Development of New Benzo[b]Thiophene‐2‐Carboxamide Derivatives as Advanced Glycation End‐Products Receptor (RAGE) Antagonists

Abstract

The activation of the receptor for advanced glycation end‐products (RAGE) induces a chronic, low‐noise inflammation responsible for the aging process, known as inflammaging. Associated with numerous pathologies such as Alzheimer's, insulin‐resistant diabetes, cardiovascular diseases, and certain cancers, RAGE has become an interesting therapeutic target in the context of aging well. To this end, we identified new benzo[b]thiophene‐2‐carboxamide derivatives as potential RAGE ligands. Herein, we developed an alternative approach to easily synthesize benzo[b]thiophene‐2‐carboxamide analogs from 5‐arylidene‐2,4‐thiazolidinedione intermediates based on the Ullmann–Goldberg coupling conditions. In light of LCMS, NMR, X‐ray, and DFT studies, a mechanism for this reaction was proposed. This novel strategy enabled us to synthesize analogs whose best molecule 3t′, with an IC₅₀ of 13.2 µM, shows similar interactions with RAGE as the reference molecule Azeliragon (13.0 µM).

A streamlined Ullmann‐Goldberg strategy enabled the synthesis of benzo[b]thiophene‐2‐carboxamides, promising ligands of the pro‐inflammatory RAGE receptor involved in inflammaging and age‐related diseases. LC‐MS, NMR, X‐ray and DFT studies elucidated the copper‐catalyzed mechanism. The lead compound, 3t’, shows inhibitory activity on the sRAGE/AGE2‐BSA interaction comparable to the reference ligand Azeliragon.

1. Introduction

While life expectancy is increasing, one of the biggest challenges of our century is to ensure independence and extend the health span of our elderlies and thus tackle both the economic and societal problems associated with it. The aging process responsible for a decrease in the body response and function was proven to be very complex with a combination of multiple highly intertwined factors. These are currently classified by the geroscience which aims to explain and predict diseases linked to aging based on the 12 hallmarks of aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient‐sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis.^{[ 1 , 2 ]} Although inflammation is a powerful defense mechanism against injury or infection, chronic low‐grade inflammation is implicated in numerous age‐related diseases: this is inflammaging.^{[ 3} , ⁴ , ^{5 ]} Moreover, the alteration of one or more of the stated aging pillars is likely to generate the presence of a high level of proinflammatory markers in old patient's blood without any apparent trigger.^{[ 1 , 2 ]} In this way, combating inflammaging means directly addressing aging rather than a single pathology. Several sources of chronic inflammation were identified such as damaged macromolecules and cells that accumulate producing damage‐associated molecular patterns (DAMPs), a dysfunction of microbiota releasing pathogen‐associated molecular patterns (PAMPs), and senescent cells secreting senescence‐associated secretory phenotype (SASP).^{[ 6 ]} These circulating molecules activate cell surface receptors known as pattern‐recognition receptors (PRRs) expressed in immune and nonimmune cells and lie behind receptor dependent intracellular biological signaling cascade resulting in massive inflammation.^{[ 7 ]} In this study, we will focus on the receptor of advanced glycation end products (RAGE), well‐known for its involvement in inflammaging.^{[ 8} , ⁹ , ^{10 ]}

Although RAGE first owes its name to its ability to bind advanced‐glycation end‐products (AGEs), this designation becomes more and more obsolete with the discovery of a large variety of other endogenous ligands including PAMPs, DAMPs, Aβ‐fibrils, HMGB1, and S100 proteins but also small molecules. To date, there are more than 30 identified RAGE ligands.^{[ 8 , 10 ]} The physiological role of RAGE remains not fully resolved even if its ability to promote proinflammatory stimuli seems to indicate that it is part of the innate immune system. Moreover, this receptor is present at low level in healthy patients and is overexpressed in numerous age‐related diseases such as cardiovascular^{[ 11 , 12 ]} or neurodegenerative diseases,^{[ 13} , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ^{19 ]} diabetes mellitus,^{[ 20 , 21 ]} lung^{[ 22 ]} and liver acute injury,^{[ 23 ]} and some cancers.^{[ 24} , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ , ^{31 ]} RAGE is a 47–55 kDa transmembrane protein from the immunoglobulin superfamily, made of a variable domain (V, 23–116 amino acid residues), a constant one domain (C1, 124–221), a constant two domain (C2, 227–317), a transmembrane domain (343–363), and finally a cytoplasmic tail (ct, 364–404).^{[ 32 ]} RAGE exists under several isoforms: the full length RAGE, dominant negative RAGE (DN‐RAGE), N‐truncated RAGE (N‐RAGE), and the soluble RAGE (sRAGE) and also as oligomers. Most of the ligands will bind to the positively charged variable domain or VC1 domain and thus activate RAGE through three different pathways. First, the mitochondrial pathway with the activation of NADPH oxidase that will produce reactive oxygen species (ROS) and cause mitochondrial dysfunction.^{[ 8 , 10 ]} Those species could either reactivate RAGE or the inflammasome. RAGE is also able to directly activate NF‐κB responsible for the transcription of proinflammatory cytokines, and it induces endoplasmic reticulum stress and activates the p21 senescence pathway.^{[ 8 , 10 ]}

