Discovery of vinyl sulfones as a novel class of neuroprotective agents toward Parkinson's disease therapy.

Article pubs.acs.org/jmc

Discovery of Vinyl Sulfones as a Novel Class of Neuroprotective Agents toward Parkinson’s Disease Therapy Seo Yeon Woo,†,∥ Ji Hyun Kim,§,∥ Mi Kyeong Moon,§ Se-Hee Han,§ Seul Ki Yeon,† Ji Won Choi,† Bo Ko Jang,† Hyo Jung Song,† Yong Gu Kang,† Jin Woo Kim,† Jaeick Lee,‡ Dong Jin Kim,† Onyou Hwang,*,§ and Ki Duk Park*,† †

Center for Neuro-Medicine, Brain Science Institute, and ‡Doping Control Center, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea § Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, 138-736, Republic of Korea S Supporting Information *

ABSTRACT: Although the etiology of Parkinson’s disease (PD) remains elusive, recent studies suggest that oxidative stress contributes to the cascade leading to dopaminergic (DAergic) neurodegeneration. The Nrf2 signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone derivatives. They exhibited a broad range of activities in inducing HO-1, whose gene expression is under the control of Nrf2. Among them, compound 12g was confirmed to activate Nrf2 and induce expression of the Nrf2-dependent antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and protein levels in DAergic neuronal cells. This was accompanied by protection of DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In addition, compound 12g effectively resulted in attenuation of the PD-associated behavioral deficits in the mouse model.

■

INTRODUCTION Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder associated with a profound loss of dopamine (DA) neurons in the substantia nigra (SN) pars compacta. Although the cause and pathogenesis of PD remain elusive, recent studies suggest that oxidative stress plays a critical role in DAergic cell death in PD.1−8 Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and cellular antioxidant defense system.2−4 ROS are likely to be elevated in the brain and neuronal cells because the excitatory amino acids and neurotransmitters produce ROS through their metabolism.8,9 The DAergic neurons are thought to be under particularly high oxidative stress due to the presence of DA.2 Furthermore, superoxide and nitric oxide produced by microglia, which can be elevated by molecules released from damaged DAergic neurons, can also contribute to oxidative stress. Therefore, attenuation of oxidative stress by induction of the antioxidant defense system can be beneficial to survival of DAergic neurons. The nuclear factor E2-related factor 2 (Nrf2) signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes such as heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamatecysteine ligase (GCL), which consists of both the modifier © 2014 American Chemical Society

(GCLM) and catalytic (GCLC) subunits, and several members of the glutathione S-transferase family.10−13 There is increasing evidence that the Nrf2 signaling is associated with PD.14−18 Expression of the Nrf2-mediated cytoprotective genes declines with age, which is the main risk factor for PD.19,20 In the few surviving DAergic neurons of post-mortem brains from PD patients, Nrf2 is localized in the nucleus and is increased compared to control subjects, suggesting an attempt to reduce oxidative stress through the Nrf2-dependent transcription of the antioxidant enzyme genes.21 In animal studies, Nrf2-deficient mice are hypersensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced neurodegeneration,14,18,19 whereas overexpression of Nrf2 is protective.19,22 These findings indicate that Nrf2 activation is a promising target for therapeutics aimed at reducing or preventing DAergic neuronal death in PD. Recently Kumar et al. reported that a chalcone derivative (1) was a potent activator of the Nrf2 signaling pathway from a study using a cell based assay.23 Chalcone contains a unique structure, α,β-unsaturated ketone, that is responsible for its various biological activities.24,25 In this study we introduced either a vinyl sulfoxide or vinyl sulfone group to the α,βunsaturated carbonyl entity of chalcone (2, 3) and initially Received: November 18, 2013 Published: January 27, 2014 1473

dx.doi.org/10.1021/jm401788m | J. Med. Chem. 2014, 57, 1473−1487

Journal of Medicinal Chemistry

Article

synthesized by the base-catalyzed Claisen−Schmidt condensation of an aldehyde and ketone in polar solvents such as ethanol or methanol. Synthesis of the compounds 9−16 followed a standard protocol (Scheme 1). Substituted benzenethiols (5) were coupled with (diethoxyphosphoryl)methyl 4-methylbenzenesulfonate (4) in the presence of base to give the sulfides (6). Oxidation of the sulfides with 1−2.2 equiv of mchloroperoxybenzoic acid (mCPBA) at −20 or 0 °C provided the sulfoxides (7) or the sulfones (8), respectively. The compounds 9−16 were obtained by utilizing Horner−Emmons olefination reaction with commercially available substituted benzaldehydes. Initial Screening against Expression of HO-1 Gene. To initially compare Nrf2 activating efficacy among various compounds, we assessed their ability to induce expression of HO-1, a major Nrf2-dependent gene.26 This was because we had set up in our laboratory a sensitive cell based assay system for detecting HO-1 protein using a sandwich enzyme-linked immunosorbent assay (ELISA). This screening system had been optimized for murine BV-2 cells, and our preliminary studies had revealed that the pattern of Nrf2-dependent HO-1 induction was similar in BV-2 cells and DAergic neuronal cells. For this reason, we used this system in the primary screening, and the results were subsequently confirmed in DAergic neuronal cells as shown below. First, to compare chalcone 1 with either the vinyl sulfoxide compound 2 or the vinyl sulfone compound 3, we prepared three compounds that included the same functional groups on ring A and ring B, respectively. Comparison of HO-1 inducing activities among the three compounds revealed that the vinyl

screened the derivatives for their capacity to induce HO-1, one of the antioxidant enzymes whose expressions are under the control of Nrf2. We found that the introduction of a vinyl sulfone group led to the most potent activities. Herein, we report the design, synthesis, and biological evaluation of a series of vinyl sulfone derivatives (3). One of the compounds with the highest potency was further evaluated for its ability to induce various antioxidant enzymes in DAergic neuronal cells and to protect these cells from oxidative insults. In addition, the compound was tested for its ability to attenuate the nigral DAergc neurodegeneration and PD-associated motor deficits in an animal model of PD.

■

RESULTS AND DISCUSSION Chemistry. We used chalcone 1, which contained an α,βunsaturated ketone moiety, as our structural template and modified the ketone position according to two categories: sulfoxide or sulfone. Chalcone compounds (1) can be readily

Scheme 1. General Procedure for the Preparation of Vinyl Sulfoxide and Vinyl Sulfone Derivatives

1474



Article

12g Activates Nrf2 and Induces NQO1, HO-1, GCL Gene Expression in DAergic Cells. CATH.a cells, a mouse DAergic neuronal cell line derived from the brain,27 were used because these cells have previously been demonstrated by us to readily induce NQO1 gene expression under a Nrf2-activating condition.28 As Nrf2 activation leads to translocation into the nucleus, where it acts as a transcription factor, we determined whether such a phenomenon occurs upon exposure to 12g. Western blot analysis on the nuclear fraction of cells treated with 12g for 3 h revealed dose-dependent increases in the amount of nuclear Nrf2 (Figure 1A). A concentration of 1 μM was sufficient to cause a statistically significant change, and 10 μM resulted in a dramatic increase of (3.5 ± 0.16)-fold. Activation of Nrf2 requires the release of its cytosolic inhibitor protein keap1, which renders Nrf2 no longer subject to degradation by the ubiquitin proteasome system.10 Therefore, the total amount of Nrf2, as well as the nuclear Nrf2, would be elevated. Western blot on total cell lysate demonstrated that Nrf2 was indeed accumulated following the 12g treatment (Figure 1B). Again, a statistically significant increase was observed at 1 μM, and further increases were observed in a dose-dependent manner. Taken together, 12g evidently led to cellular accumulation and nuclear translocation of Nrf2, indicative of activation of the transcription factor. We further asked whether the target enzyme genes’ expressions are induced, as would be expected as a result of Nrf2 activation. NQO1 activity is especially important in protection of DAergic neurons, as it removes DA quinone, the product of DA oxidation, which can damage the cell via a number of mechanisms including protein modification and ROS generation.2 RT-PCR against NQO1 on mRNA isolated from cells treated with 12g showed a dramatic and dosedependent increase. Significant induction was observed at 1 μM ((1.95 ± 0.20)-fold), and the degree of induction reached (5.67 ± 0.13)-fold at 10 μM (Figure 2A). Western blot analysis (Figure 2E) also revealed a similar pattern of elevation of the NQO1 protein level, reaching a (4.43 ± 0.04)-fold increase at 10 μM 12g. 12g also elevated HO-1 expression in DAergic neuronal cells as well. HO-1, the enzyme responsible for the conversion of heme to biliverdin and carbon monoxide, has been shown to have antioxidant and neuroprotective properties.29 A slight but statistically significant increase was observed at 1 μM 12g, and at 10 μM, over 3-fold increases were observed at both mRNA and protein levels (Figure 2B and Figure 2F). GCL is an important antioxidant enzyme because it plays a critical role in the biosynthesis of glutathione, which is the major antioxidant molecule in the cell. The enzyme consists of two subunits, the modulatory (GCLM) and catalytic (GCLC) subunits. RT-PCR against GCLM and GCLC showed that both subunits were significantly and similarly induced starting at 2 μM ((1.43 ± 0.16)- and (1.41 ± 0.01)-fold, respectively). The degrees of induction reached (2.56 ± 0.07)- and (2.64 ± 0.24)fold at 10 μM (Figure 2C and Figure 2D). Western blot analysis (Figure 2G and Figure 2H) also revealed dosedependent elevations of the GCLM and GCLC protein levels by 12g. Taken together, 12g was able to activate Nrf2 and induce expression of the antioxidant enzymes NQO1, HO-1, and GCL in DAergic neuronal cells. 12g Protects DAergic Neuronal Cells. Whether the ability of 12g to induce the antioxidant enzymes might afford neuroprotection was then assessed. For this, CATH.a cells exposed to tetrahydrobiopterin (BH4) was used, as this system

sulfone (10a) was the most potent, followed by the chalcone (1) and the vinyl sulfoxide (9a) (Table 1). Accordingly, we Table 1. Effects of Compounds 1, 9a, and 10a on Expression of the Nrf2-Dependent HO-1 Gene

a

BV-2 cells were plated in 96-well plates at 20 000 cells/well in triplicate with test compounds (20 μM) for 24 h. Protein level of HO1 was determined using a sandwich ELISA assay and expressed as % of vehicle treated control ± SEM. bVehicle: 0.04% DMSO. cSFN: sulforaphane (5 μM), a well-known potent activator of Nrf2.

subsequently synthesized 56 vinyl sulfone derivatives that included various functional groups on both ring A and ring B (Table 2, compounds 10−16). The first set of compounds contained a methoxy group attached at the para position on ring A. Although for 10a−m we did not observe a clear trend in the effect of the electronic properties of the aryl substituent (R2), substitutions on ring B with an electron-withdrawing group (CF3, F, Cl, or OCF3) led to higher activities than an electron-donating group (OMe). Next, we systematically placed a methoxy group at the 2′-, 3′-, and 4′-positions on ring A. In the three series of compounds listed in Table 2, the 2′-methoxy derivatives (12) were more active (2.4−3.8 times higher than vehicle) than the corresponding 3′- and 4′-methoxy derivatives (10 and 11) (1.2−2.6 and 1.1−2.8 times higher than vehicle, respectively). These results indicated that the position of OMe substitution on ring A was crucial for the effect on HO-1 induction activity. Among compounds 12, 12g exerted the highest activity (3.8-fold induction). When the methoxy group on ring A was replaced with hydrogen (16), halogens (13, 14), or dimethoxy group (15), the HO-1 inducing activity was similar or slightly decreased. Interestingly, in this series, we found that 2″-trifluoromethyl derivatives (13a, 14a, 15a, and 16a) showed better activities than the corresponding 3″- and 4″-trifluoromethyl derivatives (13b,c, 14b,c, 15b,c and 16b,c). These findings suggested that the substitution of an electron-withdrawing group at 2″position on ring B in the vinyl sulfone derivatives led to increased HO-1 inducing activity. A similar finding was observed in compounds 10 and 11. The above structure−activity relation study demonstrated an excellent HO-1 inducing activity of 12g. Subsequent experiments were carried out to examine whether this compound could indeed lead to Nrf2 activation and production of various antioxidant enzymes involved in cellular defense system in DAergic neuronal cells and to protection of these cells from oxidative damage. 1475



Article

Table 2. Effects of Vinyl Sulfone Derivatives on Expression of the Nrf2-Dependent HO-1 Gene

compd

R1

R2

10a 10b 10c 10d 10e 10f 10g 10h 10i 10j 10k 10l 10m 10n 11a 11b 11c 11d 11e 11f 11g 11h 11i 12a 12b 12c 12d 12e 12f

4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 4′-OMe 3′-OMe 3′-OMe 3′-OMe 3′-OMe 3′-OMe 3′-OMe 3′-OMe 3′-OMe 3′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe 2′-OMe

2″-CF3 3″-CF3 4″-CF3 2″-F 3″-F 4″-F 2″-Cl 3″-Cl 4″-Cl 2″-OMe 3″-OMe 4″-OMe 2″-OCF3 2″-NH3Cl 2″-CF3 3″-CF3 4″-CF3 2″-F 3″-F 4″-F 2″-Cl 3″-Cl 4″-Cl 2″-CF3 3″-CF3 4″-CF3 2″-F 3″-F 4″-F

HO-1 (%)a

compd

R1

R2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12g 12h 12i 13a 13b 13c 13d 13e 13f 13g 13h 13i 14a 14b 14c 14d 14e 14f 14g 14h 14i 15a 15b 15c 16a 16b 16c vehicleb SFNc

2′-OMe 2′-OMe 2′-OMe 4′-F 4′-F 4′-F 4′-F 4′-F 4′-F 4′-F 4′-F 4′-F 4′-Cl 4′-Cl 4′-Cl 4′-Cl 4′-Cl 4′-Cl 4′-Cl 4′-Cl 4′-Cl 3′,4′-OMe 3′,4′-OMe 3′, 4′-OMe H H H

