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Guaianolides and a seco-Eudesmane from the Resinous Exudates of Cushion Bush (Leucophyta brownii) and Evaluation of Their Cytostatic and Anti-inflammatory Activity Mette G. Hyldgaard,† Stig Purup,‡ Andrew D. Bond,§ Xavier C. Fretté,† Haiyan Qu,† Katrine T. Jensen,† and Lars P. Christensen*,† †

Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark ‡ Department of Animal Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark § Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom S Supporting Information *

ABSTRACT: A detailed phytochemical investigation of a dichloromethane extract of the resinous exudates of the cushion bush plant (Leucophyta brownii) resulted in the isolation of the new 8,12-guaianolides leucophytalins A (5) and B (6), the new 1,10-seco-eudesmane leucophytalin C (10), six rare 8,12-guaianolides (1−4, 7, and 8), and the xanthanolide tomentosin (9). The structures of all isolated compounds were elucidated on the basis of spectroscopic and spectrometric analyses. The structures of compounds isolated in crystalline form, including leucophytalins A and C, were further confirmed by X-ray crystallography. The crude extract exhibited moderate cytostatic activity against a breast cancer (MCF-7) and human colon cancer (HT-29) cell line with IC50 values of 9.3 and 18 μg/mL, respectively, and anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 μg/mL for thromboxane B2 and prostaglandin E2 production, respectively. The isolated compounds were evaluated for their cytostatic activity against MCF-7 and HT-29 cells (1, 3−10) and their anti-inflammatory activity against RAW 264.7 cells (1−10). All isolated compounds are most likely derived from (+)-germacrene A, and a biosynthetic pathway is proposed for these sesquiterpenoids.

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ity,22,23 and recently calocephalin was identified as a major contact allergen of cushion bush responsible for an increasing number of cases of contact dermatitis caused by this plant.2 An investigation of a CH2Cl2 extract of the resinous exudates of cushion bush revealed that the extract possesses moderate cytostatic activity against human breast cancer (MCF-7) and human colon cancer (HT-29) cells, with IC50 values of 9.3 and 18 μg/mL, respectively, as well as anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 μg/mL for thromboxane B2 (TrxB2) and prostaglandin E2 (PGE2) production, respectively. In our continuing search for cytostatic and anti-inflammatory compounds a phytochemical investigation of the resinous exudate of L. brownii was undertaken and resulted in the isolation of two new 8,12-guaianolides (5 and 6), one new 1,10-secoeudesmane (10), six known 8,12-guaianolides (1−4, 7, and 8), and one known xanthanolide (9). Herein, we report the isolation and structure elucidation of the new compounds 5, 6,

eucophyta brownii Cass. (syn. Calocephalus brownii (Cass.) F. Muell.), a shrub of the Asteraceae family belonging to the tribe Inuleae, is endemic to Australia, where it typically occurs in the southern coastal areas. L. brownii is commonly known as cushion bush and is a small, rounded shrub that is covered with leaf and stem wax, which gives the plant its characteristic silvery appearance. The plant grows in rounded bushes up to 1 m in height, consisting of silvery gray branchlets that, in summer, are topped by yellow globular flower heads. L. brownii is commonly cultivated as an ornamental plant, and it has not been used in traditional plant medicine. Previous chemical investigations of the leaf and stem wax of L. brownii have shown that the plant is a rich source for sesquiterpene lactones of the guaianolide type,1−3 such as pseudoivalin, pseudoivalin acetate, and calocephalin. Sesquiterpene lactones including those of the guaianolide type are known for their cytostatic4−12 and anti-inflammatory5,12−17 activities in vitro and in vivo. These activities may be attributed to the reactivity of their α,β-unsaturated γ-lactone moiety, which can undergo reactions with sulfhydryl groups of functional proteins via a Michael-type reaction.8,13,18−21 The reactivity of sesquiterpene lactones toward proteins also accounts for their allergenic© XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 7, 2015

A

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Figure 1. Structures of compounds 1−10 isolated from the dichloromethane extract of L. brownii.

