Synthesis of curcumin derivatives for suppression of castration-resistant prostate cancer

Abstract

In this work, 20 curcumin derivatives containing halogen atoms or nitrogen atoms were synthesized for suppression of castration-resistant prostate cancer. These curcumin derivatives were prepared by aldol condensation between the substituted benzaldehydes and acetylacetone or ethyl 3-acetyl-4-oxopentanoate. All synthesized compounds were characterized by ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, high resolution mass, ultraviolet–visible spectroscopy, fluorescence spectroscopy, and high-performance liquid chromatography. The in vitro cytotoxicity of the prepared compounds against 22Rv1 cells was evaluated by the standard MTT assay. The compounds with good inhibitory activity against 22Rv1 cells were further tested for androgen receptor suppression effect. The results show that all compounds were successfully prepared and the purities are all over 90%. Among all synthesized compounds, compound p and s exhibit better inhibitory activity against 22Rv1 cells as compared to dimethylcurcumin, compound q and r display similar inhibitory activity against 22Rv1 cells as compared to dimethylcurcumin. Compounds p-s also demonstrate enhanced androgen receptor suppression effect against 22Rv1 cells as compared to dimethylcurcumin. This work indicates that compounds p-s show promising inhibitory activity against castration-resistant prostate cancer cells by suppression of androgen receptor.

Introduction

Prostate cancer is the most frequently diagnosed cancer in males in the western countries. ¹ Androgen deprivation therapy (ADT) targeting androgens via surgical or medical castration is used as the primary therapeutic strategy for patients with advanced prostate cancer.^2,3 However, the efficacy of ADT is limited because prostate cancer cells usually progress to a castration-resistant phenotype over 1–2 years of therapy.^4,5 The newly developed anti-androgen therapies with either androgen receptor (AR) antagonists or androgen biosynthesis inhibitors are demonstrated to be beneficial.^6,7 However, these therapies might also induce drug resistance due to the alterations in AR/androgens axis. ⁸

Curcumin (Scheme 1) is a well-known compound found in turmeric. Numerous studies have reported the remarkable cancer preventive and therapeutic properties of curcumin. ⁹ In addition, various curcumin derivatives (Scheme 1) have been designed and synthesized to discover new compounds with better anticancer properties.^10,11 Among these, dimethylcurcumin (DMC, ASC-J9) is found to exhibit efficient prostate cancer cell inhibition effect.^12–14 DMC is reported to selectively promote AR degradation, resulting in the inhibition of AR transcriptional activity and prostate cancer cell growth.^15–17 In addition, DMC is proven to be effective in the control of various AR-associated diseases.^18,19 Compared with anti-androgen therapies, anti-AR therapy with DMC might better suppress castration-resistant prostate cancer progression and metastasis. ¹⁹

Scheme 1.

The chemical structure of curcumin and its derivatives.

To discovery novel curcumin derivatives with better castration-resistant prostate cancer cell inhibition effect, 20 curcumin derivatives containing halogen atoms or nitrogen atoms were synthesized by aldol condensation in the present work. The synthesized compounds were fully characterized. The in vitro cytotoxicity and AR protein suppression effect against castration-resistant prostate cancer cells of these compounds were assessed and compared with DMC.

Results and discussion

Curcumin derivatives containing halogen atoms or nitrogen atoms were prepared by an one-pot method, ²⁰ Scheme 2. Acetylacetone or ethyl 3-acetyl-4-oxopentanoate was first reacted with boric acid in N-dimethylformamide (DMF) and then reacted with substituted benzaldehydes in the presence of tributyl borate and n-butylamine. After reaction, acetic acid aqueous solution (20%) was added to afford curcumin derivatives a-t (Figure 1) as precipitates. The 2,4-diacetylaminobenzaldehyde used for preparation of compound k was synthesized by acetylation of 2,4-diaminobenzaldehyde with acetic anhydride, Scheme S1. The ethyl 3-acetyl-4-oxopentanoate used for preparation of compounds q and r was synthesized by substitution reaction between acetylacetone and ethyl chloroacetate, Scheme S2. The chemical structures of compounds a-t were confirmed by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, ¹⁹F NMR, and high-resolution mass spectrometry (HRMS). The ultraviolet–visible spectroscopy (UV-Vis) and fluorescence spectra of compounds a-t were measured to determine the maximum absorbance wavelength and maximum emission wavelength. The purities of compounds a-t are all over 90% as determined by high-performance liquid chromatography (HPLC).

Scheme 2.

Synthetic scheme of curcumin derivatives.

Figure 1.

Chemical structures of the synthesized curcumin derivatives a-t and DMC.

