Design, synthesis, and evaluation of curcumin analogues as potential inhibitors of bacterial sialidase

Abstract

Sialidases are key virulence factors that remove sialic acid from the host cell surface glycan, unmasking receptors that facilitate bacterial adherence and colonisation. In this study, we developed potential agents for treating bacterial infections caused by Streptococcus pneumoniae Nan A that inhibit bacterial sialidase using Turmeric and curcumin analogues. Design, synthesis, and structure analysis relationship (SAR) studies have been also described. Evaluation of the synthesised derivatives demonstrated that compound 5e was the most potent inhibitor of S. pneumoniae sialidase (IC₅₀ = 0.2 ± 0.1 µM). This compound exhibited a 3.0-fold improvement in inhibitory activity over that of curcumin and displayed competitive inhibition. These results warrant further studies confirming the antipneumococcal activity 5e and indicated that curcumin derivatives could be potentially used to treat sepsis by bacterial infections.

Keywords:

• Curcumin
• sepsis
• sialidase
• Nan A

1. Introduction

Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is caused by an overwhelming immune response to an existing bacterial infection¹. It commonly occurs in the ageing population and results in ∼20–30 million cases annually worldwide. Overall, sepsis remains one of the top five causes of death worldwide², also the mortality rate is ∼20–50% for severe sepsis and 40–80% for septic shock. Especially, bacterial sepsis is a major cause of mortality of hospitalised patients, thus the development of drugs for bacterial sepsis is urgently needed and many efforts have been undertaken in the medicinal and pharmaceutical industry.

The Gram-positive bacterium, Streptococcus pneumoniae, is one of the causes of sepsis. It also major human pathogen and causes a variety of diseases, including bacterial meningitis, otitis media, pneumonia, conjunctivitis^3–6. Several virulence factors contribute to colonisation and early infection processes, above all sialidases from bacteria are considered key virulence factors⁷. Sialidase removes the terminal sialic acid residues from host cell surface glycans, unmasking receptors that facilitate bacterial adherence and colonisation⁸. This process causes resistance to penicillin and other antibiotics that are used to treat S. pneumoniae infection⁹. According to known literature, all clinical isolates of S. pneumoniae have sialidases activity known to be involved in sepsis¹⁰. S. pneumoniae sialidase genes in clinical pneumococcal isolates determined that Nan A, Nan B, and Nan C are present in 100%, 96%, and 51% of these strains. Among these sialidases, Nan A has been shown to play an essential role in host–pneumococcal interactions in the respiratory tract and sepsis in mouse models^7,11,12,33. Therefore, high-affinity inhibitors that can block Nan A are potential agents for prevention and treating sepsis. In the last few years, several studies have reported the discovery of viral or bacterial sialidase inhibitors from an isolated natural product such as flavonoids, coumarins, diplacone, mimulone, pterocarpans, and phlorotannins. However, these compounds are known inhibitors as Clostridium perfringens (Cp-Nan I) or viral sialidase^9,14–17. Recently, some studies have reported to inhibition of S. pneumoniae sialidase such as diazenylaryl sulphonic acids, malabaricone C, Artocarpin, and anthraquinone glycosides^9,10,18–20. Therefore, to develop novel bacterial sialidase inhibitors, we focused on the natural product, Turmeric, because it had not yet been evaluated.

Turmeric has been used as a traditional medicine for conditions such as liver disease²¹, indigestion²², rheumatoid arthritis²³, and insect bites²⁴ and is consumed daily by millions of people for the treatment of various diseases. Curcumin is the primary component of Turmeric and has a feruloyl methane group containing methoxy, hydroxyl, and heptadienyl with a 1,3-diketone moiety. Curcumin has been extensively studied in the past few decades as an important therapeutic compound. In addition, it still receives a lot of attention for its biological properties, including anti-inflammatory, anti-viral, anti-bacterial, anti-cancer, anti-oxidant, and anti-carcinogenic activities²⁵, and its use in debilitating diseases such as Crohn’s disease, ulcerative colitis²⁶, and Alzheimer’s disease^27,28. Therefore, many studies evaluating the biological activity of curcumin have been performed and potential curcuminoids have been developed for several diseases.

In this study, we report that Turmeric and curcumin derivatives can targeting the S. pneumoniae Nan A. Designed strategies for synthesis of curcumin analogues are shown Scheme 1.

Scheme 1. Designed strategies for the synthesis of curcumin analogues.

2. Materials and methods

2.1 General

All the chemical reagents used in this work and curcumin (4a) were purchased from commercial suppliers (Aldrich, St. Louis, MO; TCI, Japan; Alfa Aesar, Haverhill, MA or Acros Organics, USA companies) and used without further purification. The ¹H and ¹³C NMR spectra were recorded using a JEOL ECA-500 spectrometer, Japan at 500 MHz and 125 MHz, respectively, with chemical shift (δ) values reported in ppm unit. Multiplicities are describes as singlet (s), doublet (d), doublet of doublet (dd), triplet (t), multiplet (m), and broad exchangeable proton (bs). High resolution mass spectra were obtained on a GC Mate 2, JEOL. A CEM Discover system (No. 908005) fitted with a temperature controller was used for microwave reactions. Irradiation was initiated at 300 W to raise the temperature to the set point (150 °C). Reactions were monitored by thin-layer chromatography (TLC) with Merck’s DC-Fertigiplatterm Kiegel 60 GE254 plates. Visualisation was accomplished with either UV light or by immersion in a solution of phosphomolybdic acid (PMA) followed by heating on a hot plate for ∼10 s. The reaction products were purified by open column chromatography using silica gel produced by Merck (Darmstadt, Germany) (Silica gel 60; 63–200 mesh, ASTM) or Cosmosil 140 C-18 OPN produced by Nacalai Tesque, Inc., USA.

2.2 Expression, purification, and preparation of S. pneumoniae Nan A

We have synthesised and expressed the full-length genes for the S. pneumoniae sialidase (Nan A) in Escherichia coli. The gene encoding Nan A (Figure S28, GenBank accession no. COT45929.1, PDB: 2VVZ) of S. pneumoniae TIGR4 was synthesised (Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany). The synthesised gene was inserted into the cloning sites of a pET151/d-TOPO vector (Invitrogen, Carlsbad, CA) containing a 6x His-tag at the C-terminus. S. pneumoniae sialidase was expressed and purified from E. coli BL21 (DE3) (HIT; Real biotech Co., Taipei, Taiwan). The purified Nan A was detected at ∼56.6 kDa with greater than 90% purity using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure S29). The purified sialidase had specific activities (K_m values) of 43.9 µM using 4-methylumbelli-feryl-α-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA; Catalogue No. M8639; Sigma) as substrate (Figure S30).

2.3 Enzyme inhibition activity

As described, the inhibitory effects of compounds on S. pneumoniae Nan A were measured using a fluorescence (FL)-based assay. In this assay, the 4-methylumbelliferyl-α-D-N-acetylneuraminic acid (Sigma Chemical Co., St. Louis, MO) was used as a substrate, and the enzyme activity was determined by measuring the increase in fluorescence by continuously monitoring the reactions at 450/40 nm with excitation at 365 nm using a SpectraMax M2e Multimode Reader (Molecular Devices Co.). The IC₅₀ values of the synthesised compounds were measured in a reaction mixture containing enzyme (final concentration of Nan A, 2.2 nM), the test compounds (from 0 to 200 µM), and 50 µM of substrate in 20 mM Sodium phosphate buffer (pH 7.5, containing 300 mM NaCl). To determine the enzyme activity, the experimental data were fit to a logistic curve with Equation (1), a time-drive protocol was used and the initial velocity was recorded over a range of concentrations, and the data were analysed using a nonlinear regression program (Sigma Plot; SPCC Inc., Chicago, IL). (1) $$\text{Inhibition} \text{activity} \left(\%\right) =100 – [\left(S\right) –(S_0)/(C -C_0\left)\right]*100,$$(1) where C is the fluorescence of the control (enzyme, buffer, and substrate) after 60 min of incubation, C₀ is the fluorescence of the control at 0 min, S is the fluorescence of the tested samples (enzyme, sample solution, and substrate) after incubation, and S₀ is the fluorescence of the tested samples at 0 min.