2″-Cl 3″-Cl 4″-Cl 2″-CF3 3″-CF3 4″-CF3 2″-F 3″-F 4″-F 2″-Cl 3″-Cl 4″-Cl 2″-CF3 3″-CF3 4″-CF3 2″-F 3″-F 4″-F 2″-Cl 3″-Cl 4″-Cl 2″-CF3 3″-CF3 4″-CF3 2″-CF3 3″-CF3 4″-CF3

257.5 213.8 153.7 174.1 203.4 177.0 157.5 124.8 168.9 125.5 145.9 132.7 212.5 140.6 251.1 204.1 147.9 148.4 136.2 159.7 284.2 111.1 227.8 328.2 256.1 357.1 372.7 353.3 259.1

6.7 6.2 25.5 20.2 19.8 1.2 22.1 14.6 8.1 13.9 21.9 17.1 35.6 7.6 56.0 25.3 16.4 10.6 7.1 14.2 12.2 1.8 5.7 31.8 39.8 11.7 11.1 23.4 19.6

HO-1 (%)a 382.2 244.7 330.4 248.1 158.2 124.7 240.7 177.4 141.3 225.3 136.8 111.8 242.5 110.8 139.8 107.4 104.9 111.2 168.6 126.6 116.7 204.1 153.0 119.1 193.2 187.8 154.7 100.0 229.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12.6 17.6 19.8 6.0 17.5 3.5 26.1 4.3 30.8 9.8 4.1 11.4 30.7 8.0 12.6 14.9 14.8 4.6 7.4 17.0 8.7 25.7 10.4 10.6 24.5 11.1 6.2

± 5.8

a

BV-2 cells were plated in 96-well plates at 20 000 cells/well in triplicate with test compounds (20 μM) for 24 h. Protein level of HO-1 was determined using a sandwich ELISA assay and expressed as % of vehicle treated control ± SEM. bVehicle: 0.04% DMSO. cSFN: sulforaphane (5 μM), a well-known potent activator of Nrf2.

has been extensively used in previous studies to examine DAergic cell death by oxidative damage.28,30−35 Co-treatment with various concentrations of 12g provided protection from the BH4-induced cell death, as demonstrated in two different cell death assays. In both ATP assay (Figure 3A) and LDH activity assay (Figure 3B), a concentration as low as 0.1 μM provided statistically significant protection, and the degree of protection was further increased at higher concentrations of 12g. Interestingly, this concentration did not result in a statistically significant increase in the antioxidant enzymes at the mRNA and protein levels (Figure 2). It is possible that the combination of small, seemingly insignificant increases of the enzymes may have been sufficient in providing cytoprotection of these cells. The cells exposed to 12g alone in the same concentration range for 72 h did not cause changes in cell viability (Figure 3C and Figure 3D), demonstrating that 12g itself has no cytotoxicity. 12g Protects the Nigral DAergic Neurons and Striatal DAergic Terminals in the PD Animal Model. Whether 12g could serve as a potential therapeutic agent for PD through neuroprotection was also tested in an animal model of PD generated by administration of the DAergic toxin MPTP. The MPTP treatment led to dramatic disappearance of the tyrosine

Figure 1. 12g induces Nrf2 nuclear translocation and elevates Nrf2 protein level. CATH.a cells were exposed to various concentrations of 12g. (A) After 3 h, the cells were harvested and the nuclear fraction was subjected to Nrf2 Western blot with B-lamin as a nuclear marker. (B) After 24 h, the cells were harvested and the cell lysate was subjected to Nrf2 Western blot with β-actin used as an internal control. All experiments were performed at least in duplicate cultures, and the data were averaged and expressed as fold induction of untreated control ± SEM: (∗) P < 0.05; (∗∗) P < 0.01 vs untreated control.

1476



Article

Figure 2. 12g induces gene expression of antioxidant enzymes in DAergic neuronal cells. CATH.a cells were exposed to various concentrations of 12g and harvested after 6 h for RT-PCR (A−D) and 24 h for Western blot (E−H) analyses. GAPDH and β-actin were used as respective internal controls. All experiments were performed at least in duplicate cultures, and the data were averaged and expressed as fold induction of untreated control ± SEM: (∗) P < 0.05; (∗∗) P < 0.01 vs untreated control.

to the vehicle-treated control (Figure 4A, left panel). Quantitative analysis revealed that the number of THimmunopositive neurons was decreased by MPTP to

Figure 3. 12g protects DAergic neuronal cells from oxidative demise. CATH.a cells were exposed to various concentrations of 12g, and cell viability was estimated by ATP assay (A, C) and LDH activity assay (B, D). (A, B) After exposure to 12g for 20 h, the cells were further treated with 200 μM BH4 for an additional 24 h. (C, D) The cells were exposed to 12g alone for 72 h (C, D). All experiments were performed at least in duplicate cultures, and the data were averaged and expressed as percent of untreated control ± SEM: (A, B) (∗∗) P < 0.01 vs BH4-treated; (C, D) the 12g alone-treated were not significantly different from the untreated controls.

Figure 4. 12g protects the nigrostriatal system against MPTP-elicited neurodegeneration in PD mouse model. (A) SN and striatal sections of mice treated with MPTP alone or with 12g (10 mg/kg) were examined for immunoreactive TH to detect DAergic neurons and fibers, respectively. Typical immunomicrographs are shown. Scale bars = 200 μm. (B) The TH-immunopositive neurons in the SN sections were counted. (C) The TH-immunopositive fibers in the striatal sections were quantitated by densitometry. The data are expressed as % vehicle-treated control. The results are shown as the mean ± SEM (n = 10 per group): (∗) P < 0.05; (∗∗) P < 0.01 vs vehicle-treated control; (#) P < 0.05; (##) P < 0.01 vs MPTP-treated.

hydroxylase (TH) immunopositive DAergic neurons in the SN. On the other hand, in the MPTP animals co-treated with 12g (10 mg/kg), the DAergic neurons remained at the level similar 1477



Article

In a vertical grid test, another behavioral test we previously demonstrated to provide a reliable measure of PD-related motor activity,36 the MPTP mice took much longer to turn around and to climb down on the vertical grid apparatus (12.4 ± 1.8 s and 22.4 ± 3.9 s, respectively) compared to the vehicletreated control (6.3 ± 0.9 s and 8.3 ± 0.7 s, respectively) (Figure 5B and Figure 5C). The animals treated with 12g significantly improved their performance on the vertical grid, as they took a shorter time to turn (4.0 ± 0.7 s) and climb down (6.3 ± 0.7 s). In addition, whereas the total time taken on the grid apparatus was much longer for the MPTP mice (34.8 ± 5.4 s) compared to the control (14.7 ± 1.4 s), the co-treatment with 12g prevented this deficit (10.3 ± 1.3 s) (Figure 5D). The number of successful steps taken on the apparatus was lower in the MPTP animals (75.4 ± 6.2%), but this also returned to the vehicle-treated control upon co-treatment with 12g (96.9 ± 5.5%) (Figure 5E). In all test analyses, the 12g-coadministered group was not significantly different from the vehicle control. Taken together, 12g was found to effectively prevent the motor deficits that are associated with PD.

approximately half of vehicle-administered control group (Figure 4B). On the other hand, the animals coadministered with 12g had the DArgic neurons whose number was not significantly different from the control. 12g alone had no apparent effect on TH-immunoreactive cells (data not shown). DAergic terminals in the striatum, the brain region to which the nigral DAergic neurons project their fibers and where DAergic neurodegeneration first occurs in PD, were also examined (Figure 4A, right panel). A dramatic decrease in the density of TH-immunoreactive fibers was noted in the striatum of the MPTP-treated animals (43% compared to vehicle-treated control by densitometric analysis), but this was prevented in the animals administered with 12g (Figure 4C). Therefore, 12g protected the nigral DAergic neurons and their terminals in the striatum in the PD animal model. On the basis of the present findings that 12g is able to induce the Nrf2 activation and expression of a number of cytoprotective enzyme genes, it is probable that this pathway played a major role in the DAergic neuroprotection. However, one cannot eliminate the possibility that pathway(s) other than the Nrf2 signaling also contributed to the protection. 12g Alleviates Motor Deficits of PD Animal Model. Whether 12g’s neuroprotective effects might be accompanied by symptomatic alleviation was tested in the same animal model. The animals were subjected to behavioral tests for evaluation of their motor activity. In hindlimb test (Figure 5A), the MPTP-treated animals in general showed weakness of postural balance and received an average score of 2.21 ± 0.21, compared to 3.58 ± 0.15 of the vehicle-treated control. In comparison, the animals treated with 12g exhibited considerable improvement, with an average score of 3.00 ± 0.22.

■

CONCLUSION We prepared and evaluated 56 vinyl sulfone compounds for their activity to induce expression of HO-1, which is an Nrf2dependent gene. Significant activity was observed for many of these compounds. Comparison of methoxy substituted aryl regioisomers (2′, 3′, 4′) on ring A demonstrated that 2′methoxy derivatives exhibited the most potent activity. The SAR indicated that electron-withdrawing group substitution on ring B led to increased activity. The compound with highest activity (12g) was demonstrated to activate Nrf2 and induce the expression of Nrf2-dependent antioxidant enzymes NQO-1, GCLC, and GLCM as well as HO-1 in DAergic neuronal cells in a dose-dependent manner. 12g significantly protected DAergic neurons from cytotoxic damage both in vitro and in vivo and attenuated the PD-associated motor deficits in MPTPinduced mouse model of PD. In conclusion, we present a novel vinyl sulfone compound with a therapeutic potential for PD.

■

EXPERIMENTAL SECTION

General Methods. Melting points were determined in open capillary tubes using an OptiMelt melting point apparatus (Stanford Research System, Inc.) and are uncorrected. NMR spectra were obtained at 400 MHz (1H) and 100 MHz (13C) using TMS as an internal standard. Chemical shifts (δ) are reported in parts per million (ppm) from tetramethylsilane (TMS). High-resolution mass spectrometry was performed on a LTQ Orbitrap (Thermo Electron Corporation) instrument. Analytical HPLC was performed using a Waters E2695 system equipped with the following column: SunFire C18 column (4.6 mm × 150 mm; 5 μm). HPLC data were recorded using following parameters: H2O/MeCN, 90/10 → 0/100 in 17 min, +3 min isocratic, flow rate of 1.0 mL/min, λ = 254 and 280 nm. Reactions were monitored by analytical thin-layer chromatography (TLC) plates (Merck, catalog no. 1.05715) and analyzed with 254 nm light. The samples were purified by column chromatography using silica gel (Merck, catalog nos. 1.07734 and 1.09385). All chemicals and solvents were reagent grade and used as obtained from commercial sources without further purification. Yields reported are for purified products and were not optimized. Compounds were checked by TLC, 1 H and 13C NMR, HRMS. The TLC, NMR, and the analytical data confirmed that the purity of the products was ≥95%. General Procedure for the Vinyl Sulfone Derivatives. To a cooled anhydrous tetrahydrofuran (THF) solution (−78 °C) of 8 was added 2 M n-BuLi solution in cyclohexane (1.05 equiv). The reaction mixture was stirred at −78 °C (1 h), and then the desired substituted

Figure 5. 12g alleviates motor deficits in MPTP-induced PD mice. Motor deficits of animals treated with MPTP alone or co-treated with 12g (10 mg/kg) were assessed by the hindlimb test (A) and by various indices on their behavior on vertical grid apparatus (B−E). The results are shown as the mean ± SEM (n = 10 per group): (∗∗) P < 0.01 vs vehicle-treated control, (#) P < 0.05; (##) P < 0.01 vs MPTP-treated. 1478



Article

111.0−113.0 °C; HPLC purity, 12.9 min, 99.3%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 9.0 Hz, 2ArH), 7.71 (d, J = 15.6 Hz, (E)isomeric CH), 7.45 (m, ArH), 7.36−7.41 (m, ArH), 7.14−7.19 (m, ArH), 7.08−7.13 (m, ArH), 6.98−7.03 (m, 2ArH, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.7, 161.5 (d, JC−F = 253.5 Hz, ArC-F), 134.3, 132.6 (d, J = 8.8 Hz), 131.9, 130.8 (d, JC−F = 8.1 Hz), 130.1, 130.0, 124.7 (d, JC−F = 3.2 Hz), 120.6 (d, JC−F = 11.3 Hz), 116.3 (d, JC−F = 21.5 Hz), 114.6 (12ArC, 2CH), 55.7 (OCH3); HRMS (M + H) + (ESI+ ) 293.0645 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene (10e). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.39 g (85%) of 10e as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 91.0−93.0 °C; HPLC purity, 12.9 min, 99.6%; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.9 Hz, 2ArH), 7.58 (d, J = 15.4 Hz, (E)isomeric CH), 7.36 (td like due to ddd, J = 5.8, 7.9 Hz, ArH), 7.24− 7.26 (m, ArH), 7.14−7.18 (m, ArH), 7.08−7.12 (m, ArH), 7.01 (d, J = 8.9 Hz, 2ArH), 6.84 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.7, 162.9 (d, JC−F = 246.2 Hz, ArC-F), 139.8, 134.7 (d, JC−F = 7.6 Hz), 131.8, 130.7 (d, JC−F = 8.2 Hz), 130.0, 129.5, 124.5 (d, JC−F = 2.0 Hz), 117.9 (d, JC−F = 21.2 Hz), 114.7 (d, JC−F = 21.7 Hz), 114.6 (12ArC, 2CH), 55.7 (OCH3); HRMS (M + H)+ (ESI+) 293.0645 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4-fluorolbenzene (10f).37 Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.38 g (83%) of 10f as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 112.0−114.0 °C; HPLC purity, 12.7 min, 97.0%;1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.8 Hz, 2ArH), 7.59 (d, J = 15.4 Hz, (E)isomeric CH), 7.46 (dd, J = 5.3, 8.8 Hz, 2ArH), 7.07 (t, J = 8.8 Hz, 2ArH), 7.01 (d, J = 8.8 Hz, 2ArH), 6.77 (d, J = 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 164.2 (d, JC−F = 251.0 Hz, ArC-F), 163.6, 140.0, 132.1, 130.5 (d, JC−F = 8.6 Hz), 129.9, 128.8 (d, JC−F = 2.8 Hz), 127.8, 116.3 (d, JC−F = 21.8 Hz), 114.6 (12ArC, 2CH), 55.7 (OCH3); HRMS (M + H)+ (ESI+) 293.0647 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2-chloromethylbenzene (10g).38 Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.36 g (76%) of 10g as a white solid. Rf = 0.30 (n-hexane/ EtOAc 3/1); mp 148.0−150.0 °C; HPLC purity, 13.6 min, 99.3%; 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 15.4 Hz, (E)-isomeric CH), 7.89 (d, J = 9.0 Hz, 2ArH), 7.50 (dd, J = 1.7, 7.9 Hz, ArH), 7.43 (dd, J = 1.3, 7.9 Hz, ArH), 7.30−7.35 (m, ArH), 7.24−7.28 (m, ArH), 7.02 (d, J = 9.0 Hz, 2ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.9, 137.4, 135.3, 131.9, 131.8, 131.0, 130.9, 130.5, 130.2, 128.3, 127.3, 114.8 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0349 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-chloromethylbenzene (10h). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.34 g (71%) of 10h as a white solid. Rf = 0.35 (n-hexane/ EtOAc 3/1); mp 71.0−72.0 °C; HPLC purity, 13.6 min, 99.1%; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.8 Hz, 2ArH), 7.56 (d, J = 15.4 Hz, (E)-isomeric CH), 7.45 (s, ArH), 7.32−7.39 (m, 3ArH), 7.02 (d, J = 8.8 Hz, 2ArH), 6.85 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.8, 139.7, 135.2, 134.4, 131.9, 130.9, 130.4, 130.1, 129.7, 128.2, 126.8, 114.8 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0349 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4-chloromethylbenzene (10i). 39 Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane

benzaldehyde (1.1 equiv) was added at −78 °C. The reaction mixture was stirred at room temperature (1 h) and then quenched with H2O (same volume of THF). The reaction mixture was diluted with EtOAc (∼200 mL) and washed with H2O (2 × ∼ 200 mL) and brine (2 × ∼200 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2. (E)-1-(2-((4-Methoxyphenyl)sulfinyl)vinyl)-2-trifluoromethylbenzene (9a). Diethyl (4-methoxyphenylsulfinyl)methylphosphonate (0.27 g, 0.88 mmol), 2 M n-BuLi solution in cyclohexane (0.49 mL, 0.92 mmol), and 2-(trifluoromethyl)benzaldehyde (0.13 mL, 0.97 mmol) gave 0.07 g (22%) of 9a as a colorless oil. Rf = 0.25 (n-hexane/ EtOAc 2/1); 1H NMR (400 MHz, CDCl3) δ 7.68−7.75 (m, ArH, (E)isomeric CH), 7.62 (d, J = 8.8 Hz, 2ArH), 7.50−7.57 (m, 2ArH), 7.44 (t like due to dd, J = 7.5 Hz, ArH), 7.02 (d, J = 8.8 Hz, 2ArH), 6.79 (d, J = 15.2 Hz, (E)-isomeric CH), 3.85 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 162.4, 138.3, 134.6, 133.2, 132.2, 131.6, 129.2, 128.5 (q, JC−F = 30.4 Hz), 128.4, 127.2, 126.3 (q, JC−F = 5.3 Hz), 124.1 (q, JC−F = 272.2 Hz, CF3), 115.2 (12ArC, 2CH), 55.7 (OCH3); HRMS (M + H)+ (ESI+) 327.0670 [M + H]+ (calcd for C16H13F3O2SH+ 327.0667). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2trifluoromethylbenzene (10a). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.49 g, 1.52 mmol), 2 M n-BuLi solution in cyclohexane (0.80 mL, 1.60 mmol), and 2(trifluoromethyl)benzaldehyde (0.22 mL, 1.67 mmol) gave 0.34 g (66%) of 10a as a white solid. Rf = 0.40 (n-hexane/EtOAc 2/1); mp 94.0−95.0 °C; HPLC purity, 13.8 min, 99.8%; 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 15.2 Hz, (E)-isomeric CH), 7.88 (d, J = 8.9 Hz, 2ArH), 7.71 (d, J = 7.6 Hz, ArH), 7.48−7.59 (m, 3ArH), 7.02 (d, J = 8.9 Hz, 2ArH), 6.82 (d, J = 15.2 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.9, 137.2, 132.5, 132.3, 131.5, 131.4, 130.3, 130.1, 129.0 (d, JC−F = 30.5 Hz), 128.3, 126.3 (q, JC−F = 5.4 Hz), 121.0 (q, JC−F = 272.4 Hz, CF3), 114.7 (12ArC, 2CH), 55.7 (OCH3); HRMS (M + H)+ (ESI+) 343.0612 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3trifluoromethylbenzene (10b). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.46 g (87%) of 10b as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 104.0−106.0 °C; HPLC purity, 13.8 min, 99.4%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 9.0 Hz, 2ArH), 7.71 (s, ArH), 7.63−7.66 (m, 2ArH, (E)-isomeric CH), 7.51−7.55 (m, ArH), 7.02 (d, J = 9.0 Hz, 2ArH), 6.92 (d, J = 15.5 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.8, 139.4, 133.4, 131.6, 131.5 (q, JC−F = 32.6 Hz), 130.2, 130.1, 129.7, 127.3 (d, JC−F = 3.2 Hz), 124.9 (d, JC−F = 3.7 Hz), 123.6 (q, JC−F = 271.0 Hz, CF3), 114.7 (12ArC, 2CH), 55.7 (OCH3); one signal was not detected and is believed to overlap with nearby peaks; HRMS (M + H)+ (ESI+) 343.0613 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4trifluoromethylbenzene (10c). Diethyl (4-methoxyphenylsulfonylmethylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.47 g (88%) of 10c as a white solid. Rf = 0.40 (n-hexane/EtOAc 3/1); mp 135.0−137.0 °C; HPLC purity, 14.0 min, 99.8%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.9 Hz, 2ArH), 7.57−7.66 (m, 4ArH, (E)-isomeric CH), 7.02 (d, J = 8.9 Hz, 2ArH), 6.93 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.9, 139.3, 136.0, 132.3 (q, JC−F = 32.5 Hz), 131.5, 130.7, 130.1, 128.7, 126.0, 123.7 (ArC, trans CH) (q, JC−F = 270.9 Hz, CF3), 114.7 (12ArC, 2CH), 55.7 (OCH3); HRMS (M + H) + (ESI +) 343.0613 [M + H] + (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2-fluorobenzene (10d). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.36 g (74%) of 10d as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 1479



Article

(0.82 mL, 1.63 mmol), and 4-chlorobenzaldehyde (0.24 g, 1.71 mmol) gave 0.43 g (89%) of 10i as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 129.0−133.0 °C (lit.39 121.0−123.0 °C); HPLC purity, 13.9 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.8 Hz, 2ArH), 7.5662−7.66 (d, J = 15.4 Hz,(E)-isomeric CH), 7.39 (d, J = 8.6 Hz, 2ArH), 7.34 (d, J = 8.6 Hz, 2ArH), 7.00 (d, J = 8.8 Hz, 2ArH), 6.82 (d, J = 15.4 Hz, (E)-isomeric CH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.8, 140.0, 137.1, 132.1, 131.2, 130.1, 129.8, 129.5, 128.7, 114.8 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0349 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2-methoxybenzene (10j). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-methoxybenzaldehyde (0.21 mL, 1.71 mmol) gave 0.44 g (91%) of 10j as a white solid. Rf = 0.25 (n-hexane/ EtOAc 3/1); mp 83.0−85.0 °C; HPLC purity, 12.8 min, 96.8%; 1H NMR (400 MHz, CDCl3) δ 7.82−7.89 (m, 2ArH, (E)-isomeric CH), 7.34−7.42 (m, 2ArH), 7.05 (d, J = 15.5 Hz, (E)-isomeric CH), 7.00 (d, J = 8.9 Hz, 2ArH), 6.90−6.97 (m, 2ArH), 3.88 (s, OCH3), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.5, 158.9, 137.6, 132.9, 132.4, 130.7, 129.9, 128.7, 121.5, 120.9, 114.6, 111.4 (12ArC, 2CH), 55.8 (OCH3), 55.6 (OCH3); HRMS (M + H)+ (ESI+) 305.0845 [M + H]+ (calcd for C16H16O4SH+ 305.0848). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-methoxybenzene (10k). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-methoxybenzaldehyde (0.21 mL, 1.71 mmol) gave 0.46 g (98%) of 10k as a yellow oil. Rf = 0.30 (n-hexane/ EtOAc 3/1); HPLC purity, 12.7 min, 97.3%; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 9.0 Hz, 2ArH), 7.59 (d, J = 15.4 Hz, (E)isomeric CH), 7.29 (t like due to dd, J = 7.9 Hz, ArH), 7.06 (d, J = 7.6 Hz, ArH), 7.01 (d, J = 9.0 Hz, 2ArH), 6.93−6.98 (m, 2ArH), 6.83 (d, J = 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3), 3.81 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.7, 160.1, 141.4, 133.9, 132.3, 130.1, 130.0, 128.3, 121.2, 117.0, 114.7, 113.4 (12ArC, 2CH), 55.8 (OCH3), 55.4 (OCH3); HRMS (M + H)+ (ESI+) 305.0845 [M + H]+ (calcd for C16H16O4SH+ 305.0848). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-4-methoxybenzene (10l). 39 Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-methoxybenzaldehyde (0.21 mL, 1.71 mmol) gave 0.43 g (91%) of 10l as a white solid. Rf = 0.20 (n-hexane/ EtOAc 3/1); mp 127.0−129.0 °C (lit.39 108.0−109.0 °C); HPLC purity, 12.6 min, 97.8%; 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 9.0 Hz, 2ArH), 7.58 (d, J = 15.4 Hz, (E)-isomeric CH), 7.42 (d, J = 8.7 Hz, 2ArH), 7.00 (d, J = 9.0 Hz, 2ArH), 6.89 (d, J = 8.7 Hz, 2ArH), 6.69 (d, J = 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3), 3.83 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.5, 162.0, 141.3, 132.8, 130.4, 129.8, 125.3, 125.2, 114.6 (12ArC, 2CH), 55.8 (OCH3), 55.5 (OCH3); one signal was not detected and is believed to overlap with nearby peaks; HRMS (M + H)+ (ESI+) 305.0847 [M + H]+ (calcd for C16H16O4SH+ 305.0848). (E)-1-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-2trifluoromethoxybenzene (10m). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2(trifluoromethoxy)benzaldehyde (0.24 mL, 1.71 mmol) gave 0.48 g (87%) of 10m as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 75.0−77.0 °C; HPLC purity, 14.0 min, 99.1%; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.9 Hz, 2ArH), 7.82 (d, J = 15.5 Hz, (E)isomeric CH), 7.54 (d, J = 7.0 Hz, ArH), 7.44 (t like due to dd, J = 7.8 Hz, ArH), 7.30 (t like due to dd, J = 7.5 Hz, 2ArH), 7.02 (d, J = 8.9 Hz, 2ArH), 6.92 (d, J = 15.5 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13 C NMR (100 MHz, CDCl3) δ 163.9, 147.7, 134.4, 132.2, 131.7, 131.3, 130.1, 128.8, 127.3, 124.3, 121.5, 120.5 (q, J = 257.5 Hz, OCF3), 114.7 (12ArC. 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 359.0565 [M + H]+ (calcd for C16H13F3O4SH+ 359.0565). (E)-2-(2-((4-Methoxyphenyl)sulfonyl)vinyl)aniline Hydrochloride (10n). Diethyl (4-methoxyphenylsulfonyl)methylphosphonate (0.30 g, 0.93 mmol), 2 M n-BuLi solution in cyclohexane

(0.49 mL, 0.98 mmol), and 2-aminobenzaldehyde (0.12 g, 1.02 mmol) gave 0.18 g (70%) of 10n as a yellow solid. Rf = 0.38 (n-hexane/EtOAc 1/3); mp 177.0−180.0 °C; HPLC purity, 11.4 min, >99.0%; 1H NMR (400 MHz, MeOD) δ 7.92 (d, J = 9.0 Hz, 2ArH), 7.82−7.87 (m, ArH, (E)-isomeric CH), 7.58 (td like due to ddd, J = 1.4, 7.6 Hz, ArH), 7.43−7.51 (m, 2ArH, (E)-isomeric CH), 7.15 (d, J = 9.0 Hz, 2ArH), 3.90 (s, OCH3); 13C NMR (100 MHz, MeOD) δ 164.2, 133.2, 132.2, 132.1, 131.2, 130.6, 129.9, 129.0, 128.4, 127.0, 123.8, 114.5 (12ArC, 2CH), 55.0 (OCH3); HRMS (M + H)+ (ESI+) 290.0854 [M + H]+ (calcd for C15H15NO3SH+ 290.0851). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-2trifluoromethylbenzene (11a). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.41 g (77%) of 11a as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 74.0−75.0 °C; HPLC purity, 14.0 min, 99.4%; 1H NMR (400 MHz, CDCl3) δ 8.05 (dd, J = 2.0, 15.2 Hz, (E)-isomeric CH), 7.72 (d, J = 7.3 Hz, ArH), 7.43−7.60 (m, 6ArH), 7.15 (ddd, J = 1.0, 2.6, 8.2 Hz, ArH), 6.83 (d, J = 15.2 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.3, 141.3, 138.5, 132.4, 132.0, 131.4, 130.6, 130.5, 129.2 (q, JC−F = 30.6 Hz), 128.4, 126.4 (d, JC−F = 5.2 Hz), 123.8 (q, JC−F = 272.2 Hz, CF3), 120.5, 120.1, 112.1 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 343.0615 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-3trifluoromethylbenzene (11b). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.43 g (80%) of 11b as a white solid. Rf = 0.40 (n-hexane/EtOAc 3/1); mp 73.0−75.0 °C; HPLC purity, 14.1 min, 99.7%; 1H NMR (400 MHz, CDCl3) δ 7.66−7.73 (m, 3ArH, (E)-isomeric CH), 7.52−7.56 (m, 2ArH), 7.45−7.49 (m, 2ArH), 7.16 (ddd, J = 0.8, 2.4, 8.2 Hz, ArH), 6.94 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.3, 141.5, 140.6, 133.3, 131.8, 131.7 (q, JC−F = 32.6 Hz), 130.7, 129.8, 129.6, 127.6, 125.1, 123.7 (q, JC−F = 270.6 Hz, CF3), 120.2, 120.1, 112.4 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 343.0619 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-4trifluoromethylbenzene (11c). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.46 g (87%) of 11c as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); mp 101.0−102.0 °C; HPLC purity, 14.1 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 15.4 Hz, (E)-isomeric CH), 7.66 (d, J = 8.5 Hz, 2ArH), 7.60 (d, J = 8.4 Hz, 2ArH), 7.53 (td like due to ddd, J = 1.4, 7.7 Hz, ArH), 7.48 (d, J = 8.0 Hz, ArH), 7.45 (t like due to dd, J = 2.1 Hz, ArH), 7.16 (ddd, J = 1.1, 2.6, 8.5 Hz, ArH), 6.95 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.3, 141.4, 140.8, 135.9, 132.7 (q, JC−F = 32.6 Hz), 130.7, 130.1, 128.9, 126.1 (d, JC−F = 3.4 Hz), 123.7 (q, JC−F = 270.8 Hz, CF3), 120.2, 120.1, 112.4 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 343.0616 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-2-fluorobenzene (11d). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.39 g (85%) of 11d as a colorless oil. Rf = 0.35 (n-hexane/EtOAc 3/1); HPLC purity, 13.1 min, 98.7%; 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 15.6 Hz, (E)-isomeric CH), 7.53 (td like due to ddd, J = 1.3, 7.8 Hz, ArH), 7.43−7.49 (m, 3ArH), 7.37−7.43 (m, ArH), 7.09−7.20 (m, 3ArH), 7.02 (d, J = 15.6 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 161.6 (d, JC−F = 253.7 Hz, ArC-F), 160.2, 141.7, 135.5, 132.9 (d, JC−F = 8.8 Hz), 130.6, 130.3, 130.1 (d, J = 8.3 Hz), 124.8, 120.6 (d, JC−F = 11.2 Hz), 120.0, 116.5 (d, JC−F = 21.5 Hz), 112.2 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 293.0647 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). 1480



