Optimization of the C2 substituents on the 1,4-bis(arylsulfonamido)naphthalene-N,N’-diacetic acid scaffold for better inhibition of Keap1-Nrf2 protein-protein interaction

Abstract

Direct inhibition of the protein-protein interaction (PPI) between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) reduces the ubiquitination and subsequent degradation of Nrf2, leading to Nrf2 accumulation in the cytosol and the nuclear translocation of Nrf2. Once inside the nucleus, Nrf2 binds to and activates the expression of antioxidant response element (ARE) genes involved in redox homeostasis and detoxification. Herein, we report a series of 1,4-bis(arylsulfonamido)naphthalene-N,N’-diacetic acid analogs with varying C2 substituents to explore the structure-activity relationships at this position of the central naphthalene core. The Keap1-binding activities were first screened with a fluorescence polarization (FP) assay followed by further evaluation of the more potent compounds using a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay. It was found that compound 24a with C2-phthalimidopropyl group was the most potent in this series showing an IC₅₀ of 2.5 nM in the TR-FRET assay with a K_i value in the subnanomolar range. Our docking study indicated that the C2-phthalimidopropyl group in compound 24a provided an extra hydrogen bonding interaction with the key residue Arg415 that may be responsible for the observed boost in binding affinity. In addition, compounds 12b, 15, and 24a were shown to activate the Nrf2 signaling pathway in NCM460D cells resulting in elevated mRNA levels of GSTM3, HMOX1 and NQO1 by 2.4–11.7 fold at 100 μM as compared to the vehicle control.

Graphical Abstract

INTRODUCTION

The imbalance between oxidants and antioxidants results in oxidative stress conditions involved in the pathogenesis of chronic inflammatory diseases, such as chronic obstructive pulmonary disorder, neurodegenerative disorders, cancer, and chronic kidney disease [1, 2]. A variety of endogenous (e.g., chemicals resulting from intracellular processes) and exogenous sources (e.g., exposure to radiation, carcinogens, and toxins) can contribute to the elevated levels of oxidants in the body. Examples of such oxidants include reactive oxygen species (ROS, e.g., hydrogen peroxide (H₂O₂), superoxide anion (O₂^·−), hydroxyl radical (·OH), and singlet oxygen (¹O₂)) and reactive nitrogen species (RNS, e.g., nitric oxide (NO) and peroxynitrate (ONO₂⁻)) [3, 4]. The antioxidant defense system is an important cytoprotective response against oxidative stress that mitigates the damaging effects of these reactive species, thereby maintaining physiological homeostasis. It mainly works through efficient catalytic detoxification of reactive species by different antioxidant enzymes [5]. The induction of a number of metabolizing and antioxidative enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), catalase, NAD(P)H quinone oxidoreductase 1 (NQO1), heme-oxygenase-1 (HO-1), thioredoxin (TRX), and glutamate-cysteine ligase (GCL), can counteract ROS/RNS and neutralize their damaging effects on cellular organelles [6–11]. These enzymes, characterized by having long half-lives, are primarily induced under the control of the Keap1-Nrf2-ARE signaling pathway [12, 13].

Kelch-like ECH-associated protein 1 (Keap1) is a cytoplasmic 69.7-kD protein that negatively regulates the activity of nuclear factor erythroid 2–related factor 2 (Nrf2) protein [2]. Keap1 contains 625 amino acid residues, 27 of which are cysteines susceptible for modification by different electrophiles and oxidants [1, 14, 15]. Nrf2 is a transcription factor that plays an essential role in inducing the expression of cytoprotective enzymes associated with antioxidant response element (ARE). There are seven highly conserved domains called Neh 1–7 in Nrf2. The ARE is located in the promoter region of numerous genes encoding cytoprotective proteins and detoxification enzymes [16].

Under normal physiological conditions, Nrf2 binds to Keap1, which promotes the ubiquitination of Nrf2 and its subsequent 26S proteasomal degradation . This would result in decreased Nrf2 protein levels in the cytoplasm [17]. Under oxidative stress conditions or in the presence of electrophiles or reactive nitrogen/oxygen species, cysteine residues on the Keap1 protein can be covalently modified or oxidized leading to the dissociation of Nrf2 from Keap1 and reduced ubiquitination/26S proteasomal degradation of Nrf2. The stabilized Nrf2 protein accumulates in the cytosol and translocates into the nucleus followed by binding with ARE-containing promoter region, activating the expression of cytoprotective enzyme-encoding genes [18, 19]. This prompted us and others to discover small molecules that can interfere directly with Keap1-Nrf2 protein–protein interaction (PPI) to induce the expression of ARE-dependent endogenous cytoprotective proteins [20–23]. Such transcriptional activation of the downstream detoxification and antioxidant genes could prevent damage caused by chronic oxidative stress and inflammation [19].

According to molecular mechanisms of action, current Keap1-Nrf2 PPI inhibitors could be categorized as either direct or indirect inhibitors [1, 2]. Indirect inhibitors (group A, Figure 1) are electrophilic modulators that interrupt Keap1-Nrf2 PPI by covalent modification of Keap1 cysteine residues located outside the Keap1-Nrf2 PPI interface [24]. Tecfidera^® (dimethyl fumarate, Figure 1), with a Michael acceptor functionality, is a well-known electrophilic Nrf2 inducer [2]. It was approved as a therapeutic agent for patients suffering from relapsing multiple sclerosis (MS) in 2013 [24, 25]. Also, sulforaphane, an isothiocyanate isolated from cruciferous vegetables such as broccoli, was shown to protect against Aβ-induced Alzheimer’s disease in acute mouse models in Y-maze and passive avoidance behavior tests [26]. Bardoxolone methyl (CDDO-Me), one of the oleanolic acid derived triterpenoids, has an α-cyano-enone that can react with cysteine residues in Keap1 leading to the activation of Nrf2 genes. A major drawback of these electrophilic indirect inhibitors is their lack of specificity that can cause off-target side effects through the modification of other cysteine-containing proteins [27].

Figure 1.

Known indirect (A) and direct inhibitors (B) of Keap1-Nrf2 PPI.

An alternative strategy to activate Nrf2 with greater target selectivity involves non-covalent inhibition of the Keap1-Nrf2 PPI. This can be achieved by directly disrupting the binding between the Kelch domain of Keap1 and the DLG and/or ETGE motifs of Nrf2 [11, 17]. The first small molecule direct inhibitor of Keap1-Nrf2 PPI reported is the tetrahydroisoquinoline (THIQ) analog 1 (LH601A, Figure 1) with an IC₅₀ of 3 μM [28]. In 2014, a symmetrical 1,4-bis(4-methoxyphenylsulfonamido)naphthalene-N,N’-diacetic acid 2a was shown to have promising inhibitory activity against Keap1-Nrf2 PPI [22, 23]. The isoquinoline bis-sulfonamides 2b [29] and 2c [30] were synthesized as isosteres of compound 2a with better metabolic stability and pharmacokinetic properties. Other Keap1-Nrf2 PPI inhibitors have been reported that encompass 3-phenylpropionic acid 3 [31] or 4-amino-1-naphthol 4a-b [32, 33] as their core scaffold. In a separate work, a molecular dissection strategy was employed on the central naphthalene ring to afford a new 1,2-xylylenediamine derivative 5 that exhibited strong potency in the FP assay and improved metabolic stability on incubation with human liver microsomes (Figure 1) [34].

Using a fluorescence polarization (FP) assay, 1,4-Bis(4-methoxyphenylsulfonamido)naphthalene-N,N’-diacetic acid 2a had shown an IC₅₀ value equal to 28.6 nM [22, 23]. The amide isostere 2d was also shown to have a potent activity on the biochemical assay (IC₅₀= 63 nM) with improved pharmacokinetic profile [20, 35]. Our previous structure-activity relationship (SAR) exploration efforts on the sulfonamide portion of this scaffold produced compound 2e with 5-chloro-3,4-ethylenedioxyphenylsulfonyl group lying on the P4/P5 sub-pockets of the Keap1 Kelch domain (Figure 2). It showed better cellular Nrf2 inducing activity with anti-inflammatory activity in microglial cells [21]. Then, we turned our attention to install different substituents on the C2 of 1,4-disubstitued phenylene/naphthalene core [36]. Our efforts led to compound 2f with a 2-(4-fluorobenzyloxy) group that was the most potent derivative in this series with an IC₅₀ of 64.5 nM in the fluorescent polarization (FP) assay and 14.2 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In cell-based assays, compound 2f significantly increased the mRNA levels of Nrf2 downstream genes (NQO1, HMOX1, and GSTM3) via Nrf2 activation. We observed that most non-electrophilic activators still have limitations including their cellular activities and drug-like properties, since most of them contain either mono- or di-carboxylate groups [20]. Thus, there is a definite need for further improvement in cellular potency and PK properties as potential preventive and/or therapeutic agents.

Figure 2.

SAR work around 1,4-bis(arylsulfonamido)naphthalene-N,N’-diacetic acid.

In this current study, we report the design, synthesis and biological evaluation of a series of 1,4-bis(arylsulfonamido)naphthalene-N,N’-diacetic acid derivatives with varying C2-substituents to explore the structure-activity relationships at the C2-position of central naphthalene core as direct inhibitors of Keap1-Nrf2 PPI. Our modeling studies suggested that a C2-alkyl/aryl substitution could additionally occupy a central binding pocket which could help improve Keap1 binding affinity as well as physicochemical and PK properties.

RESULTS AND DISCUSSION

Design strategy.

Based on a computational model developed by You and coworkers for the binding of small molecules to the Keap1 Kelch domain [22], the Nrf2-binding site on Keap1 Kelch domain contains five sub-pockets: two polar arginine-rich sub-pockets (P1 and P2), a central flat subpocket P3, and two greasy tyrosine-rich subpockets (P4 and P5). A potent inhibitor of the Keap1-Nrf2 PPI should occupy all five of these subpockets, as demonstrated by the co-crystal structure of the 1,4-diaminonaphthalene derivative 2d with Keap1 Kelch domain [20]. However, replacing the naphthalene central core with 1,4-disubstitued phenylene resulted in a 50-fold decrease in potency when compared to the naphthalene analog 2a [23]. This could be explained in terms of weaker hydrophobic contacts and the lack of π-cation interaction with Arg415 in the central P3 subpocket [23, 37]. This loss in binding affinity in 1,4-disubstitued phenylene analogs was regained by introducing an O-benzyl substituent on the 2-position. The crystal structure of one of these compounds in complex with Keap1 (6HWS) [38] shows that the π-cation interaction is maintained.

Our efforts to increase structural diversity of the central core moiety along with potential improvement of potency included the structural modifications of O-linked aromatic ring on the C2 position of 1,4-bis(arylsulfonamido)-benzene or naphthalene-N,N’-diacetic acids [36]. As a result, our SAR study led to the discovery of compound 2f with similar potency as 2a. In the same study, compounds with an O-linked aryl substituent on the naphthalene core were much more potent as Keap1-Nrf2 PPI inhibitors than those inhibitors with a phenylene core. These results suggested that the C2-substituents of these inhibitors might be occupying a previously unidentified sixth subpocket [36]. Herein, we report our subsequent SAR studies focusing on varying the substituent at the C2 position of the naphthalene core as shown in Figure 3. We also optimized the linker moiety connecting to the various aryl/alkyl substituents in order to provide some structural flexibility that might be required to map the sixth subpocket and for optimal orientation and binding affinity. Some of the C2 substituents are relatively more lipophilic and could serve to improve biological membrane permeability of this class of inhibitors containing the acetate groups.

Figure 3.

Structural modifications focused on the C2 position of compound 2a with diverse polar and nonpolar alkyl or aryl substituents

Chemistry.

Synthesis of the naphthalene analogs 12a-c with a phenyl side chain is summarized in Scheme 1. Treatment of 4-nitro-1-naphthylamine (6) with KIO₃/KI/HCl gave the 2-iodo-substituted intermediate 7 in 91% yield [39]. Compound 7 was then subjected to Suzuki cross-coupling to produce 2-aryl intermediates 8a-d. The general procedure for the Suzuki coupling involved Pd(PPh₃)₄ as the catalyst and K₂CO₃ as the base in DME/H₂O (2:1) at 80 °C [40]. Subsequently, the nitro group in compounds 8a-d was reduced using tin(II) chloride in DMF at room temperature to get 1,4-diamine intermediates 9a-d in quantitative yields. Treatment of the diamines 9a-d with p-methoxybenzenesulfonyl chloride in pyridine/DCM at room temperature gave the bis-sulfonamide intermediates 10a-d in 62–85% yield, which were then alkylated using t-butyl bromoacetate and K₂CO₃ in DMF. Removal of the t-butyl groups was done under acidic conditions using a mixture of TFA:DCM in a 1:3 ratio at room temperature to generate the final compounds 12a-c in 61–92% yield.

Scheme 1.

Synthesis of compounds 12a-c with a phenyl ring on the C2 position^a

^aReagents and conditions: (a) KI, KIO₃, HCl, MeOH, H₂O, rt, 91%%; (b) arylboronic acid, PdCl₂(PPh₃)₂, K₂CO₃, DME/H₂O, 80 °C, 24 h, 77–85%; (c) SnCl₂, DMF, rt, overnight, quant.; (d) 4-methoxybenzenesulfonyl chloride, pyridine, rt, overnight, 62–85%; (e) t-butyl bromoacetate, K₂CO₃, DMF, rt, overnight, 71–82%; (f) TFA, DCM, rt, overnight, 61–92%. ^bSee Table 1 for the chemical structures.

