthetic modification of these existing herbicidal PPO inhibitors may reveal a new strategy for the development of novel Toxoplasma antibiotics.

A previous screen of PPO-targeting herbicides identified several hit molecules that displayed moderate intracellular growth inhibition of T. gondii (Figure 1).¹⁶ Among these hit compounds was oxadiazon (1), which displayed an IC₅₀ value of 131.4 ± 3.9 μM. The derivatization of a structurally related analog, oxadiargyl (2, IC₅₀ = 20.7 ± 2.4 μM), led to two derivatives, 3 and 4 that increased the activity of the compound 20-fold over that observed for the parent compound (i.e., IC₅₀ values for 3 and 4 = 5.0 ± 1.5 and 8.3 ± 1.3 μM, respectively). These promising results led us to explore the structure–activity relationship of the aryl ring in 3.¹⁶ The results of this study are described herein.

Figure 1.

PPO herbicide oxadiazon (1) and derivatives (2–4) exhibit potent growth inhibition against Toxoplasma gondii.

Our study began with the synthesis of 18 different benzyl azide intermediates that were prepared by treatment of the commercially available bromide derivatives with sodium azide under one of two conditions (compounds S1–S18; see the SI for details).^21–23 In brief, the benzyl bromide derivatives were treated with sodium azide in an acetone–water mixture to provide a series of benzyl azide products in good to excellent yields. In addition to these derivatives, two bromomethylpyridine derivatives were treated with potassium carbonate in N,N-dimethylformamide (DMF) before the addition of sodium azide to produce the desired azidomethylpyridine products in excellent yields. With the requisite azide derivatives in hand, copper-catalyzed Huisgen 1,3-dipolar cycloaddition²⁴ “click” chemistry conditions were used to synthesize the 18-member library by coupling the azides to the commercially available oxadiargyl (2) (Figure 2).²⁵ Thus 3- and 4-methyl derivatives (5a and 5b) were prepared in 99 and 98% isolated yields. A 4-tert-butyl analog (5c) was isolated in 97% yield, whereas a series of two-, three-, and four-substituted trifluoromethyl derivatives were isolated in 97 to 98% yield (5d–5f). A series of halogenated derivatives (5g–5j) were synthesized in 91–93% yield. Other monosubstituted examples included 3-methoxy (5k, 98%), 4-acetyl (5l, 99%), 3-carboxylic acid (5m, 90%), and 2-nitro (5n, 10%) analogs. Two disubstituted examples were prepared: 5o, bearing 2-hydroxy and 5-nitro groups (99%), and 5p, bearing 2-nitro and 4-carboxylic acid substituents (95%). Finally, two azidomethyl-pyridines provided 2- and 4-pyridyl derivatives 5q (96%) and 5r (98%).

Figure 2.

Preparation of triazole-oxadiargyl library and evaluation against WT T. gondii. ¹IC₅₀ for growth inhibition of WT Toxoplasma gondii (averages ± standard deviation for three biological replicates). Structures highlighted with a light-blue background returned IC₅₀ values below 2.5 μM. ²Isolated yields. ³Three biological replicates did not converge on a solvable IC₅₀ value.

Oxadiazon derivatives 5a–5r were then evaluated for the intracellular growth inhibition of wild-type T. gondii harboring a luciferase reporter using our previously developed assay.^16,26 (See Figure S1 for a schematic representation of the assay.) The IC₅₀ values for 5a–5r are also indicated in Figure 2. Derivatization of the arene in compound 3 was generally well-tolerated in most cases, with nearly all of the new compounds exhibiting growth inhibitory effects with IC₅₀ values well below that observed for the parent oxadiazon scaffold (1, 131.4 ± 3.9 μM). Just two of the new analogs, compounds 5m and 5p, failed to return convergent IC₅₀ values below 150 μM. Nonetheless, ortho substitution in the arene appeared to be poorly tolerated, with o-CF₃ (5e), o-Br (5i), o-NO₂ (5n, 5p), and o–OH (5o) substituents all returning higher IC₅₀ values than the unsubstituted 3. With the exception of the o-bromo-substituted analog 5i (IC₅₀ = 9.2 ± 1.2 μM), all other ortho-substituted analogs were significantly less potent than 3, falling more in the range of oxadiargyl (2, 20.7 ± 2.4 μM): 20–27 μM for 5e, 5n, and 5o or >150 μM for 5p. Even positioning the basic nitrogen of the 2-pyridyl analog 5q in the pseudo-ortho position returned an IC₅₀ value of 13.5 ± 1.6 μM. In contrast, a variety of substituents were tolerated in the meta and para positions, with all remaining analogs returning IC₅₀ values below that observed for the unsubstituted 3 (with the notable exception of 5m). The p-Me (5b), p-t-Bu (5c), m-methoxy (5k), and 4-pyridyl (5r) analogs provided IC₅₀ values ranging from 2.5 to 4.6 μM. Seven analogs, m-Me (5a), m-CF₃ (5d), p-CF₃ (5f), m-Br (5g), p-Br (5h), p-Cl (5j), and p-Ac (5l), returned IC₅₀ values below 2.5 μM (analogs highlighted in blue in Figure 2, data tabulated in Table 1, column 3). These seven analogs were carried forward for further study.