Article

(E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene (11e). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.42 g (93%) of 11e as a colorless oil. Rf = 0.35 (n-hexane/EtOAc 3/1); HPLC purity, 12.9 min, 96.9%; 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 15.4 Hz, (E)-isomeric CH), 7.43−7.53 (m, 5ArH), 7.12−7.15 (m, ArH), 7.08 (t like due to dd, J = 8.6 Hz, 2ArH), 6.79 (d, J = 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.0 (d, JC−F = 246.3 Hz, ArC-F), 160.2, 141.6, 141.0, 134.6 (d, JC−F = 7.5 Hz), 130.8, 130.7, 130.6, 128.9, 124.7, 120.1 (d, JC−F = 21.2 Hz), 118.2 (d, JC−F = 21.2 Hz), 114.9 (d, JC−F = 22.1 Hz), 112.3 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 293.0650 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-4-fluorobenzene (11f). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.42 g (93%) of 11f as a colorless oil. Rf = 0.35 (n-hexane/EtOAc 3/1); HPLC purity, 13.0 min, 99.6%; 1H NMR (400 MHz, CDCl3) δ 7.63 (d, J = 15.4 Hz, (E)-isomeric CH), 7.52 (dt like due to ddd, J = 1.4, 7.8 Hz, ArH), 7.44−7.48 (m, 2ArH), 7.37 (td like due to ddd, J = 5.8, 7.8 Hz, ArH), 7.26−7.28 (m, ArH), 7.09−7.17 (m, 3ArH), 6.86 (d, J = 15.4 Hz, (E)-isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 164.4 (d, JC−F = 251.4 Hz, ArC-F), 160.2, 141.9, 141.2, 130.7 (d, JC−F = 8.7 Hz), 130.6, 128.7, 127.1, 119.9, 116.4 (d, JC−F = 21.9 Hz), 112.2 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 293.0647 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-2-chlorobenzene (11g). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.39 g (81%) of 11g as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 88.0−89.0 °C; HPLC purity, 13.9 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 15.4 Hz, (E)-isomeric CH), 7.50−7.56 (m, 2ArH), 7.43−7.48 (m, 3ArH), 7.34 (td like due to ddd, J = 1.6, 7.6 Hz, ArH), 7.25−7.29 (m, ArH), 7.15 (ddd, J = 0.8, 2.6, 8.2 Hz, ArH), 6.90 (d, J = 15.4 Hz, (E)-isomeric CH), 3.88 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.2, 141.5, 138.4, 135.3, 132.0, 130.6, 130.4, 130.0, 128.3, 127.3, 120.0, 112.3 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0350 [M + H]+ (calcd for C15H13ClO3SH+309.0352). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-3-chlorobenzene (11h). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.36 g (74%) of 11h as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); HPLC purity, 14.0 min, 98.9%; mp 71.0−72.0 °C; 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 15.4 Hz, (E)-isomeric CH), 7.50−7.53 (m, ArH), 7.43−7.47 (m, 3ArH), 7.30−7.38 (m, 3ArH), 7.14 (ddd, J = 1.0, 2.6, 8.1 Hz, ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 160.3, 141.6, 140.8, 135.3, 134.3, 131.2, 130.7, 130.5, 129.0, 128.3, 126.9, 120.2, 120.1, 112.3 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0353 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((3-Methoxyphenyl)sulfonyl)vinyl)-4-chlorobenzene (11i). Diethyl (3-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-chlorobenzaldehyde (0.24 g, 1.71 mmol) gave 0.33 g (70%) of 11i as a white solid. Rf = 0.40 (n-hexane/EtOAc 3/1); mp 85.0−87.0 °C; HPLC purity, 13.9 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 15.4 Hz, (E)-isomeric CH), 7.52 (dt like due to ddd, J = 1.3, 7.7 Hz, ArH), 7.43−7.48 (m, 3ArH), 7.31−7.40 (m, 3ArH), 7.15 (ddd, J = 1.0, 2.6, 8.2 Hz, ArH), 6.87 (d, J = 15.4 Hz, (E)isomeric CH), 3.87 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.2, 141.7, 141.0, 137.3, 130.9, 130.6, 129.8, 129.5, 127.9, 120.0, 119.9, 112.2 (12ArC, 2CH), 55.8 (OCH3); HRMS (M + H)+ (ESI+) 309.0352 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-2trifluoromethylbenzene (12a). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi

solution in cyclohexane (0.82 mL, 1.63 mmol), and 2(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.49 g (92%) of 12a as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 116.0−119.0 °C; HPLC purity, 13.4 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.08 (dd, J = 1.8, 15.2 Hz, (E)-isomeric CH), 8.04 (dd, J = 1.8, 7.8 Hz, ArH), 7.72 (d, J = 7.6 Hz, ArH), 7.65 (d, J = 7.6 Hz, ArH), 7.56−7.61 (m, 2ArH), 7.51 (t like due to dd, J = 7.6 Hz, ArH), 7.13 (t like due to dd, J = 7.4 Hz, ArH), 7.01−7.06 (m, ArH, (E)-isomeric CH), 3.95 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.6, 138.9, 135.8, 132.6, 131.6, 131.3, 130.3, 129.9, 129.3 (q, JC−F = 30.6 Hz), 128.4, 127.6, 126.4 (q, JC−F = 5.4 Hz), 123.8 (q, JC−F = 272.4 Hz, CF3), 120.7, 112.4 (12ArC, 2CH), 56.2 (OCH3); HRMS (M + H)+ (ESI+) 343.0616 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-3trifluoromethylbenzene (12b). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.51 g (96%) of 12b as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 138.0−139.0 °C; HPLC purity, 13.6 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.01 (dd, J = 1.8, 7.8 Hz, ArH), 7.63−7.72 (m, 3ArH, (E)-isomeric CH), 7.57 (ddd, J = 1.0, 1.8, 7.5 Hz, ArH), 7.52 (t like due to dd, J = 7.8 Hz, ArH), 7.19 (d, J = 15.5 Hz, (E)-isomeric CH), 7.10 (td like due to ddd, J = 1.0, 8.4 Hz, ArH), 7.01 (d, J = 8.0 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.5, 141.4, 135.8, 133.7, 131.6, 131.5 (q, JC−F = 32.6 Hz), 129.8, 129.6, 129.2, 128.1, 127.4, 125.0, 123.7 (q, JC−F = 270.8 Hz, CF3), 120.9, 112.6 (12ArC,2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 343.0618 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-4trifluoromethylbenzene (12c). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-(trifluoromethyl)benzaldehyde (0.23 mL, 1.71 mmol) gave 0.52 g (94%) of 12c as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 113.0−114.0 °C; HPLC purity, 13.7 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.03 (dd, J = 1.8, 7.7 Hz, ArH), 7.71 (d, J = 15.5 Hz, (E)-isomeric CH), 7.66 (d, J = 8.3 Hz, 2ArH), 7.58−7.62 (m, 3ArH), 7.22 (d, J = 15.5 Hz, (E)-isomeric CH), 7.13 (td like due to ddd, J = 0.8, 8.3 Hz, ArH), 7.03 (d, J = 8.3 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.5, 141.4, 136.4, 135.8, 132.4 (q, JC−F = 32.5 Hz), 129.8, 129.7, 128.8, 128.2, 126.1, 123.8 (q, JC−F = 270.6 Hz, CF3), 120.9, 112.6 (12ArC, 2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 343.0617 [M + H]+ (calcd for C16H13F3O3SH+ 343.0616). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-2-fluorobenzene (12d). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.38 g (85%) of 12d as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 110.0−113.0 °C; HPLC purity, 12.5 min, 99.0%; 1H NMR (400 MHz, CDCl3) δ 8.03 (dd, J = 1.7, 7.8 Hz, ArH), 7.78 (d, J = 15.6 Hz, (E)isomeric CH), 7.58 (ddd, J = 1.7, 7.5, 8.7 Hz, ArH), 7.48 (td like due to ddd, J = 1.7, 7.5 Hz, ArH), 7.38−7.42 (m, 1H), 7.27 (d, J = 15.6 Hz, (E)-isomeric CH), 7.18 (td like due to ddd, J = 0.9, 7.5 Hz, ArH), 7.09−7.14 (m, 2ArH), 7.02 (d, J = 8.2 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 161.4 (d, JC−F = 253.3 Hz, ArC-F), 157.4, 136.4, 135.6, 132.6 (d, JC−F = 8.8 Hz), 130.0, 129.8 (d, JC−F = 7.9 Hz), 129.5, 128.4, 124.7 (d, JC−F = 3.1 Hz), 121.0 (d, JC−F = 11.3 Hz), 120.7, 116.3 (d, JC−F = 21.6 Hz), 112.5 (12ArC,2CH), 56.2 (OCH3); HRMS (M + H) + (ESI+ ) 293.0647 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene (12e). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.37 g (82%) of 12e as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 117.0−119.0 °C; HPLC purity, 12.5 min, 99.0%; 1H NMR (400 MHz, CDCl3) δ 8.02 (dd, J = 1.7, 7.9 Hz, ArH), 7.65 (d, J = 15.5 Hz, (E)isomeric CH), 7.58 (ddd, J = 1.7, 7.4, 8.4 Hz, ArH), 7.37 (td like due to ddd, J = 5.8, 7.9 Hz, ArH), 7.28 (d, J = 7.9 Hz, ArH), 7.19 (dt like 1481



