Carbohydrate-Small Molecule Hybrids as Lead Compounds Targeting IL-6 Signaling

In the past 25 years, a number of efforts have been made toward the development of small molecule interleukin-6 (IL-6) signaling inhibitors, but none have been approved to date. Monosaccharides are a diverse class of bioactive compounds, but thus far have been unexplored as a scaffold for small molecule IL-6-signaling inhibitor design. Therefore, in this present communication, we combined a structure-based drug design approach with carbohydrate building blocks to design and synthesize novel IL-6-signaling inhibitors targeting glycoprotein 130 (gp130). Of this series of compounds, LS-TG-2P and LS-TF-3P were the top lead compounds, displaying IC50 values of 6.9 and 16 µM against SUM159 cell lines, respectively, while still retaining preferential activity against the IL-6-signaling pathway. The carbohydrate moiety was found to improve activity, as N-unsubstituted triazole analogues of these compounds were found to be less active in vitro compared to the leads themselves. Thus, LS-TG-2P and LS-TF-3P are promising scaffolds for further development and study as IL-6-signaling inhibitors.


Introduction
Carbohydrates are a class of compounds essential for numerous biological roles, such as communication, energy storage and metabolism, and protein and cell structure and function [1]. From 2000 to 2021, a total of 721 drugs have been approved by the FDA [2][3][4][5][6], but of these, only 54 were carbohydrate-containing entities [7]. Given that carbohydratecontaining drugs encompassed less than 10% of approvals during that time, it is evident that this chemical class is relatively unexplored when juxtaposed with their extensive role in biological processes [8,9]. Despite their low numbers, approved carbohydrate-based drugs have met with general success across a range of pathologies, such as viral and bacterial infections, cancer, and diabetes, with notable examples including oseltamivir (Tamiflu TM ) [10], vancomycin [11], doxorubicin [12,13], and miglitol [14] (Figure 1).
While glycomimetics base their design around specific carbohydrates in order to mimic their function in vivo, many carbohydrate-containing drugs accommodate monoand disaccharides in order to improve drug properties such as binding affinity, selectivity, and pharmacokinetics [8]. In the case of vancomycin, while its aglycon form has been shown to retain activity in vitro, the presence of its glucose and vancosamine moieties has been demonstrated to improve its in vivo properties and its ability to form homodimers [15,16]. For doxorubicin, its daunosamine unit has been shown to play a key role in its activity, improving the binding to and stability of its complex with DNA and topoisomerase II, and it has also been shown that structural modulation of this monosaccharide can affect the selectivity of the drug as a whole [8,17,18]. In general, the incorporation of carbohydrate or carbohydrate mimics into drug scaffolds can afford a number of advantages, such as Interleukin-6 is a key cytokine involved in the regulation of numerous processes within the body, including immune response, inflammatory response, and cell proliferation, and as a result of this role, its upregulation is associated with numerous disease states, such as multiple sclerosis, rheumatoid arthritis, and most types of cancer [19][20][21][22][23][24]. This cytokine acts through the formation of a hexameric complex on the surface of cells ( Figure 2) [25]. To form this complex, IL-6 and its selective IL-6 receptor (IL-6R) form a dimer, which then binds to glycoprotein 130 (gp130), a key protein ubiquitously expressed on the surface cells in the body [25,26]. Signal transduction occurs when two of these trimers form a hexamer, which modulates the intracellular domain of gp130 and activates various signaling pathways within the cell, such as the JAK-STAT3 pathway [25,27]. Currently, several monoclonalantibody-based inhibitors of IL-6 signaling have been approved for clinical use, but there are no approved small molecule agents at this time [28][29][30][31][32][33][34][35].