H3-15); 2.06 (s, H3-2′)], two oxymethines [δH 3.82 (d, J = 5.6 Hz, H-2); 4.70 (ddd, J = 11.9, 8.8, 2.8 Hz, H-8)], and one exocyclic methylene [δH 6.30 (d, J = 3.2 Hz, H-13a); 5.65 (d, J = 2.8 Hz, H-13b)] (Table 1). The 13C NMR, HSQC, and DEPT spectra of 5 revealed the presence of three methyl carbons [δC 16.3 (C-14), 22.8 (C-2′), and 25.8 (C-15)], two oxymethine carbons [δC 76.6 (C-2) and 81.4 (C-8)], and one olefinic methylene carbon [δC 122.3 (C-13)]. The 13C NMR spectrum also indicated two sp3 oxygenated tertiary carbons [δC 83.9 (C-4) and 90.8 (C-1)], one sp2 quaternary carbon [δC 139.5 (C-11)], and two ester carbonyl carbons [δC 169.9 (C12) and 172.0 (C-1′)] (Table 2). Thus, the NMR data confirmed the presence of an α,β-unsaturated γ-lactone moiety and an ester group in 5. The remaining three indices of hydrogen deficiency were due to a tricyclic core indicating a guaianolide skeleton. The 1H−1H COSY spectrum revealed three spin systems, H-5/H2-6/H-7/H-8/H2-9/H-10/H3-14, H7/H-13a, H-13b, and H-2/H2-3. The key HMBC correlations of H-8 to C-11 and C-12, H-13a, H-13b to C-6, C-7, C-11, and C-12, and H-5 to C-1, C-7, and C-10 as shown in Figure 2, in conjunction with the aforementioned COSY spin systems, confirmed the guaianolide skeleton of 5. The similarities between the NMR data of 5 and calocephalin (4) (Tables 1 and 2) suggested that 5 was a 7,8-cis-configured guaianolide, esterified with an acetyl group at C-4. The main difference between the 13C NMR spectra of 5 and 4 was that the signals of the two epoxy group carbons [δC 70.9 (C-1) and 58.3 (C-2)] in 4 were deshielded to δC 90.8 and 76.6, suggesting that the epoxy group in 4 was replaced with a 1,2-dihydroxy group in 5. This assumption was confirmed by the key HMBC correlations of H-2 to C-1, C-4, and C-5 and H3-15 to C-1, C-3, C-4, C-5, and C-1′ (Figure 2). The chemical shifts of H-7 at δH 3.21 and H-8 at δH 4.70 and the coupling constants between H-7 and H-

and 10 as well as the assessment of the cytostatic and antiinflammatory activities of the isolated compounds.

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RESULTS AND DISCUSSION Fresh aerial parts of L. brownii were extracted with CH2Cl2, and the extract was subjected to flash column chromatography over silica gel and semipreparative HPLC to yield two new 8,12guaianolides (5 and 6), one new 1,10-seco-eudesmane (10), six known 8,12-guaianolides [4α-hydroxy-5αH,10αH-1,11(13)guaidien-8β,12-olide (1), 7,24,25 4α,10β-dihydroxy-5αH1,11(13)-guaidien-8β,12-olide (2),26 4α-hydroxy-1α,2α-epoxy5αH,10αH-11(13)-guaien-8β,12-olide (3),24 calocephalin (4),1,2 pseudoivalin (7),24,25,27,28 and pseudoivalin acetate (8)1], and the known xanthanolide tomentosin (9)24,25,29−33 (Figure 1). On the basis of the accepted biosynthesis principle that H-7 in natural guaianolides is always α-oriented34 it is possible to determine the relative configuration of natural guaianolides by NMR and/or X-ray crystallography. All known compounds were characterized based on their spectroscopic and spectrometric data (1−4 and 7−9) and corroborated by single-crystal X-ray diffraction analysis (2−4, 7, and 8). 13C NMR data of compounds 3, 7, and 8 and crystal data of compounds 3, 4, 7, and 8 are reported here for the first time. Leucophytalin A (5) was obtained as white, needle-like crystals and assigned the molecular formula C17H24O6 on the basis of 13C NMR and HRESIMS data (m/z 325.1649 [M + H]+, calcd for C17H25O6 325.1651), indicating six indices of hydrogen deficiency. The IR spectrum displayed absorption bands for hydroxy groups (3425, 1372, 1153, and 1104 cm−1), an α,β-unsaturated γ-lactone group (1757 cm−1), and an acetate group (1730 and 1261 cm−1). The 1H NMR spectrum exhibited characteristic signals for one methyl doublet [δH 1.03 (d, J = 7.2 Hz, H3-14)], two methyl singlets [δH 1.45 (s, B