Article

7.50−7.60 (m, 3ArH), 7.22−7.26 (m, 2ArH), 6.82 (d, J = 15.2 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.9 (d, JC−F = 255.0 Hz, ArC-F), 138.7, 136.2 (d, JC−F = 2.3 Hz), 132.4, 131.9, 131.3, 130.8 (d, JC−F = 9.6 Hz), 130.6, 130.2, 129.2 (q, JC−F = 30.6 Hz), 128.4, 126.5 (q, JC−F = 272.4 Hz, CF3), 116.8 (d, JC−F = 22.6 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 331.0411 [M + H]+ (calcd for C15H10F4O2SH+ 331.0416). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-3-trifluoromethylbenzene (13b). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 3-(trifluoromethyl)benzaldehyde (0.24 mL, 1.77 mmol) gave 0.50 g (93%) of 13b as a white solid. Rf = 0.40 (n-hexane/ EtOAc 5/1); mp 110.0−112.0 °C; HPLC purity, 14.0 min, 99.5%; 1H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 5.0, 8.7 Hz, 2ArH), 7.73 (s, ArH), 7.70 (d, J = 15.5 Hz, (E)-isomeric CH), 7.65−7.68 (m, 2ArH), 7.55 (t like due to dd, J = 7.8 Hz, ArH), 7.24 (t like due to dd, J = 8.7 Hz, 2ArH), 6.93 (d, J = 15.5 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.9 (d, JC−F = 255.0 Hz, ArC-F), 136.4, 133.2, 131.9, 131.8 (q, JC−F = 32.6 Hz), 130.8 (d, JC−F = 9.0 Hz), 129.9, 129.5, 127.7 (d, JC−F = 3.1 Hz), 125.1 (d, JC−F = 3.5 Hz), 123.7 (q, JC−F = 270.9 Hz, CF3), 117.0, 116.8 (12ArC, 2CH); HRMS (M + H)+ (ESI+) 331.0415 [M + H]+ (calcd for C15H10F4O2SH+ 331.0416). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-4-trifluoromethylbenzene (13c). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 4-(trifluoromethyl)benzaldehyde (0.24 mL, 1.77 mmol) gave 0.49 g (92%) of 13c as a white solid. Rf = 0.50 (n-hexane/ EtOAc 5/1); mp 140.0−141.0 °C; HPLC purity, 14.1 min, 99.4%; 1H NMR (400 MHz, CDCl3) δ 7.98 (dd, J = 5.0, 8.9 Hz, 2ArH), 7.70 (d, J = 15.4 Hz, (E)-isomeric CH), 7.66 (d, J = 8.4 Hz, 2ArH), 7.60 (d, J = 8.4 Hz, 2ArH), 7.25 (t like due to dd, J = 8.6 Hz, 2ArH), 6.93 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 166.0 (d, JC−F = 255.2 Hz, ArC-F), 140.7, 136.4, 135.8, 132.8 (q, JC−F = 32.7 Hz), 130.8 (d, JC−F = 9.6 Hz), 130.0, 128.9, 126.2 (d, JC−F = 3.3 Hz), 123.7 (q, JC−F = 271.0 Hz, CF3), 116.9 (d, JC−F = 22.6 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 331.0422 [M + H]+ (calcd for C15H10F4O2SH+ 331.0416). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-2-fluorobenzene (13d). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 2-fluorobenzaldehyde (0.19 mL, 1.77 mmol) gave 0.37 g (82%) of 13d as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 72.0−74.0 °C; HPLC purity, 13.1 min, 98.8%; 1H NMR (400 MHz, CDCl3) δ 7.97 (dd, J = 5.0, 8.9 Hz, 2ArH), 7.76 (d, J = 15.4 Hz, (E)isomeric CH), 7.47 (td like due to ddd, J = 1.7, 7.6 Hz, ArH), 7.38− 7.44 (m, ArH), 7.16−7.25 (m, 3ArH), 7.12 (ddd, J = 0.9, 8.3, 10.8 Hz, ArH), 7.00 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.6 Hz, ArC-F), 161.7 (d, JC−F = 253.9 Hz, ArC-F), 136.7, 135.8, 133.0 (d, JC−F = 8.9 Hz), 130.7 (d, JC−F = 9.5 Hz), 130.2 (d, JC−F = 8.5 Hz), 124.8 (d, JC−F = 3.1 Hz), 120.6 (d, JC−F = 11.3 Hz), 116.9, 116.7, 116.4 (12ArC, 2CH); HRMS (M + H)+ (ESI+) 281.0445 [M + H]+ (calcd for C14H10F2O2SH+ 281.0448). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-3-fluorobenzene (13e). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 3-fluorobenzaldehyde (0.19 mL, 1.77 mmol) gave 0.42 g (93%) of 13e as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 52.0−54.0 °C; HPLC purity, 13.0 min, 98.4%; 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 5.0, 9.2 Hz, 2ArH), 7.64 (d, J = 15.4 Hz, (E)isomeric CH), 7.34 (td like due to ddd, J = 5.7, 8.0 Hz, ArH), 7.21− 7.28 (m, 3ArH), 7.18 (td like due to ddd, J = 2.0, 9.2 Hz, ArH), 7.13 (ddd, J = 0.8, 2.5, 8.3 Hz, ArH), 6.84 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.8 Hz, ArCF), 163.0 (d, JC−F = 246.5 Hz, ArC-F), 141.2, 136.6, 134.6, 130.9, 130.7 (d, JC−F = 9.5 Hz), 128.8, 124.8, 118.3 (d, JC−F = 21.2 Hz), 116.8 (d, JC−F = 22.5 Hz), 114.9 (d, JC−F = 22.0 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 281.0446 [M + H]+ (calcd for C14H10F2O2SH+ 281.0448). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-4-fluorobenzene (13f).37 Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g,

due to ddd, J = 2.0, 9.5 Hz, ArH), 7.08−7.16 (m, 2ArH, (E)-isomeric CH), 7.02 (d, J = 8.4 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 162.9 (d, JC−F = 246.0 Hz, ArC-F), 157.4, 141.8, 135.6, 135.0 (d, JC−F = 7.5 Hz), 130.7 (d, JC−F = 8.1 Hz), 129.5, 128.5, 128.3, 124.5, 120.8, 117.9 (d, JC−F = 21.3 Hz), 114.7 (d, JC−F = 22.0 Hz), 112.5 (12ArC, 2CH), 56.3 (OCH3); HRMS (M + H)+ (ESI+) 293.0648 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-4-fluorobenzene (12f). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.71 mmol) gave 0.39 g (87%) of 12f as a white solid. Rf = 0.35 (n-hexane/EtOAc 3/1); mp 122.0−124.0 °C; HPLC purity, 12.5 min, 98.9%;1H NMR (400 MHz, CDCl3) δ 8.02 (dd, J = 1.7, 7.8 Hz, ArH), 7.65 (d, J = 15.5 Hz, (E)isomeric CH), 7.57 (ddd, J = 1.7, 7.4, 9.1 Hz, ArH), 7.50 (dd, J = 5.3, 8.8 Hz, 2ArH), 7.01−7.13 (m, 3ArH, (E)-isomeric CH), 7.02 (d, J = 8.4 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 164.3 (d, JC−F = 251.0 Hz, ArC-F), 157.4, 142.0, 135.5, 130.6 (d, JC−F = 8.5 Hz), 129.5, 129.1, 128.5, 126.7, 120.8, 116.3 (d, JC−F = 21.9 Hz), 112.5 (12ArC, 2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 293.0648 [M + H]+ (calcd for C15H13FO3SH+ 293.0648). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-2-chlorobenzene (12g). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.44 g (91%) of 12g as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 119.0−121.0 °C; HPLC purity, 13.3 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 15.4 Hz, (E)-isomeric CH), 8.04 (dd, J = 1.7, 7.7 Hz, ArH), 7.55−7.61 (m, 2ArH), 7.43 (dd, J = 1.4, 7.7 Hz, ArH), 7.33 (dd, J = 1.7, 7.7 Hz, ArH), 7.28 (dd, J = 1.4, 7.7 Hz, ArH), 7.10−7.13 (m, ArH, (E)-isomeric CH), 7.01 (d, J = 8.4 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.3, 141.7, 136.9, 135.5, 131.3, 129.7, 129.4, 129.3, 128.3, 127.6, 120.7, 112.5 (12ArC, 12CH), 56.3 (OCH3); HRMS (M + H)+ (ESI+) 309.0352 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-3-chlorobenzene (12h). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.71 mmol) gave 0.44 g (91%) of 12h as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); mp 116.0−117.0 °C; HPLC purity, 13.4 min, 99.8%; 1H NMR (400 MHz, CDCl3) δ 8.02 (dd, J = 1.7, 7.9 Hz, ArH), 7.56−7.65 (m, ArH,(E)isomeric CH), 7.48 (s, ArH), 7.32−7.40 (m, 3ArH), 7.09−7.16 (m, ArH, (E)-isomeric CH), 7.02 (d, J = 8.3 Hz, ArH), 3.98 (s, OCH3); 13 C NMR (100 MHz, CDCl3) δ 157.5, 141.9, 137.1, 135.6, 131.5, 129.8, 129.7, 129.5, 128.6, 127.7, 120.9, 112.6(12ArC, 2CH), 56.5 (OCH3); HRMS (M + H)+ (ESI+) 309.0352[M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((2-Methoxyphenyl)sulfonyl)vinyl)-4-chlorobenzene (12i). Diethyl (2-methoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.55 mmol), 2 M n-BuLi solution in cyclohexane (0.82 mL, 1.63 mmol), and 4-chlorobenzaldehyde (0.24 g, 1.71 mmol) gave 0.44 g (91%) of 12i as a white solid. Rf = 0.45 (n-hexane/EtOAc 3/1); mp 125.0−126.0 °C; HPLC purity, 13.5 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.02 (dd, J = 1.7, 7.7 Hz, ArH), 7.64 (d, J = 15.5 Hz, (E)-isomeric CH), 7.58 (td like due to ddd, J = 1.7, 7.7 Hz, ArH), 7.43 (d, J = 8.6 Hz, 2ArH), 7.37 (d, J = 8.6 Hz, 2ArH), 7.09−7.13 (m, ArH, (E)-isomeric CH), 7.02 (d, J = 8.2 Hz, ArH), 3.97 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.4, 141.6, 135.7, 135.1, 134.7, 130.9, 130.4, 129.6, 128.6, 128.1, 126.8, 120.8, 112.6 (12ArC, 2CH), 56.4 (OCH3); HRMS (M + H)+ (ESI+) 309.0353 [M + H]+ (calcd for C15H13ClO3SH+ 309.0352). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-2-trifluoromethylbenzene (13a). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 2-(trifluoromethyl)benzaldehyde (0.23 mL, 1.77 mmol) gave 0.50 g (93%) of 13a as a white solid. Rf = 0.30 (n-hexane/ EtOAc 5/1); mp 78.0−79.0 °C; HPLC purity, 13.9 min, 99.1%; 1H NMR (400 MHz, CDCl3) δ 8.08 (dd, J = 1.8, 15.2 Hz, (E)-isomeric CH), 7.97 (dd, J = 5.0, 8.8 Hz, 2ArH), 7.72 (d, J = 7.6 Hz, ArH), 1482



Article

1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 4-fluorobenzaldehyde (0.19 mL, 1.77 mmol) gave 0.44 g (97%) of 13f as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 83.0−84.0 °C; HPLC purity, 12.9 min, 99.5%; 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 5.2, 8.8 Hz, 2ArH), 7.65 (d, J = 15.4 Hz, (E)isomeric CH), 7.49 (dd, J = 5.2, 8.8 Hz, 2ArH), 7.23 (t like due to dd, J = 8.6 Hz, 2ArH), 7.09 (t like due to dd, J = 8.6 Hz, 2ArH), 6.77 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.7 (d, JC−F = 254.5 Hz, ArC-F), 164.5 (d, JC−F = 251.6 Hz, ArC-F), 141.4, 136.8, 130.7 (d, JC−F = 8.7 Hz), 130.6 (d, JC−F = 9.5 Hz), 128.6 (d, JC−F = 2.7 Hz), 127.0, 116.7 (d, JC−F = 22.6 Hz), 116.4 (d, JC−F = 22.0 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 281.0445 [M + H]+ (calcd for C14H10F2O2SH+ 281.0448). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-2-chlorobenzene (13g). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), 2-chlorobenzaldehyde (0.20 mL, 1.77 mmol) gave 0.40 g (84%) of 13g as a white solid. Rf = 0.40 (n-hexane/EtOAc 5/1); mp 107.0−108.0 °C; HPLC purity, 13.7 min, 99.3%; 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 15.5 Hz, (E)-isomeric CH), 7.98 (dd, J = 5.1, 8.8 Hz, 2ArH), 7.51 (dd, J = 1.6, 7.7 Hz, ArH), 7.44 (dd, J = 1.1, 8.0 Hz, ArH), 7.35 (td like due to ddd, J = 1.6, 7.7 Hz, ArH), 7.22−7.29 (m, 3ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.8 Hz, ArC-F), 138.7, 136.5, 135.4, 132.1, 130.9, 130.8 (d, JC−F = 9.5 Hz), 130.5, 130.0, 128.4, 127.4, 116.8 (d, JC−F = 22.5 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 297.0149 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-3-chlorobenzene (13h). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 3-chlorobenzaldehyde (0.20 mL, 1.77 mmol) gave 0.37 g (76%) of 13h as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 107.0−109.0 °C; HPLC purity, 13.8 min, 99.4%; 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 5.0, 9.0 Hz, 2ArH), 7.61 (d, J = 15.4 Hz, (E)isomeric CH), 7.47 (s, ArH), 7.32−7.41 (m, 3ArH), 7.24 (t like due to dd, J = 9.0 Hz, 2ArH), 6.85 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.9 Hz, ArC-F), 141.0, 136.6, 135.3, 134.1, 131.2, 130.8 (d, JC−F = 9.5 Hz), 130.5, 128.9, 128.3, 127.0, 116.8 (d, JC−F = 22.6 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 297.0152 [M + H] + (calcd for C14H10ClFO2SH+ 297.0152). (E)-1-(2-((4-Fluorophenyl)sulfonyl)vinyl)-4-chlorobenzene (13i). Diethyl (4-fluorophenylsulfonyl)methylphosphonate (0.50 g, 1.61 mmol), 2 M n-BuLi solution in cyclohexane (0.85 mL, 1.69 mmol), and 4-chlorobenzaldehyde (0.25 g, 1.77 mmol) gave 0.38 g (79%) of 13i as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 134.0−136.0 °C; HPLC purity, 13.9 min, 98.9%; 1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 5.0, 8.8 Hz, 2ArH), 7.62 (d, J = 15.4 Hz, (E)isomeric CH), 7.42 (d, J = 8.6 Hz, 2ArH), 7.38 (d, J = 8.8 Hz, 2ArH), 7.23 (t like due to dd, J = 8.6 Hz, 2ArH), 6.82 (d, J = 15.4 Hz, (E)isomeric CH); 13C NMR (100 MHz, CDCl3) δ 165.8 (d, JC−F = 254.7 Hz, ArC-F), 141.2, 137.5, 136.7, 130.8 (d, JC−F = 12.0 Hz), 130.6, 129.9, 129.5, 127.9, 116.8 (d, JC−F = 22.5 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 297.0150 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-2-trifluoromethylbenzene (14a). Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 2-(trifluoromethyl)benzaldehyde (0.22 mL, 1.68 mmol) gave 0.45 g (85%) of 14a as a white solid. Rf = 0.45 (n-hexane/ EtOAc 5/1); mp 155.0−157.0 °C; HPLC purity, 14.8 min, 99.6%; 1H NMR (400 MHz, CDCl3) δ 8.06 (dd, J = 2.0, 15.2 Hz, (E)-isomeric CH), 7.89 (d, J = 8.6 Hz, 2ArH), 7.73 (d, J = 7.3 Hz, ArH), 7.51−7.59 (m, 5ArH), 6.81 (d, J = 15.2 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 140.5, 139.1, 138.7, 132.4, 131.6, 131.2, 130.7, 129.9, 129.4, 129.2 (q, JC−F = 30.5 Hz), 128.5, 126.5 (q, JC−F = 5.4 Hz), 123.8 (q, JC−F = 272.3 Hz, CF3) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 347.0114 [M + H]+ (calcd for C15H10ClF3O2SH+ 347.0120). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-3-trifluoromethylbenzene (14b). Diethyl (4-chlorophenylsulfonyl)methylphosphonate

