Structure-activity relationships of 1,4-bis(arylsulfonamido)-benzene or naphthalene-N,N'-diacetic acids with varying C2-substituents as inhibitors of Keap1-Nrf2 protein-protein interaction

Sumi Lee; Dhulfiqar Ali Abed; Mai-Uyen Nguyen; Michael P Verzi; Longqin Hu

doi:10.1016/j.ejmech.2022.114380

. Author manuscript; available in PMC: 2023 Jul 5.

Published in final edited form as: Eur J Med Chem. 2022 Apr 13;237:114380. doi: 10.1016/j.ejmech.2022.114380

Structure-activity relationships of 1,4-bis(arylsulfonamido)-benzene or naphthalene-N,N'-diacetic acids with varying C2-substituents as inhibitors of Keap1-Nrf2 protein-protein interaction

Sumi Lee ^†, Dhulfiqar Ali Abed ^†,^┴, Mai-Uyen Nguyen ^‡, Michael P Verzi ^‡,^§, Longqin Hu ^†,^§,^*

PMCID: PMC10265587 NIHMSID: NIHMS1903961 PMID: 35462166

Abstract

The Keap1-Nrf2-ARE pathway plays an important role in responding to oxidative stress and maintaining the redox homeostasis. Small molecule inhibitors targeting directly the Keap1-Nrf2 protein-protein interaction (PPI) can potentially be developed into effective preventive and therapeutic agents for numerous chronic inflammatory diseases. To improve the drug-like properties and inhibitory potency of these inhibitors, a series of 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acids with varying substituents at C-2 position of the benzene or naphthalene core were designed and synthesized. Among them, compound 12d with 2-(4-fluorobenzyloxy) group was the most potent direct inhibitor of Keap1-Nrf2 PPI 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. Moreover, cell-based biological assay showed that 12d significantly increased the mRNA levels of Nrf2 downstream genes, GSTM3, HMOX2 and NQO1, through Nrf2 activation. The discovery of the new scaffolds possessing diverse O-linked fragments at the C2 position offers opportunities to further modify the chemical structures of Keap1-Nrf2 PPI inhibitors to improve their pharmacokinetic, efficacy and safety profiles.

Keywords: Keap1, Nrf2, Keap1-Nrf2 interaction, Protein-protein interaction inhibitor, Oxidative stress, Structure-activity relationship, Structural diversity

Graphical Abstract

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INTRODUCTION

A variety of endogenous (e.g., cellular metabolism) and exogenous (e.g., radiation, chemicals, and toxins) factors are known to cause excessive production of reactive electrophilic substances, including reactive oxygen species (ROS, e.g., superoxide anion and hydroxyl radical) and reactive nitrogen species (RNS, e.g., peroxynitrite).^{[1, 2]} This results in impaired redox homeostasis between the ROS/RNS exposures and antioxidants, eventually generating oxidative stress conditions.^[2] The antioxidant defense system is an essential protective response against oxidative stress by maintaining physiological homeostasis.^[1],[3] The induction of metabolizing and antioxidative enzymes, such as glutathione S-transferase (GST), catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), NAD(P)H quinone oxidoreductase I (NQO1), heme-oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and thioredoxin (TRX), can counteract and neutralize overall ROS levels.^[4-9]

Activation of the cytoprotective enzymes could be controlled by three cellular components: Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element (ARE), providing a pathway that senses and responds to the status of oxidative stress in cells.^{[10, 11]} Under basal physiological conditions, Keap1 acts as an adaptor protein that takes part in the assembly of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex through Keap1’s BTB domain and Nrf2 through Keap1’s DGR domain. In the Keap1-Nrf2-Cul3 complex, Nrf2 becomes closer to the E3 complex, which serves as the sensor for the Nrf2 ubiquitination machine.^[12-14] Subsequently, Nrf2 is rapidly degraded by 26S proteasome, resulting in a low basal level of Nrf2. In a state of oxidative stress, oxidative and electrophilic species covalently modify cysteine residues in the BTB and IVR domains of Keap1,^{[15, 16]} potentially leading to dissociation of Cul3 (in the Cul3 dissociation model),^[17-19] disruption of Keap1-Nrf2 DLG motif interaction (in the hinge and latch model),^{[20, 21]} and/or conformational changes of Keap1 (in the conformation cycling model).^[22] These mechanisms ultimately perturb the ubiquitination of Nrf2, allowing newly formed Nrf2 to accumulate and translocate into the nucleus.^{[23, 24]} Nrf2 forms heterodimers with small Maf proteins,^{[4, 25]} and binds to ARE to activate the transcription of downstream antioxidant and detoxification genes.^[26]

Chronic oxidative stress accelerates oxidative damages to lipids, proteins and DNA in the human body, which may contribute to the pathogenesis of many chronic inflammatory diseases.^[27] Thus, Nrf2 activation through the inhibition of the Keap1-Nrf2 protein-protein interaction (PPI) has been proposed as a promising preventive and therapeutic approach for a number of diseases and conditions, including cancer, chronic obstructive pulmonary disorder (COPD), multiple sclerosis (MS), and neurodegenerative diseases.^{[16, 28-33]} The majority of Nrf2 activators are electrophilic modulators that covalently alter Keap1 cysteine residues, which eventually lead to the interruption of Keap1-Nrf2 PPI. Among the well-known Nrf2 activators are dimethyl fumarate, bardoxolone methyl (CDDO-Me), and sulforaphane (Figure 1A).^{[16, 34-36]} These covalent cysteine modifiers may not be selective and specific for Keap1 protein in whole cells, which would cause unwanted toxicity. For instance, bardoxolone methyl was under phase III clinical trial in patients with chronic kidney disease (CKD) and type 2 diabetes mellitus, but its development was halted due to adverse cardiovascular effects.^{[34, 37]} The severe safety issues may be associated with possible off-target activities, through reactions with cysteine residues in other proteins and enzymes.

Figure 1. — Structures of representative (A) indirect and (B) direct inhibitors of Keap1-Nrf2 PPI

The direct and noncovalent inhibitors of Keap1-Nrf2 PPI would have certain advantages over electrophilic compounds in terms of target selectivity. A Keap1 homodimer forms an interaction with Nrf2 through two binding sites, ETGE and DLG motifs, located in the Neh2 domain of Nrf2.^{[20, 38]} The binding of non-electrophilic small molecules to the Keap1 Kelch domain is more specific in disrupting the interactions between Keap1 and Nrf2 proteins. Recently, several small molecule inhibitors have been reported through strategies like high-throughput screening (HTS) and fragment-based drug discovery (FBDD).^{[9, 39]} As presented in Figure 1B, these direct inhibitors include tetrahydroisoquinoline (THIQ),^[40] 1,4-diaminonaphthalene,^[41-43] 3-phenylpropionic acid,^[44] 4-amino-1-naphthol,^{[45, 46]} and 1,2-xylylenediamine derivatives.^[47] Among them, the symmetric 1,4-diaminonaththalene scaffold like 2a exhibits the best potency. Although some of these non-electrophilic activators possess strong target binding affinity, their cellular activity and drug-like properties need further improvement as potential preventive and therapeutic agents.

Herein, we report the design, synthesis and evaluation of the 2-substituted derivatives on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acids 6 and 2a as inhibitors of Keap1-Nrf2 PPI.^{[41, 42]} The 2-alkoxy or aryloxy substitution occupies another binding pocket and could be optimized in terms of potency, physicochemical and PK properties. Our investigation revealed that the naphthalene scaffold 12d with 2-(4-fluorobenzyloxy) substitution led to the most potent Keap1-Nrf2 PPI inhibition in both our FP and TR-FRET assays (IC₅₀ = 64.5 nM and 14.2 nM, respectively). In addition, 12d was found to effectively stimulate transcription of Nrf2-derived genes (GSTM3, HMOX2 and NQO1), suggesting that it acts as a Nrf2 activator via the inhibition of Keap1-Nrf2 PPI. The new 2-alkoxy or aryloxy derivatives provided more structural diversity to the 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid series and serve as good leads for further optimization.

RESULTS AND DISCUSSION

Design strategy.

Computational analysis performed by You and coworkers^[48] divided the Nrf2-binding cavity of Keap1 Kelch domain into five sub-pockets: polar subpockets (P1 and P2), nonpolar subpockets (P4 and P5), and central subpocket P3.^{[48, 49]} An inhibitor that can occupy all five of these subpockets would more likely lead to potent inhibition of Keap1-Nrf2 PPI, which is demonstrated by the cocrystal structure of Keap1 Kelch domain with compound 2b as shown in Figure 2C.^[41] However, compound 6 bearing a benzene core exhibited dramatically reduced potency with an IC₅₀ value of 1.4 μM, that is about 50 fold less potent as compared to analog 2a with a naphthalene core. As reported previously, the replacement of naphthalene core with a benzene core as in 6 leads to weaker hydrophobic interactions as well as a weaker π-cation interaction with Arg415 in the P3 subpocket.^{[41, 42]} To improve the structural diversity of the core moiety without affecting potency, we were interested in replacing the outer benzene ring of the naphthalene core with varying O-linked aromatic ring fragments (Figure 2A). Importantly, the binding mode of compound 4a indicates that its p-cumenesulfonamide is inserted deep into the pore behind the P3 pocket, supporting that there is enough chemical space to introduce an additional fragment at the back of P3 pocket (Figure 2D).^{[45, 50, 51]} Most recently, Zhong and coworkers suggested that hot spot A, located in the 6 Å diameter tunnel at the center of the binding pocket, is one of the strong binding hot spots, using FTMap.^[52] Accordingly, we hypothesized C2-substituents of 1,4-bis(arylsulfonamido)benzene-N,N'-diacetic acid could play significant roles in maintaining high potency by occupying the deep hole behind the binding site. In an effort to explore the space at the back of the central P3 subpocket where the C2-fragment could reach, five compounds 7a, 7d, 7e, 7k and 7l with different size of substituents ranging from benzene to O-linked biphenyl ring were designed and docked within the binding site of the Keap1 Kelch domain (PDB code: 4XMB), as outlined in Figure 2E. As expected, it revealed that the deep back side of the P3 subpocket is wide enough to accommodate bulky moieties including small benzene rings (7a, 7d and 7e), a naphthalene (7k) and a biphenyl ring (7l). Therefore, we focused our optimization around the C2-substituents on 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acids by exploring and expanding the chemical space behind the P3 subpocket.

Figure 2. — Design strategy of 2-substituted 1,4-bis(arylsulfonamido)benzene or naphthalene-*N,N'*-diacetic acids. The Keap1 Kelch domain contains five subpockets (P1 to P5), and P3 is the central subpocket where the core moiety of ligands (represented in a blue circle) occupies. (A) Optimization process of 1,4-diaminonaphthalene derivative 2a by the introduction of a new fragment to 6. (B) Co-crystal structure of the Kelch domain in complex with compound 6 (derived from PDB code: 4IQK). (C) Co-crystal structure of the Kelch domain in complex with compound 2b (PDB code: 4XMB). (D) Docking pose of compound 4a in the active site of Keap1 Kelch domain reported by Zhuang’s group (derived from PDB code: 4IQK). (E) Docking pose of target compounds 7a, 7d, 7e, 7k, 7l with different sizes of new fragments.

Chemistry.

