UAMC-3203

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Article
Discovery of Novel, Drug-like Ferroptosis Inhibitors with In Vivo Efficacy
Lars Devisscher, Samya Van Coillie, Sam Hofmans, Dries Van Rompaey, Kenneth Goossens, Eline Meul, Louis Maes, Hans De Winter, Pieter Van
der Veken, Peter Vandenabeele, Tom Vanden Berghe, and Koen Augustyns
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01299 • Publication Date (Web): 25 Oct 2018
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Discovery of Novel, Drug-like Ferroptosis Inhibitors with In Vivo Efficacy

Lars Devisscher # a, Samya Van Coillie # b,c, Sam Hofmans a, Dries Van Rompaey a, Kenneth Goossens a, Eline Meul b,c, Louis Maes d, Hans De Winter a, Pieter Van Der Veken a, Peter Vandenabeele b,c,e, Tom Vanden Berghe # * b,c, Koen Augustyns # * a

aLaboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.

bMolecular Signalling and Cell Death unit, VIB Center for Inflammation Research, Technologiepark 927, 9052 Ghent, Belgium.

cDepartment of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Belgium.

dLaboratory for Microbiology, Parasitology and Hygiene, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.

eMethusalem Program, Ghent University, Ghent, Belgium.

# These authors contributed equally.

Keywords: ferroptosis, regulated necrosis
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ABSTRACT

Ferroptosis is an iron-catalysed, non-apoptotic form of regulated necrosis that results in oxidative lipid damage in cell membranes that can be inhibited by the radical-trapping antioxidant Ferrostatin-1 (Fer-1). Novel inhibitors derived from the Fer-1 scaffold inhibited ferroptosis potently but suffered from solubility issues. In this paper, we report the synthesis of a more stable and readily soluble series of Fer-1 analogues that potently inhibit ferroptosis. The most promising compounds (37, 38 and 39) showed an improved protection compared to Fer-1 against multi-organ injury in mice. No toxicity was observed in mice after daily injection of 39 (UAMC-3203) for 4 weeks. UAMC-3203 inserts rapidly in a phospholipid bilayer in silico, which aligns with the current understanding of the mechanism of action of these compounds. Concludingly, these analogues have superior properties compared to Fer-1, show in vivo efficacy and represent novel lead compounds with therapeutic potential in relevant ferroptosis-driven disease models.

INTRODUCTION

The classical view that a cell can undergo cell death by either one of two distinct prototypic cell death pathways, apoptosis or necroptosis, has become invalid.1 While apoptosis is carried out following a strict mechanism involving cellular proteases called caspases, necroptosis critically depends on the concerted action of several enzymes such as the receptor interacting protein kinases (RIPK) and the mixed lineage kinase domain like pseudokinase (MLKL).2–4 To date, several other forms of regulated necrosis each with their distinctive pathways and effector molecules are described.5,6

One molecule that is able to induce a form of regulated necrosis is erastin.7 Erastin interacts with mitochondrial voltage-dependent anion channels (VDACs) which induces a RAS-RAF-MEK-dependent form of cell death due to the formation of reactive oxidative species (ROS).8 Subsequently, the Stockwell lab found that erastin also inhibits the membrane-bound cystine/glutamate antiporter System Xc-, which accounts for the induction of a non-apoptotic form of cell death.9 This novel form of cell death was dubbed ferroptosis, which is characterized by iron-dependent accumulation of lipid hydroperoxides that disrupt membrane integrity leading to cell death. Ferroptosis may thus play a key role in the pathogenesis of degenerative diseases in which lipid peroxidation has been implicated. Interestingly, the execution of this form of cell death could be inhibited by the small molecule ferrostatin-1 (Fer-1, 1).9 Even though target identification of Fer-1 remains a challenge, the mechanism of action of Fer-1 was recently proposed to derive from its reactivity as a radical-trapping antioxidant.10

Unravelling the molecular pathway of ferroptosis is currently a topic of intense study. Because erastin induces ferroptosis through the inhibition of System Xc-, it can be classified as a class I ferroptosis inducer (FIN). Blockage of this antiporter impairs the cellular uptake of cystine, an essential precursor in the synthesis of the cellular antioxidant glutathione (GSH). The resulting intracellular deficit of GSH in turn triggers the accumulation of ROS, which causes cells to die by excessive oxidation of the membrane lipids (Figure 1).11 In addition to class I FINs, ferroptosis can also be induced by molecules that directly target and inactivate glutathione peroxidase 4 (GPX4) and are classified as class II FINs.12,13

Seiler et al. showed that GPX4, a specific type of GSH-dependent selenoprotein, holds an essential role in the antioxidant network of the membranes of a cell. Inducible GPX4-KO significantly increased cell death due to excessive lipid peroxidation, a hallmark feature of ferroptosis.14 GPX4 has the ability to reduce organic hydroperoxides to the corresponding alcohols while consuming GSH as a reducing agent, further implying the importance of intracellular GSH levels in ferroptosis.15 Inactivation of GPX4 either directly or indirectly resulted in the accumulation of ROS followed by lipid membrane oxidation which disrupted cell membrane integrity. These findings further solidified GPX4 as a crucial protective enzyme that supresses ferroptosis and it underlines the importance of the GSH-GPX4-axis considering redox homeostasis within the cell membrane (Figure 1).

Figure 1. Schematic representation of GPX4-dependent conversion of reactive lipid peroxides to the corresponding lipid alcohols. System Xc- facilitates the cellular uptake of cystine which is an important precursor in GSH synthesis. Cystine is reduced by cystine reductase (CR) to cysteine. Glutamate cysteine ligase (GCL) attaches a glutamate molecule to cysteine in order to generate γ-glutamyl cysteine (γGC). Addition of a glycine molecule by glutathione synthase (GS) results in the formation of a new molecule of GSH. GSH servers as an important reducing cofactor for GPX4 by catalysing the reduction of lipid peroxides to their corresponding alcohols while also forming glutathione disulfide (GSSG). Erastin inhibits the cellular uptake of cystine and thus impairs intracellular synthesis of GSH. Depletion of GSH leads to the indirect inactivation of GPX4 and the resulting accumulation of lipid peroxides disrupts membrane integrity resulting in ferroptosis. The class II FIN RSL3 acts by directly inactivating GPX4 and does not interfere with the cellular uptake of cystine or intracellular GSH synthesis.6,16

The clinical relevance of ferroptosis has been implied in various pathological settings.17 Dixon et al. first showed that Fer-1 was able to prevent glutamate-induced neurotoxicity in a model using organotypic hippocampal slice cultures.11 Another study in particular showed that ferroptosis might contribute in a whole array of diseases by showing that inhibition of ferroptosis significantly ameliorated the clinical outcome in experimental models for Huntington’s disease, periventricular leukomalacia and kidney
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dysfunction.18 Since then multiple studies have implicated an important role of ferroptosis not only in various pathological conditions like ischemia-reperfusion injury19, neuronal dysfunction20 and Alzheimer’s disease21, but also in several other biological processes such as hair follicle morphogenesis22, blood coagulation23 and in the maturation of photoreceptor cells.24

The design of novel and improved ferroptosis inhibitors is thus an interesting field of research. Fer-1 as a lead molecule itself has some unfortunate shortcomings. The presence of a labile ester moiety results in the rapid hydrolysis of Fer-1 into its inactive carboxylic acid. Multiple attempts have been made to improve the potency of this type of molecule while also improving pharmacokinetic parameters of Fer-1 (2 and 3, Figure 2).18,25 While Skouta et al. thoroughly explored the structure-activity relationship (SAR) of the Fer-1 scaffold, we previously reported that the replacement of the labile ester moiety of Fer-1 by a more stable sulfonamide group yielded promising results (4, UAMC-2418, Figure 2). This intervention significantly improved the metabolic stability while maintaining and even improving potency in a cell- based assay for ferroptosis. These molecules however displayed a poorly soluble character, which limited their use in in vivo settings.16

In this study, we expand on these earlier findings and we report the synthesis of a novel generation of ferroptosis inhibitors derived from Fer-1 with improved ADME properties and an increased solubility. The pharmacokinetic properties of the most potent molecules were characterized and the in vivo efficacy and toxicity of these molecules was investigated. Additionally, a molecular dynamics experiment was conducted as an effort to investigate the binding properties of these molecules in a phospholipid bilayer.

RESULTS AND DISCUSSION

Compound design

Our earlier published results involved a thorough exploration of the SAR which provided the following results: (1) The replacement of the labile ester moiety with a sulfonamide greatly improved stability as well as potency (2) The cyclohexyl moiety was deemed to be the most ideal substituent with regard to both potency and lipophilicity. More bulky alkyl groups tend to have a negative effect on the solubility of the compounds and smaller moieties impair the potency of the molecules. (3) The introduction of an aromatic group on the 3-amino position greatly improved potency, but also further decreased the solubility of the compounds.16

The major drawback of our previously published molecules was their poor solubility. Designing more soluble compounds is a challenging task since these molecules have to act in the highly lipophilic environment of membranes. We identified that the terminal position of the aliphatic chain on the sulfonamide moiety could be derivatized further in order to increase the solubility of these compounds. A variety of solubility improving groups were introduced at this terminal Ra position. The cyclohexylamine group was kept as in Fer-1 and the Rb position was further derivatized with benzylic and pyridinic substituents in a similar fashion to the previously reported sulfonamide analogues (Figure 3).

Chemistry

The newly developed series of inhibitors were synthesized according to the general synthetic strategy displayed in Scheme 1.

Table 1. Synthesized fer-1 analogues and their antiferroptotic activity in response to erastin-induced ferroptosis in IMR- 32 Neuroblastoma cells.

aReported IC50 values are calculated from measurements in triplicate. Additional information such as the standard error of the means can be found in the Supporting Information.

bFinal test compound concentration range between 3.125 µM and 200 µM [4 µM DMSO solution in 196 µM buffer solution (10 mM PBS pH 7.4)].

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ADME Assays

The determination of both the microsomal and plasma stability of 35, 37-39 revealed a remarkable improvement in stability when compared to Fer-1 which is unstable under nearly all conditions (Table 2 and Table 3). However, significant intraspecies differences between the four lead molecules were observed. Compounds 38 and 39 more specifically, showed a microsomal half-life (t1/2) of multiple hours across all species. Compound 39 has an impressive t1/2 when incubated with both human and rat microsomes (t1/2 = 20.5 h and t1/2 = 16.5 h respectively) but was found to be relatively less stable when incubated with murine microsomes (t1/2 = 3.46 h). In contrast, compound 38 has a microsomal t1/2 greater than 10 hours under both human (t1/2 = 17.7 h) and murine (t1/2 = 13 h) conditions but was microsomally less stable when incubated with rat microsomes (t1/2 = 2.05 h).

