Synthesis and Biological Activity of Some Bile Acid-Based Camptothecin Analogues

In an effort to decrease the toxicity of camptothecin (CPT) and improve selectivity for hepatoma and colon cancer cells, bile acid groups were introduced into the CPT 20 or 10 positions, resulting in the preparation of sixteen novel CPT-bile acid analogues. The compounds in which a bile acid group was introduced at the 20-hydroxyl group of CPT showed better cytotoxic selectivity for human hepatoma and colon cancer cells than for human breast cancer cells. Fluorescence microscopy analysis demonstrated that one compound (E2) entered human hepatoma cells more effectively than it did human breast cancer cells. Compound G4 exhibited the best anti-tumour activity in vivo. These results suggested that introduction of a bile acid group at the 20-position of CPT could decrease toxicity in vivo and improve selectivity for hepatoma cells.


Introduction
The search for effective anti-cancer drugs has led to the identification of many compounds that effectively inhibit cancer cells. However, these cytotoxic agents usually have very poor specificity, and OPEN ACCESS therefore cause systemic toxicity. Combining drugs with more specific carriers could improve targeting, increase the local drug concentration, improve cell penetration, and reduce drug toxicity. For these reasons, recent research has been focused on drug carriers. Camptothecin (CPT) is a highly effective anti-tumour and antibiotic alkaloid that was first isolated by Wall and Wani in 1958 from the extracts of Camptotheca acuminate, a tree native to China [1,2]. CPT is a neutral alkaloid that easily reacts with acids to form salts and does not readily dissolve in acid solutions, water, or common organic solvents. Moreover, the CPT α-hydroxyl lactone E-ring is unstable, and undergoes rapid hydrolysis to an inactive carboxylate form at physiological pH [3]. The open loop of CPT leads to serious side effects, which led to the termination of clinical trials on CPT. By binding to DNA topoisomerase I (Topo I), CPT increases DNA damage and triggers apoptosis in rapidly multiplying cancer cells and also in normal cells containing high levels of Topo I. Analysis of CPT structure-activity relationships has indicated that modification of the 20-hydroxyl position of CPT could eliminate intramolecular hydrogen bonding and reduce carbonyl activity. Because this modification increases carbonyl resistance to a nucleophile attack, lactone E-ring analogues of CPT show increased stability and improved anti-tumour activity [1]. Introducing changes at the CPT 10-hydroxyl position also enhances lactone stability. Moreover, this method could enhance the drug's anti-cancer activity by preventing CPT from binding with human serum albumin.
Some bile acids and their derivatives can be used as drug carriers. In the enterohepatic circulation, it enters the liver and colon cells by active transport. A large number of studies have shown that conjugating drugs with appropriate bile acid could increase their enterohepatic absorption, improve metabolic stability, enhance oral bioavailability, and reduce toxicity [4][5][6][7][8][9][10]. The latest research focuses on structure-activity relationships and the application of bile acid as a drug carrier. These studies have indicated that drugs coupled to the bile acid 3 and 24 sites did not affect the acid part of the activity [6,7], and the activities of most drugs were not affected by coupling to a bile acid [8]. Therefore, bile acids have the potential to be useful drug carriers [9].
The aim of the present study was to design a new tumour-targeting drug consisting of CPT as the cytotoxic warhead, and a bile acid as the tumour recognition moiety. We hypothesised that this could produce a new anti-tumour drug with liver and colon selectivity and reduced toxicity. We have previously synthesised CPT-bile acid analogues and evaluated their anti-tumour activities, and the research showed that bile acid linked to the 20-hydroxy position of CPT did not affect the anti-tumour activity of CPT [10]. The present study extended the above work by synthesising a series of CPT-bile acid analogues and investigating their anti-tumour activities in vitro and in vivo.
The target compounds E1-E4 were synthesised by forming an amide connecting the bile acid with CPT. The CPT was mixed with N-(tert-butoxycarbonyl) glycine in chloroform, 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDCI), and a catalyst, 4-dimethylaminopyridine (DMAP), at room temperature to yield compound camptothecin-20(S)-O-(N-(tert-butoxycarbonyl) glycine) ester (1) [11]; The amino group of compound 1 was deprotected in HCl/CH 3 OH to produce compound 2, which was then attached to activated bile acids 7-10 in the presence of triethylamine to produce compounds E1-E4 in yields of 75%-76%. The target compounds F1-F4 were synthesised using the method described above for E1-E4, but the linker chain used was D-Glu(OMe) and the yields were 64%-66%. The target compounds G1-G4 were synthesised by forming an ester bond connecting CPT and a bile acid. CPT was first converted into compound 5 at high yield (97.5%) in the presence of EDCI and DMAP at room temperature, and then the alkyl halide part of compound 5 underwent a nucleophilic substitution reaction with the bile acid carboxyl group in an alkaline environment, producing compounds G1-G4 in 62%-63% yield. This reaction occurred faster in the presence of K 2 CO 3 , but more byproduct that could not be separated easily was produced. Use of triethylamine as the catalyst resulted in a slower reaction but fewer impurities. The target compounds H1-H4 were synthesised via formation of an ester bond connecting the bile acid with 10-hydroxycamptothecine (HCPT). The HCPT hydroxyl group engaged in a nucleophile substitution reaction with 1,3-dibromopropane in dimethylsulphoxide (DMSO) at 60 °C in the presence of K 2 CO 3 , to produce compound 6 [12] Compound 6 underwent nucleophilic substitution reactions with bile acids in a similar manner to produce H1-H4 in yields of 71%-73%. The proton nuclear magnetic resonance ( 1 H-NMR), electrospray ionization-mass spectrometry (ESI-MS), and infrared (IR) spectra of these novel CPT derivatives helped confirm that all the products were consistent with their predicted structures.

