Synthesis and Evaluation of Novel Benzofuran Derivatives as Selective SIRT2 Inhibitors

Abstract

A series of benzofuran derivatives were designed and synthesized, and their inhibitory activites were measured against the SIRT1–3. The enzymatic assay showed that all the compounds showed certain anti-SIRT2 activity and selective over SIRT1 and SIRT3 with IC₅₀ (half maximal inhibitory concentration) values at the micromolar level. The preliminary structure–activity relationships were analyzed and the binding features of compound 7e (IC₅₀ 3.81 µM) was predicted using the CDOCKER program. The results of this research could provide informative guidance for further optimizing benzofuran derivatives as potent SIRT2 inhibitors.

1. Introduction

In humans, 18 histone deacetylases (HDACs) have been identified, which can be subdivided into four classes (HDAC Classes I–IV) primarily based on their homology to yeast HDACs [1,2]. Classes I, II and IV HDACs are referred to as “classical” HDACs and are dependent on Zn²⁺ for deacetylase activity. The only exception is the “unique” class III HDAC, or Sirtuins, as they require NAD⁺ (Nicotinamide adenine dinucleotide) instead of Zn²⁺ as a cofactor [3,4]. There are seven human Sirtuins (SIRT1–7) that present diversity in cellular localization and function [5]. SIRT1 is mainly a nuclear protein, while SIRT2 predominantly resides in the cytoplasm, SIRT3-5 are mitochondrial, but SIRT6 and SIRT7 are localized to the nucleus. They have been shown to have a major impact on cell cycle regulation [6], peripheral myelination [7], autophagy [8], immune and inflammatory response [9,10,11]. A dysregulation of SIRT2 activity was reported to play a critical role in the pathogenesis of cancer [12,13], neurodegenerative diseases [14], type II diabetes [15], and bacterial infections [10,11]. Therefore, SIRT2 inhibitors have been considered as candidate therapeutic agents of the diseases.

Several classes of small molecule sirtuin inhibitors have been identified so far including nicotinamide derivatives, hydroxynaphthaldehyde derivatives, thiobarbiturate derivatives, tenovin derivatives, indole derivatives and benzimidazole derivatives, and most of the sirtuin inhibitors are targeting SIRT1 and SIRT2 (Figure 1) [16,17,18,19]. Among them, Tenovin-6 was identified as an inhibitor for SIRT1 and SIRT2 at a low micromolar level, it also inhibited growth in all tumor types in the study. AGK2 (2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide) possessed selective SIRT2 inhibitory activity. EX-527 showed remarkable SIRT1 inhibition potency with an IC₅₀ (half maximal inhibitory concentration) value of 0.1 µM, structure modification of EX-527 has resulted in compound 4 with moderate SIRT1 inhibition potency and pharmacological properties, and compound Ro31-8220, a bis(indoly)maleinimides andinitially discovered as a kinase inhibitor, was found to inhibit SIRT1 and SIRT2 for IC₅₀ value of 3.5 µM and 0.8 µM in later studies. Compounds 1, 2 and 3 were reported to be moderately potent SIRT1 and SIRT2 inhibitors, and to exhibit antitumor activity on colon cancer cells and breast cancer cells. EX-527, compound 4, and Ro31-8220 were based on the indole structure, compounds 1, 2, 3 bearing benzimidazolescaffold, and they shared some similarities. AGK2 was a furan scaffold, Benzofuran is isosteric with benzimidazole and indole, based on the structural features, we envisioned that the benzofuran scaffold may be a preferable sirtuin inhibitor. Therefore, we designed and synthesized a series of novel benzofuran derivatives, in which a methoxy or fluoro group was attached onto the 6-position, and a substituented benzyl sulfoxide or sulfone was incorporated into the 2-position. Here, we presented the synthesis and enzymatic inhibitory activities against SIRT1–3 of the benzofuran derivatives. The preliminary structure–activity relationships were investigated as well.

2. Result and Discussion

2.1. Chemistry

We adopted and modified the protocol to synthesize benzofuran derivatives from previously published literature [20,21,22], the synthesis of target compounds 6a–6j and 7a–7j was depicted in Scheme 1, and their chemical structures were shown in

Table 1. The chemical structures and inhibitory activities against SIRT1–3 (silent information regulator1–3) of compounds 6a–6j and 7a–7j.

Compd.	R	R1	IC50 (µM)
			SIRT1	SIRT2	SIRT3
6a	CH3O	CH3O	>100	13.52	>100
6b	CH3O	CN	>100	15.14	>100
6c	CH3O	Br	>100	85.14	>100
6d	CH3O	F	>100	95.21	>100
6e	CH3O	CH3OCO	>100	15.68	>100
6f	F	CH3O	>100	27.89	>100
6g	F	CN	>100	32.75	>100
6h	F	Br	>100	74.93	>100
6i	F	F	>100	88.07	>100
6j	F	CH3OCO	>100	19.70	>100
7a	CH3O	CH3O	>100	7.66	>100
7b	CH3O	CN	>100	8.09	>100
7c	CH3O	Br	>100	17.76	>100
7d	CH3O	F	>100	43.93	>100
7e	CH3O	CH3OCO	>100	3.81	>100
7f	F	CH3O	>100	7.92	>100
7g	F	CN	>100	8.85	>100
7h	F	Br	>100	20.14	>100
7i	F	F	>100	51.42	>100
7j	F	CH3OCO	>100	6.16	>100
Tenovin6	-	-	37.50	15.32	89.31
AGK2	-	-	>100	1.56	52.80

.

