Ferrocenyl based pyrazoline derivatives with vanillic core: synthesis and investigation of their biological properties

Abstract

Vanillin O-alkylated derivatives and acetylferrocene reacted under Claisen–Schmidt conditions yielding the corresponding ferrocene containing chalcones in good-to-high yields. Under similar conditions, O-alkylated derivatives of acetovanillone were reacted with ferrocenylcarbaldehyde. Two series of novel N-acetyl and N-formyl pyrazoline derivatives were prepared by cyclocondensation of previously described chalcones (containing ferrocene framework and vanillic fragment) with hydrazine hydrate in acidic solvent (formic acid or acetic acid). All synthesized compounds were fully characterized by spectral and physical data and were tested for their biological activity. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration using the broth microdilution method. The activity of the synthesized compounds was compared with standard antibiotics. The most active antibacterial compounds were 1-[5-(3,4-dimethoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone (4a) and 1-[5-(4-benzyloxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone (4f); the best antifungal activity was shown by compounds of type 4. The interaction of 4a, 4f, 5a and 5e with DNA and bovine serum albumin (BSA) were investigated by fluorescence spectroscopic method. The results achieved in competitive experiments with ethidium bromide (EB) indicated that 4a and 5e have larger affinity to displace EB from the EB–DNA complex than 4f and 5a, probably through intercalation. Fluorescence spectroscopy data show that the fluorescence quenching of BSA is a result of the formation of the 4a, 4f, 5a and 5e–BSA complex species. Measured values of K_a showed that compounds which contain the acetovanillone-formyl core (5a and 5e) formed more stable complexes with BSA than compounds with the vanillin-acetyl core (4a and 4f), suggesting that 4a– and 4f–BSA are less suitable for drug–cell interactions.

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Introduction

Chalcones are an important class of organic compounds, since they often represent core structures of various natural products and pharmaceuticals. Chalcones are easily accessible compounds and can be prepared by Claisen–Schmidt condensation. Two aromatic rings (rings A and B) enables great variability of products, due to the nature of various substituents and their positions in the ring(s) which have effects on the stereochemistry and the electronic structure.¹

This unique chalcone structure, containing a planar enone system and aromatic rings offers a bifunctional site for 1,3-dinucleophiles.² For this reason, chalcones exhibit a broad spectrum of various biological activities such as antifungal,^3–5 antimicrobial,^6,7 antiprotozoal,⁸ anti-inflammatory,^9,10 anticonvulsant¹¹ or anti-cancer.^12–16

It is well known that substitution of an aromatic nucleus of tested organic compounds with a ferrocene unit can lead to products possessing unexpected therapeutic properties,^17–19 which are absent or less manifested in the parent molecule. This fact was the main driving force in the synthesis of most known ferrocene derivatives that were designed to be derivatives of known compounds that already possess desired properties.^20,21 Ferrocenyl derivatives are among the most promising compounds which can be used in microbiological research. Water-soluble ferrocenyl derivatives are more potent as drugs than water-insoluble ones. In our previous work we reported on the synthesis of different ferrocene derivatives with expressed biological activities.^15,22–24 Ferrocene derivatives containing a heterocyclic substituent are useful precursors of some new metallocene derivative drugs.

Starting from the fact that some natural products, such as curcumines, dehydrozingerone and zingerone show various bio-activities,^25–27 and the vanillin fragment is present in these molecules, we supposed that vanillin is a suitable aldehyde for chalcone formation and their further transformation. We expected that incorporation of the vanillin core and ferrocene framework into the same molecule might be promising for development of novel antimicrobial agents, expecting interesting features due to the coexistence of the two promising pharmacophores.

On the other hand, an enone system is the key part of substrates and could be easily converted into various heterocyclic derivatives such as isoxazoles, pyrimidines, 2-aminopyrimidines, thiazines, oxazines, 2-amino-3-cyano-pyridines, pyrazoles, thiazoles and pyrimidin-2-thiones,^28,29 possessing various bioactivities.^30,31 Some pyrazoline derivatives have been used as bacteriostatic, fungicidal and anticancer agents.³²

In the light of those results we wish to report on synthesis of two series of novel chalcone derivatives containing a ferrocene core and O-alkylated vanillic fragment (Scheme 1).

Scheme 1 Synthesis of chalcones 1a–f and 2a–f.

These synthesized chalcones react with hydrazine hydrate in acidic solvent, yielding a series of novel pyrazoline derivatives: N-formyl and N-acetyl (5-(4-alkoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazoles and N-formyl and N-acetyl 3-(4-alkoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazoles), Scheme 2.

Scheme 2 Synthesis of pyrazoline derivatives 3a–f, 4a–f, 5a–f and 6a–f.

All new compounds were well characterized by IR, ¹H, ¹³C NMR and MS spectroscopy and physical data, as well as evaluation of their antimicrobial activity and DNA and BSA binding study.

The obtained products were subjected to biological evaluation and fluorescence measurements. Fluorescence technology is now a very sensitive and easily available method to study intermolecular interactions and the transcriptional dynamics of the cell nucleus.³³ The interactions between DNA and various types of molecules are important in life sciences and have attracted considerable interest, because it is related to the replication and transcription of DNA in vivo, mutation of genes and related variations of species. Analysis of DNA interaction with small molecules such as drugs, organic dyes and metals, has been an intensive topic for decades, as it provides insight into the screening and design of novel and/or more efficient drugs targeting DNA, and could speed up drug discovery and development processes.³⁴ Recently, researches on the interaction between DNA and some harmful chemicals, such as environmental pollutants, pesticides, etc., has become a hot topic as a main method for the investigation of DNA damage, as well as the understanding of toxic mechanisms.^35–37 Serum albumins are the major protein constituent of blood plasma and facilitate the disposition and transport of various exogenous and endogenous ligands to the specific targets. The drugs and bioactive small molecules bind reversibly to albumin and other serum components, which then act as carriers. Consequently, it is important to study the effect of drugs on this protein.

Results and discussion

Synthesis of pyrazoline derivatives with ferrocene framework and vanillic fragment

In the Claisen–Schmidt reaction of appropriate methyl ketones and aromatic aldehydes two series of novel chalcone derivatives were prepared. A first series of ferrocene based chalcones 1 was prepared starting from acetylferrocene and O-alkylated vanillins, while the second one, 2 have reverse order of fragments, and was obtained from ferrocenyl carbaldehyde and O-alkylated acetovanillones.²⁴ Chalcones 1 and 2 react with hydrazine hydrate in acidic solvent (boiling formic acid or acetic acid) yielding a series of novel N-formyl, 3 and 5, and N-acetyl, 4 and 6 pyrazoline derivatives in 51–88% yield. All new compounds were well characterized by IR, ¹H, ¹³C NMR and MS spectroscopy and physical data.

The structures of all synthesized products 3a–f, 4a–f, 5a–f and 6a–f are presented in Table 1. Generally, good yields were achieved in all cases.

Table 1 Structures of products 3a–f, 4a–f, 5a–f and 6a–f

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Biological evaluation

The antimicrobial activities of the tested compounds (3a–f, 4a–f, 5a–f and 6a–f) against the test microorganisms are shown in Tables 2 and 3. The tested compounds inhibited all the tested bacteria (Table 2). The maximal antibacterial activity was found for 4a and 4f against Bacillus subtilis and Bacillus cereus species with MIC value of 0.156 mg mL⁻¹.

