*2.2. Biology*

#### 2.2.1. *C. elegans* Maintenance

In this paper, we used wild-type N2 nematodes which were acquired from the Caenorhabditis Genetic Center (CGC; University of Minnesota, Minneapolis, MN, USA). All nematodes were nurtured at 20 ◦C on an NGM (Nematode Growth Medium) agar plate with *Escherichia coli* OP50 as previously reported [18]

#### 2.2.2. Lifespan Assay

Lifespan assays were carried out under normal culture conditions. Age-synchronized nematodes were collected by embryo isolation and L1 arrest. The as-obtained L1 stage nematodes were grown on an NGM plate with or without of (**1**), (**2**), and (**3**). The survival rate of the test nematodes was determined using a dissecting microscope (SMZ1500, Nikon, Tokyo, Japan). Nematodes that failed to respond to prodding with the tip of a platinum wire were considered dead. Living nematodes were transferred to a fresh NGM plate every 2 days.

#### 2.2.3. Statistical Analysis

The results obtained from the lifespan assay were plotted using Kaplan–Meier analysis, and the statistical significance between each group was analyzed using the log-rank test. The mean lifespan data were presented as the mean ± standard deviation.

#### **3. Results and Discussion**

The retrosynthetic analysis for oleracones is outlined in Scheme 1. Homoisoflavone skeleton was anticipated to be e fficiently prepared via a deoxybenzoin route, one of the most popular synthetic methods used for the preparation of isoflavonoids [19]. Therefore, homoisoflavone **2** and its *O*-demethylated analog **1** were planned to be obtained from dihydrochalcone **3**. Oleracone E (**3**) was anticipated to be prepared via an aldol condensation between commercially available 4,6-dimethoxy-2-hydroxybenzophenone **4** and 2-(benzyloxy)benzaldehyde **5**.

**Scheme 1.** Retrosynthetic analysis of oleracones D (**1**), F (**2**), and E (**3**).

Our synthesis was commenced with an aldol condensation between commercially available 4,6-dimethoxy-2-hydroxybenzophenone **4** and 2-(benzyloxy)benzaldehyde **5** to give chalcone **6** (Scheme 2). Concurrent hydrogenation and hydrogenolysis of the *O*-benzyl group in chalcone **6** using 5% activated Pd on carbon under H2 atmosphere led to olereacone E (**3**) in 88% yield.

We envisaged the construction of the homoisoflavone skeleton of **1** and **2** via a selective intramolecular oxa-Michael addition reaction of an enone moiety by the phenol group at the 2"-position rather than the 2'-position of intermediate **6**, resulting from condensation reaction between **3** and a formyl reagent. According to our expectation, oleracone F (**2**) was obtained in high yield (90%) upon stirring with *<sup>N</sup>*,*<sup>N</sup>*-dimethylformamide dimethyl acetal [20] in toluene under reflux condition, and no regioisomeric by-products were observed. The high regioselectivity observed in the reaction was attributed to the enhanced nucleophilicity of the phenol group at the 2"-position compared to that at the 2'-position in **3**, which can be supported by the electron-withdrawing e ffect of the carbonyl group. Finally, selective *O*-demethylation of **2** using BCl3 afforded oleracone D (**1**) in high yield (80%). The spectral data obtained for oleracones D–F (**1**–**3**) were consistent with those previously reported data [16,17].

**Scheme 2.** Synthesis of oleracones D (**1**), F (**2**), and E (**3**).

With the oleracones D–F (**1**–**3**) in hand, we examined their effects on the longevity of nematodes. To test whether oleracones exhibit lifespan-extension activity, we performed a lifespan assay using wild-type N2 nematodes at 20 ◦C, as described previously [21]. As shown in Figure 2A,B, all of the oleracones tested could prolong the lifespan of the nematodes under standard culture conditions, although the effectiveness of each compound was somewhat variable. At the maximum concentration studied (20 μM), treatment with **3** and **2** extended the lifespan of nematodes by 13.8 and 11.8%, respectively, compared with the control, while the efficacy of **1** (4.3% extension) was inferior to that of **2** and **3**. Interestingly, the longevity effect of **1**–**3** on nematodes did not show any correlation with their radical scavenging activities (IC50 values: 11.73, 13.17, and 17.78 μM, respectively) [16,17]. These results sugges<sup>t</sup> the possibility that modulating aging-related factors may also cooperate with the direct radical scavenging activity that produces the anti-aging effect. However, detailed information on the underlying mechanism of the lifespan-extension properties of oleracone **1**–**3** remains to be defined.

**Figure 2.** The longevity effect of oleracone D–F (**1**–**3**)**.** (**A**) The lifespan curves obtained for compound-treated (20 μM) and untreated wild-type nematodes under normal culture conditions. (**B**) The change in lifespan observed for the compound-treated (20 μM) nematodes when compared to the control. (**C**) The mean lifespan of compound-treated (5, 10, and 20 μM) and untreated wild-type nematodes was calculated from the survival curves. The statistical difference between the curves was analyzed using a log-rank test. \* *p* < 0.05 compared with the vehicle alone. All experiments were repeated in triplicate.