2.4 Other sialidases assay

Clostridium perfringens sialidase (Catalogue No. 2876; Sigma) and Vibrio cholerae sialidase (Catalogue No. 72197; Sigma) activities were evaluated according to a method described by Lee et al.⁹ using 4-MUNANA as the substrate, where 10 µL of enzyme solution was mixed with 20 µL of 0.4 mM MUNANA in 20 mM sodium acetate buffer at pH 5.5 with 4 mM CaCl₂ 150 mM NaCl. For the inhibition studies, C. perfringens sialidase and 0–200 µM of the individual compounds were mixed with MUNANA at 37 °C. The production of 4-methylumbelliferone was measured by monitoring the fluorometric determination at excitation wavelength 365 nm/emission wavelength 450 nm.

2.5. Enzyme kinetic study

The inhibition mechanism was determined, and the apparent inhibition constants (K_i) for the respective sialidase (Nan A) were performed on the test compounds, for which the IC₅₀ values were below 25 µM. The test compounds were studied at three different concentrations that were chosen based on the IC₅₀ values obtained with each sialidases (∼1/2 × IC₅₀, IC₅₀, 2 × IC₅₀). The concentrations of marker substrates were chosen (∼1/4 K_m, 1/2 K_m, K_m) with regard to their Michaelis–Menten kinetics. The K_i values were calculated by nonlinear regression analysis by fitting different models of enzyme inhibition to the kinetic data using SigmaPlot Enzyme Kinetics Module 1.3 (SPSS Inc., Chicago, IL). The inhibition mechanism of the compounds was determined by comparing the statistical results, including the Akaike’s information criterion values, of different inhibition models and selecting the one with the best fit.

2.6 General procedures for the synthesis of curcumins and the characterisation of synthesised compounds

2.6.1 Synthesis of curcumin derivatives using Pabon’s reaction (compounds 3, 4b, 4e, 5a–5p, 5r, 5s)

Boron trioxide (43.44 mmol) was added to a solution of 2,4-petadione (65.16 mmol for 3, 4) or monophenyl intermediate (3, 21.7 mmol) in ethyl acetate (100 ml) at ambient temperature. After stirring for 1 h at 90 °C, the corresponding benzaldehydes (21.7 mmol) and triethyl borate (21.7 mmol) in ethyl acetate were added to the reaction mixture. The mixture was stirred for 2 h at 90 °C, then n-BuNH₂ (21.7 mmol, 1 equiv., 7% solution in ethyl acetate) was slowly added, and the mixture was stirred at 90 °C until the aldehydes disappeared on TLC monitoring. The reaction mixture was then cooled to 50 °C and 1 M HCl (aq.) was added. After stirring the mixture for an additional 1 h and cooling to room temperature, water (30 ml) and ethyl acetate (20 ml) were added. The reaction mixture was washed with water and brine until it was neutralised. The organic phase was dried over anhydrous MgSO₄ and evaporated under reduced pressure. The residue was purified using open-bed column chromatography on silica gel or Cosmosil 140 C–18 OPN to yielded curcumin derivatives.

5-Hydroxy-1,7-bis(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-trien-3-one (4b): Yield: 556 mg (23%); orange solid; mp: 182–183 °C (Lit³⁴. 181–183 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 3.83 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 5.98 (s, 1H), 6.64 (d, 1H, J = 15.5 Hz), 6.73 (d, 1H, J = 15.0 Hz), 6.97 (d, 1H, J = 1.5 Hz), 6.99 (d, 1H, J = 1.5 Hz), 7.11 (dd, 1H, J₁ = 8.0 Hz, J₂ = 2.0 Hz), 7.17 (d, 1H, J = 1.5 Hz), 7.21 (dd, 1H, J₁ = 8.5 Hz, J₂ = 1.5 Hz), 7.31 (d, 1H, J = 2.0 Hz); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.28, 55.46, 101.1, 110.3, 111.5, 111.6, 113.4, 121.7, 122.0, 122.8, 128.1, 128.5, 140.2, 140.3, 146.9, 149.7, 149.8, 151.6, 100.3, 114.1, 115.5, 183.3. HRMS (EI) m/z: [M]⁺ calcd. for C₂₁H₂₀O₆ 368.1260; Found: 368.1261.

5-Hydroxy-1,7-bis(4-hydroxyphenyl)hepta-1,4,6-trien-3-one (4e): Yield: 378 mg (15%); orange solid; mp: 234–235 °C (Lit³⁵. 232–233 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 5.95 (s, 1H), 6.63 (s, 1H), 6.66 (s, 1H), 6.86–6.89 (m, 4H), 7.53 (s, 2H), 7.55 (s, 2H), 7.56 (s, 1H), 7.59 (s, 1H), 8.99 (bs, 2H); ¹³C NMR (125 MHz, Acetone-d₆) δ 100.9, 115.9, 121.2, 126.9, 130.1, 140.2, 159.6, 183.7. HRMS (EI) m/z: [M]⁺ calcd. for C₁₉H₁₆O₄ 308.1049; Found: 308.1047.

7-(3,4-Dimethoxyphenyl)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5a): Yield: 245 mg (50%); dark orange solid; mp: 136–137 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.83 (s, 3H), 3.85 (s, 3H), 3.89 (s, 3H), 5.95 (s, 1H), 6.70 (t, 2H, J = 6.7 Hz), 6.86 (d, 1H, J = 8.0 Hz), 6.97 (d, 1H, J = 8.0 Hz), 7.15 (dd, 1H, J₁ = 8.0 Hz, J₂ = 2.0 Hz), 7.20 (dd, 1H, J₁ = 8.5 Hz, J₂ = 1.5 Hz), 7.30 (dd, 2H, J₁ = 4.7 Hz, J₂ = 1.2 Hz), 7.57 (d, 2H, J = 16.0 Hz), 8.25 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.27, 55.45, 100.9, 110.3, 110.6, 111.6, 122.0, 122.7, 123.0, 128.1, 140.2, 140.7, 147.9, 149.2, 149.7, 151.6, 183.3, 183.9. HRMS (EI) m/z: [M]⁺ calcd. for C₂₂H₂₂O₆ 382.1416; Found: 382.1416.