Thus, playing a crucial role in the inflammaging process, RAGE emerged as a highly promising therapeutic target for better aging. Unfortunately, no RAGE antagonist is currently available on the market. In fact, the molecule Azeliragon, also known as TTP488−303, developed by vTv Therapeutics failed in phase III clinical trial for lack of efficiency on patients with mild to moderate Alzheimer disease.^{[ 33 ]} The FPS‐ZM1 is another small molecule able to inhibit the interaction between RAGE and its ligands Aβ‐fibril (K_i = 25 nM), S100 (K_i = 230 nM) and HMGB1 (K_i = 148 nM), but it has never reached the clinical trials.^{[ 34 ]}

Herein, we report benzo[b]thiophene derivatives as new RAGE ligands. Heterocyclic molecules containing sulfur have been largely investigated in medicinal chemistry. Benzo[b]thiophenes are particularly of interest with FDA‐approved molecules such as Raloxifene, for the prevention and treatment of osteoporosis^{[ 35 ]}; Sertaconazole, an antifungal agent^{[ 36 ]}; Rexulti, prescribed as maintenance therapy of schizophrenia and more recently for agitation associated with dementia due to Alzheimer disease^{[ 37 , 38 ]}; and Zileuton, a treatment of asthma (Figure  1 ).^{[ 39 ]} Although benzo[b]thiophene derivatives are of great importance in medicinal chemistry, the synthesis of substituted benzo[b]thiophene carboxamides could be a limitation. As the benzo[b]thiophene‐2‐carboxylic acid, to be engaged in a peptide coupling with the desired amine, is expensive and poorly diversified, other strategies have been developed. Indeed, the obtention of 2‐substituted benzo[b]thiophene derivatives could result from the thienannulation of the easily available 2‐alkyl‐thiobenzaldehyde and chloroacetamide^{[ 40 ]} or from the nucleophilic displacement of 2‐fluoro or 2‐nitrobenzaldehyde by a methylthioglycolate.^{[ 41 ]} During efforts aimed at synthesizing thiazolidinedione derivatives, a novel and efficient synthetic route to benzo[b]thiophenes was serendipitously identified. Given the interest of this family in medicinal chemistry and due to our long‐standing interest in discovering RAGE antagonists, the compounds were tested on RAGE and preliminary in silico docking studies revealed that these benzo[b]thiophene derivatives displayed more favorable binding affinities and interaction profiles with RAGE than the initially targeted thiazolidinedione compounds.

Figure 1.

RAGE antagonists in the literature and current FDA approved drugs bearing the benzo[b]thiophene heterocycle.

A new two‐step synthetic pathway of substituted benzo[b]thiophene‐2‐carboxamides was developed from commercially available reagents and involved a low‐cost copper‐catalyzed strategy, leading to the formation of the desired amides (Scheme  1 ).

Scheme 1.

Previous work and our novel approach for the synthesis of benzo[b]thiophene‐2‐carboxamide derivatives.

This strategy in the synthesis of benzo[b]thiophene‐2‐carboxamide derivatives was chosen to produce analogs as experimental drugs permitting to analyze the structure–activity relationships of these compounds on RAGE. Their inhibitory effect of the sRAGE/ligand interaction was evaluated by ELISA assay and rationalized with docking studies. The most promising compounds were engaged in toxicity and functionality assays, and finally, their hepatic stabilities were also evaluated in the presence of liver microsomes from female mice.

2. Results and Discussion

2.1. Synthesis of Benzo[b]Thiophene‐2‐Carboxamide Derivatives

A library of the key intermediates 5‐arylidene‐2,4‐thiazolidinedione 2 was synthesized via a Knoevenagel condensation between the 2‐halobenzaldehyde of interest and the nontoxic thiazolidine‐2,4‐dione 1 in the presence of a base (Scheme  2 ). The use of sodium hydroxide was preferred to piperidine due to a better yield (e.g. 91% yield for the sodium salt of compound 2g vs. 37% for the neutral species 2g′) and a shorter reaction time (2 h vs. 16 h, respectively). This reaction only led to the formation of the thermodynamically more stable Z isomer, as described in the literature.^{[ 42} , ^{43 ]} The stereoselectivity of the reaction was confirmed by single X‐ray diffraction for compounds 2d and 2g′ (Scheme 2 and ESI, Tables S3 and S4 and Figure S16–S18, Supporting Information).

Scheme 2.

Synthesis of the 5‐aryliden‐2,4‐thiazolidinedione intermediates 2. X‐ray structure of compounds 2d and 2g; [a] isolated yields when piperidine (0.1 eq.) was used as base instead of NaOH to obtain free bases 2b′, 2d′, and 2g′; e.g. 2g vs 2g′ designates sodium salt versus free base compound.

These 5‐arylidene‐2,4‐thiazolidinedione intermediates 2 were then engaged in the second step under the Ullmann–Goldberg conditions to obtain, in one‐pot, the desired substituted benzo[b]thiophen‐2‐carboxamides 3. Firstly, several parameters of the reaction were optimized using the intermediate 2g and the 4‐bromoanisole (Table  1 ).

Table 1.

Optimization of the reaction conditions.

Entrya)	Base	Cat.	Yield 3g [%]	Yield 4g [%]
1	Cs 2 CO 3	CuI	43a)	Traces
2	Rb 2 CO 3	CuI	20a)	Traces
3	K 3 PO 4	CuI	35a)	7
4	K 2 CO 3	CuI	Traces	10
5	Cs2CO3	CuI	49/52b)	Traces
6	Cs2CO3	CuCl	44c)	_
7	Cs2CO3	CuBr	52	_
8	Cs2CO3	CuBr 2	17	Traces
9	Cs2CO3	w/o	n.d.	n.d.

