In search of high-affinity and high-selectivity inhibitors of CAs, a series of fluoro-benzenesulfonamide-based compounds were synthesized (Figure 1). The benzenesulfonamide group is important for making a coordination bond with the Zn(II). At the same time, the fluorine atoms were included to withdraw electrons from the sulfonamide group and diminish its pK_a to strengthen interaction. Surprisingly, part of the compounds exhibited extremely strong binding affinity. These compounds contained the -SO₂CH₂CH₂OCOR group that was forming an unexpected covalent bond with the protein.

Figure 1

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It has been known in organic chemistry that compounds bearing this fragment in the presence of bases can rearrange to vinyl-sulfonyl moiety which has been reported as a covalently modifying ‘warhead’ (Tsutsui et al., 1987; Alonso et al., 2003; Ichikawa et al., 1989). To the best of our knowledge, this kind of rearrangement/elimination has not been applied to enzyme covalent inhibitors. We designed benzenesulfonamides with the SO₂CH₂CH₂OCOR group that can form highly reactive electrophilic species without adding additional base by adding multiple fluorine atoms to the benzene ring. Furthermore, the rearrangement occurred only in the CA enzyme active site. The active vinyl-sulfonyl group formed via elimination reaction, which occurred easily due to the strong electron-withdrawing effect of fluorines on the benzene ring (Figure 2).

Figure 2

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The SO₂ group at the para position relative to the sulfonamide group was also necessary for a covalent bond because compounds 4 and 5 bearing the SO or S groups, respectively, did not form the covalent bond with CA isozymes. Moreover, the change of the -O-CO-R group to -NH-CO-R or -CO-O-R also prevented the formation of the covalent bond as illustrated by compounds 2, 10, 7, and 8 that did not form covalent bonds with the protein molecule.

We propose the mechanism where the covalent modification occurs via the elimination mechanism shown in Figure 2, where a basic amino acid residue removes the proton and forms the vinylsulfone moiety, which only then forms a covalent bond with the nitrogen atom of the histidine residue. To check this mechanism, two control compounds, 3 and 9, were synthesized. Both of these compounds did not form a covalent bond with the protein. In compound 9, two methyl groups located at the crucial α carbon replaced the proton that needed to be removed. In compound 3, the third methylene group prevented the β-elimination reaction. Furthermore, the concept of formation of vinylsulfone fragment was demonstrated by synthesizing compound 15 bearing the vinylsulfone itself. This compound readily formed the covalent bond with proteins.

Since it appeared that compounds bearing ester groups (e.g. compounds 17 and 20) may have too high rate of covalent bond formation leading to non-desired modification of non-targeted proteins, we have designed and synthesized compounds bearing the carbamate functional group (e.g. compounds 18 and 22). The carbamate group was more stable than the ester and thus the covalent modification reaction of the protein showed milder rate than the ester.

The cyclooctyl or cyclododecyl rings present in covalent compounds 20–24 and in non-covalent compounds 10–11 have been previously designed by our group to specifically bind to CAIX isozyme and intended not to bind to the other eleven human catalytically active CA isozymes. The ring could fit to the pocket in CAIX, but not so readily to other isozymes (Dudutienė et al., 2014; Zubrienė et al., 2017). The three-way recognition model is shown in Figure 3.

Figure 3

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The covalent bond between the compounds and protein was demonstrated by X-ray crystallography (Figure 4). The crystal structures of compounds 21, 20, and 23 with CAI, CAII, and CAIX, respectively, showed covalent bond formation between the ligands and the histidine residue of the enzymes. In CAI, the ligand forms a covalent bond with His 67, while in CAII and CAIX, the bond is made with His 64 – a residue responsible for proton shuttle function in CA isoforms (Fisher et al., 2005). The distance between the nitrogen atom of histidine and the vinyl carbon atom of the compounds was around 1.5 Å, consistent with the length of the covalent bond, and was visible with strong electron density in all three crystal structures (Figure 4A, B and C). The X-ray crystal structure refinement statistics are provided in Table 1.

Figure 4

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The tail moiety of the ligands is also coordinated by two hydrogen bonds – with Asn 62 and Gln 92 (CAII numbering) in the cases of CAII and CAIX, while in CAI, a possible hydrogen bond is formed with Gln 92 and His 64. Electron density was overall good for the whole ligand in all three crystal structures, with slightly weaker electron density observed in the hydrophobic tail region indicating higher flexibility. The hydrophobic tail part is oriented towards the active site region, which varies most between CA isozymes (the so-called ‘hot spot’ for isozyme (isoform)-selective inhibitor design Alterio et al., 2009).

There are two protein subunits in the CAI structure, the position of the modeled 21 is very similar in both. The electron density of the ligand is better in protein chain B. Sulfonamide group and modification of His67 with para-linker are visible. The fluorine atoms of the benzene ring also could be located clearly, but the electron density of the benzene ring is partially lost. This could be explained by partial occupancy of the modeled conformation of the inhibitor and by rotation of ligand fraction since the ligand is probably not fixed by the covalent bond with His67. The cyclooctyl group is visible in both subunits only partially due to the flexibility of the ring resulting in multiple conformations. This group is oriented towards the hydrophobic part of the active site, defined by Leu131, Ala135, Leu141, and Leu198.

The mass spectra of CA isoforms incubated with compound 12 showed a 319 Da shift compared to pure CA isoforms (except CAIII), equal to compound 12 molar mass without the ester group (Table 2). Moreover, after a 3-min incubation of 1:1 molar ratio of compound 12 with CAXIII protein (29,575 Da), nearly half of the protein was already covalently modified in this time as shown by an additional 29,894 Da peak (319 Da shift, Figure 5A). After 2 hr of incubation, essentially entire protein fraction was covalently modified by the compound. The presence of the minor peak at 30,213 Da (638 Da shift) in Figure 5A indicates that there is a second modification site on CAXIII protein. Figure 5—figure supplements 1–20 show MS spectra after incubation under listed conditions of various CA isozymes with inhibitors.

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The peptide mapping by digestion of the CAXIII with thrombin detected the compound 12 covalently bound exclusively to the peptide containing His64 residue (Figure 5—figure supplements 21 and 22). However, in the ¹H-¹⁵N-HSQC 2D NMR spectrum, upon incubation of CAII isozyme at 1:1 molar ratio for 1 hr with covalent compound 12, in addition to the His64 signal change, we observed a decrease of peak intensity in the N-terminal part of CAII indicating an additional minor fraction of enzyme with covalently bound 12 outside CA active site (Figure 5—figure supplements 23 and 24). Thus, ester compound 12 may be too reactive for fully specific inhibition. The non-covalent compound 6 was incubated with CAII at a tenfold surplus of the compound, but no covalent modification was detected (Figure 5—figure supplement 16).

Therefore, a series of compounds bearing the carbamate leaving group were designed (18, 19, and 22) to reduce chemical reactivity and reduce undesired reactions with non-intended groups. Compound 22 exhibited covalent modification of CAIX, but after 4 hr incubation, there was still a significant part of non-modified protein present. Thus the reaction was significantly slower than with the ester group. The MW of CAIX as calculated from the sequence was 28061.68 Da and matched closely the measured mass of 28060.32 (Figure 5B top panel and Figure 5—figure supplement 20) or 28060.45 (lower panel). The calculated MW of compound 22 was 563.1372 Da and was measured to be 564.1444 Da. The calculated mass of compound 22 without the carbamate leaving group was 426.0895 Da, while the measured difference in Figure 5B was 426.41 Da, a perfect match. Thus, compound 22 exhibited both highly specific and relatively slow modification of CAIX, with a good perspective toward drug design.

The covalent irreversible and non-covalent reversible interaction was also demonstrated by comparing the inhibition of enzymatic activity of CAIX by non-covalent compound 5 and covalent compound 20 and their possibility to be dialyzed out. Both compounds fully inhibited the enzymatic activity of CAIX at 1:1 molar ratio in the same dose-dependent manner. The resultant protein-ligand complex was then subjected to 32 hr dialysis. The CAIX complex with the non-covalent 5 regained 73% of the original enzymatic activity, while the CAIX with covalent 20 did not regain any detectable enzymatic activity (Figure 5C and D). This indicates that the covalent modification irreversibly inhibited the enzymatic activity of CAIX.

To determine the contribution of the primary sulfonamide group on the capability of making a covalent bond with CA, we synthesized a secondary sulfonamide 13 and compared it with the analogous covalent compound 12. After 2 hr of incubation at 10:1 molar surplus of secondary sulfonamide 13, the free CAII protein still dominated, indicating that only a minor fraction of the protein was covalently modified (Figure 5—figure supplement 17B). In comparison, using the same conditions, compound 12 bearing the primary sulfonamide group completely modified CAII (Figure 5—figure supplement 17C). This shows the significant effect of the primary sulfonamide group in guiding the compound into the CA active site and consequent covalent bond formation with His64 amino acid. In the absence of the guiding sulfonamide group, such as in 13, a relatively slow modification most likely occurred on nucleophilic residues different than the His64 in the protein active site. This unintended covalent modification by the secondary-sulfonamide 13 was also observed with isozymes other than CAII. Although this compound should have low affinity to all CA isozymes, it still modified CAXIII at a 10:1 compound surplus molar ratio after 2 hr incubation (Figure 5—figure supplement 18). Despite that, it is important to note that the modification most likely occurred at a different nucleophilic amino acid, not the His64 in the active site.

The presence of non-specific unintended covalent modifications prompted us to synthesize different covalently modifying groups that would be less reactive and more suitable for drug design, such as carbamate compounds 18 and 19, which showed significantly slower (at least by two orders of magnitude) covalent-modification activity compared to the ester compounds (Figure 5—figure supplement 19). Even using less reactive carbamates, CA isozymes were still able to make covalent bonds with more than one inhibitor molecule albeit in a much lower quantity.

The HeLa cell culture was grown under hypoxia and shown to express CAIX on the cell surface reaching the concentration of 2–10 nM, determined by saturating with fluorescein-labeled compound GZ19-32 as previously described (Matulienė et al., 2022). Covalent compounds were added to the cell culture at various concentrations together with 10 nM of GZ19-32 that strongly and specifically binds CAIX. The tested covalent compounds competed for the binding to the CAIX active site in a dose-dependent manner (Figure 6). At high concentrations (e.g. 100 µM, 10,000-fold surplus over CAIX and GZ19-32), the compounds completely outcompeted the CAIX-specific GZ19-32. However, at low concentrations, around 10 nM, the compounds competed with GZ19-32 depending on the compound’s chemical nature. Thus, the covalent compounds were available for binding to CAIX and, most likely, did not bind to other proteins that are expected to be present in abundant quantities on the cell surface. These other proteins certainly have His residues that would have been modified if the non-specific binding occurred.

Figure 6

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Several covalent compounds were chosen to demonstrate the importance of compound structural features for CAIX recognition in cell cultures. Two compounds 12 and 18 were para-substituted benzenesulfonamides, non-selective for CAIX. Compound 24 had a cyclododecyl amino substitution, selectively recognizing CAIX, but slightly too large for optimal binding and solubility. The 20 contains cyclooctylamine substitution at the meta position, exhibiting a high affinity for purified CAIX. Finally, 22 bore the cyclooctylamine substitution and the carbamate leaving group optimized for lower covalent modification activity compared to ester.

All tested covalent compounds competed with the fluorescein-labeled GZ19-32 for the binding to cell surface CAIX in a dose-dependent manner. Their apparent dissociation constants, as determined by the competition with GZ19-32, are listed in Table 3. The para-substituted compounds that are non-selective for CAIX, bound weaker to cell-surface CAIX than the meta substitute-bearing CAIX-selective compounds. The ester compounds bearing both para and meta substitutions designed for CAIX recognition exhibited single-digit nanomolar affinities (4.0 nM for 24 and 1.8 nM for 20). However, carbamate compound 22 exhibited the strongest affinity (300 pM) for cell-surface CAIX among all tested compounds.

The carbamate compound 22 showed the highest affinity for cell-expressed CAIX and irreversibly covalently modified the protein in the active site, thus permanently inhibiting its enzymatic activity. Therefore, this compound is a leader among tested compounds to serve as an anticancer inhibitor of CAIX, highly expressed in hypoxic solid tumors.