7-(3,4-Dimethoxyphenyl)-5-hydroxy-1-(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-trien-3-one (5b): Yield: 274 mg (56%); dark orange solid; mp: 155–158 °C (Lit³⁴. 157–158 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 3.83 (s, 3H), 3.85 (s, 3H), 3.87 (s, 3H), 5.98 (s, 1H), 6.64 (d, 1H, J = 15.5 Hz), 6.73 (d, 1H, J = 15.0 Hz), 6.98 (dd, 2H, J₁ = 8.2 Hz, J₂ = 1.2 Hz), 7.11 (dd, 1H, J₁ = 8.0 Hz, J₂ = 2.0 Hz), 7.17 (d, 1H, J = 1.5 Hz), 7.21 (dd, 1H, J₁ = 8.5 Hz, J₂ = 1.5 Hz), 7.31 (d, 1H, J = 2.0 Hz), 7.54 (d, 1H, J = 16.0 Hz), 7.58 (d, 1H, J = 16.0 Hz), 7.86 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.20, 55.46, 101.1, 110.3, 111.5, 111.6, 113.4, 121.7, 122.0, 122.8, 128.1, 128.5, 140.2, 140.3, 146.9, 149.7, 149.8, 151.6, 183.3. HRMS (EI) m/z: [M]⁺ calcd. for C₂₂H₂₂O₆ 382.1416; Found: 382.1412.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-trien-3-one (5c): Yield: 212 mg (45%); red orange solid; mp: 137–140 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.87 (s, 3H), 3.89 (s, 3H), 5.96 (s, 1H), 6.63 (d, 1H, J = 16.0 Hz), 6.69 (d, 1H, J = 15.0 Hz), 6.85 (d, 1H, J = 8.0 Hz), 6.97 (d, 1H, J = 8.5 Hz), 7.10 (dd, 1H, J₁ = 8.5 Hz, J₂ = 2.5 Hz), 7.15 (dd, 1H, J₁ = 8.0 Hz, J₂ = 2.0 Hz), 7.17 (d, 1H, J = 2.5 Hz), 7.32 (d, 1H, J = 1.5 Hz), 7.53 (d, 1H, J = 16.0 Hz), 7.57 (d, 1H, J = 16.0 Hz), 7.83 (bs, 1H), 8.18 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.44, 101.0, 110.9, 111.4, 113.5, 115.3, 121.4, 121.6, 122.0, 123.1, 140.1, 140.7, 146.8, 146.9, 147.9, 149.1, 149.2, 149.7, 183.3, 183.9. HRMS (EI) m/z: [M]⁺ calcd. for C₂₁H₂₀O₆ 368.1260; Found: 368.1262.

1-(3,4-Dihydroxyphenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5d): Yield: 100 mg (20%); orange solid; mp: 160 °C (decomposed); ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 5.94 (s, 1H), 6.58 (d, 1H, J = 13.0 Hz), 6.68 (d, 1H, J = 15.5 Hz), 6.86 (s, 2H), 7.03 (s, 1H), 7.15 (d, 1H, J = 4.0 Hz), 7.31 (s, 1H), 7.51 (d, 1H, J = 12.0 Hz), 7.56 (d, 1H, J = 16.0 Hz); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.48, 100.9, 110.6, 114.3, 115.3, 115.6, 121.2, 121.4, 121.8, 123.0, 127.3, 127.5, 140.4, 140.5, 145.5, 147.8, 147.9, 149.1, 183.0, 183.2. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₈O₁₆ 354.1103; Found: 354.1105.

1-(3,4-Dihydroxyphenyl)-5-hydroxy-7-(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-trien-3-one (5e): Yield: 127 mg (28%); brown solid; mp: 203–204 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.87 (s, 3H), 5.97 (s, 1H), 6.58 (d, 1H, J = 15.5 Hz), 6.64 (d, 1H, J = 16.0 Hz), 6.85 (s, 1H), 6.97 (d, 1H, J = 7.5 Hz), 7.04 (s, 1H), 7.11 (d, 1H, J = 7.0 Hz), 7.18 (s, 2H), 7.51 (d, 1H, J = 15.0 Hz), 7.52 (d, 1H, J = 16.0 Hz); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.46, 101.0, 111.5, 113.4, 115.5, 121.2, 121.6, 121.8, 122.0, 127.4, 128.5, 140.0, 140.6, 145.5, 146.9, 147.8, 149.7, 182.7, 183.6. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₈O₆ 354.1103; Found: 354.1104.

1-(3-Ethoxy-4-hydroxyphenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5f): Yield: 284 mg (58%); yellow solid; mp: 182–183 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 1.37 (t, 3H, J = 7.0 Hz) , 3.89 (s, 3H), 4.15 (m, 2H), 5.94 (s, 1H), 6.66 (d, 1H, J = 2.5 Hz), 6.69 (d, 1H, J = 3.0 Hz), 6.85 (d, 2H, J = 8.5 Hz), 7.15 (dd, 2H, J₁ = 10.0 Hz, J₂ = 2.5 Hz), 7.30 (dd, 2H, J₁ = 7.0 Hz, J₂ = 1.5 Hz), 7.54 (d, 1H, J = 3.0 Hz), 7.58 (d, 1H, J = 3.0 Hz), 8.21 (d, 1H, J = 10.5 Hz); ¹³C NMR (125 MHz, Acetone-d₆) δ 14.64, 55.93, 64.77, 101.3, 111.1, 112.1, 115.8, 121.9, 121.8, 123.3(4), 123.3(6), 123.4(3), 123.4(6), 127.7(4), 127.7(9), 141.0(0), 141.0(8), 147.5, 148.3, 149.5, 149.7, 184.0. HRMS (EI) m/z: [M]⁺ calcd. for C₂₂H₂₂O₆ 382.1416; Found: 382.1417.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(pyridin-3-yl)hepta-1,4,6-trien-3-one (5g): Yield: 33 mg (8%); orange solid; mp: 185 °C (decomposed); ¹H NMR (500 MHz, DMSO-d₆) δ 3.80 (s, 3H), 6.12 (s, 1H), 6.81–6.84 (m, 2H), 7.15 (d, 1H, J = 8.0 Hz), 7.22 (d, 1H, J = 16.0 Hz), 7.32 (s, 1H), 7.59 (d, 1H, J = 16.0 Hz), 7.66 (d, 1H, J = 16.0 Hz), 7.89 (d, 1H, J = 4.5 Hz), 8.62–8.69 (m, 1H), 8.77 (d, 1H, J = 3.5 Hz), 9.13 (s, 1H), 9.80 (bs, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 56.23, 102.9, 111.9, 116.2, 121.6, 124.3, 126.5, 126.7, 129.2, 133.8, 141.0, 143.1, 144.4, 144.8, 148.5, 150.4, 179.2, 187.2. HRMS (EI) m/z: [M]⁺ calcd. for C₁₉H₁₇NO₄ 323.1158; Found: 323.1156.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylhepta-1,4,6-trien-3-one (5h): Yield: 45 mg (11%); red brown solid; mp: 135–137 °C (Lit³⁶. 137–140); ¹H NMR (500 MHz, Acetone-d₆) δ 3.90 (s, 3H), 6.02 (s, 1H), 6.83 (d, 1H, J = 21.5 Hz), 6.86 (d, 1H, J = 13.0 Hz), 7.17 (dd, 1H, J₁ = 8.2 Hz, J₂ = 1.7 Hz), 7.33 (d, 1H, J = 2.5 Hz), 7.38–7.43 (m, 3H), 7.52 (d, 1H, J = 16.0 Hz), 7.63 (d, 1H, J = 8.5 Hz), 7.65 (d, 1H, J = 1.5 Hz), 7.67 (s, 1H), 8.18 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.48, 101.3, 110.7, 115.4, 121.4, 123.2, 124.3, 127.2, 128.1, 129.0, 130.0, 135.3, 139.6, 141.2, 147.9, 149.3, 182.1. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₈O₄ 322.1205; Found: 322.1205.