(0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 3-(trifluoromethyl)benzaldehyde (0.22 mL, 1.68 mmol) gave 0.50 g (95%) of 14b as a white solid. Rf = 0.50 (n-hexane/ EtOAc 5/1); mp 159.0−161.0 °C; HPLC purity, 14.9 min, >99.0%; 1 H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.7 Hz, 2ArH), 7.65−7.73 (m, 3ArH, (E)-isomeric CH), 7.53−7.57 (m, 3ArH), 6.92 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 141.1, 140.5, 138.9, 133.2, 131.9, 131.8 (q, JC−F = 32.7 Hz), 129.9, 129.4, 129.2, 127.8, 125.2, 123.6 (q, JC−F = 270.5 Hz, CF3) (12ArC, 2CH); one signal was not detected and is believed to overlap with nearby peaks; HRMS (M + H) + (ESI+ ) 347.0121 [M + H]+ (calcd for C15H10ClF3O2SH+ 347.0120). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-4-trifluoromethylbenzene (14c). Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 4-(trifluoromethyl)benzaldehyde (0.23 mL, 1.68 mmol) gave 0.49 g (91%) of 14c as a white solid. Rf = 0.55 (n-hexane/ EtOAc 5/1); mp 147.0−149.0 °C; HPLC purity, 14.9 min, 99.0%; 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.6 Hz, 2ArH), 7.65−7.73 (m, 2ArH, (E)-isomeric CH), 7.59 (d, J = 8.3 Hz, 2ArH), 7.55 (d, J = 8.6 Hz, 2ArH), 6.92 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 141.1, 140.6, 138.8, 135.7, 132.8 (q, JC−F = 32.6 Hz), 129.9, 129.8, 129.4, 128.9, 126.2 (d, JC−F = 3.4 Hz), 123.7 (q, JC−F = 270.8 Hz, CF3) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 347.0113 [M + H]+ (calcd for C15H10ClF3O2SH+ 347.0120). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-2-fluorobenzene (14d).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 2-fluorobenzaldehyde (0.18 mL, 1.68 mmol) gave 0.42 g (91%) of 14d as a white solid. Rf = 0.50 (n-hexane/EtOAc 5/1); mp 89.0−91.0 °C; HPLC purity, 14.0 min, 99.4%; 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.6 Hz, 2ArH), 7.76 (d, J = 15.6 Hz, (E)isomeric CH), 7.53 (d, J = 8.6 Hz, 2ArH), 7.46 (td like due to ddd, J = 1.6, 7.6 Hz ArH), 7.38−7.44 (m, ArH), 7.18 (td like due to ddd, J = 0.9, 7.6 Hz, ArH), 7.12 (td like due to ddd, J = 0.9, 8.3 Hz, ArH), 7.00 (d, J = 15.6 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 161.7 (d, JC−F = 254.0 Hz, ArC-F), 140.4, 139.2, 136.2, 133.1 (d, JC−F = 8.9 Hz), 130.6, 130.0, 129.8, 129.4, 124.9 (d, JC−F = 3.3 Hz), 120.5 (d, JC−F = 11.2 Hz) (12ArC, 2CH); one signal was not detected and is believed to overlap with nearby peaks; HRMS (M + H)+ (ESI+) 297.0150 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-3-fluorobenzene (14e). Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 3-fluorobenzaldehyde (0.18 mL, 1.68 mmol) gave 0.45 g (99%) of 14e as a white solid. Rf = 0.55 (n-hexane/EtOAc 5/1); mp 101.0−102.0 °C; HPLC purity, 13.9 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.7 Hz, 2ArH), 7.64 (d, J = 15.4 Hz, (E)isomeric CH), 7.54 (d, J = 8.7 Hz, 2ArH), 7.38 (td like due to ddd, J = 5.7, 8.0 Hz, ArH), 7.28 (s, ArH), 7.11−7.19 (m, 2ArH), 6.83 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 163.0 (d, JC−F = 246.4 Hz, ArC-F), 141.6, 140.3, 139.0, 134.4 (d, JC−F = 7.6 Hz), 130.8 (d, JC−F = 8.1 Hz), 129.8, 129.3, 128.5, 124.8, 118.3 (d, JC−F = 21.1 Hz), 114.9 (d, JC−F = 22.2 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 297.0156 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-4-fluorobenzene (14f).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 4-fluorobenzaldehyde (0.18 mL, 1.68 mmol) gave 0.45 g (99%) of 14f as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 117.0−120.0 °C (lit.39 100.0−102.0 °C); HPLC purity, 13.8 min, 99.7%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.6 Hz, 2ArH), 7.65 (d, J = 15.4 Hz, (E)-isomeric CH), 7.53 (d, J = 8.6 Hz, 2ArH), 7.49 (dd, J = 5.3, 8.6 Hz, ArH), 7.09 (t like due to dd, J = 8.6 Hz, 2ArH), 6.76 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 164.6 (d, JC−F = 251.9 Hz, ArC-F), 140.8, 140.3, 139.3, 130.8 (d, JC−F = 8.7 Hz), 129.8, 129.3, 128.6 (d, JC−F = 2.9 Hz), 126.8, 116.5 (d, JC−F = 22.0 Hz) (12ArC, 2CH); HRMS (M + H)+ (ESI+) 297.0155 [M + H]+ (calcd for C14H10ClFO2SH+ 297.0152). 1483



Article

+ H)+ (ESI+) 373.0723 [M + H]+ (calcd for C17H15F3O4SH+ 373.0721). ( E) - 1- ( 2- (( 3, 4 -D i m e t ho x yp h e ny l )s ul f on yl ) vi n yl ) -4 trifluoromethylbenzene (15c). Diethyl (3,4-dimethoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.42 mmol), 2 M n-BuLi solution in cyclohexane (0.75 mL, 1.49 mmol), and 4(trifluoromethyl)benzaldehyde (0.21 mL, 1.56 mmol) gave 0.50 g (94%) of 15c as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 190.0−192.0 °C; HPLC purity, 13.4 min, 99.5%; 1H NMR (400 MHz, CDCl3) δ 7.56−7.66 (m, 5ArH, (E)-isomeric CH), 7.38 (d, J = 2.0 Hz, ArH), 6.99 (d, J = 8.5 Hz, ArH), 6.94 (d, J = 15.4 Hz, (E)-isomeric CH), 3.95 (s, 2OCH3); 13C NMR (100 MHz, CDCl3) δ 152.7, 148.6, 138.4, 135.0, 131.6 (q, JC−F = 32.6 Hz), 130.7, 129.7, 127.8, 125.2, 125.1, 122.7 (q, JC−F = 270.6 Hz, CF3), 121.3, 110.2, 109.1 (12ArC, 2CH), 55.4 (2OCH3); HRMS (M + H)+ (ESI+) 373.0724 [M + H]+ (calcd for C17H15F3O4SH+ 373.0721). (E)-1-(2-((Phenyl)sulfonyl)vinyl)-2-trifluoromethylbenzene (16a). Diethyl ((phenylsulfonyl)methyl)phosphonate (0.50 g, 1.71 mmol), 2 M n-BuLi solution in cyclohexane (0.90 mL, 1.80 mmol), and 2-(trifluoromethyl)benzaldehyde (0.25 mL, 1.88 mmol) gave 0.41 g (76%) of 16a as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 72.0−74.0 °C; HPLC purity, 13.7 min, 99.7%; 1H NMR (400 MHz, CDCl3) δ 8.05 (dd, J = 1.9, 15.2 Hz, (E)-isomeric CH), 7.96 (d, J = 7.4 Hz, 2ArH), 7.72 (d, J = 7.4 Hz, ArH), 7.49−7.66 (m, 6ArH), 6.84 (d, J = 15.2 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 140.1, 138.5, 133.8, 132.4, 132.0, 131.4, 130.5, 129.5, 129.2 (q, JC−F = 30.5 Hz), 128.4, 127.9, 126.5 (12ArC, 2CH), 123.8 (q, JC−F = 272.3 Hz, CF3); HRMS (M + H)+ (ESI+) 313.0510 [M + H]+ (calcd for C15H11F3O2SH+ 312.0432). (E)-1-(2-((Phenyl)sulfonyl)vinyl)-3-trifluoromethylbenzene (16b). Diethyl ((phenylsulfonyl)methyl)phosphonate (0.50 g, 1.71 mmol), 2 M n-BuLi solution in cyclohexane (0.90 mL, 1.80 mmol), and 3-(trifluoromethyl)benzaldehyde (0.25 mL, 1.88 mmol) gave 0.48 g (89%) of 16b as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 93.0−95.0 °C; HPLC purity, 13.8 min, 98.8%; 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 7.3 Hz, 2ArH), 7.52−7.73 (m, 7ArH, (E)isomeric CH), 6.95 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 140.6, 140.3, 133.8, 133.3, 131.8, 131.7 (q, JC−F = 32.8 Hz), 129.8, 129.7, 129.6, 127.9, 127.6, 125.2 (12ArC, 2CH), 123.7 (q, JC−F = 271.0 Hz, CF3); HRMS (M + H)+ (ESI+) 313.0510 [M + H]+ (calcd for C15H11F3O2SH+ 312.0432). (E)-1-(2-((Phenyl)sulfonyl)vinyl)-4-trifluoromethylbenzene (16c).40 Diethyl ((phenylsulfonyl)methyl)phosphonate (0.50 g, 1.71 mmol), 2 M n-BuLi solution in cyclohexane (0.90 mL, 1.80 mmol), and 4-(trifluoromethyl)benzaldehyde (0.25 mL, 1.88 mmol) gave 0.50 g (94%) of 16c as a white solid. Rf = 0.40 (n-hexane/EtOAc 5/1); mp 129.0−131.0 °C (lit.40 130.0−131.0 °C); HPLC purity, 13.9 min, 99.4%; 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.1 Hz, 2ArH), 7.56−7.72 (m, 7ArH, (E)-isomeric CH), 6.95 (d, J = 15.4 Hz, (E)isomeric CH); 13C NMR (100 MHz, CDCl3) δ 140.6, 140.3, 135.9, 133.8, 132.8 (q, JC−F = 32.6 Hz), 130.2, 129.6, 128.9, 127.9, 126.2 (12ArC, 2CH), 123.7 (q, JC−F = 270.6 Hz, CF3); HRMS (M + H)+ (ESI+) 313.0510 [M + H]+ (calcd for C15H11F3O2SH+ 312.0432). HO-1 Induction Assay. BV-2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 100 IU/L penicillin, 10 μg/mL streptomycin, and 10% fetal bovine serum (FBS). The cells (2.0 × 104 cells/mL) in a 96-well plate were treated with test compounds (20 μM) for 24 h and washed. The cell pellet was combined with 50 μL of lysis solution (150 mM NaCl, 50 mM TrisHCl, pH 8.0, and 1% Nonidet-P40), incubated on ice for 20 min, and then centrifuged (3000g, 15 min). The supernatant was diluted 10 times with 50 mM Tris buffer (90 μL, pH 8.0). U-shaped 96-well plates were coated with 100 μL of mouse polyclonal HO-1 capture antibody (Enzo Life Sciences, Farmindale, NY) that had been diluted (1:250) with coating buffer (10 mM sodium phosphate, pH 7.4, and 15 mM NaCl). After incubation at room temperature overnight, the solution was replaced by 200 μL blocking buffer [10 mM sodium phosphate, pH 7.4, 15 mM NaCl, and 1% bovine serum albumin (BSA)], and the sample was incubated for 1 h. The blocking buffer was then removed, and the diluted supernatant

