Design and Synthesis of Bis-Triazole-Linked Benzenesulfonamides as Selective Carbonic Anhydrase IX/XII Inhibitors with Chemosensitizing Activity

Abstract

Carbonic anhydrase hCA IX and hCA XII are cancer-associated enzymes involved in tumor pH regulation, hypoxia-driven survival, and therapeutic resistance. Here, we report the rational design, synthesis, and biological evaluation of bis-triazole-linked benzenesulfonamides as novel carbonic anhydrase inhibitors. Several compounds exhibited potent and selective inhibition of hCA IX/XII, with compounds 2g and 4g showing nanomolar activity (hCA IX K _I = 25.1 and 28.1 nM; hCA XII K _I = 35.2 and 11.6 nM, respectively) and reduced activity against off-target isoforms hCA I/II, indicating favorable selectivity profiles. Lead compounds 2g and 4g significantly enhanced the antiproliferative effects of azacitidine and sorafenib in breast and pancreatic cancer cells under both normoxic and hypoxic conditions. Molecular modeling and simulation studies further supported productive zinc coordination and stable active-site interactions. Overall, these findings highlight bis-triazole benzenesulfonamides as promising scaffolds for targeting hCA IX/XII-expressing tumors and improving chemosensitivity to conventional chemotherapeutics to overcome cancer resistance.

1. Introduction

Carbonic anhydrases (CAs) are a family of zinc-dependent metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons, a reaction central to pH regulation and acid–base homeostasis in living systems. In humans, 15 α-class hCA isoforms have been identified, of which 12 are catalytically active, and differ markedly in tissue distribution and subcellular localization. Cytosolic, mitochondrial, membrane-associated, and secreted hCAs collectively support essential physiological processes, including respiration, electrolyte transport, biosynthesis, and the regulation of intra- and extracellular pH. While several hCA isoforms (e.g., hCA I, II, and III) are ubiquitously expressed and indispensable for normal physiology, others display highly restricted expression patterns and become selectively upregulated under pathological conditions, most notably hCA IX and hCA XII in solid tumors, thereby offering attractive opportunities for targeted therapeutic intervention. −

Tumor hypoxia is a hallmark of many solid malignancies, including breast, lung, colorectal, and pancreatic cancers, arising from rapid proliferation and insufficient vascularization. Hypoxic stress activates hypoxia-inducible factor-1α (HIF-1α), which drives metabolic reprogramming toward anaerobic glycolysis and increased production of lactic acid and CO₂. As part of this HIF-1α–driven adaptive response, cancer cells exploit specialized pH-regulating mechanisms mediated primarily by the hypoxia-inducible hCA IX and hCA XII, which maintain intracellular alkalinity while driving extracellular acidification, thereby fostering invasion, metastasis, and resistance to chemotherapy. ,

The membrane-associated hCA IX and hCA XII are strongly induced by HIF-1α and facilitate proton and bicarbonate flux across the plasma membrane, thereby maintaining tumor pH homeostasis. Importantly, hCA IX and hCA XII display limited expression in normal tissues but are highly upregulated in hypoxic tumors, positioning them as cancer-associated enzymes and validated therapeutic targets. Selective inhibition of hCA IX and hCA XII has therefore emerged as a promising strategy for disrupting hypoxic tumor metabolism while minimizing systemic off-target effects associated with inhibition of the physiologically dominant cytosolic isoforms hCA I and hCA II. ,

Beyond their metabolic functions, hCA IX and hCA XII have been directly implicated in cancer resistance to chemotherapy and radiotherapy. Under hypoxic conditions, CA-mediated extracellular acidification limits drug uptake, reduces the efficacy of weakly basic chemotherapeutics, and activates pro-survival signaling pathways, thereby promoting therapeutic failure. In particular, extracellular acidification impairs the cellular uptake of weakly basic drugs through an ion trapping mechanism, whereby protonation in the acidic tumor milieu reduces membrane permeability and leads to decreased intracellular drug accumulation. , This phenomenon is particularly relevant for drugs such as azacitidine and sorafenib, both of which exhibit weakly basic characteristics and are widely used in cancer therapy; however, their efficacy can be compromised in the acidic tumor microenvironment. Inhibition of hCA IX and hCA XII has been shown to reverse these effects, sensitizing tumor cells to conventional anticancer therapies and promoting apoptosis under hypoxia. Consequently, targeting hCA IX and hCA XII provides a dual therapeutic advantage, by simultaneously disrupting hypoxia-driven tumor metabolism and enhancing responsiveness to standard therapies, thereby addressing both intrinsic and acquired mechanisms of drug resistance

Multiple preclinical studies support this concept. The ureido-substituted sulfonamide SLC-0111 (Figure A) sensitized breast and melanoma cancer models to doxorubicin and dacarbazine, respectively, resulting in reduced tumor cell survival and increased apoptosis. In head and neck squamous cell carcinoma, hCA IX and hCA XII inhibition by SLC-0111 potentiated the anticancer activity of cisplatin, suppressing tumor growth, migration, and invasion. Similarly, cotreatment of SLC 0111 with Temozolomide reduced cell growth and induced cell cycle arrest in glioblastoma models (Figure A). Collectively, these findings establish hCA IX and hCA XII inhibition as a dual-pronged therapeutic strategy, combining direct inhibition of tumor growth and metabolic adaptation with chemosensitization that amplifies the efficacy of conventional anticancer therapies.

1.

Conceptual framework for the design of bis-triazole–linked benzenesulfonamides as hCA IX and hCA XII inhibitors, showing (A) the reported chemosensitization effect of SLC-0111 in combination with anticancer agents, (B) the chemosensitization strategy explored in this study, and (C) the molecular design rationale underlying the bis-triazole benzenesulfonamide scaffold.

From a medicinal chemistry perspective, achieving high CA isoform selectivity remains a principal challenge in the development of CA inhibitors, owing to the strong conservation of the catalytic zinc-binding site across CA isoforms. The tail approach, in which a zinc-binding sulfonamide is functionalized with flexible or bulky substituents, has emerged as a highly effective design strategy. This approach capitalizes on structural variability within the middle and rim regions of the CA active site, enabling preferential targeting of tumor-associated isoforms. A prominent example is SLC-0111, developed using the tail approach, which is currently undergoing Phase I/II clinical trials for the treatment of solid tumors and serves as a benchmark compound for next-generation hCA IX inhibitors. Building on this paradigm, extensive structure–activity relationship (SAR) studies have investigated systematic modifications of both the hydrophobic tail and the linker region of SLC-0111. These efforts include replacing the ureido spacer with enaminone, amide, or triazole-based linker, , as well as introducing diverse tail motifs such as triazoles, thiazoles, benzothiazoles, and quinoline to further enhance potency and selectivity toward tumor-associated isoforms hCA IX and hCA XII.

Building on these advances, we hypothesized that incorporating diverse triazolopyridine tails in combination with a thiomethyl 1,2,3-triazole linker within a regioisomeric benzenesulfonamide framework could enhance the potency and selectivity of hCA IX and hCA XII (Figure C). This design was further envisioned to enable effective chemosensitization of conventional chemotherapeutic agents. Herein, we report the rational design and synthesis of a focused series of bis-triazole–linked hCA IX and hCA XII inhibitors. Their enzymatic activities were comprehensively evaluated against hCA I, II, IX, and XII. Furthermore, the lead compounds were assessed as chemosensitizing agents under both normoxic and hypoxic conditions in CA-expressing breast and pancreatic cancer cell models, in combination with azacitidine and sorafenib (Figure B).

2. Results and Discussion

2.1. Design Rationale

Achieving CA isoform selectivity remains a key challenge for sulfonamide-based CA inhibitors, as off-target inhibition of physiological isoforms can result in dose-limiting side effects. To address this, we adopted a hybrid pharmacophore strategy that preserves the classical zinc-binding sulfonamide moiety while systematically modifying the linker and tail regions to exploit structural features unique to the hCA IX and hCA XII active-site environment. This approach follows the established tail-based design paradigm, in which diversification distal to the zinc-binding group promotes selective engagement of the enzyme’s variable outer regions.

Building on the tail approach, the classical 4-fluorophenyl tail of SLC-0111 can be replaced with a variety of aromatic and heteroaromatic systems to enhance interactions within the middle and rim regions of hCA isoforms, which are key determinants of isoform selectivity. Among the reported tail motifs, aryl-substituted triazoles represent a well-established class in carbonic anhydrase inhibitor design, exhibiting potent inhibition and improved selectivity toward tumor-associated isoforms. ,, In this context, a triazolopyridine scaffold was selected as a heteroaryl analogue of diaryl triazoles to further enhance interactions within the middle and rim regions of hCA IX and XII. The planar and heteroatom-rich nature of the triazolopyridine system is expected to promote π–π stacking and dipolar interactions, while the pyridine nitrogen can act as a hydrogen-bond acceptor, thereby improving binding orientation relative to simple phenyl tails or diaryl 1,2,3-triazole analogues.

In addition, the rigid triazolopyridine framework provides multiple substitution vectors, enabling systematic modulation of steric and electronic properties for the tail. To systematically evaluate these effects, a series of R₁ substituents with increasing steric demand and electronic variations, namely, methyl, phenyl, 2-chlorophenyl, and 3,5-bis­(trifluoromethyl)­phenyl, was introduced (Figure C). This progression was designed to interrogate hydrophobic subpockets at the periphery of the hCA IX and hCA XII active sites and to assess how steric bulkiness and electronic effects influence binding orientation, potency, and selectivity for hCA isoforms.

Furthermore, the classical ureido linker exemplified by SLC-0111 was replaced with a 1,4-disubstituted 1,2,3-triazole linker to modulate linker flexibility and explore alternative binding geometries. Triazoles are well-established amide/urea bioisosteres that provide increased rigidity, improved metabolic stability, and distinct dipolar interaction profiles relative to ureido linkers. , In the context of carbonic anhydrase inhibitors, such rigidity can facilitate control over the spatial orientation of the tail relative to the zinc-binding group, which is important for interactions within the outer active-site regions of hCA IX/XII. In this work, a 1,2,3-triazole linker, connected through a single methylene spacer (Figure C), was employed to balance flexibility with conformational control, thereby enabling systematic investigation of linker-driven effects on hCA IX and XII inhibition.

