INTRODUCTION
Potassium channels are transmembrane proteins that facilitate the selective passage of potassium ions across the plasma membrane following their electrochemical gradient. These channels are classified into different families, with voltage-gated potassium channels (KV) representing a major group. The KV1.x (Shaker) subfamily, the largest within this group, consists of eight members (KV1.1–KV1.8) (1, 2). KV1.3 is widely expressed throughout the human body and plays a crucial role in numerous cellular processes. It is located in the plasma membrane, the inner mitochondrial membrane (mitoKV1.3), the nuclear membrane, and the membrane of the cis-Golgi apparatus (3). KV1.3 expression has been identified in multiple cell types, including neurons, osteoclasts, epithelial cells, and immune cells, such as T- and B-lymphocytes, macrophages and microglia, where it contributes to the regulation of membrane potential and calcium signalling (2).
Given the well-established role of T-cells in the pathogenesis of autoimmune diseases, Kv1.3 has emerged as an attractive therapeutic target. It is highly expressed in effector memory T (TEM) cells, which mediate autoimmune disorders, as well as in other leukocytes involved in chronic inflammatory diseases, such as B-lymphocytes, macrophages, microglia, and dendritic cells. Inhibition of KV1.3 has been shown to induce membrane depolarisation, thereby preventing T-cell proliferation and cytokine production. Consequently, the development of selective KV1.3 inhibitors that specifically target disease-inducing TEM cells while preserving normal immune function could be beneficial in treating autoimmune and chronic inflammatory conditions, including psoriasis, multiple sclerosis, type 1 diabetes mellitus, atherosclerosis, asthma, and rheumatoid arthritis (4, 5).
KV1.3 has also been identified as a promising molecular target for anticancer therapy due to its involvement in critical cellular processes, including proliferation, calcium signalling, cell volume regulation, adhesion, migration, apoptosis, and invasion (6). Although KV1.3 is increasingly recognised as a tumour marker, a definitive pattern distinguishing its expression in cancerous versus healthy cells has not yet been established, as its levels appear to be influenced by the tumour type and disease stage (7). Nevertheless, aberrant KV1.3 expression has been observed in breast, colon, and prostate tumours, as well as in smooth muscle and skeletal muscle cancers. It is also present in mature neoplastic B-cells in chronic lymphocytic leukaemia, with a notable correlation between its expression and mitochondrial localisation (6). Potential therapeutic strategies for cancer treatment involve selectively inhibiting cancer cell proliferation or inducing apoptosis (8). Induction of apoptosis in cancer cells by KV1.3 inhibition might be considered as an effective method to selectively kill cancer cells (9). Beyond its roles in immunity and oncology, KV1.3 has been implicated in pathways regulating energy homeostasis and body weight, making it a potential target for obesity treatment (10).
One of the primary challenges in developing KV1.3 inhibitors lies in the high sequence homology among KV1.x family members, making it difficult to achieve potent and selective inhibition of KV1.3. Several KV1.3 inhibitors have been designed to specifically target KV1.3 in the plasma membrane. Among them, the psoralen derivative PAP-1 (1, Fig. 1) is currently the most potent (IC50 = 2 nmol L–1) and selective (i.e. 23-fold over KV1.5) small-molecule KV1.3 inhibitor (11). The antimycobacterial drug clofazimine (2, Fig. 1) is another well-characterised KV1.3 inhibitor (IC50 = 300 nmol L–1), displaying tenfold selectivity over KV1.1, KV1.2, KV1.5, and KV3.1 (12). Another important KV1.3 inhibitor, a benzamide derivative known as PAC (3, Fig. 1), was identified through a high-throughput screening campaign. It exhibited an IC50 of 200 nmol L–1 but lacked selectivity among KV1.x family members. In vitro functional assays demonstrated that this compound reversibly inhibits calcium-dependent T-cell activation and suppresses IL-2 production in a concentration-dependent manner without inducing cytotoxicity or affecting calcium-independent T-cell stimulation pathways (13).