(E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-2-chlorobenzene (14g).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 2-chlorobenzaldehyde (0.19 mL, 1.68 mmol) gave 0.44 g (91%) of 14g as a white solid. Rf = 0.45 (n-hexane/EtOAc 5/1); mp 139.0−141.0 °C (lit.39 139.5−140.5 °C); HPLC purity, 14.8 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 8.10 (d, J = 15.4 Hz, (E)isomeric CH), 7.90 (d, J = 8.6 Hz, 2ArH), 7.54 (d, J = 8.6 Hz, 2ArH), 7.51 (dd, J = 1.6, 7.8 Hz, ArH), 7.45 (dd, J = 1.2, 8.0 Hz, ArH), 7.35 (td like due to ddd, J = 1.6, 7.8 Hz, ArH). 7.26−7.30 (m, ArH), 6.88 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 140.4, 139.1, 139.0, 135.5, 132.2, 130.6, 130.5, 129.8, 129.7, 129.4, 128.4, 127.4 (12ArC, 2CH); HRMS (M + H)+ (ESI+) 312.9854 [M + H]+ (calcd for C14H10Cl2O2SH+ 312.9857). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-3-chlorobenzene (14h).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 3-chlorobenzaldehyde (0.19 mL, 1.68 mmol) gave 0.46 g (97%) of 14h as a white solid. Rf = 0.55 (n-hexane/EtOAc 5/1); mp 128.0−130.0 °C (lit.39 130.0−131.0 °C); HPLC purity, 14.8 min, 98.7%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.5 Hz, 2ArH), 7.62 (d, J = 15.4 Hz, (E)-isomeric CH), 7.53 (d, J = 8.5 Hz, 2ArH), 7.47 (s, ArH), 7.32−7.41 (m, 3ArH), 6.84 (d, J = 15.4 Hz, (E)isomeric CH); 13C NMR (100 MHz, CDCl3) δ 141.4, 140.4, 139.0, 135.3, 134.1, 131.3, 130.5, 129.8, 129.3, 128.6, 128.3, 127.0 (12ArC, 2CH); HRMS (M + H)+ (ESI+) 312.9851 [M + H]+ (calcd for C14H10Cl2O2SH+ 312.9857). (E)-1-(2-((4-Chlorophenyl)sulfonyl)vinyl)-4-chlorobenzene (14i).39 Diethyl (4-chlorophenylsulfonyl)methylphosphonate (0.50 g, 1.53 mmol), 2 M n-BuLi solution in cyclohexane (0.81 mL, 1.61 mmol), and 4-chlorobenzaldehyde (0.24 g, 1.68 mmol) gave 0.47 g (98%) of 14i as a white solid. Rf = 0.55 (n-hexane/EtOAc 5/1); mp 163.0−165.0 °C (lit.39 167.0−167.5 °C); HPLC purity, 14.8 min, 98.4%; 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.7 Hz, 2ArH), 7.63 (d, J = 15.4 Hz, (E)-isomeric CH), 7.53 (d, J = 8.7 Hz, 2ArH), 7.37−7.43 (m, 4ArH), 6.81 (d, J = 15.4 Hz, (E)-isomeric CH); 13C NMR (100 MHz, CDCl3) δ 141.6, 140.3, 139.1, 137.6, 130.8, 129.9, 129.8, 129.5, 129.3, 127.6 (12ArC, 2CH); HRMS (M + H)+ (ESI+) 312.9853 [M + H]+ (calcd for C14H10Cl2O2SH+ 312.9857). ( E ) - 1 -( 2- ( ( 3 , 4 - D i m e th ox y p he n y l ) s u l f o n y l ) v i n y l ) - 2 trifluoromethylbenzene (15a). Diethyl (3,4-dimethoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.42 mmol), 2 M n-BuLi solution in cyclohexane (0.75 mL, 1.49 mmol), and 2(trifluoromethyl)benzaldehyde (0.21 mL, 1.56 mmol) gave 0.37 g (69%) of 15a as a white solid. Rf = 0.25 (n-hexane/EtOAc 3/1); mp 174.0−175.0 °C; HPLC purity, 13.1 min, >99.0%; 1H NMR (400 MHz, CDCl3) δ 7.99 (dd, J = 2.1, 15.2 Hz, (E)-isomeric CH), 7.71 (d, J = 7.6 Hz, ArH), 7.49−7.60 (m, 4ArH), 7.36 (d, J = 2.1 Hz, ArH), 6.98 (d, J = 8.8 Hz, ArH), 6.81 (d, J = 15.2 Hz, (E)-isomeric CH), 3.95 (s, OCH3), 3.94 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 152.6, 148.6, 136.6, 131.6, 131.4, 130.6, 130.5, 129.4, 128.1 (q, JC−F = 30.5 Hz), 127.4, 125.4 (q, JC−F = 5.4 Hz), 122.9 (q, JC−F = 272.2 Hz, CF3), 121.2, 110.0, 119.0 (12ArC, 2CH), 55.4 (OCH3), 55.3 (OCH3); HRMS (M + H) + (ESI +) 373.0720 [M + H] + (calcd for C17H15F3O4SH+ 373.0721). ( E ) - 1 -( 2- ( ( 3 , 4 - D i m e th ox y p he n y l ) s u l f o n y l ) v i n y l ) - 3 trifluoromethylbenzene (15b). Diethyl (3,4-dimethoxyphenylsulfonyl)methylphosphonate (0.50 g, 1.42 mmol), 2 M n-BuLi solution in cyclohexane (0.75 mL, 1.49 mmol), and 3(trifluoromethyl)benzaldehyde (0.21 mL, 1.56 mmol) gave 0.46 g (87%) of 15b as a white solid. Rf = 0.30 (n-hexane/EtOAc 3/1); mp 154.0−156.0 °C; HPLC purity, 13.3 min, 99.1%; 1H NMR (400 MHz, CDCl3) δ 7.72 (s, ArH) 7.63−7.67 (m, 2ArH, (E)-isomeric CH), 7.57 (dd, J = 2.2, 8.5 Hz, ArH), 7.53 (t like due to dd, J = 7.7 Hz, ArH), 7.38 (d, J = 2.2 Hz, ArH), 6.99 (d, J = 8.5 Hz, ArH), 6.94 (d, J = 15.4 Hz, (E)-isomeric CH), 3.95 (s, 2OCH3); 13C NMR (100 MHz, CDCl3) δ 152.6, 148.5, 138.4, 135.0, 132.4, 130.7, 130.6, 130.5 (q, JC−F = 32.5 Hz), 129.1, 128.7, 126.4, 124.0, 122.6 (q, JC−F = 271.0 Hz, CF3), 121.2, 110.1, 109.0 (12ArC, 2CH), 55.3 (2OCH3); HRMS (M 1484



Article

(10 μL) combined with 90 μL of assay buffer (100 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1% BSA, and 0.1% Tween-20) was added to each well. After incubation for 1 h and four washes in washing buffer (10 mM sodium phosphate, pH 7.4, 15 mM NaCl, and 0.1% Tween-20), 100 μL of mouse polyclonal HO-1 detection antibody (Enzo Life Sciences), diluted (1:250) with assay buffer, was added. The resulting solution was incubated for 1 h and washed as before. Then 100 μL of horseradish peroxidase labeled streptavidin, diluted (1:600) with assay buffer, was added to each well, and the samples were incubated for 1 h and washed. After addition of 100 μL of Amplex-Red reagent [0.05 mM Amplex-Red (Invitrogen, Seoul, Korea), 0.0068% H2O2 in phosphate buffered saline (PBS)] and incubation for 30 min, fluorescence was measured using a fluorescence spectrophotometer (SPECTRA MAX 340 pc; Molecular Devices, Menlo Park, CA) at excitation at 530 nm and emission at 590 nm. RT-PCR. CATH.a cells were grown in RPMI 1640 containing 8% horse serum, 4% FBS, 100 IU/L penicillin, and 10 μg/mL streptomycin at 37 °C in 95% air and 5% CO2 in humidified atmosphere. The cells were treated with various concentrations of 12g and incubated for 6 h. Total RNA was isolated, and reverse transcription reactions (RT) were performed following the manufacturer’s directions (MBI Fermentas, Ontario, Canada). Polymerase chain reaction (PCR) was performed using the following primers: NQO1 (forward, 5′-CCATTCTGAAAGGCTGGTTTG-3′; reverse, 5′-CTAGCTTTGATCTGGTTGTC-3′); HO-1 (forward, 5′AGCAGGACATGGCCTTCT -3′; reverse, 5′-TCTGTCAGCATCACCTGCAG-3′); GCLC (forward, 5′-ATGACTGTTGCCAGGTGGATGAGA-3′; reverse, 5′-ACACGCCATCCTAAACAGCGATCA-3′); GCLM (forward, 5′-AGCTGGACTCTGTGATCATGGCTT-3′; reverse, 5′CAAAGGCAGTCAAATCTGGTGGCA-3′); glyceraldehyde 3-phosphate dehydrogenase (GAPDH, forward, 5′-CACCACCATGGAGAAGGCTGG-3′; reverse, 5′-TTGTCATGGATGACCTTGGCCAGG3′). GAPDH was used as an internal control. Analysis of each PCR product on 1% agarose gel showed a single band with the expected size. Densitometric analysis was performed using Image Gauge 4.0 program, and the data were normalized against GAPDH. ATP Assay. CATH.a cells were seeded on a 96-well plate at a density of 2.4 × 104 cells/well and treated with various concentrations of 12g. To estimate cell viability, ATP level in live cells was measured using CellTiter-Glo luminescent cell viability assay kit (Promega Corp., Madison, WI). The cultured cells were mixed with the reagent and lysed for 2 min, and the samples were incubated for 10 min at room temperature. Then 100 μL of mixture was transferred onto a black plate, and luminescence was measured for 1 min using a microplate luminometer (MicroLumat Plus LB96 V, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Lactate Dehydrogenase (LDH) Activity Assay. CATH.a cells were seeded on a 96-well plate at a density of 2.4 × 104 cells/well and treated with various concentrations of 12g. Aliquots (50 μL) of cell culture medium were incubated at room temperature in the presence of 0.26 mM NADH, 2.87 mM sodium pyruvate, and 100 mM potassium phosphate buffer (pH 7.4) in a total volume of 200 μL. The rate of NAD+ formation was monitored for 5 min at 2 s intervals at 340 nm using a microplate spectrophotometer (SPECTRA MAX 340 pc; Molecular Devices). Prerapartion of Proteins for Western Blot. For nuclear fractionation, CATH.a cells were treated with 12g for 3 h, harvested in PBS, and resuspended in 100 μL of cold buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM PMSF by gentle pipetting. The cells were incubated on ice for 15 min, after which 7 μL of 10% NP-40 was added and mixed for 10 s. The homogenate was then centrifuged (17800g, 2 min), and the supernatant was removed. This step was repeated once again. The nuclear pellet was resuspended in 50 μL of ice-cold buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF. The sample was mixed thoroughly and placed on a rotary shaker for 10 min. After centrifugation (17800g, 10 min), the soluble fraction containing nuclear proteins was obtained.

To obtain cell lysate, CATH.acells were treated with 12g and incubated for 24 h. The cells were washed with ice-cold PBS and lysed on ice in RIPA buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.5% NP-40) containing protease inhibitor cocktail. After centrifugation (14000g, 10 min), the supernatant was obtained. Western Blot Analyses. Protein concentrations were determined, and equal amounts of protein were separated on a 10% SDS polyacrylamide gel and transferred onto polyvinylidene difluoride− nitrocellulose filters. After treatment in blocking solution [6% skim milk in TTBS buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.4% Tween-20)] for 1 h, the membrane was incubated overnight with primary antibodies [NQO1 (Ab Frontier, Seoul, South Korea), 1:1000; HO-1 (Enzo Life Sciences, Farmingdale, NY), 1:1000; GCLC (Novus Biologicals, Littleton, CO), 1:3000; GCLM (Santa Cruz Biotechnology, Santa Cruz, CA), 1:200; β-actin (Sigma-Aldrich, St. Louis, MO), 1:20000; and lamin B (Santa Cruz Biotechnology), 1:200] at 4 °C followed by horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected by chemiluminescence. Densitometric analysis was performed using Image Gauge 4.0 program (Fujifilm, Tokyo, Japan), and the data were normalized against β-actin, used as loading control. Production of PD Animal Model and Treatment. All procedures were preapproved by the Animal Experiment Review Committee of the Asan Institute for Life Sciences and performed in compliance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (2011). All efforts were made to minimize animal suffering. Male C57Bl/6 mice weighing 23−25 g were maintained in a temperature- and humidity-controlled room with a 12 h light−dark cycle and food and water available ad libitum. The animals (n = 10 per group) were orally administered with 12g suspended in N-methyl-2-pyrrolidone and 20% Tween 80 in saline at 10 mg/kg body weight per day every 24 h for 3 consecutive days. MPTP (administered in four 20 mg/kg doses 2 h apart, dissolved in saline) was injected intraperitoneally in a single day. The first MPTP injection was made 24 h after the first 12g administration. Immunohistochemistry. The animals were sacrificed 7 days after the first MPTP injection. They were deeply anesthetized (with 15 mg/ kg tiletamine hydrochloride, 15 mg/kg zolazepam hydrochloride, and 1 mg/kg xylazine, injected intraperitoneally) and transcardially fixed in 4% paraformaldehyde as described previously.41 Brains were promptly removed and postfixed in 4% paraformaldehyde. After cryoprotection, the brain tissues were cut into 20 μm (nigral sections) or 40 μm (striatal sections), and the sections were stored in 0.08% sodium azide in PBS at 4 °C until analysis. Immunostaining of the nigral and striatal brain tissue sections was performed as described previously,42 using polyclonal antisera against TH (1:5,000; Protos, New York, NY), Vectastain ABC kit, and biotinylated secondary antibodies. The samples were visualized by incubation in 0.05% 3,3′-diaminobenzidine and 0.003% H2O2. The TH-immunopositive cells were manually counted on five sections, 80 μm apart, from all animals with the aid of a computer program (Mousotron 3.8.3, Blacksun Software). Quantitative analysis of striatal sections was performed using Image Gauge 4.0 (Fuji Photo Film Co., Tokyo, Japan) on five sections, 80 μm apart, from all animals. Hindlimb Test. At 6 days after the last injection of MPTP, the animals were subjected to the hindlimb test as previously reported.41The animals were suspended by the tail and scored on a scale of 0−4 based on the position of their hindlimbs. Each mouse received a score of 4, from which the score of 1 was deducted for each abnormal hindlimb movement of the joint or limb. Vertical Grid Test. Vertical grid test was performed as described previously.36 Prior to the drug administration, mice were allowed to acclimate to the vertical grid apparatus once a day for 3 consecutive days: they were placed at the bottom of the apparatus facing upward so that they would climb up the apparatus and then descend. On day 6, the same vertical grid trials were made and videotaped. The videos were replayed to analyze the time taken to climb down and also to make a complete turn. Data Analyses. Data are expressed as the mean ± SEM of independent experiments. Comparisons of three or more groups were 1485