Methyl 4-(4-(5-hydroxy-7–(4-hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-trien-1-yl)-2-methoxyphenoxy)-butanoate (5i): Yield: 48 mg (8%); yellow solid; mp: 100–101 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 2.04–2.09 (m, 2H), 2.51 (t, 2H, J = 7.2 Hz), 3.61 (s, 3H), 3.85 (s, 3H), 3.88 (s, 3H), 4.07 (t, 2H, J = 6.0 Hz), 5.96 (s, 1H), 6.70 (t, 2H, J = 15.7 Hz), 6.85 (d, 1H, J = 8.0 Hz), 6.97 (d, 1H, J = 7.5 Hz), 7.15 (d, 1H, J = 9.0 Hz), 7.19 (d, 1H, J = 8.0 Hz), 7.30 (s, 2H), 7.57 (d, 1H, J = 16.0 Hz), 8.29 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 24.58, 50.87, 55.37, 55.46, 67.58, 100.9, 110.6(6), 110.6(9), 112.9, 115.4, 121.4, 122.0, 122.7, 123.0, 127.2, 128.3, 140.2, 140.7, 147.9, 149.3, 149.9, 150.7, 172.9, 183.2, 184.0. HRMS (EI) m/z: [M]⁺ calcd. for C₂₆H₂₈O₈ 468.1784; Found: 468.1784.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-nitrophenyl)hepta-1,4,6-trien-3-one (5j): Yield: 249 mg (53%); orange solid; mp: 204–205 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 6.10 (s, 1H), 6.76 (d, 1H, J = 16.0 Hz), 6.87 (d, 1H, J = 8.5 Hz), 7.05 (d, 1H, J = 16.0 Hz), 7.19 (dd, 2H, J₁ = 8.2 Hz, J₂ = 2.2 Hz), 7.35 (d, 1H, J = 1.5 Hz), 7.65 (d, 1H, J = 16.0 Hz), 7.69 (d, 1H, J = 16.0 Hz), 7.93–7.96 (m, 2H), 8.25–8.28 (m, 2H), 8.31 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.48, 102.2, 110.7, 115.3, 121.6, 123.5, 124.0, 127.0, 128.2, 128.9, 136.3, 141.7, 142.1, 149.9, 148.2, 149.5, 178.8, 186.6. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₇NO₆ 367.1056; Found: 367.1056.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-(trifluoromethyl)phenyl)hepta-1,4,6-trien-3-one (5k): Yield: 114 mg (23%); yellow solid; mp: 158–160 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 6.07 (s, 1H), 6.75 (d, 1H, J = 16.5 Hz), 6.86 (d, 1H, J = 7.5 Hz), 6.98 (d, 1H, J = 16.0 Hz), 7.18 (dd, 1H, J₁ = 8.0 Hz, J₂ = 1.5 Hz), 7.34 (d, 1H, J = 1.5 Hz), 7.63 (d, 1H, J = 10.5 Hz), 7.66 (d, 1H, J = 10.5 Hz), 7.75 (d, 1H, J = 7.5 Hz), 7.89 (d, 1H, J = 7.5 Hz), 8.35 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.51, 101.8, 110.8, 115.3, 121.5, 123.4, 125.8, 126.9, 127.1, 128.5, 130.4, 137.3, 139.2, 141.8, 147.9, 149.4, 179.7, 185.9. HRMS (EI) m/z: [M]⁺ calcd. for C₂₁H₁₇F₃O₄ 390.1079; Found: 390.1080.

1-(4-Fluorophenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5l): Yield: 231 mg (53%); orange solid; mp: 146–147 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 6.00 (s, 1H), 6.72 (d, 1H, J = 16.0 Hz), 6.79 (d, 1H, J = 16.0 Hz), 6.86 (d, 1H, J = 8.5 Hz), 7.16–7.20 (m, 3H), 7.33 (d, 1H, J = 2.5 Hz), 7.59 (d, 1H, J = 3.0 Hz), 7.62 (d, 1H, J = 3.5 Hz), 7.73 (d, 1H, J = 5.0 Hz), 7.74 (d, 1H, J = 5.0 Hz), 8.27 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.48, 110.7, 115.3, 115.8, 116.0, 121.4, 123.2, 124.2, 127.2, 130.1, 130.2, 130.3, 131.8, 138.2, 141.2, 147.9, 149.2, 162.7, 164.6, 181.4, 184.5. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₇FO₄ 340.1111; Found: 340.1115.

1-(4-Bromophenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5m): Yield: 164 mg (32%); yellow solid; mp: 148–149 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 6.02 (s, 1H), 6.73 (dd, 1H, J₁ = 16.2 Hz, J₂ = 2.2 Hz), 6.85–6.88 (m, 2H), 7.17 (d, 1H, J = 7.5 Hz), 7.33 (s, 1H), 7.56–7.63 (m, 6H), 8.28 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.48, 110.7, 115.3, 110.7, 115.3, 121.5, 123.3, 123.5, 125.1, 127.2, 129.8, 132.1, 134.6, 138.0, 141.4, 147.9, 149.3, 180.7, 185.1. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₇BrO₄ 400.0310; Found: 430.0309.

N-(4–(5-Hydroxy-7–(4-hydroxy-3-methoxyphenyl)-3-oxohepta-1,4,6-trien-1-yl)phenyl)acetamide (5n): Yield: 281 mg (58%); yellow solid; mp: 183–185 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 2.07 (s, 3H), 3.89 (s, 3H), 5.98 (s, 1H), 6.69 (d, 1H, J = 10.5 Hz), 6.73 (d, 1H, J = 10.5 Hz), 6.86 (d, 1H, J = 8.5 Hz), 7.16 (dd, 1H, J₁ = 7.7 Hz, J₂ = 1.7 Hz), 7.32 (d, 1H, J = 2.5 Hz), 7.57–7.61 (m, 4H), 7.68 (s, 1H), 7.70 (s, 1H), 8.15 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 23.58, 23.62, 55.56, 101.2, 110.7, 115.4, 119.1, 119.2, 121.5, 122.7, 123.2, 127.3, 129.0, 130.1, 139.6, 141.0, 141.4, 141.5, 148.0, 149.3, 168.3, 168.4, 182.8, 184.3. HRMS (EI) m/z: [M]⁺ calcd. for C₂₂H₂₁NO₅ 379.1420; Found: 379.1419.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)hepta-1,4,6-trien-3-one (5o): Yield: 320 mg (66%); yellow solid; mp: 143–145 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.01 (s, 6H), 3.89 (s, 3H), 5.90 (s, 1H), 6.55 (d, 1H, J = 15.0 Hz), 6.67 (d, 1H, J = 16.0 Hz), 6.73 (d, 2H, J = 9.0 Hz), 6.85 (d, 1H, J = 7.5 Hz), 7.14 (dd, 1H, J₁ = 8.5 Hz, J₂ = 1.5 Hz), 7.31 (d, 1H, J = 2.0 Hz), 7.50 (d, 1H, J = 9.0 Hz), 7.55 (d, 1H, J = 1.5 Hz), 7.58 (s, 1H), 8.19 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 54.96, 55.46, 101.0, 110.6, 114.4, 115.4, 121.4, 121.9, 123.1, 127.3, 127.8, 129.9, 139.8, 140.7, 147.9, 149.2, 161.5, 183.3, 184.0. HRMS (EI) m/z: [M]⁺ calcd. for C₂₁H₂₀O₅ 352.1311; Found: 352.1314.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)hepta-1,4,6-trien-3-one (5p): Yield: 281 mg (65%); orange solid; mp: 169–170 °C (Lit³⁷. 170–172 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 5.94 (s, 1H), 6.64 (d, 1H, J = 16.0 Hz), 6.69 (d, 1H, J = 15.5 Hz), 6.84–6.88 (m, 3H), 7.15 (dd, 1H, J₁ = 8.5 Hz, J₂ = 2.5 Hz), 7.31 (d, 1H, J = 1.5 Hz), 7.53–7.56 (m, 3H), 7.59 (d, 1H, J = 3.5 Hz), 8.21 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.45, 101.9, 110.5, 115.4, 115.9, 121.2, 121.4, 123.0, 126.8, 127.3, 130.1, 140.2, 140.5, 147.9, 149.2, 159.7, 183.6. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₈O₅ 338.1154; Found: 338.1159.

