Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists © 2023 The Author(s). Published by Elsevier Ltd. Published version Leino, Teppo O.; Turku, Ainoleena; Urvas, Lauri; Adhikari, Karuna; Oksanen, Jouni; Steynen, Yana; Yli-Kauhaluoma, Jari; Xhaard, Henri; Kukkonen, Jyrki P.; Wallén, Erik A. A. Leino, T. O., Turku, A., Urvas, L., Adhikari, K., Oksanen, J., Steynen, Y., Yli-Kauhaluoma, J., Xhaard, H., Kukkonen, J. P., & Wallén, E. A.A. (2023). Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists. Bioorganic and Medicinal Chemistry, 88-89, Article 117325. https://doi.org/10.1016/j.bmc.2023.117325 2023 Bioorg. Med. Chem. 88–89 (2023) 117325 Available online 9 May 2023 0968-0896/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Azulene as a biphenyl mimetic in orexin/hypocretin receptor agonists Teppo O. Leino a , b , * , 1 , Ainoleena Turku a , c , 1 , 2 , Lauri Urvas a , d , 3 , Karuna Adhikari a , Jouni Oksanen a , Yana Steynen a , Jari Yli-Kauhaluoma a , Henri Xhaard a , Jyrki P. Kukkonen c , d , Erik A.A. Wall´ en a a Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FI-00014 University of Helsinki, Finland b Department of Chemistry and NanoScience Center, University of Jyv¨ askyl¨ a, P.O. Box 35, FI-40014 University of Jyv¨ askyl¨ a, Finland c Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 66, FI-00014 University of Helsinki, Finland d Department of Pharmacology, Faculty of Medicine, University of Helsinki, P.O. Box 63, FI-00014 University of Helsinki, Finland ARTICLE INFO Keywords: Agonist Azulene Medicinal chemistry Orexin receptor Sulfonamides ABSTRACT Azulene is a rare ring structure in drugs, and we investigated whether it could be used as a biphenyl mimetic in known orexin receptor agonist Nag 26, which is binding to both orexin receptors OX 1 and OX 2 with preference towards OX 2 . The most potent azulene-based compound was identified as an OX 1 orexin receptor agonist (pEC 50 =5.79 ±0.07, maximum response =81 ±8% (s.e.m. of five independent experiments) of the maximum response to orexin-A in Ca 2+ elevation assay). However, the azulene ring and the biphenyl scaffold are not identical in their spatial shape and electron distribution, and their derivatives may adopt different binding modes in the binding site. 1. Introduction The orexin/hypocretin system, which consists of two orexin receptors (OX 1 and OX 2 ) and their endogenous peptide-ligands orexin-A and orexin-B, 1–2 plays a part in many physiological functions, such as sleep–wake regulation, analgesia, stress response, motivational behaviour and addiction, and feeding and metabolic regulation. 3–7 Its central role in sleep-wake regulation has raised interest in orexin receptor antagonists as a novel class of hypnotics, now already realized as two compounds in the clinical use. 8–9 On the other hand, a study in a mouse model of narcolepsy has demonstrated that orexin receptor agonists may prove advantageous as a treatment for narcolepsy. 10 Furthermore, OX 1 activation is linked to anti-proliferation in some colorectal cancer cell lines suggesting that OX 1 agonists could be beneficial for cancer treatment. 11–15 Unlike antagonists, agonists have gained attention in drug discovery only recently, and a few series of potent small molecular orexin receptor agonists have been reported in the literature so far. 16–21 The first series was published in 2015 with Nag 26 (1) as the most potent compound in it (Figure 1). 16,22 Nag 26, like several other potent orexin receptor agonists, has a biphenyl scaffold. 16,19–21 As a consequence of replacing the amidesubstituted phenyl moiety of the biphenyl in Nag 26 with heteroaromatic fiveor six-membered ring, or alkyl group, the activity towards both receptor subtypes has decreased or completely abrogated. 19 The first reported orexin receptor small molecule agonists, including Nag 26, have been OX 2 -selective, 16–18 and not until 2021 a potent dual orexin receptor agonist, RTOXA-43 (2), a derivative of Nag 26, was discovered. 19 RTOXA-43 was obtained by modifying the dimethylcarbamoyl group of Nag 26 (Figure 1). Restriction of the bond rotation in Nag 26 using naphthalene in place of N-phenylethylenediamine moiety has produced a potent and OX 2 -selective agonist 3. 20 In addition, replacement of the naphthalene moiety in 3 with tetralin has led to the discovery of compound 4, which is not only a potent OX 2 but also potent OX 1 agonist. 21 The first potent OX 1 -selective agonist 5, based on compound 4 with modifications on the biphenyl part of the molecule, has been reported recently. 23 Azulene is a bicyclic aromatic hydrocarbon, which has not been intensively employed ring structure in drug molecules. We have * Corresponding author at: Department of Chemistry and NanoScience Center, University of Jyv¨ askyl¨ a, P.O. Box 35, FI-40014 University of Jyv¨ askyl¨ a, Finland. E-mail address: [email protected] (T.O. Leino). 1 These authors contributed equally to this work. 2 Present Address: Orion Pharma R&D, Espoo, Finland. 3 Present Address: Laboratoire d’Innovation Th´ erapeutique, UMR 7200 CNRS, Universit´ e de Strasbourg, Illkirch, France. Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc https://doi.org/10.1016/j.bmc.2023.117325 Received 2 February 2023; Received in revised form 26 April 2023; Accepted 5 May 2023 Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 2 previously studied the applicability of azulene structure in medicinal chemistry, and shown that azulene scaffold does not have any general liabilities to hinder its use in drug discovery. 24 Inspired by this fact, we have studied azulene-based compounds as orexin receptor ligands. 25–26 These compounds indicate a benzoyl group in the 1-position and an ester group in the 6-position of the azulene scaffold as the most important functionalities for the observed weak orexin receptor agonism, and molecular modelling suggested that the hydrogen bond acceptors of these groups lie approximately at the same distances from each other as the amide and N-sulfonyl anilide groups in the biphenyl scaffold of Nag 26. In this study, we use azulene as a substitute for the biphenyl moiety of Nag 26 to further study the applicability of azulenes as biphenyl mimetics in orexin receptor ligands. The effect of replacing the biphenyl moiety of Nag 26 with a fused bicyclic aromatic structure on orexin receptor activity is reported for the first time. 2. Results and discussion We synthesized azulene derivatives from two building blocks, A and B, which are suitable for sulfonamide coupling in the end of the synthesis (Figure 1). Building block A is a 1-sulfonate azulene with the 3and 6-positions available for functionalization and building block B is the unmodified half of Nag 26. 