**1. Introduction**

The (*E*)-1,3-diphenylpropen-1-ones, best known as chalcones, belong to the flavonoids family and are an important class of natural products across the plant kingdom [1]. Structurally, these compounds contain two aromatic rings, bonded by a three-carbon <sup>α</sup>,β unsaturated carbonyl bridge (Figure 1), which are synthesized in plants as the C15 key intermediate in the biosynthesis of the other flavonoids [2]. Flavanones are also naturally occurring compounds and are chalcones' isomeric forms. In fact, the equilibrium between chalcones and flavanones is common in nature and is regulated by chalcone-isomerase [3].

**Figure 1.** Basic chalcone structure.

Besides their natural occurrence, both chalcones and flavanones can be obtained synthetically and are often used as the preferred starting material for the synthesis of other polycyclic aromatic compounds [4]. Furthermore, they present grea<sup>t</sup> pharmacological potential with a wide variety of biological activities, including antioxidant [5], anticancer [6–8], and antimicrobial activities [9–12], and also the ability to treat cardiovascular diseases and their risk factors [13–16], among others [17].

On the other hand, cancer, neurodegenerative diseases, oxidative stress-related diseases and multi-resistant bacterial infections are, after cardiovascular diseases, the top four health problems that cause the most victims every year, leading to higher medicine consumption and putting grea<sup>t</sup> pressure on the national health systems of many countries [18–24]. These problems occur either due to a lack of effective medicines to treat diseases, such as neurodegenerative ones, or due to the increasing drug resistance presented by numerous pathogenic bacteria and by some cancers. Therefore, exploring well-known sca ffolds as lead compounds will help in the battle against diseases that a ffect humanity.

Putting together the facts stated above, the synthesis of chalcone-based functionalized derivatives remains a popular research objective. The most common and e fficient approach to obtain the chalcone nucleus is the aldol condensation of substituted acetophenones with proper substituted benzaldehydes in the presence of a base, namely sodium or potassium hydroxide [25–28]. Despite the e fficiency of this method, when planning a synthesis some drawbacks should be considered. For instance, the protection of the reagents' hydroxyl groups should be done previously, the acetophenone hydrogen α acidity should be analyzed, and by-products can be obtained if the bases are also good nucleophilic species [29,30].

In this regard, the objective of this work is to synthesize hydroxy- and/or methoxychalcones by aldol condensation, using the less common bases sodium hydride and lithium bis(trimethylsilyl)amide. Also, this work studies their antioxidant, anticholinesterasic, antibacterial and antitumor activities, aiming to establish some potential medicinal applications. Simultaneously, a structure/activity relationship was established, and the isomeric equilibrium chalcone-flavanone was also studied.

#### **2. Materials and Methods**

#### *2.1. General Methods*

The 1H, 13C, HSQC and HMBC NMR spectra were measured on Bruker AMC 300 or 500 instruments, operating at 300.13 MHz and 75.47 or 500.13 and 125.75 MHz. Chemical shifts were reported relative to tetramethysilane (TMS) in δ units (ppm) and coupling constants (*J*) in Hz. Chromatographic purifications were carried out by prep. TLC on silica gel (Merck silica gel 60 F254), the spots being visualized under a UV lamp (at 254 and/or 366 nm). Melting points were determined with a Stuart scientific SPM3 apparatus and are uncorrected. The mass spectra were acquired using ESI(+) with a Micromass Q-Tof 2TM mass spectrometer.

#### *2.2. Synthesis of Chalcones and Flavanones*

Synthesis of the compounds described below follows the general scheme outlined in 3.1 (Scheme 1).

*<sup>2</sup>-Hydroxy-4,4,6-trimethoxychalcone* **1**. Compound **1** was synthesized by mixing <sup>2</sup>-hydroxy-4,6 - dimethoxyacetophenone (661.3 mg, 3.37 mmol) dissolved in a minimum (~15 mL) amount of tetrahydrofuran (THF) with sodium hydride (NaH) (2.5 equivalents), under nitrogen atmosphere at room temperature. After 30 min of stirring, 4-methoxybenzaldehyde (1.2 equivalents) was added to the reaction mixture and allowed to react for 3 h. The product was precipitated from the reaction

mixture by pouring onto ground ice and acidifying to pH < 2 with HCl 37%. The solid was filtered and washed with water until pH > 5. The crude product was crystallized from ethanol and the desired compound **1** was obtained (928.1 mg, 88% yield).