Covalent compounds formed an irreversible covalent bond with the protein molecule. This inhibition mode may occur in two stages. In the first stage, the inhibitor interacts with the enzyme due to its affinity to the targeted enzyme. Here, the affinity is determined by the primary sulfonamide group and the hydrophobic substituent in the meta position. The compound is still able to reversibly dissociate and its non-covalent binding affinity is quantified by the dissociation constant K_d, defined as the ratio of dissociation and association rate constants k_off/k_on:

In the second stage, the pre-vinylsulfone compound is chemically transformed into the reactive vinylsulfone electrophile by a basic amino acid of the enzyme at a rate of k_trans. In the final reaction step, the vinylsulfone may form a covalent bond with the nucleophilic residue with a specific inactivation rate constant k_inact. Since the vinylsulfone is highly reactive, k_trans must be rate-limiting and accounting for the apparent inactivation rate, much slower than for vinyl sulfone 15.

It is incorrect to state covalent compound affinities in terms of a conventional dissociation constant K_d. Therefore, the apparent dissociation constant is valid only to a limited extent because if there is an irreversible chemical modification, then eventually all of the protein will be modified independent of the affinity. In our case, we can assume a rapid pre-equilibrium followed by a slow covalent modification. Therefore, relative affinity measurements are valid both by competition assay described above and the fluorescence-based thermal shift assay, described below. However, due to the interplay of kinetic and thermodynamic equilibrium contributions, the affinity measurements should still be considered with caution.

We applied the fluorescence-based thermal shift assay to determine the apparent dissociation constants K_d,app of covalent compounds to arrange them in the order of their apparent affinities (association rate constants). For example, the CAIX-specific covalent compound 22 bound with an extremely tight affinity, the apparent dissociation constant was determined to be 7.8 pM, the highest affinity among known CAIX-binding compounds. The thermal shift was over 18 °C and exhibited a typical flat dosing curve often observed for covalent compounds (Figure 7, Figure 7—figure supplements 1–9). There was a strong shift of the protein melting temperature caused by the compound but no further shift as observed in reversible non-covalent interactions. Using compound 18, we determined that the obtained apparent affinity constants were time-independent, meaning lower errors due to compound-protein incubation time and preparation for FTSA (Figure 7—figure supplements 10–15).

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The observed K_d,app values of all covalent compounds binding to CA isozymes were higher than those of its non-covalent analogs. For example, CAIX binds to the para-substituted esters 12 and 14, forming a covalent bond with the protein, up to 1000 times stronger than the non-covalent para-substituted compounds 1 and 3. Covalent compounds with meta-substituents 20–24 bind CAIX up to 10 times more strongly than their non-covalent analogs 10 and 11. The apparent dissociation constants of covalent compound binding to all 12 human catalytically active CA isozymes as determined by the fluorescence-based thermal shift assay are listed in Table 4. The apparent affinities of covalent compounds can be compared with the non-covalent reversibly-binding analog compounds and estimate the energetic contribution of the covalent bond.

As shown above, compounds with a carbamate leaving group are better than ester groups. The carbamate compounds seemed to have a good balance to enhance interaction with CAIX via a covalent bond and, at the same time, should have sufficiently low reactivity to react with any unintended proteins. It has been demonstrated that, in some cancers, CAXII isozyme is overexpressed instead of CAIX and sometimes both of these isozymes are expressed. Therefore, a dual attack on CAIX and CAXII could be beneficial over a single CAIX interaction. At the same time, inhibition of the remaining 10 CA isozymes is expected to cause more harm than benefit.

As seen on the arrows going from two left non-covalent compounds to the adjacent covalent compounds (Figure 8), there is a significant gain in affinity due to the covalent bond, at least several hundred-fold stronger binding. Second, the presence of the cyclooctyl or cyclododecyl ring at the meta-position relative to sulfonamide increased the affinity for CAIX, but – what is even more significant – greatly reduced compound affinity for non-target CAI and CAII. The covalent CAIX-targeting compounds reached the affinity of single-digit picomolar, an incredibly high value, never reached by any CAIX-binding compounds and rare among any interactions. Despite the apparent selectivity of compound 23 to CAIX compared to CAI, compound 22 is more promising as a drug candidate due to its slower covalent bond formation rate and lower associated off-target toxic effects.

Figure 8

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All recombinant human CA isozymes were produced as described previously by using either bacterial or mammalian expression system (Dudutienė et al., 2014; Mickevičiūtė et al., 2019). For isozymes possessing transmembrane parts, only catalytic domains were produced. The production of the catalytic domain of CAIX in methylotrophic yeast Pichia pastoris was performed as described in Leitans et al., 2015. Protein purity was confirmed by SDS-PAGE, and MW was confirmed by mass spectrometry.

Thermal unfolding experiments of the purified CA isozymes were carried out by a real-time PCR instrument, Rotor-Gene Q, containing six channels. Twofold serial dilutions of the 10 mM compound stock in DMSO were made by adding 10 μL of DMSO to 10 μL of each compound solution. Overall, 8 different compound concentration solutions were prepared, including the 10 mM compound stock concentration and a sample containing only DMSO, without a ligand. To prepare 12 different concentrations of the ligand, 1.5-fold serial dilutions of 10 mM compound stock were performed by adding 10 μL of DMSO to 20 μL of each compound solution (the last sample contained no ligand). Each prepared compound solution was diluted 12.5 times with the assay buffer 50 mM sodium phosphate (pH 7.0), 100 mM NaCl. The 10 μM CA isozyme solution (or 20 μM of CAIV) was prepared in the same assay buffer, which contained a reporter dye (200 μM ANS or 200 x diluted Glomelt). 5 μL of prepared CA solution with dye was added to the 100 μl of PCR tubes. Subsequently, 5 μL of the compound solution is added. The tubes are placed into the real-time thermocycler, and protein unfolding is measured by increasing the temperature from 25°C to 90°C, at the rate of 1 °C/min, and measuring the fluorescence of the dye. The raw data were analyzed to determine the T_m of the proteins. T_m values were plotted as a function of ligand concentration and the model was fitted to the dosing curves to obtain the binding affinities using Thermott software (Gedgaudas et al., 2022). Compound binding affinity and other thermodynamic data will be entered and freely available for download from the repository plbd.org (Protein-Ligand Binding Database; Lingė et al., 2023).

CAIX activity was measured in the absence and presence of the compound before and after dialysis. 1.5 µM CAIX was incubated with 15 μM 20, 15 μM 22, or 100 µM 5 for 2 hr. The solution of CAIX without a compound and the CAIX-compound complexes were then dialyzed in 25 mM Tris buffer solution (pH = 7) containing 50 mM NaCl (by changing buffer four times every 8/16 hours). CO₂ hydration velocities were measured by recording the absorbance of phenol red (final concentration 50 µM) at 557 nm using an Applied Photophysics SX.18MV-R stopped-flow spectrometer. Experiments were performed at 25 °C using 25 mM Hepes containing 50 mM NaCl, pH 7.5.

Mass spectrometry experiments were performed with an electrospray ionization time-of-flight mass spectrometer (Q-TOF). The 0.1 mg/mL CA isozyme solution was prepared in the absence or presence of compounds (1:2; 1:5, or 1:10 CA isozyme: compound molar ratio). The solution was incubated for 1 hr at room temperature before analysis. The final DMSO concentration was 1% (v/v).

All NMR spectra were recorded using a 600 MHz Bruker Avance Neo spectrometer equipped with a cryogenic probe. 2D ¹⁵N–¹H HSQC spectra of ¹⁵N labeled CAII solution (270 µM 15 N CAII, 20 mM sodium phosphate buffer, 50 mM NaCl, 5% D₂O, pH 6.8) were recorded at 25 °C using 256 increments in the indirect dimension and 8 scans. The spectra were recorded when the protein solution contained different concentrations of covalent ligand: 0.27 mM, 0.53 mM, 1.0 mM and 1.5 mM (the final DMSO concentration was 7.5%) or non-covalent ligand: 0.35 mM, 0.70 mM, 1.0 mM, 2.0 mM (the final DMSO concentration was 3%). The spectra were analyzed using Topspin 4.1.3 and CcpNMR 2.4.1 analysis software (Vranken et al., 2005).

The HPLC separation was done as previously described (Sakalauskas et al., 2022). In brief, the samples were separated and analyzed using Shimadzu UFLC system with a CMB-20A communication module, two LC20AD quaternary and isocratic pumps, a SIL-20AC autosampler, a CTO-20A column compartment and an SPD-M20A DAD detector (Shimadzu Corp., Japan). For the detection of the eluting molecules, the DAD spectra recording was set from 190 to 750 nm with a data rate of 6.25 Hz. The ACE C18-PFP HPLC separation column (100x4.6, 3 µm, Avantor) was used as a stationary phase. The HPLC grade MeCN (Fisher Scientific) and Milli-Q water (18.2 MΩ cm⁻¹, Milli-Q Plus system, Millipore Bedford, MA, USA) were used for the RP-HPLC separation.

The samples were separated using a trinary gradient consisting of ultrapure water (eluent A), MeCN (eluent B), and 1% TFA in ultrapure water (eluent C). A constant 10% flow of eluent C was used to maintain 0.1% TFA concentration in the column throughout the separation experiment. The gradient between eluents A and B was 36% (0 min), 63% (20 min), and 63% (21 min). Before each analytical run, the column equilibration (10 column volume) was performed. The column thermostat was set to 40 °C and the flow rate to 1 mL min^–1.

The 0.1 mg/ml CAXIII solution was prepared with compound 12 (molar ratio 1:2 CAXIII: 12) and filter-aided sample preparation (FASP) (Wiśniewski et al., 2009) was used for protein digestion before mass spectrometry analyses.

DDA was performed with the nanoAcquity coupled to a Synapt G2 HDMS mass spectrometer (Waters). For DDA, the instrument performed a 0.7 s MS scan (350–1350 scan range) followed by MS/MS acquisition on the top 5 ions with charge states 2+, 3+, and 4+. MS/MS scan range was 50–2000 Da, 0.6 s scan duration with exclusion after 2 MS/MS scans were acquired, and dynamic exclusion of ions within 100 mDa of the selected precursor m/z was set to 100 s.

The Progenesis QI for proteomics software (Nonlinear Dynamics), in combination with the Mascot server (2.2.07) was employed to identify peptides. The acquired raw files were imported into the Progenesis QI for proteomics software and MS2 spectra were exported directly from Progenesis in mgf format and searched using the MASCOT algorithm.

Human CAI in buffer containing 20 mM HEPES pH 7.6 and 50 mM NaCl was mixed with the 21 in DMSO in the ratio 1:1.1 and incubated for 2.5 hr at room temperature. The protein-21 complex was concentrated at 30 mg/ml, and crystallization in sitting drops was started. The crystallization solution contained 0.1 M Tris-HCl pH 8.5, 0.2 M NaCl, and 28% (w/v) PEG3350. Before cryo-cooling crystal was shortly incubated in cryo-protection buffer containing 0.1 M TrisHCl pH8.5, 15% (w/v) PEG8000, and 20% (v/v) Ethylene glycol. The synchrotron data was collected at beamline P13 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany).

Diffraction data were integrated by XDS (Kabsch, 2010), scaled using AIMLESS 0.7.4 and other CCP4 tools v. 7.1.002 (Agirre et al., 2023). The structure was solved by molecular replacement using MOLREP v.11.7.02 (Vagin and Teplyakov, 2010) and 2CAB as an initial model. The model was refined by REFMAC v. 5.8.0258 (Murshudov et al., 2011) and rebuilt in COOT v.0.9 (Emsley et al., 2010). Inhibitor model was created and minimized using AVOGADRO v. 1.2.0 (Hanwell et al., 2012).

CAII protein was concentrated at 10 mg/ml in a 20 mM Tris-HCl buffer. It was then mixed with compound 20 (5 mM final concentration) and incubated overnight at 4 °C. Previously known crystallization conditions for CAII did not yield any crystals when co-crystallizing with compound 20. Crystallization condition screening was performed using the Morpheus screen from Molecular Dimensions. After slight optimization, crystals were grown using the sitting drop technique in 0.06 M magnesium chloride hexahydrate; 0.06 M calcium chloride dihydrate; 0.1 M Tris pH 8.5; 20% v/v PEG 500 MME; 10% w/v PEG 20000 conditions diffracted at 1.4 Å resolution. The alteration in crystallization conditions was likely due to the formation of a covalent bond between the ligand and the enzyme. The dataset of the CAII-ligand complex was collected at BESSY II beamline 14.1 and processed using MOSFLM (Battye et al., 2011) and SCALA (Evans, 2006). Molecular replacement was performed using MOLREP (Vagin and Teplyakov, 2010) with 5AMD (Ivanova et al., 2015) as the initial model.