6-Methylazulene (7) 27 or the 6-carboxyazulene 25 -derived amide 8 were used as starting compounds for synthesis of building block A and the synthesis was started by introducing a trifluoroacetyl or a dimethylamide functionality into position 1 (Scheme 1b). The trifluoroacetyl group was introduced with trifluoroacetic anhydride (TFAA) in the presence of Et 3 N giving 9 and 11 in 83% and 94% yields, respectively. The dimethylamide derivative 12 was obtained in 60% yield upon heating 8 and phosgene in toluene followed by addition of dimethylamine. 26 To obtain 10, the temperature was lowered and reaction time shortened, as heating the reaction mixture led to an insoluble solid. The optimized reaction resulted in 10 in 76% yield. Introduction of a sulfonic acid group to the non-functionalized fivemembered ring of azulene failed to give the monosubstituted product, as only the 1,3-disulfonic acid derivative and unreacted starting material were obtained. However, the sulfonic acid derivatives with dimethylamide or trifluoroacetyl group in the 3-position were obtained as sodium sulfonates in good yields. Altogether four different A building blocks, 13, 14, 15 and 16, were synthesized. The synthesis of the building block B (compound 20) was started by heating 1-fluoro-3-nitrobenzene (17) in ethylenediamine, which was followed by the addition of toluoyl chloride to give 18 (Scheme 1c). In order to avoid selectivity issues in the coupling of the building blocks A and B, the amino group of 18 was protected with trifluoroacetamide using TFAA. The reduction of the nitro group of 19 gave 20 in 27% yield over three steps. Building blocks A and B were combined by first converting the sulfonate groups of 13–16 to sulfonyl chlorides by heating them in SOCl 2 (Scheme 1a). Then, the reaction between the sulfonyl chlorides and compound 20 gave 21–24 in 31%, 44%, 38% and 18% yields, respectively. The compounds were deprotected with K 2 CO 3 in water and methanol to give the final products 25, 27, 28 and 29 in 39%, 53%, 76% and 40% yield, respectively. As the building block B is the unmodified half of Nag 26, it is a useful intermediate also in the synthesis of Nag 26, which was synthesized from 20 in two steps with 30% yield. First, 20 was reacted with 5bromo-2-methoxybenzenesulfonylchloride to give the sulfonamide 30. Dimethylaminocarbonyl benzene moiety was coupled to 30 under Suzuki reaction conditions with simultaneous deprotection of the amine to obtain Nag 26. This route reduces the required synthetic steps from eleven to six compared to the original route for Nag 26. Pharmacological characterization of the resulting Nag 26 has been reported previously. 22 OX 1 and OX 2 receptor activation was evaluated first for four azulenebased analogs of Nag 26 and eight synthesis intermediates by measuring compound-mediated Ca 2+ elevation at 10 µM compound concentration in three independent experiments (Supporting Information Figure S1). Two compounds induced OX 1 -mediated Ca 2+ elevation, 27 with 80 ± Figure 1. a. Small molecule orexin receptor agonists. b. Azulene derivatives (in blue) were designed to replace the biphenyl structure of Nag 26 (in red). The synthesis of the azulene-based compounds was designed from building blocks A and B. T.O. Leino et al. Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 3 7% and 29 with 11 ±4% of the maximum response to orexin-A (Supporting Information Figure S1), while none of the compounds induced Ca 2+ response via OX 2 receptors. To further confirm that the observed responses were indeed OX 1 -mediated, we repeated the measurements using control CHO cells that do not express OX 1 receptors; no response to 27 and 29 were observed on these cells (Supporting Information Figure S2). Subsequently, we studied the ability of these 12 compounds to block the orexin-A-induced Ca 2+ signal (Supporting Information Figure S3). Compound 27 was showing OX 1 and OX 2 antagonism corresponding to ca. 4 µM and 2 µM K i values, respectively. However, as 27 also showed efficacy on OX 1 , the K i estimate on OX 1 might be misleading. On OX 2 , also 24, 25 and 29 blocked the response to 0.3 nM orexin-A at levels corresponding to approximately 5–10 µM K i . The rest of the compounds had only a minor effect on the orexin-A response indicating K i >10 µM if any. We continued with 27 assessing the concentration-dependence of the Ca 2+ elevation on OX 1 receptors; stimulation by 27 led to a similar magnitude of Ca 2+ elevation as by orexin-A, but with a notably weaker potency (Figure 2). Furthermore, the effect of 27 is approximately 10% of the effect of Nag 26 on OX 1 -receptors; pEC 50 for 27 was 5.79 ±0.07 (n =5) and for Nag 26 6.66 ±0.06 (as measured previously with identical assay set-up. 22 However, OX 2 activation differs dramatically between these two compounds – Nag 26 is also a potent OX 2 agonist (pEC 50 =7.77 ±0.07 22 ), whereas 27 has no OX 2 activity (Supporting Information Figure S1). To explain further the observed preference in efficacy of 27 towards OX 1 receptor compared to OX 2 , and corresponding structure–activity relationships, we took an advantage of a recently published cryo-EM structure of OX 2 in complex with a G protein and a small molecule agonist 31 (PDB ID: 7L1V), structurally closely related to Nag 26 (Figure 3). 