Novel 3-thio-1,2,4- and 1,2,3-triazole derivatives as allosteric glutaminase inhibitors

Abstract

Cancer cells, including those in triple-negative breast cancer (TNBC), often rely on glutamine metabolism. The enzyme glutaminase (GLS) converts glutamine to glutamate, thereby fueling the TCA cycle and affecting cell proliferation and differentiation. This metabolic dependency makes GLS a promising therapeutic target in cancer therapy, with inhibitors currently in clinical trials. Considering the key role of triazoles in medicinal chemistry and the need for the development of new drugs against cancer, we herein report the design and synthesis of a new series of 3-thio-1,2,4-triazoles from a variety of acyl hydrazines and alkyl isothiocyanates in good yields, along with 1,2,3-triazole derivatives obtained by click chemistry. A structure-activity relationship (SAR) study was performed to evaluate their potential to inhibit GLS through an enzyme inhibition assay. Among the library synthesized, compound 10k exhibited the most potent activity, exhibiting an IC₅₀ of 9 µM. To shed light on the possible mode of interaction with GLS, molecular docking was performed, and the results point to a new allosteric binding site for compound 10k.

Keywords:
Cancer; Glutaminase; Allosteric inhibitors; 3-thio-1,2,4-triazole; 1,2,3-triazoles.

INTRODUCTION

Glutaminase, a pivotal enzyme catalyzing the conversion of glutamine to glutamate, presents a critical role in cellular metabolic processes. Oncogenic transformations frequently entail an upregulation of glutaminase activity within tumor cells, thereby increasing the demand for nutrients to sustain unbridled cellular proliferation and growth. This escalated reliance on glutaminase-mediated metabolism constitutes a hallmark feature across diverse cancer types. Consequently, the targeting of glutaminase has emerged as a promising therapeutic strategy, characterized by the design of inhibitors aimed at disrupting this metabolic pathway and restraining the survival and progression of malignant cells (Wang et al., 2020). In this context, glutaminase inhibition holds a significant potential for the development of innovative and precisely targeted interventions in the realm of cancer therapeutics (Mattiuzzi, Lippi, 2019; Cyriac, Lee, 2024).

In mammals, four isoforms of the enzyme glutaminase have been identified to date, derived from two distinct but related genes. The GLS1 gene, which is regulated by the MYC oncogene, can generate two isoforms through alternative splicing, which differ only in their C-terminal regions. The longer isoform is termed Kidney-Type Glutaminase (KGA), while the shorter isoform is called Glutaminase C (GAC). GAC is the most catalytically active form and is capable of forming an oligomer resembling long fibers in the presence of an activator, inorganic phosphate (Wang et al., 2020). The GLS2 gene, regulated by the p53 protein, encodes two isoforms: Glutaminase B (GAB) and Liver-Type Glutaminase (LGA), generated from alternative transcription initiation sites. These isoforms differ in their N-terminal regions. GAB is produced by the transcription of all 18 exons of the gene, while LGA is generated from the final 71 bases of intron 1-2 and the entire sequence of exons 2-18 (Katt, Lukey, Cerione, 2017). These two variants, GAC and LGA, delineate divergent roles in glutamine metabolism across varied physiological contexts, underscoring their contributions to the intricate landscape of glutamine-associated processes in both physiological homeostasis and pathological conditions (Katt, Lukey, Cerione, 2017; Lampa et al., 2017; Yu et al., 2021).

Although the quest for glutaminase inhibitors dates back to 1968 with the identification of the first molecule, 6-diazo-5-oxo-L-norleucine (DON, 1) (Figure 1), progress in this field has not yet delivered a commercially available drug based on GLS inhibition. DON, which targets the active site of glutaminase, underwent clinical trials, however, it exhibited low selectivity due to off-target effects (Barclay, Phillipps, 1966; Catane et al., 1979; Shapiro, Clark, Curthoys, 1979). Later on, the concept of allosteric glutaminase inhibition was introduced by Newcomb and coworkers in 2002 by the discovery of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES, 2) (Figure 1). This inhibitor exhibits remarkable selectivity within the low micromolar range, and its chemical structure holds three distinct portions: Portion A (linker), encompassing an ethyl sulfide linkage; Portion B (core), comprising a 1,3,4-thiadiazole scaffold; and Portion C (side chain), consisting of a 2-phenylacetamide moiety (Newcomb, 2002). Nonetheless, BPTES also failed to enter clinical trials due to its low bioavailability.

FIGURE 1
Chemical structures of DON (1) and BPTES (2) with 3 distinct portions highlighted. Portion A in green, B in red and C in blue.

The origin of selectivity exerted by BPTES was further scrutinized, and certain aspects were proposed, including a noncompetitive inhibition (Robinson et al., 2007). Similarly, DeLaBarre and co-workers (2011) elucidated the interaction of BPTES with glutaminase through X-ray diffraction analysis (PDB: 3UO9). The authors delineated a narrow allosteric binding site wherein BPTES establishes crucial hydrogen bonds through the nitrogen atoms of the 1,3,4-thiadiazole and amide moieties, alongside covalent interactions facilitated by the central sulfur atom in the carbon chain. This interaction entails a “U” conformation adjacent to the active binding site within the dimer-dimer interface of the tetramer, culminating in a conformational change that deactivates glutaminase (DeLaBarre et al., 2011). This breakthrough provided novel insights, prompting medicinal chemists to embark on new investigations into the structure-activity relationship (SAR) of BPTES, aiming to optimize its physicochemical properties and enhance its inhibitory efficacy (Xu et al., 2019; Zimmermann, Duvall, Tsukamoto, 2019).

Subsequently, in 2012, Shukla and co-workers conducted a comprehensive synthesis of a number of truncated analogs aiming at enhancing solubility and to shed new light on their mode of interaction (Figure 2). Substitution of the 1,3,4-thiadiazole moiety (1) with thiazole (1.1) resulted in a notably diminished potency, underscoring the critical role played by the 2-nitrogen atom. Similarly, a tenfold reduction in activity was observed upon shortening the alkyl chain bridging the 1,2,4-thiadiazole core (2). Interestingly, the absence of the sulfur atom within the alkyl chain, as observed in BPTES, was tolerated. However, removal of both amide groups (3) led to a significant loss of potency. Furthermore, the presence of at least one benzyl amide group, as exemplified by analog 3.1, proved crucial for activity (Duvall et al., 2020; Shukla et al., 2012; Zimmermann, Duvall, Tsukamoto, 2019).

FIGURE 2
Structures of some BPTES derivatives prepared by Shukla et al., 2012.

Additionally, Li and co-workers, considering the solubility challenges posed by BPTES, disclosed a patent with the incorporation of polar groups and non-symmetric compounds featuring a 1,2,4-thiadiazole and a pyridazine moiety. One of the molecules designated as CB-839 (Figure 3, 4) exhibited nanomolar inhibition. Moreover, it presented improved solubility and was selected for further biological assays (Li et al., 2013). More recently, in 2020, another potent glutaminase inhibitor derived from BPTES emerged, known as IPN90060 (Figure 3, 5) (Francesco et al., 2016; Soth et al., 2020). Substitution of the 1,2,4-thiadiazole moiety with a 1,2,3-triazole core, while retaining some key structural patterns like CB-839, resulted in nanomolar inhibition. Both compounds are currently being evaluated in clinical trials (Wicker et al., 2021; Yap et al., 2021).

FIGURE 3
Chemical structures of new BPTES derivatives CB-839 (4) and IPN90060 (5).

Taken together, these findings underscore the pivotal role of the 2-nitrogen within the core structure of 1,3,4-thiadiazole in the mechanism of action against glutaminase. The potential of 1,2,4-triazole as a non-classical bioisostere warrants exploration, given its chemical similarity and the capacity of the most basic nitrogen atom to serve as a hydrogen bond acceptor. Moreover, substituting the sulfur atom with a nitrogen atom creates an additional site for investigation, as nitrogen allows for one more covalent bond, thereby expanding the scope of potential interactions.

Therefore, the 1,2,4-triazole scaffold was selected owing to its widespread application in agrochemicals and pharmaceuticals, including anticancer activity (El-Sherief et al., 2018; Sahoo, Sindhu, Sreeveena, 2019; Rathod, Kumar, 2022). Additionally, our research group recently demonstrated a robust and facile synthetic route comprising a two-step process involving condensation reaction between substituted acyl hydrazines and alkyl isothiocyanates, followed by intramolecular cyclization in basic aqueous solution, yielding these scaffolds in excellent yields in continuous flow conditions (Damião et al., 2017).

Thus, we herein explore the 1,2,4-triazole scaffold for the development of novel chemical entities for the potential selective inhibition of glutaminase (Figure 4). The synthesis of these derivatives could be readily carried out in three straightforward steps, distinguishing them significantly from more intricate inhibitors such as CB-839 and IPN60090. Furthermore, with an IC₅₀ in the low micromolar range, our results indicate that this new scaffold harbors promise for further exploration and development.

FIGURE 4
Timeline delineating key advancements in the development of glutaminase inhibitors.

