Novel thiohydantoin analogues bearing the 1-hydroxyl-2,2,2-trifluoro-1-ethyl moiety as androgen receptor inhibitors for the potential treatment of castration resistant prostate cancer

Abstract

Enzalutamide (ENT) is an approved drug for the treatment of castration resistant prostate cancer (CRPC). Despite its success, the duration of response in patients is still limited with drug resistance. More robust CRPC drugs with novel structural motifs are urgently needed. Here, we designed and synthesized a series of 1-hydroxyl or 1-amino-2,2,2-trifluoro-1-ethyl compounds as isosteres to replace the amide group of ENT. Among the compounds prepared and tested, compund 13b is 2-fold more potent than ENT against LNCaP-AR cells. Western blot analysis showed that 13b dose-dependently inhibits the expression of the prostate-specific antigen (PSA). Further in vivo efficacy studies established that 13b has anti-tumor activity with oral administration at 15 mg kg⁻¹ once daily.

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1 Introduction

Prostate cancer is the most commonly diagnosed cancer and the third-leading cause of cancer deaths in men.¹ Although approximately 75% of these patients will be cured by surgery or radiation when cancer remains within the prostate, the remainder will inevitably progress to a state of metastatic prostate cancer.² Androgen deprivation therapy or other combination therapy, through lowering serum testosterone or competitively blocking the binding of androgens to the androgen receptor (AR), are initially effective in 90% of patients at this stage.³ Nevertheless, the vast majority of these patients will eventually develop metastatic castration resistant prostate cancer (CRPC) after long term treatments.^4–6 Recent research efforts have indicated that CRPC growth is driven by AR signaling as well.⁷ Furthermore, many possible mechanisms influencing AR signaling have been suggested for the acquired resistance, among which AR mutations, androgen synthesis by prostate cancer cells, and over-expression of AR or AR co-activators have been found to directly or indirectly render AR more sensitive to low androgen concentrations or sometimes turn antagonist responses to agonistic.⁸

Enzalutamide (ENT) was approved by FDA in 2012 for the treatment of CRPC. Unlike the first-generation AR inhibitors, such as flutamide, nilutamide, and bicalutamide (Fig. 1), ENT not only binds to AR, but also reduces nuclear translocation of AR and impairs AR binding to DNA.⁹ In addition, further studies suggested that ENT possesses a higher binding affinity of AR than the first-generation AR inhibitors. Clinical studies also demonstrated unequivocally its survival benefit in men with CRPC.^10,11 Despite its success, the duration of response in patients is still limited. Recently, AR with a specific mutation (F876L) was identified to confer resistance to ENT.¹² This disappointing outlook suggests that better compounds with novel structure motif need to be developed urgently.

Fig. 1 The first and second generation AR inhibitors.

Replacement of amide group by other isosteres is a common practice in medicinal chemistry effort. Black and his colleagues from Merck reported to use trifluoroethylamine group as the isostere of amide in their searching of novel cathepsin K inhibitors.¹³ After replacement of amide with trifluoroethylamine, the potency and selectivity were improved, also the new function group are stable for P1–P2 cleavage that was observed for other amide inhibitors. Considering the major metabolic path way of ENT is through N-demthylation and amide hydrolysis,^14,15 a series of isosteres analogs bearing trifluoroethylamine or trifluoroethanol were prepared as novel AR inhibitors. At the same time, C-ring was also optimized for potentially improvement of drug resistance (Fig. 2). Here we report our progress and also the in vivo and in vitro data of compound 13b which is dose-dependently inhibiting expression of prostate-specific antigen and has anti-tumor activity at oral administration dose of 15 mg kg⁻¹ once daily.

Fig. 2 Proposed novel structure as potential AR inhibitors.

2 Results and discussion

2.1 Chemistry

Synthetic routes of evaluated compounds 13a–f were depicted in Scheme 1 as path A and path B, respectively. In path A, compounds 8a–d was obtained by coupling of bromide 7a–b with the appropriated amino-acid under Ullmann coupling reaction conditions. Then, compounds 8a–d were directly subjected to esterification without further purification to afford compounds 9a–d under conditions of K₂CO₃ and CH₃I. Finally, compounds 13a–f were obtained through the reaction between 9a–d and 10a–b.

Scheme 1 Reagents and conditions: (i) 10% CuI, 2% Cu, 20% N,N-dimethylglycine as ligand, K₂CO₃, amino-acid, DMF, 80 °C; (ii) K₂CO₃, CH₃I, DMF, rt; (iii) 10a or 10b, DMAc, 80 °C, 24 h; (iv) TMSCN, AcOH, ketone, reflux; (v) 10b, DMAc, 80 °C, 24 h, then MeOH and 1 N HCl, reflux.

In path B, the starting material 11a–b were provided from corresponding benzaldehyde according the reported procedure.¹⁵ Compounds 12a–d, then, were prepared under Strecker reaction conditions using compounds 11a–b, TMSCN and corresponding ketone as substrates. Finally, compounds 13g–j were obtained by reacting 12a–d with 10b. Other analogues, 14a–d and 15a–i, were synthesized form 13a–b by substitution, alkylation, oxidation, or hydrolysis reaction as shown in Scheme 2.

Scheme 2 Reagents and conditions: (i) Et₃N, MsCl, DCM, 0 °C, 2 h; (ii) amino or amino alcohol in THF, reflux, 12 h; (iii) NaH, MeI, DMF, rt; (iv) IBX, DMSO, rt, 12 h; (v) H₂SO₄, 100 °C, 24 h; (vi) 4 N NaOH, 30% H₂O₂, DMSO, 0 °C, 40 min.

2.2 Biology activity

The in vitro anti-proliferation activities were evaluated by using LNCaP/AR cells (prostate cancer cells overexpressing AR). In order to mimic the CRPC state, the cells were cultured in charcoal stripped serum and treated with compounds for 6 days. The results were summarized in Tables 1, 2 and 3.