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Table 2. Microsomal stability and plasma stability of compounds 1, 35 , 37-39.

aMetabolism by microsomes (CYP450 and other NADP-dependent enzymes) was monitored and expressed as half-life (h).

bPercentage of remaining parent compound.

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Table 3. Calculated Intrinsic Clearance in vitro of compounds 35, 37-39.

The Clint is calculated using the measured microsomal t1/2 and takes into account several experimental variables such as the protein concentration and the volume of incubation. The exact formula as well as the species-specific parameters can be found in the Supporting Information.

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In vivo evaluation of efficacy of novel fer-1 analogues

We have recently shown that pure cellular iron overload suffices to induce ferroptosis in IMR-32 neuroblastoma cells, which is referred to as non-canonical ferroptosis induction.26 This new concept of iron-induced ferroptosis has recently been shown by others by using FeCl2 in organotypic hippocampal slice cultures27, BAY 11-7085 in breast cancer cell lines and glioblastoma28 and FINO2 in HT1080 fibrosarcoma cells.29 Both in humans30 as well as in rodents31–33, an acute overdose of iron in vivo typically results in multiple organ failure and often death.34,35 First, we have tested the potency of our inhibitors for (NH4)2Fe(SO4)2-induced ferroptosis in vitro. Similar to erastin-induced ferroptosis, we found that our most potent inhibitors show similar or lower IC50-values than Fer-1 when inhibiting ferrous ammonium sulphate-induced ferroptosis. (See Supporting Information) Therefore, the selected compounds were assessed for their efficacy in an experimental in vivo ferroptosis model using acute iron poisoning. Intravenous injection of compounds 37, 38 and 39 in mice 15 minutes prior to iron sulphate injection significantly lowered the plasma levels of lactate dehydrogenase (LDH), a general biomarker for organ injury which is drastically elevated in response to iron poisoning. All three compounds were significantly more potent in lowering LDH levels than Fer-1 (Table 4), which illustrates the in vivo functionality of these Fer-analogues.

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Table 4. Pretreatment with the compounds significantly decreases LDH levels after acute iron poisoning.

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Vehicle, Fer-1 (1), compound 37, 38 or 39 was injected intravenously (20 µmol/kg) 15 minutes before intraperitoneal injection with 300 mg/kg iron sulfate. Two hours after IP injection, mice were sacrificed and blood was taken. Plasma levels of LDH are shown. Vehicle = 2% DMSO in 0.9% NaCl. Error represents SEM, n=5 for twice treated vehicle group, n=11-12 for FeSO4- treated groups. * = 0.01 95%, unless stated otherwise.

General procedure A

A solution containing the appropriate aliphatic or cyclic amine substituent (1 equiv.) and triethylamine (2 equiv.) in THF was added dropwise to a solution containing 4-chloro-3-nitrobenzene-1-sulfonyl chloride 5 (1 equiv.) in THF that was cooled down to -40°C. After the addition was complete, the resulting mixture was allowed to warm up to room temperature over the course of one hour. Subsequently the reaction mixture was diluted with EtOAc and washed three times with water. The organic layer was dried using anhydrous sodium sulphate before being concentrated in vacuo. The crude reaction products 6-12 were used without any further purification.

General procedure B

Intermediates 6-12 (1 equiv.) were dissolved in DMSO before potassium carbonate (2 equiv.) and cyclohexanamine (1.2 equiv.) were added. The resulting mixture was heated to a temperature of 60°C and then stirred overnight. After cooling down to room temperature, the reaction mixture was diluted with EtOAc and washed three times with water. The organic layer was dried using anhydrous sodium sulphate before being concentrated in vacuo. If deemed necessary, further purification was conducted with flash chromatography on silica gel using a gradient consisting of heptanes and EtOAc to obtain intermediates 13-19.

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General procedure C

Intermediates 13-19 (1 equiv.) were dissolved in dry MeOH and the solution was purged using argon gas. Palladium(II) hydroxide was added under inert atmosphere while continuously stirring. After the addition was complete, the resulting mixture was put under hydrogen atmosphere and left to stir overnight. The reaction mixture was filtered through a patch of Celite® and then purified by flash chromatography on silica gel using a gradient consisting of heptanes and EtOAc to yield products 20-26.

General procedure D

Compounds 20-26 (1 equiv.) were dissolved in DMF followed by the addition of potassium carbonate (1 – 3 equiv.) and the corresponding benzyl- or pyridinylderivate (1-2 equiv.). This mixture was allowed to stir at various temperatures and timespans, all of which are further specified at the corresponding compounds reported below. The crude product was then purified by either normal phase or reversed phase flash chromatography to yield compounds 30-36 and 40-46. The exact conditions of these purifications are reported below for each compound individually.

General procedure E

Compounds 21-23, 31-33 and 41-43 (1 equiv.) were dissolved in dichloromethane followed by the addition of a 4M solution of hydrochloric acid in dioxane (8 equiv.) The reaction mixture was stirred at room temperature for 2 hours. After reaction diethylether was added to the reaction mixture in order to precipitate compounds 27-29, 37-39 and 47-49. The obtained HCl-salts were subsequently washed with a minimal amount of diethylether.

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4-chloro-N-(2-(dimethylamino)ethyl)-3-nitrobenzenesulfonamide (6)

By following General Procedure A and using N,N-dimethylethane-1,2-diamine (1.38 g, 15.62 mmol) as

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nitrobenzenesulfonamide 6 (4.67 g, 15.16 mmol) was achieved. (Yield: 97%)

1H NMR (400 MHz, DMSO-d6) δ 2.07 (s, 6H), 2.25 – 2.30 (m, 2H), 2.97 (t, J = 6.5 Hz, 2H), 8.05 (d, J = 8.5 Hz, 1H), 8.12 (dd, J = 8.5, 2.2 Hz, 1H), 8.50 (d, J = 2.1 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 40.62, 44.83, 58.06, 123.88, 128.93, 131.23, 132.84, 141.20, 147.31.

tert-butyl (2-((4-chloro-3-nitrophenyl)sulfonamido)ethyl)(methyl)carbamate (7)

By following General Procedure A and using tert-butyl (2-aminoethyl)(methyl)carbamate (2.04 g, 11.72 mmol) as the corresponding amine, the formation of tert-butyl (2-((4-chloro-3- nitrophenyl)sulfonamido)ethyl)(methyl)carbamate 7 (4.58 g, 11.62 mmol) was achieved. (Yield: 99%)
1H NMR (400 MHz, Acetone-d6) δ 1.42 (s, 9H), 2.83 (d, J = 14.4 Hz, 3H), 3.20 (t, J = 6.3 Hz, 2H), 3.31 – 3.42 (m, 2H), 7.03 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 8.15 (dd, J = 8.5, 2.2 Hz, 1H), 8.45 (d, J = 2.2 Hz, 1H).
13C NMR (101 MHz, Acetone-d6) δ 27.64, 41.25, 47.95, 48.67, 78.86, 124.14, 124.22, 129.91, 131.38, 133.07, 141.36, 148.03.

tert-butyl (2-((4-chloro-3-nitrophenyl)sulfonamido)ethyl)carbamate (8)

By following General Procedure A and using tert-butyl (2-aminoethyl)carbamate (3.28 g, 20.5 mmol) as the correspoding amine, the formation of tert-butyl (2-((4-chloro-3- nitrophenyl)sulfonamido)ethyl)carbamate 8 (6.2 g, 16.32 mmol) was achieved (Yield: 84%)
1H NMR (400 MHz, Acetone-d6) δ 1.37 (s, 9H), 3.09 – 3.17 (m, 2H), 3.17 – 3.23 (m, 2H), 6.11 (t, J = 6.0 Hz, 1H), 7.02 (t, J = 5.7 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 8.14 (dd, J = 8.4, 2.2 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H).
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13C NMR (101 MHz, Acetone-d6) δ 27.74, 40.11, 43.17, 78.29, 124.27, 130.01, 131.43, 133.08, 141.28, 147.95, 156.09.

tert-butyl 4-(2-((4-chloro-3-nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate (9)

By following General Procedure A and using 4-N-(2-Aminoethyl)-1-N-Boc-piperazine (3.76 g, 16.4 mmol) as the correspoding amine, the formation of tert-butyl 4-(2-((4-chloro-3- nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate 9 (6.7 g, 16.32 mmol) was achieved (Yield: 96%)
1H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9H), 2.22 (t, J = 5.0 Hz, 4H), 2.32 (t, J = 6.5 Hz, 2H), 2.98 (q, J = 5.3 Hz, 2H), 3.20 (dd, J = 6.3, 3.6 Hz, 4H), 7.98 – 8.05 (m, 2H), 8.08 (dd, J = 8.5, 2.1 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 28.00, 43.05, 52.23, 56.69, 78.70, 123.82, 129.01, 131.24, 132.96, 141.12, 147.40, 153.72.

4-chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-nitrobenzenesulfonamide (10)

By following General Procedure A and using 2-(4-methylpiperazin-1-yl)ethanamine (2.24 g, 15.16 mmol) as the corresponding amine, the formation of 4-chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-3- nitrobenzenesulfonamide 10 (5.13 g, 14.14 mmol) was achieved. (Yield: 91%)
1H NMR (400 MHz, DMSO-d6) δ 2.09 (s, 3H), 2.12 – 2.26 (m, 8H), 2.29 (t, J = 6.5 Hz, 2H), 2.97 (t, J = 6.5 Hz, 2H), 8.02 (d, J = 8.5 Hz, 1H), 8.08 (dd, J = 8.5, 2.1 Hz, 1H), 8.45 (d, J = 2.1 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 40.03, 45.63, 52.40, 54.39, 56.86, 123.81, 128.96, 131.21, 132.91, 141.33, 147.38.

4-chloro-N-(2-morpholinoethyl)-3-nitrobenzenesulfonamide (11)

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By following General Procedure A and using 2-morpholinoethanamine (2.054 mL, 15.62 mmol) as the corresponding amine, the formation of 4-chloro-N-(2-morpholinoethyl)-3-nitrobenzenesulfonamide 11 (5.17 g, 14.78 mmol) was achieved. (Yield: 95%)
1H NMR (400 MHz, DMSO-d6) δ 2.22 – 2.27 (m, 4H), 2.30 (t, J = 6.5 Hz, 2H), 2.93 – 3.02 (m, 2H), 3.43

– 3.48 (m, 4H), 8.02 (s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 8.08 (dd, J = 8.5, 2.1 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 40.09, 52.99, 57.18, 65.92, 123.83, 128.99, 131.23, 132.94, 141.17, 147.39.