The first selective small-molecule inhibitors of IL-6 signaling were the natural product diastereomers madindoline A and B (MDL-A and MDL-B, respectively), which were isolated simultaneously in 1996 [36,37]. In later years, MDL-A was further studied in vitro and in silico, and it was determined to act via binding to the D1 domain of gp130, with several key interactions identified: hydrophobic interactions with a hydrophobic pocket, π-π interactions between its hydroxyfurindoline ring and TYR94, and hydrogen bonding with ASN92 [38][39][40]. Its docking mode has been recreated in Figure 2. Studies in vivo have also validated MDL-A's status as a lead compound for drug development targeting IL-6 signaling [38,41]. Targeting IL-6 signaling via binding to gp130-D1 is particularly advantageous as the only cytokines within the IL-6 cytokine family that require gp130-D1 for signal complex formation are IL-6 and IL-11 [25,42].
Previously, we disclosed the IL-6-signaling inhibitor MDL-101 (Figure 3), which was discovered through an effort to develop more potent analogues of MDL-A [40,43,44]. This new lead was determined to be more potent than its natural product predecessor, and was notably found to suppress T helper type 17 cell development in vitro through the inhibition of IL-6 signaling [40,43,44]. This lead compound acts via binding to gp130-D1, inhibiting the formation of the IL-6/IL-6R/gp130 hexameric signaling complex [25], and docking studies have demonstrated that MDL-101 exhibits similar interactions with gp130-D1 as MDL-A, though it also possesses a benzoyl moiety that occupies a hydrophobic subpocket adjacent to ASN92 [40]. Despite its improved activity, however, this compound possessed less-than-ideal pharmacokinetic properties, which likely arises from similar metabolic liabilities as seen with MDL-A, which is readily metabolized into its 5-hydroxy analogue [44,45]. In order to circumvent these issues and also further improve potency, the use of conformationally adaptive monosaccharides was pursued as an alternative design strategy under the premise that their multiple free hydroxyls could improve ligand binding to the flat surface of gp130, both on a conformational basis and on the basis of maximizing hydrogen bonding with surface residues. By combining data from the structure-activity relationship of MDL analogues developed en route to MDL-101, along with our previous identification of bazedoxifene as a repurposed IL-6-signaling inhibitor through multiple ligand simultaneous docking [46,47], a general, synthetically accessible core scaffold was designed for subsequent modification with various linkers and carbohydrate units ( Figure 3). In pursuit of this strategy, 15 novel, carbohydrate-containing compounds were developed and tested in vitro.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 3 new lead was determined to be more potent than its natural product predecessor, and wa notably found to suppress T helper type 17 cell development in vitro through the inhibi tion of IL-6 signaling [40,43,44]. This lead compound acts via binding to gp130-D1, inhib iting the formation of the IL-6/IL-6R/gp130 hexameric signaling complex [25], and dock ing studies have demonstrated that MDL-101 exhibits similar interactions with gp130-D as MDL-A, though it also possesses a benzoyl moiety that occupies a hydrophobic sub pocket adjacent to ASN92 [40]. Despite its improved activity, however, this compound possessed less-than-ideal pharmacokinetic properties, which likely arises from simila metabolic liabilities as seen with MDL-A, which is readily metabolized into its 5-hydrox analogue [44,45]. In order to circumvent these issues and also further improve potency the use of conformationally adaptive monosaccharides was pursued as an alternative de sign strategy under the premise that their multiple free hydroxyls could improve ligand binding to the flat surface of gp130, both on a conformational basis and on the basis o maximizing hydrogen bonding with surface residues. By combining data from the struc ture-activity relationship of MDL analogues developed en route to MDL-101, along with our previous identification of bazedoxifene as a repurposed IL-6-signaling inhibito through multiple ligand simultaneous docking [46,47], a general, synthetically accessibl core scaffold was designed for subsequent modification with various linkers and carbo hydrate units ( Figure 3). In pursuit of this strategy, 15 novel, carbohydrate-containing compounds were developed and tested in vitro. With these building blocks in hand, a series of triazole-linked compounds were sy thesized first (Scheme 2). The synthesis proceeded via amide coupling between orth meta-, and para-ethynyl anilines 8-10 with 4-(2-(piperidin-1-yl)ethoxy)benzoyl chlori (24) to afford the desired amide intermediates 11-13 in moderate to good yield. These termediates were then subjected to click chemistry using excess copper(II) sulfate to p vide the desired triazole products in moderate yield. Deprotection of the xylose-bas compounds via Zemplen deacetylation and subsequent purification via HPLC afford the free-hydroxyl products in low to decent yield as acetate salts (LS-TX-2, LS-TX-3, a LS-TX-4).