CAIX protein was concentrated to 10 mg/ml in a 20 mM Tris-HCl buffer. It was then mixed with compound 23 (5 mM final concentration) and incubated overnight at 4 °C. Crystals grew using similar co-crystallization conditions as described before (Leitans et al., 2015).

The dataset of the CAIX-ligand complex was collected at the Diamond Light Source beamline I03 and processed using XDS (Kabsch, 2010) and AIMLESS (Agirre et al., 2023). Molecular replacement was performed using MOLREP (Vagin and Teplyakov, 1997) with 8Q18 (Leitans et al., 2023) as the initial model.

Model refinement was performed with REFMAC (Murshudov et al., 2011), and the structures were visualized using COOT (Emsley et al., 2010). Ligand parameter files were generated using LIBCHECK (Lebedev et al., 2012), and the ligand was manually fitted to the electron density map in COOT (Emsley et al., 2010). The coordinates and structure factors have been deposited in the PDB. Table ST1 provides the PDB access code, data collection, and refinement statistics.

Human cervical adenocarcinoma cells (HeLa) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX (Gibco, ThermoFisher) supplemented with 10% fetal bovine serum (ThermoFisher) in a humidified atmosphere at 37 °C and 5% CO₂.

A covalent compound competition experiment with fluorescein-labeled compound GZ19-32 was conducted as described previously (Matulienė et al., 2022). In brief, HeLa cells were cultivated in DMEM in 12-well plates under hypoxic conditions (1% O₂) for 72 hr. The 10 serial twofold dilutions of covalent compounds were prepared in FluoroBrite DMEM (ThermoFisher) starting with 80 nM (1^st tube). No compound was added to the last 12th tube (it contained FluoroBrite only). Subsequently, the same volume of 20 nM GZ19-32 was added to each of the 12 tubes and mixed. Cell culture medium was removed from all 12 wells with HeLa cells and 200 µl of prepared compound mixtures were added, followed by 20 min incubation at 37 ℃ under normoxic conditions (21% O₂). Post-incubation, the compound solutions were aspirated, and the cells were washed 3 times with 400 µl of PBS. Finally, the cells were detached from the well plate surface by TrypLE (ThermoFisher), resuspended by pipetting in 200 µl of FluoroBrite DMEM, and 150 µl of the suspension was transferred to black Thermo Scientific Nunc MicroWell 96-Well Optical-Bottom Plates for fluorescence and absorbance measurements.

Compound numbering includes the intermediate compounds, and, therefore, there are two numbering systems. Compounds are marked with x to help visualize the path of synthesis and the numbered compounds are listed in the brackets. Chemical compounds may be provided upon reasonable request for research purposes if available in stock. Compound synthesis paths are shown in Figures 9—11.

Figure 9

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Figure 10

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Figure 11

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All starting materials and reagents were commercially available or prepared according to known procedures. Melting points of the compounds were determined in open capillaries on a Thermo Scientific 9100 Series and are uncorrected. ¹H and ¹³C NMR spectra were recorded on a Bruker spectrometer (400 and 100 MHz, respectively) in DMSO-D₆ or CDCl₃ using residual DMSO, CDCl₃ signals (2.50 ppm, 7.26 ppm, and 39.52 ppm, 77.16 ppm for ¹H and ¹³C NMR spectra, respectively) as the internal standard. ¹⁹F NMR spectra were recorded on a Bruker spectrometer (376 MHz) with CFCl₃ as an internal standard. TLC was performed with silica gel 60 F254 aluminum plates (Merck) and visualized with UV light. Column chromatography was performed using silica gel 60 (0.040–0.063 mm, Merck). High-resolution mass spectra (HRMS) were recorded on a Dual-ESI Q-TOF 6520 mass spectrometer (Agilent Technologies). Compound IUPAC names were generated with ChemDraw ultra 12.0.

2,3,4,5,6-pentafluorobenzenesulfonamide (2ax) was prepared according to known procedure in the literature (Dudutienė et al., 2013). To a 100 ml cooled to –10 °C temperature THF, 2,3,4,5,6-pentafluorobenzenesulfonyl chloride (1x) (5 ml; 33.70 mmol; 1 eq.) was dissolved and NH₃ (10 %) (10 ml) was slowly added dropwise while mixing as well as keeping reaction temperature under –10 °C. After reaction completion mixture was basified to pH 8–9 and left stirring for an additional hour at room temperature. The product was recrystallized from H₂O and white crystals were obtained. Yield: 5.33 g; (64%). Mp: 154–155°C (close to the value in the literature [Dudutienė et al., 2013], mp: 155–156°C).

2,3,4,5,6-pentafluoro-N-methylbenzenesulfonamide (2bx) synthesis was described in Baronas et al., 2021. To a 100 ml cooled to –10 °C temperature THF, 2,3,4,5,6-pentafluorobenzenesulfonyl chloride (1x) (2 ml; 13.47 mmol; 1 eq.) was dissolved and 2 M methylamine in methanol (0.837 g; 26.9 mmol; 2 eq.) was slowly added dropwise while mixing as well as keeping reaction temperature under –10 °C. The reaction process was monitored using TLC and after reaction completion, THF was evaporated under reduced pressure. The product was recrystallized from MeOH:H₂O (1:4) and white crystals were obtained. Yield: 2.6 g; (74%). Mp: 95–97°C (close to the value in the literature [Baronas et al., 2021], mp: 96–97°C).

2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)thio)benzensulfonamide (3ax) was prepared according to the known procedure in literature (Dudutienė et al., 2013). 2,3,4,5,6-pentafluorobenzensulfonamide (2ax) (1.000 g; 4.05 mmol; 1 eq.), 2-mercaptoethanol (0.341 ml; 4.86 mmol; 1.2 eq.), Et₃N (0.677 ml; 4.86 mmol; 1.2 eq.) were dissolved in MeOH (20 ml) and left to stir at room temperature overnight. Next morning additional 2-mercaptoethanol (0.085 ml; 1.21 mmol; 0.3 eq.) and Et₃N (0.169 ml; 1,21 mmol; 0.3 eq.) portions were added and the reaction mixture was left to stir for 2 hours. After reaction completion, the solvent was evaporated under reduced pressure and the resultant precipitate was washed with H₂O. Recrystallization was accomplished from H₂O. Yield: 1.029 g; (83%). Mp: 111–112°C (close to the value in the literature [Dudutienė et al., 2013], mp: 111–112°C).

2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)thio)-N-methylbenzenesulfonamide (3bx) was prepared according to the known procedure in the literature (Baronas et al., 2021). 2,3,4,5,6-Pentafluoro-N-methylbenzenesulfonamide (2bx) (0.500 g; 1.92 mmol; 1eq.), 2-mercaptoethanol (0.175 ml; 2.50 mmol; 1.3 eq.), Et₃N (0.349 ml, 2.50 mmol, 1.3 eq.) were dissolved in MeOH (20 ml) and left to stir at room temperature for 20 hr. After reaction completion, the solvent was evaporated under reduced pressure and the resultant precipitate was washed with H₂O. Recrystallization was accomplished from H₂O. Yield: 0.464 g; (76%). Mp: 99 °C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.62 (3H, s, SO₂NHCH₃) 3.15 (2H, t, J=6.1 Hz, SCH₂), 3.59 (2H, q, J=5.9 Hz, CH₂O), 4.94 (1H, t, J=5.4 Hz, OH), 8.42 (1H, s, NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 28.28 (SO₂NHCH₃), 36.52 (SCH₂, t, J (¹⁹F – ¹³C)=3.0 Hz), 60.66 (CH₂O), 118.23 (C1, t, J (¹⁹F – ¹³C)=15.8 Hz), 119.99 (C4, t, J (¹⁹F – ¹³C)=20.2 Hz), 143.01 (C2 and C6, ddt, ¹J (¹⁹F – ¹³C)=253.7 Hz, ²J (¹⁹F – ¹³C)=16.9 Hz, ³J (¹⁹F – ¹³C)=4.5 Hz), 146.41 (C3 and C5, ddt, ¹J (¹⁹F – ¹³C)=243.8 Hz, ²J (¹⁹F – ¹³C)=15.4 Hz, ³J (¹⁹F – ¹³C)=3.9 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –132.79 to -132.97 (2F, m), −139.08 to -139.25 (2 F, m).

2,3,5,6-tetrafluoro-4-((3-hydroxypropyl)thio)benzenesulfonamide 3cx. 2,3,4,5,6-pentafluorbenzensulfonamide (2ax) (0.400 g; 1.62 mmol), 3-mercaptopropan-1-ol (0.167 ml; 1.94 mmol; 1.2 eq.), Et₃N (0.270 ml; 1.94 mmol; 1,2 eq.) were dissolved in MeOH (15 ml) and left to stir at room temperature over night. Next morning additional 3-mercaptopropan-1-ol (0.027 ml; 0.32 mmol; 0.2 eq.) and Et₃N (0.044 ml; 0.32 mmol; 0.2 eq.) portions were added and reation mixture was left to stir for 3 hr. After reaction completion the solvent was evaporated under reduced pressure and product was purified by column chromatography silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.39. Yield 0.386 (75%). Mp: 148–149°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.65 (2H, p, J=6.3 Hz, CH₂CH₂CH₂), 3.09 (2H, t, J=7.3 Hz, CH₂S), 3.46 (2H, t, J=6.0 Hz, CH₂O), 8.39 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 30.77 (SCH₂, t, J (¹⁹F-¹³C)=3.1 Hz), 32.68 (CH₂CH₂CH₂), 58.70 (CH₂O), 118.67 (C1, t, J (¹⁹F – ¹³C)=20.4 Hz), 122.46 (C4, t, J (¹⁹F – ¹³C)=15.7 Hz), 142.54 (C2 and C6, ddt, ¹J (¹⁹F – ¹³C)=253.6 Hz, ²J (¹⁹F – ¹³C)=16.9 Hz, ³J (¹⁹F – ¹³C)=4.3 Hz), 146.48 (C3 and C5, ddt, ¹J (¹⁹F – ¹³C)=245.3 Hz, ²J (¹⁹F – ¹³C)=13.9 Hz, ³J (¹⁹F – ¹³C)=3.8 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –133.23 to -133.38 (2F, m), −139.04 to -139.19 (2 F, m). HRMS for C₉H₉F₄NO₃S₂ [(M+H)⁺]: calc. 320.0033, found 320.0028.

2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (4ax) was prepared according to the known procedure in the literature (Dudutienė et al., 2013). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)thio)benzensulfonamide (3ax) (1.542 g; 5.06 mmol; 1 eq.) was dissolved in acetic acid (30 ml) and heated at 75 °C temperature for 18 hr. H₂O₂ (30%) was added in portions (0.1 ml) every 30 min (overall 3.6 ml) until complete starting material conversion. Afterward, the solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.30). Yield: 0.768 g; (45%). Mp: 138–139°C (close to the value in the literature [Dudutienė et al., 2013], mp: 139–140°C).

Compounds 5bx and 4bx were synthesized during compound 3bx oxidation.

2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)thio)-N-methylbenzenesulfonamide (3bx) (0.300 g, 0.94 mmol) was dissolved in acetic acid (10 ml) and heated at 75 °C temperature for 21 hr. H₂O₂ (30%) was added in portions (0.1 ml) every hour (overall 2.1 ml) until complete starting material conversion. Afterward, the solvent was evaporated under reduced pressure and products were purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf_4bx=0.53, Rf_5bx=0.79).