Fig. 1. Structures of known representative KV1.3 inhibitors PAP-1 (1), clofazimine (2) and PAC (3).
Since the binding site of benzamide-based Kv1.3 inhibitors remains unidentified, we employed a ligand-based drug design strategy, utilising a 3D similarity search of previously reported benzamide inhibitors. This approach led to the discovery of a novel thiophene-based compound A, which demonstrated selectivity for Kv1.3 channels (14). Further structural modifications of compound A resulted in the development of the 3-thiophene-based inhibitor B (Fig. 2), which demonstrated an IC50 of 470 nmol L–1 and an 18-fold selectivity over related Kv1.x family channels in Xenopus laevis oocytes (15). Additional optimisation led to compound C, which inhibited Kv1.3-mediated currents in activated human T-lymphocytes with an IC50 value of 26.1 nmol L–1 (16). In this work, we further investigate the structure-activity relationship (SAR) of this series by extensively modifying the 2-methoxybenzamide moiety of the inhibitor while retaining the 3-thienyl group at position 4 of the core tetrahydropyran or cyclohexane ring (Fig. 2).
Fig. 2. Optimization of Kv1.3 inhibitor A, identified by virtual screening, to more potent analogs B and C, and further structure-activity relationship investigation in this work.
EXPERIMENTAL
Chemistry – General
The reagents and solvents used were obtained from commercial sources (i.e., Acros Organics, Sigma-Aldrich, TCI Europe, Merck, Carlo Erba, Apollo Scientific) and were used as provided. Analytical thin-layer chromatography (TLC) was performed on silica gel aluminium sheets (60 F254, 0.20 mm, Merck, Germany). Flash column chromatography was performed on silica gel 60 (particle size 0.040–0.063 mm, Merck). 1H NMR and 13C spectra were recorded at 400 and 100 MHz, respectively, on a Bruker Avance III NMR spectrometer (Bruker, USA) at 295 K. The chemical shifts (δ) are reported in ppm and are referenced to the deuterated solvent used. HRMS measurements were performed on a LC-MS/MS system (Q Executive Plus; Thermo Scientific, USA). Mass spectrometry measurements were performed on an Expression CMSL mass spectrometer (Advion, USA). Analytical reversed-phase UPLC analyses were performed using a modular system (Thermo Scientific Dionex UltiMate 3000 modular system; Thermo Fisher Scientific Inc., USA). Method: Waters Acquity UPLC® HSS C18 SB column (2.1 × 50 mm, 1.8 μm), t = 40 °C; injection volume = 5 µL; flow rate = 0.4 mL min–1; detector λ = 254 nm; mobile phase A (0.1 % trifluoroacetic acid (TFA) in water, V/V), mobile phase B acetonitrile (MeCN). Gradient: 0–2 min, 10 % B; 2–10 min, 10–90 % B; 10–12 min, 90 % B. Purities of the tested compounds were established to be ≥ 95 % at 254 nm, as determined by UPLC. The syntheses of the compounds are illustrated in Schemes 1 and 2, experimental procedures and characterisation data are provided in the Supporting Information.
Scheme 1. Synthesis of the target compounds 11a and 11b.
Scheme 2. Synthesis of the target compounds 13a–i, trans-14a–d, trans14g, trans-14i, cis-14a–d, cis-14g, cis-14i, trans-15–20 and cis-21.