As shown in Scheme 1, key intermediates 16a-y bearing two benzenesulfonamide moieties were prepared by three different routes. The biphenyl analog 16a was obtained through selective bromination of 4-nitroaniline,^[53] iron-catalyzed nitro reduction, Suzuki coupling with phenylboronic acid, and subsequent sulfonamide formation with 4-methoxy benzenesulfonyl chloride. To introduce various fragments on the core moiety (15b and 15d-o), 2-fluoro-4-nitroaniline was initially treated with piperidine, aryl or aliphatic alcohols under three reaction conditions using different bases, such as TEA, K₂CO₃ and NaH. After iron or palladium-catalyzed nitro reduction of 15b and 15d-o, 1,4-diamine intermediates were subsequently reacted with the corresponding benzenesulfonyl chloride reagents to yield the sulfonamide derivatives 16b and 16d-y. Lastly, the synthesis of N-linked analog 16c was accomplished in four steps from commercially available 4-bromo-2-fluoro-1-nitrobenzene. Treatment with N-benzylmethylamine followed by the conversion of the bromo to amino group through a copper-catalyzed amination reaction gave 15c,^[54] which was reduced by iron powder and subsequently N-sulfonylated to afford 16c. The resulting intermediates 16a-y were alkylated with ethyl bromoacetate, and then hydrolyzed under basic conditions to obtain the target compounds 7a-y.

Compound 8 with 2-phenoxymethyl substitution was prepared as shown in Scheme 2. After reducing 2-bromo-5-nitrobenzoic acid, mesylation of the generated primary alcohol and subsequent substitution with phenol produced 18. Buchwald-Hartwig amination of its bromo group with benzophenone imine gave an imine intermediate, allowing for facile reduction of the imine group under acidic conditions using sodium acetate and hydroxylamine to afford the aniline 19.^[55] Nitro reduction and reaction with 4-methoxybenzenesulfonyl chloride gave the sulfonamide derivative 20, which upon alkylation of the sulfonamides with ethyl bromoacetate and base-catalyzed hydrolysis of the ethyl esters provided the final compound 8.

As outlined in Scheme 3, the analogs 9a-b with 2-(3-pyridinyl)methoxy or 2-(4-phenoxybenzyloxy) substitution were prepared in two steps from intermediate 25. First, 4-phenoxybenzyl chloride 24, used as a building block for nucleophilic substitution, was prepared starting from 4-fluorobenzaldehyde in a three-step sequence that includes aromatic nucleophilic substitution with phenol, reduction of the aldehyde 22, and chlorination of 23. The key intermediate 25, which was synthesized by cleavage of the benzyl protecting group in 17d, was alkylated with either 3-(chloromethyl)pyridine hydrochloride or 24 to afford 26a-b. Removal of their ethyl esters by base hydrolysis gave the desired target products 9a-b.

The tetrazole 10 and (S)-methylated 11 were prepared from intermediate 16v by following the synthetic route shown in Scheme 4. Alkylation with bromoacetonitrile and cyclization of the nitrile 27 with NaN₃ yielded 10, while another compound 11 was synthesized stereospecifically through alkylation with methyl (R)-2-bromopropanoate followed by hydrolysis of the esters under basic conditions.

The synthesis of additional derivatives with benzene core (12a-c) was achieved using the synthetic route shown in Scheme 5. For the preparation of the 4-nitroaniline 31a-c, 5 or 6-methoxybenzene analogs 29a-b were first synthesized from either the methylation of 2-bromo-4-fluoro-5-nitrophenol or selective nucleophilic aromatic substitution of 5-bromo-1,3-difluoro-2-nitrobenzene in methanol. Addition of 4-fluorobenzyl alcohol fragment yielded 30a-c, which were converted to the corresponding aniline analogs 31a-c by Buchwald-Hartwig amination and subsequent imine hydrolysis.^[55] After nitro reduction, diamine intermediates were reacted with 4-toluenesulfonyl chloride to afford 32a-c. Alkylation with ethyl bromoacetate and base-promoted hydrolysis of the esters 33a-c finally yielded the target compounds 12a-c.

Compound 12d with a naphthalene core was prepared as seen in Scheme 6. First, 4-nitro-1-naphthylamine analog 35 was generated by selective bromination of 4-nitronaphthalen-1-amine with NBS^[56] and copper-catalyzed O-benzylation of 34.^[57] Nitro reduction of the resulting intermediates 35 followed by sulfonamide formation gave 36, which was further reacted with ethyl bromoacetate and hydrolyzed under basic conditions to obtain the final products 12d.

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

To evaluate the inhibitory activity of small molecules synthesized above against Keap1-Nrf2 PPI, an FP assay was carried out using the procedure reported previously by our group.^[58] Initially, the percent inhibition of a test compound was measured at three concentrations of 50, 5 and 0.5 μM. If a compound showed more than 50% inhibition at 5 μM, its IC₅₀ value was determined by performing the full dose-response curve for SAR analysis. We first focused our studies on exploring R₁ fragment on the benzene core, as shown in Table 1. The phenyl 7a showed moderate activity with an IC₅₀ value of 3.47 μM, while the piperidine 7b reached only about 50% inhibition at 5 μM, indicating a preference for the aromatic phenyl over the saturated heterocyclic ring. To increase flexibility of the C2-substituent, benzylic analogs were prepared with increasing linker length. Interestingly, the N-benzylmethylamine 7c did not afford a gain in potency, whereas the C2-benzyloxy analog 7d exhibited improved inhibitory activity by 4-fold (IC₅₀ = 0.79 μM) as compared to 7a. However, the reversed O-benzylic linker (8) resulted in 3-fold decrease in activity relative to 7d. We then prepared analogs of 7d with varying oxygen-containing linkers, and its length was changed from 0- to 3-carbon atoms. 7d-g exhibited comparable potency with IC₅₀ values ranging from 0.79 to 1.37 μM suggesting the existence of sufficient space at the back of P3 subpocket. On the other hand, varying the substitutions (7h-j) at 4-position of the benzyl fragment and its replacement with pyridine (9a) were well tolerated with similar inhibitory activity (IC₅₀ of 0.62-0.91 μM) as compared to 7d.

Table 1.

Inhibitory activity of 1,4-diamino substituted phenyl ring analogs 7a-o, 8, and 9a-b^a


compd	R₁	% inhibition			IC₅₀ (μM)^b
compd	R₁	50 μM	5 μM	0.5 μM	IC₅₀ (μM)^b
2a	-	ND	ND	ND	0.63±0.08
7a		98	75	30	3.47±1.24
7b		89	51	ND	ND
7c		101	74	14	6.17±2.70
7d		96	97	56	0.79±0.08
8		97	96	35	2.62±0.55
7e		95	93	31	1.37±0.19
7f		109	90	41	1.02±0.09
7g		103	99	48	0.95±0.16
7h		91	93	55	0.77±0.12
7i		105	88	57	0.91±0.10
7j		98	92	40	0.62±0.09
9a		98	96	48	0.76±0.15
7k		99	101	63	0.48±0.06
7l		98	92	51	0.39±0.06
7m		102	92	39	0.89±0.12
7n		98	79	41	0.79±0.12
7o		94	76	40	1.19±0.15
9b		99	98	40	0.83±0.20

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Inhibitory potency was determined by FP assay, and all experiments were performed in triplicates. ND: not determined.

IC₅₀ values are reported as an average of three replicates ± standard error of the mean (SEM).

We also replaced the benzyl group with two ring systems such as biphenyl or naphthalene rings, as outlined in Table 1. Compared to 7d with the same linker length, the naphthalene 7k and the biphenyl 7l displayed a slight improvement in inhibitory potency with similar IC₅₀ values of 0.48 and 0.39 μM, respectively. In a series of naphthalene derivatives, both 2-substituted analog 7m with a shorter linker and 1-substituted analog 7n did not provide notable changes in activity (IC₅₀ = 0.89 and 0.79 μM, respectively). However, for the biphenyl series, the shorter linker (7o) and the introduction of an oxygen between two phenyl rings (9b) led to a 3- and 2-fold reduction in activity (IC₅₀ = 1.19 and 0.83 μM, respectively) relative to 7l (IC₅₀ = 0.39 μM). Consequently, in the case of 1,4-bis(arylsulfonamido)benzene-N,N'-diacetic acids, C2-substituents generally led to improved potencies in the submicromolar range, as seen in Table 1.

Next, we turned our attention toward R₂ modification on the benzenesulfonyl moieties of 7d, as shown in Table 2. Electron-donating groups, such as 4-methyl and 4-phenyl substituents (7p and 7q), exhibited comparable or better inhibitory activities (IC₅₀ = 0.45 and 0.75 μM, respectively), along with 4-methoxy group (7d) (IC₅₀ = 0.79 μM). In contrast, electron-withdrawing groups, including 4-fluoro and 4-trifluoromethyl substituents (7r and 7s), were 3- and 2-fold less potent than 7d, except for 4-bromo substituent (7t, IC₅₀ = 0.59 μM). Additionally, we observed that the para position (7p) was preferred for PPI inhibition than the meta position (7u). Among the O-linked analogs involving a benzyl fragment (7d and 7p-u), 7p with 4-methyl substituents was the most active, which could be translated to O-linked benzene ring derivatives containing 4-fluorobenzyl (7i), naphthalene (7k), and biphenyl (7l) fragments that showed good affinities for Keap1 in Table 1. In the case of the 4-fluorobenzyl derivatives, the replacement of the methoxy groups (7i) with methyl substituents (7v) resulted in a 2-fold improvement in activity (IC₅₀ = 0.91 vs 0.45 μM). However, unlike the 4-methyl group (7v), an unfavorable impact on potency was observed with the 2,4,6-trimethylphenyl group (7w, IC₅₀ = 3.73 μM).^[46] Potencies of compounds containing the naphthalene (7k and 7x) and biphenyl (7l and 7y) moieties remained unaffected by the change, with less than 2-fold difference in IC₅₀ ranging from 0.38 to 0.44 μM.

Table 2.

Inhibitory activity of O-linked benzene ring derivatives 7p-y with various substituents^a


compd	R₁	R₂	% inhibition			IC₅₀ (μM)^b
compd	R₁	R₂	50 μM	5 μM	0.5 μM	IC₅₀ (μM)^b
2a	-	-	ND	ND	ND	0.63±0.08
7d		4-OMe	96	97	56	0.79±0.08
7p		4-Me	97	102	58	0.45±0.05
7q		4-phenyl	99	97	47	0.75±0.07
7r		4-F	103	87	41	2.41±0.46
7s		4-CF₃	90	89	32	1.76±0.30
7t		4-Br	87	91	54	0.59±0.10
7u		3-Me	93	89	43	1.00±0.16
7i		4-OMe	105	88	57	0.91±0.10
7v		4-Me	106	109	76	0.45±0.04
7w		2,4,6-Me	104	74	29	3.73±0.96
7k		4-OMe	99	101	63	0.48±0.06
7x		4-Me	109	107	68	0.38±0.06
7l		4-OMe	98	92	51	0.39±0.06
7y		4-Me	104	102	70	0.44±0.04

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Inhibitory potency was determined by FP assay, and all experiments were performed in triplicates.

IC₅₀ values are reported as an average of three replicates ± standard error of the mean (SEM).