Cytotoxicity assays were then pursued in SUM159 and MDA-MB-231 triple-negati With these building blocks in hand, a series of triazole-linked compounds were synthesized first (Scheme 2). The synthesis proceeded via amide coupling between ortho-, meta-, and para-ethynyl anilines 8-10 with 4-(2-(piperidin-1-yl)ethoxy)benzoyl chloride (24) to afford the desired amide intermediates 11-13 in moderate to good yield. These intermediates were then subjected to click chemistry using excess copper(II) sulfate to provide the desired triazole products in moderate yield. Deprotection of the xylosebased compounds via Zemplen deacetylation and subsequent purification via HPLC afforded the free-hydroxyl products in low to decent yield as acetate salts (LS-TX-2,
Cytotoxicity assays were then pursued in SUM159 and MDA-MB-231 triple-negative breast cancer cell lines [56][57][58]. To our disappointment, however, these three xylosecontaining compounds were found to be completely inactive in MTT assays against both of these cell lines at the tested concentrations (Table 1). It was then postulated that the free hydroxyls do not favorably interact with the surface of gp130-D1 as previously presumed. Drawing from previous literature and our experience with incorporation of hydrophobic moieties leading to increased activity against this target (as with MDL-101) [40,44], it was suggested that the intermediates containing protected carbohydrates could possess improved activity. Indeed, when these protected intermediates were tested in the same MTT assay conditions, activity was restored against both cell lines (Table 1). In this short series of analogues, there are some general trends that are observed, both with respect to substitution pattern and monosaccharide protecting group choice. For the protected xyloseand glucose-containing analogues, the ortho and para-substituted compounds exhibited improved activity compared to their respective meta regioisomers. Indeed, the glucosecontaining compounds are the most potent of this series, with compounds LS-TG-2P and LS-TG-4P exhibiting 6.9 and 2.5 µM activity in SUM159 cells, respectively. Among the protected fructose-containing compounds, which contain acetonide-protecting groups instead of acetyl groups, the meta regioisomer, LS-TF-3P, exhibited the best activity with an IC 50 of 16 µM in SUM159 cells. While the stereochemical arrangement of the hydroxyls of the fructose unit differs from those of xylose and glucose, it is likely that the primary driving force in the differentiation of activity patterns among regioisomers is due to the acetonide-protecting group, which forces the fructose carbohydrate unit to adopt a more rigid and bulky conformation compared its analogous xylose and glucose compounds. Additionally, despite the increase in lipophilicity due to the incorporation of protecting groups, the predicted logP values for these compounds still remain under the threshold of 5 as set by Lipinski's Rule of 5 for oral bioavailability [59]. Given that retaining protecting groups led to the recovery of activity, it was then desired to probe the effect of linker types on in vitro efficacy. A series of amide-linked fructose-containing regioisomers were then synthesized (Scheme 3). Starting with commercially available ortho-, meta-, and para-nitroanilines 14-16, amide coupling was conducted with 4-(2-(piperidin-1-yl)ethoxy)benzoyl chloride (24) to afford the desired products 17-19 in moderate to good yield. Subsequent hydrogen over palladium reduction of the nitro groups afforded the desired aniline intermediates 20-22, which were then subjected to amide coupling with fructose derivative 7 using methanesulfonyl chloride and N-methyl imidazole (procedure adapted from the literature [60]) to afford the final products in low to decent yield. While these three compounds also exhibited activity in MTT assays (see Table 1), it was apparent that these amidelinked scaffolds were less active compared to their triazole-linked analogues, save for the performance of LS-AF-4P against SUM159 cells. It is noted, though, that these amide-linked compounds displayed a different pattern of activity compared to the triazole-linked fructose compounds, LS-TF-2P, 3P, and 4P, in that the para-substituted amide-linked analogue, LS-AF-4P, was the most active, with an IC 50 of 18.1 µM against SUM159 cells. Given the overall worse performance in vitro compared to the triazole-linked analogues, however, further exploration of carbohydrate diversity within the amide-linked series was not pursued.