*<sup>2</sup>-Hydroxy-4,4,6-trimethoxychalcone* **1**: yellow crystals (ethanol); m.p. 112.4–113.6 ◦C (Lit. 111–115◦ [31]). 1H NMR (300 MHz, CDCl3) δ 14.40 (1H, s, 2-O*H*), 7.79 (2H, s br, H-α, H-β), 7.56 (2H, d, *J* = 6.8 Hz, H-2, H-6), 6.92 (2H, d, *J* = 6.8 Hz, H-3, H-5), 6.11 (1H, d, *J* = 2.4 Hz, H-3), 5.96 (1H, d, *J* = 2.4 Hz, H-5), 3.92 (3H, s, 6-OC*H*3), 3.85 (3H, s, 4-OC*H*3), 3.83 (3H, s, 4-OC*H*3); 13C NMR (75 MHz, CDCl3) δ 192.6 (*C*=O), 168.4 (C-2), 166.0 (C-4), 162.5 (C-6), 161.4 (C-4), 142.5 (C-β), 130.1 (C-2, C-6), 128.3 (C-1), 125.1 (C-α), 114.4 (C-3, C-5), 106.3 (C-1), 93.8 (C-3), 91.2 (C-5), 55.8 (6-O*C*H3), 55.6 (4-O*C*H3), 55.4 (4-O*C*H3); TOF-ESI-MS (+) *m*/*z* 315 [M+H]<sup>+</sup>, 337 [M+Na]<sup>+</sup>, 353 [M+K]<sup>+</sup>, 651 [M+Na+M]<sup>+</sup>.

*5,7-Dihydroxy-4-methoxyflavanone* **4**. The procedure to obtain this compound involved 3 different steps:


*5,7-Dihydroxy-4-methoxyflavanone* **4**. pale-yellow crystals (CHCl3); m.p. 191.7–193.3 ◦C (Lit. 193–194 ◦C [34]). 1H NMR (300 MHz, CDCl3) δ 12.05 (1H, s, 5-O*H*), 7.37 (2H, d, *J* = 8.7 Hz, H-2, H-6), 6.95 (2H, d, *J* = 8.7, H-3, H-5), 5.99 (1H, s broad, H-6), 5.98 (1H, s broad, H-8), 5.36 (1H, dd, *J* = 3.0 and 13.0 Hz, H-2), 3.83 (3H, s, 4-OC*H*3), 3.10 (1H, dd, *J* = 13.0 and 17.2 Hz, H-3a), 2.78 (1H, dd, *J* = 3.0 and 17.2 Hz, H-3b); 13C NMR (75 MHz, CDCl3) δ 196.2 (C-4), 164.8 (C-7), 164.3 (C-5), 163.3 (C-8a), 160.1 (C-4), 130.3 (C-1), 127.8 (C-2,' C-6), 114.1 (C-3, C-5), 103.1 (C-4a), 96.7 (C-6), 95.5 (C-8), 79.0 (C-2), 55.4 (4-O*C*H3), 43.1 (C-3); TOF-ESI-MS (+) *m*/*z* 287 [M+H]<sup>+</sup>, 611 [M+K+M]<sup>+</sup>.

*4-Hydroxy-5,7-dimethoxyflavanone* **5**. The synthesis of this compound also involved the 3 steps mentioned above for compound **4**.

(a) The benzylation of the hydroxyl groups: the 4-hydroxybenzaldehyde (1.3 g, 10.6 mmol) was dissolved in a minimum of dry dimethylformamide (DMF) (~15 mL), and it was mixed with K2CO3 (3 equivalents) under constant stirring. Then, benzyl bromide (1.5 equivalents) was added, and the reaction was performed at 150 ◦C under reflux for 2 h. After that, the reaction mixture

was filtered to remove the K2CO3 and washed with DMF. The filtrate was poured over crushed ice and HCl 20% added until pH < 5. The precipitated 4-benzyloxybenzaldehyde was filtered and crystallized from ethanol (1.8 g, 78%).


*4-Hydroxy-5,7-dimethoxyflavanone* **5**: 1H NMR (300 MHz, CDCl3) δ 7.29 (2H, d, *J* = 8.5 Hz, H-2, H-6), 6.86 (2H, d, *J* = 8.5 Hz, H-3and H-5), 6.13 (1H, d, *J* = 2.3 Hz, H-8), 6.07 (1H, d, *J* = 2.3 Hz, H-6), 5.34 (1H, dd, *J* = 3.0 and 12.8 Hz, H-2), 3.86 (3H, s, 5-OC*H*3), 3.81 (3H, s, 7-OC*H*3), 3.03 (1H, dd, *J* = 12.8 and 16.6 Hz, H-3a), 2.78 (1H, dd, *J* = 3.0 and 16.6 Hz, H-3b); 13C NMR (75 MHz, CDCl3) δ 190.2 (C-4), 166.2 (C-7), 165.2 (C-8a), 162.3 (C-5), 156.4 (C-4), 130.4 (C-1), 127.9 (C-2, C-6), 115.7 (C-3, C-5), 105.8 (C-4a), 93.6 (C-8), 93.1 (C-6), 78.9 (C-2), 56.1 (5-O*C*H3), 55.6 (7-O*C*H3), 45.2 (C-3); TOF-ESI-MS (+) *m*/*z* 301 [M+H]<sup>+</sup>, 323 [M+Na]<sup>+</sup>, 623 [M+Na+M]<sup>+</sup>.