Table 1 In vitro anti-proliferative activity (IC₅₀ μM) of compounds against LNCaP-AR cell^a

ID	R^1	C	IC50 (μM)
a IC50 was average of three determinations and deviation from the average was <5% of average value.
ENT	—	—	0.25
13a			0.38
14a			2.52
14b			0.35
14c			0.64
14d			0.92
13c			0.86
13g			0.98

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Table 2 In vitro anti-proliferative activity (IC₅₀ μM) of compounds against LNCaP-AR cell^a

ID	R^1	IC50 (μM)	ID	R^1	IC50 (μM)
a Control compound (IC50 of ENT is 0.25 ± 0.01 μM) used in all assays; IC50 was average of three determinations and deviation from the average was <5% of average value.
13b		0.10	15d		0.83
15a		0.15	15e		1.51
15b		0.13	15h		0.20
15c		0.44	15i		0.31

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Table 3 In vitro anti-proliferative activity (IC₅₀ μM) of compounds against LNCaP-AR cell^a

ID	C	X	R^3	IC50 (μM)	ID	C	X	R^3	IC50 (μM)
a Control compound (IC50 of ENT is 0.25 ± 0.01 μM) used in all assays; IC50 was average of three determinations and deviation from the average was <5% of average value.
13d		C	CN	9.72	13e		N	CN	0.26
13h		C	CN	8.10	13f		N	CN	5.60
13i		C	CN	7.63	15f		C	COOH	>10
13j		C	CN	5.40	15g		C	CONH2	0.86

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The hydroxyl trifluoroethyl analog compound 13a, was first prepared because of its easily synthesis, showed 0.38 μM activity against LNCaP-AR cells compared with ENT of 0.25 μM. Encourage by this result, C-ring analogs of cyclobutyl 13c and cyclohexyl 13g were also prepared and achieved sub-μM activity. However, to our surprise, the closest isostere of amide, N-methylamino trifluoroethyl analog 14a lost the activity by 10-fold. Considering the poor solubility of ENT, more hydroxyl groups were introduced. To our delight, compounds 14b, 14c and 14d were also expressed good activity with IC₅₀ from 0.35 to 0.92 μM.

Analogues with unsubstituted aromatic A-ring were also prepared in order to further study SAR (Table 2). Compound 13b represented an approximate 2-fold improvement over ENT. The second most active compound is 15b, with N–CH₃ trifluoroethyl substitutions. Compound 15e with dihydroxyl propyl amino substitution provided 1.51 μM activity against LNCaP-AR. Other compounds 15a–d, 15h–i were found to have moderate to potent activities in the range of 0.15–0.83 μM against LNCaP-AR cell line.

ARN-509, which has substituted pyridine as B-ring and cyclobutyl as C-ring is under phase III trial for CRPC,¹⁶ therefore modification of A-ring substitution and C-ring size were also conducted in this studies (Table 3). However, in comparison with compound 13b, expanded the ring size of C-ring (compounds 13d and 13h–j) was detrimental to the activity. When C-ring was cyclobutyl group and B-ring is pyridine, compound 13e regained activity with IC₅₀ of 0.26 μM. To our surprise, the IC₅₀ value of compound 13f is inferior compared with 13e. In addition, replacement of CN group with carboxyl group (compound 15f) totally lost activity and the carboxamide (compound 15g) retained some activity.

To test whether 13b has an effect on prostate-specific antigen (PSA) levels, compound 13b was verified in LNCaP-AR cells by Western blot analysis. The result (Fig. 3) showed that compound 13b inhibited the expression of PSA even at very low doses (2.5 μM) and the inhibition activity was dose-dependent. Interestingly, the result also showed that expression of AR was down-regulated, further investigation is needed to probe the mechanism.

Fig. 3 Western blot analysis of PSA expression in LNCaP-AR cells.

The in vivo anti-tumor activity of 13b was evaluated using the CPRC (LNCaP-AR) xenograft model. SCID mice were castrated and subcutaneously inoculated with LNCaP-AR cells. When the tumor volumes reached 100–200 mm³, the mice were randomly grouped (8 animals each group) and oral administrated with compound 13b at 15 mg per kg q.d, 15 mg per kg q.3d, 5 mg per kg q.3d, and for ENT at 15 mg per kg q.d for 27 days. The tumor volumes and weight were measured every 3 days (Fig. 4). Compound 13b at all dose levels inhibited the tumor proliferation compared with the control. 5 mg per kg q.3d of compound 13b have a moderate activity. 15 mg per kg q.d has similar inhibition compared ENT. The tumor inhibition rate of 13b at dose of 5 mg per kg q.3d, 15 mg per kg q.3d, 15 mg per kg q.d were 58.9%, 66.0%, 96.9%, respectively, whereas, the ENT was more than 100% (Fig. 4a). In addition, the weight change 13b, ENT treated group and control group were slightly decreased (Fig. 4b).

Fig. 4 Tumor volume and body weight changes of mice after oral administration of ENT and 13b.

3 Conclusions

In summary, we have synthesized a series of thiohydantoin analogues bearing substituted 2,2,2-trifluoro-1-phenylethanyl moiety. Compound 13b showed good cell activity against CRPC LNCaP-AR cell. Western blot analysis also verified compound 13b inhibited the expression of PSA in LNCaP-AR cells. Further evaluation in xenograft CRPC model showed compound 13b has anti-tumor activity of 96.9% inhibition at 15 mg per kg q.d. Further optimization and relevant works are in progress.

4 Experimental

4.1 General information

The ¹H and ¹³C ¹⁹F NMR spectra were recorded on Avance DMX 400 MHz NMR spectrometers (Bruker, Germany) in CDCl₃ or deuterated DMSO using TMS as internal standard. Spectra are reported as follows: chemical shift δ (ppm), (integral, multiplicity (s = singlet, br s = broad singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet), coupling constant J (Hz), assignment). ESI-HRMS spectra were recorded on a commercial apparatus and methanol was used to dissolve the sample. Reagents were purchased from commercial sources and were used as received unless mentioned otherwise. Reactions were monitored by thin layer chromatography using silica gel GF 254 plates. Column chromatography was performed on silica gel (300–400 mesh).

4.2 Experimental section

Compounds 9, 12, 13 were prepared by using a slightly modified literature-known procedure.^17–20

General procedure for the synthesis of 9. 7 (1.0 mmol), amino-acid (1.2 mmol), 10% CuI (0.1 mmol), 2% Cu (0.02 mol), 20% N,N-dimethylglycine (0.2 mmol) and K₂CO₃ (2.0 mmol) in DMF (15.0 mL) was stirred at 80 °C for 24 h, under the protection of nitrogen. After the reaction, 1 N HCl was added to adjust the PH value to 3–4. Then, the solution was partitioned between ethyl acetate and water. The organic layer was concentrated to give the crude product for the flowing reaction. A stirred solution of the crude product and K₂CO₃ (1.0 mmol) in DMF (5 mL) then, CH₃I (1 mmol) was added. After the reaction, the solution was quenched with water (25 mL). Then, aqueous residue was extracted with EA (20 mL × 3), dried over Na₂SO₄, concentrated and purified by column chromatography (PE : EA/1 : 3) to given compounds 9 (yield from 59–76%).