Article

(4) Kim, Y.; Kim, Y. K.; Hwang, O.; Kim, D. J. In Brain DamageBridging between Basic Research and Clinics; Conzalez-Quevedo, A., Ed.; InTech: Rijeka, Croatia, 2012; pp99−138. (5) de Lau, L. M. L.; Breteler, M. M. B. Epidemiology of Parkinson’s disease. Lancet Neurol. 2006, 5, 525−535. (6) Zhang, Y.; Dawson, V. L.; Dawson, T. M. Oxidative stress and genetics in the pathogenesis of Parkinson’s disease. Neurobiol. Dis. 2000, 7, 240−250. (7) Testa, C. M.; Sherer, T. B.; Greenamyre, J. T. Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures. Mol. Brain Res. 2005, 134, 109−118. (8) Uttara, B.; Singh, A. V.; Zamboni, P.; Mahajan, R. T. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr. Neuropharmacol. 2009, 7, 65−74. (9) Gilgun-Sherki, Y.; Melamed, E.; Offen, D. Oxidative stress induced-neurodegenerative stress diseases: the need for antioxidants that penetrate the blood brain barrier. Neuropharmcology 2001, 40, 959−975. (10) Wakabayashi, N.; Dinkova-Kostova, A. T.; Holtzclaw, W. D.; Kang, M.; Kobayashi, A.; Yamamoto, M.; Kensler, T. W.; Talalay, P. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteins of the Keap1 sensor modified by inducers. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2040−2045. (11) de Vries, H. E.; Witte, M.; Hondius, D.; Rozemuller, A. J. M.; Drukarch, B.; Hoozemans, J.; Horssen, J. V. Nrf2-induced antioxidant protection: a promising target to counteract ROS-mediated damage in neurodegenerative disease? Free Radical Biol. Med. 2008, 45, 1375− 1383. (12) Giudice, A.; Arra, C.; Turco, M. C. In Transcription Factors: Methods and Protocols; Higgins, P. J., Ed.; Humana Press: New York, 2010; Vol. 647, pp 37−74. (13) Sporn, M. B.; Liby, K. T. NRF2 and cancer: the good, the bad and the importance of context. Nat. Rev. Cancer 2012, 12, 564−571. (14) Kweon, M. H.; Park, Y. I.; Sung, H. C.; Mukhtar, H. The novel antioxidant 3-O-caffeoyl-1-methylquinic acid induces Nrf2-dependent phase II detoxifying genes and alters intracellular glutathione redox. Free Radical Biol. Med. 2006, 40, 1349−1361. (15) Sykiotis, G. P.; Bohmann, D. Stress-activated cap “n” collar transcription factors in aging and human disease. Sci. Signaling 2010, 3, re3. (16) Johnson, J. A.; Johnson, D. A.; Kraft, A. D.; Calkins, M. J.; Jakel, R. J.; Vargas, M. R.; Chen, P. The Nrf2-ARE pathway an indicator and modulator of oxidative stress in neurodegeneration. Ann. N.Y. Acad. Sci. 2008, 1147, 61−69. (17) Cuadrado, A.; Moreno-Murciano, P.; Pedraza-Chaverri, J. The transcription factor Nrf2 as a new therapeutic target in Parkinson’s disease. Expert Opin. Ther. Targets 2009, 13, 319−329. (18) Rojo, A. I.; Innamorato, N. G.; Martin-Moreno, A. M.; de Ceballos, M. L.; Yamamoto, M.; Cuadrado, A. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson’s disease. Glia 2010, 58, 588−598. (19) Kaidery, N. A.; Banerjee, R.; Yang, L.; Smirnova, N. A.; Hushpulian, D. M.; Liby, K. T.; Williams, C. R.; Yamamotto, M.; Kensler, T. W.; Ratan, R. R.; Sporn, M. B.; Beal, M. F.; Gazaryan, I. G.; Thomas, B. Targeting Nrf2-mediated gene transcription by extremely potent synthetic triterpenoids attenuate dopaminergic neurotoxicity in the MPTP mouse model of Parkinson’s disease. Antioxid. Redox. Signaling 2013, 18, 139−157. (20) Yang, L.; Calingasan, N. Y.; Thomas, B.; Chaturvedi, R. K.; Kiaei, M.; Wille, E. J.; Liby, K. T.; Williams, C.; Royce, D.; Risingsong, R.; Musiek, E. S.; Morrow, J. D.; Sporn, M.; Real, M. F. Neuroprotective effects of the triterpenoid, CDDO methyl amide, a potent inducer of Nrf2-mediated transcription. PLoS One 2009, 4, e5757. (21) Ramsey, C. P.; Glass, C. A.; Montgomery, M. B.; Lindl, K. A.; Ritson, G. P.; Chia, L. A.; Hamilton, R. L.; Chu, C. T.; Jordan-Sciutto, K. L. Expression of Nrf2 in neurodegenerative diseases. J. Neuropathol. Exp. Neurol. 2007, 66, 75−85.

analyzed by one-way ANOVA (analysis of variance) and post Dunnett’s multiple comparison test. Statistical tests were carried out using PRISM (GraphPad Software, San Diego, CA). p < 0.05 was considered statistically significant.

■

ASSOCIATED CONTENT

S Supporting Information *

Synthetic procedures for the preparation of 1, 4, and 6−8; 1H and 13C NMR spectra of compounds 1, 4, and 6−16 reported in this study. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*O.H.: phone, 82-2-3010-4279; fax, 82-2-3010-4248; e-mail, [email protected]. *K.D.P.: phone, 82-2-958-5132; fax, 82-2-958-5189; e-mail, [email protected]. Author Contributions ∥

S.Y.W. and J.H.K. contributed equally to the work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

This study was supported by funds from the Korea Institute of Science and Technology (KIST) (Grants 2E23870 and 2E22520, K.D.P) and by grants from the Korea Health Technology R&D Project, the Ministry of Health and Welfare (Grant HI12C1022, K.D.P), and the National Agenda Project from Korea Research Council of Fundamental Science and Technology (O.H.), and in part by the National Research Foundation of Korea (NRF-2009-0081674, D.J.K, NRF-20090081675, O.H.).

■

ABBREVIATIONS USED BH4, tetrahydrobiopterin; BSA, bovine serum albumin; DA, dopamine; DAergic, dopaminergic; DMEM, Dulbecco’s modified Eagle medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3phosphate dehydrogenase; GCL, glutamate-cysteine ligase; GCLC, glutamyl-cysteine ligase catalytic subunit; GCLM, glutamyl-cysteine ligase modifier subunit; HO-1, heme oxygenase 1; LDH, lactate dehydrogenase; mCPBA, m-chloroperoxybenzoic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO1, NAD(P)H quinone oxidoreductase; Nrf2, NfE2-related factor 2; PBS, phosphate buffered saline; PD, Parkinson’s disease; ROS, reactive oxygen species; RT-PCR, reverse transcription polymerase chain reaction; SFN, sulforaphane; SN, substantia nigra; TH, tyrosine hydroxylase

■

REFERENCES

(1) Meissner, W. G.; Frasier, M.; Gasser, T.; Goetz, C. G.; Lozano, A.; Piccini, P.; Obeso, J. A.; Rascol, O.; Schapira, A.; Voon, V.; Weiner, D. M.; Tison, F.; Bezard, E. Priorities in Parkinson’s disease research. Nat. Rev. Drug Discovery 2011, 10, 377−393. (2) Hwang, O. Role of oxidative stress in Parkinson’s disease. Exp. Neurobiol. 2013, 22, 11−17. (3) Choi, H. J.; Lee, S. Y.; Cho, Y.; No, H.; Kim, S. W.; Hwang, O. Tetrahydrobiopterin causes mitochondrial dysfunction in dopaminergic cells: implications for Parkinson’s disease. Neurochem. Int. 2006, 48, 255−262. 1486



Article

(22) Chen, P.; Vargas, M. R.; Pani, A. K.; Smeyne, R. J.; Johnson, D. A.; Kan, Y. W.; Johnson, J. A. Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson’s disease: critical role for the astrocyte. Proc. Natl. Acad. Sci.U.S.A. 2009, 106, 2933−2938. (23) Kumar, V.; Kumar, S.; Hassan, M.; Wu, H.; Thimmulappa, R. K.; Kumar, A.; Sharma, S. K.; Parmar, V. S.; Biswal, S.; Malhotra, S. V. Novel chalcone derivatives as potent Nrf2 activators in mice and human lung epithelial Cells. J. Med. Chem. 2011, 54, 4147−4159. (24) Wu, J.; Li, J.; Cai, Y.; Pan, Y.; Ye, F.; Zhang, Y.; Zhao, Y.; Yang, S.; Li, X.; Liang, G. Evaluation and discovery of novel synthetic chalcone derivatives as anti-inflammatory agents. J. Med. Chem. 2011, 54, 8110−8123. (25) Kachadourian, R.; Day, B. J.; Pugazhenti, S.; Franklin, C. C.; Genoux-Bastide, E.; Mahaffey, G.; Gauthier, C.; Pietro, A. D.; Boumendjel, A. A synthetic chalcone as a potent inducer of glutathione biosynthesis. J. Med. Chem. 2012, 55, 1382−1388. (26) Kweon, M. H.; Adhami, V. M.; Lee, J.-S.; Mukhtar, H. Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate. J. Biol. Chem. 2006, 281, 33761−33772. (27) Suri, C.; Fung, B. P.; Tischler, A. S.; Chikaraishi, D. M. Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J. Neurosci. 1993, 13, 1280−1291. (28) Han, J. M.; Lee, Y. J.; Lee, S. Y.; Kim, E. M.; Moon, Y.; Kim, H. W.; Hwang, O. Protective effect of sulforaphane against dopaminergic cell death. J. Pharmacol. Exp. Ther. 2007, 321, 249−256. (29) Jazwa, A.; Cuadrado, A. Targeting heme oxygenase-1 for neuroprotection and neuroinflammation in neurodegenerative diseases. Curr. Drug Targets 2010, 11, 1517−1531. (30) Choi, H. J.; Jang, Y. J.; Kim, H. J.; Hwang, O. Tetrahydrobiopterin is released from and causes preferential death of catecholaminergic cells by oxidative stress. Mol. Pharmacol. 2000, 58, 633−640. (31) Choi, H. J.; Kim, S. W.; Lee, S. Y.; Moon, Y. W.; Hwang, O. Involvement of apoptosis and calcium mobilization in tetrahydrobiopterin-induced dopaminergic cell death. Exp. Neurol. 2003, 181, 281−290. (32) Choi, H. J.; Lee, S. Y.; Cho, Y.; Hwang, O. JNK activation by tetrahydrobiopterin: implication for Parkinson’s disease. J. Neurosci. Res. 2004, 75, 715−721. (33) Asanuma, M.; Miyazaki, I.; Diaz-Corrales, F. J.; Miyoshi, K.; Ogawa, N.; Murata, M. Preventing effects of a novel anti-parkinsonian agent zonisamide on dopamine quinone formation. Neurosci. Res. 2008, 60, 106−113. (34) Lim, J. H.; Kim, S. S.; Boo, D. H.; No, H.; Kang, B. Y.; Kim, E. M.; Hwang, O.; Choi, H. J. Protective effect of bromocriptine against BH4-induced Cath.a cell death involving up-regulation of antioxidant enzymes. Neurosci. Lett. 2009, 451, 185−189. (35) Yoon, N. S.; Cho, Y.; Lee, S. Y.; Choi, H. J.; Hwang, O. Inactivation of aconitase by tetrahydrobiopterin in DArgic cells: relevance to PD. Exp. Neurobiol. 2010, 19, 23−29. (36) Kim, S. T.; Son, H. J.; Choi, J. H.; Ji, I. J.; Hwang, O. Vertical grid test and modified horizontal grid test are sensitive methods for evaluating motor dysfunctions in the MPTP mouse model of Parkinson’s disease. Brain Res. 2010, 1306, 176−183. (37) Xue, Q.; Mao, Z.; Shi, Y.; Mao, H.; Cheng, Y.; Zhu, C. Metalfree, one-pot highly selective synthesis of (E)-vinyl suflones and sulfoxides via addition-oxidation of thiols with alkynes. Tetrahedron. Lett. 2012, 53, 1851−1854. (38) Reddy, D. B.; Sankaraiah, B.; Balaji, T. Cyclopropanation of some α,β-unsaturated sulfones. Indian J. Chem., Sect. B 1980, 19, 563− 566. (39) Naidu, M.; Seshapathi, R.; Reddy, D. B. Synthesis of α,βethylenic sulfones. Bull. Chem. Soc. Jpn. 1975, 48, 1091−1092. (40) Wang, Z.; Pitteloud, J.-P.; Montes, L.; Rapp, M.; Derane, D.; Wnuk, S. F. Vinyl tris(trimethylsilyl)silanes: substrates for Hiyama coupling. Tetrahedron 2008, 64, 5322−5327.

(41) Son, H. J.; Lee, J. A.; Shin, N.; Choi, J. H.; Seo, J. W.; Chi, D. Y.; Lee, C. S.; Kim, E. M.; Choe, H.; Hwang, O. A novel compound PTIQ protects the nigral dopaminergic neurones in an animal model of Parkinson’s disease induced by MPTP. Br. J. Pharmacol. 2012, 165, 2213−2227. (42) Hwang, O.; Baker, H.; Gross, S.; Joh, T. H. Localization of GTP cyclohydrolase in monoaminergic but not nitric oxide-producing cells. Synapse 1998, 28, 140−153.

1487


Design, synthesis, and biological evaluation of (e)-3,4-dihydroxystyryl aralkyl sulfones and sulfoxides as novel multifunctional neuroprotective agents.

Evaluation of aminohydantoins as a novel class of antimalarial agents.

Nanobodies as novel agents for disease diagnosis and therapy.

Ecdysteroids: A novel class of anabolic agents?

Toward neuroprotective treatments of Parkinson's disease.

Preparation of Alkyl Alkynyl Sulfones and Cyclic Vinyl Sulfones from Alkynyl(aryl)iodonium Salts.

Non-feminizing estrogens: a novel neuroprotective therapy.

Discovery of Pyrazolopyridones as a Novel Class of Gyrase B Inhibitors Using Structure Guided Design.

Discovery of a Novel Class of Bicyclo[3.1.0]hexanylpiperazines as Noncompetitive Neuropeptide Y Y1 Antagonists.

Discovery of pyrazolopyridones as a novel class of noncovalent DprE1 inhibitor with potent anti-mycobacterial activity.

Regioselective synthesis of vinyl halides, vinyl sulfones, and alkynes: a tandem intermolecular nucleophilic and electrophilic vinylation of tosylhydrazones.

Depolymerizable Poly(O-vinyl carbamate-alt-sulfones) as Customizable Macromolecular Scaffolds for Mucosal Drug Delivery.

Identification of a novel class of covalent modifiers of hemoglobin as potential antisickling agents.

S100 interactions.

Synthesis and biological evaluation of adamantane-based aminophenols as a novel class of antiplasmodial agents.

Identification of metal dithiocarbamates as a novel class of antileishmanial agents.

In vitro evaluation of tetrazoles as a novel class of Antimycobacterium tuberculosis agents.

Quinolinyl Pyrimidines: Potent Inhibitors of NDH-2 as a Novel Class of Anti-TB Agents.

Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents.

Novel group-based QSAR and combinatorial design of CK-1δ inhibitors as neuroprotective agents.

A novel class of antitumour agents. II. In vitro testing.

Preparation, anti-trypanosomal activity and localisation of a series of dipeptide-based vinyl sulfones.

In vivo modification of Abeta plaque toxicity as a novel neuroprotective lithium-mediated therapy for Alzheimer's disease pathology.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.