Introduction

Oxidation processes are essential for life, nevertheless they can have a toxic potential as they generate free radicals which can cause degradation in food, chemicals, and in biological systems [1–4]. Hence, antioxidants play a central role in human health as they scavenge potentially harmful free radicals such as reactive oxygen species (ROS). Chalcones, being a subgroup of the flavonoid family, have received considerable attention as radical scavengers and show an impressive range of biological activities such as antibacterial [5, 6], antiviral [7–9], and anti-inflammatory properties [10, 11]. Polyhydroxychalcones can be considered the most active chalcones due to their ability to form stabilized phenoxy radicals [11–14]. Hwang et al. [15] have shown that the position of the hydroxyl groups on arene ring B (Fig. 1) has a major impact on radical scavenging properties of polyhydroxychalcones. While meta-substitution on ring B has no beneficial effect, ortho- and para-substitutions increase the antioxidant activity of chalcones significantly, presumably due to the ability to form quinones as oxidation products.

Fig. 1
figure 1

Proposed structure of a general M(II)–chalcone complex

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Recently, it has been shown that the antioxidant activity of polyphenols, structurally similar to polyhydroxychalcones, can be significantly enhanced by metal ion complexation [16–19]. In contrast to the interest that metal complexes of polyphenolic substances have received, little is known about the synthesis and activity of polyhydroxychalcone metal complexes, and how metal complexation would impact the antioxidant activity of these powerful secondary plant metabolites. Although in literature three metal complexes of 2'-hydroxychalcones with Zn, Cd, and Hg are reported [20], no complexes of polyhydroxychalcone have been synthesized, isolated, and characterized until now. Thus in particular the possible structure of metal ion coordination needs to be investigated. There are several options how metals can coordinate polyhydroxychalcones (see Fig. 1) [21, 22]. Both, the carbonyl moiety and the 2′-hydroxyl group of the 2′-hydroxychalcones have been suggested to coordinate a metal(II) ion [23]. Different complex formation between polyhydroxychalcones and metal ions show various affects on the electronic distribution in the molecule, resulting in possible changes of biological properties such as radical scavenging ability.

scheme 1

Copper(II) and zinc(II) are our metals of interest, since they are endogenous metals and they have shown promising features as metallodrug candidates [24, 25]. In fact Cu(II) and Zn(II) complexes of the flavonoids quercetin and rutin have been shown to greatly increase the antioxidant activity of this class of compounds [26, 27]. In many complexes Zn(II) prefers a tetrahedral coordination sphere [28] while the Cu(II) cation prefers a square planar coordination. Based on the polyhydroxychalcones scaffold other coordination geometries such as distorted octahedral geometry have been reported for copper [29]. In this communication we disclose synthesis, characterization as well as studies on antioxidant activities of four polyhydroxychalcone metal complexes.

Results and discussion

Synthesis of polyhydroxychalcone and their metal complexes

The synthesis of the polyhydroxychalcones was performed via a well-documented three-step procedure in basic condition according to [30] (see Scheme 1). First compound 1 was protected with one of the three protecting groups, methoxymethyl (MOM), tetrahydropyranyl (THP), and tert-butyldimethylsilyl-ether (t-BDMS) to get 2. Protection proceeded in all cases in moderate to good yields. Subsequently, a base-catalyzed aldol condensation reaction (Claisen–Schmidt condensation) with a protected benzaldehyde 5 or 6 was performed to obtain the protected chalcones 7 and 8. Finally, deprotection was performed to give the desired polyhydroxychalcones isoliquiritigenin (9, ISO) and butein (10, BUT).

In order to optimize the synthesis of the polyhydroxychalcones, different protecting groups have been applied in the course of the butein synthesis (Scheme 1). MOM as a protection group presents several disadvantages as it is highly carcinogenic [31], and the purification of the protected intermediate with liquid column chromatography is challenging. Nevertheless, the protection reaction of the hydroxyl group with MOM leads to a yield of 67 % for benzaldehyde and 64 % for acetophenone. Moreover, the removal of the MOM group requires drastic acidic conditions [32], which often lead to polymerization and oxidation of the desired product. t-BDMS worked better during the protection step providing similar and higher yields (64 % for the benzaldehyde and 83 % for the acetophenone). The coupling was performed using LiHMDS [33], but difficulties with the purification made isolation of the pure product problematic. Although THP is a little less efficient in protecting benzaldehyde (60 % yield) it protects the acetophenone most efficiently (89 %), and has been advantageous in all other aspects as it is easy to insert and remove. Furthermore, it is stable under the alkaline coupling conditions [30]. As a consequence THP was chosen as the protection group for the synthesis of both polyhydroxychalcones ISO and BUT.

Independently of the used protection group, all the condensation reactions worked with moderate yields of 45 and 52 %, respectively (see Table 1). For some substrates MOM-ether worked better during the deprotection step than the THP-ether [34]. Nevertheless, this strongly depended on the substrate as for some THP-ether provided better results. The overall yields for the three steps were 15 % for the MOM protecting group and 11 % for the THP protecting group (see Table 1). These overall yields are in accordance with the work published by Lee et al. [35], who obtained the final hydroxychalcone in an overall yield of 10 % when they protected the 4-hydroxy group with methoxymethyl prior to the Claisen–Schmidt condensation. It should be noted that the use of a Sephadex LH-20 for liquid column chromatography to purify the final polyhydroxychalcone increased the yield significantly compared to a silica gel-based liquid chromatography.