2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)-N-methylbenzenesulfonamide (4bx). Yield: 0.119 g; (36%). Mp: 201 °C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.65 (3H, s, SO₂NHCH₃), 3.76 (2H, t, J=5.6 Hz, SO₂CH₂), 3.86 (2H, q, J=5.2 Hz, CH₂O), 4.97 (1H, t, J=5.3 Hz, OH), 8.68 (1H, s, NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 28.31 (SO₂NHCH₃), 55.07 (SO₂CH₂), 59.54 (CH₂O), 122.97 (C1, t, J (¹⁹F – ¹³C)=15.0 Hz), 123.86 (C4, t, J (¹⁹F – ¹³C)=15.7 Hz), 143.35 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=255.2 Hz, ²J (¹⁹F – ¹³C)=15.7 Hz), 144.34 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=244.6 Hz, ²J (¹⁹F – ¹³C)=13.3 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –136.00 (2F, dd, ¹J=26.2 Hz, ²J=12.7 Hz), –136.61 to -136.77 (2F, m). HRMS for C₉H₉F₄NO₅S₂ [(M-H)^-]: calc. 349.9786, found 349.9785.

2-((2,3,5,6-Tetrafluoro-4-(N-methylsulfamoyl)phenyl)sulfonyl)ethyl acetate (5bx). Yield: 0.035 g; (9%). Mp: 143 °C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.85 (3H, s, COCH₃), 2.65 (3H, d, J=4.6 Hz, SO₂NHCH₃), 4.01 (2H, t, J=5.4 Hz, SO₂CH₂), 4.41 (2H, t, J=5.7 Hz, CH₂O), 8.73 (1H, q, J=4.6 Hz, NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.11 (COCH₃), 28.32 (SO₂NHCH₃), 55.95 (SO₂CH₂), 57.21 (CH₂O), 121.74 (C1, t, J (¹⁹F – ¹³C)=14.9 Hz), 124.46 (C4, t, J (¹⁹F – ¹³C)=16.0 Hz), 143.64 (C2 and C6, d, ¹J (¹⁹F – ¹³C)=255.6 Hz), 144.57 (C3 and C5, d, ¹J (¹⁹F – ¹³C)=264.6 Hz), 169.56 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.37 to -135.56 (2F, m), −136.09 to -136.30 (2 F, m). HRMS for C₁₁H₁₁F₄NO₆S₂ [(M-H)^-]: calc. 391.9891, found 391.9891.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl acetate (5ax). 2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.153 g; 0.45 mmol), acetic acid (0.260 ml; 4.54 mmol; 10 eq.) and one drop of H₂SO₄ (conc.) were dissolved in toluene (25 ml) and refluxed for 3 hours. The resulting mixture was cooled to 5 °C and white crystals were filtered. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.40). Yield: 0.134 g; (78%). Mp: 153–154°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.83 (3H, s, CH₃), 4.02 (2H, t, J=5.3 Hz, SO₂CH₂), 4.40 (2H, t, J=5.4 Hz, CH₂O), 8.69 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.07 (CH₃), 55.91 (SO₂CH₂), 57.28 (CH₂O), 121.33 (C1, t, J (¹⁹F – ¹³C)=15.3 Hz), 127.86 (C4, t, J (¹⁹F – ¹³C)=15.7 Hz), 142.23 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=252.0 Hz, ²J (¹⁹F – ¹³C)=20.2 Hz), 144.33 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=259.5 Hz, ²J (¹⁹F – ¹³C)=18.6 Hz), 169.52 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.60 to -135.78 (2F, m), −136.60 to -136.78 (2 F, m). HRMS for C₁₀H₉F₄NO₆S₂ [(M-H)^-]: calc. 377.9735, found 377.9735.

2,3,5,6-Tetrafluor-4-[2-(acetil)propilsulfonil]benzensulfonamidas (5cx). 2,3,5,6-tetrafluoro-4-((3-hydroxypropyl)thio)benzenesulfonamide (3cx) (0.378 g; 0.95 mmol) was dissolved in acetic acid (10 ml) and heated at 75 °C temperature for 18 hr. H₂O₂ (30%) was added in portions (0.1 ml) every 30 min (overall 2.1 ml) until complete starting material conversion. Afterward, the solvent was evaporated under reduced pressure, and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.64). Yield: 0.216 g; (58%). Mp: 188 °C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.00 (3H, s, CH₃), 2.03 (2H, p, J=7.8 Hz, CH₂CH₂CH₂) 3.66 (2H, t, J=7.8 Hz CH₂SO₂), 4.08 (2H, t, J=6.4 Hz, CH₂O), 8.64 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.66 (CH₃), 21.39 (CH₂CH₂CH₂), 53.71 (SO₂CH₂), 61.58 (CH₂O), 120.30 (C1, t, J (¹⁹F – ¹³C)=15.0 Hz), 127.74 (C4, t, J (¹⁹F – ¹³C)=15.4 Hz), 142.99 (C2 and C6, d, J (¹⁹F – ¹³C)=254.1 Hz), 144.46 (C3 and C5, d, J (¹⁹F – ¹³C)=261.0 Hz), 170.39 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135,62–-135,82 (2F, m), −136,35–-136,58 (2 F, m). HRMS for C₁₁H₁₁F₄NO₆S₂ [(M-H)^-]: calc. 391.9891, found 391.891.

2-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl propionate (5dx). 2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.152 g; 0.451 mmol), propionic acid (2 ml) and three drops of H₂SO₄ (conc.) were dissolved in toluene (25 ml) and refluxed for 1 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromotography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.74). Yield: 0.0448 g; (25%). Mp: 109–111°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 0.93 (3H, t, J=7.5 Hz, CH₂CH₃), 2.08 (2H, q, J=7.5 Hz, CH₂CH₃), 4.03 (2H, t, J=5.6 Hz, SO₂CH₂), 4.42 (2H, t, J=5.6 Hz, CH₂O), 8.69 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 8.63 (CH₃), 26.36 (CH₂CH₃), 56.00 (SO₂CH₂), 57.22 (CH₂O), 121.32 (C1, t, J (¹⁹F – ¹³C)=14.6 Hz), 127.85 (C4, t, J (¹⁹F – ¹³C)=15.2 Hz), 142.90 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=256.1 Hz, ²J (¹⁹F – ¹³C)=18.8 Hz), 144.36 (C2 and C5, dd, ¹J (¹⁹F – ¹³C)=259.1 Hz, ²J (¹⁹F – ¹³C)=20.4 Hz), 172.82 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.61 to -135.83 (2F, m), −136.58 to -136.79 (2 F, m). HRMS for C₁₁H₁₁F₄NO₆S₂ [(M-H)^-]: calc. 391.9891, found 391.9892.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl pivalate (5ex). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.050 g; 0.015 mmol), pivalic acid (0.076 g; 0.074 mmol; 5 eq.) and three drops of H₂SO₄ (conc.) were dissolved in toluene (10 ml) and refluxed for 30 min. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.62). Yield: 0.018 g; (29%). Mp: 67–68°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.02 (9H, s, C(CH₃)₃), 4.04 (2H, t, J=5.4 Hz, SO₂CH₂), 4.42 (2H, t, J=5.5 Hz, CH₂O), 8.70 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 26.46 (C(CH₃)₃), 38.00 (C(CH₃)₃), 56.32 (SO₂CH₂), 57.35 (CH₂O), 121.12 (C1, t, J (¹⁹F – ¹³C)=14.9 Hz), 127.94 (C4, t, J (¹⁹F – ¹³C)=15.4 Hz), 143.01 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=257.3 Hz, ²J (¹⁹F – ¹³C)=13.4 Hz), 144.35 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=256.9 Hz, ²J (¹⁹F – ¹³C)=16.7 Hz), 176.84 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.72 to -135.90 (2F, m), −136.46 to -136.63 (2 F, m). HRMS for C₁₃H₁₅F₄NO₆S₂ [(M-H)^-]: calc. 420.0204, found 420.0202.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl 2-phenylacetate (5fx). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.041 g; 0.122 mmol; 1 eq.), phenylacetic acid (0.075 g; 0.551 mmol; 5 eq.) and three drops of H₂SO₄ (conc.) were dissolved in toluene (15 ml) and refluxed for 1 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.71). Yield: 0.0282 g; (51%). Mp: 77–79°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 3.49 (2H, s, OC(O)CH₂), 4.05 (2H, t, J=5.5 Hz, SO₂CH₂), 4.46 (2H, t, J=5.5 Hz, CH₂O), 7.19 (2H, d, J=6.9 Hz, phenyl), 7.26 (1H, t, J=7.2 Hz, phenyl), 7,31 (2H, t, J=7.1 Hz, phenyl), 8.69 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 40.68 (COCH₂), 55.96 (SO₂CH₂), 57.55 (CH₂O), 121.24 (C1, t, J (¹⁹F – ¹³C)=14.8 Hz), 126.95 (C4 of phenyl), 127.90 (C4, t, J (¹⁹F – ¹³C)=15.7 Hz), 128.36 (phenyl), 142.96 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=259.0 Hz, ²J (¹⁹F – ¹³C)=17.7 Hz), 144.33 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=254.2 Hz, ²J (¹⁹F – ¹³C)=18,6 Hz), 170.50 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.53 to -135.70 (2F, m), −136.43 to -136.60 (2 F,m). HRMS for C₁₆H₁₃F₄NO₆S₂ [(M-H)^-]: calc. 454.0048, found 454.0044.

Methyl 3-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)thio)propanoate (6x). 2,3,4,5,6-Pentafluorbenzensulfonamide (2ax) (0.675 g; 2.73 mmol), methyl 3-mercaptopropanoate (0.394 ml, 3.55 mmol, 1.3 eq.), Et₃N (0.456 ml, 3.28 mmol, 1.2 eq.) were dissolved in MeOH (15 ml) and left to stir at room temperature for 0.5 hr. After reaction completion, the solvent was evaporated under reduced pressure and the resultant precipitate was washed with H₂O. Recrystallization was accomplished from H₂O. Yield: 0.921 g; (97%). Mp: 124–125°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.66 (2H, t, J=6.8 Hz, CH₂C(O)), 3.22 (2H, t, J=6.8 Hz, SCH₂), 3.56 (3H, s, CH₃O), 8.43 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 29.22 (SCH₂, t, J (¹⁹F – ¹³C)=2.8 Hz), 34.42 (CH₂C(O)O), 51.54 (CH₃O), 117.71 (C1, t, J (¹⁹F – ¹³C)=20.5 Hz), 122.86 (C4, t, J (¹⁹F – ¹³C)=15.6 Hz), 142.52 (C2 and C6, ddt, ¹J (¹⁹F – ¹³C)=253.7 Hz, ²J (¹⁹F – ¹³C)=16.8 Hz, ³J (¹⁹F – ¹³C)=4.4 Hz), 146.70 (C3 and C5, ddt, ¹J (¹⁹F – ¹³C)=243.6 Hz, ²J (¹⁹F – ¹³C)=15.8 Hz, ³J (¹⁹F – ¹³C)=3.4 Hz), 171.35 (C(O)O). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –132.67 to -132.83 (2F, m), −139.06 to -139.23 (2 F, m). HRMS for C₁₀H₉F₄NO₄S₂ [(M-H)^-]: calc. 345.9836, found 345.9836.