In parallel, we also explored different orientations of the sulfonamide group (ortho, meta, para) relative to the appended tail, as well as the effect of para-methyl substitution on the meta-benzenesulfonamides (Figure C). Although sulfonamide coordination to the catalytic Zn²⁺ ion is conserved across CA isoforms, changes in regiochemistry can markedly influence the overall binding topology of the inhibitor within the active site. This regioisomeric strategy was employed to exploit subtle structural differences between the active-site boundaries of hCA IX and hCA XII and those of the cytosolic isoforms (hCA I and hCA II). Such regioisomeric variations can alter the projection of the linker and tail toward the enzyme surface, thereby modulating both CA binding affinity and isoform selectivity. By integrating N-substituted triazolopyridine-based tail diversification, a thiomethyl 1,2,3-triazole linker, and systematic sulfonamide regioisomerization, we generated a focused library of bis-triazole–linked benzenesulfonamides to interrogate steric, hydrophobic, and electronic determinants of CA inhibition. This multifaceted design strategy was pursued to discover novel and potent hCA IX and hCA XII inhibitors incorporating bis-triazole-linked benzenesulfonamide scaffold.

2.2. Synthesis of Bis-triazole–Linked Benzenesulfonamides

The synthetic route was developed to provide efficient and flexible access to a series of bis-triazole–linked benzenesulfonamides. A convergent, modular strategy was employed in which azido-benzenesulfonamides and propargylated triazolopyridine derivatives were prepared independently and coupled via copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC). This approach enabled straightforward assembly of the target compounds while allowing independent variation of sulfonamide regiochemistry and triazolopyridine R₁ substitution, thereby facilitating systematic SAR analysis across the compound series.

The azido-benzenesulfonamide building blocks (1–4) were synthesized from commercially available sulfanilamide derivatives via a two-step diazotization–azidation sequence (Scheme ). Briefly, the corresponding sulfanilamides were treated with NaNO₂ in concentrated HCl at 0 °C to generate the diazonium intermediates, which were immediately quenched with sodium azide to afford the desired azido-benzenesulfonamides. This approach enabled the preparation of four regioisomeric azido-benzenesulfonamides (1–4), differing in the position of the sulfonamide group relative to the azide functionality (ortho, meta, meta with para methyl substitution, and para).

1. Synthetic Route to Bis-Triazole–Linked Benzenesulfonamides .

^a Reagents and conditions: (I) R₁NCS, EtOH, reflux, 3–4 h; then NaOH (2 N), 100 °C, 2–3 h; acidification with HCl (pH 5–7). (II) Propargyl bromide, K₂CO₃, DMF or acetone, 0 °C → rt → 70 °C, 4 h. (III) NaNO₂, HCl, THF/H₂O, 0 °C; then NaN₃, 0 °C → rt, overnight. (IV) CuSO₄, sodium ascorbate, DMF/H₂O, 70 °C, 6 h.

In parallel, the propargylated triazolopyridine derivatives were prepared via a concise two-step synthetic sequence (Scheme ). Isonicotinic acid hydrazide was reacted under reflux in ethanol with a series of substituted isothiocyanates, namely methyl isothiocyanate, phenyl isothiocyanate, 2-chlorophenyl isothiocyanate, and 3,5-bis­(trifluoromethyl)­phenyl isothiocyanate, to afford the corresponding N-R₁-2-(pyridin-4-yl)­hydrazine-1-carbothioamide (see Supporting Information, pp ), thereby introducing the desired R₁ substituents. Subsequent treatment of these intermediates with aqueous sodium hydroxide under reflux, followed by neutralization with dilute hydrochloric acid, yielded the triazolopyridine thiols (a–d). Alkylation of thiols a–d with propargyl bromide in the presence of potassium carbonate under a nitrogen atmosphere furnished the propargylated triazolopyridine derivatives (e–h). Installation of the terminal propargyl functionality provided the requisite handle for subsequent CuAAC assembly.

The final target compounds were assembled via CuAAC reaction between the azido-benzenesulfonamides − and the propargylated triazolopyridine derivatives (e-h). The reactions were carried out with CuSO₄ and sodium ascorbate to generate the active Cu­(I) catalyst in situ. Heating the reaction mixtures to 70 °C ensured efficient cycloaddition, affording the desired bis-triazole–linked benzenesulfonamides (1e–h, 2e–h, 3e–h, and 4e–h) in good to excellent yields (Scheme ).

2.3. Carbonic Anhydrase Assays

Given the established role of hCA IX and hCA XII in tumor-associated pH regulation and their frequent overexpression in solid tumors, the synthesized compounds were evaluated for inhibitory activity against the four isoforms hCA (I, II, IX, XII). Evaluation of the cytosolic isoforms hCA I and hCA II enabled assessment of off-target inhibition and definition of isoform selectivity, a key determinant of the safety and therapeutic index of sulfonamide-based CA inhibitors

2.3.1. Inhibition of Cytosolic Isoforms (hCA I and hCA II)

All synthesized compounds were evaluated for inhibitory activity against the physiological isoforms hCA I and hCA II, using acetazolamide (AAZ, K _I = 250 nM against hCA I and 12.0 nM against hCA II) as a reference inhibitor. Structure–activity relationship (SAR) analysis focused on the effects of sulfonamide regiochemistry, triazolopyridine tail substitution (R₁), and R₂ modification on off-target inhibition.

2.3.1.1. Impact of Sulfonamide Regioisomerization

The positional variation of the sulfonamide group exerted a pronounced impact on the inhibition of hCA I and hCA II across all compound series. In the methyl-substituted series (R₁ = CH₃, 1–4e), the para isomer 4e displayed the highest activity against both hCA I and hCA II (K _I = 815 nM and 32.8 nM, respectively), whereas the corresponding ortho analogue 1e showed negligible activity (K _I > 100,000 nM for hCA I, 2468 nM for hCA II) (Table ). This trend generally holds across the other series (f-h), with the para-isomers (4g–h) displaying the strongest inhibition, followed by the meta-analogues, while the ortho isomers were uniformly the weakest. Overall, inhibition of the physiological isoforms followed the trend para > meta ≫ ortho, indicating that ortho sulfonamide placement effectively suppresses off-target hCA I and hCA II inhibition. While this substitution pattern is advantageous for minimizing activity against physiological isoforms, it provides a useful contrast point for assessing potency trends toward the tumor-associated isoforms discussed below.

1. Inhibition Data of Physiological hCA Isoforms I, II, and Cancer-Associated hCA IX and hCA XII Using AAZ as a Reference Drug.

	K I (nM)
Comp.	R1	R2	SO2NH2	hCA I	hCA II	hCA IX	hCA XII
1e	CH3	H	o	>100,000	2468	843	628
2e	CH3	H	m	2327	214	87.6	65.3
3e	CH3	CH3	m	5861	426	104	89.7
4e	CH3	H	p	815	32.8	21.3	44.6
1f	Ph	H	o	>100,000	3516	802	714
2f	Ph	H	m	1749	358	69.3	42.8
3f	Ph	CH3	m	6583	517	57.0	59.4
4f	Ph	H	p	944	51.8	49.4	14.3
1g	2-ClPh	H	o	>100,000	2149	723	798
2g	2-ClPh	H	m	1652	312	25.1	35.2
3g	2-ClPh	CH3	m	5990	558	96.5	21.8
4g	2-ClPh	H	p	744	47.9	28.1	11.6
1h	3,5(CF3)2Ph	H	o	>100,000	5068	1258	911
2h	3,5(CF3)2Ph	H	m	3056	183	74.6	63.2
3h	3,5(CF3)2Ph	CH3	m	5361	504	111	46.8
4h	3,5(CF3)2Ph	H	p	870	26.8	39.5	55.8
AAZ	-	-	-	250	12.0	25.0	5.7

^aMean from 3 different assays, by a stopped-flow technique (errors were in the range of ± 5–10% of the reported values)

2.3.1.2. Impact of Triazole R₁-Group Substitution

Analysis of R₁ substitution within each sulfonamide regiochemical series revealed further SAR insights for inhibition of hCA I and hCA II. Among the para-substituted analogues (4e–h), the 3,5-bis­(trifluoromethyl)­phenyl derivative 4h exhibited the strongest inhibition of hCA II (K _I = 26.8 nM), whereas the methyl-substituted analogue 4e showed the highest activity toward hCA I (K _I = 815 nM). Within the meta-substituted series (2e–h), the 2-chlorophenyl analogue 2g was the most active inhibitor of hCA I (K _I = 1652 nM), while the 3,5-bis­(trifluoromethyl)­phenyl derivative 2h displayed the strongest hCA II inhibition (K _I = 183 nM). In contrast, the ortho-substituted analogue 1h bearing a 3,5-bis­(trifluoromethyl)­phenyl tail exhibited minimal activity against both hCA I and hCA II (K _I > 100,000 nM and 5068 nM, respectively; Table ). These results indicate that, within the para- and meta-substituted series, off-target inhibition of hCA I and hCA II varies with R₁ identity and does not follow a simple trend based on tail bulk or electronic character. In contrast, the ortho-substituted analogues consistently exhibit low hCA I and hCA II inhibition that is largely insensitive to changes in the R₁ substituent.

2.3.1.3. Effect of para-Methyl Substitution (R₂)

Comparison of meta-benzenesulfonamide-substituted compounds (2e–h) with their corresponding para-methyl analogs (3e–h) revealed a consistent reduction in inhibitory potency against both hCA I and hCA II upon introduction of the additional methyl group. For example, compound 3e (K _I = 5861 nM for hCA I, 426 nM for hCA II) was significantly less potent than its unmethylated counterpart 2e (K _I = 2327 nM for hCA I, 214 nM for hCA II). This trend was maintained across all R₁ substituents, suggesting that para-methyl substitution introduces steric or conformational constraints that disfavor productive binding within the active sites of hCA I and hCA II, thereby reducing off-target inhibition of physiological CA isoforms.

2.3.2. Inhibition of Tumor-Associated Isoforms (hCA IX and hCA XII)

Most of the synthesized compounds exhibited strong inhibitory activity against the tumor-associated isoforms hCA IX and hCA XII, the primary therapeutic targets in hypoxic solid tumors. Accordingly, a detailed SAR analysis was conducted to identify the structural determinants governing potency and isoform selectivity toward these clinically relevant enzymes.