Table 1.

Evaluation of Selected Oxadiazon Derivatives against Wild-Type, Knockout, and Complemented Strains of Toxoplasma gondii

compound	Ar	WT∷Nluc (μM)a	Δppo∷Nluc (μM)	ΔppoPPO∷Nluc (μM)
5a	3-MeC6H4	1.8 ± 0.1	34.6 ± 8.9	0.9 ± 0.5
5d	3-CF3C6H4	1.4 ± 0.2	21.0 ± 4.9	1.0 ± 0.4
5f	4-CF3C6H4	1.0 ± 0.3	28.4 ± 6.3	0.7 ± 0.1
5g	3-BrC6H4	1.3 ± 0.3	31.5 ± 8.0	1.5 ± 0.5
5h	4-BrC6H4	2.4 ± 0.2	25.8 ± 4.8	1.7 ± 0.2
5j	4-ClC6H4	2.1 ± 0.3	28.8 ± 6.3	1.1 ± 0.4
5l	4-AcC6H4	1.9 ± 0.5	31.9 ± 7.7	1.6 ± 0.5

^aIC₅₀ values for intracellular growth inhibition (averages ± standard deviation for three biological replicates).

The seven most potent analogs arising from this study were next evaluated for intracellular growth inhibition of a protoporphyrinogen oxidase (PPO) knockout mutant carrying a luciferase reporter, Δppo∷Nluc,¹⁶ to demonstrate that the potent growth inhibition observed against wild-type T. gondii resulted primarily from PPO inhibition (Table 1, column 4). The compounds were found to be approximately 10 to 30 times less potent against this knockout mutant, underscoring that the compounds likely inhibit PPO as the major mode of action. Furthermore, potent activity was restored against the strain complemented with Toxoplasma PPO, ΔppoPPO∷Nluc (Table 1, column 5). Taken together, these results clearly suggest that the oxadiazon analogs prepared in this study elicit their potent intracellular growth inhibition mainly through interaction with PPO, leading to disruption of heme biosynthesis. In a final series of experiments, we evaluated the eight most potent compounds (5a, 5d, 5f–h, 5j, and 5l) for cytotoxicity against human foreskin fibroblast (HFF) cells using a standard alamarBlue cell viability assay. Gratifyingly, all seven compounds exhibited no detectable cytotoxicity against mammalian cells up to 100 μM.

In conclusion, a simple Huisgen 1,3-dipolar cycloaddition strategy was employed to generate a small library of oxadiazon derivatives that exhibit potent intracellular growth inhibition against Toxoplasma gondii without any detectable cytotoxicity to mammalian cells. Seven compounds (5a, 5d, 5f–h, 5j, and 5l) exhibited potent inhibitory activity with IC₅₀ values ranging between 1 and 2.4 μM. An assessment of these compounds against the PPO-knockout and the corresponding complementation strains of T. gondii confirms that the mode of action of this class of compounds is likely due to the inhibition of the PPO component of the heme biosynthesis pathway. The current efforts are focused on further structure–activity relationship studies that will explore the incorporation of various aliphatic chains in place of the arene (cf. 4, Figure 1) and evaluate the influence of structural modulation of the four-substituent (i.e., t-butyl in 1-5) in the 1,3,4-oxadiazolin-5-one heterocycle of the scaffold. Additionally, we are beginning to explore the utility of oxadiazon derivatives for treating T. gondii infections in relevant animal models for human disease. The results of these studies will be reported in due course.