4-chloro-N-(2-methoxyethyl)-3-nitrobenzenesulfonamide (12)

By following General Procedure A and using 2-methoxyethanamine (1.42 g, 16.4 mmol) as the correspoding amine, the formation of 4-chloro-N-(2-methoxyethyl)-3-nitrobenzenesulfonamide 12 (4.2 g, 14.25 mmol) was achieved (Yield: 91%)
1H NMR (400 MHz, Acetone-d6) δ 3.18 (s, 3H), 3.22 (t, J = 5.3 Hz, 2H), 3.38 – 3.45 (m, 2H), 6.86 (s, 1H), 7.93 (d, J = 8.5 Hz, 1H), 8.14 (dd, J = 8.5, 2.2 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H).
13C NMR (101 MHz, Acetone-d6) δ 42.95, 57.83, 70.75, 124.28, 129.97, 131.50, 132.95, 141.48, 147.79.

4-(cyclohexylamino)-N-(2-(dimethylamino)ethyl)-3-nitrobenzenesulfonamide (13)

By following General Procedure B and using 4-chloro-N-(2-(dimethylamino)ethyl)-3- nitrobenzenesulfonamide 6 (4.665 g, 15.16 mmol) as the staring material, the formation of 4- (cyclohexylamino)-N-(2-(dimethylamino)ethyl)-3-nitrobenzenesulfonamide 13 (4.72 g, 12.74 mmol) was achieved. (Yield: 84%)
1H NMR (400 MHz, DMSO-d6) δ 1.26 – 1.32 (m, 1H), 1.41 – 1.51 (m, 4H), 1.61 – 1.68 (m, 1H), 1.71 – 1.80 (m, 2H), 1.96 – 2.03 (m, 2H), 2.09 (s, 6H), 2.29 (t, J = 6.8 Hz, 2H), 2.85 (t, J = 6.8 Hz, 2H), 3.70 –

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13C NMR (101 MHz, DMSO-d6) δ 23.97, 24.92, 31.72, 40.58, 45.00, 50.63, 58.02, 115.89, 126.04, 126.17, 129.47, 133.34, 145.80.

tert-butyl (2-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)ethyl)(methyl)carbamate (14)

By following General Procedure B and using tert-butyl (2-((4-chloro-3- nitrophenyl)sulfonamido)ethyl)(methyl)carbamate 7 (4.58 g, 11.62 mmol) as the starting material, the formation of tert-butyl (2-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)ethyl)(methyl)carbamate 14 (3.46 g, 7.58 mmol) was achieved. (Yield: 65%)
1H NMR (400 MHz, DMSO-d6) δ 1.22 – 1.30 (m, 1H), 1.36 (s, 9H), 1.39 – 1.48 (m, 4H), 1.56 – 1.65 (m, 1H), 1.71 (dd, J = 9.1, 4.7 Hz, 2H), 1.92 – 2.01 (m, 2H), 2.75 (s, 3H), 2.85 (t, J = 6.7 Hz, 2H), 3.18 (t, J = 6.7 Hz, 2H), 3.68 – 3.79 (m, 1H), 7.32 (d, J = 9.2 Hz, 1H), 7.69 (s, 1H), 7.78 (dd, J = 9.3, 2.3 Hz, 1H),
8.28(d, J = 7.8 Hz, 1H), 8.44 (d, J = 2.3 Hz, 1H).

13C NMR (101 MHz, DMSO-d6) δ 24.44, 25.41, 28.45, 32.22, 41.02, 48.73, 51.16, 79.04, 116.52, 126.49, 126.54, 130.08, 133.74, 146.37, 155.07.

tert-butyl (2-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)ethyl)carbamate (15)

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nitrophenyl)sulfonamido)ethyl)carbamate 8 (3 g, 7.90 mmol) as the starting material, the formation of tert- butyl (2-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)ethyl)carbamate 15 (3.3 g, 7.46 mmol) was achieved. (Yield: 94%)
1H NMR (400 MHz, Acetone-d6) δ 1.24 – 1.33 (m, 1H), 1.37 (s, 9H), 1.43 – 1.58 (m, 4H), 1.62 – 1.72 (m, 1H), 1.72 – 1.84 (m, 2H), 2.07 – 2.13 (m, 2H), 2.79 (s, 1H), 3.01 (q, J = 6.3 Hz, 2H), 3.17 (q, J = 6.0 Hz,

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2H), 6.03 (s, 1H), 6.58 (t, J = 6.1 Hz, 1H), 7.29 (dd, J = 9.4, 2.9 Hz, 1H), 7.84 (dd, J = 9.2, 2.4 Hz, 1H), 8.37 (d, J = 7.7 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H).
13C NMR (101 MHz, Acetone-d6) δ 24.24, 25.23, 27.66, 32.19, 40.07, 43.11, 51.19, 78.04, 115.56, 126.51, 126.69, 130.33, 133.45, 146.34, 155.96.

tert-butyl 4-(2-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate (16)
By following General Procedure B and using tert-butyl 4-(2-((4-chloro-3- nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate 9 (6.7 g, 14.92 mmol) as the starting material, the
formation of tert-butyl 4-(2-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)ethyl)piperazine-1- carboxylate 16 (6.5 g, 12.7 mmol) was achieved. (Yield: 85%)
1H NMR (400 MHz, DMSO-d6) δ 1.38 (s, 9H), 1.40 – 1.47 (m, 5H), 1.56 – 1.65 (m, 1H), 1.68 – 1.77 (m, 2H), 1.92 – 1.99 (m, 2H), 2.22 (t, J = 5.0 Hz, 4H), 2.32 (t, J = 6.7 Hz, 2H), 2.86 (t, J = 6.7 Hz, 2H), 3.22 (t, J = 5.0 Hz, 4H), 3.73 (d, J = 9.3 Hz, 1H), 7.22 (s, 1H), 7.33 (d, J = 9.3 Hz, 1H), 7.81 (ddd, J = 9.3, 2.3, 0.7 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 23.97, 24.91, 28.00, 31.71, 42.79, 50.63, 52.29, 56.58, 78.66, 115.95, 126.04, 126.17, 129.48, 133.36, 145.81, 153.70.

4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-nitrobenzenesulfonamide (17)

By following General Procedure B and using 4-chloro-N-(2-(4-methylpiperazin-1-yl)ethyl)-3- nitrobenzenesulfonamide 10 (5.1 g, 14.06 mmol) as the starting material, the formation of 4- (cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-nitrobenzenesulfonamide 17 (5.04 g, 11.84 mmol) was achieved. (Yield: 84%)

1H NMR (400 MHz, DMSO-d6) δ 1.21 – 1.31 (m, 1H), 1.37 – 1.47 (m, 4H), 1.57 – 1.65 (m, 1H), 1.67 – 1.76 (m, 2H), 1.91 – 1.99 (m, 2H), 2.09 (s, 3H), 2.12 – 2.26 (m, 8H), 2.29 (t, J = 6.8 Hz, 2H), 2.84 (t, J =
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6.7 Hz, 2H), 3.68 – 3.78 (m, 1H), 7.31 (d, J = 9.4 Hz, 1H), 7.80 (dd, J = 9.2, 2.3, 0.6 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 2.3 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 23.96, 24.91, 31.73, 40.03, 45.69, 50.62, 52.51, 54.50, 56.77, 115.90, 126.03, 126.27, 129.50, 133.33, 145.81.

4-(cyclohexylamino)-N-(2-morpholinoethyl)-3-nitrobenzenesulfonamide (18)

By following General Procedure B and using 4-chloro-N-(2-morpholinoethyl)-3- nitrobenzenesulfonamide 11 (5.13 g, 14.66 mmol) as the starting material, the formation of 4- (cyclohexylamino)-N-(2-morpholinoethyl)-3-nitrobenzenesulfonamide 18 (5.3 g, 12.85 mmol) was achieved. (Yield: 88%)

1H NMR (400 MHz, DMSO-d6) δ 1.21 – 1.32 (m, 1H), 1.36 – 1.48 (m, 4H), 1.57 – 1.65 (m, 1H), 1.66 – 1.76 (m, 2H), 1.90 – 1.99 (m, 2H), 2.23 – 2.28 (m, 4H), 2.30 (t, J = 6.7 Hz, 2H), 2.85 (t, J = 6.7 Hz, 2H), 3.49 (t, J = 4.6 Hz, 4H), 3.69 – 3.77 (m, 1H), 7.32 (d, J = 9.3 Hz, 1H), 7.81 (dd, J = 9.1, 2.2, 0.6 Hz, 1H),
8.29(d, J = 7.8 Hz, 1H), 8.44 (d, J = 2.3 Hz, 1H).

13C NMR (101 MHz, DMSO-d6) δ 23.97, 24.92, 31.71, 40.11, 50.63, 53.08, 57.11, 66.00, 115.94, 126.05, 126.15, 129.48, 133.33, 145.81.

4-(cyclohexylamino)-N-(2-methoxyethyl)-3-nitrobenzenesulfonamide (19)

By following General Procedure B and using 4-chloro-N-(2-methoxyethyl)-3-nitrobenzenesulfonamide 12 (3 g, 10.18 mmol) as the starting material, the formation of 4-(cyclohexylamino)-N-(2-methoxyethyl)- 3-nitrobenzenesulfonamide 19 (2.27 g, 6.36 mmol) was achieved. (Yield: 62.5%)

1H NMR (400 MHz, DMSO-d6) δ 1.18 – 1.33 (m, 1H), 1.36 – 1.51 (m, 4H), 1.54 – 1.66 (m, 1H), 1.67 –

1.78(m, 2H), 1.88 – 2.04 (m, 2H), 2.90 (q, J = 5.6 Hz, 2H), 3.17 (s, 3H), 3.29 – 3.31 (m, 2H), 3.66 – 3.80 (m, 1H), 7.31 (d, J = 9.3 Hz, 1H), 7.69 (t, J = 5.8 Hz, 1H), 7.80 (dd, J = 9.2, 2.3 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.44 (d, J = 2.2 Hz, 1H).

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13C NMR (101 MHz, DMSO-d6) δ 24.45, 25.42, 32.23, 42.61, 51.14, 58.33, 70.98, 116.39, 126.53, 126.79, 130.03, 133.82, 146.33.