Methyl 3-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)sulfonyl)propanoate (7x). Methyl 3-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)thio)propanoate (6x) (0.043 g; 0.12 mmol) was dissolved in acetic acid (5 ml) and heated at 75 °C temperature for 4.5 hr. H₂O₂ (30%) was added in portions (0.1 ml) every 1.5 hr (overall 0.3 ml) until complete starting material conversion. Afterwards, the solvent was evaporated under reduced pressure and the product was recrystallized from H₂O. Yield: 0.032 g; (70%). Mp: 164 °C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.84 (2H, t, J=7.2 Hz, CH₂C(O)), 3.59 (3H, s, CH₃O), 3.85 (2H, t, J=7.2 Hz, SO₂CH₂), 8.67 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 26.93 (CH₂C(O)), 51.98 (CH₃O), 52.51 (SO₂CH₂), 120.14 (C1, t, J (¹⁹F – ¹³C)=15.0 Hz), 127.89 (C4, t, J (¹⁹F – ¹³C)=15.6 Hz), 143.01 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=259.7 Hz, ²J (¹⁹F – ¹³C)=16.7 Hz), 144.56 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=254.3 Hz, ²J (¹⁹F – ¹³C)=14.9 Hz), 170.12 (C(O)O). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135,68 -: –135,88 (2F, m), –136,56 -: –136,76 (2F, m). HRMS for C₁₀H₉F₄NO₆S₂ [(M-H)^-]: calc. 377.9735, found 377.9735.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)thio)ethyl acetate (8x). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)thio)benzenesulfonamide (3ax) (0.104 g; 0.30 mmol; 1eq.), acetic acid (10 ml) and two drops of H₂SO₄ (conc.) were dissolved in toluene (35 ml) and refluxed for 2 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromotography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.76). Yield: 0.078 g; (65%). Mp: 108–110°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.90 (3H, s, CH₃), 3.29 (2H, t, J=5.9 Hz, SCH₂), 4.16 (2H, t, J=5.3 Hz, CH₂O), 8.44 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.27 (CH₃), 32.56 (SCH₂, t, J (¹⁹F – ¹³C)=2.8 Hz), 63.15 (CH₂O), 117.89 (C4, t, J (¹⁹F – ¹³C)=20.5 Hz), 122.94 (C1, t, J (¹⁹F – ¹³C)=15.6 Hz), 142.48 (C3 and C5, ddt, ¹J (¹⁹F – ¹³C)=253.7 Hz, ²J (¹⁹F – ¹³C)=17.2 Hz, ³J (¹⁹F – ¹³C)=4.4 Hz), 146.72 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=240.8 Hz, ²J (¹⁹F – ¹³C)=13.9 Hz), 169.95 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –132.44 to -132.69 (2F, m), −139.10 to -139.30 (2 F, m). HRMS for C₁₀H₉F₄NO₄S₂ [(M-H)^-]: calc. 345.9836, found 345.9838.

2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfinyl)benzenesulfonamide (9x). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)thio)benzenesulfonamide (3ax) (0.095 g; 0.31 mmol; 1eq.), H₂O₂ (0.2 ml; 30%) were dissolved in acetic acid (4 ml) and left to stir at room temperature for 20 hr (after 2 hr additional portion of H₂O₂ (0.2 ml; 30%) was added). Afterward, the solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.13). Yield: 0.081 g; (81%). Mp: 160–161°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 3.39–3.49 (1H, m, SOCH₂), 3.59 (1H, dt, ¹J=13.2 Hz, ²J=4.0 Hz, SOCH₂), 3.84 (2H, q, J=4.8 Hz, CH₂OH), 5.18 (1H, t, J=5.0 Hz), 8.55 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 54.20 (SO₂CH₂), 56.90 (CH₂O), 125.50 (C1, t, J=15.4 Hz), 126.09 (C4, t, J=17.6 Hz), 142.42 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=247.8 Hz, ²J (¹⁹F – ¹³C)=16.4 Hz), 144.34 (C3 and C5, ddt, ¹J (¹⁹F – ¹³C)=252.3 Hz, ²J (¹⁹F – ¹³C)=15.7 Hz, ³J (¹⁹F – ¹³C)=5.9 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –137.61 to -137.80 (2F, m), −139.07 to -139.26 (2 F, m). HRMS for C₈H₇F₄NO₄S₂ [(M+H)⁺]: calc. 321.9825, found 321.9824.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)sulfinyl)ethyl acetate (10x). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfinyl)benzenesulfonamide (9x) (0.081 g; 0.22 mmol), acetic acid (2 ml) and 3 drops of H₂SO₄ (conc.) were dissolved in toluene (30 ml) and refluxed for 1 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.36). Yield: 0.023 g; (25%). Mp: 132–133°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.96 (3H, s, CH₃), 3.68–3.81 (2H, m, SO₂CH₂), 4.36–4.49 (2H, m, CH₂O), 8.58 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.30 (CH₃), 52.52 (SO₂CH₂), 57.06 (CH₂O), 125.33 (C1, t, J (¹⁹F – ¹³C)=17.1 Hz), 125.80 (C4, t, J (¹⁹F – ¹³C)=16.0 Hz), 142.47 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=251.9 Hz,²J (¹⁹F – ¹³C)=15.3 Hz), 144.35 (C3 and C5, ¹J (¹⁹F – ¹³C)=252.7 Hz, ²J (¹⁹F – ¹³C)=14.9 Hz), 169.92 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –137.51 to -137.68 (2F, m), −138.94 to -139.10 (2 F, m). HRMS for C₁₀H₉F₄NO₅S₂ [(M-H)^-]: calc. 361.9786, found 361.9794.

2,3,5,6-Tetrafluoro-4-(vinylsulfonyl)benzenesulfonamide (11x). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (3ax) (0.200 g; 0.592 mmol; 1 eq.), thionyl chloride (0.064 ml; 0.85 mmol; 1.5 eq.), Et₃N (0.006 ml; 0.43 mmol; 0.75 eq.) were dissolved in MeCN (2 ml) and left to stir at room temperature for 52 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.64). Yield: 0.173 g; (92%). Mp: 174–176°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 6.54 (1H, d, J=17.0 Hz, CHCH₂), 6.57 (1H, d, J=23.6 Hz, CHCH₂), 7.36 (1H, dd, ¹J=16.3 Hz, ²J=9.8 Hz, CHCH₂), 8.63 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 121.79 (C1, t, J (¹⁹F – ¹³C)=14.2 Hz), 128.17 (C4, t, J (¹⁹F – ¹³C)=15.7 Hz), 133,34 (CH₂), 138,12 (CH), 143.45 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=259.0 Hz, ²J (¹⁹F – ¹³C)=11.1 Hz) 144.49 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=256.0 Hz, ²J (¹⁹F – ¹³C)=14.8 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.89 to -136.11 (2F, m), −136.43 to -136.72 (2 F, m). HRMS for C₈H₅F₄NO₄S₂ [(M-H)^-]: calc. 317.9523, found 317.9524.

2,3,5,6-Tetrafluoro-4-((1-hydroxy-2-methylpropan-2-yl)thio)benzenesulfonamide (12x). 2,3,4,5,6-pentafluorobenzensulfonamide (2ax) (0.753 g; 3.04 mmol; 1 eq.), 2-mercapto-2-methylpropan-1-ol (0.388 g; 3.66 mmol; 1.2 eq.), Et₃N (0.510 ml; 3.66 mmol; 1.2 eq.) were dissolved in MeOH (10 ml) and left to stir at room temperature overnight. Afterward, the solvent was evaporated under reduced pressure and the resultant precipitate was washed with H₂O. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.61). Yield: 0.690 g; (68%). Mp: 146–147°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.22 (6H, s, CH₃), 3.39 (2H, d, J=5.5 Hz, CH₂OH), 5.12 (1H, s, OH), 8.44 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 25.29 (CH₃), 55.27 (CH₂OH), 69.96 (SC(CH₃)₂), 115.08 (C1, t, J (¹⁹F – ¹³C)=22.2 Hz), 124.57 (C4, t, J (¹⁹F – ¹³C)=15.6 Hz), 142.53 (C2 and C6, ddt, ¹J (¹⁹F – ¹³C)=254.5 Hz, ²J (¹⁹F – ¹³C)=17.5 Hz, ³J (¹⁹F – ¹³C)=4.1 Hz), 148.32 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=244.0 Hz, ²J (¹⁹F – ¹³C)=14.6 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –128.45 to -128.60 (2F, m), −138.67 to -138.84 (2 F, m). HRMS for C₁₀H₁₁F₄NO₃S₂ [(M+H)⁺]: calc. 356.0009, found 355.9999.

Compounds 13x and 14x were synthesized during 12x oxidation.

2,3,5,6-Tetrafluoro-4-((1-hydroxy-2-methylpropan-2-yl)thio)benzenesulfonamide (12x) (0.676 g, 2.03 mmol) was dissolved in acetic acid (10 ml) and heated at 75 °C temperature for 2.5 hr. H₂O₂ (30%) was added in portions (0.2 ml) every ~0.3 H (overall 0.8 ml) until complete starting material conversion. Afterward, the solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf_{14x (9)}=0.66, Rf_13x=0.55).

2,3,5,6-Tetrafluoro-4-((1-hydroxy-2-methylpropan-2-yl)sulfonyl)benzenesulfonamide (13x). Yield: 0.145 g; (20%). Mp: 210–211°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.33 (6H, s, (CH₃)₂), 3.69 (2H, d, J=5.3 Hz, CCH₂), 5.22 (1H, t, J=4.2 Hz, OH), 8.61 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 17.35 ((CH₃)₂), 63.92 (CH₂O), 67.74 (SO₂C(CH₃)₂), 120.08 (C1, t, J (¹⁹F – ¹³C)=14.6 Hz), 127.79 (C4, t, J (¹⁹F – ¹³C)=15.7 Hz), 142.83 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=252.0 Hz, ²J (¹⁹F – ¹³C)=15.7 Hz), 144.98 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=257.4 Hz, ²J (¹⁹F – ¹³C)=17.7 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –132.27 to -132.44 (2F, m), −137.02 to -137.18 (2 F, m). HRMS for C₁₀H₁₁F₄NO₅S₂ [(M-H)⁺]: calc. 366.0088, found 366.0088.

2-Methyl-2-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)sulfonyl)propyl acetate (14x). Yield: 0.153 g; (19%). Mp: 177–178°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.41 (6H, s, (CH₃)₂), 1.87 (3H, s, C(O)CH₃), 4.29 (2H, s, CCH₂), 8.64 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 17.36 ((CH₃)₂), 19.92 (C(O)CH₃), 65.31 (CH₂O), 65.76 (SO₂C(CH₃)₂), 118.79 (C1, t, J (¹⁹F – ¹³C)=14.7 Hz), 128.33 (C4, t, J (¹⁹F – ¹³C)=15.8 Hz), 143.12 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=258.8 Hz, ²J (¹⁹F – ¹³C)=13.9 Hz), 145.06 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=255.9 Hz, ²J (¹⁹F – ¹³C)=13.9 Hz), 169.41 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –132.52 to -132.70 (2F, m), −136.40 to -136.57 (2 F, m). HRMS for C₁₂H₁₃F₄NO₆S₂ [(M-H)^-]: calc. 406.0048, found 406.0052.

N-(2-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)thio)ethyl)acetamide (15x) was prepared according to known procedure in the literature (Baronas et al., 2021). 2,3,4,5,6-Pentafluorbenzensulfonamide (2ax) (2.320 g; 9.39 mmol), N-(2-mercaptoethyl)acetamide (1.300 ml; 13.1 mmol; 1.4 eq.), Et₃N (1.960 ml; 14.1 mmol; 1,5 eq.) were dissolved in MeOH (25 ml) and left to stir at room temperature for 2 hr. Afterward, additional portions of N-(2-mercaptoethyl)acetamide (0.150 ml; 1.51 mmol, 0.16 eq.) and Et₃N (0.150 ml; 1.08 mmol; 0.11 eq.) were added. The reaction mixture was left to stir further for an additional 1 hr. After reaction completion, the solvent was evaporated under reduced pressure and the product was purified by recrystallization from MeOH/H₂O 1:6 mixture obtaining white crystals. Yield 2.562 (78%). Mp: 169–170°C. (Baronas et al., 2021), mp: 169–171°C.

N-(2-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl)acetamide (16x) was prepared as previously described (Petrosiute et al., 2024). N-(2-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)thio)ethyl)acetamide (15x) (2.592 g; 7.48 mmol; 1 eq.) was dissolved in acetic acid (70 ml) and heated at 75 °C temperature for 10 hr. H₂O₂ (30%) was added by portions (0.1 ml) every 30 min (overall 5 ml) until complete starting material conversion. Afterwards, the solvent was evaporated under reduced pressure and the product was purified by recrystallization in MeOH/H₂O 1:4 mixture obtaining white crystals. Yield: 1.576 g; (56%). Mp: 224–225°C, mp: 224–225°C.

N'-((4-bromophenyl)sulfonyl)-N,N-dimethylformimidamide (18x) was prepared according to the known procedure in literature (Dudutienė et al., 2013). N,N-Dimethylformamide dimethyl acetal (0.303 g; 2.54 mmol; 1.2 eq.) was dissolved in MeCN (7 ml) and added dropwise to a mixture of 4-bromobenzenesulfonamide (17x) (0.500 g; 2.12 mmol; 1 eq.) in MeCN (3 ml). The reaction mixture was left to stir at room temperature for 1 hr and afterward, the solvent was evaporated under reduced pressure and the resultant precipitate was washed with H₂O. Yield: 0.562 g; (91%). Mp: 142–143°C (close to the value in the literature [Dudutienė et al., 2013], mp: 141–143°C).

