Lead Optimization of Androgen Receptor-HSP27 Disrupting Agents in Glioblastoma

Abstract

Glioblastoma (GBM) is the most common malignant brain tumor with poor prognosis under the current standard treatment. It is critical to develop new approaches to selectively battle the disease. GBM sex differences suggest that an androgen receptor (AR) is a potential therapeutic target to treat AR-overexpressed GBM. Heat shock 27 kDa protein (HSP27) is a well-documented chaperone protein that stabilizes AR. Inhibition of HSP27 leads to AR degradation, indicating that HSP27 inhibitors could suppress AR activity in GBM. We have identified a lead HSP27 inhibitor that could induce AR degradation. Lead optimization resulted with two new derivatives (compounds 4 and 26) showing potent anti-GBM activity and improved drug distribution in comparison to the lead compound. Compounds 4 and 6 exhibit IC₅₀s of 35 and 23 nM, respectively, to inhibit cell proliferation and also show significant activity to decrease the tumor growth in vivo.

Graphical Abstract

1. INTRODUCTION

Glioblastoma (GBM) is the most common primary malignant brain tumor, with about 12,000 cases diagnosed in the United States annually.^1,2 The prognosis of GBM remains poor, despite the advancement of new surgical techniques and chemotherapy.³ The median length of survival after a diagnosis is about 15–18 months.⁴ The current protocol for treatment in GBM patients includes standard surgery, accurately measured gamma knife radiotherapy, or temozolomide (TMZ, an oral alkylating chemotherapy agent).⁵ However, tumor recurrence happens within a couple months after these standard treatments. Therefore, new molecular targets are increasingly essential to design and develop more effective small molecules for GBM treatment.

Epidemiological studies have demonstrated that the incidence of GBM in men is 1.6-fold higher than that in women, indicating that there is a sex difference in GBM.^6–8 Also, researchers have shown that androgen plays an important role in the development of GBM.⁹ Androgens contribute to the development of a male phenotype via activation of an androgen receptor (AR), which is also responsible for prostate cancer progression.¹⁰ Recently, a few studies demonstrated that AR is overexpressed in GBM and androgens enhance the GBM growth.^11–13 Thus, AR has the potential as a new molecular target for GBM treatment.^14,15 Androgens promote AR dimerization and nuclear translocation to promote gene expression via an androgen-response element (ARE). It was shown that, in AR-overexpressed GBM cell lines, testosterone could increase cell proliferation, migration, and invasiveness.¹⁶ Seviteronel, a nonsteroidal antiandrogenic agent, has been investigated in a clinical trial for AR-overexpressed GBM patients.¹⁷ Additionally, AR antagonists enzalutamide and bicalutamide inhibit the proliferation of GBM cells in vitro and in vivo.^12,15 However, mutated AR (AR-V7) that lacks the ligand binding domain has been identified in about 30% of the GBM patient specimens in a study.¹² The high ratio of AR mutation in GBM suggests that AR antagonists in GBM treatment might be with limited efficiency. Therefore, developing alternative drug candidates to suppress AR activity in GBM could be a more promising approach.

Heat shock proteins including heat shock protein 27 kDa (HSP27) play important roles in the folding, degradation, activation, and translocation of their client proteins.¹⁸ Overexpression of HSP27 enhances the stability of its client proteins and prevents cell apoptosis from a variety of stressors.¹⁹ It is well known that AR is one of the client proteins of HSP27, which escorts AR translocation into the nucleus to trigger ARE gene regulation.^19,20 HSP27 is responsible for both AR stability and translocation, which makes it an ideal target to disrupt the AR signaling pathway. Currently, the available approach to target HSP27 is antisense oligonucleotides (ASO), or short interfering RNA (siRNA), which have demonstrated acceptable efficacy to reduce HSP27 expression in in vitro and in vivo in prostate cancer.^21,22 However, ASO and siRNA are not applicable to GBM because of the poor blood brain barrier (BBB) permeability. Thus, small-molecule HSP27 inhibitors could be a promising strategy to induce AR degradation in GBM, due to their ability to pass BBB.

Previously, we identified compound I that could abolish AR via inhibiting HSP27 (Figure 1) in GBM cells. It selectively inhibits the proliferation of AR-overexpressed GBM cells in vitro and in vivo.¹¹ Also, it reduces the AR expression in the homogenized tumor samples in the harvested xenograft treated with compound I.¹¹ The results demonstrate that compound I is a potential new drug candidate to treat AR-overexpressed GBM. However, the pharmacokinetic study shows that the area under the concentration–time curve (AUC) of compound I in brain tissue is only about 2% of the AUC in plasma, and the half-life is only about 1.5 h.²³ The result indicates that compound I could cross the BBB, but with a limited permeability. In this study, we set out to lead optimize compound I to generate more desirable drug candidates. Forty two new derivatives are synthesized based on compound I as the lead. Two compounds are identified to have better in vivo activity than compound I and also exhibit longer half-life and slightly improved permeability to BBB.

Figure 1.

New derivative design.

2. RESULTS AND DISCUSSION

2.1. Lead Optimization and Summary of the Structure–Activity Relationship (SAR).

Our previous study identified compound I (Figure 1, N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido) phenyl)-4-methoxybenzamide), with a high safety profile and AR targeting efficacy, showing potent anti-glioblastoma activity in vitro and in vivo.¹¹ In the current study, our goal is to optimize compound I to improve the pharmacokinetic profile, aim to improve BBB penetration and the half-life to a comparable level as temozolomide. A total of 42 new derivatives were synthesized by modifying the circled moiety in Figure 1. We have explored different structures at the R₁ domain, including aromatic rings with electron withdraw or donating substitutes, and aliphatic groups. The synthesis is illustrated in Scheme 1.

Scheme 1. Synthesis of Compounds 1–42^a.

^a Reagents and conditions: (a) 2,5-dimethoxybenzyl chloride, DMF, K₂CO₃; (b) methyl sulfonyl chloride, NaH, DMF; (c) NH₄Cl, Zn, H₂O, THF; (d) R₁COCl, K₂CO₃, DMF; (e) R₂C₂O₂Cl₂, DMF, K₂CO₃; (f) NH₄Cl, Zn, H₂O, THF; (g) R₃C₂O₂Cl₂, DMF, K₂CO₃.

All of the derivatives were examined for growth inhibition of AR-overexpressed GBM cell lines U87 and T98G.¹¹ The compounds were also evaluated for their AR downregulation effect in these GBM cells. Compounds showing the best potency in the library both in cell proliferation inhibition and AR down regulation will be proceeded into in vivo study. This criteria of selection is based on the compound potency and selectivity to AR-overexpressed GBM cells. The results are summarized in Table 1 and Figure 2. More specifically in SAR, electron withdraw groups on the benzamide moiety reduce the activity, indicated by a nitro substitute as compounds 3, 13, 16, and 23 showing IC₅₀s above 1 μM. Similarly, trifluoromethyl and fluoro substitutes such as compounds 1, 7, 18, 24, and 33 exhibit the same trend. Cyano group-substituted compounds 2 and 14 all have IC₅₀s above 1 μM. Heterocyclic and alkyl moieties lead to lower activity too, indicated by compounds 30, 35, and 36. The dimers 37, 38, 39, 40, and 42 show poor activity. On the other hand, electron donating groups in the benzamide moiety contribute to the activity. Compounds 4, 5, 6, 8, 11, 12, 17, 19, 25, 26, and 29 have the IC₅₀s lower than 1 μM in both GBM cell lines. Meanwhile, compounds 4, 13, 15, 19, 26, 33, 34, 35, 36, and 37 show significant AR downregulation activity at 100 nM in T98G cells (Figure 2). To further evaluate the AR regulation effect of the compound, they are retested at 50 nM, and the results are exhibited in Figure 2F. Compounds 4 and 26 have potent AR downregulation activity at 50 nM in T98G cells, similar to compound I. Also, the IC₅₀s of compounds 4, 8, 19, and 26 are lower than 0.1 μM in the GBM cells. Based on the cell growth inhibition potency and activity to abolish AR in the Western blotting assay, compounds 4 and 26 are the most potent. Both compounds have a dual ring structural moiety, which is speculated to be the key structure for the biological activity.

Table 1.

