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Article Cite This: J. Med. Chem. 2019, 62, 811−830

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Optimization of Vinyl Sulfone Derivatives as Potent Nuclear Factor Erythroid 2‑Related Factor 2 (Nrf2) Activators for Parkinson’s Disease Therapy Ji Won Choi,†,∇ Siwon Kim,†,‡,∇ Jong-Hyun Park,† Hyeon Jeong Kim,†,§ Su Jeong Shin,† Jin Woo Kim,† Seo Yeon Woo,† Changho Lee,∥ Sang Moon Han,⊥ Jaeick Lee,⊥ Ae Nim Pae,†,‡,# Gyoonhee Han,§ and Ki Duk Park*,†,‡,#

J. Med. Chem. 2019.62:811-830. Downloaded from pubs.acs.org by MIDWESTERN UNIV on 01/26/19. For personal use only.

†

Convergence Research Center for Diagnosis, Treatment & Care System of Dementia, Korea Institute of Science & Technology (KIST), Seoul 02792, Republic of Korea ‡ Division of Bio-Med Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea § Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea ∥ Division of Functional Food Research, Korea Food Research Institute, Wanju-gun, Jeollabuk-do 55365, Republic of Korea ⊥ Doping Control Center, KIST, Seoul 02792, Republic of Korea # KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 02447, Republic of Korea S Supporting Information *

ABSTRACT: We previously developed a novel series of vinyl sulfones as nuclear factor erythroid 2-related factor 2 (Nrf2) activators with therapeutic potential for Parkinson’s disease (PD). However, the previously developed lead compound (1) exhibited undesirable druglike properties. Here, we optimized vinyl sulfones by introducing nitrogen heterocycles to improve druglike properties. Among the synthesized compounds, 17e was the most promising drug candidate with good druglike properties. Compound 17e showed superior effects on Nrf2 activation in cell-based assays compared to compound 1 (17e: half-maximal effective concentration (EC50) = 346 nM; 1: EC50 = 530 nM). Compound 17e was further confirmed to induce expression of Nrf2-dependent antioxidant enzymes at both mRNA and protein levels. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of PD, 17e significantly attenuated loss of tyrosine hydroxylase-immunopositive dopaminergic neurons, suppressed microglial activation, and alleviated PDassociated motor dysfunction. Thus, 17e is a novel Nrf2 activator with excellent druglike properties and represents a potential therapeutic candidate for PD.

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INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease and mainly affects the motor system. The incidence of PD is about 1−4% of populations over 60 years of age, and main clinical features often include tremors, rigidity, posture instability, akinesia, and cognitive impairment.1,2 The major pathophysiology of PD is loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc).3,4 Hence, most treatment strategies focus on increasing dopamine levels, typically using L-3,4-dihydroxyphenylalanine (L-DOPA) as a precursor, to alleviate symptoms. However, LDOPA only ameliorates motor symptoms for a short period of time and can cause side effects, such as L-DOPA-induced dyskinesia, when taken for long periods.5−7 Therefore, it is important to develop new therapies that prevent the death of DAergic neurons in PD-affected brain areas and increase dopamine levels.3,8,9 © 2018 American Chemical Society

Unfortunately, the exact mechanism of DAergic neuronal cell death in PD patients remains unclear, but many recent studies have shown that oxidative stress plays an important role.10−12 Oxidative stress is caused by an imbalance in the production of reactive oxygen species and cellular antioxdant defense systems.13−16 Thus, upregulation of endogenous antioxidant defense genes is an attractive strategy for the treatment of various brain diseases associated with oxidative stress, including Alzheimer’s disease, PD, and epilepsy.17−20 The Kelch-like ECH-associated protein 1 (Keap1)−nuclear factor erythroid 2-related factor 2 (Nrf2) system is the main signaling pathway involved cellular defense against oxidative stress. Nrf2 is a transcription factor that regulates oxidative stress by inducing expression of antioxidant enzyme genes, such as heme oxygenase-1 (HO-1), reduced nicotinamide Received: October 1, 2018 Published: December 12, 2018 811

DOI: 10.1021/acs.jmedchem.8b01527 J. Med. Chem. 2019, 62, 811−830

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Compounds 5−8

adenine dinucleotide phosphate (NAD(P)H) quinone oxidoreductase 1, glutamate−cysteine ligase (GCL), and several members of the glutathione S-transferase family.21,22 Under normal conditions, Nrf2 binding to the repressor protein Keap1 is maintained at low levels in the cytoplasm through Keap1-dependent ubiquitination and proteasomal degradation. Under conditions of oxidative stress, Nrf2 is released from Keap1 and translocates into the nucleus, where it binds the promoter region of the antioxidant response element that is associated with expression of antioxidant genes.17−20 Therefore, targeting the Keap1−Nrf2 signaling pathway has been considered for the reduction or prevention of neuronal cell death, with Nrf2 activation representing a promising avenue for PD therapy. In our previous studies23,24 we developed a series of neuroprotective vinyl sulfones, which targeted the Keap1− Nrf2 signaling pathway. In particular, the lead compound (1) was found to exhibit the most potent Nrf2 activation and subsequent dose-dependent induction of Nrf2-dependent antioxidant enzyme expression. Compound 1 also exhibited promising efficacy in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model but had poor druglike properties, such as low solubility, metabolic stability, cytochrome P (CYP) inhibition, and human ether-a-go-gorelated gene (hERG) safety.

In the present study, we attempted to improve the druglike properties of compound 1 by introducing nitrogen heterocycles because nitrogen heterocycles are among the most significant structural components of pharmaceuticals and 59% of unique small-molecule drugs in U.S. Food and Drug Administration-approved drugs contain a nitrogen heterocycle.25,26 In addition, recent studies suggested that a morpholine moiety seemed to improve druglike properties of molecules.27,28 Herein, the design, synthesis, and biological evaluation of a series of vinyl sulfone derivatives with heterocycles is described. The in vitro absorption, distribution, metabolism, and excretion/toxicity (ADME/Tox) profiles of synthesized compounds as well as their ability to induce expression of various antioxidant enzyme genes was also evaluated. Furthermore, the selected compound was tested in an MPTP-induced PD mouse model to assess its ability to protect DAergic neurons from cytotoxic damage and attenuate PD-associated behavioral deficits. 812



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Scheme 2. Synthesis of Compounds 11 and 12

Scheme 3. Synthesis of Compounds 17−19

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RESULTS AND DISCUSSION

we substituted a pyridine group as a nitrogen heterocycle instead of the benzyl group in the ring B of the lead compound 1. Synthesis of the pyridinyl vinyl sulfones (5a−o, 6a−i, 7a−c, 8a−d, 11a−d, and 12) followed standard protocols (Schemes 1 and 2). Synthesis began with commercially available benzenethiols coupled with (diethoxyphosphoryl)methyl-4methylbenzenesulfonate (2) in the presence of Cs2CO3 to give substituted sulfides (3 and 9). Oxidation of the substituted sulfides with a 2.2 equiv of m-chloroperoxybenzoic acid at room temperature provided the desired sulfones (4 and 10).

Chemical Synthesis. We used our previously developed lead compound (1), which contained an α,β-unsaturated sulfone moiety, as our structural template and prepared various derivatives by introducing nitrogen heterocycles, which are among the most significant structural components of pharmaceuticals.25,26 According to the synthetic methods, the synthesized derivatives were divided into three scaffolds: pyridinyl, morpholinyl, and piperazinyl vinyl sulfones. First, 813



Article

Scheme 4. Synthesis of Compounds 23

corresponding 2- and 4-methoxy derivatives (5a−e and 5k−o, respectively; 3-OMe > 2-OMe > 4-OMe). In our previous study, 2-methoxy compounds, including 1, were the most active; however, in the present study, 3-methoxy compounds in which a pyridine ring was introduced as the ring B were more Nrf2-active. Next, we examined whether Nrf2 activation was improved by introduction of an o-pyridine versus a m- or p-pyridine. mPyridine derivatives (6) were generally less potent than opyridine derivatives (5) with respect to Nrf2 activation. On the other hand, while p-pyridine derivatives (7) showed a significant increase in Nrf2 activation, they were also more cytotoxic than both o- and m-pyridine derivatives (Table 3). In addition, conversion of the pyridine in ring B to a pyrimidine (8) had little effect on Nrf2 activation ability. Next, we prepared compounds 11 and 12 to investigate whether introduction of a pyridine group into ring A would enhance the potency of Nrf2 activation. Among this series, electronwithdrawing groups were inserted into the pyridine (11c and 12) and exerted significant Nrf2 activation effects (11c: EC50 = 0.124 μM; 12: EC50 = 0.202 μM). To improve the solubility and other druglike properties of potent compounds, we synthesized the salt forms of each by adding morpholine or piperazine groups. On the basis of the above-described results of Nrf2 activation efficacy and cell viability, we prepared a series of compounds in which the opyridine group was maintained in ring B and the morpholine or piperazine group was introduced into ring A instead of the methoxy group. Unlike the above results showing the highest Nrf2 activity when a methoxy group was introduced at 3position of ring A (5, 3-OMe > 2-OMe > 4-OMe), the most potent Nrf2 activating effect was observed when the propylmorpholine group was introduced at the 4-position of ring A (17, 17e > 17c > 17b, Table 2). Among the compounds 17, 17e, and 17f included electron-withdrawing groups (F or Cl) on the pyridine exerted remarkable Nrf2 activation effects as compared to the other 17 series (17e: EC50 = 0.346 μM; 17f: EC50 = 0.246 μM). We further investigated whether replacement of the o-pyridine group of compound 17 by either m-pyridine (18) or benzene groups (19) affects the efficacy of Nrf2 activation. Compounds 18a and 18b exhibited significantly lower activity than compound 17, and most of the compounds included benzene groups as ring B instead of opyridine group (19) showed no activity. Finally, on the basis of the potency of morpholine compounds with 3′-F or 3′-Cl on opyridine (17e and 17f), we attempted to introduce a 4-

The Horner−Emmons olefination reaction with commercially available formylpyridine gave the final compounds 5−8, 11, and 12. Second, we further introduced a morpholinyl group into ring A to improve microsomal stability while increasing solubility through the HCl salt form. The synthetic pathway used to prepare morpholinyl vinyl sulfones (17a−f, 18a, 18b, and 19a−f) is provided in Scheme 3. First, commercially available hydroxylbenzenethiols were coupled with (diethoxyphosphoryl)methyl-4-methylbenzenesulfonate (2) in the presence of Cs2CO3 to give the sulfides (13). Oxidation of the sulfides with a 2.2 equiv of m-chloroperoxybenzoic acid at room temperature provided the corresponding sulfones (14). The sulfones were coupled with 3-morpholinoprorpyl-4methylbenzene-sulfonate (15) in the presence of K2CO3 to incorporate a morpholine group in the sulfones (16). The final compounds (17−19) were synthesized using the Horner− Emmons olefination reaction with commercially available formylpyridine. Preparation of the piperazinyl vinyl sulfones (23a and 23b) followed a standard protocol (Scheme 4). The intermediate (14c) was coupled with ethyl-2-bromoacetate in the presence of K2CO3 to give compound 20. The ester of 20 was converted to its free acid form (21) using stoichiometric amounts of NaOH. Coupling of 21 and 1-methylpiperazine using carbonyldiimidazole gave 22. The final compounds (23) were obtained in salt forms by the Horner−Emmons olefination followed by addition of excess 4.0 M HCl. Evaluation of the Synthesized Compounds for Nrf2 Activation. To evaluate the Nrf2 activating ability of all of the synthesized compounds, we assessed their ability to release Nrf2 from Keap1 and translocate Nrf2 into the nucleus using our previously reported cell-based assay system.29 The potency of Nrf2 activation is shown in Tables 1 and 2 as half-maximal effective concentration (EC50) values. The optimization process was designed to improve the potency and ADME/ Tox profiles of lead compound 1. First, we introduced opyridine as a heterocycle into ring B of 1 (5a), but this reduced Nrf2 activation by 17-fold (1: EC50 = 0.53 μM; 5a: EC50 = 9.07 μM). However, addition of a Cl group to o-pyridine (5b, 5c, and 5e) enhanced the Nrf2 activation effect relative to 5a; 5e had its Cl group in the ortho position, as in 1, and induced the highest Nrf2 activity (5e: EC50 = 0.142 μM). Substitution with a F instead of Cl group in compound 5e resulted in a 6fold reduction in Nrf2 activation (5d: EC50 = 0.877 μM). Next, we systematically placed a methoxy group at the 2-, 3-, and 4positions of ring A. In these three series of compounds, the 3methoxy derivatives (5f−j) were more Nrf2-active than the 814



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Table 1. Effects of Synthesized Compounds 5−12 on Nrf2 Activation

a

The Keap1−Nrf2 functional assay was performed using a PathHunter U2OS Keap1−Nrf2 nuclear translocation cell line (93-0821C3, DiscoveRx). U2OS cells were plated at 13 000 cells/well in triplicate with various compound concentrations for 6 h. The activation-dependent nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation was determined using a cell-based functional assay, with mean ± standard error of the mean halfmaximal effective concentration (EC50) values. bCompound 1 developed in previous study as an Nrf2 activator.23 cSFN, sulforaphane: a wellknown potent activator of Nrf2.

compounds containing 4-morpholine or 4-piperazine, those with a 3′-Cl on o-pyridine (17f and 23b) showed lower cell viability than 17e and 23a which contained a 3′-F at 50 μM (Table 3). In our previous study,23 we compared Nrf2 activating efficacy among the synthesized compounds by determining the ability to induce expression of HO-1, a major Nrf2-dependent gene, using a sandwich enzyme-linked immunosorbent assay (ELISA). To confirm the effects of the present class of molecules on Nrf2 activation, we also performed an ELISA

piperazine group instead of 4-morpholine. Interestingly, the 4piperazinyl compound with 3′-Cl on o-pyridine (23b) had 3.7 times higher Nrf2 activation efficacy than that with a 3′-F (23a). On the basis of the ability of Nrf2 to translocate, compounds 5e, 5h, 5i, 5j, 5o, 6d, 6f, 8b, 11c, 11d, 12, 17e, 17f, and 23b were identified as the most potent Nrf2 activators (Tables 1 and 2). Most of the synthesized compounds were not cytotoxic at 10 μM, whereas compounds of series 7 containing ppyridine on the right ring were cytotoxic. Among the 815



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Table 2. Effects of Synthesized Compounds 17−23 on Nrf2 Activation

a

U2OS cells were plated at 13 000 cells/well in triplicate with various compound concentrations for 6 h. Activation-dependent nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation was determined using a cell-based functional assay mean ± standard error of the mean halfmaximal effective concentration (EC50) values.