Table 1 Comparison of protecting groups for synthesis of butein
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The two complexes [Cu(ISO)2] and [Cu(BUT)2] were prepared by mixing copper(II) acetate with (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (9, ISO) and (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-2-propen-1-one (10, BUT), respectively, in a 1:2 (M, M = metal: L, L = ligand) molar ratio. The reactions were carried out in methanol as solvent.

The complexes [Zn(ISO)2] and [Zn(BUT)2] were prepared by mixing zinc(II) chloride with (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-prop-2-en-1-one (9, ISO) and (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (10, BUT) in a 1:2 (M:L) molar ratio. The reactions were carried out in acetonitrile. Four new Cu(II) and Zn(II) complexes of polyhydroxychalcones termed [Cu(ISO)2], [Cu(BUT)2], [Zn(ISO)2], and [Zn(BUT)2] have been obtained with yields of about 50 %.

Analysis of the polyhydroxychalcone metal complexes

The polyhydroxychalcones remain structurally unchanged in the metal complexes, as exemplarily shown by NMR data of compound 9, ISO. In particular, the metal complexes exist in the E-form, which was confirmed by the characteristic coupling constant between the α and β protons of the double bond [see 1H NMR of (9, ISO), J = 15.4 Hz, Fig. 3, ratio 10:0]. Despite several attempts we were not able to obtain single crystals of [Cu(ISO)2], [Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2].

Stoichiometry of the metal complexes using Job’s plot

Studies to explore the stoichiometry between the polyhydroxychalcones and Zn2+ and Cu2+ were performed using Job’s plot on the basis of 1H NMR measurements [36]. The product concentration of the metal ion and the shift difference of some indicative protons was plotted against the molar fraction (see Fig. 2). Chemical shift changes of H-5′ of 9, ISO (6.42 ppm) were measured as a function of molar fraction. The rather large deviation in the region of the low isoliquiritigenin (9, ISO) concentration is caused by quite moderate S/N ratio and in particular by reasonable line broadening. The latter is likely caused by a higher amount of compound 9 being involved in complex formation with Cu(II) bearing unpaired electrons. The maximum at a mole fraction of ca. 0.66 is, however, even detectable in this case and indicates a 1:2 stoichiometry of the investigated complex (Fig. 2). Similar measurements with Zn(II) provide comparable results with an even better S/N ratio and less line broadening. Hence, isoliquiritigenin (9, ISO) coordinates to Zn2+ and Cu2+ in a ratio of 2:1 ISO:metal.

Fig. 2
figure 2

Job’s plot determined from the chemical shift changes of H-5′ of isoliquiritigenin (9, ISO) with Cu(OAc)2 in methanol-d 4 . Summed concentration of isoliquiritigenin (9, ISO) and Cu(OAc)2 is 13 mM

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The predominately shifted signals are attributed to the hydrogen of the double bond (Hα, Hβ) and aromatic H (H3′, H5′, and H6′) on ring A (see Figs. 1 and 3), which confirm the keto function and the hydroxyl group at position C-2′ to be very likely involved in the coordination process. The NMR complexation studies give further insights on the side where the complexation occurs by showing a more distinct line broadening of signals from protons influenced by the metal ions (Fig. 3). These are in particular H5′ and H6′, as the hydroxyl group C-2′ ring A is involved in binding.

Fig. 3
figure 3

1H NMR titration of the Job’s plot of isoliquiritigenin (9, ISO) and Zn2+ with increasing amount of Zn2+ from the bottom to the top (methanol-d 4 ). Colored indications highlight additional small signals attributed to H6′, Hβ, and Hα (color figure online)

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Addition of Zn(OAc)2 to the free ligand leads to the formation of some new small peaks which are shifted into a more shielded area in the 1H NMR (Fig. 3), likely indicating a side reaction induced by the complexation.

In all attempts to record a Job’s plot with butein (10, BUT) the resulting complexes were not completely soluble and a pale yellow precipitate was formed after certain time. Hence no 1H NMR spectra for the complex between butein and zinc as well as between butein and copper can be presented. Additionally 13C NMR studies have been carried out to investigate possible coordination modes but the analysis did not show any significant shift originating from metal complexation. These observations are in accordance with previous published data where the NMR spectra of the ligand in the presence of the metal do not show any differences from the pure ligand spectrum [37]. A possible explanation is that the butein–metal complex is removed from the solution due to precipitation.

Mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is performed on the Cu2+ and Zn2+ complexes of butein (10, BUT) and isoliquiritigenin (9, ISO), respectively. The ESI spectra show a typical isotope splitting matching the masses of ligand to metal complexes with a stoichiometry of 2:1, confirming their existence.