The in vitro cytotoxicity of compounds a-t against 22Rv1 cells were studied by a standard MTT method. DMC, which is an AR degradation enhancer, exhibits efficient inhibitory effect on androgen-responsive and androgen-independent prostate cancer cells. Therefore, DMC was used as a positive control here. The compounds with different concentrations were incubated with 22Rv1 cells for 48 h and 72 h. All tested compounds exhibit dose-dependent cytotoxicity against 22Rv1 cells. The average half maximum inhibitory concentrations of compounds a-t and DMC against 22Rv1 cells were presented in Table 1.

Table 1.

Average half maximum inhibitory concentration (IC₅₀) of DMC and a-t against 22Rv1 cells.

Compound no.	IC50 (μg/mL)
	48 h	72 h
a	8.791	8.516
b	10.727	8.602
c	13.322	11.700
d	8.966	8.653
e	16.849	8.695
f	92.945	92.934
g	51.458	25.600
h	33.730	33.448
i	25.323	16.044
j	8.574	5.510
k	90.657	47.449
l	27.421	11.253
m	54.392	13.220
n	19.774	20.097
o	8.897	6.538
p	3.423	1.710
q	4.355	4.150
r	6.981	3.558
s	3.464	2.601
t	29.090	4.758
DMC	4.029	4.116

As for curcumin derivatives containing halogen atoms, compounds c and d with 3-fluoro-4-methoxy and 3-methoxy-4-fluoro substituents on benzene rings exhibit enhanced cytotoxicity against 22Rv1 cells as compared to compounds h and i with 3-bromo-4-methoxy and 3-methoxy-4-bromo substituents on benzene rings, compounds h and i exhibit higher cytotoxicity against 22Rv1 cells than compounds f and g with 3-chloro-4-methoxy and 3-methoxy-4-chloro substituents on benzene rings. These results indicate that the activity of the halogen substituents ranks as F > Br > Cl. Compared with Cl and Br, F can form hydrogen bonding interaction with amino acids due to its strong electronegativity and small atomic radius. Halogens can form halogen-bonding interaction with amino acids. The strength of halogen-bonding interaction is correlated with the size of the halogen atoms. The halogen atom with larger size usually displays stronger halogen bonding interaction. This might explain the activity of F > Br > Cl. The above results also indicate that 3-halo-4-methoxy substituents exhibit higher activity than the corresponding 3-methoxy-4-halo substituents. Compounds a and b exhibits similar cytotoxicity against 22Rv1 cells as compared to compound d, suggesting that 3,4-di(difluoromethoxy), 3,4-difluoro, and 3-fluoro-4-methoxy substituents have similar activity. Compound e display enhanced cytotoxicity against 22Rv1 cells as compared to compounds f and g, suggesting that 2-chloro-3,4-dimethoxy substituent shows higher activity than 3-chloro-4-methoxy and 3-methoxy-4-chloro substituents. The activity of the halogen containing substituents ranks as 3,4-di(difluoromethoxy), 3-fluoro-4-methoxy, 3,4-difluoro > 3-methoxy-4-fluoro, 2-chloro-3,4-dimethoxy > 3-bromo-4-methoxy > 3-methoxy-4-bromo > 3-chloro-4-methoxy > 3-methoxy-4-chloro.

Among the prepared curcumin derivatives j-p that contain nitrogen atoms, compounds j, o, and p exhibit significantly higher cytotoxicity against 22Rv1 cells than compounds k-n, and compound p exhibits the highest cytotoxicity against 22Rv1 cells. It is worth noting that the cytotoxicity of compound p against 22Rv1 cells is also higher than DMC. The activity of the nitrogen containing substituents ranks as 3-methylsulfonamide > 4-methylsulfonamide, 4-acetylamino > 4-(4-morpholinyl), 4-dimethylamino > 4-(N-methyl-N-(2-hydroxyethyl)amino) > 2,4-diacetylamino.

Compound q and s exhibit enhanced cytotoxicity against 22Rv1 cells as compared to compound o and j, respectively, whereas compound r and t exhibit reduced cytotoxicity against 22Rv1 cells as compared to compound p and DMC, respectively. These results indicate that the influence of the α-substituted β-diketone structure on 22Rv1 inhibitory activity is complicated and might vary with the different substituents on the benzene rings. It is worth noting that compound s exhibits enhanced cytotoxicity, and compounds q and r exhibit similar cytotoxicity against 22Rv1 cells as compared to DMC.

Taken together, these results demonstrate that compounds p-s exhibit enhanced cytotoxicity against 22Rv1 cells as compared to other prepared compounds. Among these compounds, compound p and s exhibit better inhibitory activity against 22Rv1 cells as compared to DMC and compound q and r show similar inhibitory activity against 22Rv1 cells as compared to DMC.