As a means of assessing the effect of incorporating carbohydrates on in vitro activity, three N-unsubstituted triazole compounds were synthesized using a modified click reaction adapted from the literature (Scheme 4) [61,62]. In this procedure, alkynes 11-13 were reacted with azidomethanol (formed in situ from sodium azide and formaldehyde) under mildly acidic click conditions, after which the hydroxymethyl group was removed under basic conditions to afford the desired N-unsubstituted triazoles LS-T-2, LS-T-3, and LS-T-4 in low yield (Note: Compounds LS-T-3 and LS-T-4 were purified via HPLC to afford their acetate salt). These compounds were then subjected to MTT assays (Table 2). When controlled for substitution pattern, these compounds were generally less potent compared to their carbohydrate-containing companions in SUM159 cell lines. Key exceptions to this rule include ortho analogues LS-TF-2P and LS-AF-2P, which were weaker than LS-T-2, as well as para analogues LS-TF-4P and LS-AF-4P, which were weaker than LS-T-4. Unexpectedly, however, among all compounds disclosed herein, only LS-TG-2P exhibited better activity against MDA-MB-231 cells than LS-T-4.
x FOR PEER REVIEW 8 of 33  With these in vitro results in hand, two compounds, LS-TF-3P and LS-TG-2P, were chosen for further analysis. LS-TG-4P was not chosen as LS-TG-2P performed better against  With these in vitro results in hand, two compounds, LS-TF-3P and LS-TG-2P, were chosen for further analysis. LS-TG-4P was not chosen as LS-TG-2P performed better against MDA-MB-231 cells, and the former also tended to plateau in cell viability assays rather than kill one-hundred percent of cells. These two lead compounds were then assayed against PC3 and LNCaP prostate cancer cell lines (CCK-8 assay). It was anticipated that these compounds would display different activity against PC3 cells compared to LNCaP cells since the former produces IL-6, while the latter does not [63]. The results of viability assays against these cells are shown in Table 3. Similar to the results seen in the breast cancer MTT data, LS-TG-2P was found to be comparably active to the positive control, bazedoxifene, while LS-TF-3P was shown to be less potent. As anticipated, both bazedoxifene and LS-TF-3P exhibited less potent activity in LNCaP cell lines compared to their PC3 results. While the  With these in vitro results in hand, two compounds, LS-TF-3P and LS-TG-2P, were chosen for further analysis. LS-TG-4P was not chosen as LS-TG-2P performed better against MDA-MB-231 cells, and the former also tended to plateau in cell viability assays rather than kill one-hundred percent of cells. These two lead compounds were then assayed against PC3 and LNCaP prostate cancer cell lines (CCK-8 assay). It was anticipated that these compounds would display different activity against PC3 cells compared to LNCaP cells since the former produces IL-6, while the latter does not [63]. The results of viability assays against these cells are shown in Table 3. Similar to the results seen in the breast cancer MTT data, LS-TG-2P was found to be comparably active to the positive control, bazedoxifene, while LS-TF-3P was shown to be less potent. As anticipated, both bazedoxifene and LS-TF-3P exhibited less potent activity in LNCaP cell lines compared to their PC3 results. While the former has been reported to have no antiproliferative effect in LNCaP cell lines, it was tested at much lower concentrations than in the present study [64]. LS-TG-2P, however, had nearly equivalent activity in both prostate cancer cell lines. In both MTT and CCK-8 assays, this compound was observed to form aggregates at high concentrations, so it is possible that part of its cytotoxicity at higher doses is due to these aggregates rather than its binding to gp130-D1.
