Synthesis, Characterization and Cytotoxicity of New Rotundic Acid Derivatives

Rotundic acid (RA, 1), a natural compound, exhibits potent tumor cell growth inhibiting properties. To date there are no reports on derivatives of RA. Furthermore, the 28-COOH position of RA might make it unstable and induced serious gastrointestinal side effects when it was applied in vivo. Therefore, in order to explore and make use of this compound, eight new amino acid derivatives of RA at the 28-COOH position were synthesized and evaluated for their cytotoxicities in vitro on three tumor cell lines including A375, HepG2 and NCI-H446. As a result, a few of these new amino acid derivatives showed stronger cytotoxicity. Compound 5a was found to have the best inhibition activity on the three tested human tumor cell lines with IC50 values of less than 10 μM compared with RA treatment. Meanwhile, the cytotoxicity of compound 6b was significantly higher than that of RA on the A375 cell line and almost the same as RA on the HepG2 and NCI-H446 cell lines. Hence, compounds 5a and 6b may serve as potential lead compounds for the development of new anti-tumor drugs.


Introduction
Rotundic acid (RA, 1, Figure 1) belongs to the pentacyclic triterpenoid family and is mainly found in Ilex rotunda, Ilex purpurea, Ilex integra and other Aquifoliaceae plants which are widely distributed in China [1][2][3][4]. RA was also isolated from Mussaenda Pubescens and Guettarda platypoda of the Rubiaceae family [5,6]. Olea europaea and Planchonella duclitan, which are part of the Oleaceae and Sapotaceae families, respectively, also contain RA [7,8]. Although there are sufficient sources for extraction of RA in China, as mentioned above, there are still few reports on its bioactivity because of little interest from pharmacological researchers. In our open patent, a considerable amount of RA was isolated and purified from I. rotunda [9]. Moreover, Xu et al. demonstrated that RA, as one of many isolated compounds, showed anti-cancer activity [10]. Li et al. also reported that RA showed cytotoxicity, with IC 50 values of 21.8 μM and 9.5 μM when it was applied on the HT29 and MCF-7 cell lines, respectively [8]. However, they did not continue to pay much attention to this compound. Since RA might be a potential native anticancer drug with sufficient sources, our research group has investigated and applied for a series of patents regarding RA and its derivatives during the past few years to explore and make use of this compound [11][12][13][14][15]. It has been widely reported that compounds with free carboxylic acids might be unstable during metabolic processes and further induce serious gastrointestinal side effect in humans. Although RA presented potential anti-tumor activity, RA with its free carboxylic acid might have the same problems when administrated in vivo [16]. Currently, structure modification is considered to be an effective method to produce lead compounds to enhance the activity and avoid possible side effects. Moreover, the structure of RA is comparatively simple, with a few active positions available for modification. These chemical modifications could be controlled easily, which would make it possible to explore new compounds with better anti-tumor activities. In this work, we carried out structure modification at the 28-COOH position of RA to improve the bioactivity of RA according to the theory of medicinal chemistry and with the experience of structural modification of pentacyclic triterpenoids [17][18][19][20].
Amino acids, as the basis of all metabolic cycles, are the essential compounds responsible for all life. There is a sizeable amount of literature that shows that tumor cells require larger quantities of amino acids than normal cells in the body [21,22]. Hence, in theory the selectivity of a drug for tumor cells may improve when amino acids are introduced into the drug's molecular structure. Many researchers have given much attention to the investigation of the bioactivities of amino acid drugs. Recently, many anti-tumor drugs have exhibited increased selectivity of tumor cells after undergoing amino acid modification. Their anti-tumor activities have been markedly improved and the toxicity on normal cells was lowered [23][24][25].
To the best of our knowledge, there are few reports on the bioactivity of RA and no reports on its derivatives. Therefore, the objective of our present study was to investigate the synthesis, characterization, and cytotoxicity of some new RA derivatives produced via introduction of amino acid groups. Their structures were elucidated on the basis of spectroscopic assays such as IR, MS, 1 H-NMR and 13 C-NMR. The MTT assay was employed to screen their cytotoxicity on the A375, HepG2 and NCI-H446 human cell lines.

