Introduction

Alzheimer’s disease (AD), known as the most prevalent type of dementia, is a progressive neurodegenerative disease which causes symptoms of memory loss and cognitive impairment, eventually lead to the loss of cognitive function [1]. AD, which affected 36 million people worldwide in recent years, has placed a heavy burden on family and decreased the quality of life in patients, but even worse, the numbers of AD patients are estimated to increase up to 114 million by 2050 [2]. Therefore, scientific researchers have put massive efforts to better understanding of the pathogenesis of the disease as well as developing effective therapeutic agents to be able to prevent or to cure AD [3,4,5,6]. However, to date, no satisfactory treatment has been proved to prevent or cure AD. For this reason, there is an urgent need for developing new drugs being able to limiting and stopping the process of AD.

Although AD pathogenesis is multifaceted and difficult to pinpoint, genetic and pathological evidence strongly supports that amyloid-beta (Aβ) plays an early and vital role in AD [7]. An alternative hypothesis indicates that Aβ can cause cellular toxicity, which ultimately results in neuronal dysfunction and death. Therefore, development of active compounds to improve Aβ-associated neurotoxicity has been taken up as a promising therapeutic strategy against AD [8].

Marine nature products (MNPs) have drawn the attention of both medicinal chemists and pharmacologists for several decades, due to their chemical diversities and various biological activities as well. MNPs have played and continued playing a vital role in the development of new drugs and drug candidates [9,10,11,12]. Marine alkaloids exhibit various kinds of pharmacological activities such as antitrypanosomal, antibacterial, antimalarial, anti-infective activities, and so on [13, 14]. Our group has long engaged in isolation, synthesis, and biological evaluation of MNPs from the South China Sea marine organisms for many years, and in the course of these efforts, recently, two novel alkaloids, phidianidines A (1a) and B (1b) (Fig. 1), have been isolated from the opisthobranch mollusk Phidiana militaris. These two alkaloids, featured by the first identified MNPs bearing an uncommon 1,2,4-oxadiazole moiety, exhibited promising antitumor activity against C6 and HeLa cells with IC50 values of 0.64 and 0.14 µM, respectively [15]. Phidianidines have attracted many researchers’ interests due to their unique structure and excellent bioactivity [16, 17]. In addition, phidianidine A was found to be a novel potential ligand of CXCR4, a chemokine receptor deeply involved in HIV infection, rheumatoid arthritis, cancer development/progression, and metastasization [18]. Interestingly, phidianidines were found to be selective inhibitors of dopamine transporter (DAT) as well as partial agonist of the μ opioid receptor, these results enlightening us that they can be utilized for developing new ligands or ligand analogs for central nervous system (CNS) targets. Recently, a series of phidianidine-based derivatives have been synthesized and some derivatives exhibited promising neuroprotective effects against amyloid-beta 25–35 (Aβ25–35)-, hydrogen peroxide (H2O2)-, or oxygen-glucose deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells [19]. These results provoked our great interest in further exploring neuroprotective activities of phidianidine-based compounds. In this communication, the neuroprotective effects against Aβ25–35-induced toxicity in SH-SY5Y cells of new benzothiazol-based 1,3,4-oxadiazole derivatives are reported.

Fig. 1
figure 1

Structures of phidianidines A and B

Full size image

Results and discussion

Chemistry

Analysis of the structural characteristic of phidianidines A and B, as shown in Fig. 2, reveals that their structures contained three partials, namely moiety A, B, and C. The moiety A, the indole motif is an important pharmacophore widely presented in numerous bioactive natural products or drugs [20]. The moiety B, the 1,2,4-oxadiazole ring has been incorporated in drug discovery programs as an essential element of pharmacophore in contribution of ligand binding [21, 22]. Our previous preliminary structure–activity relationships (SAR) study about the moiety C suggested that the guanidine structure was not required for neuroprotective activity [19], while the different substituted benzene rings which replaced the guanidine moiety play a crucial role for their bioactivity.

Fig. 2
figure 2

Structural modifications

Full size image

Benzothiazole (BTA) scaffold, the main moiety of traditional amyloid-binding dyes-thioflavin-T (ThT) [23], exhibited strong affinity for amyloid fibrils and was used as promising imaging agents targeting to Aβ plaques [24,25,26]. In addition, compounds contain BTA moiety are quite capable of inhibiting Aβ aggregation [27,28,29] as well as Aβ induced neurotoxicity [30, 31]. In another hand, oxadiazoles are a class of heterocyclic compounds with broad spectrum of biological activities [32,33,34,35,36], specially the 1,3,4-oxadiazole derivatives displayed excellent affinity for Aβ aggregates [37] including excellent affinity for Aβ aggregates [38, 39]. All this evidences suggest that combining 1,3,4-oxadiazole fragment with BTA moiety might be a potential therapeutic strategy for AD. In this work, 1,3,4-oxadiazole was introduced to moiety B to replace 1,2,4-oxadiazole. Substituted benzylthio group or different alkylthio groups were reserved as moiety C. Accordingly, we designed a series of novel phidianidines analogs 2 bearing different terminal groups (Fig. 2).