Growth Inhibitory Effects of the New Compounds in GBM Cells

No.	Compound Structure	U87 (1C50 μM)	T98G (1C50 μM)
1		2.65±0.50	1.04±0.29
2		2.88±0.82	0.79±0.26
3		>10	0.57±0.17
4		0.12±0.045	0.035±0.011
5		0.15±0.074	0.097±0.036
6		0.21±0.096	0.13±0.044
7		1.5±0.55	4.77±2.85
8		0.046±0.022	0.048±0.018
9		1.87±0.83	3,85±2.18
10		>10	1.91±0.95
11		0.22±0.11	0.09±0.03
12		0.208±0.091	0.077±0.029
13		>10	1.19±0.521
14		1.889±0.619	5.026±3.271
15		0.530±0.267	2.674±1.096
16		2.038±0.936	5.069±1.410
17		0.155±0.071	0.148±0.058
18		1.706±0.893	2.363±0.554
19		0.092±0.032	0.029±0.009
20		3.829±2.328	0.0384±0.012
21		8.313±4.629	0.220±0.099
22		>10	0.065±0.024
23		2.630±0.966	3.688±1.394
24		2.085±0.840	>10
25		0.998±0.440	0.423±0.151
26		0.067±0.025	0.023±0.0007
27		3.367±1.685	0.218±0.079
28		0.476±0.231	2.382±0.846
29		0.091±0.036	0.213±0.094
30		2.043±0.921	1.186±0.413
31		6.907±5.179	0.455±0.189
32		>10	0.422±0.152
33		2.490±1.085	0.899±0.377
34		1.189±0.445	0.306±0.110
35		>10	3.430±0.814
36		>10	3.965±1.098
37		>10	>10
38		>10	>10
39		5.232±1.323	2.276±0.655
40		>10	5.468±1.030
41		2.064±0.513	3.609±1.777
42		>10	>10

Figure 2.

AR downregulation effect of compounds 1–42 at 100 nM for 12 h in T98G cells (A–E). The AR downregulation effect of the promising compounds at 50 nM for 12 h in T98G cells (F). The proteins are analyzed by Western blotting with specific antibodies, and the results are representative images and quantification. Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared to the DMSO treatment group by the unpaired t-test.

2.2. Compounds 4 and 26 Inhibit the Chaperone Activity of HSP27.

The lead compound I was identified to inhibit HSP27 from our previous study.¹¹ We expect that the new derivatives could inhibit HSP27 chaperone function too. It is well documented that HSP27 chaperone function is critical for stabilizing its client proteins, including AR.^18,24,25 To determine the in vitro chaperone activity, insulin is often used as a substrate protein to mimic the protein aggregation. HSP27 is used as the chaperone to prevent aggregation. Inhibition of HSP27 could change the insulin aggregation, which is used as the readout for compound testing.¹¹ In the current study, dithiothreitol (DTT) is used to denature insulin, which induces insulin B chain aggregation. α-Crystallin is the chaperone function domain of HSP27, which is used in this study to perform the chaperone assay.²⁶ The aggregated insulin could be determined by the absorbance at 400 nm. The potency of compounds 4 and 26 to modulate the in vitro chaperone activity of HSP27 is evaluated by monitoring the DTT-induced insulin aggregation in the presence of α-crystalline with or without compounds 4 and 26. The results are showed in Figure 3. α-Crystallin exhibits significant potency against DTT-induced insulin aggregation. Compounds 4 and 26 do not interfere with DTT-induced insulin aggregation, while the chaperone activity of α-crystalline is inhibited with the presence of compound 4 or 26. The results indicate that both compounds 4 and 26 significantly inhibit the in vitro chaperone function of HSP27. Particularly, compound 4 shows much better potency than compound 26 and inhibits the chaperone function more than 50%, which is also more potent than compound I in previous study.¹¹

Figure 3.

Inhibition of compounds 4 and 26 (10 μM) on chaperone function. α-Crystallin loses the activity to prevent DTT-induced insulin B chain aggregation in the presence of compound 4 (Figure 3A) or 26 (Figure 3B). The kinetics of the DTT-induced insulin B chain aggregation is monitored in the absence of a chaperone protein or in the presence of a chaperone protein with or without compound 4 or 26. The mixture of insulin and DTT with or without other components with the assay buffer is incubated for 80 min at 37 °C, and the absorbance at 400 nm is measured. The compound at this concentration or below does not interfere with DTT and insulin interaction. The results are representative of three independent experiments, each curve is measured in triplicate, and the mean is used to generate the curve. The representative one of the three experiments is presented. The statistical analysis is performed for the end reading of the curve with the unpaired t-test, **p < 0.01, with compound 4 or 26 versus without compound 4 or 26.

2.3. Compounds 4 and 26 Dose-Dependently Decrease AR and Mutated AR Protein Levels in GBM Cells.

It is reported that AR is one of the most common client proteins of HSP27, and knocking down HSP27 with siRNA could induce AR degradation in prostate cancer.²⁷ Our hypothesis is that eliminating AR via inhibiting HSP27 by small molecules such as our compounds in the current study with potential to cross the BBB could be promising for GBM treatment. The results in Figure 4 show that compounds 4 and 26 have such potential. Both compounds 4 and 26 could significantly downregulate the AR protein level at 25, 50, and 100 nM in T98G and U87 cells. Moreover, compounds 4 and 26 also could downregulate mutated AR (AR-V7) in T98G cells. The results demonstrate the advantage to target AR with our compounds compared with AR antagonists, since both wild type and mutated ARs could be all downregulated by disrupting HSP27 chaperone function. So far, small-molecule HSP27 inhibitor compounds 4 and 26 could inhibit GBM cell proliferation potently and downregulate the AR protein level effectively.

Figure 4.

AR and/or mutated AR (AR-V7) are abolished by compound 4 or 26 in T98G and U87 cells. AR and AR-V7 expressions are analyzed by Western blotting after the compound treatment. The experiment is repeated three times independently, and the representative image and quantification are shown. Data are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01 compared to the DMSO treatment group by the unpaired t-test.

2.4. AR Downregulation Effect of Compounds 4 and 26 in Living Cells.

We also examine the AR distribution in U87 cells and T98G cells after the treatment with both compounds 4 and 26, respectively. Immunofluorescence imaging is used to visualize the AR protein in living cells. The results show that the fluorescence intensities of AR are decreased with the treatment of compounds 4 and 26 in both cell lines compared to the control (Figure 5), which is consistent to the Western blotting results. Additionally, the result shows that AR distribution in the treatment groups is confined to close proximity to the nucleus, indicating that free cytoplasmic AR has decreased due to degradation. Furthermore, some ARs in the nucleus of the cells after the treatment still remain, indicating that the degradation of AR through inhibition of HSP27 chaperone activity happens in the cytosol of the cells. When AR is located within the cell nucleus, its interaction with HSP27 is dissociated; therefore, the compounds are unable to affect AR without HSP27 present. The results provide another indirect evidence that the compounds induce AR degradation via HSP27 inhibition.

Figure 5.

Expression and location of AR with DMSO or compounds treatment in T98G cells and U87 cells with natural expression of AR. The cells are analyzed by the immunofluorescence assay.

2.5. Compounds 4 and 26 Significantly Suppress the GBM Growth and AR Level in the Xenograft Tumor Model.

Both compounds 4 and 26 have demonstrated potent in vitro activity. Next, to determine the in vivo activity of the compounds, a xenograft tumor model is established by subcutaneously injecting U87 cells (T98G cells failed to develop the tumor) into the left and right flank of nude mice. Compounds 4 and 26 are administrated with intraperitoneal injection (IP) every 2 days for 2 weeks, until the tumor size of the control group reached 1500 mm³. The mouse body weight and tumor size are measured and recorded when the compounds are applied. The tumor images are exhibited in Figure 6A, and the tumor size is significantly decreased in the treatment groups, and compound 26 is more effective than compound 4. In Figure 6B, nude mice body weight is not affected by both compounds, indicating low toxicity of the agents. The tumor size during treatment course also shows a significant decrease in both compound 4 and 26 groups, compared to the treatment group in Figure 6C. Tumor weight is recorded at the end of the experiment, and results show that compounds 4 and 26 significantly decrease the tumor weight compared to the control group in Figure 6D. To determine if the compounds could abolish AR in vivo, the AR protein level in tumors is examined via Western blotting (Figure 6E). The results exhibit that the AR protein level is significantly decreased in both compound 4 and 26 groups, compared to the control group. The results indicate that both compounds 4 and 26 exhibit potent in vivo activity, providing the solid evidence that they could be promising new drug candidates to treat AR-overexpressed GBM.

Figure 6.

In vivo efficacy of compounds 4 and 26 in human GBM. Nude mice bearing xenograft are treated with compound 4 or 26 at 20 mg/kg with IP injection every other day. Tumor images (A, n = 6); body weight (B, n = 4); tumor size (C, n = 6, data are expressed as mean ± SD, compound 4 *p < 0.05, compound 26 ^#p < 0.05 compared to the DMSO group); tumor weight (D, n = 6, data are expressed as mean ± SD, compound 4 *p < 0.05, compound 26 ^#p < 0.05 compared to the DMSO group), and AR expression in tumor is analyzed by Western blotting, as shown by representative images and by quantification (E, n = 4, data are expressed as mean ± SD, compound 4 **p < 0.01, compound 26 ^##p < 0.01 compared to the DMSO group).