19 and 23) showed outstanding microsomal stability. On the basis of these results, compound 17e was selected for its excellent Nrf2 activation potency, safety, and stability for further ADME/Tox studies (Table 4). The plasma stability of 17e in both humans and rats was also excellent, with 98.2 and 90.2% of the parent compound remaining after a 30 min incubation, respectively (Tables 4 and S2 in Supporting Information). In the hERG channel-binding assay, 17e exerted low inhibitory effects against hERG, which produces an important cardiac ion channel subunit protein, indicating that it is unlikely to cause human cardiotoxicity (half-maximal inhibitory concentration (IC50): 112.1 μM, Table 4 and Figure S1 in the Supporting Information). To predict the blood−brain barrier permeability of 17e, we performed a parallel artificial membrane permeability assayblood−brain barrier test using a commercial kit. In this test, permeability (Pe, cm/s) was divided into two classifications: central nervous system-positive (Pe > 10 × 10−6) and -negative (Pe < 10 × 10−6). The permeability of 17e was found to be favorable for a central nervous system (CNS) drug (Pe: 15.6 × 10−6, Table S3 in the Supporting Information). Finally, the Ames test was used to test mutagenesis. The fold induction with 17e was lower than baseline in both TA98 and TA100 strains and did not induce reverse mutation (Table 4 and Figure S2 in the Supporting Information). In addition, the physicochemical properties of 17e were confirmed to be compatible with beneficial druglike properties (17e solubility: >30 mg/mL vs 1 solubility: 10 μM). However, the CYP 2C19 inhibition issue was addressed by introducing bulky groups, such as morpholine or piperazine, into ring A (17−19, and 23); these compounds also had good Nrf2 activation effects. The stability of each synthesized compound was determined from the percentage of parent compound remaining after a 30 min incubation with human liver microsomes (Table 3). The previous lead compound (1) showed was metabolically unstable, with only 20% of the parent compound remaining after incubation with human liver microsomes. Although inclusion of a pyridine group (5−8) did not produce a clear trend regarding microsomal stability, the most potent Nrf2inducing compounds (5h: EC50 = 0.132 μM; 5j: EC50 = 0.098 μM; 6d: EC50 = 0.178 μM) exhibited unfavorable microsomal stabilities (9.6, 22, and 18%, respectively). In contrast, most compounds containing morpholine or piperazine groups (17− 816


100.0 100.0 100.0 91.9 98.4 82.7 91.2 81.8 100.0 93.2 100.0 90.7 89.0 89.7 97.5 68.5 100.0 100.0 84.3 91.9 90.0 84.0 95.5 87.9 78.6 34.2

47.8 18.9 7.40 19.5 11.1 7.8 2.35 3.33 5.6 3.40 9.70 1.16 1.90 1.50 3.80 100.0 20.1 29.0 13.0 22.0 0.9 6.6 4.0 3.6 14.2 1.8

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 6a 6b 6c 6d 6e 6f 6g 6h 6i 7a 7b

817

97.7 100.0 73.6 100.0 100.0 100.0 100.0 39.5 97.2 31.9 100.0 100.0 59.0 100.0 100.0 49.4 100.0 100.0 100.0 100.0 100.0 76.5 100.0 100.0 52.2 44.4

2C9

72.5 69.2 82.4 85.2 78.5 84.1 82.7 80.8 86.6 81.3 82.8 87.8 75.6 83.5 88.2 88.7 54.0 82.0 46.3 69.7 76.8 75.1 91.8 64.7 53.4 36.0

1A2 38.4 64.6 83.1 75.9 48.6 83.7 36.7 40.6 68.8 38.0 82.9 70.0 85.8 70.4 91.8 33.2 75.7 92.0 56.3 63.3 44.3 80.5 96.4 98.2 85.0 49.7

3A4 91 nde nde nde nde 74 34 9.6 31 22 56 nde 6.0 1.8 2.0 97 nde 77 18 75 29 nde nde 46 nde 72

microsomal stabilityb (%, human) 100 ± 0.1, 100 ± 2.9 100 ± 2.9, 80.5 ± 0.8 100 ± 1.5, 92.6 ± 0.6 100 ± 1.2, 99.2 ± 1.3 100 ± 2.7, 100 ± 3.2 100 ± 2.0, 98.9 ± 1.3 100 ± 2.3, 89.3 ± 2.4 100 ± 3.1, 67.6 ± 1.4 100 ± 0.8, 97.4 ± 0.3 100 ± 2.9, 82.2 ± 4.3 100 ± 1.7, 100 ± 1.2 97.0 ± 0.9, 85.0 ± 1.0 100 ± 2.0, 84.1 ± 0.8 94.2 ± 0.3, 86.6 ± 1.7 94.7 ± 0.1, 89.8 ± 2.0 100 ± 2.1, 92.0 ± 3.7 100 ± 0.5, 81.9 ± 1.8 99.7 ± 1.0, 87.5 ± 0.9 100 ± 3.2, 99.8 ± 2.0 100 ± 0.6, 90.8 ± 0.8 100 ± 0.4, 78.8 ± 0.8 99.5 ± 3.8, 85.8 ± 1.5 100 ± 2.3, 76.3 ± 0.2 100 ± 2.4, 95.7 ± 0.3 96.8 ± 0.4, 78.9 ± 0.4 84.3 ± 1.0, 21.3 ± 0.1

cell viabilityc at 10 and 50 μM (%) 7c 8a 8b 8c 8d 11a 11b 11c 11d 12 17a 17b 17c 17d 17e 17f 18a 18b 19a 19b 19c 19d 19e 19f 23a 23b 1e

comp 0.1 80.5 16.9 57.1 57.8 47.4 37.4 24.8 33.7 22.6 87.4 68.7 63.1 87.7 77.4 53.6 93.6 80.1 43.6 43.0 59.8 56.2 63.8 82.8 83.9 71.9 5.4

2C19 23.0 86.6 96.4 89.8 86.6 91.1 99.1 74.0 84.2 72.2 86.7 66.7 96.0 99.3 99.7 89.6 87.9 91.7 89.6 71.9 70.3 83.6 46.8 57.7 93.0 89.9 88.1

2D6 38.8 100.0 100.0 87.5 100.0 100.0 100.0 100.0 92.0 100.0 87.9 100.0 100.0 90.7 100.0 100.0 93.0 100.0 100.0 78.4 60.8 97.6 84.9 80.1 100.0 73.4 100.0

2C9 65.9 81.6 57.5 85.9 63.0 47.1 74.9 71.5 8.8 65.1 97.3 53.2 98.1 92.3 87.2 98.6 86.3 77.8 95.1 93.1 89.7 93.6 88.0 87.1 95.6 93.9 41.7

1A2

CYP enzyme activitiesa (%)

76.9 84.4 78.5 86.9 90.7 98.8 98.8 75.2 74.3 78.3 91.8 62.8 80.7 84.8 97.4 100.0 79.9 100.0 56.6 53.2 83.0 89.7 91.3 100.0 96.2 91.1 42.3

3A4 nde 98 nde 96 94 71 91 97 73 98 nde nde 48 84 84 nde 71 nde 82 nde nde nde nde nde 84 50 20

microsomal stabilityb (%, human)

71.0 ± 1.2, 69.0 ± 1.5 100 ± 2.4, 97.9 ± 3.0 100 ± 1.3, 79.9 ± 0.8 100 ± 3.0, 100 ± 4.6 100 ± 1.0, 100 ± 0.5 100 ± 0.9, 93.4 ± 0.7 100 ± 2.0, 98.6 ± 0.5 100 ± 2.5, 100 ± 3.1 100 ± 1.6, 100 ± 0.8 100 ± 1.1, 84.1 ± 0.1 100 ± 1.2, 87.8 ± 1.3 100 ± 2.0, 92.2 ± 0.1 100 ± 1.6, 92.6 ± 0.8 96.3 ± 2.5, 93.0 ± 1.1 100 ± 1.3, 99.0 ± 1.5 100 ± 2.9, 83.4 ± 0.2 88.2 ± 1.9, 80.1 ± 0.3 84.5 ± 2.2, 80.8 ± 0.3 91.0 ± 0.7, 74.3 ± 1.3 100 ± 1.2, 71.6 ± 0.9 94.5 ± 0.1, 78.5 ± 0.4 77.4 ± 3.1, 70.2 ± 0.5 100 ± 1.4, 82.4 ± 0.1 100 ± 0.8, 90.4 ± 3.0 100 ± 0.8, 100 ± 0.3 100 ± 1.0, 88.5 ± 0.6 100 ± 1.4, 82.5 ± 1.1

cell viabilityc at 10 and 50 μM (%)

Cytochrome P450 (CYP) inhibition assay was performed using a P450-Glo assay system (Promega). bIn vitro microsomal stability of the synthesized compound; % remaining was determined after 30 min incubation with human microsomes. The % of parent compound remaining was calculated by comparing peak areas. cCell viability was determined using Ez-Cytox. BV2 cells (10 000 cells/well) were treated with test compounds (10 or 50 μM) and incubated for 24 h before EZ-cytox assay. All experiments were performed in triplicate cultures, and the data were averaged and expressed as percent of untreated control ± standard error of the mean. dnd, not determined. eCompound 1 developed in a previous study.23

a

2D6

2C19

comp

CYP enzyme activitiesa (%)a

Table 3. CYP Inhibition, Microsomal Stability, and Cell Viability Tests of the Synthesized Compounds

Journal of Medicinal Chemistry Article



Article

Table 4. Summary of 17e Propertiesa

aqueous solubility

>30 mg/mL

pKa, log P (c log P) activation of the Nrf2 response (EC50, μM) cell viability in 50 μM (%) CYP inhibition (% of control activity) human microsomal stability (% remaining) PAMPA-BBB (Pe, cm/s) plasma stability (% remaining) hERG toxicity (IC50, μM) mutagenic toxicity (TA98, TA100, fold induction over the negative control)

7.58, 2.29 (2.49) 0.346 ± 0.050 99.0 ± 1.5 77.4 (2C19), 99.7 (2D6), 96.7 (2C9), 87.2 (1A2), 97.4 (3A4), 84.0 15.63 (CNS+) 98.2 (human), 90.2 (rat) 112.1 ± 13.5 negative

a All experiments were performed in triplicate except for microsomal and plasma stability tests. The predicted pKa and c log P values were calculated using ChemDraw v15.1. PAMPA-BBB, parallel artificial membrane permeability assay-blood−brain barrier; CYP, cytochrome P; hERG, human ether-a-go-go-related gene; Nrf2, nuclear factor (erythroid-derived 2)-like 2; IC50, half-maximal inhibitory concentration; Pe, permeability.

Figure 1. 17e induces nuclear translocation of Nrf2. (a) The Keap1−Nrf2 functional assay of engineered U2OS cells treated with various concentrations of 17e for 6 h. Signal was detected as relative luminescence units of the saturated concentration using a chemiluminescent substrate. Cell viability assay of BV2 cells treated with various concentrations of 17e for 24 (b) or 6 h (c, d). The amount of nuclear and total Nrf2 was measured by Western blot with lamin B1 as a nuclear marker; β-actin was used as an internal control. All experiments were performed in triplicate. Data are shown as mean ± standard error of the means and analyzed by Student’s t-test; * P < 0.05, ** P < 0.01, *** P < 0.001 vs the vehicletreated control group.

translocation of Nrf2 and subsequent expression of various antioxidant enzymes in BV2 cells.24 Initial assessment of the cytotoxic potential of 17e in BV2 cells showed no apparent cytotoxicity up to 100 μM (Figure 1b). To evaluate its ability to translocate Nrf2 into the nucleus, BV2 cells were treated

with various concentrations of 17e for 6 h before measuring the nuclear Nrf2 levels by Western blot. Nrf2 protein levels in the nucleus were significantly increased in a dose-dependent manner (2-fold at 0.1 μM; 9-fold at 10 μM; Figure 1c). In several studies, Nrf2 activation has been shown without 818



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Figure 2. 17e dose-dependently induced expression of antioxidant enzymes in BV2 cells. (a, b) BV2 cells were treated with various concentrations of 17e for 24 h and then subjected to Western blot analysis of HO-1, GCLM, and GCLC using β-actin as an internal control. (c) BV2 cells were treated with various concentrations of 17e for 6 h, and relative mRNA levels of Nrf2-dependent genes were measured by RT-qPCR; hypoxanthineguanine phosphoribosyltransferase was used as an internal control. All experiments were performed in triplicate. Data are shown as mean ± standard error of the means and analyzed by Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001 vs the vehicle-treated control group.

Figure 3. 17e attenuates motor abnormalities in MPTP-induced PD mice. (a) Experimental protocols for vertical grid and coat-hanger behavioral tests. (b) Time to turn and total time were measured in the vertical grid test. (c) Coat-hanger test scores measured according to the position of the mouse. Data are shown as means ± standard error of the mean (n = 10 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 vs the MPTP-treated group.

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Figure 4. 17e protects DAergic neurons and reduces microglial activation in MPTP-induced PD mice. (a) Representative immunohistochemical staining (brown, TH) in the striatum and SNpc. Bar graphs summarize TH-immunopositive density and cell numbers. (b) Representative double immunofluorescent staining in the SNpc (green, TH; red, Iba-1). Bar graph summarizing analysis of Iba-1 immunofluorescence intensity. Data are shown as the mean ± standard error of the mean (n = 6 per group); *** P < 0.001 vs the MPTP-treated group.

was able to induce nuclear translocation of Nrf2 and upregulate expression of HO-1 and GCL enzymes in BV2 cells. 17e Alleviates Movement Abnormalities in Acute MPTP-Induced PD Mice. We previously reported synthesis of potent Nrf2 activators that exhibited positive therapeutic effects in an MPTP-induced mouse model of PD;23,24 MPTP treatment causes PD-like motor dysfunctions in mice. Here, we investigated whether 17e can alleviate movement abnormalities in MPTP-induced PD mice as well. The motor activity of MPTP-treated animals can be evaluated by vertical grid and coat-hanger behavioral tests (Figure 3a). In the vertical grid test (Figure 3b), MPTP injection significantly delayed the time to turn and total time (time to turn: 4.2 ± 0.5 s; total time: 9.4 ± 0.6 s) compared to saline-injected mice (time to turn: 2.3 ± 0.2 s; total time: 6.2 ± 0.4 s), whereas 17e treatment significantly alleviated abnormal movement caused by MPTP (time to turn: 2.6 ± 0.2 s; total time: 7.4 ± 0.5 s). In the coathanger test (Figure 3a), saline-injected mice climbed the coathanger and reached the safety zone (score: 4.5 ± 0.2), whereas MPTP-injected mice generally stayed at the edge of the coathanger and failed to climb (score: 2.7 ± 0.1). On the other hand, MPTP-injected mice treated with 17e showed dramatic improvement in the ability to climb to the top of the coathanger and reach the safety zone (score: 4.1 ± 0.3). Taken together, these behavioral results suggest that 17e could effectively alleviate movement disorders related to PD. 17e Protects DAergic Neurons and Attenuates Microglial Activation against MPTP in the Striatum and SNpc. We next assessed whether 17e could protect against DAergic neurotoxicity in MPTP-induced PD mice. First, immunohistochemical staining for tyrosine hydroxylase (TH) in the nigrostriatal pathway was performed (Figure 4a). TH is known to be involved in motor movement and is used as

degradation by the Keap1-mediated ubiquitin proteasome system when released from the cytosolic inhibitor Keap1.30,31 Therefore, Western blot with whole cell lysate was performed to determine whether Nrf2 activation by 17e increased the total amount of Nrf2 in BV2 cells. As shown in Figure 1d, total Nrf2 levels were elevated in a dose-dependent manner, indicating accumulation of Nrf2 with 17e treatment. Next, we investigated whether expression of target antioxidant enzymes was induced upon as a result of Nrf2 activation. HO-1 is major Nrf2-dependent enzyme that converts heme to biliverdin and CO, which is known to have antioxidant and neuroprotective properties.32 Western blot analysis of 17e-treated cells showed a dose-dependent increase in the amount of HO-1. A concentration of 0.3 μM was sufficient to cause a statistically significant change in HO-1 protein levels, and 10 μM 17e resulted in a 2.9 ± 0.1-fold increase (Figure 2a,b). Real-time quantitative polymerase chain reaction (RT-qPCR) assay of HO-1 mRNA isolated from BV2 cells treated with 17e revealed a dramatic and dose-dependent increase. Significant induction was observed at 1 μM (2.7 ± 0.1-fold), reaching 7.5 ± 0.5-fold with 10 μM 17e (Figure 2c). GCL is an important antioxidant enzyme responsible for the biosynthesis of glutathione, a major antioxidant enzyme in cells that consists of a modulatory (GCLM) and catalytic (GCLC) subunit. Western blot analysis (Figure 2a,b) revealed dosedependent elevation of both GCLM and GCLC protein levels, reaching 12.4 ± 0.5-fold and 2.7 ± 0.2-fold increases with 10 μM 17e, respectively. RT-qPCR assay of GCLM and GCLC showed that both were significantly upregulated with 0.3 μM 17e treatment (1.7 ± 0.2-fold and 1.2 ± 0.1-fold, respectively), and reached 5.3 ± 0.4-fold and 2.1 ± 0.1-fold induction, respectively, with 10 μM 17e (Figure 2c). Taken together, 17e 820