3-amino-4-(cyclohexylamino)-N-(2-(dimethylamino)ethyl)benzenesulfonamide (20)

By following General Procedure C and using 4-(cyclohexylamino)-N-(2-(dimethylamino)ethyl)-3- nitrobenzenesulfonamide 13 (4.72 g, 12.74 mmol) as the starting material, 3-amino-4-(cyclohexylamino)- N-(2-(dimethylamino)ethyl)benzenesulfonamide 20 (2.2 g, 6.46 mmol) was generated. (Yield: 51%)

1H NMR (400 MHz, DMSO-d6) δ 1.13 – 1.25 (m, 3H), 1.29 – 1.43 (m, 2H), 1.63 (dt, J = 12.6, 3.6 Hz, 1H), 1.74 (dt, J = 13.2, 3.7 Hz, 2H), 1.92 – 1.99 (m, 2H), 2.06 (s, 6H), 2.23 (t, 2H), 2.70 – 2.75 (m, 2H), 3.24 – 3.33 (m, 1H), 4.85 (d, J = 7.5 Hz, 1H), 4.95 (s, 2H), 6.50 (d, J = 9.0 Hz, 1H), 6.81 – 6.86 (m, 1H), 6.90 – 6.95 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 24.66, 25.55, 32.53, 40.63, 45.08, 50.69, 58.00, 107.91, 111.49, 117.19, 125.77, 134.44, 138.06.

tR 1.32 min, MS (ESI) m/z 341 [M +H] (100%)

tert-butyl (2-((3-amino-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate (21)

By following General Procedure C and using tert-butyl (2-((4-(cyclohexylamino)-3- nitrophenyl)sulfonamido)ethyl)(methyl)carbamate 14 (3.46 g, 7.58 mmol) as starting material, tert-butyl (2-((3-amino-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate 21 (1.98 g, 4.64 mmol) was generated. (Yield: 61.2%)
1H NMR (400 MHz, DMSO-d6) δ 1.13 – 1.27 (m, 3H), 1.33 – 1.40 (m, 11H), 1.63 (dt, J = 12.7, 3.7 Hz, 1H), 1.74 (dt, J = 13.3, 3.6 Hz, 2H), 1.92 – 2.00 (m, 2H), 2.71 – 2.80 (m, 5H), 3.15 (t, J = 6.9 Hz, 2H), 3.23
– 3.33 (m, 1H), 4.86 (d, J = 7.5 Hz, 1H), 4.96 (s, 2H), 6.50 (d, J = 8.9 Hz, 1H), 6.90 – 6.95 (m, 2H), 7.11 (s, 1H).

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13C NMR (101 MHz, DMSO-d6) δ 24.66, 25.55, 27.99, 32.52, 34.83, 40.66, 48.24, 50.68, 78.51, 107.86, 111.38, 117.14, 125.72, 134.48, 138.09, 154.57.

tert-butyl (2-((3-amino-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)carbamate (22)

By following General Procedure C and using tert-butyl (2-((4-(cyclohexylamino)-3- nitrophenyl)sulfonamido)ethyl)carbamate 15 (2.7 g, 6.1 mmol) as starting material, tert-butyl (2-((3- amino-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)carbamate 22 (1.5 g, 3.64 mmol) was generated. (Yield: 59.6%)
1H NMR (400 MHz, DMSO-d6) δ 1.15 – 1.27 (m, 3H), 1.36 (s, 11H), 1.55 – 1.68 (m, 1H), 1.70 – 1.78 (m, 2H), 1.92 – 2.03 (m, 2H), 2.67 (q, J = 6.6, 6.1 Hz, 2H), 2.95 (q, J = 6.7 Hz, 2H), 3.24 – 3.30 (m, 1H), 4.83 (d, J = 7.5 Hz, 1H), 4.93 (s, 2H), 6.50 (d, J = 8.8 Hz, 1H), 6.71 (d, J = 6.4 Hz, 1H), 6.88 – 6.94 (m, 2H), 6.97 – 7.05 (m, 1H).
13C NMR (101 MHz, DMSO-d6) δ 25.15, 26.04, 28.66, 31.78, 33.05, 42.87, 51.22, 78.20, 108.42, 111.97, 117.68, 126.27, 134.95, 138.65, 155.95.

tert-butyl 4-(2-((3-amino-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate (23)
By following General Procedure C and using tert-butyl 4-(2-((4-(cyclohexylamino)-3- nitrophenyl)sulfonamido)ethyl)piperazine-1-carboxylate 16 (6.5 g, 12.7 mmol) as starting material, tert- butyl 4-(2-((3-amino-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate 23 (3.9 g, 8.1 mmol) was generated. (Yield: 63.7%)
1H NMR (400 MHz, DMSO-d6) δ 1.12 – 1.27 (m, 3H), 1.29 – 1.36 (m, 2H), 1.39 (s, 9H), 1.58 – 1.68 (m, 1H), 1.68 – 1.78 (m, 2H), 1.90 – 2.01 (m, 2H), 2.21 (t, J = 5.3 Hz, 4H), 2.30 (dd, J = 7.7, 6.2 Hz, 2H), 2.77 (dt, J = 7.7, 6.0 Hz, 2H), 3.24 (t, J = 5.1 Hz, 4H), 3.27 – 3.32 (m, 1H), 4.85 (d, J = 7.5 Hz, 1H), 4.95 (s, 2H), 6.50 (d, J = 9.0 Hz, 1H), 6.81 (t, J = 5.8 Hz, 1H), 6.93 (dq, J = 4.3, 2.2 Hz, 2H).

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13C NMR (101 MHz, DMSO-d6) δ 24.65, 25.55, 28.01, 30.65, 32.52, 42.98, 50.70, 52.33, 56.54, 78.65, 107.92, 111.50, 117.24, 125.69, 134.47, 138.09, 153.71.

3-amino-4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)benzenesulfonamide (24)

By following General Procedure C and using 4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)- 3-nitrobenzenesulfonamide 17 (5.0 g, 11.75 mmol) as starting material, 3-amino-4-(cyclohexylamino)-N- (2-(4-methylpiperazin-1-yl)ethyl)benzenesulfonamide 24 (3.00 g, 7.58 mmol) was generated. (Yield: 64.5%)
1H NMR (400 MHz, DMSO-d6) δ 1.11 – 1.26 (m, 3H), 1.29 – 1.43 (m, 2H), 1.58 – 1.67 (m, 1H), 1.69 –

1.78(m, 2H), 1.93 – 1.99 (m, 2H), 2.11 (s, 3H), 2.14 – 2.35 (m, 10H), 2.68 – 2.77 (m, 2H), 3.23 – 3.34 (m, 1H), 4.86 (d, J = 7.5 Hz, 1H), 4.96 (s, 2H), 6.49 (d, J = 9.0 Hz, 1H), 6.81 (t, J = 5.8 Hz, 1H), 6.90 – 6.94 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 24.66, 25.55, 32.52, 40.10, 45.71, 50.68, 52.53, 54.59, 56.67, 107.90, 111.46, 117.19, 125.63, 134.45, 138.06.

tR 1.29 min, MS (ESI) m/z 396 [M +H] (100%)

3-amino-4-(cyclohexylamino)-N-(2-morpholinoethyl)benzenesulfonamide (25)

By following General Procedure C and using 4-(cyclohexylamino)-N-(2-morpholinoethyl)-3- nitrobenzenesulfonamide 18 (5.3 g, 12.85 mmol) as starting material, 3-amino-4-(cyclohexylamino)-N-(2- morpholinoethyl)benzenesulfonamide 25 (3.4 g, 8.89 mmol) was generated. (Yield: 69.2%)
1H NMR (400 MHz, DMSO-d6) δ 1.13 – 1.28 (m, 3H), 1.29 – 1.43 (m, 2H), 1.63 (dt, J = 12.5, 3.8 Hz, 1H), 1.70 – 1.79 (m, 2H), 1.92 – 1.99 (m, 2H), 2.23 – 2.32 (m, 6H), 2.77 (dt, J = 7.6, 5.9 Hz, 2H), 3.23 – 3.34 (m, 1H), 3.49 – 3.52 (m, 4H), 4.84 (d, J = 7.5 Hz, 1H), 4.95 (s, 2H), 6.50 (d, J = 9.0 Hz, 1H), 6.82 (t, J = 11.7, 5.9 Hz, 1H), 6.92 – 6.95 (m, 2H).

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13C NMR (101 MHz, DMSO-d6) δ 24.65, 25.55, 32.52, 39.79, 50.69, 53.12, 57.06, 66.06, 107.90, 111.51, 117.24, 125.68, 134.46, 138.10.

tR 1.31 min, MS (ESI) m/z 383 [M +H] (100%)

3-amino-4-(cyclohexylamino)-N-(2-methoxyethyl)benzenesulfonamide (26)

By following General Procedure C and using 4-(cyclohexylamino)-N-(2-methoxyethyl)-3- nitrobenzenesulfonamide 19 (3.5 g, 9.79 mmol) as starting material, 3-amino-4-(cyclohexylamino)-N-(2- methoxyethyl)benzenesulfonamide 26 (2.13 g, 6.51 mmol) was generated. (Yield: 66.5%)
1H NMR (400 MHz, DMSO-d6) δ 1.15 – 1.25 (m, 3H), 1.28 – 1.43 (m, 2H), 1.58 – 1.67 (m, 1H), 1.69 –

1.79(m, 2H), 1.91 – 2.02 (m, 2H), 2.81 (q, J = 6.1 Hz, 2H), 3.19 (s, 3H), 3.26 – 3.33 (m, 3H), 4.82 (d, J = 7.5 Hz, 1H), 4.92 (s, 2H), 6.50 (d, J = 8.9 Hz, 1H), 6.92 (s, 1H), 6.94 (d, J = 2.2 Hz, 1H), 7.03 (t, J = 6.1 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 25.14, 26.05, 33.04, 42.53, 51.21, 58.36, 71.08, 108.44, 112.05, 117.71, 126.45, 134.94, 138.64.

tR 1.58 min, MS (ESI) m/z 328 [M +H] (100%)

3-amino-4-(cyclohexylamino)-N-(2-(methylamino)ethyl)benzenesulfonamide hydrochloride (27)

By following General Procedure E using tert-butyl (2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate 21 (0.1 g, 0.23 mmol) as starting material
to afford the desired 3-amino-4-(cyclohexylamino)-N-(2-(methylamino)ethyl)benzenesulfonamide hydrochloride 27 (0.024 g, 0.067 mmol). (Yield: 28.6%) The reported NMR spectrum is that of the hydrochloric acid form of 27.