2.3.2.1. Impact of Sulfonamide Regioisomerization

Sulfonamide regiochemistry exerted a decisive influence on the inhibition of both hCA IX and hCA XII. Across all substitution series, para-substituted sulfonamide derivatives (4e–h) consistently displayed the highest potency, followed by meta-analogues (2e–h), whereas ortho-substituted compounds (1e–h) showed markedly reduced activity (Figure ). For example, within the methyl-substituted triazolopyridine series (e), the para isomer 4e potently inhibited hCA IX and hCA XII (K _I = 21.3 nM, 44.6 nM, respectively), in sharp contrast to the corresponding ortho analogue 1e, which showed substantially weaker activity (K _I = 843 nM, 628 nM, respectively) (Table ). This regioisomeric preference was consistently observed across all R₁ substitution series (e–h), following the potency order para (4e–h) > meta (2e–h) ≫ ortho (1e–h).

2.

Structure–activity relationship summary for bis-triazole–linked benzenesulfonamides inhibitors of hCA IX and hCA XII.

2.3.2.2. Impact of Triazole R₁-Group Substitution

Variation of the triazolopyridine R₁ substituent further modulated inhibitory potency toward hCA IX and hCA XII within each sulfonamide regiochemical series (Figure ). Comparison of the most potent para- and meta-substituted analogues reveals a clear and instructive SAR trend driven by the nature of the R₁ substituent. Increasing tail bulk from methyl (4e) to phenyl (4f) reduced hCA IX potency (K _I = 21.3 → 49.4 nM) while concomitantly enhancing inhibition of hCA XII (K _I = 44.6 → 14.3 nM). Notably, the introduction of an ortho-chlorinated phenyl group proved particularly advantageous. The 2-chlorophenyl derivative 4g restored hCA IX potency (K _I = 28.1 nM) while further improving hCA XII inhibition (K _I = 11.6 nM). In contrast, further increasing lipophilicity and steric bulk by incorporating a 3,5-bis­(trifluoromethyl)­phenyl group (4h) reduced activity toward both isoforms (K _I = 39.5 and 55.8 for hCA IX and XII, respectively), suggesting that excessive bulk compromises productive binding within the CA active-site rim.

A particularly striking trend emerged in the meta-sulfonamide series. Here, the 2-chlorophenyl analogue 2g maintained potent inhibition of both hCA IX (K _I = 25.1 nM) and hCA XII (K _I = 35.2 nM), approaching the potency of the corresponding para-analogues despite the generally reduced activity of meta-substituted analogs. This preservation of high potency is notable, as meta-sulfonamide derivatives exhibit substantially weaker inhibition of physiological hCA I and hCA II compared to para-analogues. Collectively, these results identify the 2-chlorophenyl triazolopyridine tail as a uniquely favorable substituent that not only enhances inhibition of tumor-associated hCA IX and hCA XII but also retains this potency within a meta-sulfonamide framework that minimizes off-target activity. This unexpected convergence of potency and selectivity highlights the 2-chlorophenyl motif as a key driver of the optimized isoform selectivity profile observed in this series.

2.3.2.3. Effect of para-Methyl Substitution (R ₂ )

Introduction of a para-methyl substituent (R₂) exerted a differential and isoform-dependent effect on inhibition of the tumor-associated CA isoforms (Figure ). For hCA IX, para-methylation generally reduced potency, as illustrated by compound 3e (K _I = 104 nM), which was less active than its unmethylated analogue 2e (K _I = 87.6 nM). In contrast, para-methylated 3g and 3h exhibited improved hCA XII potency (K _I = 21.8 and 46.8 nM, respectively) relative to their corresponding nonmethylated analogues 2g and 2h (K _I = 35.2 and 63.2 nM, respectively) (Table ). Taken together with the previously discussed data showing that para-methyl substitution markedly reduces activity against hCA I and hCA II, these results indicate that para-methyl substitution can be strategically employed to fine-tune CA isoform selectivity, enhancing potency toward hCA XII while minimizing inhibition of physiological isoforms.

2.3.3. Selectivity Profile

To assess the therapeutic potential of the most potent hCA IX and hCA XII inhibitors among the synthesized series, selectivity indices (SI) were calculated as the ratio of inhibition constants for the physiological isoforms to those for the tumor-associated isoform $\mathrm{SI}=\frac{K_{\mathrm{I}}\mathrm{physiological}\mathrm{isoform}}{K_{\mathrm{I}}\mathrm{tumor}-\mathrm{associated\; isoform}}$ (Table ). Higher SI values indicate a preferential targeting of hCA IX and hCA XII while limiting off-target CA inhibition.

2. Selectivity Indices of Compounds 4e–g and 2g for Tumor-Associated hCA IX and hCA XII over Cytosolic hCA I and hCA II.

	hCA I/IX	hCA II/IX	hCA I/XII	hCA II/XII
4e	38.2	1.5	18.2	0.7
4f	19.1	1.0	66.0	3.6
2g	65.8	12.4	46.9	8.8
4g	26.4	1.7	64.1	4.1
4h	22.0	0.6	15.5	0.4
AAZ	10	0.48	43.8	2.1

In line with the SAR trends, several compounds exhibited improved selectivity for the tumor-associated isoform hCA IX over the physiological isoform hCA I when benchmarked against the reference carbonic anhydrase inhibitor AAZ. Among these, compound 2g showed the highest hCA I/IX selectivity (SI ∼ 66), representing an approximately 6.5-fold enhancement relative to AAZ (SI ∼ 10). Para-sulfonamide analogues 4e and 4g also exceeded the reference, displaying hCA I/IX selectivity indices of approximately 38 and 26, corresponding to ∼3.8-fold and ∼2.6-fold improvements, respectively.

Selectivity against hCA II proved more difficult to achieve across the series; however, compound 2g again demonstrated a pronounced advantage. Specifically, 2g retained an hCA II/IX selectivity index of approximately 12, in sharp contrast to the subunity selectivity of AAZ (SI = 0.48), amounting to a ∼25-fold relative improvement. A comparable trend was observed for hCA II/XII discrimination, where 2g exhibited an SI of ∼9, exceeding that of AAZ (SI ≈ 2.1) by approximately 4-fold. In comparison, para-substituted compounds such as 4e and 4g showed weaker discrimination against hCA II, consistent with their increased off-target inhibition of cytosolic isoforms.

Collectively, these findings demonstrate that meta-sulfonamide regiochemistry combined with a 2-chlorophenyl triazolopyridine tail confers a substantial and consistent selectivity advantage over the clinically used CAI AAZ. While multiple analogues improve hCA IX selectivity over hCA I, compound 2g uniquely achieves concurrent gains across both hCA IX and hCA XII relative to hCA I and hCA II, firmly establishing it as the most advanced and well-balanced lead in this series.

2.4. Validation of hCA IX Expression under Hypoxia

MDA-MB-231 breast cancer and PANC-1 pancreatic cancer cells were selected for cellular studies based on literature reports describing hypoxia-induced expression of tumor-associated carbonic anhydrases, particularly hCA IX and hCA XII. − To experimentally confirm the relevance of hCA IX as a molecular target within our study, hCA IX protein expression was evaluated in both cell lines following exposure to normoxic and hypoxic conditions.

As shown in Figure , hypoxic treatment resulted in a statistically significant increase in hCA IX protein levels relative to normoxic controls (p < 0.0001), confirming successful induction of hypoxia-associated signaling in both MDA-MB-231 and PANC-1cells. Importantly, this experiment was intended to validate the hypoxic environment rather than to compare cell lines. Based on this experimental confirmation, together with prior reports of pronounced hCA IX and hCA XII expression, MDA-MB-231 and PANC-1 cells were subsequently employed for cytotoxicity and chemosensitization studies under normoxic and hypoxic conditions.

3.

Quantification of hCA IX protein expression by ELISA in PANC-1 and MDA-MB-231 cultured under normoxic (20% O₂, 5% CO₂) or hypoxic (1% O₂, 5% CO₂, 94% N) conditions for 48 h. The data represent a representative experiment. Statistical significance was determined using Student’s t-test versus the corresponding normoxic control (****p < 0.0001).

2.5. Chemosensitization by Compounds 4g and 2g with Azacitidine and Sorafenib

To investigate the cytotoxicity and chemosensitization potential of the synthesized compounds, compounds 2g and 4g were selected based on their potent inhibitory activity and favorable selectivity profiles toward the tumor-associated isoforms hCA IX and hCA XII. Their chemosensitization potential was evaluated in combination with azacitidine (AZA) or sorafenib (SOR) in MDA-MB-231 and PANC-1 cells under normoxic and hypoxic conditions using the MTT assay (Figure A–D).

4.

Effects of compounds 4g and 2g alone or in combination with azacitidine (AZA) and sorafenib (SOR) on cell viability under normoxic and hypoxic conditions. (A–B) represent MTT assay results for MDA-MB-231 cells treated with compound 4g, while (C–D) show results for PANC-1 cells treated with compound 2g. Cells were treated with 4g or 2g (100 μM), AZA (25 μM), SOR (25 μM), or their combinations ((4g + AZA), (4g + SOR), (2g + AZA), (2g + SOR)) for 48 h under normoxic and hypoxic conditions. Statistical comparisons between normoxia and hypoxia at each treatment condition were performed using a paired t test. Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control.

In MDA-MB-231 cells under normoxia (Figure A), treatment with 4g, AZA, or SOR alone significantly reduced cell viability relative to untreated controls. Among the single agents, AZA exhibited the strongest antiproliferative activity, followed by 4g and SOR. Notably, cotreatment with 4g + AZA produced a further significant reduction in cell viability compared to either monotherapy, consistent with the chemosensitizing effect of 4g. In contrast, the 4g + SOR combination did not result in a statistically significant enhancement of cytotoxicity beyond that observed with the individual treatments.