DMC is reported to suppress AR-related signal pathways by degradation of AR, resulting in suppression effect on castration-resistant prostate cancer (CRPC) cell proliferation. Accordingly, compounds p-t were evaluated regarding AR protein suppression effect. As shown in Figure 2, compounds p-s all exhibit enhanced AR suppression effect as compared to DMC, suggesting that compounds p-s could inhibit 22Rv1 cell growth by suppression of AR. Compound t exhibits no AR suppression effect, indicating that compound t might suppress 22Rv1 cell growth by other pathways.

Figure 2.

AR protein suppression effect of compounds p-t and DMC (2 μg/mL).

Conclusion

In summary, curcumin derivatives containing halogen atoms or nitrogen atoms were prepared for castration-resistant prostate cancer cell inhibition. Among compounds a-i, the activity of the halogen containing substituents on the benzene rings ranks as 3,4-di(difluoromethoxy), 3-fluoro-4-methoxy, 3,4-difluoro > 3-methoxy-4-fluoro, 2-chloro-3,4-dimethoxy > 3-bromo-4-methoxy > 3-methoxy-4-bromo > 3-chloro-4-methoxy > 3-methoxy-4-chloro. Among compounds j-p, the activity of the nitrogen-containing substituents on the benzene rings ranks as 3-methylsulfonamide > 4-methylsulfonamide, 4-acetylamino > 4-(4-morpholinyl), 4-dimethylamino > 4-(N-methyl-N-(2-hydroxyethyl)amino) > 2,4-diacetylamino. The effect of the α-substituted β-diketone structure on 22Rv1 inhibitory activity might vary with the different substituents on the benzene rings. Among all synthesized compounds, compound p and s exhibit better inhibitory activity against 22Rv1 cells as compared to DMC, compound q and r display similar inhibitory activity against 22Rv1 cells as compared to DMC. Compounds p-s also exhibit enhanced AR suppression effect against 22Rv1 cells as compared to DMC. Compounds p-s show significant potential for the inhibition of castration-resistant prostate cancer cells by suppression of AR.

Materials and methods

Materials

Acetylacetone (99%), boric acid (99%), tributyl borate (98%), n-butylamine (98%), substituted benzaldehydes (95%-99%), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, 98%) were purchased from Aladdin Reagent Co., Ltd. (China). Roswell Park Memorial Institute 1640 (RPMI1640) medium, fetal bovine serum (FBS), penicillin-streptomycin solution, trypsin-EDTA (0.25%) were purchased from Fisher Scientific International, Inc. (USA). All other chemicals were of HPLC grade or analytical grade and used as received.

Synthesis of 2,4-diacetylaminobenzaldehyde

2,4-Diacetylaminobenzaldehyde was synthesized by the reaction between 2,4-diaminobenzaldehyde and acetic anhydride in the presence of triethylamine, Scheme S1. Briefly, 2,4-diaminobenzaldehyde (544 mg, 4 mmol) was dissolved in 30 mL of dichloromethane. To the solution, acetic anhydride (2.04 g, 20 mmol) and triethylamine (1.2 g, 12 mmol) were added and the mixture was refluxed for 48 h. After that, the reaction mixture was evaporated to remove the solvent, and ethanol was added to precipitate the product. The precipitate was isolated by filtration, washed with ethanol for three times and dried under vacuum afford 2,4-diacetylaminobenzaldehyde as a faint yellow solid (yield: 92%). ¹H NMR (400 MHz, DMSO-D₆): δ (ppm) 10.90 (s, 1H), 10.42 (s, 1H), 9.79 (s, 1H), 8.42 (d, J = 1.8 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.66 (dd, J = 8.5 Hz, 1.8 Hz, 1H), 2.13 (s, 3H), 2.06 (s, 3H). ¹³C NMR (100 MHz, DMSO-D₆): δ (ppm) 193.8, 169.8, 169.7, 146.1, 141.6, 136.3, 119.1, 113.8, 109.8, 25.1, 24.8. HRMS: m/z of [M+Na]⁺ calculated for C₁₁H₁₂N₂O₃: 243.0746, found: 243.0746. m.p.: 232.8–234.4 °C. HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 254 nm): Retention time 2.997 min, estimated purity 98.1%.

Synthesis of ethyl 3-acetyl-4-oxopentanoate

Ethyl 3-acetyl-4-oxopentanoate was synthesized by the reaction between ethyl chloroacetate and acetylacetone in the presence of potassium carbonate,^21,22 Scheme S2. In brief, potassium carbonate (13.8 g, 100 mmol) was dispersed in 60 mL of ethanol. To the mixture, acetylacetone (10.0 g, 100 mmol) was added. Then, ethyl chloroacetate (13.5 g, 110 mmol) dissolved in 20 mL of ethanol was dropwise added. The resulting mixture was heated to 60 °C and stirred for 24 h. The solvent was removed by evaporation under vacuum and deionized water was added to the residues to form two phases. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate for three times. The obtained organic phases were combined and dried overnight with anhydrous manganese sulfate. The crude product was obtained by removing the solvent by evaporation under vacuum, which was further purified by column chromatography (eluent: ethyl acetate/petroleum ether = 1:8, v/v) to afford ethyl 3-acetyl-4-oxopentanoate as a pale yellow oil (yield: 71%). HRMS: m/z of [M+Na]⁺ calculated for C₉H₁₄O₄: 209.0790, found: 209.0789.

