Synthesis and Biological Activity of 23-Hydroxybetulinic Acid C-28 Ester Derivatives as Antitumor Agent Candidates

23-Hydroxybetulinic acid (1) served as the precursor for the synthesis of C-28 ester derivatives. The target compounds were evaluated in vitro for their antitumor activities against five cell lines (A549, BEL-7402, SF-763, B16 and HL-60). Among the obtained compounds, 6i had the most potent antitumor activity, with the IC50 values of 8.35 µM in HL-60 cells and showed similar antitumor activity as cyclophosphamide in H22 liver tumor and as 5-fluorouracil in B16 melanoma in vivo.

The chemical structure of 23-hydroxybetulinic acid is similar to that of betulinic acid, and they also have the similar pharmaceutical activities [10]. In our previous study, many derivatives of 23-hydroxybetulinic acid showed antitumor activity in different cell lines in vitro and compound 3 ( Figure 1) displayed stronger antitumor activity in mice H22 and B16 than betulinic acid or 23-hydroxybetulinic acid [11]. It was shown that the polarity and length of the chain in C-28 had an important impact on the antitumor activity. These results motivated us to undertake further modifications of the 28-carboxylic acid moiety of lead compound 1. In this paper, we wish to report a series of 28-carboxylic acid modified 23-hydroxybetulinic acid ester derivatives and their antitumor activity. The preliminary structure-activity relationships are also discussed.

Chemistry
For the synthetic experiments, the starting material 23-hydroxybetulinic acid (1) was isolated from the root of Pulsatilla chinensis. As described in Scheme 1, several derivatives of 23-hydroxybetulinic acid were synthesized. Compounds 5a-d were readily prepared in two steps starting from compound 1. Treatment of 5a-d with anhydrides R 2 (CO) 2 O, such as succinic anhydride, glutaric anhydride, maleic anhydride and phthalic anhydride, in the presence of DMAP in CH 2 Cl 2 gave compounds 6a-n in the yields of 53%-76%.

Biological Studies
The MTT (3-(4,5)dimethylthiahiazo (-z-y1) -3,5-diphenyltetrazoliumromide) assay results (Table 1) showed that most of the 23-hydroxybetulinic acid derivatives displayed better cytotoxic activities against the tested cells than betulinic acid and 23-hydroxybetulinic acid, especially compounds 6f, 6h, and 6i with IC 50 values ranging from 8.35 to 28.78 μM. As the assay results showed, C-28 ester derivatives with carboxylic acid substituents at the end of the C-28 side chain and whose C-28 side chain had appropriate length and flexibility exhibited stronger cytotoxicity. The activities of compounds 6k-n were weaker compared with 6f, 6h and 6i. It was possible due to the alkyne group in the C-28 side chain blocking the bending and rotation of the compounds.
Compound 6i was chosen to evaluated its antitumor activities in mice in vivo based on its significant efficiency in vitro. As illustrated in Tables 2 and 3, in H22 mice, compound 6i exhibited stronger activity than 23-hydroxybetulinic acid and comparable activity to cyclophosphamide, which was used as a positive control. In the B16 group, compound 6i also showed more potent activity than 23-hydroxybetulinic acid but somewhat weaker than 5-fluorouracil.

General
Melting points were obtained on a MEL-TEMP II melting-point apparatus and are uncorrected. IR were recorded on Nicolet Impact 410 or Bruker FT-IR TENSOR27 instruments. 1 H-NMR spectra were recorded on a Bruker-ACF-300 (chemical shifts are expressed as values relative to TMS as internal standard). HR-MS were obtained using a Agilent QTOF 6520 instrument.

Pharmacology
In vitro, the cytotoxic activities of 23-hydroxybetulinic acid, betulinic acid and all derivatives were determined by the MTT cytotoxicity assay, which was performed in 96-well plates. The tumor cell line panel consisted of A549 (human lung carcinoma), BEL-7402 (human hepatoma), SF-763 (human cerebroma), B16 (mice melanoma), HL-60 (human leukaemia) (final concentration in the growth medium was 2~4 × 10 4 /mL). MTT solution (20 L/well) was added after cells were treated with drug for 48 h, and cells were incubated for a further 4 h at 37 °C. The purple formazan crystals were dissolved in 150 L DMSO. After 5 min, the plates were read on an automated microplate spectrophotometer at 570 nm. Assays were performed in triplicate in three independent experiments. The concentration required for 50% inhibition of cell viability (IC 50 ) what was calculated. In all of these experiments, three replicate wells were used to determine each point [11].
In vivo, ICR female mice with body weight of 18-22 g were transplanted with H22 and B16 subcutaneously into the right axilla according to protocols of transplant tumor research. After 24 h of tumor transplantation, mice were weighed, and each model group was at random divided into 4 groups, each of which had 10 mice in H22 and B16 group. The groups with 23-OH betulinic acid and 6i were administered intraperitoneously 25 mg/kg in a vehicle of 20% DMSO/80% saline. respectively. The positive control group was treated with cyclophosphamide (30 mg/kg) in H22 group and 5-fluorouracil (25 mg/kg) in B16 group through intravenous injection in a vehicle of 20% DMSO/80% saline. The negative control group received 0.9% normal saline through intravenous injection. All test compounds were given through injections 24 h after tumor transplantation (or inoculation). Treatments were done at a frequency of intravenous or intraperitoneal injection one dose per day for a total of four consecutive days in H22 group and for a total of 11 consecutive days in B16 group. After the treatments, all mice were killed and weighed simultaneously, and then segregated and weighed the tumor [11]. Tumor inhibitory ratio was calculated by the following formula and perform T test: Tumor inhibitory ratio (%) = (1-average tumor weight of treated group/average tumor weight of control group) × 100%

Conclusions
In summary, a series of novel of 23-hydroxybetulinic acid C-28 ester derivatives were synthesized and tested for their in vitro cytotoxic activities against five human tumor cell lines. Most of the compounds showed moderate potent cytotoxic activities on all the tested cells. The results of preliminary biological activity showed that three compounds (6f, 6h, 6i) possessed impressive cytotoxicities. Compound 6i was chosen to evaluate its antitumor activities in vivo. Compound 6i showed similar antitumor activity as cyclophosphamide in H22 and as 5-fluorouracil in B16. Further structure modification and SAR studies of antitumor 23-hydroxybetulinic acid derivatives are in progress in our laboratory and the results will be reported in due course [12].