General procedure for the synthesis of 12. The mixture of 11 (1.0 mmol), TMSCN (2.0 mmol), ketone (4.0 mmol) and 5 mL AcOH was stirred in a sealed tube at 80 °C for 24 h. The solution was neutralized with aqueous sodium hydrogen carbonate. Then, the mixture was extracted with ethyl acetate (20 mL × 3). The combined organics were dried over Na₂SO₄, filtered, concentrated and purified by column chromatography (PE : EA/1 : 2) to give 12 (yield from 58–75%).

General procedure for the synthesis of 13a–f. To the mixture of compound 9a (53 mg, 0.17 mmol) and compound 10b (79 mg 0.34 mmol) in DMAc (10 mL), the mixture was heated to 80 °C for 24 h. After the reaction, water (30 mL) was added. The resulting mixture was extracted with ethyl acetate, washed with brine, dried over sodium sulfate, concentrated and purified by column chromatography (PE : EA/1 : 1) to give a brown solid as crude product, which was purified by preparative chromatography to give compound 13a as a white solid (32 mg, 37% yield).

Compounds 13b–f were synthesized by a similar procedure as described for compound 13a.

4-(3-(3-Fluoro-4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-4,4-di-methyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)ben-zonitrile (13a). ¹H NMR (400 MHz, CDCl₃) δ 7.97 (dd, J = 9.0, 4.9 Hz, 2H), 7.87–7.76 (m, 2H), 7.21 (dd, J = 8.3, 1.7 Hz, 1H), 7.12 (td, J = 9.7, 1.9 Hz, 1H), 5.43 (q, J = 6.3 Hz, 1H), 3.88–3.45 (m, 1H), 1.61 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.85, 174.78, 160.31 (d, J = 248.6 Hz), 137.18 (d, J = 9.2 Hz), 137.00, 135.41, 134.61 (q, J = 33.1 Hz), 132.28, 130.24 (d, J = 4.5 Hz), 127.15 (q, J = 4.8 Hz), 125.94 (d, J = 3.5 Hz), 123.93 (q, J = 281.1 Hz), 123.81 (d, J = 13.2 Hz), 121.87 (q, J = 283.3 Hz), 117.43 (d, J = 23.8 Hz), 114.83, 110.18, 66.75, 65.62 (q, J = 33.5 Hz), 23.69. ¹⁹F NMR (376 MHz, CDCl₃) δ −62.38, −78.42, −113.83. HRMS (EI) calcd for C₂₁H₁₄F₇N₃O₂S [M + H]⁺: 506.0768, found: 506.0769.
4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-hydroxyeth-yl)phenyl)imidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (13b). ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 9.2 Hz, 2H), 7.84 (dd, J = 8.3, 1.6 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 5.28–5.02 (m, 1H), 2.87 (s, 1H), 1.61 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.88, 174.96, 165.29, 137.09, 136.00, 135.77, 135.32, 133.62 (q, J = 34.1 Hz), 132.26, 129.76, 129.07, 127.16 (q, J = 5.1 Hz), 124.04 (q, J = 280.1 Hz), 121.89 (q, J = 273.3 Hz), 114.83, 110.19, 71.97 (q, J = 32.1 Hz), 66.60, 23.73. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.97, −78.16. HRMS (EI) calcd for C₂₁H₁₅F₆N₃O₂S [M + H]⁺: 488.0862, found: 488.0869.
4-(5-(3-Fluoro-4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-2-(trifluoromethyl)benzoni-trile (13c). ¹H NMR (400 MHz, CDCl₃) δ 7.98 (dd, J = 8.4, 5.0 Hz, 2H), 7.93–7.79 (m, 2H), 7.24 (dd, J = 8.4, 1.9 Hz, 1H), 7.14 (dd, J = 9.9, 1.9 Hz, 1H), 5.52 (q, J = 6.3 Hz, 1H), 2.88 (ddd, J = 5.3, 3.5, 2.5 Hz, 1H), 2.70 (ddd, J = 8.9, 6.6, 3.4 Hz, 2H), 2.56 (ddd, J = 20.0, 10.0, 2.4 Hz, 2H), 2.36–2.15 (m, 1H), 1.83–1.64 (m, 1H). ¹³C NMR (101 MHz, CDCl3) δ 179.86, 174.51, 160.54 (d, J = 252.3 Hz), 137.52 (d, J = 9.6 Hz), 136.88, 135.28, 133.68 (d, J = 33.5 Hz), 132.11, 130.39 (d, J = 4.1 Hz), 127.04 (q, J = 5.0 Hz), 126.43 (d, J = 3.1 Hz), 124.38 (q, J = 280.6 Hz), 123.57 (d, J = 13.1 Hz), 121.73 (q, J = 282.6 Hz), 117.89 (d, J = 23.4 Hz), 114.78, 110.24, 67.51, 65.83 (q, J = 32.1 Hz), 31.69, 13.67. ¹⁹F NMR (376 MHz, CDCl3) δ −61.98, −78.38, −113.55. HRMS (EI) calcd for C₂₂H₁₄F₇N₃O₂S [M + H]⁺: 518.0768, found: 518.0772.
4-(8-Oxo-6-thioxo-5-(4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-5,7-diazaspiro[3.4]octan-7-yl)-2-(trifluoromethyl)benzonitri-le (13d). ¹H NMR (400 MHz, CDCl₃) δ 7.98 (dd, J = 5.1, 3.2 Hz, 2H), 7.90–7.83 (m, 1H), 7.74 (t, J = 9.1 Hz, 2H), 7.43–7.34 (m, 2H), 5.15 (q, J = 6.6 Hz, 1H), 3.11–2.76 (m, 1H), 2.75–2.63 (m, 2H), 2.64–2.50 (m, 2H), 2.34–2.16 (m, 1H), 1.70 (dtt, J = 10.5, 7.7, 5.1 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 179.91, 174.77, 137.04, 136.16, 135.72, 135.23, 133.61 (q, J = 34.1 Hz), 132.16, 130.20, 129.25, 127.07 (q, J = 5.1 Hz), 124.03 (q, J = 280.1 Hz), 121.89 (q, J = 273.3 Hz), 114.84, 110.12, 72.10 (q, J = 32.1 Hz), 67.45, 31.59, 13.70. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.96, −78.09. HRMS (EI) calcd for C₂₂H₁₅F₆N₃O₂S [M + H]⁺: 500.0862, found: 500.0866.
5-(8-Oxo-6-thioxo-5-(4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolino-nitrile (13e). ¹H NMR (400 MHz, DMSO) δ 9.23 (s, 1H), 8.77 (d, J = 1.9 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.03 (s, 1H), 5.49–5.19 (m, 1H), 2.72–2.59 (m, 2H), 2.50–2.38 (m, 2H), 2.07–1.90 (m, 1H), 1.66–1.43 (m, 1H). ¹³C NMR (101 MHz, DMSO) δ 180.05, 174.96, 154.07, 137.59, 136.19, 136.03 (q, J = 4.2 Hz), 133.82, 130.31, 129.51, 129.24 (q, J = 34.1 Hz), 129.20 (q, J = 2.0 Hz), 125.43 (q, J = 277.1 Hz), 122.04 (q, J = 273.3 Hz), 114.73, 70.41 (q, J = 31.5 Hz), 67.98, 31.49, 13.86. ¹⁹F NMR (376 MHz, DMSO) δ −60.83, −76.56. HRMS (EI) calcd for C₂₁H₁₄F₆N₄O₂S [M + H]⁺: 501.0814, found: 501.0815.
5-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-hydro-xyeth-yl)phenyl)imidazolidin-1-yl)-3-(trifluoromethyl)picolinonitrile (13f). ¹H NMR (400 MHz, CDCl₃) δ 9.08 (dd, J = 12.6, 2.2 Hz, 1H), 8.37 (t, J = 3.9 Hz, 1H), 7.67 (dd, J = 18.2, 8.3 Hz, 2H), 7.45–7.31 (m, 2H), 5.13 (q, J = 6.6 Hz, 1H), 1.63 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.08, 174.67, 152.34, 135.96, 135.72, 134.16 (q, J = 4.2 Hz), 132.41, 130.54 (q, J = 34.3 Hz), 129.96, 129.67, 129.15, 124.04 (q, J = 283.1 Hz), 121.26 (q, J = 270.9 Hz), 113.75, 71.99 (q, J = 26.3 Hz), 66.81, 23.76. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.87, −78.12. HRMS (EI) calcd for C₂₀H₁₄F₆N₄O₂S⁺ [M + H]⁺: 489.0814, found: 489.0815.