Patch-clamp electrophysiology
Mouse L929 fibroblasts stably expressing mKv1.3, were a gift from Dr. K. George Chandy (University of California, Irvine, USA). All experiments were conducted with an EPC-10 amplifier (HEKA, Germany) in the whole-cell configuration with a holding potential of –80 mV. Pipette resistances averaged around 2.5 MΩ. Compound solutions were prepared fresh in Na+ Ringer from 10 mmol L–1 stock solutions in DMSO directly before the experiments. For current measurements, we used an internal pipette solution containing 160 mmol L–1 KF, 2 mmol L–1 MgCl2, 10 mmol L–1 HEPES, and 10 mmol L–1 EGTA, with a pH of 7.2 and an osmolarity of ~300 mOsm. Sodium Ringer was used as an external solution containing the following: 160 mmol L–1 NaCl, 4.5 mmol L–1 KCl, 2 mmol L–1 CaCl2, 1 mmol L–1 MgCl2, and 10 mmol L–1 HEPES, with a pH of 7.4 and an osmolarity of ~300 mOsm. Currents were elicited with a 200-ms voltage step to + 40 mV, followed by 45 seconds of holding at a resting membrane potential of –80 mV. A use-dependence protocol, whereby cells were pulsed to 40 mV every 1 second, was used prior to the step protocol for the compound to ensure that channel kinetics were as expected. If currents exceeded 2 nA, 60–80 % series resistance compensation was used. Concentration-dependent current inhibition, measured as reduction of area under the current curve, was fitted with the Hill equation using GraphPad Prism 8 (GraphPad Software, USA). All data points represent at least 3 independent experiments and are presented as mean ± standard deviation (SD). IC50 values are reported with 95 % confidence intervals (CI).
RESULT AND DISCUSSION
Chemistry
Based on our previously published compounds (15, 16), we further investigated the SAR for KV1.3 inhibition (Fig. 2). We synthesised compounds 11a and 11b, in which the 3-thiophene and tetrahydropyran moieties were retained, whereas the 2-methoxyphenyl group of compound B was replaced with either a 2-methoxycyclohexyl or 3-methoxythiophenyl moiety, respectively. Next, the tetrahydropyran ring was substituted with a cyclohexane ring bearing keto, hydroxy, or carbamate groups. Compounds 13a–i contain a ketone at the 4-position of the cyclohexane ring and feature various modifications of the 2-methoxybenzamide moiety. Reduction of the ketone in compounds 13a–h produced the corresponding hydroxy analogues: trans-14a–d, cis-14a–d, trans-14g, cis-14g, trans-14i, and cis-14i. These hydroxy compounds were then further modified to generate methylcarbamate-, propylcarbamate-, and 3-methoxypropylcarbamate-containing derivatives 15–21. All hydroxy- and carbamate-substituted compounds were obtained as diastereomeric mixtures, which were subsequently separated into their cis- and trans-isomers using column chromatography. The synthetic routes for these new compounds are outlined in Schemes 1 and 2.
In Scheme 1, thiophene-3-acetonitrile was reacted with 1-chloro-2-(2-chloroethoxy)ethane in the presence of sodium hydride (NaH, 60 % dispersion in mineral oil) under an argon atmosphere at room temperature, yielding the intermediate 4-(thiophen-3-yl)tetrahydro-2H-pyran-4-carbonitrile (4). The nitrile group was subsequently reduced using lithium aluminium hydride (LiAlH4) in tetrahydrofuran (THF) to produce the primary amine 9. This amine was then reacted with either 4-methoxythiophene-3-carbonyl chloride or 2-methoxycyclohexane-1-carbonyl chloride to afford the final compounds 11a and 11b.
For the synthesis of cyclohexane-based analogues (Scheme 2), thiophene-3-acetonitrile was refluxed in tert-butanol and subjected to a double Michael addition in the presence of methyl acrylate and benzyl trimethylammonium hydroxide (Triton B), yielding the diester intermediate 5. In the following step, compound 5 was deprotonated using potassium tert-butoxide and underwent Dieckmann condensation to form the 4-heteroaryl-4-cyano-2-carbomethoxycyclohexanone derivative 6. The 2-carboxymethyl group was then removed by heating at 100 °C in a mixture of 10 % sulfuric acid and glacial acetic acid, producing 4-oxo-1-(thiophen-3-yl)cyclohexane-1-carbonitrile (7). The ketone group of 7 was protected with ethylene glycol, forming compound 8. Subsequent reduction of the nitrile group using LiAlH4 in THF provided the primary amine 10, which was then acylated with various acyl chlorides to produce intermediates 12a–i. Deprotection of the ketone yielded compounds 13a–i. Selective reduction of the ketone group in 13a–i using sodium borohydride (NaBH4) produced diastereomeric mixtures of alcohols 14a–i, which were separated into their cis- and trans-isomers by column chromatography. The resulting hydroxy analogues were reacted with 4-nitrochloroformates to form 4-nitrophenyl carbonate intermediates, which were subsequently treated with various primary amines to yield novel carbamate derivatives trans-15, trans-16, trans-17, trans-18, trans-19, trans-20, and cis-21.