28 OX 2 shares 80% sequence identity with OX 1 in the TM region and has nearly identical ligand binding site (only 2–4 differing amino acid residues, depending on how the binding site is framed). We built a homology model of OX 1 based on the cryo-EM structure of OX 2 and docked 27 to the OX 1 model; the conformation and the interactions formed by the highest scoring binding mode of 27 were similar to those of 31 in the OX 2 structure (Figure 3 a and b). In the 27 docking pose, the sulfonamide core of 27 forms two hydrogen bonds with the upward-facing Gln 3.32 (for the amino acid numbering, see references 29,30 ), while the surrounding ring structures Scheme 1. Synthesis of azulene-based target agonists via building blocks A and B, and a novel synthetic route for Nag 26. Reagents and conditions: (a) TFAA, Et 3 N, DCM, 0 ◦C, 1 h, 83% (9), 94% (11); (b) i) Phosgene, toluene, rt or 80 ◦C, 1.5–5.5 h, ii) Dimethylamine, THF, 0 ◦C, 30 min, 76% (10), 60% (12); (c) i) Sulfur trioxide pyridine complex, benzene, 85 ◦C, 3.5 h, ii) NaOH, H 2 O, EtOH, 30 ◦C, 2 h, 84% (13), 88% (14), 53% (15), 77% (16); (d) i) Ethylenediamine, 100 ◦C, 23 h, ii) mToluoyl chloride, N,N-diisopropylethylamine (DIPEA), DCM, 0 ◦C, 2 h, 54%; (e) TFAA, Et 3 N, THF, 0 ◦C, 1 h, 99%; (f) H 2 , Pd/C, EtOH, rt, 4.5 h, 51%; (g) i) Building block A, SOCl 2 , 67 ◦C, 15 min, ii) 20, Et 3 N, DCM, rt, 19–22 h, 31% (21), 44% (22), 38% (23), 18% (24); (h) K 2 CO 3 , MeOH, H 2 O, rt, 3 h, 39% (25), 53% (27), 76% (28), 40% (29); (i) 5-Bromo-2-methoxybenzenesulfonyl chloride, Et 3 N, DCM, rt, 48 h, 57%; (j) 3-(N,N-Dimethylaminocarbonyl)benzeneboronic acid, Pd(dppf)Cl 2 , Na 2 CO 3 , H 2 O, 1,4-dioxane, 100 ◦C, 48 h, 53%. T.O. Leino et al. Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 4 are lined by Pro 3.29 , Met 4.64 , Ser 2.61 and Val 2.64 . The ability to stabilize Gln 3.32 in the upward conformation was shown to be crucial for agonist activity on OX 2 receptors. 28 Notably, only compounds with a sulfonamide group have been reported as potent small molecule orexin receptor agonists so far. 16–21 The hydrophobic bottom of the binding cavity is occupied by the amide-linked tolyl, while the amide carbonyl lays within a hydrogen-bonding distance from His 7.39 . Compounds with the trifuoroacetyl protecting group of the secondary amine (as in 22 and 24) were inactive, suggesting that limited space is available in the bottom of the binding site. The azulene moiety of 27 is located near TM2 and TM7, surrounded by residues His 7.39 , Tyr 7.32 , Val 2.64 , and Ser 2.61 . The methyl group in the 6-position of the azulene occupies a nook between Ser 2.61 and His 7.39 . Ser 2.61 is one of the few positions where the ligand binding site of OX 1 differs from that of OX 2 ; OX 2 has a threonine in this position, thus leaving less room between TM2 and TM7. This likely explains the difference between the orexin receptor subtypes in efficacy of 27, as the methyl group would not fit between TM2 and TM7 in OX 2 as it does in OX 1 (Figure 3c). Furthermore, when replacing the methyl group in the 6position of azulene with a dimethylamide group (compound 29), the OX 1 agonist activity decreases dramatically, as there is likely no room for a larger functional group between the helices. Even more drastic drop in the activity is observed when the dimethylamide group in the 3position of azulene was replaced with a trifluoroacetyl group, as both trifluoroacetyl derivatives 25 and 28 were inactive. Previously reported modifications at a certain position of Nag 26 structure have resulted in either OX 2 selective agonists, or agonists with equal effect on both orexin receptors. 19–21 Identification of OX 1 selectivity has required the modification of the structure of Nag 26 on both ends of the molecule – at first, a tetralin-type dual-orecin receptor agonist 4 was developed, 21 and further replacement of the biphenyl structure of 4 with anisole or cinnamide derivative has led to compounds preferentially activating OX 1 at moderate potency. 23 Optimization of the compounds has resulted in the first potent OX 1 selective agonist 5. Interestingly, replacing the biphenyl moiety of Nag 26 itself with cinnamide derivative has led to complete loss of both OX 1 and OX 2 activity, 19 suggesting that the preference towards OX 1 in compound 5 and its analogs is not dependent solely on the modification of the biphenyl structure. Thus, the azulene-based derivative 27 reported herein is the first analog of Nag 26 with preference towards OX 1 , which has been reached by modifying only the biphenyl structure. As such, compound 27 contributes to the establishment of structure–activity relationships related to subtype selectivity between the orexin receptors and development of OX 1 -selective agonists, although the potency of the compound has yet remained moderate. 3. Conclusions In conclusion, our results demonstrate that the azulene ring can mimic a biphenyl moiety in orexin receptor agonists. Both types of orexin receptor agonists, those bearing biphenyl as well as those bearing Figure 2. Ca 2+ responses to orexin-A (filled triangles), Nag 26 (filled squares) and 27 (filled circles) on CHO-hOX 1 cells. The responses were normalized to baseline separately for each independent sample before averaging. N =5. Nag 26 graph is reproduced from data originally published by Rinne et al. 22 for easier comparison. Figure 3. a. Binding mode of agonist 31 (cyan) in complex with OX 2 (PDB ID: 7L1V). b. Docking pose of 27 (bright yellow) in complex with OX 1 homology model. c. Superimposition of 27-OX 1 complex to OX 2 structure. Red dashes mark the possible clash between 27 and OX 2 . Receptors are shown as cartoon and the binding site residues as sticks (white and pink for OX 1 and OX 2 , respectively). Color code is as follows: blue, nitrogen; red, oxygen; yellow, sulfur; pink (OX 2 ) / white (OX 1 ) / cyan (31) / bright yellow (27), carbon. Gray dashes indicate possible hydrogen bonds. d. 2D comparison of Nag 26 (1), 27 and 31. Analogous parts of the compounds are marked with same colors. T.O. Leino et al. Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 5 azulene scaffold, have been shown to exhibit good efficacy on orexin receptors. While the efficacy of the azulene-based OX 1 agonist 27 is close to that of orexin-A, the potency of 27 remains moderate. However, this study was based on a limited number of compounds, and thus further compound optimization could improve the potency. As the majority of the currently known small molecule orexin receptor agonists are OX 2 -selective, 27 poses a promising starting point for developing OX 1 -selective tool compounds for pharmacological research to validate the significance of OX 1 modulation in vivo. Understanding the structure–activity relationships for OX 1 -selective agonists could also open other therapeutic opportunities in conditions such as colon cancer. 