Bioactivity-Guided Isolation of Totarane-Derived Diterpenes from Podocarpus neriifolius and Structure Revision of 3-Deoxy-2α-hydroxynagilactone E

Abstract Bioactivity-guided phytochemical investigation of Podocarpus neriifolius D. Don. (Podocarpaceae) has led to the isolation of one new (2) and three known (1, 3, and 4) B-type podolactones, along with three totarane-type diterpenes (5-7). Their structures were determined by interpretation of High Resolution ElectroSpray Ionization Mass Spectrometry (HRESIMS) and 1D and 2D NMR data, and comparison with the values reported in the literature. The structure of compound 1, previously identified as 3-deoxy-2α-hydroxynagilactone E (8), was revised as its 2β-epimer, which has been reported recently as a new compound. All of the isolates were evaluated for their antiproliferative activity against a panel of four human cancer cell lines, namely, ovarian (OVCAR3), breast (MDA-MB-231), colon (HT-29), and melanoma (MDA-MB-435), and compounds 1 and 3 were found to be cytotoxic with IC50 values in the low micromolar range for most of the cell lines used. The major compound, inumakilactone A (3), was further tested in vivo using the HT-29, MDA-MB-435, and OVCAR3 cells in a murine hollow fiber model, for the first time. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-0198-x) contains supplementary material, which is available to authorized users.


Introduction
Podocarpus neriifolius D. Don (Podocarpaceae) is a tree growing in south Asian countries, such as Nepal and Vietnam, in Eastern China, and in the Pacific Islands [1]. While the wood of this plant is used as timber for furniture and in paper-making, its edible fruits are consumed 1 3 raw or cooked, and decoctions from its leaves are used in folk medicine to relieve rheumatism and painful joints [2]. Plants of the genus Podocarpus have been reported to exhibit a variety of biological activities ranging from plant-growth regulation [3,4] to antibacterial [5] and antiproliferative [6][7][8][9] effects. These activities have been attributed mainly to their chemotaxonomic markers, the nor-and bisnorditerpene dilactones, referred to as the podolactones or nagilactones [10,11]. The podolactones exist in three main classes (types A-C) depending on the conjugated system between the B and C rings. As such, type A possesses a [8(14), 9(11)-dienolide] moiety, while types B and C are characterized by the presence of [7α,8αepoxy-9(11)-enolide] and [7(8), 9(11)-dienolide] groups, respectively ( Fig. 1) [7,10,12]. P. neriifolius produces several podolactones including the cytotoxic nagilactone C [13], the sulfur containing derivatives, podolactones C and D [14][15][16], and more recently a new cyclopeptide, neriitide A and the lignan, neriilignan, were reported from the leaves of this plant [17] (Fig. 1). In a continuing effort to discover potential lead anticancer agents from natural sources as part of a multidisciplinary program project grant [18], a root sample of P. neriifolius was investigated, resulting in the purification of one new (2) and three known (1, 3 and 4) B-type podolactones, as well as three known totarane-type diterpenes (5)(6)(7). The isolation and structure determination of the obtained isolates, along with their antiproliferative properties against a panel of four human cancer cell lines (ovarian, breast, colon, and melanoma) are reported herein. Moreover, for the first time, in vivo evaluation of the major isolate, inumakilactone A (3), in a murine hollow fiber assay was conducted and described in the present study.