Compound 12d with 4-phthalimidophenyl group at the 2-position of the naphthalene central core was synthesized as shown in Scheme 2. Treatment of 10d with TFA/DCM (1:3) at room temperature followed by the addition of phthalic anhydride in acetic acid under reflux afforded compound 10e in 65% yield. Alkylation of 10e with t-butyl bromoacetate afforded the dialkylated analog 11d which was followed by acidic deprotection to remove the t-butyl groups furnishing the desired product 12d in 60% yield.

Scheme 2.

Synthesis of compound 12d with 4-phthalimidophenyl group on the C2 position^a

^aReagents and conditions: (a) TFA, DCM, rt, overnight, quantitative for 10d and 60% for 12d; (b) phthalic anhydride, acetic acid, reflux, overnight, 65%; (c) t-butyl bromoacetate, K₂CO₃, DMF, rt, overnight, 61%.

The synthesis of 2-alkyl substituted naphthalene scaffold is shown in Scheme 3 via the key intermediates 13a-c which were obtained through Heck coupling of 2-iodo-4-nitro-1-naphthylamine (7) with an acrylate or acrylamide in 77–83% yield. The general procedure for the Heck coupling involved the use of Pd(OAc)₂, tri(o-tolyl)phosphine, and TEA in ACN at 90 °C with either t-butyl acrylate for compound 13a or acrylamides for 13b-c [41] in 77–83% yield. Subsequently, both the alkene double bond and nitro group were reduced using H₂ (1 atm) and 10% Pd/C in THF to get 1,4-diamine compounds 14a-c in quantitative yield. Sulfonamide intermediates 15a-c were synthesized using 4-methoxybenzenesulfonyl chloride in pyridine as a base and solvent, which is followed by alkylation using t-butyl bromoacetate/bromoacetamide in the presence of K₂CO₃ in DMF at room temperature to generate the dialkylated products 16a-c in 71–82% yield. Treatment of the alkylated intermediates 16a-c with TFA/DCM mixture at room temperature gave the target compounds 17a-c in 68–78% yield.

Scheme 3.

Synthesis of compounds 17a-c with an alkyl substituent on the C2 position^a

^aReagents and conditions: (a) t-butyl acrylate for 13a/acrylamide for 13b-c, Pd(OAc)₂, P(o-tolyl)₃, TEA, ACN, 90 °C, 77–83%; (b) H₂ (1 atm), 10% Pd/C, THF, rt, 8 h, quant.; (c) 4-methoxybenzenesulfonyl chloride, pyridine, rt, 8 h, 61–74%; (d) bromoacetamide for 16a; t-butyl bromoacetate for 16b-c, K₂CO₃, DMF, rt, overnight, 71–82%; (e) TFA, DCM, rt, overnight, 68–78%. ^bSee Table 1 for the chemical structures.

Continuation of our efforts to install amide, imide or amine functionalities linked via an alkyl spacer to the 2-position on the naphthalene scaffold is shown in Scheme 4. Compound 18 containing a propanoic acid moiety was synthesized from 15a through the removal of the t-butyl group under acidic conditions in 89% yield. The derivative 19 with propanamide side chain was obtained in 84% yield relying on amide coupling of the carboxylic group with ammonia in DCM using PyAOP in TEA and DMF. Alkylation of the sulfonamide NHs with t-butyl bromoacetate furnished compound 20 which was subjected to acidic deprotection yielding the target compound 21 in 75% yield. The 3-napthalenylpropanoic acid 18 underwent reduction with lithium aluminum hydride (LAH) in anhydrous THF under reflux generating the alcohol intermediate. Alkylation for this alcohol derivative was done selectively on its sulfonamide NHs using t-butyl bromoacetate and K₂CO₃ in DMF at room temperature to yield the dialkylated intermediate 22 in 51% yield. The hydroxyl group on the side chain of compound 22 was activated for substitution by treatment with tosyl chloride to afford the O-tosyl analog in 91% yield. Treatment of the tosylated intermediate with phthalimide potassium salt or N-methylbenzyl amine in K₂CO₃/DMF at room temperature generated compounds 23a and 23b. The final step involved acid-mediated removal of the t-butyl group yielding the diacidic compounds 24a and 24b in 68% yield.

Scheme 4.

Synthesis of compounds 21 and 24a-b with alkyl substituent on the C2 position^a

^aReagents and conditions: (a) TFA, DCM, rt, overnight, 73–89%; (b) NH₃, PyAOP, TEA, DCM, DMF, rt, overnight, 84%; (c) t-butyl bromoacetate, K₂CO₃, DMF, rt, overnight, 72% for 20; 51% for 22; (d) lithium aluminum hydride (LAH), THF, reflux; (e) TsCl, pyridine, DCM, rt, overnight, 91%; (f) phthalimide potassium salt (23a) or N-methylbenzylamine (23b) , K₂CO₃, DMF, rt, 3 h, 84% for 23a; 69% for 23b. ^bSee Table 2 for the chemical structure.

The synthetic pathway of analog 29 with a thioether-linked phenyl group on the 2-position of naphthalene scaffold is outlined in Scheme 5. The previously reported 1,4-bis(4-methoxyphenylsulfonamido)naphthalene intermediate 25 [21] was used as the starting point for the synthesis of this analog. Treatment of 1,4-bis-p-methoxybenzenesulfonamide 25 with ceric ammonium nitrate in acetonitrile gave the p-quinoneimine intermediate 26 in quantitative yield [42]. Then, treatment of compound 26 with benzenethiol via a Michael addition-type reaction allowed the installation of the phenylthio group on the C2 position of the naphthalene core as shown in intermediate 27 in 68% yield. The addition reaction was accomplished in toluene using DIPEA as the base to produce the thioether analog 27 in 84% yield. Alkylation of intermediate 27 with ethyl bromoacetate in DMF at room temperature followed by the removal of the ethyl protecting groups from compound 28 under basic conditions produced final compound 29 in 68% yield.

Scheme 5.

Synthesis of compounds 29 with thioether-linked phenyl group on the C2 position^a

^aReagents and conditions: (a) ceric ammonium nitrate, ACN, rt, overnight, quant.; (b) thiophenol, DIPEA, toluene, rt, 8 h, 68%; (c) ethyl bromoacetate, K₂CO₃, DMF, rt, overnight, 69%; (e) NaOH, EtOH/H₂O, rt, 8 h, 68%.

Inhibition of Keap1-Nrf2 PPI and structure-activity relationships (SAR).

The activity of all target compounds was initially evaluated using an FP assay. The results from the FP assay are summarized in Tables 1–2. With the exception of the analog with basic tertiary amine, 24b, all target compounds with substitution at the C2-position were as potent as compound 2a. With the FP assay conditions used to evaluate the potency of our compounds, we were having difficulty differentiating inhibitors with an IC₅₀ value of around 50–100 nM. Therefore, a more sensitive time-resolved fluorescence energy transfer (TR-FRET) assay was employed to differentiate among these potent inhibitors [43]. In order to evaluate the inhibitory potency of our more potent compounds, we performed the TR-FRET assay recently developed by our group, under the optimized conditions that used 0.5 nM Tb-anti-his-antibody, 5 nM Keap1 Kelch domain protein, 25 nM fluorescein-labeled Nrf2 peptide probe, and 1% DMSO [43]. From the results shown in Table 1, introduction of a phenyl group on the C2-position of the naphthalene core as in 12a did not adversely affect the binding affinity when compared with the unsubstituted 2a. This could be useful means of increasing the lipophilicity of this series that could ultimately help improve the overall permeability of these dicarboxylate inhibitors. We were interested in exploring the nature of the substituents that could be installed on that phenyl group and how they would affect their binding affinity to the Kelch domain of Keap1. Introduction of an electron-donating methoxy group as in 12b or an electron-withdrawing cyano as in 12c at the para position of the C2-phenyl side chain did not significantly affect the binding affinity as compared to the unsubstituted analog 12a. Surprisingly, introduction of a bulky phthalimide group at the para-position of the C2-phenyl group as in 12d results in a comparable inhibitory potency to 12a. Moreover, the insertion of a thioether linker between the phenyl group and the C2 position of the naphthalene core as in 29 was well tolerated as seen in the results from both the FP and TR-FRET assays. The inhibition constant (K_i) was calculated from the TR-FRET results and listed in Table 1. All compounds included in Table 1 had similar K_i values relative to compound 2a with the exception of compound 12b with the 4-methoxyphenyl group at the C2 position of the naphthalene ring

Table 1.

The inhibitory activities of different 2-substituted naphthalene analogs (compounds 12a-d and 29)

Compd	R	FP assay IC50 (nM)a	TR-FRET IC50 (nM)a	Ki (nM)b
2a (LH762)	H	110 ± 7	7.5 ± 0.39	2.3
12a (LH890)		96 ± 6	7.7 ± 0.13	2.4
12b (LH924)		85 ± 6	13.1 ± 0.9	4.9
12c (LH935)		117 ± 7	9.2 ± 0.32	3.1
12d (LH936)		90 ± 6	7.1 ± 0.57	2.1
29 (LH913)		85 ± 5	5.8 ± 0.28	1.5

^aIC₅₀ values are calculated as an average of three replicate measurements ± SEM

^bK_i is calculated from the TR-FRET IC₅₀ for better comparison

Table 2.

The inhibitory activities of different 2-substituted naphthalene analogs (compounds 17a-d, 21, and 24a-b).

Compd	R1	R2	FP assay IC50 (nM)a	TR-FRET IC50 (nM)a	Ki (nM)b
2a (LH762)	H	COOH	110 ± 7	7.5 ± 0.39	2.3
17a (LH914)		CONH2	162 ± 10	22.7 ± 1.8	9.3
17b (LH915)		COOH	102 ± 7	12.6 ± 0.18	4.6
17c (LH916)		COOH	87 ± 6	6.9 ± 0.55	2.0
21 (LH927)		COOH	108 ± 7	11.7 ± 0.68	4.2
24a (LH918)		COOH	77 ± 5	2.5 ± 0.18	< 0.1
24b (LH919)		COOH	276 ± 17	73.6 ± 16.6	32.9

^aIC₅₀ values are calculated as an average of three replicate measurements ± SEM

^bK_i is calculated from the TR-FRET IC₅₀ for better comparison

From the data shown in Table 2, introduction of a propanamide group on the C2-position as in 21 exhibited a near 2-fold drop in inhibitory activity as indicated by the K_i value from the TR-FRET assay, as compared to 2a without any substitution at the C2-position. Introduction of a large isopropyl on the C2-propanamide as in 17b did not adversely affect its binding to Keap1 kelch domain (17b vs 21). Compound 17a containing a propanoic acid fragment resulted in a 3-fold decrease in potency relative to 2a as indicated by TR-FRET IC₅₀ and K_i. But, this decrease in binding affinity could be attributed to the amide groups on the R₂ of 17a rather than the propanoic acid fragment, as seen in the previous results of 2a and 2d [20]. Additionally, the replacement of the isopropyl secondary amide group in 17b with the morpholine amide as in 17c showed 2-fold increase in binding affinity as indicated by the TR-FRET K_i. The introduction of phthalimidopropyl group at the C2 of the naphthalene core generated the most active compound 24a in this series with TR-FRET IC₅₀ value of 2.5 nM and a K_i value less than 0.1 nM. Compound 24a demonstrated a 3-fold enhancement of potency over compound 2a with no substituent at C2. Removal of the carbonyl groups in the imide group of 24a and breaking the ring structure of the phthalimide ring produced compound 24b containing a tertiary amine moiety. When compared to 24a, this structural change resulted in more than 20-fold reduction in potency obtained from TR-FRET assay. This signified the importance of the carbonyl imide group on the side chain or the deleterious effect of potential repulsive forces created by introduction of basic amine on the side chain.

In summary and as shown in Figure 4, compound 24a with a phthalimidopropyl group at the C2-position of the naphthalene core was found to be the most potent inhibitor of this series with a FP IC₅₀ of 77 ± 5 nM, TR-FRET IC₅₀ of 2.5 ± 0.18 nM and K_i less than 0.1 nM. There are several structural features that could be extracted from this series: a) substitution at C2-position of the naphthalene core is well tolerated, b) there is an increase in potency for specific analogs, and c) only side chain with a basic center showed diminishing binding affinity.

Figure 4.

Dose response curve of FP and TR-FRET assay for compound 24a vs 2a

Docking study.

Our SAR study above indicated that only compound 24a with a phthalimidopropyl group is more active than 2a in the TR-FRET assay, while the other C2-substitutions led to similar or reduced binding affinity. Therefore, docking studies were used to investigate the role of the phthalimidopropyl fragment in enhancing the binding affinity of 24a to the target Keap1 protein. Compounds 2a and 24a were docked into the co-crystal structure of 2d with the Kelch domain of Keap1 (PDB: 4XMB). As seen in Figure 5, both naphthalene derivatives occupy five subpockets and have a similar binding mode, including hydrogen bonds with four polar residues (Arg380, Arg483, Asn414 and Arg415), and a π-π stacking interaction with Tyr572. Interestingly, compound 24a displays stronger binding interactions via additional hydrogen bonds to the key residue Arg415, and one of which is particularly associated with the phthalimide group connected to the C2-position of the naphthalene core. Therefore, the docking study supports that the C2 substitution in 24a plays a significant role in binding to the target Keap1 protein through an additional hydrogen bonding interaction with Arg415, thereby contributing to improved inhibitory activity in the TR-FRET assay.

Figure 5.

Binding modes of compounds 2a (A) and 24a (B) docked into the Kelch domain of Keap 1 (derived from PDB code: 4XMB). The surface of keap1 was colored based on the partial charge (indicated in blue for the most polar areas, and red for the most lipophilic areas). Red dashed lines represent hydrogen bonds, and yellow lines represent π-π stacking or π-cation interactions.