^a) Reaction carried out with 2g (1.69 mmol, 1 eq.), 4‐bromoanisole (1.69 mmol, 1 eq.), CuI (0.84 mmol, 0.5 eq.), N,N′‐DMEDA (2.03 mmol, 1.2 eq.), and Cs₂CO₃ (5.07 mmol, 3 eq.);

^b) 4‐Bromoanisole was introduced in excess (3.38 mmol, 2 eq.);

^c) Isolated yield obtained with a CuI of 99.998% purity vs 98% purity when not stated; Cat. = catalyst; n.d. = not detected; w/o = without catalyst.

After this study, the desired compound 3g was obtained in 52% yield when the reaction was carried out using CuI as catalyst, N,N′‐DMEDA as ligand, and Cs₂CO₃ as base in the weakly polar dry toluene at 110 °C under nitrogen atmosphere. It is worth noticing that when 2 equivalents of the bromoaryl were engaged, a second coupling on the amide was observed. In the meantime, bases like K₂CO₃ seemed to favor the formation of a subproduct identified as thioacrylamide derivative 4g. The latter also appeared in a smaller extent with Cs₂CO₃. The presence of this subproduct may increase purification difficulty by flash chromatography. One hypothesis regarding the mechanism of the reaction was a copper‐catalyzed ring opening of the compound 2 to form an acyl isocyanate intermediate that will, after recyclization into the benzo[b]thiophene moiety and a decarboxylation, undergo an Ullmann–Goldberg coupling with the bromoaryl present in the reaction medium. In Ullmann–Goldberg reactions, the choice of the bidentate and diamine ligands is of great importance, and also Buchwald et al. reported that an excess of ligands over copper is favorable by preventing a double coordination of amide on the reactive copper which would lead to the deactivation of the latter.^{[ 44 ]} Hence, we worked in this study with a ratio CuI/ligand of 1 : 2.4. A short selection of five ligands was tested to understand the influence of the amine or diamine and between primary, secondary and tertiary amines (Scheme  3A). Interestingly, the desired benzo[b]thiophene 3a was only obtained with the ligand N,N′‐DMEDA 6. If the tertiary amines 7, 8, and 9 did not promote the reaction, the primary amine 5 was highly reactive toward the 4‐bromoanisole in these conditions and was completely consumed in a side Ullmann coupling reaction to afford the compound 11 (Scheme 3B). However, the formation of the 6‐chlorobenzo[b]thiophene‐2‐carboxamide intermediate 10 was still observed. This finding suggests that the reaction occurs in two steps: first, the formation of intermediate 10 via ring opening of the thiazolidinedione, decarbonylation, and copper catalyzed intramolecular S‐arylation, followed by a classical Ullmann–Goldberg coupling reaction on the amide group with the bromoaryl present in the reaction medium. In this case, the second step was prevented due to the total reagent consumption of 5 (Scheme 3B). A mechanistic study will be investigated and described later in this report.

Scheme 3.

A. Ligands tested for the formation of benzo[b]thiophene‐2‐carboxamide: the N,N‐dimethylethylenediamine 5, N,N′‐dimethylethylenediamine 6, N,N,N′,N′‐tetramethylethylenediamine 7, tris[2‐(2‐methoxyethoxy)ethyl]amine 8, 1,10‐phenantroline 9; B. Reaction of the arylidene‐2,4‐thiazolidinedione 2g and 4‐bromoanisole under Ullmann conditions with the ligand 5.

Then, we wanted to highlight the difference in reactivity due to the halide in ortho position of the aryliden‐2,4‐thiazolidinedione intermediate 2 (Table  2 ). We noted a slight improvement of the isolated yield of compound 3 with X₁ = I or Br compared to X₁ = Cl with 29, 31, and 18% yield, respectively. Not surprisingly, the compound 3 was not observed with X₁ = F, to the benefit of the thioacrylamide derivative 4 formation. However, entries 1 and 3 also suggested an influence of the arylidene‐2,4‐thiazolidinedione 2 substituents in the reactivity. Hence, the scope of this reaction was investigated with differently substituted intermediates 2. In all cases, the desired benzo[b]thiophene‐2‐carboxamide was obtained (Scheme  4 ). The substitution of the benzene moiety of 2 with chlorine atom, either in the para or meta position seemed to favor the reaction compared to the unsubstituted intermediate 2a with respectively, 48, 42, and 18% yield. The effect of steric hindrance remained insignificant with methoxy groups in both meta and para positions which allow the synthesis of compound 3i with 24% yield, quite like the yield obtained with other electron‐donating groups such as methyl or N,N‐dimethyl for compounds 3h and 3j with 28% and 27% yield, respectively. Thus, even if higher yields were obtained with electron‐withdrawing groups, electron‐donating groups were still tolerated with more moderate yield. This finding is interesting given that few examples of inactivated aryl chlorides engaged in copper‐catalyzed reactions are described in the literature.^{[ 45 , 46 ]} However, copper catalyzed intramolecular O‐arylation or aryl amidation with aryl chlorides were previously reported for the benzo[d]oxazoles^{[ 47 ]} or indoline‐1‐carbaldehyde^{[ 48 ]} synthesis but was never, to our knowledge, extended to the intramolecular S‐arylation.

Table 2.

Influence of the halogen atom on the ortho position of the arylidene‐2,4‐thiazolidinedione intermediates 2.

Entry	X1	X2	Yield 3 [%]	Yield 4 [%]
1	Cl	Cl	48	Traces
2	F	F	0	23
3	Cl	H	18	0
4	Br	H	31	0
5	I	H	29	0

Scheme 4.