2.6. Compounds 4 and 26 Distribution in Brain Tissue.

Compounds 4 and 26 inhibit GBM tumor growth in the mouse xenograft model, but it is unclear if the compounds could cross BBB. Therefore, the pharmacokinetics of compounds 4 and 26 in mouse plasma and brain tissue was analyzed via LC–MS/MS (Figure S1, supporting material). The mice are treated with the compounds and then euthanized at different time points, and the serum is collected first; then, the brain tissue is collected after a perfusion procedure. The drug concentrations in serum and brain tissue of compounds 4 and 26 at different time point are exhibited in Figure 7. The pharmacokinetic parameters are listed as mean ± SD in Table 2. The results show that compounds 4 and 26 are rapidly absorbed and distributed. The apparent half-life (t_1/2) values of compounds 4 and 26 are 1.49 and 6.28 h, respectively. The results suggest that compound 26 lasted longer in the animals, which might be correlated to the better in vivo anti-tumor activity. The AUC of compound 4 in mouse plasma reaches 32.13 μg·h/mL, and the AUC of it in brain tissue is 0.62 μg·h/g (about 2% of the amount in serum), indicating that compound 4 can pass BBB and accumulate in the brain tissue (Figure 7A,B). The AUC of compound 26 in mouse plasma reaches 80.84 μg·h/mL, which is much higher than compound 4. The AUC of compound 26 in brain tissue is 2.44 μg·h/g (about 3% of the concentration in blood), suggesting that the BBB crossing efficiency is slightly better than compound 4 (Figure 7C,D). However, the BBB crossing efficiency of both compounds 4 and 26 is not significant like TMZ (30% permeability), and more lead optimization is needed for this group of compounds.

Figure 7.

Plasma and brain tissue concentration–time profiles of compounds 4 (A, B) and 26 (C, D) with IP administration in mice. (mean ± SD, n = 4).

Table 2.

Noncompartmental Pharmacokinetic Parameters of Compounds 4 and 26 in Mouse after IP Administration (n = 4, Mean ± SD)

	value
	compound 4		compound 26
pharmacokinetic parameters	plasma	brain	plasma	brain
t1/2	1.49 ± 0.42 (h)	0.57 ± 0.30 (h)	6.28 ± 1.39 (h)	8.66 ± 3.81 (h)
Cmax	20.20 ± 8.10 (μg/mL)	0.68 ± 0. 25 (μg/g)	30.62 ± 6.99 (μg/mL)	0.76 ± 0.19 (μg/g)
AUC0–24h	20.02 ± 5.62 (μg⋅h/mL)	0.31 ± 0.05 (μg ⋅ h/g)	61.92 ± 15.47 (μg⋅h/mL)	1.34 ± 0.31 (μg ⋅ h/g)
AUC0-∞h	32.13 ± 5.59 (μg⋅h/mL)	0.62 ± 0.24 (μg ⋅ h/g)	80.84 ± 12.78 (μg⋅h/mL)	2.44 ± 0.53 (μg ⋅ h/g)

2.7. Compounds 4 and 26 Show a Great Therapeutic Index.

To further examine the toxicity of the compounds, we treated mice with a much higher dose of the compounds and evaluated the toxicity profile including tissue damage and hematological changes. C57BL/6 mice are treated with compounds 4 and 26, respectively, at 100 mg/kg per day for 10 days. The toxicity of compounds 4 and 26 is determined by the mice body weight change, tissue staining, and hematological change. (1) Condition observation: all of the groups of mice do not show any sign of toxicity, such as dehydration, acute pain, or distress. (2) Body weight: it is monitored and recorded every day, and the results are shown in Figure 8A, and there is no growth rate difference between compounds 4 and 26 compared to the control group. (3) Hematology: the hematological results of all the mice are listed in Table S2 (supporting material S2). There is no change in the profile of the blood compared with the control group. (4) Tissue staining: at the end of the study, the mice are euthanized; then, the tissues (kidney, spleen, lung, liver, and heart) are collected and rinsed with PBS and then fixed with 4% formalin for 24 h at room temperature. After tissues are sliced and stained with hematoxylin and eosin, the images are taken from the microscope, and the results are exhibited in Figure 8B. Compared to the control group, there are no substantial histological abnormalities such as inflammation and premature cell death in compound 4 and 26 groups. In the kidney, normal histology of the glomerulus and tubules is found and there are no necrosis and glomerular atrophy and other inflammatory changes in drug treatment groups; in the spleen, intact and distinct spleen follicles with clear white pulp, red pulp, and marginal zone are visualized in the control and drug treatment groups; in the lung, there is no severe injury or fibrosis observed in all the groups; in the liver, the sample shows normal hepatic architecture with central vein and surrounding hepatocytes, sinusoids and nucleus; in the heart, there are no obvious infarcts, inflammatory cell infiltration, rupture, or necrosis of myocardial cells. Overall, there is no abnormal physical condition observed in compound 4 and 26 treatment groups; the body weight growth rate in drug treatment groups is also similar to the control group, indicating that compounds 4 and 26 do not cause toxicity in mice.

Figure 8.

Body weight (A) and histopathological examination of the kidney, spleen, lung, liver, and heart H&E staining (scale bar, 200 μm) after compound 4 or 26 treatment (B). Data expressed with mean ± SD (n = 3).

3. CONCLUSIONS

GBM is the most common and aggressive brain tumor, with very poor prognosis.² Recent studies demonstrate that AR is a potential therapeutic target for the treatment of GBM.^12,28,29 In our previous study, compound I has been identified as a promising drug candidate for AR-overexpressed GBM, and it could suppress GBM in vitro and in vivo by abolishing AR.¹¹ However, the pharmacokinetic profile of compound I still needs improvement to move forward in the drug development pipeline, due to the limited BBB permeability and short half-life.²³

Based on the structure of compound I, new analogs are developed in this study aiming to optimize the biological activity or pharmacokinetic profile. Compounds 4 and 26 within the new library exhibit potent in vitro anti-glioblastoma activity, including nanomolar level IC₅₀s and AR downregulation effects in GBM cells, indicating that the anti-glioblastoma activity is similar to the lead compound I. Both compounds 4 and 26 inhibit α-crystallin chaperone activity with better potency than compound I. It has been well documented that HSP27 stabilizes AR and escorts its translocating into nucleus, triggers ARE activation and cell proliferation.^18,22,26,30 Via inhibiting HSP27, compounds 4 and 26 downregulated AR and AR-V7 proteins in GBM cells. The immunofluorescence assay results indicate that the AR level is decreased in cytosol with the presence of compounds 4 and 26. This result solidifies our hypothesis that the compounds induce AR degradation by disrupting HSP27-AR complex outside the nucleus. The in vivo study reveals that compounds 4 and 26 inhibit the growth of tumor xenograft and abolish the AR in the tumor tissues as well. Furthermore, the pharmacokinetic studies indicate that compounds 4 and 26 could be distributed rapidly and are able to cross BBB. Compared to compound I, these two compounds have slightly higher permeability from serum into brain tissue, indicating that dual ring benzamide moieties seem helpful to enhance the permeability and half-life. Furthermore, these two compounds do not exhibit any in vivo toxicity even at much higher doses. Overall, this study reveals that compounds 4 and 26 show a better drug-like profile compared to the lead compound. This work provides a meaningful insight for further structural modification to retain or improve the potency and BBB permeability of the compound. In the future, more new derivatives will be prepared based on the structure of compounds 4 and 26. Also, the GBM intracranial brain tumor model will be applied to validate the drug efficacy in vivo.

4. MATERIALS AND METHODS

4.1. Reagents.

Thiazolyl blue tetrazolium bromide (98%, P31B064) was from Alfa Aesar. Insulin (91077C) and α-crystallin (C4163) were from Sigma-Aldrich. Dithiothreitol (DTT) (EC# 222–468-7) was from Amresco. DMEM (10–013-CV) and RPMI 1640 (10–040-CV) were from Corning. FBS (S11150) was from Atlanta Biologicals. Pen/Strep solution (725–100p) was from Cleveland Clinic. Anti-β-actin antibody (4967S), anti-HSP27 antibody (2402S), anti-androgen receptor antibody (5153S), anti-AR-V7 antibody (68492S), and anti-rabbit IgG (7074S) were from Cell Signaling Technology. Bovine serum albumin (2905-OP) was from Millipore Sigma. RIPA (prod# 89900), protease inhibitor cocktail (prod# 1861278), anti-rabbit Alexa Fluor 488 secondary antibody (A-21206), anti-mouse Alexa Fluor 594 secondary antibody (SA5–10168), and the chemiluminescent substrate (34577) were from Thermo Scientific. The BCA protein assay kit (prod# K813–2500-1) was from Biovision. Nonfat dry milk (B51–0500) was from Rockland. Hematoxylin solution (Harris, 95057–844) and eosin solution (95057–848) were from VWR Internationals. All the other chemicals are analytical grade.