The synthetic route of compounds 2 is shown in Scheme 1. First, commercially available 1,3-benzothiazole-2-thiol (3) was reacted with ethyl chloroacetate to produce ester 4 in 95% yield [40]. Then, treatment of the ester 4 with 80% hydrazine hydrate in refluxing EtOH gave hydrazide 5 in 80% yield [41]. Furthermore, the key intermediate 6 was formed via reactions of hydrazide 5 with carbon disulfide in the presence of KOH under refluxing EtOH in 70% yield [41]. Finally, the intermediate 6 was converted into 2 in good yield by reacting with different substituted benzyl bromide in the presence of K2CO3 using CH3CN as solvent.

scheme 1

Pharmacology

The influence of the compounds on cell viabilities of SH-SY5Y with or without Aβ25–35 exposure was measured. The active neuroprotective compounds (10 μM) did not cause notable changes in cell viability of SH-SY5Y cells, when incubating with the cells for 24 h (Fig. 3). These compounds exhibited significant neuroprotective effects against Aβ25–35-induced neurotoxicity in SH-SY5Y cells, and the screening data are shown in Table 1. SH-SY5Y cells exposed to 10 μM Aβ25–35 for 24 h resulted in decrease cell viability (63.2%) as compared with control. As listed in the table, compounds 2a–2m (except for 2k) markedly attenuated Aβ25–35-induced cytotoxicity at the concentration of 10 μM with cell viabilities ranged from 74.6 to 95.7%, while compounds 2n–2t showed no activity. All the test compounds at 1 μM did not show beneficial effects against Aβ25–35-induced neurotoxicity. As a positive control, EGCG showed 72.3 and 90.7% of cell viability at 1 and 10 μM, respectively. Compound 2h bearing bromo group at the 4-position of the benzene ring showed the strongest activity (95.7% of cell viability at 10 μM), better than the positive control EGCG. In addition, the neuroprotective effects of 2-chlorine compound 2e (87.7% of cell viability at 10 μM), 2-fluoro-4-bromo compound 2i (89.1% of cell viability at 10 μM), and 4-methoxy compound 2m (87.7% of cell viability at 10 μM) were comparable with EGCG. Furthermore, a significant change in cell viability of SH-SY5Y cells was not observed when the cells were incubated with 10 μM of active compounds for 24 h in Aβ25–35-free condition (Fig. 3). This result indicates that the neuroprotective activity of compounds may not be attributed to the promotion of cell proliferation.

Fig. 3
figure 3

Effects of compounds alone on cell viability of SH-SY5Y cells without Aβ25–35 exposure. The cell viability was expressed as percentage of the untreated control (100%). Values are mean ± SEM of at least three independent experiments

Full size image
Table 1 Protective effects of compounds against Aβ25–35-induced neurotoxicity in SH-SY5Y cells
Full size table

A primary structure–activity relationship (SAR) study showed that the R group in moiety C is essentially important for the bioactivity. In fact, most synthesized compounds bearing benzene rings as R groups exhibited potent neuroprotective activity against Aβ25–35-induced neurotoxicity, while compounds with R group bearing alkyl or ester showed no activity. Compared with compound 2a, the introduction of fluoro groups in the benzene ring (2c, 2d) decreased the neuroprotective activity. Coincidentally, the unanimous conclusion was reached from the comparison of 2-chloro compound 2e and 2-chloro-4-fluoro compound 2f as well as the comparison of 4-bromo compound 2h and 2-fluoro-4-bromo compound 2i. Interestingly, trifluoromethyl substitution at C-4 position in the benzene ring (2k) led to the loss of neuroprotective activity, while 2-trifluoromethyl compound 2j exhibited neuroprotective activity (78.2% of cell viability at 10 μM), indicating that the CF3 group at C-2 position might be helpful for the neuroprotective activity. However, the opposite result has been observed for the 4-bromo substituted compound 2h, which showed the highest activity (95.7% of cell viability at 10 μM) among all the tested compounds. Such result suggested that the activity of the compounds should not only be influenced by the position of the substituted group (e.g., 4-CF3 vs. 2-CF3), but also by the group itself (e.g., 4-CF3 vs. 4-Br). Compound 2n, bearing a bulky sulfoxide group at 4-position in the benzene ring, was lack of neuroprotective activity probably due to the strong steric hindrance. In addition, the replacement of electron-withdrawing groups (trifluoromethyl, sulfoxide, or halogen groups) with electron-releasing groups (such as methoxyl group) provided the corresponding 2m, which still showed potent neuroprotective activity (87.7% of cell viability at 10 μM), indicating that electronic effect of the substitution in 4-position might not be a key factor in their neuroprotective activity against Aβ25–35-induced neurotoxicity.

