**1. Introduction**

*Callicarpa* (family Lamiaceae) is a genus of about 190 species of herbaceous plants. The plant is geographically found throughout east and southeast Asia, Australia, Madagascar, southeast North America, and South America [1]. Folkloric usage of various parts of *Callicarpa* includes preparations used as fish poisons [2,3], insect repellents [1], and for some medical indications [3]. The phytochemical investigation of this genus has resulted in the identification of diterpenoids, phenylethanoids, phenypropanoids, and flavonoids. These components display various biological effects, such as anti-inflammatory [4–6], anti-platelet aggregation [7], hemostatic [8], antioxidative [9,10], cytotoxic [6,11,12], and neuroprotective [13], antitubercular [14], hepatoprotective [15,16], antimicrobial [17], anti-arthritic [18], as well as analgesic properties [19]. From the above-mentioned phytochemical and biological studies, we know this genus may offer a rich supply of bioactive phytochemicals. Because the phytochemical profile of the Taiwanese endemic plant *Callicarpa hypoleucophylla* has never been analyzed, we carried out an investigation of the constituents and bioactivity of *C. hypoleucophylla*. A meticulous separation of an ethanolic extract of *C. hypoleucophylla* led to the isolation of two new clerodane-type diterpenoids that we named callihypolins A and B (**1** and **2**), together with seven known analogues (**3**–**9**). The anti-inflammatory evaluation of these isolates is also presented in this paper.

#### **2. Results and Discussion**

The leaves and twigs of *C. hypoleucophylla* were extracted with 95% ethanol; the yielded extracts were suspended in H2O and extracted with ethyl acetate (EtOAc). The EtOAc-soluble part was further partitioned with hexanes/methanol (MeOH)/H2O (4:3:1) to obtain a MeOH layer. The MeOH layer was subjected to extensive chromatography by normal- and reversed-phase HPLC, using a normal-phase silica gel open column and a Sephadex LH-20 resin column, supplying callihypolins A and B (**1** and **2**) as well as seven known compounds (4a*R*,5*S*,6*R*,8a*R*)-5-[2-(2,5-dihydro-5-methoxy-2-oxofuran-3-yl)ethyl]-3,4,4a,5,6,7,8,8a-octahydro-5,6,8atrimethylnaphthalene-1-carboxylic acid (**3**) [20], patagonic acid (**4**) [21], limbatolide F (**5**) [22], limbatolide A (**6**) [23], methyl (4a*R*,5*S*,6*R*,8*S*,8a*R*)-3,4,4a,5,6,7,8,8a-octahydro-8-hydroxy-5,6,8a-trimethyl-5-[2-(2-oxo-2,5-dihydrofuran-3-yl)ethyl]naphthalene-1-carboxylate (**7**) [24], clerodermic acid (**8**) [25], and visclerodol acid (**9**) [26] (Figure 1).