Based on the known metabolic stability of fluorine substitution and the chemical structure of 7v, one of the most potent inhibitors, we sought to explore the core moiety and the acetate group (X). As shown in Table 3, bioisosteric replacement of acetates with tetrazoles (10)^[59] and the addition of (S)-methyl groups to α-carbon of acetates (11)^[47] resulted in a significant reduction in inhibitory potency by 8- and 3-fold, respectively. Interestingly, the presence of an extra methoxy (12a-b) or 4-fluorobenzyloxy (12c) group at the 5- or 6-position of the 1,4-diamino substituted phenyl ring bearing 2-(4-fluorobenzyloxy) group led to a dramatic drop-off in potency, whereas the O-linked naphthalene analog 12d exhibited a remarkable improvement on the inhibitory activity against Keap1-Nrf2 PPI with an IC₅₀ value of 64.5 nM. Overall, our SAR studies demonstrated that 12d was the most active direct inhibitor among the 1,4-diamino substituted aromatic ring analogs with O-linked fragments.

Table 3.

Activity of 5 or 6-substituted benzene ring (12a-c) and naphthalene (12d) analogs^a


compd	Core	X	% inhibition			IC₅₀ (μM)^b
compd	Core	X	50 μM	5 μM	0.5 μM	IC₅₀ (μM)^b
2a			ND	ND	ND	0.63±0.08
7v			106	109	76	0.45±0.04
10			95	60	16	3.92±0.58
11			98	78	ND	1.27±0.22
12a			74	41	26	ND
12b			75	45	7	ND
12c			58	19	10	ND
12d			101	103	ND	0.065±0.004

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Inhibitory potency was determined by FP assay, and all experiments were performed in triplicates. ND: not determined.

IC₅₀ values are reported as an average of three replicates ± standard error of the mean (SEM).

Biological evaluation of compound 12d in the TR-FRET assay.

To gain a better understanding of the inhibitory activity of 12d against Keap1-Nrf2 PPI, we further evaluated 12d using a time-resolved fluorescence energy transfer (TR-FRET) assay,^[60] In the FP assay, the lower limit for IC₅₀ is 50 nM, which is half of the Keap1 Kelch domain protein concentration used (100 nM). Accordingly, we developed a more sensitive TR-FRET assay with a lower limit of detection (IC₅₀ = 2.5 nM), as a tool to differentiate potent direct inhibitors with nanomolar activity, like 12d. Briefly, His-tagged Keap1 Kelch domain, terbium-labeled anti-His antibody, and a fluorescein-labeled 9-mer Nrf2 peptide amide derived from Nrf2 ETGE motif (FITC-LDEETGEFL-NH₂) were used together in the presence of varying concentrations of 12d or a positive control (2a). The distance-dependent transfer of excited state energy from the donor Tb to acceptor fluorescein was detected by measuring the time-resolved fluorescence intensities at 520 nm and 495 nm, providing a measure of competitive inhibitory activity (IC₅₀). Compound 12d was found to have 2-fold greater potency with an IC₅₀ value of 14.2 nM than the reference compound 2a (IC₅₀ = 28.5 nM), demonstrating the 2-(4-fluorobenzyloxy) fragment of 12d was involved in binding to Keap1 Kelch domain and could more effectively disrupt the Keap1-Nrf2 PPI (Figure 3). In addition to improving the binding affinity as measured in our TR-FRET assay, the 4-fluorobenzyl substituent at the C2-position of the naphthalene core can also improve the lipophilicity of 12d as indicated by a significant increase in its cLogP from 3.03 for 2a to 6.08 for 12d. More relevant to cellular permeability and bioavailability is the significant improvement in the LogD at physiologically relevant pH: 0.33 for 12d vs −2.93 for 2a at pH 7.4 and 1.94 for 12d vs −1.38 for 2a at pH 6.5.

Figure 3. — Concentration-response curves of 2a and **12d**; (A) FP assay, (B) TR-FRET assay. (C) IC₅₀ values of 2a and **12d** in FP and TR-FRET assays. The IC₅₀ values are reported as an average of three replicates ± standard error of the mean (SEM).

Effects of compounds 7v and 12d on the transcription of Nrf2-ARE regulated genes.

Additionally, we examined the ability of the potent Keap1-Nrf2 PPI inhibitors 7v and 12d to induce the expression of Nrf2-ARE-controled genes in NCM460D cells. As shown in Figure 4, the naphthalene analog 12d significantly increased the transcription of Nrf2 target genes, GSTM3, HMOX1 and NQO1, while compound 7v with the benzene ring scaffold has no effects on the upregulation of the Nrf2-mediated genes. Particularly, the naphthalene 12d possessing the O-linked benzyl fragment was found to enhance the mRNA levels of NQO1, in comparison to the potent direct inhibitor 2a, reaching a 15-fold change in NQO1 expression at 100 μM.

Figure 4. — Expression of Nrf2 target genes by the potent Keap1-Nrf2 PPI inhibitors in NCM460D cells. Cells were treated with test compounds 7v and **12d** (100, 10 and 1 μM), 0.1 % DMSO as a negative control, and two positive controls 2a (100, 10 and 1 μM) and CDDO (100 and 10 nM). After 24 h of incubation, the mRNA levels of *GSTM3*, *HMOX1* or *NQO1* were determined by RT-PCR analysis. *GAPDH* and *HPRT* were used as endogenous controls to normalize the target gene expression. The data are expressed as the fold change, relative to untreated DMSO control.

Molecular docking analysis.

In order to identify the role of O-linked fragments connected to core structure in the Keap1 Kelch domain binding site, we selected active compounds 2a, 7v and 12d, and performed molecular docking studies based on the co-crystal structure of 2b bound to the Kelch domain of Keap1 protein (PDB code: 4XMB).^[61] Similar to the reference 2a (Figure 5A), the other O-linked derivatives occupy all of the binding subpockets on the Keap1 Kelch domain. As expected, the binding mode of 7v reveals that the 4-fluorobenzyl alcohol is inserted deeply into the central solvent-exposed channel (Figure 5B). Introduction of the fragment indeed appears to make hydrophobic contacts at the back of P3 subpocket, given that 7v shows enhanced activity relative to known compound 6. However, the benzene ring core (7v) is found to be less potent than the naphthalene scaffold (2a), probably due to the reduced hydrophobic interactions and the loss of π-cation interaction with Arg415. In addition, one carboxylic group of 7v displays no hydrogen bonds and electrostatic interactions with Arg483 in the P1 subpocket. Interestingly, the docked binding pose of O-linked naphthalene scaffold 12d indicates a different spatial orientation in the P3 central pocket (Figure 5C). The 4-fluorobenzyl alcohol moiety is located toward hydrophobic residue of Tyr334 in the P5 subpocket, while the naphthalene core is positioned toward the pore of P3 subpocket. It is likely that the strong preference for hydrophobic contact and π-cation interactions of the naphthalene core with Arg415 in the P3 pocket makes the O-linked fragment oriented forward. Since compound 12d is 2-fold more potent than 2a in our TR-FRET assay, the 2-(4-fluorobenzyl) group is expected to form additional hydrophobic interactions with Keap1 Kelch domain. According to our docking studies, the 2-(4-fluorobenzyl) fragment is positioned near the P5 region occupied by Tyr334 and Phe557 to provide the additional boost in binding affinity.

Figure 5. — Binding modes of compounds (represented in cyan sticks) bound to the Keap1 Kelch domain using molecular docking (derived from PDB code: 4XMB); (A) 2a, (B) 7v, (C) **12d**. The surface of Keap1 was colored as the partial charge (represented in red for the most lipophilic areas, and blue for polar areas). Red dashed lines represent hydrogen bonds, and yellow lines represent π-π stacking or π-cation interactions.

CONCLUSION

In conclusion, a series of 1,4-bis(arylsulfonamido)benzene or naphthalene-N,N'-diacetic acid derivatives including 2-alkoxy or aryloxy fragments was designed based on the analysis of co-crystal structure of the Keap1 Kelch domain with the non-electrophilic inhibitor 2b. First, structure-activity relationship (SAR) studies around the 2-O-linked fragments (R₁) exhibited a two-fold boost to the binding affinity as shown in our TR-FRET assay as well as enhanced inhibition of Keap1-Nrf2 protein-protein interaction in cellular assays resulting in increased expression of Nrf2-ARE-controled genes. In the case of the benzene core, the addition of 2-O-linked moieties such phenyl (7d and 7h-j), pyridinyl (9a), naphthalenyl (7k and 7m-n), and biphenyl (7l) resulted in improved inhibitory activity with IC₅₀ values in the submicromolar range (0.39-0.91 μM) in our FP assay in comparison to lead 6 without 2-subsituents. In the case of the naphthalene scaffold, 2a and 12d exhibit a similar trend in the TR-FRET assay with 2-fold increase in inhibitory potency with the 2-(4-fluorobenzyloxy) group (IC₅₀ = 28.5 vs 14.2 nM). However, potencies of the benzene core with an oxygen-containing linker remained limited due to its relatively weaker inhibition as compared to potent naphthalene analog 2a without a linker. Generally, the small molecule direct inhibitors such as those found in this paper interrupted the interactions between Keap1 and Nrf2 proteins with the following order of potency: naphthalene core with 2-O-linked fragment (12d) > naphthalene core without 2-O-linked fragment (2a) > benzene core with 2-O-linked fragment > benzene core without 2-O-linked fragment (6). These results were supported by our molecular docking studies. Different from 6, the 2-O-linked benzyl group of 7v occupied additional binding sites behind the P3 subpocket, leading to increased binding affinity via more hydrophobic interactions. When compared to the benzene ring scaffold, the naphthalene core structure played a more critical role in tightly binding to the Keap1 Kelch domain through strong hydrophobic and π-cation interactions with Arg415, enabling the formation of a more stable complex. Therefore, 1,4-diaminonaphthalene derivatives 2a and 12d displayed potent inhibitory activity of Keap1-Nrf2 PPI in our FP and TR-FRET assays. In particular, compound 12d with 2-(4-fluorobenzyloxy) group was shown to be the most potent inhibitor against Keap1-Nrf2 PPI, probably as a result of hydrophobic interactions of the 4-fluorobenzyl fragment toward the nonpolar P5 subpocket. Based on our docking studies, the 4-fluorobenzyl group of 7v and that of 12d occupied completely the opposite sides of the corresponding core moiety, as the 2-substituent of 12d is forced to project toward the P5 subpocket by the strong interactions of its naphthalene core in the P3 pocket. Surprisingly, the introduction of extra fragments such as methoxy (12a-b) and 4-fluorobenzyloxy (12c) substituents into the benzene core resulted in significantly reduced inhibitory activities. The additional moieties appeared to have a negative effect on forming a stable complex with regards to their proper occupation of all five pockets of the Keap1 Kelch domain. The most potent inhibitor 12d was further evaluated in a cell-based assay. The cellular activity data revealed that the naphthalene scaffold 12d is a promising Nrf2 activator, exhibiting strong induction of Nrf2-dependent gene NQO1 at 100 μM than 2a. Although further studies are needed for the development of Nrf2 activators through direct inhibition of the Keap1-Nrf2 PPI, the new 2-O-linked naphthalene core scaffold offers an additional site for structural diversity and optimization.

EXPERIMENTAL

General Chemistry.

