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Article

Synthesis, Selective Cytotoxic Activity against Human Breast Cancer MCF7 Cell Line and Molecular Docking of Some Chalcone-Dihydropyrimidone Hybrids

by
Eduardo B. Mass
1,
Carolina A. de Lima
2,
Marcelo G. M. D’Oca
3,
Juliana M. Sciani
4,
Giovanna B. Longato
2,† and
Dennis Russowsky
1,*,†
1
Laboratório de Sínteses Orgânicas, Instituto de Química, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, Porto Alegre CEP 91501-970, RS, Brazil
2
Laboratório de Pesquisa em Farmacologia Molecular e Compostos Bioativos–Prédio VII, Universidade São Francisco, Avenida São Francisco de Assis 218, Bragança Paulista CEP 12916-900, SP, Brazil
3
Departamento de Química, Universidade Federal do Paraná, Centro Politécnico, Av. Coronel Francisco H. Santos 100, Curitiba CEP 81531-980, PR, Brazil
4
Laboratório Multidisciplinar de Pesquisas–Prédio V, Universidade São Francisco, Av. São Francisco de Assis 218, Bragança Paulista CEP 12916-900, SP, Brazil
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Drugs Drug Candidates 2022, 1(1), 3-21; https://doi.org/10.3390/ddc1010002
Submission received: 10 October 2022 / Revised: 17 November 2022 / Accepted: 23 November 2022 / Published: 1 December 2022
(This article belongs to the Section Medicinal Chemistry and Preliminary Screening)
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Versions Notes

Abstract

:
Designed Chalcone-Dihydropyrimidinone hybrid compounds were synthesized expeditiously. The hybridization was performed through the Copper-catalyzed Alkyne-Azide Cycloaddition (CuAAC) from the propargyloxy chalcones and azido-dihydropyrimidinones. The hybrid products were prepared in five steps with a 30–48% overall yield. Most of the compounds showed selective cytotoxicity and lower IC50 values (<10 µM) against MCF-7 (breast adenocarcinoma) cancer. Cytotoxicity was also observed against OVCAR-3 (ovary, adenocarcinoma), NCI/ADR-RES (ovary, multidrug-resistant adenocarcinoma), and U-251 (brain, glioblastoma) cell lines. The potency of the most active hybrids 9d, 9g, and 9h was greater than the individual parental compounds, suggesting the effectiveness of molecular hybridization on the cytotoxicity. Compounds 9d, 9g, and especially 9h showed high selectivity for breast cancer cells (MCF-7) regarding human keratinocytes (HaCaT). Molecular docking calculations for the 9d, 9g, and 9h hybrids in the active site of estrogen supported the hypothesis that the compounds act as ER-α antagonists, disrupting the cell proliferation process of MCF-7, corroborating the potency and selectivity observed for this tumoral cell line.

1. Introduction

Molecular hybridization is a strategy that consists of the combination of two or more pharmacophores through a covalent bond [1]. The hybrid molecules can affect different factors of the same disease, counterbalancing side effects, interacting with multiple targets (multifunctional), or amplifying the potency [2,3]. This approach has increasingly become a paradigm for the pharmaceutical industry not only in terms of financial costs but also due to the reduction of side effects caused by unwanted drug interactions in drug cocktails [4,5,6].
Many successful examples of highly bioactive compounds can be found in the literature due to the hybridization protocol of different pharmacophores, including natural-based hybrid molecules [7,8]. Remarkably antiproliferative activities [9], leishmanicidal [10], or inhibitory activity against acetylcholinesterase enzyme [11] are some of the prominent reported cases.
One interesting scaffold to explore in the design of hybrid compounds is the chalcone moiety. Chalcones are a group of plant-derived polyphenolic compounds belonging to the flavonoids family. The structural variety and plethora of biological activities of chalcones were exhaustively discussed in recent reviews [12,13,14]. Additionally, they are known to express high anticancer activity, notably as inhibitors of tubulin polymerization by binding in the colchicine site [15,16]. Over the last few years, intense research on chalcone hybrid compounds has been reported. New biologically active chalcone hybrids were synthesized by connecting different counterparts such as coumarins [17], sulfonamides [18], anthraquinones [19], indolizines [20], quinazolines [21,22], cinchonas [23], quinolines [24], pyrazolines [25] and benzimidazoles [26] among others. Despite the availability of different methods for preparing chalcones, they can be easily synthesized directly via base-catalyzed Claisen–Schmidt condensation from acetophenones and benzaldehydes [27]. Among the available methods to prepare the chalcone hybrids [28,29], the CuAAC (Copper Catalyzed Alkyne-Azide Cycloaddition) strategy [30,31] is an easy way to link the chalcone part to another molecular unity. The CuAAC methodology produces a 1,2,3-triazole heterocycle as the linker between the two individual parts as shown in the hybrids Chalcone-Benzyl (I) [32] Chalcone β-Lactam (II) [33] and Chalcone-Quinoline (III) [34] respectively (Figure 1).
Other privileged scaffolds in Medicinal Chemistry are the dihydropyrimidin-2-(thi)ones (DHPMs) also due to various biological activities they present. Monastrol (IV), a dihydropyrimidin-2-thione discovered by Mayer in early 1999s [35], became a relevant non-natural small molecule able to disturb the mitosis process by inhibiting the mitotic kinesin Eg5, a motor protein responsible for the bipolar spindle formation without affecting the tubulin dynamics [36]. Several recent reviews covered the synthesis and biological activities [37,38,39].
The DHPMs have been used as a template for developing diverse new anticancer hybrid molecules. The hybrids Monastrol-Fatty acids (V, cytotoxic against glioblastomas) [40] and DHPM-perillyl alcohol (VI, cytotoxic against OVCAR-3, ovary cell line) were investigated by our group [9]. More recently, a fluorescent dihydropyrimin-2-thinone-arylbenzoxazole hybrid (VII) was identified as promising against prostate cells (PC3 cell line). Due to the strong fluorescence of this compound, it was possible to observe their accumulation in the endoplasmic reticulum and the nuclei of cancer cells (Figure 2) [41].
Inspired by these observations, we propose a new design for hybrid molecules based on the combination of chalcones and DHPMs, in our quest for potential antitumoral activity. For this purpose, we installed an alkyne group in the chalcone component and an azide group in the DHPM. The CuAAC protocol was chosen to connect both pharmacophores via a triazole linkage. The unique properties of the triazole linker bring stability to the hybrid molecule adding the advantage of being a bioisostere, notably as an amide function [42].

2. Results and Discussion

2.1. Syntehsis of Chalcone-DHPM Hybrids

The propargyloxy chalcones were prepared according to the synthetic route shown in Scheme 1. The hydroxy benzaldehydes 1a–e were alkylated with propargyl bromide to afford derivatives 2a–e in 82–95% yields [43]. The aldehydes 2a–e were further condensed with acetophenone (3) to give access to chalcones 4a–e in 67–82% yield (Scheme 1) [44].
In parallel, the chloro-DHPMs 7a,b were prepared via multicomponent Biginelli reaction from benzaldehydes 1f–g, ethyl 4-chloroacetoacetate (5), and urea (6) [45]. The Biginelli compounds 7a–c were obtained in 67–72% yield. Next, they were converted to azido-DHPMs 8a,b in excellent yields, 82–91% in the reaction with NaN3 (Scheme 2) [46].
After that, the chalcones 4a–e and DHPM 8a,b were hybridized through the CuAAC methodology in the presence of CuSO4·5H2O/sodium ascorbate system to generate in situ the active Cu(I) specie for the alkyne-azide 1,3-dipolar cycloaddition reaction [47]. The combination of each chalcone with all DHPMs afforded a small library of ten Chalcone-DHPM hybrids 9a–j (Scheme 3). After purification by column chromatography, the hybrids were obtained in reasonable-to-good yields. All hybrids were fully characterized by conventional spectroscopic methods, including HRMS (High-Resolution Mass Spectrometry) for novel compounds.
The appearance of a singlet signal around 8.39–9.35 ppm in the 1H NMR spectra, assigned as the hydrogen of the triazole ring, confirms its formation as a linker. The results are shown in Table 1 below.