**Figure 1.** Structures of compounds **1**–**9** isolated from *Callicarpa hypoleucophylla*.

The molecular formula of compound **1** was established to be C21H28O6 on the basis of the [M + Na]<sup>+</sup> peak at *m*/*z* 399.17785 (calcd. 399.17781 for C21H28O6Na) obtained from high-resolution electrospray ionization mass spectrometry (HRESIMS) (Figure S13). The IR absorption bands of compound **1** indicated the presence of hydroxy (3451 cm<sup>−</sup>1), α,β-unsaturated-γ-lactone (1739 cm<sup>−</sup>1), and carboxyl (1678 cm−1) functionalities. The 13C and distortionless enhancement by polarization transfer (DEPT)-135 NMR data (Figure S2) showed the presence of 21 carbons divided into 7 quaternary carbons (including 3 carbonyls), 5 methines, 5 methylenes, and 4 methyls. The 1H (Figure S1) and 13C NMR signals of compound 1 showed some characteristic peaks such as an olefinic methine singlet (δ<sup>H</sup> 5.96, δ<sup>C</sup> 126.6, C-3), two tertiary methyls (δ<sup>H</sup> 1.33, δ<sup>C</sup> 14.0, Me-19; δ<sup>H</sup> 0.84, δ<sup>C</sup> 17.3, Me-20), a secondary methyl (δ<sup>H</sup> 0.90, *J* = 6.8 Hz, δ<sup>C</sup> 15.3, Me-17), as well as a butenolide unit (δ<sup>C</sup> 134.0, C-13; δ<sup>H</sup> 7.09, 1 H, quin, *J* = 1.7 Hz, δ<sup>C</sup> 143.9, C-14; δ<sup>H</sup> 4.77, 1H, dd, *J* = 3.8, 1.7 Hz, δ<sup>C</sup> 70.2, C-15; δ<sup>C</sup> 174.0, C-16). The above NMR data indicated that the structure of compound **1** was similar to that of dichrocephnoid E [27], a clerodane diterpenoid, except for a methylene corresponding to C-6 that was replaced by an oxymethine (δ<sup>H</sup> 3.84, δ<sup>C</sup> 72.5) and an additional methoxy (δ<sup>H</sup> 3.81, δ<sup>C</sup> 52.8) present in compound **1**. The whole structure of compound 1 was then determined, starting from characteristic signals, by means of correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC) NMR correlations (Figures S3–S5). The COSY spectrum (Figure 2) showed cross-peaks with signals at H-1 (δ<sup>H</sup> 2.43, 2.56)/H-10 (δ<sup>H</sup> 2.00); H-6 (δ<sup>H</sup> 3.84)/H-7 (δ<sup>H</sup> 1.61, 1.70)/H-8 (δ<sup>H</sup> 1.76)/Me-17 (δ<sup>H</sup> 0.90); H-14 (δ<sup>H</sup> 7.09)/H-15 (δ<sup>H</sup> 4.77). Moreover, the key HMBC correlations (Figure 2) of H-1 with C-2, H-3 with C-1, C-4, C-5, and C-18; Me-19 with C-4, C-5, C-6, and C-10, Me-17 with C-7, C-8, and C-9, Me-20 with C-9, C-10, and C-11, and methoxy proton with C-18 led to the construction of the decalin core of compound **1**, including a hydroxy group at C-6 and a methyl ester substituted at C-4. The linkage between C-12 and butanolide via C-13 was established by comparing the corresponding NMR data with those of similar analogues and confirmed by mass spectrometry analysis [22,24,27]. The planar structure of compound **1** is represented in Figure 2. The relative stereochemistry of compound **1** was deduced from nuclear overhauser effect spectroscopy (NOESY) correlations (Figure 2 and Figure S6) and by comparison of its spectroscopic data with those of clerodane analogues. The NOESY experiment showed correlations of H-6 (δ<sup>H</sup> 3.84)/H-10 (δ<sup>H</sup> 2.00)/H-8 (δ<sup>H</sup> 1.76), which indicated protons located on the β face of the molecule. On the other hand, Me-20 presented NOESY correlations with Me-19 and Me-17, but neither Me-19 nor Me-20 correlated with H-10, suggesting that compound **1** is an *ent*-clerodane-type molecule with *trans*-decalin core [28]. The *trans* A/B ring junction was also evidenced by the carbon chemical shifts of C-19 (δ<sup>C</sup> 14.0) and C-20 (δ<sup>C</sup> 17.3) [29–31]. Thus, these correlations indicated that the hydroxy group at C-6 had an α-configuration, as confirmed by the coupling constants of H-6 with H-7α (*J* = 12.6 Hz) and H-7β (*J* = 4.4 Hz) [32,33]. All the spectral data appeared thus to be in agreement with the structure and stereochemistry of compound **1**.

