**4. Conclusions**

Hydroxylated chalcone/flavanone derivatives were synthesized using the less common bases NaH or LiHMDS. Overall, it was shown that the use of NaH is efficient (~80% yield in 3–4 h) for the synthesis of chalcones not hydroxylated other than at C-2 position. However, if the desired derivatives are polyhydroxylated chalcones, the use of LiHMDS is preferable, because the conversion rate is good and it is a one-pot procedure that avoids the protection and subsequent deprotection steps, a time-consuming procedure that also significantly decreases the total yield. Although the aim of this work was to obtain the compounds for biological evaluation, we sugges<sup>t</sup> microwave or ultrasound irradiation as a better source of energy to increase the compounds' yields, an aspect that is important for their future applications.

Some of the compounds synthesized presented interesting results in their bioactivities, with chalcone **6** being the most active compound in terms of antioxidant, anti-butyrylcholinesterase and antimicrobial activity. Its isomer, flavanone **7**, showed activity against both acetylcholinesterase and butyrylcholinesterase, which is an interesting result since this dual inhibition is a much-appreciated feature in Alzheimer's disease therapy. Also, it is interesting that these compounds can be obtained in the one-pot methodology using LiHMDS.

Some important structure/activity relationships were established for all the activities tested, and the most important are highlighted and summarized in Figure 8.

**Figure 8.** Summary of the most relevant structure/activity relationships established.

As shown in Figure 8, the free hydroxyl groups are essential to increase antioxidant, anti-butyrylcholinesterase and antimicrobial activities. Not only their presence but also their number increases the compounds' activity, while methoxyl groups decrease it. The flavanone scaffold increases the acetylcholinesterase inhibitory activity, while for the butyrylcholinesterase inhibition, the chalcone scaffold appears to be better. This is another interesting result because it is known that these compounds are isomers, and in biological systems they can exist in a controlled equilibrium. Regarding the antimicrobial activity, it is possible to detect that the cyclization into flavanone has no effect against the *M. luteus* strain, whereas against *B. subtilis*, the flavanone decreases the antibacterial activity. So, it seems that the <sup>α</sup>,β unsaturated carbonyl bridge linking rings A and B is important to inhibit the growth of the *B. subtilis* strain. Finally, it can be established that the presence of methoxyl groups in both chalcones and flavanones increases the antitumor activity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3417/9/14/2846/s1, Figure S1: 1H NMR spectrum of compound 1; Figure S2: 13C NMR spectrum of compound 1; Figure S3: HSQC NMR spectrum of compound 1; Figure S4: HMBC NMR spectrum of compound 1; Figure S5: ESI(+) Mass spectrum of compound 1; Figure S6: 1H NMR spectrum of compound 4; Figure S7: 13C NMR spectrum of compound 4; Figure S8: HSQC NMR spectrum of compound 4; Figure S9: HMBC NMR spectrum of compound 4; Figure S10: Antioxidant activity (%) presented by the compounds tested at three concentrations in the DPPH assay; Figure S11: Antioxidant activity (%) presented by the compounds tested at three concentrations in the ABTS assay; Figure S12: BuChE inhibitory activity (%) presented by the compounds tested at three concentrations; Figure S13: Inhibition of M. luteus growth (%) of the compounds tested at three concentrations; Figure S14: Inhibition of B. subtilis growth (%) of the compounds tested at three concentrations; Figure S15: Inhibition of A549 cell-line growth (%) of the compounds tested at three concentrations

**Author Contributions:** Conceptualization and methodology, A.M.L.S., M.C.B. and D.C.G.A.P.; formal analysis and investigation, G.P.R.; writing—original draft preparation, G.P.R.; writing—review and editing, A.M.L.S., M.C.B., A.M.S.S. and D.C.G.A.P.; supervision, A.M.L.S. and M.C.B.

**Funding:** This research was funded by FCT—Fundação para a Ciência e a Tecnologia, the European Union, QREN, FEDER, COMPETE, by funding the cE3c centre (FCT Unit funding (Ref. UID/BIA/00329/2013, 2015–2018) and UID/BIA/00329/2019) and the QOPNA research unit (project FCT UID/QUI/00062/2019).

**Acknowledgments:** Thanks are due to the University of Azores and University of Aveiro.

**Conflicts of Interest:** The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