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Table 1. 1H NMR Data of Compounds 4−6 (400 MHz, δ in ppm)a 4b

position 2 3

5b

3.49, br s α. 2.55, d (15.5)

9

β. 1.79, m 2.79, dd (13.0, 2.9) α. 1.86, ddd (14.2, 2.9, 2.6) β. 1.49, ddd (14.2, 13.0, 12.5) 3.25, ddddd (12.5, 7.4, 2.6, 2.0, 1.8) 4.70, ddd (12.2, 7.4, 5.1) α. 2.04, m

10 13a 13b 14 15 2′

β. 1.74, m 2.07, m 6.32, d (2.0) 5.77, d (1.8) 0.91, d (7.2) 1.47, s 1.98, s

5 6

7 8

6c

3.82, d (5.6) α. 2.39, dd (16.0, 5.6) β. 2.30, d (16.0) 2.52, brd (10.8) α. 1.96, m

5.20, d (4.8) α. 1.91, dd (16.0, 4.8) β. 2.35, d (16.0) 2.55, d (10.0) α. 2.37, m

β. 1.82, m

β. 1.71, m

3.21, ddddd (11.8, 8.8, 6.0, 3.2, 2.8) 4.70, ddd (11.9, 8.8, 2.8) α. 1.60, ddd (13.9, 2.8, 1.6) β. 1.90 m 2.18, m 6.30, d (3.2) 5.65, d (2.8) 1.03, d (7.2) 1.45, s 2.06, s

3.29, ddddd (11.8, 8.8, 5.8, 3.2, 2.8) 4.72, ddd (12.1, 8.8, 3.2)d α. 1.59, ddd (14.1, 3.2, 1.2) β. 1.80, m 2.38, m 6.18, d (3.2) 5.71, d (2.8) 1.03, d (7.2) 1.33, s 1.90, s

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations for compound 5.

which indicated no β-effect, and therefore the hydroxy group at C-1 must be β-oriented.25 Thus, the structure of 5 was established as 4α-acetoxy-1β,2α-dihydroxy-5αH,10αH-11(13)guaien-8β,12-olide, which was also confirmed by X-ray diffraction analysis of 5 (Figure 3). Compound 5 was assigned the trivial name leucophytalin A.

a

J values (Hz) are given in parentheses. Assignments based on 1H−1H COSY, NOESY, HSQC, and HMBC spectroscopic data. bRecorded in CDCl3. cRecorded in methanol-d4. dPartly overlapped with solvent. Coupling pattern and constants confirmed in CDCl3.

8 (J = 8.8 Hz) as well as the coupling constants between H-7 and H-13a, H-13b (J = 3.2 and 2.8 Hz) confirmed the cisconfiguration and the β-orientation of the γ-lactone ring.35−37 The NOESY correlations between H-5 (δH 2.52)/H-6α (δH 1.96), H-5/H-7, H-5/H-10 (δH 2.18), and H-10/H-8 indicated that they were cofacial and were arbitrarily assigned as αoriented. As a consequence, the NOESY correlations between H-2 (δH 3.82)/H-14 and H-2/H-15 revealed that they were cofacial and β-oriented (Figure 2). Finally, the chemical shift of H-5 in 5 was shielded compared to H-5 in 4 by 0.27 ppm,

Figure 3. Crystal structure of compound 5 (ORTEP diagram) with displacement ellipsoids at 50% probability level for non-H atoms.