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1H NMR (400 MHz, DMSO-d6) δ 1.14 – 1.26 (m, 3H), 1.29 – 1.45 (m, 2H), 1.57 – 1.67 (m, 1H), 1.69 –

1.79(m, 2H), 1.92 – 1.99 (m, 2H), 2.18 (s, 3H), 2.46 (t, J = 6.6 Hz, 2H), 2.72 (t, J = 6.6 Hz, 2H), 3.23 – 3.37 (m, 1H), 4.85 (d, J = 7.5 Hz, 1H), 4.95 (s, 2H), 6.47 – 6.53 (m, 1H), 6.90 – 6.95 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 24.66, 25.55, 32.53, 35.53, 42.02, 50.40, 50.69, 107.89, 111.47, 117.17, 125.77, 134.42, 138.04.

tR 1.26 min, MS (ESI) m/z 327 [M +H] (100%)

3-amino-N-(2-aminoethyl)-4-(cyclohexylamino)benzenesulfonamide hydrochloride (28)

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1H NMR (400 MHz, DMSO-d6) δ 1.10 – 1.44 (m, 7H), 1.58 – 1.69 (m, 1H), 1.69 – 1.79 (m, 2H), 1.93 – 2.00 (m, 2H), 2.51 – 2.55 (m, 2H), 2.66 (t, J = 6.3 Hz, 2H), 4.85 (d, J = 7.4 Hz, 1H), 4.95 (s, 2H), 6.50 (d, J = 9.0 Hz, 1H), 6.88 – 6.97 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 24.67, 25.55, 32.54, 41.13, 45.87, 50.68, 107.87, 111.46, 117.16, 125.88, 134.40, 138.02.

tR 1.17 min, MS (ESI) m/z 313 [M +H] (100%)

3-amino-4-(cyclohexylamino)-N-(2-(piperazin-1-yl)ethyl)benzenesulfonamide (29)

By following General Procedure E using tert-butyl 4-(2-((3-amino-4-

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yl)ethyl)benzenesulfonamide 29 (0.120 g, 0.289 mmol). (Yield: 69.1%) The reported NMR spectrum is that of the free base of 29.
1H NMR (400 MHz, DMSO-d6) δ 1.14 – 1.26 (m, 4H), 1.29 – 1.43 (m, 2H), 1.58 – 1.68 (m, 1H), 1.68 – 1.78 (m, 2H), 1.91 – 2.03 (m, 2H), 2.13 – 2.21 (m, 4H), 2.22 – 2.29 (m, 2H), 2.62 (t, J = 4.9 Hz, 4H), 2.74 (t, J = 7.1 Hz, 2H), 3.26 – 3.30 (m, 1H), 4.86 (d, J = 7.5 Hz, 1H), 4.96 (s, 2H), 6.50 (d, J = 9.0 Hz, 1H), 6.74 – 6.84 (m, 1H), 6.87 – 6.96 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 24.66, 25.55, 32.52, 45.38, 50.68, 53.96, 57.41, 107.89, 111.47, 117.20, 125.60, 134.45, 138.07.

tR 1.17 min, MS (ESI) m/z 381 [M +H] (100%)

3-(benzylamino)-4-(cyclohexylamino)-N-(2-(dimethylamino)ethyl)benzenesulfonamide (30)

By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-(dimethylamino)ethyl)benzenesulfonamide 20 (0.300 g, 0.881 mmol, 1equiv.), bromomethylbenzene (0.105 mL, 0.881 mmol, 1 equiv.) and potassium carbonate (0.122 g, 0.881 mmol, 1 equiv.). This mixture was allowed to stir for 15 minutes at room temperature. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated, lyophilisated and purified further using reverse phase flash chromatography (Water/MeOH) to afford the desired 3-(benzylamino)-4- (cyclohexylamino)-N-(2-(dimethylamino)ethyl)benzenesulfonamide 30 (0.151 g, 0.351 mmol) (Yield: 40%)
1H NMR (400 MHz, Acetone-d6) δ 1.18 – 1.48 (m, 5H), 1.61 – 1.70 (m, 1H), 1.74 – 1.82 (m, 2H), 2.01 – 2.09 (m, 2H), 3.32 (s, 6H), 3.35 – 3.42 (m, 1H), 3.50 (t, J = 5.9 Hz, 2H), 3.79 (t, J = 6.0 Hz, 2H), 4.76 – 4.82 (m, 2H), 4.97 (s, 2H), 6.58 (d, J = 8.4 Hz, 1H), 7.18 (dd, J = 8.4, 2.2 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.47 – 7.57 (m, 3H), 7.75 – 7.78 (m, 2H).

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13C NMR (101 MHz, Acetone-d6) δ 25.85, 26.66, 33.72, 38.36, 50.91, 52.23, 64.79, 68.57, 109.18, 114.01, 119.81, 126.66, 128.97, 129.83, 131.29, 134.32, 135.67, 140.63.

tR 1.51 min, MS (ESI) m/z 431 [M +H] (100%)

tert-butyl (2-((3-(benzylamino)-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate (31)
By following General Procedure D, the reaction mixture was prepared using tert-butyl (2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate 21 (0.100 g, 0.234 mmol, 1 equiv.), bromomethylbenzene (0.028 mL, 0.234 mmol, 1 equiv.) and potassium carbonate (0.032 g, 0.234 mmol, 1 equiv.). This mixture was allowed to stir for 45 minutes at 40°C. The mixture was diluted with water and washed with EtOAc. The organic layer was concentrated to afford tert-butyl (2-((3-(benzylamino)-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate 31 which was introduced in the next step without further purification.

tert-butyl (2-((3-(benzylamino)-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)carbamate (32)

By following General Procedure D, the reaction mixture was prepared using tert-butyl (2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)carbamate 22 (0.200 g, 0.485 mmol, 1equiv.), bromomethylbenzene (0.058 mL, 0.485 mmol, 1 equiv.) and potassium carbonate (0.080 g, 0.485 mmol, 1 equiv.). This mixture was allowed to stir for 60 minutes at 40°C. The mixture was diluted with water and washed with EtOAc. The organic layer was concentrated to afford tert-butyl (2-((3-(benzylamino)-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)carbamate 32 which was introduced in the next step without further purification.

tert-butyl 4-(2-((3-(benzylamino)-4-(cyclohexylamino)phenyl)sulfonamido)ethyl)piperazine-1- carboxylate (33)
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By following General Procedure D, the reaction mixture was prepared using tert-butyl 4-(2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate 23 (0.300 g, 0.623 mmol, 1equiv.), bromomethylbenzene (0.074 mL, 0.623 mmol, 1 equiv.) and potassium carbonate (0.172 g, 0.623 mmol, 1 equiv.). This mixture was allowed to stir for 60 minutes at 40°C. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated and purified further using normal phase flash chromatography (Heptane/Ethyl acetate) to afford the desired tert-butyl 4-(2-((3-(benzylamino)-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate 33 (0.135 g, 0.236 mmol) (Yield: 37.9%)
1H NMR (400 MHz, DMSO-d6) δ 1.14 – 1.30 (m, 3H), 1.32 – 1.38 (m, 2H), 1.39 (s, 9H), 1.60 – 1.69 (m, 1H), 1.70 – 1.80 (m, 2H), 1.94 – 2.05 (m, 2H), 2.10 – 2.19 (m, 4H), 2.21 (t, J = 6.8 Hz, 2H), 2.55 – 2.64 (m, 2H), 3.17 – 3.28 (m, 4H), 3.29 – 3.35 (m, 1H), 4.32 (d, J = 5.4 Hz, 2H), 5.13 (d, J = 7.2 Hz, 1H), 5.69 (t, J = 5.6 Hz, 1H), 6.55 (d, J = 8.5 Hz, 1H), 6.73 (d, J = 2.1 Hz, 1H), 6.78 (t, J = 5.8 Hz, 1H), 6.98 (dd, J = 8.3, 2.1 Hz, 1H), 7.21 – 7.41 (m, 5H).
13C NMR (101 MHz, DMSO-d6) δ 24.67, 25.57, 28.01, 32.46, 42.67, 43.67, 46.89, 50.85, 52.20, 56.43, 78.66, 107.49, 117.22, 125.80, 126.77, 127.33, 128.28, 134.49, 138.33, 139.42, 153.70.

3-(benzylamino)-4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)benzenesulfonamide (34) By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)benzenesulfonamide 24 (0.200 g, 0.506 mmol, 1equiv.), bromomethylbenzene (0.060 mL, 0.506 mmol, 1 equiv.) and potassium carbonate (0.070 g, 0.506 mmol, 1 equiv.). This mixture was allowed to stir for 10 minutes at room temperature. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated, lyophilisated and purified further using reverse phase flash chromatography (Water/MeOH) to afford the desired 3- (benzylamino)-4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)benzenesulfonamide 34 (0.066 g, 0.136 mmol) (Yield: 26.9%)

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1H NMR (400 MHz, Methanol-d4) δ 1.21 – 1.35 (m, 3H), 1.36 – 1.52 (m, 2H), 1.66 – 1.75 (m, 1H), 1.81 (dt, J = 13.3, 3.4 Hz, 2H), 2.00 – 2.12 (m, 2H), 2.53 (dd, J = 6.6, 5.2 Hz, 2H), 2.62 (ddd, J = 13.2, 9.7, 2.7 Hz, 2H), 2.78 (dt, J = 13.5, 3.7 Hz, 2H), 2.94 – 3.06 (m, 5H), 3.26 – 3.30 (m, 2H), 3.35 – 3.41 (m, 1H), 3.43
– 3.56 (m, 2H), 4.62 (s, 2H), 6.63 (d, J = 8.4 Hz, 1H), 7.18 – 7.25 (m, 2H), 7.50 – 7.64 (m, 5H).

13C NMR (101 MHz, Methanol-d4) δ 26.28, 27.05, 34.10, 41.03, 47.05, 52.70, 56.40, 61.04, 69.95, 109.77, 114.53, 120.83, 127.11, 128.24, 130.36, 131.95, 134.46, 135.33, 141.30.

tR 1.48 min, MS (ESI) m/z 486 [M +H] (100%)

3-(benzylamino)-4-(cyclohexylamino)-N-(2-morpholinoethyl)benzenesulfonamide (35)

By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-morpholinoethyl)benzenesulfonamide 25 (0.300 g, 0.784 mmol, 1equiv.), bromomethylbenzene (0.094 mL, 0.784 mmol, 1 equiv.) and potassium carbonate (0.108 g, 0.784 mmol, 1 equiv.). This mixture was allowed to stir for 20 minutes at 40°C. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated, lyophilisated and purified further using reverse phase flash chromatography (Water/MeOH) to afford the desired 3-(benzylamino)-4-(cyclohexylamino)- N-(2-morpholinoethyl)benzenesulfonamide 35. (0.065 g, 0.138 mmol.) (Yield: 17.5%)
1H NMR (400 MHz, Acetone-d6) δ 1.21 – 1.33 (m, 3H), 1.38 – 1.51 (m, 2H), 1.64 – 1.70 (m, 1H), 1.74 – 1.83 (m, 2H), 2.05 – 2.13 (m, 2H), 2.16 – 2.20 (m, 4H), 2.28 (t, J = 6.6, 5.8 Hz, 2H), 2.76 (q, J = 6.4, 5.7 Hz, 2H), 3.39 – 3.49 (m, 1H), 3.52 (t, 4H), 4.40 (d, J = 5.4 Hz, 2H), 4.65 (d, J = 7.2 Hz, 1H), 4.84 (t, J = 5.5 Hz, 1H), 5.57 (t, J = 5.6 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 2.1 Hz, 1H), 7.18 (dd, J = 8.3, 2.1 Hz, 1H), 7.24 – 7.30 (m, 1H), 7.32 – 7.38 (m, 2H), 7.40 – 7.44 (m, 2H).
13C NMR (101 MHz, Acetone-d6) δ 25.76, 26.68, 33.74, 40.37, 48.73, 52.26, 54.01, 57.47, 67.31, 109.35, 110.47, 119.54, 127.76, 127.89, 128.55, 129.31, 136.11, 140.37, 140.61.