Cytokine selectivity assays were then conducted in order to validate the selective inhibition of IL-6 signaling. Monitoring downstream STAT3 or STAT1 phosphorylation provides a quantitative assessment of the inhibition of each of the indicated signaling pathways. While LIF and OSM are members of the IL-6 cytokine family, they do not require a hexameric assembly containing two gp130 units for signaling, and therefore their signaling should not be inhibited by compounds targeting gp130-D1 [65]. IFN-γ, however, does not signal via gp130, and is included to better monitor off-target activity. While not a direct means of assessing ligand-gp130-D1 binding, this provides a good determination of selectivity for this target as only IL-6 and IL-11 require binding to the D1 domain of gp130 to facilitate signaling [25,42]. The results of these assays can be seen in Figure 5. From these data, it is evident that both LS-TG-2P and LS-TF-3P selectively inhibit IL-6 signaling at lower concentrations. The latter exhibits some off-target effects at higher concentrations, but this provides a good springboard for further lead development.
These two lead compounds were docked to gp130-D1 using GlideSP, along with LS-T-4, in order to better rationalize their activity ( Figure 6). While the docking scores are not significantly different from the compounds discussed in Figure 4, some conclusions are able to be drawn from the proposed binding modes. For LS-TG-2P, the acetyl groups afford additional hydrogen bonding with ASN92. LS-TF-3P, however, exhibits hydrogen bonding between THR97 and both its monosaccharide and its triazole moieties. Several hydrophobic residues are adjacent to THR97 (PRO14, VAL15, and ILE99), and this could form a small hydrophobic patch that favorably interacts with its acetonide-protecting groups as well. Finally, LS-T-4 displays a favorable binding mode, exhibiting key aromatic interactions with TYR94 as well as hydrogen bonding with THR97. The scores of these three compounds do not correlate well with activity, however, so further modelling is warranted to better understand the relationship between various functionalities of this class of compounds and activity, which would prompt better-directed analogue design. not signal via gp130, and is included to better monitor off-target activity. While not a direct means of assessing ligand-gp130-D1 binding, this provides a good determination of selectivity for this target as only IL-6 and IL-11 require binding to the D1 domain of gp130 to facilitate signaling [25,42]. The results of these assays can be seen in Figure 5. From these data, it is evident that both LS-TG-2P and LS-TF-3P selectively inhibit IL-6 signaling at lower concentrations. The latter exhibits some off-target effects at higher concentrations, but this provides a good springboard for further lead development. These two lead compounds were docked to gp130-D1 using GlideSP, along with LS-T-4, in order to better rationalize their activity ( Figure 6). While the docking scores are not significantly different from the compounds discussed in Figure 4, some conclusions are able to be drawn from the proposed binding modes. For LS-TG-2P, the acetyl groups afford additional hydrogen bonding with ASN92. LS-TF-3P, however, exhibits hydrogen bonding between THR97 and both its monosaccharide and its triazole moieties. Several hydrophobic residues are adjacent to THR97 (PRO14, VAL15, and ILE99), and this could form a small hydrophobic patch that favorably interacts with its acetonide-protecting groups as well. Finally, LS-T-4 displays a favorable binding mode, exhibiting key aromatic interactions with TYR94 as well as hydrogen bonding with THR97. The scores of these three compounds do not correlate well with activity, however, so further modelling is warranted to better understand the relationship between various functionalities of this class of compounds and activity, which would prompt better-directed analogue design. Overall, carbohydrate incorporation into an optimized IL-6-signaling inhibitor scaffold was pursued as an alternative means of improving compound activity, leading to the development of lead compounds