2.2. Cytotoxicity of Chalcone-DHPM Hybrids

The cytotoxicity of hybrids 9a–j was evaluated against a panel of human cancer cell lines through the MTT assay [48]. Doxorubicin (DOXO) was used as the positive control. The results are presented in Table 2 below.
As shown in Table 2, breast adenocarcinoma (MCF-7) was the most sensitive cell line to hybrid compounds treatment, and IC50 values were very low, ranging from 4.7 to 14.6 µM. Besides being potent for MCF-7, compounds 9f and 9g were the most potent (<15 µM) for glioblastoma (U-251), and compounds 9a, 9b, and 9f presented low IC50 values (<15 µM) for ovarian adenocarcinoma (OVCAR-3). Compounds 9a and 9b presented lower IC50 values (6.4 and 14.5 µM, respectively) than the positive control doxorubicin (22.6 µM) for NCI/ADR-RES. This result is very relevant because this is an ovarian adenocarcinoma cell line that expresses the phenotype of resistance to multiple drugs, characterized by the high expression of glycoprotein-P (P-gp), an energy-dependent transmembrane efflux pump responsible for modulating multidrug resistance (MDR) [49]. Further studies with these molecules must be conducted on this cell line to investigate their role in MDR reversion.
The IC50 values of all hybrid compounds against non-tumoral cell line HaCaT were higher than the doxorubicin (IC50 = 1.6 µM). Furthermore, compound 9h presented a great cytotoxic effect against MCF-7 cells (IC50 = 5.8 µM) with no toxic effects for HaCaT (IC50 > 100 µM). The IC50 value of hybrid 9h against HaCaT is 62.9 times greater than the one found for doxorubicin. It might indicate a preference for this compound for tumoral cell lines and less toxicity against healthy cells. Considering that compounds 9d and 9g presented the lowest IC50 values for the most sensitive cell line MCF-7 and that compound 9h was not cytotoxic for HaCaT, these molecules were chosen for a comparative cytotoxicity profile study with their parental molecules.

2.3. Comparative Cytotoxicity Profile of Hybrids and Their Parental Molecules

In order to investigate the effect of molecular hybridization on the cytotoxicity, the chalcones 4b–d, and DHPMs 8a,b (as constituent parts of the chosen molecules 9d, 9g, and 9h) were screened separately against U-251, MCF-7, and NCI/ADR-RES tumoral cell lines, as well as HaCaT.
As can be seen in Table 3, hybrids 9d and 9g were more cytotoxic than their parental chalcone and DHPM for all the cell lines evaluated, including the non-tumoral HaCaT. Hybridization turned molecules very potent for breast adenocarcinoma MCF-7 cell line (IC50 value of 4.72 and 4.89 µM) differing from their parental chalcone 4d e 4b and DHPM 8a e 8b, which showed the following IC50 values: >342, 80.33, >332 and >255 µM, respectively. Although the IC50 of 9d and 9g was high for NCI/ADR-RES (89.79 and 39.62 µM, respectively), it is noteworthy that the hybridization also induced a better sensitization of this resistant ovarian adenocarcinoma cell line in comparison to their parental chalcone (>342 and >381 µM, respectively) and DHPM (>332 and >255 µM, respectively).
Hybrid compound 9h was also very potent for breast adenocarcinoma MCF-7 cell line (IC50 value of 5.81 µM) differing from their parental chalcone 4c (IC50 value > 381 µM) and DHPM 8b (IC50 value > 255 µM). Interestingly, different from 9d and 9g, hybrid 9h was not cytotoxic for HaCaT non-tumoral cell line at low concentrations (Table 3). The cytotoxicity for the non-tumoral cell line observed for parental chalcone 4c was reduced with the hybridization process.
Table 3 contains information about the hybrids’ selective index (SI). All of them were selective for breast adenocarcinoma MCF-7, with SI greater than 2, as recommended by the National Cancer Institute [50], suggesting a selective cytotoxic effect for these tumor cells in detriment of healthy ones with hybrid compound 9h presenting a more pronounced SI value (17.33) for MCF-7. This result is very interesting, considering that although substantial progress has been made in cancer chemotherapy, intolerable side effects frequently occur due to the unsatisfactory selectivity of chemotherapeutic agents between tumoral and non-tumoral cells. Hence, the development of new agents with selective chemotherapeutic effects is highly desired [51]. In this regard, hybrid 9h deserves further investigation due to its preference for this tumoral cell line.
The achievement of hybrid compounds from chalcones and dihydropyrimidinones with greater cytotoxicity than their parental compounds is strong evidence of the benefits brought by the molecular hybridization strategy. The fact that hybridization does not always produce greater cytotoxicity than their compounds appears to be more a function of the cell susceptibility of each cell line than the hybrid itself.
The great selectivity of hybrids for MCF-7 breast cancer cells has aroused interest in investigating the action of these molecules on α- and β-estrogen receptors (ERα and ERβ, respectively), highly present in this cell line [52,53], classified as luminal [54], which would exert a blocking effect on the growth of breast cancer tumor. The activation of ERα is responsible for the increase of breast tumor cell proliferation, while ERβ has antiproliferative properties. Therefore, compounds that could act as ERα antagonists and/or ERβ agonists for estrogen-based neoplasia control would have positive effects [55].
To gain preliminary information on this assumption, the flexible molecular docking calculation was performed for the most potent active compounds 9d, 9g, and 9h at the active site of ERα and Erβ. The results show that the compounds 9d, 9g, and 9h were positioned at the same site of an endogenous ligand for ERα (estradiol), with stable interaction, high binding energy and short distance to the amino acids from the pocket (Table 4), which would disturb the action of proliferative molecules, impairing the cell growth, as shown by the cell culture experiments. Besides Thr347, Asp351, and Glu353, binding residues of agonist, the hybrids could bind to Leu 346, indicating more stability to the protein. Moreover, the hybrids docked in the same position as the H3B-9224 antagonist, used as a template for the receptor antagonism, confirming our hypothesis of the participation of ERα receptors as a mechanism of action of hybrids (Figure 3).
On the other hand, the hybrids could bind to the ERβ receptor but not at the active site (Figure 4), not characterizing an agonist or antagonist action, although with high binding energy (Table 5). This data reinforces the antiproliferative activity of the hybrids as an antagonist of ERα receptors only, promoting a reduction of cell viability, and not an agonist of the antiproliferative receptor ERβ.

3. Materials and Methods

3.1. Chemistry

The reagents were purchased from commercial sources and used without further purification, except ethyl acetate and hexanes, which were purified by simple distillation. Column chromatography was performed using a Silica Gel 60 Å (ACROS Organics, 0.035–0.070 mm). The reactions were monitored using thin-layer chromatography (TLC) performed with plates containing silica gel (Merck 60GF245), and the spots were visualized under UV light.
The NMR spectra were recorded using Varian VNMRS 300 spectrometer (1H at 300 MHz and 13C at 75 MHz) or Bruker (1H at 400 MHz and 13C at 100 MHz in CDCl3 or DMSO-d6 as the solvent). The chemical shifts (δ) are reported in ppm units downfield from tetramethylsilane (TMS), which was used as an internal standard. The coupling constants (J) are reported in Hz and refer to peak multiplicities. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectra were collected using Bruker Alpha-P equipment. The melting points were obtained on a Buchi Melting Point M-565 apparatus with a non-calibrated thermometer.
The 1H NMR and 13C NMR data and spectra of compounds can be visualized in Supplementary Materials (Figures S1–S41).

3.1.1. General Procedure for Synthesis of Propargyloxy Benzaldehydes (2a–e) [43]

In a 50 mL round bottom flask charged with 25 mL of acetone were added 10 mmol of hydroxybenzaldehyde (1a–e), 20 mmol (2.764 g) of K2CO3, and 15 mmol (1.60 mL) of propargyl bromide (80% solution in toluene). The resulting solution was stirred under reflux for 2–4 h. After the consumption of aldehyde (monitored by TLC), the crude mixture was filtered, and the volatiles was retired under a vacuum. The products were sufficiently pure (by 1H NMR) to proceed without further purification.