MATERIAL AND METHODS

Material

Starting materials, reagents and solvents were obtained from commercial sources and used as received unless otherwise specified. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 plates under UV light (254 nm). Flash chromatography was performed using silica gel 60 (230-400 mesh, Sigma-Aldrich). Organic solutions were concentrated under reduced pressure on a Büchi or Heidolph rotary evaporator. Column chromatography was performed on a Biotage Isolera flash chromatography system using SNAP KP-Sil columns. ¹H NMR spectra were recorded on either Bruker Avance-400, Bruker Avance-500, or Bruker Avance-600 instruments and are reported relative to residual solvent: CHCl₃ (δ 7.26) or DMSO-d6 (δ 2.50). ¹³C NMR spectra were recorded on the same instruments and are reported relative to CHCl₃ (δ 77.16) or DMSO-d6 (δ 39.52). Data for ¹H NMR are reported as follows: chemical shift (multiplicity, coupling constant in Hz, integration). Multiplicities are reported as follows: s = singlet, br s = broad singlet, d = doublet, t = triplet, tt = triplet of triplets, q = quartet, qu = quintet, sext = sextet, dd = doublet of doublets, ddd = doublet of doublets of doublets, m = multiplet, bs = broad signal. NMR spectra were processed using MestReNova NMR Processor version 12.0.4. Melting points were recorded on a Mettler Toledo MP50 benchtop melting point system with a heating rate of 5 °C.min^-1 and are uncorrected. High-resolution mass spectrometry in electrospray ionization mode (HRMS (ESI+)) was performed on a BRUKER Impact II mass spectrometer, and the data processed using the Bruker Compass Data Analysis 4.3 software (Bruker Daltonics). IUPAC names of the compounds were generated using ChemBioDraw Ultra 14.0.

Experimental section

General procedures A and B: Synthesis of 1-alkyl-1,2,4-triazole-3-thiols:

Procedure A

Aryl/acyl-hydrazine (1.0 eq., 10 mmol) and alkyl/ aryl isothiocyanate (1.0 eq., 10 mmol) were added to a 100 mL round-bottomed flask containing 50 mL of MeOH. The mixture was heated under refluxing conditions for 2 hours. Precipitation of a white solid may occur during the reaction. Upon completion, as confirmed by TLC analysis, the reaction mixture was concentrated under vacuum without additional purification for further use.

Procedure B

To the crude obtained in procedure A, a fresh aqueous NaOH (1M) solution (30 mL) was added and the mixture heated under reflux for 4 hours. The reaction was monitored by TLC analysis (UV 254 nm (rf = 0.375; 3:1 Hex:EtOAc). After completion, the volatiles were removed under vacuum, the crude was cooled and acidified to pH = 1 with an aqueous HCl (1M) solution. The precipitates formed were filtered under vacuum using a Büchner funnel to afford the desired thiols (21-92%).

4,5-Diphenyl-4H-1,2,4-triazole-3-thiol (8a): Compound 8a was prepared according to the general procedures A-B. White solid (Yield: 88%). M.p. = 280 - 282 °C. ¹H NMR (500 MHz, DMSO-d6): δ 14.13 (br s, 1H), 7.50 - 7.38 (m, 3H), 7.45 - 7.38 (m, 1H), 7.34 (dd, J = 7.8, 2.0 Hz, 4H), 7.33 - 7.23 (m, 2H). ¹³C NMR (126 MHz, DMSO-d6): δ 168.7 (C), 150.6 (C), 134.6 (C), 130.4 (CH), 129.5 (CH), 129.4 (2×CH), 128.8 (2×CH), 128.6 (2×CH), 128.3 (2×CH), 125.8 (C).

4-(3-Methoxyphenyl)-5-phenyl-4H-1,2,4-triazole-3-thiol (8b): Compound 8b was prepared according to the general procedures A-B. White solid (Yield: 91%). M.p. = 192 - 194 °C. ¹H NMR (500 MHz, DMSO-d6): δ 14.10 (br s, 1H), 7.44 - 7.40 (m, 1H), 7.40 - 7.36 (m, 1H), 7.36 - 7.32 (m, 4H), 7.05 (ddd, J = 8.3, 2.5, 0.9 Hz, 1H), 7.00 (t, J = 2.2 Hz, 1H), 6.87 (ddd, J = 7.8, 1.9, 0.9 Hz, 1H), 3.73 (s, 3H). ¹³C NMR (126 MHz, DMSO-d6): δ 168.5 (C), 159.6 (C), 150.5 (C), 135.7 (C), 130.4 (CH), 130.1 (CH), 128.6 (2×CH), 128.2 (2×CH), 125.9 (C), 120.8 (CH), 114.9 (CH), 114.9 (CH), 55.5 (CH₃).

4-Phenyl-4H-1,2,4-triazole-3-thiol (8c): Compound 8c was prepared according to the general procedures A-B. White solid (Yield: 92%) and obtained as a tautomeric mixture (80:20 thione/thiol). ¹H NMR (500 MHz, DMSO-d6, major tautomer): δ 13.93 (br s, 1H), 8.70 (s, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.56 (t, J = 7.7 Hz, 2H), 7.49 (t, J = 7.4 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d6, major tautomer): δ 166.2 (C), 142.1 (CH), 134.2 (C), 129.1 (2×CH), 128.8 (CH), 125.8 (2×CH).

4-(Naphthalen-1-yl)-5-phenyl-4 H -1,2,4- triazole-3-thiol (8d): Compound 8d was prepared according to the general procedures A-B. White solid (Yield: 80%). ¹H NMR (500 MHz, DMSO-d6): δ 14.29 (br s, 1H), 8.11 (dd, J = 7.5, 1.9 Hz, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.68 - 7.61 (m, 2H), 7.58 (ddd, J = 8.2, 6.7, 1.4 Hz, 1H), 7.53 (ddd, J = 8.2, 67, 1.4 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.33 - 7.27 (m, 1H), 7.27 - 7.15 (m, 4H). ¹³C NMR (126 MHz, DMSO-d6): δ 169.3 (C), 151.1 (C), 133.8 (C), 130.9 (C), 130.5 (CH), 130.3 (CH), 129.5 (C), 128.6 (2×CH), 128.5 (CH), 128.2 (CH), 127.7 (CH), 127.5 (2×CH), 126.8 (CH), 125.7 (CH), 122.1 (CH).

4,5-Bis(3-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (8e): Compound 8e was prepared according to the general procedures A-B. White solid (Yield: 67%) ¹H NMR (400 MHz, CDCl₃): δ 11.92 (br s, 1H), 7.37 (t, J = 8.1 Hz, 1H), 7.22 - 7.14 (m, 1H), 7.00 (ddd, J = 8.5, 2.6, 0.9 Hz, 1H), 6.97 (br s, 1H), 6.96 - 6.88 (m, 2H), 6.85 - 6.75 (m, 2H), 3.77 (s, 3H), 3.67 (s, 3H). ¹³C NMR (101 MHz, CDCl₃): δ 160.4 (C), 159.5 (C), 135.3 (C), 130.4 (CH), 129.7 (CH), 126.8 (C), 120.6 (CH), 120.3 (CH), 117.1 (CH), 115.9 (CH), 113.9 (CH), 113.0 (CH), 55.7 (CH₃), 55.3 (CH₃).

4-Cyclopropyl-5-(3-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (8f): Compound 8f was prepared according to the general procedures A-B. White solid (Yield: 35%). ¹H NMR (400 MHz, DMSO-d6): δ 13.77 (br s, 1H). 7.44 (t, J = 7.9 Hz, 1H), 7.35 - 7.27 (m, 2H), 7.12 (dd, J = 9.0, 2.3 Hz, 1H), 3.81 (s, 3H), 3.31 (1H), 1.09 - 0.81 (m, 2H), 0.62 - 0.49 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 169.0 (C), 159.0 (C), 152.0 (C), 129.7 (CH), 127.3 (C), 120.9 (CH), 116.1 (CH), 114.0 (CH), 55.3 (CH₃), 26.3 (CH), 9.0 (2×CH₂).

4-(3,5-Bis(trifluoromethyl)phenyl)-5-phenyl-4H-1,2,4-triazole-3-thiol(8g):Compound8gwas prepared according to the general procedures A-B. Whitesolid(Yield:21%).¹HNMR(500MHz, DMSO-d6): δ 14.29 (br s, 1H), 8.26 (s, 2H), 8.26 (br s, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.37 (t, J = 7.7 Hz, 2H), 7.36 - 7.30 (m, 2H). ¹³C NMR (125 MHz, DMSO-d6): δ 168.3 (C), 150.5 (C), 136.3 (C), 130.9 (q, J = 33.7 Hz, 2×C), 130.6 (CH), 130.5 (q, J = 4.7 Hz, 2×CH), 128.8 (2×CH), 128.6 (2×CH), 125.4 (C), 123.2 (q, J = 3.9 Hz, CH), 122.7 (q, J = 273.0 Hz, 2×CF₃).

4-Benzyl-5-phenyl-4H-1,2,4-triazole-3-thiol (8h): Compound 8h was prepared according to the general procedures A-B. White solid (Yield: 63%). ¹H NMR (400 MHz, DMSO-d6): 12.5 (s, 1H), 7.51-7.48 (m, 1H), 7.42-7.38 (m, 4H), 7.30-7.24 (m, 3H), 7.13-7.12 (m, 2H), 5.38 (s, 2H). ¹³C NMR (100 MHz, DMSO-d6): 168.7 (C), 162.7 (C), 132.2 (C), 131.2 (C), 129.1 (CH), 128.9 (CH), 128.8 (CH), 128.1 (CH), 127.2 (CH), 125.8 (CH) e 48.1 (CH2).

4-Phenyl-5-(pyridin-2-yl)-4H-1,2,4-triazole-3-thiol (8i): Compound 8i was prepared according to the general procedures A-B. White solid (Yield: 35%). ¹H NMR (500 MHz, DMSO-d6): δ 14.17 (br s, 1H), 8.39 (dd, J = 4.8, 1.3 Hz, 1H), 7.89 (td, J = 7.9, 1.8 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.40 (ddd, J = 7.9, 4.8, 1.2 Hz, 1H), 7.20 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 3.78 (s, 3H). ¹³C NMR (126 MHz, DMSO-d6): δ 169.3 (C) , 159.2 (C), 149.8 (C), 149.3 (CH), 145.3 (C), 137.3 (CH), 129.6 (2×CH), 127.7 (C), 125.0 (CH), 124.1 (CH), 113.9 (2×CH), 55.3 (CH₃).