Conclusion

In the present work, a series of novel benzothiazol-based 1,3,4-oxadiazole derivatives were designed and synthesized, and their neuroprotective effects against Aβ25–35-induced neurotoxicity in SH-SY5Y cells were evaluated. Based on the preliminary SAR analysis, most of the target compounds containing benzothiazol-based 1,3,4-oxadiazole skeleton showed potential neuroprotective activities. In particular, compound 2h exhibited the neuroprotective activity superior to the positive control EGCG. While the neuroprotective activity of compound 2e, 2i, and 2m was comparable with EGCG. The preliminary SAR study indicated that benzene ring played a pivotal role in neuroprotective activity and the position of substitution was important for activity. Bromo substitution at 4-position improved the neuroprotective activity. In addition, groups contain fluoro on the benzene ring which was not conducive to the activity. The pharmacological data obtained here may be useful for the design of novel neuroprotective compounds with the skeleton of benzothiazol-based 1,3,4-oxadiazole derivatives. Further studies to improve the neuroprotective effect and in vivo bioassay of this class of compounds are in progress.

Experimental

The starting materials and reagents, purchased from commercial suppliers, were used without further purification. All solvents used for the reactions were dried prior to use according to standard procedures. All primary reagents were commercially available. Anal. TLC: pre-coated G60 F-254 silica gel plates (SiO2; Yan Tai Zi Fu Chemical Group Co.). Column chromatography (CC): silica gel (SiO2, 200–300 mesh; Qing Dao Hai Yang Chemical Group Co.), solvents were of analytical grade. NMR spectra: Bruker Avance spectrometer (400 MHz for 1H and 100 or 150 MHz for 13C), using the residual CHCl3 signal (δ(H) = 7.26 ppm) as an internal standard for 1H NMR and CDCl3 signal (δ(C) = 77.0 ppm) for 13C NMR; δ in ppm, J in Hz. ESI–MS: Q-TOF Micro LC/MS–MS mass spectrometer. EI-MS: Finnigan-MAT-95 mass spectrometer.

Methyl 2-(benzo[d]thiazol-2-ylthio)acetate (4)

To a solution of 3.34 g 1,3-benzothiazol-2-ylthiol 3 (20.00 mmol), triethylamine (25.00 mmol) in EtOH was added 5 cm3 ethyl chloroacetate (25.00 mmol). The reaction mixture was refluxed in 20 cm3 EtOH for 3 h. After removal of EtOH under reduced pressure, the resulting mixture was recrystallized with EtOH to obtain white crystals (3.80 g, 75.10%). The spectra of acetate 4 were similar to the Ref. [39].

2-(Benzo[d]thiazol-2-ylthio)acetohydrazide (5)

Compound 4 (3.80 g, 15.02 mmol) and 2.4 cm3 80% hydrazine hydrate (40.00 mmol) in 30 cm3 EtOH were refluxed for 14 h. After removal of solvent under reduced pressure, the resulting mixture was recrystallized with EtOH to obtain white crystals (3.30 g, 84.83%). The spectra of hydrazide 5 were similar to Ref. [41].

5-[(Benzo[d]thiazol-2-ylthio)methyl]-1,3,4-oxadiazole-2-thiol (6)

A mixture of 3.30 g hydrazide 5 (12.74 mmol), 1.70 g KOH (30.00 mmol), and 2 cm3 CS2 (30 mmol) was refluxed in 20 cm3 EtOH for 18 h. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and 30 cm3 ethyl acetate was added and neutralized to slight acidity (pH ~6) by HCl. Then, the resulting mixture was extracted with ethyl acetate and purified by silica-gel chromatography (EtOAc/hexanes, 1:4) to yield the product as an pale yellow powder (2.6 g, 72.87%). The spectra of thiol 7 were similar to the Ref. [42].

General procedure for preparation of compounds 2

A mixture of 84 mg thiol 6 (0.30 mmol), 0.42 g K2CO3 (3.00 mmol), and bromide (0.60 mmol) was stirred for 4 h in CH3CN at room temperature. The reaction solution was concentrated under reduced pressure, and was extracted with CH2Cl2. The organic layer was concentrated and was purified by silica gel column chromatography to afford benzothiazol-based 1,3,4-oxadiazole derivatives 2a–2t, respectively.