Article

General Procedure for the Compounds. To a cooled anhydrous tetrahydrofuran solution (−78 °C) of phosphonate derivatives (4, 10, 16 and 22) was added 2 M n-BuLi solution in cyclohexane (1.1 equiv). The reaction mixture was stirred at −78 °C (1 h) and then the desired substituted benzaldehyde (1.1 equiv) was added at −78 °C. The reaction mixture was stirred at room temperature (1 h) and then quenched with H2O (∼50 mL). 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 with anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography on SiO2. In the final step, the production rates of the (E) and (Z)-isomers are changed according to the reaction temperature. At room temperature, the (E)-isomer is predominantly formed and at very low temperatures (−78 °C), the (Z)-isomer is also formed. In our previous study,23 we observed that each isomer of vinyl sulfone showed a distinct Rf value difference on TLC, and thus could be separated by silica column chromatography. The major product of vinyl sulfone derivatives synthesized at room temperature is the (E)-isomer, which has a lower Rf value than the (Z)-isomer on TLC. The (E)-isomer showed a different J-value from the (Z)-isomer in the NMR spectrum ((E): J = 14−16 Hz, (Z): J = 8−10 Hz) and was confirmed when it was not overlapped with other aromatic peaks. Among the final compounds, the morpholinyl and piperazinyl compounds (17−19 and 23), were prepared the salt forms by addition of 4.0 M HCl to EtOAc solutions. Synthetic procedures for all intermediates were reported in the Supporting Information. (E)-2′-(2-((2-Methoxyphenyl)sulfonyl)vinyl)pyridine (5a). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.55 mmol), 2.0 M n-BuLi solution in cyclohexane (0.85 mL, 1.71 mmol), and 2-pyridinecarboxaldehyde (0.19 mL, 1.71 mmol) gave 0.34 g (83%) of 5a as a white solid; Rf = 0.45 (n-hexane/EtOAc 1/1); mp: 123−124 °C; 1H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 4.2 Hz, ArH), 8.02 (dd, J = 1.5, 7.8 Hz, ArH), 7.73 (td, J = 1.5, 7.6 Hz, ArH), 7.67 (s, (E)-isomeric 2H), 7.42−7.59 (m, ArH), 7.41 (d, J = 7.7 Hz, ArH), 7.27−7.31 (m, ArH), 7.10 (t, J = 7.6 Hz, ArH), 7.01 (d, J = 8.4 Hz, ArH), 3.97 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.7, 151.6, 150.4, 141.6, 137.1, 135.7, 131.6, 129.9, 128.3, 125.4, 124.9, 120.8, 112.5, 56.4 (OCH3); HPLC purity: 12.1 min, 98.2%; HRMS (M + H)+ (positive electrospray ionization (ESI+)) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2′-Chloro-6′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (5b). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 6-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.22 g (93%) of 5b as a white powder; Rf = 0.34 (n-hexane/EtOAc 1/1); mp: 104−105 °C; 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (t, J = 7.7 Hz, ArH), 7.88 (m, 2ArH), 7.57−7.75 (m, 2ArH, (E)-isomeric 2H), 7.29 (d, J = 8.3 Hz, ArH), 7.20 (t, J = 8.0 Hz, ArH), 3.94 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 157.7, 152.0, 151.0, 141.6, 140.5, 136.7, 132.6, 129.3, 127.7, 126.5, 125.2, 121.3, 114.0, 57.0 (OCH3); HPLC purity: 14.4 min, 95.8%; HRMS (M + H)+ (ESI+) 310.0295 [M + H]+ (calcd for C14H12ClNO3S+ 310.0299). (E)-5′-Chloro-2′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (5c). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 5-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.16 g (68%) of 5c as a white powder; Rf = 0.40 (n-hexane/EtOAc 2/1); mp: 153−154 °C; 1 H NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 2.4 Hz, ArH), 8.06 (dd, J = 2.4, 8.4 Hz, ArH), 7.87−7.91 (m, 2ArH), 7.68−7.75 (m, ArH, (E)-isomeric 2H), 7.29 (d, J = 8.4 Hz, ArH), 7.19 (t, J = 7.5 Hz, ArH), 3.94 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 157.6, 149.9, 149.4, 141.0, 137.6, 136.7, 133.0, 131.9, 129.1, 127.9, 127.1, 121.2, 113.9, 56.9 (OCH3); HPLC purity: 14.3 min, 99.0%; HRMS (M + H)+ (ESI+) 310.0292 [M + H]+ (calcd for C14H12ClO3SH+ 310.0299). (E)-3′-Fluoro-2′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (5d). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)-

a marker for DAergic neurons. Although the striatal THimmunopositive nerve fibers significantly decreased after MPTP injection (18.8 ± 3.5%) compared to controls, they increased 3-fold with 17e treatment (59.6 ± 5.7%). THimmunostaining in the SNpc showed similar results as the substantial reduction in TH-immunopositivity with MPTP (32.0 ± 2.6%) was significantly moderated by 17e (76.7 ± 2.2%). These results indicate that 17e elicits neuroprotective effects against DAergic neuronal cell death in PD. Ionized calcium-binding adapter molecule 1 (Iba-1) immunofluorescent staining in the SNpc was also performed to determine whether 17e suppressed microglial activation related to neuroinflammation. In MPTP-injected mice, the immunofluorescence intensity of Iba-1 was 3-fold higher than that of saline-injected mice (Figure 4b). However, treatment of MPTP-injected mice with 17e dramatically reduced Iba-1 immunoreactivity, indicating that 17e suppresses microglial activation. Double immunofluorescent staining for TH and Iba-1 (Figure 4b) confirmed the neuroprotective and antiinflammatory effects of 17e.

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CONCLUSIONS We previously developed a novel neuroprotective compound (1) that induces Nrf2 activity and subsequent expression of antioxidant genes, but exhibits unfavorable druglike properties. In the present study, we improved the druglike properties and Nrf2 activation potency of compound 1 by introducing morpholine and pyridine groups. Further examination of these compounds revealed that 17e dose-dependently induced optimal Nrf2 activity, nuclear translocation, and related upregulation of antioxidant enzymes (HO-1, GCLM, and GCLC), as well as excellent druglike properties. In MPTPinduced PD mice, treatment with 17e was neuroprotective, alleviating loss of DAergic neurons and preventing microglia activation. These effects were accompanied by improvement of movement ability. In conclusion, 17e represents a novel therapeutic agent with potential for treating PD.

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EXPERIMENTAL SECTION

General Methods. All chemicals, solvents, and reagents were obtained from commercial sources as reagent grade without further purification. Yields reported are for purified products and were not optimized. Synthesized compounds were checked by thin-layer chromatography (TLC) and 1H and 13C nuclear magnetic resonance, melting point, high-resolution mass spectrometry (HRMS), and highperformance liquid chromatography (HPLC) analyses. Reactions were monitored by analytical thin-layer chromatography plates (Merck, Cat No. 1.05715) and analyzed by ultraviolet light at 254 nm. The reactions were purified by column chromatography using silica gel (Merck, Cat Nos. 1.07734 and 1.09385). Melting points were measured in open capillary tubes using OptiMelt melting point equipment (Stanford Research Systems, Inc.). Nuclear magnetic resonance spectra were recorded at 400 MHz (1H)/100 MHz (13C) or 300 MHz (1H)/75 MHz (13C) using Bruker spectrometers. Chemical shifts (δ) were reported in ppm downfield from tetramethylsilane. HPLC analysis was performed using a Waters E2695 system equipped with a Capcell Pak C18 column (4.6 mm × 75 mm; 3 μm particle size). HPLC data were recorded using the following parameters: H2O/acetonitrile, 100/0 → 0/100 in 20 min, +5 min isocratic, flow rate of 0.5 mL/min, λ = 254 and 280 nm. All compounds were >95% pure. High-resolution MS was performed with electron scatter ionization on an LTQ Orbitrap (Thermo Electron Corp.) instrument. 821



Article

phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.46 mL, 0.85 mmol), and 3-fluoropyridine-2-carboxaldehyde (0.11 g, 0.85 mmol) gave 0.17 g (77%) of 5d as a white powder; Rf = 0.23 (n-hexane/EtOAc 1/1); mp: 119−120 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 4.4 Hz, ArH), 7.86−7.91 (m, 2ArH), 7.67−7.76 (m, ArH, (E)-isomeric 2H), 7.59−7.64 (m, ArH), 7.31 (d, J = 8.4 Hz, ArH), 7.20 (t, J = 7.6 Hz, ArH), 3.95 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 158.0 (d, JC−F = 261.4 Hz), 157.1, 146.5 (d, JC−F = 4.8 Hz), 138.6 (d, JC−F = 10.9 Hz), 136.3, 133.5, 132.3 (d, JC−F = 4.3 Hz), 128.7, 127.8 (d, JC−F = 4.8 Hz), 127.0, 124.8 (d, JC−F = 18.9 Hz), 120.8, 113.4, 56.5 (OCH3); HPLC purity: 7.3 min, 99.1%; HRMS (M + H)+ (ESI+) 294.0590 [M + H]+ (calcd for C14H12FNO3SH+ 294.0595). (E)-3′-Chloro-2′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (5e). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 3-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.18 g (75%) of 5e as a pale yellow powder; Rf = 0.32 (n-hexane/EtOAc 1/1); mp: 113−114 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.62−8.63 (m, ArH), 8.08 (dd, J = 1.2, 8.2 Hz, ArH), 7.96 (d, J = 14.8 Hz, (E)-isomeric H), 7.90 (dd, J = 1.5, 7.8 Hz, ArH), 7.74−7.76 (m, ArH), 7.70 (d, J = 14.9 Hz, (E)-isomeric H), 7.52−7.55 (m, ArH), 7.30 (d, J = 8.4 Hz, ArH), 7.20 (t, J = 7.6 Hz, ArH), 3.95 (s, OCH3); 13C NMR (100 MHz, DMSOd6) δ 157.6, 149.2, 147.6, 138.9, 136.9, 136.6, 133.9, 132.6, 129.3, 127.5, 127.4, 121.3, 113.9, 57.0 (OCH3); HPLC purity: 10.9 min, 98.5%; HRMS (M + H)+ (ESI+) 310.0294 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-2′-(2-((3-Methoxyphenyl)sulfonyl)vinyl)pyridine (5f). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.30 g, 0.93 mmol), 2.0 M n-BuLi solution in cyclohexane (0.51 mL, 1.02 mmol), and 2-pyridinecarboxaldehyde (0.11 mL, 1.02 mmol) gave 0.24 g (93%) of 5f as a brown oil; Rf = 0.26 (n-hexane/EtOAc 2/1); 1 H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 4.1 Hz, ArH), 7.72−7.76 (m, ArH), 7.64 (d, J = 14.9 Hz, (E)-isomeric H), 7.54 (d, J = 7.7 Hz, ArH), 7.40−7.47 (m, 3ArH, (E)-isomeric H), 7.26−7.31 (m, ArH), 7.13 (m, ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.1, 151.0, 150.3, 141.4, 140.5, 137.1, 131.8, 130.5, 125.5, 125.0, 120.2, 120.1, 112.2, 55.7 (OCH3); HPLC purity: 4.4 min, 97.3%; HRMS (M + H)+ (ESI+) 276.0685 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2′-Chloro-6′-(2-((3-methoxyphenyl)sulfonyl)vinyl)pyridine (5g). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 6-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.22 g (94%) of 5g as a white powder; Rf = 0.31 (n-hexane/EtOAc 2/1); mp: 101−103 °C; 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (t, J = 7.7 Hz, ArH), 7.84 (d, J = 7.5 Hz, ArH), 7.73 (d, J = 15.0 Hz, (E)-isomeric H), 7.67 (d, J = 15.1 Hz, (E)-isomeric H), 7.31−7.62(m, 5ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 160.2, 151.9, 151.0, 141.5, 141.5, 139.8, 133.2, 131.4, 126.5, 125.2, 120.6, 120.1, 112.7, 56.2 (OCH3); HPLC purity: 12.6 min, 99.8%; HRMS (M + H)+ (ESI+) 310.0296 [M + H]+ (calcd for C14H13ClNO3SH+ 310.0299). (E)-5′-Chloro-2′-(2-((3-methoxyphenyl)sulfonyl)vinyl)pyridine (5h). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.29 g, 0.90 mmol), 2.0 M n-BuLi solution in cyclohexane (0.50 mL, 0.99 mmol), and 5-chloropyridine-2carboxaldehyde (0.14 g, 0.99 mmol) gave 0.22 g (78%) of 5h as a white powder, Rf = 0.40 (n-hexane/EtOAc 2/1); mp: 92−93 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (dd, J = 1.0, 4.4 Hz, ArH), 8.07 (dd, J = 1.2, 8.2 Hz, ArH), 7.93 (d, J = 14.8 Hz, (E)-isomeric H), 7.80 (d, J = 14.8, (E)-isomeric H), 7.50−7.62 (m, 4ArH), 7.33 (m, ArH), 3.87 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 159.7, 148.6, 147.0, 140.7, 138.4, 135.0, 134.0, 132.2, 131.0, 127.0, 120.2, 119.6, 112.1, 55.8 (OCH3); HPLC purity: 15.9 min, 99.5%; HRMS (M + H)+ (ESI+) 310.0290 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-3′-Fluoro-2′-(2-((3-methoxyphenyl)sulfonyl)vinyl)pyridine (5i). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)-