For [Cu(ISO)2] these peaks can be found at m/z = 573.0612 for 63Cu and at 575.0604 for 65Cu (see Fig. 4). The peak corresponding to the free ligand was observed at m/z = 255.0664 in negative mode and also the adduct of two isoliquiritigenin molecules [(2 ISO-H+)] is present at m/z = 511.1391. In the case of butein the peaks of the copper adduct are present at 603.0362 for 63Cu and 605.0404 for 65Cu (see Fig. 4). These masses are attributed to the formula [Cu(C15H10O5)2]2+ indicating that the butein coordinates the copper ions in the oxidized form thereby explaining the loss of two Hs and we assumed that the catechol functionality on the B ring is oxidized to the o-chinone (catecholase activity, E.C. 1.10.3.1). Oxidation of flavonols may occur after copper complexation [38, 39]. The presence of the two peaks at m/z = 271.0599 (C10H15O5+H+) and 293.0422 (C10H15O5+Na+) results from the oxidized butein.

Fig. 4
figure 4

Electrospray ionization mass spectra of Cu2+/polyhydroxychalcones 1:2 system for [Cu(ISO)2] (a) in negative mode, [Cu(BUT)2] (b) in positive mode. The spectra a and b show the typical isotopic spectral pattern of the experimentally observed copper complexes Cu/L 1:2 in comparison with the corresponding calculated one (blue). The spectra c and d show the typical isotopic spectral pattern of the experimentally observed zinc complexes Zn/L 1:2 in comparison with the corresponding calculated one (blue) (color figure online)

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For [Zn(ISO)2] the peaks confirming a stoichiometry of two ligands per one zinc ion are found at m/z = 575.0676 (63Zn2+) and 577.0656 for 65Zn2+, while for [Zn(BUT)2] these peaks are observed at m/z = 607.0576 for 63Zn2+ and 609.0564 for 65Zn2+. The mass spectra did not show higher coordinated metal complexes of stoichiometry 3:1 or 4:1 (ligand to metal). There is no peak detectable, indicating a 1:1 stoichiometry of ligand to metal.

IR spectroscopy

Infrared spectra of the Cu2+ and Zn2+ compounds were recorded and compared to the free ligands butein (10, BUT) and isoliquiritigenin (9, ISO). The intensity of the OH-vibration at 3300 cm−1 is reduced and broadened for both copper complexes, indicating that one or more hydroxyl groups could be interacting with the metal center [29]. Interestingly, the effect is more pronounced in the case of butein possessing the catechol type functionality on ring B. There is a shift of the carbonyl stretch from 1640 to 1610 cm−1 for both ligands in the presence of copper, meaning that the bond strength of the C=O bond is weakened. This is a strong indication for a participation of the carbonyl moiety in the complexation process [40]. Additionally, a new band is observed at 500 cm−1 in the spectra of the two metal complexes. In both cases these bands are attributed to the ν (Cu–O). This result is in accordance with the disclosure of McWhinnie [41], who has previously assigned the copper oxygen vibration at 500 cm−1 for related hydroxy-bridged copper compounds.

The IR spectra of the two polyhydroxychalcones with Zn2+ are quite complex as they in general exhibit a large number of bands of varying intensities. The IR spectra of isoliquiritigenin with Zn2+ do not show any significant shift compared to the free ligand, while the butein–Zn2+ complex shows a shift of the C=O stretching mode from 1630 to 1610 cm−1, indicating coordination of carbonyl oxygen to metal ion [42–44]. In combination with the results obtained from our 1H NMR titration studies, the IR data support the formation of the complex on the 2′-hydroxy/carbonyl side.

Free radical scavenging ability of metal chalcone complexes

The antioxidant activity of the metal polyhydroxychalcones complexes has been evaluated applying a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay test, which is one of the most common methods used to determine radical scavenging ability. The DPPH test was chosen as it is a non-enzymatic method widely used to provide basic information on the capability of compounds in scavenging free radicals. It has already been proven to be a reliable method for flavonoids and other organic compounds. The test is performed in methanol because it is well known that DPPH is not soluble in water [45–47]. ESI, NMR, and IR spectroscopic investigations on the complexes point towards metal coordination at the keto enol moiety of the polyhydroxychalcones. Thus, the hydroxy groups are able to participate in the free radical scavenging activity. The activity of the metal complexes of isoliquiritigenin (9, ISO) with copper and zinc and of butein (10, BUT) with copper have been measured and compared with the one of the free ligands. Catechol has been used as a positive control and studies were performed on the system catechol-Cu2+ and catechol-Zn2+ as the free radical scavenging activity of phenols is not always enhanced upon metal binding [48]. Blanks of the sole metals Cu2+ and Zn2+ have also been carried out, too. The results are summarized in Table 2.

Table 2 The percentage of inhibition of the free DPPH radical in the presence of different Me-polyhydroxychalcone systems at 1.5 molar ratio (mol Me-polyhydroxychalcone/mol DPPH) for the studies with isoliquiritigenin (9, ISO) and the Me-isoliquiritigenin system and 0.75 molar ratio for the butein (10, BUT) and the Cu-butein system
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Cu2+ and Zn2+ ions coordinated to the keto enol functionality of isoliquiritigenin (9, ISO) alter the antioxidant activity of the chalcone. In fact at the final molar ratio of 1.5 (0.8 mM of ligand and 0.4 mM of metal) the metal complexes [Cu(ISO)2] and [Zn(ISO)2] show 72 and 88 % inhibition of the DPPH radical, whereas the sole ligand shows 10 % inhibition. A similar behavior is observed for butein (10, BUT) when complexating with Cu2+ (Table 2). The scavenging effect of the [Cu(ISO)2] was higher than that one of the sole ligand ISO. For [Cu(BUT)2] the free radical scavenging activity could only be observed up to a 0.75 molar ratio with 66 % inhibition in the presence of copper while the crude butein (10, BUT) shows only 19 % inhibition. In the preparation of the system butein/zinc a red precipitate was immediately formed, therefore it was not possible to perform the measurements. Pekal et al. [49] provided the antioxidant properties of a Cu2+/quercetin complex and Bratu et al. [26] on a Zn2+/rutin complex using the DPPH method. Both studies conclude that the radical scavenging activity of the metal flavonoid-complexes is higher than the one of the ligands alone. The results obtained support the mechanism suggested by Souza et al. [21]. The higher antioxidant activity of the complexes is due to the acquisition of additional superoxide dismutating centers, which causes an increase of the molecule’s ability to stabilize unpaired electrons and therefore to scavenge free radicals.