2-[[5-(Phenylmethylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2a, C17H13N3OS3)

Yield 44.0%; white solid; m.p.: 96–100 °C; R f = 0.44 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.96–7.89 (m, 1H, Ar–H), 7.80 (d, J = 8.0 Hz, 1H, Ar–H), 7.52–7.43 (m, 1H, Ar–H), 7.43–7.25 (m, 6H, Ar–H), 4.82 (s, 2H, –CH2–), 4.44 (s, 2H, –CH2–) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.1 (C), 164.0 (C), 163.2 (C), 152.7 (C), 135.7 (C), 135.3 (C), 129.1 (CH), 128.8 (CH), 128.1(CH), 126.3 (CH), 124.8 (CH), 122.0 (CH), 121.2 (CH), 36.7(CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H13N3OS3 (M+) 371.0221, found 371.0220.

2-[[5-[(3-Methylphenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2b, C18H15N3OS3)

Yield 83.1%; yellow solid; m.p.: 58–68 °C; R f = 0.42 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.95–7.90 (m, 1H, Ar–H), 7.80–7.76 (m, 1H, Ar–H), 7.50–7.42 (m, 1H, Ar–H), 7.38–7.30 (m, 1H, Ar–H), 7.24–7.14 (m, 3H, Ar–H), 7.13–7.07 (m, 1H, Ar–H), 4.81 (s, 2H, –CH2–), 4.40 (s, 2H, –CH2–), 2.34 (s, 3H, –CH3) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.2 (C), 163.9 (C), 163.2 (C), 152.7 (C), 138.6 (C), 135.6 (C), 135.2 (C), 129.8 (CH), 128.9 (CH), 128.7 (CH), 126.3 (CH), 126.2 (CH), 124.8 (CH), 122.0 (CH), 121.2 (CH), 36.75 (CH2), 26.2 (CH2), 21.4 (CH3) ppm; HREI-TOF: m/z calcd. for C18H15N3OS3 (M+) 385.0377, found 385.0370.

2-[[5-[(3-Fluorophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2c, C17H12FN3OS3)

Yield 94.3%; yellow solid; m.p.: 91–96 °C; R f = 0.38 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.92 (d, J = 8.1 Hz, 1H, Ar–H), 7.80 (d, J = 8.0 Hz, 1H, Ar–H), 7.51–7.43 (m, 1H, Ar–H), 7.41–7.32 (m, 3H, Ar–H), 7.04–6.95 (m, 2H, Ar–H), 4.82 (s, 2H, –CH2–), 4.40 (s, 2H, –CH2–) ppm; 13C NMR (151 MHz, CDCl3): δ = 164.9 (C), 164.1 (C), 163.1 (C), 162.5 (d, J = 246.0 Hz, C), 152.7 (C), 135.7 (C), 131.3 (d, J = 3.3 Hz, C), 130.9 (d, J = 8.3 Hz, CH), 126.3 (CH), 124.8 (CH), 122.0 (CH), 121.21 (CH), 115.7 (d, J = 21.0 Hz, CH), 35.9 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H12FN3OS3 (M+) 389.0127, found 389.0125.

2-[[5-[(3,4-Difluorophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2d, C17H11F2N3OS3)

Yield 57.3%; white solid; m.p.: 85–92 °C; R f = 0.36 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.91 (d, J = 8.1 Hz, 1H, Ar–H), 7.80 (d, J = 8.0 Hz, 1H, Ar–H), 7.51–7.43 (m, 1H, Ar–H), 7.40–7.33 (m, 1H, Ar–H), 7.28–7.21 (m, 1H, Ar–H), 7.16–7.01 (m, 2H, Ar–H), 4.82 (s, 2H, –CH2–), 4.37 (s, 2H, –CH2–) ppm; 13C NMR (151 MHz, CDCl3): δ = 164.5 (C), 164.2 (C), 163.1 (C), 152.7 (C), 151.0 (t, J = 13.0 Hz, C), 149.3 (t, J = 13.0 Hz, C), 135.7 (C), 132.6 (t, J = 4.5 Hz, C), 126.3 (CH), 125.3 (dd, J = 6.4, 3.7 Hz, CH), 124.9 (CH), 122.0 (CH), 121.2 (CH), 118.2 (d, J = 17.9 Hz, CH), 117.5 (d, J = 17.5 Hz, CH), 35.6 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H11F2N3OS3 (M+) 407.0032, found 407.0028.