Table 2. 13C NMR Data of Compounds 3−8 (100 MHz, δ in ppm)a,b position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′

3c 72.4, 59.7, 40.6, 78.2, 54.9, 30.6, 41.9, 79.1, 35.3, 30.0, 140.1, 169.5, 123.3, 17.2, 23.5,

4c C CH CH2 C CH CH2 CH CH CH2 CH C C CH2 CH3 CH3

70.9, 58.3, 40.8, 89.1, 50.9, 30.5, 41.7, 79.0, 34.8, 29.4, 140.5, 169.7, 123.6, 17.5, 22.2, 170.7, 22.7,

5c C CH CH2 C CH CH2 CH CH CH2 CH C C CH2 CH3 CH3 C CH3

90.8, 76.6, 45.0, 83.9, 49.4, 23.0, 41.4, 81.4, 33.3, 33.8, 139.5, 169.9, 122.3, 16.3, 25.8, 172.0, 22.8,

6d C CH CH2 C CH CH2 CH CH CH2 CH C C CH2 CH3 CH3 C CH3

105.7, 70.8, 46.7, 90.4, 55.2, 24.6, 42.4, 82.5, 34.4, 35.9, 141.3, 171.9, 123.3, 19.3, 23.3, 172.7, 22.2,

C CH CH2 C CH CH2 CH CH CH2 CH C C CH2 CH3 CH3 C CH3

7c 123.4, 39.1, 27.9, 79.3, 53.0, 25.1, 42.4, 79.7, 36.6, 137.0, 138.8, 170.3, 121.7, 21.7, 21.1,

8c C CH2 CH2 C CH CH2 CH CH CH2 C C C CH2 CH3 CH3

123.9, 35.3, 28.3, 87.3, 52.0, 25.3, 42.1, 79.5, 36.5, 134.9, 138.7, 170.1, 121.8, 22.0, 17.4, 170.4, 22.1,

C CH2 CH2 C CH CH2 CH CH CH2 C C C CH2 CH3 CH3 C CH3

a

Assignments are based on HSQC and HMBC NMR spectra. bMultiplicity obtained from the DEPT spectrum. cRecorded in CDCl3. dRecorded in methanol-d4. C

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Epoxides react readily with aqueous acid to produce transdiols, and it has previously been demonstrated that epoxides may be hydrolyzed to the corresponding diols during separation; thus, trans-diols could be artifacts.38 Nucleophilic attack by water of the epoxide in 4 will occur at the most substituted position (C-1), resulting in inversion of configuration in accordance with the α-orientation of the epoxide in 4 and the β-orientation of the hydroxy group at C-1 in 5. Compound 5 was, however, present in the fresh prepared extract, as shown by analytical HPLC and LC-MS, and is considered therefore not to be an artifact. Consequently, 4 must be the biosynthetic precursor of 5. Leucophytalin B (6) was obtained as a colorless oil and assigned the molecular formula C17H22O5 on the basis of 13C NMR and HRESIMS data (m/z 329.1363 [M + Na]+, calcd for C17H22O5Na, 329.1365), indicating seven indices of hydrogen deficiency, the same as compound 4. The IR spectrum of 6 was similar to that of 4, displaying absorption bands for an α,βunsaturated γ-lactone group (1756 cm−1) and an ester group (1716 and 1263 cm−1). The similarities between the 1H and 13 C NMR data of 6 and 4 (Tables 1 and 2) suggested that 6 was a 7,8-cis-configured guaianolide containing an acetoxy and an epoxy group in the cyclopentane moiety. This was confirmed by the 1H−1H COSY spectrum showing the same key spin systems as in compounds 4 and 5 (Figure 4).