4.2. Chemistry.

Chemicals are commercially available and used as received without further purification unless otherwise noted. Thin-layer chromatography (TLC) is performed on silica gel TLC plates with a fluorescence indicator at 254 nm (Analytikjena). Mass spectra are obtained on a Shimadzu single quad mass spectrometer at Cleveland State University MS Facility Center. All NMR spectra are recorded on a Bruker 400 MHz spectrometer using DMSO-d₆ as the solvent.

Reversed-phase HPLC analysis of compounds is conducted on a Beckman HPLC system with an autosampler. The chromatographic separation is performed on a Kinetex C₁₈ column (50 mm × 2.1 mm, 1.3 μm) with a mobile phase consisting of acetonitrile–0.1% formic acid and water (80:20, v/v) at a flow rate of 0.3 mL/min. The temperature of the column is maintained at 36 °C. The injection volume is 5.0 μL. The reaction procedure is illustrated in Scheme 1. The final step of the reaction and the product characterization are listed below. The original spectrum of the compounds is exhibited in supporting material S3. All compounds are >95% purity by HPLC analysis.

4.2.1. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3,5-bis(trifluoromethyl) Benzamide (1).

Yield 32%, white powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.60 (s, 1H), 9.06 (s, 1H), 8.62 (s, 2H), 8.40 (s, 1H), 7.66 (s, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.30 (m, 2H), 6.98 (d, J = 8.9 Hz, 1H), 6.88 (d, J = 3.9 Hz, 1H), 5.11 (s, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 2.90 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 163.00, 153.68, 153.01, 150.97, 138.22, 137.48, 131.47, 131.14, 130.80, 129.04, 128.35, 125.73, 124.95, 122.23, 115.20, 113.79, 113.33, 112.08, 106.04, 65.52, 56.24, 55.91. ESI-MS calculated for (C₂₅H₂₂F₆N₂O₆S) [M − H]⁻: m/z 591; molecular weight (calculated from the structure): 592.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.85 min, purity: >95%.

4.2.2. 4-Cyano-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (2).

Yield 14%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.52 (s, 1H), 9.00 (s, 1H), 8.12 (m, 2H), 8.05 (m, 2H), 7.69 (s, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.41 (s, 1H), 7.26 (d, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 5.10 (s, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 2.90 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.58, 153.67, 153.03, 150.96, 139.28, 138.60, 132.95, 128.98, 128.46, 125.76, 121.88, 118.77, 115.20, 114.40, 113.77, 113.03, 112.06, 105.79. ESI-MS calculated for (C₂₄H₂₃N₃O₆S) [M − H]⁻: m/z 480; molecular weight (calculated from the structure): 481.52. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.3. 4-Chloro-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-nitrobenzamide (3).

Yield 41%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.58 (s, 1H), 9.04 (s, 1H), 8.64 (s, 1H), 8.28 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.65 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.41 (s, 1H), 7.28 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.81 (m, 1H), 5.10 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 162.89, 153.68, 153.02, 150.95, 135.24, 133.30, 132.45, 128.60, 125.75, 125.30, 115.18, 113.77, 112.06, 105.86, 56.25, 55.92. ESI-MS calculated for (C₂₄H₂₄N₂O₈S) [M − H]⁻: m/z 534; molecular weight (calculated from the structure): 535.95. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.4. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzo[d][1,3]dioxole-5-carboxamide (4).

Yield 39%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.10 (s, 1H), 8.95 (s, 1H), 7.69 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.41 (d, J = 8.9 Hz, 1H), 7.28 (s, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 8.9 Hz, 1H), 5.10 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.94, 153.70, 153.07, 151.01, 125.86, 123.33, 115.25, 113.83, 112.88, 112.11, 108.42, 108.15, 105.72, 102.31, 65.44, 56.27, 55.94. ESI-MS calculated for (C₂₄H₂₄N₂O₈S) [M − H]⁻: m/z 499; molecular weight (calculated from the structure): 500.52. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.5. 3,5-Dichloro-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (5).

Yield 39%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.45 (s, 1H), 9.02 (s, 1H), 7.99 (s, 2H), 7.89 (s, 1H), 7.65 (s, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.24 (m, 2H), 6.98 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 6.8 Hz, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 163.13, 153.68, 153.01, 150.98, 138.43, 134.81, 131.48, 128.38, 126.95, 125.75, 121.98, 115.21, 113.80, 113.09, 112.09, 105.86, 65.47, 56.26, 55.92. ESI-MS calculated for (C₂₃H₂₂Cl₂N₂O₆S) [M − H]⁻: m/z 523; molecular weight (calculated from the structure): 525.40. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.6. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-iodobenzamide (6).

Yield 25%, yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.34 (s, 1H), 8.99 (s, 1H), 8.30 (s, 1H), 7. 98 (d, J = 7.8 Hz, 2H), 7.65 (s, 1H), 7.40 (dd, J = 2, 8.5 Hz, 1H), 7.39 (d, J = 2.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H), 6.99 (d, 1H), 6.90 (d, J = 8.9 Hz, 1H), 5.08 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.86 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.34, 153.68, 152.93, 150.96, 140.61, 137.30, 136.36, 131.06, 127.63, 115.20, 113.74, 113.08, 112.06, 95.16, 65.46, 56.25, 55.92. ESI-MS calculated for (C₂₃H₂₃IN₂O₆S) [M − H]⁻: m/z 581; molecular weight (calculated from the structure): 582.41. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.7. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-fluoro-4-(trifluoromethyl)benzamide (7).

Yield 23%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.62 (s, 1H), 9.01 (s, 1H), 8.01 (m, 2H), 7.55 (m, 2H), 7.29 (d, 3H), 6.99 (d, J = 9.0 Hz, 1H), 6.86 (m, 1H), 5.08 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H), 2.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 161.62, 153.67, 152.79, 150.97, 136.11, 129.62, 127.23, 126.03, 125.67, 115.17, 113.70, 112.64, 112.05, 105.65, 65.48, 26.25, 55.90. ESI-MS calculated for (C₂₄H₂₂F₄N₂O₆S) [M − H]⁻: m/z 541; molecular weight (calculated from the structure): 542.50. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.8. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-iodobenzamide (8).

Yield 45%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.34 (s, 1H), 9.00 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7. 77 (d, J = 8.5 Hz, 2H), 7.68 (s, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.40 (s, 1H), 7.27 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.08 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.87 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.22, 153.67, 153.03, 150.96, 138.74, 137.75, 134.62, 130.05, 128.38, 125.83, 121.82, 115.19, 113.75, 112.97, 112.05, 105.78, 99.86, 65.43, 56.24, 55.91. ESI-MS calculated for (C₂₃H₂₃IN₂O₆S) [M − H]⁻: m/z 581; molecular weight (calculated from the structure): 582.41. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.9. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-ethylbenzamide (9).

Yield 30%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.34 (s, 1H), 8.98 (s, 1H), 7.91 (d, J = 8.1 Hz, 2H), 7. 72 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.29 (s, 1H), 7.28 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.89 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 3.30 (d, 2H), 2.87 (s, 3H), 2.70 (t, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.22, 153.67, 153.03, 150.96, 138.74, 137.75, 134.62, 130.05, 128.38, 125.83, 121.82, 115.19, 113.75, 112.97, 112.05, 105.78, 99.86, 65.43, 56.24, 55.91. ESI-MS calculated for (C₂₅H₂₈N₂O₆S) [M − H]⁻: m/z 483; molecular weight (calculated from the structure): 484.57. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.10. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-(dimethylamino)benzamide (10).

Yield 20%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.34 (s, 1H), 8.95 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7. 73 (s, 1H), 7.40 (m, 1H), 7.29 (s, 1H), 7.18 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.90 (m, 1H), 6.78 (d, J = 8.9 Hz, 2H), 5.08 (s, 2H), 3.79 (s, 3H), 3.74 (s, 3H), 3.01 (s, 6H), 2.86 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 1 165.65, 153.67, 153.16, 152.92, 150.96, 139.83, 129.60, 128.66, 125.89, 121.33, 120.65, 115.21, 113.74, 112.65, 112.03, 111.24, 105.43, 65.35, 56.23, 55.91. ESI-MS calculated for (C₂₅H₂₉N₃O₆S) [M − H]⁻: m/z 498; molecular weight (calculated from the structure): 499.58. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.11. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3,4-dimethoxybenzamide (11).