Table 2 Antibacterial activity of compounds 3a–f, 4a–f, 5a–f and 6a–f; values of MIC given as mg mL⁻¹. The values are means of three replicates ± standard deviation. In all cases, the standard deviation is ±0.001

Test compounds	Staphylococcus aureus	Bacillus subtilis	Basillus cereus	Escherichia coli	Proteus mirabilis
3a	1.25	0.312	0.625	2.5	2.5
3b	1.25	0.312	0.312	1.25	2.5
3c	1.25	0.312	0.625	2.5	2.5
3d	2.5	0.625	1.25	2.5	5
3e	2.5	1.25	1.25	5	2.5
3f	1.25	0.625	0.312	1.25	2.5
4a	0.625	0.156	0.312	1.25	1.25
4b	2.5	0.625	1.25	2.5	5
4c	2.5	0.625	0.312	1.25	1.25
4d	1.25	0.625	0.312	2.5	2.5
4e	0.625	0.312	0.312	1.25	1.25
4f	0.625	0.156	0.156	0.625	1.25
5a	1.62	0.81	0.81	3.25	1.62
5b	7.5	3.25	1.62	15	7.5
5c	1.62	0.81	0.81	3.25	1.62
5d	3.25	1.62	1.62	7.5	3.25
5e	3.25	1.62	0.81	7.5	3.25
5f	15	3.25	3.25	15	15
6a	3.25	1.62	0.81	3.25	3.25
6b	3.25	1.62	1.62	7.5	7.5
6c	3.25	1.62	1.62	7.5	3.25
6d	7.5	3.25	3.25	15	7.5
6e	3.25	0.81	0.81	7.5	3.25
6f	3.25	1.62	0.81	7.5	7.5
Streptomycin	0.031	0.016	0.016	0.062	0.062

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Table 3 Antifungal activity of compounds 3a–f, 4a–f, 5a–f and 6a–f; values of MIC given as mg mL⁻¹. The values are means of three replicates ± standard deviation. In all cases, the standard deviation is ±0.002^a

Test compounds	Mucor mucedo	Trichoderma viride	Aspergillus niger	Candida albicans	Penicillium italicum
a (/) – no obvious effects.
3a	2.5	2.5	5	1.25	5
3b	2.5	5	10	2.5	5
3c	1.25	5	5	1.25	10
3d	2.5	5	10	1.25	5
3e	5	5	5	2.5	10
3f	1.25	2.5	5	1.25	5
4a	1.25	2.5	5	0.625	2.5
4b	5	2.5	5	1.25	5
4c	2.5	2.5	5	0.625	2.5
4d	1.25	5	10	0.625	2.5
4e	1.25	2.5	2.5	1.25	2.5
4f	1.25	2.5	5	0.625	2.5
5a	7.5	3.25	15	1.62	15
5b	15	15	/	7.5	/
5c	7.5	3.25	7.5	1.62	93
5d	15	7.5	/	3.25	/
5e	15	7.5	/	3.25	/
5f	/	15	/	7.5	/
6a	15	7.5	15	1.62	15
6b	/	15	/	3.25	/
6c	15	7.5	/	3.25	/
6d	/	15	/	3.25	/
6e	7.5	7.5	/	1.62	/
6f	15	7.5	/	3.25	/
Ketoconazole	0.156	0.078	0.078	0.039	0.156

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These compounds also inhibited the growth of tested fungi but at slightly higher concentrations. Tested components 6b, 6d, 5b, 5d, 5e and 5f acted selectively on the tested fungi (Table 3), while other components inhibited growth of all tested fungi. The strongest antifungal activity was manifested by 4a, 4d, 4c and 4f components against Candida albicans (MIC values of 0.625 mg mL⁻¹).

The most sensitive, among the bacteria, were Bacillus subtilis and Bacillus cereus species while among the fungi, the most sensitive appeared to be Candida albicans.

The antimicrobial activity was compared with the standard antibiotics, streptomycin (for bacteria) and ketoconazole (for fungi). In a negative control, DMSO had no inhibitory effect on the tested organisms.

In these experiments, the compounds examined at the same concentrations showed a slightly stronger antibacterial than antifungal activity. These results could be expected due to the fact that numerous tests proved that bacteria are more sensitive to antibiotics compared to fungi. The reason for different sensitivities between fungi and bacteria can be found in different permeabilities of the cell wall. The cell wall of the Gram-positive bacteria consists of peptidoglycans (murein) and teichoic acids, while the cell wall of Gram-negative bacteria consists of lipopolysaccharides and lipopoliproteins,³⁸ whereas, the cell wall of fungi consists of polysaccharides such as chitin and glucan.³⁹

Fluorescence measurements

Florescence quenching on EB–DNA. An intermolecular deactivation of EB–DNA (complex formed between ethidium bromide and DNA) was investigated in the presence of selected quenchers 4a, 4f, 5a and 5e. We used the molecular fluorophore EB, which when bound to DNA, shows intense fluorescence light due to its strong intercalation between the adjacent DNA base pairs.⁴⁰ The molecules of 4a, 4f, 5a and 5e were selected because they are exhibit most pronounced biological activities (Tables 2 and 3).

The fluorescence emission titrations were recorded in the range of 550–740 nm at 25 °C. After the addition of 4a, 4f, 5a and 5e into the EB–DNA solution the mixtures were incubated for 4 h at 37 °C prior to measurements. A significant decrease in the intensity of the florescence emission band (at 614, 610, 611 and 613 nm, respectively) was observed. The observed quenching of EB–DNA indicates a competition between the added compounds and EB in binding to DNA.

The fluorescence quenching of quenchers was described by means of the Stern–Volmer eqn (1),⁴¹ with the dependence of I₀/I on [Q] examined (Fig. 1).

I₀/I = 1 + k_qτ₀[Q] = 1 + K_SV[Q] (1)

In eqn (1) I₀ and I are the emission intensities in the absence and presence of the quencher, [Q] is the total concentration of the quencher, k_q is the bimolecular quenching rate constant, and τ₀ is the average lifetime of DNA in the absence of quencher (10⁻⁸ s). K_sv is the Stern–Volmer quenching constant whose values were obtained from the slopes of the plots of I₀/I vs. [Q]. Emission spectra of 4a, 4f, 5a or 5e (Fig. 1 and S1†) clearly shows that fluorescence intensity of EB–DNA decreases continuously with increasing concentration of quencher.

Fig. 1 Top: emission spectra of EB bound to DNA in the absence (black lines) and presence of compounds 4a and 5e. The red lines denote solutions: buffer + quencher. [EB] = 25 μM, [DNA] = 25 μM; [4a] = 0–25 μM and [5e] = 0–17.2 μM; pH = 7.4; λ_ex = 520 nm. Bottom: Stern–Volmer plots of I₀/I versus [Q].

Quenching parameters are presented in Table 4. As can be seen, 4a and 5e showed higher K_sv values [(2.5 ± 0.1) × 10⁴ and (2.9 ± 0.2) × 10⁴ M⁻¹, respectively] than 4f and 5a. This finding indicates their high affinity and efficiency to substitute EB from the EB–DNA complex.