oriented (Figure 4). On that basis the structure of 6 was established as 2α-acetoxy-1α,4α-epoxy-5αH,10αH-11(13)guaien-8β,12-olide and given the trivial name leucophytalin B. Leucophytalin B is the first example of a guaianolide with a C1−O−C-4 ether bridge. Leucophytalin C (10) was obtained as colorless needle-like crystals and assigned the molecular formula C15H24O3 on the basis of 13C NMR and HRESIMS data (m/z 253.1806 [M + H]+, calcd for C15H25O3, 253.1804 and m/z 275.1618 [M + Na]+, calcd for C15H24O3Na, 275.1623), indicating four indices of hydrogen deficiency. The IR spectrum displayed absorption bands of a primary hydroxy group (3376 and 1061 cm−1) and a carboxylic acid (1713 and 1402 cm−1) function. The 1H NMR spectrum of 10 exhibited characteristic signals for two methyl groups [δH 0.96 (d, J = 7.0 Hz, H3-15); 1.60 (s, H3-14)], one oxygenated methylene [δH 4.15 (s, H2-12)], and one exocyclic methylene [δH 5.06 (d, J = 1.0 Hz, H-13a); 4.91 (s, H-13b)] (Table 3). This was confirmed by the 13C NMR, DEPT, and Table 3. 1H NMR (400 MHz) and 13C NMR (100 MHz) Data of Compound 10 in CDCl3a,b position 1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 4. Key 1H−1H COSY, HMBC, and NOESY correlations for compound 6.

14 15

δC (ppm), type 179.7, 32.4, 29.0, 34.3, 131.3, 29.1,

C CH2 CH2 CH C CH2

37.7, CH 28.5, CH2 33.0, 126.7, 153.8, 65.2, 108.0,

CH2 C C CH2 CH2

18.6, CH3 18.9, CH3

δH (ppm) (J in Hz) 2.25, td (7.7, 2.8) 1.64, m 2.72, ddq (8.0, 7.0, 7.0) α. 2.07, m β. 1.69, m 2.12, m α. 1.79, dddd (11.9, 3.0, 2.8, 2.5) β. 1.40, dddd (11.9, 11.9, 11.9, 6.0) 2.06, m

4.15, 5.06, 4.91, 1.60, 0.96,

s d (1.0) s s d (7.0)

a Assignments are based on 1H−1H COSY, NOESY, HSQC, and HMBC NMR spectra. bMultiplicity obtained from the DEPT spectrum.

Furthermore, the cis-configured α,β-unsaturated γ-lactone moiety in 6 was confirmed by the key HMBC correlations of H-13a, H-13b to C-7, C-11, and C-12, by the chemical shifts of H-7 (δH 3.29) and H-8 (δH 4.72), and by the coupling constants between H-7 and H-8 (J = 8.8 Hz) and H-7 and H13a, H-13b (J = 3.2 and 2.8 Hz).35−37 The main difference between the 13C NMR data of 6 and 4 (Table 2) was that the signals of the oxymethine at C-2 [δC 58.3] and the sp3 oxygenated tertiary carbon at C-1 [δC 71.9] in 4 were deshielded to δC 70.8 and 105.7, respectively, in 6. These observations suggested that the acetoxy group was located at C2 and that the epoxy group was located between C-1 and C-4. The position of the acetoxy group at C-2 in 6 was substantiated by the HMBC correlations of H-2 to C-1, C-4, and C-5 and H315 to C-3, C-4, and C-5 and the chemical shift of H-2 (δH 3.49, br s) in 4 being deshielded to δH 5.20 (d, J = 4.8 Hz) in 6 (Table 1).39,40 The NOESY correlations between H-5 (δH 2.55)/H-6α (δH 2.37) and H-5/H-7 indicated that H-5 was αoriented, whereas the NOESY correlations between H-2/H-14 (δH 1.03), H-2/H-3β (δH 2.35), H-3β/H-6β (δH 1.71), and H3β/H3-15 (δH 1.90) indicated that H-2 and H3-15 were β-

HSQC spectra of 10, showing the presence of two methyl carbons [δC 18.6 (C-14), 18.9 (C-15)], one oxygenated methylene [δC 65.2 (C-12)], and one terminal olefinic carbon [δC 108.0 (C-13)]. The 13C NMR spectrum of 10 also indicated four sp2 carbons comprising three olefinic quaternary carbons [δC 126.7 (C-10), 131.3 (C-5), and 153.8 (C-11)] and one carboxylic acid carbon [δC 179.7 (C-1)] (Table 3). The remaining index of hydrogen deficiency was due to a monocyclic core indicating a 1,10-seco-eudesmane skeleton containing a hydroxycarbonyl and a primary hydroxy group, which was confirmed by the 1H−1H COSY and HMBC spectra. The 1H−1H COSY spectrum revealed three spin systems consisting of (i) H2-12/H2-13, (ii) H2-6/H-7/H2-8/H2-9, and (iii) H3-15/H-4/H2-3/H2-2, as well as the key HMBC correlations of H2-13 to C-7, C-11, and C-12, H2-8 to C-6, C-7, C-9, and C-10, H-4 to C-2, C-3, C-5, C-6, C-10, C-14, and C-15, and H2-2 to C-1 as shown in Figure 5. Hence, the 2D NMR data established the positions of the hydroxycarbonyl and D