4- Cyclopropyl-5-phenyl-4H-1,2,4-triazole-3-thiol (8j): Compound 8j was prepared according to the general procedures A-B. White solid (Yield: 71%). ¹H NMR (400 MHz, DMSO-d6): δ 13.8 (s, 1H), 7.77-7.75 (m, 2H), 7.57-7.51 (m, 3H), 3.29 (m, 1H), 0.96-0.92 (m, 2H),0.58-0.55 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 169.0 (C), 152.1 (C), 130.3 (C), 128.6 (CH), 128.4 (CH), 126.1 (CH), 26.3 (CH₂), 9.0 (CH).

4-(4-Methoxyphenyl)-5-(pyridin-2-yl)-4H-1,2,4-triazole-3-thiol (8k): Compound 8k was prepared according to the general procedures A-B. White solid (Yield: 35%). ¹H NMR (500 MHz, DMSO-d6): δ 14.17 (br s, 1H), 8.39 (dd, J = 4.8, 1.3 Hz, 1H), 7.89 (td, J = 7.9, 1.8 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.40 (ddd, J = 7.9, 4.8, 1.2 Hz, 1H), 7.20 (d, J = 8.9 Hz, 2H), 6.96 (d, J = 8.9 Hz, 2H), 3.78 (s, 3H). ¹³C NMR (125 MHz, DMSO-d6): δ 169.3 (C), 159.2 (C), 149.8 (C), 149.3 (CH), 145.3 (C), 137.3 (CH), 129.6 (2×CH), 127.7 (C), 125.0 (CH), 124.1 (CH), 113.9 (2×CH), 55.3 (CH₃).

Procedure for the synthesis of 5-phenyl-4H-1,2,4-triazole-3-thiol (8l):

In a 20 mL microwave vial, ammonium thiocyanate (2.66 g, 5 eq., 35 mmol) and benzoyl hydrazine (973 mg, 1 eq., 7.5 mmol) were dissolved in 10 mL of water. Concentrated HCl (770 µL, 1.34 eq., 9.38 mmol) was then added, and the tube was sealed to allow complete homogenization. The mixture was heated in the microwave oven at 130 ºC for 45 minutes at 900 rpm. After completion of the reaction, the crude product was neutralized to pH = 7 using an aqueous NaOH (1M) solution, cooled, and filtered. The resulting brown filter cake was utilized in the subsequent step without further purification.

The filter cake obtained previously and an aqueous NaOH (1M) solution (10 mL) were added to a 20 mL microwave vial. The vial was sealed and mixed until complete homogenization. Next, the reaction mixture was heated under microwave irradiation at 130 ºC for 45 minutes at 900 rpm. After completion, the mixture was cooled, acidified to pH = 2 with an aqueous HCl (1M) solution. The solid obtained was filtered under vacuum with a Büchner funnel to afford the desired product.

5- Phenyl-4H-1,2,4-triazole-3-thiol (8l): Compound 8l was prepared according to the procedure for the synthesis of 5-phenyl-4H-1,2,4-triazole-3-thiol (8l). White solid (Yield: 56%). ¹H NMR (400 MHz, DMSO-d6): δ 13.8 (s, 1H), 13.6 (s, 1H), 7.91-7.89 (m, 2H),7.51-7.50 (m, 3H). ¹³C NMR (100 MHz, DMSO-d6): δ 167.0 (C), 150.2 (C), 130.6 (C), 129.0 (CH), 125.6 (CH),125.4 (CH).

General procedure C: Synthesis of symmetric analogues:

In a 5 mL round-bottomed flask, 1.0 mL of DMF, the desired 4,5-aryl-1,2,4-triazole-3-thiol (1.1 eq., 0.484 mmol), and potassium tert-butoxide (56 mg, 1.1 eq., 0.484 mmol) were combined. To this suspension, 1,3-dibromopropane (22.6 µL, 0.5 eq., 0.22 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Upon completion, the crude reaction mixture was diluted with 15 mL of ethyl acetate, washed with water (3 × 10 mL) and brine (3 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude was purified by column chromatography (SiO₂, Rf = 0.25; eluent: 95:5 ethyl acetate:methanol).

1,3-Bis((4-cyclopropyl-5-(3-methoxyphenyl)-4H-1,2,4-triazol-3-yl) thio) propane (10a): Compound 10a was prepared according to the general procedure C. White solid (Yield: 35%). ¹H NMR (500 MHz, CDCl₃): δ 7.36 (t, J = 8.0 Hz, 2H), 7.34 - 7.28 (m, 4H), 7.00 (dd, J = 7.6, 2.1 Hz, 2H), 3.84 (s, 6H), 3.52 (t, J = 7.0 Hz, 4H), 3.18 (tt, J = 7.2, 3.8 Hz, 2H), 2.41 (quint, J = 7.0 Hz, 2H), 1.15 - 1.00 (m, 4H), 0.79 - 0.64 (m, 4H). ¹³C NMR (126 MHz, CDCl₃): δ 159.5 (2×C), 156.2 (2×C), 154.0 (2×C), 129.4 (2×CH), 128.6 (2×C), 121.0 (2×CH), 115.6 (2×CH), 113.9 (2×CH), 55.4 (2×CH₃), 30.70 (2×CH₂), 29.1 (CH₂), 25.7 (2×CH), 9.3 (4×CH₂). HRMS (ESI+): m/z calculated for C₂₇H₃₁N₆O₂S₂⁺ [M+H]⁺ 535.1944, found 535.1962.

1,3-Bis((5-(3-methoxyphenyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)propane (10b): Compound 10b was prepared according to the general procedure C White solid (Yield: 46%) ¹H NMR (400 MHz, CDCl₃): δ 7.53 - 7.44 (m, 6H), 7.25 - 7.20 (m, 4H), 7.13 (t, J = 8.0 Hz, 2H), 6.99 (dd, J = 2.6, 1.5 Hz, 2H), 6.89 (dd, J = 7.8, 1.3 Hz, 2H), 6.85 (ddd, J = 8.4, 2.6, 1.0 Hz, 2H), 3.64 (s, 6H), 3.35 (t, J = 7.0 Hz, 4H), 2.31 (quint, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃): δ 159.4 (2×C), 154.8 (2×C), 152.5 (2×C), 134.3 (2×C), 130.0 (4×CH), 129.9 (2×CH), 129.5 (2×CH), 127.7 (2×C), 127.4 (4×CH), 120.4 (2×CH), 116.3 (2×CH), 112.9 (2×CH), 55.2 (2×CH₃), 31.0 (2×CH₂), 28.9 (CH₂). HRMS (ESI+): m/z calculated for C₃₃H₃₁N₆O₂S₂⁺ [M+H]⁺ 607.1944, found 607.1956.

1,3-Bis((4-phenyl-4H-1,2,4-triazol-3-yl) thio) propane (10c): Compound 10c was prepared according to the general procedure C. White solid (Yield: 42%). ¹H NMR (500 MHz, CDCl₃): δ 8.23 (s, 2H), 7.53 - 7.42 (m, 6H), 7.31 (d, J = 6.2 Hz, 4H), 3.31 (t, J = 7.0 Hz, 4H), 2.24 (quint, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 150.4 (2×C), 144.3 (2×CH), 133.2 (2×C), 129.8 (4×CH), 129.6 (2×CH), 125.1 (4×CH), 31.1 (2×CH₂), 28.6 (CH₂). HRMS (ESI+): m/z calculated for C₁₉H₁₉N₆S₂⁺ [M+H]⁺ 395.1107, found 395.1111.

1,3-Bis((4-phenyl-5-(pyridin-2-yl)-4H-1,2,4-triazol-3-yl) thio) propane (10d): Compound 10d was prepared according to the general procedure C White solid (Yield: 35%). ¹H NMR (500 MHz, CDCl₃): δ 8.26 (dd, J = 4.8, 1.4 Hz, 2H), 8.04 (d, J = 8.0 Hz, 2H), 7.71 (td, J = 7.8, 1.8 Hz, 2H), 7.48 - 7.38 (m, 6H), 7.25 - 7.20 (m, 4H), 7.17 (ddd, J = 7.8, 4.8, 1.4 Hz, 2H), 3.37 (t, J = 7.0 Hz, 4H), 2.32 (quint, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 154.0 (2×C), 153.7 (2×C), 148.9 (2×CH), 146.7 (2×C), 136.6 (2×CH), 135.0 (2×C), 129.3 (2×CH), 129.3 (4×CH), 127.3 (4×CH), 123.8 (2×CH), 123.6 (2×CH), 30.9 (2×CH₂), 28.8 (CH₂). HRMS (ESI+): m/z calculated for C₂₉H₂₅N₈S₂⁺ [M+H]⁺ 549.1638, found 549.1651.

1,3-Bis((4-(naphthalen-1-yl)-5-phenyl-4H-1,2,4-triazol-3-yl) thio) propane (10e): Compound 10e was prepared according to the general procedure C White solid (Yield: 34%). Product was obtained as a diastereoisomeric mixture. ¹H NMR (500 MHz, CDCl₃): δ 8.01 (d, J = 8.3 Hz, 2H), 7.94 (d, J = 8.2 Hz, 2H), 7.58 - 7.50 (m, 4H), 7.50 - 7.44 (m, 2H), 7.39 (ddd, J = 7.3, 4.2, 1.1 Hz, 2H), 7.34 (d, J = 7.1 Hz, 4H), 7,30 (d, J = 8.4 Hz, 2H), 7.21 (t, J = 7.4 Hz, 2H), 7.11 (t, J = 7.7 Hz, 4H), 3.39 - 3.10 (m, 4H), 2.24 (sept, J = 7.2 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 155.7 (2×C), 153.5 (2×C), 134.3 (2×C), 130.8 (2×CH), 130.6 (2×C), 129.7 (2×C), 129.7 (2×CH), 128.6 (2×CH), 128.5 (4×CH), 128.3 (2×CH), 127.4 (4×CH), 127.3 (2×CH), 126.7 (2×C), 126.5 (2×CH), 125.5 (2×CH), 121.9 (2×CH), 31.0 (2×CH₂), 28.7 (CH₂). HRMS (ESI⁺): m/z calculated for C₃₉H₃₁N₆S₂⁺ [M+H]⁺ 647.2046, found 647.2021.