4. Experimental section 4.1. Chemistry General information: All compounds were characterized by NMR spectroscopy using a Bruker Avance III 400 MHz spectrometer or a Varian Mercury 300 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) relative to the residual solvent signals: CDCl 3 7.26 and 77.16 ppm, DMSO‑d 6 2.50 and 39.52 ppm, acetone‑d 6 2.05 and 29.84 ppm, CD 3 OD 3.31 and 49.00 ppm for 1 H and 13 C NMR, respectively. The progress of the reactions was monitored by thin-layer chromatography on silica gel 60-F 254 plates. When a product was purified by flash chromatography, silica gel (SiO 2 ) 60 (230–400 mesh) or silica gel with a Biotage SP1 purification system (SNAP 10, 25, or 50 g cartridges) was used. Mass spectrometric analysis were performed with a Waters Synapt G2 HDMS mass spectrometer using electrospray ionization (ESI). The purity was determined by UPLC-MS with diode-array detector. The purity of all biologically tested compounds was 95% or higher. N,N-Dimethylazulene-6-carboxamide (8). Azulene-6-carboxylic acid (0.25 g, 1.4 mmol) was dissolved in DMF (5.5 mL). The blue solution was cooled to 0 ◦C and EDC⋅HCl (0.30 g, 1.6 mmol) and HOBt⋅H 2 O (0.24 g, 1.6 mmol) were added. The reaction mixture was stirred at 0 ◦C for 15 min and at room temperature for 1 h. A 2 M solution of dimethylamine in THF (1.8 mL, 3.6 mmol) was added and the stirring was continued at room temperature for 1 h. H 2 O (40 mL) was added to the reaction mixture, and it was extracted with EtOAc (80 mL). The organic phase was washed with a 1 M aqueous solution of HCl (40 mL) and a saturated aqueous solution of NaHCO 3 (40 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide a green solid. The crude product was purified by flash chromatography (EtOAc) to give 8 as a green, amorphous solid (0.23 g, 81%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.39–8.36 (m, 2H), 7.97–7.96 (m, 1H), 7.45–7.44 (m, 2H), 7.15–7.12 (m, 2H), 3.16 (s, 3H), 2.94 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 172.9, 144.9, 140.1, 138.5, 136.3, 120.8, 119.4, 39.5, 35.4. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 13 H 14 NO 200.1075; Found 200.1077. 2,2,2-Trifluoro-1-(6-methylazulen-1-yl)ethan-1-one (9). TFAA (0.46 mL, 3.3 mmol) was added dropwise to a solution of 6-methylazulene (0.43 g, 3.0 mmol) and Et 3 N (0.84 mL, 6.0 mmol) in anhydrous DCM (8.0 mL) under argon on ice bath. The resulting mixture was stirred at 0 ◦C for 1 h. Then H 2 O (60 mL) was added and stirring was continued for an additional 5 min. DCM (60 mL) was added and phases were separated. The organic phase was washed with H 2 O (3 ×40 mL) and brine (40 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide a red solid. The crude product was purified by flash chromatography (n-heptane/EtOAc 9:1) to give 9 as a red, amorphous solid (0.60 g, 83%). 1 H NMR (300 MHz, CDCl 3 ) δ 9.72 (d, J =10.2 Hz, 1H), 8.39 (d, J =10.2 Hz, 1H), 8.26–8.23 (m, 1H), 7.67 (d, J =10.5 Hz, 1H), 7.57 (d, J =10.2 Hz, 1H), 7.23 (d, J =4.2 Hz, 1H), 2.78 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 176.0 (q, J =33.5 Hz), 153.6, 146.0, 142.7, 139.8 (q, J =3.8 Hz), 138.9, 138.2, 133.0, 131.9, 119.6, 117.6, 117.6 (q, J = 290.2 Hz), 28.3. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 13 H 10 OF 3 239.0684; Found 239.0690. N,N,6-Trimethylazulene-1-carboxamide (10). A 20% solution of phosgene in toluene (9.3 mL, 18 mmol) was added to a solution of 6methylazulene (0.50 g, 3.5 mmol) in anhydrous toluene (0.90 mL) under argon. The resulting dark blue solution was stirred at room temperature for 1.5 h and then solvents were evaporated. The purple residue was dissolved in anhydous THF (6.0 mL) under argon. The purple solution was cooled to 0 ◦C and a 2 M solution of dimethylamine in THF (8.8 mL) was slowly added. The reaction mixture was stirred at 0 ◦C for 30 min. The mixture was diluted with DCM (60 mL) and washed with a 1 M aqueous solution of HCl (30 mL). The aqueous phase was extracted with DCM (3 ×60 mL +1 ×30 mL). The organic phases were combined, washed with a saturated aqueous solution of NaHCO 3 (100 mL) and H 2 O (100 mL), dried over anhydrous Na 2 SO 4, filtered, and evaporated to provide a purple oil. The crude product was purified by flash chromatography (n-heptane/EtOAc 1:4) to give 10 as a purple oil (0.57 g, 76%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.62 (d, J =10.0 Hz, 1H), 8.25 (d, J =10.0 Hz, 1H), 7.82 (d, J =4.0 Hz, 1H), 7.25–7.19 (m, 3H), 3.15 (s, 6H), 2.67 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 169.4, 150.7, 140.6, 137.0, 136.9, 136.1, 135.5, 126.8, 126.4, 122.8, 116.7, 37.6* 28.3 (*Methyl carbons of amide only visible in HSQC). HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 14 H 16 NO 214.1232; Found 214.1233. N,N-Dimethyl-1-(2,2,2-trifluoroacetyl)azulene-6-carboxamide (11). TFAA (0.30 mL, 2.3 mmol) was added dropwise to a solution of 8 (0.15 g, 0.75 mmol) and Et 3 N (0.32 mL, 2.3 mmol) in anhydrous DCM (2.1 mL) under argon on ice bath. The resulting mixture was stirred at 0 ◦C for 1 h. Then H 2 O (15 mL) was added and stirring was continued for an additional 5 min. DCM (45 mL) was added and phases were separated. The organic phase was washed with H 2 O (2 ×30 mL) and brine (30 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide a purple solid. The crude product was purified by flash chromatography (n-heptane/EtOAc 2:3) to give 11 as purple, amorphous solid (0.21 g, 94%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.87 (d, J =10.4 Hz, 1H), 8.58 (d, J =10.0 Hz, 1H), 8.42–8.40 (m, 1H), 7.72 (dd, J =10.2 Hz, 1.4 Hz, 1H), 7.63 (dd, J =10.0 Hz, 1.6 Hz, 1H) 7.38 (d, J =4.4 Hz, 1H), 3.20 (s, 3H), 2.95 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 176.2 (q, 2 J C,F = 34.3 Hz), 171.0, 148.3, 146.8, 143.6, 142.2 (q, 3 J C,F =3.7 Hz), 139.3, 139.1, 129.1, 128.0, 120.7, 118.3, 117.4 (q, 1 J C,F =292.9 Hz), 39.5, 35.5. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 15 H 13 NO 2 F 3 296.0898; Found 296.0901. N 1 ,N 1 ,N 6 ,N 6 -Tetramethylazulene-1,6-dicarboxamide (12). A 20% solution of phosgene in toluene (1.7 mL, 3.3 mmol) was added to a solution of 8 (0.13 g, 0.65 mmol) in anhydrous toluene (5.0 mL) under argon. The resulting dark blue solution was heated at 80 ◦C for 5.5 h and then solvents were evaporated. The purple residue was dissolved in anhydous THF (1.1 mL) under argon. The purple solution was cooled to 0 ◦C and a 2 M solution of dimethylamine in THF (1.6 mL) was slowly added. The reaction mixture was stirred at 0 ◦C for 30 min. The mixture was diluted with DCM (30 mL) and washed with a 1 M aqueous solution of HCl (15 mL), a saturated aqueous solution of NaHCO 3 (15 mL) and H 2 O (15 mL), dried over anhydrous Na 2 SO 4, filtered, and evaporated to provide a dark blue solid. The crude product was purified by flash chromatography (manual gradient of EtOAc → EtOAc/MeOH 97:3) to give 12 as a dark blue, amorphous solid (0.11 g, 60%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.74 (d, J =10.0 Hz, 1H), 8.40 (d, J =10.0 Hz, 1H), 7.97 (d, J =3.6 Hz, 1H), 7.35 (d, J =4.0 Hz, 1H), 7.26–7.24 (m, 2H), 3.16 (m, 9H), 2.92 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 172.3, 168.8, 146.2, 141.7, 137.9, 137.9, 137.6, 136.6, 124.3, 122.9, 122.7, 118.0, 39.5, 35.4. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 16 H 19 N 2 O 2 271.1447; Found 271.1447. General procedure for the synthesis of compounds 13, 14, 15, 16. A mixture of the appropriate azulene derivative and sulfur trioxide pyridine complex (3 equiv) in anhydrous benzene (1.5–10 mL) under argon was heated at 85 ◦C for 3.5 h. Solvents were evaporated and the residue was dissolved in EtOH (2.2–15 mL). A 1 M aqueous solution of NaOH (2.2–15 mL) was added and the resulting mixture was stirred at 30 ◦C for 2 h. n-Butanol and brine were added to the reaction mixture. Phases were separated and the aqueous phase was extracted with several T.O. Leino et al. Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 6 times of n-butanol. The organic phases were combined and dried over anhydrous Na 2 SO 4 , filtered and evaporated to provide a crude product, which was purified by flash chromatography. Sodium 6-methyl-3-(2,2,2-trifluoroacetyl)azulene-1-sulfonate (13). Compound 9 (0.48 g, 2.0 mmol) gave a red solid, which after flash chromatography (manual gradient of MeCN/EtOH 9:1 → 3:2) yielded 13 as a pink, amorphous solid (0.57 g, 84%). 1 H NMR (400 MHz, CD 3 OD) δ 9.79 (d, J =10.4 Hz, 1H), 9.30 (d, J =10.8 Hz, 1H), 8.49–8.48 (m, 1H), 7.97–7.94 (m, 2H), 2.86 (s, 3H). 13 C NMR (101 MHz, CD 3 OD) δ 176.7 (q, J =33.8 Hz), 158.1, 145.0, 141.6, 140.9, 140.0, 139.6 (q, J = 3.9 Hz), 136.0, 135.1, 134.3, 118.7 (q, J =292.5 Hz), 114.9, 28.2. HRMS (ESI-TOF) m/z: [M – Na] - Calcd for C 13 H 8 O 4 F 3 S 317.0096; Found 317.0099. Sodium 3-(dimethylcarbamoyl)-6-methylazulene-1-sulfonate (14). Compound 10 (0.54 g, 2.5 mmol) gave a purple solid, which after flash chromatography (manual gradient of MeCN/EtOH 4:1 → 3:2) yielded 14 as a purple, amorphous solid (0.70 g, 88%). 1 H NMR (400 MHz, CD 3 OD) δ 9.15 (d, J =10.0 Hz, 1H), 8.59 (d, J =10.4 Hz, 1H), 8.10 (s, 1H), 7.57–7.54 (m, 1H), 7.52–7.49 (m, 1H), 3.15 (s, 6H), 2.75 (s, 3H). 13 C NMR (101 MHz, CD 3 OD) δ 170.6, 154.7, 139.3, 138.6, 138.2, 136.2, 135.8, 131.1, 129.9, 129.7, 120.8, 40.1, 35.8, 28.2. HRMS (ESI-QTOF) m/z: [M – Na +2H] + Calcd for C 14 H 16 NO 4 S 294.0800; Found 294.0800. Sodium 6-(dimethylcarbamoyl)-3-(2,2,2-trifluoroacetyl)azulene-1-sulfonate (15). Compound 11 (0.23 g, 0.78 mmol) gave a red solid, which after flash chromatography (manual gradient of MeCN → MeCN/EtOH 4:1) yielded 15 as a red, amorphous solid (0.16 g, 53%). 1 H NMR (400 MHz, CD 3 OD) δ 9.98 (d, J =10.4 Hz, 1H), 9.51 (d, J =10.4 Hz, 1H), 8.67–8.65 (m, 1H), 7.98–7.93 (m, 2H), 3.19 (s, 3H), 2.97 (s, 3H). HRMS (ESI-QTOF) m/z: [M – Na +2H] + Calcd for C 15 H 13 NO 5 F 3 S 376.0467; Found 376.0466. Sodium 3,6-bis(dimethylcarbamoyl)azulene-1-sulfonate (16). Compound 12 (0.099 g, 0.37 mmol) gave a blue solid, which after flash chromatography (MeCN/EtOH 8:2) yielded 16 as a blue, amorphous solid (0.11 g, 77%). 1 H NMR (400 MHz, CD 3 OD) δ 9.34 (d, J =10.0 Hz, 1H), 8.76 (d, J =10.0 Hz, 1H), 8.27 (s, 1H), 7.55–7.52 (m, 1H), 7.49–7.47 (m, 1H), 3.19–3.14 (m, 9H), 2.96 (s, 3H). 13 C NMR (101 MHz, CD 3 OD) δ 173.5, 170.0, 148.7, 140.3, 139.3, 139.1, 138.3, 137.2, 132.9, 125.8, 125.7, 122.6, 39.7, 35.5. HRMS (ESI-QTOF) m/z: [M – Na +2H] + Calcd for C 16 H 19 N 2 O 5 S 351.1015; Found 351.1015. 3-Methyl-N-[2-[(3-nitrophenyl)amino]ethyl]benzamide (18). A mixture of 1-fluoro-3-nitrobenzene (1.1 mL, 10 mmol) and ethylenediamine (7.5 mL) was heated at 100 ◦C for 23 h under argon. The red reaction mixture was quenched with a saturated aqueous solution of NaHCO 3 (70 mL) and it was extracted with DCM (3 ×40 mL). The organic phases were combined, washed with brine (2 ×50 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide a red solid. The solid was dissolved in anhydrous DCM (40 mL) under argon and DIPEA (3.2 mL, 19 mmol) was added. The resulting mixture was cooled to 0 ◦C and m-toluoyl chloride (1.2 mL, 9.3 mmol) was added dropwise over 5 min. The reaction mixture was stirred at 0 ◦C for 2 h and then it was diluted with DCM (40 mL). The organic phase was washed with a saturated aqueous solution of NaHCO 3 (3 ×40 mL) and brine (30 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide an orange solid. The crude product was purified by flash chromatography (n-heptane/(EtOAc:EtOH 3:1) 1:1 → 2:3) to give 18 as a yellow, amorphous solid (1.6 g, 54%). 1 H NMR (400 MHz, acetone‑d 6 ) δ 7.96 (s, 1H), 7.70–7.65 (m, 2H), 7.45 (t, J =2.2 Hz, 1H), 7.42–7.39, (m, 1H), 7.35–7.31 (m, 3H), 7.10–7.07 (m, 1H), 5.94–5.90 (m, 1H), 3.69–3.64 (m, 2H), 3.50–3.45 (m, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, acetone‑d 6 ) δ 168.2, 150.9, 150.4, 138.8, 135.8, 132.6, 130.8, 129.1, 128.6, 125.1, 119.1, 111.2, 106.4, 44.0, 39.7, 21.31. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 16 H 18 N 3 O 3 300.1348; Found 300.1351. 3-Methyl-N-[2-[2,2,2-trifluoro-N-(3-nitrophenyl)acetamido] ethyl]benzamide (19). TFAA (0.75 mL, 5.4 mmol) was added dropwise over 5 min to a solution of 18 (1.6 g, 5.4 mmol) and Et 3 N (0.75 mL, 5.4 mmol) in anhydrous THF (36 mL) under argon on ice bath. The resulting mixture was stirred at 0 ◦C for 1 h. A 1 M aqueous solution of HCl (60 mL) was added and the resulting suspension was stirred at 0 ◦C for 20 min. The reaction mixture was extracted with DCM (100 mL). The phases were separated and the organic phase was washed with H 2 O (60 mL) and brine (60 mL), dried over anhydrous Na 2 SO 4 , filtered and evaporated to provide a brownish solid. The crude product was purified by flash chromatography (DCM/MeOH 99:1 → 9:1) to give 19 as a yellow, amorphous solid (2.1 g, 99%). 1 H NMR (400 MHz, (CDCl 3 ) δ 8.30 (d, J =8.4 Hz, 1H), 8.23–8.22 (m, 1H), 7.75–7.73 (m, 1H), 7.67–7.63 (m, 1H), 7.55 (s, 1H), 7.51–7.48 (m, 1H), 7.32–71 (m, 2H), 6.66 (s, 1H), 4.11–4.06 (m, 2H), 3.76–3.75 (m, 2H), 2.39 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 168.4, 157.9 (q, 2 J C,F =36.9 Hz), 148.9, 140.2, 138.8, 134.9, 133.8, 132.8, 130.8, 128.7, 127.8, 124.5, 123.9, 123.6, 116.1 (q, 1 J C,F =289.3 Hz), 51.4, 38.0, 21.5. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 18 H 17 N 3 O 4 F 3 396.1171; Found 396.1171. N-[2-[N-(3-Aminophenyl)-2,2,2-trifluoroacetamido]ethyl]-3methylbenzamide (20). Compound 19 (2.1 g, 5.3 mmol) was dissolved in EtOH (45 mL) and Pd/C (0.21 g) was added. The resulting black mixture was stirred under H 2 at room temperature for 4 h. The reaction mixture was filtered through a small pad of Celite with EtOH. Solvents were evaporated to provide a yellow solid. After two flash chromatographic purifications (n-heptane/(EtOAc:EtOH 3:1) 9:1 → 2:3 and DCM/ MeOH 99:1 → 19:1) 20 was obtained as yellow, amorphous solid (1.0 g, 51%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.58 (s, 1H), 7.53–7.51 (m, 1H), 7.31–7.30 (m, 2H), 7.17 (t, J =8.0 Hz, 1H), 6.80–6.78 (m, 1H), 6.70–6.68 (m, 1H), 6.61–6.60 (m, 1H), 6.56–6.55 (m, 1H), 4.02–3.99 (m, 2H), 3.74–3.70 (m, 2H), 2.39 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 168.1, 158.6 (q, 2 J C,F =36.2 Hz), 147.9, 139.8, 138.6, 134.1, 132.5, 130.5, 128.7, 127.9, 123.9, 117.6, 116.5 (q, 1 J C,F =289.1 Hz), 115.9, 114.2, 51.0, 38.8, 21.5. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 18 H 19 N 3 O 2 F 3 366.1429; Found 366.1429. General procedure for the synthesis of compounds 21, 22, 23, 24. A mixture of the appropriate azulene derivative (1–2 equiv) and SOCl 2 (0.80–2.0 mL) was heated at 67 ◦C for 15 min. SOCl 2 was evaporated and the residue was dissolved in anhydrous DCM (1–5 mL) under argon. Compound 20 (1 equiv) and Et 3 N (4 equiv) were added, and the resulting mixture was stirred at room temperature for 19–22 h. A 1 M aqueous solution of HCl (5.0–10 mL) was added and then the mixture was stirred for 5 min. The mixture was diluted with DCM (15–50 mL) and the organic phase was washed with a 1 M aqueous solution of HCl (3 ×10–50 mL) and brine (10–50 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide a crude product, which was purified by flash chromatography. 3-Methyl-N-[2-[2,2,2-trifluoro-N-[3-[[6-methyl-3-(2,2,2-trifluoroacetyl)azulene]-1-sulfonamido]phenyl]acetamido]ethyl] benzamide (21). Compound 13 (0.20 g, 0.60 mmol) and 20 (0.11 g, 0.30 mmol) gave a dark red solid, which after flash chromatography (DCM/MeOH 99:1) yielded 21 as an orange, amorphous solid (0.062 g, 31%). 1 H NMR (400 MHz, acetone‑d 6 ) δ 9.80 (d, J =10.4 Hz, 1H), 9.42 (s, 1H), 9.20 (d, J =10.4 Hz, 1H), 8.45–8.43 (m, 1H), 8.15 (d, J =10.8 Hz, 1H), 8.09 (d, J =10.4 Hz, 1H), 7.76–7.72 (m, 1H), 7.57–7.53 (m, 2H), 7.31–7.22 (m, 6H), 3.84 (t, J =5.6 Hz, 2H), 3.47–3.43 (m, 2H), 2.89 (s, 3H), 2.35 (s, 3H). HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 31 H 26 N 3 O 5 F 6 S 666.1497; Found 666.1497. N,N,6-Trimethyl-3-[N-[3-[2,2,2-trifluoro-N-[2-(3-methylbenzamido)ethyl]acetamido]phenyl]sulfamoyl]azulene-1-carboxamide (22). Compound 14 (0.32 g, 1.0 mmol) and 20 (0.18 g, 0.50 mmol) gave a brown solid, which after flash chromatography (manual gradient of EtOAc/DCM/Toluene 40:59:1 → EtOAc) yielded 22 as a red solid (0.14 g, 44%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.91 (d, J =10.4 Hz, 1H), 8.63 (d, J =10.0 Hz, 1H) 8.29 (s, 1H), 7.97 (s, 1H), 7.57 (s, 1H), 7.50 (d, J =6.8 Hz, 1H), 7.42–7.23 (m, 6H), 7.18–7.11 (m, 2H), 6.89–6.88 (m, 2H), 3.73–3.69 (m, 2H), 3.38–3.34 (m, 2H), 3.10–2.97 (m, 6H), 2.66 (s, 3H), 2.35 (s, 3H). HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 32 H 32 N 4 O 5 F 3 S 641.2045; Found 641.2045. N,N-Dimethyl-1-[N-[3-[2,2,2-trifluoro-N-[2-(3T.O. Leino et al. Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 7 methylbenzamido)ethyl]acetamido]phenyl]sulfamoyl]-3-(2,2,2trifluoroacetyl)azulene-6-carboxamide (23). Compound 15 (0.15 g, 0.38 mmol) and 20 (0.069 g, 0.19 mmol) gave a red solid, which after two flash chromatographic purifications (manual gradient of DCM → DCM/MeOH 49:1 and DCM/EtOAc 5:5 → 3:7) yielded 23 as a red solid (0.053 g, 38%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.95 (d, J =10.4 Hz, 1H), 9.27 (d, J =10.4 Hz, 1H), 8.59–5.51 (m, 2H), 7.86 (d, J =10.0 Hz, 1H), 7.74 (d, J =10.0 Hz, 1H), 7.46 (s, 1H), 7.42–7.40 (m, 1H), 7.26–7.22 (m, 4H), 7.07 (s, 1H), 6.10–6.99 (m, 1H), 6.76 (t, J =5.0 Hz, 1H), 3.87–3.85 (m, 2H), 3.54–3.52 (m, 2H), 3.18 (s, 3H), 2.86 (s, 3H), 2.32 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) 176.4 (q, 2 J C,F =35.3 Hz), 170.0, 168.5, 157.9 (q, 2 J C,F =36.3 Hz), 150.7, 145.3, 143.7 (q, 3 J C,F =3.1 Hz), 141.8, 141.4, 139.6, 139.4, 138.7, 138.2, 133.7, 132.7, 132.0, 131.1, 130.7, 128.7, 127.7, 125.2, 125.0, 124.0, 122.7, 122.0, 116.8 (q, 1 J C,F =292.6 Hz), 116.1 (q, 1 J C,F =289.2 Hz), 115.9, 50.9, 39.3, 38.2, 35.6, 21.4. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 33 H 29 N 4 O 6 F 6 S 723.1712; Found 723.1714. N 1 ,N 1 ,N 6 ,N 6 -Tetramethyl-3-[N-[3-[2,2,2-trifluoro-N-[2-(3methylbenzamido)ethyl]acetamido]phenyl]sulfamoyl]azulene1,6-dicarboxamide (24). Compound 16 (0.097 g, 0.26 mmol) and 20 (0.095 g, 0.26 mmol) gave a blue solid, which after flash chromatography (manual gradient of DCM → DCM/MeOH 24:1) yielded 24 as a purple solid (0.032 g, 18%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.10 (d, J = 10.0 Hz, 1H), 8.83 (d, J =9.2 Hz, 1H), 8.39 (s, 1H), 8.10 (s, 1H), 7.57 (s, 1H), 7.46–7.39 (m, 3H), 7.29–7.27 (m, 2H), 7.24–7.16 (m, 2H), 7.02–6.92 (m, 3H), 3.76–3.73 (m, 2H), 3.40–3.37 (m, 2H), 3.15–2.86 (m, 12H), 2.37 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 170.9, 168.3, 167.1, 157.5 (q, 2 J C,F =36.2 Hz), 148.8, 140.8, 139.6, 139.5, 138.9, 138.7, 138.5, 137.9, 137.4, 134.0, 132.5, 130.3, 128.6, 128.0, 126.8, 126.8, 124.8, 124.1, 122.8, 122.8, 122.2, 121.8, 116.1 (q, 1 J C,F =289.3 Hz), 50.8, 39.4, 37.9, 35.5, 21.5. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 34 H 35 N 5 O 6 F 3 S 698.2260; Found 698.2259. General procedure for the synthesis of compounds 25, 27, 28, 29. An appropriate azulene derivative was suspended in a mixture of MeOH (5.5–22 mL) and H 2 O (0.55–2.2 mL). K 2 CO 3 (30 equiv) was added and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAc (60 mL) and washed with H 2 O (2 ×30 mL) and a saturated aqueous solution of NaHCO 3 (30 mL), dried over anhydrous Na 2 SO 4 , filtered and evaporated to provide a crude product, which was purified by flash chromatography. 3-Methyl-N-[2-[[3-[[6-methyl-3-(2,2,2-trifluoroacetyl)azulene]-1-sulfonamido]phenyl]amino]ethyl]benzamide (25). Compound 21 (0.052 g, 0.078 mmol) gave a brown solid, which after flash chromatography (n-heptane/(EtOAc:EtOH 3:1) 3:2) yielded 25 as a an orange, amorphous solid (0.017 g, 39%). 