Solubility and kinetic stability of [Cu(ISO)2], [Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2]

The synthesized complexes [Cu(ISO)2], [Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2] are insoluble in water only. Solubility is achieved in the presence of organic solvents. In fact the four complexes are soluble in methanol and dimethyl sulfoxide. 35 % of water is the maximum of water that is tolerated in the presence of dimethyl sulfoxide and methanol until a precipitate is formed.

Several methods for a spectrophotometric determination of the stability of metal complexes of flavonoids and related structures have been described in the literature. We chose the method described by Ferrari et al. [50] because the reproducibility and accuracy are satisfactory, and it has already been used to assess the kinetic stability of  a similar class of natural compounds such as curcumin derivatives. We tested the stability of the complexes under physiological conditions. Figures S1 and S2, reported in the Supplementary Material, show the decomposition kinetics profile of the four complexes and their linear fittings. Table S1 in the supporting information summarizes the hyperbolic function parameters (a and b) together with their reciprocal values and statistical parameters R 2. The reciprocal of a (1/a) is the percentage of residual compounds that remains intact under physiological conditions. The value (−1/b) is related to the degradation rate: the higher the value the faster the degradation process. [Zn(ISO)2] is characterized by the lowest values of 1/a and −1/b. In fact it decomposes up to ∼40 % in 16 h, and a similar value of ∼45 % is observed for [Cu(ISO)2]. [Cu(BUT)2] and [Zn(BUT)2] posses the highest values for 1/a which are 0.64 and 0.74, respectively. They are the most stable compounds, as after 8 h only 20 and 30 % degraded vs. 40 and 45 % of [Zn(ISO)2] and [Cu(ISO)2], respectively. This data points towards a relationship between the aromatic substituents and the kinetics profiles. In particular, the presence of an ortho/para substitution on the B ring seems to be fundamental in order to achieve stability. This is probably due to the possibility to form an intramolecular hydrogen bond, while the hydroxyl group on the A ring looks to be of minor importance. Therefore, the substitution pattern on the aromatic rings seems to be a key factor in slowing the degradation process of this class of metal complexes. At physiological pH, they have a kinetic stability comparable with other metal complexes of isoflavonoids [49].

Conclusion

In this work, two polyhydroxychalcones isoliquiritigenin (ISO) and butein (BUT) were synthesized by Claisen condensation and characterized applying different spectroscopic and spectrometric techniques. The two chalcones have been reacted with Cu2+ and Zn2+ ions leading to the coordination compounds: [Cu(ISO)2], [Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2].

The Job’s plot for the system isoliquiritigenin/metal(II) (metal = Cu2+, Zn2+) showed that isoliquiritigenin (9, ISO) forms a complex in solution with stoichiometry two to one, with the metal coordinating the keto function and the hydroxyl group at position C-2′. The IR and the mass spectra confirmed that both isoliquiritigenin and butein form stable complexes with copper and zinc. The metals coordinate through the carbonyl function and the hydroxyl function in ortho-position on ring A, again indicating that zinc and copper coordinate the polyhydroxychalcones with a ligand/metal stoichiometry of 2:1.

Finally, the antioxidant activity of the metal polyhydroxychalcones was determined with the DPPH test. The investigated complexes [Cu(ISO)2], [Zn(ISO)2], and [Zn (BUT)2] possess higher antioxidant activity than the free ligands isoliquiritigenin and butein.

Experimental

Chemicals were purchased from Sigma Aldrich, they were of reagent grade and used without any further purification. All NMR spectra were recorded in the NMR Core Facility of the University of Vienna with a Bruker Avance III 500 MHz NMR spectrometer at 500.32 (1H), 125.81 (13C), in CDCl3 or methanol-d 4, at ambient temperature. The splitting of proton resonances in the 1H NMR spectra are defined as s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublets of doublets, t = triplet, and m = multiplet. Numbering of carbon atoms and proton refers to Fig. 1 and Scheme 1. Elemental analyses were carried out in the Mikroanalytisches Laboratorium of the University of Vienna using an “EA 1108 CHNS-O” elemental analyzer by Carlo Erba Instruments. Electrospray ionization mass spectra were recorded in the Massenspektrometriezentrum of the University of Vienna with a Bruker Esquire 3000 with an orthogonal ESI source with MeOH/ACN as solvent, the molecular mass was determined in the positive as well as in the negative mode. The spectrophotometer Shimadzu UV 1800 has been used to recorder the UV–Vis spectra. The infrared spectra were recorded with an infrared spectrometer Bruker Tensor 27 FTIR equipped with a globar MIR light source, a KBr beam splitter, and a DLaTGS detector. Sample and background spectra were averaged from 100 scans at 4 cm−1 resolution. Undiluted sample powder was pressed on the diamond window of a Harrick MVP 2 diamond ATR accessory. Background spectra were obtained from the empty ATR unit. Data handling was performed with OPUS 5.5 software (Bruker Optik GmbH, 2005).