Synthesis of curcumin derivatives

Briefly, 10 mmol of acetylacetone (or ethyl 3-acetyl-4-oxopentanoate) and 5 mmol of boric acid were added to 10 mL of DMF and the resulting mixture was heated to 80 °C and stirred for 20 min. Then, 20 mmol of tributyl borate and 20 mmol of substituted benzaldehyde were added and stirred at 80° for 30 min. After that, 10 mmol of n-butylamine was added and the resulting mixture was stirred at 80° for 1–3 h. After reaction, 50 mL of acetic acid aqueous solution (20%) was added to the reaction mixture and stirred overnight. The formed precipitate was isolated by filtration and purified by recrystallization in methanol.

a Yield: 23%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.77 (s, 2H), 7.70 (dd, J = 8.5 Hz, 2.0 Hz, 2H), 7.66 (d, J = 16.0 Hz, 2H), 7.42 (d, 8.5 Hz, 2H), 7.30 (t, J_FH = 23 Hz, 2H), 7.27 (t, J_FH = 23 Hz, 2H), 7.02 (d, J = 16.0 Hz, 2H), 6.23 (s, 1H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.5, 143.5, 142.3, 138.9, 133.4, 127.3, 126.0, 121.0 (d, J_FC = 50 Hz), 118.9 (d, J_FC = 25 Hz), 116.8 (d, J _FC = 25 Hz), 114.8 (d, J_FC = 25 Hz), 102.6. ¹⁹F NMR (470 MHz, DMSO-D₆): δ (ppm) -82.0, -82.4. HRMS: m/z of [M+Na]⁺ calculated for C₂₃H₁₆F₈O₆: 563.0717, found: 563.0699. UV-Vis (CH₃CN): Maximum absorbance wavelength 392 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 453 nm. m.p.: 112.4–113.5 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 8.493 min, estimated purity 99.5%.

b Yield: 28%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.92 (m, 2H), 7.56–7.69 (m, 4H), 7.52 (q, J = 8.5 Hz, 2H), 7.01 (d, J = 16.0 Hz, 2H), 6.12 (s, 1H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.5, 152.0, 151.1, 150.0, 149.3, 138.7, 133.1, 126.6, 126.0, 118.5 (d, J_FC = 17.5 Hz), 117.2 (d, J_FC = 17.5 Hz), 102.8. ¹⁹F NMR (470 MHz, DMSO-D₆): δ (ppm) –135.5, –135.6, –137.9, –137.9. HRMS: m/z of [M+Na]⁺ calculated for C₁₉H₁₂F₄O₂: 371.0671, found: 371.0677. UV-Vis (CH₃CN): Maximum absorbance wavelength 387 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 449 nm. m.p.: 122.6–125.0 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 12.601 min, estimated purity 97.7%.

c Yield: 45%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.70 (dd, J_FH = 12.5 Hz, 2.0 Hz, 2H), 7.58 (d, J = 16.0 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.22 (t, J_FH = 8.5 Hz, 2H), 6.88 (d, J = 16.0 Hz, 2H), 6.06 (s, 1H), 3.89 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 153.0, 151.1, 149.4 (d, J_FC = 12.5 Hz), 139.6, 128.4 (d, J_FC = 5 Hz), 126.8, 123.7, 115.2 (d, J_FC = 17.5 Hz), 114.3, 102.2, 55.6. ¹⁹F NMR (470 MHz, DMSO-D₆): δ (ppm) -134.8. HRMS: m/z of [M+Na]⁺ calculated for C₂₁H₁₈F₂O₄: 395.1071, found: 395.1078. UV-Vis (CH₃CN): Maximum absorbance wavelength 406 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 479 nm. m.p.: 167.6–168.3 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 9.359 min, estimated purity 99.8%.

d Yield: 28%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.63 (d, J = 16.0 Hz, 2H), 7.57 (dd, J = 8.5 Hz, 1.5 Hz, 2H), 7.24–7.36 (m, 4H), 6.97 (d, J = 16.0 Hz, 2H), 6.17 (s, 1H), 3.91 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.7, 154.2, 152.2, 148.0 (d, J_FC = 10 Hz), 140.1, 132.3 (d, J_FC = 5 Hz), 124.8, 122.4 (d, J_FC = 12.5 Hz), 116.8 (d, J_FC = 17.5 Hz), 113.5, 102.1, 56.6. ¹⁹F NMR (470 MHz, DMSO-D₆): δ (ppm) -131.7. HRMS: m/z of [M+Na]⁺ calculated for C₂₁H₁₈F₂O₄: 395.1071, found: 395.1078. UV-Vis (CH₃CN): Maximum absorbance wavelength 396 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 453 nm. m.p.: 162.4–163.4 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 9.493 min, estimated purity 99.8%.