1-(4-(Dimethylamino)phenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5r): Yield: 220 mg (48%); red solid; mp: 174–175 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.01 (s, 6H), 3.89 (s, 3H), 5.90 (s, 1H), 6.55 (d, 1H, J = 15.0 Hz), 6.67 (d, 1H, J = 16.0 Hz), 6.73 (d, 2H, J = 9.0 Hz), 6.82 (d, 1H, J = 7.5 Hz), 7.14 (dd, 1H, J₁ = 8.5 Hz, J₂ = 1.5 Hz), 7.31 (d, 1H, J = 2.0 Hz), 7.50 (d, 2H, J = 9.0 Hz), 7.55 (d, 1H, J = 1.5 Hz), 7.58 (s, 1H), 8.19 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 39.36, 55.52, 100.8, 110.6, 111.9, 115.3, 118.7, 121.5, 122.8, 127.4, 130.0, 139.8, 141.3, 147.8, 148.8, 152.0, 182.0, 184.4. HRMS (EI) m/z: [M]⁺ calcd. for C₂₂H₂₃NO₄ 365.1627; Found: 365.1624.

5-Hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-(piperidin-1-yl)phenyl)hepta-1,4,6-trien-3-one (5s): Yield: 56 mg (12%); red solid; mp: 204–205 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 1.61–1.63 (m, 6H), 3.22–3.31 (m, 4H), 3.89 (s, 3H), 5.92 (s, 1H), 6.58 (d, 1H, J = 15.5 Hz), 6.67 (d, 1H, J = 15.5 Hz), 6.85 (d, 1H, J = 8.0 Hz), 6.93 (d, 2H, J = 9.5 Hz), 7.14 (dd, 1H, J₁ = 8.2 Hz, J₂ = 1.7 Hz), 7.31 (d, 1H, J = 2.0 Hz), 7.50 (d, 2H, J = 9.5 Hz), 7.54 (d, 1H, J = 5.0 Hz), 7.57 (d, 1H, J = 4.5 Hz), 8.19 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 24.22, 25.36, 48.70, 55.46, 100.81, 110.58, 114.7, 115.3, 119.7, 121.5, 122.9, 124.3, 127.4, 129.8, 140.1, 140.7, 147.9, 149.1, 153.0, 182.9, 184.3. HRMS (EI) m/z: [M]⁺ calcd. for C₂₅H₂₇NO₄ 405.1940; Found: 405.1943.

2.6.2 Synthesis of 1,7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (4c)

Curcumin (1.0 g, 2.71 mmol) was dissolved in dry acetone (30 ml) then anhydrous K₂CO₃ (1.12 g, 8.14 mmol) and CH₃I (1.69 ml, 27.14 mmol) were added. The reaction mixture was refluxed for 24 h and monitored by TLC. The reaction mixture was then cooled to ambient temperature and filtrated. The resulting filtrate was evaporated under reduced pressure and ethyl acetate (30 ml) and water (20 ml) were added. The organic layer was washed with water and brine, and then dried over anhydrous MgSO₄. After evaporating under reduced pressure, the residue was purified using a Cosmosil 140 C-18 OPN column (CH₃CN:H₂O = 3:2 (v/v)) to yield 1,7-bis(3, 4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (4c, 150 mg, 14%) and 1-(3,4-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5a, 210 mg, 20%) as orange solids.

1,7-Bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (4c): Yield: 150 mg (14%); orange solid; mp: 134–136 °C (Lit³⁸. 132–133 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 3.84 (s, 6H), 3.85 (s, 6H), 5.97 (s, 1H), 6.70 (s, 1H), 6.73 (s, 1H), 6.97 (s, 1H), 6.98 (s, 1H), 7.11 (s, 1H), 7.21 (s, 1H), 7.29 (s, 2H), 7.57 (s, 1H), 7.60 (s, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.30, 101.2, 110.4, 111.6, 122.0, 122.8, 123.0, 123.7, 128.1, 140.4, 142.8, 149.8, 151.6, 183.6. HRMS (EI) m/z: [M]⁺ calcd. for C₂₃H₂₄O₆ 396.1573; Found: 396.1572.

2.6.3 Synthesis of 1,7-bis(3,4-dihydroxyphenyl)hepta-1,6-diene-3,5-dione (4d)³⁴

Curcumin (0.3 g, 0.81 mmol) was suspended in dry CH₂Cl₂ (30 ml), then stirred for 10 min at –20 °C under N₂ (g) atmosphere. Tribromoborane (0.25 ml ×5 times) was slowly added. The reaction mixture was allowed to warm up to ambient temperature and stirred overnight. Therefore, the reaction mixture was slowly poured into the saturated NaHCO₃ solution (30 ml) with ice powder, and then stirred for 2 h. The water layer was separated and acidified with 1 M HCl (aqueous), then extracted with ethyl acetate (30 ml ×3 times). The organic layer was washed with water and brine, and then dried over anhydrous MgSO₄. After evaporating the solvent under reduced pressure, the residue was applied to the top of an open-bed silica gel column (n-hexane: ethyl acetate: MeOH =60:38:2 (v/v)) to yield 1,7-bis(3,4-dihydroxyphenyl)hepta-1,6-diene-3,5-dione (4d, 27 mg, 10%) as an orange amorphous solid.

1,7-bis(3,4-dihydroxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one (4d): Yield: 27 mg (10%); orange solid; mp: 300 °C (decomposed, Lit³⁹. 304–305); ¹H NMR (500 MHz, Acetone-d₆) δ 5.95 (d, 1H, J = 6.0 Hz), 6.58 (dd, 2H, J₁ = 15.5 Hz, J₂ = 6.0 Hz), 6.85 (s, 2H), 7.04 (s, 2H), 7.16 (d, 2H, J = 3.0 Hz), 7.51 (dd, 2H, J₁ = 15.2 Hz, J₂ = 6.2 Hz), 8.39 (bs, 4H); ¹³C NMR (125 MHz, Acetone-d₆) δ 100.9, 114.4, 115.6, 121.2, 121.8, 127.5, 140.5, 145.5, 147.9, 183.5. HRMS (EI) m/z: [M]⁺ calcd. for C₁₉H₁₆O₆ 340.0947; Found: 340.0949.

2.6.4 Synthesis of 2–(3-hydroxy-4-methoxybenzylidene)-6–(3-(3-hydroxy-4-methoxyphenyl)acryloyl)cyclohexa-n-1-one (4f)

2-Acetyl-cyclohexan-1-one (0.3 g, 2.14 mmol), boron trioxide (0.14 g, 2.14 mmol), morpholine (0.037 ml, 0.42 mmol), acetic acid (0.024 ml, 0.42 mmol), and vanillin (0.651 g, 4.28 mmol) were placed in a capped vial without solvent at ambient temperature. The resulting mixture was irradiated in a microwave oven (300 W output, 1378 KPa) at 150 °C for 40 min until the 2-acetyl-cyclohexan-1-one or vanillin was consumed. The reaction was monitored by TLC. After the reaction vial cooled, the product was dissolved in MeOH. The reaction mixture was evaporated under reduced pressure, then purified further by silica gel column chromatography (n-hexane: ethyl acetate: MeOH =60:38:2 (v/v)) to give 2-(3-hydroxy-4-methoxybenzylidene)-6-(3-(3-hydroxy-4-methoxyphenyl)acryloyl)cyclohexan-1-one (4f, 390 mg, 45%) as an orange red solid.