METHODS

General Methods: Chemistry.

¹H and ¹³C NMR spectra were obtained using Bruker Avance 300 and Bruker Avance 500 MHz spectrometers. Chemical shifts are reported in parts per million (ppm). Spectra are referenced to residual solvent peaks. Melting points were determined using an SRS Digimelt MPA160 apparatus and are uncorrected. Infrared spectroscopy data were collected using a Shimadzu IRAffinity-1S instrument (with a MIRacle 10 single reflection ATR accessory) operating over the range of 400 to 4000 cm⁻¹. Flash column chromatography was carried out using ZEOCHEM silica gel (40–63 μm). Analytical and preparative thin-layer chromatography (TLC) were performed on Sorbtech silica G TLC plates. All nonaqueous reactions were performed under an inert atmosphere of nitrogen in flame-dried glassware containing a stir bar unless otherwise noted. Acetone, DMF, dichloromethane (DCM), methanol, and pyridine were obtained from commercial sources and dried following standard distillation procedures. All other solvents were obtained from commercial sources and used without drying unless otherwise noted. All water and aqueous solutions were made using deionized (DI) water.

Mammalian Cell and Parasite Culture.

Toxoplasma gondii parasites were passaged in HFFs (ATCC, SCRC-1041), which were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% cosmic calf serum (D10 medium) at 37 °C with 5% CO₂.

Evaluation of Inhibition Potency of Oxidiargyl-Triazole Derivatives by Using the Bioluminescence-based Growth Assay.

The freshly lysed parasites were harvested by membrane filtration, as previously reported.^16,26 The 1500 WT∷Nluc and ΔppoPPO∷Nluc or 7500 Δppo∷Nluc parasites were inoculated in each well in 96-well white plates for the bioluminescence-based growth assay, as previously described.²⁶ In brief, the parasites were allowed to invade the host cells for 4 h before noninfected parasites were washed away followed by medium replacement with fresh D10 medium supplemented with various concentrations of PPO inhibitors, starting at 100 μM with three-fold serial dilution in a total of 10 concentrations. The well not including inhibitors served as the reference control for normalization. The parasites were grown in the drug-containing medium for 96 h before bioluminescence determination, as previously described.^16,26 The assay was repeated in three independent biological replicates with two technical replicates for each biological replicate.

Toxicity Quantification of Oxidiargyl-Triazole Derivatives for HFFs by Using the alamarBlue Assay.

HFFs were plated in 96-well clear plates and allowed to become confluent monolayers. The PPO inhibitors are soluble in dimethyl sulfoxide (DMSO), so 100 mM stock solutions of each compound were prepared. These DMSO stocks were diluted 1000-fold upon direct addition to the growth medium for the assay. After this addition, the culture medium either remained completely clear or cleared and remained clear upon gentle mixing by pipetting. The PPO inhibitors were then added to each well at the same dilutions mentioned in the drug potency assay and kept in the medium for 96 h at 37 °C with 5% CO₂ followed by medium replacement with 0.004% (m/v) resazurin in regular D10 medium for 4 h at 37 °C with 5% CO₂ before absorbance detection at 570 and 600 nm by a BioTek H1 hybrid plate reader. The cell viability was calculated using the equation previously reported.¹⁶

General Procedure for the Synthesis of Oxadiargyl-Triazole Derivatives 5a–5r.

Oxadiargyl (50 mg, 0.145 mmol, 1.0 equiv) was dissolved in DCM/H₂O, 1:1 (0.5 mL) in a 20 mL round-bottomed flask. The appropriate azide (0.175 mmol, 1.2 equiv) was added followed by anhydrous CuSO₄ (1 mg, 0.007 mmol, 0.05 equiv) and sodium ascorbate (4 mg, 0.02 mmol, 0.15 equiv). The resulting solution was then vigorously stirred at room temperature for 4 h. Upon completion, the reaction was diluted with DCM (5 mL) and water (5 mL). The organic layer was dried over sodium sulfate and concentrated to provide the crude product as a colored oil. The isolate was purified by silica gel flash column chromatography under the following gradient: hexanes (50 mL), ethyl acetate/hexanes (1:9; 150 mL), ethyl acetate/hexanes (2:8; 150 mL), ethyl acetate/hexanes (3:7; 150 mL), ethyl acetate/hexanes (2:5; 150 mL), and ethyl acetate/hexanes (1:1; 150 mL) to afford an opaque white oil/foam (10–99% isolated yield).