Table 4 Stern–Volmer constants (K_sv) and correlation coefficients (R) for quenchers 4a, 4f, 5a and 5e

Quencher	Ksv/M^−1	R
4a	(2.5 ± 0.1) × 10^4	0.992
4f	(1.2 ± 0.1) × 10^3	0.989
5a	(2.1 ± 0.1) × 10^3	0.990
5e	(2.9 ± 0.2) × 10^4	0.994

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Interaction with bovine serum albumin

Bearing in mind that the efficiency of drugs strongly depends on their protein binding ability, we decided to investigate the affinity of compounds 4a, 4f, 5a and 5e to bind to bovine serum albumin (BSA). This task was realized by means of fluorescence emission titration of BSA with quenchers 4a, 4f, 5a and 5e in the range of 325–415 nm with an incubation time of 2 h at 37 °C. The characteristic BSA emission line (black line, Fig. 2) was centered at 355 nm and after addition of appropriate quenchers it was hypsochromic shifted (≈5 nm). This shift indicates that the 4a–, 4f–, 5a– and 5e–BSA complex species were formed and changed the polarity of the microenvironment in the vicinity of Trp-214.^42,43 As can been seen from examples, which are presented on Fig. 2 and S2,† the fluorescence intensity of BSA continually decreased with increasing concentration of compounds. Linearity of the plots from Fig. 2 and S2† indicates the participation of predominantly one type of quenching in the interaction of BSA with the selected compounds, either dynamic or static.⁴⁴

Fig. 2 Emission spectra of BSA in the absence (black lines) and presence of compounds 4a and 5e. The red lines denote solutions: buffer + quencher. [BSA] = 1.2 μM; [4a] and [5e] = 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 μM; pH = 7.4; λ_ex = 295 nm. Plots of log [(I₀ − I/I)] vs. log [Q].

The fluorescence quenching data were analyzed by using eqn (2):⁴⁵

log (I₀ − I/I) = log K_a + n log [Q] (2)

where I₀ and I are the emission intensities in the absence and presence of the quencher, K_a is the binding constant for 4a, 4f, 5a or 5e–protein interaction, n is the number of binding sites per BSA molecule, and [Q] is the concentration of the quencher. The plots of log [(I₀ − I)/I] vs. log [Q] were depicted (Fig. 2 and S2†). Values of K_a and n were obtained from the intercept and slope of the obtained straight lines. The values of binding parameters for 4a–, 4f–, 5a– and 5e–BSA complexes are given in Table 5.

Table 5 Binding parameters (K_a and n) and the correlation coefficient (R) for interaction of quenchers with BSA

Quencher	Ka/M	n	R
4a	(1.3 ± 0.1) × 10^4	1.2	0.995
4f	(1.9 ± 0.1) × 10^4	1.1	0.989
5a	(2.3 ± 0.2) × 10^6	2.1	0.991
5e	(3.3 ± 0.2) × 10^6	1.9	0.993

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Based on the values of n (Table 5) we can conclude that quenchers with vanillin-acetyl (4a and 4f) and acetovanillone–formyl fragment (5a and 5e) bind to BSA in the molar ratios of 1 : 1 and 2 : 1, respectively. The K_a values followed the order 5a, 5e, 4f, 4a. The larger K_a value for 5a–BSA and 5e–BSA than for 4a–BSA and 4f–BSA complex species clearly shows that these complexes are more stable, which means that 4a– and 4f–BSA complexes are less suitable for drug–cell interactions.

Experimental

All solvents (CH₂Cl₂, toluene and EtOAc), substrate (ferrocene, vanillin and acetovanillone), calf-thymus DNA (CT-DNA), BSA (bovine serum albumin) and ethidium bromide (EB) were purchased from Sigma. Phosphate buffered saline (PBS) tablets were purchased from Fisher BioReagents. The used O-alkyl vanillic aldehydes were synthesized according to the previously described methodology.^46,47

Melting points (mps) were determined on a Mel-Temp apparatus and are uncorrected. Thin-layer chromatography (TLC) was carried out on 0.25 mm Sigma-Aldrich coated silica gel plates (60F-254) using eluent toluene : EtOAc (4 : 1) mixture as a mobile phase and anisaldehyde spray solution or UV light for visualization. The IR spectra were recorded by a Perkin-Elmer Spectrum One FT-IR spectrometer on KBr pellets. The NMR spectra of all compounds were measured in CDCl₃ with TMS as internal standard on a Varian Gemini 200 MHz NMR spectrometer (¹H at 200 and ¹³C at 50 MHz); the only exception was compound 5e which was measured in DMSO-d₆. Abbreviations for the NMR signal that were used are: s = singlet, d = doublet, t = triplet, m = multiplet, and dd = doublet of doublet. All NMR (¹H and ¹³C) spectral data are given in ESI.† Mass spectrometry was performed on a Waters Micromass ZQ mass spectrometer and MassLynx software for control and data processing. Electrospray ionization in the positive mode was used. The electrospray capillary was set at 4.3 kV and the cone at 40 V. The ion source temperature was set at 125 °C and the nitrogen flow rates were 400 L h⁻¹ and 50 L h⁻¹, for desolvation and cone gas flow, respectively. The collision energy was 40 eV. The RF-1501 PC spectrofluorometer (Shimadzu, Japan) for fluorescence measurements was used.

The following strains of bacteria were used as test organisms in this study: Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Bacillus cereus (ATCC 10987), Escherichia coli (ATCC 25922) and Proteus mirabilis (ATCC 29906). All of the bacteria used were obtained from the American Type Culture Collection (ATCC). The bacterial cultures were maintained on Müller–Hinton agar substrates (Torlak, Belgrade). The strains of fungi used as test organisms were: Aspergillus niger (ATCC 16888), Candida albicans (ATCC 10259), Penicillium italicum (ATCC 10454), Mucor mucedo (ATCC 20094) and Trichoderma viride (ATCC 13233). All of the fungi were from the American Type Culture Collection (ATCC). The fungal cultures were maintained on potato dextrose (PD) agar, except for Candida albicans that was maintained on Sabouraud dextrose (SD) agar (Torlak, Belgrade). All of the cultures were stored at 4 °C and subcultured every 15 days.

Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37 °C on Müller–Hinton agar substrates and brought up by dilution according to the 0.5 McFarland standard to approximately 108 CFU mL⁻¹. Suspensions of fungal spores were prepared from freshly mature (3- to 7-day-old) cultures that grew at 30 °C on a PD agar substrate. The spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometrically at 530 nm, and were then further diluted to approximately 106 CFU mL⁻¹ according to the procedure recommended by NCCLS.⁴⁸

Minimal inhibitory concentration (MIC)

The minimal inhibitory concentration (MIC) was determined by the broth microdilution method using 96-well micro-titer plates.⁴⁹ Starting solutions of tested compounds were obtained by measuring a certain quantity of the compounds and dissolving it in 5% DMSO. Twofold serial dilutions of tested compounds were made in the concentration range from 20 to 0.0195 mg mL⁻¹ (for the tested compounds 3a–f and 4a–f) and 30–0.0125 mg mL⁻¹ (for the tested compounds 5a–f and 6a–f) in sterile 96-well plates containing Müller–Hinton broth for bacterial cultures and a SD broth for fungal cultures. Then, a 10 μL of diluted microbial suspension was added to each well and finally, 10 μL of resazurin solution was also added to each well. Resazurin is an oxidation–reduction indicator used for the evaluation of microbial growth. It is a blue non-fluorescent dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. Results were read visually. The boundary dilution without any changing of resazurin color was defined as the MIC for the tested microorganism at a given concentration. As a positive control of growth inhibition, streptomycin was used in the case of bacteria and ketoconazole in the case of fungi. A 5% DMSO solution was used as a negative control for the influence of the solvents.