**Figure 2.** COSY (bold bond), selected HMBC (arrow), and NOESY (left-right arrow) correlations of compound **1**.

Callihypolin B (**2**) was isolated as a yellow oil. It possesses the molecular formula C22H32O5, corresponding to seven indices of hydrogen deficiency, as determined by the HRESIMS ion at *m*/*z* 399.21419 [M + Na]<sup>+</sup> (calcd. 399.21420) (Figure S14) and 13C NMR data. The IR spectrum revealed the presence of ester (1768 cm<sup>−</sup>1) and conjugated carbonyl (1682 cm−1) groups. The 1H NMR data of compound **2** (Table 1, Figure S7) demonstrated the presence of one ethoxy [δ<sup>H</sup> 3.94 (m) and 3.74 (m); 1.27 (t, *J* = 7.1 Hz)], one secondary methyl [δ<sup>H</sup> 0.81 (d, *J* = 6.2 Hz)], two tertiary methyls (δ<sup>H</sup> 0.76 and 1.23), and two olefinic methines [δ<sup>H</sup> 6.85 (m), and 6.76 (d, *J* = 1.2)], together with one hemiacetal methine [δ<sup>H</sup> 5.79 (brd, *J* = 1.2)]. The 13C NMR and DEPT spectra (Table 1, Figure S8) of compound **2** showed the presence of 22 carbon signals ascribable to 4 methyls, 7 methylenes (of which one was oxygenated), 2 olefinic methines, 3 aliphatic methines, 2 aliphatic quaternary carbons, 2 olefinic quaternary carbons, and 2 carbonyl carbons. Two carbonyls and two C=C double bonds accounted for four indices of hydrogen deficiency, so the remaining three indices suggested that compound **2** was a tricyclic compound. In the 1H-1H COSY spectrum (Figure S9), the correlations of H2-1/H2-2/H2-3, H2-6/H2-7/H-8/Me-17, H2-11/H2-12, H-14/H-15, and H2-1- /Me-2 were used to establish the presence of five fragments, as shown in Figure 3. In the HMBC spectrum (Figure 3, Figure S11), the cross-peaks of H-3 with C-4 and C-18; of Me-19 with C-4, C-5, C-6, and C-10; and of H-10 with C-1 and C-5 revealed the presence of a cyclohexene ring (ring A), in which a carboxyl group and Me-19 were attached to C-4 and C-5, respectively. The presence of a cyclohexane ring (ring B) with Me-20 attached at C-9 was elucidated by the HMBC correlations of Me-20 to C-8, C-9, and C-10, as well as of H-10 to C-9. Additionally, both H3-20 and H-10 showed correlations with C-11 and indicated the linkage between ring B and C-11 via C-9. The HMBC cross-peaks of H-14 to C-13 (δ<sup>C</sup> 139.0) and C-16 (δ<sup>C</sup> 171.5); H-15 (δ<sup>H</sup> 5.79) to C-16 and C-1- (δ<sup>C</sup> 66.0), as well as H2-12 to C-13 and C-16, revealed the presence of an α,β-unsaturated γ-lactone ring with an ethoxy group located at C-15. Thus, the planar structure of compound **2** could be established. The stereochemistry of compound **2** was determined by its NOESY spectrum, relative NMR data, and circular dichroism spectrum. The NOESY experiments (Figure 3 and Figure S12) carried out on compound **2** showed correlations of Me-19/Me-20/Me-17, and H-6β (δ<sup>H</sup> 2.44)/H-10/H-8, whereas no correlation was revealed between H-10 and Me-19. These data, as well as the carbon chemical shift of Me-19 at δ<sup>C</sup> 20.5 [29], indicated that compound **1** is characterized by a type TC clerodane skeleton under a chair conformation of ring B [34], a *trans* relationship between rings A and B, α-orientations of Me-17, Me-19, and Me-20, and β-orientation of H-10. The ethoxy group attached at C-15 in the butenolide moiety was assigned to the α-face by comparison with the circular dichroism (CD) data of known butenolides and by applying the octant rule. The CD spectrum showed a negative Cotton effect near 243 nm (π-π\*) and supported the *S* configuration of C-15 [31,35,36]. Thus, the structure and stereochemistry of compound **2** were clearly determined.