phosphonate (0.70 g, 2.17 mmol), 2.0 M n-BuLi solution in cyclohexane (1.20 mL, 2.39 mmol), and 3-fluoropyridine-2-carboxaldehyde (0.31 g, 2.39 mmol) gave 0.50 g (43%) of 5i as a white powder; Rf = 0.33 (n-hexane/EtOAc 2/1); mp: 113−114 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 4.4 Hz, ArH), 7.88 (t, J = 9.2 Hz, ArH), 7.76 (d, J = 15.2 Hz, (E)-isomeric H), 7.71 (d, J = 14.8 Hz, (E)-isomeric H), 7.49−7.63 (m, 4ArH), 7.31 (m, ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 159.7, 158.1 (d, JC−F = 261.8 Hz), 146.3 (d, JC−F = 4.7 Hz), 140.8, 138.5 (d, JC−F = 10.9 Hz), 132.8 (d, JC−F = 4.2 Hz), 132.7, 131.0, 127.9 (d, JC−F = 4.6 Hz), 124.8 (d, JC−F = 18.8 Hz), 120.2, 119.6, 112.1, 55.7 (OCH3); HPLC purity: 14.9 min, 99.7%; HRMS (M + H)+ (ESI+) 294.0590 [M + H]+ (calcd for C14H12FNO3SH+ 294.0595). (E)-3′-Chloro-2′-(2-((3-methoxyphenyl)sulfonyl)vinyl)pyridine (5j). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 3-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.20 g (84%) of 5j as pale yellow powder; Rf = 0.28 (n-hexane/EtOAc 2/1); mp: 89−90 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.59−8.61 (m, ArH), 8.07 (dd, J = 1.2, 8.2 Hz, ArH), 7.93 (d, J = 14.8 Hz, (E)-isomeric H), 7.80 (d, J = 14.8 Hz, (E)-isomeric H), 7.50−7.62 (m, 4ArH), 7.32−7.34 (m, ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 160.2, 149.1, 147.5, 141.2, 138.9, 135.5, 134.5, 132.7, 131.5, 127.5, 120.8, 120.1, 112.6, 56.3 (OCH3); HPLC purity: 11.3 min, 99.5%; HRMS (M + H)+ (ESI+) 310.0292 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-2′-(2-((4-Methoxyphenyl)sulfonyl)vinyl)pyridine (5k). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.55 mmol), 2.0 M n-BuLi solution in cyclohexane (0.85 mL, 1.71 mmol), and 2-pyridinecarboxaldehyde (0.19 mL, 1.71 mmol) gave 0.36 g (85%) of 5k as a pale yellow solid; Rf = 0.45 (n-hexane/EtOAc 1/2); mp: 97−98 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.61−8.62 (m, ArH), 7.86−7.92 (m, 3ArH), 7.78−7.81 (m, ArH), 7.65 (d, J = 15.1 Hz, (E)-isomeric H), 7.60 (d, J = 15.1 Hz, (E)-isomeric H), 7.42−7.46 (m, ArH), 7.16−7.21 (m, 2ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.7, 151.2, 150.3, 139.5, 137.0, 132.4, 131.7, 130.1, 125.3, 124.9, 114.6, 55.7 (OCH3); HPLC purity: 12.6 min, 99.0%; HRMS (M + H)+ (ESI+) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2′-Chloro-6′-(2-((4-methoxyphenyl)sulfonyl)vinyl)pyridine (5l). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 6-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.16 g (66%) of 5l as a white powder; Rf = 0.28 (n-hexane/EtOAc 2/1); mp: 118−120 °C; 1 H NMR (400 MHz, DMSO-d6) δ 7.96 (t, J = 7.8 Hz, ArH), 7.89 (d, J = 8.9 Hz, 2ArH), 7.81 (d, J = 7.4 Hz, ArH), 7.64 (d, J = 15.1 Hz, (E)-isomeric H), 7.56−7.60 (m, ArH, (E)-isomeric H), 7.18 (d, J = 8.9 Hz, 2ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 164.0, 152.0, 151.0, 141.5, 138.6, 134.0, 131.6, 130.4, 126.3, 125.0, 115.5, 56.3 (OCH3); HPLC purity: 13.3 min, 98.0%; HRMS (M + H)+ (ESI+) 310.0296 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-5′-Chloro-2′-(2-((4-methoxyphenyl)sulfonyl)vinyl)pyridine (5m). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 5-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.14 g (58%) of 5m as a white solid; Rf = 0.40 (n-hexane/EtOAc 2/1); mp: 138.0−139.7 °C; 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 2.4 Hz, ArH), 8.05 (dd, J = 8.9 Hz, ArH), 7.85−7.89 (m, 3ArH), 7.67 (d, J = 15.1 Hz, (E)-isomeric H), 7.62 (d, J = 15.1 Hz, (E)-isomeric H), 7.18 (d, J = 8.9 Hz, ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 163.9, 149.9, 149.2, 139.1, 137.6, 133.3, 132.9, 131.7, 130.3, 126.8, 115.4, 56.3 (OCH3); HPLC purity: 11.7 min, 99.0%; HRMS (M + H)+ (ESI+) 310.0293 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-3′-Fluoro-2′-(2-((4-methoxyphenyl)sulfonyl)vinyl)pyridine (5n). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)822



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phosphonate (0.75 g, 2.33 mmol), 2.0 M n-BuLi solution in cyclohexane (1.28 mL, 2.56 mmol), and 3-fluoropyridine-2-carboxaldehyde (0.32 g, 2.56 mmol) gave 0.46 g (68%) of 5n as a pale yellow powder; Rf = 0.28 (n-hexane/EtOAc 1/1); mp: 84−85 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 4.4 Hz, ArH), 7.85−7.92 (m, 3ArH), 7.66 (s, (E)-isomeric 2H), 7.57−7.61 (m, ArH), 7.17− 7.26 (m, 2ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 163.8, 158.3 (d, JC−F = 263.8 Hz), 145.9 (d, JC−F = 5.0 Hz), 139.8 (d, JC−F = 11.2 Hz), 133.9 (d, JC−F = 4.4 Hz), 132.0, 131.5, 130.2, 126.5 (d, JC−F = 4.2 Hz), 124.1 (d, JC−F = 19.0 Hz), 114.7, 55.7 (OCH3); HPLC purity: 8.5 min, 98.9%; HRMS (M + H)+ (ESI+) 294.0588 [M + H]+ (calcd for C14H12FNO3SH+ 294.0595). (E)-3′-Chloro-2′-(2-((4-methoxyphenyl)sulfonyl)vinyl)pyridine (5o). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.42 mL, 0.85 mmol), and 3-chloropyridine-2carboxaldehyde (0.12 g, 0.85 mmol) gave 0.14 g (58%) of 5o as a white powder; Rf = 0.24 (n-hexane/EtOAc 2/1); mp: 134−135 °C; 1 H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 3.6 Hz, ArH), 8.06 (dd, J = 1.1, 8.2 Hz, ArH) 7.92 (d, J = 8.9 Hz, 2ArH), 7.87 (d, J = 14.8 Hz, (E)-isomeric H), 7.70 (d, J = 14.8 Hz, (E)-isomeric H), 7.50−7.53 (m, ArH), 7.18 (d, J = 8.9 Hz, 2ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 164.0, 149.0, 147.6, 138.9, 135.3, 134.3, 132.5, 131.3, 130.5, 127.4, 115.5, 56.4 (OCH3); HPLC purity: 12.2 min, 98.0%; HRMS (M + H)+ (ESI+) 310.0294 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-3′-(2-((2-Methoxyphenyl)sulfonyl)vinyl)pyridine (6a). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 3-pyridinecarboxaldehyde (0.09 g, 0.85 mmol) gave 0.22 g (97%) of 6a as a white solid; Rf = 0.25 (n-hexane/EtOAc 1/3); mp: 128−129 °C; 1H NMR (400 MHz, CDCl3) δ 8.74 (d, J = 2.1 Hz, ArH), 8.63 (dd, J = 1.6, 4.8 Hz, ArH), 8.03 (dd, J = 4.7, 7.8 Hz, ArH), 7.81 (dd, J = 1.9, 7.9 Hz, ArH), 7.69 (d, J = 15.6 Hz, (E)-isomeric H), 7.59 (m, ArH), 7.35 (m, ArH), 7.23 (d, J = 15.6 Hz, (E)-isomeric H), 7.12 (t, J = 8.0 Hz, ArH), 7.03 (d, J = 8.3 Hz, ArH), 3.98 (s, OCH3); 13 C NMR (100 MHz, CDCl3) δ 157.5, 151.7, 150.0, 139.7, 135.8, 134.9, 129.7, 129.4, 128.9, 128.3, 123.9, 121.0, 112.6, 56.5; HPLC purity: 4.4 min, 98.0%; HRMS (M + H)+ (ESI+) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2′-Methoxy-3′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (6b). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 2-methoxypyridine-3carboxaldehyde (0.12 g, 0.85 mmol) gave 0.17 g (72%) of 6b as a white powder; Rf = 0.26 (n-hexane/EtOAc 1/2); mp: 123−125 °C; 1 H NMR (400 MHz, CDCl3) δ 8.02−8.13 (m, 2ArH), 7.74 (d, J = 15.5 Hz, (E)-isomeric H), 7.51−7.69 (m, ArH), 7.56 (td, J = 1.6, 7.6 Hz, ArH), 7.41 (d, J = 15.5 Hz, (E)-isomeric H), 7.10 (t, J = 7.6 Hz, ArH), 7.01 (d, J = 8.3 Hz, ArH), 6.74−6.95 (m, ArH), 4.03 (s, OCH3), 3.97 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 162.2, 157.5, 149.0, 139.5, 138.1, 135.5, 129.7, 129.6, 128.8, 120.8, 117.2, 116.6, 112.5, 56.3, 53.9; HPLC purity: 8.2 min, 97.1%; HRMS (M + H)+ (ESI+) 306.0786 [M + H]+ (calcd for C15H15NO4SH+ 306.0795). (E)-2′-Chloro-3′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (6c). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 2-chloropyridine-3carboxaldehyde (0.12 g, 0.85 mmol) gave 0.16 g (68%) of 6c as a white powder; Rf = 0.25 (n-hexane/EtOAc 1/1); mp: 154−155 °C; 1 H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 1.2 Hz, ArH), 7.98−8.02 (m, ArH, (E)-isomeric H), 7.87 (dd, J = 1.7, 7.7 Hz, ArH), 7.59 (td, J = 1.6, 8.4 Hz, ArH), 7.30 (m, ArH), 7.08−7.16 (m, ArH, (E)-isomeric H), 7.02 (d, J = 8.4 Hz, ArH), 3.97 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.6, 151.5, 151.2, 138.0, 136.9, 136.0, 131.9, 130.0, 128.3, 127.7, 123.0, 121.0, 112.6, 56.4; HPLC purity: 5.4 min, 99.8%; HRMS (M + H)+ (ESI+) 310.0292 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-2′,6′-Dichloro-3′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyridine (6d). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)-

phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 2,6-dichloropyridine-3carboxaldehyde (0.16 g, 0.83 mmol) gave 0.25 g (94%) of 6d as a white solid; Rf = 0.45 (n-hexane/EtOAc 1/1); mp: 136−138 °C; 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 5.3 Hz, ArH), 8.05 (dd, J = 1.5, 7.9 Hz, ArH), 7.82 (d, J = 15.8 Hz, (E)-isomeric H), 7.62 (m, ArH), 7.47 (d, J = 15.8 Hz, (E)-isomeric H), 7.37 (d, J = 5.3 Hz, ArH), 7.13 (t, J = 7.6 Hz, ArH), 7.04 (d, J = 8.4 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.6, 151.8, 149.5, 145.6, 136.8, 136.1, 134.3, 130.0, 127.6, 127.4, 124.7, 121.0, 112.5, 56.4; HPLC purity: 4.6 min, 99.0%; HRMS (M + H)+ (ESI+) 343.9906 [M + H]+ (calcd for C14H11Cl2NO3SH+ 343.9909). (E)-3′-(2-((3-Methoxyphenyl)sulfonyl)vinyl)pyridine (6e). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.55 mmol), 2.0 M n-BuLi solution in cyclohexane (0.85 mL, 1.71 mmol), and 3-pyridinecarboxaldehyde (0.18 g, 1.71 mmol) gave 0.42 g (85%) of 6e as a pale yellow oil; Rf = 0.45 (n-hexane/EtOAc 1/3); mp: 52−53 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.91 (d, J = 1.8 Hz, ArH), 8.61 (d, J = 4.7 Hz, ArH), 8.18 (d, J = 8.1 Hz, ArH), 7.81 (d, J = 15.4 Hz, (E)-isomeric H), 7.71 (d, J = 15.4 Hz, (E)-isomeric H), 7.59 (m, ArH), 7.45−7.51 (m, 2ArH), 7.41 (m, ArH), 7.31 (m, ArH), 3.85 (s, CH3); 13C NMR (100 MHz, CDCl3) δ 160.2, 151.8, 150.0, 141.3, 138.7, 134.8, 130.6, 129.6, 128.3, 123.9, 120.2, 120.0, 112.2, 55.8; HPLC purity: 11.2 min, 96.4%; HRMS (M + H)+ (ESI+) 276.0685 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2′-Chloro-3′-(2-((3-methoxyphenyl)sulfonyl)vinyl)pyridine (6f). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 2-chloropyridine-3carboxaldehyde (0.12 g, 0.85 mmol) gave 0.19 g (78%) of 6f as a pale yellow powder; Rf = 0.34 (n-hexane/EtOAc 1/1); mp: 101−103 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.49 (dd, J = 1.7, 4.7 Hz, ArH), 8.35 (dd, J = 1.7, 8.0 Hz, ArH), 7.88 (d, J = 15.4 Hz, (E)isomeric H), 7.78 (d, J = 15.4 Hz, (E)-isomeric H), 7.61 (t, J = 7.9 Hz, ArH), 7.51−7.54 (m, 2ArH), 7.41−7.44 (m, ArH), 7.33 (dd, J = 1.9, 8.0 Hz, ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.2, 152.2, 150.7, 141.4, 138.4, 136.2, 133.3, 131.6, 127.5, 124.2, 120.5, 119.9, 112.5, 56.3; HPLC purity: 5.0 min, 98.8%; HRMS (M + H)+ (ESI+) 310.0294 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-3′-(2-((4-Methoxyphenyl)sulfonyl)vinyl)pyridine (6g). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.30 g, 0.93 mmol), 2.0 M n-BuLi solution in cyclohexane (0.51 mL, 1.02 mmol), and 3-pyridinecarboxaldehyde (0.11 g, 1.02 mmol) gave 0.23 g (89%) of 6g as a white powder; Rf = 0.26 (n-hexane/EtOAc 1/3); mp: 111−112 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 1.7 Hz, ArH), 8.61 (dd, J = 1.4, 4.7 Hz, ArH), 8.16−8.18 (m, ArH), 7.85 (d, J = 8.9 Hz, 2ArH), 7.74 (d, J = 15.5 Hz, (E)-isomeric H), 7.65 (d, J = 15.5 Hz, (E)-isomeric H), 7.44−7.47 (m, ArH), 7.19 (d, J = 8.9 Hz, 2ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 163.8, 151.9, 150.7, 138.2, 135.7, 132.2, 131.2, 130.1, 129.0, 124.4, 115.4, 56.3; HPLC purity: 4.6 min, 99.7%; HRMS (M + H)+ (ESI+) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2′-Chloro-3′-(2-((4-methoxyphenyl)sulfonyl)vinyl)pyridine (6h). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 2-chloropyridine-3carboxaldehyde (0.12 g, 0.85 mmol) gave 0.18 g (77%) of 6h as a white solid; Rf = 0.28 (n-hexane/EtOAc 1/1); mp: 146−148 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.46 (dd, J = 1.3, 4.4 Hz, ArH), 8.33 (m, ArH), 7.87 (d, J = 8.8 Hz, 2ArH), 7.81 (d, J = 15.4 Hz, (E)isomeric H), 7.73 (d, J = 15.4 Hz, (E)-isomeric H), 7.49−7.52 (m, ArH), 7.19 (d, J = 8.8 Hz, 2ArH), 3.87 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 164.0, 152.0, 150.7, 138.3, 134.9, 134.1, 131.5, 130.3, 127.6, 124.2, 115.5, 56.3; HPLC purity: 5.4 min, 99.2%; HRMS (M + H) + (ESI+) 310.0293 [M + H]+ (calcd for C14H12ClNO3SH+ 310.0299). (E)-2′-Chloro-5′-(2-((4-methoxyphenyl)sulfonyl)vinyl)pyridine (6i). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.55 mmol), 2.0 M n-BuLi solution in 823