Preparation of RA
The procedure reported by Xu et al., for isolating RA from I. rotunda [10] was followed. Briefly, the barks of I. rotunda were shade-dried, ground, and extracted with refluxing 80% EtOH. The EtOH extract was evaporated ubder vacuum to obtain the total saponins fraction. The air-dried and powdered total saponins were hydrolyzed by 4% NaOH in 30% EtOH and purified by recrystallization to prepare RA. The purity of RA used was ≥98% (HPLC assay). The extraction yield of RA in our study was much higher, up to 100 mg/g, which made it suitable for industry production.
It has been broadly reported that amino acid modification could enhance the anticancer activities of original compounds. Zhuo et al. demonstrated that amino acid derivatives of 5-fluorouracil had higher anti-tumor activity with lower toxicity; some of them reached 90% inhibition rate in Ehrlich carcinoma or sarcoma in mice [30][31][32][33][34]. Sun et al. designed and synthesized a series of amino acid conjugates of 3-oxooleanolic acid, and determined their anti-tumor activities in vitro. Preliminary anti-tumor bioassayd showed that conjugates with higher water solubility retained anti-tumor activity [35]. In the present study, we modified the 28-COOH position of RA whereby eight new compounds were obtained. Since the amino acid modification might enhance the antitumor activities of original compound as reported, the pharmacological activity of RA and its eight derivatives were tested in the following study.

Biological Activity
In the present study, three types of human cancer cell lines including A375 (human malignant melanoma cells), HepG2 (human hepatoma cells) and NCI-H446 (human small cell lung cancer) were used to observe the cytotoxicity of RA (as a positive control) and its derivatives 4a-7a, 4b-7b. Antiproliferative effects were determined with the MTT assay [36]. Each experiment was repeated at least three times. The results are shown in Table 1 and Figure 2. Notes: Data are represented in mean ± SD; n = 3. a IC 50 values more than 100 µM are indicated as >100,* p < 0.05 vs. RA.  Table 1, RA showed significant IC 50 values of 16.58, 7.33, 11.40 μM on A375, HepG2, and NCI-H446, respectively, which is consistent with the previous results by Li et al. [8] and Xu et al. [10], who investigated the cytotoxicity of RA on HeLa, MDA-MB-435, CNE1, HT29b, MCF-7c etc. Based on the cytotoxicity of RA, we tested the cytotoxicity of the compounds 4a-7a, 4b-7b. The result demonstrated that compounds 4a and 5a presented more potent anti-tumor activity on the A375, HepG2 and NCI-H446 cell lines compared to compounds 4b and 5b. Interestingly, when comparing compounds 6a and 7a to compounds 6b and 7b, the results were just the opposite. The compounds 6b and 7b presented more potent anti-tumor activity on the A375, HepG2 and NCI-H446 cell lines compared to compounds 6a and 7a ( Table 1). The results might be explained by a steric hindrance arising from the conjugation of an amino group at C-28.When the group is an amino acid methyl ester, the activity of a small steric group is more potent than that of a large one, but when the group is an amino acid, a large steric hindrance is more effective than a small one. In addition, we can see from Table 1 that the cytotoxicity of 6b was similar to that of the RA treatment on the HepG2 and NCI-H446 cell lines, and significantly higher than the RA treatment on the A375 cell line (8.03μM vs. 16.58μM). Also, the IC 50 of 6b on HepG2 was also less than 10 μM (6.11 μM), but it was not significantlt different when compared to the IC 50 of RA. These results demonstrate that compound 6b might be a potential anticancer drug and this will require further investigation. Furthermore, as shown in Figure 2, the inhibitory rates of compound 5a on the three cell lines were significantly increased at low concentration (5.0 and 10.0 μM) compared to RA treatment in a dose dependent manner. The IC 50 of compound 5a was significantly less than RA treatment group (<10 μM), which indicated that compound 5a could be a candidate for the development of new anticancer drugs. It was well known that hydroxyl groups can increase water solubility which might enhance the activity of the compound (the IC 50 values of compound 5a on the three cell lines were 5.99, 3.41, and 3.84 μM, respectively). However, further investigation of compound 5a still needs to be conducted and could include studying its anticancer activity in vivo.