Effects of compounds 12b, 12c, 12d, 17c and 24a on the transcription of Nrf2-ARE regulated genes.

Additionally, we tested the ability of selected members of our potent Keap1-Nrf2 PPI inhibitors in NCM460D cells to induce the expression of Nrf2-ARE-controled genes. As shown in Figure 6, all test compounds (12b, 12d, 17c, and 24a) led to the significant induction of the Nrf2 target genes (HMOX1, GSTM3, and NQO1 in comparison to GAPDH and HPRT, two housekeeping genes) at 100 μM, except for 12c. It should be noted that the concentrations used to test these compounds in cellular assays remain high suggesting that membrane permeability needs further improvement in future studies.

Figure 6.

Nrf2 target gene expression level by the potent Keap1-Nrf2 PPI inhibitors.

Particularly, the naphthalene analogue 12b with 4-methoxyphenyl group at the C2 position showed significant increase in the transcription of Nrf2 target genes (GSTM3, HMOX1 and NQO1) at 100 μM. The three target genes were up-regulated in the range of 8–12 fold increase compared with the DMSO-treated cells, and even more active in all three target genes than 2a. On the other hand, a significant drop in the transcription of those targeted genes on replacing the methoxy with cyano group was observed as in compound 12c. There was no induction of any of the measured target genes even at 100 μM concentration of our inhibitor 12c. Interestingly, the phthalimido group on the p-position of the phenyl moiety on the side chain as in compound 12d showed 3–4 fold increase in mRNA levels of the three genes even at 10 μM, as compared to the control cells. Furthermore, compound 24a with a phthalimidopropyl group showed a similar trend in up-regulation of mRNA levels of GSTM3, HMOX1 and NQO1 by 2–4 fold at 10 μM compared with the DMSO-treated cells. When normalized by the expression of the internal control HPRT, the phthalimide analogs 12d and 24a displayed more active cellular activities in all the Nrf2 target genes than 2a, suggesting that the C2-substitutions containing a phthalimide could contribute to increased cellular activities through their higher lipophilicities.

CONCLUSION

Recently, there have been multiple and diverse direct and indirect inhibitors of Keap1-Nrf2 PPI reported in the literature [1, 11]. In the present study, we carried out a detailed SAR study of well-known symmetrical inhibitor 2a by exploring the nature of the substituent at the C2-position of the central naphthalene core. Using a TR-FRET assay, we were able to further differentiate the activity of the potent C2 substituted naphthalene derivatives and confirmed their nanomolar to subnanomolar inhibitory activities. Compound 24a with phthalimidopropyl group installed on the C2 position showed a 3-fold boost in TR-FRET IC₅₀ value with a K_i less than 0.1 nM. Using docking simulation, compound 24a bound to the Keap1 protein in a comparable manner as compound 2a with an additional hydrogen bonding interaction with Arg415 via its phthalimido group. Cellular activities of 12b, 12d and 24a were shown to be superior to or equally as active as compound 2a, in inducing the mRNA levels of NQO1, HMOX1 and GSTM3. These results showed that these asymmetric 1,4-bis(arylsulfonamido)naphthalene-N,N’-diacetic acid analogs could serve as new leads for future optimization.

Experimental Section

1. General Chemistry

All reagents used in the synthetic procedures were purchased as ACS grade and used directly without further pretreatment/purification, while all solvents were obtained as either ACS reagent or HPLC grade and used as received unless otherwise specified. Reactions progress were monitored by analytical thin-layer chromatography (TLC) using aluminum backed Silica G DC Kieselgel 60 F254-coated TLC plates (Merck or Sigma-Aldrich). TLC plates were visualized under ultraviolet light (UV) illumination and/or by using TLC stain such as iodine or potassium permanganate coupled with heating. Analytical liquid chromatography/mass spectrometry (LC/MS) system was also utilized for monitoring of reaction progress. The data were obtained using the Shimadzu 2010 LC-MS system, and/or Agilent 1200 HPLC system combined with an Agilent 6140 single quadrupole MS system (Santa Clara, CA), operating a multimode source. LC-MS system used an Inertsil ODS-3 C18 column (3 mm × 33 mm, 3 μM) which was kept at a temperature of 40 °C. Mobile phase A was water/0.1% formic acid, while mobile phase B was methanol/0.1% formic acid. The applied gradient program involved a flow rate of 0.8 mL/min with a linear increase in mobile phase B from 10% to 90% over 5 minutes. The eluted peaks were monitored using UV absorbance at 280 nm. Compound purification using combiflash column chromatography was performed on a Teledyne ISCO Companion using hexane, ethyl acetate, methanol, and dichloromethane as mobile phases with pre-packed RediSep normal phase silica cartridges (230–400 mesh) as the stationary phase. A VirTis freeze dryer (SP Scientific, Warminster, PA) was used to carry out lyophilization of the final compounds. ¹H NMR spectra (400 MHz) and ¹³C NMR spectra (100 MHz) were recorded on Bruker 400 MHz Multinuclear NMR spectrometer (Billerica, MA) using CDCl₃, methanol-d₄, acetone-d₆, and DMSO-d₆. NMR data were reported as follows: chemical shift in parts per million (ppm) relative to the nondeuterated residual solvent signals, and coupling constant (J values) in Hertz (Hz). In the NMR tabulation, spin multiplicities are indicated as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and brs (broad singlet). High-resolution mass spectrometry (HRMS) experiments were conducted by the Center for Integrative Proteomics Research (CIPR) at Rutgers University. Compound solutions (1 μg/mL) were directly infused using the Thermo LTQ Orbitrap Velos ETD with Dionex Ultimate 3000 nanoflow 2D LC. Waters ACQUITY UPLC system was used to determine purities for each final compound and found to be ≥95% with exception of compound 12d (94.60%) and compound 17a (94.49%).

2. Synthetic Procedure and Characterization of Compounds

2.1. General procedure for Suzuki coupling (Method A)

2-Iodo-4-nitronaphthalen-1-amine (1 equiv.) was charged into a Schlenk tube under argon atmosphere followed by addition of phenylboronic acid (1.2 equiv.), PdCl₂(PPh₃)₂ (0.02 equiv.), DME: H₂O (1:1), and K₂CO₃ (2 equiv.). After that, the reaction flask was degassed and refilled with argon for three cycles. The reaction mixture was stirred at 80 °C for overnight. After the reaction was completed as indicated by TLC and LC-MS, water was added to the reaction mixture followed by extraction with ethyl acetate. The organic phase was then separated and washed with saturated brine solution. The ethyl acetate solution was then dried by passing over anhydrous Na₂SO₄ and the volume was concentrated under vacuum. The obtained residue was purified on Teledyne ISCO combiflash using normal phase pre-packed silica gel columns and hexane/ethyl acetate as mobile phase.

2.2. General procedure for reduction of nitro group (Method B1):

The nitro-containing compound (1 mmol) was dissolved in THF/EtOH (1:1) mixture in 50 ml rounded-bottom flask. The reaction vessel was then degassed and refilled with nitrogen for three cycles. To the reaction mixture, a catalytic equivalent of 10% Pd/C (5 mg) was added, then reaction was purged with hydrogen for additional last time. The reaction was stirred under hydrogen atmosphere (1 atm; using a hydrogen balloon) for 8 hours. After that, the reaction mixture was then filtered through a celite pad which helps in removal of the palladium catalyst. The filtrate was concentrated under reduced pressure to produce the desired amino product. The crude product was used directly in the subsequent step with no need for further purification.

2.3. General procedure for reduction of nitro group (Method B2):

The nitro containing starting material (1 equiv.) was dissolved in DMF (1 mL for 1 mmole starting material) and then tin(II) chloride monohydrate (SnCl₂.H₂O) (5 equiv.) was slowly added with stirring at room temperature. Then, the reaction mixture was stirred at room temperature overnight. The reaction was monitored with TLC and LC-MS and on complete conversion, the reaction mixture was diluted with ethyl acetate. The organic phase was then washed with sodium bicarbonate (saturated solution), separated, dried over anhydrous sodium sulfate and then concentrated under vacuum. The crude product was used in the following step with no need for further purification.

2.4. General procedure for synthesis of sulfonamides derivatives (Method C1)

To a mixture of a diamine intermediate (1 mmol, 1 equiv.) in DCM (5 mL), triethylamine (5 mmol, 5 equiv.), was added. After that, a solution of 4-methoxybenzenesulfonyl chloride (2.2 mmol, 2.2 equiv.) in DCM (5 mL) was added portion-wise at a slow rate over 5 minutes at 0 °C under nitrogen atmosphere. The reaction mixture was warmed to room temperature and stirring was continued at the same temperature for overnight. The reaction was monitored with TLC and LC-MS. After the reaction was complete, the crude mixture was diluted with DCM followed by washing with 1 N HCl and saturated brine (NaCl) solutions. The organic layer was separated, dried over anhydrous Na₂SO₄ and then concentrated under vacuum. The obtained residue was purified by flash column chromatography using gradient mobile phase composed of hexane/ethyl acetate.

2.5. General procedure for synthesis of sulfonamides (Method C2)

1,4-Diaminonaphthalene (1 mmol, 1 equiv.) was dissolved in pyridine (5 mL) followed by dropwise addition of 4-methoxybenzenesulfonyl chloride (2.2 mmol, 2.2 equiv.) solution in DCM (5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 8 h under nitrogen atmosphere. Upon completion as indicated by TLC and LC-MS, the crude mixture was diluted with DCM, and then shaken against 1 N HCl and saturated NaCl solutions. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under vacuum. The obtained residue was purified by flash column chromatography using eluting mobile phase starting from 0–100% ethyl acetate in hexane.

2.6. General procedure for synthesis of alkylated sulfonamides (Method D)

To a solution of a sulfonamide intermediate (1 mmol, 1 equiv.) and in DMF (3 mL), potassium carbonate (10 mmol, 10 equiv.) was added followed by addition of either alkyl bromoacetate or bromoacetamide (5 mmol, 5 equiv.). Then, the reaction suspension was stirred at room temperature overnight. The reaction was monitored by TLC and LC-MS. On complete conversion, the crude mixture was diluted with ethyl acetate, and then washed with 1 N HCl and saturated brine solutions. The organic phase was separated, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Using 0–50% ethyl acetate in hexane as eluent, the obtained residue was purified using flash column chromatography.

2.7. General procedure for basic deprotection of ethyl esters (method E1)

To a solution of an alkylated sulfonamide intermediate in EtOH (2 mL), 4 N NaOH in water (4 equiv.) was added. After that, the reaction mixture stirred at room temperature for 8 hours. Upon completion, the crude mixture was concentrated under vacuum and then acidified with 1 N HCl solution. The aqueous suspension was then extracted with ethyl acetate. The organic phase was then separated, and then washed with saturated brine solutions. The organic layer was collected, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The obtained residue was then crystallized using ethyl acetate/hexane and washed with petroleum ether to get the final product in pure form.

2.8. General procedure for acidic deprotection of tert-butyl esters (method E2)

The alkylated sulfonamide intermediate was dissolved in DCM (0.5 mL) followed by slow addition of a TFA/DCM mixture (1:2, 1.5 mL). Then, the reaction mixture was warmed and stirred at room temperature for 1 hour. Upon complete conversion, the crude mixture was concentrated under vacuum. The obtained oily residue was crystallized using ethyl acetate/hexane or diethyl ether to get the final diacidic product in pure form.

2.9. General procedure for Heck coupling (method F)

2-Iodo-4-nitronaphthalen-1-amine (1 equiv.) was loaded in a Schlenk tube equipped with a magnetic stir bar under argon atmosphere followed by addition of acrylate/acrylamide (1.4 equiv.), Pd(OAc)₂ (0.06 equiv.), P(o-tolyl) (0.12 equiv.) and triethylamine (1.4 equiv.) in acetonitrile (4 mL). The reaction vessel was evacuated and refilled with argon three times and then refluxed for 24 hours under argon atmosphere. The reaction was monitored with TLC and LC-MS. On completion, the reaction mixture was diluted with ethyl acetate and then washed with saturated brine solution. The organic phase was then separated and then dried over anhydrous Na₂SO₄, followed by concentration under vacuum. The crude product purified by combiflash ISCO using a gradient of hexane/ethyl acetate to get the desired product in fairly-pure form.

2-Iodo-4-nitronaphthalen-1-amine (7)

To a stirred solution of 4-nitronaphthalene-1-amine (2.0 g, 10.6 mmol) in methanol/water mixture (10 mL/60 mL), potassium iodide (1.17 g, 7.1 mmol) and potassium iodate (0.76 g, 3.05 mmol) were added and the mixture was stirred at room temperature for 10 min. Then, dilute HCl (1.2 mL diluted to10 mL) was added during a period of 1 hour, and the reaction stirred overnight at room temperature. Upon completion, the reaction mixture was filtered, and the yellow solid washed with water, dilute sodium thiosulfate solution and dried to get the desired product as a yellow solid 3.06 g yield: (91%); ¹H NMR (400 MHz, CDCl₃) δ 8.90 (d, 1 H, J = 8.8 Hz), 8.75 (s, 1 H), 7.83 (d, 1 H, J = 8.8 Hz), 7.76−7.72 (m, 1 H), 7.60−7.56 (m, 1 H), 5.44 (br, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 149.3, 136.8, 130.1, 127.1, 125.1, 121.6, 121.0.