The mixture of compounds 1a–1b, ethoxycarbonylhydrazine and concentrated HCl in ethanol were heated to give rise to the intermediates 2a–2b. 1,2,3-thiadiazoles 3a–3b were obtained from 2a–2b with subsequent treatment with thionyl chloride. In the presence of potassium carbonate, the key intermediate benzofuran-2-thiols 4a–4b were prepared by the intramolecular cyclization reaction, which were further transformed into compounds 5a–5j by the alkylation with 4-substituted benzyl bromide, the oxidation of 5a–5j with 1.1 equivalents m-chloroperoxybenzoic acid (m-CPBA) at 0 °C to generate the corresponding sulfoxides 6a–6j or with an excess of m-CPBA at r.t. to form the sulfone target compounds 7a–7j in good yield (over 90%).

During the course of compounds 3a–3b synthesis, we initially used the reaction conditions according to the reference [22], and the mixture of compounds 2a–2b and thionyl chloride was heated in chloroform for 1–2 h at 60 °C to afford compounds 3a–3b. We found that compounds 3a–3b, particularly 3a, had poor yield about 30%. Thus, we tried to optimize the reaction conditions as shown in Scheme 1. In this condition, the solvent was the mixture of chloroform and DMF (5:1), the reaction started and finished by releasing heat with regulation of the rate of dropping thionyl chloride to giving rise to the intermediates 3a–3b over 90% yield. Then, 3a–3b reacted with potassium carbonate and 4-substituted benzyl bromide, forming the key intermediates 5a–5j and quantitative reaction in one-pot reaction. In fact, compared with the method of reference, our approach was more efficient and concise.

2.2. Sirtuin-Inhibitory Activity Evaluation

All target compounds were screened against human recombinant SIRT1-3 using AMC (Coumarin)-tagged deacetylation substrates [23]. Tenovin-6 is one of the few sirtuin inhibitors that displayed antitumor activities in a mouse xenograft model. AGK2 is one of the most selective SIRT2 inhibitors to date, and reduced α-synuclein-induced cytotoxicity in a Parkinson’s disease model. Thus, they were used as the standard control for SIRT1–3 assays. IC₅₀ values were determined for all of the compounds.

To explore the SAR (Structure activity relationships) of the substituted benzofuran core, the electron donating groups (methoxy-) and electron withdrawing groups (fluoro-) were chosen as the substituent attached on the 6-position of benzofuran moiety. A substituted benzyl sulfoxide or sulfone was incorporated into the 2-position. Substitution of benzenering was based on previous benzimidazole work [19], and those that showed high sirtuin inhibitory activities were substituted at the para position of benzene ring. Therefore, methoxyl, cyano, bromo, fluoro and methoxycarbonyl were added at the 4-position. In vitro screening on all of the target compounds 6a–6j and 7a–7j exhibited selective SIRT2 inhibitory activities with IC₅₀ varied from 3.81 µM to 95.21 µM compared to SIRT1 and SIRT3 with IC₅₀ more than 100 µM. Compound 7e (IC₅₀ 3.81 µM) was the most potent SIRT2 inhibitor, being more potent than Tenovin-6 (IC₅₀ 15.32 µM), and SIRT2 activity dose-response profiles for compound 7e are shown in Figure 2. Compounds 7a–7j with benzyl sulfone scaffold possessed the better SIRT2 inhibitory activities in comparison with the corresponding benzyl sulfoxide compounds 6a–6j, which are chiral sulfones. Chirality has revealed to be crucial for other SIRT inhibitors as EX-527, in view of the structural differences and the general activity trend of such compounds. We speculate that the SIRT-inhibitory activity of them is related to chirality, and this deserves to be studied further. Among compounds 7a–7j, most compounds showed good potency, and they had IC₅₀ values at single digit micromolar level (IC₅₀ 3.81–8.85 µM). The inhibitory activity of the 4-substituted derivatives on benzene rings increased in the order of CH₃OCO- > CH₃O- > CN- > Br- > F-. It was noted that 4-broro substituted compounds (7c, 7h) had somewhat lower activity (IC₅₀ 17.76, 20.14 µM), and 4-fluoro substituted compounds (7d, 7i) displayed the weakest inhibition on SIRT2 (IC₅₀ 43.93, 51.42 µM) in this series. Collectively, it suggested that the 4-halogen substituent of the benzene ring might be unfavorable for potency. In addition, when the methoxyl group is substituted for the benzofuran moiety as R-group, and compounds 7a–7e possessed the better inhibitory activity than that of compounds 7f–7j with a fluoro group. This result gave an indication that the inhibitory effect was enhanced when an electron donating groups was added to the benzofuran core. Compounds 6a–6j have a similar structure–activity relationship to compounds 7a–7j.