General
Reagent-grade chemicals and solvents were obtained from commercial suppliers. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 983G spectrometer. NMR spectra were measured in C 5 D 5 N on a Bruker AM-400 spectrometer, using TMS as an internal standard. NMR experiments included the HMQC and HMBC pulse sequences. Coupling constants (J values) are given in Hz, and a MS Agilent 1100 Series LC/MSD ion-trap mass spectrometer was used to record the ESI-MS and HR-ESI-MS. All solvents were freshly distilled and dried prior to use, according to the standard procedures. All chemicals were purchased from Sigma Chemicals Ltd. The human hematoma cell line (HepG2), human malignant melanoma cell line (A375) and human small cell lung cancer cell line (NCI-H446) were purchased from Jilin Provincial Tumor Hospital. (1) The barks (1.0 kg) of I. rotunda were shade-dried, ground, and extracted with refluxing 80% EtOH successively (8 L, 3 h, two times). The EtOH extract was evaporated in vacuo to yield total saponins (100 g). The air-dried and powdered total saponins (100.0 g) were refluxed with 4% NaOH in 30% EtOH (5.0 L) at 100 °C for 4 h. The mixture was then cooled to room temperature and extracted with EtOAc  (2) RA (1.02 mmol) was dissolved in pyridine (20 mL), then acetic anhydride (10 mL) was added to mixture which was stirred at 80 °C for 16 h. The solvent was removed under reduced pressure using a rotary evaporator. The residue was washed with water, and evaporated to dryness.

General Procedure for the Preparation of N-[3β,23-diacetoxy-19α-hydroxy urs-12-en-28-oyl]amino acid methyl esters 4a-7a
To a solution of compound 2 (2.33 mmol) in CH 2 Cl 2 (25 mL) added oxalyl chloride (2 mL) and the mixture was stirred at an ice-water bath for 1 h, then further stirred at room temperature for 24 h. The mixture was concentrated to dryness under reduced pressure (30 °C). CH 2 Cl 2 was added to the residue three times (each time 50 mL), then the concentrated to dryness to yield crude 3,23-O-diacetylursolyl chloride 3. Next in an ice-water bath, glycine methyl ester hydrochloride (12 mmol, which was dissolved in 60 mL CH 2 Cl 2 and 6 mL triethylamine) was added to a CH 2 Cl 2 solution (90 mL) of 3 (2.33 mmol) The reaction mixture was stirred in the ice-water bath for 0.5 h, and then stirred at room temperature for 24 h, and then washed in turn with 2.5% hydrochloric acid, water, and saturated sodium chloride solution (each liquid three times, each time 50 mL). Then the reaction mixture was treated with anhydrous sodium sulfate, filtered, concentrated, and then dried to yield a light yellow solid that was recrystallized from 95% ethanol (400 mL) to yield a white solid. The solid was purified on a silica gel column with petroleum ether and ethyl acetate as eluents to yield white needles.

In Vitro Anti-tumor Assays
Aliquots (200 µL) of 5 × 10 3 cells per mL of A375, HepG2 and NCI-H446 cells were seeded in 96 well flat-bottomed plates in DMEM medium containing 10% FBS and a penicillin-streptomycin mixture at 37 °C in a humidified atmosphere of 5% CO 2 . The test drugs were dissolved in DMSO. The incubation medium was replaced with each test medium giving a final concentration of 5-40 µmol/L of test compounds and no drug in 2 µL DMSO over 24 h. The ability of the drug to inhibit cellular growth was determined by performing the MTT assay. Each experiment was performed in six wells, and all the experiments involving a control (DMSO only). The drug treatments were performed separately three times. All data are presented as mean ± standard deviations (S.D.). Statistical significance of the differences between groups was assessed by Student's t-test.

Conclusions
To date, though RA has been reported to show cytotoxicity, there are no reports on chemical modification of RA and the bioactivity of its derivatives. In this work, based on our previous investigation of RA, eight novel amino acid derivatives of RA were synthesized and their anti-tumor activities were tested in vitro for the first time. These eight compounds showed different cytotoxicities on the three tested tumor cell lines. Especially, compound 5a possesses better activity than RA, with 2.76-, 2.14-, 2.96-fold more potent activities than RA, respectively. The cytotoxicity of compound 5a was more sensitive than RA, with an IC 50 value of less than 10 μM on all three cell lines. Compound 6b showed equivalent activity to RA on the HepG2 and NCI-H446 cell lines, and higher cytotoxicity on the A375 cell line. Compounds 5a and 6b may thus serve as potential lead compounds for the development of new anticancer drugs. More derivative synthesis and further biological evaluations are currently in progress and will be reported in due course.