2-[[5-[(2-Chlorophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2e, C17H12ClN3OS3)

Yield 64.2%; white solid; m.p.: 66–75 °C; R f = 0.34 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.89 (d, J = 8.0 Hz, 1H, Ar–H), 7.75 (d, J = 7.6 Hz, 1H, Ar–H), 7.51 (dd, J = 7.5, 1.7 Hz, 1H, Ar–H), 7.46–7.39 (m, 1H, Ar–H), 7.38–7.27 (m, 2H, Ar–H), 7.20 (td, J = 7.7, 1.8 Hz, 1H, Ar–H), 7.14 (td, J = 7.5, 1.3 Hz, 1H, Ar–H), 4.79 (s, 2H, –CH2–), 4.51 (s, 2H, –CH2–) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.0 (C), 164.1 (C), 163.2 (C), 152.6 (C), 135.6 (C), 134.27 (C), 133.5 (C), 131.4 (CH), 129.7 (CH), 129.6 (CH), 127.0 (CH), 126.3 (CH), 124.8 (CH), 121.9 (CH), 121.2 (CH), 34.5 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H12ClN3OS3 (M+) 404.9831, found 404.9815.

2-[[5-[(2-Chloro-4-fluorophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2f, C17H11ClFN3OS3)

Yield 92.4%; yellow solid; m.p.: 66–71 °C; R f = 0.43 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.91 (d, J = 8.1, 1H, Ar–H), 7.79 (d, J = 8.0, 1H, Ar–H), 7.55 (dd, J = 8.6, 6.0 Hz, 1H, Ar–H), 7.48–7.42 (m, 1H, Ar–H), 7.35 (td, J = 7.7, 1.2 Hz, 1H, Ar–H), 7.14 (dd, J = 8.4, 2.6 Hz, 1H, Ar–H), 6.90 (td, J = 8.3, 2.6 Hz, 1H, Ar–H), 4.82 (s, 2H, –CH2–), 4.50 (s, 2H, –CH2–) ppm; 13C NMR (126 MHz, CDCl3): δ = 164.5 (C), 163.8 (C), 162.7 (C), 161.8 (d, J = 249.5 Hz, C), 152.2 (C), 135.2 (C), 134.7 (d, J = 10.4 Hz, C), 132.1 (d, J = 8.9 Hz, C), 129.2 (d, J = 3.5 Hz, CH), 125.9 (CH), 124.4 (CH), 121.5 (CH), 120.8 (CH), 116.8 (d, J = 25.0 Hz, CH), 113.8 (d, J = 21.0 Hz, CH), 33.4 (CH2), 25.7 (CH2) ppm; HREI-TOF: m/z calcd. for C17H11ClFN3OS3 (M+) 422.9737, found 422.9717.

2-[[5-[(3-Bromophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2g, C17H12BrN3OS3)

Yield 61.5%; yellow oil; R f = 0.35 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.91 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H, Ar–H), 7.56 (s, 1H, Ar–H), 7.51–7.38 (m, 2H), Ar–H, 7.38–7.30 (m, 2H, ArH, Ar–H), 7.16 (t, J = 7.8 Hz, 1H, Ar–H), 4.81 (s, 2H, –CH2–), 4.37 (s, 2H, –CH2–) ppm; 13C NMR (151 MHz, CDCl3): δ = 164.6 (C), 164.2 (C), 163.1 (C), 152.7 (C), 137.8 (C), 135.7 (C), 132.0 (CH), 131.2 (CH), 130.3 (CH), 127.8 (CH), 126.3 (CH), 124.8 (CH), 122.7 (C), 122.0 (CH), 121.2 (CH), 35.9 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H12BrN3OS3 (M+) 448.9326, found 448.9317.

2-[[5-[(4-Bromophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2h, C17H12BrN3OS3)

Yield 85%; white solid, m.p.: 96–100 °C; R f = 0.35 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.94–7.88 (m, 1H, Ar–H), 7.79 (dd, J = 8.0, 0.6 Hz, 1H, Ar–H), 7.50–7.39 (m, 3H, Ar–H), 7.38–7.33 (m, 1H, Ar–H), 7.30–7.23 (m, 2H, Ar–H), 4.81 (s, 2H, –CH2), 4.36 (s, 2H, –CH2) ppm; 13C NMR (101 MHz, CDCl3): δ = 164.7 (C), 164.1 (C), 163.1 (C), 152.7 (C), 135.6 (C), 134.6 (C), 131.0 (CH), 130.8 (CH), 126.4 (CH), 124.9 (CH), 122.2 (C), 122.0 (CH), 121.2 (CH), 36.0 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H12BrN3OS3 (M+) 448.9326, found 448.9331.

2-[[5-[(4-Bromo-3-fluorophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2i, C17H11BrFN3OS3)

Yield 78.3%; yellow solid; m.p.: 82–85 °C; R f = 0.40 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.90 (d, J = 8.1 Hz, 1H, Ar–H), 7.78 (d, J = 8.0 Hz, 1H, Ar–H), 7.50–7.42 (m, 1H, Ar–H), 7.39–7.31 (m, 2H, Ar–H), 7.25–7.15 (m, 2H, Ar–H), 4.80 (s, 2H, –CH2–), 4.38 (s, 2H, –CH2–) ppm; 13C NMR (151 MHz, CDCl3): δ = 164.6 (C), 164.3 (C), 163.1 (C), 160.6 (d, J = 252.0 Hz, C), 152.7 (C), 135.6 (C), 132.3 (d, J = 3.8 Hz, CH), 127.6 (d, J = 3.7 Hz, CH), 126.3 (CH), 124.84 (CH), 122.6 (d, J = 9.5 Hz) (C), 122.4 (d, J = 15.0 Hz, C), 122.0(CH), 121.2 (CH), 119.3 (d, J = 64.0 Hz, CH), 29.5 (d, J = 2.8 Hz, CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C17H11BrFN3OS3 (M+) 466.9232, found 466.9221.