2-(Prop-2-ynil-1-oxy)benzaldehyde (2a)

White solid (m.p. 65 °C, lit. 66–68 °C [43]), 95% yield. 1H NMR (400 MHz, CDCl3) δ 10.49 (s, 1H), 7.87 (dd, 1H, J = 7.8 and 2.0 Hz), 7.57 (ddd, 1H, J = 8.5, 7.3 and 2.0 Hz), 7.15–7.06 (m, 2H), 4.84 (d, 2H, J = 2.3 Hz), 2.57 (t, 1H, J = 2.3 Hz). 13C NMR (100 Hz, CDCl3) δ 189.4, 159.6, 135.6, 128.4, 125.4, 121.6, 113.1, 77.6, 76.4, 56.3.

3-(Prop-2-ynil-1-oxy)benzaldehyde (2b)

Colorless oil [43], 82% yield. 1H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 7.53–7.45 (m, 3H), 7.27–7.23 (m, 1H), 4.76 (d, 2H, J = 2.3 Hz), 2.56 (t, 1H, J = 2.3 Hz). 13C NMR (100 Hz, CDCl3) δ 191.7, 157.9, 137.6, 130.0, 123.9, 121.9, 113.4, 77.8, 76.1, 55.8.

4-(Prop-2-ynil-1-oxy)benzaldehyde (2c)

White solid (m.p. 70 °C, lit. 73 °C [43]), 90% yield. 1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 7.87 (d, 2H, J = 8.8 Hz), 7.08 (d, 2H, J = 8.8 Hz), 4.77 (d, 2H, J = 2.5 Hz), 2.58 (t, 1H, J = 2.5 Hz). 13C NMR (100 Hz, CDCl3) δ 190.7, 162.4, 131.9, 130.6, 115.2, 77.5, 76.3, 55.9.

3-Methoxy-4-(prop-2-ynil-1-oxy)benzaldehyde (2d)

White solid (m.p. 90 °C, lit. 86 °C [56]), 90% yield. 1H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 7.45 (dd, 1H, J = 8.0 and 1.8 Hz), 7.44 (d, 1H, J = 1.8 Hz), 7.15 (d, 1H, J = 8.0 Hz), 4.86 (d, 2H, J = 2.3 Hz), 3.94 (s, 3H), 2.57 (t, 1H, J = 2.3 Hz). 13C NMR (100 Hz, CDCl3) δ 190.9, 152.1, 150.0, 130.9, 126.2, 112.6, 109.5, 77.5, 76.7, 56.6, 56.0.

4-Methoxy-3-(prop-2-ynil-1-oxy)benzaldehyde (2e)

White solid (m.p. 74 °C, lit. 74–75 °C [57]), 89% yield. 1H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H), 7.53 (d, 1H, J = 1.5 Hz), 7.51 (dd, 1H, J = 8.2 and 1.8 Hz), 7.00 (d, 1H, J = 8.1 Hz), 4.81 (d, 2H, J = 2.3 Hz), 3.95 (s, 3H), 2.54 (t, 1H, J = 2.3 Hz). 13C NMR (75 MHz, CDCl3) δ 190.6, 154.9, 147.3, 129.9, 127.3, 112.0, 110.9, 77.7, 76.4, 56.6, 56.2.

3.1.2. General Procedure for Synthesis of Chalcones (4a–e) [44]

In a 25 mL round bottom flask charged with 4 mL of ethanol, was added 2.5 mmol of the propargyloxy benzaldehydes (2a–e), and 2.6 mmol of acetophenone (3, 0.3124 g). The mixture was stirred until a clear solution. After, 0.3 mL of a 1M solution of NaOH was added. The reaction was stirred for about 1–2 h and quenched with 10 g of ice water. After, the crude mixture was extracted with 10 mL of CH2Cl2 three times. The organic phases were combined, washed with 20 mL of brine, and dried with MgSO4.
After filtration, the solvent was evaporated under a vacuum. The resultant solid product was purified by silica gel column chromatography (hexane/ethyl acetate 80/20, v/v).

1-Phenyl-3-(2-prop-2-ynil-1-oxyphenyl)-(2E)-propen-1-one (4a)

Yellow solid, (m.p. 64 °C, lit. 63–64.6 °C [58]), 82% yield. 1H NMR (400 MHz, CDCl3) δ 8.10 (d, 1H, J = 15.9 Hz), 8.06-8.01 (m, 2H), 7.67 (d, 1H, J = 15.9 Hz), 7.65 (dd, 1H, J = 7.8 and 1.8 Hz), 7.61–7.54 (m, 1H), 7.52–7.46 (m, 2H), 7.38 (ddd, 1H, J = 8.5, 7.2, and 1.8 Hz), 7.07–7.03 (m, 2H), 4.80 (d, 2H, J = 2.5 Hz), 2.56 (t, 1H, J = 2.5 Hz). 13C NMR (100 MHz, CDCl3) δ 191.0, 158.7, 140.0, 138.4, 132.6, 131.5, 129.5, 128.5, 124.5, 123.3, 121.7, 112.7, 78.2, 76.0, 56.2.

1-Phenyl-3-(3-prop-2-ynil-1-oxyphenyl)-(2E)-propen-1-one (4b)

Yellow solid (m.p. 54 °C, Iit. 55 °C [59]), 67% yield. 1H NMR (400 MHz, CDCl3) δ 8.04–7.99 (m, 2H), 7.77 (d, 1H, J = 15.6 Hz), 7.62–7.57 (m, 1H), 7.51 (d, 2H, J = 15.6 Hz), 7.53–7.48 (m, 2H), 7.39–7.33 (m, 1H), 7.31–7.27 (m, 1H), 7.25–7.23 (m, 1H), 7.04 (ddd, 1H, J = 8.1, 2.5 and 0.8 Hz), 4.74 (d, 2H, J = 2.3 Hz), 2.56 (t, 1H, J = 2.3 Hz). 13C NMR (100 MHz, CDCl3) δ 190.5, 144.5, 138.1, 136.3, 132.9, 130.0, 128.6, 128.5, 122.5, 121.9, 117.1, 114.6, 78.2, 75.9, 55.9.

1-Phenyl-3-(4-prop-2-ynil-1-oxyphenyl)-(2E)-propen-1-one (4c)

Yellow solid (m.p. 72 °C, 70–72 °C [60]), 71% yield. 1H NMR (400 MHz, CDCl3) δ 8.04–7.98 (m, 2H), 7.78 (d, 1H, J = 15.6 Hz), 7.61 (d, 2H, J = 8.6 Hz), 7.61–7.54 (m, 1H), 7.50 (t, 2H, J = 7.0 Hz), 7.43 (d, 1H, J = 15.6 Hz), 7.02 (d, 2H, J = 8.6 Hz), 4.74 (d, 2H, J = 2.3 Hz), 2.55 (t, 1H, J = 2.3 Hz). 13C NMR (100 MHz, CDCl3) δ 190.6, 159.5, 144.4, 138.4, 132.6, 130.2, 128.6, 128.5, 128.4, 120.3, 115.3, 78.0, 76.0, 55.9.

1-Phenyl-3-(3-methoxy-4-prop-2-ynil-1-oxyphenyl)-(2E)-propen-1-one (4d)

Yellow solid (m.p. 110 °C, lit. 108 °C [34]), 60% yield. 1H NMR (400 MHz, CDCl3) δ 8.01 (d, 2H, J = 7.3 Hz), 7.76 (d, 1H, J = 15,6 Hz), 7.58 (t, 1H, J = 7.3 Hz), 7.50 (t, 2H, J = 7.3 Hz), 7.41 (d, 1H, J = 15.8 Hz), 7.23 (dd, 1H, J = 8.3 and 1,5 Hz), 7.18 (d, 1H, J = 1.5 Hz), 7.06 (d, 1H, J = 8.3 Hz), 4.82 (d, 2H, J = 2.3 Hz), 3.94 (s, 3H), 2.55 (t, 1H, J = 2.3 Hz). 13C NMR (75 MHz, CDCl3) δ 190.5, 149.7, 149.0, 144.7, 138.3, 132.6, 129.0, 128.5, 128.4, 122.5, 120.5, 113.7, 110.7, 77.9, 76.2, 56.6, 55.94.