e Yield: 24%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.88 (d, J = 16.0 Hz, 2H), 7.77 (d, J = 9.0 Hz, 2H), 7.17 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 16.0 Hz, 2H), 6.15 (s, 1H), 3.91 (s, 6H), 3.77 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.3, 155.5, 145.4, 135.7, 129.0, 125.8, 125.23, 124.1, 112.4, 102.8, 60.6, 56.8. HRMS: m/z of [M+Na]⁺ calculated for C₂₃H₂₂Cl₂O₆: 487.0691, found: 487.0671. UV-Vis (CH₃CN): Maximum absorbance wavelength 406 nm. Fluorescence spectrum (CH₃CN, excitation at 420 nm): Maximum emission wavelength 492 nm. m.p.: 176.8–179.1 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 360 nm): Retention time 14.519 min, estimated purity 98.4%.

f Yield: 68%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.91 (d, J = 1.7 Hz, 2H), 7.70 (dd, J = 8.5 Hz, 1.6 Hz, 2H), 7.58 (d, J = 16.0 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 16.0 Hz, 2H), 6.07 (s, 1H), 3.92 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 156.5, 139.3, 129.8, 129.7, 128.9, 123.7, 122.3, 113.5, 102.3, 56.9. HRMS: m/z of [M+Na]⁺ calculated for C₂₁H₁₈Cl₂O₄: 427.0480, found: 427.0488. UV-Vis (CH₃CN): Maximum absorbance wavelength 405 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 481 nm. m.p.: 144.3–147.4 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 12.318 min, estimated purity 98.2%.

g Yield: 82%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.65 (d, J = 16.0 Hz, 2H), 7.52 (d, J = 1.6 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 7.33 (dd, J = 8.3 Hz, 1.7 Hz, 2H), 7.04 (d, J = 16.0 Hz, 2H), 6.19 (s, 1H), 3.93 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 155.3, 140.0, 135.5, 130.7, 125.63, 123.5, 122.1, 112.5, 102.5, 56.7. HRMS: m/z of [M+Na]⁺ calculated for C₂₁H₁₈Cl₂O₄: 427.0480, found: 427.0494. UV-Vis (CH₃CN): Maximum absorbance wavelength 395 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 456 nm. m.p.: 165.3–169.9 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 13.986 min, estimated purity 99.6%.

h Yield: 55%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 8.04 (s, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 16.0 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 6.92 (d, J = 16.0 Hz, 2H), 6.07 (s, 1H), 3.91 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 157.3, 139.2, 132.9, 130.3, 129.4, 123.7, 113.3, 111.9, 102.3, 57.0. HRMS: m/z of [M (⁷⁹Br: ⁸¹Br = 1:1)+Na]⁺calculated for C₂₁H₁₈Br₂O₄: 516.94, found: 516.9454. UV-Vis (CH₃CN): Maximum absorbance wavelength 405 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 494 nm. m.p.: 132.2–133.3 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 14.629 min, estimated purity 98.6%.

i Yield: 54%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.66 (d, J = 2.8 Hz, 2H), 7.64 (d, J = 5.0 Hz, 2H), 7.48 (d, J = 1.3 Hz, 2H), 7.28 (dd, J = 8.3 Hz, 1.3 Hz, 2H), 7.07 (d, J = 16.0 Hz, 2H), 6.21 (s, 1H), 3.93 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 156.2, 140.1, 136.2, 133.8, 125.7, 122.6, 113.4, 112.3, 102.5, 56.9. HRMS: m/z of [M (⁷⁹Br: ⁸¹Br = 1:1)+Na]⁺ calculated for C₂₁H₁₈Br₂O₄: 516.94, found: 516.9473. UV-Vis (CH₃CN): Maximum absorbance wavelength 400 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 465 nm. m.p.: 173.6–176.5 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 16.804 min, estimated purity 99.9%.

j Yield: 57%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 10.19 (s, 2H), 7.67 (s, 8H), 7.58 (d, J = 16.0 Hz, 2H), 6.83 (d, J = 16.0 Hz, 2H), 6.12 (s, 1H), 2.08 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 169.2, 141.7, 140.4, 129.8, 129.7, 123.0, 119.4, 101.9, 24.6. HRMS: m/z of [M+Na]⁺ calculated for C₂₃H₂₂N₂O₄: 413.1477, found: 413.1482. UV-Vis (CH₃CN): Maximum absorbance wavelength 415 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 505 nm. m.p.: 271.5–272.4 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 3.501 min, estimated purity 98.6%.