General procedure for the synthesis of 13g–j. To the mixture of compound 12a (54 mg, 0.17 mmol) and compound 10b (79 mg, 0.34 mmol) in DMAc (10 mL), the mixture was heated to 80 °C for 24 h, and then MeOH (2 mL) and 1 N HCl (1 mL) added in the mixture. The reaction mixture was stirred at 90 °C for 2 h. After completion of the reaction (TLC), water (30 mL) were added. The resulting mixture was extracted with ethyl acetate, washed with brine, dried over sodium sulfate, concentrated and purified by column chromatography (PE : EA/1 : 1) to give a brown solid as crude product, which was purified by preparative chromatography to give compound 13g as a white solid (26 mg, 28% yield).

Compounds 13h–j were synthesized by a similar procedure as described for compound 13g.

4-(1-(3-Fluoro-4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.5]decan-3-yl)-2-(trifluoromethyl)benzo-nitrile (13g). ¹H NMR (400 MHz, CDCl₃) δ 7.99 (t, J = 10.4 Hz, 1H), 7.94 (d, J = 1.7 Hz, 1H), 7.89–7.78 (m, 2H), 7.17–7.10 (m, 1H), 7.05 (dd, J = 9.9, 1.9 Hz, 1H), 5.61–5.38 (m, 1H), 3.03–2.80 (m, 1H), 2.26–1.98 (m, 4H), 1.87–1.64 (m, 5H), 1.17–1.00 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 180.12, 173.49, 160.36 (d, J = 251.3 Hz), 137.10 (d, J = 10.0 Hz), 136.95, 135.25, 133.32 (q, J = 33.8. Hz), 132.31, 130.06 (d, J = 4.6 Hz), 127.22 (q, J = 3.2 Hz), 127.98 (d, J = 3.1 Hz), 123.86 (q, J = 280.1 Hz), 123.52 (d, J = 14.0 Hz), 122.36 (q, J = 283.3 Hz), 118.26 (d, J = 23.3 Hz), 114.80, 110.19, 67.73, 65.97 (q, J = 61.4 Hz), 32.82, 23.86, 20.90. ¹⁹F NMR (376 MHz, CDCl₃) δ −62.04, −78.37, −113.90. HRMS (EI) calcd for C₂₄H₁₈F₇N₃O₂S [M + H]⁺: 546.1081, found: 546.1086.
4-(4-Oxo-2-thioxo-1-(4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-1,3-diazaspiro[4.4]nonan-3-yl)-2-(trifluoromethyl)benzonitrile (13h). ¹H NMR (400 MHz, CDCl₃) δ 7.98 (dd, J = 4.7, 3.2 Hz, 2H), 7.90–7.83 (m, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 5.25–5.05 (m, 1H), 3.07–2.49 (m, 1H), 2.34 (dt, J = 13.2, 6.2 Hz, 2H), 2.23–2.11 (m, 2H), 1.94–1.82 (m, 2H), 1.66–1.44 (m, 3H). ¹³C NMR (101 MHz, CDCl3) δ 180.19, 176.05, 137.19, 136.74, 135.58, 135.20, 133.61 (q, J = 37.0 Hz), 132.19, 130.21, 129.09, 127.12 (q, J = 4.8 Hz), 123.73 (q, J = 279.6 Hz), 121.84 (q, J = 270.7 Hz), 114.83, 110.17, 75.27, 72.10 (q, J = 32.3 Hz), 36.16, 25.12. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.96, −78.19. HRMS (EI) calcd for C₂₃H₁₇F₆N₃O₂S [M + H]⁺: 514.1018, found: 514.1023.
4-(4-Oxo-2-thioxo-1-(4-(2,2,2-trifluoro-1-hydroxyethyl)phenyl)-1,3-diazaspiro[4.5]decan-3-yl)-2-(trifluoromethyl)benzonitr-ile (13i). ¹H NMR (400 MHz, CDCl₃) δ 8.05–7.90 (m, 2H), 7.83 (dd, J = 8.2, 1.7 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.31 (t, J = 9.3 Hz, 2H), 5.12 (q, J = 6.6 Hz, 1H), 3.28–2.83 (m, 1H), 2.19–2.00 (m, 4H), 1.70 (dd, J = 17.4, 7.1 Hz, 5H), 1.17–1.00 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 180.07, 173.81, 137.12, 135.76, 135.21, 133.57 (q, J = 34.0 Hz), 132.36, 130.69, 128.99, 127.32 (q, J = 5.0 Hz), 124.05 (q, J = 278.0 Hz), 121.90 (q, J = 273.0 Hz), 114.86, 110.08, 72.05 (q, J = 32.0 Hz), 67.62, 32.75, 23.88, 20.69. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.94, −78.07. HRMS (EI) calcd for C₂₄H₁₉F₆N₃O₂S [M + H]⁺: 528.1175, found: 528.1183.
4-(7,7-Dimethyl-4-oxo-2-thioxo-1-(4-(2,2,2-trifluoro-1-hydro-xyeth-yl)phenyl)-1,3-diazaspiro[4.5]decan-3-yl)-2-(trifluoromethyl)benz-onitrile (13j). ¹H NMR (400 MHz, CDCl₃) δ 8.04–7.89 (m, 2H), 7.90–7.77 (m, 1H), 7.75–7.64 (m, 2H), 7.28 (t, J = 6.1 Hz, 2H), 5.27–5.01 (m, 1H), 3.17–2.69 (m, 1H), 2.36–2.20 (m, 1H), 2.21–2.10 (m, 1H), 1.98–1.86 (m, 1H), 1.73–1.60 (m, 3H), 1.57–1.46 (m, 2H), 1.22 (d, J = 14.6 Hz, 3H), 0.98–0.90 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 180.04, 174.36, 137.23, 135.66, 135.17, 133.57 (q, J = 34.0 Hz), 132.40, 131.28, 128.98, 127.32 (q, J = 5.0 Hz), 124.05 (q, J = 281.2 Hz), 121.89 (q, J = 272.5 Hz), 114.84, 110.11, 72.09 (q, J = 32.5 Hz), 68.81, 43.20, 37.41, 35.03, 32.48, 32.19, 26.36, 17.95. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.96, −78.04. HRMS (EI) calcd for C₂₆H₂₃F₆N₃O₂S [M + H]⁺: 556.1488, found: 556.1494.