1 H NMR (400 MHz, DMSO‑d 6 ) δ 10.19 (s, 1H), 9.69 (d, J =10.4 Hz, 1H), 9.17 (d, J =10.4 Hz, 1H), 8.41 (t, J =1.8 Hz, 1H), 8.38–8.37 (m, 1H), 8.18–8.15 (m, 1H), 8.11–8.08 (m, 1H), 7.63–7.59 (m, 2H), 7.34–7.32 (m, 2H), 6.83 (t, J =8.0 Hz, 1H), 6.34 (t, J =2.0 Hz, 1H), 6.24–6.22 (m, 2H), 5.73 (t, J =5.6 Hz, 1H), 3.31–3.27 (m, 2H), 3.03–2.99 (m, 2H), 2.84 (s, 3H), 2.35 (s, 3H). 13 C NMR (101 MHz, DMSO‑d 6 ) δ 174.5 (q, 2 J C,F =33.5 Hz), 166.5, 159.2, 149.3, 143.2, 140.4, 140.2 (q, 3 J C,F =4.1 Hz), 139.9, 138.3, 138.2, 137.5, 137.1, 135.9, 134.4, 131.7, 129.5, 128.2, 127.7, 125.0, 124.3, 116.8 (q, 1 J C, F =293.6 Hz), 113.4, 107.9, 107.9, 103.9, 42.3, 38.5, 27.8, 20.9. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 29 H 27 N 3 O 4 F 3 S 570.1674; Found 570.1679. N,N,6-Trimethyl-3-[N-[3-[[2-(3-methylbenzamido)ethyl] amino]phenyl]sulfamoyl]azulene-1-carboxamide (27). Compound 22 (0.11 g, 0.17 mmol) gave a red solid, which after flash chromatography (manual gradient of DCM → DCM/MeOH 97:3) yielded 27 as a red, amorphous solid (0.049 g, 53%). 1 H NMR (400 MHz, DMSO‑d 6 ) δ 9.97 (s, 1H), 9.04 (d, J =10.0 Hz, 1H), 8.64 (d, J =10.4 Hz, 1H), 8.44 (s, 1H), 8.00 (s, 1H), 7.67–7.61 (m, 4H), 7.33 (s, 2H), 6.79 (t, J =7.8 Hz, 1H), 6.36 (s, 1H), 6.23–6.17 (m, 2H), 5.69–5.67 (m, 1H), 3.34 (m, 2H), 3.02–2.91 (m, 8H), 2.72 (s, 3H), 2.35 (s, 3H). 13 C NMR (101 MHz, DMSO‑d 6 ) δ 166.5, 166.1, 155.0, 149.2, 138.9, 138.7, 138.4, 137.5, 136.5, 136.4, 135.1, 134.4, 131.7, 130.5, 130.1, 129.3, 128.2, 127.7, 124.3, 121.1, 120.9, 107.4, 107.1, 103.4, 42.4, 38.6, 27.6, 20.9. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 30 H 33 N 4 O 4 S 545.2222; Found 545.2222. N,N-Dimethyl-1-[N-[3-[[2-(3-methylbenzamido)ethyl]amino] phenyl]sulfamoyl]-3-(2,2,2-trifluoroacetyl)azulene-6-carboxamide (28). Compound 23 (0.038 g, 0.053 mmol) gave a red solid, which after flash chromatography (manual gradient of DCM → DCM/ MeOH 49:1) yielded 28 as a red, amorphous solid (0.025 g, 76%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.81 (d, J =10.4 Hz, 1H), 9.07 (d, J =10.0 Hz, 1H), 8.65–8.64 (m, 1H), 7.98 (s, 1H), 7.71–7.68 (m, 1H), 7.60–7.57 (m, 1H), 7.51 (s, 1H), 7.46–7.44 (m, 1H), 7.24–7.17 (m, 2H), 7.01–6.98 (m, 1H), 6.85 (t, J =8.0 Hz, 1H), 6.40–6.38 (m, 1H), 6.27–6.24 (m, 2H), 3.50–3.46 (m, 2H), 3.15 (s, 3H), 3.11–3.08 (m, 2H), 2.84 (s, 3H), 2.29 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 176.4 (q, 2 J C,F =35.1 Hz), 170.1, 168.9, 150.23, 148.9, 145.0, 143.6 (q, 3 J C,F =3.6 Hz), 141.6, 139.6, 138.5, 137.5, 134.0, 132.5, 131.7, 130.7, 130.1, 128.5, 127.9, 125.9, 124.2, 116.8 (q, 1 J C, F =291.9 Hz), 115.7, 111.5, 111.5, 110.6, 106.6, 44.0, 39.4, 39.4, 35.5, 21.4. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 31 H 30 N 4 O 5 F 3 S 627.1889; Found 627.1887. N 1 ,N 1 ,N 6 ,N 6 -Tetramethyl-3-[N-[3-[[2-(3-methylbenzamido) ethyl]amino]phenyl]sulfamoyl]azulene-1,6-dicarboxamide (29). Compound 24 (0.029 g, 0.042 mmol) gave a purple solid, which after flash chromatography (manual gradient of DCM → DCM/MeOH 24:1) yielded 29 as a purple, amorphous solid (0.010 g, 40%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.02 (d, J =10.4 Hz, 1H), 8.81 (d, J =10.0 Hz, 1H), 8.13 (s, 1H), 7.78–7.74 (m, 1H), 7.64 (s, 1H), 7.59–7.58 (m, 1H), 7.36–7.31 (m, 3H), 7.26–7.23 (m, 2H), 6.83 (t, J =8.0 Hz, 1H), 6.43 (d, J =7.6 Hz, 1H), 6.21 (d, J =8.0 Hz, 1H), 5.89 (s, 1H), 3.28–3.26 (m, 2H), 3.14 (s, 6H), 2.91–2.84 (m, 8H), 2.35 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 171.0, 168.7, 167.3, 148.8, 148.5, 140.8, 139.4, 139.0, 138.4, 138.1, 138.0, 137.6, 134.3, 132.4, 129.9, 128.4, 128.1, 126.4, 124.4, 122.6, 122.6, 111.9, 110.7, 106.3, 44.0, 39.4, 39.3, 35.5, 21.5. HRMS (ESIQTOF) m/z: [M +H] + Calcd for C 32 H 36 N 5 O 5 S 602.2437; Found 602.2437. N-[2-[N-[3-[(5-Bromo-2-methoxyphenyl)sulfonamido] phenyl]-2,2,2-trifluoroacetamido]ethyl]-3-methylbenzamide (30). 5-Bromo-2-methoxybenzenesulfonyl chloride (0.78 g, 2.7 mmol) was added to a yellow solution of 20 (1.0 g, 2.7 mmol) and Et 3 N (0.76 mL, 5.4 mmol) in anhydrous DCM (11 mL) under argon. The resulting mixture was stirred at room temperature for 48 h. A 1 M aqueous solution of HCl (20 mL) was added and the reaction mixture was stirred at room temperature for an additional 15 min. The mixture was extracted with DCM (30 mL). The organic phase was washed with a 1 M aqueous solution of HCl (20 mL) and brine (20 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide an orange oil. The crude product was purified by flash chromatography (n-heptane/(EtOAc:EtOH 3:1) 9:1 → 2:3) to give 30 as a yellowish, amorphous solid (0.96 g, 57%). 1 H NMR (300 MHz, CDCl 3 ) δ 7.91 (d, J =2.4 Hz, 1H), 7.56–7.48 (m, 4H), 7.32–7.20 (m, 4H), 7.09 (s, 1H), 7.00 (d, J =7.8 Hz, 1H), 6.84 (d, J =9.0 Hz, 1H), 6.63–6.60 (m, 1H), 3.96–3.93 (m, 5H), 3.65–3.60 (m, 2H), 2.39 (s, 3H). 13 C NMR (75 MHz, CDCl 3 ) δ 168.2, 158.2 (q, J =35.9 Hz), 155.4, 139.6, 138.7, 138.0, 137.9, 134.1, 133.5, 132.6, 130.7, 128.7, 127.8, 127.5, 125.1, 124.0, 122.1, 120.9, 116.2 (q, J =286.8 Hz), 114.2, 112.9, 56.9, 51.1, 38.3, 21.5. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 25 H 24 N 3 O 5 BrF 3 S 614.0572; Found 614.0576. 4′-Methoxy-N,N-dimethyl-3′-[N-[3-[[2-(3-methylbenzamido) ethyl]amino]phenyl]sulfamoyl][1,1′-biphenyl]-3-carboxamide (1). A mixture of 30 (0.95 g, 1.6 mmol), 3-(N,N-dimethylaminocarbonyl)benzene boronic acid (0.36 g, 1.9 mmol), Pd(dppf)Cl 2 (0.057 g, 0.078 mmol) and Na 2 CO 3 (0.66 g, 6.2 mmol) in 1,4-dioxane (4.8 mL) and H 2 O (4.8 mL) was heated at 100 ◦C for 48 h. The reaction mixture was filtered through a small pad of Celite with EtOAc (150 mL). The organic phase was washed brine (40 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to provide a brown oil. After two flash chromatographic purifications (n-heptane/(EtOAc:EtOH 3:1) 9:1 → EtOAc: T.O. Leino et al. Bioorganic & Medicinal Chemistry 88–89 (2023) 117325 8 EtOH 3:1 and EtOAc) 1 was obtained as an off-white, amorphous solid (0.48 g, 53%). 1 H NMR (400 MHz, DMSO‑d 6 ) δ 9.79 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 7.99 (d, J =2.4 Hz, 1H), 7.89 (dd, J =8.8, 2.4 Hz, 1H), 7.67–7.64 (m, 2H), 7.62–7.61 (m, 1H), 7.58–7.57 (m, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.36–7.32 (m, 3H), 7.27 (d, J =8.8 Hz, 1H), 6.87 (d, J =8.0 Hz, 1H), 6.42–6.41 (m, 1H), 6.33–6.31 (m, 1H), 6.22 (dd, J =8.0, 1.6 Hz, 1H), 5.75 (t, J =5.8 Hz, 1H), 3.94 (s, 3H), 3.36–3.33 (m, 2H), 3.08–3.03 (m, 2H), 2.99 (s, 3H), 2.90 (s, 3H), 2.34 (s, 3H). 