Gubič Š , Montalbano A , Sala C , Becchetti A , Hendrickx LA , Van Theemsche KM , Pinheiro-Junior EL , Altadonna GC , Peigneur S , Ilaš J , Labro AJ , Pardo LA , Tytgat J , Tomašič T , Arcangeli A , Peterlin Mašič L .

             calcium-activated potassium channel activity
             T cell proliferation
             T cell activation
             lymphocyte activation

	Graphical abstract
	Fig. 1. Examples of known KV1.3 inhibitors [20,24,25].
	Fig. 2. Design of new thiophene-based KV1.3 inhibitors.
	Scheme 1. Synthesis of hydroxyl- and oxo-based 3-thiophene and 2-thiophene KV1.3 inhibitorsa. a Reagents and conditions: (i) (a) methyl acrylate, benzyltrimethylammonium hydroxide, tert-butanol, reflux, 4 h (80–81%); (b) potassium tert-butoxide, anhydrous THF, 0 °C, reflux, 5 h, room temperature (rt), overnight (60–77%); (ii) 10% sulfuric acid, glacial acetic acid, 100 °C, 24 h (50–62%); (iii) ethane-1,2-diol, p-toluenesulfonic acid (PTSA), toluene, 140 °C, overnight (96–98%); (iv) LiAlH4, anhydrous THF, 0 °C, rt, overnight (96–97%); (v) (a) appropriate benzoic acid, oxalyl chloride, dichloromethane (DCM), 2 drops N,N-dimethylformamide (DMF), rt, overnight (100%); (b) 2-methoxybenzoyl chloride or appropriate benzoyl chloride, Et3N, DCM, rt, overnight (89–95%); (vi) water, pyridinium p-toluenesulfonate, acetone, reflux, overnight (75%); (vii) sodium borohydride, anhydrous THF, 0 °C, rt, overnight (trans 34–43% and cis 31–57%).
	Scheme 2. Synthesis of new alcohol and carbamate derivatives of 2-thiophene-based KV1.3 inhibitorsa. a Reagents and conditions: (i) (a) 2-methoxybenzoic acid or furan-3-carboxylic acid or appropriate benzoic acid, oxalyl chloride, DCM, 2 drops DMF, rt, overnight (100%); (b) 2-methoxybenzoyl chloride or furan-3-carbonyl chloride or appropriate benzoyl chloride, Et3N, DCM, rt, overnight (57–96%); (3) water, pyridinium p-toluenesulfonate, acetone, reflux, overnight (46–75%); (ii) sodium borohydride, anhydrous THF, 0 °C, rt, overnight (cis 16–34% and trans 24–43%); (iii) (a) 4-nitrophenyl chloroformate, Et3N, DCM, rt, overnight (52%); (b) methyl amine or other appropriate amine, DCM, rt, overnight (85–92%).
	Scheme 3. Synthesis of carbamate derivatives of 3-thiophene-based KV1.3 inhibitorsa. a Reagents and conditions: (i) (a) 4-nitrophenyl chloroformate, Et3N, DCM, rt, overnight (52%); (b) appropriate amine, DCM, rt, overnight (81–96%); (ii) (a) methanesulfonyl chloride, Et3N, DCM, ice bath, 2 h (100%); (b) sodium azide, DMF, 100 °C, overnight (50–57%); (c) H2(g), Pd/C, methanol (MeOH), rt, overnight (86%); (d) 4 M HCl in 1,4-dioxane, MeOH, rt, 1 h (34–42%).
	Fig. 3. Percentages of inhibition produced by the application of reference compounds (blue), type-I 2-thiophene based compounds (orange) and type-II 3-thiophene based compounds (green) on PBMC PHA-activated T-lymphocytes at 5 μM. ns = not significant.
	Fig. 4. Effect on intracellular Ca2+ levels. Flow cytometry analysis of Fluo-4 fluorescence on CD3+ PBMC. (A) Mean Fluo-4 fluorescence from 3 replicates was normalized to that of the untreated control (Ctrl) for each individual experiment (n = 4). The error bars represent the standard deviation. After activation, cells were treated with the described compounds [5 μM] for 2 h. (B) Representative histograms showing Fluo-4 fluorescence in CD3+ lymphocytes treated with the different compounds and a graph showing a comparison among the different treatment conditions.
	Fig. 5. (A) Bar graph showing the number of T-cell CFUs formed after PHA activation. PBMC were treated with the different drugs [30 μM] together with PHA for 5 days before counting the colonies. Bars represent the mean of 3 experiments, and standard deviation is indicated. (B) Representative images of colonies from A. The arrow shows a representative colony. Images were taken with a bright-field microscope.
	Fig. 6. Flow cytometry cell cycle analysis of PBMC after 5 days of PHA stimulation and treatment with the described drugs [30 μM]. (A) Bars represent the mean of the percentage of cycling cells (S + G2+M) from 3 experiments. Error bars represent the standard deviation between replicates. Unpaired t-test was performed to compare drug-treated and untreated (Ctrl) samples (*: p < 0.05; **: p < 0.005. (B) Representative histograms showing activated T-lymphocytes at different stages of cell cycle progression, grouped in G0/G1 and in cycling (cell cycle phases S + G2+M) as shown in A.