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ArH), 7.55 (d, J = 15.8 Hz, (E)-isomeric H), 7.15 (t, J = 7.6 Hz, ArH), 7.05 (d, J = 8.3 Hz, ArH), 3.98 (s,OCH3); 13C NMR (100 MHz, CDCl3) δ 161.2, 157.6, 157.2, 138.1, 136.3, 132.1, 130.1, 127.2, 126.1, 121.1, 112.5, 56.5 (OCH3); HPLC purity: 5.0 min, 98.3%; HRMS (M + H) + (ESI+) 344.9867 [M + H]+ (calcd for C13H10Cl2N2O3SH+ 344.9862). (E)-5′-(2-((3-Methoxyphenyl)sulfonyl)vinyl)pyrimidine (8c). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.30 g, 0.93 mmol), 2.0 M n-BuLi solution in cyclohexane (0.51 mL, 1.02 mmol), and pyrimidine-5-carboxaldehyde (0.11 g, 1.02 mmol) gave 0.10 g (37%) of 8c as a white powder; Rf = 0.46 (n-hexane/EtOAc 1/ 2); mp: 123−125 °C; 1H NMR (400 MHz, CDCl3) δ 9.21 (s, ArH), 8.89 (s, 2ArH), 7.88 (d, J = 8.8 Hz, 2ArH), 7.58 (d, J = 15.6 Hz, (E)isomeric H), 7.02−7.06 (m, 2ArH, (E)-isomeric H), 3.89 (OCH3); 13 C NMR (100 MHz, CDCl3) δ 160.3, 159.9, 156.0, 140.7, 134.9, 131.6, 130.7, 126.7, 120.4, 120.1, 112.4, 55.8; HPLC purity: 4.5 min, 97.2%; HRMS (M + H)+ (ESI+) 277.0640 [M + H]+ (calcd for C13H12N2O3SH+ 277.0641). (E)-5′-(2-((4-Methoxyphenyl)sulfonyl)vinyl)pyrimidine (8d). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.55 mmol), 2.0 M n-BuLi solution in cyclohexane (0.85 mL, 1.71 mmol), and pyrimidine-5-carboxaldehyde (0.18 mL, 1.71 mmol) gave 0.37 g (86%) of 8d as a white powder; Rf = 0.36 (n-hexane/EtOAc 1/ 2); mp: 194−195 °C; 1H NMR (400 MHz, CDCl3) δ 9.21 (s, ArH), 8.89 (s, 2ArH), 7.88 (d, J = 8.8 Hz, 2ArH), 7.58 (d, J = 15.6 Hz, (E)isomeric H), 7.04 (m, 2ArH, (E)-isomeric H), 3.89 (OCH3); 13C NMR (100 MHz, CDCl3) δ 164.1, 159.7, 155.9, 133.8, 132.3, 130.9, 130.3, 126.8, 114.9, 55.8; HPLC purity: 4.3 min, 99.3%; HRMS (M + H)+ (ESI + ) 277.0640 [M + H]+ (calcd for C13 H12N2 O3SH+ 277.0641). (E)-2-((2′-Methoxystyryl)sulfonyl)pyridine (11a). Diethyl ((pyridine-2-ylsulfonyl)methyl)phosphonate (0.30 g, 1.02 mmol), 2.0 M n-BuLi solution in cyclohexane (0.56 mL, 1.12 mmol), and 2methoxybenzaldehyde (0.15 g, 1.12 mmol) gave 0.22 g (75%) of 11a as a white powder; Rf = 0.40 (n-hexane/EtOAc 1/1); mp: 74−75 °C; 1 H NMR (400 MHz, DMSO-d6) δ 8.78 (d, J = 4.4 Hz, ArH), 7.12− 8.19 (m, 2ArH), 7.90 (d, J = 15.6 Hz, (E)-isomeric H), 7.72−7.78 (m, 2ArH), 7.54 (d, J = 15.6 Hz, (E)-isomeric H), 7.48 (m, ArH), 7.13 (d, J = 8.4 Hz, ArH), 7.01 (t, J = 7.5 Hz, ArH), 3.90 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 159.0, 158.9, 150.4, 141.1, 138.1, 132.7, 131.1, 126.9, 125.3, 121.9, 121.3, 120.8, 111.3, 55.5; HPLC purity: 4.6 min, 99.8%; HRMS (M + H)+ (ESI+) 276.0685 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-2-((2′-Fluorostyryl)sulfonyl)pyridine (11b). Diethyl ((pyridine-2-ylsulfonyl)methyl)phosphonate (0.30 g, 1.02 mmol), 2.0 M nBuLi solution in cyclohexane (0.56 mL, 1.12 mmol), and 2fluorobenzaldehyde (0.14 g, 1.12 mmol) gave 0.25 g (92%) of 11b as a white powder; Rf = 0.30 (n-hexane/EtOAc 3/2); mp: 95−96 °C; 1 H NMR (400 MHz, DMSO-d6) δ 8.80 (d, J = 4.5 Hz, ArH), 8.15− 8.21 (m, 2ArH), 7.95 (t, J = 7.4 Hz, ArH), 7.78 (d, J = 15.5 Hz, (E)isomeric H), 7.74−7.78 (m, ArH), 7.69 (d, J = 15.6 Hz, (E)-isomeric H), 7.56 (m, ArH), 7.28−7.38 (m, 2ArH); 13C NMR (100 MHz, CDCl3) δ 161.6 (d, JC−F = 254.4 Hz), 158.4, 150.4, 138.3, 138.1 (d, JC−F = 2.0 Hz), 133.0 (d, JC−F = 8.9 Hz), 130.5 (d, JC−F = 2.5 Hz), 127.5 (d, JC−F = 8.6 Hz), 127.2, 124.7 (d, JC−F = 3.6 Hz), 120.0, 120.6 (d, JC−F = 11.4 Hz), 116.4 (d, JC−F = 21.4 Hz); HPLC purity: 11.2 min, 96.8%; HRMS (M + H)+ (ESI+) 264.0486 [M + H]+ (calcd for C13H10FNO2SH+ 264.0489). (E)-2-((2′-Fluorostyryl)sulfonyl)-3-(trifluoromethyl)pyridine (11c). Diethyl (((3-(trifluoromethyl)pyridin-2-yl)sulfonyl)methyl)phosphonate (0.40 g, 1.11 mmol), 2.0 M n-BuLi solution in cyclohexane (0.61 mL, 1.22 mmol), and 2-fluorobenzaldehyde (0.15 g, 1.12 mmol) gave 0.28 g (70%) of 11c as a white powder; Rf = 0.39 (n-hexane/EtOAc 1/1); mp: 130−131 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 4.2 Hz, ArH), 8.57 (d, J = 7.0 Hz, ArH), 7.96−8.03 (m, 2ArH), 7.90 (d, J = 15.7 Hz, (E)-isomeric H), 7.74 (d, J = 15.7 Hz, (E)-isomeric H), 7.56−7.62 (m, ArH), 7.32−7.40 (m, 2ArH); 13C NMR (100 MHz, DMSO-d6) δ 161.2 (d, JC−F = 251.3 Hz), 156.8, 152.7, 138.6 (q, JC−F = 5.4, 10.9 Hz), 136.1 (d, JC−F = 4.0 Hz), 134.2 (d, JC−F = 9.1 Hz), 130.6 (d, JC−F = 1.6 Hz), 129.2 (d, JC−F

cyclohexane (0.85 mL, 1.71 mmol), and 2-fluoropyridine-5-carboxaldehyde (0.21 g, 1.71 mmol) gave 0.32 g (71%) of 6i as a white solid; Rf = 0.24 (n-hexane/EtOAc 3/1); mp: 124−125 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 1.8 Hz, ArH), 8.40 (td, J = 2.3, 8.3 Hz, ArH), 7.84 (d, J = 8.8 Hz, 2ArH), 7.71 (d, J = 15.6 Hz, (E)isomeric H), 7.67 (d, J = 15.6 Hz, (E)-isomeric H), 7.28−7.30 (m, ArH), 7.19 (d, J = 8.8 Hz, 2ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 164.5 (d, JC−F = 243.5 Hz), 163.9, 148.5 (d, JC−F = 15.6 Hz), 139.7 (d, JC−F = 8.7 Hz), 136.1, 131.4, 130.1, 130.0, 126.7 (d, JC−F = 4.8 Hz), 114.8, 110.3 (d, JC−F = 37.6 Hz), 55.8; HPLC purity: 4.9 min, 99.1%; HRMS (M + H)+ (ESI+) 294.0593 [M + H]+ (calcd for C14H12FNO3SH+ 294.0595). (E)-4′-(2-((2-Methoxyphenyl)sulfonyl)vinyl)pyridine (7a). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 4-pyridinecarboxaldehyde (0.09 g, 0.85 mmol) gave 0.23 g (99%) of 7a as a pale gray solid; Rf = 0.20 (n-hexane/EtOAc 1/3); mp: 107−108 °C; 1H NMR (400 MHz, CDCl3) δ 8.67 (d, J = 6.0 Hz, 2ArH), 8.02 (dd, J = 1.5, 7.8 Hz, ArH), 7.58−7.64 (m, 2ArH, (E)isomeric H), 7.34 (d, J = 6.0 Hz, 2ArH), 7.31 (d, J = 15.6 Hz, (E)isomeric H), 7.13 (t, J = 7.7 Hz, ArH), 7.03 (d, J = 8.3 Hz, ArH), 3.98 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 157.6, 150.9, 140.3, 140.2, 136.0, 132.0, 129.9, 127.9, 122.2, 121.0, 112.6, 56.5; HPLC purity: 4.8 min, 98.8%; HRMS (M + H)+ (ESI+) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-4′-(2-((3-Methoxyphenyl)sulfonyl)vinyl)pyridine (7b). Diethyl (((3-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 4-pyridinecarboxaldehyde (0.09 g, 0.85 mmol) gave 0.12 g (56%) of 7b as a black oil; Rf = 0.31 (n-hexane/EtOAc 1/1); 1H NMR (300 MHz, DMSO-d6) δ 8.66 (dd, J = 1.6, 4.5 Hz, ArH), 7.94 (d, J = 15.5 Hz, (E)-isomeric H), 7.58−7.71 (m, 4ArH, (E)-isomeric H), 7.49−7.53 (m, ArH), 7.41−7.43 (m, ArH), 7.30−7.34 (m, ArH), 3.86 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 160.2, 150.8, 140.9, 139.6, 139.3, 132.2, 130.7, 122.1, 120.3, 120.1, 112.4, 55.8; HPLC purity: 4.6 min, 99.0%; HRMS (M + H)+ (ESI+) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-4′-(2-((4-Methoxyphenyl)sulfonyl)vinyl)-3-fluorobenzene (7c). Diethyl (((4-methoxyphenyl)sulfonyl)methyl)phosphonate (0.25 g, 0.77 mmol), 2.0 M n-BuLi solution in cyclohexane (0.43 mL, 0.85 mmol), and 4-pyridinecarboxaldehyde (0.09 g, 0.85 mmol) gave 0.12 g (56%) of 7c as a orange solid; Rf = 0.32 (n-hexane/EtOAc 1/3); mp: 97−99 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.65 (d, J = 4.8 Hz, 2ArH), 7.86−7.90 (m, 2ArH, (E)isomeric H), 7.70 (d, J = 4.9 Hz, 2ArH), 7.60 (d, J = 15.4 Hz, (E)isomeric H), 7.20 (d, J = 8.4 Hz, 2ArH), 3.87 (s, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 163.9, 150.9, 140.2, 138.7, 133.9, 131.7, 130.3, 123.0, 115.5, 56.3; HPLC purity: 4.6 min, 98.7%; HRMS (M + H)+ (ESI+) 276.0687 [M + H]+ (calcd for C14H13NO3SH+ 276.0689). (E)-5′-(2-((2-Methoxyphenyl)sulfonyl)vinyl)pyrimidine (8a). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.70 g, 2.17 mmol), 2.0 M n-BuLi solution in cyclohexane (1.20 mL, 2.39 mmol), and pyrimidine-5-carboxaldehyde (0.26 g, 2.39 mmol) gave 0.43 g (72%) of 8a as a white powder; Rf = 0.24 (n-hexane/EtOAc 1/ 2); mp: 170−171 °C; 1H NMR (400 MHz, CDCl3) δ 9.22 (s, ArH), 8.87 (s, 2ArH), 8.03 (dd, J = 1.6, 7.8 Hz, ArH), 7.65 (d, J = 15.7 Hz, (E)-isomeric H), 7.60−7.64 (m, ArH), 7.34 (d, J = 15.7 Hz, (E)isomeric H), 7.14 (t, J = 7.8 Hz, ArH), 7.05 (d, J = 8.3 Hz, ArH), 3.99 (s, OCH3); 13C NMR (100 MHz, CDCl3) δ 159.7, 157.4, 155.9, 136.0, 135.7, 131.3, 129.8, 127.6, 127.1, 121.0, 112.5, 56.4; HPLC purity: 4.3 min, 99.3%; HRMS (M + H)+ (ESI+) 277.0642 [M + H]+ (calcd for C13H12N2O3SH+ 277.0641). (E)-4′,6′-Dichloro-5′-(2-((2-methoxyphenyl)sulfonyl)vinyl)pyrimidine (8b). Diethyl (((2-methoxyphenyl)sulfonyl)methyl)phosphonate (0.35 g, 1.03 mmol), 2.0 M n-BuLi solution in cyclohexane (0.57 mL, 1.13 mmol), and 4,6-dichloropyrimidine-5carboxaldehyde (0.20 g, 1.13 mmol) gave 0.21 g (68%) of 8b as a white powder; Rf = 0.34 (n-hexane/EtOAc 1/2); mp: 150−151 °C; 1 H NMR (400 MHz, CDCl3) δ 8.73 (s, ArH), 8.04 (dd, J = 1.6, 7.8 Hz, ArH), 7.78 (d, J = 15.8 Hz, (E)-isomeric H), 7.61−7.65 (m, 824