4-((2-Hydroxyethyl)thio)benzenesulfonamide (19x) was prepared according to known procedure in literature (Dudutienė et al., 2013). 2-Mercaptoethanol (0.161 g; 2.04 mmol; 2 eq.) was added dropwise to a suspension of NaH (0.089 g; 55% oil dispersion; 2.04 mmol; 2 eq.) in DMF (1 ml). After gas emission was complete, N'-((4-bromophenyl)sulfonyl)-N,N-dimethylformamidine (18x) (0.300 g; 1.02 mmol; 1eq.) was added and the reaction mixture was heated for 1 hr at 95 °C temperature. Subsequently, DMF was removed under reduced pressure, and the resultant precipitate was dissolved in a mixture of MeOH (1 ml) /NaOH solution (10%; 1 ml) and refluxed for another hour. MeOH was evaporated under reduced pressure and the resultant suspension was diluted with H₂O, washed with petrol ether, and acidified with HCl (10 %). Afterwards reaction mixture was extracted with EtOAc (3x10 ml), collected organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.27). Yield 0.149 g; (63%). Mp: 110–111°C (close to the value in the literature [Dudutienė et al., 2013], mp: 111–112°C).

Compounds 20x and 21x (Singh et al., 2012) were synthesized during 19x oxidation.

4-((2-Hydroxyethyl)thio)benzenesulfonamide (19x) (0.050 g, 0.21 mmol) was dissolved in acetic acid (2 ml) and heated at 75 °C temperature for 5 hr. H₂O₂ (30%) was added in portions (0.1 ml) every hour (overall 0.5 ml) until the complete starting material conversion. Afterward, the solvent was evaporated under reduced pressure and products were purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf_20x=0.09, Rf_{21x (6)}=0.34).

4-((2-Hydroxyethyl)sulfonyl)benzenesulfonamide 20x. Yield: 0.032 g; (57%). Mp: 153–154°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 3.54 (2H, t, J=6.2 Hz SO₂CH₂), 3.70 (2H, t, J=6.0 Hz, CH₂O), 7.68 (2H, s, SO₂NH₂), 8.04 (2H, d, J=8.7 Hz, ArH), 8.10 (2H, d, J=8.8 Hz, ArH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 54.98 (SO₂CH₂), 57.50 (CH₂O), 126.47 (C1), 128.65 (C4), 142.97 (C2 and C6), 148.40 (C3 and C5). HRMS for C₈H₁₁NO₅S₂ [(M-H)^-]: calc. 264.0006, found 264.0009.

2-((4-Sulfamoylphenyl)sulfonyl)ethyl acetate (21x). Yield: 0.004 g; (6%). Mp; 129–130°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.68 (3H, s, COCH₃), 3.82 (2H, t, J=5.6 Hz, SO₂CH₂), 4.27 (2H, t, J=5.6 Hz, CH₂O), 7.68 (2H, s, SO₂NH₂), 8.07 (2H, d, J=8.7 Hz, ArH), 8.12 (2H, d, J=8.7 Hz, ArH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.10 (CH₃CO), 53.73 (SO₂CH₂), 57.49 (CH₂O), 126.57 (C1), 128.78 (C4), 142.49 (C2 and C6), 148.64 (C3 and C5), 169.59 (OC(O)). HRMS for C₁₅H₁₂F₄N₂O₆S₂ [(M-H)^-]: calc. 306.0112, found 306.0116.

N,N-dimethyl-N'-((2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)sulfonyl)phenyl)sulfonyl)formamidine (22x). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.160 g; 0.047 mmol) and N,N-dimethyl-formamide dimethyl acetal (0.076 g; 0.074 mmol; 5 eq.) were dissolved in acetonitrile (10 ml) and left to stir at room temperature for 1 hr. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc, Rf = 0.65). Yield: 0.155 g; (83%). Mp: 188–189°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.99 (3H, s, NCH₃), 3.23 (3H, s, NCH₃), 3.71 (2H, t, J=5.3 Hz, SO₂CH₂), 3.85 (2H, q, J=5.3 Hz, CH₂O), 4.98 (1H, t, J=5.2 Hz, OH), 8.30 (1H, s, NCH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 35.64 (NCH₃), 41.36 (NCH₃), 55.07 (SO₂CH₂), 59.53 (CH₂OH), 122.46 (C1, t, J (¹⁹F – ¹³C)=15.1 Hz), 126.45 (C4, t, J (¹⁹F – ¹³C)=15.2 Hz), 143.00 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=253.7 Hz, ²J (¹⁹F – ¹³C)=17.8 Hz), 144.23 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=252.2 Hz, ²J (¹⁹F – ¹³C)=16.3 Hz), 160.93 (NCHN). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –136.20 to -136.38 (2F, m), −136.72 to -136.91 (2 F, m). HRMS for C₁₁H₁₂F₄N₂O₅S₂ [(M+H)⁺]: calc. 393.0197, found 393.0199.

2-((4-(N-((dimethylamino)methylene)sulfamoyl)–2,3,5,6-tetrafluorophenyl)sulfonyl)ethyl phenylcarbamate (23ax). N,N-dimethyl-N'-((2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)sulfonyl) phenyl)sulfonyl)formamidine (22x) (0.068 g; 0.17 mmol; 1 eq.) and phenyl isocyanate (0.028 ml; 0.26 mmol; 1.5 eq.) were dissolved in toluene (10 ml) and refluxed at boiling point for 14 hr. Additional phenyl isocyanate portions (0.057 ml; 0.052 mmol; 3 eq.) were added after 2 hr and 7 hr, respectively. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.43). Yield: 0.072 g; (81%). Mp: 177–178°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.96 (3H, s, NCH₃), 3.22 (3H, s, NCH₃), 4.04 (2H, t, J=5.3 Hz, SO₂CH₂), 4.51 (2H, t, J=5.4 Hz, CH₂O), 7.00 (1H, t, J=7.3 Hz, CH (C4) of phenyl), 7.27 (2H, t, J=7.8 Hz, CH (C3 and C5) of phenyl), 7.39 (2H, d, J=7.6 Hz, CH (C2 and C6) of phenyl), 8.27 (1H, s, NCHN), 9.62 (1H, s, OC(O)NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 35.61 (NCH₃), 41.34 (NCH₃), 56.36 (SO₂CH₂), 57.38 (CH₂O), 118.54 (C4 of phenyl), 121.10 (C1, t, J (¹⁹F – ¹³C)=14.9 Hz), 122.75 (C3 and C5 of phenyl), 126.95 (C4, t, J (¹⁹F – ¹³C)=15.1 Hz), 128.70 (C2 and C6 of phenyl), 138.54 (C1 of phenyl), 143.29 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=262.1 Hz, ²J (¹⁹F – ¹³C)=22.0 Hz), 144.25 (C5 and C3, dd, ¹J (¹⁹F – ¹³C)=247.2 Hz, ²J (¹⁹F – ¹³C)=13.0 Hz), 152.64 (OC(O)NH), 160.89 (NCHN). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.99 to -136.16 (2F, m), −136.28 to -136.47 (2 F, m). HRMS for C₁₈H₁₇F₄N₃O₆S₂ [(M+H)⁺]: calc. 512.0568, found 512.0571.

2-((4-(N-((dimethylamino)methylene)sulfamoyl)–2,3,5,6-tetrafluorophenyl)sulfonyl)ethyl (4-methoxyphenyl)carbamate (23bx). N,N-dimethyl-N'-((2,3,5,6-tetrafluoro-4-((2-hydroxyethyl) sulfonyl) phenyl)sulfonyl)formamidine (22x) (0.050 g; 0.13 mmol; 1 eq.), 4-methoxyphenyl isocyanate (0.025 ml; 0.19 mmol; 1.5 eq.), dibutyltin dilaurate (0.015 ml; 0.026 mmol; 0.2 eq.) were dissolved in acetonitrile (3 ml) and left to stir at room temperature for 5 d. An additional 4-methoxyphenyl isocyanate portion (0.025 ml; 0.19 mmol; 1.5 eq.) was added after 2 d. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.41). Yield: 0.008 g; (12%). Mp: 167–168°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 2.96 (3H, s, NCH₃), 3.21 (3H, s, NCH₃), 3.70 (3H, s, OCH₃), 4.03 (2H, t, J=5.4 Hz, SO₂CH₂), 4.49 (2H, t, J=5.3 Hz, CH₂O), 6.85 (2H, d, J=8.8 Hz, CH (C2 and C6) of phenyl), 7.28 (2H, d, J=7.2 Hz, CH (C3 and C6) of phenyl), 8.27 (1H, s, NCHN), 9.41 (1H, s, OC(O)NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 35.58 (NCH₃), 41.34 (NCH₃), 55.14 (OCH₃), 56.40 (SO₂CH₂), 57.31 (CH₂O), 113.91 (C3 and C5 of phenyl), 120.25 (C2 and C6 of phenyl), 121.13 (C1, t, J (¹⁹F – ¹³C)=14.7 Hz), 126.91 (C4), 128.63 (C1 of phenyl), 143.34 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=250.0 Hz, ²J (¹⁹F – ¹³C)=11.2 Hz), 144.16 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=242.7 Hz, ²J (¹⁹F – ¹³C)=10.3 Hz), 152.79 (OC(O)NH), 155.03 (C4 of phenyl), 160.90 (NCHN). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –135.95 to -136.16 (2F, m), −136.30 to -136.55 (2 F,m). HRMS for C₁₉H₁₉F₄N₃O₇S₂ [(M+Na)⁺]: calc. 564.0493, found 564.0501.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl phenylcarbamate (24ax) (Figure 11). 2-((4-(N-((dimethylamino)methylene)sulfamoyl)–2,3,5,6-tetrafluorophenyl)sulfonyl)ethyl phenylcarbamate (23ax) (0.030 g; 0.059 mmol; 1eq.) and three drops of HCl (conc.) were dissolved in toluene (3 ml) and refluxed at boiling point for 18 hr. After 5 hr, additional 10 drops of HCl (conc.) were added. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.59). Yield: 0.007 g; (26%). Mp: 204–205°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 4.06 (2H, t, J=5.4 Hz, SO₂CH₂), 4.52 (2H, t, J=5.5 Hz, CH₂O), 7.00 (1H, t, J=7.3 Hz, CH (C4) of phenyl), 7.26 (2H, t, J=7.9 Hz, (C3 and C5) of phenyl), 7.41 (2H, d, J=7.9 Hz, CH (C2 and C6) of phenyl), 8.60 (2H, s, SO₂NH₂), 9.65 (1H, s, OC(O)NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 56.39 (SO₂CH₂), 57.26 (CH₂O), 118.51 (C4 of phenyl), 121.18 (C1, t, J (¹⁹F – ¹³C)=13.1 Hz), 122.72 (C3 and C5 of phenyl), 127.86 (C4, t, J (¹⁹F – ¹³C)=15.0 Hz), 128.71 (C2 and C6 of phenyl), 138.57 (C1 of phenyl), 143.12 (C2 and C6, dd, ¹J (¹⁹F – ¹³C)=257.4 Hz, ²J (¹⁹F – ¹³C)=17.7 Hz), 144.26 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=259.2 Hz, ²J (¹⁹F – ¹³C)=19.5 Hz), 152.65 (OC(O)NH). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –136.18 to -136.49 (4F, m). HRMS for C₁₅H₁₂F₄N₂O₆S₂ [(M-H)^-]: calc. 455.0000, found 455.0001.