4-Nitro-2-phenylnaphthalen-1-amine (8a)

Prepared using the method described in the general procedure for Suzuki coupling (Method A) to get the desired compound as a yellow solid; 449 mg, yield 85; ¹H NMR (400 MHz, CDCl₃) δ 9.00 (d, 1 H, J = 8.8 Hz), 8.99 (s, 1 H), 7.90 (d, 1 H, J = 8.8 Hz), 7.76−7.72 (m, 1 H), 7.61−7.43 (m, 6 H), 5.11 (br, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ 146.3, 137.5, 136.4, 130.0, 129.9, 129.6, 129.6, 128.4, 127.1, 126.4, 124.9, 122.3, 121.6, 119.5.

2-(4-Methoxyphenyl)-4-nitronaphthalen-1-amine (8b)

Prepared using the method described in the general procedure for Suzuki coupling (Method A) to get the desired compound as a yellow solid; 232 mg, yield 79%; ¹H NMR (400 MHz, DMSO-d₆) δ 8.90 (d, 1 H, J = 8.8 Hz), 8.47 (d, 1 H, J = 8.4 Hz), 8.23 (s, 1 H), 7.76−7.72 (m, 1 H), 7.56 (t, 1 H, J = 8.0 Hz), 7.42 (d, 2 H, J = 8.0 Hz), 7.07 (d, 2 H, J = 8.8 Hz), 7.00 (br, 2 H), 3.81 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 158.8, 149.7, 132.3, 130.8, 130.5, 130.0, 129.6, 126.8, 125.4, 123.9, 123.3, 121.3, 117.4, 114.6, 55.1.

4-(1-Amino-4-nitronaphthalen-2-yl)benzonitrile (8c)

Prepared using the method described in the general procedure for Suzuki coupling (Method A) to get the desired compound as dark brown solid; 453 mg, yield 77%; ¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (d, 1 H, J= 8.8 Hz), 8.49 (d, 1 H, J = 8.8 Hz), 8.23 (s, 1 H), 7.97 (d, 2 H, J = 8.4 Hz), 7.81−7.77 (m, 1 H), 7.72 (d, 2 H, J = 8.4 Hz), 7.61−7.57 (m, 1 H), 7.22 (br, 2 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 149.3, 142.5, 132.7, 130.3, 130.2, 126.8, 125.4, 123.7, 123.0, 121.2, 118.6, 115.3, 109.9.

tert-Butyl (4-(1-amino-4-nitronaphthalen-2-yl)phenyl)carbamate (8d)

Prepared using the method described in the general procedure for Suzuki coupling (Method A) to get the desired compound as a yellow solid; 585 mg, yield 77%; ¹H NMR (400 MHz, CDCl₃) δ 8.97 (d, 1 H, J = 8.8 Hz), 8.36 (s, 1 H), 7.88 (d, 1 H, J = 8.8 Hz), 7.71−7.67 (m, 1 H), 7.56−7.50 (m, 3 H), 7.39 (d, 1 H, J = 8.4 Hz), 6.71 (s, 1 H), 5.15 (br, 2 H), 1.54 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 152.9, 146.7, 138.5, 136.0, 131.9, 130.2, 130.0, 129.8, 127.0, 126.3, 124.7, 122.1, 121.7, 119.6, 119.0, 81.1, 28.5.

2-Phenylnaphthalene-1,4-diamine (9a)

Prepared using the method described in the general procedure for nitro group reduction (Method B2) to get the desired compound as dark yellow solid; 344 mg, quantitative; ¹H NMR (400 MHz, CDCl₃) δ 7.94−7.89 (m, 2 H), 7.52−7.47 (m, 6 H), 7.40−7.36 (m, 1 H), 6.72 (s, 1 H), 3.82 (br, 4 H); ¹³C NMR (100 MHz, CDCl₃) δ 129.7, 129.0, 127.3, 125.7, 125.2, 122.3, 121.9.

2-(4-Methoxyphenyl)naphthalene-1,4-diamine (9b)

Prepared using the method described in the general procedure for nitro group reduction (Method B2) to get the desired compound as a yellow solid; 261 mg, quantitative; ¹H NMR (400 MHz, DMSO-d₆) δ 7.90−7.85 (m, 2 H), 7.53−7.46 (m, 3 H), 7.41 (d, 2 H, J = 8.8 Hz), 7.00 (d, 2 H, J = 8.8 Hz), 3.85 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 158.8, 149.7, 132.3, 130.8, 130.5, 130.0, 129.6, 126.8, 125.4, 123.9, 123.3, 121.3, 117.4, 114.6, 55.1.

4-(1,4-Diaminonaphthalen-2-yl)benzonitrile (9c)

Prepared using the method described in the general procedure for nitro group reduction (Method B2) to get the desired compound as dark solid; 378 mg, quantitative; ¹H NMR (400 MHz, CDCl₃) δ 7.92−7.87 (m, 2 H), 7.74 (d, 2 H, J = 8.4 Hz), 7.64 (d, 2 H, J= 8.4 Hz), 7.55−7.52 (m, 2 H), 6.23 (s, 1 H), 3.87 (br, 4 H); ¹³C NMR (100 MHz, CDCl₃) δ 159.1, 143.7, 131.1, 131.0, 130.8, 125.8, 125.3, 125.2, 123.4, 122.4, 122.1, 121.6, 114.78, 114.7, 55.7.

tert-Butyl (4-(1,4-diaminonaphthalen-2-yl)phenyl)carbamate (9d)

Prepared using the method described in the general procedure for nitro group reduction (Method B2) to get the desired compound as an off-white solid; 519 mg, yield quantitative; ¹H NMR (400 MHz, CDCl₃) δ 7.92−7.86 (m, 2 H), 7.53−7.40 (m, 6 H), 3.86 (br, 4 H), 1.55 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 153.8, 137.2, 134.7, 134.1, 131.4, 130.0, 125.3, 125.0, 124.8, 124.5, 122.7, 122.0, 121.6, 118.9, 113.3, 80.5, 28.3.

N,N’-(2-Phenylnaphthalene-1,4-diyl)bis(4-methoxybenzenesulfonamide) (10a)

Using general procedure (method C2) starting from 1 mmole of 9a to obtain 489 mg (yield 85%); ¹H NMR (400 MHz, DMSO-d₆) δ 10.16 (s, 1 H), 9.76 (s, 1 H), 8.14−8.11 (m, 2 H), 7.64 (d, 2 H, J = 8.8 Hz), 7.52−7.50 (m, 2 H), 7.24−7.18 (m, 2 H), 7.09−7.03 (m, 2 H), 6.89 (s, 1 H), 6.70 (d, 2 H, J = 8.8 Hz), 3.82 (s, 3 H), 3.80 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.5, 161.8, 138.8, 138.7, 133.9, 132.5, 132.4, 131.1, 129.4, 129.2, 129.0, 128.0, 127.8, 126.9, 126.6, 126.6, 126.1, 125.3, 125.2, 123.2, 114.3, 113.8, 55.7, 55.5.

N,N’-(2-(4-Methoxyphenyl)naphthalene-1,4-diyl)bis(4-methoxybenzenesulfonamide) (10b)

Prepared using the method described in the general procedure for synthesis of sulfonamides (method C2) starting from 1 mmole of 9b to get the desired compound as a beige solid; 472 mg, yield 78%; ¹H NMR (400 MHz, CDCl₃) δ 8.50 (d, 1H, J = 8.4 Hz), 7.68−7.66 (m, 2 H), 7.55 (t, 1H, J = 7.2 Hz), 7.48−7.44 (m, 1 H), 7.24 (s, 1 H), 7.07−7.05 (m, 3 H), 6.98 (s, 1 H), 6.84−6.82 (m, 2 H), 6.77−6.70 (m, 4 H), 6.61 (d, 2H, J = 8.8 Hz), 3.85 (s, 3 H), 3.83 (s, 3 H), 3.79 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 163.3, 162.8, 158.9, 136.8, 133.0, 131.4, 130.8, 130.6, 130.4, 129.8, 129.6, 29.0, 128.4, 127.1, 127.0, 126.8, 126.5, 124.1, 120.9, 114.2, 114.1, 113.8, 55.6, 55.6, 55.3.

N,N’-(2-(4-Cyanophenyl)naphthalene-1,4-diyl)bis(4-methoxybenzenesulfonamide) (10c)

Prepared using the method described in the general procedure for synthesis of sulfonamides (method C2) starting from 1 mmole of 9c to obtain the desired compound as a yellow solid; 408 mg, yield 68%; ¹H NMR (400 MHz, CDCl₃) δ 8.30−8.28 (m, 1 H), 7.84−7.81 (m, 1 H), 7.69 (d, 2 H, J = 8.8 Hz), 7.52−7.45 (m, 5 H), 7.29 (s, 1 H), 7.16−7.14 (m, 4 H), 6.95 (br, 1 H), 6.83 (d, 2 H, J = 9.2 Hz), 6.64 (d, 2 H, J = 8.8 Hz), 3.87 (s, 3 H), 3.79 (s, 3 H); ¹³C NMR (100 MHz, CDCl₃) δ 163.6, 163.4, 163.3, 162.8, 143.5, 136.6, 133.4, 132.9, 132.6, 1310.2, 130.6, 130.3, 130.1, 129.9, 129.7, 129.6, 129.1, 128.8, 127.8, 127.8, 126.8, 125.8, 122.8, 121.4, 118.7, 114.4, 114.3, 114.2, 114.1, 111.3, 55.9, 55.8,

tert-Butyl (4-(1,4-bis((4-methoxyphenyl)sulfonamido)naphthalen-2-yl)phenyl)carbamate (10d)

Prepared using the method described in the general procedure for synthesis of sulfonamides (method C2) starting from 1 mmole of 9d to get the desired compound as an off-white solid; 421 mg, yield 62%; ¹H NMR (400 MHz, MeOH-d₄) δ 8.31 (d, 1 H, J = 8.4 Hz), 8.04 (d, 1 H, J = 8.4 Hz), 7.75 (d, 1 H, J = 8.8 Hz), 7.64 (s, 1 H), 7.62 (d, 1 H, J = 8.8 Hz), 7.51−7.41 (m, 2 H), 7.18 (d, 1 H, J = 8.8 Hz), 7.11−7.09 (m, 2 H), 7.00 (s, 1 H), 6.89 (d, 4 H, J = 9.2 Hz), 6.61 (d, 2 H, J = 9.2 Hz), 3.82 (s, 3 H), 3.81 (s, 3 H), 1.55 (s, 9 H); ¹³C NMR (100 MHz, MeOH-d₄) δ 162.9, 162.4, 137.9, 132.8, 132.2, 131.6, 130.8, 129.3, 129.2, 128.4, 126.6, 126.5, 126.1, 125.23, 124.9, 122.2, 118.0, 113.8, 113.5, 80.1, 55.2, 55.1, 27.9.

Di-tert-butyl 2,2’-((2-phenylnaphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (11a)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.19 mmole of 10a to get the desired compound as oily product; 127 mg, yield 82%; ¹H NMR (400 MHz, CDCl₃) δ 8.24 (t, 1 H, J = 8.0 Hz), 8.15 (t, 1 H, J = 8.4 Hz), 7.72 (d, 1 H, J = 8.8 Hz), 7.63 (t, 2 H, J = 8.8 Hz), 7.55−7.50 (m, 2 H), 7.40−7.29 (m, 6 H), 7.08−7.02 (m, 1 H), 6.95− 6.85 (m, 4 H), 4.39−4.32 (m, 3 H), 4.16−3.94 (m, 3 H), 3.93−3.86 (m, 6 H), 1.18−1.14 (m, 3 H), 1.09−1.02 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 168.9, 168.8, 163.4, 163.4, 139.6, 138.6, 136.7, 136.3, 134.0, 132.6, 132.2, 131.6, 131.4, 130.8, 130.7, 130.6, 129.5, 128.3, 128.1, 128.1, 127.9, 127.4, 126.9, 126.8, 126.4, 124.2, 124.1, 114.1, 113.8, 61.6, 61.4, 55.8, 54.4, 53.7, 53.3, 14.3, 14.1, 14.0.

Di-tert-butyl 2,2’-((2-(4-methoxyphenyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (11b)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.2 mmole of 10b to get the desired compound as oily product; 123 mg, yield 74%; ¹H NMR (400 MHz, CDCl₃) δ 8.22−8.17 (m, 2 H), 7.74−7.69 (m, 1 H), 7.65−7.63 (m, 2 H), 7.61−7.47 (m, 2 H), 7.36 (t, 1H, J = 8.0 Hz), 7.30 (d, 1H, J = 8.4 Hz), 7.23 (d, 1H, J = 8.4 Hz), 7.08−7.05 (m, 1 H), 6.94−6.85 (m, 6 H), 4.49−4.20 (m, 3 H), 3.89−3.81 (m, 9 H), 3.77−3.60 (m, 1 H), 1.33 (s, 9 H), 1.23−1.22 (m, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.8, 167.6, 167.6, 163.34, 163.3, 163.2, 163.1, 162.6, 159.4, 159.3, 139.9, 139.2, 136.8, 136.8, 136.1, 135.9, 134.2, 134.1, 132.5, 132.4, 132.3, 132.2, 131.5, 131.4, 131.3, 131.0, 130.6, 130.5, 130.3, 130.0, 127.1, 127.0, 126.8, 126.7, 124.2, 114.1, 113.8, 113.7, 82.4, 82.2, 77.5, 77.2, 76.8, 55.7, 55.4, 55.2, 54.5, 54.2, 54.2, 28.0, 27.9.