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(E)-4-(3-(3-((2-(3′-Fluoropyridin-2′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine (17c). Diethyl (((3-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.98 g, 2.27 mmol), 2.0 M n-BuLi solution in cyclohexane (1.25 mL, 2.50 mmol), 3-fluoropyridine-2-carboxaldehyde (0.31 g, 2.50 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.57 g (46%) of 17c as a pale yellow oil; salt is not formed because salt free 17c is oil form; Rf = 0.50 (EtOAc/MeOH 4/1); 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 4.4 Hz, ArH), 7.91 (d, J = 15.0 Hz, (E)isomeric H), 7.42−7.55 (m, 4ArH, (E)-isomeric H), 7.33−7.37 (m, ArH), 7.14 (dd, J = 2.1, 8.2 Hz, ArH), 4.08 (t, J = 6.3 Hz, CH2), 3.71−3.73 (m, 2CH2), 2.46−2.54 (m, 3CH2), 1.95−2.02 (m, CH2); 13 C NMR (100 MHz, CDCl3) δ 159.8, 158.4 (d, JC−F = 245.9 Hz), 145.9 (d, JC−F = 5.1 Hz), 141.1, 139.7 (d, JC−F = 44.8 Hz), 133.3 (d, JC−F = 4.4 Hz), 132.9, 130.5, 126.7 (d, JC−F = 4.4 Hz), 124.1 (d, JC−F = 18.9 Hz), 120.6, 120.1, 112.8, 66.9, 66.7, 55.3, 53.7, 26.2; HPLC purity: 4.3 min, 97.3%; HRMS (M + H)+ (ESI+) 407.1431 [M + H]+ (calcd for C20H23FN2O4SH+ 407.1435). (E)-4-(3-(4-((2-(3′-Fluoropyridin-2′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (17d). Diethyl (((4-(3-morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.53 mmol), 2.0 M n-BuLi solution in cyclohexane (0.84 mL, 1.68 mmol), 3-fluoropyridine-2-carboxaldehyde (0.21 g, 1.68 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.57 g (92%) of 17d as a white powder; Rf = 0.35 (EtOAc/MeOH 4/ 1); mp: 218−219 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, HCl), 8.63 (d, J = 4.3 Hz, ArH), 7.81−7.93 (m, 4ArH), 7.67 (d, J = 15.1 Hz, (E)-isomeric H), 7.61 (d, J = 15.1 Hz, (E)-isomeric H), 7.45−7.48 (m, ArH), 7.18 (d, J = 8.8 Hz, 2ArH), 4.19 (t, J = 5.9 Hz, CH2), 3.79−4.05 (m, 2CH2), 3.44 (d, J = 12.2 Hz, CH2), 3.05−3.26 (m, 2CH2), 2.20−2.26 (m, CH2); 13C NMR (100 MHz, DMSO-d6) δ 162.9, 150.7, 149.9, 139.6, 138.8, 133.2, 132.1, 130.3, 126.1, 126.0, 115.9, 66.2, 63.6, 53.7, 51.5, 23.2; HPLC purity: 4.2 min, 98.3%; HRMS (M + H) + (ESI+) 389.1526 [M + H]+ (calcd for C20H24N2O4SH+ 389.1530). (E)-4-(3-(4-((2-(3′-Fluoropyridin-2′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (17e). Diethyl (((4-(3-morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.53 mmol), 2.0 M n-BuLi solution in cyclohexane (0.84 mL, 1.68 mmol), 3-fluoropyridine-2-carboxaldehyde (0.21 g, 1.68 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.57 g (92%) of 17e as a white powder; Rf = 0.35 (EtOAc/MeOH 4/ 1); mp: 229−230 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, HCl), 8.50 (d, J = 3.8 Hz, ArH), 7.93 (d, J = 8.6 Hz, 2ArH), 7.85− 7.90 (m, ArH), 7.67 (s, (E)-isomeric 2H), 7.58−7.62 (m, ArH), 7.19 (d, J = 8.7 Hz, 2ArH), 4.20 (t, J = 5.8 Hz, CH2), 3.95−3.98 (m, CH2), 3.77−3.83 (m, CH2), 3.43−3.47 (m, CH2), 3.24 (s, CH2), 3.03−3.11 (m, CH2), 2.20−3.24 (m, CH2); 13C NMR (75 MHz, MeOD-d4) δ 164.6, 160.2 (d, JC−F = 261.9 Hz), 147.5 (d, JC−F = 4.8 Hz), 140.0 (d, JC−F = 11.7 Hz), 134.3, 134.2 (d, JC−F = 4.9 Hz), 131.9, 131.4, 129.4 (d, JC−F = 5.1 Hz), 126.6 (d, JC−F = 19.2 Hz), 116.9, 66.8, 65.2, 56.1. 53.3, 24.6; HPLC purity: 4.4 min, 98.6%; HRMS (M + H)+ (ESI+) 407.1431 [M + H]+ (calcd for C20H23FN2O4SH+ 407.1435). (E)-4-(3-(4-((2-(3′-Chloropyridin-2′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (17f). Diethyl (((4(3-morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.36 g, 0.83 mmol), 2.0 M n-BuLi solution in cyclohexane (0.46 mL, 0.91 mmol), 3-chloropyridine-2-carboxaldehyde (0.13 g, 0.91 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.35 g (98%) of 17f as a pale yellow powder; Rf = 0.31 (EtOAc/MeOH 9/ 1); mp: 210−211 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.40 (s, HCl), 8.59 (dd, J = 1.3, 4.5 Hz, ArH), 8.06 (dd, J = 1.4, 8.2 Hz, ArH), 7.93 (d, J = 8.9 Hz, 2ArH), 7.87 (d, J = 14.8 Hz, (E)-isomeric H), 7.70 (d, J = 14.8 Hz, (E)-isomeric H), 7.51−7.54 (m, ArH), 7.19 (d, J = 8.9 Hz, 2ArH), 4.20 (t, J = 6.0 Hz, CH2), 3.82−3.97 (m, 2CH2), 3.44 (d, J = 12.2 Hz, CH2), 3.21−3.26 (m, CH2), 3.02−3.11 (m, CH2), 2.21−2.28 (m, CH2); 13C NMR (75 MHz, DMSO-d6) δ 162.4, 148.5, 147.0, 138.3, 134.6, 133.8, 132.0, 131.0, 130.0, 126.9, 115.4, 65.6, 63.0, 53.1, 50.9, 22.6; HPLC purity: 4.4 min, 98.2%; HRMS (M

= 5.8 Hz), 128.4, 125.7 (d, JC−F = 3.4 Hz), 123.2 (q, JC−F = 35.2 Hz), 122.6 (q, JC−F = 272.1 Hz), 120.4 (q, JC−F = 43.6 Hz), 116.7 (d, JC−F = 21.3 Hz); HPLC purity: 16.4 min, 98.1%; HRMS (M + H)+ (ESI+) 332.0359 [M + H]+ (calcd for C14H9F4NO2SH+ 332.0363). (E)-4-((2′-Fluorostyryl)sulfonyl)pyridine (11d). Diethyl ((pyridine-4-ylsulfonyl)methyl)phosphonate (0.30 g, 0.83 mmol), 2.0 M nBuLi solution in cyclohexane (0.46 mL, 0.91 mmol), and 2fluorobenzaldehyde (0.12 g, 0.91 mmol) gave 0.23 g (71%) of 11d as a white powder; Rf = 0.35 (n-hexane/EtOAc 1/1); mp: 68−69 °C; 1 H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 6.0 Hz, 2ArH), 7.88− 7.93 (m, 3ArH), 7.80 (d, J = 15.5 Hz, (E)-isomeric H), 7.73 (d, J = 15.5 Hz, (E)-isomeric H), 7.55−7.60 (m, ArH), 7.29−7.38 (m, 2ArH); 13C NMR (100 MHz, CDCl3) δ 161.7 (d, JC−F = 254.6 Hz), 151.3, 148.7, 138.2, 133.4 (d, JC−F = 8.9 Hz), 130.7 (d, JC−F = 2.2 Hz), 128.6 (d, JC−F = 9.0 Hz), 124.8 (d, J = 3.5 Hz), 120.7, 120.1 (d, JC−F = 11.3 Hz), 116.5 (d, JC−F = 21.6 Hz); HPLC purity: 9.8 min, 97.5%; HRMS (M + H)+ (ESI+) 264.0484 [M + H]+ (calcd for C13H10FNO2SH+ 264.0489). (E)-3′-(Fluoro-2-(2-((3-trifluoromethyl)pyridin-2-yl)sulfonyl)vinyl)pyridine (12). Diethyl (((3-(trifluoromethyl)pyridin-2-yl)sulfonyl)methyl)phosphonate (0.30 g, 0.83 mmol), 2.0 M n-BuLi solution in cyclohexane (0.46 mL, 0.91 mmol), and 3fluoropyridine-2-carboxaldehyde (0.11 g, 0.91 mmol) gave 0.26 g (75%) of 12 as a white powder; Rf = 0.40 (n-hexane/EtOAc 1/1); mp: 126−127 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 4.5 Hz, ArH), 8.58−8.60 (m, 2ArH), 7.90−8.03 (m, 2ArH, (E)-isomeric H), 7.79 (d, J = 15.2 Hz, (E)-isomeric H), 7.64−7.68 (m, ArH); 13C NMR (75 MHz, DMSO-d6) δ 160.8 (d, JC−F = 261.9 Hz), 156.0, 152.3, 146.5 (d, JC−F = 4.7 Hz), 138.3, 138.2 (d, JC−F = 21.9 Hz), 134.1, 131.3 (d, JC−F = 4.1 Hz), 128.2 (d, JC−F = 4.7 Hz), 128.0, 127.5, 124.9 (d, JC−F = 18.8 Hz), 122.7 (d, JC−F = 35.2 Hz); HPLC purity: 13.3 min, 98.0%; HRMS (M + H)+ (ESI+) 333.0310 [M + H]+ (calcd for C13H8F4N2O2SH+ 333.0315). (E)-4-(3-(4-((2-(Pyridin-2′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (17a). Diethyl (((2-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (1.10 g, 2.66 mmol), 2.0 M n-BuLi solution in cyclohexane (1.46 mL, 2.93 mmol), 2-pyridinecarboxaldehyde (0.31 g, 2.93 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.91 g (87%) of 17a as a white powder; Rf = 0.29 (CH2Cl2/MeOH 19/1); mp: 227−229 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, HCl), 8.67 (d, J = 4.6 Hz, ArH), 7.89−7.98 (m, 3ArH), 7.70−7.74 (m, ArH, (E)isomeric 2H), 7.49 (t, J = 5.0 Hz, ArH), 7.28 (d, J = 8.4 Hz, ArH), 7.21 (t, J = 7.6 Hz, ArH), 4.27 (t, J = 5.8 Hz, 2ArH), 3.95−4.05 (m, 2CH2), 3.80−3.86 (m, CH2), 3.41 (d, J = 12.2 Hz, CH2), 3.28 (s, CH2), 2.23−2.27 (m, CH2); 13C NMR (100 MHz, DMSO-d6) δ 157.0, 151.5, 150.3, 141.3, 136.9, 135.4, 131.8, 129.7, 128.1, 125.3, 124.8, 120.5, 113.1, 67.2, 67.0, 55.2, 53.7, 26.2; HPLC purity: 4.3 min, 98.2%; HRMS (M + H)+ (ESI+) 389.1523 [M + H]+ (calcd for C20H24N2O4SH+ 389.1530). (E)-4-(3-(2-((2-(3′-Fluoropyridin-2′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (17b). Diethyl (((2-(3-morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.70 g, 1.60 mmol), 2.0 M n-BuLi solution in cyclohexane (0.88 mL, 1.76 mmol), 3-fluoropyridine-2-carboxaldehyde (0.22 g, 1.76 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.64 g (98%) of 17b as a white powder; Rf = 0.31 (EtOAc/MeOH 9/ 1); mp: 86−88 °C; 1H NMR (400 MHz, CDCl3) δ 12.94 (s, HCl), 8.37 (d, J = 4.0 Hz, ArH), 7.91 (d, J = 7.7 Hz, ArH), 7.75 (d, J = 15.2 Hz, (E)-isomeric H), 7.64 (d, J = 15.2 Hz, (E)-isomeric H), 7.50 (t, J = 7.4 Hz, ArH), 7.43 (t, J = 9.2 Hz, ArH), 7.30−7.34 (m, ArH), 7.05 (t, J = 7.6 Hz, ArH), 6.90 (d, J = 8.3 Hz, ArH), 4.20−4.23 (m, 2CH2), 3.90 (d, J = 12.5 Hz, CH2), 3.33−3.43 (m, 2CH2), 2.92−2.95 (m, CH2), 2.52 (s, CH2); 13C NMR (100 MHz, CDCl3) δ 158.6 (d, JC−F = 263.3 Hz), 156.7, 143.7, 138.3 (d, d, JC−F = 14.5 Hz), 136.7 (d, JC−F = 4.7 Hz), 136.5, 130.2, 129.9, 127.8 (d, JC−F = 5.1 Hz), 127.2, 121.6, 113.6, 67.2, 66.5, 63.7, 55.4, 52.3, 23.6; HPLC purity: 4.4 min, 97.5%; HRMS (M + H)+ (ESI+) 407.1430 [M + H]+ (calcd for C20H23FN2O4SH+ 407.1435). 825