Scope of copper‐catalyzed reaction with substituted sodium (Z)‐5‐(2‐chlorobenzylidene)‐2,4‐dioxothiazolidin‐3‐ide 2 and the 4‐bromoanisole; the structures of compounds 3g and 3i were confirmed by X‐Ray diffraction analysis (see ESI, Table S4 and Figure S19, S20, S22a, S22a, Supporting Information); in this series, 3g vs 3g′ designates the S‐arylation and mono Ullmann–Goldberg coupling product versus the S‐arylation and double Ullmann–Goldberg coupling product obtained in the reaction with the same bromoaryl.

The substrate scope of the transformation was further explored with the most reactive intermediate sodium (Z)‐5‐(2,4‐dichlorobenzylidene)−2,4‐dioxothiazolidin‐3‐ide 2g (Scheme  5A). The reaction of 2g with aryl bromide bearing various substituents at ortho, meta, and/or para positions successfully resulted in the desired benzo[b]thiophene‐2‐carboxamide 3. However, the steric hindrance generated with a methoxy group on the ortho position on 3o seemed to decrease the reactivity of the coupling compared to the compounds 3g and 3p with 26%, 48%, and 72%, respectively. This drop in reactivity for compound 3o is coupled with the formation of the thioacrylamide derivative 4o in sufficient quantity to isolate it by flash chromatography. The structure of molecule 4o was confirmed by single X‐ray diffraction (Scheme 5A and ESI, Tables S3 and S4 and Figure S25–28, Supporting Information). On the other hand, the influence of the substituents electronic properties was difficult to rationalize. The introduction of the aryl bromide in excess could in some cases lead to the disubstituted amide. The structures of compounds 3p′ and 3q′ were confirmed by single X‐ray diffraction (Scheme 5 and ESI, Tables S3 and S4 and Figures S22–S24, Supporting Information). These disubstituted amides could also be obtained in two steps, from the isolated monosubstituted benzo[b]thiophene‐2‐carboxamide. As shown in Scheme 5B, the compound 3u resulted from the Ullmann–Goldberg coupling reaction of 3,4,5‐trimethoxybromoaryl and the previously synthesized 3t in the same conditions with 86% yield.

Scheme 5.

A. Scope of the reaction; in this series, compounds 3 vs 3′ designates the S‐arylation and mono Ullmann–Goldberg coupling product versus the S‐arylation and double Ullmann–Goldberg coupling product obtained in the reaction with the same bromoaryl. B. Insertion of a second chain on the amide via an Ullmann–Goldberg coupling reaction in the same conditions with different bromoaryl derivative; C. Cyclization step from a N‐substituted intermediate 12a or 12b; (*) yield obtained with only 1 equivalent of bromoaryl.

Interestingly, N‐substituted 5‐arylidene‐2,4‐thiazolidinedione with aryl or benzyl group 12 could also undergo the intramolecular cross coupling reaction to give compounds 3g and 3v. The one‐pot strategy appeared to be advantageous for the synthesis of compound 3g.

To generalize the procedure to the synthesis of benzo[b]furan and indole, the Ullmann–Goldberg coupling conditions were applied on the oxazolidine‐2,4‐dione and hydantoin analogs of the 5‐arylidene‐2,4‐thiazolidinedione 2. As shown in Scheme  6a, difficulties were encountered in the synthesis of compound 15. In fact, the dehydration step was not successful, leading to the obtention of the intermediate 14. The latter failed to give the desired product 15 when engaged in a mixture of P₂O₅/methanesulfonic acid (1:10 and 1:2), heated in the presence of H₂SO₄ or when treated with Martin's sulfurane dehydrating agent (5 eq.). However, the intermediate 14 was still engaged under the Ullmann–Goldberg conditions and provided only traces of the mono‐ and disubstituted benzo[b]furane‐2‐carboxamide compounds 16 and 16′, respectively. A single crystal X‐ray analysis confirmed the structure of compound 16′ (Scheme 6a and ESI, Tables S3 and S4 and Figures S19 and S21, Supporting Information). In the same coupling conditions, the hydantoin derivatives 17 did not react (Scheme 6b).

Scheme 6.

Investigation of the synthesis of benzo[b]furan and indole compounds from the oxygen and nitrogen analogs of 5‐arylidene‐2,4‐thiazolidine intermediates 15 and 17, respectively; compound 16 vs 16′ designates the O‐arylation and mono Ullmann–Goldberg coupling product versus the O‐arylation and double Ullmann–Goldberg coupling product obtained in the reaction with the same bromoaryl.

As copper‐catalyzed conditions were described for intramolecular cyclization of aryl chlorides leading to the formation of benzo[d]oxazoles^{[ 47 ]} or cyclic aryl ether,^{[ 49 ]} we tried out our conditions on the 2‐chlorophenylacetone 18, 2‐(2‐chlorophenyl)ethanol 20 and the 2,4‐dichlorophenethylamine 22. We successfully obtained the 2‐methylbenzofuran 19 and the 2,3‐dihydrobenzofuran 21, but unsurprisingly, the intramolecular cyclization did not occur with the amino derivative 22 (Scheme  7 ).

Scheme 7.

Application of reaction conditions on 2‐chlorophenylacetone 18, 2‐(2‐chlorophenyl)ethanol 20 and 2,4‐dichlorophenethylamine 22; (*) conversion rate obtained by ¹H NMR.

2.2. Investigation of the Reaction Mechanism

To highlight the intermediates formed during this reaction and to be able to propose a mechanism, LCMS and NMR studies and DFT calculations were carried out.