Di-tert-butyl 2,2’-((2-(4-cyanophenyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (11c)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.2 mmole of 10c to get the desired compound as oily product; 118 mg, yield 71%; ¹H NMR (400 MHz, CDCl₃) δ 8.16−8.05 (m, 2 H), 7.69−7.63 (m, 4 H), 7.57−7.38 (m, 6 H), 7.14−7.08 (m, 1 H), 6.91−6.83 (m, 4 H), 4.57−4.48 (m, 1 H), 4.29−4.12 (m, 2 H), 3.92−3.84 (m, 6 H), 3.80−3.76 (m, 1 H), 1.34−1.33 (m, 9 H), 1.24 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.7, 167.1, 167.0, 163.5, 163.4, 143.9, 143.7, 138.8, 138.2, 137.3, 136.0, 135.8, 133.9, 133.8, 133.0, 132.9, 131.9, 131.8, 131.6, 131.4, 130.7, 130.6, 130.5, 130.4, 130.1, 127.9, 127.7, 127.5, 126.8, 126.4, 124.3, 118.8, 114.1, 113.9, 111.9, 111.7, 82.6, 82.5, 55.8, 55.8, 55.3, 54.8, 54.0, 53.9, 28.0, 27.9.

2,2’-((2-Phenylnaphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (12a)

Prepared using the method described in the general procedure for hydrolysis of ethyl ester to get di-acidic compounds (Method E1) (basic hydrolysis) starting from 0.05 mmole of 11a to get the desired compound as a white solid; 32 mg, yield 92%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.27(d, 2 H, J = 7.6 Hz), 7.62 (d, 2 H, J = 7.2 Hz), 7.55−749 (m, 3 H), 7.42−7.27 (m, 6 H), 7.05 (d, 2 H, J = 7.2 Hz), 6.95 (s, 1 H), 6.87−6.86 (m, 1 H), 6.70−6.69 (m, 1 H), 4.43−4.4.24 (m, 2H), 3.90 (s, 3 H), 3.87 (s, 2 H), 3.85 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.0, 170.0, 169.7, 169.6, 162.9, 162.8, 162.7, 139.6, 138.8, 138.6, 138.4, 136.6, 136.5, 135.4, 134.9, 133.7, 133.5, 132.3, 131.3, 131.0, 130.7, 130.2, 130.1, 129.9, 129.2, 129.0, 129.0, 127.8, 127.6, 127.4, 126.6, 126.5, 126.2, 124.4, 124.1, 114.3, 114.0, 55.7, 53.3, 53.0; HRMS (ESI) calcd for C₃₄H₃₀N₂O₁₀S₂ [M + H]⁺ 691.1415; found 691.1414.

2,2’-((2-(4-Methoxyphenyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (12b)

Prepared using the method described in the general procedure for removal of acid sensitive protecting group (Method E2) starting from 0.033 mmole of 11b to get the desired compound as a yellow solid; 17 mg, yield 71%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.30 (br, 1 H), 8.24 (d, 1 H, J = 6.8 Hz), 7.62 (d, 2 H, J = 7.2 Hz), 7.54−7.51 (m, 3 H), 7.40 (t, 1 H, J = 6.0 Hz), 7.17 (s, 2 H), 7.05 (d, 2 H, J = 7.6 Hz), 6.99 (d, 2 H, J = 6.4 Hz), 6.96 (s, 1 H), 6.87 (d, 2 H, J = 6.8 Hz), 4.44 −3.89 (m, 4 H), 3.85 (s, 3 H), 3.83 (s, 3 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 169.0, 168.5, 162.6, 162.4, 158.4, 135.9, 134.7, 133.2, 131.5, 131.2, 130.6, 130.4, 129.7, 129.5, 129.4, 129.4, 125.9, 125.7, 125.6, 123.5, 113.8, 113.5, 112.9, 55.2, 55.2, 54.7, 52.7; HRMS (ESI) calcd for C₃₅H₃₂N₂O₁₁S₂ [M + H]⁺ 721.1520; found 721.1532.

2,2’-((2-(4-Cyanophenyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (12c)

Prepared using the method described in the general procedure for removal of acid sensitive protecting group (Method E2) starting from 0.03 mmole of 11c to get the desired compound as brown solid; 13 mg, yield 61%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.30 (d, 1 H, J = 8.5 Hz), 8.22 (d, 1 H, J = 8.5 Hz), 7.74 (d, 2 H, J= 8.0 Hz), 7.62−7.56 (m, 3 H), 7.47−7.44 (m, 5 H), 7.05 (d, 2 H, J = 7.0 Hz), 6.96 (d, 3 H, J = 9.0 Hz),4.45−4.30 (m, 4 H), 3.90 (s, 3 H), 3.85 (s, 3 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 168.8, 168.2, 162.5, 162.4, 142.9, 137.7, 136.3, 134.5, 132.7, 132.0, 130.7, 130.5, 129.7, 129.4, 129.3, 129.0, 126.1, 126.0, 125.3, 123.7, 117.7, 113.6, 113.4, 110.0, 55.1, 55.1, 52.5; HRMS (ESI) calcd for C₃₅H₂₉N₃O₁₀S₂ [M + H]⁺ 716.1367; found 716.1373.

N,N’-(2-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)naphthalene-1,4-diyl)bis(4-methoxybenzenesulfonamide) (10e)

Compound 10d (0.33 mmole) was subjected to removal of acid sensitive protecting group (Method E2) to get the desired compound as oily liquid in quantitative yield. The obtained 1ry amine compound was mixed with phthalic anhydride (50 mg, 0.33 mmol) in acetic acid (3 mL) and the reaction mixture was refluxed for overnight. After completion, acetic acid removed, and the product purified by ISCO chromatography using ethyl acetate/hexane to get 154 mg, yield 65%; ¹H NMR (400 MHz, DMSO-d₆) δ 10.21 (s, 1 H), 9.84 (s, 1 H), 8.21−8.16 (m, 2 H), 8.02−8.01 (m, 2 H), 7.94−7.93 (m, 2 H), 7.66 (d, 2 H, J = 8.8 Hz), 7.55−7.52 (m, 2 H), 7.37 (d, 2 H, J = 8.0 Hz), 7.19 37 (d, 2 H, J = 8.0 Hz), 7.08 37 (d, 4 H, J = 8.8 Hz), 6.95 (s, 1 H), 6.80 37 (d, 2 H, J = 8.8 Hz), 3.81 (s, 3 H), 3.74 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.1, 162.6, 162.0, 138.5, 138.0, 134.8, 133.8, 132.7, 131.9, 131.5, 131.1, 131.0, 130.9, 129.6, 129.5, 129.3, 128.3, 126.8, 126.7, 126.6, 126.3, 125.4, 125.1, 123.5, 123.3, 114.3, 114.1, 55.7, 55.4.

Di-tert-butyl 2,2’-((2-(4-(1,3-dioxoisoindolin-2-yl)phenyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl)) diacetate (11d)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.1 mmole of 10e to get the desired compound as oily product; 58 mg, yield 61%; ¹H NMR (400 MHz, CDCl₃) δ 8.32−8.29 (m, 2 H), 8.01−7.99 (m, 2 H), 7.85−7.82 (m, 2 H), 7.70 (d, 1 H, J = 8.8 Hz), 7.65 (d, 1 H, J = 8.8 Hz), 7.56 (d, 2 H, J = 8.8 Hz), 7.51−7.39 (m, 6 H), 7.06−6.89 (m, 5 H), 4.48−4.26 (m, 3 H), 3.90−3.85 (m, 6 H), 3.78−3.75 (m, 1 H), 1.34−1.33 (m, 9 H), 1.24 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 167.3, 167.3, 163.5, 163.3, 138.9, 138.5, 137.2, 134.7, 134.2, 133.0, 131.9, 131.8, 131.5, 130.8, 130.7, 130.5, 130.3, 130.2, 127.4, 127.2, 127.0, 126.0, 125.9, 124.5, 123.9, 114.1, 114.0, 82.5, 82.3, 55.8, 55.7, 28.0, 27.9.

2,2’-((2-(4-(1,3-Dioxoisoindolin-2-yl)phenyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (12d)

Prepared using the method described in the general procedure for removal of acid sensitive protecting group (Method E2) starting from 0.03 mmole of 11d to get the desired compound as a yellow solid; 15 mg, yield 60%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.34 (d, 2 H, J = 8.0 Hz), 8.03−8.01 (m, 2 H), 7.96−7.94 (m, 2 H), 7.63 (d, 2 H, J = 8.5 Hz), 7.58 (t, 1 H, J = 7.0 Hz), 7.48−7.43 (m, 7 H), 7.08 (d, 2 H, J = 8.5 Hz), 7.08 (d, 2 H, J = 8.5 Hz), 7.00 (s, 1 H), 4.47−4.33 (m, 4 H), 3.87 (s, 3 H), 3.86 (s, 3 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 169.1, 168.5, 166.3, 162.7, 162.6, 137.9, 136.3, 134.7, 134.2, 133.1, 132.0, 131.2, 130.7, 130.2, 129.6, 129.6, 129.2, 126.0, 125.9, 125.7, 123.9, 122.9, 113.9, 113.8, 55.4, 55.2, 52.9; HRMS (ESI) calcd for C₄₂H₃₃N₃O₁₂S₂ [M + H]⁺ 836.1578; found 836.1587.

tert-Butyl (E)-3-(1-amino-4-nitronaphthalen-2-yl)acrylate (13a)

Prepared using the method described in the general procedure for Heck coupling (Method F) starting from 2.0 mmole of compound 7 to get the desired compound as brown solid; 521 mg, yield 83%; ¹H NMR (400 MHz, CDCl₃) δ 8.88 (d, 1 H, J = 8.8 Hz), 8.54 (s, 1 H), 7.88 (d, 1 H, J = 8.4 Hz), 7.80 (d, 1 H, J = 15.6 Hz), 7.74−7.70 (m, 1 H), 7.60−7.56 (m, 1 H), 6.45 (d, 1 H, J = 15.6 Hz), 5.35 (br, 2 H), 1.56 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 147.4, 137.1, 136.5, 130.6, 127.6, 126.9, 126.5, 125.0, 122.6, 122.4, 121.6, 111.5, 81.2, 28.4.

(E)-3-(1-Amino-4-nitronaphthalen-2-yl)-N-isopropylacrylamide (13b)

Prepared using the method described in the general procedure for Heck coupling (Method F) starting from 2.0 mmole of compound 7 to get the desired compound as a beige solid; 468 mg, yield 78%; ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (d, 1 H, J = 8.0 Hz), 8.59 (s, 1 H), 8.46 (d, 1 H, J = 8.8 Hz), 7.99 (d, 1 H, J = 7.6 Hz), 7.86 (d, 1 H, J = 15.6 Hz), 7.76−7.73 (m, 3 H), 7.58−7.54 (m, 1 H), 6.64 (d, 1 H, J = 15.6 Hz), 3.99−3.97 (m, 1 H), 1.13 (d, 6 H, J = 6.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.1, 150.7, 132.9, 132.5, 130.5, 127.1, 126.7, 125.9, 124.0, 123.5, 122.7, 122.0, 110.3, 22.5.

(E)-3-(1-Amino-4-nitronaphthalen-2-yl)-1-morpholinoprop-2-en-1-one (13c)

Prepared using the method described in the general procedure for Heck coupling (Method F) starting from 2.0 mmole of compound 7 to get the desired compound as an off-white solid; 504 mg, yield 77%; ¹H NMR (400 MHz, DMSO-d₆) δ 8.72−7.70 (m, 2 H), 8.46 (d, 1 H, J = 8.8 Hz), 7.96 (d, 1 H, J = 15.2 Hz), 7.74 (t, 1 H, J = 8.0 Hz), 7.69 (br, 2 H), 7.56 (t, 1 H, J = 8.0 Hz), 3.74 (br, 2 H), 3.61 (br, 6 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.7, 150.4, 135.8, 133.4, 130.3, 127.0, 126.9, 125.8, 123.8, 123.2, 121.9, 117.9, 110.3, 66.3, 45.6, 42.0.

tert-Butyl 3-(1,4-diaminonaphthalen-2-yl)propanoate (14a)

Prepared using the method described in the general procedure for nitro group reduction (Method B1) starting from 1.5 mmole of compound 13a to get the desired compound as an off-white solid in quantitative yield; ¹H NMR (400 MHz, CDCl₃) δ 7.86−7.81 (m, 2 H), 7.48−7.41 (m, 2 H), 6.60 (s, 1 H), 3.94 (br, 2 H), 3.76 (br, 2 H), 2.93 (t, 2 H, J = 8.0 Hz), 2.59 (t, 2 H, J = 7.6 Hz), 1.44 (s, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 172.8, 134.3, 131.9, 125.2, 125.2, 124.3, 124.1, 121.7, 121.5, 120.7, 113.1, 80.6, 35.4, 28.1, 27.0.

3-(1,4-Diaminonaphthalen-2-yl)-N-isopropylpropanamide (14b)

Prepared using the method described in the general procedure for nitro group reduction (Method B1) starting from 1.5 mmole of compound 13b to get the desired compound as a beige solid in quantitative yield; ¹H NMR (400 MHz, CDCl₃) δ 7.81−7.78 (m, 2 H), 7.45−7.40 (m, 2 H), 6.52 (s, 1 H), 5.37 (br, 1 H), 4.03−3.96 (br, 2 H), 3.75 (br, 2 H), 2.95 (t, 2 H, J = 7.6 Hz), 2.43 (t, 2 H, J = 7.6 Hz), 0.98 (d, 6 H, J= 6.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 171.8, 134.3, 132.1, 125.3, 124.5, 124.2, 121.7, 121.6, 120.9, 113.4, 41.5, 37.0, 27.5, 22.7.