As mentioned above (Figure 1), compounds 1, 2 and 3 bearing the benzimdazole scaffold were generally unselective with regards to SIRT1 and SIRT2 with inhibition potency. The sirtuin inhibitors of compound 4, Ro31–8220, and EX-527 with indole moiety were shown to have certain selectivity for SIRT1 and SIRT2. We found that most of the N-containing heterocycles target SIRT1 and SIRT2, while benzofurans and furan (AGK2) preferentially target only SIRT2.

To explore potential binding modes of these novel benzofuran derivatives, molecular docking was performed using CDOCER protocol integrated in Accelrys Discovery Studio Client 2016 (Accelrys Software Inc., San Diego, CA, USA). The coordinates of the X-ray co-crystal structure of ADP-ribose (ADPr) with SIRT2 (PDB entry code: 3ZGV, X-ray resolution = 2.30 Å), reported by Moniot et al. in 2013 [24], was employed for docking the representative compound 7e (the most active compound). The binding modes of the benzofuran derivatives were somewhat similar and exemplified by compound 7e, as shown in Figure 3, and compound 7e could nicely situate in the binding pocket and took a very similar binding pose with the ADPr.

The docking analysis reveals that key interactions of compound 7e with the active site of SIRT2 are the hydrogen bonds as well as the π–π stacking interactions. The benzofuran moieties involved in potential π-π stacking with Tyr104. The oxygen atom of benzofuran moiety interacted with Gln167 and Arg97 via a strong hydrogen bond as we intended. The co-crystal structures of known SIRT2 inhibitors have demonstrated that the hydrogen bonds between Arg97 and Gln167 are crucial to inhibit the function of SIRT2 [25,26,27]. Similarly, our docking results have also shown hydrogen bond interactions with Gln167 and Arg97. Therefore, the benzofuran moiety played a critical role in the potency. It was noticed that the sulfone oxygen on the 2-position of the benzofuran scaffold formed strong hydrogen bonds with Ala85, Thr262, Ser263. Other hydrogen bonds that could be observed include Gln167 and Gln265, and these suggested that the oxygen atom on the 2-position was benefical for the binding. On the other hand, the binding pattern suggests that the configuration of the 2-position oxygen atom determines the intensity of the hydrogen bond. In fact, the corresponding compound 6e bearing a sulfoxide group showed a poorer inhibition effect (IC₅₀ 15.68 µM) than that of compound 7e. Similarly, all of the other compounds with a sulfoxide group had lower SIRT2 inhibitory activities, in comparison with the corresponding sulfone compounds in Table 1. The results prove our speculation that the chirality of sulfoxide affects its inhibitory activity. In addition, the 6-methoxyl oxygen on the benzofuran core also formed a hydrogen bond with Arg97, and the 4-carboxylate oxygen of benzene ring formed hydrogen bonds with Lys287 and Asn286. These may add more favorable binding affinity.

3. Experimental Section

3.1. Chemistry

Commercial grade reagents and solvents were used without further purification. Melting points were measured on a Fisher Scientific (Waltham, MA, USA) micro melting point apparatus and are uncorrected. ¹H-NMR spectra was recorded on a Varian Mercury 400 MHz (Palo Alto, CA, USA) spectrometer with tetramethylsilane (TMS) as the internal standard for ¹H. Chemical shifts are reported in δ (ppm) units. ¹³C-NMR spectra were recorded using a Bruker 600 MHz NMR (Billerica, MA, USA) spectrometer. High-resolution mass spectra (HRMS) were obtained on a 9.4T Q-q-FTMSApex Qespectrometer (Karlsruhe, Germany). All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) on pre-coated silica gel G plates (Qingdao Marine Chemical Factory, Qingdao, China) at 254 nm under a UV lamp. Column chromatography separations were performed with silica gel (200–400 mesh).

3.2. Synthesis of Compounds 6a–6j

6-Methoxy-2-((4-methoxybenzyl)sulfinyl)benzofuran (6a). To a stirred solution of 5a (400 mg, 1.33 mmol) in DCM (dichloromethane) (15.0 mL) and cooled in an ice bath, m-CPBA (296 mg, 1.47 mmol) was added slowly at 0 °C. The reaction mixture was stirred at 0 °C for 30 min. The reaction mixture was quenched with aqueous saturated NaHCO₃ (10 mL). The aqueous layer was then extracted with DCM (3 × 10 mL) and the combined organic layer washed with brine (20 mL), dried (Na₂SO₄) and concentrated in vacuo. The crude product was purified by column. Chromatography on silica gel eluting with petroleum ether/ethyl acetate/dichloromethane(v:v = 4:1:0.6) to yield the product (399 mg, 95%) as white flocky solid; m.p. 129–130 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.45 (d, J = 8.8 Hz, 1H), 7.10 (s, 1H), 7.03 (t, J = 8.4 Hz, 3H), 6.94 (dd, J₁ = 8.4 Hz, J₂ = 1.9 Hz, 1H), 6.76 (d, J = 8.42 Hz), 4.53 (d, J = 12.4 Hz, 1H), 4.40 (d, J = 12.4 Hz, 1H), 3.90 (s, 3H), 3.75 (s, 3H) (Supplementary Figure S1). ¹³C NMR (151 MHz, CDCl₃) δ (ppm): 159.93, 159.71, 157.73, 151.73, 131.32, 122.53, 120.96, 119.70, 114.20, 113.57, 95.92, 58.68, 55.75, 55.21 (Supplementary Figure S2). HRMS (ESI (electrospray ionization)) Calcd. for C₁₇H₁₆NaO₄S [M + Na]⁺: 339.0667; Found: 339.0665.