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well as TrxB2 and PGE2 production induced by LPS in macrophages. The known 8,12-guaianolides 3, 4, 7, and 8 were found to be the most active in inhibiting the proliferation of MCF-7 and HT-29 cells, with IC50 values in the range 2.1−3.4 μg/mL (6.9−13.7 μM) and 1.5−4.9 μg/mL (4.8−18.6 μM), respectively (Table 4). The same 8,12-guaianolides also exhibited the strongest anti-inflammatory activity inhibiting TrxB2 and PGE2 production stimulated by LPS in RAW 264.7 cells, with IC50 values in the range 0.8−3.2 μg/mL (2.6−12.9 μM) and 0.2−1.1 μg/mL (0.7−3.5 μM), respectively (Table 5). The compounds with less cytostatic activity, i.e., 5, 6, and 10, were also among those compounds displaying less antiinflammatory activity (Tables 4 and 5). Compound 9 was previously shown to exert anti-inflammatory activities through the inhibition of inflammatory mediators (NO, iNOS, PGE2, COX-2, TNF-α, and IL-6) by regulating NF-κB activation and phosphorylation of p38/JNK kinases in RAW 264.7 cells at concentrations of 0.999) the quantity of compounds

Scientific, Denmark), equipped with a PDA detector and a Foxy Jr. fraction collector (Teledyne Isco Inc., Lincoln, NE, USA). TLC analysis was run on silica gel 60 F254 plates (layer thickness 0.25 mm, particle size 10−12 μm; Merck, Germany), and compounds were visualized by exposure to UV light at 254 nm and spraying with a solution of vanillin in 5% H2SO4. Flash column chromatography (CC) was performed using silica gel 60 (0.04−0.063 mm, 230−400 mesh ASTM; Merck). Plant Material. Plants of L. brownii were obtained from Florashop, Staurbyskov Garden Centre ApS., Staurbyskovvej, 5500 Middelfart, Denmark, in September 2011 and were authenticated by Dr. Martin Jensen, Department of Food Science − Plant, Food & Sustainability, Aarhus University, Aarslev, Denmark. A voucher specimen (No. 2012C01) is deposited at the Department of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark. Extraction and Isolation. Whole fresh aerial parts of 25 plants (1.4 kg) were extracted with CH2Cl2 (16 L) at room temperature for 2 h, with manual shaking every 15 min. The extract (9.0 g) was subjected to silica gel flash CC (330 g, 230−400 mesh, 10 × 23 cm) and eluted with a step gradient of n-hexane−EtOAc (500 mL, 9:1; 200 mL, 4:1; 200 mL, 7:3; 400 mL, 3:2; 600 mL, 1:1; 400 mL, 2:3; 200 mL, 3:7; 200 mL, 1:4; 200 mL, 1:9; 500 mL, 0:1) and finally with 600 mL of MeOH to give 10 fractions (Fr1−Fr10) based on TLC and analytical HPLC analysis (CH3CN−H2O, 10:90 (0 min); 35:65 (20− 30 min); 50:50 (40−60 min); 10:90 (62−72 min), flow rate 1 mL/ min, detection wavelengths λ 210, 230, and 254 nm). Fr5 (1.54 g) was F