2-((2,3,5,6-Tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl (4-methoxyphenyl)carbamate (24bx). 2-((4-(N-((dimethylamino)methylene)sulfamoyl)–2,3,5,6-tetrafluorophenyl)sulfonyl)ethyl (4-methoxyphenyl)carbamate (23bx) (0.006 g; 0.011 mmol; 1 eq.) and three drops of HCl (conc.) were dissolved in MeOH (3 ml) and refluxed for 31 hr. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃, (1:1), Rf = 0.57). Yield: 0.002 g; (37%). Mp: 188–189°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 3.70 (3H, s, OCH₃), 4.05 (2H, t, J=5.4 Hz, SO₂CH₂), 4.49 (2H, t, J=5.4 Hz, CH₂O), 6.85 (2H, d, J=8.9 Hz, phenyl), 7.30 (2H, d, J=7.5 Hz, phenyl), 8.61 (2H, s, SO₂NH₂), 9.45 (1H, s, OC(O)NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 55.16 (OCH₃), 56.44 (SO₂CH₂), 57.23 (CH₂O), 113.94 (C3 and C5 of phenyl), 120.14 (C2 and C6 of phenyl), 121.23 (C1), 127.85 (C4, t, J (¹⁹F – ¹³C)=12.2 Hz), 143.06 (C2 and C6, d, J (¹⁹F – ¹³C)=255.9 Hz), 144.26 (C3 and C5, dd, ¹J (¹⁹F – ¹³C)=259.6 Hz, ²J (¹⁹F – ¹³C)=19.2 Hz), 152.82 (OC(O)NH), 155.01 (C4 of phenyl). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –136.22 to -136.49 (4F, m). HRMS for C₁₆H₁₄F₄N₂O₇S₂ [(M-H)^-]: calc. 485.0106, found 485.0105.

3-(Cyclooctylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (25ax) was prepared according to the known procedure in literature (Dudutienė et al., 2014). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.549 g; 1.63 mmol; 1 eq.) and cyclooctylamine (0.446 ml; 3.26 mmol; 2 eq.) were dissolved in DMSO (2 ml) and left to stir at room temperature overnight. After full starting material conversion reaction mixture was washed with brine (10 ml) and extracted with EtOAc (3x15 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.52). Yield 0.380 g; (52%). Mp: 90–91°C (close to the value in the literature [Dudutienė et al., 2014], mp: 89–90°C).

3-(Cyclododecylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (25bx) was prepared according to known procedure in literature (Dudutienė et al., 2015). 2,3,5,6-Tetrafluoro-4-((2-hydroxyethyl)sulfonyl)benzensulfonamide (4ax) (0.300 g 0.89 mmol; 1 eq.) and cyclododecylamine (0.326 g; 1.78 mmol; 2 eq.) were dissolved in DMSO (2 ml) and left to stir at room temperature for 2 hrs. After full starting material conversion reaction mixture was washed with brine (10 ml) and extracted with EtOAc (3x15 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.60). Yield: 0.278 g; (62%). Mp: 143–145°C (close to the value in the literature [Dudutienė et al., 2015], mp: 143–144°C).

N'-((3-(Cyclooctylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)phenyl)sulfonyl)-N,N-dimethylformamidine (26x). N,N-dimethyl-N'-((2,3,5,6-tetrafluoro-4-((2-hydroxyethyl)sulfonyl) phenyl)sulfonyl)formamidine (22x) (0.100 g; 0.25 mmol; 1 eq.) and cyclooctylamine (0.070 ml; 0.51 mmol; 2 eq.) were dissolved in DMSO (1 ml) and left to stir at room temperature for 1.5 hr. The reaction mixture was washed with brine (5 ml) and extracted using EtOAc (3x10 ml). The collected organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc, Rf = 0.64) and yellow oil was obtained. Yield: 0.062 g; (49%). ¹H NMR (400 MHz, DMSO-d₆, δ): 1.40–1.70 (12H, m, cyclooctane), 1.76–1.87 (2H, m, cyclooctane), 2.97 (3H, s, NCH₃), 3.21 (3H, s, NCH₃), 3.64 (2H, t, J=5.4 Hz, SO₂CH₂), 3.73 (1H, br.s, NHCH(CH₂)₂), 3.81 (2H, q, J=5,4 Hz, CH₂O), 4.98 (1H, t, J=5.2 Hz, OH), 6.60 (1H, d, J=8.2 Hz, NHCH(CH₂)₂), 8.28 (1H, s, NCHN). ¹³C NMR (100 MHz, DMSO-d₆, δ): 22.89 (cyclooctane), 25.11 (cyclooctane), 26.65 (cyclooctane), 32.27 (cyclooctane), 35.48 (NCH₃), 41.20 (NCH₃), 55.02 (CH₂O), 55.31 (CH of cyclooctane, d, J (¹⁹F – ¹³C)=11.1 Hz), 59.52 (SO₂CH₂, d, J (¹⁹F – ¹³C)=2.5 Hz), 116.70 (C1, dd, ¹J (¹⁹F – ¹³C)=12.8 Hz, ²J (¹⁹F – ¹³C)=5.5 Hz), 126.05 (C4, dd, ¹J (¹⁹F – ¹³C)=18.2 Hz, ²J (¹⁹F – ¹³C)=13.9 Hz), 134.49 (C3, d, J (¹⁹F – ¹³C)=16.1 Hz), 136.97 (C6, d, J (¹⁹F – ¹³C)=246.7 Hz), 144.26 (C2, d, ¹J (¹⁹F – ¹³C)=252.5 Hz), 145.62 (C5, dd, ¹J (¹⁹F – ¹³C)=246.0 Hz, ¹J (¹⁹F – ¹³C)=12.4 Hz), 160.75 (NCHN). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –124.67 (1F, br.s), –134.26 (1F, dd, J¹=27.3 Hz, J² = 12.2 Hz), –150.64 (1F, dd, J¹=27.3 Hz, J² = 6.7 Hz). HRMS for C₁₉H₂₈F₃N₃O₅S₂ [(M+H)⁺]: calc. 500.1495, found 500.1493.

2-((2-(Cyclooctylamino)–3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)ethyl acetate (27ax). 3-(Cyclooctylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (25ax) (0.040 g; 0.090 mmol), acetic acid (1.2 ml) and two drops of H₂SO₄ (conc.) were dissolved in toluene (10 ml) and refluxed for 1 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromotography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.78). Yield: 0.0203 g; (46%). Mp: 96 °C. ¹H NMR (400 MHz, CDCl₃, δ): 1.45–1.73 (12H, m, cyclooctane), 1.83–1.91 (2H, m, cyclooctane), 1.93 (3H, s, CH₃), 3.66 (2H, t, J=5.7 Hz, SO₂CH₂), 3.88 (1H, s, cyclooctane), 4.48 (2H, t, J=5.7 Hz, CH₂O), 5.65 (2H, s, SO₂NH₂), 6.84 (2H, d, J=6.7 Hz, NH). ¹³C NMR (100 MHz, CDCl₃, δ): 20.46 (CH₃), 23.38 (cyclooctane), 25.54 (cyclooctane), 27.31 (cyclooctane), 33.04 (cyclooctane), 56.24 (cyclooctane, d, J (¹⁹F-¹³C)=11.7 Hz), 56.43 (SO₂CH₂, d, J (¹⁹F-¹³C)=4.0 Hz), 57.39 (CH₂O), 115.10 (C1, dd, ¹J (¹⁹F-¹³C)=12.7 Hz, ²J (¹⁹F-¹³C)=5.9 Hz), 126.63 (C4, dd, ¹J (¹⁹F-¹³C)=17.1 Hz, ²J (¹⁹F-¹³C)=13.3 Hz), 135.85 (C3, d, J (¹⁹F-¹³C)=13.6 Hz), 136.57 (C6, ddd, ¹J (¹⁹F-¹³C)=248.33 Hz, ²J (¹⁹F-¹³C)=17.8 Hz, ³J (¹⁹F-¹³C)=3.9 Hz), 144.36 (C2, d, ¹J (¹⁹F-¹³C)=252.7 Hz), 146.28 (C5, dd, ¹J (¹⁹F-¹³C)=252.8 Hz, ²J (¹⁹F-¹³C)=15.8 Hz, ³J (¹⁹F-¹³C)=4.5 Hz), 170.29 (OC(O)). ¹⁹F NMR (376 MHz, CDCl₃, δ): –125.63 (1F, dd, ¹J=12.4 Hz, ²J=8.8 Hz), –132.86 (1F, dd, ¹J=25.7 Hz, ²J=12.5 Hz), –151.63 (1F, dd, ¹J=25.8 Hz, ²J=8.8 Hz). HRMS for C₁₈H₂₅F₃N₂O₆S₂ [(M+H)⁺]: calc. 487.1179, found 487.1179.

2-((2-(Cyclododecylamino)–3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)ethyl acetate (27bx). 3-(Cyclododecylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (25bx) (0.102 g; 0.204 mmol), acetic acid (4 ml) and three drops of H₂SO₄ (conc.) were dissolved in toluene (25 ml) and refluxed for 4 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.86). Yield: 0.052 g; (47%). Mp: 138–140°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.21–1.46 (20H, m, cyclododecane), 1.55–1.65 (2H, m, cyclododecane), 1.81 (3H, s, OC(O)CH₃), 3.78 (1H, s, cyclododecane), 3.92 (2H, t, J=4.9 Hz, SO₂CH₂), 4.36 (2H, t, J=4.9 Hz, CH₂O), 6.52 (1H, d, J=8.6 Hz, NH), 8.38 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 20.04 (CH₃), 20.45 (cyclododecane), 22.60 (cyclododecane), 22.71 (cyclododecane), 23.73 (cyclododecane), 23.89 (cyclododecane), 30.08 (cyclododecane), 52.84 (CH of cyclododecane, d, J=11.6 Hz), 55.66 (SO₂CH₂, d, J=2.3 Hz), 57.65 (CH₂O), 115.34 (C1, dd, ¹J (¹⁹F – ¹³C)=13.2 Hz, ²J (¹⁹F – ¹³C)=5.1 Hz), 127.71 (C4, dd, ¹J (¹⁹F – ¹³C)=18.1 Hz, ²J (¹⁹F – ¹³C)=13.9 Hz), 135.13 (C3, d, ¹J (¹⁹F – ¹³C)=15.3 Hz), 136.67 (C6, d, ¹J (¹⁹F – ¹³C)=253.6 Hz), 143.95 (C2, d, ¹J (¹⁹F – ¹³C)=252.9 Hz), 145.58 (C5, dd, ¹J (¹⁹F – ¹³C)=246.4 Hz, ²J (¹⁹F – ¹³C)=13.7 Hz), 169.54 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –124.88 (1F, s), –134.22 (1F, dd, ¹J=26.8 Hz, ²J=12.5 Hz), –150.76 (1F, dd, ¹J=26.8 Hz, ²J=6.7 Hz). HRMS for C₂₂H₃₃F₃N₂O₆S₂ [(M+H)⁺]: calc. 543.1805, found 543.1803.

2-((2-(Cyclooctylamino)–3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)ethyl propionate (28ax). 3-(Cyclooctylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (25ax) (0.100 g; 0.225 mmol), propionic acid (3 ml) and three drops of H₂SO₄ (conc.) were dissolved in toluene (25 ml) and refluxed for 3 hr. The resulting mixture was washed with brine (3x10 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.89) and yellow oil was obtained. Yield: 0.0313 g; (28%). ¹H NMR (400 MHz, CDCl₃, δ): 1.06 (3H, t, J=7.6 Hz, CH₂CH₃), 1.45–1.74 (12H, m, cyclooctane), 1.83–1.92 (2H, m, cyclooctane), 2.17 (2H, q, J=7.6 Hz, CH₂CH₃), 3.66 (2H, t, J=5.8 Hz, SO₂CH₂), 3.88 (1H, s, CH of cyclooctane), 4.49 (2H, t, J=5.8 Hz, CH₂O), 5.55 (2H, s, SO₂NH₂), 6,85 (1H, d, J=8.0 Hz, NH). ¹³C NMR (100 MHz, CDCl₃, δ):): 8.88 (CH₂CH₃), 23.40 (cyclooctane), 25.56 (cyclooctane), 27.19 (CH₂CH₃), 27.31 (cyclooctane), 33.06 (cyclooctane), 56.25 (CH of cyclooctane, d, J (¹⁹F-¹³C)=11.7 Hz), 56.58 (SO₂CH₂, d, J (¹⁹F-¹³C)=3.9 Hz), 57.28 (CH₂O), 115.18 (C1, dd, ¹J (¹⁹F-¹³C)=13.3 Hz, ²J (¹⁹F-¹³C)=6.5 Hz), 126.63 (C4, dd, ¹J (¹⁹F-¹³C)=16.8 Hz, ²J (¹⁹F-¹³C)=13.4 Hz), 135.89 (C3, d, J (¹⁹F-¹³C)=12.9 Hz), 136.59 (C6, ddd, ¹J (¹⁹F-¹³C)=248.3 Hz, ²J (¹⁹F-¹³C)=17.4 Hz, ³J (¹⁹F-¹³C)=4.3 Hz), 144.39 (C2, d, ¹J (¹⁹F-¹³C)=253.3 Hz, ²J (¹⁹F-¹³C)=3.7 Hz), 146.30 (C5, ddd, ¹J (¹⁹F-¹³C)=253.2 Hz, ²J (¹⁹F-¹³C)=16.5 Hz, ³J (¹⁹F-¹³C)=5.0 Hz), 173.73 (OC(O)). ¹⁹F NMR (376 MHz, CDCl₃, δ): –125.61 (1F, dd, ¹J=12.4 Hz, ²J=8.9 Hz), –132.79 (1F, dd, ¹J=25.8 Hz, ²J=12.5 Hz), –151.60 (1F, dd, ¹J=25.8 Hz, ²J=8.8 Hz). HRMS for C₁₉H₂₇F₃N₂O₆S₂ [(M+H)⁺]: calc. 501.1335, found 501.1339.