Yield 27%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.34 (s, 1H), 8.99 (s, 1H), 7.71 (s, 1H), 7. 65 (d, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.40 (d, J = 10.6 Hz, 1H), 7.38 (s, 1H), 7.29 (d, J = 8.6 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H) 6.99 (d, J = 8.9 Hz, 1H), 6.87 (d, 1H), 5.08 (s, 2H), 3.85 (s, 6H), 3.79 (s, 3H), 3.74 (s, 3H), 2.87 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.37, 153.67, 153.11, 152.21, 150.96, 148.82, 139.29, 128.56, 127.30, 125.83, 121.47, 121.19, 115.23, 113.76, 112.96, 112.05, 111.59, 111.38, 105.73, 65.40, 56.23, 56.18, 56.13, 55.92. ESI-MS calculated for (C₂₅H₂₈N₂O₈S) [M − H]⁻: m/z 515; molecular weight (calculated from the structure): 516.57. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.12. 4-Chloro-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (12).

Yield 55%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.36 (s, 1H), 9.00 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.69 (s, 1H), 7.42 (d, J = 8.7 Hz, 2H), 7.40 (s, 1H), 7.22 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.9 Hz, 1H), 6.88 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.86, 153.67, 153.07, 150.96, 138.88, 136.97, 133.96, 130.08, 128.96, 128.51, 125.80, 121.59, 115.19, 113.76, 112.96, 112.05, 105.74, 65.42, 56.24, 55.91. ESI-MS calculated for (C₂₃H₂₃ClN₂O₆S) [M − H]⁻: m/z 489; molecular weight (calculated from the structure): 490.96. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.13. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-nitrobenzamide (13).

Yield 42%, yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.36 (s, 1H), 9.04 (s, 1H), 8.40 (d, J = 8.8 Hz, 2H), 8.20 (d, J = 8.7 Hz, 2H), 7.69 (s, 1H), 7.45 (d, J = 10.4 Hz, 1H), 7.42 (s, 1H), 7.28 (d, J = 12.8 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.88 (m, 1H), 5.10 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.31, 153.67, 153.02, 150.96, 149.68, 140.92, 138.52, 138.52, 129.67, 128.42, 125.76, 124.04, 122.01, 115.19, 113.76, 113.08, 112.05, 105.84, 65.47, 56.25, 55.91. ESI-MS calculated for (C₂₃H₂₃N₃O₈S) [M − H]⁻: m/z 500; molecular weight (calculated from the structure): 501.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.14. 3-Cyano-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (14).

Yield 44%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.36 (s, 1H), 9.04 (s, 1H), 8.27 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.79 (dd, J = 2, 7.8 Hz, 1H), 7.67 (s, 1H), 7.43 (d, J = 10.4 Hz, 1H), 7.29 (d, J = 16.1 Hz, 1H), 7.28 (s, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.10 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.07, 153.69, 153.05, 150.96, 138.60, 136.27, 135.53, 132.98, 131.72, 130.33, 128.47, 125.77, 121.89, 118.78, 115.18, 113.76, 112.98, 112.05, 112.01, 105.74, 65.46, 56.25, 55.92. ESI-MS calculated for (C₂₄H₂₃N₃O₆S) [M − H]⁻: m/z 480; molecular weight (calculated from the structure): 481.52. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.15. 3-Bromo-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (15).

Yield 24%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.36 (s, 1H), 9.04 (s, 1H), 8.15 (s, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.67 (s, 1H), 7.52 (dd, J = 3, 7.9 Hz 1H), 7.29 (s, 1H), 7.25 (d, J = 8.5 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.38, 153.67, 153.03, 150.97, 138.67, 137.42, 134.84, 131.15, 130.67, 128.39, 127.33, 125.81, 122.15, 121.84, 115.21, 113.77, 113.01, 112.06, 105.81, 65.45, 56.24, 55.92. ESI-MS calculated for (C₂₃H₂₃BrN₂O₆S) [M − H]⁻: m/z 535; molecular weight (calculated from the structure): 535.41. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.16. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-nitrobenzamide (16).

Yield 47%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.63 (s, 1H), 9.04 (s, 1H), 8.80 (s, 1H), 8.46 (m, 2H), 7.86 (t, 1H), 7.68 (s, 1H), 7.45 (d, J = 6.6 Hz, 1H), 7.28 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 163.78, 153.68, 150.97, 148.25, 138.50, 136.63, 134.65, 130.70, 128.41, 126.72, 125.78, 122.85, 122.00, 115.21, 113.78, 113.17, 112.07, 105.94, 65.49, 56.25, 55.92. ESI-MS calculated for (C₂₃H₂₃N₃O₈S) [M − H]⁻: m/z 500; molecular weight (calculated from the structure): 501.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.17. 4-Bromo-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (17).

Yield 40%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.37 (s, 1H), 9.01 (s, 1H), 7.91 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.68 (s, 1H), 7.42 (d, J = 10.7 Hz, 1H), 7. 27 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.98, 153.67, 153.06, 150.96, 138.87, 134.32, 131.90, 130.25, 128.51, 125.91, 125.79, 121.61, 115.19, 113.75, 112.96, 112.04, 105.74, 65.43, 56.24, 55.91. ESI-MS calculated for (C₂₃H₂₃BrN₂O₆S) [M − H]⁻: m/z 535; molecular weight (calculated from the structure): 535.41. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.18. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-2,4-bis(trifluoromethyl)benzamide (18).

Yield 31%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.75 (s, 1H), 9.01 (s, 1H), 8.24 (m, 2H), 7.99 (d, J = 7.8 Hz, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 7.26 (m, 3H), 6.99 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.65, 153.66, 153.18, 151.04, 140.14, 138.33, 131.10, 130.77, 130.57, 130.31, 128.66, 127.58, 127.26, 125.68, 124.96, 124.75, 123.94, 122.24, 122.10, 115.30, 113.89, 112.35, 112.08, 105.13, 65.47, 56.23, 55.91. ESI-MS calculated for (C₂₅H₂₂F₆N₂O₆S) [M − H]⁻: m/z 591; molecular weight (calculated from the structure): 592.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.19. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-(methylthio)benzamide (19).

Yield 60%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.23 (s, 1H), 9.00 (s, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.70 (s, 1H), 7.41 (m, 3H), 7.29 (s, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.80 (s, 3H), 3.73 (s, 3H), 2.87 (s, 3H), 2.55 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.33, 153.67, 153.10, 150.96, 143.72, 139.17, 131.10, 128.62, 128.56, 125.82, 125.37, 121.31, 115.20, 113.75, 112.89, 112.03, 105.67, 65.40, 56.23, 55.91, 14.60. ESI-MS calculated for (C₂₄H₂₆N₂O₆S₂) [M − H]⁻: m/z 501; molecular weight (calculated from the structure): 502.60. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.20. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-ethoxybenzamide (20).

Yield 30%, white powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.26 (s, 1H), 9.00 (s, 1H), 7.72 (s, 1H), 7.44 (m, 4H), 7. 82 (s, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m 1H), 5.08 (s, 2H), 4.12 (m, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.87 (s, 3H), 1.37 (t, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.67, 158.93, 153.67, 150.98, 139.06, 136.60, 130.05, 128.51, 125.82, 120.22, 118.13, 115.23, 113.97, 113.77, 112.94, 112.05, 105.73, 65.41, 63.79, 56.24, 55.92, 15.08. ESI-MS calculated for (C₂₅H₂₈N₂O₇S) [M − H]⁻: m/z 499; molecular weight (calculated from the structure): 500.57. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.21. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-methoxybenzamide (21).

Yield 29%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.27 (s, 1H), 9.00 (s, 1H), 7.70 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.44 (m, 3H), 7.29 (d, J = 2.9 Hz, 1H), 7.21 (d, J = 8.6 Hz, 1H), 7.18 (d, J = 8.1 Hz 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.08 (s, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 3.73 (s, 3H), 2.87 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.69, 159.68, 153.67, 153.08, 150.97, 139.06, 136.66, 130.04, 128.53, 125.81, 121.43, 120.29, 117.81, 115.23, 113.76, 113.45, 112.96, 112.04, 105.74, 65.41, 56.23, 55.91, 55.83. ESI-MS calculated for (C₂₄H₂₆N₂O₇S) [M − H]⁻: m/z 485; molecular weight (calculated from the structure): 486.54. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.22. 3-Chloro-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (22).

