Anatolicin, a Highly Potent and Selective Cytotoxic Sesquiterpene Coumarin from the Root Extract of Heptaptera anatolica

Seven known sesquiterpene coumarins and a new sesquiterpene coumarin, anatolicin (8), were isolated from the dichloromethane extract of the roots of Heptaptera anatolica. Structures of these compounds were elucidated based on their spectral properties. While some of these sesquiterpene coumarins showed modest cytotoxic activity against COLO205, KM12, A498, UO31, and TC32 cancer cell lines, selective cytotoxicity of anatolicin (8) and 14′-acetoxybadrakemin (7) were observed at nanomolar level against the UO31 kidney cancer cell line.


Introduction
Cancer is the second leading cause of death worldwide according to WHO statistics [1]. Cancer is a highly complex disease that evolves through the acquisition of multiple biological capabilities by normal cells to become cancer cells [2]. In order to develop new cancer medicines, it is crucial to recognize the various pathways that provide immortality to cancer cells [3]. Natural products represent a major and highly diverse source for the development of novel cancer treatments [4]. As part of our continuing studies on potential anticancer phytochemicals from Apiaceae family, we report here the cytotoxic compounds of Heptaptera anatolica (Boiss.) Tutin.
Oleo-gum-resins obtained from the Ferula (Apiaceae) species have been used for the treatment of various tumors since ancient times; Dioscorides refers to the use of such drugs in the De Materia Medica [5], and Avicenna describes the application of Khiltit (the oleo-gum-resin of Ferula foetida) in the treatment of tumors, by direct application to the tumor after cutting it open, as described in the Al-Qanun fi al-Tibb (the Canon of Medicine) [6]. The sesquiterpene ethers found in the oleo-gum-resins of Ferula species are mainly responsible for their cytotoxic activities, in addition, they show many biological activities such as antiviral, antibacterial, antileishmanial, anti-inflammatory and P-glycoprotein inhibitory activities [7].
Heptaptera Marg. & Reuter is a small genus of the Apiaceae family represented by six species [8]. The majority of Heptaptera species are distributed throughout the countries of the Middle East (i.e., Turkey, Iran, Iraq, and Syria), except for Heptaptera angustifolia (Bertol.) Tutin and H. colladonioides Marg. & Reuter which are endemic to Italy and Greece, respectively. Characteristic secondary metabolites of Heptaptera (syn. Colladonia) sp. are sesquiterpene ethers of umbelliferone [9][10][11], in which the sesquiterpene moiety is either an acyclic sesquiterpene as in the case of umbelliprenin (1), or is a bicyclic drimane derivative, e.g., badrakemin (4). Similar sesquiterpene coumarins are also known from Ferula species, however, several drimane sesquiterpene ethers isolated from Heptaptera are biogenetically oxygenated at the C-14 position that is a specific chemotaxonomic character of this genus and not been found in the sesquiterpene ethers isolated from Ferula species. In order to evaluate the cytotoxic activities of sesquiterpene coumarin ethers where C-14 is oxygenated and related, compounds, we decided to investigate the dichloromethane root extract of H. anatolica.

Discussion
The molecular formula of anatolicin was determined as C 24 H 28 O 5 by the 13 C-NMR data (  (4) (Table 2) with the exception of a missing methyl signal of the drimane sesquiterpene moiety, the presence of a tertiary aldehyde group signal at δ 9.76 ppm strongly suggesting that it was biogenetically oxidized to an aldehyde group in 8. The strong anisotropic deshielding effect of the aldehyde carbonyl group on the chemical shifts of H-3 and H-5 protons (i.e., 0.70 and 0.34 ppm, respectively) in anatolicin (8) in comparison with those observed in badrakemin (4) suggests that one of the methyl groups located on the C-4 position (i.e., C-13 or C-14 ) of the drimane skeleton must be biogenetically oxidized to an aldehyde group. The 2D HMBC spectra data (Table 1 and Figure 2) also confirm this observation.  Clear interactions observed between the aldehyde proton and C-13′ and H-6α proton (Figure 3) in the 2D NOESY spectrum of anatolicin indicate that the C-14′ axillary methyl group on the C-4′ of the drimane skeleton was biogenetically oxidized to an aldehyde carbonyl, thus, anatolicin is an oxidized C-14′ derivative of badrakemin (4). The C-14′ oxidation pattern observed in 6, 7, and 8 is in agreement with the biogenetic oxidation pattern of the sesquiterpene coumarin ethers isolated from Heptaptera species. Although C-14′ hydroxymethylene derivatives of drimane sesquiterpene coumarin ethers have been reported previously [9][10][11], anatolicin (8) is the first drimane sesquiterpene coumarin ether with an aldehydic functional group.  Clear interactions observed between the aldehyde proton and C-13 and H-6α proton (Figure 3) in the 2D NOESY spectrum of anatolicin indicate that the C-14 axillary methyl group on the C-4 of the drimane skeleton was biogenetically oxidized to an aldehyde carbonyl, thus, anatolicin is an oxidized C-14 derivative of badrakemin (4). The C-14 oxidation pattern observed in 6, 7, and 8 is in agreement with the biogenetic oxidation pattern of the sesquiterpene coumarin ethers isolated from Heptaptera species. Although C-14 hydroxymethylene derivatives of drimane sesquiterpene coumarin ethers have been reported previously [9][10][11], anatolicin (8) is the first drimane sesquiterpene coumarin ether with an aldehydic functional group. Clear interactions observed between the aldehyde proton and C-13′ and H-6α proton (Figure 3) in the 2D NOESY spectrum of anatolicin indicate that the C-14′ axillary methyl group on the C-4′ of the drimane skeleton was biogenetically oxidized to an aldehyde carbonyl, thus, anatolicin is an oxidized C-14′ derivative of badrakemin (4). The C-14′ oxidation pattern observed in 6, 7, and 8 is in agreement with the biogenetic oxidation pattern of the sesquiterpene coumarin ethers isolated from Heptaptera species. Although C-14′ hydroxymethylene derivatives of drimane sesquiterpene coumarin ethers have been reported previously [9][10][11], anatolicin (8) is the first drimane sesquiterpene coumarin ether with an aldehydic functional group.  The sesquiterpene coumarin ethers isolated from Heptaptera anatolica were tested against COLO205, KM12, A498, UO31, and TC32 cancer cell lines, and the cytotoxic activities observed with these compounds are shown in Table 3. While most of the sesquiterpene coumarin ethers were active against the UO31 kidney cancer cell line, 14 -acetoxybadrakemin (7) and anatolicin (8) were the most active compounds with 17 and 24 nM IC 50 values, respectively. Colladonin (5) was also active against the UO31 cell line with a 0.75 µM IC 50 value and, furthermore, showed similar activity against the KM12 colon cancer cell line, was moderately active against the COLO205 and A498 cell lines, and weakly active against the TC32 cell line. In addition, badrakemin (4) also showed strong activity against the UO31 cell line and moderate activity against the KM12 and A498 cell lines.