Analytical Data for Oxadiargyl-Triazole Derivatives 5a–5r.

5a.

3-(((5-[(3-Methyl)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one. Compound 5a was obtained as an opaque viscous oil in 99% yield using S1 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.40 (s, 9H), 2.36 (s, 3H), 5.30 (s, 2H), 5.52 (s, 2H), 7.09 (m, 2H), 7.18 (d, 1H, J = 7.6), 7.26 (s, 1H), 7.28 (t, 1H, J = 7.6), 7.53 (s, 1H), 7.61 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 21.3, 27.0, 33.0, 54.4, 63.8, 113.9, 123.0, 124.0, 125.2, 125.2, 128.9, 129.0, 129.6, 131.4, 134.2, 139.0, 143.1, 152.1, 153.0, 163.6; IR: (neat) ν (cm⁻¹): 2974, 1778, 1616, 1489, 1423, 1327, 1246, 1188, 1123, 1042, 988, 941, 737, 594; HRMS: [M + H]⁺ calcd for C₂₃H₂₄Cl₂N₅O₃: 488.1256, Found m/z 488.1256; R_f 0.47 (1:1 ethyl acetate/hexanes, UV).

5b.

3-(((5-[(4-Methyl)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one. Compound 5b was obtained as a solid in 98% yield using S2 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 2.38 (s, 3H), 5.29 (s, 2H), 5.52 (s, 2H), 7.21 (s, 4H), 7.25 (s, 1H), 7.53 (s, 1H), 7.59 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 21.2, 27.0, 33.0, 54.2, 63.7, 113.8, 122.9, 123.9, 125.1, 128.2, 129.8, 131.3, 131.4, 131.4, 138.9, 143.1, 152.1, 152.9, 163.6; IR: (neat) ν (cm⁻¹): 3040, 1778, 1616, 1493, 1427, 1327, 1254, 1192, 1126, 1092, 1053, 1015, 907, 806, 748, 671; HRMS: [M + H]⁺ calcd for C₂₃H₂₄Cl₂N₅O₃: 488.1256, Found m/z 488.1257; Melting point: 138 °C; R_f 0.61 (2:1 ethyl acetate/hexanes; UV).

5c.

3-(((5-[(4-tert-Butyl)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5c was obtained as a white foam in 97% yield using S3 as the azide reagent. ¹H NMR: (300 MHz, CDCl₃) δ 1.33 (s, 9H), 1.40 (s, 9H), 5.30 (s, 2H), 5.53 (s, 2H), 7.23 (m, 1H), 7.26 (m, 2H), 7.41 (m, 1H), 7.44 (m, 1H), 7.53 (s, 1H), 7.61 (s, 1H); ¹³C{¹H} NMR: (75 MHz, CDCl₃) δ 27.0, 31.2, 33.0, 34.7, 54.0, 63.8, 113.9, 123.0, 124.0, 125.2, 126.1, 128.0, 131.3, 131.4, 143.0, 152.0, 152.1, 153.0, 163.7; IR: (neat) ν (cm⁻¹): 2963, 1778, 1616, 1489, 1424, 1327, 1246, 1192, 1126, 1045, 988, 941, 806, 671; HRMS: [M + H]⁺ calcd for C₂₆H₃₀Cl₂N₅O₃: 530.1726, Found m/z 530.1725; R_f 0.56 (1:1 ethyl acetate/hexanes, UV).

5d.

3-(((5-[(3-(Trifluoromethyl)methyl]-1H-1,2,3-triazol-4-yl)methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one (4.24): Compound 5d was obtained as a white solid in 97% yield using S4 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 3H), 5.32 (s, 2H), 5.62 (s, 2H), 7.24 (s, 1H), 7.46 (d, 2H, J = 2.3), 7.53 (m, 2H), 7.58 (s, 1H), 7.64 (d, 1H, J = 7.7), 7.69 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 53.7, 63.7, 113.8, 123.2, 124.0, 124.7 (q, J_C,F = 3.6), 125.2, 125.7 (q, J_C,F = 3.6), 129.8, 131.2 (q, J_C,F = 32.6), 131.3, 131.4, 131.5, 131.8, 135.4, 143.5, 152.1, 152.8, 163.6; IR: (neat) ν (cm⁻¹): 2978, 1775, 1620, 1593, 1489, 1327, 1246, 1123, 1045, 991, 802, 752, 702, 660; HRMS: [M + H]⁺ calcd for C₂₃H₂₁Cl₂F₃N₅O₃: 542.0974, Found m/z 542.0977; Melting point: 67 °C; R_f 0.5 (2:1 ethyl acetate/hexanes, UV).