Fluorescence experiments

A fresh solution of CT-DNA and EB in doubly distilled water was prepared in 10 mM Tris-HCl/100 mM NaCl buffer at pH = 7.4. The DNA solution gave a ratio of UV absorbance at 260 and 280 nm (A₂₆₀/A₂₈₀) of ca. 1.8–1.9, indicating that the DNA was sufficiently free of proteins. CT-DNA concentration was measured by the UV absorbance at 260 nm (ε = 6600 M⁻¹ cm⁻¹).⁵⁰ BSA solution (12 μM) in 10 mM PBS buffer at pH = 7.4 was prepared and stored in the dark at 4 °C, no longer than 3 days. The 4a–, 4f–, 5a– or 5e–BSA complexes were prepared by independently incubating a constant amount of BSA with increasing amounts of the quenchers 4a, 4f, 5a or 5e. A dilution series of DMSO solutions of quenchers (for BSA 0.1–1 mM or for DNA 0.46–17.2 and 0.25–2.5 mM) was prepared and for each data point 0.03 mL of the solution of quenchers were added into 3 mL, to give final concentration of quenchers of 1–10, 4.6–17.2 and 2.5–25 μM. The addition of constant volume of quenchers to the BSA solution avoided complications due to dilution effects. The emission spectra of the solutions that contain only compounds 4a, 4f, 5a or 5e (at the highest concentration) were also recorded. Appropriate blanks (0.03 mL DMSO + 2.97 mL buffer) were used to correct the fluorescence background. All values of absorbance measured at 220–600 nm for solutions that contained only 4a, 4f, 5a or 5e are sufficiently low (<0.05)⁵¹ to avoid inner filtering effects and therefore corrections were not necessary. To evaluate the quenching effect of DMSO; the effect of BSA (or DNA) dilution by buffer titration was evaluated and was compared to the effect of dilution with DMSO. It was observed that DMSO had the same effect on BSA (or DNA) fluorescence as the buffer dilution effect. The native structure of BSA and DNA is retained in the presence of low concentrations of DMSO (<5%).^52–54 Thus, the effect of DMSO on the 4a, 4f, 5a and 5e interactions with BSA (or DNA) can be considered negligible in the used amount.

Synthetic procedure

General procedure for synthesis of chalcones. The corresponding aldehyde and acetyl-derivative were dissolved in 50 mL of warm ethanol and the mixture was stirred for 10 min and 2 mL of 40% NaOH was slowly added. In all cases the ferrocene component should be used in 20% excess relative to the vanillic component (10 mmol) and the reaction mixture was stirred overnight at 50 °C. Crushed ice, 100 g, was added into beaker and the reaction mixture was poured onto this with stirring. The products in some cases could be isolated by filtration, otherwise it is necessary to extract with toluene or dichloromethane (3 × 50 mL). The organic layer was washed with water (2 × 50 mL), brine (2 × 50 mL) and dried over anhydrous Na₂SO₄. Most of the solvent was evaporated at reduced pressure and the crude concentrated solution was filtered through a short column of silica gel. Solvent was evaporated under reduced pressure and residue, if necessary, was chromatographed on a silica gel column using toluene–ethyl acetate (8 : 2) mixture as eluent for separation of reaction products. Yields of compounds from series 1 and 2 were 59–85% and 70–98% respectively.²⁴

General procedure for synthesis of N-formyl and N-acetyl pyrazoline derivatives. To a stirred solution of chalcones 1a–f or 2a–f (2 mmol), in the corresponding acid (formic or acetic acid, 15 mL) was added hydrazine monohydrate (2 mL) and the reaction mixture was heated to reflux for 5 h. The solvent was evaporated under reduced pressure and cold water (50 mL) was added to the yellow residue. Products were extracted from the reaction mixture with toluene or toluene–EtOAc (95 : 5) mixture. After solvent evaporation the oily residue was dissolved in diethyl ether from which products 1a–f crystallized on standing in a deep freeze.

All synthesized compounds were well soluble in common organic solvents. Compound 4a, as a representative sample, was tested for its stability in DMSO-d₆, as this solvent is used for sample preparations in biological experiments. No visible changes were observed in NMR experiments performed at 18, 24 or 48 h.