Synthesis of the polyhydroxychalcones

(E)-1-(2,4-Dihydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one and (E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-2-propen-1-one were synthesized performing a Claisen–Schmidt condensation between the benzaldehyde and acetophenone derivatives according to the previously published procedure [30]. The 3,4-dihydro-α-pyran is used to protect the hydroxyl group present on the aromatic ring before performing the aldol reaction in basic condition. The yields of each reaction are summarized in Table 1. The synthesized compounds were characterized by 1H NMR, mass spectrometry, and IR.

2-Hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)acetophenone (2a, C13H16O4)

2,4-Hydroxyacetophenone (1, 6.60 mmol) and pyridinium para-toluenesulfonate (0.16 mmol) were dissolved in 30 cm3 of dichloromethane and the solution was stirred at room temperature. Subsequently, 3,4-dihydro-α-pyran (19.73 mmol) was added dropwise. The reaction was stirred for 24 h and the progress was monitored by TLC. Then the mixture was washed with water, dried over Na2SO4, concentrated under vacuum, and purified by flash column chromatography (hexane/ethyl acetate 9:2) to obtain pure 2a. 1H NMR (500 MHz, CDCl3): δ = 12.62 (s, 1H, Ar–OH), 7.65 (d, J = 8.8 Hz, 1H, H-6), 6.63 (d, J = 2.5 Hz, 1H, H-3), 6.57 (dd, J = 8.8, 2.5 Hz, 1H, H-5), 5.49 (t, J = 3.1 Hz, 1H, H-7), 3.85 (ddd, J = 11.3, 9.9, 3.1 Hz, 1H, H-11a), 3.66–3.61 (m, 1H, H-11b), 2.58 (s, 3H, H-α), 2.04–1.95 (m, 1H, H-8a), 1.90–1.85 (m, 1H, H-8b), 1.75–1.65 (m, 2H, H-9a, H-9b), 1.65–1.59 (m, 1H, H-10a), 1.58–1.52 (m, 1H, H-10b) ppm; 13C NMR (125 MHz, CDCl3): δ = 202.6 (C=O), 164.8 (C-2), 163.1 (C-4), 132.3 (C-6), 114.5 (C-1), 108.5 (C-5), 103.4 (C-3), 96.1 (C-7), 63.0 (C-11), 30.6 (C-8), 30.0 (C-α), 25.4 (C-10), 19.7 (C-9) ppm; HRMS (ESI-MS): m/z = 259.0931 ([M+Na]+), 237.1452 ([M+H]+, calcd. for C13H16O4 237.1121).

4-(Tetrahydro-2H-pyran-2-yloxy)benzaldehyde (5a, C12H14O3)

4-Hydroxybenzaldehyde (6.60 mmol) and pyridinium para-toluenesulfonate (0.16 mmol) were dissolved in 30 cm3 of dichloromethane and the solution was stirred at room temperature. Subsequently, 3,4-dihydro-α-pyran (19.73 mmol) was added dropwise. The reaction was stirred for 24 h and the progress was monitored by TLC. Then the mixture was washed with water, dried over Na2SO4, concentrated under vacuum, and purified by flash column chromatography (hexane/ethyl acetate 7:3) to obtain pure 5a. 1H NMR (500 MHz, CDCl3): δ = 9.97 (s, 1H, C=O), 7.87–7.81 (dd, J = 8.9, 2.0 Hz, 2H, H-2, H-6), 7.20–7.13 (dd, J = 8.9, 2.0 Hz, 2H, H-3, H-5), 5.55 (t, J = 3.1 Hz, 1H, H-7), 3.93–3.82 (m, 1H, H-11a), 3.64 (m, 1H, H-11b), 2.08–1.96 (m, 1H, H-8a), 1.93–1.82 (m, 2H, H-8b, H-9a), 1.80–1.60 (m, 2H, H-9b, 10a), 1.58–1.50 (m, 1H, H-10b) ppm; 13C NMR (125 MHz, CDCl3): δ = 191.1 (C=O), 161.7 (C-4), 132.4 (C-2,6), 131.9 (C-1), 115.9 (C-3,5), 96.1 (C-7), 63.0 (C-11), 30.0 (C-8), 25.3 (C-10), 19.6 (C-9) ppm; HRMS (ESI-MS): m/z = 229.0829 ([M+Na]+, calcd. for C12H14O3Na 229.0835).