5e.

3-(((5-[(2-(Trifluoromethyl)methyl]-1H-1,2,3-triazol-4-yl)methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5e was obtained as a white foam in 97% yield using S5 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 5.33 (s, 2H), 5.78 (s, 2H), 7.21 (d, 1H, J = 7.7), 7.26 (s, 1H), 7.48 (t, 1H, J = 7.7), 7.54 (s, 1H), 7.56 (t, 1H, J = 7.6), 7.67 (s, 1H), 7.75 (d, 1H, J = 7.8); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 50.3, 63.7, 113.8, 123.0, 123.5, 124.0, 125.2, 126.3 (q, J_C,F = 5.5), 127.9 (q, J_C,F = 30.7), 128.9, 130.2, 131.4, 131.5, 132.8, 143.4, 152.1, 152.9, 163.7; IR: (neat) ν (cm⁻¹): 2978, 1778, 1616, 1489, 1423, 1311, 1246, 1118, 1038, 991, 941, 833, 768, 752, 594; HRMS: [M + H]⁺ calcd for C₂₃H₂₁Cl₂F₃N₅O₃: 542.0974, Found m/z 542.0977; R_f 0.59 (1:1 ethyl acetate/hexanes, UV).

5f.

3-(((5-[(4-(Trifluoromethyl)methyl]-1H-1,2,3-triazol-4-yl)methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5f was obtained as a white foam in 98% yield using S6 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 5.33 (s, 2H), 5.63 (s, 2H), 7.24 (s, 1H), 7.40 (d, 2H, J = 8.0), 7.54 (s, 1H), 7.66 (d, 2H, J = 8.0); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 53.6, 63.6, 113.8, 122.7, 123.2, 124.1, 124.8, 125.2, 126.2 (q, J_C,F = 3.6), 128.3, 131.0 (q, J_C,F = 32.8), 138.3, 143.5, 152.1, 152.8, 163.7; IR: (neat) ν (cm⁻¹): 3148, 2974, 1775, 1616, 1493, 1427, 1323, 1277, 1254, 1126, 1053, 1015, 910, 810, 752, 683, 671; HRMS: [M + H]⁺ calcd for C₂₃H₂₁Cl₂F₃N₅O₃: 542.0974, Found m/z 542.0981; Melting point: 146 °C; TLC: R_f 0.51 (2:1 ethyl acetate/hexanes, UV).

5g.

3-(((5-[(3-Bromo)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5g was obtained as an opaque viscous oil in 99% yield using S7 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 5.32 (s, 2H), 5.53 (s, 2H), 7.21 (d, 1H, J = 8.0), 7.25 (s, 1H), 7.28 (t, 1H, J = 7.8), 7.46 (s, 1H), 7.51 (d, 1H, J = 8.0), 7.54 (s, 1H), 7.66 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 53.5, 63.7, 113.8, 123.1, 123.1, 124.0, 125.1, 126.6, 130.8, 131.1, 131.1, 131.5, 132.1, 136.5, 143.4, 152.1, 152.8, 163.7; IR: (neat) ν (cm⁻¹): 2974, 1778, 1616, 1485, 1424, 1327, 1285, 1246, 1188, 1123, 1042, 991, 941, 833, 748, 729, 667; HRMS [M + H]⁺ calcd for C₂₂H₂₁BrCl₂N₅O₃: 552.0205, Found m/z 552.0205; R_f 0.33 (1:1 ethyl acetate/hexanes, UV).

5h.