All solvents and reagents were purchased from commercial sources and used as received unless specified otherwise. All moisture sensitive reactions were carried out using dry solvents under nitrogen atmosphere. Analytical thin layer chromatography was carried out using aluminum-backed Silica G TLC plates coated with DC Kieselgel 60 F254 (Merk or Sigma-Aldrich). Plates were visualized using ultraviolet light (254 nm) and/or by staining with potassium permanganate followed by heating. Flash chromatography was carried out on a Teledyne ISCO CombiFlash Companion system with prepacked Teledyne ISCO RediSep normal phase silica cartridges (230-400 mesh), eluting with a 0–100% ethyl acetate/ hexane or 0-20% methanol/dichloromethane gradient. ¹H and ¹³C NMR spectra were recorded at 400 MHz and 100 MHz, respectively, at ambient temperature, using a Bruker Avance III 400 MHz Multinuclear NMR spectrometer. Chemical shifts (δ) are expressed in parts per million (ppm) relative to a solvent signal as an internal reference (CDCl₃, MeOH-d₄, or DMSO-d₆). 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). Coupling constants (J) are reported in hertz (Hz). Analytical liquid chromatography/ mass spectrometry (LC/MS) were performed on an Agilent 1200 Infinity high-performance LC (HPLC) system coupled to an Agilent 6410 single quadrupole mass spectrometer operating a multimode source, with an Inertsil ODS-3 C18 column (3 mm × 33 mm, 3 μM) at a temperature of 40 °C. The mobile phases A and B were water with 0.1% formic acid and methanol with 0.1% formic acid, respectively. The ratio of mobile phase B was increased linearly from 5% to 90% over 5 minutes, at a flow rate of 0.8 mL/min, monitoring UV absorbance at 280 nm. High-resolution mass spectra (HRMS) experiments were conducted by Center for Integrative Proteomics Research (CIPR) at Rutgers University. 1 μg/mL solutions were directly infused using the Thermo LTQ Orbitrap Velos ETD with Dionex Ultimate 3000 nano-flow 2D LC. Purities of all final compounds were determined to be ≥95% by UPLC analysis on a Waters Acquity UPLC^™ system.

2-Phenyl-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7a).

To a solution of 17a (34 mg, 0.05 mmol) in methanol (0.3 mL) was added sodium hydroxide (10 mg, 0.24 mmol) in water (0.1 mL) at room temperature. The reaction mixture was stirred at 60 °C overnight. After the reaction was completed as monitored by TLC or LCMS, the crude mixture was concentrated under reduced pressure and the resulting residue dissolved in water and then washed with dichloromethane. The aqueous phase was acidified with 6 N HCl to pH around 1 and extracted with dichloromethane. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The resulting solid was dissolved with the minimum amount of dichloromethane and triturated with hexane. The product was collected by filtration as a white solid (21 mg, 67%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.60 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.40-7.32 (m, 4H), 7.22-7.16 (m, 3H), 7.06-6.99 (m, 5H), 4.43 (s, 2H), 3.90 (s, 2H), 3.89 (s, 3H), 3.87 (s, 3H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.1, 171.7, 165.0, 164.9, 143.6, 141.4, 139.6, 138.0, 133.0, 132.7, 132.2, 131.23, 131.19, 130.0, 129.3, 129.1, 128.9, 128.6, 115.3, 115.2, 115.0, 56.30, 56.27, 53.12, 53.06; LC/MS (ESI⁻) m/z 639.0 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₀H₂₈N₂O₁₀S₂ [M − H]⁻ 639.1113; found 639.1118. R_t: 4.11 min, HPLC purity: 99.0%.

2-(Piperidin-1-yl)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7b).

Prepared as described in the experimental procedure of 7a from intermediate 17b to obtain the title compound as a white solid (19 mg, 25%); ¹H NMR (400 MHz, DMSO-d₆) δ 7.74 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 9.2 Hz, 2H), 7.05 (d, J = 9.2 Hz, 2H), 6.85 (dd, J = 8.4, 2.4 Hz, 1H), 6.71 (d, J = 2.4 Hz, 1H), 4.53 (s, 2H), 4.35 (s, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 2.58-2.54 (m, 4H), 1.48-1.41 (m, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.1, 169.9, 162.8, 162.6, 149.7, 139.7, 132.2, 132.1, 131.1, 130.1, 129.6, 121.9, 120.3, 114.32, 114.27, 55.7, 52.5, 52.0, 49.3, 25.9, 23.6; LC/MS (ESI⁻) m/z 646.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₂₉H₃₃N₃O₁₀S₂ [M − H]⁻ 646.1535; found 646.1541. R_t: 3.61 min, HPLC purity: 99.6%.

2-(Benzyl(methyl)amino)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7c).

Prepared as described in the experimental procedure of 7a from intermediate 17c to obtain the title compound as a white solid (53 mg, 66%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.76 (brs, 2H, CO₂H), 7.73 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 7.34-7.28 (m, 3H), 7.12 (d, J = 7.6 Hz, 2H), 7.15 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 4.53 (s, 2H), 4.33 (s, 2H), 4.02 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 2.40 (s, 3H); ¹³C NMR (100 MHz, MeOH-d₄) δ 173.4, 171.9, 165.3, 165.0, 147.3, 142.8, 134.9, 134.1, 131.9, 131.5, 131.3, 131.1, 131.0, 129.8, 127.0, 123.8, 115.5, 115.3, 114.4, 62.8, 56.33, 56.31, 53.2, 52.8, 42.1; LC/MS (ESI⁻) m/z 682.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₂H₃₃N₃O₁₀S₂ [M − H]⁻ 682.1535; found 682.1525. R_t: 3.88 min, HPLC purity: 98.1%.

2-Benzyloxy-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7d).

Prepared as described in the experimental procedure of 7a from intermediate 17d to obtain the title compound as a white solid (21 mg, 46%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.60 (d, J = 9.2 Hz, 2H), 7.50 (d, J = 8.8 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.33-7.31 (m, 3H), 7.10-7.07 (m, 2H), 7.03 (d, J = 9.2 Hz, 2H), 6.87 (d, J = 2.4 Hz, 1H), 6.82-6.78 (m, 3H), 4.59 (s, 2H), 4.41 (s, 2H), 4.31 (s, 2H), 3.87 (s, 3H), 3.80 (s, 3H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.7, 172.3, 165.0, 164.6, 156.3, 142.9, 137.3, 135.2, 132.8, 131.3, 130.5, 129.5, 129.1, 128.3, 127.7, 120.9, 115.2, 115.0, 114.4, 71.3, 56.3, 56.1, 30.7; LC/MS (ESI⁻) m/z 669.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₁H₃₀N₂O₁₁S₂ [M − H]⁻ 669.1218; found 669.1224. R_t: 4.17 min, HPLC purity: 98.2%.

2-Phenoxy-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7e).

Prepared as described in the experimental procedure of 7a from intermediate 17e to obtain the title compound as a white solid (61 mg, 55%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.61 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.25 (dd, J = 8.0, 8.0 Hz, 2H), 7.12 (t, J = 8.0 Hz, 1H), 6.99-6.96 (m, 3H), 6.91 (d, J = 8.8 Hz, 2H), 6.51 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 2.0 Hz, 1H), 4.43 (s, 2H), 4.31 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.5, 172.0, 164.9, 164.8, 155.8, 155.5, 142.6, 135.5, 132.7, 131.2, 131.0, 130.9, 130.8, 128.9, 125.6, 122.9, 120.5, 117.5, 115.3, 115.2, 56.3, 56.2, 52.9, 52.4; LC/MS (ESI⁻) m/z 655.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₀H₂₈N₂O₁₁S₂ [M − H]⁻ 655.1062; found 655.1068. R_t: 4.10 min, HPLC purity: 99.1%.

2-Phenethoxy-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7f).

Prepared as described in the experimental procedure of 7a from intermediate 17f to obtain the title compound as a white solid (60 mg, 68%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.58 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 7.30-7.26 (m, 3H), 7.21-7.20 (m, 1H), 7.09 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 9.2 Hz, 2H), 6.96 (d, J = 9.2 Hz, 2H), 6.78 (dd, J = 8.8, 2.0 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 4.41 (s, 2H), 4.05 (s, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 3.62 (t, J = 6.8 Hz, 2H), 2.54 (t, J = 6.8 Hz, 2H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.7, 172.2, 164.9, 164.6, 156.1, 142.7, 138.9, 135.1, 133.0, 131.2, 130.7, 129.9, 129.6, 127.7, 127.4, 120.9, 115.2, 115.0, 113.9, 69.8, 56.31, 56.28, 53.2, 51.7, 35.9; LC/MS (ESI⁻) m/z 683.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₂H₃₂N₂O₁₁S₂ [M − H]⁻ 683.1375; found 683.1382. R_t: 4.27 min, HPLC purity: 97.0%.

2-(3-Phenylpropoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7g).

Prepared as described in the experimental procedure of 7a from intermediate 17g to obtain the title compound as a white solid (15 mg, 52%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.92 (brs, 2H, CO₂H), 7.61 (d, J = 9.2 Hz, 2H), 7.49 (d, J = 9.2 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.32-7.28 (m, 2H), 7.22-7.18 (m, 1H), 7.12 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 9.2 Hz, 2H), 7.00 (d, J = 9.2 Hz, 2H), 6.81 (dd, J = 8.4, 2.0 Hz, 1H), 6.58 (d, J = 2.0 Hz, 1H), 4.40 (s, 2H), 4.22 (s, 2H), 3.82 (s, 3H), 3.73 (s, 3H), 3.42-3.40 (m, 2H), 2.41 (t, J = 7.8 Hz, 2H), 1.52-1.47 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 170.0, 162.8, 162.5, 154.5, 141.2, 140.9, 133.4, 131.5, 129.9, 129.6, 129.1, 128.3, 128.2, 125.8, 125.3, 118.9, 114.3, 114.0, 111.3, 66.9, 55.7, 55.5, 51.8, 50.5, 31.3, 29.9; LC/MS (ESI⁻) m/z 697.2 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₃H₃₄N₂O₁₁S₂ [M − H]⁻ 697.1531; found 697.1525. R_t: 4.39 min, HPLC purity: 97.6%.

2-(4-Methoxybenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7h).

Prepared as described in the experimental procedure of 7a from intermediate 17h to obtain the title compound as a white solid (66 mg, 67%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (brs, 2H, CO₂H), 7.62 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 9.2 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 9.2 Hz, 2H), 6.83 (dd, J = 8.4, 2.4 Hz, 2H), 6.77 (d, J = 2.4 Hz, 1H), 4.51 (s, 2H), 4.41 (s, 2H), 4.16 (s, 2H), 3.84 (s, 3H), 3.80 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 169.9, 162.8, 162.4, 158.9, 154.4, 140.9, 133.2, 131.2, 129.9, 129.6, 129.0, 128.7, 127.7, 125.6, 119.1, 114.3, 113.9, 113.6, 111.9, 69.3, 55.7, 55.5, 55.0, 51.8, 50.4; LC/MS (ESI⁻) m/z 699.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₂H₃₂N₂O₁₂S₂ [M − H]⁻ 699.1324; found 699.1329. R_t: 4.13 min, HPLC purity: 96.6%.

2-(4-Fluorobenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7i).

Prepared as described in the experimental procedure of 7a from intermediate 17i to obtain the title compound as a white solid (56 mg, 44%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (brs, 2H, CO₂H), 7.62 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.17-7.08 (m, 6H), 6.86-6.83 (m, 3H), 6.78 (d, J = 2.0 Hz, 1H), 4.58 (s, 2H), 4.41 (s, 2H), 4.19 (s, 2H), 3.84 (s, 3H), 3.75 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 170.0, 162.8, 162.4, 154.3, 141.0, 133.4, 132.1, 131.2, 129.9, 129.7, 129.1 (J_C,F = 8 Hz), 129.0, 125.7, 119.3, 115.0 (J_C,F = 21 Hz), 114.3, 113.9, 111.9, 68.8, 55.7, 55.5, 51.8, 50.6; LC/MS (ESI⁺) m/z 689.2 [M + H]⁺; HRMS (ESI⁺) m/z calcd for C₃₁H₂₉FN₂O₁₁S₂ [M + H]⁺ 689.1270; found 689.1281. R_t: 4.19 min, HPLC purity: 95.1%.