2-((2-(Cyclododecylamino)–3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)ethyl propionate (28bx). 3-(Cyclododecylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)benzenesulfonamide (25bx) (0.0377 g; 0.075 mmol), propionic acid (2 ml) and three drops of H₂SO₄ (conc.) were dissolved in toluene (10 ml) and refluxed for 1 hr. The resulting mixture was washed with brine (2x5 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, CHCl₃, Rf = 0.20). Yield: 0.0248 g; (59%). Mp: 110–112°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 0.91 (3H, t, J=7.5 Hz, CH₂CH₃), 1.23–1.44 (20H, m, cyclododecane), 1.54–1.64 (2H, m, cyclododecane), 2.05 (2H, q, J=7.6 Hz, CH₂CH₃), 3.78 (1H, s, cyclododecane), 3.94 (2H, t, J=5.2 Hz, SO₂CH₂), 4.37 (2H, t, J=5.0 Hz, CH₂O), 6.52 (1H, d, J=8.2 Hz, NH), 8.40 (2H, s, SO₂NH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ): 8.56 (CH₂CH₃), 20.44 (cyclododecane), 22.58 (cyclododecane), 22.70 (cyclododecane), 23.71 (cyclododecane), 23.87 (cyclododecane), 26.36 (CH₂CH₃), 30.08 (cyclododecane), 52.82 (CH of cyclododecane, d, J=11.6 Hz), 55.76 (SO₂CH₂), 57.59 (CH₂O), 115.32 (C1, t, J (¹⁹F – ¹³C)=7.0 Hz), 127.67 (C4, t, J (¹⁹F – ¹³C)=14.2 Hz), 135.11 (dd, ¹J (¹⁹F – ¹³C)=13.9 Hz, ²J (¹⁹F – ¹³C)=2.4 Hz), 136.65 (C6, d, ¹J (¹⁹F – ¹³C)=252.8 Hz), 143.98 (C2, d, ¹J (¹⁹F – ¹³C) 253.0 Hz,), 145.56 (C5, dd, ¹J (¹⁹F – ¹³C)=249.8 Hz, ²J (¹⁹F – ¹³C)=15.4 Hz), 172.82 (OC(O)). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –124.86 (1F, s), –134.21 (1F, dd, ¹J=26.9 Hz, ²J=12.4 Hz), –150.79 (1F, dd, ¹J=26.9 Hz, ²J=6.6 Hz). HRMS for C₂₃H₃₅F₃N₂O₆S₂ [(M+H)⁺]: calc. 557.1961, found 557.1966.

2-((2-(Cyclooctylamino)–4-(N-((dimethylamino)methylene)sulfamoyl)–3,5,6-trifluorophenyl) sulfonyl)ethyl phenylcarbamate (29x). N'-((3-(cyclooctylamino)–2,5,6-trifluoro-4-((2-hydroxyethyl)sulfonyl)phenyl)sulfonyl)-N,N-dimethylformamidine (26x) (0.038 g; 0.08 mmol; 1 eq.) and phenyl isocyanate (0.025 ml; 0.23 mmol; 3 eq.) were dissolved in toluene (7 ml) and refluxed at boiling point for 28 hr. Additional phenyl isocyanate portions (0.025 ml; 0.023 mmol; 3 eq.) were added after 10 hours and 24 hours respectively. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.65). Yield: 0.018 g; (38%). Mp: 177–178°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.33–1.63 (12H, m, cyclooctane), 1.71–1.82 (2H, m, cyclooctane), 2.95 (3H, s, NCH₃), 3.20 (3H, s, NCH₃), 3.69 (1H, br.s, CH of cyclooctane), 3.95 (2H, t, J=5.0 Hz, SO₂CH₂), 4.46 (2H, t, J=5.2 Hz, CH₂O), 6.58 (1H, d, J=8.2 Hz, NHCH(CH₂)₂), 6.99 (1H, t, J=7.3 Hz, CH (C4) of phenyl), 7.26 (2H, t, J=7.8 Hz, CH (C3 and C5) of phenyl), 7.42 (2H, d, J=6.6 Hz, CH (C2 and C6) of phenyl), 8.27 (1H, s, NCHN), 9.66 (1H, s, C(O)NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 22.83 (cyclooctane), 25.03 (cyclooctane), 26.54 (cyclooctane), 32.31 (cyclooctane), 35.46 (NCH₃), 41.20 (NCH₃), 55.27 (CH of cyclooctane, d, J=11.3 Hz), 56.14 (SO₂CH₂, d, J=2.4 Hz), 57.50 (CH₂O), 115.03 (C1, dd, ¹J (¹⁹F – ¹³C)=13.2 Hz, ²J (¹⁹F – ¹³C)=5.9 Hz), 118.48 (C4 of phenyl), 122.64 (C3 and C5 of phenyl), 126.57 (C4, dd, ¹J (¹⁹F – ¹³C)=18.2 Hz, ²J (¹⁹F – ¹³C)=14.1 Hz), 128.62 (C2 and C6 of phenyl), 134.55 (C3, d, J (¹⁹F – ¹³C)=14.5 Hz), 137.09 (C6, ddd, ¹J (¹⁹F – ¹³C)=241.5 Hz, ²J (¹⁹F – ¹³C)=17.6 Hz, ³J (¹⁹F – ¹³C)=5.0 Hz), 138.68 (C1 of phenyl), 144.29 (C2, dd, ¹J (¹⁹F – ¹³C)=253.2 Hz, ²J (¹⁹F – ¹³C)=21.3 Hz), 145.66 (C5, ddd, ¹J (¹⁹F – ¹³C)=250.5 Hz, ²J (¹⁹F – ¹³C)=17.2 Hz, ³J (¹⁹F – ¹³C)=4.0 Hz), 152.75 (OC(O)NH), 160.76 (NCHN). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –124.61 (1F, s), –134.25 (1F, dd, ¹J=27.4 Hz, ²J=12.3 Hz), 150.25 (1F, dd, ¹J=27.7 Hz, ²J=6.7 Hz). HRMS for C₂₆H₃₃F₃N₄O₆S₂ [(M-H)^-]: calc. 617.1721, found 617.1723.

2-((2-(Cyclooctylamino)–3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)ethyl phenylcarbamate (30x). 2-((2-(Cyclooctylamino)–4-(N-((dimethylamino)methylene)sulfamoyl)–3,5,6-trifluorophenyl) sulfonyl)ethyl phenylcarbamate (29x) (0.015 g; 0.024; 1 eq.) and three drops of HCl (conc.) were dissolved in MeOH (3 ml) and refluxed at boiling point for 29 hr. The solvent was evaporated under reduced pressure and the product was purified by column chromatography (silica gel, EtOAc/CHCl₃ (1:1), Rf = 0.88). Yield: 0.004 g; (29%). Mp: 174–175°C. ¹H NMR (400 MHz, DMSO-d₆, δ): 1.36–1.63 (12H, m, cyclooctane), 1.75–1.85 (2H, m, cyclooctane), 3.72 (1H, br.s, CH of cyclooctane), 3.97 (2H, t, J=5.3 Hz, SO₂CH₂), 4.46 (2H, t, J=5.4 Hz, CH₂O), 6.62 (1H, d, J=8.3 Hz, NHCH(CH₂)₂), 6.99 (1H, t, J=7.3 Hz, C4 of phenyl), 7.26 (2H, t, J=7.8 Hz, C3 and C5 of phenyl), 7.43 (2H, d, J=8.0 Hz, C2 and C6 of phenyl), 8.32 (2H, s, SO₂NH₂), 9.67 (1H, s, OC(O)NH). ¹³C NMR (100 MHz, DMSO-d₆, δ): 22.74 (cyclooctane), 24.89 (cyclooctane), 26.66 (cyclooctane), 32.15 (cyclooctane), 55.39 (CH of cyclooctane, d, J=11.2 Hz), 56.14 (SO₂CH₂, d, J=2.8 Hz), 57.45 (CH₂O), 114.93 (C1, d, J (¹⁹F – ¹³C)=12.6 Hz), 118.45 (C4 of phenyl), 122.64 (C3 and C5 of phenyl), 127.61 (C4, dd, ¹J (¹⁹F – ¹³C)=18.6 Hz, ²J (¹⁹F – ¹³C)=14.2 Hz), 128.65 (C2 and C6 of phenyl), 134.66 (C3, dd, ¹J (¹⁹F – ¹³C)=14.2 Hz, ²J (¹⁹F – ¹³C)=2.7 Hz), 138.68 (C1 of phenyl), 144.10 (C2, d, J (¹⁹F – ¹³C)=18.2 Hz), 145.59 (C5, ddd, ¹J (¹⁹F – ¹³C)=250.0 Hz, ²J (¹⁹F – ¹³C)=19.3 Hz, ³J (¹⁹F – ¹³C)=3.0 Hz), 152.75 (OC(O)NH). ¹⁹F NMR (376 MHz, DMSO-d₆, δ): –124.89 (1F, s), –134.36 (1F, dd, ¹J=27.0 Hz, ²J=12.2 Hz), –150.47 (1F, dd, ¹J=27.0 Hz, ²J=6.4 Hz). HRMS for C₂₃H₂₈F₃N₃O₆S₂ [(M+H)⁺]: calc. 564.1444, found 564.1444.

N-(2-((2-(cyclooctylamino)–3,5,6-trifluoro-4-sulfamoylphenyl)sulfonyl)ethyl)acetamide (31x) was prepared according to the known procedure in the literature (Petrosiute et al., 2024). N-(2-((2,3,5,6-tetrafluoro-4-sulfamoylphenyl)sulfonyl)ethyl)acetamide (16x) (1.39 g; 3.67 mmol; 1 eq.) and cyclooctylamine (1.01 ml; 7.35 mmol; 2 eq.) were dissolved in DMSO (3 ml) and left stirring at room temperature overnight. After full starting material conversion reaction mixture was washed with brine (10 ml) and extracted with EtOAc (3x15 ml). The organic phase was dried using anhydrous Na₂SO₄ and evaporated under reduced pressure. The product was purified by column chromatography (silica gel, EtOAc Rf = 0.53). Yield 0.900 g; (50%). Mp: 161–162°C (close to the value in the literature [Petrosiute et al., 2024], mp: 162–163°C).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This research was funded by the grant from Research Council of Lithuania No S-MIP-22–35. Access to the EMBL beamline P13 at PETRA III (DESY) and has been supported by iNEXT-Discovery, project number 871037, funded by the Horizon 2020 program of the European Commission. This research was also supported within the framework of the European Union’s Recovery and Resilience Mechanism project No.5.2.1.1.i.0/2/24/I/CFLA/001 "Consolidation of the Latvian Institute of Organic Synthesis and the Latvian Biomedical Research and Study Centre".

1. Sent for peer review: July 19, 2024
2. Preprint posted: August 6, 2024
3. Reviewed Preprint version 1: September 26, 2024
4. Reviewed Preprint version 2: November 11, 2024
5. Version of Record published: December 17, 2024

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