3-(((5-[(4-Bromo)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5h was obtained as a white solid in 99% yield using S8 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.40 (s, 9H), 5.32 (s, 2H), 5.52 (s, 2H), 7.17 (d, 2H, J = 8.4), 7.24 (s, 1H), 7.53 (s, 1H), 7.54 (d, 2H, J = 8.4), 7.63 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 53.6, 63.7, 113.9, 123.0, 123.1, 124.1, 125.2, 129.7, 131.4, 131.5, 132.4, 133.3, 143.4, 152.1, 152.8, 163.6; IR: (neat) ν (cm⁻¹): 2978, 1786, 1624, 1489, 1427, 1327, 1246, 1126, 1045, 1003, 941, 903, 876, 802, 748, 729; HRMS: [M + H]⁺ calcd for C₂₂H₂₁BrCl₂N₅O₃: 552.0205, Found m/z 552.0199; Melting point: 138 °C; R_f 0.5 (1:1 ethyl acetate/hexanes, UV).

5i.

3-(((5-[(2-Bromo)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5i was obtained as an opaque viscous oil in 91% yield using S9 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 5.33 (s, 2H), 5.69 (s, 2H), 7.18 (dd, 1H, J = 7.6, 1.3), 7.26 (m, 2H), 7.33 (t, 1H, J = 7.0), 7.54 (s, 1H), 7.64 (d, 1H, J = 8.0), 7.74 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 32.9, 54.0, 63.8, 113.9, 123.4, 123.5, 124.5, 124.0, 125.2, 128.3, 130.4, 130.5, 131.4, 133.3, 133.9, 143.1, 152.1, 152.9, 163.6; IR: (neat) ν (cm⁻¹): 2974, 1778, 1616, 1489, 1424, 1327, 1246, 1188, 1123, 1042, 988, 941, 748, 667; HRMS: [M + H]⁺ calcd for C₂₂H₂₁BrCl₂N₅O₃: 552.0205, Found m/z 552.0198; R_f 0.43 (1:1 ethyl acetate/hexanes, UV).

5j.

3-(((5-[(4-Chloro)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5j was obtained as a white foam in 91% yield using S10 as the azide reagent. ¹H NMR: (300 MHz, CDCl₃) δ 1.40 (s, 9H), 5.32 (s, 2H), 5.53 (s, 2H), 7.24 (m, 3H), 7.37, (m, 2H), 7.54 (s, 1H), 7.63 (s, 1H); ¹³C{¹H} NMR: (75 MHz, CDCl₃) δ 27.0, 33.0, 53.6, 63.7, 113.8, 123.0, 124.1, 125.2, 129.4, 129.5, 131.4, 131.5, 132.8, 135.0, 143.3, 152.1, 152.8, 163.7; IR: (neat) ν (cm⁻¹): 2974, 1778, 1616, 1493, 1327, 1254, 1196, 1126, 1096, 1053, 1015, 941, 910, 802, 671; HRMS: [M + H]⁺ calcd for C₂₂H₂₁Cl₃N₅O₃: 508.0710, Found m/z 508.0708; R_f 0.38 (1:1 ethyl acetate/hexanes, UV).

5k.

3-(((5-[(3-Methoxy)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5k was obtained as an opaque viscous oil in 98% yield using S11 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 3.80 (s, 3H), 5.30 (s, 2H), 5.53 (s, 2H), 6.82 (t, 1H, J = 1.9), 6.87 (d, 1H, J = 7.6), 6.90 (dd, 1H, J = 5.2, 2.5), 7.25 (s, 1H), 7.30 (t, 1H, J = 7.9), 7.53 (s, 1H), 7.63 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 54.3, 55.3, 63.7, 113.7, 113.8, 114.8, 120.3, 123.1, 124.0, 125.2, 130.3, 131.4, 135.7, 143.2, 152.1, 152.9, 160.1, 163.6; IR: (neat) ν (cm⁻¹): 2970, 1790, 1597, 1489, 1427, 1246, 1188, 1123, 1092, 1038, 980, 903, 799, 748; HRMS: [M + H]⁺ calcd for C₂₃H₂₄Cl₂N₅O₄: 504.1205, Found m/z 504.1209; R_f 0.40 (1:1 ethyl acetate/hexanes, UV).

5l.