General Experimental Procedures
Melting points were determined on a Reichert microscope equipped with a Kofler heating stage and were uncorrected (Wien, Austria). Optical rotations were measured using a Perkin-Elmer Model 241 polarimeter in a 100 × 2 mm cell (units 10 −1 deg cm 2 g −1 ) in dichloromethane. IR spectra (neat) were recorded on a Perkin-Elmer FT-IR Spectrometer, SPECTRUM 2000 (Waltham, MA, USA). NMR spectra of anatolicin (8) were acquired on a Bruker Avance III spectrometer (Billerica, MA, USA) operating at 600 MHz for 1 H and 150 MHz for 13 C and equipped with a 3 mm cryogenically cooled probe. 1 H-NMR spectrum of compound 1 was acquired on a Bruker Avance III spectrometer (Rheinstetten, Germany) operating at 500 MHz and equipped with a 5 mm indirect observation probe and 1 H-NMR spectra of compounds 2-7 were acquired on a Varian (Agilent) Mercury Spectrometer (Palo Alto, CA, USA) operating at 400 MHz and equipped with a 5 mm probe. 1 H and 13 C spectra were referenced to the residual deuterated solvent peaks. HRESIMS data were acquired on an Agilent 6530 Accurate Mass Q-TOF instrument (Santa Clara, CA, USA) and on a Triple TOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA). Initial purification of the dichloromethane extract was carried out on a Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) column. Further purification of column fractions was performed using silica gel F 254 PLC plates (1 mm thickness) (Merck KGaA, Darmstadt, Germany).

Plant Material
The roots of Heptaptera anatolica were collected from the Izmir province in Western Anatolia in June 2013 and identified by Prof. A. Duran. A voucher specimen (A. Duran 9703) was deposited in the Herbarium of Selcuk University, Faculty of Sciences, Department of Biology.

Cytotoxic Activity Assay
The primary assay used for this study was a two-day, two-cell-line XTT bioassay [16], an in vitro antitumor colorimetric assay developed by the MTP Assay Development and Screening Section. Renal cancer cell lines used were UO31 and A498. Cells were harvested and plated (45 µL) at a seeding density of 1.5 × 10 4 cells per well for both the UO31 and A498 cell line into a 384-well "assay plate" and then incubated for 24 h. DMSO solutions of the test materials (8 µL) were diluted 1:25 with medium (192 µL) and then subjected to five 2:1 serial dilutions (100 µL each) on a 96-well plate. Duplicate 40 µL aliquots of each sample concentration were then transferred to a 384-well "dilution plate", which could accommodate the duplicate samples from two 96-well plates. A 5 µL aliquot of each solution on the dilution plate was transferred to the cell cultures on the 384-well assay plate to give a final volume of 50 µL and a DMSO concentration of 0.4%. Cells were incubated for 48 h at 37 • C in the presence of the test samples and then treated with the tetrazolium salt XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide). Viable cells reduced the XTT to a colored formazan product, and after an additional 4 h incubation period, the amount of formazan produced was quantified by absorption at 450 nm using the absorption at 650 nm as a reference. Sanguinarine was used on each plate as a positive control. Similar methodology and seeding densities were used for COLO205, KM12 (colon), A673, and TC32 (Ewing sarcoma) cell lines.

Conclusions
Investigation of the dichloromethane extract of the roots of Heptaptera anatolica yielded seven known and one new sesquiterpene coumarin. Although cytotoxic activity of sequiterpene coumarins were described in the literature earlier [7], high potency and selectivity of C-3 -β-hydroxy and C-14 -acetoxy or C-14 -keto derivatives (i.e., 14 -acetoxybadrakemin (7) and anatolicin (8)) toward UO31 kidney cancer cell line had not been reported previously.