3-(1,4-diaminonaphthalen-2-yl)-1-morpholinopropan-1-one (14c)

Prepared using the method described in the general procedure for nitro group reduction (Method B1) starting from 1.5 mmole of compound 13c to get the desired compound as a beige solid in quantitative yield.

tert-Butyl 3-(1,4-bis((4-methoxyphenyl)sulfonamido)naphthalen-2-yl)propanoate (15a)

Prepared using the method described in the general procedure for synthesis of sulfonamides (method C1) starting from 1.0 mmole of compound 14a to get the desired compound as an off-white solid; 464 mg, yield 74%; ¹H NMR (400 MHz, DMSO-d₆) δ 10.03 (s, 1 H), 9.69 (s, 1 H), 7.96 (d, 1 H, J = 8.4 Hz), 7.66 (d, 1 H, J = 7.6 Hz), 7.60 (d, 2 H, J = 8.8 Hz), 7.47 (d, 2 H, J = 8.4 Hz), 7.33 (t, 1 H, J = 8.0 Hz), 7.23 (t, 1 H, J = 7.6 Hz), 7.02 (d, 2 H, J = 8.0 Hz), 6.98 (d, 2 H, J = 8.8 Hz), 6.94 (s, 1 H). 3.80 (s, 3 H), 3.78 (s, 3 H), 2.69 (br, 2 H), 2.14 (br, 2 H), 1.39 (s, 9 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.2, 162.4, 162.4, 137.5, 132.6, 132.5, 132.2, 131.3, 129.0, 128.9, 128.6, 127.9, 125.9, 125.3, 124.3, 124.2, 123.1, 114.3, 114.2, 79.7, 55.6, 35.2, 27.7, 26.5.

3-(1,4-Bis((4-methoxyphenyl)sulfonamido)naphthalen-2-yl)-N-isopropylpropanamide (15b)

Prepared using the method described in the general procedure for synthesis of sulfonamides (method C1) starting from 1.0 mmole of compound 14b to get the desired compound as a beige solid; 416 mg, yield 68%; ¹H NMR (400 MHz, DMSO-d₆) δ 7.93 (d, 1 H, J = 8.4 Hz), 7.74 (d, 1 H, J = 8.4 Hz), 7.64−7.59 (m, 3 H), 7.47−7.44 (m, 2 H), 7.33−7.23 (m, 2 H), 7.04−6.97 (m, 5 H), 3.80 (s, 4 H), 3.77 (s, 3 H), 2.60 (br, 2 H), 2.13 (t, 2 H, J = 7.6 Hz), 1.01 (d, 6 H, J = 6.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 162.4, 162.4, 138.1, 132.5, 132.2, 132.1, 131.5, 129.0, 128.7, 128.6, 127.8, 125.8, 125.2, 124.5, 124.3, 122.9, 114.3, 114.2, 55.6, 55.6, 36.1, 26.7, 22.3.

N,N’-(2-(3-Morpholino-3-oxopropyl)naphthalene-1,4-diyl)bis(4-methoxybenzene-sulfonamide) (15c)

The diamine compound 14c (1.0 mmole) was dissolved in pyridine using general procedure (method C1) to get the title compound, 317 mg, yield 65%; ¹H NMR (400 MHz, DMSO-d₆) δ 10.06 (s, 1 H), 9.75 (s, 1 H), 7.99 (d, 1 H, J = 8.4 Hz), 7.75 (d, 1 H, J = 8.4 Hz), 7.65 (d, 2 H, J = 8.8 Hz), 7.48 (d, 2 H, J = 8.8 Hz), 7.37−7.33 (m, 1 H), 7.29−7.27 (m, 1 H), 7.04−6.98 (m, 5 H), 3.81 (s, 3 H), 3.78 (s, 3 H), 3.53−3.52 (m, 4 H), 3.43−3.32 (m, 4 H), 2.65 (br, 2 H), 2.31 (t, 2 H, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.1, 162.5, 162.4, 138.0, 133.6, 132.3, 132.2, 131.5, 129.1, 128.8, 128.7, 128.0, 125.9, 125.4, 124.5, 124.2, 123.1, 114.3, 114.2, 66.0, 55.7, 55.7, 45.3, 41.6, 33.2, 26.6.

tert-Butyl 3-(1,4-bis((N-(2-amino-2-oxoethyl)-4-methoxyphenyl)sulfonamido)naphthalen-2yl)propanoate (16a)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.1 mmole of compound 15a to get the desired compound as oily product; 61 mg, yield 82%; ¹H NMR (400 MHz, CDCl₃) δ 8.08−7.95 (m, 1 H), 7.66−7.59 (m, 4 H), 7.37−7.30 (m, 1 H), 7.16−7.15 (m, 2 H), 6.99−6.55 (m, 7 H), 5.97−5.88 (m, 2 H), 4.48−4.20 (m, 4 H), 3.88−3.84 (m, 6 H), 3.21−2.82 (2 H), 2.55−2.26 (m, 2 H), 1.46 (s, 9 H); ¹³C NMR (100 MHz) (CDCl₃) δ 171.9, 171.8, 170.5, 170.3, 170.2, 163.7, 162.6, 140.6, 137.8, 137.6, 134.8, 134.7, 132.1, 132.0, 132.0, 131.8, 130.6, 130.5, 130.4, 130.3, 130.1, 129.9, 128.6, 128.4, 128.3, 127.2, 126.6, 124.3, 114.4, 114.4, 114.3, 80.7, 80.6, 55.7, 36.4, 36.3, 35.9, 31.6, 31.4, 28.1.

Di-tert-butyl 2,2’-((2-(3-(isopropylamino)-3-oxopropyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (16b)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.1 mmole of compound 15b to get the desired compound as oily product; 60 mg, yield 71%; ¹H NMR (400 MHz, CDCl₃) δ 8.32 (d, 1 H, J = 8.4 Hz), 7.67−7.60 (m, 3 H), 7.52−7.18 (m, 4 H), 7.04−6.86 (m, 5 H), 6.38−6.30 (m, 1 H), 4.72−4.4.02 (m, 5 H), 3.91 (s, 1 H), 3.84 (s, 3 H), 3.47−3.40 (m, 1 H), 2.77−2.70 (m, 1 H), 2.20−2.07 (m, 2 H), 1.47−1.33 (m, 18 H), 1.17−1.11 (m, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.4, 168.8, 167.4, 163.6, 163.5, 140.70, 138.0, 135.0, 132.7, 131.0, 130.4, 130.3, 130.0, 128.8, 127.5, 126.6, 125.4, 124.2, 114.4, 114.3, 114.2, 82.7, 82.6, 82.4, 55.9, 55.7, 54.5, 54.1, 41.2, 39.2, 29.5, 28.1, 28.1, 28.0, 22.9, 22.8, 22.7.

Di-tert-butyl 2,2’-((2-(3-morpholino-3-oxopropyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (16c)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.1 mmole of compound 15c to get the desired compound as oily product; 60 mg, yield 71%; ¹H NMR (400 MHz, CDCl₃) δ 8.18−8.13 (m, 1 H), 7.67−7.60 (m, 4 H), 7.42−7.36 (m, 2 H), 7.26−7.11 (m, 2 H), 6.95−6.86 (m, 4 H), 4.46−4.14 (m, 4 H), 3.86 (s, 3 H), 3.85 (s, 3 H), 3.75−3.01 (m, 10 H), 2.72−2.25 (m, 2 H), 1.40−1.32 (m, 18 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.0, 170.8, 167.5, 167.4, 167.3, 163.3, 163.2, 163.1, 162.4, 140.7, 140.5, 137.4, 137.4, 135.3, 135.2, 132.4, 132.2, 132.0, 131.9, 130.6, 130.5, 130.2, 130.1, 130.0, 129.8, 129.1, 129.0, 126.8, 126.7, 126.3, 126.2, 124.6, 124.5, 124.1, 114.0, 113.9, 82.1, 82.0, 66.9, 66.8, 55.5, 54.4, 54.3, 53.8, 53.8, 46.1, 46.0, 41.9, 34.0, 33.9, 27.8, 27.7, 20.9.

3-(1,4-Bis((N-(2-amino-2-oxoethyl)-4-methoxyphenyl)sulfonamido)naphthalen-2-yl)propanoic acid (17a)

Prepared using the method described in the general procedure for removal of acid-sensitive protecting group (Method E2) starting from 0.033 mmole of compound 16a to get the desired compound as a yellow solid; 18 mg, yield 78%. ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.18 (d, 1 H, J = 6.4 Hz), 7.86−7.83 (m, 1 H), 7.64−7.59 (m, 4 H), 7.44−7.41 (m, 1 H), 7.32−7.29 (m, 1 H), 7.08−7.06 (m, 3 H), 7.01 (d, 1 H, J = 6.8 Hz), 6.84 (br, 4 H), 4.35−4.23 (m, 4 H), 3.87 (s, 3 H), 3.86 (s, 3 H), 2.96−2.90 (br, 1 H), 2.75 (br, 1 H), 2.26 (br, 2 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 172.7, 168.5, 168.4, 162.6, 162.6, 139.3, 136.8, 134.4, 132.3, 131.3, 130.9, 129.6, 129.5, 129.4, 129.4, 129.3, 128.1, 125.7, 125.6, 125.1, 125.0, 124.5, 124.0, 114.0, 113.9, 113.9, 113.8, 55.3, 55.3, 53.9, 53.8, 33.7, 26.1; HRMS (ESI) calcd for C₃₁H₃₂N₄O₁₀S₂ [M + H]⁺ 685.1633; found 685.1628.

2,2’-((2-(3-(Isopropylamino)-3-oxopropyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (17b)

Prepared using the method described in the general procedure for removal of acid-sensitive protecting group (Method E2) starting from 0.036 mmole of compound 16b to get the desired compound as an off-white solid; 19 mg, yield 71%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.18 (d, 1 H, J = 8.5 Hz), 7.94 (d, 1 H, J = 9.0 Hz), 7.61−7.59 (m, 4 H), 7.43 (t, 1 H, J = 7.0 Hz), 7.36−7.33 (m, 1 H), 7.13 (s, 1 H), 7.09−7.08 (m, 3 H), 7.03 (d, 2 H, J= 9.0 Hz), 4.39−4.38 (m, 4 H), 3.87−3.86 (m, 6 H), 2.93−2.87 (m, 1 H), 2.71−2.65 (m, 1 H), 2.19 (br, 2 H), 1.09−1.06 (m, 6 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 169.7, 169.1, 162.7, 162.7, 139.9, 136.9, 134.3, 132.5, 131.1, 130.5, 129.5, 129.4, 128.3, 125.8, 125.1, 124.5, 124.0, 114.0, 114.0, 55.3, 53.1, 52.8, 35.7, 26.7, 21.8; HRMS (ESI) calcd for C₃₄H₃₇N₃O₁₁S₂ [M + H]⁺ 728.1942; found 728.1928.

2,2’-((2-(3-Morpholino-3-oxopropyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (17c)

Prepared using the method described in the general procedure for removal of acid sensitive protecting group (Method E2) starting from 0.033 mmole of compound 16c to get the desired compound as a pink solid; 17 mg, yield 68%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 12.37 (br, 2 H), 8.18 (d, 1 H, J = 6.8 Hz), 7.83 (d, 1 H, J = 6.8 Hz), 7.62−7.58 (m, 4 H), 7.43 (t, 1 H, J = 5.6 Hz), 7.32 (t, 1 H, J = 5.6 Hz), 7.14 (s, 1 H), 7.07 (d, 2 H, J = 5.6 Hz), 7.02 (d, 2 H, J = 7.2 Hz), 4.43− 4.34 (m, 4 H), 3.87 (s, 3 H), 3.86 (s, 3 H), 3.58 (t, 4 H, J = 4.0 Hz), 3.43 (t, 4 H, J = 3.6 Hz), 2.94−2.92 (m, 1 H), 2.83−2.77 (m, 1 H), 2.40 (br, 2 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 169.4, 168.8, 162.6, 162.5, 139.6, 136.7, 134.1, 132.1, 131.0, 130.2, 129.4, 129.4, 129.3, 128.1, 125.6, 124.9, 124.2, 123.8, 113.8, 113.7, 65.5, 55.2, 55.2, 52.8, 52.6, 32.0, 26.2; HRMS (ESI) calcd for C₃₅H₃₇N₃O₁₂S₂ [M + H]⁺ 756.1891; found 756.1877.

3-(1,4-Bis((4-methoxyphenyl)sulfonamido)naphthalen-2-yl)propanoic acid (18)

Prepared using the method described in the general procedure for removal of acid sensitive protecting group (Method E2) starting from 0.2 mmole of compound 15a to get the title compound as an off-white solid; 102 mg, yield 89%; ¹H NMR (400 MHz, MeOH-d₄) δ 7.96 (d, 1 H, J = 8.4 Hz), 7.75 (d, 1 H, J = 8.4 Hz), 7.60 (d, 2 H, J = 8.8 Hz), 7.49 (d, 2 H, J = 9.2 Hz), 7.33−7.29 (m, 1 H), 7.24−7.20 (m, 1 H), 7.03 (s, 1 H), 6.95−6.90 (m, 4 H), 3.83 (s, 3 H), 3.81 (s, 3 H), 2.80 (br, 2 H), 2.36 (t, 2 H, J = 8.0 Hz); ¹³C NMR (100 MHz, MeOH-d₄) δ 176.6, 164.7, 164.6, 139.1, 134.3, 133.9, 133.4, 132.3, 130.7, 130.6, 130.3, 129.6, 127.2, 126.6, 125.9, 125.6, 124.0, 115.2, 115.1, 56.2, 56.1, 35.4, 27.6.