Results and Discussion
The bioactivity-guided fractionation of the cytotoxic ethyl acetate-soluble extract (IC 50 = 4.3 µg/mL against the HT-29 human colon cancer cells) from the root sample of Podocarpus neriifolius led to the isolation of seven compounds including a new B-type podolactone glucoside (2) and six known diterpenoids (1, 3-7), which were identified by spectroscopic data interpretation and comparison with published values (Fig. 2).
Compound 1 was obtained as a white amorphous powder, and its positive HRESIMS displayed a sodiated molecular ion peak at m/z 371.1469, equivalent to C 19 H 24 NaO 6 + (calcd. for C 19 H 24 NaO 6 + , 371.1465). Preliminary interpretation of its 1D and 2D NMR spectroscopic data led to the assignment of its corresponding planar structure as a B-type podolactone [10,19]. A subsequent literature search for this chemical structure revealed that the collected 1 H and 13 C NMR data for 1 matched with those of 3-deoxy-2α-hydroxynagilactone E (8), previously isolated from Podocarpus nagi [20]. However, careful inspection of the 1 H NMR data measured in methanol-d 4 revealed that the splitting pattern and coupling constants (J) values of H-2 (dddd, J = 13.5, 8.5, 7.4, 4.9 Hz) shared a close similarity with those of the revised C-type podolactone 2β-hydroxynagilactone F (H-2, dddd, J = 12.9, 9.1, 7.2, 5.1 Hz) [21], and suggested a comparable 2β configuration of the hydroxy group substitution at C-2. A very recent study on P. nagi reporting the X-ray structure of 3-deoxy-2β-hydroxynagilactone E confirmed this assertion [11]. While this compound was reported as a new molecule in this latter publication, its NMR data matched both the initial report [20], compound 8, and the present data (Tables 1  and 2). Thus the structure of compound 8 as reported therein [20] should be revised as 3-deoxy-2β-hydroxynagilactone E (1).
Moreover, 20 carbon signals corresponding to the 2-O-substituted type-B podolactone were observed in the 13 C NMR spectrum (Table 2). Accordingly, compound 2 could be assigned as a glucosylated B-type podolactone with a glucopyranosyl moiety attached either to the A ring or at C-14, since C-6, C-7, and C-11 were protonated. However, the presence of signals at δ H 1.14 (d, J = 6.7 Hz), 1.00 (d, J = 6.7 Hz), and 1.93 (m) corresponding to an isopropyl side chain at C-14, indicated that the glucose unit must be linked to the A ring of the podolactone core ( Fig. 1, Tables 1 and  2). Comparison of the 13 C NMR spectroscopic data of 1 and 2 showed a β-d-glycosylation shift of +8 ppm at C-2 (63.5 ppm in 1 vs. 71.4 ppm in 2), and further shift values of − 2.0 and − 3.9 ppm for C-1 (40.4 ppm in 1 to 38.4 ppm in 2) and C-3 (38.3 ppm in 1 to 34. 3 ppm in 2), respectively [23]. Key HMBC correlations, including a cross-peak between H-2 and C-1′ confirmed the above conclusions on the attachment of the β-d-glucopyranosyl unit at C-2 (Fig. 3). Thus, the structure of compound 2 was assigned as a C-2 glycosylated derivative of 1, named nagilactone G-2β-O-β-d-glucoside. In addition to compounds 1 and 2, two known B-type podolactones, inumakilactone A (3) [8] and makilactone E (4) [19], along with three known totarane-type diterpenes, inumakiols D (5) and E (6), and 4β-carboxy-19-nor-totarol (7) [8] were isolated and identified during the present study.
All these isolates were evaluated for their antiproliferative activity against four human cancer cell lines, namely, HT-29 (colon), MDA-MB-231 (breast), OVCAR3 (ovarian), and MDA-MB-435 (melanoma). Compounds 1 and 3 exhibited moderate potency across all four cell lines (Table 3), whereas the remaining compounds were inactive (IC 50 > 10 μM). The antiproliferative activity of the podolactones isolated was consistent with the reported SAR studies where the aglycones but not their glucoside derivatives proved to be active [7,11]. Inumakilactone A (3) which was isolated in a larger quantity than 1, was further assessed for its in vivo antitumor efficacy in a hollow fiber assay using the three human cancer cell lines, HT-29, MDA-MB-435, and OVCAR3 [24,25]. Test mice within the treatment groups were initially administered 1 and 2 mg/kg doses of 3, but following signs of toxicity, the dosages were reduced to half in each group. Nevertheless, compound 3 did not display any significant effect on cell survival for all three cell lines tested when compared to the vehicle control. While inumakilactone A (3) has been previously reported as an antiproliferative compound in vitro [7], this is the first report of its in vivo evaluation in the murine hollow fiber model using the abovementioned human cancer cell lines.