3a 5-(3,4-Dimethoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 57%; orange oil; IR (KBr): 2931, 1668, 1594, 1517, 1411, 1359, 1309, 1259, 1235, 1139, 1025, 819 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 3.04 (dd, J = 17.6, 4.6 Hz, 1H), 3.69 (dd, J = 17.4, 11.4, 1H), 3.87 (d, J = 6.4 Hz, 6H), 4.15 (s, 5H), 4.42–4.45 (m, 2H), 4.57 (dt, J = 2.4, 1.4 Hz, 1H), 4.68 (dt, J = 2.6, 1.2 Hz, 1H), 5.44 (dd, J = 11.4, 4.4 Hz, 1H), 6.77–6.84 (m, 3H), 8.89 (d, J = 1.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 30.8, 43.9, 55.9, 58, 67.3, 67.8, 69.2, 69.4, 70.6, 70.7, 74.5, 108.9, 111.6, 117.5, 133.4, 148.7, 149.4, 157.9, 159.5 (CO). ESI-MS (40 eV): m/z (%) = 418.07 (100%) [M]⁺.
3b 5-(4-Ethoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 63%; orange oil; IR (KBr): 2929, 1671, 1594, 1516, 1415, 1360, 1308, 1259, 1233, 1140, 1121, 1034, 825 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.43 (t, J = 7.0 Hz, 3H), 3.03 (dd, J = 17.4, 4.4 Hz, 1H), 3.68 (dd, J = 17.4, 11.6, 1H), 3.87 (s, 3H), 4.06 (q, J = 7.0 Hz, 2H), 4.15 (s, 5H), 4.42–4.44 (m, 2H), 4.56 (dt, J = 2.6, 1.4 Hz, 1H), 4.68 (dt, J = 2.6, 1.2 Hz, 1H), 5.44 (dd, J = 11.4, 4.4 Hz, 1H), 6.77–6.83 (m, 3H), 8.89 (d, J = 1.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 14.7, 43.9, 56, 58, 64.4, 67.3, 67.8, 69.4, 70.5, 70.7, 74.5, 84.1, 109.1, 113.1, 117.5, 133.3, 148, 149.8, 157.9, 159.5 (CO). ESI-MS (40 eV): m/z (%) = 432.27 (100%) [M]⁺.
3c 5-(4-Isopropoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 54%; light orange, mp = 55–57 °C; IR (KBr): 3086, 2973, 2926, 1671, 1591, 1512, 1412, 1359, 1307, 1258, 1231, 1138, 1106, 1032, 821 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.34 (d, J = 6.2 Hz, 6H), 3.03 (dd, J = 17.4, 4.4 Hz, 1H), 3.68 (dd, J = 17.4, 11.6, 1H), 3.85 (s, 3H), 4.14 (s, 5H), 4.41–4.43 (m, 2H), 4.44–4.51 (m, 1H), 4.56 (dt, J = 2.6, 1.4 Hz, 1H), 4.69 (dt, J = 2.6, 1.2 Hz, 1H), 5.44 (dd, J = 11.2, 4.2 Hz, 1H), 6.79–6.89 (m, 3H), 8.93 (d, J = 0.8 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 22, 43.9, 56, 58, 67.3, 67.8, 69.4, 70.5, 70.7, 71.5, 74.5, 109.5, 116.1, 117.4, 133.7, 147.1, 150.8, 158, 159.5 (CO). ESI-MS (40 eV): m/z (%) = 446.14 (100%) [M]⁺.
3d 5-(3-Methoxy-4-propoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 64%; light orange, mp = 52–54 °C; IR (KBr): 2935, 2874, 1671, 1515, 1410, 1358, 1308, 1259, 1139, 1034, 818 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.01 (t, J = 7.4 Hz, 3H), 1.78–1.89 (m, 2H), 3.03 (dd, J = 17.4, 4.4 Hz, 1H), 3.68 (dd, J = 17.6, 11.6, 1H), 3.86 (s, 3H), 3.94 (t, J = 6.8 Hz, 2H), 4.15 (s, 5H), 4.42–4.44 (m, 2H), 4.56 (dt, J = 2.4, 1.4 Hz, 1H), 4.68 (dt, J = 2.4, 1.2 Hz, 1H), 5.44 (dd, J = 11.4, 4.6 Hz, 1H), 6.77–6.87 (m, 3H), 8.89 (d, J = 0.8 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 10.4, 22.4, 30.8, 43.9, 56.1, 58, 67.3, 67.8, 69.4, 70.5, 70.6, 70.7, 74.5, 109.3, 113.3, 117.5, 133.3, 148.3, 149.9, 157.9, 159.5 (CO). ESI-MS (40 eV): m/z (%) = 446.14 (100%) [M]⁺.
3e 5-(4-Butoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 77%; orange oil; IR (KBr): 2930, 2873, 1672, 1516, 1411, 1358, 1308, 1259, 1234, 1139, 1029, 824 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 0.95 (t, J = 7.4 Hz, 3H), 1.41–1.52 (m, 2H), 1.72–1.83 (m, 2H), 3.03 (dd, J = 17.6, 4.6 Hz, 1H), 3.68 (dd, J = 17.6, 11.6, 1H), 3.86 (s, 3H), 3.98 (t, J = 6.8 Hz, 2H), 4.15 (s, 5H), 4.42–4.44 (m, 2H), 4.56 (dt, J = 2.4, 1.4 Hz, 1H), 4.68 (dt, J = 2.6, 1.4 Hz, 1H), 5.44 (dd, J = 11.2, 4.4 Hz, 1H), 6.77–6.83 (m, 3H), 8.89 (d, J = 1.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 13.8, 19.2, 31.2, 44, 56.2, 58.1, 67.3, 67.8, 68.8, 69.4, 70.6, 70.7, 74.6, 84.1, 109.4, 113.4, 117.6, 133.3, 148.4, 149.9, 157.9, 159.5 (CO). ESI-MS (40 eV): m/z (%) = 460.23 (100%) [M]⁺.
3f 5-(4-Benzyloxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 81%; light orange, mp = 149–150 °C; IR (KBr): 3089, 2924, 2858, 1664, 1601, 1514, 1416, 1358, 1310, 1256, 1229, 1169, 1137, 1014 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 3.01 (dd, J = 17.6, 4.6 Hz, 1H), 3.66 (dd, J = 17.6, 11.6 Hz, 1H), 3.88 (s, 3H), 4.13 (s, 5H), 4.41–4.43 (m, 2H), 4.55 (dt, J = 2.4, 1.4 Hz, 1H), 4.66 (dt, J = 2.4, 1.2 Hz, 1H), 5.11 (s, 2H), 5.43 (dd, J = 11.6, 4.8 Hz, 1H), 6.77–6.87 (m, 3H), 7.26–7.39 (m, 5H), 8.88 (d, J = 1.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 43.9, 56.1, 57.9, 67.3, 67.8, 69.4, 70.5, 70.7, 71.1, 74.5, 109.4, 114.5, 117.5, 127.2, 127.7, 128.4, 133.9, 136.9, 147.9, 150.1, 157.9, 159.4 (CO). ESI-MS (40 eV): m/z (%) = 494.15 (100%) [M]⁺.
4a 1-[5-(3,4-Dimethoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 56%; light orange, mp = 78–79 °C; IR (KBr): 2926, 1653, 1596, 1516, 1413, 1308, 1259, 1235, 1138, 1020, 822 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 2.36 (s, 3H), 2.96 (dd, J = 17.4, 4.2 Hz, 1H), 3.62 (dd, J = 17.4, 11.6, 1H), 3.85 (d, J = 9.2 Hz, 6H), 4.12 (s, 5H), 4.38–4.41 (m, 2H), 4.53 (dt, J = 2.4, 1.4 Hz, 1H), 4.67 (dt, J = 2.6, 1.2 Hz, 1H), 5.48 (dd, J = 11.4, 4.0 Hz, 1H), 6.75–6.80 (m, 3H); ¹³C NMR (50 MHz, CDCl₃): δ 21.8, 43.7, 55.9, 58.9, 67.1, 67.6, 69.3, 70.2, 70.4, 75.4, 108.9, 111.6, 117.2, 134.7, 148.4, 149.3, 155.8, 168.2 (CO). ESI-MS (40 eV): m/z (%) = 432.14 (100%) [M]⁺.
4b 1-[5-(4-Ethoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 64%; light orange, mp = 125–126 °C; IR (KBr): 2931, 1662, 1600, 1591, 1520, 1409, 1311, 1260, 1236, 1139, 1036, 824 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.42 (t, J = 7.0 Hz, 3H), 2.37 (s, 3H), 2.96 (dd, J = 17.2, 4.0 Hz, 1H), 3.61 (dd, J = 17.4, 11.6, 1H), 3.87 (s, 3H), 4.05 (q, J = 7.0 Hz, 2H), 4.12 (s, 5H), 4.37–4.41 (m, 2H), 4.53 (dt, J = 2.4, 1.4 Hz, 1H), 4.68 (dt, J = 2.6, 1.4 Hz, 1H), 5.49 (dd, J = 11.4, 4.0 Hz, 1H), 6.73–6.85 (m, 3H); ¹³C NMR (50 MHz, CDCl₃): δ 14.7, 21.8, 43.7, 55.9, 58.9, 64.3, 67.1, 67.7, 69.3, 70.2, 70.4, 75.3, 109.3, 113.1, 117.2, 134.6, 147.7, 149.6, 155.9, 168.2 (CO). ESI-MS (40 eV): m/z (%) = 446.14 (100%) [M]⁺.