2-(4-Methylbenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7j).

Prepared as described in the experimental procedure of 7a from intermediate 17j to obtain the title compound as a white solid (29 mg, 87%); ¹H NMR (400 MHz, CDCl₃) δ 7.59 (d, J = 9.2 Hz, 2H), 7.47-7.43 (m, 3H), 7.11 (d, J = 7.6 Hz, 2H), 6.98-6.95 (m, 3H), 6.90 (d, J = 8.8 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 6.59 (dd, J = 8.4, 2.4 Hz, 2H), 4.62 (s, 2H), 4.33 (s, 2H), 4.28 (s, 2H), 3.84 (s, 3H), 3.76 (s, 3H), 2.35 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.2, 163.4, 163.1, 155.1, 141.4, 138.1, 134.0, 132.5, 131.3, 130.2, 129.7, 129.3, 127.5, 127.4, 126.6, 119.3, 114.2, 113.9, 113.8, 70.4, 55.8, 55.6, 52.2, 51.0, 21.3; LC/MS (ESI⁻) m/z 683.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₂H₃₂N₂O₁₁S₂ [M − H]⁻ 683.1375; found 683.1381. R_t: 4.31 min, HPLC purity: 95.6%.

2-(Naphthalen-2-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7k).

Prepared as described in the experimental procedure of 7a from intermediate 17k to obtain the title compound as a white solid (59 mg, 67%); ¹H NMR (400 MHz, DMSO-d₆) δ 7.95-7.92 (m, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.57-7.53 (m, 3H), 7.43 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 9.2 Hz, 1H), 7.21 (dd, J = 8.4, 1.2 Hz, 1H), 7.05 (d, J = 9.2 Hz, 2H), 6.87-6.85 (m, 2H), 6.76 (d, J = 9.2 Hz, 2H), 4.77 (s, 2H), 4.43 (s, 2H), 4.26 (s, 2H), 3.82 (s, 3H), 3.54 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 170.0, 162.8, 162.3, 154.5, 141.0, 133.5, 132.6, 132.5, 131.3, 129.9, 129.7, 129.0, 127.9, 127.6, 126.3, 126.2, 125.7, 125.5, 124.9, 114.3, 113.9, 111.9, 69.6, 55.8, 55.3, 51.8, 50.6; LC/MS (ESI⁻) m/z 719.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₅H₃₂N₂O₁₁S₂ [M − H]⁻ 719.1375; found 719.1382. R_t: 4.46 min, HPLC purity: 97.6%.

2-([1,1'-Biphenyl]-4-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7l).

Prepared as described in the experimental procedure of 7a from intermediate 17l to obtain the title compound as a white solid (65 mg, 81%); ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (d, J = 7.6 Hz, 2H), 7.63-7.61 (m, 4H), 7.51-7.37 (m, 6H), 7.12 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.87-6.84 (m, 3H), 6.81 (d, J = 2.4 Hz, 1H), 4.65 (s, 2H), 4.42 (s, 2H), 4.23 (s, 2H), 3.82 (s, 3H), 3.69 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 170.0, 162.8, 162.4, 154.4, 141.0, 139.7, 139.5, 135.0, 133.4, 131.2, 129.9, 129.7, 129.0, 127.5, 127.4, 126.6, 126.5, 125.6, 119.3, 114.3, 114.0, 111.9, 69.2, 55.7, 55.4, 51.8, 50.6; LC/MS (ESI⁻) m/z 745.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₇H₃₄N₂O₁₁S₂ [M − H]⁻ 745.1531; found 745.1538. R_t: 4.61 min, HPLC purity: 96.8%.

2-(Naphthalen-2-yloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7m).

Prepared as described in the experimental procedure of 7a from intermediate 17m to obtain the title compound as a white solid (32 mg, 27%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.87 (brs, 2H, CO₂H), 7.94 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.57-7.48 (m, 3H), 7.45 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.76 (dd, J = 8.8, 2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 4.38 (s, 2H), 4.31 (s, 2H), 3.75 (s, 3H), 3.66 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 169.9, 162.64, 162.57, 153.4, 151.9, 140.8, 133.9, 133.5, 131.1, 130.2, 129.9, 129.8, 129.3, 127.7, 127.1, 126.9, 126.8, 125.3, 120.9, 119.5, 115.3, 115.0, 114.2, 114.1, 55.7, 55.5, 51.6, 51.3; LC/MS (ESI⁻) m/z 705.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₄H₃₀N₂O₁₁S₂ [M − H]⁻ 705.1218; found 705.1224. R_t: 4.39 min, HPLC purity: 97.5%.

2-(Naphthalen-1-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7n).

Prepared as described in the experimental procedure of 7a from intermediate 17n to obtain the title compound as a white solid (84 mg, 82%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.74 (brs, 2H, CO₂H), 7.99-7.97 (m, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.79-7.77 (m, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.58-7.55 (m, 2H), 7.47-7.43 (m, 2H), 7.39 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 6.95-6.93 (m, 2H), 6.72 (d, J = 8.8 Hz, 2H), 5.03 (s, 2H), 4.46 (s, 2H), 4.15 (s, 2H), 3.80 (s, 3H), 3.59 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 169.9, 162.8, 162.3, 154.5, 141.0, 133.3, 133.0, 131.22, 131.15, 130.1, 129.9, 129.7, 128.9, 128.5, 128.2, 126.3, 125.9, 125.8, 125.2, 125.0, 123.1, 120.1, 114.3, 113.8, 112.1, 67.8, 55.7, 55.3, 51.8, 50.6; LC/MS (ESI⁻) m/z 719.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₅H₃₂N₂O₁₁S₂ [M − H]⁻ 719.1375; found 719.1382. R_t: 4.39 min, HPLC purity: 97.8%.

2-([1,1'-Biphenyl]-4-yloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7o).

Prepared as described in the experimental procedure of 7a from intermediate 17o to obtain the title compound as a white solid (68 mg, 74%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.91 (brs, 2H, CO₂H), 7.66 (d, J = 7.6 Hz, 2H), 7.58-7.47 (m, 9H), 7.37 (t, J = 7.2 Hz, 1H), 7.03-7.01 (m, 3H), 6.96 (d, J = 8.8 Hz, 2H), 6.59 (d, J = 8.8 Hz, 2H), 6.45 (d, J = 2.0 Hz, 1H), 4.34 (s, 2H), 4.33 (s, 2H), 3.75 (s, 3H), 3.73 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 169.9, 162.7, 162.6, 153.8, 153.2, 140.7, 139.2, 136.0, 133.8, 131.1, 129.8, 129.4, 129.2, 129.0, 127.9, 127.3, 127.2, 126.4, 121.7, 119.2, 115.6, 114.4, 114.2, 55.60, 55.57, 51.7, 51.3; LC/MS (ESI⁻) m/z m/z 731.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₆H₃₂N₂O₁₁S₂ [M − H]⁻ 731.1375; found 731.1381. R_t: 4.55 min, HPLC purity: 99.5%.

2-Benzyloxy-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7p).

Prepared as described in the experimental procedure of 7a from intermediate 17p to obtain the title compound as a white solid (20 mg, 70%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (brs, 2H, CO₂H), 7.56 (d, J = 8.0 Hz, 2H), 7.39-7.36 (m, 5H), 7.33-7.32 (m, 3H), 7.14 (d, J = 8.0 Hz, 2H), 7.02-7.00 (m, 2H), 6.84 (d, J = 8.4 Hz, 1H), 6.77 (s, 1H), 4.54 (s, 2H), 4.42 (s, 2H), 4.22 (s, 2H), 2.40 (s, 3H), 2.28 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.1, 169.8, 154.3, 143.6, 143.0, 140.8, 136.7, 135.8, 135.5, 133.2, 129.6, 129.2, 128.1, 127.7, 127.3, 126.8, 125.5, 119.3, 112.0, 69.4, 51.8, 50.6, 21.0, 20.9; LC/MS (ESI⁻) m/z 637.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₁H₃₀N₂O₉S₂ [M − H]⁻ 637.1320; found 637.1326. R_t: 4.44 min, HPLC purity: 98.3%.

2-Benzyloxy-1,4-bis(4-phenylphenylsulfonamido)benzene-N,N'-diacetic acid (7q).

Prepared as described in the experimental procedure of 7a from intermediate 17q to obtain the title compound as a white solid (25 mg, 87%); ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.56-7.53 (m, 5H), 7.44-7.35 (m, 10H), 7.19-7.11 (m, 3H), 6.94 (d, J = 2.0 Hz, 1H), 6.92 (d, J = 6.4 Hz, 2H), 6.69 (dd, J = 8.4, 2.0 Hz, 1H), 4.56 (s, 2H), 4.34 (s, 2H), 4.32 (s, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.3, 170.6, 155.0, 146.1, 145.4, 141.3, 139.1, 139.0, 138.3, 137.1, 135.4, 134.4, 129.2, 129.0, 128.7, 128.50, 128.46, 128.4, 128.1, 127.9, 127.5, 127.3, 127.2, 127.10, 127.06, 126.5, 119.6, 113.8, 70.2, 52.4, 50.9; LC/MS (ESI⁻) m/z 761.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₄₁H₃₄N₂O₉S₂ [M − H]⁻ 761.1633; found 761.1628. R_t: 5.05 min, HPLC purity: 95.0%.

2-Benzyloxy-1,4-bis(4-fluorophenylsulfonamido)benzene-N,N'-diacetic acid (7r).

Prepared as described in the experimental procedure of 7a from intermediate 17r to obtain the title compound as a white solid (32 mg, 54%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.79 (brs, 2H, CO₂H), 7.76 (dd, J = 8.4, 5.2 Hz, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.56 (dd, J = 8.4, 5.2 Hz, 2H), 7.45-7.37 (m, 3H), 7.34-7.32 (m, 2H), 7.15-7.08 (m, 2H), 7.06-7.04 (m, 2H), 6.88 (dd, J = 8.4, 1.6 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H), 4.61 (s, 2H), 4.46 (s, 2H), 4.25 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.1, 169.8, 163.3 (J_C,F = 30 Hz), 154.4, 140.8, 135.9, 135.7, 134.7, 133.4, 130.5 (J_C,F = 10 Hz), 129.8 (J_C,F = 10 Hz), 129.6, 128.2, 127.8, 126.9, 126.0, 119.7, 116.4 (J_C,F = 22 Hz), 115.9 (J_C,F = 23 Hz), 114.3, 112.1, 69.6, 52.0, 50.7; LC/MS (ESI⁻) m/z 645.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₂₉H₂₄F₂N₂O₉S₂ [M − H]⁻ 645.0819; found 645.0826. R_t: 4.32 min, HPLC purity: 98.7%.

2-Benzyloxy-1,4-bis(4-(trifluoromethyl)phenylsulfonamido)benzene-N,N'-diacetic acid (7s).