tR 1.66 min, MS (ESI) m/z 473 [M +H] (100%)

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3-(benzylamino)-4-(cyclohexylamino)-N-(2-methoxyethyl)benzenesulfonamide (36)

By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-methoxyethyl)benzenesulfonamide 26 (0.300 g, 0.916 mmol, 1 equiv.), bromomethylbenzene (0.110 mL, 0.916 mmol, 1 equiv.) and potassium carbonate (0.127 g, 0.916 mmol, 1 equiv.). This mixture was allowed to stir for 4 hours at 60°C. The mixture was diluted with EtOAc and washed with water. The organic layer was dried using anhydrous sodium sulfate, concentrated and purified further using preperative HPLC (Water/Acetonitrile) to afford the desired 3-(benzylamino)-4- (cyclohexylamino)-N-(2-methoxyethyl)benzenesulfonamide 36 (0.020 g, 0.048 mmol) (Yield: 5.23%)
1H NMR (400 MHz, Methanol-d4) δ 1.22 – 1.36 (m, 3H), 1.36 – 1.53 (m, 2H), 1.66 – 1.77 (m, 1H), 1.83 (dt, J = 13.1, 3.7 Hz, 2H), 2.11 (dd, J = 13.3, 3.4 Hz, 2H), 2.72 (t, J = 5.6 Hz, 2H), 3.23 (d, J = 3.5 Hz, 5H), 3.37 – 3.42 (m, 1H), 4.40 (s, 2H), 6.62 – 6.67 (m, 1H), 6.89 (d, J = 2.1 Hz, 1H), 7.15 (dd, J = 8.4, 2.1 Hz, 1H), 7.21 – 7.27 (m, 1H), 7.32 (ddd, J = 7.6, 6.8, 1.2 Hz, 2H), 7.39 (ddt, J = 7.7, 1.5, 0.7 Hz, 2H).
13C NMR (101 MHz, Methanol-d4) δ 26.29, 27.10, 34.11, 43.57, 48.89, 52.82, 58.84, 72.03, 109.62, 110.66, 119.71, 127.10, 128.04, 128.60, 129.54, 136.35, 140.80, 141.25.

tR 2.18 min, MS (ESI) m/z 418 [M +H] (100%)

3-(benzylamino)-4-(cyclohexylamino)-N-(2-(methylamino)ethyl)benzenesulfonamide hydrochloride (37)
By following General Procedure E using tert-butyl (2-((3-(benzylamino)-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate 31 (0.300 g, 0.581mmol) as starting
material to afford the desired 3-(benzylamino)-4-(cyclohexylamino)-N-(2- (methylamino)ethyl)benzenesulfonamide hydrochloride 37 (0.075 g, 0.166 mmol). (Yield: 28.5%) The reported NMR spectrum is that of the free base of 37.

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1H NMR (400 MHz, DMSO-d6) δ 1.14 – 1.29 (m, 3H), 1.30 – 1.44 (m, 2H), 1.59 – 1.69 (m, 1H), 1.70 – 1.82 (m, 2H), 1.95 – 2.03 (m, 2H), 2.15 (s, 3H), 2.37 (t, J = 6.4 Hz, 2H), 2.55 (t, J = 6.4 Hz, 2H), 3.30 – 3.42 (m, 1H), 4.32 (d, J = 5.3 Hz, 2H), 5.14 (d, J = 7.2 Hz, 1H), 5.69 (t, J = 5.6 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 2.1 Hz, 1H), 6.96 (dd, J = 8.3, 2.1 Hz, 1H), 7.20 – 7.43 (m, 5H).
13C NMR (101 MHz, DMSO-d6) δ 24.68, 25.57, 32.47, 35.50, 41.90, 46.91, 50.30, 50.85, 107.43, 117.16, 125.92, 126.78, 127.37, 128.29, 134.50, 138.32, 139.40.

tR 1.82 min, MS (ESI) m/z 417 [M +H] (100%)

N-(2-aminoethyl)-3-(benzylamino)-4-(cyclohexylamino)benzenesulfonamide hydrochloride (38)

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hydrochloride 38 (0.034 g, 0.078 mmol). (Yield: 26.1%) The reported NMR spectrum is that of the hydrochloric salt of 38.
1H NMR (400 MHz, DMSO-d6) δ 1.12 – 1.29 (m, 3H), 1.31 – 1.45 (m, 2H), 1.64 (m, 1H), 1.70 – 1.82 (m, 2H), 1.94 – 2.06 (m, 2H), 2.38 – 2.45 (m, 2H), 2.45 – 2.49 (m, 2H), 3.25 – 3.41 (m, 1H), 4.32 (d, J = 5.3 Hz, 2H), 5.13 (d, J = 7.2 Hz, 1H), 5.68 (t, J = 5.6 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 6.96 (dd, J = 8.4, 2.1 Hz, 1H), 7.20 – 7.30 (m, 1H), 7.30 – 7.42 (m, 4H).
13C NMR (101 MHz, DMSO-d6) δ 24.69, 25.57, 32.49, 41.16, 46.07, 46.92, 50.86, 107.43, 117.14, 126.08, 126.78, 127.38, 128.30, 134.48, 138.29, 139.39.

tR 1.81 min, MS (ESI) 403 m/z [M +H] (100%)

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1H NMR (400 MHz, DMSO-d6) δ 1.15 – 1.29 (m, 4H), 1.31 – 1.45 (m, 2H), 1.59 – 1.68 (m, 1H), 1.70 –

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8.4Hz, 1H), 6.72 (d, J = 2.1 Hz, 1H), 6.74 – 6.80 (m, 1H), 6.97 (dd, J = 8.3, 2.1 Hz, 1H), 7.20 – 7.29 (m, 1H), 7.30 – 7.40 (m, 4H).
13C NMR (101 MHz, DMSO-d6) δ 24.68, 25.57, 32.46, 39.73, 45.38, 46.88, 50.86, 53.89, 57.34, 107.48, 117.20, 125.76, 126.76, 127.33, 128.28, 134.49, 138.33, 139.40.

tR 1.44 min, MS (ESI) m/z 472 [M +H] 100(%)

4-(cyclohexylamino)-N-(2-(dimethylamino)ethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide (40)
By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-(dimethylamino)ethyl)benzenesulfonamide 20 (0.293 g, 0.861 mmol, 1equiv.), 4- (bromomethyl)pyridine hydrobromide (0.218 g, 0.861 mmol, 1 equiv.) and potassium carbonate (0.119 g, 0.861 mmol, 1 equiv.). This mixture was allowed to stir for 30 minutes at room temperature. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated, lyophilisated and purified further using reverse phase flash chromatography (Water/MeOH) to afford the desired 4-

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(cyclohexylamino)-N-(2-(dimethylamino)ethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide 40 (0.170 g, 0.394 mmol) (Yield: 45.8 %)
1H NMR (400 MHz, Methanol-d4) δ 1.23 – 1.35 (m, 3H), 1.37 – 1.50 (m, 2H), 1.70 (dt, J = 12.8, 3.6 Hz, 1H), 1.81 (dt, J = 13.3, 3.6 Hz, 2H), 2.02 – 2.10 (m, 2H), 3.19 (s, 6H), 3.34 – 3.39 (m, 1H), 3.43 (t, J = 6.2 Hz, 2H), 3.59 (t, J = 6.3 Hz, 2H), 4.72 (s, 2H), 6.63 (d, J = 9.1 Hz, 1H), 7.21 – 7.25 (m, 2H), 7.63 – 7.66 (m, 2H), 8.68 – 8.71 (m, 2H).
13C NMR (101 MHz, Methanol-d4) δ 26.30, 27.05, 34.08, 38.44, 51.70, 52.69, 64.96, 67.94, 109.97, 114.39, 121.01, 125.56, 129.35, 135.46, 138.05, 141.76, 151.35.

tR 1.35 min, MS (ESI) m/z 432 [M +H] (100%)

tert-butyl (2-((4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)phenyl)sulfonamido)ethyl)(methyl)carbamate (41)
By following General Procedure D, the reaction mixture was prepared using tert-butyl (2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)(methyl)carbamate 21 (0.250 g, 0.586 mmol, 1 equiv.), 4- (bromomethyl)pyridine hydrobromide (0.148 g, 0.586 mmol, 1 equiv.) and potassium carbonate (0.081 g, 0.586 mmol, 1 equiv.). This mixture was allowed to stir for 40 minutes at 40°C. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated and purified further using normal phase flash chromatography (Heptane/Ethyl acetate) to afford the desired tert-butyl (2-((4- (cyclohexylamino)-3-((pyridin-4-ylmethyl)amino)phenyl)sulfonamido)ethyl)(methyl)carbamate 41 (0.070 g, 0.135 mmol) (Yield: 23.1 %). The compound was immediately introduced in the next step.

tert-butyl (2-((4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)phenyl)sulfonamido)ethyl)carbamate (42)

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By following General Procedure D, the reaction mixture was prepared using tert-butyl (2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)carbamate 22 (0.400 g, 0.970 mmol, 1 equiv.), 4- (bromomethyl)pyridine hydrobromide (0.490 g, 1.939 mmol, 2 equiv.) and potassium carbonate (0.402 g, 2.91 mmol, 3 equiv.). This mixture was allowed to stir for 60 minutes at 40°C. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated and purified further using normal phase flash chromatography (Heptane/Ethyl acetate) to afford the desired tert-butyl (2-((4- (cyclohexylamino)-3-((pyridin-4-ylmethyl)amino)phenyl)sulfonamido)ethyl)carbamate 42 (0.200 g, 0.387 mmol) (Yield: 41.0 %).
1H NMR (400 MHz, Acetone-d6) δ 1.27 – 1.36 (m, 3H), 1.40 (s, 9H), 1.43 – 1.51 (m, 2H), 1.63 – 1.72 (m, 1H), 1.74 – 1.83 (m, 2H), 2.12 (d, J = 3.9 Hz, 2H), 2.79 (q, J = 6.3 Hz, 2H), 3.08 (q, J = 6.2 Hz, 2H), 3.38 – 3.51 (m, 1H), 4.46 (d, J = 5.4 Hz, 2H), 4.57 – 4.66 (m, 1H), 4.93 – 5.06 (m, 1H), 5.92 (s, 1H), 6.03 – 6.12 (m, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 2.1 Hz, 1H), 7.20 (dd, J = 8.3, 2.1 Hz, 1H), 7.38 (dd, J = 4.7, 1.6 Hz, 2H), 8.47 – 8.59 (m, 2H).
13C NMR (101 MHz, Acetone-d6) δ 24.79, 25.76, 27.73, 32.88, 43.09, 46.67, 51.38, 78.00, 108.73, 109.76, 119.09, 122.44, 127.37, 134.81, 139.94, 148.69, 149.78, 155.96.