1,3-Bis((4-(3-methoxyphenyl)-5-(pyridin-2-yl)-4H-1,2,4-triazol-3-yl) thio) propane (10f): Compound 10f was prepared according to the general procedure C. White solid (Yield: 46%). ¹H NMR (500 MHz, CDCl₃): δ 8.32 (dd, J = 4.8, 1.2 Hz, 2H), 8.01 (dd, J = 7.8, 1.2 Hz, 2H), 7.71 (td, J = 7.8, 1.8 Hz, 2H), 7.33 (t, J = 8.1 Hz, 2H), 7.19 (ddd, J = 7.5, 4.8, 1.2 Hz, 2H), 6.98 (ddd, J = 8.4, 2.6, 0.9 Hz, 2H), 6.80 (dd, J = 7.8, 0.9 Hz, 2H), 6.78 (t, J = 2.2 Hz, 2H), 3.78 (s, 6H), 3.38 (t, J = 7.0 Hz, 4H), 2.33 (quint, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 160.1 (2×C), 154.0 (2×C), 153.7 (2×C), 149.1 (2×CH), 146.8 (2×C), 136.6 (2×CH), 135.9 (2×C), 130.0 (2×CH), 123.9 (2×CH), 123.6 (2×CH), 119.5 (2×CH), 115.1 (2×CH), 113.1 (2×CH), 55.6 (2×CH₃), 31.0 (2×CH₂), 28.8 (CH₂). HRMS (ESI+): m/z calculated for C₃₁H₂₉N₈O₂S₂⁺ [M+H]⁺ 609.1849, found 609.1826.

1,3-Bis((4-(4-methoxyphenyl)-5-(pyridin-2-yl)-4H-1,2,4-triazol-3-yl) thio) propane (10g): Compound 10g was prepared according to the general procedure C. White solid (Yield: 58%). ¹H NMR (500 MHz, CDCl₃): δ 8.32 (ddd, J = 4.8, 1.8, 0.9 Hz, 2H), 8.01 (dd, J = 8.0, 1.1 Hz, 2H), 7.70 (td, J = 7.8, 1.8 Hz, 2H), 7.18 (ddd, J = 7.8, 4.8, 1.1 Hz, 2H), 7.15 (d, J = 8.9 Hz, 4H), 6.93 (d, J = 8.9 Hz, 4H), 3.85 (s, 6H), 3.38 (t, J = 7.0 Hz, 4H), 2.32 (p, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 160.0 (2×C), 154.2 (2×C), 149.0 (2×CH), 146.9 (2×C), 136.6 (2×CH), 128.6 (4×CH), 127.5 (2×C), 123.8 (2×CH), 123.6 (2×CH), 114.4 (4×CH), 55.5 (2×CH₃), 30.9 (2×CH₂), 28.8 (CH₂). HRMS (ESI+): m/z calculated for C₃₁H₂₉N₈O₂S₂⁺ [M+H]⁺ 609.1849, found 609.1855.

1,3-Bis((4-benzyl-5-phenyl-4H-1,2,4-triazol-3-yl) thio) propane (10h): Compound 10h was prepared according to the general procedure C. White solid (Yield: 44%). ¹H NMR (400 MHz, DMSO-d6): δ 7.52-7.50 (m, 4H), 7.46-7.38 (m, 6H), 7.33-7.28 (m, 6H), 6.99-6.97 (m, 4H), 5.2 (s, 4H), 3.3 (t, J = 6.98 Hz, 2H), 2.2 (qu, J = 6.98 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 156.4 (C), 151.8 (C), 135.2 (CH), 130.3 (CH), 128.3 (CH), 127.1 (CH), 126.4 (CH), 48.0 (CH₂), 31.9 (CH₂), 29.2 (CH₂). HRMS (ESI+): m/z calculated for C₃₃H₃₀N₆S₂H⁺ [M+H]⁺: 575.20461, found 575.20496.

1,3-Bis((4,5-bis(3-methoxyphenyl)-4H-1,2,4-triazol-3-yl)thio)propane (10i): Compound 10i was prepared according to the general procedure C. Yellow oil (Yield: 42%).¹H NMR (500 MHz, CDCl₃): δ 7.37 (t, J = 8.0 Hz, 2H), 7.13 (t, J = 8.0 Hz, 2H), 7.05 (dd, J = 2.6, 1.5 Hz, 2H), 7.01 (ddd, J = 8.4, 2.6, 0.9 Hz, 2H), 6.91 (dd, J = 7.7, 1.5 Hz, 2H), 6.86 (ddd, J = 8.3, 2.6, 0.9 Hz, 2H), 6.80 (ddd, J = 7.7, 2.2, 0.9 Hz, 2H), 6.76 (t, J = 2.2 Hz, 2H), 3.77 (s, 6H), 3.66 (s, 6H), 3.36 (t, J = 7.0 Hz, 4H), 2.31 (quint, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 160.6 (2×C), 159.4 (2×C), 154.7 (2×C), 152.5 (2×C), 135.2 (2×C), 130.7 (2×CH), 129.5 (2×CH), 127.8 (2×C), 120.3 (2×CH), 119.5 (2×CH), 116.2 (2×CH), 115.6 (2×CH), 113.0 (2×CH), 112.8 (2×CH), 55.6 (2×CH₃), 55.2 (2×CH₃), 31.0 (2×CH₂), 28.8 (CH₂). HRMS (ESI+): m/z calculated for C₃₅H₃₅N₆O₄S₂⁺ [M+H]⁺ 667.2156, found: 667.2150.

1,3-Bis((4-(3-methoxyphenyl)-5-phenyl-4H-1,2,4-triazol-3-yl) thio) propane (10j): Compound 10j was prepared according to the general procedure C. White solid (Yield: 57%). ¹H NMR (500 MHz, CDCl₃): δ 7.43 (d, J = 7.1 Hz, 4H), 7.40 - 7.30 (m, 4H), 7.30 - 7.19 (m, 4H), 7.02 (dd, J = 8.5, 2.5 Hz, 2H), 6.80 (dd, J = 7.8, 2.0 Hz, 2H), 6.76 (t, J = 2.3 Hz, 2H), 3.78 (s, 6H), 3.38 (t, J = 7.0 Hz, 4H), 2.32 (quint, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 160.6 (2×C), 154.9 (2×C), 152.5 (2×C), 135.2 (2×C), 130.7 (2×CH), 129.7 (2×CH), 128.5 (4×CH), 128.1 (4×CH), 126.7 (2×C), 119.5 (2×CH), 115.6 (2×CH), 113.0 (2×CH), 55.6 (2×CH ), 31.0 (2×CH ), 28.8 (CH ). HRMS (ESI+): m/z calculated for C₃₃H₃₁N₆O₂S₂ ⁺ [M+H]⁺ 607.1944, found 607.1990.

1,3-Bis((4,5-diphenyl-4H-1,2,4-triazol-3-yl) thio) propane (10k): Compound 10k was prepared according to the general procedure C. White solid (Yield: 73%). ¹H NMR (500 MHz, CDCl₃): δ 7.53 - 7.46 (m, 6H), 7.42 - 7.37 (m, 4H), 7.35 - 7.29 (m, 2H), 7.28 - 7.25 (m, 4H), 7.24 - 7.19 (m, 4H), 3.37 (t, J = 7.0 Hz, 4H), 2,32 (quint, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 155.0 (2×C), 152.5 (2×C), 134.3 (2×C), 130.0 (4×CH), 129.9 (2×CH), 129.7 (2×CH), 128.5 (4×CH), 128.2 (4×CH), 127.4 (4×CH), 126.7 (2×C), 31.1 (2×CH₂), 28.9 (CH₂). HRMS (ESI+): m/z calculated for C₃₁H₂₇N₆S₂⁺ [M+H]⁺ 547.1733, found: 547.1729.

Procedureforthesynthesisof5,5’-((oxybis(propane-3,1-diyl)) bis(sulfanediyl)) bis(4-(3-methoxyphenyl)-3-phenyl-4H-1,2,4-triazole) (11):

To a 5 mL vial, 1 mL of H₂O, thiol 8b (283 mg, 1 mmol, 1 eq.) and NaOH (50 mg, 1.2 mmol, 1.2 eq.) were added. Then, 1,3-dibromopropane (142 µL, 1.5 mmol, 1.5 eq.) was added and the resulting solution heated to 100 ºC for 80 minutes. The reaction was neutralized with an aqueous HCl (1M) solution and extracted with EtOAc (4 × 10 mL). The organic phase was dried with anhydrous sodium sulfate, concentrated under vacuum and the crude was purified by column chromatography (SiO₂, Hex:EtOAc 20-100%).