6-Methoxy-2-((4-cyanobenzyl)sulfinyl)benzofuran (6b). Following the preparation protocol of compound 6a, starting from compound 5b (280 mg, 0.95 mmol), the title compound 6b was obtained as white crystals (268 mg, 91%); m.p. 137–138 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.55 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.26–7.22 (m, 2H), 7.07 (s, 1H), 7.02 (s, 1H), 6.98–6.94 (m, 1H), 4.53 (d, J = 12.8 Hz, 1H), 4.47 (d, J = 12.8 Hz, 1H), 3.90 (s, 3H) (Supplementary Figure S3). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 160.16, 157.83, 150.92, 134.54, 132.34, 130.85, 122.68, 119.43, 118.27, 113.82, 113.74, 112.41, 95.89, 58.79, 55.78 (Supplementary Figure S4). HRMS (ESI) Calcd. for C₁₇H₁₄NO₃S [M + H]⁺: 312.0694; Found: 312.0687.

6-Methoxy-2-((4-bromobenzyl)sulfinyl)benzofuran (6c). Following the preparation protocol of compound 6a, starting from compound 5c (400 mg, 1.33 mmol), the title compound 6c was obtained as white crystals (430 mg, 94%); m.p. 146–147 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.46 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.08 (s, 1H), 7.04–6.97 (m, 3H), 6.94 (dd, J₁ = 8.4 Hz, J₂ = 1.6 Hz, 1H), 4.49 (d, J = 12.4 Hz, 1H), 4.39 (d, J = 12.8 Hz, 1H), 3.90 (s, 3H) (Supplementary Figure S5). ¹³C-NMR (151 MHz, CDCl₃) δ: 160.06, 157.78, 151.26, 131.91, 131.70, 128.15, 122.79, 122.64, 119.56, 113.74, 113.71, 95.90, 58.55, 55.77. HRMS (ESI) Calcd. for C₁₆H₁₄BrO₃S [M + H]⁺: 364.9847, Found: 364.9840.

6-Methoxy-2-((4-fluorobenzyl)sulfinyl)benzofuran (6d). Following the preparation protocol of compound 6a, starting from compound 5d (420 mg, 1.46 mmol), the title compound 6d was obtained as white crystals (408 mg, 92%); m.p. 104.5–105.5 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.45 (d, J = 8.8 Hz, 1H), 7.13–7.07 (m, 3H), 7.01 (s, 1H), 6.97–6.90 (m, 3H), 4.51 (d, J = 12.8 Hz, 1H), 4.41 (d, J = 12.8 Hz, 1H), 3.90 (s, 3H) (Supplementary Figure S6). ¹³C-NMR (151 MHz, CDCl₃) δ: 162.78 (d, J = 246.8 Hz), 160.01, 157.76, 151.39, 131.8 (d, J = 8.3 Hz), 124.98 (d, J = 3.0 Hz), 122.58, 119.58, 115.77 (d, J = 21.6 Hz), 113.66, 113.62, 95.89, 58.37, 55.75 (Supplementary Figure S7). HRMS (ESI) Calcd. for C₁₆H₁₄FO₃S [M + H]⁺: 305.0647; Found: 305.0638.

6-Methoxy-2-((4-methoxycarbonylbenzyl)sulfinyl)benzofuran (6e). Following the preparation protocol of compound 6a, starting from compound 5e (328 mg, 1.00 mmol), the title compound 6e was obtained as white crystals (319 mg, 93%); m.p. 157–158 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.91 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.8 Hz, 1H), 7.20 (d, J =8.0 Hz, 2H), 7.09 (s, 1H), 7.00 (s, 1H), 6.97–6.91 (m, 1H), 4.59 (d, J = 12.4 Hz, 1H), 4.49 (d, J = 12.8 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H) (Supplementary Figure S8). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 166.51, 160.09, 157.80, 151.18, 134.19, 130.17, 130.13, 129.91, 122.65, 119.51, 113.77, 113.75, 95.90, 59.02, 55.77, 52.18 (Supplementary Figure S9). HRMS (ESI) Calcd. for C₁₈H₁₇O₅S [M + H]⁺: 345.0796, Found: 345.0791.

