Synthesis and Antitumor Activities of Phenanthrene-Based Alkaloids

A series of phenanthrene-based tylophorine derivatives (PBTs) were synthesized and their cytotoxic activities against the H460 human large-cell lung carcinoma cell line were evaluated. Among these compounds, N-(3-hydroxy-2,6,7-tri-methoxyphenanthr-9-ylmethyl)-l-prolinol (5a), and N-(3-hydroxy-2,6,7-trimethoxy-phenanthr-9-ylmethyl)-l-valinol (9) exhibited good activities, with IC50 values of 11.6 and 6.1 μM, respectively.


Introduction
Phenanthroindolizidine alkaloids have been of considerable interest as anticancer agents because of their exceptionally potent antitumor activity [1]. Based on recent studies, the molecular target of phenanthroindolizidine analogues may be novel and different from the targets of known anticancer drugs [2]. Over the past decades, the synthesis and biological activities of varied 2,3,6,7-functionalized phenanthroindolizidine alkaloids have been reported. The substituents mainly include functionalities such as hydroxyl, methoxyl, and hydrogen [3][4][5]. The structure activity relationships of phenanthroindolizidine alkaloids were summarized by Gao et al. in 2007 [3]. The degree of cytotoxicity was dependent on the type and pattern of substitution on the phenanthrene ring. In 2006, Wei et al. [6] found that phenanthrene-based tylophorine derivatives (PBTs), which were phenanthroindolizidine derivatives resulting from opening of the indolizidine ring, exhibited significant cytotoxic activity. Further work from the same group resulted in the synthesis of a series of PBTs with different substituents at C-9, which indicated that a five-or six-carbon distance between the nitrogen and a terminal polar substituent in the C-9 chain is quite favorable for cytotoxic activity [7]. They also reported that antitumor activities of PBTs were induced by inhibition of the activation of Akt and NF-κB signaling pathway in tumor cells [8,9].
As part of our studies on antitumor agents derived from natural products, we have designed and synthesized a series of PBT analogues to investigate the effects of oxygenic functional groups on the phenanthrene ring and C-9 side chain on cytotoxic activity. The present paper reports the synthesis of these PBTs (Scheme 1) and evaluation of their cytotoxic activities against the H460 human large-cell lung carcinoma cell line in vitro (Table 1). Adriamycin used as positive control, IC 50 = 1.72 μM.

Chemical synthesis
The synthetic routes to the PBTs are depicted in Scheme 1. Perkin condensation [10] involving 4-hydroxy-3-methoxy-or 3,4-dimethoxybenzaldehyde and 3,4-dimethoxyphenylacetic acid in the presence of Ac 2 O and Et 3 N afforded the cinnamic acid derivatives 2a (R = Ac) or 2b (R = Me) [11][12][13][14]. By treatment with oxalyl chloride [15], the acids 2a or 2b gave an acid chloride intermediate which could be converted to the corresponding aromatic amides 3a (R = Ac) or 3b (R = Me) [10] by using L-proline methyl ester hydrochloride. Through oxidation-coupling with ferric trichloride [11] as the catalyst, the amides were efficiently cyclized to deliver 9-amido-substituted phenanthrenes 4a (R = Ac) and 4b (R = Me) [10,16], respectively. Finally, the two amides were reduced with LiAlH 4 [17] to give 5a (R = OH) and 5b (R = Me) [18,19] [21] , 5a was converted to 10 and 11, or 12. Compound 7 was synthesized from 2a and L-valine methyl ester hydrochloride following the same synthetic route as described for 4a. In our synthetic route, the key step is the reduction of the aromatic amides with aluminum and boron hydrides. In the reduction process we tried LiAlH 4 /THF [17], NaBH 4 -MeSO 3 H/DMSO [22] and NaBH 4 -I 2 /THF [23] systems to reduce compound 7. First, reduction with the NaBH 4 -MeSO 3 H/DMSO system did not afford the target product, but rather a series of small amounts of unidentified products. Then, by using LiAlH 4 as the catalyst, the 3-acetoxyl on the phenanthrene ring and the ester group in the amido side chain of 7 were reduced to hydroxyl and hydroxymethyl groups, respectively, as shown for compound 8, but the carbonyl linkage was not reduced. Finally, under the catalysis of NaBH 4 -I 2 /THF system, 8 was successfully reduced to 9 in a satisfactory yield of 78%. However, compounds 4a and 4b were completely reduced using the LiAlH 4 /THF system in one step to give 5a and 5b in consistently high yields of 90% and 93%, respectively.