Yield 20%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.25 (s, 1H), 8.99 (s, 1H), 7.49 (m, 4H), 7.28 (d, J = 2.9 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.16 (m, 1H), 7.00 (d, J = 8.9 Hz 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.47, 153.67, 153.03, 150.96, 137.23, 133.69, 131.95, 130.91, 128.41, 127.83, 126.96, 125.81, 115.19, 113.75, 113.00, 112.05, 105.79, 65.43, 56.24, 55.91. ESI-MS calculated for (C₂₃H₂₃ClN₂O₆S) [M − H]⁻: m/z 489; molecular weight (calculated from the structure): 490.96. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.23. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-methyl-3-nitrobenzamide (23).

Yield 20%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.51 (s, 1H), 9.03 (s, 1H), 8.58 (s, 1H), 8.22 (d, J = 8.0 Hz 1H), 7.70 (m, 2H), 7.44 (d, J = 8.0 Hz 1H), 7.28 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H), 2.61 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 163.62, 153.68, 153.03, 150.96, 136.85, 134.13, 133.58, 132.59, 125.79, 123.99, 115.19, 113.77, 113.11, 112.06, 105.88, 65.46, 56.24, 55.92, 20.02. ESI-MS calculated for (C₂₄H₂₅N₃O₈S) [M − H]⁻: m/z 514; molecular weight (calculated from the structure): 515.54. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.24. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-(trifluoromethyl)benzamide (24).

Yield 39%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.41 (s, 1H), 9.01 (s, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.68 (s, 1H), 7.54 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.27 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.79, 153.68, 153.06, 150.99, 138.85, 134.41, 130.51, 128.45, 125.79, 121.65, 121.17, 115.22, 113.81, 112.96, 112.09, 105.76, 65.46, 56.26, 55.92. ESI-MS calculated for (C₂₄H₂₃F₃N₂O₆S) [M − H]⁻: m/z 539; molecular weight (calculated from the structure): 524.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.72 min, purity: >95%.

4.2.25. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-(trifluoromethyl)benzamide (25).

Yield 37%, white powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.51 (s, 1H), 9.03 (s, 1H), 8.28 (m, 2H), 7.98 (d, J = 7.7 Hz, 1H), 7.80 (m, 1H), 7.566 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.28 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.46, 153.68, 152.96, 150.97, 136.15, 132.29, 130.23, 129.83, 129.51, 128.67, 128.21, 125.83, 124.70, 124.66, 115.21, 113.78, 113.18, 112.07, 105.99, 65.48, 56.25, 55.92. ESI-MS calculated for (C₂₄H₂₃F₃N₂O₆S) [M − H]⁻: m/z 523; molecular weight (calculated from the structure): 524.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.71 min, purity: >95%.

4.2.26. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-2-naphthamide (26).

Yield 36%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.50 (s, 1H), 9.02 (s, 1H), 8.59 (s, 1H), 8.09 (m, 4H), 7.75 (s, 1H), 7.65 (m, 2H), 7.50 (dd, J = 3, 6.6 Hz 1H), 7.29 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.11 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 2.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 166.04, 153.68, 153.13, 150.99, 139.17, 134.78, 132.57, 132.54, 129.43, 128.52, 128.44, 128.37, 128.16, 127.37, 125.84, 124.68, 121.46, 115.23, 113.78, 112.94, 112.08, 105.73, 65.46, 56.26, 55.93. ESI-MS calculated for (C₂₇H₂₆N₂O₆S) [M − H]⁻: m/z 505; molecular weight (calculated from the structure): 506.57. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.27. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3,4,5-trimethoxybenzamide (27).

Yield 55%, white powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.17 (s, 1H), 8.97 (s, 1H), 7.68 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.25 (m, 4H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.89 (s, 6H), 3.79 (s, 3H), 3.73 (s, 6H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.43, 153.65, 153.10, 151.02, 140.91, 138.91, 130.32, 128.36, 125.77, 121.55, 115.30, 113.85, 113.24, 112.11, 105.99, 105.83, 65.47, 60.63, 56.63, 56.25, 55.91. ESI-MS calculated for (C₂₆H₃₀N₂O₉S) [M − H]⁻: m/z 545; molecular weight (calculated from the structure): 546.59. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.28. 3. ,4-Dichloro-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (28).

Yield 33%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.44 (s, 1H), 9.03 (s, 1H), 8.22 (d, J = 2.0 Hz, 1H), 7.96 (m, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.40 (m, 1H), 7.25 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 163.62, 153.67, 153.02, 150.98, 138.57, 135.52, 134.94, 131.79, 131.26, 130.03, 128.50, 128.41, 125.75, 121.83, 115.21, 113.80, 112.09, 105.84, 65.47, 56.25, 55.92. ESI-MS calculated for (C₂₃H₂₂Cl₂N₂O₆S) [M − H]⁻: m/z 523; molecular weight (calculated from the structure): 525.40. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.71 min, purity: >95%.

4.2.29. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-methylbenzamide (29).

Yield 22%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.22 (s, 1H), 8.99 (s, 1H), 7.88 (d, J = 8.1 Hz, 2H), 7.71 (s, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 3.0 Hz, 1H), 7.21 (d, J = 8.6 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.08 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.87 (s, 3H), 2.40 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.81, 153.67, 153.08, 151.00, 142.18, 139.19, 132.36, 129.40, 128.48, 128.13, 125.83, 121.31, 115.24, 113.81, 112.90, 112.09, 105.72, 65.43, 56.25, 55.92, 21.48. ESI-MS calculated for (C₂₄H₂₆N₂O₆S) [M − H]⁻: m/z 469; molecular weight (calculated from the structure): 470.54. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.72 min, purity: >95%.

4.2.30. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)hexanamide (30).

Yield 55%, yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.94 (s, 1H), 8.93 (s, 1H), 7.53 (s, 1H), 7.25 (d, J = 2.8 Hz, 1H), 7.15 (s, 2H), 6.99 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.04 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.84 (s, 3H), 2.29 (t, 2H), 1.59 (m, 2H), 1.29 (m, 4H), 0.88 (m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 171.86, 153.66, 153.26, 150.98, 139.38, 128.82, 125.82, 120.66, 115.19, 113.78, 112.07, 111.61, 104.43, 65.35, 56.25, 55.91, 36.88, 31.34, 25.22, 22.35, 14.31. ESI-MS calculated for (C₂₂H₃₀N₂O₆S) [M − H]⁻: m/z 449; molecular weight (calculated from the structure): 450.55. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.31. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-[1,1′-biphenyl]-4-carboxamide (31).

Yield 36%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.35 (s, 1H), 9.00 (s, 1H), 8.06 (d, J = 8.3 Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.76 (m, 3H), 7.53 (m, 2H), 7.45 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.10 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.59, 153.69, 153.10, 151.00, 143.68, 139.56, 139.13, 134.01, 129.55, 128.82, 128.65, 128.50, 127.40, 127.07, 125.84, 115.24, 113.80, 112.92, 112.09, 105.73, 65.45, 56.26, 55.93. ESI-MS calculated for (C₂₉H₂₈N₂O₆S) [M − H]⁻: m/z 531; molecular weight (calculated from the structure): 532.61. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.69 min, purity: >95%.

4.2.32. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-3-methylbenzamide (32).

Yield 46%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.26 (s, 1H), 8.98 (s, 1H), 7.76 (m, 3H), 7.42 (m, 3H), 7.28 (s, 1H), 7.22 (d, J = 8.8 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 166.74, 153.46, 152.85, 151.21, 138.62, 138.54, 134.83, 132.98, 128.98, 128.35, 127.97, 125.55, 125.08, 121.53, 115.49, 114.12, 113.29, 112.34, 106.14, 65.54, 56.26, 55.85, 21.29. ESI-MS calculated for (C₂₄H₂₆N₂O₆S) [M − H]⁻: m/z 469; molecular weight (calculated from the structure): 470.54. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.71 min, purity: >95%.

4.2.33. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-4-(trifluoromethoxy)benzamide (33).

Yield 38%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.40 (s, 1H), 8.99 (s, 1H), 8.08 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.7 Hz, 1H), 7.27 (m, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 164.79, 153.68, 153.05, 150.99, 138.80, 134.41, 130.50, 128.41, 125.80, 121.72, 121.17, 119.18, 115.22, 113.81, 112.97, 112.09, 105.77, 65.46, 56.26, 55.92. ESI-MS calculated for (C₂₄H₂₃F₃N₂O₇S) [M − H]⁻: m/z 539; molecular weight (calculated from the structure): 540.51. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.77 min, purity: >95%.

4.2.34. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)-2-methylbenzamide (34).

Yield 23%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.31 (s, 1H), 8.93 (s, 1H), 7.68 (s, 1H), 7.44 (m, 2H), 7.30 (m, 3H), 7.20 (d, J = 7.9 Hz, 1H), 7.00 (d, J = 8.6 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 168.30, 153.66, 153.13, 151.04, 137.56, 135.69, 131.00, 128.43, 127.65, 126.09, 125.86, 115.31, 113.83, 112.30, 112.08, 105.19, 65.43, 56.25, 55.92, 19.77. ESI-MS calculated for (C₂₄H₂₆N₂O₆S) [M − H]⁻: m/z 469; molecular weight (calculated from the structure): 470.54. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.73 min, purity: >95%.