4c 1-[5-(4-Isopropoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 51%; orange oil; IR (KBr): 2975, 2930, 1655, 1508, 1412, 1308, 1259, 1230, 1137, 1106, 1030, 824 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.33 (d, J = 6.0 Hz, 6H), 2.38 (s, 3H), 2.97 (dd, J = 17.2, 4.0 Hz, 1H), 3.62 (dd, J = 17.2, 11.4 Hz, 1H), 3.85 (s, 3H), 4.12 (s, 5H), 4.39–4.41 (m, 2H), 4.43–4.49 (m, 1H), 4.53 (dt, J = 2.6, 1.2 Hz, 1H), 4.68 (dt, J = 2.6, 1.2 Hz, 1H), 5.49 (dd, J = 11.6, 4.2 Hz, 1H), 6.72–6.87 (m, 3H); ¹³C NMR (50 MHz, CDCl₃): δ 21.9, 22.1, 43.8, 56.1, 58.9, 67.1, 67.7, 69.3, 70.3, 70.4, 71.5, 75.4, 109.8, 116.2, 117.3, 135.1, 146.8, 150.7, 155.9, 168.3 (CO). ESI-MS (40 eV): m/z (%) = 460.22 (100%) [M]⁺.
4d 1-[5-(3-Methoxy-4-propoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 68%; light orange, mp = 87–89 °C; IR (KBr): 2973, 2931, 1662, 1600, 1591, 1520, 1410, 1311, 1260, 1236, 1139, 1036, 824 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.00 (t, J = 7.6 Hz, 3H), 1.82 (sx, J = 7.2 Hz, 2H), 2.37 (s, 3H), 2.97 (dd, J = 17.4, 4.2 Hz, 1H), 3.61 (dd, J = 17.4, 11.6 Hz, 1H), 3.86 (s, 3H), 3.93 (t, J = 6.8 Hz, 2H), 4.12 (s, 5H), 4.38–4.41 (m, 2H), 4.53 (dt, J = 2.4, 1.4 Hz, 1H), 4.68 (dt, J = 2.6, 1.2 Hz, 1H), 5.49 (dd, J = 11.4, 4.0 Hz, 1H), 6.73–6.85 (m, 3H); ¹³C NMR (50 MHz, CDCl₃): δ 10.3, 21.8, 22.4, 43.7, 56.1, 58.9, 67.1, 67.6, 69.3, 70.2, 70.4, 70.6, 75.4, 109.6, 113.4, 117.3, 134.6, 148.1, 149.7, 155.8, 168.2 (CO). ESI-MS (40 eV): m/z (%) = 460.11 (100%) [M]⁺.
4e 1-[5-(4-Butoxy-3-methoxyphenyl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 67%; red-orange, mp = 50–51 °C; IR (KBr): 3087, 2956, 2933, 1658, 1593, 1515, 1411, 1310, 1258, 1234, 1138, 1028, 820 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 0.94 (t, J = 7.4 Hz, 3H), 1.40–1.48 (m, 2H), 1.75–1.82 (m, 2H), 2.37 (s, 3H), 2.97 (dd, J = 17.4, 4.2 Hz, 1H), 3.62 (dd, J = 17.4, 11.4 Hz, 1H), 3.86 (s, 3H), 3.97 (t, J = 6.8 Hz, 2H), 4.13 (s, 5H), 4.38–4.41 (m, 2H), 4.54 (dt, J = 2.4, 1.4 Hz, 1H), 4.68 (dt, J = 2.6, 1.4 Hz, 1H), 5.49 (dd, J = 11.4, 4.0 Hz, 1H), 6.73–6.85 (m, 3H); ¹³C NMR (50 MHz, CDCl₃): δ 13.8, 19.1, 21.9, 31.2, 43.8, 56.1, 58.9, 67.1, 67.7, 68.8, 69.3, 70.2, 70.4, 75.4, 109.6, 113.4, 117.3, 134.7, 148.1, 149.8, 155.8, 168.2 (CO). ESI-MS (40 eV): m/z (%) = 475.01 (100%) [M + 1]⁺.
4f 1-[5-(4-Benzyloxy-3-methoxyphenyl)-3-ferrocenyl-4,5- dihydro-1H-pyrazol-1-yl]ethanone. Yield 81%; light orange, mp = 132–133 °C; IR (KBr): 3031, 2927, 1651, 1594, 1508, 1497, 1415, 1310, 1259, 1172, 1137, 1011, 819 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 2.36 (s, 3H), 2.95 (dd, J = 17.4, 4.2 Hz, 1H), 3.60 (dd, J = 17.4, 11.6 Hz, 1H), 3.88 (s, 3H), 4.11 (s, 5H), 4.38–4.40 (m, 2H), 4.53 (dt, J = 2.4, 1.4 Hz, 1H), 4.66 (dt, J = 2.6, 1.4 Hz, 1H), 5.10 (s, 2H), 5.48 (dd, J = 11.6, 4.2 Hz, 1H), 6.68–6.85 (m, 3H), 7.25–7.41 (m, 5H); ¹³C NMR (50 MHz, CDCl₃): δ 21.8, 43.7, 56.1, 58.9, 67.1, 67.6, 69.3, 70.2, 70.4, 71.1, 75.4, 109.6, 114.5, 117.2, 127.2, 127.7, 128.4, 135.3, 137.1, 147.7, 149.9, 155.8, 168.2 (CO). ESI-MS (40 eV): m/z (%) = 508.12 (100%) [M]⁺.
5a 3-(3,4-Dimethoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 60%; ochre, mp = 214–216 °C; IR (KBr): 3084, 2924, 1655, 1603, 1519, 1435, 1362, 1269, 1245, 1143, 1024, 818 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 3.52 (dd, J = 17.2, 4.4 Hz, 1H), 3.70 (dd, J = 17.2, 10.6 Hz, 1H), 3.96 (d, J = 3.0 Hz, 6H), 4.05–4.07 (m, 1H), 4.13–4.15 (m, 1H), 4.15 (s, 5H), 4.17–4.20 (m, 1H), 4.43–4.46 (m, 1H), 5.42 (dd, J = 11.0, 4.2 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.19–7.27 (m, 1H), 7.48 (d, J = 2.0 Hz, 1H), 8.81 (d, J = 0.8 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 39.9, 54.5, 56, 65.6, 68.3, 68.4, 68.6, 69.7, 86.8, 108.6, 110.7, 120.5, 124, 149.4, 151.4, 155.7, 160 (CO). ESI-MS (40 eV): m/z (%) = 418.12 (100%) [M]⁺.
5b 3-(4-Ethoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 51%; ochre, mp = 144–146 °C; IR (KBr): 3084, 2935, 1665, 1600, 1515, 1428, 1355, 1258, 1240, 1150, 1029, 827 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.51 (t, J = 7.0 Hz, 3H), 3.52 (dd, J = 17.2, 4.2 Hz, 1H), 3.69 (dd, J = 17.4, 10.8 Hz, 1H), 3.95 (s, 3H), 4.05–4.07 (m, 1H), 4.11–4.15 (m, 2H), 4.15 (s, 5H), 4.17–4.19 (m, 2H), 4.43–4.45 (m, 1H), 5.41 (dd, J = 10.6, 4.2 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 7.17–7.27 (m, 1H), 7.47 (d, J = 2.0 Hz, 1H), 8.80 (d, J = 0.8 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 14.6, 39.9, 54.5, 56.1, 64.4, 65.6, 68.4, 68.6, 69.7, 86.8, 108.8, 111.8, 120.5, 123.8, 149.6, 150.8, 155.7, 159.9 (CO). ESI-MS (40 eV): m/z (%) = 432.00 (100%) [M]⁺.
5c 3-(4-Isopropoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 70%; ochre, mp = 127–128 °C; IR (KBr): 3086, 2931, 1675, 1662, 1598, 1513, 1430, 1362, 1261, 1242, 1139, 1028, 828 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.42 (d, J = 6.0 Hz, 6H), 3.51 (dd, J = 17.4, 4.6 Hz, 1H), 3.69 (dd, J = 17.4, 10.8 Hz, 1H), 3.93 (s, 3H), 4.05–4.07 (m, 1H), 4.12–4.15 (m, 1H), 4.15 (s, 5H), 4.17–4.19 (m, 1H), 4.43–4.46 (m, 1H), 4.57–4.69 (m, 1H), 5.41 (dd, J = 9.8, 4.4 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 7.16–7.26 (m, 1H), 7.46 (d, J = 2.0 Hz, 1H), 8.80 (d, J = 1.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 21.9, 39.9, 54.5, 56.1, 65.6, 68.4, 68.6, 69.7, 71.4, 86.8, 109.3, 114.4, 120.4, 123.9, 149.9, 150.5, 155.7, 159.9. ESI-MS (40 eV): m/z (%) = 446.01 (100%) [M]⁺.
5d 3-(3-Methoxy-4-propoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 55%; ochre, mp = 162 °C; IR (KBr): 3080, 2936, 1660, 1600, 1516, 1428, 1354, 1258, 1241, 1147, 1028, 832 cm⁻¹; ¹H NMR (200 MHz, CDCl3): δ 1.07 (t, J = 7.4 Hz, 3H), 1.86–1.97 (m, 2H), 3.52 (dd, J = 17.2, 4.2 Hz, 1H), 3.69 (dd, J = 17.4, 10.8 Hz, 1H), 3.95 (s, 3H), 4.01–4.07 (m, 3H), 4.12–4.15 (m, 1H), 4.15 (s, 5H), 4.17–4.19 (m, 1H), 4.43–4.45 (m, 1H), 5.41 (dd, J = 9.8, 4.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.17–7.26 (m, 1H), 7.47 (d, J = 2.0 Hz, 1H), 8.80 (d, J = 1.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 10.4, 22.4, 39.9, 54.5, 56.1, 65.6, 68.4, 68.6, 69.7, 70.5, 86.9, 109.1, 112.1, 120.5, 123.8, 149.7, 151.1, 155.8, 160. ESI-MS (40 eV): m/z (%) = 446.01 (100%) [M]⁺.