3-(((5-[(4-Ethanone-phenyl)methyl]-1H-1,2,3-triazol-4-yl)methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5l was obtained as a white solid in 99% yield using S12 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39 (s, 9H), 2.62 (s, 3H), 5.33 (s, 2H), 5.63 (s, 2H), 7.24 (s, 1H), 7.36 (s, 1H), 7.37 (s, 1H), 7.53 (s, 1H), 7.67 (s, 1H), 7.97 (s, 1H), 7.99 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 53.8, 63.7, 113.8, 123.2, 124.1, 125.2, 128.1, 129.2, 131.4, 131.5, 137.4, 139.3, 152.1, 152.8, 163.7, 197.3; IR: (neat) ν (cm⁻¹): 2982, 1778, 1682, 1613, 1493, 1431, 1258, 1192, 1126, 1092, 1049, 1007, 907, 806, 748, 691, 594; HRMS [M + H]⁺ calcd for C₂₄H₂₄Cl₂N₅O₄: 516.1205, Found m/z 516.1200; Melting point: 162 °C; R_f 0.47 (2:1 ethyl acetate/hexanes, UV).

5m.

3-(((5-[(3-Benzoic acid)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5m was obtained as a white solid in 90% yield using S13 as the azide reagent. ¹H NMR: (500 MHz, DMSO-d₆) δ 1.32 (s, 9H), 5.28 (s, 2H), 5.73 (s, 2H), 5.76 (s, 2H), 7.52 (t, 1H, J = 7.5), 7.58 (d, 1H, J = 8.0), 7.79 (s, 1H), 7.91 (s, 1H), 7.93 (m, 2H), 8.37 (s, 1H); ¹³C NMR: (125 MHz, DMSO-d₆) δ 27.0, 32.9, 52.9, 55.4, 63.3, 123.4, 124.4, 125.8, 129.3, 129.5, 129.6, 131.1, 131.7, 132.0, 132.9, 136.9, 142.3, 152.1, 153.1, 153.3, 167.3; IR: (neat) ν (cm⁻¹): 3044, 2924, 1771, 1701, 1616, 1493, 1554, 1412, 1327, 1258, 1192, 1126, 1092, 1018, 907, 806, 729, 648; HRMS: [M + H]⁺ calcd for C₂₃H₂₂Cl₂N₅O₅: 518.0998, Found m/z 518.099; Melting point: 115 °C; R_f 0.38 (4:1 ethyl acetate/hexanes with 1% trifluoroacetic acid; UV).

5n.

3-(5-((1-(2-Nitro-benzyl)-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5n was obtained as a dark-brown solid in 10% isolated yield using S14 as the azide reagent. ¹H NMR: (500 MHz, DMSO-d₆) δ 1.32 (s, 9H), 5.32 (s, 2H), 5.99 (s, 2H), 7.10 (d, 1H, J = 7.5), 7.65 (t, 1H, J = 7.5), 7.75 (t, 1H, J = 7.5), 7.78 (s, 1H), 7.89 (s, 1H), 8.16 (d, 1H, J = 8.0), 8.33 (s, 1H); ¹³C NMR: (125 MHz, DMSO-d₆) δ 27.0, 32.9, 50.5, 63.3, 115.7, 123.5, 124.5, 125.6, 126.4, 130.2, 130.7, 131.1, 132.0, 134.8, 142.3, 148.1, 152.1, 153.3, 163.3; IR: (neat) ν (cm⁻¹): 1771, 1528, 1416, 1246, 1188, 1126, 864, 791, 725, 667; HRMS: [M + H]⁺ calcd for C₂₂H₂₁Cl₂N₆O₅: 519.0951; Found m/z 519.0943; Melting point: 144 °C; R_f 0.45 (4:1 ethyl acetate/hexanes, UV).

5o.

3-(((5-[(2-Hydroxy-5-nitrophenyl)methyl]-1H-1,2,3-triazol-4-yl)methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5o was obtained as a yellow solid in 88% yield using S15 as the azide reagent. ¹H NMR: (500 MHz, DMSO-d₆) δ 1.32 (s, 9H), δ 5.27 (s, 2H), δ 5.64 (s, 2H), δ 7.04 (d, 1H, J = 9.0), δ 7.79 (s, 1H), δ 7.88 (s, 1H), δ 8.10 (s, 1H, J = 2.5), δ 8.14 (dd, 1H, J = 9.0, 3.0), δ 8.30 (s, 1H); ¹³C NMR: (125 MHz, DMSO-d₆) δ 27.0, 32.9, 48.3, 63.2, 115.6, 116.2, 123.4, 124.4, 126.1, 126.7, 131.1, 132.0, 139.9, 141.9, 152.1, 153.3, 162.3, 163.3; IR: (neat) ν (cm⁻¹): 3136, 2967, 2635, 1778, 1593, 1493, 1343, 1292, 1242, 1126, 1092, 1053, 1018, 934, 910, 845, 752, 637; HRMS: [M + H]⁺ calcd for C₂₂H₂₁Cl₂N₆O_6: 535.0900, Found m/z 535.0890; Melting point: 159 °C; R_f 0.50 (4:1 ethyl acetate/hexanes, UV).

