2.4.3. Ferric Reducing Power Assay

The results of the investigation of the antioxidant capacity of the compounds tested by the ferric reducing power method are presented in Table 8. All the tested compounds possessed the ability to reduce iron(III) to iron(II). The α-acylamino ketone **7a** was a better iron(III) reducer (absorbance at 700 nm, *A*<sup>700</sup> of 0.0722 ± 0.0013) than the other new derivatives, followed by three compounds, which showed similar values of the ferric reducing power, in descending order: **6** (*A*<sup>700</sup> = 0.0461 ± 0.0088), **8b** (*A*<sup>700</sup> = 0.0439 ± 0.0057), and **7b** (*A700* = 0.0437 ± 0.0105). Of the compounds tested, *N*-acyl-*L*-valine **5** was the least active tested compound (*A*<sup>700</sup> = 0.0224 ± 0.0019) and *L*-valine had the best antioxidant activity (*A*<sup>700</sup> = 0.0854 ± 0.0051). None of the activities of the analyzed compounds were comparable to those of the AA, BHA, and BHT positive controls.



## *2.5. Daphnia magna Toxicity Bioassay*

The results of the *Daphnia magna* toxicity test are presented in Table 9, and the lethality curves are shown in Figure 4. At 24 h, the highest toxicity was induced by **7a** and **7b**, and the compounds **5**, **6**, **8a**, and **8b** were non-toxic on *D. magna* at the tested concentrations. At 48 h, the most toxic compound was **7a**. At the lowest concentration, its lethality was 65%; therefore, the LC50 being lower than 2 μg/mL, the estimated value by extrapolation is 0.21 μg/mL. The compounds **5**, **6**, **7b**, and **8a** induced medium to high toxicity, their LC50 values varying from 21.07 to 54.62 μg/mL. Except for **7a**, all compounds induced lower toxicity than the key starting material **3**. The compound **8b** at the highest concentration (50 μg/mL) induced a lethality of 25%, and at all other concentrations, the lethality varies from 0 to 10%. The predicted LC50 values were significantly lower than those obtained experimentally, except for compound **3**.

**Tested Compound Predicted LC50 <sup>1</sup> (48 h) (**μ**g/mL) L % Max (48 h) <sup>2</sup> Determined LC50 (24 h) (**μ**g/mL) 95% CI <sup>3</sup> of LC50 (24 h) (**μ**g/mL) Determined LC50 (48 h) (**μ**g/mL) 95% CI of LC50 (48 h) (**μ**g/mL) 5** 0.51 60 ND <sup>4</sup> \* ND \* 43.5 ND \*\* **6** 0.33 70 ND \* ND \* 31.25 22.32 to 43.75 **7a** 0.11 100 42.93 38.14 to 48.31 ND \*\*\* ND \*\* **7b** 0.11 90 58.83 46.28 to 74.78 21.07 12.92 to 34.35 **8a** 0.1 60 ND \* ND \* 54.62 41.73 to 71.48 **8b** 0.04 30 ND \* ND \* ND \* ND \* *L*-Valine (Control 1) 1078.3 20 ND \* ND \* ND \* ND \* **3** (Control 2) 11.8 <sup>100</sup> 31.11 ND \*\* 1.144 0.13 to 9.93

**Table 9.** *Daphnia magna* toxicity test results.

<sup>1</sup> 50% lethal concentration; <sup>2</sup> maximum lethality induced at 48 h; <sup>3</sup> 95% confidence interval; <sup>4</sup> not determined because of the results obtained; \* lethality ranged between 0 and 40%; \*\* 95% CI could not be determined due to the results; \*\*\* lethality was higher than 50% at all concentrations.

**Figure 4.** *Daphnia magna* lethality curves for the tested compounds: (**a**) **5**; (**b**) **6**; (**c**) **7a**; (**d**) **7b**; (**e**) **8a**; (**f**) **8b**; (**g**) *L*-valine (control 1); (**h**) **3** (control 2).