6-Fluoro-2-((4-methoxybenzyl)sulfinyl)benzofuran (6f). Following the preparation protocol of compound 6a, starting from compound 5f (350 mg, 1.22 mmol), the title compound 6f was obtained as white crystals (330 mg, 89%); m.p. 140–141 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.53 (dd, J₁ = 8.4 Hz, J₂ = 5.2 Hz, 1H), 7.32 (dd, J₁ = 8.8 Hz, J₂ = 1.6 Hz, 1H), 7.12–7.01 (m, 4H), 6.80–6.74 (m, 2H), 4.50 (d, J = 12.8 Hz, 1H), 4.38 (d, J = 12.8 Hz, 1H), 3.76 (s, 3H) (Supplementary Figure S10). ¹³C-NMR (151 MHz, CDCl₃) δ: 162.08 (d, J = 245.6 Hz), 159.81, 156.40 (d, J = 13.5 Hz), 154.24, 131.32, 122.89 (d, J = 10.2 Hz), 122.83, 120.57, 114.22, 112.74, 112.73 (d, J = 24.3 Hz), 99.72 (d, J = 26.7 Hz), 58.90, 55.23 (Supplementary Figure S11). HRMS (ESI) Calcd. for C₁₆H₁₃FNaO₃S [M + Na]⁺: 327.0467, Found: 327.0463.

6-Fluoro-2-((4-cyanobenzyl)sulfinyl)benzofuran (6g). Following the preparation protocol of compound 6a, starting from compound 5g (240 mg, 0.85 mmol), the title compound 6g was obtained as white crystals (268 mg, 95%); m.p. 105.5–107 °C;¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.59–7.53 (m, 3H), 7.31 (dd, J₁ = 8.4 Hz, J₂ = 1.2 Hz, 1H), 7.28–7.22 (m, 2H), 7.13–7.04 (m, 2H), 4.52 (d, J = 12.8 Hz, 1H), 4.45 (d, J = 12.8 Hz, 1H) (Supplementary Figure S12). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 162.19 (d, J = 246.3 Hz), 156.46 (d, J = 13.4 Hz), 153.47, 134.14, 132.32, 130.84, 123.07 (d, J = 10.2 Hz), 122.59, 118.19, 113.06 (d, J = 24.3 Hz), 112.91, 112.54, 99.71 (d, J = 26.7 Hz), 58.83 (Supplementary Figure S13). HRMS (ESI) Calcd. for C₁₆H₁₁FNO₂S [M + H]⁺: 300.0494, Found: 300.0488.

6-Fluoro-2-((4-bromobenzyl)sulfinyl)benzofuran (6h). Following the preparation protocol of compound 6a, starting from compound 5h (490 mg, 1.45 mmol), the title compound 6h was obtained as white crystals (462 mg, 90%); m.p. 137–138.5 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.55 (dd, J = 8.8 Hz, 5.6 Hz, 1H), 7.41–7.36 (m, 2H), 7.31 (dd, J₁ = 8.8 Hz, J₂ = 2.0 Hz, 1H), 7.13–7.06 (m, 2H), 6.99 (d, J = 8.4 Hz, 2H), 4.47 (d, J = 12.8 Hz, 1H), 4.37 (d, J = 12.8 Hz, 1H) (Supplementary Figure S14). ¹³C-NMR (151 MHz, CDCl₃) δ: 162.17 (d, J = 245.9 Hz), 156.45 (d, J = 13.4 Hz), 153.80, 131.95, 131.69, 127.78, 123.07, 122.98 (d, J = 5.7 Hz), 122.72, 112.92 (d, J = 24.2 Hz), 112.91, 99.74 (d, J = 26.9 Hz), 58.70 (Supplementary Figure S15). HRMS (ESI) Calcd. for C₁₅H₁₀BrFNaO₂S [M + Na]⁺: 374.9466; Found: 374.9457.

6-Fluoro-2-((4-fluorobenzyl)sulfinyl)benzofuran (6i). Following the preparation protocol of compound 6a, starting from compound 5i (480 mg, 1.74 mmol), the title compound 6i was obtained as white crystals (489 mg, 96%); m.p. 107.5–109 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.54 (dd, J₁ = 8.4 Hz, J₂ = 5.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.13–7.04 (m, 4H), 6.95 (d, J = 8.4 Hz, 2H), 4.49 (d, J = 12.8 Hz, 1H), 4.40 (d, J = 12.8 Hz, 1H) (Supplementary Figure S16). ¹³C-NMR (151 MHz, CDCl₃) δ(ppm): 162.87 (d, J = 246.9 Hz), 162.13 (d, J = 245.8 Hz), 156.44 (d, J = 13.5 Hz), 153.95, 131.84 (d, J = 8.3 Hz), 124.63 (d, J = 2.6 Hz), 122.96 (d, J = 10.2 Hz), 122.74, 115.81 (d, J = 21.6 Hz), 112.94, 112.78, 99.72 (d, J = 26.7 Hz), 58.55 (Supplementary Figure S17). HRMS (ESI) Calcd. for C₁₅H₁₁F₂O₂S [M + H]⁺: 293.0447; Found: 293.0439.