2-[[5-[[2-(Trifluoromethyl)phenyl]methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2j, C18H12F3N3OS3)

Yield 83.5%; white solid; m.p.: 95–98 °C; R f = 0.30 (petroleum ether:acetone 3:1); 1H NMR (400 MHz): δ = 7.91 (d, J = 8.1 Hz, 1H, Ar–H), 7.79 (d, J = 7.9 Hz, 1H, Ar–H), 7.69 (dd, J = 18.9, 7.6 Hz, 2H, Ar–H), 7.52-7.31 (m, 4H, Ar–H), 4.83 (s, 2H, –CH2–), 4.64 (s, 2H, –CH2–) ppm; 13C NMR (151 MHz): δ = 165.1 (C), 164.3 (C), 163.1 (C), 152.7 (C), 135.6 (C), 134.3 (C), 132.4 (CH), 132.0 (CH), 128.7 (q, J = 30.0 Hz, C), 128.4 (CH), 126.4 (q, J = 5.5 Hz, CH), 126.3 (CH), 124.8 (CH), 124.2 (d, J = 273.0 Hz, CF3), 122.0 (CH), 121.2 (CH), 33.2 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C18H12F3N3OS3 (M+) 439.0095, found 439.0096.

2-[[5-[[4-(Trifluoromethyl)phenyl]methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2k, C18H12F3N3OS3)

Yield 83.5%; yellow solid; m.p.: 90–96 °C; R f = 0.31 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.91 (d, J = 8.1 Hz, 1H, Ar–H), 7.80 (d, J = 8.0 Hz, 1H, Ar–H), 7.54 (q, J = 8.4 Hz, 4H, Ar–H), 7.47 (t, J = 7.7 Hz, 1H, Ar–H), 7.36 (t, J = 7.6 Hz, 1H, Ar–H), 4.82 (s, 2H, –CH2–), 4.46 (s, 2H, –CH2–) ppm; 13C NMR (151 MHz, CDCl3): δ = 164.5 (C), 164.3 (C), 163.1 (C), 152.7 (C), 139.7 (C), 135.6 (C), 130.3 (q, J = 33.0 Hz, C), 129.4 (CH), 126.3 (CH), 125.7 (q, J = 33.0 Hz, CH), 124.9 (CH), 123.9 (d, J = 270.0 Hz, CF3), 122.0 (CH), 121.2 (CH), 35.9 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C18H12F3N3OS3 (M+) 439.0095, found 439.0096.

2-[[5-[(4-Nitrophenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2l, C17H12N4O3S3)

Yield 71.3%; white solid; m.p.: 98–102 °C; R f = 0.20 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.94–7.88 (m, 1H), 7.82–7.76 (m, 1H, Ar–H), 7.51–7.38 (m, 3H, Ar–H), 7.38–7.33 (m, 1H, Ar–H), 7.30–7.24 (m, 2H, Ar–H), 4.81 (s, 2H, –CH2–), 4.36 (s, 2H, –CH2–) ppm; 13C NMR (101 MHz, CDCl3): δ = 164.5 (C), 164.1 (C), 163.0 (C), 152.6 (C), 147.5 (C), 143.2 (C), 135.6 (C), 130.0 (CH), 126.4 (CH), 124.9 (CH), 123.9 (CH), 121.9 (CH), 121.35 (CH), 35.5 (CH2), 26.1 (CH2) ppm; HREI-TOF: m/z calcd. for C17H12N4O3S3 (M+) 416.0072, found 416.0068.

2-[[5-[(4-Methoxyphenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2m, C18H15N3O2S3)

Yield 63.2%; yellow solid; m.p.: 58–67 °C; R f = 0.34 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.92 (dd, J = 8.2, 0.5 Hz, 1H, Ar–H), 7.79 (dd, J = 8.0, 0.6 Hz, 1H, Ar–H), 7.51–7.42 (m, 1H, Ar–H), 7.39–7.25 (m, 3H, Ar–H), 6.87–6.79 (m, 2H, Ar–H), 4.81 (s, 2H, –CH2–), 4.39 (s, 2H, –CH2–), 3.79 (s, 3H, –CH3) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.2 (C), 163.8 (C), 163.2 (C), 159.4 (C), 152.7 (C), 135.6 (C), 130.3 (CH), 127.2 (C), 126.3 (CH), 124.8 (CH), 121.9 (CH), 121.2 (CH), 114.1 (CH), 55.3 (CH3), 36.4 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C18H15N3O2S3 (M+) 401. 0326, found 401.0318.