5 , 5 ’ - ( ( O x y bi s( pr o pa n e - 3 , 1 - di y l ) ) bis(sulfanediyl)) bis(4-(3-methoxyphenyl)-3-phenyl-4H-1,2,4-triazole) (11): Compound 11 was prepared according to the procedure for the synthesis of 5,5’-((oxybis(propane-3,1-diyl)) bis(sulfanediyl)) bis(4-(3-methoxyphenyl)-3-phenyl-4H-1,2,4-triazole) (11). Colorless oil (Yield: 22%). ¹H NMR (500 MHz, CDCl₃): δ 7.44 (d, J = 7.9 Hz, 2H), 7.39 - 7.32 (m, 5H), 7.32 - 7.25 (m, 5H), 7.02 (dd, J = 8.5, 2.8 Hz, 1H), 6.92 (dd, J = 8.5, 2.5 Hz, 1H), 6.84 - 6.77 (m, 3H), 6.77 - 6.71 (m, 1H), 4.02 (t, J = 6.8 Hz, 2H), 3.77 (s, 3H), 3.75 (s, 3H), 3.36 (t, J = 7.0 Hz, 2H), 2.71 - 2.60 (m, 4H), 2.21 - 2.13 (m, 2H), 2.12 - 2.06 (m, 2H). ¹³C NMR (126 MHz, CDCl₃): δ 160.5 (C), 160.3 (C), 154.9 (C), 153.4 (C), 152.7 (C), 144.5 (C), 135.3 (C), 134.8 (C), 130.7 (CH), 130.1 (CH), 130.0 (CH), 129.7 (CH), 128.6 (2×CH), 128.5 (2×CH), 128.1 (2×CH), 127.8 (2×CH), 126.7 (C), 126.7 (C), 119.5 (CH), 115.6 (CH), 114.8 (CH), 113.0 (CH), 112.7 (CH), 55.6 (CH₃), 55.5 (CH₃), 44.7 (CH₂), 31.2 (CH₂), 30.6 (CH₂), 29.1 (CH₂), 29.0 (CH₂), 28.7 (CH₂). HRMS (ESI+): m/z calculated for C₃₆H₃₇N₆O₃S₂⁺ [M+H]⁺ 665.2363, found 665.2395.

Procedure for the synthesis of non-symmetric analogue (13):

Compound 8a (1 mmol, 1.1 eq., 253 mg) was dissolved in anhydrous DMF (1 mL), followed by the addition of t-BuOK (112 mg, 1 mmol, 1 eq.). After 30 minutes, 1,2-dibromoethane (431 µL, 5 mmol, 5 eq.) was added to the suspension, and the mixture was allowed to react at room temperature for an additional 30 minutes until all starting material was consumed. Subsequently, the reaction mixture was diluted with 10 mL of ethyl acetate and the combined organic phase was washed with water (4 × 10 mL) and brine (4 × 10 mL). The organic layer was then dried over anhydrous Na₂SO₄, concentrated under vacuum, and the product was purified by flash column chromatography (hexane:ethyl acetate, 5-80%). Despite instability observed during NMR analysis, the bromo derivative was used directly in the subsequent step.

In a 5 mL flask containing anhydrous DMF (1 mL), the bromide derivative 12 (1.0 mmol, 1 eq., 72 mg), t-BuOK (112 mg, 1.0 mmol, 1 eq.), and thiol 8g (389mg, 1.0 mmol, 1.0 eq.) were combined. The mixture was allowed to react at room temperature for 30 minutes until all starting material was consumed. Subsequently, the reaction mixture was diluted with 10 mL of ethyl acetate, and the combined organic phase was washed with water (4 × 10 mL) and brine (4 × 10 mL). The organic layer was then dried over anhydrous Na₂SO₄, concentrated under vacuum, and the product was purified by flash column chromatography (ethyl acetate:methanol, 0-10%).

4-(3,5-Bis(trifluoromethyl)phenyl)-3-((2-((4,5-diphenyl-4H-1,2,4-triazol-3-yl) thio) ethyl) thio)-5-phenyl-4H-1,2,4-triazole (13): Compound 13 was obtained according to the procedure for the synthesis of non-symmetric analogue. White solid (Yield: 60%). ¹H NMR (400 MHz, CDCl₃): δ 8.01 (br s, 1H), 7.74 (s, 2H), 7.55 - 7.47 (m, 3H), 7.44 - 7.38 (m, 3H), 7.37 - 7.32 (m, 5H), 7.31 - 7.27 (m, 2H), 7.25 (dd, J = 7.8, 2.0 Hz, 2H), 3.85 - 3.72 (m, 4H). ¹³C NMR (101 MHz, CDCl₃): δ 155.1 (C), 155.0 (C), 152.0 (C), 151.5 (C), 135.7 (C), 134.1 (C), 133.6 (q, J = 34.6 Hz, 2×C), 130.5 (CH), 130.0 (2×CH), 130.0 (CH), 129.8 (CH), 128.9 (2×CH), 128.5 (2×CH), 128.4 (2×CH), 128.1 (2×CH), 127.7 (q, J = 3.2 Hz, 2×CH), 127.3 (2×CH), 126.5 (C), 125.5 (C), 123.5 (q, J = 4.0 Hz, CH), 122.3 (q, J = 273.3 Hz, 2×CF₃), 32.3 (CH₂), 31.7 (CH₂). HRMS (ESI+): m/z calculated for C₃₂H₂₃F₆N₆S₂⁺ [M+H]⁺ 669.1324, found 669.1318.

General procedure D: Synthesis of propargyl derivatives:

3- Thiol-1,2,4-triazoles (8j, 8l or 5-amine-1,3,4-thiadiazole-2-thiol) (1 eq., 2 mmol) and potassium carbonate (415 mg, 1.5 eq., 3 mmol) were added to a round-bottomed flask containing anhydrous THF (10 mL). After complete homogenization, propargyl bromide (245 µL, 1.1 eq., 2.2 mmol) was added and the reaction mixture was kept at room temperature for 2 hours. Next, the reaction mixture was diluted in EtOAc (10 mL) and the combined organic phase was washed with water (3 × 10 mL) and brine (10 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum. and the product was purified by flash column chromatography (Hex:EtOAc, 2:1).

4- Cyclopropyl-3-phenyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazole (20a): Compound 20a was prepared according to the general procedure D. White solid (Yield: 57%). ¹H NMR (400 MHz, DMSO-d6): δ 7.80-7.78 (m, 2H), 7.52-7.44 (m, 3H), 4.11 (d, J = 2.67 Hz, 2H), 3.53 (m, 1H), 3.20 (t, J = 2.67 Hz, 1H), 1.02-0.98 (m, 2H), 0.62-0.58 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 155.0 (C), 151.5 (C), 129.7 (CH), 128.4 (CH), 128.3 (CH), 127.2 (CH), 79.6 (CH), 25.5 (CH₂), 20.0 (CH), 8.66 (CH).

3-Phenyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazole (20b): Compound 20b was prepared according to the general procedure D. White solid (Yield: 77%). ¹H NMR (400 MHz, DMSO-d6): δ 14.50 (br s, 1H), 7.99-7.96 (m, 2H), 7.53-7.50 (m, 3H), 4.01 (d, J = 2.2 Hz, 2H), 3.19 (t, J = 2.3 Hz, 1H). This compound was unstable under NMR analysis. HRMS (ESI+): m/z calculated for C₁₁H₉N₃SH⁺ [M+H]⁺: 216.05899, found 216.05874.

5-(Prop-2-yn-1-ylthio)-1,3,4-thiadiazol-2-amine (22): Compound 22 was prepared according to the general procedure D. Yellow powder (Yield: 82%). ¹H NMR (400 MHz, DMSO-d6): δ 7.4 (s, 2H); 3.8 (d, J = 2.6 Hz, 2H), 3.29 (t, J = 2.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d6): δ 170.5 (C), 148.3 (C), 79.7 (C), 75.0 (CH), 22.8 (CH₂). HRMS (ESI+): m/z calculated for C₅H₅N₃S₂H⁺ [M+H]⁺: 171.99977, found 171.99956.

Procedure for the synthesis of 2-phenyl-N-(5-(prop-2-yn-1-ylthio)-1,3,4-thiadiazol-2-yl)acetamide (23):

In a round-bottomed flask, 5-(prop-2-yn-1-ylthio)-1,3,4-thiadiazol-2-amine (22) (282 mg, 1 eq., 1.65 mmol) and dry Et₃N (345 µL, 1.5 eq., 2.47 mmol) in 8.5 mL of dry THF. The crude was cooled to 0 ºC then phenylacetyl chloride (436 µL, 2.0 eq., 3.46 mmol) was added dropwise. After completion of the reaction, the crude was diluted in 15 mL of EtOAc and was washed with water (3 × 10 mL) and brine (10 mL). The organic layer was dried over anhydrous Na₂SO₄, concentrated under vacuum and the crude was used in the next step without any further purification.

2-Phenyl-N-(5-(prop-2-yn-1-ylthio)-1,3,4-thiadiazol-2-yl)acetamide (23): Compound 23 was prepared according to the procedure for the synthesis of 2-phenyl-N-(5-(prop-2-yn-1-ylthio)-1,3,4-thiadiazol-2-yl)acetamide. White solid (Yield: 63%). HRMS (ESI+): m/z calculated for C₅H₅N₃S₂H⁺ [M+H]⁺: 423,10563, found 423,10536.

General procedure E: Synthesis of 1,2,3-triazole analogues (21a-b, 24):

In a round-bottomed flask, propargyl thiol derivatives (20a-b or 23) (1 eq., 0.975 mmol), sodium azide (69.8 mg, 1.1 eq.; 1.07 mmol), benzyl bromide (142 µL, 1.2 eq., 1.17 mmol) and DIPEA (214 µL, 1.26 eq., 1.23 mmol) were added to a water:MeOH mixture (2:1) forming a suspension. In another flask, copper sulfate (46.7 mg, 30 mol%) and sodium ascorbate (116 mg, 60 mol%) were added to DMF (7 mL),) and the resulting solutions were purged for 1 hour with nitrogen in an ultrasound bath. Next, the DMF solution was carefully added with a syringe to the suspension and the resulting reaction mixture was kept under stirring at room temperature for 4 hours. After completion of the reaction, the crude was diluted with brine (25 mL) and washed with EtOAc (3 × 20 mL). The combined organic layers were washed with water (3 × 15 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, concentrated under vacuum and the crude was purified by flash column chromatography (EtOAc for 21a; 1:1 Hex:EtOAc for 21b). The crude product was solubilized in NaOH (1M), acidified to pH = 7 with HCl (1M), then filtered under vacuum to afford compound 24.