1-Phenyl-3-(4-methoxy-3-prop-2-ynil-1-oxyphenyl)-(2E)-propen-1-one (4e)

Yellow solid (m.p. 103 °C), 62% yield. 1H NMR (40 MHz, CDCl3) δ 8.03–8.00 (m, 2H), 7.77 (d, 1H, J = 15.6 Hz), 7.62–7.56 (m, 1H), 7.54–7.48 (m, 2H), 7.40 (d, 1H, J = 15.6 Hz), 7.35 (d, 1H, J = 2.0 Hz), 7.32–7.28 (m, 1H), 6.93 (d, 1H, J = 8.3 Hz), 4.83 (d, 2H, J = 2.3 Hz), 3.93 (s, 3H), 2.57 (t, 1H, J = 2.3 Hz). 13C NMR (100 Hz, CDCl3) δ 190.5, 151.9, 146.8, 144.7, 138.3, 132.6, 128.5, 128.4, 127.7, 124.1, 120.2, 113.5, 111.6, 78.1, 76.3, 56.8, 55.9. ATR-FTIR (νmax, cm−1) 3277, 3001, 2907, 2831, 2130, 1658, 1513, 1013. HRMS (ESI) m/z calc. for [C19H16O3 + Na]+: 315.0992; obs. 315.0993.

3.1.3. General Procedure for the Synthesis of Chloro-DHPMs (7a,b) [45]

In a 50 mL round-bottom flask, were added, in this order, 5 mmol of the benzaldehydes 1f,g, 5 mmol (0.503 mL) of ethyl 4-chloroacetoacetate (6), 7.5 mmol (0.4505 g) of urea (7) and 12.5 mL of acetic acid. The mixture was stirred under reflux for 48 h. After cooling, the solution was slowly poured into an Erlenmeyer flask containing 100 mL of water, and a solid appeared. The solid was filtrated on a Büchner funnel and washed with water (25 mL), saturated NaHCO3 solution (25 mL), and water again (25 mL). The solid was dried under reduced pressure.

Ethyl 6-(2-chloromethyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (7a)

Pale yellow solid (m.p. 200 °C, lit. 226 °C [50]), 72% yield. 1H NMR (300 MHz, DMSO-d6) δ 9.24 (ls, 1H), 7.63 (ls, 1H), 7.35–7.21 (m, 5H), 5.22 (d, 1H, J = 2.9 Hz), 4.77 (d, 1H, J = 10,6 Hz), 4.61 (d, 1H, J = 10,6 Hz), 4.06 (q, 2H, J = 7.0 Hz), 1.12 (t, 1H, J = 7.0 Hz). 13C NMR (100 MHz, DMSO-d6) δ 164.2, 152.1, 146.0, 144.0, 128.5, 127.6, 126.3, 101.8, 60.0, 53.9, 39.2, 13.9.

Ethyl 6-chloromethyl-4-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (7b)

Dark yellow solid (m.p. 244 °C), 70% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (ls, 1H), 7.82 (ls, 1H), 6.57 (s, 2H), 5.15 (d, 1H, J = 3.3 Hz), 4.80 (d, 1H, J = 10.6 Hz), 4.64 (d, 1H, J = 10.6 Hz), 4.08 (q, 2H, J = 7.0 Hz), 3.73 (s, 6H), 3.64 (s, 3H), 1.14 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, DMSO-d6) δ 172.1, 164.3, 152.9, 152.1, 146.3, 139.4, 137.0, 103.5, 101.4, 60.0, 55.8, 53.8, 39.4, 21.1, 14.0. ATR-FTIR (νmax, cm−1 3318, 3210, 3098, 2936, 1686, 1643, 1220, 1123, 737. HRMS (ESI) m/z calc. for [C17H21ClN2O6 + Na]+: 407.0980; obs. 407.0986.

3.1.4. General Procedure for the Synthesis of Azido-DHPMs (8a,b) [46]

A 50 mL round bottom flask was charged with 7.5 mL acetone and 3 mL deionized water. Next, 3 mmol of chloro-DHPM (7a,b) and 3.6 mmol (0.2340 g) of sodium azide were added at room temperature. The mixture was heated at 60 °C until the consumption of the Cl-DHPM (monitored by TLC). Acetone was evaporated under a vacuum, and the resulting solid was washed with water (25 mL), filtered on a Büchner funnel, and dried at reduced pressure.

Ethyl 6-(azidomethyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (8a)

Yellow solid (m.p. 111 °C, lit. 110 [61]), 88% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.47 (ls, 1H), 7.88 (ls, 1H), 7.37–7.31 (m, 2H), 7.29–7.23 (m, 3H), 5.21 (d, 1H, J = 3.3 Hz), 4.86 (d, 1H, J = 12.8 Hz), 4.35 (d, 1H, J = 12.8 Hz), 4.03 (q, 2H, J = 7.0 Hz), 1.11 (t, 1H, J = 7.0 Hz). 13C NMR (100 MHz, DMSO-d6) δ 164.7, 152.0, 144.4, 144.1, 128.6, 127.7, 126.4, 102.4, 60.1, 54.1, 48.0, 13.9.

Ethyl 6-(azidomethyl)-4-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (8b)

Pale yellow solid (mp 177 °C), 91% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.45 (ls, 1H), 7.83 (d, 1H, J = 2.8 Hz), 6.57 (s, 2H), 5.19 (d, 1H, J = 3.0 Hz), 4.50 (d, 1H, J = 12.8 Hz), 4.31 (d, 1H, J = 12.8 Hz), 4.07 (qd, 2H, J = 7.0 and 1.8 Hz), 3.73 (s, 6H), 3.63 (s, 3H), 1.13 (t, 1H, J = 7.0 Hz). 13C NMR (100 MHz, DMSO-d6) δ 172.1, 164.3, 152.9, 152.1, 146.3, 139.4, 137.0, 103.5, 101.4, 60.0, 55.8, 53.8, 39.4, 21.1, 14.0. ATR-FTIR (νmax, cm−1) 3321, 3209, 3116, 2939, 2095, 1683, 1224, 1125. HRMS (ESI) m/z calc. for [C17H21N5O6 + Na]+: 414.1384, obs. 414.1386.

3.1.5. General Procedure for the Synthesis of Chalcone-DHPM Hybrids (9a–j) [47]

In a 25 mL round bottom flask was placed 2 mL of CH2Cl2 and 2 mL of water. Then, 0.2 mmol of azido-DHPM (8a,b) was added, followed by 0.2 mmol of propargyloxy chalcone (4a–e), 0.02 mmol (0.0050 g) of CuSO4·5H2O and 0.02 mmol (0.0040 g) of sodium ascorbate. The reaction was stirred, at room temperature, until consumption of starting materials (monitored by TLC). After, 4 mL of EDTA aqueous solution (0.1 M) was added and steered for 5 min. The crude aqueous mixture was extracted with 5 mL of CH2Cl2 three times. The organic phases were combined, washed with 10 mL of saturated NaCl solution, and dried over MgSO4. After filtration, the solvent was removed under reduced pressure. The resulting solid was purified by silica gel column chromatography (ethyl acetate/hexanes, 70/30, v/v).

Ethyl 6-((4-((2-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9a)

Yellow solid (m.p. 109 °C), 75% yield. 1H NMR (400 MHz, CDCl3) δ 9.00 (ls, 1H), 8.09 (d, 1H, J = 15,6 Hz), 8.04 (s, 1H), 7.96 (d, 2H, J = 7.3 Hz), 7.63 (d, 1H, J = 7.5 Hz), 7.62 (d, 1H, J = 15.8 Hz), 7.57 (t, 1H, J = 7.3 Hz), 7.47 (d, 2H, J = 7.3 Hz), 7.37 (d, 1H, J = 7.5 Hz), 7.21 (s, 5H), 7.08 (d, 1H, J = 8.3 Hz), 7.02 (t, 1H, J = 7.5 Hz), 6.33 (ls, 1H), 5.85 (d, 1H, J = 14.6 Hz), 5.59 (d, 1H, J = 14.6 Hz), 5.38 (d, 1H, J = 2.5 Hz), 5.29 (s, 2H), 4.10 (q, 2H, J = 7.0 Hz), 1.13 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.8, 164.7, 157.3, 152.9, 143.6, 142.8, 141.2, 140.0, 138.1, 132.8, 131.9, 129.0, 128.7, 128.5, 128.5, 128.1, 126.6, 124.2, 124.1, 122.4, 121.3, 112.6, 104.5, 62.6, 60.8, 55.6, 47.7, 13.9. ATR-FTIR (νmax, cm−1) 3238, 3143, 2935, 1688, 1653, 1217, 1098, 1005. HRMS (ESI) m/z calc. for [C32H29N5O5 + Na]+: 586.2061; obs. 586.2061.