tert-butyl 4-(2-((4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate (43)
By following General Procedure D, the reaction mixture was prepared using tert-butyl 4-(2-((3-amino-4- (cyclohexylamino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate 23 (0.400 g, 0.830 mmol, 1 equiv.), 4-(bromomethyl)pyridine hydrobromide (0.420 g, 1.661 mmol, 2 equiv.) and potassium carbonate (0.344 g, 2.491 mmol, 3 equiv.). This mixture was allowed to stir for 2 hours at 40°C. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated and purified further using normal phase flash chromatography (Heptane/Ethyl acetate) to afford the desired tert-butyl 4-(2-((4-

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(cyclohexylamino)-3-((pyridin-4-ylmethyl)amino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate 43 (0.160 g, 0.279 mmol) (Yield: 33.6 %).
1H NMR (400 MHz, DMSO-d6) δ 1.24 (s, 3H), 1.39 (s, 11H), 1.62 – 1.69 (m, 1H), 1.72 – 1.79 (m, 2H), 1.97 – 2.06 (m, 2H), 2.13 (t, J = 5.0 Hz, 4H), 2.18 (t, J = 6.8 Hz, 2H), 2.53 – 2.59 (m, 2H), 3.18 – 3.28 (m, 4H), 3.38 (s, 1H), 4.38 (d, J = 5.5 Hz, 2H), 5.10 (d, J = 7.2 Hz, 1H), 5.82 (t, J = 5.7 Hz, 1H), 6.57 (d, J =
8.5Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.78 (t, J = 5.8 Hz, 1H), 6.98 (dd, J = 8.3, 2.1 Hz, 1H), 7.30 – 7.37 (m, 2H), 8.47 – 8.55 (m, 2H).
13C NMR (101 MHz, Acetone-d6) δ 25.73, 26.69, 28.62, 33.76, 40.60, 44.38, 47.46, 52.28, 53.29, 57.03, 79.53, 109.68, 110.56, 119.88, 123.32, 127.91, 135.61, 140.74, 149.65, 150.68, 154.97.

4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-((pyridin-4- ylmethyl)amino)benzenesulfonamide (44)
By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)benzenesulfonamide 24 (0.300 g, 0.758 mmol, 1equiv.), 4-(bromomethyl)pyridine hydrobromide (0.0.192 g, 0.758 mmol, 1 equiv.) and potassium carbonate (0.105 g, 0.758 mmol, 1 equiv.). This mixture was allowed to stir for 15 minutes at room temperature. The mixture was diluted with water and washed with EtOAc. The aqueous layer was concentrated, lyophilisated and purified further using reverse phase flash chromatography (Water/MeOH) to afford the desired 4-(cyclohexylamino)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-((pyridin-4- ylmethyl)amino)benzenesulfonamide 44 (0.170 g, 0.349 mmol) (Yield: 46.1 %)
1H NMR (400 MHz, Methanol-d4) δ 1.22 – 1.34 (m, 3H), 1.36 – 1.50 (m, 2H), 1.69 (dt, J = 12.8, 3.8 Hz, 1H), 1.80 (dt, J = 13.2, 3.7 Hz, 2H), 1.99 – 2.10 (m, 2H), 2.52 (t, J = 6.0 Hz, 2H), 2.58 – 2.67 (m, 2H), 2.71
– 2.79 (m, 2H), 3.00 (t, J = 6.0 Hz, 2H), 3.05 (s, 3H), 3.34 – 3.40 (m, 3H), 3.55 – 3.64 (m, 2H), 4.74 (s, 2H), 6.63 (d, J = 8.5 Hz, 1H), 7.21 (dd, J = 8.4, 2.3 Hz, 1H), 7.29 (d, J = 2.2 Hz, 1H), 7.69 – 7.71 (m, 2H), 8.70 – 8.73 (m, 2H).

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13C NMR (101 MHz, Methanol-d4) δ 26.32, 27.06, 34.12, 41.03, 46.96, 52.71, 56.22, 61.67, 109.75, 114.37, 120.85, 127.09, 129.54, 135.48, 137.79, 141.25, 151.31.

tR 1.35 min, MS (ESI) m/z 487 [M +H] (100%)

4-(cyclohexylamino)-N-(2-morpholinoethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide (45) By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-morpholinoethyl)benzenesulfonamide 25 (0.200 g, 0.523 mmol, 1equiv.), 4- (bromomethyl)pyridine hydrobromide (0.246 g, 1.046 mmol, 2 equiv.) and potassium carbonate (0.217 g, 1.569 mmol, 3 equiv.). This mixture was allowed to stir for 2 hours at 60°C. The mixture was diluted with EtOAc and washed with water. The organic layer was dried using anhydrous sodium sulfate, concentrated and purified further using normal phase flash chromatography on silica gel (Heptanes/EtOAc/MeOH) to

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ylmethyl)amino)benzenesulfonamide 45 (0.065 g, 0.137 mmol) (Yield: 26.3 %)

1H NMR (400 MHz, Methanol-d4) δ 1.24 – 1.36 (m, 3H), 1.39 – 1.52 (m, 2H), 1.71 (dt, J = 13.1, 3.6 Hz, 1H), 1.82 (dt, J = 13.2, 3.7 Hz, 2H), 2.06 – 2.14 (m, 2H), 2.25 – 2.30 (m, 6H), 2.73 (t, J = 6.5 Hz, 2H), 3.39 (tt, J = 10.4, 3.7 Hz, 1H), 3.59 (t, J = 4.7 Hz, 4H), 4.46 (s, 2H), 6.66 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 2.1 Hz, 1H), 7.19 (dd, J = 8.4, 2.1 Hz, 1H), 7.41 – 7.44 (m, 2H), 8.43 – 8.46 (m, 2H).
13C NMR (101 MHz, Methanol-d4) δ 26.28, 27.11, 34.13, 40.59, 47.50, 52.81, 54.44, 58.28, 67.60, 109.80, 110.24, 120.25, 124.15, 126.87, 135.74, 141.26, 150.13, 152.08.

tR 1.34 min, MS (ESI) m/z 474 [M +H] (100%)

4-(cyclohexylamino)-N-(2-methoxyethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide (46)

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By following General Procedure D, the reaction mixture was prepared using 3-amino-4- (cyclohexylamino)-N-(2-methoxyethyl)benzenesulfonamide 26 (0.400 g, 1.222 mmol, 1 equiv.), 4- (bromomethyl)pyridine hydrobromide (0.618 g, 2.443 mmol, 2 equiv.) and potassium carbonate (0.506 g, 3.66 mmol, 3 equiv.). This mixture was allowed to stir for 4 hours at 60°C. The mixture was diluted with EtOAc and washed with water. The organic layer was dried using anhydrous sodium sulfate, concentrated and purified further using normal phase flash chromatography on silica gel (Heptanes/EtOAc) to afford the desired 4-(cyclohexylamino)-N-(2-methoxyethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide 46 (0.212 g, 0.507 mmol) (Yield: 41.5 %)
1H NMR (400 MHz, DMSO-d6) δ 1.18 – 1.36 (m, 3H), 1.36 – 1.51 (m, 2H), 1.63 – 1.73 (m, 1H), 1.73 –

1.85(m, 2H), 1.98 – 2.10 (m, 2H), 2.64 (q, J = 6.0 Hz, 2H), 3.19 (s, 3H), 3.22 (t, J = 6.0 Hz, 2H), 3.39 (ddt, J = 8.9, 6.0, 3.1 Hz, 1H), 4.45 (s, 2H), 5.14 (s, 1H), 5.89 (s, 1H), 6.61 (d, J = 8.4 Hz, 1H), 6.65 (d, J = 2.1 Hz, 1H), 7.00 – 7.09 (m, 2H), 7.40 – 7.50 (m, 2H), 8.50 – 8.66 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 25.11, 26.05, 32.97, 42.41, 46.25, 51.38, 58.30, 70.96, 108.15, 108.28, 118.08, 123.09, 126.62, 134.50, 139.04, 149.18, 150.64.

tR 1.37 min, MS (ESI) m/z 419 [M +H] (100%)

4-(cyclohexylamino)-N-(2-(methylamino)ethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide hydrochloride (47)
By following General Procedure E using tert-butyl (2-((4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)phenyl)sulfonamido)ethyl)(methyl)carbamate 41 (0.070 g, 0.135 mmol) as starting
material to afford the desired 4-(cyclohexylamino)-N-(2-(methylamino)ethyl)-3-((pyridin-4- ylmethyl)amino)benzenesulfonamide hydrochloride 47 (0.059 g, 0.130 mmol). (Yield: 96%).The reported NMR spectrum is that of the free base of 47.