Under hypoxic conditions (Figure B), a broadly similar response pattern was observed, although overall viability was reduced relative to normoxia. Compounds 4g and SOR exhibited comparable cytotoxic effects under hypoxia, whereas the antiproliferative activity of AZA alone was markedly attenuated, consistent with reduced efficacy of weakly basic agents in an acidified extracellular environment. Notably, cotreatment of AZA + 4g resulted in a significant reduction in cell viability relative to the hypoxic control and produced the lowest viability among all treatment groups. The preservation of AZA cytotoxicity in the presence of 4g is consistent with the proposed inhibition of hypoxia-induced carbonic anhydrases hCA IX and hCA XII, which regulate extracellular acidification and influence drug availability under hypoxic conditions.

To further validate this behavior in a distinct carbonic anhydrase–expressing tumor model and using an alternative lead compound, the chemosensitization potential of compound 2g was evaluated in PANC-1 pancreatic cancer cells (Figure C–D). Under normoxic conditions (Figure C), SOR displayed the weakest cytotoxic activity among the tested agents, consistent with the trend observed in MDA-MB-231 cells. In contrast to MDA-MB-231 cells, however, cotreatment of SOR with 2g resulted in a significant enhancement of cytotoxicity, indicating a cell-line-dependent chemosensitizing effect. Treatment with 2g or AZA alone significantly reduced cell viability, and both combination treatments (2g + AZA and 2g + SOR) produced a further decrease in viability, with the 2g + AZA combination yielding the strongest antiproliferative response.

Under hypoxic conditions (Figure D), all single agents retained significant antiproliferative activity. Notably, both 2g + AZA and 2g + SOR combinations resulted in a further reduction in cell viability relative to the corresponding monotherapies, demonstrating that the chemosensitizing effect of 2g is preserved under hypoxia. In contrast to MDA-MB-231 cells, the 2g + SOR combination remained effective in PANC-1 cells under hypoxic conditions, highlighting tumor-type-specific differences in combination response.

Overall, these results demonstrate that compound 4g preferentially sensitizes MDA-MB-231 cells to AZA, whereas compound 2g enhances the responsiveness of PANC-1 cells to both AZA and SOR under normoxic and hypoxic conditions. These chemosensitization effects are consistent with inhibition of tumor-associated carbonic anhydrases, which play a central role in regulating extracellular acidification within the tumor microenvironment. By disrupting carbonic anhydrase–mediated pH homeostasis, compounds 4g and 2g likely alleviate extracellular acidification that limits the uptake and efficacy of AZA and SOR, thereby enhancing chemosensitivity under both normoxic and hypoxic conditions.

2.6. Morphological Evidence for Chemosensitization

To provide phenotypic support for the chemosensitization observed in viability assays, cellular morphology was examined by inverted light microscopy under normoxic and hypoxic conditions. Under normoxia, untreated MDA-MB-231 and PANC-1 cells exhibited a typical adherent morphology with high cell density. In contrast, hypoxic exposure resulted in reduced cell density, cell shrinkage, and irregular morphology, consistent with hypoxia-induced cellular stress (Figure S90). Treatment with 4g (MDA-MB-231) and 2g (PANC-1) produced modest morphological changes when administered as single agents, characterized primarily by partial cell rounding and limited detachment, with more pronounced effects under hypoxia. In contrast, combination treatments with standard chemotherapeutic agents resulted in markedly enhanced morphological disruption. Specifically, 4g + AZA in MDA-MB-231 cells and 2g + AZA in PANC-1 cells caused extensive loss of cell adhesion, pronounced rounding, and widespread structural collapse relative to either treatment alone. These combination-dependent morphological effects were consistently amplified under hypoxic conditions and closely mirrored the enhanced cytotoxic and chemosensitizing responses observed in the MTT assay. Collectively, the morphological data provide visual confirmation that inhibition of tumor-associated carbonic anhydrases potentiates chemotherapy-induced cellular damage, under both normoxia and hypoxia (Figure ).

5.

Morphological effects of hCA IX/XII inhibitors and combination treatments under normoxic and hypoxic conditions. Representative inverted light microscope images (10× magnification) of MDA-MB-231 and PANC-1 cells following 48 h treatment with compounds 4g or 2g, azacitidine (AZA), sorafenib (SOR), and their combinations under normoxic and hypoxic conditions. Panels A–B show MDA-MB-231 cells under normoxia (A) and hypoxia (B), while panels C–D show PANC-1 cells under normoxia (C) and hypoxia (D). Scale bars represent 100 μm for all panels.

2.7. Apoptosis Induction by Lead Compounds (2g and 4g)

To elucidate the cellular basis of the antiproliferative activity of our lead compounds, apoptosis was evaluated in MDA-MB-231 and PANC-1 cells using Annexin V–FITC/propidium iodide (PI) staining (Figure ). In MDA-MB-231 cells (Figure A), treatment with compound 4g induced a clear redistribution of the cell population from viability toward apoptosis compared with untreated controls. This shift was characterized by a significant reduction in the viable cell fraction accompanied by a corresponding increase in apoptotic cells, indicating effective induction of programmed cell death. Similarly, in PANC-1 cells (Figure B), exposure to compound 2g resulted in a pronounced decrease in the viable cell population and a concomitant increase in apoptotic cells relative to control conditions. These data demonstrate that 2g efficiently promotes apoptosis in PANC-1 cells. Collectively, these results show that the lead compounds 4g and 2g induce apoptosis in breast and pancreatic cancer cell lines, respectively. The observed induction of programmed cell death provides mechanistic support for their antiproliferative and cytotoxic activities and is consistent with their proposed mode of action involving inhibition of tumor-associated carbonic anhydrase isoforms hCA IX and hCA XII.

6.

Apoptosis analysis of MDA-MB-231 and PANC-1 cells. Annexin V–FITC/propidium iodide (PI) staining was used to assess viable and apoptotic cell populations following 48 h treatment with compound 4g in MDA-MB-231 cells (A) and compound 2g in PANC-1 cells (B). The data represent a representative experiment.

2.8. In Silico Studies

2.8.1. Molecular Modeling

Molecular docking and MD simulations were used to investigate how 4g binds to the catalytic domains of hCA IX (PDB: 5FL4) and hCA XII (PDB: 1JD0). The best protein–ligand complexes, referred to as 5FL4 and 4g and 1JD0 & 4g, displayed favorable Glide XP G-scores (−5.77 and −7.89, respectively) and low eModel values (−62.8 and −84.9), indicating well-stabilized poses within the active site (Figure A–F).

7.

Molecular modeling of 4g in hCA IX and hCA XII. (A) Docking pose of 4g within the hCA IX active site (5FL4). (B) Docking pose of 4g within the hCA XII active site (1JD0). (C) Time evolution of protein Cα RMSD (blue) and ligand RMSD relative to the protein (Lig fit Prot, red) for the 5FL4 and 4g complex over 200 ns. (D) Corresponding RMSD plots for the 1JD0 and 4g complex. (E) Two-dimensional interaction diagram of 4g in hCA IX, highlighting the contacts that are maintained throughout the 200 ns trajectory. (F) Two-dimensional interaction diagram of 4g in hCA XII, showing the interactions that persist during the 200 ns simulation.

2.8.1.1. Molecular Docking Analysis of 4g within hCA IX

Molecular docking of 4g into hCA IX (5FL4) placed the deprotonated sulfonamide in the canonical zinc-binding position at the bottom of the conical cavity (Figure A,E). The sulfonamide nitrogen completes the tetrahedral coordination sphere of the catalytic Zn²⁺ together with His94, His96, and His119. One sulfonamide oxygen points toward the gatekeeper region and accepts a hydrogen bond from Thr200, whereas the sulfonamide NH is oriented toward Thr200/Thr201, forming the typical polar clamp at the base of the pocket. The phenyl ring of the benzenesulfonamide occupies a narrow hydrophobic niche, defined primarily by Leu199 and nearby nonpolar residues. Moving toward the solvent-exposed region, the triazole linker and central scaffold cross the middle of the cavity and contact Leu91, Gln92, and Val121. The terminal triazolopyridine group extends to the rim: the pyridine nitrogen hydrogen bonds to the backbone of Val130 and lies close to Asp131 and Arg129, which provide additional electrostatic stabilization. At the entrance of the pocket, the tail also interacts with Trp210 and Val142. Overall, the docked pose suggests that 4g spans the full length of the hCA IX active site from the Zn²⁺ center to the outer rim.

The MD trajectory starting from this pose confirms that the complex remains stable over 200 ns (Figure C). The protein Cα RMSD rises rapidly during the initial equilibration, after which it fluctuates smoothly around ∼1.4 Å (typically staying between 1.1 and 1.8 Å), indicating that the catalytic domain preserves its overall fold. The ligand RMSD with respect to the protein frame (Lig fit Prot) starts at ∼0.6 Å, increases to about 1.4–1.5 Å within the first 10 ns, and then briefly relaxes to values close to 1.0 Å. Around 40–60 ns it undergoes a clear shift and, for the remainder of the simulation, oscillates around a plateau in the 5.0–6.5 Å range, with an average close to 5.5 Å (Figure C, red trace). This behavior is consistent with a rearrangement of the distal rings of 4g within the relatively open rim region, while the metal-binding headgroup remains anchored. Throughout the trajectory, the sulfonamide ring and the adjacent linker remain in the same hydrophobic pocket and maintain contacts with Leu91, Gln92, and Val121. The triazolopyridine tail continues to sample the Val130/Asp131/Arg129 area and repeatedly engages with Trp210 and Val142. These results indicate that the sulfonamide headgroup remains stably coordinated within the catalytic site, while the triazolopyridine tail retains conformational flexibility and adapts to the rim region through dynamic interactions.

2.8.1.2. Molecular Docking Analysis of 4g within hCA XII

Docking of 4g into hCA XII (1JD0) produced a very similar arrangement at the catalytic center (Figure B,F). The deprotonated sulfonamide nitrogen again coordinates Zn²⁺ together with His94, His96, and His119, and one sulfonamide oxygen forms a strong hydrogen bond with the Thr199-equivalent gatekeeper residue. The sulfonamide NH is directed toward the same region, giving a polar network that closely matches the one observed in hCA IX. The upper part of the cavity, however, differs between the two isoforms. In hCA XII, the conical pocket narrows and becomes more hydrophobic. The sulfonamide ring and the adjacent linker are tightly packed against a Leu/Val-rich region (including Leu198/Leu199 and Val121), leading to a slightly more compact conformation of 4g. The distal triazolopyridine tail lies closer to the protein surface and makes fewer long-range contacts with rim residues than in hCA IX.