LS-TG-2P and LS-TF-3P, which exhibited 6.9 and 16 µ M activity, respectively, against SUM159 breast cancer cell lines in vitro. LS-TG-2P was demonstrated to have single-digit micromolar activity against prostate cancer cell lines as well, though its activity could be skewed by aggregation at higher concentrations. Cytokine selectivity assays indicate that both of these compounds act as selective inhibitors of IL-6 signaling at lower concentrations. Furthermore, these compounds were more active than their carbohydrate-deficient analogues, demonstrating the viability of this strategy as a means of improving compound activity against flat, difficult-to-target proteins such as gp130. Further tests of these lead compounds are ongoing in order to further validate their activity and mechanism of action, as are drug development efforts focused on improving their drug properties and further increasing their potency. Overall, carbohydrate incorporation into an optimized IL-6-signaling inhibitor scaffold was pursued as an alternative means of improving compound activity, leading to the development of lead compounds LS-TG-2P and LS-TF-3P, which exhibited 6.9 and 16 µM activity, respectively, against SUM159 breast cancer cell lines in vitro. LS-TG-2P was demonstrated to have single-digit micromolar activity against prostate cancer cell lines as well, though its activity could be skewed by aggregation at higher concentrations. Cytokine selectivity assays indicate that both of these compounds act as selective inhibitors of IL-6 signaling at lower concentrations. Furthermore, these compounds were more active than their carbohydrate-deficient analogues, demonstrating the viability of this strategy as a means of improving compound activity against flat, difficult-to-target proteins such as gp130. Further tests of these lead compounds are ongoing in order to further validate their activity and mechanism of action, as are drug development efforts focused on improving their drug properties and further increasing their potency.

Chemistry-General Information
All reactions were carried out under air unless stated otherwise. Reagents and solvents were purchased from commercial vendors and used without further purification. All reactions were monitored using thin layer chromatography (silica gel 60 F254 pre-coated aluminum plates). For non-UV active compounds, visualization of TLC spots was conducted using a 5% H 2 SO 4 in ethanol stain. Purification was conducted using automated flash column chromatography (Biotage Isolera One Purification System) or High-Performance Liquid Chromatography (Dionex UltiMate 3000 with a pump and DAD-3000 in-line Diode Array Detector; reverse-phase C18 Luna, 5 µM, 100 Å, 250 × 21.2 mm column).
NMR spectra were obtained using the Bruker Avance NEO-600 ( 1 H NMR: 600 MHz; 13 C NMR: 151 MHz). All spectra are visualized using MestReNova 11.0, and all structures shown were drawn using ChemDraw 18.1. The following solvents were used for obtaining spectral data, and their corresponding reference peaks are shown here: CDCl 3 ( 1 H NMR: 7.26 ppm; 13 C NMR: 77.16 ppm) and DMSO-d6 ( 1 H NMR: 2.50 ppm, 13 C NMR: 39.52 ppm). All peaks were referenced either to these solvent peaks or to TMS ( 1 H NMR: 0.00 ppm, 13 C NMR: 0.00 ppm). All NMR experiments were conducted at room temperature. For 1 H NMR, multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, quint = quintet, and m = multiplet. Multiplets containing chemically inequivalent protons are further annotated as overlapping signals (os).
Low-resolution mass spectra for certain known compounds were obtained from the University of Florida Department of Medicinal Chemistry 3200 QTrap LC/MS/MS spectrometer via direct injection, and high-resolution mass spectra were obtained from the Mass Spectrometry Facility within the University of Florida Chemistry Department Agilent 6220 or Agilent 6230 Time-of-Flight Spectrometer with Electrospray Ionization or from the University of Florida Department of Medicinal Chemistry Thermo Scientific™ Q Exactive Focus mass spectrometer with Dionex™ Ultimate™ RSLC 3000 UHPLC system, equipped with H-ESI II probe on Ion Max API Source.