4.2.35. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)thiophene-2-carboxamide (35).

Yield 53%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.27 (s, 1H), 8.98 (s, 1H), 8.02 (s, 1H), 7.88 (d, J = 4.6 Hz, 1H), 7.64 (s, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.25 (m, 3H), 7.00 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.80 (s, 3H), 3.74 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 160.34, 153.68, 150.98, 140.38, 138.52, 132.48, 129.63, 128.54, 128.40, 125.83, 121.74, 115.23, 113.80, 112.92, 112.06, 105.73, 65.45, 56.25, 55.92. ESI-MS calculated for (C₂₁H₂₂N₂O₆S₂) [M − H]⁻: m/z 461; molecular weight (calculated from the structure): 462.54. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.72 min, purity: >95%.

4.2.36. N-(3-((2,5-Dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)isoxazole-5-carboxamide (36).

Yield 48%, yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.79 (s, 1H), 9.03 (s, 1H), 8.82 (s, 1H), 7.65 (s, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.26 (m, 3H), 7.00 (d, J = 8.6 Hz, 1H), 6.87 (m, 1H), 5.09 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 162.90, 154.52, 153.64, 152.91, 152.33, 151.04, 137.40, 128.18, 125.65, 122.46, 115.28, 113.88, 113.38, 112.15, 107.32, 106.21, 65.55, 56.27, 55.90. ESI-MS calculated for (C₂₀H₂₁N₃O₇S) [M − H]⁻: m/z 446; molecular weight (calculated from the structure): 447.46. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.67 min, purity: >95%.

4.2.37. N1,N5-Bis(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)glutaramide (37).

Yield 25%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.97 (s, 2H), 8.89 (s, 2H), 7.55 (s, 1H), 7.25 (s, 2H), 7.17 (s, 4H), 7.00 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H), 5.04 (s, 4H), 3.78 (s, 6H), 3.73 (s, 6H), 2.85 (s, 6H), 2.38 (m, 4), 1.91 (m, 2); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 171.37, 153.66, 153.25, 150.97, 139.33, 128.79, 125.81, 120.73, 115.19, 113.78, 112.07, 111.66, 104.47, 65.37, 56.25, 55.91, 36.06, 21.31. ESI-MS calculated for (C₃₇H₄₄N₄O₁₂S₂) [M − H]⁻: m/z 799; molecular weight (calculated from the structure): 800.90. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.70 min, purity: >95%.

4.2.38. 2,2′-Oxybis(N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)acetamide) (38).

Yield 18%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.77 (s, 2H), 8.84 (s, 2H), 7.39 (s, 2H), 7.23 (s, 2H), 7.14 (s, 2H), 7.00 (m, 4H), 6.88 (s, 2H), 5.04 (s, 4H), 3.78 (s, 6H), 3.72 (s, 6H), 3.67 (s, 4H), 3.59 (s, 4H), 2.85 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 168.55, 153.64, 153.11, 150.96, 125.76, 115.19, 113.76, 112.31, 112.03, 71.28, 65.40, 56.22, 55.89. ESI-MS calculated for (C₃₆H₄₂N₄O₁₃S₂) [M − H]⁻: m/z 801; molecular weight (calculated from the structure): 802.87. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.39. Ethane-1,2-diylbis((3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)carbamate) (39).

Yield 24%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.80 (s, 2H), 8.86 (s, 2H), 7.39 (s, 2H), 7.22 (s, 2H), 7.15 (d, J = 7.5 Hz, 2H), 7.03 (m, 4H), 6.87 (s, 2H), 5.04 (s, 4H), 4.35 (s, 3H), 3.78 (s, 6H), 3.72 (s, 6H), 2.85 (s, 6H), 2.51 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 153.73, 153.61, 153.46, 151.05, 139.05, 129.01, 125.71, 120.36, 115.31, 113.86, 112.09, 110.93, 103.81, 65.34, 63.20, 56.24, 55.89. ESI-MS calculated for (C₃₆H₄₂N₄O₁₄S₂) [M − H]⁻: m/z 817; molecular weight (calculated from the structure): 818.87. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.65 min, purity: >95%.

4.2.40. (Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)bis((3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)carbamate) (40).

Yield 19%, pale gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.77 (s, 2H), 8.85 (s, 2H), 7.38 (s, 2H), 7.23 (s, 2H), 7.15 (d, J = 7.9 Hz, 2H), 7.00 (m, 4H), 6.87 (m, 2H), 5.03 (s, 4H), 4.21 (s, 4H), 3.77 (s, 6H), 3.72 (s, 6H), 3.67 (s, 4H), 3.59 (s, 4H), 2.84 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 153.87, 153.64, 153.49, 151.04, 139.20, 129.04, 125.76, 120.27, 115.31, 113.84, 112.08, 110.82, 103.71, 70.20, 69.16, 65.34, 64.07, 56.24, 55.90. ESI-MS calculated for (C₄₀H₅₀N₄O₁₆S₂) [M − H]⁻: m/z 905; molecular weight (calculated from the structure): 906.97. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.68 min, purity: >95%.

4.2.41. 4-Amino-N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide (41).

Yield 60%, pale yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 9.82 (s, 1H), 8.92 (s, 1H), 7.72 (d, J = 8.7 Hz, 3H), 7.41 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 2.8 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.87 (m, 1H), 6.60 (d, J = 8.5 Hz, 2H), 5.78 (s, 2H), 5.07 (s, 2H), 3.79 (s, 3H), 3.73 (s, 3H), 2.86 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 165.75, 153.67, 153.19, 152.72, 150.95, 139.94, 129.63, 128.70, 125.90, 121.37, 120.54, 115.21, 113.71, 113.03, 112.57, 112.01, 105.36, 65.35, 56.21, 55.90. ESI-MS calculated for (C₂₃H₂₅N₃O₆S) [M − H]⁻: m/z 470; molecular weight (calculated from the structure): 471.53. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.65 min, purity: >95%.

4.2.42. 4,4′-((2,2′-Oxybis(acetyl))bis(azanediyl))bis(N-(3-((2,5-dimethoxybenzyl)oxy)-4-(methylsulfonamido)phenyl)benzamide) (42).

Yield 60%, gray powder. ¹H NMR (400 MHz, DMSO-d₆) δ_H 10.36 (s, 1H), 10.20 (s, 1H), 8.98 (s, 2H), 7.99 (d, J = 8.6 Hz, 3H), 7.84 (d, J = 8.6 Hz, 3H), 7.70 (m, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 6.87 (m, 2H), 5.09 (s, 4H), 4.35 (s, 3H), 3.79 (s, 6H), 3.73 (s, 6H), 2.88 (s, 6H); ¹³C NMR (100 MHz, DMSO-d₆) δ_C 169.07, 165.31, 153.68, 153.08, 150.98, 141.84, 130.06, 129.10, 125.86, 119.35, 115.23, 113.78, 112.90, 112.08, 105.73, 89.07, 71.35, 65.44, 56.25, 55.92. ESI-MS calculated for (C₅₀H₅₂N₆O₁₅S₂) [M − H]⁻: m/z 1039; molecular weight (calculated from the structure): 1041.11. HPLC mobile phase: 80% ACN with 0.1% formic acid, retention time 0.65 min, purity: >95%.

4.3. Cell Culture.

T98G, A172, U87, and U251 cells are from ATCC. The cells are maintained in RPMI1640 or DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a humidified incubator with 5% CO₂ at 37 °C.

4.4. Cell Viability Analysis.

The MTT assay is used to evaluate the effect of HSP27 inhibitors on the growth of T98G and U87 cells in eight replications. Three thousand cells per well are seeded with RPMI1640 or DMEM in 96-well flat-bottomed plates for 24 h and are then exposed to various concentrations of test compounds dissolved into DMSO (highest final concentration, 0.1%) in the medium for 48 h. Controls receive DMSO at the same concentration as that in highest does drug-treated cells. Cells are incubated in 100 μL of 1 mg/mL MTT reagent diluted in a fresh media at 37 °C for 2 h. Supernatants are removed from the wells, and the precipitated MTT dye is dissolved in 200 μL/well DMSO. Absorbance at 570 nm is determined on a SpectraMax Plus 384 spectrophotometer (Molecular Devices).

4.5. Experimental Animals.

Male C57BL/6 mice and nude mice are purchased from Taconic lab. Mice are housed in Plexiglas cages, kept on a 12/12 h light–dark cycle, and receive food and water in a temperature- and humidity-controlled environment. All the experimental procedures involving animals are performed in accordance with the guide for the Care and Use of The Cleveland State University (CSU) Institutional Animal Care and Use Committee (IACUC).