6-Fluoro-2-((4-methoxycarbonylbenzyl)sulfinyl)benzofuran (6j). Following the preparation protocol of compound 6a, starting from compound 5j (310 mg, 0.95 mmol), the title compound 6j was obtained as white crystals (308 mg, 95%); m.p. 137–138 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.92 (d, J = 8.0 Hz, 2H), 7.53 (dd, J₁ = 8.8 Hz, J₂ = 5.2 Hz, 1H), 7.31 (dd, J₁ = 8.8 Hz, J₂ = 1.2 Hz, 1H), 7.22–7.17 (m, 2H), 7.09 (td, J₁ = 9.0 Hz, J₂ = 2.2 Hz, 1H), 7.04 (s, 1H), 4.57 (d, J = 12.6 Hz, 1H), 4.47 (d, J = 12.4 Hz, 1H), 3.89 (s, 3 H) (Supplementary Figure S18). ¹³C-NMR (151 MHz, CDCl3) δ: 166.44, 162.17 (d, J = 246.0 Hz), 156.44 (d, J = 13.5 Hz), 153.68, 133.78, 130.28, 130.11, 129.90, 123.02 (d, J = 10.2 Hz), 122.65, 112.91 (d, J = 24.2 Hz), 112.90, 99.72 (d, J = 26.9 Hz), 59.12, 52.18 (Supplementary Figure S19). HRMS (ESI) Calcd. for C₁₇H₁₄FO₄S [M + H]⁺: 333.0596; Found: 333.0587.

3.3. Synthesis of Compounds 7a–7j

6-Methoxy-2-((4-methoxybenzyl)sulfonyl)benzofuran (7a). To a stirred solution of compound 5a (400 mg, 1.33 mmol) in DCM (20.0 mL) and m-CPBA (675 mg, 3.34 mmol) was added slowly at r.t. The mixture was stirred at r.t for 30 min. The reaction mixture was quenched with aqueous saturated NaHCO₃ (10 mL). The aqueous layer was then extracted with CH₂Cl₂ (3 × 10 mL) and the combined organic layer washed with brine (20 mL), dried (Na₂SO₄) and concentrated in vacuo. The crude was purified by silica gel column chromatography (ether/ethyl acetate 2:1) to afford the title compound 7a (413 mg, 93%). white flocky solid; m.p. 114–115 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.49 (d, J = 8.8 Hz, 1H), 7.20 (s, 1H), 7.10 (d, J = 8.4 Hz, 2H), 7.05 (s, 1H), 6.97 (dd, J₁ = 8.8 Hz, J₂ = 2.0 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 4.45 (s, 2H), 3.89 (s, 3H), 3.77 (s, 3H) (Supplementary Figure S20). ¹³C-NMR (151 MHz, CDCl₃) δ 160.83, 160.17, 157.72, 147.54, 131.91, 123.39, 119.10, 118.94, 115.79, 114.54, 114.24, 95.79, 60.91, 55.77, 55.24 (Supplementary Figure S21). HRMS (ESI) Calcd. for C₁₇H₁₆NaO₅S [M + Na]⁺: 355.0616; Found: 355.0613.

6-Methoxy-2-((4-cyanobenzyl)sulfonyl)benzofuran (7b). Following the preparation protocol of compound 7a, starting from compound 5b (650 mg, 2.20 mmol), the title compound 7b was obtained as white solid (668 mg, 93%); m.p. 160–161 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.59(d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 7.6 Hz, 2H), 7.27–7.23 (m, 1H), 7.04–6.97 (m, 2H), 4.55 (s, 2H), 3.90 (s, 3H) (Supplementary Figure S22). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 161.22, 157.83, 146.82, 132.56, 132.45, 131.44, 123.59, 118.62, 118.08, 116.37, 114.96, 113.15, 95.7361.18, 55.82 (Supplementary Figure S23). HR-MS (ESI) Calcd. for C₁₇H₁₃NNaO₄S [M + Na]⁺: 350.0463, Found: 350.0456.

6-Methoxy-2-((4-bromobenzyl)sulfonyl)benzofuran (7c). Following the preparation protocol of compound 7a, starting from compound 5c (500 mg, 1.43 mmol), the title compound 7c was obtained as white solid (530 mg, 97%); m.p. 133–134 °C;¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.51 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 7.08–7.03 (m, 3H), 6.98 (dd, J₁ = 8.8 Hz, J₂ = 2.0 Hz, 1H), 4.46 (s, 2H), 3.89 (s, 3H) (Supplementary Figure S24). ¹³C-NMR (151 MHz, CDCl3) δ (ppm): 161.02, 157.78, 147.08, 132.24, 132.01, 126.36, 123.56, 123.51, 118.77, 116.13, 114.76, 95.76, 60.88, 55.79; HR-MS (ESI) Calcd. for C₁₆H₁₃BrNaO₄S [M + Na]⁺: 402.9615, Found:402.9610.

6-Methoxy-2-((4-fluorobenzyl)sulfonyl)benzofuran (7d). Following the preparation protocol of compound 7a, starting from compound 5d (580 mg, 2.01 mmol), the title compound 7d was obtained as white solid (601 mg, 93%); m.p. 99–100 °C;¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.50 (d, J = 8.4 Hz, 1H), 7.22 (s, 1H), 7.20–7.14 (m, 2H), 7.04 (s, 1H), 7.01–6.94 (m, 3H), 4.48 (s, 2H), 3.89 (s, 3H) (Supplementary Figure S25). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 163.19(d, J = 247.5 Hz), 160.98, 157.76, 147.19, 132.48 (d, J = 8.4 Hz), 123.46, 123.19 (d, J = 3.0 Hz), 118.81, 116.02, 115.89 (d, J = 21.6 Hz), 114.70, 95.77, 60.70, 55.79 (Supplementary Figure S26). HR-MS (ESI) Calcd. for C₁₆H₁₃FNaO₄S [M + Na]⁺: 343.0416, Found: 343.0410.