3,4-Bis(tetrahydro-2H-pyran-2-yloxy)benzaldehyde (6a, C17H22O5)

3,4-Dihydroxybenzaldehyde (6.60 mmol) and pyridinium para-toluenesulfonate (0.16 mmol) were dissolved in 30 cm3 of dichloromethane and the solution was stirred at room temperature. Subsequently, 3,4-dihydro-α-pyran (39.46 mmol) was added dropwise. The reaction was stirred for 24 h and the progress was monitored by TLC. Then the mixture was washed with water, dried over Na2SO4, concentrated under vacuum, and purified by flash column chromatography (hexane/ethyl acetate 9:2) to obtain 6a as a mixture of stereoisomers. 1H NMR (500 MHz, CDCl3): δ = 9.87 (s, 1H, CHO), 7.50–7.49 (d, J = 8.0 Hz, 1H, H-6), 7.25 (s, 1H, H-2), 7.07–7.05 (d, J = 8.2 Hz, 1H, H-5), 5.55 (t, J = 2.7 Hz, 2H, H-7, H-7′), 3.93–3.82 (m, 2H, H-11a, H-11′a), 3.64 (m, 2H, H-11b, H-11b), 2.08–1.96 (m, 2H, H-8a, H-8a′), 1.93–1.82 (m, 4H, H-8b, H-8b′, H-9a, H-9a′), 1.80–1.60 (m, 4H, H-9b, H-9b′, H-10a, H-10a′), 1.58–1.50 (m, 2H, H-10b, 10b′) ppm; 13C NMR (125 MHz, CDCl3): δ = 190.6 (C=O), 153.1 (C-4), 146.9 (C-3), 129.8 (C-1), 123.9 (C-6), 115.8 (C-5), 115.2 (C-2), 98.4 (C-7), 98.9 (C-7′), 63.7 (C-11), 63.3 (C-11′), 30.1 (C-8), 30.1 (C-8′), 25.4 (C-10), 25.2 (C-10′), 19.4 (C-9), 18.8 (C-9′) ppm; HRMS (ESI-MS): m/z = 329.1356 ([M+Na]+), 307.1534 ([M+H]+, calcd. for C17H22O5 307.1461).

(E)-1-[2-Hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)phenyl]-3-[4-(tetrahydro-2H-pyran-2-yloxy)phenyl]prop-2-en-1-one (7a, C25H28O6)

Compound 2a (4.24 mmol) and 8.48 mmol of 5a were dissolved in 10 cm3 of methanol at 35 °C under reflux. Subsequently, a solution of 20 cm3 of methanol containing barium hydroxide octahydrate (16.96 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred for 24 h and the progress was monitored by TLC. Then the mixture was concentrated under vacuum, quenched with HCl (0.1 M), and extracted with ethyl acetate. The organic layer was separated, dried over Na2SO4, and then concentrated under vacuum. The reaction mixture was purified by flash column chromatography (hexane/ethyl acetate 7:3) to obtain pure 7a. 1H NMR (500 MHz, methanol-d 4): δ = 8.07 (d, J = 8.8 Hz, 1H, H-6′), 7.85 (d, J = 15.4 Hz, 1H, H-β), 7.73 (d, J = 8.8 Hz, 2H, H-2, H-6), 7.70 (d, J = 15.4 Hz, 1H, H-α), 7.14 (d, J = 8.8 Hz, 2H, H-3, H5), 6.67 (dd, J = 8.8, 2.5 Hz, 1H, H-5′), 6.62 (d, J = 2.5 Hz, 1H, H-3′), 5.58 (t, J = 3.1 Hz, 1H, H-7), 5.53 (t, J = 3.1 Hz, 1H, H-7′), 3.92–3.80 (m, 2H, H-11a, H-11a′), 3.69–3.59 (m, 2H, H-11b, H-11b′), 2.07–1.96 (m, 2H, H-8a, H-8a′), 1.93–1.77 (m, 4H, H-8b, H-8′b, H-9a, H9a′), 1.76–1.55 (m, 4H, H-9b, H-9b′, H-10a, H-10a′), 1.66–1.54 (m, 2H, H-10b, H-10b′) ppm; 13C NMR (125 MHz, methanol-d 4): δ = 192.0 (C=O), 164.7 (C-4′), 163.5 (C-2′), 159.3 (C-4), 144.3 (C-β), 132.9 (C-6′), 132.2 (C-2, C-6), 128.2 (C-1), 118.1 (C-α), 116.7 (C-5), 116.5 (C-3), 114.4 (C-1′), 108.5 (C-5′), 104.0 (C-3′), 96.1 (C-7′), 94.7 (C-7), 62.9 (C-11′), 62.1 (C-11), 30.6 (C-8′), 29.9 (C-8), 25.4 (C-10′), 24.9 (C-10), 19.7 (C-9′), 18.5 (C-9) ppm; HRMS (ESI-MS): m/z = 447.1771 ([M+Na]+), 425.1956 ([M+H]+, calcd. for C25H28O6 425.1880).

(E)-3-[3,4-Bis(tetrahydro-2H-pyran-2-yloxy)phenyl]-1-[2-hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)phenyl]prop-2-en-1-one (8a, C30H36O8)

Compound 2a (4.24 mmol) and 8.48 mmol of 6a were dissolved in 10 cm3 of methanol at 35 °C under reflux. Subsequently, a solution of 20 cm3 of methanol containing barium hydroxide octahydrate (16.96 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred for 24 h and the progress was monitored by TLC. Then it was concentrated in vacuum, quenched with HCl (0.1 M), and extracted with ethyl acetate. The organic layer was separated, dried over Na2SO4, and then concentrated under vacuum. The residue yielded the crude chalcone 8a as a yellow powder which was a mixture of stereoisomers and was used for the next step without any further purification. NMR data have not been recorded from the unpurified reaction mixture. HRMS (ESI-MS): m/z = 547.2291 ([M+Na]+), 525.2472 ([M+H]+).