4.6. Maximum-Tolerated Dose (MTD) Study.

Twelve C57BL/6 mice are randomly divided into three groups. Mice are injected with a vehicle (DMSO) or compounds (100 mg/kg in PBS) by intraperitoneal injection (IP) daily. Mice are euthanized after 10 days treatment. Body weights were determined at the start dosing day and the end dosing day.

4.6.1. Hematological Analysis.

Nonfasted blood samples are collected from the heart immediately for hematological analysis after the euthanizing. Hematology analysis is performed with a hematology analyzer—Element HT5 (Heska Corporation, USA).

4.6.2. Hematoxylin and Eosin (H&E) Staining.

Five types of tissues (Heart, liver, spleen, lung, and kidney) are collected. Then, the tissues are fixed in 10% neutral buffered formalin (10 mL of formalin per cm³ tissue). Tissues are cut and transferred into cassettes and then are immersed into increasing concentration ethanol to remove the water and formalin (dehydration). Xylene is used to remove the alcohol and allow infiltration with paraffin wax. Then, tissues are embedded into paraffin wax. Tissues are cut at 4 μm with microtome (sectioning). Generally, tissue slides are transparent when unstained. H&E staining is used to provide visualization to tissue sections for evaluation. The protocol is slightly modified from Wang’s method.³¹

4.7. Western Blotting.

Cells are cultured in 6-well culture plates, incubated with DMSO or inhibitors, and then lysed with RIPA supplemented with a protease inhibitor cocktail. After incubating the cells on ice for 10 min, lysates are collected into a 1.5 mL centrifuge tube; then, the supernatant would be collected after centrifuged at 10,000g for 10 min. Protein concentrations are determined by the BCA protein assay kit. Fifty micrograms of total protein lysate for each sample are boiled with 1× loading buffer for 10 min. Samples are then separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane is blocked for 2 h with 5% nonfat milk in 1× TBS-T (150 mM NaCl, 10 mM Tris, pH 7.4, 0.1% Tween 20) at room temperature and then incubated with primary antibody at 4 °C overnight. After the membrane is incubated with the primary antibody, washed three times with 1× TBS-T for 10 min each wash, and then incubated with the secondary antibody for 60 min at room temperature, the membrane is washed three times again for 10 min each time with 1× TBS-T. Eventually, the membranes are incubated with a SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the protocol of the manufacturer.

4.8. HSP27 Chaperone Activity Assay.

Twenty four microliters of 1 mg/mL insulin stock solution are added to the single well of 384 well plate; 3 μL of 5 mg/mL α-crystallin (a segment of HSP27 responsible for the chaperone function of HSP27) and 71 μL PBS with three different concentrations (10, 100, and 1000 nM) of compounds dissolved inside are added as well. The mixture is thoroughly mixed and incubated at 37 °C for 5 min; then, 2 μL of 1 M DTT in water is added to initiate the insulin aggregation. The mixture of insulin in the absence or presence of α-crystallin with 0.1% DMSO is used as the control. The absorbance at 400 nm is monitored every 3 min continuously for 2 h using a Molecular Devices SpectraMax Microplate reader.

4.9. Immunofluorescence Assay.

U87 and T98G cells are seeded in 6-well plates. Cover slips are placed into the wells and the cells could attach naturally. After 24 h, cells are treated with compounds 4 and 26 at 50 nM for 12 h, respectively. Then, cells are washed with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin for 30 min. Several washing steps with PBS occur in between fixation, permeabilization, and blocking. Then, it is incubated with the AR primary antibody for 1 h, followed by washing with PBS, and then incubated with fluorescein-labeled secondary antibodies for 1 h. DAPI (1 μg/mL) is added and incubated for 10 min to stain the nucleus. Images are visualized and analyzed using an ECHO Revolve microscope.

4.10. In Vivo Xenograft Study.

U87 cells are resuspended in sterile PBS (100 μL) and injected (5 × 10⁶ cells/injection) subcutaneously at the left and right flank of the nude mice (5–6 weeks, n = 4/group, two tumors per mouse and eight tumors per group). Tumors and body weight are monitored with Vernier calipers three times weekly, and tumor volume is calculated via the following formula: V = 2/3 d₁ × d₂², where d₁ is the larger diameter and d₂ is the smaller diameter. When the tumor volume reaches approximately 50 mm³, mice are injected with a vehicle (DMSO) or compound 4 or 26 (20 mg/kg in PBS) by intraperitoneal injection (IP) three times weekly for 14 days, and the tumor size is monitored and measured at the same time. In the end, mice are euthanized by exposure to excess CO₂ and the tumors are removed and weighted. Then, every two tumors from every single one mouse are mixed and homogenized with RIPA buffer (protein inhibitors and PMSF were added) to prepare the tumor lysates after tumors are weighted.

4.11. Determination of Anti-Glioblastoma Agents in Mouse Plasma and Brain.

4.11.1. HPLC-MS/MS Conditions.

The HPLC-MS/MS method is performed with a Shimadzu UPLC system (Columbia, MD), which consists of a Prominence DGU-20A_3R inline degasser, two LC-30 AD pumps, a SIL-30 AC autosampler, and a CBM-20A controller. The chromatographic separation is performed on a Kinetex C₁₈ column (50 mm × 2.1 mm, 1.3 μm) with a mobile phase consisting of acetonitrile–0.1% formic acid and water (50:50, v/v) at a flow rate of 0.3 mL/min. The temperature of the column is maintained at 36 °C. The injection volume is 5.0 μL. Mass spectrometric detection is operated on an AB Sciex Qtrap 5500 mass spectrometer (Toronto, Canada) with negative electrospray ionization mode. The multiple reaction monitoring (MRM) function is used for quantification with the transitions of compound 4, 26, and internal standard (IS) compound I, which are detected at m/z 501.2 → 151.1, m/z 507.2 → 151.1, and m/z 487.1 → 151.1, respectively (product ion spectrums are exhibited in supplemental material S1). The optimized ion source parameters are set as follows: ion spray voltage, 2000 V; ion source temperature, 550 °C; nebulization gas 40 psi; auxiliary gas, 40 psi; curtain gas, 30 psi. Compound parameters are as follows: compound 4: declustering potential, 40 V; entrance potential, 5 V; collision energy, 23 V; collision entrance potential, 15 V. Compound 26: declustering potential, 40 V; entrance potential, 5 V; collision energy, 25 V; collision entrance potential, 15 V. Compound I: declustering potential, 35 V; entrance potential, 5 V; collision energy, 30 V; collision entrance potential, 15 V.

4.11.2. Preparation of Standards and Samples.

The stock solutions are prepared by dissolving compounds 4, 26, and I in methanol at 1.0 mg/mL. Then, the stock solution of compound 4 or 26 is serially diluted with methanol into a concentration gradient: 1.0, 2.0, 5.0, 10, 20, 50, 100, 200, 500, 1000 ng/mL. Also, a 500 ng/mL working solution of compound I (IS) is prepared in methanol from the stock solution. All the solutions are stored at 4 °C in the dark. A simple protein precipitation method is applied to prepare the samples of compound 4 or 26 in mouse plasma or brain homogenate (0.4 g brain tissue mix with 2 mL of PBS). Briefly, 100 μL of each sample, 40 μL of compound I (IS, 500 ng/mL), and 800 μL of methanol are combined in a 1.5 mL tube. Then, it is vortexed and centrifuged at 12,000g for 5 min. The supernatant is collected and then transferred into a new 1.5 mL tube. The liquid is dried by a nitrogen blowing instrument. The residue is stored at −80 °C and dissolved with 100 μL 50% acetonitrile before analysis. At the end, a noncompartmental modeling method was applied for PK model building.

4.12. Statistical Analysis.

Statistical and graphical information is determined using GraphPad Prism software (GraphPad Software Incorporated) and Microsoft Excel (Microsoft Corporation). Gray values from Western blot are determined via Quantity One Software (Bio-Rad). The determination of IC₅₀s is performed using nonlinear regression analysis. Statistically significant differences are calculated with the two-tailed unpaired Student’s t-test and p values reported at 95% confidence intervals.

ABBREVIATIONS

AR

androgen receptor

ASO

antisense oligonucleotides

BBB

blood brain barrier

FBS

fetal bovine serum

cAMP

cyclic adenosine monophosphate

COX-2

cyclooxygenase 2

GBM

glioblastoma

HSP27

heat shock protein 27 kDa

IP

intraperitoneal injection

NF-κB

nuclear factor κB

PROTAC

proteolysis targeting chimera

siRNA

short interfering RNA

TMZ

temozolomide