Ethyl 6-((4-((3-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3- triazol-1-yl)methyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9b)

Yellow solid (m.p. 91 °C), 70% yield. 1H NMR (400 MHz, CDCl3) δ 8.35 (ls, 1H), 8.02 (d, 1H, J = 7.3 Hz), 7.91 (s, 1H), 7.76 (d, 1H, J = 15.6 Hz), 7.59 (t, 1H, J = 7.3 Hz), 7.55–7.47 (m, 3H), 7.36–7.21 (m, 7H), 7.06–7.01 (m, 1H), 6.08 (ls, 1H), 5.82 (d, 1H, J = 14.6 Hz), 5.64 (d, 1H, J = 14.9 Hz), 5.40 (d, 1H, J = 2.5 Hz), 5.22 (s, 2H), 4.10 (q, 2H, J = 7.0 Hz), 1.15 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.4, 164.8, 158.5, 152.4, 144.5, 142.7, 141.4, 138.0, 136.3, 132.8, 130.0, 128.8, 128.6. 128.5, 128.3, 126.6, 124.0, 122.4, 121.7, 116.9, 114.4, 104.4, 61.9, 60.9, 55.6, 47.9, 13.9. ATR-FTIR (νmax, cm−1) 3226, 3114, 2956, 1693, 1656, 1221, 1094, 1014. HRMS (ESI) m/z calc. for [C32H29N5O5 + Na]+: 586.2061; obs. 586.2064.

Ethyl 6-((4-((4-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9c)

Yellow solid (m.p. 111 °C), 78% yield. 1H NMR (400 MHz, CDCl3) δ 8.43 (ls, 1H), 8.03-7.98 (m, 2H), 7.90 (s, 1H), 7.77 (d, 1H, J = 15.6 Hz), 7.61–7.55 (m, 3H), 7.52–7.47 (m, 2H), 7.42 (d, 1H, J = 15.6 Hz), 7.29–7.20 (m, 5H), 7.01 (d, 1H, J = 8.8 Hz), 6.10 (ls, 1H), 5.77 (d, 1H, J = 14.6 Hz), 5.65 (d, 1H, J = 14.6 Hz), 5.40 (d, 1H, J = 2.8 Hz), 5.22 (s, 2H), 4.09 (q, 2H, J = 7.0 Hz), 1.15 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.5, 164.8, 160.2, 152.4, 144.4, 143.7, 142.7, 141.4, 138.4, 132.6, 130.2, 128.8, 128.7. 128.4, 128.3, 128.1, 126.6, 124.1, 120.0, 115.2, 104.4, 61.9, 60.9, 55.6, 49.0, 13.9. ATR-FTIR (νmax, cm−1) 3238, 3131, 2957, 1695, 1643, 1211, 1171, 1016. HRMS (ESI) m/z calc. For [C32H29N5O5 + Na]+: 586.2061; obs.586.2062.

Ethyl 6-((2-methoxy-4-((4-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9d)

Yellow solid (m.p. 107 °C), 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.48 (ls, 1H), 8.03–7.99 (m, 2H), 7.95 (s, 1H), 7.74 (d, 1H, J = 15.6 Hz), 7.61–7.56 (m, 1H), 7.58 (t, 2H, J = 7.3 Hz), 7.39 (d, 1H, J = 15.6 Hz), 7.29–7.18 (m, 6H), 7.14 (d, 1H, J = 1.7 Hz), 7.07 (d, 1H, J = 8.3 Hz), 6.06 (ls, 1H), 5.82 (d, 1H, J = 14.6 Hz), 5.60 (d, 1H, J = 14.6 Hz), 5.38 (d, 1H, J = 2.5 Hz), 5.28 (s, 2H), 4.09 (q, 2H, J = 7.0 Hz), 3.87 (s, 3H), 1.14 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.6, 164.8, 152.4, 149.9, 149.5, 144.9, 143.7, 142.8, 141.4, 138.4, 132.7, 128.8, 128.6. 128.5, 128.3, 126.7, 124.5, 122.8, 120.4, 113.6, 110.7, 104.3, 62.8, 60.9, 55.9, 55.7, 47.9, 14.0. ATR-FTIR (νmax, cm−1) 3299, 3133, 2937, 1694, 1651, 1221, 1096, 1013. HRMS (ESI) m/z calc. for [C33H31N5O6 + Na]+: 616.2167; obs. 616.2175.

Ethyl 6-((3-methoxy-4-((3-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-phenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9e)

Yellow solid (m.p. 112 °C), 75% yield. 1H NMR (300 MHz, CDCl3) δ 8.39 (ls, 1H), 8.04 (d, 2H, J = 7.2 Hz), 7.96 (s, 1H), 7.76 (d, 1H, J = 15.8 Hz), 7.61–7.54 (m, 1H), 7.54–7.47 (m, 2H), 7.47–7.40 (m, 2H), 7.31–7.20 (m, 6H), 6.89 (d, 1H, J = 8.5 Hz), 6.03 (ls, 1H), 5.85 (d, 1H, J = 14.5 Hz), 5.59 (d, 1H, J = 14.5 Hz), 5.38 (d, 1H, J = 2.6 Hz), 5.30 (s, 2H), 4.09 (q, 2H, J = 7.0 Hz), 3.85 (s, 3H), 1.14 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.4, 164.7, 152.5, 15.8, 147.8, 144.6, 143.6, 142.8, 141.4, 138.3, 132.5, 128.7, 128.5, 128.4, 128.1, 127.8. 126.6, 124.5, 124.1, 120.1, 113.1, 111.5, 104.3, 62.9, 60.7, 55.8, 55.5, 47.8, 13.8. ATR-FTIR (νmax, cm−1) 3232, 3114, 2937, 1693, 1650, 1226, 1094, 1012. HRMS (ESI) m/z calc. for [C33H31N5O6 + Na]+: 616.2167; obs. 616.2174.

6-((4-((2-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidin-(1H)-2-one-5 carboxylate (9f)

Yellow solid (m.p. 107 °C), 72% yield. 1H NMR (300 MHz, CDCl3) δ 9.39 (ls, 1H), 8.14 (d, 1H, J = 15,6 Hz), 8.14 (s, 1H), 7.96 (d, 2H, J = 7.5 Hz), 7.65 (d, 1H, J = 7.0 Hz), 7.58 (d, 1H, J = 15.6 Hz), 7.56 (t, 1H, J = 7.8 Hz), 7.47 (t, 2H, J = 7.8 Hz), 7.39 (t, 1H, J = 7.3 Hz), 7.07 (d, 1H, J = 8.3 Hz), 7.03 (t, 1H, J = 7.5 Hz), 6.56 (ls, 1H), 6.41 (s, 2H), 5.91 (d, 1H, J = 14.1 Hz), 5.57 (d, 1H, J = 14.3 Hz), 5.34 (d, 1H, J = 2.3 Hz), 5.27 (s, 2H), 4.11 (q, 2H, J = 7.0 Hz), 3.77 (s, 3H), 3.66 (s, 6H), 1.15 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.7, 164.7, 157.2, 153.3, 153.0, 143.6, 141.3, 139.8, 138.4, 138.0, 137.6, 132.9, 132.1, 128.6, 128.6, 128.5, 124.4, 124.0, 122.1, 121.5, 112.6, 104.2, 103.5, 62.8, 60.8, 60.7, 55.9, 55.9, 47.8, 14.0. ATR-FTIR (νmax, cm–1) 3214, 3097, 2936, 1693, 1649, 1217, 1126, 1000. HRMS (ESI) m/z calc. for [C35H35N5O8 + Na]+: 676.2378; obs. 676.2380.