2.2.1. LCMS Studies

The reaction between the intermediate 2g and the 5‐bromo‐1,2,3‐trimethoxybenzene introduced in excess (2 eq.) in the previously defined standard conditions was monitored by LCMS at 0.25, 1, 2, 3, 4, and 6 h of reaction. After 2 h, the UV spectra indicated that the intermediate 2g reacted fully for the benefit of the benzo[b]thiophene‐2‐carboxamide intermediate 10, which was then consumed to form the monosubstituted compound 3p (see ESI, Figures S2,3,5,Supporting Information)). The absorbance peak corresponding to compound 3l decreased in turn to form the disubstituted benzo[b]thiophene 3p′ (see ESI, Figures S2–4, Supporting Information)). It was also from the second hour of reaction that the thioacrylamide derivative appeared under a peak with retention time of 9.33 min (see ESI, Figures S2,6, Supporting Information)). The latter reached a maximum after 3 h and then decreased in favor of the absorbance peaks at 9.44 and 9.54 min (see ESI, Figure S2, Supporting Information). These two new signals may correspond to the formation of a chromene and 2‐quinolone derivatives. Although these compounds could not have been isolated in this case, such derivatives have already been identified when the thioacrylamide 4p was engaged into the copper‐catalyzed system (see ESI, Scheme S1, Supporting Information). From the LCMS study, it was also interesting to notice in the total ion chromatogram (TIC) a non‐UV‐visible compound corresponding to the 1,3‐dimethyl‐2‐imidazolidinone 25, a by‐product of the reaction (see ESI, Figure S8, Supporting Information).

2.2.2. NMR Studies

To corroborate the LCMS results, the formation of the potential intermediate 10 (Scheme 3) was monitored by NMR spectroscopy. Firstly, we studied the influence of the catalytic system on the ¹H and ¹³C NMR spectra of the 5‐arylidene‐2,4‐thiazolidinedione 2g. A strong broadening of the proton spectrum signals was observed in the presence of the CuI/N,N′‐DMEDA mixture, making impossible the attribution of the signals to the protons of the molecule (see ESI, Figure S10, Supporting Information). On the other hand, the ¹³C NMR spectra of the intermediate 2g in the presence of the catalytic system revealed a broadening and/or a chemical shift of carbon signals varying in intensity according to their involvement in the formation of a complex. As shown in Figure  2 (see ESI Figure S11, Supporting Information for a zoom in), the carbons of the thiazolidine‐2,4‐dione ring are highly affected by the new chemical environment with an important broadening of the signal of the carbons C2, C4, and C5. Interestingly, the carbon C8 bearing a chlorine atom in ortho position appeared to be more affected by a broadening of its signal (Δν ≈ 11.8 Hz) than the other phenyl carbons (Δν ≈ 5–8 Hz). Then, it was worth noticing that a more important deshielding occurred to the signal corresponding to the ethylene carbon C6 and to the quaternary carbons C2 and C5 with +1.25 ppm, +1.83 ppm, and +1.42 ppm, respectively, compared to the signals of the carbons from the phenyl ring (between +0.11 and +0.18 ppm). These observations seemed to point out that the carbons C2, C4, C5, C6, and C8 were highly influenced by the catalytic system and thus were likely to be close to the chelation site of the copper.

Figure 2.

Superposition of 13C NMR spectra of compound 2g (in blue) and of compound 2g in the presence of cuI (0.5 eq.) and N,N′‐DMEDA (1.2 eq.) in DMSO‐d6 at 298 K.

Taking these findings into account and the oxygen and sulfur atoms propensity to be coordinated with the copper, we proposed the formation of the copper complex 26 (Figure  3 ). Then, we carried out an ex‐situ NMR kinetic monitoring of the reagent 2g when engaged with the catalytic system CuI/N,N′‐DMEDA in deuterated DMSO‐d ₆ at 100 °C to highlight its consumption and the formation of new species. First, with the disappearance of the signal of the carbon C6, we noticed a complete consumption of compound 2g after 60 min (Figure  4 ). A contrario, traces of the signal at 163.5 ppm of the amide bond carbon (in gray) of compound 10, started to be visible after 15 min and continued to intensify until 120 min of reaction. It was interesting to notice that while the signal of carbon C6 of compound 2g was no longer visible after 120 min, signals at chemical shifts very similar to carbons C7, C10, C8, C12, C9, and C11 were present on the spectra (in cyan boxes). Very weak signals in the range from 160 to 190 ppm were also present on the ¹³C NMR spectra of the 15‐ and 60‐min samples, before disappearing at 120 min. This evolution of signals as a function of time reaction is characteristic of an intermediate and a signal at 168.4 ppm suggested an opened form with an amide group. This observation could be consistent with a ring opening of the thiazolidinedione, resulting in the formation of a new complex with copper. Unfortunately, this intermediate could not be isolated or formed in situ in sufficient quantities for characterization.

Figure 3.

Proposition of a copper complex formed in the reaction mixture under cuI/N,N′‐DMEDA conditions.

Figure 4.

1D ¹³C{¹H} spectra of the ex‐situ NMR monitoring of compound 10 formation after 5, 15, 60 and 120 min of reaction. Are represented in gray the carbon of the amide bond of compound 10; in dotted line the signal corresponding to the carbon atoms of the subproduct 25.

Finally, the NMR monitoring confirmed the formation of the subproduct 25 with the appearance of a signal at 161.8 ppm, which, according to a 2D ¹H‐¹³C HMBC NMR map is correlated with two singlets at 3.22 and 2.65 ppm of the proton spectra integrating, respectively, for 6 and 4 protons (see ESI, Figure S12, Supporting Information).