Determination of KV1.3 inhibition
To expand the library of 2-methoxybenzamides, a series of new KV1.3 inhibitors was synthesised and evaluated for their inhibitory activity at 1 and 10 μmol L–1 using whole-cell patch-clamp electrophysiology (Tables I–III). Replacement of the 2-methoxyphenyl moiety in compound B (Fig. 2) with a 2-methoxycyclohexyl, as in 11b, resulted in loss of inhibitory activity, while compound 11a with a 3-methoxythiophen-4-yl group showed full inhibition of KV1.3 at 10 µmol L–1 (Table I). Within the ketone series, compound 13a, featuring a 5-fluoro substituent on the 2-methoxyphenyl ring, exhibited the highest potency, achieving 49 % inhibition at 1 μmol L–1 and complete block at 10 μmol L–1. Substitution with either a 4-methyl (13c) or 4-methoxy (13f) group on the 2-methoxyphenyl ring was better tolerated than a 5-methyl substituent (13b), which showed reduced activity. Strong inhibition was also observed when the 2-methoxyphenyl group was replaced with a 3-methoxythiophen-4-yl (13d) or a 2-ethoxyphenyl group (13h). In contrast, compounds 13e (bearing a methoxycyclohexyl group) and 13g (with a furan-3-yl substituent) only weakly inhibited KV1.3 at 10 µmol L–1 (Table I).
Table I. KV1.3 inhibitory activity of new analogues 11a, 11b and 13a–h, manually patch-clamped to determine the percentage of inhibition at 1 and 10 μmol L–1
| Compd. | Structure | Average block at 1 μmol L–1 (%) | Average block at 10 μmol L–1 (%) |
|---|---|---|---|
| 11a | 60 | 100 | |
| 11b | 0 | 22 | |
| 13a | 49 | 100 | |
| 13b | 9 | 63 | |
| 13c | 32 | 89 | |
| 13d | 25 | 92 | |
| 13e | 4 | 25 | |
| 13f | 27 | 85 | |
| 13g | 0 | 20 | |
| 13h | 29 | 88 |
n = 3
In the hydroxy series (14a–d,g,i), an SAR pattern similar to that observed for the ketones (13a–h) was evident. Notably, the cis-isomers consistently exhibited more potent Kv1.3 inhibition than their trans counterparts, regardless of the substitution on the 2-methoxyphenyl ring. Among the hydroxy derivatives, the most potent compounds were cis-14c, cis-14d, and cis-14i, bearing 4-methyl-2-methoxyphenyl, 3-methoxythiophen-4-yl, and 2-methoxythiophen-3-yl substituents, respectively. Each of these compounds achieved complete Kv1.3 inhibition at 1 μmol L–1 (Table II). In the carbamate series (Table III), the 3-methoxypropyl analog of compound C (trans-15) also demonstrated full Kv1.3 inhibition at 1 μmol L–1. Consistent with findings from our previous study (16), the trans-carbamates generally exhibited stronger inhibitory activity than their cis counterparts. For example, trans-17 inhibited Kv1.3 by 87 % at 1 μmol L–1, whereas cis-21 showed only 21 % inhibition at the same concentration. Other potent inhibitors included the propylcarbamates trans-16 and trans-18, analogues of 14d and 14a, respectively, both of which showed complete inhibition at 1 μmol L–1 (Table III). Based on this preliminary screening, the most promising compounds, cis-14c, cis-14d, cis-14i, trans-15, trans-16, trans-17, and trans-18, were selected for further evaluation in concentration-response experiments (Table IV).