k Yield: 46%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 10.20 (s, 2H), 9.88 (s, 2H), 7.82 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 16.0 Hz, 4H), 7.53 (d, J = 8.5 Hz, 2H), 6.82 (d, J = 16.0 Hz, 2H), 6.10 (s, 1H), 2.11 (s, 6H), 2.07 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.7, 169.2, 169.2, 141.8, 138.3, 136.1, 127.7, 124.1, 123.4, 116.7, 102.5, 24.6, 23.8. HRMS: m/z of [M+Na]⁺ calculated for C₂₇H₂₈N₄O₆: 527.1907, found: 527.1910. UV-Vis (CH₃CN): Maximum absorbance wavelength 332 nm. Fluorescence spectrum (CH₃CN, excitation at 420 nm): Maximum emission wavelength 539 nm. m.p.: 240.8–246.4 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 2.488 min, estimated purity 94.9%.

l Yield: 30%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.45–7.60 (m, 6H), 6.73 (m, 4H), 6.61 (d, J = 15.7 Hz, 1H), 6.51 (d, J = 15.7 Hz, 1H), 5.97 (s, 1H), 3.00 (s, 12 H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.4, 152.1, 141.2, 141.0, 130.5, 130.4, 122.6, 119.0, 117.5, 112.4, 112.3, 100.4, 40.2. HRMS: m/z of [M+Na]⁺ calculated for C₂₃H₂₆N₂O₂: 385.1892, found: 385.1899. UV-Vis (CH₃CN): Maximum absorbance wavelength 479 nm. Fluorescence spectrum (CH₃CN, excitation at 480 nm): Maximum emission wavelength 609 nm. m.p.: 136.6–140.5 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 500 nm): Retention time 12.062 min, estimated purity 99.4%.

m Yield: 15%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.52 (d, J = 8.5 Hz, 4H), 6.74 (d, J = 8.5 Hz, 4H), 6.58 (d, J = 15.7 Hz, 2H), 5.95 (s, 1H), 4.75 (t, J = 4.5 Hz, 2H), 3.56 (q, J = 5.5 Hz, 4H), 3.47 (t, J = 5.5 Hz, 4H), 3.01 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.4, 151.2, 141.0, 130.6, 122.2, 118.7, 112.1, 100.9, 58.6, 54.4, 39.2. HRMS: m/z of [M+Na]⁺ calculated for C₂₅H₃₀N₂O₄: 445.2103, found: 445.2109. UV-Vis (CH₃CN): Maximum absorbance wavelength 484 nm. Fluorescence spectrum (CH₃CN, excitation at 480 nm): Maximum emission wavelength 598 nm. m.p.: 179.5–181.6 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 500 nm): Retention time 3.853 min, estimated purity 95.6%.

n Yield: 42%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.59 (d, J = 8.5 Hz, 4H), 7.55 (d, J = 15.7 Hz, 2H), 6.99 (d, J = 8.5 Hz, 4H), 6.71 (d, J = 15.7 Hz, 2H), 6.03 (s, 1H), 3.74 (t, J = 4.5 Hz, 8H), 3.24 (t, J = 4.5 Hz, 8H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.5, 152.8, 140.7, 130.3, 125.3, 120.7, 114.8, 66.4, 47.7. HRMS: m/z of [M+Na]⁺ calculated for C₂₇H₃₀N₂O₄: 469.2103, found: 469.2083. UV-vis (CH₃CN): Maximum absorbance wavelength 455 nm. Fluorescence spectrum (CH₃CN, excitation at 450 nm): Maximum emission wavelength 604 nm. m.p.: 268.6–270.3 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 450 nm): Retention time 8.072 min, estimated purity 99.2%.

o Yield: 52%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 10.13 (s, 2H), 7.71 (d, J = 8.5 Hz, 4H), 7.61 (d, J = 16.0 Hz, 2H), 7.26 (d, J = 8.5 Hz, 4H), 6.86 (d, J = 16.0 Hz, 2H), 6.16 (s, 1H), 3.08 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.6, 140.9, 140.2, 130.2, 130.2, 123.4, 119.3, 101.9. HRMS: m/z of [M+Na]⁺ calculated for C₂₁H₂₂N₂O₆S₂: 485.0817, found: 485.0835. UV-Vis (CH₃CN): Maximum absorbance wavelength 403 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 482 nm. m.p.: 237.5–239.4 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 2.677 min, estimated purity 96.6%.