2.2.3. DFT Studies

To better understand the mechanisms of the reaction that could explain the observed differences in reactivity between the chlorinated compound 27a and the fluorinated one 27b (Scheme  8 ), we analyzed the processes involved using computational methods. The study was conducted at the M06‐2X/LanL2DZ level of theory. We first examined the cyclization reaction of the halogenated adducts in the presence of Cu(TMEDA) (see ESI, Section 2.4.2., Supporting Information Total energy and Cartesian coordinates). For the different (Cl or F) adducts, we identified four types of copper coordination with the reagent, resulting from the approach of Cu(TMEDA) (see ESI, Section 2.4.2., Supporting Information, Total energy and Cartesian coordinates). Surprisingly, all the formed precomplexes exhibit lower stability than the isolated reactants. The most stable precomplex is situated 9.2 kcal mol⁻¹ (Cl) and 8.2 kcal mol⁻¹ (F) above the energy of the reactants. The energy barrier associated with the cyclization of adducts 27a and 27b are similar, 25.4 and 25.9 kcal mol⁻¹, respectively. However, notable differences emerge when considering the products: 27a leads to a final compound with a stability of 32.0 kcal mol⁻¹ relative to the reactants, whereas 27b shows only a stability gain of 4.4 kcal mol⁻¹. These results align perfectly with the synthesis observations, indicating that the chlorinated reagent clearly follows an irreversible mechanistic pathway, while the fluorinated reagent does not display a marked preference for either reactants or products, leading to a reversible reaction. Finally, it is important to note that the anticipated four‐center transition state, designated as 28a/b, was observed in both reaction pathways. In the case of intramolecular cyclization, this transition state involves the formation of a four‐membered cycle between copper, chlorine, sulfur, and carbon.

Scheme 8.

Proposed mechanism for the synthesis of benzo[b]thiophene‐2‐carboxamide derivatives 3 from 5‐arylidene‐2,4‐thiazolidinedione 2 in the presence of a bromoaryl (1 eq.), CuI (0.5 eq.), N,N′‐DMEDA (1.2 eq.) and Cs₂CO₃ (3 eq.); a. Formation of the key intermediate A; b. The Ullmann–Goldberg coupling reaction of the intermediate A to synthesize the desired product 3; c. Thioacrylamide derivatives 4 formation from intermediate 27 (27a when X = Cl and 27b when X = F) and two successive copper‐catalyzed coupling reactions.

To get further insights into the experimental results, we conducted a computational study of the reaction between 27a or 27b and bromobenzene (see ESI, Section 2.4.2., Supporting Information, Total energy and Cartesian coordinates). The resulting Van der Waals complexes between the fluorinated species and the bromobenzene, as well as the chlorinated one, were found to be 5.9 and 5.7 kcal mol⁻¹ more stable than the reactants, respectively. The activation energies were determined to be 32.7 for 27a and 32.6 kcal mol⁻¹ for 27b, which aligns with the requirement for elevated temperatures (T = 110 °C). Notably, the fluorinated product exhibited substantial, with an energy of—30.8 kcal mol⁻¹, indicating that its formation is effectively irreversible.

As a result, the chlorinated species 27a forms a cycloadduct due to a favorable reaction pathway and a stable product. The feasibility of the intramolecular cyclization precludes any interaction between the bromoaryl and reagent 27a, as its formation directly leads to the cycloadduct. On the other hand, the reaction involving 27b clearly indicates a reversible reaction. As a result, the solution contains a balanced mixture of 27b and the corresponding cycloadduct. The approach of the bromoaryl within the solvation sphere of 27b lead to the formation of a significantly more stable compound. Thus, as this reaction is irreversible, it results in a shift of the previous equilibrium in favor of 27b, resulting in a gradual disappearance of the cycloadduct, which aligns well with the experimental observations.

2.2.4. Proposed Mechanism

In light of all these studies, we proposed a plausible mechanism for the formation of benzo[b]thiophene‐2‐carboxamide regarding the one‐pot strategy presented herein. This mechanism could be separated in two major steps. First is the formation of the key intermediate A (Scheme 8a) and then a classic Ullmann–Goldberg catalytic cycle between the amide and the bromoaryl (Scheme 8b). As suggested by the NMR study of the influence of the catalytic system on the intermediate 2g, the complex 26 could be formed in the reaction mixture. This copper complex would then activate the carbon C2 for a nucleophilic attack of the diamine ligand present in excess. A second intramolecular nucleophilic substitution would result in the release of compound 25, identified in both LCMS and NMR monitoring of the reaction, and the formation of a new copper‐complex 27. According to the mechanistic study of Lefèvre et al. on the copper‐catalyzed C‐N and C–O bond formation with a phenyl chloride, the complex 27 is likely to form a three‐center transition state 28 to undergo the intramolecular oxidative addition.^{[ 50 ]} Then, the complex 29 could undergo a reductive elimination to give the key intermediate A. Finally, the latter could be engaged in a classic Ullmann–Goldberg catalytic cycle with the oxidative addition of the bromoaryl followed by the reductive elimination of the desired product 3. In the presence of an excess of reactive bromoaryl, the compound 3 could undergo a new Ullmann–Goldberg coupling reaction to form the disubstituted compound 3′. In the case of less reactive aryl chloride or for aryl fluoride, the intramolecular oxidative addition would not occur, and the thiol could react directly with the bromoaryl present in the reaction media via an Ullmann coupling as well as the amide group to form the corresponding thioacrylamide 4 (Scheme 8c).