Prepared as described in the experimental procedure of 7a from intermediate 17s to obtain the title compound as a white solid (25 mg, 64%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.87-7.85 (m, 4H), 7.70 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 1H), 7.31-7.29 (m, 3H), 7.04-7.01 (m, 2H), 6.91-6.87 (m, 2H), 4.54 (s, 2H), 4.49 (s, 2H), 4.37 (s, 2H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.2, 171.9, 156.3, 145.0, 143.9, 142.6, 136.9, 135.7, 135.4, 129.8, 129.5, 129.23, 129.17, 128.4, 127.7, 127.2, 127.0, 121.6, 114.7, 71.3, 53.5, 52.2; LC/MS (ESI⁻) m/z 745.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₁H₂₄F₆N₂O₉S₂ [M − H]⁻ 745.0755; found 745.0764. R_t: 4.78 min, HPLC purity: 97.9%.

2-Benzyloxy-1,4-bis(4-bromophenylsulfonamido)benzene-N,N'-diacetic acid (7t).

Prepared as described in the experimental procedure of 7a from intermediate 17t to obtain the title compound as a white solid (31 mg, 56%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.88 (brs, 2H, CO₂H), 7.81 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.52 (d, J = 7.6 Hz, 2H), 7.43-7.39 (m, 3H), 7.36-7.34 (m, 3H), 7.07-7.02 (m, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.76 (s, 1H), 4.54 (s, 2H), 4.46 (s, 2H), 4.25 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.1, 169.8, 154.4, 140.7, 138.7, 137.5, 135.6, 135.5, 132.3, 131.9, 129.4, 128.7, 128.3, 127.8, 127.3, 126.94, 126.85, 125.6, 120.1, 112.2, 69.6, 52.0, 50.7; LC/MS (ESI⁺) m/z 767.0 [M(2⁷⁹Br,) + H]⁺, 769.0 [M(⁷⁹Br, ⁸¹Br) + H]⁺, 771.0 [M(2⁸¹Br) + H]⁺ ; HRMS (ESI⁺) m/z calcd for C₂₉H₂₄Br₂N₂O₉S₂ [M(2⁷⁹Br,) + H]⁺ 766.9324, [M(⁷⁹Br, ⁸¹Br) + H]⁺ 768.9303 and [M(2⁸¹Br) + H]⁺ 770.9283; found 766.9380, 768.9356 and 770.9332. R_t: 4.79 min, HPLC purity: 97.0%.

2-Benzyloxy-1,4-bis(3-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7u).

Prepared as described in the experimental procedure of 7a from intermediate 17u to obtain the title compound as a white solid (29 mg, 81%); ¹H NMR (400 MHz, CDCl₃) δ 7.50 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 8.8 Hz, 2H), 7.37-7.27 (m, 7H), 7.17 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.00-6.98 (m, 3H), 6.55 (dd, J = 8.4, 2.4 Hz, 1H), 4.59 (s, 2H), 4.31 (s, 2H), 4.29 (s, 2H), 2.35 (s, 3H), 2.18 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 171.3, 170.5, 155.0, 141.3, 139.7, 139.3, 139.0, 138.3, 135.6, 134.3, 134.0, 133.5, 128.9, 128.54, 128.48, 128.13, 128.10, 127.6, 127.0, 126.6, 124.9, 124.5, 119.3, 114.1, 70.2, 52.4, 50.9, 21.3, 21.2; LC/MS (ESI⁻) 637.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₁H₃₀N₂O₉S₂ [M − H]⁻ 637.1320; found 637.1311. R_t: 4.47 min, HPLC purity: 96.0%.

2-(4-Fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7v).

Prepared as described in the experimental procedure of 7a from intermediate 17v to obtain the title compound as a white solid (66 mg, 70%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.83 (brs, 2H, CO₂H), 7.56 (d, J = 7.6 Hz, 2H), 7.40-7.35 (m, 5H), 7.18-7.14 (m, 4H), 7.08-7.05 (m, 2H), 6.84 (d, J = 8.4 Hz, 1H), 6.78 (s, 1H), 4.55 (s, 2H), 4.43 (s, 2H), 4.20 (s, 2H), 2.40 (s, 3H), 2.29 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.3, 169.9, 160.5, 154.3, 143.7, 143.1, 140.9, 136.7, 135.5, 133.3, 132.1 (J_C,F = 3 Hz), 129.6, 129.3, 129.1 (J_C,F = 8 Hz), 127.4, 126.8, 125.6, 119.3, 115.0 (J_C,F = 22 Hz), 112.0, 68.8, 51.8, 50.6, 21.0, 20.9; LC/MS (ESI⁻) m/z 655.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₁H₂₉FN₂O₉S₂ [M − H]⁻ 655.1226; found 655.1221. R_t: 4.45 min, HPLC purity: 97.2%.

2-(4-Fluorobenzyloxy)-1,4-bis(2,4,6-trimethylphenylsulfonamido)benzene-N,N'-diacetic acid (7w).

To a solution of 17w (102 mg, 0.12 mmol) in dichloromethane (2 mL) was added trifluoroacetic acid (0.5 mL) at room temperature. The reaction mixture was stirred at room temperature for overnight. After the reaction was completed, the crude mixture was added to water_, and extracted with dichloromethane. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatograph (0-10% methanol/ dichloromethane) to give the product as a pale yellow solid (79 mg, 89%); ¹H NMR (400 MHz, CDCl₃) δ 11.22 (brs, 2H, CO₂H), 7.45 (d, J = 8.4 Hz, 1H), 7.08-7.00 (m, 4H), 6.87 (s, 2H), 6.72 (s, 2H), 6.64-6.61 (m, 2H), 4.56 (s, 6H), 2.36 (s, 6H), 2.30 (s, 3H), 2.28 (s, 3H), 2.18 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 176.6, 175.6, 162.8 (J_C,F = 246 Hz) 155.4, 143.4, 142.3, 140.7, 140.4, 134.5, 133.6, 132.1, 131.9, 131.8, 131.3, 129.5 (J_C,F = 8 Hz), 125.8, 121.4, 115.7 (J_C,F = 21 Hz), 112.6, 69.9, 50.8, 50.2, 23.3, 23.0, 21.2, 21.1; LC/MS (ESI⁻) m/z 711.2 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₅H₃₇FN₂O₉S₂ [M − H]⁻ 711.1852; found 711.1847. R_t: 5.07 min, HPLC purity: 97.2%.

2-(Naphthalen-2-ylmethoxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7x).

Prepared as described in the experimental procedure of 7a from intermediate 17x to obtain the title compound as a white solid (59 mg, 46%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.83 (brs, 2H, CO₂H), 7.96-7.93 (m, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.56-7.54 (m, 5H), 7.41-7.37 (m, 3H), 7.33 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.87-6.85 (m, 2H), 4.74(s, 2H), 4.44 (s, 2H), 4.27 (s, 2H), 2.37 (s, 3H), 2.09 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 169.9, 154.4, 143.7, 143.0, 140.9, 136.7, 135.5, 133.5, 133.4, 132.6, 132.5, 129.6, 129.2, 127.84, 127.81, 127.6, 127.4, 126.8, 126.3, 126.1, 125.7, 125.5, 124.9, 119.3, 112.0, 69.6, 51.9, 50.6, 21.0, 20.8; LC/MS (ESI⁻) m/z 687.2 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₅H₃₂N₂O₉S₂ [M − H]⁻ 687.1476; found 687.1473. R_t: 4.72 min, HPLC purity: 99.9%.

2-([1,1'-Biphenyl]-4-ylmethoxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7y).

Prepared as described in the experimental procedure of 7a from intermediate 17y to obtain the title compound as a white solid (52 mg, 58%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (brs, 2H, CO₂H), 7.71 (d, J = 7.6 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.50 (dd, J = 7.6, 7.6 Hz, 2H), 7.41-7.38 (m, 6H), 7.17 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 6.85 (dd, J = 8.4, 2.4 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 4.61 (s, 2H), 4.44 (s, 2H), 4.25 (s, 2H), 2.38 (s, 3H), 2.26 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 169.9, 154.4, 143.7, 143.1, 140.9, 139.7, 139.5, 136.7, 135.5, 135.0, 133.3, 129.6, 129.3, 129.0, 127.5, 127.42, 127.35, 126.8, 126.6, 126.4, 125.6, 119.3, 112.0, 69.2, 51.8, 50.6, 21.0; LC/MS (ESI⁻) m/z 713.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₇H₃₄N₂O₉S₂ [M − H]⁻ 713.1633; found 713.1630. R_t: 4.86 min, HPLC purity: 99.5%.

2-(Phenoxymethyl)-1,4-bis(4-methyoxyphenylsulfonamido)benzene-N,N'-diacetic acid (8).

Prepared as described in the experimental procedure of 7a from intermediate 21 to obtain the title compound as a white solid (44 mg, 72%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.53 (d, J = 8.8 Hz, 4H), 7.40 (d, J = 2.4 Hz, 1H), 7.26 (dd, J = 8.0, 8.0 Hz, 2H), 7.09 (dd, J = 8.8, 2.4 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.95-6.91 (m, 5H), 6.77 (d, J = 8.8 Hz, 1H), 5.51-5.48 (m, 1H), 5.16-5.13 (m, 1H), 4.44-4.40 (m, 1H), 4.37 (s, 2H), 4.16-4.12 (m, 1H), 3.85 (s, 3H), 3.82 (s, 3H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.1, 171.7, 165.1, 164.8, 160.0, 142.1, 141.6, 138.1, 131.4, 131.2, 131.1, 130.5, 130.4, 128.6, 128.2, 122.0, 116.0, 115.9, 115.4, 115.2, 67.1, 56.3, 56.3, 54.1, 53.1; LC/MS (ESI⁻) m/z 699.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₁H₃₀N₂O₁₁S₂ [M − H]⁻ 669.1218; found 669.1225. R_t: 4.25 min, HPLC purity: 98.2%.

2-(Pyridin-3-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (9a).

Prepared as described in the experimental procedure of 7a from intermediate 26a to obtain the title compound as a white solid (7 mg, 45%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.71 (brs, 2H, CO₂H), 8.54 (d, J = 4.4 Hz, 1H), 8.33 (s, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.40-7.35 (m, 4H), 7.10 (d, J = 8.8 Hz, 2H), 6.87-6.80 (m, 4H), 4.67 (s, 2H), 4.43 (s, 2H), 4.21 (s, 2H), 3.85 (s, 3H), 3.76 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.2, 169.8, 162.8, 162.3, 154.3, 149.0, 148.2, 141.0, 134.7, 133.3, 131.4, 131.1, 129.9, 129.6, 128.9, 125.7, 123.3, 119.3, 114.3, 113.8, 111.9, 67.2, 55.7, 55.4, 51.7, 50.6; LC/MS (ESI⁻) m/z 670.0 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₀H₂₉N₃O₁₁S₂ [M − H]⁻ 670.1171; found 670.1167. R_t: 3.15 min, HPLC purity: 99.0%.

2-(4-Phenoxybenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (9b).