2–(1-Hydroxy-3-(4-hydroxy-3-methoxyphenyl)allylidene)-6-(4-hydroxy-3-methoxybenzylidene)cyclohexan-1-one (4f): Yield: 390 mg (45%); orange red solid; mp: 182–183 °C (Lit⁴⁰. 175–176 °C); ¹H NMR (500 MHz, MeOH-d₄) δ 1.75–1.80 (m, 2H), 2.67 (t, 2H, J = 6.2 Hz), 2.77 (t, 2H, J = 5.5 Hz), 3.86 (s, 3H), 3.90 (s, 3H), 6.80 (d, 1H, J = 3.5 Hz), 6.82 (d, 1H, J = 4.0 Hz), 6.95 (dd, 1H, J₁ = 8.5 Hz, J₂ = 1.5 Hz), 7.03 (d, 1H, J = 1.5 Hz), 7.07 (d, 1H, J = 15 Hz), 7.13 (dd, 1H, J₁ = 8.2 Hz, J₂ = 2.2 Hz), 7.22 (d, 1H, J = 2.5 Hz), 7.58 (s, 1H), 7.63 (d, 1H, J = 15.5 Hz); ¹³C NMR (125 MHz, MeOH-d₄) δ 22.90, 24.03, 27.08, 55.06, 108.3, 110.6, 113.5, 114.8, 115.2, 117.3, 122.9, 123.8, 127.4, 128.2, 130.6, 133.2, 142.5, 147.1, 147.4, 148.0, 149.2, 177.6, 185.7. HRMS (EI) m/z: [M]⁺ calcd. for C₂₄H₂₄O₆ 408.1573; Found: 408.1572.

2.6.5 Synthesis of 1-(4-aminophenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1, 6-diene-3,5-dione (5q)

Compound 5n (100 mg, 0.26 mmol) was dissolved in tetrahydrofuran (5 ml), then ethanol (20 ml) and 1 M HCl (aq., 20 ml) were added. The reaction mixture was stirred at reflux for 22 h and monitored by TLC, then cooled to ambient temperature and evaporated under reduced pressure. Ethyl acetate (20 ml) was added to the residue and the mixture was neutralised using NaHCO₃ (aq, sat.). The organic phase was washed with water (20 ml ×3 times) and brine, dried over MgSO₄, and the solvent was evaporated under reduced pressure. The residue was applied to the top of an open-bed silica gel column (n-hexane: ethyl acetate: methanol =60:35:5 (v/v)) to yield 1-(4-aminophenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5q, 75 mg, 85%) as a red solid.

1-(4-Aminophenyl)-5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5q): Yield: 75 mg (85%); red solid; mp: 99–100 °C; ¹H NMR (500 MHz, Acetone-d₆) δ 3.89 (s, 3H), 5.29 (bs, 2H), 5.89 (s, 1H), 6.51 (d, 1H, J = 15.5 Hz), 6.65–6.68 (m, 3H), 6.85 (d, 1H, J = 8.5 Hz), 7.13 (dd, 1H, J₁ = 8.0 Hz, J₂ = 2.0 Hz), 7.30 (d, 1H, J = 2.0 Hz), 7.39 (d, 2H, J = 9.5 Hz), 7.52 (d, 1H, J = 2.5 Hz), 7.55 (d, 1H, J = 2.5 Hz), 8.22 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.46, 100.6, 110.5, 114.2, 115.3, 118.6, 120.0, 121.4, 122.9, 123.4, 127.4, 129.2, 130.2, 139.9, 141.4, 147.9, 149.0, 151.3, 182.4, 184.8. HRMS (EI) m/z: [M]⁺ calcd. for C₂₀H₁₉NO₄ 337.1314; Found: 337.1316.

2.6.6 Synthesis of 1,7-bis(3-hydroxy-4-methoxyphenyl)heptane-3,5-dione (6)

Pd-C (10%, 50 mg) was added to a solution of curcumin (0.5 g, 0.27 mmol) in methanol (10 ml). After degassing, the mixture was stirred at 0 °C for 10 min, and then hydrogenated in a hydrogen atmosphere (balloon) for 12 h. The mixture was filtered through a Celite 545 and the solvent was evaporated under reduced pressure. The residue was applied to the top of an open-bed silica gel column (CH₂Cl₂:MeOH gradient elution) to yield 1,7-bis(3-hydroxy-4-methoxyphenyl)heptane-3,5-dione (6, 140 mg, 28%) as a white solid.

1,7-Bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (6): Yield: 140 mg (28%); white solid; mp: 94–96 °C (Lit⁴¹. 95–97 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 2.55 (t, 2H, J₁ = 7.5 Hz, J₂ = 8.5 Hz), 2.76–2.80 (m, 6H), 3.78 (s, 6H), 5.62 (s, 1H), 6.59–6.64 (m, 2H), 6.68–6.70 (m, 2H), 6.78 (d, 1H, J = 1.5 Hz), 6.81 (d, 1H, J = 1.5 Hz); ¹³C NMR (125 MHz, Acetone-d₆) δ 30.99, 40.03, 45.07, 55.34, 99.49, 111.8, 114.8, 120.6, 132.2, 144.9, 147.5, 197.6. HRMS (EI) m/z: [M]⁺ calcd. for C₂₁H₂₄O₆ 372.1573; Found: 372.1574.

2.6.7 Synthesis of (3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-1,4-phenylene) diacetate (7)

N,N-dimethylaminopyridine (0.13 g, 1.08 mmol) was added to a solution of curcumin (2 g, 5.42 mmol) in anhydrous pyridine (10 ml) at ambient temperature. Acetic anhydride (1.02 ml, 10.8 mmol) was dropped into the reaction mixture in a 0 °C ice bath, and then stirred overnight at ambient temperature. The reaction mixture was then poured into crashed ice and extracted with EtOAc (3 × 30 ml). The organic layer was washed with water, dried over anhydrous Na₂SO₄, and the solvent was evaporated under reduced pressure. The crude product was purified by flash column chromatography (CH₂Cl₂) to yield di-(3,5-dioxohepta-1,6-diene-1,7-diyl)bis(2-methoxy-1,4-phenylene) diacetate⁷ (1.9 g, 77%) as a yellow solid.

3-Hydroxy-5-oxohepta-1,3,6-triene-1,7-diyl)bis(2-methoxy-4,1-phenylene) diacetate (7): Yield: 1.9 g (77%); yellow solid; mp: 158–159 °C (Lit³⁸. 156–158); ¹H NMR (500 MHz, Acetone-d₆) δ 2.23 (s, 6H), 3.86 (s, 6H), 6.04 (s, 1H), 6.82 (s, 1H), 6.86 (s, 1H), 7.08 (s, 1H), 7.10 (s, 1H), 7.24 (d, 1H, J = 2.5 Hz), 7.26 (d, 1H, J = 1.5 Hz), 7.41 (d, 2H, J = 2.0 Hz); ¹³C NMR (125 MHz, Acetone-d₆) δ 19.65, 55.54, 101.6, 111.7, 121.1, 123.3, 124.4, 134.0, 139.8, 141.7, 151.8, 168.0, 183.6. HRMS (EI) m/z: [M]⁺ calcd. for C₂₅H₂₄O₈ 352.1472; Found: 352.1472.

2.6.8 Synthesis of 4,4'-((1H-pyrazole-3,5-diyl)bis(ethene-1,2-diyl))bis(2-methoxyphenol) (8)

Hydrazine hydrate (0.07 ml, 0.81 mmol) was added to a solution of curcumin (0.2 g, 0.54 mmol) in glacial acetic acid at room temperature, then the mixture was stirred for 2 h under reflux and the solvent was evaporated under reduced pressure. EtOAc (30 ml) was added to the residue and the organic layer was washed with water and brine, and then dried over anhydrous MgSO₄. After the solvent was evaporated under reduced pressure, the residue was applied to the top of an open-bed silica gel column (CH₂Cl₂: MeOH =95:5 (v/v)) to yield 4,4'-((1H-pyrazole-3, 5-diyl)bis(ethene-1,2-diyl))bis(2-methoxyphenol) (8, 130 mg, 68%) as an ivory solid.