General procedure for the synthesis of 14a–d and 15a–e. At 0 °C, DCM solution of 13a (505 mg, 1 mmol), Et₃N (151 mg, 1.5 mmol) and MsCl (137 mg, 1.2 mmol) were added in this order. The reaction was stirred at room temperature for 30 min, and concentrated under vacuum to give the crude product for the flowing reaction. A stirred solution of the crude product and aqueous ammonia in THF was refluxed overnight, and concentrated under vacuum to give the crude product. Purification by flash chromatography afforded the products 14a (410 mg, 79% yield).

Compounds 14b–d and 15b–e were synthesized by a similar procedure as described for compound 14a.

4-(3-(3-Fluoro-4-(2,2,2-trifluoro-1-(methylamino)ethyl)phenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)ben-zonitrile (14a). ¹H NMR (400 MHz, CDCl₃) δ 8.04–7.91 (m, 2H), 7.89–7.78 (m, 1H), 7.68 (t, J = 7.9 Hz, 1H), 7.22–7.16 (m, 1H), 7.16–7.05 (m, 1H), 4.75–4.38 (m, 1H), 2.49 (s, 3H), 1.70 (ddd, J = 11.6, 10.6, 7.5 Hz, 1H), 1.62 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.78, 174.59, 162.34 (d, J = 251.6 Hz), 136.89, 136.80 (d, J = 9.8 Hz), 135.29, 133.66 (q, J = 33.2 Hz), 132.13, 130.13 (d, J = 4.0 Hz), 127.08 (q, J = 4.1 Hz), 126.02 (d, J = 3.8 Hz), 125.07 (q, J = 277.2 Hz), 123.59 (q, J = 14.0 Hz) 121.84 (q, J = 273.1 Hz), 117.49 (q, J = 23.9 Hz), 114.76, 110.36, 66.59, 58.65 (q, J = 27.5 Hz), 34.84, 23.81. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.88, −78.43, −78.45, −113.50, −113.52. HRMS (EI) calcd for C₂₂H₁₇F₇N₄OS [M + H]⁺: 519.1084, found: 519.1093.
4-(3-(3-Fluoro-4-(2,2,2-trifluoro-1-((2-hydroxyethyl)amino)ethyl) phenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoro-methyl)benzonitrile (14b). ¹H NMR (400 MHz, CDCl₃) δ 8.04–7.91 (m, 2H), 7.85 (t, J = 10.9 Hz, 1H), 7.68 (t, J = 7.9 Hz, 1H), 7.18 (t, J = 9.0 Hz, 1H), 7.12 (d, J = 9.9 Hz, 1H), 4.67 (q, J = 7.0 Hz, 1H), 3.91–3.51 (m, 2H), 3.13–2.51 (m, 2H), 2.28–1.90 (m, 2H), 1.62 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.81, 174.58, 161.20 (d, J = 251.0 Hz), 136.92, 136.91 (d, J = 9.9 Hz), 135.31, 133.69 (q, J = 33.1 Hz), 132.18, 130.05 (d, J = 4.0 Hz), 127.10 (q, J = 4.8 Hz), 126.08 (d, J = 3.0 Hz), 124.93 (q, J = 281.1 Hz), 123.84 (d, J = 13.8 Hz), 121.85 (q, J = 273.1 Hz), 117.58 (d, J = 24.3 Hz), 114.76, 110.36, 66.62, 61.26, 56.93 (q, J = 24.3 Hz), 49.51, 23.81. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.94, −73.19, −113.75. HRMS (EI) calcd for C₂₃H₁₉F₇N₄O₂S [M + H]⁺: 549.1190, found: 549.1191.
4-(3-(3-Fluoro-4-(2,2,2-trifluoro-1-((1-hydroxy-2-methylpro-pan-2-yl)amino)ethyl)phenyl)-4,4-dimethyl-5-oxo-2-thioxoi-midazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (14c). ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.6 Hz, 2H), 7.82–7.74 (m, 1H), 7.59–7.45 (m, 2H), 7.23 (dd, J = 18.1, 4.6 Hz, 2H), 4.32–4.11 (m, 1H), 3.41–3.24 (m, 1H), 3.22–3.07 (m, 1H), 2.14–1.85 (m, 1H), 1.69–1.61 (m, 1H), 1.53 (s, 6H), 0.95 (d, J = 11.9 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.80, 174.58, 161.35 (d, J = 257.0 Hz), 136.92, 136.69 (d, J = 10.0 Hz), 135.30, 133.65 (q, J = 33.0 Hz), 132.15, 130.05 (d, J = 4.0 Hz), 127.09 (q, J = 5.0 Hz), 126.79 (d, J = 14.1 Hz), 126.13 (d, J = 4.0 Hz), 124.81 (q, J = 280.3 Hz), 121.85 (q, J = 273.1 Hz), 117.53 (d, J = 24.0 Hz), 114.76, 110.33, 69.35, 66.54, 54.91, 51.21 (q, J = 29.3 Hz), 24.81, 23.93, 23.82. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.95, −74.66, −114.24. HRMS (EI) calcd for C₂₅H₂₃F₇N₄O₂S [M + H]⁺: 577.1503, found: 577.1504.
4-(3-(4-(1-((2,3-Dihydroxypropyl)amino)-2,2,2-trifluoroethyl)-3-fluorophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (14d). ¹H NMR (400 MHz, CDCl₃) δ 8.10–7.91 (m, 2H), 7.90–7.74 (m, 1H), 7.74–7.58 (m, 1H), 7.18 (t, J = 9.6 Hz, 1H), 7.13 (d, J = 10.0 Hz, 1H), 4.73–4.51 (m, 1H), 3.92–3.69 (m, 2H), 3.67–3.52 (m, 1H), 2.92–2.83 (m, 1H), 2.82–2.70 (m, 1H), 2.49–2.05 (m, 2H), 1.62 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.82, 174.55, 161.20 (d, J = 261.1 Hz), 137.03 (d, J = 8.5 Hz), 136.99, 136.89, 135.31, 133.69 (q, J = 33.4 Hz), 132.17, 129.94 (d, J = 5.1 Hz), 127.11 (q, J = 5.1 Hz), 126.14 (d, J = 4.1 Hz), 124.84 (q, J = 277.3 Hz), 119.15 (q, J = 270.6 Hz), 117.68 (d, J = 24.1 Hz), 114.74, 110.41, 70.15 (d, J = 30.2 Hz), 66.62, 65.02 (d, J = 12.3 Hz), 57.20 (q, J = 30.3 Hz), 50.21, 23.83. ¹⁹F NMR (377 MHz, CDCl₃) δ −61.99, −73.69, −73.71, −73.72, −113.60, −113.61, −113.66, −113.67. HRMS (EI) calcd for C₂₄H₂₁F₇N₄O₃S [M + H]⁺: 579.1295, found: 579.1299.