Prepared as described in the experimental procedure of 7a from intermediate 26b to obtain the title compound as a white solid (15 mg, 33%); ¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.40-7.34 (m, 3H), 7.14 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 7.02-7.00 (m, 2H), 6.94-6.92(m, 5H), 6.75 (d, J = 8.8 Hz, 2H), 6.52 (d, J = 7.6 Hz, 1H), 4.65 (s, 2H), 4.42 (s, 2H), 4.39 (s, 2H), 3.86 (s, 3H), 3.77 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 164.5, 158.7, 158.5, 156.3, 142.9, 135.2, 132.8, 132.1, 131.3, 131.0, 130.6, 130.1, 127.8, 124.6, 120.7, 120.1, 120.0, 119.5, 115.2, 115.0, 114.5, 70.8, 56.3, 56.1, 53.2, 51.9; LC/MS (ESI⁻) m/z 761.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₈H₃₆N₂O₁₂S₂ [M − H]⁻ 761.1480; found 761.1476. R_t: 4.61 min, HPLC purity: 97.0%.

N,N'-bis((1H-tetrazol-5-yl)methyl)-2-(4-fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)benzene (10).

To a solution of 27 (40 mg, 0.06 mmol) in N,N-dimethylformamide (1 mL) were added sodium azide (25 mg, 0.39 mmol) and acetic acid (3 drops) at room temperature. The reaction mixture was stirred at 120 °C for 6 hours. After the reaction was completed, the crude mixture was added to water_, and extracted with ethyl acetate. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatograph (0-10% methanol/dichloromethane) to give the product as a white solid (14 mg, 30%); ¹H NMR (400 MHz, MeOH-d₄) δ 7.40 (d, J = 8.4 Hz, 2H), 7.36-7.33 (m, 4H), 7.21 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.13-7.03 (m, 4H), 6.76 (d, J = 2.4 Hz, 1H), 6.67 (dd, J = 8.4, 2.4 Hz, 1H), 5.12 (s, 2H), 5.10 (s, 2H), 4.50 (s, 2H), 2.43 (s, 3H), 2.36 (s, 3H); ¹³C NMR (100 MHz, MeOH-d₄) δ 162.8, 156.6, 155.6, 146.1, 145.4, 142.1, 137.3, 135.2, 134.7, 133.1, 130.9, 130.7 (J_C,F = 4 Hz), 130.6, 129.1, 128.6, 127.2, 121.4, 116.2 (J_C,F = 22 Hz), 114.8, 70.8, 45.5, 44.2, 21.62, 21.58; LC/MS (ESI⁺) m/z 705.2 [M + H]⁺; HRMS (ESI⁺) m/z calcd for C₃₁H₂₉FN₁₀O₅S₂ [M + H]⁺ 705.1821; found 705.1822. R_t: 4.38 min, HPLC purity: 95.1%.

(2S,2'S)-2,2'-((2-((4-Fluorobenzyl)oxy)-1,4-phenylene)bis(tosylazanediyl))dipropionic acid (11).

Prepared as described in the experimental procedure of 7a from intermediate 28 to obtain the title compound as a white solid (28 mg, 44%); ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 7.6 Hz, 2H), 7.47-7.45 (m, 1H), 7.42 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 7.2 Hz, 2H), 7.16-7.12 (m, 6H), 6.90-6.80 (m, 2H), 4.77-4.74 (m, 1H), 4.57-4.46 (m, 3H), 2.36 (s, 3H), 2.30 (s, 3H), 1.10 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 174.8, 163.7, 161.2, 156.7, 143.7, 143.2, 138.2, 137.3, 136.8, 134.6, 131.6, 129.2, 129.1, 129.0, 127.9, 127.7, 123.9, 116.6, 115.4, 115.1, 69.5, 57.8, 57.7, 21.4, 21.3, 16.9, 15.9; LC/MS (ESI⁻) m/z 683.2 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₃H₃₃FN₂O₉S₂ [M − H]⁻ 683.1539; found 683.1545. R_t: 4.55 min, HPLC purity: 100.0%.

2-(4-Fluorobenzyloxy)-5-methoxy-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (12a).

Prepared as described in the experimental procedure of 7a from intermediate 35a to obtain the title compound as a beige solid (16 mg, 79%); ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.28-7.24 (m, 2H), 7.15 (s, 1H), 7.12-7.08 (m, 2H), 7.01 (s, 1H), 6.97 (d, J = 7.2 Hz, 4H), 4.62 (s, 2H), 4.41 (s, 2H), 4.38 (s, 2H), 3.29 (s, 3H), 2.42 (s, 3H), 2.33 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.19, 170.15, 161.7 (J_C,F = 241 Hz), 149.1, 147.8, 143.34, 143.25, 136.5, 132.4 (J_C,F = 3 Hz), 129.30, 129.25, 129.2 (J_C,F = 9 Hz), 127.7, 127.2, 127.0, 117.7, 116.8, 114.9 (J_C,F = 21 Hz), 69.2, 55.6, 51.0, 21.0, 20.9; LC/MS (ESI⁻) m/z 685.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₂H₃₁FN₂O₁₀S₂ [M − H]⁻ 685.1331; found 685.1338. R_t: 4.47 min, HPLC purity: 97.2%.

2-(4-Fluorobenzyloxy)-6-methoxy-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (12b).

Prepared as described in the experimental procedure of 7a from intermediate 35b to obtain the title compound as a white solid (35 mg, 37%); ¹H NMR (400 MHz, CDCl₃) δ 7.59 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.229-7.19 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.04-7.00 (m, 2H), 6.56 (s, 1H), 6.41 (s, 1H), 4.85-4.76 (m, 2H), 4.43 (s, 2H), 4.21-4.10 (m, 2H), 3.51 (s, 3H), 2.44 (s, 3H), 2.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.6, 170.4, 162.8 (J_C,F = 247 Hz), 157.7, 156.7, 144.6, 144.4, 142.3, 135.9, 135.5, 130.9 (J_C,F = 2 Hz), 129.8, 129.6, 129.5, 128.1, 128.0, 116.2, 115.7 (J_C,F = 21 Hz), 106.5, 105.4, 70.7, 56.4, 53.6, 52.1, 21.71, 21.66; LC/MS (ESI⁻) m/z 685.1 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₂H₃₁FN₂O₁₀S₂ [M − H]⁻ 685.1331; found 685.1327. R_t: 4.47 min, HPLC purity: 96.0%.

2,6-Bis(4-fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (12c).

Prepared as described in the experimental procedure of 7a from intermediate 35c to obtain the title compound as a white solid (25 mg, 45%); ¹H NMR (400 MHz, CDCl₃) δ 7.58 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.29-7.26 (m, 2H), 7.21-7.18 (m, 4H), 7.04-6.99 (m, 6H), 6.56 (s, 2H), 4.84-4.75 (m, 4H), 4.41 (s, 2H), 4.15 (s, 2H), 2.43 (s, 3H), 2.28 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.1, 162.9 (J_C,F = 246 Hz), 156.8, 144.6, 144.5, 142.3, 135.7, 135.5, 130.7, 129.8, 129.6 (J_C,F = 8 Hz), 128.1, 116.5, 115.8 (J_C,F = 21 Hz), 106.5, 70.8, 52.2, 52.0, 21.8, 21.7; LC/MS (ESI⁻) m/z 779.2 [M − H]⁻; HRMS (ESI⁻) m/z calcd for C₃₈H₃₄F₂N₂O₁₀S₂ [M − H]⁻ 779.1550; found 779.1545. R_t: 4.82 min, HPLC purity: 95.4%.

2-(4-Fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)naphthalene-N,N'-diacetic acid (12d).

Prepared as described in the experimental procedure of 7a from intermediate 35d to obtain the title compound as a white solid (38 mg, 77%); ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (brs, 2H, CO₂H), 8.74-8.66 (m, 1H), 8.31-8.21 (m, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.47-7.41 (m, 1H), 7.37-7.33 (m, 3H), 7.20-7.14 (m, 4H), 7.10 (d, J = 8.0 Hz, 1H), 7.03-6.99 (m, 1H), 6.97-6.69 (m, 1H), 6.96-6.93 (m, 1H), 4.56-3.79 (m, 4H), 4.40-4.35 (m, 1H), 4.19-4.08 (m, 1H), 2.33-2.25 (m, 6H); ¹³C NMR (100 MHz, MeOH-d₄) δ 172.5, 172.0, 163.9 (J_C,F = 243 Hz), 153.2, 153.0, 145.6, 145.5, 145.0, 139.4, 139.3, 137.8, 137.6, 137.2, 137.2, 136.7, 136.6, 133.2 (J_C,F = 4 Hz), 130.6, 130.5 (J_C,F = 10 Hz), 130.3 (J_C,F = 9 Hz), 129.5, 129.2, 128.9, 127.9, 127.50, 127.45, 126.0, 125.9, 124.3, 118.5, 118.4, 116.2 (J_C,F = 22 Hz), 116.1 (J_C,F = 22 Hz), 70.9, 70.8, 54.1, 53.7, 21.7, 21.64, 21.57, 21.5; LC/MS (ESI⁺) m/z 707.3 [M + H]⁺; HRMS (ESI⁺) m/z calcd for C₃₅H₃₁FN₂O₉S₂ [M + H]⁺ 707.1528; found 707.1540. R_t: 4.51 min, HPLC purity: 95.4%.

Fluorescence Polarization Competition Assay.

To determine the inhibitory potency of the Keap1-Nrf2 interaction, the FP assay was performed in a 384-well non-bonding surface (NBS) black plate (Corning, 3575). All FP assay data were recorded using a PerkinElmer Wallac Victor 3V microplate reader equipped with excitation (485 nm) and emission (535 nm) filters appropriate for the label detected. The assay buffer solution was 10 mM HEPES pH 7.4 buffer containing 3.4 mM EDTA, 150 mM NaCl, and 0.005% Tween-20. The fluorescent probe used for the FP assay was 9-mer Nrf2 ETGE motif derived peptide, FITC-LDEETGEFL-NH2. In each well, 10 μL of test compounds in 20 % DMSO/HEPES buffer were incubated with 10 μL of 400 nM Keap1 Kelch domain (final concentration, 100 nM) in HEPES assay buffer for 30 minutes at room temperature. Then 20 μL of 20 nM FITC- 9mer Nrf2 amide (final concentration, 10 nM) in the assay buffer was dispensed to each well and incubated again for 1 hour at room temperature. For an evaluation of % inhibition at a single concentration of 50, 5, 0.5 μM, 200, 20, 2 μM of samples in 20 % DMSO/HEPES buffer were prepared, respectively. For determination of IC₅₀ value, compounds were prepared in a ten-point dilution with 20 % DMSO/HEPES buffer. All experiments were performed in triplicates, and all plates were centrifuged at 2500 rpm for 2 minutes at room temperature before and after incubation. Subsequently, the fluorescence polarization was determined by measuring the parallel and perpendicular fluorescence intensity (F║ and F┴) with respect to the linearly polarized excitation light. The % inhibition at each concentration of compounds was calculated according to the method previously reported by our group.⁵⁸ The IC₅₀ values were determined by fitting the % inhibition data to a four-parameter logistic fit using Sigma Plot 12.3 software.

Time-Resolved Fluorescence Resonance Energy Transfer Assay.