4,4'-(1H-Pyrazole-3,5-diyl)bis(ethene-1,2-diyl))bis(2-methoxyphenol) (8): Yield: 130 mg (68%); ivory solid; mp: 220–222 °C (Lit. 218 °C); ¹H NMR (500 MHz, Acetone-d₆) δ 3.86 (s, 6H), 6.65 (s, 1H), 6.79 (s, 1H), 6.81 (s, 1H), 6.96 (d, 1H, J = 3.0 Hz), 6.98 (d, 1H, J = 1.5 Hz), 7.00 (s, 1H), 7.09 (s, 1H), 7.12 (s, 1H), 7.17 (d, 2H, J = 2.5 Hz), 8.21 (bs, 1H); ¹³C NMR (125 MHz, Acetone-d₆) δ 55.38, 99.21, 109.0, 115.2, 115.8, 120.3, 129.2, 129.9, 146.9, 147.1, 147.8. HRMS (EI) m/z: [M]⁺ calcd. for C₂₁H₂₀N₂O₄ 364.1423; Found: 364.1423.

3. Results and discussion

3.1 Chemistry

The synthetic route of the curcumin derivatives is shown in Schemes 2–4. The 6-(3, 4-substituted-phenyl)-hex-5-ene-2,4-diones (3) were prepared using acetylacetone with 1 equivalent of corresponding aldehydes by Pabon’s method. From this reaction, the symmetrical curcumins, 4a–4e, were obtained as by-products (Scheme 2)³⁹. The symmetrical curcumin derivatives, 4, were synthesised by combining 1 with 2 equivalents of corresponding aldehydes 2, except of compounds 4c and 4d owing to trace yields. The synthesis of 4c and 4d is shown in Scheme 4. Compound 4f, which has an inserted cyclohexyl group in 1,3-diketone, was prepared by combining 2-acetylcyclohexanone with vanillin under microwave irradiation⁴⁰.

Scheme 2. Reagent and conditions for synthesis of 3: (i) 1, B₂O₃, ethyl acetate, 90 °C; (ii) 2, n-(BuO)₃B, ethyl acetate, 90 °C; (iii) n-BuNH₂, ethyl acetate, 90 °C; (iv) hydrochloric acid (1M, aq.), 50 °C; synthesis of 4: (i) 3, B₂O₃, ethyl acetate, 90 °C; (ii) 2, n-(BuO)₃B, ethyl acetate, 90 °C; (iii) n-BuNH₂, ethyl acetate, 90 °C; (iv) hydrochloric acid (1M, aq.), 50 °C; synthesis of 4f: 1, 2, B₂O₃, morpholine, AcOH, 40 min, microwave irradiation (300 W).

Scheme 3. Reagent and conditions for synthesis of asymmetrical curcumin derivatives 5: (i) 3, B₂O₃, ethyl acetate, 90 °C; (ii) 2, n-(BuO)₃B, ethyl acetate, 90 °C; (iii) n-BuNH₂, ethyl acetate, 90 °C; (iv) hydrochloric acid (1M, aq.), 50 °C.

Scheme 4. Reagent and conditions for synthesis of curcumin derivatives 4c, 4d, and 6–8: (A) 4a, CH₃I, K₂CO₃, acetone, reflux; (B): 4a, BBr₃, –78 °C to ambient temperature, N₂ (g), H₂O; (C) 4a, H₂/Pd-C (10 wt. % of palladium), MeOH, 0 °C to ambient temperature; (D) 4a, Ac₂O, DMAP, pyridine, ambient temperature; (E) 4a, NH₂NH₂^.H₂O, AcOH, reflux.

Twenty asymmetrical curcumin derivatives, 5, were prepared using Pabon’s reaction with 3 and the corresponding aldehydes (Scheme 3)³⁹. Compound 5q was synthesised by deacetylation of 5n using 1 M HCl aqueous solution.

Symmetrical curcumin derivatives 4c, 4d, and 6–8 were prepared by treating the corresponding reagent with curcumin (Scheme 4). Compound 4c was prepared with MeI/K₂CO₃ in acetone under reflux conditions and compound 4d was produced by a demethylation reaction after treating tribromoborane with dry dichloromethane⁴¹. Tetrahydrocurcumin (6) was prepared by hydrogenation using 10% Pd-C as a catalyst⁴² and acetylated curcumin 7 was prepared using acetic anhydride with DMAP as a catalyst with anhydrous pyridine³⁴. Compound 8 was prepared by condensation the 1, 3-diketone in curcumin with hydrazine hydrate under reflux condition²⁸. The structures of all compounds were characterised using ¹H NMR, ¹³C NMR, and EI-HRMS.

3.2 Biological evaluation

Hydrolytic activity of S. pneumoniae Nan A was confirmed using DANA (Neu5Ac2en). The IC₅₀ value of DANA with respect to S. pneumoniae sialidase inhibition was 4.8 ± 1.1 µM¹². To identify a sialidase inhibitor of S. pneumoniae, the inhibitory activity of Turmeric ethanol extract and its three major components was compared. For the methanol extract, the Nan A activity was 88% at 30 µg/mL. The sialidase inhibitory activity of the major components of Turmeric was as follows; curcumin (4a, IC₅₀ = 0.6 ± 0.1 µM), demethoxycurcumin (5p, IC₅₀ = 0.6 ± 0.2 µM), and bisdemthoxycurcumin (4e, IC₅₀ = 4.0 ± 1.2 µM).

Among these compounds, 4e, in which two of the methoxy groups were removed from curcumin showed diminished inhibitory activity. Based on this result, we predicted that curcumin derivatives would have inhibitory activity against sialidase from S. pneumoniae Nan A and that the methoxy group played an important role in the inhibitory activity. Therefore, we modified curcumin and evaluated the inhibitory effects on S. pneumoniae Nan A (Table 1).

Table 1. Inhibitory effects of synthesised compounds against S. pneumoniae Nan A.

Compounds	IC50 (μM)^a	Compounds	IC50 (μM)
4a	0.6 ± 0.1	5i	1.0 ± 0.5
4b	1.0 ± 0.4	5j	0.9 ± 0.3
4c	3.4 ± 1.0	5k	1.0 ± 0.1
4d	1.5 ± 0.7	5l	1.8 ± 0.2
4e	0.4 ± 1.2	5m	1.0 ± 0.2
4f	4.7 ± 0.5	5n	1.8 ± 0.4
5a	1.5 ± 0.7	5o	1.2 ± 0.1
5b	0.6 ± 0.1	5p	0.6 ± 0.2
5c	1.0 ± 0.4	5q	1.6 ± 0.1
5d	0.7 ± 0.2	5r	1.5 ± 0.2
5e	0.2 ± 0.1	5s	1.5 ± 0.1
5f	1.5 ± 0.8	6	82.1 ± 7.6
5g	4.4 ± 0.1	7	2.6 ± 0.6
5h	7.1 ± 0.1	8	6.2 ± 1.5

^a IC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss; all compounds were examined in a set of triplicates experiment.

To evaluate the functionalisation of the curcumin skeleton to find a suitable backbone. Hydrogenation of the heptadienyl group (6, IC₅₀ = 82.1 ± 7.6 µM) resulted in significantly diminished inhibitory activity. Next, to confirm the phenyl group at the 7-position, inhibitory activity after the addition of a pyridinyl (5g) or phenyl ring (5h) was compared. The results indicated that the pyridinyl group (5 g, IC₅₀ = 4.4 ± 0.1 µM) was more effective than the phenyl group (5 h, IC₅₀ =7.1 ± 0.1 µM), but both compounds showed lower inhibitory activity than curcumin. To investigate the effect of 1,3-diketone, 4-hydroxy or 3-methoxy groups, synthesised and compared with six kinds of curcumins. First, to investigate the effect of the 1, 3-diketone moiety, pyrazole (8, IC₅₀ = 6.2 ± 1.5 µM) or carbocyclic 1, 3-diketone (4f, IC₅₀ = 4.7 ± 0.5 µM) was substituted. Second, to investigate the effect of the 4-hydroxyl groups in curcumin, acetyl (7, IC₅₀ = 2.6 ± 0.6 µM) or methyl groups (4c, IC₅₀ = 3.4 ± 1.0 µM) were substituted and conversion of the hydroxy at the para position to ester (5i, IC₅₀ = 1.0 ± 0.5 µM). Third, to investigate the 3-methoxy groups, elimination of the 3-methoxy groups (4d, IC₅₀ = 1.5 ± 0.7 µM) and conversion of the methoxy at the meta position to ethoxy (5f, IC₅₀ = 1.5 ± 0.8 µM) were compared. Inhibitory activity of these compounds was diminished than curcumin (4a). This suggested that 1,3-diketone and a heptadienyl group were essential functional groups for sialidase inhibition and either a methoxy or a hydroxyl group was required.