6-Methoxy-2-((4-methoxycarbonylbenzyl)sulfonyl)benzofuran (7e). Following the preparation protocol of compound 7a, starting from compound 5e (292 mg, 0.89 mmol), the title compound 7e was obtained as light white solid (293 mg, 92%); m.p. 175–176 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.94 (d, J = 8.4 Hz, 2H), 7.49(d, J = 8.8 Hz, 1H), 7.29–7.25 (m, 2H), 7.19 (d, J = 0.8 Hz, 1H), 7.04 (d, J = 1.6 Hz, 1H), 6.98 (dd, J₁ = 8.8 Hz, J₂ =2.0 Hz, 1H), 4.56 (s, 2H), 3.90 (s, 3H), 3.89 (s, 3H) (Supplementary Figure S27). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 166.41, 161.05, 157.77, 147.00, 132.25, 130.73, 129.92, 123.51, 118.73, 116.17, 114.77, 114.69, 95.76, 61.26, 55.79, 52.24 (Supplementary Figure S28). HR-MS (ESI) Calcd. for C₁₈H₁₇O₆S [M + H]⁺: 361.0745, Found: 361.0737.

6-Fluoro-2-((4-methoxybenzyl)sulfonyl)benzofuran (7f). Following the preparation protocol of compound 7a, starting from compound 5f (400 mg, 1.39 mmol), the title compound 7f was obtained as light white solid (410 mg, 92%); m.p. 119–120.5 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.60 (dd, J₁ = 8.8 Hz, J₂ = 5.6 Hz, 1H), 7.30 (dd, J₁ = 8.0 Hz, J₂ = 2.0 Hz, 1H), 7.26 (d, J = 1.2 Hz, 1H), 7.16–7.08 (m, 3H), 6.80 (d, J = 8.8 Hz, 2H), 4.47 (s, 2H), 3.77 (s, 3H) (Supplementary Figure S29). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 162.85 (d, J = 247.5 Hz), 160.27, 156.31 (d, J = 13.8 Hz), 149.69, 131.90, 124.00 (d, J = 10.4 Hz), 122.07, 118.71, 115.27, 114.30, 113.57 (d, J = 24.5 Hz), 100.02 (d, J = 26.9 Hz), 60.88, 55.26 (Supplementary Figure S30). HR-MS (ESI) Calcd. for C₁₆H₁₃FNaO₄S [M + Na]⁺: 343.0416, Found: 343.0409.

6-Fluoro-2-((4-cyanobenzyl)sulfonyl)benzofuran (7g). Following the preparation protocol of compound 7a, starting from compound 5g (160 mg, 0.57 mmol), the title compound 7g was obtained as white solid (177 mg, 99%); m.p. 178–179.5 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.66–7.57 (m, 3H), 7.35 (d, J = 8.4 Hz, 2H), 7.31–7.28 (m, 2H), 7.16 (td, J₁ = 8.8 Hz, J₂ = 2.0 Hz, 1H), 4.58 (s, 2H) (Supplementary Figure S31). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 163.12 (d, J = 248.7 Hz), 156.39 (d, J = 13.7 Hz), 149.01, 132.51, 132.15, 131.45, 124.27 (d, J = 10.4 Hz), 121.79, 117.98, 115.80, 114.00 (d, J = 24.5 Hz), 113.33, 100.18 (d, J = 27.0 Hz), 61.03 (Supplementary Figure S32). HR-MS (ESI) Calcd. for C₁₆H₁₀FNNaO₃S [M + Na]⁺: 338.0263, Found: 338.0258.

6-Fluoro-2-((4-bromobenzyl)sulfonyl)benzofuran (7h). Following the preparation protocol of compound 7a, starting from compound 5h (188 mg, 0.56 mmol), the title compound 7h was obtained as white solid (188 mg, 91%); m.p. 167–168 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.62 (dd, J₁ = 8.8 Hz, J₂ = 5.6 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.33–7.27 (m, 2H), 7.15 (td, J₁ = 9.2 Hz, J₂ = 2.4 Hz, 1H), 7.10–7.04 (m, 2H), 4.48 (s, 2H) (Supplementary Figure S33). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 163.00 (d, J = 248.1 Hz), 156.35 (d, J = 13.8 Hz), 149.25, 132.22, 132.10, 125.98, 124.16 (d, J = 10.4 Hz), 123.74, 121.93, 115.59, 113.79 (d, J = 24.5 Hz), 100.06 (d, J = 26.9 Hz), 60.79 (Supplementary Figure S34). HR-MS (ESI) Calcd. for C₁₅H₁₀BrFNaO₃S [M + Na]⁺: 390.9415, Found: 390.9411.