The MD simulation shows that 1JD0-4g complex is also stable over 200 ns (Figure D). The Cα RMSD starts around 0.9 Å, slowly increases during the first 60–70 ns, and then fluctuates between roughly 1.4 and 1.9 Å, with a mean of ∼1.6 Å. This pattern reflects modest breathing motions while the overall architecture of the catalytic domain remains intact. The Lig fit Prot trace reveals a three-stage behavior. For the first 50–60 ns, the ligand RMSD remains very low, typically 0.3–0.7 Å, indicating that 4g stays almost identical to its docked conformation. Between approximately 60 and 150 ns, the RMSD shifts to an intermediate band around 0.7–1.2 Å, which can be interpreted as a gentle adjustment of the tail within the compact upper pocket. After ∼150–160 ns, the ligand RMSD increases further and reaches a second plateau in the 5.0–6.4 Å range toward the end of the simulation (Figure D, red trace). As in hCA IX, this late plateau reflects a larger reorientation of the distal rings inside the rim, while the Zn²⁺ coordination and the Thr199 hydrogen bond remain conserved.

Throughout the trajectory, the sulfonamide ring and linker remain anchored in the same hydrophobic environment. In contrast, the tail of 4g explores a smaller volume than in hCA IX because of the tighter pocket. The solvent network in this region is less extensive and less dynamic, in line with the reduced solvent accessibility of the hCA XII rim. Taken together, the docking poses and MD trajectories show that 4g behaves as a typical Zn²⁺-binding sulfonamide inhibitor in both hCA IX and hCA XII. The deprotonated sulfonamide nitrogen coordinates the metal together with the three histidines, and the sulfonamide oxygen and NH consistently interact with the Thr gatekeeper at the base of the cavity. These headgroup interactions are preserved throughout the 200 ns simulations, as reflected by the low protein RMSD values and by the fact that the ligand rearrangements occur without disrupting the Zn²⁺–sulfonamide–Thr motif. The main isoform-dependent difference lies in the dynamics of the tail within the rim region. In hCA IX (5FL4 and 4g), the broader and more solvent-exposed upper pocket allows the distal rings to reorganize relatively early and to adopt a new orientation about 5–6 Å away from the starting pose, while still engaging Val130/Asp131/Arg129 and Trp210/Val142. In hCA XII (1JD0 and 4g), the narrower hydrophobic pocket constrains the tail more strongly; the ligand remains close to its initial conformation for a longer time and only later reaches a rearranged state of similar magnitude. These findings suggest that, while the sulfonamide headgroup remains firmly anchored at the catalytic site, the triazolopyridine tail undergoes more restricted conformational adaptation within the narrower and more hydrophobic rim region of hCA XII.

2.8.2. Predicted Physicochemical and ADMET Parameters

In addition to biochemical potency, physicochemical and ADMET parameters were preliminarily calculated in silico to support compound selection. The most active hCA IX/XII inhibitors (2g, and 4g) were analyzed using ADMETlab 3.0 and compared with SLC-0111. The selected compounds were characterized by calculated descriptors relevant to oral drug development, including molecular weight, lipophilicity, topological polar surface area (tPSA), aqueous solubility, and hydrogen-bonding capacity. The predicted lipophilicity and polar surface area values for (2g, and 4g) suggest a reasonable balance between polarity and lipophilicity. Overall, these parameters were within commonly accepted ranges for drug-like molecules and were comparable to those predicted for SLC-0111 (Figure S95). In addition, predicted ADMET-related parameters, including absorption and general toxicity indices, were consistent with acceptable drug-like profiles. However, these results are derived from computational models and should be interpreted with caution, as experimental validation will be required to confirm these properties.

3. Conclusion

In this study, we have identified bis-triazole–linked benzenesulfonamides as a novel chemotype for selective inhibition of the tumor-associated carbonic anhydrase isoforms hCA IX and hCA XII. Guided by a rational design strategy integrating sulfonamide regioisomerization, triazolopyridine tail diversification, and a nonclassical ring-closed triazole linker, we delineated key structural features governing potency and isoform selectivity. Lead compounds, particularly 2g and 4g, achieved low-nanomolar inhibition of hCA IX and hCA XII while substantially sparing the physiological isoforms hCA I and hCA II, defining a favorable selectivity index. Importantly, enzymatic efficacy translated into functional anticancer activity, as these inhibitors exhibited intrinsic cytotoxicity in CA-expressing breast and pancreatic cancer cell lines models. In addition, they significantly enhanced the efficacy of azacitidine and sorafenib and induced apoptotic cell death, supporting their potential to overcome tumor-associated resistance mechanisms. Computational analyses, including docking and molecular dynamics simulations, demonstrated that the ligand maintains a stable coordination geometry with the catalytic zinc ion while engaging key residues within the hCA IX active site. This predicted binding mode aligns with and strengthens the proposed mechanism of action. Collectively, these results position bis-triazole benzenesulfonamides as compelling lead candidates for the development of hCA-targeted combination therapies and provide a robust framework for further optimization toward in vivo validation and clinical translation in hCA-driven solid tumors.

4. Experimental Part

4.1. ChemistryGeneral Information

All commercially available reagents and solvents were obtained from TCI, Wako, or Sigma-Aldrich and were used as received without additional purification. Nuclear magnetic resonance (NMR) spectra were recorded at 298 K on Bruker Ascend 400 spectrometers (¹H NMR at 400 MHz and ¹³C NMR at 100 MHz). Chemical shifts (δ) are reported in parts per million (ppm) relative to the residual solvent signal and coupling constants (J) are expressed in hertz (Hz). Signal multiplicities are designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and ddd (doublet of doublet of doublets), tdd (triplet of doublet of doublets), dtd (doublet of triplets of doublets). High-resolution mass spectra were acquired using a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer. The purity of all final compounds was verified to be ≥95% by analytical HPLC.

4.2. General Synthetic Procedures for Azido-benzenesulfonamides (1–4)

The appropriate sulfanilamide (1.0 equiv, 15.0 mmol) was dissolved in THF (20 mL) and cooled to 0 °C. Concentrated HCl (8.0 mL) was added dropwise, followed by slow addition of an aqueous solution of NaNO₂ (1.2 equiv, 18.0 mmol in 10 mL H₂O) at 0 °C. The reaction mixture was stirred at 0 °C for 40–60 min to generate the corresponding diazonium intermediate. A precooled aqueous solution of NaN₃ (2.0 equiv, 30.0 mmol in 10 mL H₂O) was then added dropwise, and the mixture was stirred at 0 °C for an additional 30 min before warming to room temperature and stirring overnight to afford the azido-benzenesulfonamide. The reaction was diluted with water and extracted three times with ethyl acetate. Combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Crude products were purified by recrystallization or column chromatography, as appropriate. Caution: Diazonium salts and organic azides are potentially explosive and should be handled on a small scale using appropriate safety precautions.

4.2.1. 2-Azidobenzenesulfonamide (1)

The crude product was recrystallized from acetone/hexane to afford the desired compound as a yellow solid. Yield 55%. ¹H NMR (400 MHz, DMSO-d ₆) δ 7.84 (dd, J = 7.9, 1.4 Hz, 4H), 7.65 (td, J = 8.0, 1.5 Hz, 4H), 7.50 (dd, J = 8.0, 0.8 Hz, 4H), 7.35 (s, 7H), 7.31 (td, J = 7.8, 1.0 Hz, 5H).¹³C NMR (100 MHz, DMSO-d ₆) δ 137.4, 134.0, 128.4, 125.2, 121.0. HRMS-ESI calcd for C₆H₆O₂N₄NaS [M + Na]⁺ 221.01037; found, 221.01045.

4.2.2. 3-Azidobenzenesulfonamide (2)

The crude product was recrystallized from acetone/hexane to afford the desired compound as a yellow solid. Yield 89%. ¹H NMR (400 MHz, DMSO-d ₆) δ 7.62–7.58 (m, 2H), 7.50 (dd, J = 2.5, 1.3 Hz, 1H), 7.48 (s, 2H), 7.37–7.33 (m, 1H).¹³C NMR (100 MHz, DMSO-d ₆) δ 146.2, 140.8, 131.3, 22.9, 122.5, 116.5. HRMS-ESI calcd for C₆H₆O₂N₄NaS [M + Na]⁺ 221.01037; found, 221.01039.

4.2.3. 5-Azido-2-methylbenzenesulfonamide (3)

The crude product was recrystallized from acetone/hexane to afford the desired compound as a yellow solid. Yield 75%. ¹H NMR (400 MHz, DMSO-d ₆) δ 7.52 (s, 3H), 7.50 (d, J = 2.5 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.25 (dd, J = 8.1, 2.5 Hz, 2H), 2.55 (s, 5H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 143.9, 137.8, 134.4, 132.9, 122.7, 117.9, 19.6. HRMS-ESI calcd for C₇H₈O₂N₄NaS [M + Na]⁺ 235.02602; found, 235.02602.

4.2.4. 4-Azidobenzenesulfonamide (4)

Compound 4 was separated using column chromatography on silica gel using hexane/ethyl acetate (2:1 v/v) as eluent to afford the desired compound as a yellow solid. Yield 23%. ¹H NMR (400 MHz, DMSO-d ₆) δ 7.84 (d, J = 8.7 Hz, 1H), 7.37 (s, 1H), 7.30 (d, J = 8.6 Hz, 1H).¹³C NMR (100 MHz, DMSO-d ₆) δ 143.4, 141.0, 128.1, 120.0. HRMS-ESI calcd for C₆H₆O₂N₄NaS [M + Na]⁺ 221.01037; found, 221.01041.

4.3. General Synthetic Procedures for Triazolo-Pyridines Thiols (a–d)

Isonicotinic acid hydrazide (1.0 equiv, 20 mmol) and the appropriate isothiocyanate (1.0 equiv, 20 mmol) were dissolved in ethanol and heated under reflux for 3–4 h. Upon cooling to room temperature, the resulting precipitate was collected by filtration, washed with cold ethanol, and dried. The N-R₁-2-(pyridin-4-yl)­hydrazine-1-carbothioamide intermediates were then dissolved in 2N NaOH (2.0 equiv) and refluxed at 100 °C for 2–3 h to promote cyclization. After cooling in an ice bath, the reaction mixture was carefully neutralized with dilute HCl (pH 5–7), leading to precipitation of the product. The solid was filtered, washed with water and ether, and dried. When necessary, further purification was achieved by column chromatography or recrystallization from acetone/hexane to afford the corresponding triazolopyridine thiols (a–d).