1-Benzyl-4-(((4-cyclopropyl-5-phenyl-4H-1,2,4-triazol-3-yl) thio) methyl)-1H-1,2,3-triazole (21a): Compound 21a was prepared according to the general procedure E. Pale yellow solid (Yield: 40%). ¹H NMR (400 MHz, DMSO-d6): δ 8.1 (s, 1H), 7.8-7.7 (m, 2H), 7.5-7.4 (m, 3H), 7.4-7.3 (m, 5H), 5.6 (s, 2H), 4.6 (s, 2H), 3.4-3.3 (m, 2H), 0.97-0.94 (m, 2H), 0.55-0.52 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 155.8 (C), 152.4 (C), 143.1 (CH), 136.0 (CH), 129.7 (CH), 128.8 (CH), 128.5 (CH), 128.4 (CH), 128.1 (CH), 127.9 (CH), 127.2 (CH), 123.9 (CH), 52.8 (CH₂), 26.4 (CH), 25.5 (CH₂), 8.70 (CH₂). HRMS (ESI₊): m/z calculated for C₂₁H₂₁N₆S⁺ [M+H]⁺: 389.15429, found 389.15409.

1-Benzyl-4-(((5-phenyl-4H-1,2,4-triazol-3-yl) thio)methyl)-1H-1,2,3-triazole (21b): Compound 21b was prepared according to the general procedure E. White solid (Yield: 40%). ¹H NMR (400 MHz, DMSO-d6): δ 14.5 (br s, 1H), 8.0 (s, 1H), 7.97-7.96 (m, 2H), 7.51-7.23 (m, 8H), 5.5 (s, 2H), 4.5 (s, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 135.39 (CH), 128.38 (CH), 128.1 (CH), 127.4 (CH), 127.2 (CH), 125.4 (CH), 122.9 (CH), 52.1 (CH₂).

N-(5-(((1-Benzyl-1H-1,2,3-triazol-4-yl) methyl) thio)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (24): Compound 24 was prepared according to the general procedure E. White solid (Yield: 27%). ¹H NMR (400 MHz, DMSO-d6): δ 13.0 (s, 1H), 7.40-7.38 (m, 4H), 7.30-7.27 (m, 5H), 7.16-7.14 (m, 2H), 5.40 (s, 2H), 4.53 (s, 2H), 3.95 (s, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 169.6 (C), 161.0 (C), 159.6 (C), 143.8 (CH), 134.5 (CH), 133.6 (CH), 129.5 (CH), 129.2 (CH), 129.0 (CH), 128.9 (CH), 128.0 (CH), 127.7 (CH), 122.6 (CH), 54.2 (CH₂), 42.8 (CH₂), 28.6 (CH₂). HRMS (ESI+): m/z calculated for C₂₀H₁₇N₆OS₂H⁺ [M+H]⁺: 423.10563, found 423.10536.

General procedure F: Synthesis of azide-3-thio-1,2,4-triazole derivatives (17a-b):

To a round-bottomed flask, anhydrous DMF (800 µL), the desired thiol (8j or 8l) (1 eq.; 0.224 mmol) and t-BuOK (25.9 mg, 1 eq., 0.224 mmol) were added and stirred at room temperature under nitrogen atmosphere. In another flask, 3-azide-bromopropane (16) was solubilized in 200 µL of anhydrous DMF and this solution was added slowly with syringe to the first flask and the reaction mixture was heated to 40 ºC for 4 hours. After completion, the reaction mixture was diluted with EtOAc (15 mL), washed with water (3 × 10 mL) and brine (10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated under vacuum. The crude was purified by flash column chromatography (rf = 0.37, 1:1 Hex:EtOAc).

3-((2-Azidoethyl) thio)-4-cyclopropyl-5-phenyl-4H-1,2,4-triazole (17a): Compound 17a was prepared according to the general procedure F. Pale yellow solid (Yield: 76%). ¹H NMR (400 MHz, DMSO-d6): δ 7.76-7.74 (m, 2H), 7.46-7.45 (m, 2H), 3.49 (t, J = 6.6 Hz, 2H), 3.49 (t, J = 7.02 Hz, 2H), 3.19-3.15 (m, 1H), 2.16 (m, 2H), 1.07-1.03 (m, 2H), 0.71-0.68 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 156.2 (C), 153.6 (C), 129.6 (CH), 128.4 (CH), 127.2 (CH), 49.9 (CH₂), 28.8 (CH₂), 28.6 (CH₂), 25.5 (CH), 9.11 (CH₂). HRMS (ESI+): m/z calculated for C₁₄H₁₆N₆SH⁺ [M+H]⁺: 301.12299, found 301.12274.

3-((2-Azidoethyl)thio)-5-phenyl-4H-1,2,4-triazole (17b): Compound 17b was prepared according to the general procedure F. White solid (Yield: 67%). ¹H NMR (400 MHz, DMSO-d6): δ 13.10 (br s, 1H), 7.94-7.21 (m, 2H), 7.42-7.35 (m, 3H), 3.41 (t, J³ = 6.5 Hz, 2H), 3.22 (t, J³ = 7.00 Hz, 2H), 2.00 (qu, J³ = 7.00 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 159.3 (C), 157.8 (C), 130.4 (CH), 129.0 (CH), 128.0 (CH), 127.4 (CH), 126.6 (CH), 49.9 (CH₂), 29.8 (CH₂), 24.1 (CH₂). HRMS (ESI+): m/z calculated for C₁₁H₁₂N₆SH⁺ [M+H]⁺: 261.09169, found 261.09179.

General procedure G: Synthesis of 1,2,3-triazole derivatives (19a-b):

To a round-bottomed flask, propargyl thiol derivative (17a-b) (1 eq., 1.32 mmol) and DIPEA (291 µL, 1.26 eq., 1.67 mmol) were added to a water:MeOH mixture (2:1) forming a suspension. In another flask, copper sulfate (63 mg, 30 mol%) and sodium ascorbate (157 mg, 60 mol%) were added to DMF (7 mL), and the solution was purged for 1 hour with nitrogen gas in an ultrasound bath. Next, the DMF solution was carefully added with a syringe to the suspension and the reaction mixture was kept under stirring at room temperature for 4 hours. After completion, the reaction mixture was diluted with brine (25 mL) and washed with EtOAc (3 × 20 mL). The organic layer was washed with water (3 × 15 mL) and brine (20 mL). The organic phase was dried over anhydrous Na₂SO₄, concentrated and the crude containing 19a-b. The crude product was solubilized in NaOH (1M), acidified to pH = 7 with HCl (1M), then filtered under vacuum to afford compound 19a-b.

N-benzyl-1-(3-((5-phenyl-4H-1,2,4-triazol-3-yl) thio)propyl)-1H-1,2,3-triazole-4-carboxamide (19a): Compound 19a was prepared according to the general procedure G. White solid (Yield: 30%). ¹H NMR (400 MHz, DMSO-d6): δ 14.11 (s, 1H), 9.0 (t, J = 6.4 Hz, 1H), 8.6 (s, 1H), 7.94-7.92 (m, 2H), 7.49-7.20 (m, 9H), 4.56 (t, J = 7.0 Hz, 2H), 4.46 (d, J = 6.4, 1H), 3.11 (m, 2H), 2.30 (qu, J = 7.0 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 159.7 (C), 142.8 (C), 139.6 (C), 129.1 (CH), 128.2 (CH), 127.3 (CH), 126.7 (CH), 126.6 (CH), 126.0 (CH), 48.6 (CH₂), 41.9 (CH₂), 30.0 (CH₂). HRMS (ESI+): m/z calculated for C₂₁H₂₁N₇OSH⁺ [M+H]⁺: 420.16011, found 420.15983.

N-benzyl-1-(3-((4-cyclopropyl-5-phenyl-4H-1,2,4-triazol-3-yl) thio) propyl)-1H-1,2,3-triazole-4-carboxamide (19b): Compound 19b was prepared according to the general procedure G. White solid (Yield: 28%). ¹H NMR (400 MHz, DMSO-d6): δ 8.2 (s, 1H), 7.76-7.74 (m, 2H), 7.48-7.46 (m, 4H), 7.36-7.27 (m, 5H), 4.65 (t, J = 6.6 Hz, 2H), 4.61 (t, J = 7.0 Hz, 2H), 3.3 (t, J = 6.8 Hz, 2H), 3.17-3.14 (m, 1H), 2.6 (qu, J = 7.0 Hz, 2H), 1.07-1.02 (m, 2H), 0.72-0.68 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 160.0 (C), 143.5 (C), 138.0 (C), 130.0 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.0 (CH), 127.2 (CH), 125.8 (CH), 49.3 (CH₂), 43.3 (CH₂), 29.9 (CH₂), 28.5 (CH₂), 25.8 (CH₂), 9.30 (CH₂). HRMS (ESI+): m/z calculated for C₂₄H₂₅N₇OSH⁺ [M+H]⁺: 461.19476, found 461.19363.

Procedure for the synthesis of BPTES derivative (15):

Compound 14 (153 mg, 1 eq., 0.5 mmol) and Et₃N (174 µL, 2.5 eq., 1.25 mmol) were added to DMF (1.5 mL). The suspension was cooled down to 0 ºC, then phenyl acetyl chloride (165 µL, 2.5 eq, 1.25 mmol) was added dropwise. After the addition, the reaction mixture was heated up at 100 ºC for 2 hours. After completion, the reaction mixture was cooled to room temperature diluted with EtOAc (15 mL). The organic phase was washed with water (3 × 10 mL) and brine (10 mL), dried over anhydrous Na₂SO₄ and concentrated under vacuum. The crude product was solubilized in NaOH (1M), acidified to pH = 7 with HCl (1M), then filtered under vacuum to afford compound 15.