6-Fluoro-2-((4-fluorobenzyl)sulfonyl)benzofuran (7i). Following the preparation protocol of compound 7a, starting from compound 5i (400 mg, 1.44 mmol), the title compound 7i was obtained as white solid (421 mg, 94%); m.p. 144–145 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.61 (dd, J₁ = 8.8 Hz, J₂ = 5.2 Hz, 1H), 7.32–7.25 (m, 2H), 7.21–7.10 (m, 3H), 6.98 (t, J = 8.8 Hz, 2H), 4.50 (s, 2H) (Supplementary Figure S35). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 163.26 (d, J = 248.1 Hz), 162.96 (d, J = 247.8 Hz), 156.34 (d, J = 13.7 Hz), 149.36, 132.49 (d, J = 8.4 Hz), 124.10 (d, J = 10.4 Hz), 122.83 (d, J = 2.0 Hz), 121.96, 115.98 (d, J = 21.8 Hz), 115.48, 113.74 (d, J = 24.6 Hz), 100.04 (d, J = 26.9 Hz), 60.63 (Supplementary Figure S36). HR-MS (ESI) Calcd. for C₁₅H₁₀F₂NaO₃S [M + Na]⁺: 331.0216, Found: 331.0208.

6-Fluoro-2-((4-methoxycarbonylbenzyl)sulfonyl)benzofuran (7j). Following the preparation protocol of compound 7a, starting from compound 5j (290 mg, 0.92 mmol), the title compound 7j was obtained as white solid (282 mg, 88%); m.p. 160–161 °C; ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.95 (d, J = 8.4 Hz, 2H), 7.60 (dd, J₁ = 8.8 Hz, J₂ = 5.2 Hz, 1H), 7.32–7.23 (m, 4H), 7.14 (td, J₁ = 9.2 Hz, J₂ = 2.0 Hz, 1H), 4.58 (s, 2H), 3.90 (s, 3H) (Supplementary Figure S37). ¹³C-NMR (151 MHz, CDCl₃) δ (ppm): 166.34, 163.01 (d, J = 248.1 Hz), 156.34 (d, J = 13.7 Hz), 149.17, 131.86, 130.91, 130.72, 129.98, 124.16 (d, J = 10.4 Hz), 121.88, 115.63, 113.80 (d, J = 24.6 Hz), 100.07 (d, J = 27.0 Hz), 61.18, 52.28 (Supplementary Figure S38). HR-MS (ESI) Calcd. for C₁₇H₁₄FO₅S [M + H]⁺: 349.0546, Found: 349.0539.

Note: Except for target compounds, the synthesis and characterization of all other compounds mentioned in the Scheme 1 were described in the supporting information.

3.4. Biological Evaluation

3.4.1. Expression and Purification of SIRT1−3

Human Sirtuin 1 (GenBank accession no. NM_012238), full length; human Sirtuin 2 (GenBank accession no. NM_012237), full length; human SIRT3 (GenBank accession no. NM_012239), 118−399. These three genes were cloned into pET21a (+). The recombinant proteins with an N-terminal fusion His₆ tag were induced by IPTG (Isopropyl β-d-1-thiogalactopyranoside) and expressed in Escherichia coli BL21 (DE3) cells. The purification was performed by using a Qiagen Ni-NTA column (Duesseldorf, Germany). An SIRT1/2/3 enzymatic assay was performed according to the reference. In brief, the SIRT activity assay was performed using substrate peptide Ac-Arg-His-Lys-[Lys-(Ac)]-AMC. Costar^® EIA/RIA plate (Corning Incorporated, Corning, NY, USA, #3693) was used in this assay.

3.4.2. SIRT1-3 Assay

The stand control compounds and target compounds were tested against human recombinant SIRT1−3 using AMC-tagged deacetylation substrates.

The assay procedure involves two steps [23]. In the first step, the inhibition assay was run in 60 µL assay buffer (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂ and 1 mg/mL BSA (Bovine albumin), 500 μM NADH (Nicotinamide adenine dinucleotide), 50 μM peptide substrate) and 1.0 µg recombinant SIRT protein. The assay was incubated at 37 °C for 2 h. Then, a 60 µL second batch assay buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, containing 10 µL trypsin (60,000 BAEE/mL) and 2 µL SIRT inhibitor nicotinamide (120 mM) were added. The reaction wells were mixed and incubated for another 37 °C for 20 min. The fluorescence was measured on a fluorometric reader (PerkinElmer EnSpire, Waltham, MA, USA) with excitation at 355 nm and emission at 460 nm. The IC₅₀ calculation was calculated using GraphPad Prism Software (GraphPad Prism 5, Manufacturer, La Jolla, CA, USA). For each concentration, at least two wells were performed to calculate the average parameter.

4. Conclusions

In conclusion, we have discovered a series of novel benzofuran derivatives containing benzyl sulfoxide or benzyl sulfone scaffold. The enzymatic assay was performed against SIRT1–3 and all of the target compounds displayed potent selective SIRT2 inhibitory activity with IC₅₀ values at the micromolar level. The preliminary SAR and the binding features of the benzofuran derivatives were analyzed. It has been demonstrated that benzyl sulfone moiety could be useful in developing potent SIRT2 inhibitors, and the benzofuran core could serve as an appropriate scaffold for discovering structurally novel and drug-like SIRT2 inhibitors. These results will be the foundation for further developments to achieve more potent benzofuran derivatives as selective SIRT2 inhibitors.