General Experimental Procedures
The optical rotation was measured on a modular circular polarimeter (MCP) 150 (software version 1.50; Anton Paar OptoTec GmbH, Seelze-Letter, Germany). A Hitachi U-2910 spectrophotometer (Hitachi High-Technologies Corporation, Tokyo, Japan) was utilized to obtain UV/vis data. Highresolution mass spectra were collected with a LTQ Orbit-rap™ (Thermo Fisher Scientific Inc., Bremen, Germany) equipped with ITMS and FTMS analyzers, covering a mass range of m/z 50-4000, and with resolution ranging from 7500-100,000, operated in the positive-ion mode using

Extraction, Isolation, and Structure Determination
The air-dried powdered root sample (100 g) of P. neriifolius was extracted by exhaustive percolation in methanol. Evaporation of this percolate in vacuo resulted in a crude MeOH extract (6.1 g), which was re-suspended in a hydromethanolic solution and further partitioned with hexanes and subsequently with EtOAc. The three obtained extracts, namely, hexanes (D1, 296 mg), aqueous (D2, 3.0 g), and EtOAc (D3, 2.6 g) partitions were evaluated for their cytotoxicity in vitro, and the active (IC 50 < 20 µg/mL) EtOAc partition in having exhibited an IC 50 value of 4.3 µg/mL was further

Antiproliferative Evaluation Using Cancer Cell Lines
Preliminary cytotoxicity screening of plant extracts against the human colon cancer cell line HT-29, and subsequent in vitro evaluation of the isolated compounds against four human cancer cell lines, including HT-29, MDA-MB-231 (breast), MDA-MB-435 (melanoma), and OVCAR3 (ovarian), were performed following previously reported protocols [25,26].

In Vivo Hollow Fiber Assay
Immunodeficient NCr nu/nu mice (7-weeks-old) were procured from Taconic Biosciences (Rensselaer, NY, USA) and housed in microisolation cages at room temperature and with a relative humidity of 50-60% under 12:12 h light-dark cycle. All animal procedures were performed following approval by the University of Illinois at Chicago (UIC) Animal Care and Use Committee (protocol number 16-035), and the mice were treated according to the institutional guidelines for animal care. The antitumor activity of inumakilactone A (3) against OVCAR3, HT-29, and MDA-MB-435, was evaluated in vivo using an established hollow fiber assay procedure described previously [24,25,27]. Briefly, cells were first cultured in hollow fibers 2 days (OVCAR3 cells, 4 × 10 6 cells/mL) and 1 day (HT-29, 1 × 10 6 cells/mL and MDA-MB-435, 2.5 × 10 6 cells/mL) prior to insertion. Inumakilactone A (3) was dissolved in DMSO and further diluted with 60% PEG 300 and 30% water. The immunodeficient NCr nu/nu mice were divided into four groups, including a paclitaxel positive control group (n = 2), a negative vehicle group (n = 6), and inumakilactone A (3) treatment groups receiving 1 mg/kg (n = 6) or 2 mg/kg (n = 3). On day 0, hollow fibers containing the human cancer cells were implanted in the abdominal cavity of the mice. The animals were then injected i.p. once daily for four days (day 3 through day 6) with vehicle, paclitaxel, or 3. Each mouse was weighed daily during the study. Doses were reduced to half after one animal from each treatment group died and the rest exhibited signs of toxicity after the second injection. The remaining mice were sacrificed on day 7. The fibers were removed, and viable cell mass was measured by a modified MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Statistical analysis was performed using ANOVA.