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Journal of Natural Products

Article

[M + H − HOAc − 2 H2O]+ (100); HRESIMS m/z 325.1649 (calcd for C17H25O6, 325.1651). Leucophytalin B [2α-acetoxy-1α,4α-epoxy-5αH,10αH-11(13)guaien-8β,12-olide] (6): colorless oil; [α]25D +25 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 205 (3.75) nm; IR (KBr) νmax 3435, 2951, 1756, 1716, 1657, 1372, 1263 cm−1; 1H NMR (400 MHz, CDCl3) and 13 C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; APCIMS m/z 307 [M + H]+ (4), 264 [M + H − Ac]+ (8), 247 [M + H − HOAc]+ (100), 229 [M + H − HOAc − H2O]+ (63); HRESIMS m/z 307.1548 (calcd for C17H23O5, 307.1546) and m/z 329.1363 (calcd for C17H22O5Na, 329.1365). Leucophytalin C [12-hydroxy-7αH-1,10-seco-eudesma5(10),11(13)-dien-1-oic acid] (10): colorless, needle-like crystals; mp 109−110 °C; [α]25D +93 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 205 (3.68) nm; IR (KBr) νmax 3376, 2964, 2912, 1713, 1654, 1462, 1402, 1203, 1061, 1046, 893 cm−1; 1H NMR (400 MHz, CDCl3) and 13 C NMR (100 MHz, CDCl3) data, see Table 3; APCIMS m/z 253 [M + H]+ (46), 235 [M + H − H2O]+ (100), 217 [M + H − 2 H2O]+ (26); HRESIMS m/z 253.1806 (calcd for C15H25O3, 253.1804) and m/z 275.1618 (calcd for C15H24O3Na, 275.1623). General X-ray Experimental Data. Crystal structures of compounds 2−5, 7, 8, and 10 were determined using data collected at 180 K on a Nonius KappaCCD diffractometer using Mo Kα radiation (λ = 0.7107 Å). The crystal structure of 2 has been reported previously.26 H atoms on C atoms were placed in idealized positions. H atoms on O atoms were located in Fourier maps, and their positions were refined, with O−H distances restrained to 0.85(1) Å for 3, 5, and 7. In the absence of significant anomalous scattering effects, absolute structures were not determined. Crystal data for 3: C15H20O4, Mr = 264.31, monoclinic space group P21, a = 6.2688(2) Å, b = 32.9821(11) Å, c = 6.4770(3) Å, β = 90.904(1)°, V = 1339.01(9) Å3, Z = 4, ρcalcd = 1.311 g cm−3, μ = 0.094 mm−1, 6966 data collected with θ < 26.4°, R = 0.0431 [I > 2σ(I)], Rw = 0.1088 (all data) for 2636 unique data and 355 refined parameters. CCDC 1049970. Crystal data for 4: C17H22O5, Mr = 306.35, orthorhombic space group P212121, a = 6.4175(1) Å, b = 13.4673(3) Å, c = 18.1921(5) Å, V = 1572.28(6) Å3, Z = 4, ρcalcd = 1.294 g cm−3, μ = 0.095 mm−1, 12 686 data collected with θ < 27.1°, R = 0.0398 [I > 2σ(I)], Rw = 0.0850 (all data) for 2002 unique data and 201 refined parameters. CCDC 1049971. Crystal data for 5: C17H24O6, Mr = 324.36, monoclinic space group P21, a = 6.1603(2) Å, b = 15.3222(9) Å, c = 8.6223(6) Å, β = 90.131(3)°, V = 813.85(9) Å3, Z = 2, ρcalcd = 1.324 g cm−3, μ = 0.100 mm−1, 5953 data collected with θ < 27.5°, R = 0.0393 [I > 2σ(I)], Rw = 0.0924 (all data) for 1865 unique data and 219 refined parameters. CCDC 1049972. Crystal data for 7: C15H20O3, Mr = 248.31, triclinic space group P1, a = 6.8116(2) Å, b = 12.1630(3) Å, c = 13.5671(4) Å, α =112.835(2)°, β = 103.767(2)°, γ = 91.324(2)°, V = 997.77(5) Å3, Z = 3, ρcalcd = 1.240 g cm−3, μ = 0.085 mm−1, 12 983 data collected with θ < 25.0°, R = 0.0365 [I > 2σ(I)], Rw = 0.0805 (all data) for 3469 unique data and 506 refined parameters. CCDC 1049973. Crystal data for 8: C17H22O4, Mr = 290.35, orthorhombic space group P212121, a = 9.3366(3) Å, b = 10.0176(3) Å, c = 16.1750(5) Å, V = 1512.85(8) Å3, Z = 4, ρcalcd = 1.275 g cm−3, μ = 0.090 mm−1, 7783 data collected with θ < 25.0°, R = 0.0303 [I > 2σ(I)], Rw = 0.0722 (all data) for 1533 unique data and 193 refined parameters. CCDC 1049974. Crystal data for 10: C15H24O3, Mr = 252.34, orthorhombic space group P212121, a = 5.9977(2) Å, b = 12.2726(4) Å, c = 19.9741(7) Å, V = 1470.24(9) Å3, Z = 4, ρcalcd = 1.140 g cm−3, μ = 0.078 mm−1, 7308 data collected with θ < 25.0°, R = 0.0347 [I > 2σ(I)], Rw = 0.0868 (all data) for 1500 unique data and 174 refined parameters. CCDC 1049975. Supplementary data of compounds 3−5, 7, 8, and 10 in CIF format can be obtained free of charge from the Cambridge Crystallographic Data Centre via https://summary.ccdc.cam.ac.uk/structure-summaryform.