2-[[5-[(4-Methylsulfonylphenyl)methylthio]-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2n, C18H15N3O3S4)

Yield 89.0%; white solid; m.p.: 120–123 °C; R f = 0.44 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.94–7.83 (m, 3H, Ar–H), 7.82–7.75 (m, 1H, Ar–H), 7.60 (d, J = 8.4 Hz, 2H, Ar–H), 7.49–7.41 (m, 1H, Ar–H), 7.38–7.31 (m, 1H, Ar–H), 4.80 (s, 2H, –CH2–), 4.45 (s, 2H, –CH2–), 3.02 (s, 3H, –CH3) ppm; 13C NMR (151 MHz, CDCl3): δ = 164.4 (C), 164.2 (C), 163.1 (C), 152.7 (C), 142.2 (C), 140.1 (C), 135.6 (C), 130.1 (CH), 127.8 (CH), 126.4 (CH), 124.9 (CH), 121.9 (CH), 121.3 (CH), 44.5 (CH3), 35.7 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C18H15N3O3S4 (M+) 448.9996, found 448.9993.

2-[[5-(Butylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2o, C14H15N3OS3)

Yield 78.1%; yellow solid; m.p.: 45–52 °C; R f = 0.42 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.90 (d, J = 8.1 Hz, 1H, Ar–H), 7.76 (d, J = 8.0 Hz, 1H, Ar–H), 7.48–7.39 (m, 1H, Ar–H), 7.35–7.25 (m, 1H, ArH), 4.80 (s, 2H, –CH2–), 3.18 (t, J = 8.0 Hz, 2H, –CH2–), 1.78–1.65 (m, 2H, –CH2–), 1.47–1.34 (m, 2H, –CH2–), 0.90 (t, J = 7.4 Hz, 3H, –CH3) ppm; 13C NMR (151 MHz, CDCl3): δ = 165.8 (C), 163.7 (C), 163.2 (C), 152.7 (C), 135.6 (C), 126.3 (CH), 124.8 (CH), 121.9 (CH), 121.2 (CH), 32.2 (CH2), 31.2 (CH2), 26.3 (CH2), 21.7 (CH2), 13.5 (CH3) ppm; HREI-TOF: m/z calcd. for C14H15N3OS3 (M+) 337.0377, found 337.0377.

2-[[5-(2-Bromopropylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2p, C13H12BrN3OS3)

Yield 66.3%; yellow oil; R f = 0.30 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.92 (d, J = 8.0 Hz, 1H, Ar–H), 7.79 (d, J = 8.0 Hz, 1H, Ar–H), 7.46 (ddd, J = 8.2, 7.3, 1.2 Hz, 1H, Ar–H), 7.35 (ddd, J = 8.4, 7.3, 1.1 Hz, 1H, Ar–H), 4.82 (s, 2H, –CH2–), 3.49 (t, J = 6.2 Hz, 2H, –CH2–), 3.35 (t, J = 6.9 Hz, 2H, –CH2–), 2.37–2.28 (m, 2H, –CH2–) ppm; 13C NMR (126 MHz, CDCl3): δ = 164.6 (C), 163.6 (C), 162.7 (C), 152.2 (C), 135.2 (C), 125.9 (CH), 124.4 (CH), 121.5 (CH), 120.8, (CH), 31.1 (CH2), 30.9 (CH2), 30.2 (CH2), 25.8 (CH2) ppm; HREI-TOF: m/z calcd. for C13H12BrN3OS3 (M+) 400.9326, found 400.9334.

2-[[5-(3-Bromobutylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2q, C14H14BrN3OS3)

Yield 69.0%; yellow oil; R f = 0.31 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.95–7.89 (m, 1H, Ar–H), 7.82-7.76 (m, 1H, Ar–H), 7.51–7.41 (m, 1H, Ar–H), 7.39–7.31 (m, 1H, Ar–H), 4.82 (s, 2H, –CH2–), 3.39 (t, J = 6.3, 2H, –CH2–), 3.22 (t, J = 6.9 Hz, 2H, –CH2–), 2.06–1.86 (m, 4H, –CH2CH2–) ppm; 13C NMR (126 MHz, CDCl3): δ = 164.9 (C), 163.5 (C), 162.7 (C), 152.3 (C), 135.2 (C), 125.9 (CH), 124.4 (CH), 121.5 (CH), 120.8 (CH), 32.1 (CH2), 31.1 (CH2), 30.8 (CH2), 27.4 (CH2), 25.8 (CH2) ppm; HREI-TOF: m/z calcd. for C14H14BrN3OS3 (M+) 414.9482, found 414.9482.