Pharmacology
We tested the biological activities of these novel CPT-bile acid analogues in vitro and in vivo. We also studied their ability to enter cells and their stability in plasma. The in vitro anti-tumour activities of the CPT-bile acid analogues were evaluated against a human colon cancer cell line (HCT-116), a human hepatoma cell line (SMMC-7721), and a human breast cancer cell line (MCF-7). These studies resulted in the selection of compound E2 for subsequent examination of its entry into SMMC-7721 and MCF-7 cells. In addition, the in vivo anti-tumour activities of compounds E2, G4, and H2 against a mouse liver adenocarcinoma model (H 22 ) were investigated. The stability of these novel CPT-bile acid derivatives in serum was measured using high-performance liquid chromatography (HPLC).

MTT Test
The results of the in vitro anti-tumour studies are summarized in Table 1. These results showed that although CPT showed similar toxicity towards the three cell lines, and the cells showed different sensitivities to the novel analogues. The majority of the compounds were more toxic to the human colon cancer and hepatoma cell lines (HCT-116 and SMMC-7721) than to the human breast cancer cell line (MCF-7). This indicated selectivity of the prepared analogues with bile acids at the CPT 20hydroxyl position for colon cancer and hepatoma cells in Table 1. With regard to the SMMC-7721 cell line, compound G4 showed the highest cytotoxic activity (IC 50 = 0.14 µM), four times more effective than CPT. For the HCT-116 cell line, compounds H2 and H3 showed the highest cytotoxicity (IC 50 = 0.8 µM), similar to that of CPT. For the MCF-7 cell line, compound H2 showed the highest cytotoxicity (IC 50 = 2.5 µM), which was still much less effective than CPT (IC 50 = 0.57). E series were generally more cytotoxic to the HCT-116 and SMMC-7721 cell lines according to Table 1. The activities of G1-G4 and E1-E4 showed that the use of amide or ester bonds did not affect the cytotoxic activity of the analogues. It is entirely possible that binding of bile acids at CPT's 10hydroxyl group contributed to decreased selectivity for colon cancer and hepatoma cells.