Ethyl 6-((4-((3-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9g)

Yellow solid (m.p. 103 °C), 70% yield. 1H NMR (400 MHz, CDCl3) δ 8.42 (ls, 1H), 8.05–7.99 (m, 2H), 7.97 (s, 1H), 7.76 (d, 1H, J = 15.6 Hz), 7.62–7.56 (m, 3H), 7.56–7.48 (m, 3H), 7.36–7.30 (t, 1H, J = 8.1 Hz), 7.27–7.23 (m, 2H), 7.05–7.01 (m, 1H), 6.43 (s, 1H), 6.07 (ls, 1H), 5.91 (d, 1H, J = 14.4 Hz), 5.58 (d, 1H, J = 14.4 Hz), 5.37 (d, 1H, J = 2.5 Hz), 5.21 (s, 2H), 4.13 (q, 2H, J = 7.1 Hz), 3.79 (s, 3H), 3.74 (6H, s), 1.18 (t, 3H, J = 7.1 Hz). 13C NMR (100 MHz, CDCl3) δ 190.4, 164.7, 158.5, 153.4, 152.4, 144.4, 143.9, 141.5, 138.3, 138.0, 137.8, 136.4, 132.8, 130.0, 128.6, 128.5, 124.1, 122.5, 121.7, 116.9, 114.3, 104.1, 103.4, 61.9, 60.9, 60.7, 56.0, 55.8, 48.1, 14.0. ATR-FTIR (νmax, cm−1) 3228, 3102, 2939, 1693, 1653, 1222, 1093, 1013. HRMS (ESI) m/z calc. for [C35H35N5O8 + Na]+: 672.2378; obs. 672.2373.

Ethyl 6-((4-((4-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9h)

Yellow solid (m.p. 109 °C), 62% yield. 1H NMR (400 MHz, CDCl3) δ 8.56 (ls, 1H), 8.05–8.01 (m, 2H), 7.98 (s, 1H), 7.79 (d, 1H, J = 15.6 Hz), 7.63–7.58 (m, 1H), 7.60 (t, 2H, J = 8.8 Hz), 7.52 (t, 2H, J = 7.0 Hz), 7.44 (d, 1H, J = 15.6 Hz), 7.02 (d, 2H, J = 8.8 Hz), 6.44 (s, 1H), 6.17 (ls, 1H), 5.94 (d, 1H, J = 14.3 Hz), 5.57 (d, 1H, J = 14.3 Hz), 5.38 (d, 1H, J = 2.8 Hz), 5.22 (s, 2H), 4.14 (q, 2H, J = 7.0 Hz), 3.82 (s, 3H), 3.76 (s, 6H) 1.19 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.5, 164.7, 160.1, 153.5, 152.3, 144.4, 143.7, 141.5, 138.3, 138.2, 137.8, 132.6, 130.2, 128.5, 128.4, 128.2, 124.2, 120.1, 115.1, 104.1, 103.4, 61.8, 60.9, 60.8, 56.0, 55.8, 48.1, 14.0. ATR-FTIR (νmax, cm−1) 3229, 3104, 2936, 2831, 1696, 1653, 1217, 1119, 1000. HRMS (ESI) m/z calc. for [C 35 H 35 N 5 O 8 + Na]+: 676.2378, obs. 676.2377.

Ethyl 6-((3-methoxy-4-((4-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy)methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxyphenyl-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9i)

Yellow solid (m.p. 102 °C), 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.49 (ls, 1H), 8.04–8.00 (m, 2H), 7.97 (s, 1H), 7.74 (d, 1H, J = 15.6 Hz), 7.61–7.56 (m, 1H), 7.54–7.48 (m, 2H), 7.40 (d, 1H, J = 15.6 Hz), 7.20 (dd, 1H, J = 8.4 and 1.9 Hz), 7.14 (d, 1H, J = 1.8 Hz), 7.06 (d, 1H, J = 8.3 Hz), 6.42 (s, 2H), 6.08 (ls, 1H), 5.85 (d, 1H, J = 14.5 Hz), 5.59 (d, 1H, J = 14.5 Hz), 5.34 (d, 1H, J = 2.5 Hz), 5.26 (s, 2H), 4.12 (q, 2H, J = 7.0 Hz), 3.88 (s, 3H), 3.80 (s, 3H), 3.74 (s, 6H), 1.17 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3) δ 190.5, 164.7, 153.4, 152.4, 149.8, 149.5, 144.7, 143.5, 141.5, 138.3, 138.3, 137.8, 132.6, 128.7, 128.5, 128.4, 124.7, 128.5, 128.4, 124.7, 122.8, 120.4, 113.6, 110.7, 104.1, 103.4, 62.7, 60.8, 60.7, 56.0, 55.8, 48.0. ATR-FTIR (νmax, cm−1) 3246, 3107, 2929, 2851, 1696, 1653, 1222, 1125, 1000. HRMS (ESI) m/z calc. for [C36H37N5O9 + Na]+: 706.2483; obs. 706.2482.

Ethyl 6-((3-methoxy-4-((3-(3-oxo-3-phenylprop-1-(E)-en-1-yl)phenoxy) methyl)-(1H)-1,2,3-triazol-1-yl)methyl)-4-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidin-(1H)-2-one-5-carboxylate (9j)

Yellow solid (m.p. 114 °C), 85% yield. 1H NMR (400 MHz, CDCl3) δ 8.43 (ls, 1H), 8.03–7.98 (m, 2H), 7.90 (s, 1H), 7.77 (d, 1H, J = 15.6 Hz), 7.61–7.55 (m, 1H), 7.53–7.47 (m, 2H), 7.45–7.38 (m, 2H), 7.25 (dd, 1H, J = 8.3 and 2.0 Hz), 6.89 (d, 1H, J = 8.3 Hz), 6.43 (s, 2H), 6.10 (ls, 1H), 5.84 (d, 1H, J = 14.4 Hz), 5.61 (d, 1H, J = 14.4 Hz), 5.34 (d, 1H, J = 2.5 Hz), 5.27 (s, 2H), 4.12 (q, 2H, J = 7.2 Hz), 3.85 (s, 3H), 3.79 (s, 3H), 3.73 (s, 6H), 1.17 (t, 3H, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ 190.5, 164.8, 153.4, 152.3, 151.9, 147.8, 144.7, 143.8, 141.6, 138.4, 138.3, 137.7, 132.7, 128.6, 128.5, 127.9, 124.7, 124.1, 120.2, 113.2, 111.6, 104.1, 103.4, 63.0, 60.9, 60.8, 56.1, 55.9, 55.9, 48.0, 14.1. ATR-FTIR (νmax, cm−1) 3235, 3116, 2940, 1690, 1649, 1221, 1122, 1013. HRMS (ESI) m/z calc. for [C36H37N5O9 + Na]+: 706.2483; obs. 706.2473.

3.2. Cell Culture and MTT Assay

The cytotoxic activity of compounds was evaluated in a panel of commercial human cell lines purchased from the American Type Culture Collection–ATCC: five tumoral (U-251–brain, glioblastoma; MCF-7–breast, adenocarcinoma; NCI/ADR-RES–ovary, multidrug-resistant adenocarcinoma; OVCAR-3–ovary, adenocarcinoma, and HT-29–colorectal, adenocarcinoma) and one non-tumoral (HaCaT–keratinocyte).
For this assay, the colorimetric method 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, MTT (Sigma-Aldrich, Burlington, MA, USA) was used to indirectly evaluate cell viability by the mitochondrial enzymatic activity of living cells [62]. The cell suspensions were prepared in RPMI-1640 (Sigma-Aldrich, Burlington, MA, USA) medium containing 5% FBS (LGC Biotecnologia, Cotia, SP, Brazil) and 1% PS (LGC Biotecnologia, Cotia, SP, Brazil). One hundred μL of cell suspension containing 5000 cells were inoculated per well into 96-well plates and incubated for 24 h at 37 °C in a 5% CO2 atmosphere and humidity. After 24 h, samples were diluted in DMSO and added to the cells at concentrations ranging from 2.5–381 μM (100 μL/well) in triplicate and then incubated for 48 h at 37 °C in a 5% CO2 atmosphere and humidity. As a positive control, the chemotherapy drug doxorubicin hydrochloride, doxo (Eurofarma, São Paulo, SP, Brazil), was used at concentrations of 0.3–18.4 μM (100 μL/well) in triplicate. The final DMSO concentration (less than 0.25%) did not affect cell viability [63]. After 48 h of treatment, the treated cells were then stained with MTT. The absorbance data were analyzed and compiled in the graphs plotting the percentage of viable cells with the sample concentration. The IC50 (half maximal inhibitory concentration) values were calculated, which refers to the concentration of samples required to decrease in 50% cell viability. Hybrids and their parental molecules were also evaluated for the selectivity index (SI), which reflects the differential cytotoxicity of a compound against tumor and normal cells. The greater the SI value of a compound, the more selective it is. This study obtained SI from the following formula: IC50 of the non-tumoral cell line (HaCaT)/IC50 of tumor cell lines. For this analysis, a SI value greater than or equal to 2.0 was adopted as significant, as previously described by the NCI, USA (National Cancer Institute, Bethesda, MD, USA) [50].