N,N’-((Propane-1,3-diylbis(sulfanediyl)) bis(1,3,4-thiadiazole-5,2-diyl))bis(2- phenylacetamide) (15): Compound 15 was prepared according to the procedure for the synthesis of BPTES derivative (15). White solid (Yield: 27%). ¹H NMR (400 MHz, DMSO-d6): δ 12.8 (s, 2H), 7.34-7.24 (m, 10H), 3.8 (s, 4H), 3.31 (t, J = 7.05 Hz, 4H), 2.08 (qu, J = 7.05 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6): δ 169.7 (C); 159.0 (C); 158.0 (C); 134.5 (CH); 129.3 (CH); 128.4. (CH); 126,9 (CH); 41.5 (CH₂); 32.2 (CH₂), 28.9 (CH₂). HRMS (ESI⁺) m/z calculated C₂₀H₁₇N₆OS₂H⁺ [M+H]⁺: 423.10563, found 423.10536.

GLS/GLS2-GDH and GDH activity assays

To determine the IC₅₀ values of the compounds, serial dilutions of the compounds (in 1% DMSO) were mixed with 10 nM of purified enzyme in 50 mM Trisacetate (pH 8.6), 0.2 mM EDTA, 2 mM NAD⁺, 0.6 U of GDH, 20 mM K₂HPO₄, and 7.5 mM glutamine. The GDH cross enzyme activity assay was performed under the same experimental conditions, removing the murine GAC enzyme (construct ∆1-127 (Pavlova, Thompson, 2016)) from the assay, and replacing the glutamine substrate by the GDH-specific substrate, glutamate, also at a final concentration of 7.5 mM. For the GLS2 reaction, the reaction contained GLS2 at 5 nM and 3 U of GDH. Readings were performed on a PerkinElmer EnSpire 2300 multilabel plate reader at 340 nm. The percent activity was calculated based on the DMSO control reaction (100% activity). Adjustment of inhibitor dose-response curves was performed with the program GraphPad Prism v8.0 (GraphPad Software, USA) using the log (inhibitor) vs normalized response (variable slope) function. The inhibitor dose-response curves were adjusted with LL.4 model from drm r-package (Heng, Gryncel, Kantrowitz, 2009). R-squared values obtained from drm (Heng, Gryncel, Kantrowitz, 2009) models using R2nls function from aomisc r-package. Standard deviations from fitted models were obtained using coeftest function from lmtest r-package. IC50 confidence interval were obtained using function ED from drm (Heng, Gryncel, Kantrowitz, 2009; Costa et al., 2023).

Molecular docking analysis

The tridimensional structures of the analogous were built by modifying the crystallographic structure of BPTES complexed to human glutaminase (PDB: 3UO9). Compounds were drawn in the software Discovery Studio Visualizer (BIOVIA, Dassault Systèmes, Discovery Studio Visualizer, Release 2016. San Diego, CA, USA, 2016.). The resulting 3D structures were then optimized using the Merck Molecular Force Field (Halgren, 1996). Since the structure of glutaminase complexed with BPTES is only available with a resolution of 2.30 Å, the crystallographic model can display some missing amino acid side chains. Consequently, the DockPrep modul of UCFS Chimera was used to complete these structures (Pettersen et al., 2004), as well as to attribute appropriate charges to the amino acid residues. The protein structure was loaded in GOLD 5.6 for molecular docking simulations (Jones et al., 1997), which were performed inside of a radius sphere of 12 Å from the geometric center of the crystallographic inhibitor.

RESULTS AND DISCUSSION

Chemical synthesis

In this study, we aimed at devising two new series of BPTES analogues with potential glutaminase inhibition activity. In the first series, we replaced the BPTES thiadiazol heterocycle with the 3-thio-1,2,4-triazole core (compounds 9-15). Furthermore, we examined alterations at positions 4 and 5 of the triazole ring, as well as variations in linker nature and lengths. The design of our second series was inspired by the structure of compound 5 (IPN90060).

Figure 5 depicts the synthesis of compounds 9, 10a-k, 11, 13, and 15. The reaction between acyl hydrazine (6) and alkyl isothiocyanates (7) yielded thiol intermediates 8a-k in good to excellent yields. Compound 9 was synthesized from the respective thiol intermediate and 1,2-dibromoethane. Similarly, compounds 10a-k were obtained under analogous conditions using different thiols and 1,3-dibromopropane.

FIGURE 5
Synthesis of 3-thio-1,2,4-triazoles through bisalkylation strategy. Reagents and conditions: (a) i. MeOH, reflux, 2 h ii. NaOH, reflux, 2-4 h; (b) DMF, t-ButOK, 1,2-dibromoethane, r.t., 30 min; (c) DMF, t-ButOK, 1,3-dibromopropane, r.t., 30 min (35-73%); (d) DMF, t-BuOK, 1,3-dibromopropane, H₂O, reflux, NaOH, 80 min (22%); (e) DMF, t-ButOK, 1,2-dibromoethane, r.t., 30 min (22%); (f) KOH, 1,3-dibromopropane, EtOH, reflux, 3 h (68%); (g) Et₃N, DMF, 100 ºC, 4 h (27%).

Compound 11 was synthesized using 1,3-dibromopropane, although the addition of water was required to construct the alkyl ether linker chain. Compound 13 was prepared from intermediate 12, 1,2-dibromoethane and the appropriate thiol. Compound 15 was synthesized to assess the impact of the presence of sulfide groups, as proposed by Shukla et al. 2012 (Tomi, Al-Daraji, Aziz, 2015).

Based on the glutaminase inhibition of compound 5, several analogs were synthesized through click chemistry reactions to introduce a 1,2,3-triazole moiety into the second series of analogues. Figure 6 outlines the synthesis of compounds 19a-b, 21a-b, and 24. Compounds 17a-b were obtained by reacting 1 equivalent of previously prepared 3-azide-1-bromopropane under conditions described in the literature (Agnew et al., 2009) with potassium tert-butoxide. The azide group in compounds 17a-b was converted into the 1,2,3-triazole scaffold via click chemistry (copper catalyzed azide alkyne cycloaddition reactions) in the presence of N-benzyl propiolamide 18, which was synthesized according to the procedure described in the literature (Lanz, Riedl, 2015), resulting in the desired compounds 19a-b. Additionally, thiols 8a-b were alkylated with propargyl bromide to yield compounds 20a-b. The alkyne group in these compounds was converted to compounds 21a-b under click chemistry conditions. Similarly, 23 was prepared by alkylating the existing thiol group, followed by acylation under conditions described in the literature (Jäger, Heitzer, 1981), and subsequently converted into the 1,2,3-triazole 24.

FIGURE 6
Synthesis of 1,2,3-triazole derivatives. Reagents and conditions: (a) i. R-NCS or NH₄SCN, H₂O, 130 ºC (MW), 45 min, ii. NaOH (1M), 130 ºC (MW), 45 min, (b) DMF, t-ButOK, 16, 40 ºC, 3 h, (67-76%); (c) CuSO₄, Sodium ascorbate, DIPEA, 18, H₂O:MeOH (1:2), DMF, 4 h, (28-30%); (d) Propargyl bromide, K₂CO₃, THF, r.t., 2 h (57-78%); (e) CuSO₄, Sodium ascorbate, DIPEA, NaN₃, benzyl bromide, H₂O:MeOH (1:2), DMF, 4 h, (28-40%); (f) Phenyl acetyl chloride, Et₃N, DMF, r.t.; 3 h (63%).

Structure-activity relationship (SAR) evaluation

Our work was motivated by previous results obtained through the screening of several compounds against glutaminase (Figure 7) (Damião, Marçon, Pastre, 2020). Compound 25 exhibited potential as a new scaffold due to the incorporation of a nitrogen heterocycle linked to an N-aryl-carboxamide and the presence of a sulfur atom, which could serve as a site for a carbon chain linker. These features align with the known mode of interaction of BPTES with glutaminase. Furthermore, although the biological assay results were in the low micromolar range, several noteworthy observations were made, particularly regarding the comparison of the potential effects of p-OMe- (25.1) and m-OMe-substituted (25.2) compounds, where the inhibition index was nearly halved.

FIGURE 7
Structure of compound 25 and derivatives and their IC₅₀ in glutaminase inhibition assays.

In addition to these preliminary findings and considering the mode of interaction of BPTES at the dimer-dimer interface of glutaminase, we proposed novel derivatives for further investigation. Our study was divided into two phases: in the first phase, we explored the synthesis of a BPTES derivative with dithiopropyl as a linker, the effects of varying linker lengths (region A of BPTES), the significance of substituents in region B, and finally the potential incorporation of a 1,2,3-triazole moiety (Table I). In the second phase, we selected the 3-thio-1,2,4-triazole scaffold present in analog 10b to synthesize additional analogues, aiming to maximize the inhibitory potency, further modifying substituents at regions B and explore region C (Table II).

TABLE I
Evaluation of linker length (Region A) and new scaffolds (Region B) for GLS inhibition

Compound	Structure a	IC50 (µM)
CB-839		0.3
9		> 200
10a		37
10b		13
11		130
13		73
14		> 200
15		1.2
19a		> 400
19b		34
21a		> 400
21b		> 400
24		32

• ^a
  Respective region colors are as follows: Region A, Region B and Region C.

TABLE II
Exploration of regions B and C on new GLS inhibitors and their biological evaluation

Compound	Structure a	IC50 (µM)
10c		> 200
10d		> 200
10e		>200
10f		43
10g		39
10h		32
10i		16
10j		15
10k		9

• ^a
  Respective region colors are as follows: Region A, Region B and Region C.

Through structural analysis, compound 15, presenting the greatest structural resemblance to BPTES 2 with 5 atoms in the linker, exhibited enhanced inhibition. The two sulfide groups in the linker attached to the heterocyclic moiety could account for the improved activity probably due to non-covalent sulfur interactions. On the other hand, when the linker was shortened to 2 atoms in compound 24, a loss of activity was observed. Comparing compounds 9 and 13, which feature a thioethane linker, with compounds 10a-b, which possess a thiopropane linker, it is evident that linkers with two carbon atoms cannot allow for an adequate fit into the allosteric site, resulting in higher IC₅₀ values. In the same way, the thiomethane linker present in compounds 21a-b led to diminished activity in comparison to compounds 19a-b, possessing dithiopropane as linker. Furthermore, 3,3’-oxybis(propane-1-thiol) as linker, containing nine atoms, resulted in decreased glutaminase inhibition for compound 11 when compared to its analogue 10b containing a dithiopropane linker.