4.3.1. 4-Methyl-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol (a)

The crude product was filtered, washed with water and ether, and dried to afford the desired compound as a white solid. Yield 90%. ¹H NMR (400 MHz, DMSO-d ₆) δ 14.15 (s, 1H), 8.78 (d, J = 6.1 Hz, 2H), 7.76 (d, J = 6.1 Hz, 2H), 3.59 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 168.6, 150.9, 149.9, 134.0, 122.9, 32.2. HRMS-ESI calcd for C₈H₉N₄S [M + H]⁺ 193.05424; found, 193.05431.

4.3.2. 4-Phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol (b)

The crude product was filtered, washed with water and ether, and dried to afford the desired compound as a white solid. White solid. Yield 73%. ¹H NMR (400 MHz, DMSO-d ₆) δ 14.38 (s, 1H), 8.55 (d, J = 5.6 Hz, 2H), 7.51 (d, J = 2.1 Hz, 3H), 7.47–7.34 (m, 2H), 7.23 (d, J = 5.7 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 169.7, 150.5, 148.9, 134.6, 133.6, 130.3, 130.0, 129.1, 122.4. HRMS-ESI calcd for C₁₃H₁₁N₄S [M + H]⁺ 255.06989; found, 255.06993.

4.3.3. 4-(2-Chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol (c)

The crude product was filtered, washed with water and ether, and recrystallized from acetone/hexane to afford the desired compound as a white solid. Yield 53%. ¹H NMR (400 MHz, DMSO-d ₆) δ 14.52 (s, 1H), 8.60 (d, J = 6.0 Hz, 2H), 7.79–7.73 (m, 1H), 7.72–7.68 (m, 1H), 7.66–7.56 (m, 2H), 7.26 (d, J = 6.2 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 169.8, 150.9, 148.6, 133.3, 132.7, 132.2, 132.1, 132.0, 130.9, 129.2, 121.5. HRMS-ESI calcd for C₁₃H₁₀N₄ClS [M + H]⁺ 289.03092; found, 289.03098.

4.3.4. 4-(3,5-Bis­(trifluoromethyl)­phenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol (d)

The crude product was collected by filtration, washed with water, and purified by flash column chromatography on silica gel using hexane/ethyl acetate (2:1, v/v) as eluent to afford the desired compound as an off-white solid. Yield 75%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.58 (dd, J = 4.5, 1.6 Hz, 2H), 8.29 (s, 1H), 8.28 (s, 2H), 7.28 (dd, J = 4.5, 1.6 Hz, 2H).¹³C NMR (100 MHz, DMSO-d ₆) δ 169.4, 150.5, 148.9, 136.2, 133.3, 132.2, 131.8, 131.5, 131.2, 130.7, 127.2, 124.4, 124.0, 122.9, 121.7, 119.0. HRMS-ESI calcd for C₁₅H₉N₄F₆S [M + H]⁺ 391.04466; found, 391.04495.

4.4. General Procedure for Propargylated Triazolopyridines (e–h)

In a dry round-bottom flask, the appropriate triazolopyridine thiol (a–d, 1.0 equiv) and K₂CO₃ (1.2 equiv) were suspended in anhydrous DMF or acetone under a nitrogen atmosphere and stirred at room temperature for 1 h to allow complete deprotonation. The reaction mixture was cooled to 0 °C, and propargyl bromide (1.2 equiv) was added dropwise. After stirring at 0 °C for 10 min, the mixture was warmed to room temperature and stirred for an additional 20 min, then heated to 70 °C for 4 h. Upon completion, the reaction was cooled to room temperature, diluted with water (50 mL), and extracted with dichloromethane (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (1:1), followed by a gradient to 100% ethyl acetate, to afford the corresponding propargylated triazolopyridine derivatives as solids.

4.4.1. 4-(4-Methyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-3-yl)­pyridine (e)

Light brown solid. Yield 58%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.78 (d, J = 5.4 Hz, 2H), 7.77 (d, J = 5.7 Hz, 2H), 4.02 (d, J = 2.3 Hz, 2H), 3.74 (s, 3H), 3.31–3.18 (t, 1H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.6, 150.8, 150.4, 134.4, 122.3, 79.5, 75.1, 32.3, 21.8. HRMS-ESI calcd for C₁₁H₁₁N₄S [M + H]⁺ 231.06989; found, 231.06999.

4.4.2. 4-(4-Phenyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-3-yl)­pyridine (f)

Light brown solid. Yield 73%. Compound f was separated using column chromatography with gradient ethyl acetate: Hexane (1/5 v/v) ∼ (0/1 v/v) respectively. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.57 (d, J = 5.3 Hz, 6H), 7.60 (d, J = 6.3 Hz, 9H), 7.48 (d, J = 6.0 Hz, 6H), 7.31 (d, J = 5.5 Hz, 6H), 4.02 (d, J = 2.1 Hz, 6H), 3.25 (s, 4H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.0, 152.3, 150.6, 134.3, 133.8, 131.0, 130.6, 128.1, 122.1, 79.8, 75.1, 21.3. HRMS-ESI calcd for C₁₆H₁₃N₄S [M + H]⁺ 293.08554; found, 293.08560.

4.4.3. 4-(4-(2-Chlorophenyl)-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-3-yl)­pyridine (g)

Light brown solid. Yield 80%. Compound g was separated using column chromatography with gradient ethyl acetate: Hexane (1/5 v/v) ∼ (0/1 v/v) respectively. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.59 (dd, J = 4.5, 1.6 Hz, 2H), 7.81 (dd, J = 7.7, 1.6 Hz, 1H), 7.76 (dd, J = 8.0, 1.4 Hz, 1H), 7.69 (td, J = 7.8, 1.8 Hz, 1H), 7.63 (td, J = 7.6, 1.6 Hz, 1H), 7.32 (dd, J = 4.5, 1.7 Hz, 2H), 4.03 (t, J = 2.5 Hz, 2H), 3.21 (t, J = 2.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.0, 152.4, 150.8, 134.0, 133.4, 131.4, 131.3, 131.0, 130.9, 129.7, 121.4, 79.7, 75.1, 21.4. HRMS-ESI calcd for C₁₆H₁₂N₄ClS [M + H]⁺ 327.04657; found, 327.04654.

4.4.4. 4-(4-(3,5-Bis­(trifluoromethyl)­phenyl)-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-3-yl)­pyridine (h)

Light brown solid. Yield 82%. Compound h was separated using column chromatography with gradient ethyl acetate: Hexane (1/5 v/v) ∼ (0/1 v/v) respectively.¹H NMR (400 MHz, DMSO-d ₆) δ 8.59 (d, J = 6.0 Hz, 2H), 8.38 (s, 3H), 7.33 (dd, J = 4.5, 1.6 Hz, 2H), 3.99 (d, J = 2.6 Hz, 2H), 3.18 (t, J = 2.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.2, 151.8, 150.6, 135.6, 133.9, 132.9, 132.5, 132.2, 131.9, 129.9, 129.9, 127.0, 124.8, 124.8, 124.3, 122.6, 121.6, 118.9, 79.6, 75.3, 22.2. HRMS-ESI calcd for C₁₈H₁₁N₄F₆S [M + H]⁺ 429.06031.; found, 429.06058.

4.5. General Synthetic Procedures of Bis-triazole–Linked Benzenesulfonamides

The appropriate azidobenzensulfonamide (1–4, 1.1 mmol) and propargylated triazolopyridine derivative (e–h, 1.0 mmol) were dissolved in DMF (5 mL) and stirred at room temperature. In a separate vessel, CuSO₄ (0.2 mmol) and sodium ascorbate (0.4 mmol) were dissolved in water (2.5 mL) and sonicated until a homogeneous orange solution formed. This freshly generated Cu­(I) catalyst solution was added dropwise to the reaction mixture at room temperature under a nitrogen atmosphere. The resulting mixture was heated to 70 °C and stirred for 6 h. After completion, the reaction was cooled to room temperature, diluted with ethyl acetate (50 mL), and washed with 5% aqueous ammonia (2 × 20 mL) to remove residual copper, followed by water (2 × 30 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by precipitation from acetone, with the addition of hexane added gradually, to afford the corresponding bis-triazole benzenesulfonamides as solids.

4.5.1. 2-(4-(((4-Methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (1e)

Pale yellow solid. Yield 75%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.76 (d, J = 4.0 Hz, 2H), 8.31 (s, 1H), 8.11 (dd, J = 6.0, 3.4 Hz, 1H), 7.84–7.76 (m, 2H), 7.74 (dd, J = 4.5, 1.6 Hz, 2H), 7.56 (dd, J = 5.9, 3.3 Hz, 1H), 4.56 (s, 2H), 3.64 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.3, 151.1, 150.2, 142.4, 138.8, 134.4, 133.2, 133.0, 130.5, 128.7, 128.4, 126.5, 122.1, 32.0, 28.10. HRMS-ESI calcd for C₁₇H₁₆O₂N₈NaS₂ [M + Na]⁺ 451.07298; found, 451.07336.

4.5.2. 3-(4-(((4-Methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (2e)

Pale yellow solid. Yield 59%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.89 (s, 1H), 8.76 (d, J = 1.9 Hz, 2H), 8.39 (t, J = 1.8 Hz, 1H), 8.10 (ddd, J = 8.1, 2.1, 0.9 Hz, 1H), 7.96–7.84 (m, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.75 (d, J = 5.7 Hz, 2H), 7.61 (s, 2H), 4.59 (s, 2H), 3.67 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.4, 151.1, 150.3, 145.8, 144.2, 136.5, 134.3, 130.8, 125.5, 122.9, 122.2, 120.0, 117.2, 32.0, 27.5. HRMS-ESI calcd for C₁₇H₁₆O₂N₈NaS₂ [M + Na]⁺ 451.07298; found, 451.07339.