5p.

3-(((5-[(2-Nitro-benzoic acid)methyl]-1H-1,2,3-triazol-4-yl)methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5p was obtained as a yellow solid in 95% yield using S16 as the azide reagent. ¹H NMR: (500 MHz, DMSO-d₆) δ 1.32 (s, 9H), 5.33 (s, 2H), 6.05 (s, 2H), 7.07 (d, 1H, J = 8.0), 7.78 (s, 1H), 7.89 (s, 1H), 8.17 (dd, 1H, J = 8.0, 1.5), 8.34 (s, 1H); ¹³C NMR: (125 MHz, DMSO-d₆) δ 27.0, 32.9, 50.5, 63.3, 115.7, 123.5, 124.5, 125.9, 126.5, 130.5, 131.1, 132.0, 133.9, 134.8, 142.4, 147.7, 152.1, 153.3, 163.3, 165.9; IR: (neat) ν (cm⁻¹): 2967, 1775, 1713, 1620, 1535, 1489, 1404, 1250, 1192, 1126, 1045, 814, 745; HRMS: [M + H]⁺ calcd for C₂₃H₂₁Cl₂N₆O₇: 563.0849, Found m/z 563.0840; Melting point: 144 °C; R_f 0.53 (4:1 ethyl acetate/hexanes, UV).

5q.

3-(((5-[(2-Pyridine)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5q was obtained as a white solid in 96% yield using S17 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.39, (s, 9H), 5.33 (s, 2H), 5.69 (s, 2H), 7.21 (d, 1H, J = 7.8), 7.26 (s, 1H), 7.27 (m, 1H), 7.53 (s, 1H), 7.70 (td, 1H, J = 7.8, 1.8), 7.88 (s, 1H); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 55.8, 63.7, 113.8, 122.5, 123.5, 123.8, 124.0, 125.2, 131.5, 137.5, 143.2, 149.9 152.1, 152.9, 154.1, 163.6; IR: (neat) ν (cm⁻¹): 2974, 1775, 1616, 1489, 1427, 1400, 1327, 1285, 1250, 1123, 1092, 1042, 995, 941, 907, 837, 752, 598; HRMS: [M + H]⁺ calcd for C₂₁H₂₁Cl₂N₆O₃: 475.1052, Found m/z 475.1052; Melting point: 105 °C; R_f 0.44 (99:1 ethyl acetate/triethylamine, UV).

5r.

3-(((5-[(4-Pyridine)methyl]-1H-1,2,3-triazol-4-yl)-methoxy)-2,4-dichlorophenyl)-5-(tert-butyl)-1,3,4-oxadiazol-2(3H)-one: Compound 5r was obtained as a light-brown foam in 98% yield using S18 as the azide reagent. ¹H NMR: (500 MHz, CDCl₃) δ 1.40 (s, 9H), 5.36 (s, 2H), 5.59 (s, 2H), 7.13 (d, 2H, J = 5.9), 7.24, (s, 1H), 7.55 (s, 1H), 7.71 (s, 1H) 8.65 (d, 2H, J = 6.0); ¹³C{¹H} NMR: (125 MHz, CDCl₃) δ 27.0, 33.0, 52.9, 63.6, 113.8, 122.1, 123.4, 124.1, 125.2, 131.4, 131.6, 143.2, 143.7, 150.6, 152.1, 152.7, 163.7; IR: (neat) ν (cm⁻¹): 2974, 1775, 1601, 1489, 1416, 1327, 1246, 1192, 1123, 1042, 991, 941, 799, 667; HRMS: [M + H]⁺ calcd for C₂₁H₂₁Cl₂N₆O₃: 475.1052, Found m/z 475.1061; R_f 0.2 (99:1 ethyl acetate/triethylamine, UV).

ABBREVIATIONS

PPO

protoporphyrinogen oxidase

DMF

N,N-dimethylformamide

DCM

dichloromethane

HFF

human foreskin fibroblast