2.3. Structure‐Activity Relationship

Evaluation of benzo[b]thiophene derivatives for their inhibitory effect of the sRAGE/ligand interaction via ELISA assays. The previously synthesized molecules were tested on commercially available ELISA plates at 100 µM concentration to establish a structure–activity relationship and identify the more promising molecules able to block the sRAGE/ligand interaction. In fact, this competitive ELISA test provides the inhibition percentage of the interaction between the known RAGE ligand (AGE2‐BSA) and sRAGE induced by the molecule of interest. As shown in Table  3 , the inhibition effect of the benzo[b]thiophene derivatives 3 on the target interaction highly depends on the substituents. The presence of electron donating groups on the benzo[b]thiophene ring induced a blocking effect of the sRAGE/ligand interaction compared to electron withdrawing group or unsubstituted aryl. Nevertheless, a chlorine atom on the position 8 of the benzo[b]thiophene increases the inhibition compared to the substitution of position 7, with respectively 29% inhibition for the molecule 3g against 45% inhibition for compound 3f. Regarding the substitution of the amide, the presence of an aryl substituted in para position with a methyl 3l, methoxy 3g, or a cyano 3n group enables an inhibition of 9, 29, or 26%, respectively. However, unsubstituted aryl in compound 3k or a substitution with a bisphenyl group 3 m failed to inhibit the AGE2‐BSA/sRAGE interaction. The most promising side chains were the 1‐chloro‐4‐phenoxybenzene 3r, the 1‐fluoro‐4‐phenoxybenzene 3s, and the phenoxyethylpyrrolidine 3t with up to 77, 52, and 89% inhibition, respectively. Increased side chain flexibility and insertion of a pyrrolidine ring are conducive to disrupting the sRAGE/ligand interaction. It was also interesting to notice that, compared to analog 3t (89% inhibition, IC₅₀ of 52.5 µM), the introduction of a second chain with the molecule 3t’ improved the effect with 97% inhibition and an IC₅₀ value of 13.2 µM, therefore similar to Azeliragon with an IC₅₀ value of 13.0 µM, used here as a positive reference. The disymmetrization of the side chain for the compound 3u, decreases the disrupting effect of the AGE2‐BSA/sRAGE interaction with 49% inhibition. The thioacrylamide, 2‐quinolone, and chromene derivatives did not show any effect on the interaction of AGE2‐BSA with sRAGE (see ESI, Table S2, Supporting Information).

Table 3.

Structure‐activity relationship activity of benzo[b]thiophene derivatives 3.

Compounda)	Structure	% inhibition at 100 µM
3a		0
3f		45
3g		29
3h		37
3i		n.t.
3j		59
3k		0
3l		9
3m		0
3n		26
3o		99
3p		0
3q		0
3r		77
3s		52
3t		89 (52.5)
3v		25
3l′		0
3p′		0
3q′		0
3t′		97 (13.2)
3u		49

^a) % interaction evaluated at 100 µM by competitive ELISA test, with Azeliragon as reference; () correspond to the IC50 value; n.t. = not tested.

Docking and NMR studies. After a redocking validation step of our methodology, interactions between the benzo[b]thiophene carboxamide derivatives and the domain VC1 of RAGE were evaluated in silico on the crystal structures available on the protein data bank (PDB 3O3U and 6XQ9).^{[ 51 , 52 ]} Interestingly, they interacted with the same hydrophobic pocket on the variable domain identified by Matsumoto et al. as the AGEs binding site (Figure  5a).^{[ 51 ]} Van der Waals interactions were dominant with a high proportion of proline, leucine and valine amino acids (Pro45, Pro46, Leu49, Val63, Leu64, Pro66, Val78, Leu79, and Pro80). Moreover, a possible hydrogen bond was also identified between the Trp51 nitrogen atom of the amide bond and the oxygen of the molecule 3t phenoxybenzene. A fine‐tuning of the second chain inserted on the amide group plays a crucial role enhancing the contact area with the receptor leading to a significant decrease of the binding energy (−9.52 kcal mol⁻¹ for compound 3t′ vs −7.65 kcal mol⁻¹ for compound 3t). These results were consistent with the higher inhibition percentage of the molecule 3t′ than 3t observed on the ELISA assays. Despite a different orientation of the benzo[b]thiophene moiety in the cavity, it binds to the same pocket for both mono‐ and disubstituted molecules represented in orange and magenta on Figure 5, respectively. The importance of this central core was also highlighted by STD experiment using NMR spectroscopy. Despite a very poor solubility in the buffer medium, interactions between the benzo[b]thiophene and pyrrolidine ring protons with the sRAGE were observed after 46 h of acquisition (see ESI, Figure S15, Supporting Information).

Figure 5.

a) Surface and cartoon representation of a part of the variable domain of RAGE (PDB 3O3U). In white the hydrophobic cavity, in yellow the most implicated amino acids in the binding with AGE‐BSA, and in cyan the weakly implicated amino acids in the binding of AGE‐BSA according to Matsumoto et al. studies.^{[ 28 ]} Area represented in: orange the first cluster of compound 3t with a binding energy of −7.65 kcal mol⁻¹; in magenta, the first cluster of compound 3t′ with a binding energy of −9.52 kcal mol⁻¹. b) Zoom in on the amino acids implicated in the interaction. In red the distance for the potential hydrogen bound between the nitrogen of Trp51 and the oxygen of the phenoxybenzene of compound 3t (2.68 Å).