4.5.3. 2-Methyl-5-(4-(((4-methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (3e)

Pale yellow solid. Yield 63%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.76 (d, J = 3.8 Hz, 2H), 8.32 (s, 1H), 8.15–8.04 (m, 1H), 7.80 (dd, J = 6.0, 3.3 Hz, 2H), 7.74 (dd, J = 4.5, 1.6 Hz, 2H), 7.61–7.55 (m, 1H), 7.53 (s, 2H), 4.55 (s, 2H), 3.64 (s, 3H), 3.30 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.3, 151.2, 150.2, 142.4, 138.9, 134.4, 133.2, 133.1, 130.6, 128.8, 128.4, 126.5, 122.2, 32.0, 28.1. HRMS-ESI calcd for C₁₈H₁₈O₂N₈NaS₂ [M + Na]⁺ 465.08863; found, 465.08899.

4.5.4. 4-(4-(((4-Methyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (4e)

Pale yellow solid. Yield 70%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.84 (s, 1H), 8.79 (d, J = 4.4 Hz, 2H), 8.15–8.06 (m, 2H), 8.04–7.96 (m, 2H), 7.75 (d, J = 4.1 Hz, 2H), 7.53 (s, 2H), 4.60 (s, 2H), 3.66 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.9, 151.7, 150.8, 144.7, 143.4, 138.9, 134.8, 128.0, 122.6, 120.8, 32.5, 28.0. HRMS-ESI calcd for C₁₇H₁₆O₂N₈NaS₂ [M + Na]⁺ 451.07298; found, 451.07324.

4.5.5. 2-(4-(((4-Phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (1f)

Pale yellow solid. Yield 73%. ¹H NMR (400 MHz, Acetone-d ₆) δ 8.55 (d, J = 2.1 Hz, 2H), 8.34 (s, 1H), 8.19 (dd, J = 7.8, 1.5 Hz, 1H), 7.83 (dtd, J = 26.7, 7.6, 1.3 Hz, 2H), 7.64 (tdd, J = 7.8, 6.4, 1.4 Hz, 4H), 7.48 (dd, J = 7.5, 1.9 Hz, 2H), 7.34 (d, J = 5.4 Hz, 2H), 6.71 (s, 2H), 4.64 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.0, 150.4, 142.6, 139.4, 133.9, 133.7, 133.6, 131.1, 131.0, 130.6, 129.4, 129.0, 128.1, 127.2, 40.7, 40.5, 40.2, 40.0, 39.8, 39.6, 39.4, 27.4. HRMS-ESI calcd for C₂₂H₁₉O₂N₈S₂ [M + H]⁺ 491.0669; found, 491.0693.

4.5.6. 3-(4-(((4-Phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (2f)

Pale yellow solid. Yield 82%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.84 (s, 1H), 8.57 (d, J = 1.6 Hz, 2H), 8.35 (t, J = 1.8 Hz, 1H), 8.11 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 7.92 (ddd, J = 7.8, 1.6, 1.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.66–7.52 (m, 5H), 7.47 (dt, J = 4.0, 2.6 Hz, 2H), 7.30 (d, J = 6.0 Hz, 2H), 4.61 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.4, 150.1, 145.7, 144.1, 136.6, 133.8, 133.3, 130.9, 130.5, 130.1, 127.5, 125.5, 123.0, 122.2, 121.5, 117.1, 26.7. HRMS-ESI calcd for C₂₂H₁₉O₂N₈S₂ [M + H]⁺ 491.0669; found, 491.0696.

4.5.7. 2-Methyl-5-(4-(((4-phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (3f)

Pale yellow solid. Yield 80%.¹H NMR (400 MHz, DMSO-d ₆) δ 8.77 (s, 1H), 8.58 (d, J = 2.0 Hz, 2H), 8.34 (d, J = 2.2 Hz, 1H), 7.97 (dd, J = 8.2, 2.3 Hz, 1H), 7.70–7.52 (m, 6H), 7.47 (dd, J = 7.8, 1.5 Hz, 2H), 7.30 (d, J = 4.0 Hz, 2H), 4.59 (s, 2H), 2.65 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.9, 150.6, 144.5, 144.0, 136.9, 134.7, 134.3, 133.8, 131.0, 130.6, 128.1, 123.4, 122.6, 122.0, 119.1, 27.2, 19.9. HRMS-ESI calcd for C₂₃H₂₀O₂N₈NaS₂ [M + Na]⁺ 527.10428; found, 527.10455.

4.5.8. 4-(4-(((4-Phenyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (4f)

Pale yellow solid. Yield 82%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.84 (s, 1H), 8.53 (d, J = 31.5 Hz, 2H), 8.17–8.06 (m, 2H), 8.07–7.90 (m, 2H), 7.63–7.54 (m, 3H), 7.53 (s, 2H), 7.47 (dt, J = 3.9, 2.6 Hz, 2H), 7.30 (d, J = 5.4 Hz, 2H), 4.60 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.9, 150.6, 144.6, 144.4, 138.9, 134.3, 133.8, 131.0, 130.6, 128.1, 128.0, 122.7, 122.0, 120.8, 27.2. HRMS-ESI calcd for C₂₂H₁₉O₂N₈S₂ [M + H]⁺ 491.0669; found, 491.0706.

4.5.9. 2-(4-(((4-(2-Chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (1g)

Pale yellow solid. Yield 76%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.60 (d, J = 5.2 Hz, 2H), 8.38 (s, 1H), 8.10 (dd, J = 6.0, 3.4 Hz, 1H), 7.79 (ddd, J = 10.9, 8.1, 3.2 Hz, 4H), 7.68 (td, J = 7.8, 1.6 Hz, 1H), 7.64–7.58 (m, 2H), 7.57 (s, 2H), 7.31 (d, J = 6.0 Hz, 2H), 4.64 (d, J = 4.5 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.6, 152.2, 150.3, 141.9, 138.9, 133.5, 133.2, 133.1, 132.8, 131.0, 130.8, 130.7, 130.6, 130.5, 129.2, 128.8, 128.5, 126.7, 120.7, 27.0. HRMS-ESI calcd for C₂₂H₁₇O₂N₈Cl NaS₂ [M + Na]⁺ 547.04966; found, 547.05005.

4.5.10. 3-(4-(((4-(2-Chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (2g)

Off-white solid. Yield 86%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.83 (s, 1H), 8.60 (d, J = 5.0 Hz, 2H), 8.34 (t, J = 1.8 Hz, 1H), 8.10 (ddd, J = 8.0, 2.1, 0.8 Hz, 1H), 7.81 (t, J = 8.1 Hz, 2H), 7.76 (dd, J = 8.0, 1.4 Hz, 1H), 7.68 (td, J = 7.8, 1.7 Hz, 1H), 7.62 (dd, J = 7.7, 1.5 Hz, 1H), 7.59 (s, 2H), 7.31 (dd, J = 4.6, 1.5 Hz, 2H), 4.64 (s, 2H).¹³C NMR (100 MHz, DMSO-d ₆) δ 152.4, 152.3, 150.4, 145.8, 143.9, 136.5, 133.5, 132.8, 131.0, 130.9, 130.8, 130.7, 130.4, 129.2, 125.5, 123.0, 122.2, 120.7, 117.1, 26.9. HRMS-ESI calcd for C₂₂H₁₇O₂N₈Cl NaS₂ [M + Na]⁺ 547.04966; found, 547.04993.

4.5.11. 5-(4-(((4-(2-Chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)-2-methylbenzenesulfonamide (3g)

Pale yellow solid. Yield 51%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.75 (s, 1H), 8.60 (d, J = 5.2 Hz, 2H), 8.32 (d, J = 2.4 Hz, 1H), 7.96 (dd, J = 8.2, 2.4 Hz, 1H), 7.81 (dd, J = 7.7, 1.7 Hz, 1H), 7.76 (dd, J = 8.0, 1.4 Hz, 1H), 7.68 (td, J = 7.8, 1.7 Hz, 1H), 7.61 (ddd, J = 7.6, 4.0, 1.5 Hz, 4H), 7.31 (dd, J = 4.6, 1.6 Hz, 2H), 4.62 (s, 2H), 2.65 (s, 3H).

¹³C NMR (100 MHz, DMSO-d ₆) δ 152.4, 152.3, 150.3, 150.2, 143.8, 143.5, 136.4, 134.2, 133.8, 133.3, 132.8, 131.0, 130.8, 130.7, 130.4, 129.2, 122.9, 122.1, 118.6, 26.9, 19.4. HRMS-ESI calcd for C₂₃H₁₉O₂N₈Cl NaS₂ [M + Na]⁺ 561.06531; found, 561.06580.

4.5.12. 4-(4-(((4-(2-Chlorophenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (4g)

Yellow solid. Yield 65%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.84 (s, 1H), 8.60 (d, J = 3.5 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 8.8 Hz, 2H), 7.79 (ddd, J = 21.3, 7.9, 1.4 Hz, 2H), 7.65 (dtd, J = 27.1, 7.6, 1.5 Hz, 2H), 7.53 (s, 2H), 7.31 (d, J = 5.9 Hz, 2H), 4.64 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.4, 152.3, 150.4, 143.9, 143.9, 138.4, 133.5, 132.8, 131.0, 130.8, 130.7, 130.4, 129.2, 127.5, 122.2, 120.7, 120.3, 40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 26.9. HRMS-ESI calcd for C₂₂H₁₇O₂N₈Cl NaS₂ [M + Na] ⁺ 547.04966; found, 547.04987.

4.5.13. 2-(4-(((4-(3,5-Bis­(trifluoromethyl)­phenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (1h)

Pale yellow solid. Yield 81%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.61 (d, J = 5.4 Hz, 2H), 8.41 (s, 1H), 8.36 (s, 2H), 8.34 (s, 1H), 8.15–8.04 (m, 1H), 7.86–7.76 (m, 2H), 7.62–7.57 (m, 1H), 7.56 (s, 2H), 7.32 (dd, J = 4.6, 1.5 Hz, 2H), 4.62 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.5, 151.8, 150.1, 141.9, 138.9, 135.2, 133.5, 133.2, 133.1, 132.3, 132.0, 131.6, 131.3, 130.6, 129.4, 129.4, 128.7, 128.4, 126.7, 126.6, 124.4, 124.4, 124.4, 124.3, 124.3, 123.8, 122.0, 121.1, 118.4, 27.6. HRMS-ESI calcd for C₂₄H₁₆O₂N₈F₆ NaS₂ [M + Na]⁺ 649.06340; found, 649.06366.