(E)-1-(2,4-Dihydroxyphenyl)-3-(4-hydroxyphenyl)-prop-2-en-1-one (9, ISO, C15H12O4)

7a (1.26 mmol) was dissolved in 50 cm3 of methanol. Subsequently pyridinium para-toluenesulfonate (0.062 mmol) was added and the reaction was stirred under reflux at 50 °C and the progress was monitored by TLC. Then the reaction mixture was directly concentrated under vacuum and purified by flash column chromatography (dichloromethane/methanol 9.5:0.5). 1H NMR (500 MHz, methanol-d 4): δ = 7.98 (d, J = 9.1 Hz, 1H, H-6′), 7.80 (d, J = 15.4 Hz, 1H, H-β), 7.62 (d, J = 8.9 Hz, 2H, H-2, H-6), 7.60 (d, J = 15.4 Hz, 1H, H-α), 6.87 (d, J = 8.9 Hz, 2H, H-3, H-5), 6.42 (dd, J = 8.8, 2.5 Hz, 1H, H-5′), 6.29 (d, J = 2.5 Hz, 1H, H-3′) ppm; 13C NMR (125 MHz, methanol-d 4): δ = 193.7 (C=O), 167.6 (C-4′), 166.5 (C-2′), 161.7 (C-4), 145.8 (C-β), 133.5 (C-6′), 131.9 (C-2, C-6), 128.0 (C-1), 118.5 (C-α), 117.0 (C-3, C-5), 114.8 (C-1′), 109.2 (C-5′), 103.9 (C-3′) ppm; FT-IR (KBr): \(\bar{v}\) = (C–OH) 3300, (C=O) 1640, (C=C) 1600, 1587, 1520 cm−1; HRMS (ESI-MS): m/z = 279.0627 ([M+Na]+), 257.0807 ([M+H]+, calcd. for C15H12O4 257.0730).

(E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one (10, BUT, C15H12O5)

8a (1.26 mmol) was dissolved in 50 cm3 of methanol. Subsequently pyridinium para-toluenesulfonate (0.062 mmol) was added and the reaction was stirred under reflux at 50 °C and the progress was monitored by TLC. Then the reaction mixture was directly concentrated under vacuum and purified by column flash chromatography (Sephadex LH-20, dichloromethane/methanol 9.5:0.5). 1H NMR (500 MHz, methanol-d 4): δ = 7.95 (d, J = 9.2 Hz, 1H, H-6′), 7.77 (d, J = 15.4 Hz, 1H, H-β), 7.53 (d, J = 15.4, 1H, H-α), 7.19 (d, J = 2.2 Hz, 1H, H-2), 7.12 (dd, J = 8.0, 2.2 Hz, 1H, H-6), 6.84 (d, J = 8.0 Hz, 1H, H-5), 6.42 (dd, J = 8.8, 2.5 Hz, 1H, H-5′), 6.29 (d, J = 2.5 Hz, 1H, H-3′) ppm; 13C NMR (125 MHz, methanol-d 4): δ = 191.9 (C=O), 166.0 (C-4′), 164.4 (C-2′), 148.4 (C-5), 145.3 (C-4), 144.5 (C-β), 131.8 (C-6′), 126.9 (C-1), 122.1 (C-6), 116.8 (C-α), 115.1 (C-5), 114.2 (C-2), 113.1 (C-1′), 107.7 (C-5′), 102.4 (C-3′) ppm; FT-IR (KBr): \(\bar{v}\) = (C–OH) 3300, (C=O) 1640, (C=C) 1600, 1587, 1520 cm−1; HRMS (ESI-MS): m/z = 293.1756 ([M−2H++Na+]), 271.0617 ([M−H]+, calcd. for C15H11O5 271.0611).

Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one]copper ([Cu(ISO)2], C30H22CuO8)

ISO (0.78 mmol) was dissolved in 15 cm3 of methanol under reflux. Subsequently sodium methoxide (0.8 mmol) was added to the reaction mixture. After a few minutes a solution of 15 cm3 methanol containing copper acetate monohydrate (0.35 mmol) was added dropwise and the color of the reaction mixture changed from orange to red-brown. The reaction mixture was stirred over night under reflux at 35 °C, then raised to room temperature and a brown precipitate was formed, which was filtered, washed with diethyl ether, and analyzed via mass spectrometry and IR spectroscopy. Yield: 52 %; FT-IR (KBr): \(\bar{v}\) = (C–OH) 3300, (C=O) 1610, (C=C) 1600, 1587, 1520, (Cu–O) 500 cm−1; HRMS (ESI-MS): m/z = 573.0612 [2 ISO + 63Cu2+ + e−], 575.0604 ([2 ISO + 65Cu2+ + e−], calcd. for C30H22CuO8 573.0605).

Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one]copper ([Cu(BUT)2], C30H20CuO10)

BUT (0.36 mmol) was dissolved in 5 cm3 of methanol at reflux. Subsequently a solution containing 35 mg of copper acetate monohydrate (0.17 mmol) was added dropwise. The solution was stirred under reflux for 1 h, then a red/brown precipitate was formed, which was filtered, washed with diethyl ether, and analyzed via mass spectrometry and IR spectroscopy. Yield: 50 %; FT–IR (KBr): \(\bar{v}\) = (C–OH) 3250, (C=O) 1610, (C=C) 1600, 1570, 1490, (Cu–O) 500 cm−1; HRMS (ESI-MS): m/z = 603.0362 ([2 BUT–4H + 63Cu2+], calcd. for C30H20CuO10 603.034), 605.0404 [2 BUT–4H + 65Cu2+].

Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one]zinc ([Zn(ISO)2], C30H22ZnO8)

ISO (0.195 mmol) was dissolved in 5 cm3 of acetonitrile. After a few minutes a solution of 2.5 cm3 acetonitrile containing zinc chloride (0.09 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. The solvent was removed under reduced pressure at room temperature to half of the initial volume. After a few hours an orange precipitate was formed, which was filtered, washed with diethyl ether, dried in air, and analyzed via mass spectrometry and IR spectroscopy. Yield: 53 %; FT–IR (KBr): \(\bar{v}\) = (C–OH) 3300, (C=O) 1610, (C=C) 1600, 1587, 1520, 500 (Cu–O) cm−1; HRMS (ESI-MS): m/z = 575.0676 ([2 ISO + 63Zn2+ + H]+, calcd. for [C30H22ZnO8 + H]+ 575.0679), 577.0656 ([2 ISO +65Zn2+ + H])+.

Bis[(E)-1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)prop-2-en-1-one]zinc ([Zn(BUT)2], C30H22ZnO8)

BUT (0.2 mmol) was dissolved in 5 cm3 of acetonitrile. After a few minutes a solution of 2.5 cm3 acetonitrile containing zinc chloride (0.09 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. After few hours a yellow precipitate was formed, which was filtered, washed with diethyl ether, dried in air, and analyzed via mass spectrometry and IR spectroscopy. Yield: 50 %; FT–IR (KBr): \(\bar{v}\) = (C–OH) 3300, (C=O) 1610, (C=C) 1600, 1587, 1520 (Cu–O) 500 cm−1; HRMS (ESI-MS): m/z = 607.0576 ([2 BUT + 63Zn2+ + H]+, calcd. for [C30H22ZnO8 + H]+ 607.0577), 609.0564 [2 ISO + 65Cu2+ + H].

Spectroscopic data: Job’s plot

For the Job’s plot (method of continuous variations) stock solutions of the polyhydroxychalcones 9 and 10 and Cu(OAc)2, Zn(OAc)2 were prepared in methanol-d 4. For each polyhydroxychalcone/divalent cations system, eleven 5 mm high-precision NMR sample tubes were filled with 0.6 cm3 of the compounds in the ratios [cation]/[polyhydroxychalcone] = 0:10, 1:9 up to 9:1, and 10:0, respectively. The total concentration was kept constantly at 13 mM (600 mm3).

Antioxidant activity (DPPH radical scavenging method)

The antioxidant activity test is conducted according to the method described by Ferrari et al. [48]. The test is based on the usage of 2,2-diphenyl-1-picrylhydrazyl, which is a nitrogen-centered stable radical that gives a specific absorption with a maximum at 517 nm. The free radical species DPPH reacts with the antioxidant compound and is consequently reduced to the non-radical species which produces a change in absorption [51]. The resulting decrease in absorbance was used to quantify the antioxidant activity. 1 cm3 of 6 × 10−5 mM DPPH radical solution is prepared in methanol and mixed with a variable amount (10, 25, 50, 75 mm3, etc.) of a methanolic solution containing the system metal-chalcone 1.2 mM. Immediately the absorbance of the mixture is measured every second up to 30 min at 517 nm. For the baseline control 1 cm3 of methanol was used. The percentage of inhibition of the DPPH radical was calculated for each sample referring to the following formula in accordance to Ref. [48]:

$$\% In = \frac{{A_{0} - A_{t} }}{{A_{0} }} \times 100 ,$$

where A 0 is the absorbance of the control (DPPH radical) at time 0 and A t is the absorbance of the mixture DPPH-antioxidant at time t (30 min). All determinations were performed in triplicate and the values of absorbance were corrected considering the factor of dilution.

Solubility and kinetic stability

To test the solubility of [Cu(ISO)2], [Zn(ISO)2], [Cu(BUT)2], and [Zn(BUT)2] in water, we used the method of the shaken-flask. 100 mm3 stock solution of each compound in methanol (52 mM) were mixed in a vessel with regular addition of 10 mm3 of water and then shaken. The procedure was repeated until the formation of precipitate was observed.

The chemical stability of the four complexes was investigated at 37 °C in the darkness applying UV–Vis spectroscopy by measuring the change in absorbance in the 200–600 nm range over an overall period of 16 h. Then 50 µM solutions of the complexes were prepared in DMSO and solubilized in a 0.1 M TRIS–HCl buffer (pH 7.4). Spectra were recorded every 30 min. All profiles were linearized by a hyperbolic function (Eq. 1), which represents an empirical model that well describes drug decomposition or release [51]:

$$\frac{t}{{f_{\% } }} = at + b ,$$
(1)

where f % is the fraction of residual compound at time t (min) expressed as a percentage referred to starting concentration at time zero.