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1H NMR (400 MHz, DMSO-d6) δ 1.21 – 1.35 (m, 3H), 1.38 – 1.51 (m, 2H), 1.65 – 1.74 (m, 1H), 1.77 –

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13C NMR (101 MHz, DMSO-d6) δ 24.64, 25.56, 32.46, 35.50, 41.86, 45.70, 50.26, 50.84, 107.50, 107.68, 117.48, 122.29, 125.96, 134.04, 138.42, 148.82, 149.52.

tR 1.19 min, MS (ESI) m/z 418 [M +H] (100%)

N-(2-aminoethyl)-4-(cyclohexylamino)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide hydrochloride (48)
By following General Procedure E using tert-butyl (2-((4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)phenyl)sulfonamido)ethyl)carbamate 42 (0.050 g, 0.124 mmol) as starting material to
afford the desired 4 N-(2-aminoethyl)-4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)benzenesulfonamide hydrochloride 48 (0.039 g, 0.089 mmol). (Yield: 71.1%).The reported NMR spectrum is that of the free base of 48.
1H NMR (400 MHz, Methanol-d4) δ 1.22 – 1.39 (m, 3H), 1.40 – 1.54 (m, 2H), 1.68 – 1.79 (m, 1H), 1.80 – 1.88 (m, 2H), 2.06 – 2.16 (m, 2H), 2.84 (t, J = 6.7 Hz, 2H), 3.16 (t, J = 6.7 Hz, 2H), 3.35 – 3.46 (m, 1H), 4.48 (s, 2H), 6.67 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 2.1 Hz, 1H), 7.18 (dd, J = 8.4, 2.1 Hz, 1H), 7.35 – 7.55 (m, 2H), 8.29 – 8.51 (m, 2H).
13C NMR (101 MHz, Methanol-d4) δ 26.26, 27.10, 34.13, 44.24, 47.52, 51.57, 52.80, 109.76, 110.22, 120.20, 124.10, 127.31, 135.77, 141.24, 150.09, 150.11, 152.10.

tR 1.24 min, MS (ESI) m/z 404 [M +H] (100%)

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4-(cyclohexylamino)-N-(2-(piperazin-1-yl)ethyl)-3-((pyridin-4-ylmethyl)amino)benzenesulfonamide hydrochloride (49)
By following General Procedure E using tert-butyl 4-(2-((4-(cyclohexylamino)-3-((pyridin-4- ylmethyl)amino)phenyl)sulfonamido)ethyl)piperazine-1-carboxylate 43 (0.150 g, 0.262 mmol) as starting material to afford the desired 4-(cyclohexylamino)-N-(2-(piperazin-1-yl)ethyl)-3-((pyridin-4- ylmethyl)amino)benzenesulfonamide hydrochloride 49 (0.075 g, 0.147 mmol). The reported NMR spectrum is that of the free base of 49.
1H NMR (400 MHz, DMSO-d6) δ 1.20 – 1.54 (m, 6H), 1.66 – 1.74 (m, 1H), 1.76 – 1.86 (m, 2H), 2.01 – 2.11 (m, 2H), 2.13 – 2.26 (m, 6H), 2.57 – 2.61 (m, 2H), 2.66 (t, J = 4.7 Hz, 4H), 3.22 – 3.33 (m, 1H), 4.43 (d, J = 5.5 Hz, 2H), 5.18 (d, J = 7.2 Hz, 1H), 5.90 (t, J = 5.7 Hz, 1H), 6.62 (d, J = 8.5 Hz, 1H), 6.65 (d, J = 2.1 Hz, 1H), 6.81 (t, J = 5.5 Hz, 1H), 7.03 (dd, J = 8.3, 2.1 Hz, 1H), 7.36 – 7.44 (m, 2H), 8.54 – 8.59 (m, 2H).
13C NMR (101 MHz, DMSO-d6) δ 24.63, 25.55, 32.45, 45.41, 45.66, 50.84, 53.94, 57.31, 107.51, 107.72, 117.52, 122.26, 125.82, 134.03, 138.42, 148.82, 149.52.

tR 1.12 min, MS (ESI) m/z 473 [M +H] (100%)

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Inhibition of erastin- of ferrous ammonium sulphate-induced ferroptosis in IMR-32 neuroblastoma cells

In order to determine IC50-values, human neuroblastoma cells (IMR-32) were seeded in a 96-well plate at a density of 25,000 cells/well. The next day, the cells were pretreated for 1h (in triplicates) with a 1/3 dilution series of ferrostatin-1 analogues ranging from 5μM to 0.68nM and Sytox Green (1.6 μM) and a 1/2 dilution series of ferrostatin-1 analogues ranging from 200 nM to 0.78nM and Sytox Green (1.6 μM) for erastin and ferrous ammonium sulphate respectively. After stimulating the cells with erastin (10μM) or ferrous ammonium sulphate (600 µM), the plate was transferred to a temperature- and CO2-controled FLUOstar Omega fluorescence plate reader (BMG Labtech). Sytox Green intensity was measured after 13h using an excitation filter of 485 nm and an emission filter of 520 nm. In each setup, Triton-X100 (0.05%) was used to induce lyses of the cells in 6 wells/plate, and was used as 100% cell death reference. The percentage of the cell death was calculated by the formula ((AVG[erastin] – AVG[background]) /
(AVG[Triton-X100] – AVG[background])) × 100. Cell death percentage was plotted in GraphPad Prism 6, and IC50-values were calculated using a sigmoidal dose-response (variable slope) curve.
In vivo mouse studies

Mice were bred and housed under SPF conditions in individually ventilated cages at the VIB Inflammation Research Center in conventional, temperature-controlled animal facilities with a 14/10-hour light/dark cycle. Water and feed were provided ad libitum. Iron poisoning experiments were performed with C57BL/6N mice from Janvier Labs, toxicity of compound 39 was assessed in wild type mice derived from a Gpx4 fl/fl breeding. The present studies in animals were reviewed and approved by the Ethical committee on laboratory animal experiments of Ghent University, Faculty of Sciences, Ghent, Belgium.

Acute iron poisoning

All mice treated with iron sulfate received an intraperitoneal injection of 300 mg/kg body weight FeSO4.7H2O dissolved in sterile 0.9% NaCl or vehicle (0.9% NaCl). The injection volume was 200

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μL/20 g body weight. Vehicle solution (2% DMSO) or compound was administered at a concentration of 2 mM (in 0.9% NaCl containing 2% DMSO; 200 μL / 20 g body weight) by intravenous injection 15 minutes before IP injection with FeSO4.7H2O. Two hours after iron sulfate injection, mice were anesthetized with isoflurane and blood was sampled. Hereafter, mice were sacrificed by cervical dislocation. LDH levels in plasma were measured in the clinical lab of Ghent University Hospital by COBAS 8000 (Roche).

Repeated daily injection of compound 39

Vehicle solution (2% DMSO) or compound was administered daily at a concentration of 2 mM (in 0.9% NaCl containing 2% DMSO; 200 μL / 20 g body weight) by intraperitoneal injection. Body temperature and weight were monitored daily. On day 28 the mice were anesthetized with isoflurane and blood was sampled. Hereafter, mice were sacrificed by cervical dislocation. ALT, AST and LDH levels in plasma were measured in the clinical lab of Ghent University Hospital by COBAS 8000 (Roche). CK, creatinine, urea and troponin T were determined at the lab for medical analysis CRI (https://www.cri.be/).

Bilayer insertion of UAMC-3203 occurs on the nanosecond scale

Parameters for alpha-tocopherol and UAMC3203 were generated using the Automated Topology Builder webserver after an optimization at the B3LYP/6-31G* level.37 Charges were fitted to the elec-trostatic potential using the Merz-Singh-Kollman scheme. Water molecules were treated with the SPC water model. The system was treated using the GROMOS 54A7 force field.38 The pre-equilibrated hydrated 128 POPC lipid box was taken from Poger and Mark.39 A 2 fs timestep was adopted, with all bonds constrained to their equilibrium lengths using LINCS.40 Non-bonded interactions were truncated at 1.4 nm. PME was used to account for electrostatic interactions beyond the cut-off. The system was energy- minimised with the steepest descent algorithm until the maximum force exerted on any atom was less than 1000.0 kJ/mol.nm. The system was equilibrated in the NPT ensemble for 10 ns using a semi-isotropic Berendsen41 barostat with a reference pressure of 1 bar and a coupling time of 0.5 ps, while the

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temperature was controlled through one velocity-rescaling thermostat42 coupled to the membrane and one thermostat coupled to water and the ligand with a reference temperature of 303K and a coupling time of 0.1ps. Three separate production simulations with a length of 300 ns were per-formed following three separate 10 ns equilibrations. Pressure control was achieved through a semi-isotropic Parrinello-Rahman barostat with a coupling time of 5 ps.43 All simulations were performed using Gromacs 2016.5.44 The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation – Flanders (FWO) and the Flemish Government – department EWI.

AUTHOR INFORMATION

Corresponding Authors

* Koen Augustyns, Laboratory of Medicinal Chemistry (UAMC), University of Antwerp, Universiteitsplein 1, 2610 Wilrijk. Belgium, E-mail: [email protected]. Phone: +3232652717.

* Tom Vanden Berghe, Molecular Signalling and Cell Death unit, VIB Center for Inflammation Research, Technologiepark 927, 9052 Zwijnaarde, Belgium, E-mail: [email protected], Phone: +3293313721,

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

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We would like to thank Sophie Lyssens and Ingmar Stuyver for excellent technical assistance with the determination of both the in vitro ADME parameters and the kinetic solubility. Also An Matheeussen’s work determining the in vivo characteristics deserves special recognition. This research was funded by the Fonds Wetenschappelijk Onderzoek (FWO) Flanders by means of a personal grant appointed to Lars Devisscher under grant agreements no. 11Z815N and 11Z817N. This research was also financed by the FWO under grant agreements no. G078713N and G0B7118N, and by the FWO Excellence of Science program under the grant agreement no. G0G6618N (EOS ID 30826052). Sam Hofmans is paid by the University of Antwerp as a researcher to work in the Laboratory of Medicinal Chemistry. The Laboratory of Medicinal Chemistry is partner of the Antwerp Drug Discovery Network (www.addn.be). Research in the P.V. unit is supported by Belgian grants (Interuniversity Attraction Poles, IAP 7/32), Flemish grants (Research Foundation Flanders, FWO G.0875.11, FWO G.0973.11, FWO G.0A45.12, FWO G.0172.12, FWO G.0787.13N, FWO G.0C31.14N, FWO KAN 31528711, FWO KAN 1504813N and Foundation against Cancer 2012-188), Ghent University grants (MRP, GROUP-ID consortium) and grants from Flanders Institute for Biotechnology (VIB). P.V. holds a Methusalem grant (BOF09/01M00709) from the Flemish Government.

ABBREVIATIONS

TNF, Tumour Necrosis Factor; RIPK, Receptor-Interacting Protein Kinase; MLKL, Mixed-Lineage Kinase Like pseudokinase; VDAC, Voltage-Dependent Anion Channel; Fer-1, ferrostatin-1; ROS, Reactive Oxygen Species; GSH, glutathione:; GPX4, Glutathione peroxidase 4; SAR, Structure-Activity Relationship; CR, cystine reductase; GCL, glutamate cysteine lipase; GC, -glutamyl cysteine; GS, glutathione synthase; GSSG, glutathione disulfide; FIN, Ferroptosis Inducer.

ANCILLIARY INFORMATION

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Supporting Information. Detailed protocols, extended data series, molecular formula strings, additional charts and graphs for in vitro ADME analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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TABLE OF CONTENTS GRAPHIC

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H
N

NH2

1
(Fer-1)

O

O

H
N

HN

O
S
N
O
H
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(UAMC-3203)

N

NH
Microsomal stability t1/2(Human) = 20.5 h t1/2(Rat) = 16.5 h t1/2(Mouse) = 3.5 h

Plasma recovery after 6h
%(Human) = 84.2
%(Rat) = 85.8

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Ferroptosis IC50 = 33 nM
Ferroptosis IC50 = 12 nM
%(Mouse) = 100

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