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+ H)+ (ESI+) 423.1136 [M + H]+ (calcd for C20H23ClN2O4SH+ 423.1140). (E)-4-(3-(4-((2-(2′-Chloropyridine-3′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (18a). Diethyl (((4-(3-morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.43 mmol), 2-chloropyridine-3-carboxaldehyde (0.34 g, 2.43 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.71 g (76%) of 18a as a pale pink powder; Rf = 0.35 (EtOAc/ Acetone 4/1); mp: 221−223 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, HCl), 8.48 (d, J = 4.7 Hz, ArH), 8.34 (d, J = 7.8 Hz, ArH), 7.88 (d, J = 8.6 Hz, 2ArH), 7.83 (d, J = 15.2 Hz, (E)-isomeric H), 7.73 (d, J = 15.4 Hz, (E)-isomeric H), 7.48−7.54 (m, ArH), 7.21 (d, J = 8.8 Hz, 2ArH), 4.21 (t, J = 5.9 Hz, CH2), 3.97 (d, J = 11.7 Hz, CH2), 3.82 (t, J = 11.9 Hz, CH2), 3.44−3.47 (m, CH2), 3.23−3.28 (m, CH2), 3.04−2.11 (m, CH2), 2.22−2.25 (m, CH2); 13C NMR (100 MHz, DMSO) δ 163.0, 152.0, 150.7, 138.3, 135.0, 134.1, 131.7, 130.4, 127.6, 124.3, 116.0, 66.2, 64.0, 53.7, 51.5, 23.2; HPLC purity: 4.0 min, 97.8%; HRMS (M + H)+ (ESI+) 423.1136 [M + H]+ (calcd for C20H23ClN2O4SH+ 423.1140). (E)-4-(3-(4-((2-(6′-Fluoropyridine-3′-yl)vinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (18b). Diethyl (((4-(3-morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.14 mmol), 2.0 M n-BuLi solution in cyclohexane (0.63 mL, 1.25 mmol), 6-fluoropyridine-3-carboxaldehyde (0.16 g, 1.25 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.42 g (79%) of 18b as a white powder; Rf = 0.35 (EtOAc/MeOH 99/1); mp: 187−188 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, HCl), 8.61 (s, ArH), 8.38−8.42 (m, ArH), 7.85 (d, J = 8.8 Hz, 2ArH), 7.72 (d, J = 15.5 Hz, (E)-isomeric H), 7.67 (d, J = 15.6 Hz, (E)isomeric H), 7.28−7.30 (m, ArH), 7.19 (d, J = 8.8 Hz, 2ArH), 4.19 (t, J = 5.8 Hz, CH2), 3.96 (d, J = 12.3 Hz, CH2), 3.79 (t, J = 12.0 Hz, CH2), 3.2 (s, CH2), 3.03−3.11 (m, CH2), 2.21 (s, CH2), 2.22−2.25 (m, CH2); 13C NMR (75 MHz, MeOD-d4) δ 165.9 (d, JC−F = 240.9 Hz), 164.2, 149.8 (d, JC−F = 15.1 Hz), 142.0 (d, JC−F = 8.9 Hz), 137.8, 133.7, 131.7, 131.2, 128.8 (d, JC−F = 4.7 Hz), 116.4, 111.3 (d, JC−F = 37.7 Hz), 66.6, 65.1, 55.9, 53.3, 24.7; HPLC purity: 4.4 min, 97.1%; HRMS (M + H)+ (ESI+) 407.1433 [M + H]+ (calcd for C20H23FN2O4SH+ 407.1435). (E)-4-(3-(4-((2′-Chlorostyrylvinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (19a). Diethyl (((4-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.42 mmol), 2-chlorobenzaldehyde (0.34 g, 2.42 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.71 g (72%) of 19a as a white powder; Rf = 0.38 (EtOAc/MeOH 19/1); mp: 242−243 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.30 (s, HCl), 7.82−7.92 (m, 3ArH, (E)-isomeric H), 7.72 (d, J = 15.4 Hz, (E)isomeric H), 7.58 (dd, J = 1.2, 8.0 Hz, ArH), 7.48 (td, J =1.6, 7.4 Hz, ArH), 7.18−7.42 (m, 2ArH), 4.20 (t, J = 6.0 Hz, CH2), 3.97 (d, J = 10.7 Hz, CH2), 3.80−3.97 (m, CH2), 3.45 (d, J = 12.1 Hz, CH2), 3.22−3.25 (m, CH2), 3.03−3.10 (m, CH2), 2.22−2.26 (m, CH2); 13C NMR (75 MHz, DMSO-d6) δ 162.3, 135.4, 134.0, 132.5, 131.7, 130.6, 130.1, 130.0, 129.7, 128.8, 127.8, 115.4, 65.7, 63.1, 53.2, 51.0, 22.7; HPLC purity: 4.5 min, 97.8%; HRMS (M + H)+ (ESI+) 422.1181 [M + H]+ (calcd for C21H24ClNO4SH+ 422.1187). (E)-4-(3-(4-((3′-Chlorostyrylvinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (19b). Diethyl (((4-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.42 mmol), 3-chlorobenzaldehyde (0.34 g, 2.42 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.91 g (89%) of 19b as a white powder; Rf = 0.35 (EtOAc/MeOH 19/1); mp: 225−227 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, HCl), 7.84−7.89 (m, 2ArH, (E)-isomeric H), 7.68−7.73 (m, 2ArH), 7.58 (d, J = 15.4 Hz, (E)-isomeric H), 7.43−7.52 (m, 2ArH), 7.19 (d, J = 8.8 Hz, 2ArH), 4.20 (t, J = 5.8 Hz, CH2), 3.95 (d, J = 11.7 Hz, CH2), 3.81 (t, J = 11.8 Hz, CH2), 3.45 (d, J = 11.9 Hz, CH2), 3.24 (s, CH2), 3.03−3.11 (m, CH2), 2.21−2.25 (m, CH2); 13C NMR (75 MHz, DMSO-d6) δ 162.2, 139.2, 134.7, 133.8, 132.1, 130.7, 130.5, 130.4,

129.6, 128.2, 127.7, 115.3, 65.7, 63.1, 53.2, 51.0, 22.7; HPLC purity: 4.3 min, 98.9%; HRMS (M + H)+ (ESI+) 422.1180 [M + H]+ (calcd for C21H24ClNO4SH+ 422.1187). (E)-4-(3-(4-((4′-Chlorostyrylvinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (19c). Diethyl (((4-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.42 mmol), 4-chlorobenzaldehyde (0.34 g, 2.42 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.94 g (90%) of 19c as a white powder; Rf = 0.33 (EtOAc/MeOH 19/1); mp: 241−242 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, HCl), 7.85 (d, J = 8.8 Hz, 2ArH), 7.77 (d, J = 8.5 Hz, 2ArH), 7.63 (d, J = 15.4 Hz, (E)-isomeric H), 7.58 (d, J = 15.6 Hz, (E)-isomeric H), 7.50 (d, J = 8.5 Hz, 2ArH), 7.18 (d, J = 8.8 Hz, 2ArH), 4.18 (t, J = 5.9 Hz, CH2), 3.95 (d, J = 11.6 Hz, CH2), 3.81 (t, J = 11.8 Hz, CH2), 3.44 (d, J = 11.3 Hz, CH2), 3.23 (s, CH2), 3.06−3.08 (m, CH2), 2.22 (s, CH2); 13C NMR (75 MHz, DMSO-d6) δ 162.2, 139.5, 135.5, 132.2, 131.4, 130.6, 129.5, 129.0, 115.3, 65.7, 63.1, 53.2, 51.0, 22.7; one signal was not detected and is believed to overlap with a nearby peak; HPLC purity: 4.2 min, 99.7%; HRMS (M + H)+ (ESI+) 422.1183 [M + H]+ (calcd for C21H24ClNO4SH+ 422.1187). (E)-4-(3-(4-((2′-Fluorostyrylvinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (19d). Diethyl (((4-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.42 mmol), 2-fluorobenzaldehyde (0.34 g, 2.42 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.81 g (82%) of 19d as a white powder; Rf = 0.38 (EtOAc/MeOH 19/1); mp: 247−248 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, HCl), 7.83−7.89 (m, 3ArH), 7.64 (d, J = 15.6 Hz, (E)-isomeric H), 7.59 (d, J = 15.6 Hz, (E)-isomeric H), 7.49−7.55 (m, ArH), 7.17−7.35 (m, 2ArH), 7.18 (d, J = 8.9 Hz, 2ArH), 4.19 (t, J = 5.9 Hz, CH2), 3.97 (d, J = 12.2 Hz, CH2), 3.74 (t, J = 12.1 Hz, CH2), 3.46 (d, J = 11.6 Hz, CH2), 3.23−3.28 (m, CH2), 3.04−3.12 (m, CH2), 2.20 (s, CH2); 13C NMR (75 MHz, DMSO-d6) δ 162.1, 160.4 (d, JC−F = 250.1 Hz), 133.3 (d, JC−F = 8.9 Hz), 132.5 (d, JC−F = 4.4 Hz), 131.8, 131.1 (d, JC−F = 4.6 Hz), 129.6 (d, JC−F = 7.0 Hz), 125.1, 120.1 (d, JC−F = 11.2 Hz), 116.2 (d, JC−F = 21.4 Hz), 115.4, 65.6, 63.2, 53.2, 51.0, 22.8; one signal was not detected and is believed to overlap with a nearby peak; HPLC purity: 4.7 min, 98.4%; HRMS (M + H)+ (ESI+) 406.1479 [M + H]+ (calcd for C21H24FNO4SH+ 406.1483). (E)-4-(3-(4-((3′-Fluorostyrylvinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (19e). Diethyl (((4-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.42 mmol), 3-fluorobenzaldehyde (0.34 g, 2.42 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.89 g (89%) of 19e as a white powder; Rf = 0.35 (EtOAc/MeOH 19/1); mp: 220−221 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.77 (s, HCl), 7.85 (d, J = 8.9 Hz, 2ArH), 7.57−7.78 (m, 2ArH, (E)-isomeric 2H), 7.31− 7.50 (m, ArH), 7.27−7.31 (m, ArH), 7.18 (d, J = 8.9 Hz, 2ArH), 4.19 (t, J = 5.8 Hz, CH2), 3.96 (d, J = 12.1 Hz, CH2), 3.77 (t, J = 11.8 Hz, CH2), 3.45 (d, J = 11.9 Hz, CH2), 3.25 (s, CH2), 3.06−3.09 (m, CH2), 2.20 (s, CH2); 13C NMR (75 MHz, MeOD-d6) δ 162.3 (d, JC−F = 242.4 Hz), 162.1, 139.4 (d, JC−F = 2.6 Hz), 134.9 (d, JC−F = 8.1 Hz), 133.2, 132.1, 131.0 (d, JC−F = 8.4 Hz), 130.7, 130.2, 129.5, 125.3 (d, JC−F = 2.6 Hz), 117.6 (d, JC−F = 21.2 Hz), 115.3, 114.9 (d, JC−F = 22.1 Hz), 65.6, 63.1, 53.1, 51.0, 22.7; HPLC purity: 4.7 min, 96.6%; HRMS (M + H) + (ESI+) 406.1479 [M + H]+ (calcd for C21H24FNO4SH+ 406.1483). (E)-4-(3-(4-((4′-Fluorostyrylvinyl)sulfonyl)phenoxy)propyl)morpholine Hydrochloride (19f). Diethyl (((4-(3morpholinopropoxy)phenyl)sulfonyl)methyl)phosphonate (0.96 g, 2.20 mmol), 2.0 M n-BuLi solution in cyclohexane (1.21 mL, 2.42 mmol), 4-fluorobenzaldehyde (0.34 g, 2.42 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.79 g (78%) of 19f as a white powder; Rf = 0.33 (EtOAc/MeOH 19/1); mp: 221− 223 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, HCl), 7.76−7.87 (m, 4ArH), 7.60 (d, J = 15.4 Hz, (E)-isomeric H), 7.53 (d, J = 15.4 Hz, (E)-isomeric H), 7.25−7.30 (m, 2ArH), 7.16−7.20 (m, 2ArH), 826



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4.18 (t, J = 6.0 Hz, CH2), 3.96 (d, J = 12.7 Hz, CH2), 3.78−3.82 (m, CH2), 3.45 (d, J = 12.4 Hz, CH2), 3.24 (s, CH2), 3.06−3.08 (m, CH2), 2.19−2.23 (m, CH2); 13C NMR (75 MHz, MeOD-d6) δ 163.8 (d, JC−F = 249.5 Hz), 162.1, 139.7, 132.4, 131.3 (d, JC−F = 8.8 Hz), 129.4, 129.1, 128.5, 116.0 (d, JC−F = 21.9 Hz), 115.3, 115.2, 65.6, 63.2, 53.2, 51.0, 22.8; one signal was not detected and is believed to overlap with a nearby peak; HPLC purity: 4.3 min, 96.4%; HRMS (M + H)+ (ESI+) 406.1480 [M + H]+ (calcd for C21H24FNO4SH+ 406.1483). (E)-2-(4-((2-(3′-Fluoropyridin-2′-yl)vinyl)sulfonyl)phenoxy)1-(4-methylpiperazin-1-yl)ethan-1-one Hydrochloride (23a). Diethyl (((4-(2-(4-methylpiperazine-1-yl)2-oxoethoxy)phenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.11 mmol), 2.0 M n-BuLi solution in cyclohexane (0.61 mL, 1.22 mmol), 3-fluoropyridine-2carboxaldehyde (0.15 g, 1.22 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.51 g (91%) of 23a as a white powder; Rf = 0.35 (EtOAc/acetone 4/1); mp: 153−154 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.72 (s, HCl), 8.49 (d, J = 3.7 Hz, ArH), 7.84−7.90 (m, 3ArH), 7.74 (s, (E)-isomeric 2H), 7.57−7.60 (m, ArH), 7.19 (d, J = 8.6 Hz, 2ArH), 5.04−5.17 (m, CH2), 4.36 (d, J = 13.1 Hz, CH), 4.00 (d, J = 13.4 Hz, CH), 3.60−3.63 (m, CH), 3.42 (s, CH2), 3.13−3.16 (m, CH2), 2.97 (s, CH), 2.76 (s, CH3); 13C NMR (100 MHz, DMSO-d6) δ 165.9, 162.8, 158.5 (d, JC−F = 261.4 Hz), 146.8 (d, JC−F = 4.8 Hz), 139.1 (d, JC−F = 11.0 Hz), 134.0 (d, JC−F = 4.0 Hz), 132.1, 131.7, 130.3, 128.2 (d, JC−F = 4.5 Hz), 125.2 (d, JC−F = 18.8 Hz), 116.2, 66.8, 66.1, 52.3 (d, JC−F = 15.6 Hz), 42.4, 41.4, 38.6; HPLC purity: 4.5 min, 98.7%; HRMS (M + H)+ (ESI+) 420.1384 [M + H]+ (calcd for C20H22FN3O4SH+ 420.1388). (E)-2-(4-((2-(3′-Chloropyridin-2′-yl)vinyl)sulfonyl)phenoxy)1-(4-methylpiperazin-1-yl)ethan-1-one Hydrochloride (23b). Diethyl (((4-(2-(4-methylpiperazine-1-yl)2-oxoethoxy)phenyl)sulfonyl)methyl)phosphonate (0.50 g, 1.11 mmol), 2.0 M n-BuLi solution in cyclohexane (0.61 mL, 1.22 mmol), 3-chloropyridine-2carboxaldehyde (0.17 g, 1.22 mmol), and 4.0 M hydrogen chloride solution in dioxane (2.0 equiv) gave 0.45 g (85%) of 23b as a white powder; Rf = 0.38 (EtOAc/acetone 4/1); mp: 148−149 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, HCl), 8.59 (d, J = 4.0 Hz, ArH), 8.06 (d, J = 8.1 Hz, ArH), 7.86−7.91 (m, 2ArH, (E)-isomeric H), 7.72 (d, J = 14.8 Hz, (E)-isomeric H), 7.51−7.54 (m, ArH), 7.18 (d, J = 8.8 Hz, 2ArH), 5.03−5.15 (m, CH2), 4.37 (d, J = 13.4 Hz, CH), 4.40 (d, J = 13.6 Hz, CH), 3.43−3.57 (m, CH, CH2), 3.09−3.12 (m, CH2), 2.96 (s, CH), 2.77 (s, CH3); 13C NMR (100 MHz, DMSO-d6) δ 165.9, 162.9, 149.1, 147.6, 138.9, 135.2, 134.4, 132.5, 131.6, 130.3, 127.4, 116.2, 99.1, 66.0, 52.5, 42.5; two signals were not detected and believed to overlap with a nearby solvent peak; HPLC purity: 4.5 min, 98.3%; HRMS (M + H)+ (ESI+) 436.1087 [M + H]+ (calcd for C20H22ClN3O4SH+ 436.1092). Cell Culture. Engineered U2OS Keap1−Nrf2 nuclear translocation cell lines (93-0821C3, DiscoveRX, Fremont, CA) were grown with an AssayComplete cell culture kit (92-3103G, DiscoveRx) at 37 °C in a humidified atmosphere containing 5% CO2. BV2 cells were grown in RPMI-1640 (Gibco, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (Atlas Biologicals, Fort Collins, CO), 100 U/mL penicillin, 10 μg/mL streptomycin, and 2 mM L-glutamine (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. Keap1−Nrf2 Functional Assay. The Keap1−Nrf2 functional assay was performed using a PathHunter U2OS Keap1−Nrf2 nuclear translocation cell line (93-0821C3, DiscoveRx) according to the manufacturer’s instructions. Briefly, this Nrf2 activation system was designed to measure a nontranscriptional response of activationdependent Nrf2 translocation to the nucleus using a principal enzyme fragment complementation (EFC) technology. This EFC technology utilized a genetically engineered β-galactosidase enzyme consisting of two fragments: (a) an Nrf2-tagged enzyme donor and (b) an enzyme acceptor, which is localized to the nucleus. The activation of the Keap1−Nrf2 protein complex resulted in the formation of the functional β-galactosidase in the nucleus, the activity of which can be quantitatively detected using a chemiluminescent substrate. The engineered U2OS cells (1.5 × 104 cells/well) were seeded on a white 96-well plate and incubated overnight at 37 °C in a humidified