**Figure 3.** COSY (bold bond), selected HMBC (arrow), and NOESY (left-right arrow) correlations of compound **2**.


**Table 1.** 1H and 13C NMR Data of compounds **1** and **2** in CDCl3.

*<sup>a</sup>* 1H and 13C-NMR were measured at 600 and 150 MHz. *<sup>b</sup>* 1H and 13C-NMR were measured at 400 and 100 MHz.

Compounds **1**–**9** were evaluated for their inhibitory activities on superoxide anion generation and elastase release in formyl-methionyl-leucyl-phenylalanine (fMLF)/cytochalasin (CB)-induced human neutrophils. The formyl peptide fMLF in combination with the priming agent CB serves as a stimulator that mimics the over-activation of neutrophils by a pathogen or an immune system reaction [37]. As shown in Table 2, compounds **2**–**4** exerted anti-inflammatory activity by suppressing superoxide anion generation and elastase release. The positive control genistein, which acts via inhibition of protein tyrosine kinases, showed a profound effect on the respiratory burst (89% inhibition of superoxide generation) and only a mild effect on degranulation (22.8% inhibition of elastase release). Among the tested samples, the new compound **2** showed the best activity, suppressing 32.2% of superoxide generation and 17.6% of elastase release. To exclude possible toxicity to the cells, the lactate dehydrogenase (LDH) release assay was employed, and none of the tested clerodane diterpenoids resulted toxic to human neutrophils (Figure 4). Clerodane diterpenes with an open lactone ring at C16 were previously reported to exert inhibitory effects on the function of neutrophils activated by fMLF/CB, including respiratory burst [38] and degranulation [39]. Thus, our results well correlate with the anti-inflammatory effects of previously isolated clerodane diterpenes and indicate the potential of the new compounds for the development of anti-inflammatory drugs targeting neutrophils.



Percentage of inhibition (Inh %) at 10 μM concentration. Results are presented as mean ± S.E.M. (n = 4–5); \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared with the control (solvent). <sup>a</sup> Genistein served as a positive control.

**Figure 4.** Compounds **1**–**9** do not cause LDH release in human neutrophils. Human neutrophils were incubated with DMSO (as a control) or compounds **1**–**9** (10 μM) for 15 min. Cytotoxicity was evaluated by LDH release. All data are presented as the means ± S.E.M. (*n* = 3).

#### **3. Experimental**

#### *3.1. General*

Silica gel 60 (Merck) was used for open-column chromatography (CC). Luna C18 (5 m, 250 × 10 mm, Phenomenex), Luna CN (5 m, 250 10 mm, Phenomenex), and Luna phenyl-hexyl (5 m, 250 × 10 mm, Phenomenex) semi-preparative columns were used for high-performance liquid chromatography (HPLC). HPLC used a Shimadzu LC-10AT pump with an SPD-20A UV-Vis detector. The UV spectra were obtained by using a Jasco UV-530 ultraviolet spectrophotometer (Jasco, Tokyo, Japan), whereas the IR spectra were obtained on a Jasco FT-IR-4600 spectrophotometer (Jasco, Tokyo, Japan). Optical rotations were measured with a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). NMR spectra were obtained using JEOL JNM ECS 400 MHz (JEOL, Tokyo, Japan) and Varian 600 MHz NMR spectrometers (Varian, Palo Alto, CA, USA). ESI–MS data were collected on a VG Biotech Quattro 5022 mass spectrometer (VG Biotech, Altrincham, UK). High-resolution ESI–MS data were obtained with a Bruker APEX II spectrometer (Bruker, Bremen, Germany). Circular dichroism spectra were recorded on a JASCO J-810 spectrophotometer (Jasco, Tokyo, Japan).