13 C NMR (101 MHz, DMSO‑d 6 ) δ 169.8, 166.5, 156.2, 149.2, 138.5, 138.4, 137.5, 137.4, 134.4, 133.0, 131.7, 131.2, 129.4, 129.1, 128.1, 128.0, 127.7, 127.3, 127.1, 125.8, 124.6, 124.3, 113.6, 107.3, 107.3, 103.3, 56.4, 42.4, 39.0, 38.6, 34.7, 20.9. HRMS (ESI-QTOF) m/z: [M +H] + Calcd for C 32 H 35 N 4 O 5 S 587.2328; Found 587.2328. 4.2. Pharmacological analysis Materials: Human orexin-A was from NeoMPS (Strasbourg, France), N-(2-methyl-6-benzoxazolyl)-N′-1,5-naphthyridin-4-yl urea (TCS-1102) and N-biphenyl-2-yl-1-[[(1-methyl-1H-benzimidazol-2-yl)sulfanyl] acetyl]-L-prolinamide (SB334867) from Tocris Bioscience (Bristol, UK), and probenecid from Sigma–Aldrich (St.Louis, MO, USA). Cell culture and media: CHO-K1 cells expressing human OX 1 and OX 2 receptors cells (CHO-hOX 1 and CHO-hOX 2 , respectively), 31–32 and control CHO-K1 cells (not expressing orexin receptors,ctrl CHO cells) were cultured in Ham’s F12 medium (Gibco/Life Technologies, Paisley, UK) +supplements on plastic cell culture dishes (56 cm 2 bottom area; Greiner Bio-One GmbH, Frickenhausen, Germany) as previously described. 33–34 Black, clear-bottom half-area Cellstar μ Clear 96-well cell culture plates (Greiner Bio-One GmbH; Frickenhausen, Germany), coated with polyethyleneimine (25 μ g/mL for 1 h at 37 ◦C; SigmaAldrich, St. Louis, MO, USA), were used for Ca 2+ measurements. Hepes-buffered medium (HBM) consisting of 137 mM NaCl, 5 mM KCl, 1.2 mM MgCl 2 , 0.44 mM KH 2 PO 4 , 4.2 mM NaHCO 3 , 1 mM CaCl 2 , 10 mM glucose, 20 mM HEPES and 0.1% (w/v) stripped bovine serum albumin (adjusted to pH 7.4 with NaOH) was used as the basic experimental medium. Ca 2þ elevation: The compound-induced Ca 2+ elevations were measured as described before. 35 In brief, CHO-hOX 1 , CHO-hOX 2 or control CHO cells were plated on Cellstar plates, 1.5 ×10 4 cells per well. After 24-hour incubation, the cell culture medium was exchanged to the loading solution consisting of FLIPR Calcium 4 Assay Kit (Molecular Devices, Sunnyvale, CA, USA) dissolved in and diluted with HBM +1.0 mM probenecid. After subsequent 60 min incubation at 37 ◦C, intracellular Ca 2+ levels were measured at 37 ◦C using a FlexStation 3 fluorescence plate reader (Molecular Devices, excitation at 485 nm, emission at 525 nm). The wells were measured for 150 s with 30 s of baseline before stimulation and measured approximately every 1.3 s. After the measurement, 0.3 nM orexin-A was added to the wells and the plate was re-measured as described above. Data analysis: Data were visualized using Graph Pad Prism 8 and analysed using Microsoft Excel software. Concentration–response curves were fit using three-parameter nonlinear regression equation. The data points represent the mean ±s.e.m. of 3–5 individual experiments (biological replicates) performed typically in triplicates (technical replicates) unless otherwise stated. All presented Ca 2+ responses were separately normalized to the control maximum orexin-A or ATP responses for each independent experiment before averaging. The background response (vehicle addition) was subtracted from all data values (both in the single-concentration screen and concentration–response curves) prior to the analysis. Crude estimates of the K i values were calculated from the inhibition-% values by first estimating the IC 50 - values and then converting these to K i utilizing the Cheng–Prusoff equation with cooperativity coefficients. The rational of this follows the Equation (1), which also lies behind the Cheng–Prusoff equation. (100 −%inhibition)responsenoinhibitor =[orexin −A]n× (100 −%inhibition) [orexin −A]n+EC50n =responsewithinhibitor =[orexin −A]n×100 [orexin −A]n+EC50n(1+[I] Ki) (1) 4.3. Computational studies Homology modeling: Our goal was to build a model of OX 1 in an active, agonist-bound, conformation suitable for a subsequent docking study with a small molecule agonist. Thus, we used the active-state cryoEM structure of OX 2 (PDB ID: 7L1V) as a template. The binding sites of OX 1 and OX 2 are nearly identical as there are only 2–4 conservative differences among the binding site residues, depending on how the site is framed (S/T 2.61 , S/T 23.49 , A/T 3.33 , S/T 45.48 ). Prior to building the models, the ligand and residues within 5 Å of the ligand in the OX 2 structure were refined using Prime minimization 36–37 (Schr¨ odinger Release 2020–4). Based on this minimized OX 2 structure, ten models of OX 1 were built consisting of residues Trp47 1.33 -Trp243 5.66 and Glu286 6.23 -Ala372 8.57 using MODELLER 10.1 38 with default settings. The Nand C-termini and the ICL3 were not modeled in the cryo-EM structure 7L1V, so they were not included in our models. The model used for the docking study was selected based on the similarity of the binding site residue conformers between the template and the model to facilitate the docking. Molecular docking of 27: The homology model selected for docking was first preprocessed by Protein Preparation Wizard of Schr¨ odinger Maestro (Schr¨ odinger Release 2020–4) with default parameters, and the hydrogen bond assignments were optimized. Control agonist 31 was docked to the refined OX 2 cryo-EM structure and 27 to the OX 1 homology model using Glide SP 39–41 with default settings and a grid defined by the location of the agonist 31 in the OX 2 complex. The docking poses were analyzed visually in Schr¨ odinger Maestro, and further visualization and figure preparation was carried out with PyMOL 2.3.3 and Gimp 2.10.14. 27 docking poses and homology models are provided as supplementar y files. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements T.O.L. acknowledges the Doctoral Programme in Drug Research of the University of Helsinki and the Academy of Finland (grant no. 330800); A.T. acknowledges the Finnish Cultural Foundation and the Orion Research Foundation; and J.P.K. acknowledges the Magnus Ehrnrooth Foundation, the Liv & H¨ alsa Foundation and Finska l¨ akares¨ allskapet for financial support. Marja Peltola, Santeri Suokas and MaijuK. Rinne are acknowledged for assistance with the experiments. We would like to thank Nina Sipari from Viikki Metabolomics Unit (Helsinki Institute of Life Science, University of Helsinki; Biocenter Finland) for her expertise with the LC-MS analyses. CSC – IT Center for Science is thanked for the computational resources. T.O. Leino et al.