Table II.KV1.3 inhibitory activity of new analogues 14a-d,g,i, manually patch-clamped to determine the percentage of inhibition at 1 and 10 μmol L–1
n = 3
Table III. KV1.3 inhibitory activity of new analogues 15–21, manually patch-clamped to determine the percentage of inhibition at 1 and 10 μmol L–1
| Compd. | Structure | Average block at 1 μmol L–1 (%) | Average block at 10 μmol L–1 (%) |
|---|---|---|---|
| trans- 15 | 100 | n.t. | |
| trans- 16 | 100 | n.t. | |
| trans- 17 | 87 | n.t. | |
| trans- 18 | 100 | n.t. | |
| trans- 19 | 7 | 66 | |
| trans- 20 | 0 | 21 | |
| cis- 21 | 21 | 83 |
n = 3; n.t. – not tested
The potency of compounds cis-14c, cis-14d, and cis-14i was assessed by determining their IC50 values for KV1.3 inhibition and compared to the reference compounds cis-D (IC50 = 226 nmol L–1 and trans-D (IC50 = 2.2 μmol L–1). Among the tested compounds, cis-14i showed the highest potency, with an IC50 of 326 nmol L–1, followed closely by cis-14d (IC50 = 346 nmol L–1) and cis-14c (IC50 = 505 nmol L–1). Although all three compounds displayed submicromolar IC50 values, they were less potent than cis-D. The potency of compounds trans-15, trans-16, trans-17, and trans-18 was compared to the parent compound trans-C (IC50 = 0.23 μmol L–1). Among these, trans-18 exhibited the highest potency, with an IC50 of 122 nmol L–1, followed by trans-16 with an IC50 of 166 nmol L–1. While both compounds were less potent than trans-C, they still retained significant inhibitory activity. trans-15 and trans-17 were less potent than trans-16 and trans-18, though their inhibitory potency remained within the submicromolar range. These results suggest that the 2-methoxyphenyl ring, as present in compound trans-C, represents an optimal substituent for KV1.3 inhibition at this position. However, small modifications, such as those present in trans-16 and trans-18, are well tolerated in maintaining strong inhibitory potency.
Table IV. IC50 values determined on KV1.3 channels
CONCLUSIONS
In this study, a ligand-based design approach was used for the optimisation of novel thiophene-based Kv1.3 inhibitors, building on a previously reported benzamide scaffold. Structural modifications focused on the 2-methoxybenzamide moiety and the core tetrahydropyran or cyclohexane ring, while retaining the 3-thienyl group, enabled the identification of several potent inhibitors. Structure–activity relationship analysis revealed that cis-isomers in the hydroxy series consistently exhibited stronger Kv1.3 inhibition than their trans counterparts, with cis-14i, cis-14d, and cis-14c emerging as the most effective (IC50 = 326–505 nmol L–1). In the carbamate series, trans-isomers showed superior activity, with trans-18 and trans-16 achieving IC50 values of 122 and 166 nmol L–1, respectively. While none of the new compounds outperformed the most potent reference inhibitors, several analogues exhibited submicromolar potency. These findings provide valuable insights into the SAR of Kv1.3 inhibitors and highlight promising candidates for further development.
Acknowledgment. – This research was funded by the Slovenian Research Agency (ARIS) grant numbers J7-4635 (MitoCan) and P1-0208. J.A.N. was supported by a National Institute of General Medical Sciences-funded Pharmacology Training Program [T32GM099608].
Conflicts of interest. – The authors declare no conflict of interest.
Authors contributions. – Conceptualization, L.P.M. and T.T.; methodology, M.F., Š.P., and J.A.N.; analysis M.F., Š.P., and J.A.N.; investigation, M.F., Š.P., and J.A.N.; writing, original draft preparation, M.F. and T.T.; writing, review and editing, Š.P., J.A.N., H.W., L.P.M., and T.T. All authors have read and agreed to the published version of the manuscript.