2-[[5-(Methoxycarbonylmethylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2r, C13H11N3OS3)

Yield 85.0%; yellow oil; R f = 0.19 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.91 (d, J = 8.1 Hz, 1H, Ar–H), 7.78 (d, J = 8.0 Hz, 1H, Ar–H), 7.49–7.41 (m, 1H, Ar–H), 7.38–7.31 (m, 1H, Ar–H), 4.82 (s, 2H, –CH2–), 4.04 (s, 2H, –CH2–), 3.76 (s, 3H, –CH3) ppm; 13C NMR (126 MHz, CDCl3): δ = 167.3 (CO), 163.9 (C), 163.7 (C), 162.6 (C), 152.2 (C), 135.2 (C), 125.9 (CH), 124.4 (CH), 121.5 (CH), 120.8 (CH), 52.8 (CH3), 33.6 (CH2), 25.7 (CH2) ppm; HREI-TOF: m/z calcd. for C14H14BrN3O3S3 (M+) 352.9963, found 352.9950.

2-[[5-(Ethoxycarbonylmethylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2s, C14H13N3O3S3)

Yield 81.7%; yellow oil; R f = 0.20 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.91 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H, Ar–H), 7.49–7.42 (m, 1H, Ar–H), 7.38–7.30 (m, 1H, Ar–H), 4.82 (s, 2H, –CH2–), 4.22 (q, J = 7.1 Hz, 2H, –CH2–), 4.03 (s, 2H, –CH2–), 1.27 (t, J = 7.1 Hz, 3H, –CH3) ppm; 13C NMR (126 MHz, CDCl3): δ = 166.8 (CO), 163.8 (C), 163.8 (C), 162.6 (C), 152.2 (C), 135.2 (C), 125.9 (CH), 124.4 (CH), 121.5 (CH), 120.8 (CH), 62.0 (CH2), 33.8 (CH2), 25.7 (CH2), 13.6 (CH3) ppm; HREI-TOF: m/z calcd. for C14H13N3O3S3 (M+) 367.0119, found 367.0128.

2-[[5-(4-Pentenylthio)-1,3,4-oxadiazol-2-yl]methylthio]benzothiazole (2t, C15H15N3O3S3)

Yield 80.2%; yellow oil; R f = 0.40 (petroleum ether:acetone 3:1); 1H NMR (400 MHz, CDCl3): δ = 7.96–7.87 (m, 1H), 7.76 (d, J = 8.0 Hz, 1H, Ar–H), 7.49–7.40 (m, 1H, Ar–H), 7.37–7.29 (m, 1H, Ar–H), 5.82–5.66 (m, 1H, –CH=), 5.12–4.92 (m, 2H, –C=CH2), 4.80 (s, 2H, –CH2–), 3.18 (t, J = 7.3 Hz, 2H, –CH2–), 2.15 (q, J = 7.1 Hz, 2H, –CH2–), 1.85 (qui, J = 7.3 Hz, 2H, –CH2–) ppm; 13C NMR (101 MHz, CDCl3): δ = 165.6 (C), 163.7 (C), 163.2 (C), 152.7 (C), 136.8 (CH), 135.6 (C), 126.3 (CH), 124.8 (CH), 121.9 (CH), 121.2 (CH), 115.9 (=CH2), 32.3 (CH2), 31.7 (CH2), 28.2 (CH2), 26.2 (CH2) ppm; HREI-TOF: m/z calcd. for C15H15N3O3S3 (M+) 349.0377, found 349.0380.

Neuroprotection activity against Aβ25–35-induced neurotoxicity in SH-SY5Y cells

SH-SY5Y cells were high passages from American Type Culture Collection (ATCC) and maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were seeded into multiwell plates at a density of 2–2.5×105 cells per cm3 in MEM/F12 medium (Gibco), supplemented with 10% heat-inactivated bovine calf serum, 100 U/cm3 penicillin, and 100 μg/cm3 streptomycin. Experiments were carried out 24 h after cells were seeded. Stock solution of Aβ25–35 (Sigma, 1 mM) was prepared in phosphate buffer saline (PBS) with 4% dimethylsulfoxide (DMSO) and stored at −20°C, and diluted to 0.1 mM with PBS before application to cultures. All the test compounds were dissolved in DMSO to 10 mM as a stock solution, stored at −20°C, and diluted with MEM/F12 medium before usage. After pretreatment with the compounds for 2 h, 10 μM Aβ25–35 were added to SH-SY5Y cell cultures for 24 h. Assays for cell viability were performed 24 h after cultured in fresh medium.