4.5.14. 3-(4-(((4-(3,5-Bis­(trifluoromethyl)­phenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (2h)

Pale yellow solid. Yield 88%. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.85 (s, 1H), 8.61 (d, J = 4.4 Hz, 2H), 8.42 (d, J = 4.6 Hz, 3H), 8.34 (t, J = 1.7 Hz, 1H), 8.10 (dd, J = 8.1, 1.2 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.60 (s, 2H), 7.32 (d, J = 6.0 Hz, 2H), 4.62 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.6, 152.0, 150.2, 145.8, 143.9, 136.6, 135.2, 133.4, 132.4, 132.0, 131.7, 131.4, 130.9, 129.4, 129.3, 126.6, 125.5, 124.5, 124.4, 124.4, 123.8, 123.0, 122.3, 121.9, 121.1, 118.4, 117.1, 27.3. HRMS-ESI calcd for C₂₄H₁₆O₂N₈F₆ NaS₂ [M + Na]⁺ 649.06340; found, 649.06360.

4.5.15. 5-(4-(((4-(3,5-Bis­(trifluoromethyl)­phenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)-2-methylbenzenesulfonamide (3h)

Pale yellow solid. Yield 90%.¹H NMR (400 MHz, Acetone-d ₆) δ 8.63 (s, 1H), 8.58 (d, J = 2.8 Hz, 2H), 8.41 (d, J = 2.3 Hz, 1H), 8.37 (s, 1H), 8.33 (s, 2H), 7.97 (dd, J = 8.2, 2.3 Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 5.0 Hz, 2H), 6.88 (s, 2H), 4.68 (s, 2H), 2.74 (s, 3H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 152.7, 152.0, 150.3, 143.7, 143.5, 136.4, 135.2, 134.2, 133.8, 133.1, 132.4, 132.0, 131.7, 131.3, 129.3, 126.5, 124.5, 124.4, 124.4, 123.8, 122.8, 122.2, 121.1, 118.6, 118.4, 27.3, 19.3. HRMS-ESI calcd for C₂₅H₁₈O₂N₈F₆ NaS₂ [M + Na]⁺ 663.07905; found, 663.07916.

4.5.16. 4-(4-(((4-(3,5-Bis­(trifluoromethyl)­phenyl)-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)­thio)­methyl)-1H-1,2,3-triazol-1-yl)­benzenesulfonamide (4h)

Pale yellow solid. Yield 58%. Compound 4h was separated using column chromatography with gradient ethyl acetate: Hexane (1/2; v/v) ∼ (0:1) respectively.¹H NMR (400 MHz, DMSO-d ₆) δ 9.11 (s, 1H), 9.02 (dd, J = 4.5, 1.6 Hz, 2H), 8.80 (s, 1H), 8.76 (s, 2H), 8.55 (s, 4H), 7.83 (dd, J = 4.5, 1.7 Hz, 2H), 7.22 (s, 2H), 5.12 (s, 2H). ¹³C NMR (100 MHz, DMSO-d ₆) δ 153.0, 152.4, 152.4, 150.7, 144.4, 144.4, 138.9, 135.7, 133.9, 132.8, 132.5, 132.2, 131.8, 129.9, 129.8, 128.0, 125.0, 124.9, 124.9, 124.3, 122.7, 122.4, 121.6, 120.7, 27.9. HRMS-ESI calcd for C₂₄H₁₆O₂N₈F₆ NaS₂ [M + Na]⁺ 649.06340; found, 649.06360.

4.6. Carbonic Anhydrase Inhibition Assay

An applied photophysics stopped-flow instrument has been used for assaying the CA-catalyzed CO₂ hydration activity. Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at an absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and 20 mM Na₂SO₄ (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor, at least six traces of the initial 5–10% of the reaction were applied to determine the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of the inhibitor (0.1 mM) were prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature before assay to allow for the formation of the E-I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier, , and represent the mean from at least three different determinations. The enzyme concentrations were in the range 6–14 nM. All hCA isoforms were recombinant ones obtained in-house as reported earlier.

4.7. Determination of CA IX Protein Levels by ELISA

MDA-MB-231 and A549 cells were seeded in 24-well plates at 5 × 10⁴ cells/well and incubated for 48 h at 37 °C in a humidified atmosphere with 5% CO₂. In parallel, cells were cultured under normoxic (20% O₂) and hypoxic (1% O₂) conditions without compound exposure to validate hypoxia induction by assessing CA IX protein expression, which increases under hypoxia. After incubation, cells were rinsed with ice-cold PBS, scraped, and centrifuged at 12,000 rpm for 20 min at 4 °C. Pellets were resuspended in ice-cold RIPA lysis buffer (Santa Cruz Biotechnology, USA) with protease inhibitors, incubated on ice for 30 min with intermittent vortexing, and centrifuged again to collect supernatants containing soluble proteins. Protein concentrations were then measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). CA9 protein levels were quantified using a CA9 ELISA kit (E2273Hu, Bioassay Technology Laboratory, Shanghai, China) per the manufacturer’s instructions. Absorbance was measured at 450 nm using an Epoch Microplate Spectrophotometer (BioTek, USA).

4.8. Cell Viability Assay

The cytotoxic and chemosensitizing effects of the synthesized compounds 2g and 4g were evaluated using the MTT assay, which measures mitochondrial activity in viable cells. MDA-MB-231 and PANC-1 cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and allowed to attach for 24 h. Cells were then treated with 4g or 2g (100 μM), azacitidine (AZA, 25 μM), sorafenib (SOR, 25 μM), or their combinations ((4g + AZA), (4g + SOR), (2g + AZA), (2g + SOR)) and incubated for 48 h under normoxic (20% O₂, 5% CO₂) or hypoxic (1% O₂, 5% CO₂, 94% N₂) conditions. For the MDA-MB-231 cell line, treatments included 4g, AZA, SOR, and combinations (4g + AZA) and (4g + SOR), whereas for the PANC-1 cell line, treatments included 2g, AZA, SOR, and combinations (2g + AZA) and (2g + SOR). Following treatment, 20 μL of MTT solution (1 mg/mL) was added to each well and incubated for 3 h at 37 °C. The resulting formazan crystals were dissolved in 150 μL of DMSO, and absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to untreated control cells.

4.9. Molecular Docking and Dynamics

The crystal structures of the catalytic domains of hCA IX in complex with a thiophene-2-sulfonamide inhibitor (PDB ID: 5FL4) and hCA XII in complex with acetazolamide (PDB ID: 1JD0) were taken from the Protein Data Bank. Protein structures were prepared with the Protein Preparation Wizard (Schrödinger Suite 2025–4). Missing atoms and side chains were added, bond orders were checked, and hydrogens were added at physiological pH. Histidine protonation states were adjusted to preserve the His94–His96–His119 Zn²⁺ coordination motif. Nonessential water molecules were removed, whereas the catalytic Zn²⁺ ion was retained. The prepared structures were then energy-minimized with positional restraints using the OPLS4 force field to remove local steric clashes while keeping the backbone conformation close to the experimental one. Compound 4g was constructed in Maestro and prepared using LigPrep with the OPLS4 force field. Relevant tautomers and protonation states were generated at pH 7.4 ± 0.5 with Epik, with particular attention to the sulfonamide group. In line with the known mechanism of CA inhibition, the deprotonated sulfonamide form of 4g was selected for all docking and MD simulations, as this is the species that coordinates the Zn²⁺ ion. For docking, receptor grids were generated in Glide for hCA IX and hCA XII by centring the grid box on the cocrystallized inhibitor in each structure. The grid dimensions were chosen to include the Zn²⁺ site, the conical cavity and the rim region. Docking was performed in Glide XP mode with flexible sampling of 4g and a rigid protein backbone. Poses were ranked by GlideScore and inspected to confirm correct Zn²⁺–sulfonamide coordination, hydrogen bonding to the Thr-type gatekeeper residues, and a reasonable orientation of the distal rings in the rim. As a validation step, the cocrystallized ligands in 5FL4 and 1JD0 were redocked and their experimental poses were reproduced with low RMSD values.

4.10. Molecular Dynamics (MD) Simulations

The best-scoring poses of 4g in hCA IX and hCA XII were used as starting structures for 200 ns MD simulations. Each complex was solvated in an orthorhombic box of TIP3P water, with a sufficient distance between the protein surface and the box edges. Counterions were added to neutralize the system and mimic physiological ionic strength. Simulations were performed in Desmond using the OPLS4 force field for both the protein and ligand. After energy minimization, the systems were equilibrated under NVT and then NPT conditions, with restraints on the protein’s heavy atoms gradually released. Production runs were performed in the NPT ensemble at 300 K using a Nose–Hoover thermostat and an appropriate barostat. Long-range electrostatics were treated by the particle-mesh Ewald method, and standard cut-offs were used for short-range interactions. Trajectory analysis was performed using Desmond tools and in-house scripts. For each system, the time evolution of protein backbone Cα RMSD relative to the minimized starting structure was used to assess overall stability. Ligand RMSD was calculated relative to the protein frame (Lig fit Prot) and relative to the initial ligand conformation to follow the motion and internal rearrangements of 4g in the binding pocket.

Glossary

Abbreviations Used

AAZ

acetazolamide

AZA

azacitidine

CAI(s)

carbonic anhydrase inhibitor(s)

ELISA

enzyme-linked immunosorbent assay

FITC

fluorescein isothiocyanate

hCA

human carbonic anhydrase

K _I

inhibition constant

PI

propidium iodide

SAR

structure–activity relationship

SI

selectivity index

SOR

sorafenib

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c00547.

• NMR spectra, ESI-MS data, HPLC purity chromatograms, and predicted physicochemical and ADMET parameters (PDF)

• Molecular formula strings (CSV)

• 1JD0 4g (PDB)

• 5FL4 4g (PDB)

All the authors contributed to the preparation of the manuscript and approved the final version.

The authors declare no competing financial interest.