4-(3-(4-(1-Amino-2,2,2-trifluoroethyl)phenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (15a). ¹H NMR (400 MHz, CDCl₃) δ 8.12–7.93 (m, 2H), 7.91–7.76 (m, 1H), 7.74–7.55 (m, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.66–4.40 (m, 1H), 1.93–1.77 (m, 2H), 1.60 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.86, 174.90, 137.15, 137.09, 135.66, 135.27, 133.59 (q, J = 33.1 Hz), 132.24, 129.84, 129.53, 127.14 (q, J = 4.3 Hz), 125.37 (q, J = 280.6 Hz), 121.89 (q, J = 272.7 Hz), 114.83, 110.22, 66.53, 57.51 (q, J = 31.2 Hz), 23.75. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.95, −76.41. HRMS (EI) calcd for C₂₁H₁₆F₆N₄OS [M + H]⁺: 487.1022, found: 487.1025.
4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-(methyl-amino)ethyl)phenyl)imidazolidin-1-yl)-2-(trifluoromethyl)ben-zonitrile (15b). ¹H NMR (400 MHz, CDCl₃) δ 8.05–7.95 (m, 2H), 7.89–7.82 (m, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.40–7.32 (m, 2H), 4.23–4.07 (m, 1H), 2.47 (s, 3H), 1.61 (d, J = 2.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.82, 174.92, 137.11, 135.99, 135.71, 135.26, 133.57 (q, J = 33.8 Hz), 132.24, 130.06, 129.85, 127.12 (q, J = 5.1 Hz), 125.14 (q, J = 280.2 Hz), 121.89 (q, J = 272.5 Hz), 114.83, 110.21, 66.53, 66.02 (q, J = 32.1 Hz), 34.85, 23.76. ¹⁹F NMR (377 MHz, CDCl₃) δ −61.98, −73.81. HRMS (EI) calcd for C₂₂H₁₈F₆N₄OS [M + H]⁺: 501.1178, found: 501.1188.
4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-((2-hydroxy-ethyl)amino)ethyl)phenyl)imidazolidin-1-yl)-2-(trifluoromethyl) benzonitrile (15c). ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 8.4 Hz, 2H), 7.92–7.77 (m, 1H), 7.62 (t, J = 13.0 Hz, 2H), 7.42–7.27 (m, 2H), 4.28 (q, J = 7.2 Hz, 1H), 3.85–3.51 (m, 2H), 2.97–2.65 (m, 2H), 2.01 (s, 2H), 1.61 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.83, 174.89, 137.07, 136.11, 135.77, 135.26, 133.60 (q, J = 33.2 Hz), 132.23, 129.97, 129.93, 127.14 (q, J = 4.8 Hz), 125.13 (q, J = 281.2 Hz), 121.88 (q, J = 273.2 Hz), 114.82, 110.23, 66.54, 64.06 (q, J = 28.6 Hz), 61.37, 49.41, 23.77. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.96, −73.78. HRMS (EI) calcd for C₂₃H₂₀F₆N₄O₂S [M + H]⁺: 531.1284, found: 531.1288.
4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-((1-hydroxy-2-methylpropan-2-yl)amino)ethyl)phenyl)imida-zolidin-1-yl)-2-(trifluoromethyl)benzonitrile (15d). ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.6 Hz, 2H), 7.82–7.74 (m, 1H), 7.51 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 4.40–4.15 (m, 1H), 3.39–3.23 (m, 1H), 3.24–3.09 (m, 1H), 2.15–1.87 (m, 1H), 1.79–1.64 (m, 2H), 1.53 (s, 6H), 0.95 (d, J = 11.9 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.80, 174.88, 139.13, 137.06, 135.48, 135.25, 133.60 (q, J = 33.1 Hz), 132.18, 129.96, 129.51, 127.13 (q, J = 4.9 Hz), 125.26 (q, J = 279.3 Hz), 121.88 (q, J = 273.4 Hz), 114.81, 110.24, 69.46, 66.47, 58.32 (q, J = 29.6 Hz), 55.04, 25.05, 24.27, 23.80. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.97, −74.32. HRMS (EI) calcd for C₂₅H₂₄F₆N₄O₂S [M + H]⁺: 559.1597, found: 559.1600.
4-(3-(4-(1-((2,3-Dihydroxypropyl)amino)-2,2,2-trifluoroethyl)phen-yl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl)-2-(trifluorometh-yl)benzonitrile (15e). ¹H NMR (400 MHz, CDCl₃) δ 8.06–7.94 (m, 2H), 7.88–7.80 (m, 1H), 7.66–7.55 (m, 2H), 7.41–7.32 (m, 2H), 4.25 (qd, J = 7.1, 2.3 Hz, 1H), 3.87–3.76 (m, 1H), 3.73 (dt, J = 11.3, 3.3 Hz, 1H), 3.60 (ddd, J = 11.5, 6.3, 5.4 Hz, 1H), 2.84 (dt, J = 12.0, 3.6 Hz, 1H), 2.77–2.67 (m, 1H), 2.20 (s, 1H), 1.75–1.64 (m, 2H), 1.61 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.84, 174.93, 137.03, 135.90, 135.77, 135.26, 133.63 (q, J = 33.3 Hz), 132.19, 130.02, 129.88, 127.13 (q, J = 4.8 Hz), 124.83 (q, J = 281.3 Hz), 118.27 (q, J = 278.3 Hz), 114.79, 110.29, 70.21 (d, J = 36.2 Hz), 66.54, 65.12 (d, J = 8.1 Hz), 64.33 (q, J = 28.2 Hz), 50.37 (d, J = 45.3 Hz), 23.79. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.97, −73.75, −73.78. HRMS (EI) calcd for C₂₄H₂₂F₆N₄O₃S [M + H]⁺: 561.1390, found: 561.1390.
4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-hydroxyeth-yl)phenyl)imidazolidin-1-yl)-2-(trifluoromethyl)benzoic acid (15f). In a 20 mL tube sealing, 13b (49 mg, 0.1 mmol), concentrated H₂SO₄ (5 mL), were added. The mixture was sealed and stirred for 24 h at 100 °C. After the reaction, the mixture was poured into 100 mL ice water and saturated sodium bicarbonate was added to adjust pH to 4. The mixture was washed with EA (30 mL × 3) and the organic layer was separated and dried over Na₂SO₄. After filtration and evaporation, the resulting crude product was purified by column chromatography with petrol ether as eluent to afford 15f (43 mg, 84% yield).