p Yield: 73%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 9.91 (s, 2H), 7.64 (d, J = 16.0 Hz, 2H), 7.50 (m, 4H), 7.43 (t, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 16.0 Hz, 2H), 6.35 (s, 1H), 3.06 (s, 6H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 183.5, 140.5, 139.5, 136.2, 130.5, 125.3, 124.1, 122.1, 119.6, 102.2. HRMS: m/z of [M+Na]⁺ calculated for C₂₁H₂₂N₂O₆S₂: 485.0817, found: 485.0799. UV-Vis (CH₃CN): Maximum absorbance wavelength 380 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 451 nm. m.p.: 252.8–254.9 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 2.827 nm, estimated purity 95.7%.

q Yield: 52%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 10.17 (s, 1H), 10.14 (s, 1H), 7.74 (t, J = 8.5 Hz, 4H), 7.68 (d, J = 16.0 Hz, 1H), 7.65 (d, J = 15.5 Hz, 1H), 7.25 (t, J = 8.5 Hz, 4H), 7.22 (d, J = 15.5 Hz, 1H), 7.10 (d, J = 16.0 Hz, 1H), 4.98 (t, J = 7.3 Hz, 1H), 4.10 (q, J = 7.0 Hz, 1H), 4.05 (q, J = 7.0 Hz, 1H), 3.87 (s, 1H), 3.08 (s, 3H), 3.07 (s, 3H), 2.92 (d, J = 7.3 Hz, 1H), 1.17 (td, J = 7.0 Hz, 2.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 194.4, 183.8, 172.4, 171.6, 143.7, 141.6, 141.4, 140.9, 130.7, 130.5, 130.4, 129.5, 124.5, 120.0, 119.2, 119.1, 106.2, 60.9, 69.7, 58.0, 32.8, 31.3, 14.6, 14.5. HRMS: m/z of [M+Na]⁺ calculated for C₂₅H₂₈N₂O₈S₂: 571.1185, found: 571.1173. UV-Vis (CH₃CN): Maximum absorbance wavelength 424 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 501 nm. m.p.: 159.4–160.4 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 2.809 min, estimated purity 99.5%.

r Yield: 45%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 9.89 (s, 2H), 7.72 (d, J = 16.0 Hz, 1H), 7.67 (d, J = 15.5 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.42 (q, J = 8.3 Hz, 2H), 7.23–7.32 (m, 3H), 7.11 (d, J = 16.0 Hz, 1H), 5.12 (t, J = 7.3 Hz, 1H), 4.12 (q, J = 7.0 Hz, 1H), 4.06 (q, J = 7.0 Hz, 1H), 3.87 (s, 1H), 3.06 (s, 3H), 3.05 (s, 3H), 2.94 (d, J = 7.3 Hz, 1H), 1.17 (td, J = 7.0 Hz, 2.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 194.8, 183.8, 172.2, 171.5, 143.8, 141.8, 139.5, 139.5, 136.4, 135.7, 130.5, 130.4, 126.4, 124.3, 124.1, 122.7, 122.0, 120.5, 120.3, 106.7, 61.0, 60.8, 57.7, 32.8, 31.4, 14.6, 14.5. HRMS: m/z of [M+K]⁺ calculated for C₂₅H₂₈N₂O₈S₂: 587.0924, found: 587.1116. UV-Vis (CH₃CN): Maximum absorbance wavelength 398 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 473 nm. m.p.: 176.6–181.2 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 2.972 min, estimated purity 99.7%.

s Yield: 55%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 10.19 (s, 2H), 7.60–7.75 (m, 10H), 7.20 (d, J = 15.5 Hz, 1H), 7.08 (d, J = 16.0 Hz, 1H), 4.95 (t, J = 7.3 Hz, 1H), 4.10 (q, J = 7.0 Hz, 1H), 4.05 (q, J = 7.0 Hz, 1H), 3.86 (s, 1H), 2.91 (d, J = 7.3 Hz, 1H), 2.08 (s, 3H), 2.07 (s, 3H), 1.16 (t, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 194.4, 183.8, 169.2, 143.9, 142.3, 141.8, 130.2, 130.0, 129.1, 124.1, 119.6, 119.3, 60.8, 24.6, 14.6, 14.5. HRMS: m/z of [M+Na]⁺ calculated for C₂₇H₂₈N₂O₆: 499.1845, found: 499.1853. UV-vis (CH₃CN): Maximum absorbance wavelength 424 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 505 nm. m.p.: 216.4–217.5 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 2.981 min, estimated purity 95.4%.