2.4. Cytotoxicity Evaluation

Several teams have shown that invalidation of the Ager gene, encoding the RAGE receptor, protects against certain age‐related pathologies (Alzheimer's, cardiovascular diseases, nephropathies),^{[ 8 , 10 , 53 , 54 ]} and low‐grade sustained sterile inflammation also known as inflammaging.^{[ 53 , 54 ]} Skeletal muscle is also impacted by aging (sarcopenia) and differentiated C2C12 and AB678 muscle cells express RAGE.^{[ 55 ]} Therefore, we evaluated on‐target cell toxicity in relevant cells.

MTS cell viability assays on immortalized mouse myoblast cell line (C2C12) showed no cytotoxicity after 72 h for compounds 3o, 3p′, 3r, and 3s at 100 µM. These results were confirmed on immortalized human myoblast cell line (AB678) except that we noticed a higher sensitivity of AB678 cells than C2C12 cells for compounds 3r and 3s at 100 µM. We observed for molecules 3r and 3s an AB678 cell viability rate of less than 40% at 100 µM against 79% and 76%, respectively, at 10 µM on C2C12 cell lines. However, for molecules 3t and 3t′, carrying the phenoxyethylpyrrolidine side, only 2% and 5% of cell viability, respectively, was observed at 100 µM in C2C12. The cytotoxicity of molecules 3t and 3t’ was confirmed at 10 and 100 µM on AB678 cell line. Nevertheless, it is worth noticing that reference RAGE antagonist Azeliragon showed cytotoxicity on both cell lines tested, at 10 µM, in the same conditions (Figure  6 ).

Figure 6.

Effects of RAGE‐targeting compounds on muscle cell viability. A) Cell viability in C2C12 cells. Azeliragon (10 µM), compound 3t (100 µM), and compound 3t′ (100 µM) were significantly cytotoxic. B) Cell viability in AB678 cells. Azeliragon (10 µM), compounds 3s (100 µM), 3r (100 µM), and 3t and 3t′ (10 and 100 µM) induced significant cell death. n = 3 independent experiments, *p < 0.05 vs. control, one‐way ANOVA.

Microsomal studies. The most promising molecules 3t and 3t′ were subjected to microsomal stability test carried out by the Engineer Analytical Chemistry ADME platform of University of Lille on a pool of female mouse microsomes. Both molecules 3t and 3t′ showed a good stability in the presence of mouse female liver microsomes with a Cl_int of 25 µL/min/mg and 8 µL min mg⁻¹, respectively, and a half‐life largely higher than 40 min (see ESI for the report).

3. Conclusions

We have demonstrated a novel strategy that provides substituted benzo[b]thiophene‐2‐carboxamide derivatives from 5‐arylidene‐2,4‐thiazolidinedione intermediates. The optimized conditions of the catalytic system (CuI/N,N′‐DMEDA) in the presence of Cs₂CO₃ enabled the intramolecular S‐arylation with aryl chlorides with good tolerance toward inactivated aryl chlorides. This copper‐catalyzed reaction was then successfully generalized on the cyclization of 2‐chlorophenylacetone and 2‐(2‐chlorophenyl)ethanol. Mechanistical investigations through LCMS, NMR, X‐ray, and DFT studies allowed us to propose a two‐step mechanism with, first, the formation of a caesium benzo[b]thiophene‐2‐carbonylamide intermediate that will then undergo an Ullmann–Goldberg catalytic cycle to provide the desired product. This copper‐catalyzed strategy also enabled the formation of chromene and 2‐quinolone derivatives in a very efficient manner from thioacrylamides.

The synthesized molecules were then evaluated by competitive ELISA assays on the sRAGE. The most promising compound 3t′ achieved an IC₅₀ value of 13.2 µM, like the positive reference Azeliragon (13.0 µM). Nevertheless, as Azeliragon, this molecule has cytotoxic effects. The interactions with the receptor were evidenced by STD experiment and rationalized by docking studies. Hence, this work enables the identification of promising benzo[b]thiophene derivatives and mark an important starting point for the elaboration of new drug candidates for pathological aging. In fact, these molecules shown a very good stability in the presence of mouse female liver microsomes. Fine‐tuning of the side chain of the benzo[b]thiophene derivatives should enable to increase the shape complementarity with the hydrophobic pocket of the variable domain of RAGE as well as improving the pharmacokinetic properties.

Conflict of Interest

There are no conflicts to declare.

Author Contributions

Lisa Bonin: design of the study, data curation, formal analysis, investigation, methodology (docking, organic synthesis, LCMS and STD experiments), software, visualization, writing—original draft. Matthieu Hedouin: data curation, formal analysis, DFT studies. Christophe Furman: performed ELISA assays. Ophélie Not: performed cytotoxicity assays. Steve Lancel: data curation, supervision cytotoxicity, funding acquisition, resources. Mona Bensalah: investigation, methodology on myoblast cell lines. Gael Coadou: supervision, docking. Eric Boulanger: resources, supervision. Sergiu Shova: data curation, X‐Ray experiments. Hassan Oulyadi: conceptualization of the DFT and NMR studies, formal analysis, resources, supervision, validation. Alina Ghinet: Conceptualization, data curation, formal analysis, funding acquisition, project administration, resources, supervision, validation, writing—contribution to original draft and review & editing.

Supporting information

Supplementary Material

Bonin Lisa, Hedouin Matthieu, Furman Christophe, Not Ophélie, Lancel Steve, Bensalah Mona, Coadou Gael, Boulanger Eric, Shova Sergiu, Oulyadi Hassan, Ghinet Alina, ChemMedChem 2026, 21, e202500503, 10.1002/cmdc.202500503

Data Availability Statement

The data supporting this article have been included as part of the Supplementary Information. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.