5e 3-(4-Butoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 57%; ochre, mp = 154–155 °C; IR (KBr): 2927, 1663, 1601, 1517, 1429, 1362, 1260, 1243, 1148, 1031, 837 cm⁻¹; ¹H NMR (200 MHz, DMSO-d₆): δ 0.94 (t, J = 7.4 Hz, 3H), 1.39–1.49 (m, 2H), 1.66–1.76 (m, 2H), 3.48–3.62 (m, 1H), 3.70–3.91 (m, 1H), 3.82 (s, 3H), 3.95–4.06 (m, 3H), 4.12–4.15 (m, 1H), 4.16–4.24 (m, 1H), 4.19 (s, 5H), 5.33 (dd, J = 10.2, 4.2 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 7.36–7.40 (m, 2H), 8.73 (d, J = 0.4 Hz, 1H); ¹³C NMR (50 MHz, DMSO-d₆): δ 13.8, 18.9, 30.9, 54.3, 55.8, 65.8, 67.9, 68, 68.1, 68.7, 69.7, 87, 121, 123.6, 149.2, 150.6, 156.6, 159.6 (CO). ESI-MS (40 eV): m/z (%) = 460.11 (100%) [M]⁺.
5f 3-(4-Benzyloxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazole-1-carbaldehyde. Yield 78%; ochre, mp = 172–174 °C; IR (KBr): 3088, 2933, 1655, 1599, 1515, 1429, 1312, 1264, 1240, 1150, 1030, 835 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 3.49 (dd, J = 17.4, 4.6 Hz, 1H), 3.67 (dd, J = 17.2, 10.6 Hz, 1H), 3.97 (s, 3H), 4.03–4.05 (m, 1H), 4.12–4.14 (m, 1H), 4.14 (s, 5H), 4.16–4.19 (m, 1H), 4.42–4.45 (m, 1H), 5.23 (s, 2H), 5.40 (dd, J = 10.6, 4.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 8.2, 2.0 Hz, 1H), 7.31–7.49 (m, 6H), 8.79 (d, J = 0.8 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 39.9, 54.5, 56.1, 65.6, 68.4, 68.6, 69.7, 70.9, 86.8, 109.1, 113.2, 120.4, 124.4, 127.2, 128, 128.6, 136.5, 149.9, 150.5, 155.6, 159.9. ESI-MS (40 eV): m/z (%) = 494.03 (100%) [M]⁺.
6a 1-[3-(3,4-Dimethoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 57%; light ochre, mp = 198–199 °C; IR (KBr): 3091, 2930, 1645, 1601, 1517, 1435, 1407, 1358, 1320, 1263, 1245, 1143, 1026, 838 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 2.32 (s, 3H), 3.48 (dd, J = 17.2, 4.2 Hz, 1H), 3.64 (dd, J = 17.2, 10.6 Hz, 1H), 3.96 (d, J = 3.0 Hz, 6H), 4.00–4.03 (m, 1H), 4.09–4.12 (m, 1H), 4.14 (s, 5H), 4.15–4.17 (m, 1H), 4.48–4.49 (m, 1H), 5.48 (dd, J = 10.6, 4.2 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.23 (dd, J = 8.4, 2.0 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 21.9, 39.6, 55.4, 56, 65.6, 68.1, 68.3, 68.5, 87.5, 108.6, 110.7, 120.4, 124.5, 149.3, 151.2, 153.8, 168.6, (CO). ESI-MS (40 eV): m/z (%) = 432.00 (100%) [M]⁺.
6b 1-[3-(4-Ethoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 56%; ochre, mp = 220–222 °C; IR (KBr): 3085, 2979, 1646, 1600, 1517, 1465, 1407, 1319, 1265, 1244, 1149, 1028, 839 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.51 (t, J = 7.0 Hz, 3H), 1.74 (s, 3H), 3.48 (dd, J = 17.2, 4.4 Hz, 1H), 3.64 (dd, J = 17.2, 10.4 Hz, 1H), 3.95 (s, 3H), 4.00–4.02 (m, 1H), 4.10–4.12 (m, 1H), 4.14 (s, 5H), 4.14–4.18 (m, 3H), 4.48–4.49 (m, 1H), 5.48 (dd, J = 10.4, 4.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.23 (dd, J = 8.4, 2.0 Hz, 1H), 7.46 (d, J = 2.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 14.7, 21.9, 39.6, 55.3, 56.1, 64.4, 65.6, 68.1, 68.3, 68.5, 70.2, 87.5, 108.9, 111.9, 120.4, 124.4, 149.6, 150.6, 153.9, 168.6, (CO). ESI-MS (40 eV): m/z (%) = 446.14 (100%) [M]⁺.
6c 1-[3-(4-Isopropoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 60%; light yellow, mp = 184–185 °C; IR (KBr): 3081, 2937, 1650, 1598, 1513, 1467, 1431, 1404, 1317, 1264, 1243, 1149, 1028, 953, 820 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.42 (d, J = 6.0 Hz, 6H), 2.32 (s, 3H), 3.48 (dd, J = 17.4, 4.4 Hz, 1H), 3.64 (dd, J = 17.4, 10.6 Hz, 1H), 3.94 (s, 3H), 4.01–4.02 (m, 1H), 4.10–4.12 (m, 1H), 4.15 (s, 5H), 4.15–4.17 (m, 1H), 4.48–4.49 (m, 1H), 4.57–4.69 (m, 1H), 5.48 (dd, J = 10.6, 4.4 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 7.21 (dd, J = 8.2, 2.0 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 21.9, 39.6, 55.3, 56.1, 65.6, 68.1, 68.3, 68.5, 70.2, 71.4, 87.6, 109.5, 114.6, 120.3, 124.5, 149.7, 150.5, 153.9, 168.6, (CO). ESI-MS (40 eV): m/z (%) = 460.36 (100%) [M]⁺.
6d 1-[3-(3-Methoxy-4-propoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 68%; light ochre, mp = 200–202 °C; IR (KBr): 3075, 2938, 1645, 1599, 1516, 1466, 1435, 1407, 1320, 1265, 1245, 1151, 1027, 1016, 841 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.07 (t, J = 7.4 Hz, 3H), 1.75–1.96 (m, 2H), 2.32 (s, 3H), 3.48 (dd, J = 17.2, 4.2 Hz, 1H), 3.64 (dd, J = 17.2, 10.6 Hz, 1H), 3.95 (s, 3H), 4.04 (t, J = 6.8 Hz, 2H), 4.08–4.11 (m, 2H), 4.15 (s, 5H), 4.16–4.17 (m, 1H), 4.48–4.49 (m, 1H), 5.47 (dd, J = 10.6, 4.2 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.2, 2.0 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 10.4, 21.9, 22.4, 39.6, 55.2, 56.2, 65.6, 68.1, 68.3, 68.6, 70.1, 70.5, 87.6, 109.2, 112.2, 120.4, 124.3, 149.7, 150.9, 153.9, 168.6, (CO). ESI-MS (40 eV): m/z (%) = 460.11 (100%) [M]⁺.
6e 1-[3-(4-Butoxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 79%; ochre, mp = 158 °C; IR (KBr): 3084, 2958, 2933, 1661, 1601, 1516, 1460, 1423, 1318, 1258, 1242, 1147, 1027, 827 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 1.00 (t, 7.4 Hz, 3H), 1.47–1.58 (m, 2H), 1.83–1.91 (m, 2H), 2.32 (s, 3H), 3.48 (dd, J = 17.2, 4.2 Hz, 1H), 3.64 (dd, J = 17.2, 10.6 Hz, 1H), 3.94 (s, 3H), 4.00–4.01 (m, 1H), 4.08 (t, J = 6.8 Hz, 2H), 4.08–4.12 (m, 1H), 4.14 (s, 5H), 4.14–4.17 (m, 1H), 4.48–4.49 (m, 1H), 5.48 (dd, J = 10.8, 4.2 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.2, 2.0 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H); ¹³C NMR (50 MHz, CDCl₃): δ 13.8, 19.2, 21.9, 31.1, 39.6, 55.2, 56.2, 65.6, 68.1, 68.3, 68.5, 68.8, 70.1, 87.6, 109.2, 112.1, 120.4, 124.3, 149.7, 150.9, 153.9, 168.6, (CO). ESI-MS (40 eV): m/z (%) = 474.11 (100%) [M]⁺.
6f 1-[3-(4-Benzyloxy-3-methoxyphenyl)-5-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl]ethanone. Yield 88%; ochre, mp = 165 °C; IR (KBr): 3078, 2879, 1655, 1600, 1517, 1457, 1428, 1316, 1242, 1146, 1026, 838 cm⁻¹; ¹H NMR (200 MHz, CDCl₃): δ 2.31 (s, 3H), 3.46 (dd, J = 17.2, 4.2 Hz, 1H), 3.62 (dd, J = 17.2, 10.6 Hz, 1H), 3.97 (s, 3H), 3.97–3.99 (m, 1H), 4.09–4.11 (m, 1H), 4.13 (s, 5H), 4.13–4.16 (m, 1H), 4.47–4.49 (m, 1H), 5.22 (s, 2H), 5.46 (dd, J = 10.6, 4.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 8.4, 2.0 Hz, 1H), 7.31–7.47 (m, 6H); ¹³C NMR (50 MHz, CDCl₃): δ 21.9, 31.5, 55.3, 56.1, 65.6, 68.1, 68.3, 68.5, 70.2, 70.9, 87.5, 109.2, 113.3, 120.2, 124.9, 127.2, 127.9, 128.6, 136.6, 149.9, 150.3, 153.8, 168.6. ESI-MS (40 eV): m/z (%) = 508.12 (100%) [M]⁺.