¹H NMR (400 MHz, DMSO) δ 8.06 (d, J = 1.7 Hz, 1H), 8.02–7.92 (m, 1H), 7.88 (dd, J = 8.2, 1.7 Hz, 1H), 7.68 (t, J = 9.4 Hz, 2H), 7.48 (t, J = 10.3 Hz, 2H), 5.52–5.13 (m, 1H), 1.52 (s, 6H). ¹³C NMR (101 MHz, CDCl3) δ 185.74, 180.49, 172.60, 142.00, 141.17, 140.70, 138.48, 135.54, 134.91, 133.94, 132.82 (q, J = 2.9 Hz), 132.08 (q, J = 32.1 Hz), 130.17 (q, J = 282.7 Hz), 128.37, (q, J = 273.0 Hz), 75.18 (q, J = 29.8 Hz), 71.42, 28.23. ¹⁹F NMR (376 MHz, CDCl₃) δ −53.41, −71.81. HRMS (EI) calcd for C₂₁H₁₆F₆N₂O₄S [M + H]⁺: 507.0808, found: 507.0817.

4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-hydroxyeth-yl)phenyl)imidazolidin-1-yl)-2-(trifluoromethyl)benza-mide (15g). To a mixture of 13b (49 mg, 0.1 mmol) and sodium hydroxide (4.0 M in water, 0.25 mL, 1.0 mmol) in DMSO (2.5 mL) was added hydrogen peroxide (30% aqueous solution, 0.05 mL, 0.49 mmol). The reaction mixture was heated to 35 °C. After 40 minutes, 20 mL H₂O was added, the mixtures was extracted with ethyl acetate. The organic layer was separated and dried over Na₂SO₄. After filtration and evaporation, the resulting crude product was purified by column chromatography with petrol ether as eluent to afford 15g (39 mg, 77% yield).

¹H NMR (400 MHz, DMSO) δ 8.19–8.10 (m, 1H), 8.04–7.91 (m, 1H), 7.87–7.78 (m, 1H), 7.70 (dd, J = 16.7, 7.3 Hz, 4H), 7.46 (d, J = 8.2 Hz, 2H), 7.09–6.91 (m, 1H), 5.37–5.24 (m, 1H), 1.51 (s, 6H). ¹³C NMR (101 MHz, DMSO) δ 181.07, 175.85, 168.88, 137.55, 137.21, 136.47, 134.84, 133.55, 130.18, 129.51, 129.17, 127.84 (q, J = 5.1 Hz), 126.53, 125.43 (q, J = 283.1 Hz), 123.81 (q, J = 273.3 Hz), 70.47 (q, J = 21.1 Hz), 66.62, 23.51. ¹⁹F NMR (376 MHz, DMSO) δ −57.99, −76.56. HRMS (EI) calcd for C₂₁H₁₇F₆N₃O₃S [M + H]⁺: 506.0968, found: 506.0975.