3-(1,4-Bis((4-methoxyphenyl)sulfonamido)naphthalen-2-yl)propanamide (19)

To a stirred solution of compound 18 (96 mg, 0.128 mmol) in DCM (2 ml): DMF (0.2 ml), PyAOP (93 mg, 0.18 mmol), TEA (95 uL, 0.7 mmol) were added at 0 °C under nitrogen. Then, NH₃/ DCM (1.5 N, 100 uL) was added and reaction stirred at 0 °C for 10 min. and then room temperature for 3 hours Upon completion, the reaction diluted with ethyl acetate and washed with 1 N HCl solution, saturated brine, dried over Na₂SO₄, and the organic layer removed under reduced pressure. The crude product purified by ISCO using ethyl acetate/hexane to get the desired product as a beige solid (63 mg, yield 84%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.38 (s, 1 H), 10.01 (s, 1 H), 8.28 (d, 1 H, J = 8.0 Hz), 8.07 (d, 1 H, J = 8.4 Hz), 7.95 (d, 2 H, J = 8.8 Hz), 7.79 (d, 2 H, J = 8.8 Hz), 7.67−7.56 (m, 3 H), 7.36−7.30 (m, 5 H), 7.17 (s, 1 H), 4.14 (s, 3 H), 4.10 (s, 3 H), 3.67 (br, 3 H), 2.83 (br, 1 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.6, 162.4, 162.4, 138.1, 132.4, 132.2, 132.1, 131.4, 129.0, 128.7, 128.6, 127.8, 125.8, 125.2, 124.5, 124.1, 122.9, 114.3, 114.2, 55.6, 55.6, 35.6, 26.4.

(Di-tert-butyl 2,2’-((2-(3-amino-3-oxopropyl) naphthalene-1,4-diyl) bis (((4-methoxyphenyl) sulfonyl) azanediyl)) diacetate) (20)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.05 mmole of compound 19 to get the desired compound as oily product; 29 mg, yield 72%; ¹H NMR (400 MHz, CDCl₃) δ 8.49−8.45 (m, 1 H), 7.84 (d, 2 H, J = 8.8 Hz), 7.80−7.77 (m, 2 H), 7.67−7.59 (m, 1 H), 7.45−7.44 (m, 2 H), 7.39−7.32 (m, 1 H), 7.17−7.14 (m, 2 H), 7.07−7.04 (m, 2 H), 4.81−4.55 (m, 2 H), 4.41−4.29 (m, 2 H), 4.09 (s, 3 H), 4.03 (s, 3 H), 3.81−3.73 (m, 1 H), 3.13−3.04 (m, 1 H), 2.47−2.42 (m, 2 H), 1.64−1.59 (m, 9 H), 1.53−1.51 (m, 9 H); ¹³C NMR (100 MHz, CDCl₃) δ 174.9, 174.8, 168.8, 168.4, 167.6, 167.4, 163.7, 140.9, 140.7, 138.0, 138.0, 134.9, 132.7, 132.4, 130.8, 130.5, 130.4, 130.4, 130.0, 129.8, 128.9, 128.8, 127.4, 127.2, 126.7, 126.4, 125.4, 124.8, 123.9, 114.4, 114.3, 55.9, 55.8, 54.4, 54.1, 38.2, 37.7, 28.2, 28.1, 28.0.

2,2’-((2-(3-Amino-3-oxopropyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (21)

The final diacidic compound 21 was prepared from the dialkylated analog 20 (0.04 mmole) as described in the general procedure for removal of acid sensitive protecting group (Method E2) to get the title compound as an off-white solid; 21 mg, yield 75%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.18 (d, 1 H, J = 6.8 Hz), 7.90 (d, 1 H, J = 6.8 Hz), 7.62−7.59 (m, 4 H), 7.44 (t, 1 H, J = 6.0 Hz), 7.34 (t, 1 H, J = 6.0 Hz), 7.15 (s, 1 H), 7.10 (d, 2 H, J = 6.8 Hz), 7.03 (d, 2 H, J = 6.8 Hz), 6.63 (br, 2 H), 4.40 (s, 4 H), 3.89 (s, 3 H), 3.87 (s, 3 H), 2.99−2.95 (m, 2 H), 2.74−2.68 (m, 2 H), 2.23(br, 2 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 172.6, 169.0, 168.9, 162.7, 162.6, 139.9, 136.8, 134.2, 132.3, 131.1, 130.4, 129.4, 129.4, 129.3, 128.2, 125.7, 125.0, 124.4, 123.9, 113.9, 113.9, 55.3, 55.3, 53.0, 52.7, 35.2, 26.5. HRMS (ESI) calcd for C₃₁H₃₁N₃O₁₁S₂ [M + H]⁺ 686.1473; found 686.1471.

Di-tert-butyl 2,2’-((2-(3-hydroxypropyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (22)

To a stirred solution of 18 (100 mg, 0.18 mmol) in anhydrous THF (2 ml ) under nitrogen at 0 °C, lithium aluminium hydride (LAH) (20 mg, 0.53 mmol) was added at room temperature and the mixture refluxed for 8 hours. Upon completion, the reaction quenched with methanol and diluted with ethyl acetate, washed with bine, dried over anhydrous Na₂SO₄, and the organic layer removed and concentrated under vaccum. The crude product was then diluted with DMF and using general procedure (method B) to give the desired dialkylated product as oily product 71 mg, yield 51%; ¹H NMR (400 MHz, CDCl₃) δ 8.18−8.10 (m, 1 H), 7.70−7.66 (m, 2 H), 7.64−7.61 (m, 2 H), 7.54−7.37 (m, 2 H), 7.32−7.21 (m, 1 H), 7.19−7.14 (m, 1 H), 6.96−6.93 (m, 2 H), 6.90−6.86 (m, 2 H), 4.56−4.43 (m, 2 H), 4.31−4.17 (m, 2 H), 3.87−3.85 (m, 6 H), 3.62−3.56 (m, 2 H), 3.29−3.21 (m, 1 H), 2.59−2.52 (m, 1 H), 1.87−1.58 (m, 2 H), 1.43−1.34 (m, 18 H); ¹³C NMR (100 MHz, CDCl₃) δ 168.2, 168.0, 167.8, 167.7, 163.5, 163.4, 141.5, 141.4, 137.4, 137.3, 135.4, 135.4, 133.0, 132.8, 132.0, 131.8, 131.1, 131.0, 130.5, 130.5, 130.5, 130.4, 130.3, 129.6, 129.3, 127.1, 126.9, 126.5, 126.3, 124.7, 124.7, 124.6, 124.5, 114.1, 82.6, 82.4, 82.4, 62.2, 55.8, 54.6, 54.1, 33.3, 33.1, 31.7, 28.1, 28.0, 28.0, 22.7.

Di-tert-butyl 2,2’-((2-(3-(1,3-dioxoisoindolin-2-yl)propyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (23a)

The O-tosyl analog was prepared as described in the general procedure for synthesis of sulfonamides (method C1) to get the desired compound (di-tert-butyl 2,2’-((2-(3-(tosyloxy)propyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate) as brown solid; 67 mg, yield 91%; ¹H NMR (400 MHz, CDCl₃) δ 8.22−8.14 (m, 1 H), 7.78 (d, 2 H, J = 8.4 Hz), 7.68−7.52 (m, 4 H), 7.46−719 (m, 5 H), 7.07−6.84 (m, 5 H), 4.50−4.38 (m, 2 H), 4.28−4.01 (m, 4 H), 3.89−3.85 (m, 6 H), 3.56−2.47 (m, 4 H), 2.44 (s, 3 H), 1.40−1.34 (m, 18 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.7, 167.5, 163.5, 144.8, 140.7, 137.6, 135.2, 135.1, 133.2, 132.7, 132.2, 131.0, 130.4, 130.3, 130.3, 130.0, 129.7, 129.2, 128.8, 128.0, 127.8, 127.2, 127.0, 126.5, 126.4, 124.9, 124.7, 124.5, 114.2, 82.2, 70.4, 55.9, 55.7, 54.3, 54.0, 30.1, 28.0, 28.0, 27.6, 21.7.

To a stirred solution of the O-tosyl analog (30 mg, 0.03 mmol) in 1 ml DMF, phthalimide potassium salt (9 mg, 0.045mmol) and K₂CO₃ (12 mg, 0.06 mmol) were added. The reaction mixture was stirred at room tempreture for 3 hours. After the reaction was complete as shown by TLC and LC-MS, the mixture diluted with ethyl acetate and washed with brine, dried over anhydrous Na₂SO₄. The organic layer was separated and then concentrated under vacuum. The crude mixture was purified by ISCO using ethyl acetate/hexane as mobile phase gradient to get the desired product in 24 mg, yield 84%; ¹H NMR (400 MHz, CDCl₃) δ 8.22−8.16 (m, 1 H), 7.87−7.32 (m, 11 H), 7.08−7.05 (m, 1 H), 6.93−6.87 (m, 4 H), 4.51−4.16 (m, 4 H), 3.87−3.73 (m, 6 H), 3.72−3.62 (m, 2 H), 3.08−3.00 (m, 1 H), 2.85−2.81 (m, 1 H), 2.49−2.41 (m, 1 H), 1.74−1.66 (m, 1 H), 1.39−1.32 (m, 18 H); ¹³C NMR (100 MHz, CDCl₃) δ 171.2, 168.3, 168.3, 167.8, 167.7, 167.6, 167.6, 163.4, 140.6, 137.6, 135.4, 135.3, 134.5, 133.9, 133.7, 133.3, 132.4, 132.2, 132.0, 131.2, 130.4, 130.3, 130.0, 129.1, 128.9, 127.2, 127.0, 126.6, 126.3, 125.1, 124.8, 123.2, 123.1, 114.2, 114.0, 82.1, 55.7, 55.6, 54.7, 54.5, 54.3, 54.2, 38.1, 32.0, 22.8, 21.1.

Di-tert-butyl 2,2’-((2-(3-(benzyl(methyl)amino) propyl) naphthalene-1,4-diyl) bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetate (23b)

To a stirred solution of the O-tosyl analog (30 mg, 0.03 mmol) in 1 ml DMF, N-benzylmethylamine (5 uL, 0.045mmol) and K₂CO₃ (12 mg, 0.06 mmol) were added. After that, the reaction mixture stirred at room tempreture for 3 hours. Then, the mixture diluted with ethyl acetate and washed with saturated brine solution. The organic phase was then collected, dried over anhydrous Na₂SO₄, concentrated under reduced pressure. The crude mixture was purified by ISCO using ethyl acetate/hexane mobile phase to get the desired product in 18 mg, yield 69%; ¹H NMR (400 MHz, CDCl₃) δ 8.15 (d, 1 H, J = 8.8 Hz), 7.66 (d, 2 H, J = 8.8 Hz), 7.61 (d, 2 H, J = 8.8 Hz), 7.46−7.28 (m, 7 H), 724−7.17 (m, 2 H), 7.00−6.92 (m, 2 H), 6.88−6.85 (m, 2 H), 4.47−4.12 (m, 4 H), 3.85−3.81 (m, 6 H), 3.56−3.51 (m, 2 H), 2.85−2.79 (m, 2 H), 2.59−2.39 (m, 2 H), 2.27−2.07 (m, 2 H), 1.60 (s, 3 H), 1.42−1.35 (m, 18 H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 167.2, 167.1, 162.8, 162.8, 129.8, 129.0, 128.7, 128.0, 126.5, 125.9, 125.7, 124.1, 113.6, 81.8, 55.1, 54.1, 53.5, 27.5, 27.4.

2,2’-((2-(3-(1,3-Dioxoisoindolin-2-yl) propyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (24a)

Prepared using the method described in the general procedure for hydrolysis of t-butyl ester to get di-acidic compounds (Method E2) starting from 0.03 mmole of compound 23a to get the desired compound as a pink solid; 17 mg, yield 68%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.22 (d, 1 H, J = 6.8 Hz), 7.93 (d, 1 H, J = 6.8 Hz), 7.90−7.84 (m, 4 H), 7.58 (d, 2 H, J = 7.2 Hz), 7.56 (d, 2 H, J = 7.2 Hz), 7.46−7.43 (m, 1 H), 7.37−7.34 (m, 1 H), 7.08 (s, 1 H), 7.01 (d, 4 H, J = 6.8 Hz), 4.41 (s, 2 H), 4.33 (s, 2 H), 3.86 (s, 3 H), 3.76 (s, 3 H), 3.57 (t, 2 H, J = 6.0 Hz), 2.76 (t, 2 H, J = 6.0 Hz), 1.72 (t, 2 H, J = 6.0 Hz); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 168.8, 168.8, 167.1, 162.5, 162.47, 139.4, 136.7, 134.1, 133.6, 132.3, 131.2, 131.0, 130.2, 129.3, 129.2, 129.2, 127.9, 125.6, 125.0, 124.3, 123.9, 122.3, 113.8, 113.7, 55.2, 55.1, 55.0, 55.0, 52.8, 52.7, 37.0, 27.9; HRMS (ESI) calcd for C₃₉H₃₅N₃O₁₂S₂ [M + H]⁺ 802.1735; found 802.1733.