Table 4. Cytostatic Activity of Compounds Isolated from L. brownii on Cancer Cells of Breast (MCF-7) and Colon (HT29) Origin IC50 values in μg/mL (μM)a compound

MCF-7

HT-29

1 3 4 5 6 7 8 9 10 extract

6.0 (24.2) 3.2 (12.1) 2.1 (6.9) 8.8 (27.2) >20 (>65) 3.4 (13.7) 2.5 (8.0) 6.6 (26.6) >20 (>79) 9.3

7.0 (28.2) 4.9 (18.6) 4.3 (14.1) 9.5 (29.3) >20 (>65) 4.5 (18.1) 1.5 (4.8) 14 (56.5) >20 (>79) 18

a IC50 values in μg/mL and in parentheses in μM were determined by treatment of cells with different concentrations of the compounds. Compound 2 was not available, at the time of testing for cytostatic activity, and was not tested for this activity. 17β-Estradiol in two concentrations was used as positive and negative controls in MCF-7 cells. The low concentration of 17β-estradiol (0.5 × 10−10 M) increased proliferation by a factor 2.65 ± 0.5 (mean of 4 assays), while the high concentration (0.5 × 10−4 M) decreased proliferation by 0.51 ± 0.05 (mean of 4 assays). Fetal calf serum used as a positive control in HT-29 cells increased cell proliferation a factor 3.79 ± 0.4 (mean of 4 assays).

Table 5. Anti-inflammatory Activity of Compounds Isolated from L. brownii on Thromboxane B2 (TrxB2) and Prostaglandin E2 (PGE2) Production Stimulated by LPS (50 ng/mL) in RAW 264.7 Cells IC50 values in μg/mL (μM)a compound

TrxB2

PGE2

1 2 3 4 5 6 7 8 9 10 extract

4.4 (17.7) 9.9 (37.5) 3.1 (11.7) 2.0 (6.5) 11.2 (34.6) 11.8 (38.6) 3.2 (12.9) 0.8 (2.6) 3.9 (15.7) >20 (>79) 3.9

1.6 (6.5) 5.6 (21.2) 0.8 (3.0) 0.2 (0.7) 3.4 (10.5) 5.0 (16.3) 0.8 (3.2) 1.1 (3.5) 1.8 (7.3) >20 (>79) 6.1

IC50 values in μg/mL and in parentheses in μM were determined by treatment of cells with different concentrations of the compounds. Polymyxin B (1 μg/mL) used as a positive control decreased the TrxB2 and PGE2 production to 21% and 8% of the LPS stimulation, respectively (mean of 4 assays). Hydrocortisone (10 μM) as a positive control decreased the TrxB2 and PGE2 production to 42% and 21% of the LPS stimulation, respectively (mean of 3 assays). a

1−10 in mg/g extract was determined as follows: 1, 22.1 mg/g; 2, 3.7 mg/g; 3, 14.0 mg/g; 4, 49.4 mg/g; 5, 7.2 mg/g; 6,