Design, synthesis and biological evaluation of novel betulinic acid derivatives

Abstract

Background: Tumor, is one of the major reason for human death, due to its widespread occurrence. Betulinic acid derivatives have attracted considerable attention as cancer chemopreventive agents and also as cancer therapeutics. Many of its derivatives inhibit the growth of human cancer cell lines by triggering apoptosis. With this background, we planned to synthesize a series of betulinic acid derivatives to assess their antiproliferation efficacy on human cancer cell lines.

Results: A series of novel betulinic acid derivatives were designed and synthesized as highlighted by the preliminary antitumor evaluation against MGC-803, PC3, A375, Bcap-37 and A431 human cancer cell lines in vitro. The pharmacological results showed that some of the compounds displayed moderate to high levels of antitumor activities with most of new exhibiting higher inhibitory activities compared to BA. The IC50 values of compound 3c on the five cancer cell lines were 2.3, 4.6, 3.3, 3.6, and 4.3 μM, respectively. Subsequent fluorescence staining and flow cytometry analysis (FCM) indicated that compound 3c could induce apoptosis in MGC-803 and PC3 cell lines, and the apoptosis ratios reached the peak (37.38% and 33.74%) after 36 h of treatment at 10 μM.

Conclusions: This study suggests that most of betulinic acid derivatives could inhibit the growth of human cancer cell lines. Furthermore, compound 3c could induce apoptosis of cancer cells.

Scheme 1

Synthetic route to BA derivatives. Reagents and conditions: (i) BrCH₂CH₂Br, BrCH₂CH₂CH₂Br, or BrCH₂(CH₂)₂CH₂Br, K₂CO₃, DMF, r.t.; (ii) amine, K₂CO₃, DMF, r.t.

Scheme 2

Synthetic route to BA derivatives. Reagents and conditions: (i) piperazine, K₂CO₃, DMF, 80 °C; (ii) EDCI, HOBT, R³COOH, DCM, r.t.

Figure 1

The AO/EB staining of compound 3c in tumor cells. For MGC-803 group, A: negative control; B and C: treated with HCPT and BA (10 μM each) as positive for 48 h; D, E, and F: treated with compound 3c (5 μM) for 12 h, 24 h, and 48 h, respectively. For PC3 cells group, A’: negative control; B’ and C’: treated with HCPT and BA (10 μM each) as positive for 48 h; D’, E’, and F’: treated with compound 3c (5 μM) for 12 h, 24 h, and 48 h, respectively.

Figure 2

The Hoechst 33258 staining of compound 3c in tumor cells. For MGC-803 group, A: negative control; B and C: treated with HCPT and BA (10 μM each) as positive for 48 h; D, E, and F: treated with compound 3c (5 μM) for 12 h, 24 h, and 48 h, respectively. For PC3 cells group, A’: negative control; B’ and C’: treated with HCPT and BA (10 μM each) as positive for 48 h; D’, E’, and F’: treated with compound 3c (5 μM) for 12 h, 24 h, and 48 h, respectively.

Figure 3

The TUNEL assay of compound 3c in tumor cells. For MGC-803 group, A: negative control; B and C: treated with HCPT and BA (10 μM each) as positive for 48 h; D, E, and F: treated with compound 3c (5 μM) for 12 h, 24 h, and 48 h, respectively. For PC3 cells group, A’: negative control; B’ and C’: treated with HCPT and BA (10 μM each) as positive for 48 h; D’, E’, and F’: treated with compound 3c (5 μM) for 12 h, 24 h, and 48 h, respectively.

Figure 4

Annexin V/PI apoptosis ratio detection assay. The appearance of apoptosis cells was detected by flow cytometry using Annexin V/PI staining in the figure. (A, B, and C) MGC-803 cells were treated with HCPT (10 μM) from 12 to 36 h; (D, E, and F) MGC-803 cells were treated with compound 3c (10 μM) from 12 to 36 h; (G, H, and I) PC3 cells were treated with HCPT (10 μM) from 12 to 36 h; (J, K, and L) PC3 cells were treated with compound 3c (10 μM) from 12 to 36 h.

Figure 5

The apoptosis ratios of MGC-803 and PC3 cells treated with compound 3c assessed by FCM. These cells were treated with HCPT (10 μM) and compound 3c (10 μM) for 12, 24, and 36 h. The apoptosis ratio includes the early and late apoptosis ratios.