2,2’-((2-(3-(Benzyl(methyl)amino)propyl)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (24b)

Prepared using the method described in the general procedure for hydrolysis of t-butyl ester to get di-acidic compounds (Method E2) starting from 0.03 mmole of compound 23b to get the desired compound as a yellow solid; 16 mg, yield 68%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.03 (d, 1 H, J = 6.8 Hz), 7.61 (d, 2 H, J = 7.2 Hz), 7.56−7.52 (m, 5 H), 7.47−7.45 (m, 3 H), 7.38 (t, 1 H, J = 6.0 Hz), 7.32 (s, 1 H), 7.26 (t, 1 H, J = 6.4 Hz), 7.06 (d, 2 H, J = 7.2 Hz), 6.99 (d, 2 H, J = 7.2 Hz), 4.49−4.46 (m, 1 H), 4.28−4.26 (m, 3 H), 3.84 (s, 3 H), 3.82 (s, 3 H), 3.09−3.07 (m, 2 H), 3.01−2.94 (m, 1 H), 2.69−2.68 (m, 1 H), 2.67 (s, 3 H), 1.94−1.90 (m, 2 H); ¹³C NMR (125 MHz 100 °C, DMSO-d₆) δ 168.8, 168.7, 162.5, 162.3, 139.1, 136.6, 134.0, 131.6, 130.7, 130.2, 130.0, 129.5, 129.3, 129.1, 128.7, 128.2, 128.0, 125.6, 124.9, 123.6, 113.7, 113.6, 58.2, 55.1, 55.0, 54.4, 52.8, 52.3, 27.6, 23.3; HRMS (ESI) calcd for C₃₉H₄₁N₃O₁₀S₂ [M + H]⁺ 776.2306; found 776.2308.

N,N’-((1Z,4Z)-naphthalene-1,4-diylidene)bis(4-methoxybenzenesulfonamide) (26)

Ceric ammonium nitrate (219 mg, 0.4 mmol) was added to a solution of 1,4-bis(4-methoxyphenylsulfonamido)naphthalene intermediate 25 (99 mg, 0.2 mmol) in acetonitrile (20 mL). After that, the reaction mixture was stirred for overnight at room temperature and then checked by TLC and LC-MS. Upon completion, the reaction mixture was filtered and the yellow solid product was collected. The obtained solid was washed with diethyl ether and left to dry to get the pure product yellow solid (99 mg, quantitative); ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 2 H), 8.20−8.18 (m, 2 H), 8.00 (d, 4 H, J = 8.8 Hz), 7.61−7.58 (m, 2 H), 7.05 (d, 2 H, J = 8.8 Hz), 3.90 (s, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ) 163.8, 161.4, 133.3, 132.9, 132.4, 130.6, 129.9, 127.0, 114.5, 55.9.

N,N’-(2-(Phenylthio)naphthalene-1,4-diyl)bis(4-methoxybenzenesulfonamide) (27)

To a suspension of compound 26 (40 mg, 0.08 mmol) in toluene (1 mL), thiophenol (11 uL, 0.1 mmol) and DIPEA (29 uL, 0.1 mmol) were added. The reaction mixture was stirred at room temperature for 8 hours and checked by TLC and LC-MS. After the reaction was finished, the reaction mixture was filtered and the crude product was purified by ISCO using ethyl acetate/hexane solvent system to yield the desired compound as a pink solid in 33 mg, yield 68%; ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (s, 1 H), 9.95 (s, 1 H), 8.00 (d, 1 H, J = 8.0 Hz), 7.70 (d, 1 H, J = 8.4 Hz), 7.57 (d, 2 H, J = 8.8 Hz), 7.41−7.3 (m, 7 H), 7.12−7.10 (m, 2 H), 6.98 (d, 2 H, J = 8.8 Hz), 6.91 (d, 2 H, J = 8.8 Hz), 6.77 (s, 1 H), 3.79 (s, 3 H), 3.77 (s, 3 H); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.5, 162.3, 135.6, 133.5, 133.0, 132.8, 132.3, 131.4, 129.5, 129.0, 128.9, 128.6, 128.1, 128.0, 127.8, 126.8, 125.9, 124.2, 123.2, 122.1, 114.3, 114.2, 55.6.

Diethyl 2,2’-((2-(phenylthio)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl)) diacetate (28)

Prepared using the method described in the general procedure for synthesis of alkylated sulfonamides (method D) starting from 0.05 mmole of compound 27 to get the desired compound as oily product; 27 mg, yield 69%; ¹H NMR (400 MHz, CDCl₃) δ 8.47−8.38 (m, 1 H), 8.08−7.95 (m, 1 H), 7.72−7.68 (m, 2 H), 7.55−7.45 (m, 4 H), 7.31−7.27 (m, 3 H), 7.14−7.13 (m, 2 H), 7.12 (s, 1 H), 6.92−6.89 (m, 2 H), 6.83 (d, 1 H, J = 8.8 Hz), 6.67 (d, 1 H, J = 8.8 Hz), 4.89−4.83 (m, 1 H), 4.58−4.35 (m, 1 H), 4.22−3.99 (m, 6 H), 3.88−3.81 (m, 6 H), 1.25−1.09 (m, 6 H); ¹³C NMR (100 MHz, CDCl₃) δ 169.1, 168.5, 163.6, 163.3, 163.2, 138.3, 137.9, 137.6, 135.2, 134.9, 134.0, 132.2, 131.3, 131.1, 130.9, 130.8, 130.7, 130.5, 130.3, 130.1, 129.6, 129.5, 128.0, 127.8, 127.7, 127.7, 127.6, 127.5, 126.8, 126.4, 123.9, 114.2, 114.2, 114.1, 61.7, 61.6, 61.6, 61.5, 55.8, 55.7, 53.3, 53.2, 52.9, 14.2, 14.1.

2,2’-((2-(Phenylthio)naphthalene-1,4-diyl)bis(((4-methoxyphenyl)sulfonyl)azanediyl))diacetic acid (29)

Prepared using the method described in the general procedure for removal of base-sensitive protecting group (Method E1) starting from 0.039 mmole of compound 28 to get the desired compound as a yellow solid; 19 mg, yield 68%; ¹H NMR (500 MHz, 100 °C, DMSO-d₆) δ 8.49 (d, 1 H, J = 6.4 Hz), 8.09 (d, 1 H, J = 6.4 Hz), 7.66 (d, 2 H, J = 6.8 Hz), 7.58−7.46 (m, 5 H), 7.35−7.34 (m, 3 H), 7.16−7.15 (m, 2 H), 7.06 (d, 2 H, J = 7.2 Hz), 6.93−6.92 (m, 2 H), 4.71−4.17 (m, 4 H), 3.87 (s, 6 H); ¹³C NMR (125 MHz, 100 °C, DMSO-d₆) δ 169.0, 168.7, 163.0, 162.6, 137.5, 134.0, 131.2, 130.8, 129.8, 129.2, 129.1, 127.5, 126.2, 126.2, 126.0, 123.5, 114.0, 55.4, 55.4, 52.6, 52.3; HRMS (ESI) calcd for C₃₄H₃₀N₂O₁₀S₃ [M + H]⁺ 723.1135; found 723.1133.

3. Fluorescence Polarization Competition Assay

A Wallac Victor 3V plate reader (PerkinElmer, Shelton, CT) was used to perform fluorescence polarization assay using excitation at 484 nm and emission at 535 nm as we reported previously [44]. FP measurement was done using Corning 384-well plate (product #3575) and 40 μL of assay solution was added in each well. The assay buffer used consists of 10 mM HEPES buffer, 150 mM NaCl, 0.005% Tween-20, and 50 mM EDTA, with pH adjusted to 7.4. The fluorescent probe, used in this assay, is composed of fluorescein linked to 9-mer Nrf2 ETGE motif derived peptide, FITC-LDEETGEFL-NH₂. The final total volume is 40 μL in each well. As a result, the solution in each well consisted of Keap1 Kelch domain protein (400 nM, 10 μL), 20 μL FITC-9mer Nrf2 peptide amide (20 nM), and 10 μL of serial dilutions of the inhibitor compound in triplicate. Then, the assay plate was centrifuged at 2000 rpm for 2 min, covered, and incubated with shaking at 700 rpm for 30 min at room temperature. After the incubation, the plate was centrifuged again at 2000 rpm for 2 min prior to FP signal measurements. The FP signal was measured the parallel (F_║) and perpendicular fluorescence (F_┴) intensity with respect to the linearly polarized excitation light. The IC₅₀ values for each inhibitors was derived using Sigma Plot 12.3 from the sigmoidal plot of percent inhibition against inhibitor concentration.

4. Time-Resolved Fluorescence Resonance Energy Transfer Assay

TR-FRET measurement was accomplished using Corning 384-well white plate (product #3574) on a Wallac Victor 3V multi-label plate reader (PerkinElmer, Shelton, CT). Each well being loaded with 20 μL of assay solution as we reported previously [43]. The assay buffer used was made using 10 mM HEPES buffer, 150 mM NaCl, 0.005% Tween-20, and 0.5 mM EDTA with pH adjusted to 7.4. Terbium labeled-anti-His antibody (purchased from Thermo Fisher Scientific) was used as obtained from the provider after dividing into small aliquots. FITC-LDEETGEFL-NH₂ was used as the fluorescent probe used in this assay. The inhibitor compound (volume equal to 0.2 μL) was delivered to each well serial dilutions in DMSO ranging from 1 nM to 100 μM. Keap1 Kelch domain protein and terbium labeled anti-His antibody with were mixed in a 1:1 ratio and preincubated with shaking at 300 rpm for 30 min at room temperature. After that, 10 μL of the protein/antibody mixture solution was added to each well which already had the inhibitor solution. The solution mixture (inhibitor/protein/antibody) was further incubated with shaking at 300 rpm for 30 min at room temperature. After that, 9.8 μL of fluorescein labeled-9mer Nrf2 peptide amide was added to each well following by incubation at 300 rpm for 60 minutes at room temperature. The final volume in each well consists of 20 μL consisted of Keap1 (5 nM), Terbium labeled anti-HIS antibody (0.5 nM), fluorescein-labeled-9mer Nrf2 peptide amide (25 nM), and triplicates of serial dilutions from inhibitor compound. All plates were centrifuged at 2000 rpm at room temperature for 2 minutes before and after incubation cycle. The excitation was done at 340 nm, and then the TR-FRET was determined on a Victor 3V microplate reader. The fluorescence ratio was calculated employing the following equation; (fluorescence intensity of 520 nm)/(fluorescence intensity of 495 nm) × 10,000. Sigma Plot 12.3 was used to derive the IC₅₀ value from the sigmoidal plot of the fluorescence ratio value vs the inhibitor concentration. The K_i value was calculated using two equations, as seen in the paper previously published by our group [43].

5. Molecular docking simulation

Molecular docking study was performed using the Autodock 4.0 software. The X-ray crystal structure of the small molecule inhibitor 2d with Keap1 Kelch domain, obtained from the RCSB protein data bank (4XMB), was utilized. As for protein preparation, missing hydrogen atoms were added, all water molecules were eliminated from the protein structure, and Kollman charges were assigned using Autodock program. The ligand structures were built in MOE and prepared for docking job using Autodock. The docking grid was placed in a central position on the binding pocket of the ligand. The number of grid points was set to X, Y, Z = 40, 40, 40 in each direction with spacing of 0.375 Å resolution. The prepared ligands structures were docked into the Keap1 protein active binding sites. Lamarckian genetic algorithm was selected as a docking parameter with the number of GA runs was set to 100. All other additional parameters were kept to default values. The binding affinity were reported in kcal/mol, and the docked conformations were ranked based on the corresponding binding energy. For the validation of the docking protocol, the prepared ligand was re-docked using the same methodology, and compared with the corresponding co-ordinates obtained from the crystal structure. The top-scoring docked pose was found to be similar with the crystal structure of the ligand and having a low RMSD value less than 2 Å. This should support the evidence that the docking method was reliable for running docking simulation of other ligands and get an estimate for their binding affinity to the Keap1 domain.

6. Cell culture, RNA quantification, cDNA synthesis, and RT-qPCR

Cell culture using NCM460D cells, drug treatment, RNA extraction, cDNA synthesis, and RT-qPCR were performed similarly as we reported previously [36].

Highlights:

• C2 Substituted 1,4-bis(arylsulfonamido)naphthalene were synthesized

• The Keap1-binding activities were screened with FP and TR-FRET assays

• Compound 24a with C2-phthalimidopropyl group was the most potent analog

• Compound 24a provided an extra hydrogen bonding interaction with Arg415

• Compounds 12b, 15, and 24a activate the Nrf2 signaling pathway in NCM460D cells

ABBREVIATIONS

Aβ

amyloid-β

ARE

antioxidant response element

Cul3

cullin3

DCM

dichloromethane

DMF

N,Ndimethylformamide

DMSO

dimethyl sulfoxide

FDA

food and drug administration

FITC

fluorescein isothiocyanate

FP

fluorescence polarization

GST

glutathione S-transferase

HEPES

4-(2-hydroxyethyl)1-piperazineethanesulfonic acid

HO-1

heme-oxygenase-1

HRMS

high resolution mass spectrometry

iNOS

inducible nitric oxide synthase

IFN

interferon

IL

interleukin

Keap1

Kelch-like ECH-associated protein 1

LC-MS

liquid chromatography mass spectrometry

Maf

musculoaponeurotic fibrosarcoma

MS

multiple sclerosis

Neh

Nrf2-ECH homology

NQO1

NAD(P)H:quinone oxidoreductase 1

Nrf2

nuclear factor erythroid 2-related factor 2

PPI

protein−protein interaction

qRT-PCR

quantitative real-time polymerase chain reaction

RNS

reactive nitrogen species

ROS

reactive oxygen species

SAR

structure–activity relationship

TEA

triethylamine

TFA

trifluoroactic acid

TFAA

trifluoroacetic anhydride

THF

tetrahydrofuran

TNF

tumor necrosis factor

TR-FRET

time-resolved fluorescence resonance energy transfer