Based on these results, we substituted a methoxy or hydroxyl group at the para- or meta- position of the 1,7-diphenylhepta-1,6-diene-3,5-dione backbone. To confirm the positional tendency of the methoxy and hydroxyl groups, we reacted it with feruloyl (3a) or isoferuloyl (3b) acetone or hispolon (3c) with the corresponding aldehyde 2. Because a methoxy or hydroxyl group was essential for sialidase inhibition, compounds 4c and 4d were excluded from consideration. As shown in Table 1, IC₅₀ values ranged from 0.2–1.5 µM, indicating there was no positional tendency for inhibition (4a, 4 b, and 5a–5e in Table 1). Among the examined compounds, 5e, containing the catechol with the isoferuloyl moiety, was the most potent inhibitor (IC₅₀ = 0.2 ± 0.1 µM), with a 3.0-fold improvement in inhibitory activity over that of curcumin. Thereafter, to investigate the electronic effect, we substituted an electron-donating group (EDG) or electron-withdrawing group (EWG) into the para position of the phenyl rings, including nitro (5j), trifluoromethyl (5k), fluoro (5l), bromo (5m), acetamido (5n), methoxy (5o), hydroxyl (5p), amino (5q), N, N-dimethylamino (5r), and piperidinyl (5s) groups. The IC₅₀ values ranged from 0.6–1.8 µM and the electronic effect did not influence in Nan A inhibition. Based on these observations, we investigated the kinetic mechanisms of inhibitors with IC₅₀ values of 25 µM or less. We selected the major components of Turmeric (4a, 4e, 5q) and compound 5e as the most potent inhibitors for the kinetic study. We found that the major components of Turmeric (4a, 4e, 5q) showed noncompetitive inhibition characteristics with a K_i of 1.3 µM (4a), 1.2 µM (4e), and 0.8 µM (5q), respectively. Conversely, compound 5e exhibited a potent competitive inhibition against Nan A with a K_i of 0.14 µM (Figure 1).

Figure 1. Graphical determination of the inhibition type for compounds 4a, 4e, 5q, and 5e. Lineweaver–Burk (A–D) plots for the inhibitory activity of compounds 4a, 4e, 5q, and 5e, respectively, against S. pneumoniae Nan A hydrolysis activity in the presence of different substrate concentrations.

Synthesised compounds were then evaluated for their inhibitory effect on sialidase from Vibrio cholerae and Clostridium perfringens, which also release sialidase and play a role in the pathogenesis. The inhibitory assay results are summerised in Table 2.

Table 2. Inhibitory effects of curcumin derivatives in V. cholera and C. perfringens sialidase.

Compound	IC50 (μM)^a		Compound	IC50 (μM)
	V. cholerae	C. perfringens		V. cholerae	C. perfringens
4a	5.3 ± 0.7	1.6 ± 0.4	5g	43.6 ± 0.1	32.9 ± 5.1
4b	7.6 ± 0.5	3.2 ± 0.4	5i	9.5 ± 1.5	3.3 ± 0.4
4c	21.5 ± 7.0	13.1 ± 2.2	5j	6.9 ± 1.8	1.6 ± 0.5
4d	11.9 ± 2.0	6.1 ± 0.8	5n	6.8 ± 0.8	5.9 ± 0.8
4e	15.1 ± 0.1	11.0 ± 0.8	5o	4.2 ± 0.8	2.8 ± 0.3
4f	24.9 ± 1.8	13.4 ± 0.1	5p	4.3 ± 0.7	2.1 ± 0.4
5a	11.7 ± 0.2	6.7 ± 0.5	5q	2.9 ± 0.9	1.7 ± 0.1
5b	9.2 ± 0.2	6.7 ± 0.8	5r	1.9 ± 0.2	1.3 ± 0.2
5c	7.7 ± 0.7	3.5 ± 0.2	6	NA^b	269.4 ± 31.2
5d	2.7 ± 0.8	1.0 ± 0.1	7	37.5 ± 3.5	13.8 ± 0.6
5e	4.8 ± 1.5	0.5 ± 0.07	8	41.6 ± 18.7	9.5 ± 0.1
5f	6.7 ± 0.6	2.9 ± 0.5

^a IC₅₀ values of compounds represent the concentration that caused 50% enzyme activity loss; all compounds were examined in a set of triplicates experiment.

^b No activity.

Similar to the above results, methoxy or hydroxyl, heptadienyl and α, β-unsaturated ketone groups played important roles in the inhibitory activity. Although the position of the methoxy and hydroxyl group did not influence the inhibition of the above enzymes, the inhibitory effect increased with the greater substitution of hydroxyl groups in the phenyl rings. Thereafter, we evaluated inhibitor activity to confirm the electronic effect. C. perfringens sialidase inhibitory activity was not affected by the electronic effect. Among the examined compounds, 5e (IC₅₀ = 0.5 ± 0.07 µM), containing the catechol moiety, was the most potent inhibitor of C. perfringens and displayed a 3.2-fold improvement over curcumin. Conversely, substitution of an electron donating groups at the para position of the phenyl group resulted in better potency than the substitution of an electron withdrawing groups against V. cholera sialidase. Among the examined compounds, compound 5r (IC₅₀=1.9 ± 0.2 µM), containing the N, N-dimethylamino group, was the most potent and displayed a 2.7-fold improvement in inhibitory activity over that of curcumin. The IC₅₀ values for sialidase inhibitory activity were 2.7–43.6 µM in V. Choleara and 0.5–269.4 µM in C. perfringens.

4. Conclusions

In conclusion, we developed, for the first time, inhibitors of sialidase from S. pneumoniae Nan A, V. cholerae and C. perfringens using curcumin derivatives. Of the 28 compounds synthesised, 7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(3-hydroxy-4-methoxyphenyl)hepta-1,4,6-trien-3-one (5e), which is novel compound contained isoferuloyl and catechol moieties, was the most potent inhibitors of S. pneumoniae (IC₅₀ = 0.2 ± 0.1 µM) and C. perfringens (IC₅₀ = 0.5 ± 0.07 µM). All major Turmeric components showed noncompetitive inhibition, but compound 5e exhibited competitive inhibition against S. pneumoniae Nan A. In the case of V. cholerae sialidase, 7-(4-(dimethylamino)phenyl)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)hepta-1,4,6-trien-3-one (5r), containing N, N-dimethylamino as an electron-donating group, was the most potent inhibitor (IC₅₀ = 1.9 ± 0.2 µM). The SAR analysis suggested that the α, β-unsaturated ketone, heptadienyl and either the methoxy or hydroxy group in curcumin was required for sialidase inhibition. These results indicated that curcumin analogues may potentially be used for sepsis caused by bacterial sialidase. Further in vivo evaluation of compound 5e and 5r will be performed in our laboratory.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by the KRIBB Research Initiative Program and part of the project titled “Development of the control of aquaculture infectious disease using natural resources,” funded by the Ministry of Oceans and Fisheries, Republic of Korea. J-. Y. Park and H-. J. Kwon were supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning [NRF-2016R1C1B2015753]. Also, this study was supported by Korea Coast Guard.