3.3. Molecular Docking

The hybrids were drawn, converted to a PDB file, and then set to rotatable bonds to get flexible docking. ER receptors α and β were selected in the Protein Data Bank (PDB) by their resolution (1.68 and 2.20 Å, respectively) and binding to an antagonist and agonist. For ER-α, it was chosen PDB code 6CHZ and considered the binding pocket of three residues (Thr347, Asp351, and Glu353), the position of the endogenous agonist. For Erβ, the protein 3OLS PDB code was selected and considered the binding pocket Glu305, Arg346, and Phe356. Moreover, hybrids were superimposed onto co-crystallized proteins containing endogenous agonists to check the positioning.
Missing atoms, chain breaks, and water molecules were removed, and hydrogens were added for a pH of 7.0. The grid was centered within the protein’s active site (subunit A) with a size of 22 × 24 × 28 Å in the x, y, and z axes, respectively.
Ligands and proteins were subjected to analysis by AutoDock Vina to get the binding energy (kcal/mol) and to Chimera 1.15 software for distances calculation, and amino acid binds determination.

4. Conclusions

A series of 10 hybrid molecules based on chalcones and dihydropyrimidinones were designed and readily synthesized using simple reactions such as the Claisen–Schmidt condensation, multicomponent Biginelli reaction, and copper-catalyzed Huisgen reaction. Different hybrids showed low IC50 values for different cancer cell lines, such as ovary adenocarcinoma (including one resistant), glioblastoma, and breast adenocarcinoma cell lines. It is noteworthy that all hybrids exhibited powerful cytotoxicity against luminal breast adenocarcinoma MCF-7.
The cytotoxicity of parentals (chalcone and dihydropyrimidinone, respectively) was always smaller than the cytotoxicity of the most potent hybridized compounds 9d, 9g, and 9h, suggesting that potency may be the result of molecular hybridization. Additionally, the hybrids were more selective to breast cancer cell lines than healthy cell lines, which is desirable for a drug candidate. In this sense, compound 9h showed the highest SI, deserving a further complementary study.
In silico studies denoted that the hybrid compounds 9d, 9g, and 9h are positioned on the active site of the ERα or in its surroundings, acting as antagonist molecules that can, in turn, disrupt or hinder the cell proliferation process. These findings deeply encouraged us to proceed with further investigations to elucidate the mechanism of action against luminal breast cancers.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ddc1010002/s1, Figure S1: 1H NMR (400 MHz, CDCl3) of 4a. Figure S2: 13C NMR (100 MHz, CDCl3) of 4a. Figure S3: 1H NMR (400 MHz, CDCl3) of 4b. Figure S4: 13C NMR (100 MHz, CDCl3) of 4b. Figure S5: 1H NMR (400 MHz, CDCl3) of 4c. Figure S6: 13C NMR (400 MHz, CDCl3) of 4c. Figure S7: 1H NMR (400 MHz, CDCl3) of 4d. Figure S8: 13C NMR (75 MHz, CDCl3) of 4d. Figure S9: 1H NMR (400 MHz, CDCl3) of 4e. Figure S10: 13C NMR (400 MHz, CDCl3) of 4e. Figure S11: 1H NMR (400 MHz, DMSO-d6) of 7a. Figure S12: 13C NMR (100 MHz, DMSO-d6) of 7a. Figure S13: 1H NMR (400 MHz, DMSO-d6) of 7b. Figure S14: 13C NMR (100 MHz, DMSO-d6) of 7b. Figure S15: 1H NMR (400 MHz, DMSO-d6) of 7a. Figure S16: 13C NMR (100 MHz, DMSO-d6) of 8a. Figure S17: 1H NMR (100 MHz, DMSO-d6) of 8b. Figure S18: 13C NMR (100 MHz, DMSO-d6) of 8b. Figure S19: 1H NMR (400 MHz, CDCl3) of 9a. Figure S20: 13C NMR (100 MHz, CDCl3) of 9a. Figure S21: 1H NMR (400 MHz, CDCl3) of 9b. Figure S22: 13C NMR (100 MHz, CDCl3) of 9b. Figure S23: 1H NMR (400 MHz, CDCl3) of 9c. Figure S24: 13C NMR (100 MHz, CDCl3) of 9c. Figure S25: 1H NMR (400 MHz, CDCl3) of 9d. Figure S26: 13C NMR (100 MHz, CDCl3) of 9d. Figure S27: 1H NMR (400 MHz, CDCl3) of 9e. Figure S28: 13C NMR (100 MHz, CDCl3) of 9e. Figure S29: 1H NMR (400 MHz, CDCl3) of 9f. Figure S30: 13C NMR (100 MHz, CDCl3) of 9f. Figure S31: 1H NMR (400 MHz, CDCl3) of 9g. Figure S32: 13C NMR (100 MHz, CDCl3) of 9g. Figure S33: 1H NMR (400 MHz, CDCl3) of 9h. Figure S34: 13C NMR (100 MHz, CDCl3) of 9h. Figure S35: 1H NMR (400 MHz, CDCl3) of 9i. Figure S36: 13C NMR (100 MHz, CDCl3) of 9i. Figure S37: 1H NMR (400 MHz, CDCl3) of 9j. Figure S38: 13C NMR (100 MHz, CDCl3) of 9j. Figure S39: Cell viability of parentals 4d and 8a and hybrid 9d. Figure S40: Cell viability of parentals 4b and 8b and hybrid 9g. Figure S41: Cell viability of parentals 4c and 8b and hybrid 9h.