Conversely, replacing the 1,3,4-thiadiazole scaffold in compound 24 for a 1,2,4-triazole moiety in compounds 21a-b, along with the exclusion of an amide group, which may be crucial for accommodating the molecule in the allosteric site, resulted in lower inhibition. Moreover, the replacement of 1,2,4-triazole in 9 for 1,2,3-triazole moiety in 19b improved the inhibition, which could be correlated to a lesser steric demand and the presence of acyl benzyl amide. Additionally, removing the cycloalkyl substituent in compound 19b leading to compound 19a significantly decreased the IC₅₀, possibly due to the poor aqueous solubility of compound 19a, leading to precipitation during biological assays. Indeed, 3-thio-1,2,4-triazole analogs 10a-b, showed a good inhibition value and were tolerated when compared to 15. Removal of all benzylamide groups of compound 15 resulted in total loss of activity of compound 14, in agreement with previous literature (Duvall et al., 2020).

Although compound 15 had better inhibition throughout the first series, it presented solubility issues during biologic assays. Moreover, a thorough investigation of BPTES structure through the synthesis of close analogues had already been previously reported in the literature (Robinson et al., 2007; Shukla et al., 2012). On the other hand, there are no reports of GLS inhibitors containing the 1,2,4-triazole scaffold, which warrants continued structural exploration in regions B and C to evaluate the influence that aromatic substituents might have. Also, analog 10b presents 5 atoms in the linker, keeps sulfur atoms close to the triazole ring, and no solubility issues were observed during the biological assays. Thus, a second series of compounds was synthesized based on the structure of compound 10b for further studies. The results are presented in Table II.

Given the narrowness of the allosteric site of glutaminase, substituents with increased steric bulk may hinder the accommodation of the inhibitor. While substituents with moderate electronic density, such as phenyl in region B, were well tolerated and exhibited a favorable activity index, an attenuation of this value was observed with larger substituents. For instance, the introduction of a benzyl group (in compound 10h) resulted in a decreased activity, which was exacerbated with the incorporation of a larger group such as naphthyl (in compound 10e), leading to almost complete loss of activity. Similarly to what was observed for compound 14, when phenyl groups from region C were completely removed (compound 10c), total loss of activity was observed.

Furthermore, the inclusion of 2-pyridyl in region C was well tolerated, particularly when OMe groups were attached to phenyl groups in region B, resulting in comparable inhibition values regardless of the position of the substituents (compounds 10f and 10g). However, upon complete removal of the OMe groups (compound 10d), a decrease in solubility was observed, adversely affecting biological assays. Conversely, 3-OMe groups attached to phenyl rings in regions B and C (compound 10i) or only in the region C (compound 10j) were well tolerated. Nevertheless, when 3-OMe were completely removed, an enhancement in inhibition was noted (compound 10k).

Molecular docking of compounds

As described by DeLaBarre and coworkers (2011), BPTES showed relevant interactions at the dimer interface of glutaminase, resulting in a conformation change that inhibits glutaminase’s activity. Thus, following our biological evaluation, we investigated the possible binding modes of some of the synthesized compounds with the glutaminase enzyme through molecular docking. By using the crystallographic pose of BPTES as a reference, the docking poses of these compounds were superposed, for which the binding modes are depicted in Figure 8 and the corresponding interactions with glutaminase in Figure 9.

FIGURE 8
(A) BPTES in monomer-monomer interface and poses obtained through molecular docking of glutaminase for compounds (B) 10a, (C) 10h, (D) 10i and (E) 10k.

FIGURE 9
Two-dimensional diagrams for the poses of ligand-glutaminase interactions generated from 3-D models using PoseView (Stierand, Rarey, 2010). A) BPTES, B) 10a, C) 10h, D) 10i and E) 10k. Dashed black lines correspond to hydrogen bonds, green dashed lines to π-π or cation-π interactions and the solid green to hydrophobic interactions.

Compound 10a displayed a similar interaction mode to BPTES at the allosteric site of glutaminase (Figure 9, B and A respectively), which seems coherent since the smaller cyclopropyl groups would impose less conformational hindrance when compared to the phenyl groups in 10h, 10i or 10k, for example. As far as the interactions are concerned, BPTES (Figure 9A) displays six intermolecular hydrogen bonds (~ 5 Kcal/ mol each), two π-π stackings (~ 3 kcal.mol^-1 each) (Sinnokrot, Valeev, Sherrill, 2002) and two Van der walls interactions (~ 1 kcal.mol^-1 each) (Nolan, Singh, McCurdy, 2000). Therefore, despite the similar binding mode, 10a (Figure 9A) would perform less interactions (i.e., two H-bonds, five π-π stackings and two Van der walls interactions), which also seems coherent with the observed IC₅₀ values (0.15 vs. 37 µM, respectively).

All the remaining studied compounds, 10h, 10i and 10k only partially interact with the BPTES ligand site, also accessing a contiguous hydrophobic pocket at the glutaminase enzyme (Figure 8, C to E respectively). For compound 10h (IC₅₀ = 32 µM), only half of the structure is predicted to interact with glutaminase tetramer, performing three π-π stackings and four Van der walls interactions (Figure 9C) while for compound 10i (IC₅₀ = 16 µM), two H-bonds, three π-π stackings and five Van der walls interactions were observed (Figure 9D). It is worth nothing that the most potent of the synthesized compounds (10k, IC₅₀ = 9 µM) is also the one displaying the highest number of predicted interactions, namely two H-bonds, three π-π stackings and eight Van der walls interactions (Figure 8E), but still, with less pronounced stronger interactions than in BPTES (Figure 9A).

Compound 10a showed a very similar interaction mode to BPTES at the allosteric site of glutaminase. On the other hand, compound 10k, which exhibited the lowest IC₅₀ among the compounds evaluated, only partially overlaps the BPTES structure, and is predicted to explore adjacent pockets in the glutaminase enzyme. Similar results were observed for 10i and 10h, where only a fraction of the BPTES ligand site is explored for interactions with the target. Those results obtained for compounds 10k, 10h and 10i suggested a new allosteric site.

CONCLUSION

Our study aims at evaluating the structure of BPTES and propose structure-activity relationship (SAR) investigations using analogues containing 1,2,3-triazole and 3-thio-1,2,4-triazole cores. Three distinct regions have been identified: Region A (linker), Region B (core), and Region C (side chain). In Region A, the optimal linker length, as described in the literature, consists of five atoms with a central sulfur atom in the chain. Supporting previous findings, we found that a five-atom linker chain is the most effective; additionally, the presence of two sulfur atoms in proximity to the heterocyclic core was well tolerated. The choice of linker length significantly impacted inhibitory activity, as demonstrated by the comparison of compounds with varying linker lengths: compound 9 (four-atom linker, IC₅₀ > 200 µM) to compound 10a (five-atom linker, IC₅₀ = 37 µM) and compound 11 (nine-atom linker, IC₅₀ = 130 µM) to compound 10b (five-atom linker, IC₅₀ = 13 µM). In Region B, we observed a good tolerance for 1,2,3-triazole and 1,2,4-triazole cores as replacements for the 1,2,4-thiadiazole core, although the inhibition values remained in the low micromolar range. Furthermore, inhibitory activity was notably sensitive to substituents at the N4 position; increased steric hindrance correlated with decreased activity, as shown by the following trend: phenyl group 10k (IC₅₀ = 9 µM) > benzyl group 10h (IC₅₀ = 32 µM) > naphthyl group 10e (IC₅₀ = 163 µM), although the presence of para group in 10g (IC₅₀ = 39 µM) or meta group in 10f (IC₅₀ = 43 µM) were well tolerated. In Region C, the presence of a phenyl group proved necessary; complete removal resulted in a total loss of activity, as evidenced by compound 10c (IC₅₀ = >200 µM) and compound 14 (IC₅₀ > 200 µM) when compared to compounds 10k (IC₅₀ = 9 µM) and 15 (IC₅₀ = 1.2 µM), respectively. Even though some compounds exhibited inhibition values in the low micromolar range, molecular docking analyses revealed the potential for some compounds to target a new allosteric site. This suggests the existence of alternative allosteric inhibition mechanisms beyond those delineated for BPTES and CB-839, thereby opening new opportunities for allosteric modulation of glutaminase. Thus, our findings expand the application of 1,2,3-triazole and introduce the 1,2,4-triazole core as a bioisostere of 1,3,4-thiadiazole with significant activity against the glutaminase enzyme. Given the enzyme’s pivotal role in breast cancer treatment, these findings hold substantial promise. Additionally, the optimized structure, exhibiting the most potent activity against isolated glutaminase with an IC₅₀ of approximately 9 µM, is depicted in Figure 10.

FIGURE 10
Optimized structure from the study.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial support from the São Paulo Research Foundation - FAPESP (JCP, 2014/26378-2 and 2021/06661-5; MCFCBD, 2015/18572-6; RKEC, 2016/09077-4; BNS, 2021/13736-1) and the Brazilian National Council for Scientific and Technological Development - CNPq (160907/2021-7, LCF). We also thank LNBio for access to core facilities and financial support.

DATA AVAILABILITY STATEMENT

All data is available within the article or its supplementary materials.

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Edited by

• Associate Editor:
  João Paulo Fernandes

Publication Dates

• Publication in this collection
  31 July 2026
• Date of issue
  2026

History

• Received
  26 Nov 2024
• Accepted
  07 Mar 2025