Biological activity
The synthesized PBTs were screened for in vitro cytotoxic activity against the H460 human largecell lung carcinoma cell line with an MTT assay procedure [24]. Adriamycin was used as the reference compound. Table 1 summarizes the structures of 5a, 5b, 8-12 and their cytotoxicities (IC 50 ) against the H460 cell line. All of them exhibited diminished cytotoxic activities to some extent, in comparison to their parent natural products (e.g., tylophorine, IC 50 0.5~1.7 μM) [25] .
The results indicate that cytotoxicities of PBTs were influenced by the presence of oxygenic functional substituents on the phenanthrene, and amino acid side chains at the C-9 position of phenanthrene and the linkage between the nitrogen and the phenanthrene. Replacing the 3-methoxyl on the phenanthrene skeleton with a hydroxyl increased the cytotoxic activity, as shown in the comparison of 5b (3-methoxyl, IC 50 53.8 μM) and 5a (3-hydroxyl, IC 50 11.6 μM). Thus, a hydroxyl at the phenanthrene C-3 is quite favorable for cytotoxic activity. In the prolinol side chain, when the terminal hydroxyl was converted to an acetate ester, sulfate ester and chloride, the cytotoxic activities were significantly diminished, it may relate to the removal of the terminal hydrogen bond effect on the assumed biological target [7]. Conserving the C-3 hydroxyl substitution on the phenanthrene skeleton of 5a and opening the pentacyclic prolinol resulted in a more active compound 9 (IC 50 6.1μM). This result indicates that changing the cyclic side chain to an acyclic one may increase the flexibility of the C-9 side chain, which favors attaining an optimal conformation for binding to an assumed biological target. The amide derivative 8 exhibited very low activity (IC 50 68.1μM), in agreement with the report of Wei et al. [6].

General
Melting points of the synthesized compounds were determined on a Digital Melting Point Apparatus XT4A and are uncorrected. IR spectra were recorded with a Perkin-Elmer model 298 spectrometer (KBr). 1D NMR spectra were recorded on Bruker ARX-300 or Bruker AV-600 spectrometers. ESIMS were recorded with a Finnigan LCQ mass spectrometer and EIMS were measured by an Agilent/HP HP5973 and 6890 GC/MSD. Column chromatography was carried out on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). The MTT assay was recorded on a microplate reader (KHB ST-360, SH Kehua Laboratory System Co., Ltd., Shanghai, China). Thin-layer chromatography (TLC) was performed on TLC silica gel 60 F254 plates (0.5 mm, Merck). Chemicals from Sinopharm Chemical Reagent Co. were used without further purification.

Cytotoxic assay
Cytotoxicities were determined by MTT method [24] using human large-cell lung carcinoma cell line (H460) grown in RPM1-1640 medium plus 10% heat-inactived fetal bovine serum. The assays were performed in 96-well microtiter plates. Compounds 5a, 5b, 8-12 were dissolved in DMSO and diluted to six different concentrations (10, 3.3, 1.0, 0.33, 0.10, and 0.033 mM) respectively, and each solution was ten-fold diluted to six different concentrations using culture medium (1.0, 0.33, 0.10, 0.033, 0.010, and 0.0033 mM), then 10 µL of each solution was added to 90 µL (about 5,000 cells) culture medium wells. After incubation at 37 °C for 72 hours, 10 µL of MTT (5 mg/mL) was added to each well and incubated for four hours, and then liquid in the wells was removed. DMSO (150 µL) was added to each well. The absorbance was recorded on a microplate reader at wavelength of 590 nm, and the IC 50 was defined as 50% reduction of absorbance in the control assay. Compound 5a, 5b, 8-12 showed cytotoxicity against human tumor cell line H460 with IC 50 value of 11.6, 53.8, 68.1, 6.1, 46.8, 53.4 and 62.9 μM, respectively. Adriamycin, with an IC 50 value of 1.72 μM, was used as positive control.

Conclusions
In conclusion, we have synthesized a series of PBT analogues by a facile route and evaluated their in vitro cytotoxic activities against human large-cell lung carcinoma cell line (H460). On this basis, the SAR analysis and potential antitumor activities of these alkaloids are under investigation. Compounds 5a and 9 with a substitution of hydroxyl at position 3 and a hydroxyl at C-9 side chain terminus were the most effective cytotoxic compounds. These results may be helpful for the design of future PBT antitumor reagents, and offer potential application in the discovery of antitumor drugs.