Author Contributions

Conceptualization, D.R.; methodology, E.B.M. and C.A.d.L.; formal analysis and investigation, D.R., M.G.M.D., G.B.L. and J.M.S.; writing—original draft preparation, E.B.M. and D.R.; writing—review and editing, D.R. and G.B.L.; funding acquisition, D.R. and G.B.L. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge the Fundação de Apoio à Pesquisa do Estado do Rio Grande do Sul (FAPERGS, grant no. 19/2551-0001767-7), Fundação de Apoio à Pesquisa do Estado de São Paulo (FAPESP, grant no. 2016/06137-5) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant no. 312452/2017-9). E.B.M. and C.A.d.L. acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the fellowships.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Ddc 01 00002 g001 550
Figure 1. Structures of hybrid Chalcone compounds I–III.
Figure 1. Structures of hybrid Chalcone compounds I–III.
Ddc 01 00002 g001
Ddc 01 00002 g002 550
Figure 2. Monastrol (IV) and bioactive DHPM hybrids V–VII.
Figure 2. Monastrol (IV) and bioactive DHPM hybrids V–VII.
Ddc 01 00002 g002
Ddc 01 00002 sch001 550
Scheme 1. Preparation of propargyloxy chalcones 4a–e.
Scheme 1. Preparation of propargyloxy chalcones 4a–e.
Ddc 01 00002 sch001
Ddc 01 00002 sch002 550
Scheme 2. Preparation of azido-dihydropyrimidinones 8a,b.
Scheme 2. Preparation of azido-dihydropyrimidinones 8a,b.
Ddc 01 00002 sch002
Ddc 01 00002 sch003 550
Scheme 3. Preparation of Chalcone-Dihydropyrimidinones 9a–j.
Scheme 3. Preparation of Chalcone-Dihydropyrimidinones 9a–j.
Ddc 01 00002 sch003
Ddc 01 00002 g003 550
Figure 3. Molecular docking of hybrids to ERα receptor (6CHZ PDB code). (A) Hybrid 9d and antagonist H3B-9224 to show the entire protein and positioned molecules–the same pattern was observed for the other hybrids; (B) Detail of the hybrid 9d; (C) Hybrid 9g; (D) Hybrid 9h. The hybrids are shown in blue, the ERα receptor in green, and the antagonist is used as a model in magenta.
Figure 3. Molecular docking of hybrids to ERα receptor (6CHZ PDB code). (A) Hybrid 9d and antagonist H3B-9224 to show the entire protein and positioned molecules–the same pattern was observed for the other hybrids; (B) Detail of the hybrid 9d; (C) Hybrid 9g; (D) Hybrid 9h. The hybrids are shown in blue, the ERα receptor in green, and the antagonist is used as a model in magenta.
Ddc 01 00002 g003
Ddc 01 00002 g004 550
Figure 4. Molecular docking of hybrids to ERβ receptor (3OLS PDB code). (A) Hybrid 9d, (B) Hybrid 9g, and (C) Hybrid 9h, in blue. The Erβ receptor (3OLS PDB code) is in green, and the agonist estradiol is used as a model in magenta.
Figure 4. Molecular docking of hybrids to ERβ receptor (3OLS PDB code). (A) Hybrid 9d, (B) Hybrid 9g, and (C) Hybrid 9h, in blue. The Erβ receptor (3OLS PDB code) is in green, and the agonist estradiol is used as a model in magenta.
Ddc 01 00002 g004
Table
Table 1. Synthesis of hybrids chalcone-DHPM 9a–j.
Table 1. Synthesis of hybrids chalcone-DHPM 9a–j.
EntryHybrid CompoundsH-Triazole (ppm)Yield (%) a
19aDdc 01 00002 i0019.0075
29bDdc 01 00002 i0028.3570
39cDdc 01 00002 i0038.4378
49dDdc 01 00002 i0048.4890
59eDdc 01 00002 i0058.3975
69fDdc 01 00002 i0069.3972
79gDdc 01 00002 i0078.4270
89hDdc 01 00002 i0088.5662
99iDdc 01 00002 i0098.4990
109jDdc 01 00002 i0108.4385
a Yields correspond to the purified compounds.
Table
Table 2. IC50 values of hybrid compounds 9a–j against different cell lines a.
Table 2. IC50 values of hybrid compounds 9a–j against different cell lines a.
HybridU-251MCF-7NCI/ADR-RESOVCAR-3HT-29HaCaT
9a27.0 ± 11.06.2 ± 3.46.4 ± 3.49.9 ± 3.518.6 ± 8.76.7 ± 3.0
9b26.1 ± 8.25.3 ± 2.714.5 ± 5.59.4 ± 3.443.8 ± 12.822.0 ± 3.9
9c65.1 ± 11.011.5 ± 5.154.8 ± 9.670.6 ± 12.1134.1 ± 33.722.0 ± 3.9
9d74.6 ± 17.54.7 ± 2.589.8 ± 12.1109.7 ± 31.8109.7 ± 46.517.5 ± 2.3
9e24.6 ± 6.612.3 ± 3.426.8 ± 7.215.3 ± 3.441.1 ± 7.912.0 ± 2.2
9f10.9 ± 4.17.5 ± 2.740.4 ± 11.212.5 ± 4.725.1 ± 12.56.4 ± 3.0
9g13.3 ± 3.84.9 ± 1.439.6 ± 7.917.9 ± 5.893.9 ± 29.815.1 ± 4.1
9h70.4 ± 9.55.8 ± 2.6>153.036.4 ± 7.2>153.0100.7 ± 13.0
9i104.1 ± 33.25.3 ± 2.5>146.323.5 ± 6.0103.4 ± 18.134.8 ± 4.8
9j128.7 ± 35.514.6 ± 4.5>146.388.9 ± 11.0>146.369.3 ± 7.9
DOXO>18.40.4 ± 0.022.6 ± 3.911.6 ± 5.1>18.41.6 ± 0.0
a Values of IC50 are expressed in µM, triplicate. U-251 (brain, glioblastoma), MCF-7 (breast, adenocarcinoma), NCI/ADR-RES (ovary, multidrug-resistant adenocarcinoma), OVCAR-3 (ovary, adenocarcinoma), HT-29 (colorectal, adenocarcinoma) and HaCaT (normal human keratinocytes). Doxo: doxorubicin.
Table
Table 3. Comparative IC50 (µM) and selectivity index (SI) values.
Table 3. Comparative IC50 (µM) and selectivity index (SI) values.
Cell LinesChalconeDHPMHybridSelectivity Index (SI)
4d8a9d
(4d + 8a)		9d
U-251251.51>33274.620.23
MCF-7>342>3324.723.71
NCI/ADR-RES>342>33289.790.19
HaCaT97.05>33217.52-
4b8b9g
(4b + 8b)		9g
U-25124.47>25513.301.14
MCF-780.33>2554.893.10
NCI/ADR-RES>381>25539.620.38
HaCaT23.33>25515.14-
4c8b9h
(4c + 8b)		9h
U-251126.50>25570.371.43
MCF-7>381>2555.8117.33
NCI/ADR-RES>381>255>153-
HaCaT37.59>255100.7-
Table
Table 4. Molecular interactions between hybrids and the amino acid residues in ER-α (6CHZ) obtained by molecular docking a.
Table 4. Molecular interactions between hybrids and the amino acid residues in ER-α (6CHZ) obtained by molecular docking a.
HybridBinding Energy (kcal/mol)Binding InteractionBond Lenght (Å)
9d−10.32Leu3466.96
Thr347a3.93
Asp351a3.29
Glu353a8.27
9g−12.02Leu3466.33
Thr347a4.05
Asp351a3.68
Asn5322.36
Leu5362.50
9h−7.52Leu3464.86
Thr347a5.35
Asp351a4.27
a Binding pocket: Thr347, Asp351, Glu353 for the endogenous agonist.
Table
Table 5. Molecular interactions between hybrids and the amino acid residues in ER-β1 (3OLS) obtained by molecular docking a.
Table 5. Molecular interactions between hybrids and the amino acid residues in ER-β1 (3OLS) obtained by molecular docking a.
HybridBinding Energy (kcal/mol)Binding InteractionBond Lenght (Å)
9d−9.38Cys5302.38
Ser5379.88
Asp3514.46
9g−7.85Cys5302.59
Ser5377.26
Leu5362.36
9h−8.51Cys5304.24
Ser5377.01
Leu5367.62
a Binding pocket: Glu305, Arg346, Phe356 for the endogenous agonist.
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Mass, E.B.; de Lima, C.A.; D’Oca, M.G.M.; Sciani, J.M.; Longato, G.B.; Russowsky, D. Synthesis, Selective Cytotoxic Activity against Human Breast Cancer MCF7 Cell Line and Molecular Docking of Some Chalcone-Dihydropyrimidone Hybrids. Drugs Drug Candidates 2022, 1, 3-21. https://doi.org/10.3390/ddc1010002

AMA Style

Mass EB, de Lima CA, D’Oca MGM, Sciani JM, Longato GB, Russowsky D. Synthesis, Selective Cytotoxic Activity against Human Breast Cancer MCF7 Cell Line and Molecular Docking of Some Chalcone-Dihydropyrimidone Hybrids. Drugs and Drug Candidates. 2022; 1(1):3-21. https://doi.org/10.3390/ddc1010002

Chicago/Turabian Style

Mass, Eduardo B., Carolina A. de Lima, Marcelo G. M. D’Oca, Juliana M. Sciani, Giovanna B. Longato, and Dennis Russowsky. 2022. "Synthesis, Selective Cytotoxic Activity against Human Breast Cancer MCF7 Cell Line and Molecular Docking of Some Chalcone-Dihydropyrimidone Hybrids" Drugs and Drug Candidates 1, no. 1: 3-21. https://doi.org/10.3390/ddc1010002

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