TR-FRET assay was performed in a 384-well non-bonding surface (NBS) white plate (Corning, 3574). All TR-FRET assay data were recorded on a PerkinElmer Wallac Victor 3V microplate reader using an excitation wavelength of 340 nm with filters detecting the fluorescent emission signals of terbium at 495 nm and fluorescein at 520 nm. The assay buffer solution was 10 mM HEPES pH 7.4 buffer containing 0.5 mM EDTA, 150 mM NaCl, and 0.005% Tween-20. Terbium labeled-anti-His antibody was purchased from Thermo Fisher Scientific. The fluorescent probe used for the TR-FRET assay was 9-mer Nrf2 ETGE motif derived peptide, FITC-LDEETGEFL-NH2. Test compounds were serially diluted in neat DMSO by a semi-log dilution (100 μM to 1 nM), and added to the assay well plate. His-tagged Keap1 Kelch domain (20 nM) and Terbium-anti-His antibody (2 nM) diluted with the assay buffer were mixed with a ratio of 1:1, and then pre-incubated for 30 minutes at room temperature. Ten microliters of a mixture of the protein (final, 5 nM) and the antibody (final, 0.5 nM) were dispensed to each well where 0.2 μL of compound (final, 1 μM to 0.01 nM) in DMSO was added in advance. After incubation for 30 minutes at room temperature, 9.8 μL of 51 nM FITC-9mer Nrf2 peptide amide (final, 25 nM) in the assay buffer was added and incubated again for 1 hour at room temperature. All experiments were performed in triplicates, and all plates were centrifuged at 2500 rpm for 2 minutes at room temperature before and after incubation. Subsequently, the TR-FRET fluorescence emission intensities at two different nanometers were measured using the microplate reader, and then their ratio was calculated according to the following equation: (intensity of 520nm)/(intensity of 495 nm) x 10,000. The IC₅₀ values were determined by fitting the 520 nm/495 nm ratio data to a four-parameter logistic fit using Sigma Plot 12.3 software.

Cell culture and treatment

NCM460D cells were cultured in 300 μL RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were seeded at 75% confluency in three 48-well plates for ~90% confluency on the day of treatment. On the day of treatment, each well was treated with test compounds (100, 10, and 1 μM), 0.1% DMSO control a negative control, and positive controls 2a (100, 10, and 1 μM) and CDDO (100 and 10 nM), except for one plate with 1% DMSO replacing 1 μM compound 2. The well plate was swirled and mixed for 2-3 minutes, and then incubated for 24 hours at 37°C. Media was aspirated prior to collecting the treated NCM460D cells in 500 μL of Trizol.

RNA quantification, cDNA synthesis, and RT-qPCR

RNA was extracted from NCM460D cells in Trizol using chloroform-based methods and following the instructions from the RNeasy Micro Kit (Qiagen, catalog number 74004). For cDNA, 500 ng of RNA was measured by nanodrop and processed per the SuperScript III first-strand synthesis protocol provided by Invitrogen (Thermo Fisher Scientific, catalog number 18080051). RT-qPCR was conducted with the ABI PRISM 7900HT Sequence Detection System for SYBR green (Thermo Fisher Scientific, catalog number 4309155) and reaction mix containing a 0.5 μM final primer concentration.

Molecular docking simulation.

To analyze the interactions of designed compounds with the active site of Keap1 Kelch domain, molecular docking simulation was performed using the Autodock4.0 software.^[62] The co-crystal structure of compound 2b bound to Keap1 protein was obtained from the RCSB protein data bank (4XMB).^[61] For protein preparation, all water molecules were removed from the structure file, missing hydrogen atoms were added, and Kollman charges were calculated using Autodock tool. The ligand structures were built in MOE and prepared for docking using Autodock. The docking grid was centered on the observed binding pocket of the ligand, and the number of grid points in each direction was set to X, Y, Z = 44, 40, 40 with spacing of 0.375 Å resolution. The prepared ligands were docked into the binding sites of the Keap1 kelch domain. For a docking parameter file, Lamarckian genetic algorithm was used,^[63] the number of GA runs was 100, and all other parameters were set to default values. The results of the docking experiments were reported in kcal/mol, and the docked conformations were ranked according to their binding energy. To validate the docking method, the prepared compound 2b was re-docked using the same method, and then compared with the corresponding X-ray crystal structure. As a result, the top-scoring docked pose was similar to the crystal structure of the ligand with a low RMSD value of 0.94 Å. This demonstrated that the docking method was reliable protocol that could be used for docking simulation of other compounds with the Keap1 Kelch domain.

Supplementary Material

Supplementary Doc

NIHMS1903961-supplement-Supplementary_Doc.docx^{(10.4MB, docx)}

ACKNOWLEDGMENT

We thank the Center for Integrative Proteomics Research (CIPR), Rutgers University for performing the HRMS experiments.

Funding Sources

This work was supported in part by the National Cancer Institute [R01CA133791] and the Rutgers TechAdvance Grant [TA2019-0300].

ABBREVIATIONS

ACN: acetonitrile
AcOH: acetic acid
ARE: antioxidant response element
BINAP: 2,2′-bis (diphenylphosphino)-1,1′-binaphthalene
BTB: broad-complex, Tramtrack and Bric-a-Brac
CDDO-Me: bardoxolone methyl
COPD: chronic obstructive pulmonary disease
Cul3: cullin3
DCM: dichloromethane
DGR: double glycine repeat
DMF: N,N-dimethylformamide
DMSO: dimethyl sulfoxide
FITC: fluorescein isothiocyanate
FP: fluorescence polarization
GCL: glutamate-cysteine ligase
GCLM: glutamate-cysteine ligase modifier
GPx: glutathione peroxidase
GST: glutathione S-transferase
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HO-1: heme-oxygenase-1
HRMS: high resolution mass spectrometry
HTS: high throughput screening
IVR: intervening region
Keap1: Kelch-like ECH-associated protein 1
LC-MS: liquid chromatography mass spectrometry
Maf: musculoaponeurotic fibrosarcoma
MD: molecular dynamics
MM-GBSA: molecular mechanics generalized Born surface area
MS: multiple sclerosis
MsCl: mesyl chloride
MW: molecular weight
NBS: N-bromosuccinimide
Neh: Nrf2-ECH homology
NQO1: NAD(P)H:quinone oxidoreductase 1
Nrf2: nuclear factor erythroid 2-related factor 2
PDB: Protein Data Bank
PK: pharmacokinetics
PPI: protein–protein interaction
RNS: reactive nitrogen species
ROS: reactive oxygen species
SAR: structure–activity relationship
SOD: superoxide dismutase
TEA: triethylamine
THF: tetrahydrofuran
THIQ: tetrahydroisoquinoline
TR-FRET: time-resolved fluorescence resonance energy transfer
TRX: thioredoxin

Footnotes

Supporting Information. The following Supporting Information is available:

Synthetic procedure and characterization of intermediates
Table S1 original data of activity in FP assay for final compounds
1H and 13C NMR spectra of final compounds
UPLC analysis and purity spectra of final compounds

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PERMALINK

Structure-activity relationships of 1,4-bis(arylsulfonamido)-benzene or naphthalene-N,N'-diacetic acids with varying C2-substituents as inhibitors of Keap1-Nrf2 protein-protein interaction

Sumi Lee

Dhulfiqar Ali Abed

Mai-Uyen Nguyen

Michael P Verzi

Longqin Hu

Abstract

Graphical Abstract

INTRODUCTION

Figure 1.

RESULTS AND DISCUSSION

Design strategy.

Figure 2.

Chemistry.

Scheme 1. Synthesis of various 1,4-diamino substituted benzene ring derivatives 7a-ya,b.

Scheme 2. Synthesis of compound 8 with 2-phenoxymethyl substitutiona.

Scheme 3. Synthesis of O-linked benzene ring derivatives 9a-b from key intermediate 25a.

Scheme 4. Synthesis of analogs 10 and 11 including tetrazole and α-methyl acetatea.

Scheme 5. Synthesis of 5 or 6-substituted benzene ring (12a-c) analogsa.

Scheme 6. Synthesis of naphthalene (12d) analoga.

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

Table 1.

Table 2.

Table 3.

Biological evaluation of compound 12d in the TR-FRET assay.

Figure 3.

Effects of compounds 7v and 12d on the transcription of Nrf2-ARE regulated genes.

Figure 4.

Molecular docking analysis.

Figure 5.

CONCLUSION

EXPERIMENTAL

General Chemistry.

2-Phenyl-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7a).

2-(Piperidin-1-yl)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7b).

2-(Benzyl(methyl)amino)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7c).

2-Benzyloxy-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7d).

2-Phenoxy-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7e).

2-Phenethoxy-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7f).

2-(3-Phenylpropoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7g).

2-(4-Methoxybenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7h).

2-(4-Fluorobenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7i).

2-(4-Methylbenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7j).

2-(Naphthalen-2-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7k).

2-([1,1'-Biphenyl]-4-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7l).

2-(Naphthalen-2-yloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7m).

2-(Naphthalen-1-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7n).

2-([1,1'-Biphenyl]-4-yloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (7o).

2-Benzyloxy-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7p).

2-Benzyloxy-1,4-bis(4-phenylphenylsulfonamido)benzene-N,N'-diacetic acid (7q).

2-Benzyloxy-1,4-bis(4-fluorophenylsulfonamido)benzene-N,N'-diacetic acid (7r).

2-Benzyloxy-1,4-bis(4-(trifluoromethyl)phenylsulfonamido)benzene-N,N'-diacetic acid (7s).

2-Benzyloxy-1,4-bis(4-bromophenylsulfonamido)benzene-N,N'-diacetic acid (7t).

2-Benzyloxy-1,4-bis(3-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7u).

2-(4-Fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7v).

2-(4-Fluorobenzyloxy)-1,4-bis(2,4,6-trimethylphenylsulfonamido)benzene-N,N'-diacetic acid (7w).

2-(Naphthalen-2-ylmethoxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7x).

2-([1,1'-Biphenyl]-4-ylmethoxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (7y).

2-(Phenoxymethyl)-1,4-bis(4-methyoxyphenylsulfonamido)benzene-N,N'-diacetic acid (8).

2-(Pyridin-3-ylmethoxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (9a).

2-(4-Phenoxybenzyloxy)-1,4-bis(4-methoxyphenylsulfonamido)benzene-N,N'-diacetic acid (9b).

N,N'-bis((1H-tetrazol-5-yl)methyl)-2-(4-fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)benzene (10).

(2S,2'S)-2,2'-((2-((4-Fluorobenzyl)oxy)-1,4-phenylene)bis(tosylazanediyl))dipropionic acid (11).

2-(4-Fluorobenzyloxy)-5-methoxy-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (12a).

2-(4-Fluorobenzyloxy)-6-methoxy-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (12b).

2,6-Bis(4-fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)benzene-N,N'-diacetic acid (12c).

2-(4-Fluorobenzyloxy)-1,4-bis(4-methylphenylsulfonamido)naphthalene-N,N'-diacetic acid (12d).

Fluorescence Polarization Competition Assay.

Time-Resolved Fluorescence Resonance Energy Transfer Assay.

Cell culture and treatment

RNA quantification, cDNA synthesis, and RT-qPCR

Molecular docking simulation.

Supplementary Material

ACKNOWLEDGMENT

Funding Sources

ABBREVIATIONS

Footnotes

REFERENCES

Associated Data

Scheme 1. Synthesis of various 1,4-diamino substituted benzene ring derivatives 7a-y^a,b.

Scheme 2. Synthesis of compound 8 with 2-phenoxymethyl substitution^a.

Scheme 3. Synthesis of O-linked benzene ring derivatives 9a-b from key intermediate 25^a.

Scheme 4. Synthesis of analogs 10 and 11 including tetrazole and α-methyl acetate^a.

Scheme 5. Synthesis of 5 or 6-substituted benzene ring (12a-c) analogs^a.

Scheme 6. Synthesis of naphthalene (12d) analog^a.