*<sup>2</sup>,4,4-Trihydroxychalcone* **6** *and <sup>4</sup>,7-dihydroxyflavanone* **7**. The <sup>2</sup>,4-dihydroxyacetophenone (226.7 mg) was dissolved in dried toluene and mixed with 10 mL of a 1 mol.dm−<sup>3</sup> solution of LiHMDS (6.6 equivalents), under nitrogen atmosphere, at room temperature. After 30 min, 4-hydroxybenzaldehyde (1.2 equivalents) was added, and the reaction was stirred for 5 days. The reaction was finished, poured over crushed ice and acidified to pH < 2 with HCl 37%. The mixture was extracted with CH2Cl2 and purified by TLC, using a mixture of hexane and ethyl acetate (1:1) as eluent (twice). Compounds <sup>2</sup>,4,4-trihydroxychalcone **6** and <sup>4</sup>,7-dihydroxyflavanone **7** were obtained as yellow amorphous powder, respectively, 18.2 mg (5%) and 8.2 mg (2%). Approximately 80% of the starting acetophenone was also recuperated.

*<sup>2</sup>,4,4-Trihydroxychalcone* **6**: 1H NMR (300 MHz, acetone-d6) δ 13.69 (1H, s, 2-O*H*), 8.12 (1H, d, *J* = 8.9 Hz, H-6), 7.85 (1H, d, *J* = 15.4 Hz, H-β), 7.77 (1H, d, *J* = 15.4 Hz, H-α), 7.75 (2H, d, *J* = 8.6 Hz, H-2, H-6), 6.94 (2H, d, *J* = 8.6 Hz, H-3, H-5), 6.48 (1H, dd, *J* = 2.4 and 8.9 Hz, H-5), 6.38 (1H, d, *J* = 2.4 Hz, H-3); 13C-NMR (75 MHz, acetone-d6) δ 192.6 (*C*=O), 167.6 (C-4), 166.2 (C-2), 161.1 (C-4), 145.0 (C-β), 133.2 (C-6), 131.7 (C-2, C-6), 127.4 (C-1), 118.2 (C-α), 116.8 (C-3, C-5), 114.2 (C-1), 108.9 (C-5), 103.7 (C-3); TOF-ESI-MS (+) *m*/*z* 257 [M+H]<sup>+</sup>, 279 [M+Na]<sup>+</sup>, 535 [M+Na+M]<sup>+</sup>, 551 [M+K+M]<sup>+</sup>.

*<sup>4</sup>,7-Dihydroxyflavanone* **7**: 1H NMR (300 MHz, acetone-d6) δ 9.53 (1H, s, 7-O*H*), 8.60 (1H, s, 4-O*H*), 7.74 (1H, d, *J* = 8.6 Hz, H-5), 7.42 (2H, d, *J* = 8.6 Hz, H-2, H-6), 6.91 (2H, d, *J* = 8.6 Hz, H-3, H-5), 6.59 (1H, dd, *J* = 2.3 and 8.6 Hz, H-6), 6.43 (1H, d, *J* = 2.3 Hz, H-8), 5.46 (1H, dd, *J* = 2.8 and 13.1 Hz, H-2), 3.06 (1H, dd, *J* = 13.1 and 16.7 Hz, H-3a); 2.68 (1H, dd, *J* = 2.8 and 16.7 Hz, H-3b); 13C-NMR (75 MHz, acetone-d6) δ 190.5 (C-4), 165.2 (C-7), 164.5 (C-8a), 158.6 (C-4), 131.2 (C-1), 129.4 (C-5), 128.9 (C-2, C-6), 116.1 (C-3, C-5), 115.1 (C-4a), 111.2 (C-6), 103,6 (C-8), 80.5 (C-2), 44.6 (C-3); TOF-ESI-MS (+) *m*/*z* 257 [M+H]<sup>+</sup>, 279 [M+Na]<sup>+</sup>, 513 [M+H+M]<sup>+</sup>, 535 [M+Na+M]<sup>+</sup>, 551 [M+K+M]<sup>+</sup>.