4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoro-1-methoxyeth-yl)phenyl)imidazolidin-1-yl)-2-(trifluoromethyl)benzo-nitrile (15h). In a 25 mL round-bottomed flask, 13b (49 mg, 0.1 mmol), DMF (5 mL), and NaH 60% (12 mg, 0.3 mmol) were added. The mixture was stirred and simultaneously CH₃I (12.3 mg 0.3 mmol) was added then, the reaction was stirred for 2 h at room temperature. After the reaction, 20 mL saturated NH₄Cl was added, the mixtures was extracted with ethyl acetate. The organic layer was separated and dried over Na₂SO₄. After filtration and evaporation, the resulting crude product was purified by column chromatography with petrol ether as eluent to afford 15h (40 mg, 77% yield).

¹H NMR (400 MHz, CDCl₃) δ 8.22–7.93 (m, 2H), 7.86–7.84 (m, 1H), 7.63–7.57 (m, 2H), 7.39–7.34 (m, 2H), 4.61 (dd, J = 11.0, 4.6 Hz, 1H), 3.52–3.48 (m, 3H), 1.61 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 179.83, 174.88, 137.11, 136.19, 135.26, 133.52 (q, J = 33.0 Hz), 132.26, 129.82, 129.57, 127.15 (q, J = 4.8 Hz), 123.64 (q, J = 281.0 Hz), 121.85 (q, J = 276.3 Hz), 114.84, 110.20, 80.79 (q, J = 31.3 Hz), 66.56, 58.99, 23.75. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.96, −62.02, −76.36, −76.47. HRMS (EI) calcd for C₂₂H₁₇F₆N₃O₂S [M + H]⁺: 502.1018, found: 502.1020.

4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(4-(2,2,2-trifluoroacetyl)phenyl) imidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (15i). In a 25 mL round-bottomed flask, 13b (49 mg, 0.1 mmol), DMSO (5 mL), and IBX (84 mg, 0.3 mmol) were added. The mixture was stirred for 12 h at room temperature. After the reaction, the mixture was poured over and filtered through a silica gel pad under vacuum the filtrate was added with EA (15 mL) and washed with water (20 mL × 3) and saturated brine (15 mL × 3). The organic layer was separated and dried over Na₂SO₄. After filtration and evaporation, the resulting crude product was purified by column chromatography with petrol ether as eluent to afford 15i (45 mg, 90% yield).

¹H NMR (400 MHz, CDCl₃) δ 8.24 (dd, J = 22.9, 8.3 Hz, 2H), 7.99 (dd, J = 9.2, 4.7 Hz, 2H), 7.92–7.77 (m, 1H), 7.58 (dd, J = 22.9, 8.6 Hz, 2H), 1.79–1.52 (m, 6H). ¹³C NMR (101 MHz, CDCl3) δ 179.73, 179.49 (q, J = 35.1 Hz), 174.43, 141.68, 136.84, 135.35, 133.69 (q, J = 34.9 Hz), 132.18, 131.66, 130.62, 130.55, 127.07 (q, J = 5.1 Hz), 121.86 (q, J = 273.4 Hz), 116.46 (q, J = 290.1 Hz), 114.76, 110.42, 66.76, 23.95. ¹⁹F NMR (376 MHz, CDCl₃) δ −61.97, −71.55. HRMS (EI) calcd for C₂₁H₁₃F₆N₃O₂S [M + H]⁺: 486.0705, found: 486.0708.

4.3 Cell proliferation inhibition assay

Cell viability was determined using the CCK8 assay method. The cells were seeded in a 96-well plate at 3 × 10³ cells per well for 24 hours (37 °C, 5% CO₂), and an equal volume of medium containing increasing concentration of inhibitors was added to each well. After a 5 days incubation, CCK8 reagent was added for 1.5 hours. The light absorption (OD) of the 96-well plate was measured at 490 nm using a SpectraMAX M5 microplate spectrophotometer (Molecular Devices). All experiments were performed in triplicate. The percentage of viable cells was calculated and compared with that of the control cells, the half maximal inhibitory concentration (IC₅₀) was calculated by GraphPad Prism5 software.

4.4 Western blot analysis

Cells were treated with a series of concentrations of 13b and ENT for 5 days at 37 °C, then the cells were harvested, washed in ice-cold PBS, analyzed with RIPA buffer (10 mM Tris–HCl (pH 7.8), 1% NP40, 0.15 M NaCl, 1 mM EDTA, 10 μM aprotinin, 1 mM NaF and 1 mM Na₃VO₄), protease inhibitors, phosphatase cocktails A and B, and PMSF (1 mM). Protein concentration was determined by the BCA Protein Assay Kit (beyotime#p0012s), the sample proteins were separated by 10% SDS-PAGE gel and transferred onto 0.2 um polyvinylidene difluoride membranes (millpore#ISEQ00010), then incubated overnight at 4 °C with the AR or PSA antibody overnight at the indicated concentrations in 5% BSA/TBST buffer with gently shaking, then washing with 1 × TBS/T 3 times and followed by incubation for 1.5 hour with a 1/5000 dilution of secondary HRP antibody in 5% nonfat milk/TBST. The target blots were detected with a chemiluminescence system.

4.5 In vivo model

The animal studies were carried out under protocols approved by the animal protection law of the People's Republic of China Care and Use Committees. All rodent studies were also carried out in accordance with the guidelines of Institutional Animal Care and Treatment Committee of Sichuan University in China. The male CB17 SCID mice were purchased (Beijing HFK Bioscience Co. ltd., Beijing, China). LNCap-AR cells were harvested during the exponential-growth phase, washed twice with serum-free medium, and re-suspended at a concentration of 1 × 10⁷ mL⁻¹. One hundred microliters of the cell suspension was injected subcutaneously into the hind flank of each male CB17 SCID mouse (6–7 weeks old) after castrated 2 days. The tumors were allowed to grow to 100–150 mm³, at which point the mice were randomized into 5 groups (8 mice for each group). The mice were dosed orally with 13b (15 mg per kg q.d, 15 mg per kg q.3d, or 5 mg per kg q.3d), vehicle, and the reference compound ENT (15 mg per kg q.d). Tumor growth and body weight were measured every 3 days using vernier calipers for the duration of the treatment. The volume was calculated as follows: tumor volume = a × b²/2 (a, long diameter; b, short diameter).

Acknowledgements

This work is financially supported by Hinova Pharmaceuticals Inc. and also partially supported by National Natural Science Foundation of China (No. 81472418, 81672951). We also thank the State Key Laboratory of Biotherapy and Cancer Center of Sichuan University for providing analytical service.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra02142a

‡ These authors contributed equally.

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