Conclusions

Two series of chalcones, 1 and 2, with ferrocenyl framework were prepared by Claisen–Schmidt reaction and reacted with hydrazine in boiling formic or acetic acid. By this way new N-formyl (compounds 3 and 5) and N-acetyl (compounds 4 and 6) pyrazoline derivatives were prepared. All described compounds were prepared in good to fairly good yields.

It is known that properties of chalcone derivatives depends on structure of ring A and ring B. The vanillic core give us a opportunity to tune their structure and properties by changing O-alkyl group in p-position. Pyrazoline derivatives prepared from chalcones with different neighbouring groups (A and B) connected to enone system provide us with two similar series of compounds with quite different electronic environment. From biological experimental results is evident that the structure differences induce various biological activities.

Biological evaluations for the 3a–f, 4a–f, 5a–f, 6a–f were investigated via treatment of a series of bacteria and fungi strains. The strongest antibacterial activity was found in compounds 4a and 4f, which in low concentrations inhibited all the species of bacteria. The lowest measured MIC value was 0.156 mg mL⁻¹ for 4a and 4f related to the Bacillus subtilis species. Based on the MIC value, the compounds 4a, 4c and 4f showed the strongest antifungal activity, and the most sensitive among the fungi were Candida albicans.

DNA binding study showed that all investigated compounds (4a, 4f, 5a and 5e) in the presence of ethidium bromide (EB) partially replace EB from the EB–DNA complex species, and quench fluorescence intensity. The measured Stern–Volmer quenching constants followed the order 5e, 4a, 5a and 4f, and indicate their high affinity and efficiency to substitute EB from EB–DNA complexes. Binding experiments for 4a, 4f, 5a and 5e with BSA showed that 5a–BSA and 5e–BSA had larger K_a values than 4a–BSA and 4f–BSA complex species which means that 4a– and 4f–BSA complexes are less suitable for drug–cell interactions.

Acknowledgements

The authors are grateful to the Ministry of Education, Science and Technological Development of the Republic of Serbia for financial support (Grants 172034, 172011 and 173032).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Characterization of compounds 3–6(a–f), copies of ¹H and ¹³C NMR spectra. See DOI: 10.1039/c6ra18977f

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