atmosphere with 5% CO2. The cells were then incubated with test compounds in the dark at room temperature for 6 h. Subsequently, the cells were incubated for 1 h in the same environment with working detection reagent solution. Then, luminescence was detected using a SpectraMax i3 microplate reader (Molecular Devices, Sunnyvale, CA). Cell Viability Assay. Cell viability was assessed using an Ez-Cytox cell viability assay kit (DoGenBio, Cheongju, South Korea) according to the manufacturer’s protocol. This assay is based on a water-soluble tetrazolium salt method. BV2 cells (1.0 × 104 cells/well) were seeded on a 96-well plate in 100 μL of medium and incubated overnight at 37 °C in a humidified atmosphere with 5% CO2. The cells were then treated with various concentrations of the synthesized compounds for 24 h at 37 °C. Then, 10 μL of Ez-Cytox reagent was added to the wells and incubated for 1 h at 37 °C. Cell viability was then determined by measuring the absorbance at 450 nm using a SpectraMax i3 microplate reader. CYP Inhibition Assay. CYP inhibition was measured using a P450-Glo screening system (Promega Corp.) according to the manufacturer’s protocols (Promega Technical Bulletin, P450-Glo Assays, 2009). Briefly, the CYP enzyme and substrate were mixed in KH2PO4 buffer (100 mM, pH 7.4) with or without test compounds, and the reaction was initiated by addition of the NADPH regeneration system (containing NADP+, MgCl2, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase). After incubation for 10−30 min at 37 °C (different incubation times depending on the CYP isotype), the reconstituted luciferin detection reagent was added to stop the reaction and generate the luminescent signal. After incubation for 20 min to stabilize the luminescence, signals were detected using a SpectraMax i3 microplate reader; inhibition of each CYP isotype by test compounds was expressed as the percentage of activity versus control. Microsomal Stability. Human liver microsomes (0.5 mg/mL) were preincubated with compounds (1 μM) in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 5 min before adding the NADPH regeneration buffer (containing NADP+, MgCl2, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase). The incubation was started by treating the NADPH regeneration buffer and terminated with chlorpropamide in acetonitrile after 30 min at 37 °C. Precipitated proteins were removed by centrifugation for 5 min at 14 000g at 4 °C. The supernatant was injected into a liquid chromatography-MS/MS system and analyzed using a Shimadzu Nexera XR system and TSQ vantage (Thermo). The HPLC column was a Luna C18 column (2.0 × 50 mm2, 3 μm particle size; Phenomenex), and the mobile phase was distilled water (A) containing 0.1% formic acid and acetonitrile (B) containing 0.1% formic acid. Data analysis was performed using Xcalibur (version 1.6.1). Percentage of remaining compound was calculated by comparing the peak area. RT-qPCR. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) and converted to cDNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). RT-qPCR was performed using 50 ng of cDNA and the iQ-SYBR Green Supermix (Bio-Rad). Nrf2-dependent antioxidant genes were amplified using the following primers: GCLC (forward: 5′-TGTGGTATTCGTGGTACTGCTC3′, reverse: 5′-GGGCCACTTTCATGTTCTCG-3′); GCLM (forward: 5′-GGAGCTTCGGGACTGTATCC-3′, reverse: 5′TCGGGATTTATCTTC TCCACTGC-3′); HO-1 (forward: 5′CAGCCACACAGCACTATG-3′, reverse: 5′-GCAATC TTCTTCAGGACCT-3′); hypoxanthine-guanine phosphoribosyltransferase (forward: 5′-CAGGAGAGAAAGATGTGATTGA TA-3′, reverse: 5′-GCCAACACTGCTGAAACA-3′); hypoxanthine-guanine phosphoribosyltransferase was used as an internal control. All RTqPCR reactions were performed on a Bio-Rad CFX Connect RealTime PCR Detection system. The 2ΔΔCt method was used to calculate fold changes in gene expression. Western Blot. Nuclear Lysate Extraction. Nuclear lysates were extracted by a previously described method.14 BV2 cells (3 × 106 cells/plate) were seeded onto a culture dish and treated with various concentrations of 17e for 6 h. Then, the cells were harvested in icecold phosphate-buffered saline (PBS) and resuspended in 400 μL of 827



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cytoplasmic extract buffer containing 10 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES, pH 7.9), 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The resuspended cells were incubated at 4 °C for 20 min. Then, 28 μL of 10% NP40 was added and the cells were vortexed vigorously for 15 s. After centrifuging for 2 min at 17 800g and 4 °C, the cytoplasmic supernatant was transferred to a new tube. This process was repeated to wash the nuclear pellet; the supernatant was discarded. The nuclear pellet was subsequently resuspended in 100 μL of nuclear extract buffer containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride and incubated at 4 °C for 20 min. During this incubation, the pellet was vortexed for 10 s in 5 min intervals. The lysates were then centrifuged for 10 min at 17 800g and 4 °C, and the nuclear supernatant was transferred to a new tube. The lysates were stored at −80 °C until used for analysis. Whole Cell Lysate Extraction. To extract the whole cell lysate, the cells (5 × 105 cells/well) were seeded onto a six-well plate and treated with various concentrations of 17e for 24 h. Then, the cells were harvested in radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO) containing a protease inhibitor cocktail and incubated at 4 °C for 30 min. The lysates were then centrifuged for 20 min at 15 814g and 4 °C before being transferred to a new tube. The lysates were stored at −80 °C until used for analysis. Lysate concentrations were determined by bicinchoninic acid method, and equal amounts of protein (10−20 μg) diluted in 5× sample buffer were separated on a 10% sodium dodecyl sulfatepolyacrylamide gel, followed by transfer onto a poly(vinylidene difluoride) membrane (Millipore, Billerica, MA). Membranes were incubated with blocking solution containing 4% nonfat dried milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The membranes were then rinsed three times with TBST and immunoblotted overnight at 4 °C with primary antibodies specific for Nrf2 (1:500; Cell Signaling Technology, Inc., Danvers, MA), GCLC (1:1000; Novus Biologicals, Littleton, CO), GCLM (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (1:2000; Santa Cruz Biotechnology), HO-1 (1:1000; Enzo Life Science, Ann Arbor, MI), and lamin B1 (1:4000; Bioworld Technology, St. Louis Park, MN). Then, membranes were rinsed three times with TBS-T and treated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:10000; GeneTex, Irvine, CA) for 1 h at room temperature. Subsequently, the membranes were rinsed three times with TBS-T and treated with the D-Plus enhanced chemiluminescence Pico system (Dongin Biotech, Seoul, South Korea) for 3 min. Immunoblotted proteins were detected with an Amersham Imager 600 (GE Healthcare, Arlington Heights, IL). Densitometric analyses were performed using ImageJ software (National Institutes of Health), and the data were normalized to lamin B1 or β-actin as loading controls. PD Animal Model and Treatment. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (Publication No. 8023, revised 1978) and the guidelines of the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology (Seoul, South Korea). Male 10 week old C57BL/6 mice (weight, 23− 26 g) were purchased from Daehan Biolink (Eumseong, South Korea). Food and water were given ad libitum, and mice were housed in the animal facility of the Korea Institute of Science and Technology on a standard 12 h light/12 h dark cycle. Vehicle for oral administration was used as a solution containing 10% N-methyl-2pyrrolidone and 20% Tween-80 in distilled water. Vehicle or 17e (20 mg/(kg day)) dissolved in the same vehicle solution was orally administered for 3 days; on the second day, MPTP-HCl (20 mg/kg) or saline was intraperitoneally injected four times at 2 h intervals to create the MPTP-induced PD mouse models (n = 10 per group). Six days after MPTP injection, the mice were sacrificed for further study. Behavioral Tests. Vertical Grid Test. All of the mice were handled daily and trained on the vertical grid apparatus for 3 days before oral

drug administration. The mice were allowed to move freely on the apparatus for acclimation and trained to turn around and climb down within 15 s when placed on the top position of the apparatus with their head facing up. After 6 days of MPTP injection, vertical grid testing was conducted to evaluate motor function. The test was conducted twice and videotaped to measure the time to turn, total time, and time to climb down after turn. Coat-Hanger Test. The mice were placed on the center of the coathanger and allowed to climb into the safety zone for 3 min. The position of the mouse on the hanger was scored: 0, falling off the hanger within 20 s; 1, hanging on and moving from the starting position of the hanger; 2, reaching the side of the hanger; 3, climbing the top of one side of the hanger; 4, moving toward the top of the hanger; and 5, reaching the safety zone by climbing the hook of the hanger. The mice did not receive a score if they immediately fell off the hanger. The test was conducted twice. Immunohistochemistry. After anesthetized, the mice were transcardially perfused using cold saline and 4% paraformaldehyde. The brains were removed and subsequently postfixed in paraformaldehyde at 4 °C overnight, followed by transfer to 30% sucrose solution for 48 h for cryoprotection. Then, the brains were mounted onto optimum cutting temperature embedding solution. The striatum and SNpc were serially cut in 35 or 30 μm sections in an HM525 NX Cryostat (Thermo Shandon Ltd., Cambridge, U.K.) and then stored in a cryoprotectant containing 30% glycerol, 30% ethylene glycol, and 10% 0.2 M phosphate buffer at 4 °C. Immunostaining was performed by the free-floating method. The brain sections were rinsed in PBS and then incubated in 3% H2O2 for 15 min at room temperature. Subsequently, the sections were treated with serum-free proteinblocking buffer (Agilent, Santa Clara, CA) for 15 min, followed by incubation with a primary anti-TH antibody (1:1000; Pel-Freez Biologicals, Rogers, AZ) overnight at 4 °C. On the next day, the sections were washed three times with PBS and treated with a Polink2 Plus Horseradish Peroxidase Broad secondary antibody for mouse and rabbit (GBI Labs, Mukilteo, WA) in accordance with the manufacturer’s instructions. Immunoreactivity was visualized by application of 3,3′-diaminobenzidine. The striatum and SNpc were imaged using an Olympus IX51 microscope (Olympus, Tokyo, Japan) to measure the optical density and count the number of TH-positive cells. Double Immunofluorescent Staining. SNpc sections were rinsed in PBS with 0.3% Triton X-100 (PBS-T) and incubated in a blocking solution (1% bovine serum albumin in PBS-T) for 2 h. Then, the sections were cotreated at 4 °C (16 h) with Iba-1 (1:800; Wako, Osaka, Japan) and TH (1:800; Millipore, Burlington, MA) primary antibodies. Then, the sections were washed three times with PBS-T before coincubating with antirabbit Alexa Fluor 594 IgG (1:1000) and antimouse Alexa Fluor 488 IgG (1:1000) for 2 h at room temperature. Finally, all sections were washed three times with PBS-T and mounted on slides using fluorescent mounting medium (Agilent). Fluorescent images were obtained with a CELENAS Digital Imaging System microscope (Logos Biosystems, Anyang, South Korea). Statistical Analysis. All experimental results are presented as the mean ± standard error of the mean. Statistical analyses were computed by GraphPad Prism 7.0 software (San Diego, CA). Differences between groups were measured using an unpaired twotailed Student’s t-test or one-way analysis of variance, followed by Tukey’s or Dunnett’s multiple comparison test. P < 0.05 was considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01527. Additional ADME/Tox results of compound 17e; result graph of hERG fluorescence polarization assay for 17e (Figure S1); result graph of Ames test for 17e (Figure 828



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S2); the ability to induce HO-1 by ELISA assay for compound 5f, 5n, 6b, 7a, 7b, 17d, 17e, 18b, 19a, 19b, 19c, 19d, 19e, and 19f (Table S1); plasma stability of 17e (Table S2); PAMPA-BBB data for 17e (Table S3); synthetic methods and data for the intermediates 2, 3, 4, 9, 10, 13, 14, 15, 16, 20, 21, 22, supporting methods, and 1H and 13C NMR spectra of the final compounds 5, 6, 7, 8, 11, 12, 17, 18, 19, 23 (PDF)

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Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +82-2-9585132. Fax: +822-9585189. ORCID

Jaeick Lee: 0000-0001-7548-0297 Ki Duk Park: 0000-0002-7753-214X Author Contributions ∇

J.W.C and S.K. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a National Research Council of Science & Technology grant by the South Korean government (MSIP, No. CRC-15-04-KIST), the National Research Foundation of Korea (NRF-2018M3A9C8016849), and Main Research Program (E0164503-01) of the Korea Food Research Institute.

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ABBREVIATIONS ADME/Tox, absorption, distribution, metabolism, and excretion/toxicity; CNS, central nervous system; CYP, cytochrome P; DAergic, dopaminergic; EC50, half-maximal effective concentration; GCL, glutamate−cysteine ligase; GCLC, glutamate−cysteine ligase catalytic subunit; GCLM, glutamate−cysteine ligase modifier subunit; HO-1, heme oxygenase 1; hERG, human ether-a-go-go-related gene; HPLC, highperformance liquid chromatogtraphy; Iba-1, ionized calciumbinding adapter molecule 1; IC50, half-maximal inhibitory concentration; Keap1, Kelch-like ECH-associated protein 1; LDOPA, L-3,4-dihydroxyphenylalanine; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; MS, mass spectrometry; Nrf2, nuclear factor (erythroid-derived 2)-like 2; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered salineTriton X-100; PD, Parkinson’s disease; Pe, permeability (cm/ s); RT-qPCR, real-time quantitative polymerase chain reaction; SNpc, substantia nigra pars compacta; TBS-T, Trisbuffered saline-Tween-20; TH, tyrosine hydroxylase

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REFERENCES

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