t Yield: 69%. ¹H NMR (500 MHz, DMSO-D₆): δ (ppm) 7.67 (d, J = 15.0 Hz, 1H), 7.64 (d, J = 15.0 Hz, 1H), 7.39 (dd, J = 8.5 Hz, 1.7 Hz, 2H), 7.31 (td, J = 7.5 Hz, 1.7 Hz, 2H), 7.16 (dd, J = 16.0 Hz, 2.8 Hz, 2H), 7.03 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 4.96 (t, J = 7.3 Hz, 1H), 4.11 (q, J = 7.0 Hz, 1H), 4.05 (q, J = 7.0 Hz, 1H), 3.90 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 2.93 (d, J = 7.3 Hz, 1H), 1.17 (q, J = 7.0 Hz, 3H). ¹³C NMR (125 MHz, DMSO-D₆): δ (ppm) 194.1, 183.8, 172.6, 171.7, 151.9, 151.5, 149.4, 144.7, 142.4, 128.2, 127.4, 124.4, 123.7, 123.7, 118.9, 112.1, 112.0, 111.3, 110.8, 105.9, 60.8, 60.7, 58.2, 56.1, 56.1, 56.0, 32.7, 31.4, 14.6, 14.5. HRMS: m/z of [M+Na]⁺ calculated for C₂₇H₃₀O₈: 505.1838, found: 505.1835. UV-vis (CH₃CN): Maximum absorbance wavelength 432 nm. Fluorescence spectrum (CH₃CN, excitation at 360 nm): Maximum emission wavelength 520 nm. m.p.: 148.5–153.0 °C (MeOH). HPLC (CH₃CN/H₂O = 80/20, 1 mL/min, 420 nm): Retention time 5.512 min, estimated purity 92.9%.

Characterization

The NMR spectra were recorded on a on a NMR spectrometer (AVANCE III, Bruker) using dimethyl sulfoxide-D6 (DMSO-D₆) as the solvent and tetramethylsilane (TMS) as an internal reference. The HRMS spectra were recorded on an Agilent Technologies 6200 series TOF/6500 series Q-TOF B.06.01 (B6157) instrument (CA, USA). The ultraviolet-visible (UV-Vis) absorbance spectra and fluorescence spectra were recorded on a microplate reader (Spark, Tecan). The HPLC measurement was performed on an Agilent 1260 Infinity HPLC system. The melting point was recorded on a B-545 melting point apparatus (Buchi, Switzerland).

Cell culture

Human prostate cell line 22Rv1 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). 22Rv1 cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 units/mL penicillin and 100 g/mL streptomycin at 37 °C under 5% carbon dioxide atmosphere. 22Rv1 cells are androgen-responsive and androgen-independent human prostate cell line, which express endogenous AR.

In vitro cytotoxicity against 22Rv1 cells

The in vitro cytotoxicity of the curcumin derivatives against 22Rv1 cells was assessed by MTT assay. In briefly, 22Rv1 cells were seeded on 96-well plates in quadruplicates at a cell density of 5 × 10³ cells per well. The curcumin derivatives were dissolved in DMSO and diluted with RPMI1640 medium to the desired concentrations. After incubation with curcumin derivatives with different concentrations for 48 h or 72 h, the cell viability of each group was determined by standard MTT assay. Three separated experiments were performed.

AR protein suppression against 22Rv1 cells

22Rv1 cells were seeded in six-well plates in triplicates at a density of 2 × 10⁵ cells per well. The cells were treated with different compounds (2 μg/mL) for 24 h. Then, the culture medium was removed, and the cells were washed three times with phosphate-buffered saline (PBS; pH 7.4, 6.7 mM). After that, 150 μL RIPA Pyrolysis liquid with 1% protease inhibitor was used to lyse 22Rv1 cells. The lysates we collected into 1.5 mL centrifuge tube and bathed in ice for 30 min and then centrifuged at 12,000 r/min for 5 min. The supernatant was collected as the total protein solution. After quantification, 25% loading buffer was added into protein solution. The total protein solutions were stored at −80 °C for further use.

Equal protein amounts with denaturation treatment were loaded and separated in 5–15% SDS-PAGE acrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. These membranes were blocked with 5% defatted milk powder in tris-buffered saline containing 0.1% Tween-20 for 1.5 h. The membranes were then incubated with 5 mL primary antibodies to AR and β-actin at 4 °C overnight. After that, the membranes were washed three times and probed with horseradish peroxidase-conjugated goat anti-mouse secondary antibody at room temperature for 1.5 h. The protein bands were detected and imaged with the immobilon western chemiluminescent horseradish peroxidase (HRP) substrate on a chemiluminescence imaging analysis system (Tanon-5200Multl, China).

Supplemental Material

sj-docx-1-chl-10.1177_17475198241230042 – Supplemental material for Synthesis of curcumin derivatives for suppression of castration-resistant prostate cancer

Supplemental material, sj-docx-1-chl-10.1177_17475198241230042 for Synthesis of curcumin derivatives for suppression of castration-resistant prostate cancer by Shunze Gong, Chixiang Yuan, Hang Hu and Defeng Xu in Journal of Chemical Research