NMR spectra were obtained using the Bruker Avance NEO-600 ( 1 H NMR 13 C NMR: 151 MHz). All spectra are visualized using MestReNova 11.0, and al shown were drawn using ChemDraw 18.1. The following solvents were used ing spectral data, and their corresponding reference peaks are shown here: NMR: 7.26 ppm; 13 C NMR: 77.16 ppm) and DMSO-d6 ( 1 H NMR: 2.50 ppm, 13 C N ppm). All peaks were referenced either to these solvent peaks or to TMS ( 1 H ppm, 13 C NMR: 0.00 ppm). All NMR experiments were conducted at room te For 1 H NMR, multiplicities are reported as follows: s = singlet, d = doublet, t = tr = quintet, and m = multiplet. Multiplets containing chemically inequivalent p further annotated as overlapping signals (os).
Low-resolution mass spectra for certain known compounds were obtaine University of Florida Department of Medicinal Chemistry 3200 QTrap LC/M trometer via direct injection, and high-resolution mass spectra were obtaine Mass Spectrometry Facility within the University of Florida Chemistry Depa ilent 6220 or Agilent 6230 Time-of-Flight Spectrometer with Electrospray Io from the University of Florida Department of Medicinal Chemistry Thermo Sc Exactive Focus mass spectrometer with Dionex™ Ultimate™ RSLC 3000 UHP equipped with H-ESI II probe on Ion Max API Source.

General Procedure for the Preparation of 4-(2-piperidin-1-yl)ethoxy)benzoyl chloride (24):
To an oven-dried 50 mL round bottom flask was added 4-(2-piperidin-1-yl)ethoxy)benzoic acid hydrochloride (1.8269 g, 6.393 mmol, 1.0 eq), which was suspended in 2.5 mL thionyl chloride. Anhydrous DMF (3 drops, catalytic) was added, and the mixture was refluxed for 2 h. The solvent was then evaporated to afford the desired product as a pale yellow solid in quantitative yield, which was taken directly to the next step.

General Procedure for the Preparation of 4-(2-piperidin-1-yl)ethoxy)benzoyl chloride (24):
To an oven-dried 50 mL round bottom flask was added 4-(2-piperidin-1-yl)ethoxy)benzoic acid hydrochloride (1.8269 g, 6.393 mmol, 1.0 eq), which was suspended in 2.5 mL thionyl chloride. Anhydrous DMF (3 drops, catalytic) was added, and the mixture was refluxed for 2 h. The solvent was then evaporated to afford the desired product as a pale yellow solid in quantitative yield, which was taken directly to the next step.
Molecules 2023, 28, x FOR PEER REVIEW 13 of 33 General Procedure for the Preparation of 4-(2-piperidin-1-yl)ethoxy)benzoyl chloride (24): To an oven-dried 50 mL round bottom flask was added 4-(2-piperidin-1-yl)ethoxy)benzoic acid hydrochloride (1.8269 g, 6.393 mmol, 1.0 eq), which was suspended in 2.5 mL thionyl chloride. Anhydrous DMF (3 drops, catalytic) was added, and the mixture was refluxed for 2 h. The solvent was then evaporated to afford the desired product as a pale yellow solid in quantitative yield, which was taken directly to the next step.

General Procedure for the Preparation of 4-(2-piperidin-1-yl)ethoxy)benzoyl chlo ride (24):
To an oven-dried 50 mL round bottom flask was added 4-(2-piperidin-1-yl)eth oxy)benzoic acid hydrochloride (1.8269 g, 6.393 mmol, 1.0 eq), which was suspended in 2.5 mL thionyl chloride. Anhydrous DMF (3 drops, catalytic) was added, and the mixtur was refluxed for 2 h. The solvent was then evaporated to afford the desired product as a pale yellow solid in quantitative yield, which was taken directly to the next step.