Fluorescence and Phase Contrast Microscopy
Fluorescence and phase contrast images of SMMC-7721 cells treated with CPT and E2 were obtained using an inverted fluorescence microscope with ultraviolet light. Compound E2 was selected for this experiment, because E2 showed good cytotoxic activity towards SMMC-7721 and low cytotoxicity in MCF-7 cells. Inverted fluorescence microscopy ( Figure 1) indicated that E2 accumulated in the SMMC-7721 cells, as evidenced by the blue-green fluorescence that increased in a concentration-dependent manner. In contrast, cells exposed to CPT showed no distinct fluorescence. The cause of the accumulated blue-green fluorescence in SMMC-7721 is structure of CPT in E2, so at same concentration, E2 showed an better affinity for SMMC-7721 cell than CPT. Moreover, MCF-7 cells exposed to either E2 or CPT were also imaged (data not shown), but no fluorescent signal in MCF-7 cells was observed with either treatment, so E2 does not have a good affinity for MCF-7 cells. This result was consistent with our MTT test data. These results suggested that the introduction of DCA by an amide at the CPT 20-hydroxyl position greatly increased the activity of the new compound entering into SMMC-7721 cells.

Lactone Stability
To measure the stability of these novel CPT-bile acid derivatives, Compounds E2, F2, G2, and H2 were subjected to rat blood serum stability studies (Table 1). Half-life of the tested analogues was all >24 h in rat blood serum, and the lactone stability of the tested analogues was improved significantly compared to CPT (half-life of CPT is 2.3 h).

In Vivo Anti-Tumour Efficacy Studies
Preliminary anti-tumour activity studies of E2, G4, and H2 were evaluated in vivo in a mouse liver adenocarcinoma model (H 22 ) and compared with CPT. An in vivo toxicity study showed that the maximum tolerated dose (MTD) values of these CPT-bile acid analogues were >60 mg/kg (data not shown). Three doses of 15 mg/kg, 30 mg/kg, and 45 mg/kg were therefore selected for treatment of the mice by intraperitoneal (i.p.) injection every two days (three doses in all). The data in Table 2 show that the novel CPT-bile acid analogues had significant anti-tumour activity and the low weight loss, compared to CPT group. G4, with a tumour inhibitory rate (TIR) of 71.12% at 57.63 µmol/kg (higher than the TIR of 27.45 µmol/kg CPT), showed the best anti-tumour activity. The dose of 27.45 µmol/kg CPT is maximum dose which the mouse can tolerate in this project. Moreover, the effects of G4, E2 and H2 all showed better dose-dependency over the range tested. Although E2 had a lower in vitro IC 50 value than CPT in SMMC-7721 cells, it showed poorer anti-tumour activity in vivo. Interestingly, in compound G4, CPT and CDCA are joined by an ester bond, and E2 is a conjugate which CPT jointed with DCA by an amide, but G4 showed superior in vivo anti-tumour activity than E2.

In Vivo Anti-Tumour Activity
Anti-tumour activity screens of CPT-bile acid analogues were conducted in KM mice with H 22 mouse liver tumours. Briefly, female mice weighing 18-22 g were subcutaneously inoculated at the right flank with a tumour cell suspension (1 × 10 6 cells) in 0.2 mL of phosphate-buffered saline. The mice were divided into subgroups 24 h after inoculation. These groups (n = 4) were treated with either vehicle (control), CPT, or the CPT-bile acid analogues. Mice received i.p. treatments once every two days. When the tumour weight of the control group reached 1.0-1.5 g, the mice were killed and the TIR (%) calculated.

Conclusions
Sixteen different CPT-bile acid analogues were synthesised using different bile acids and linker chains. To explore the effects of this on CPT activity, the anti-tumour activities of these analogues were evaluated in vitro and in vivo. Most of the CPT-bile acid analogues showed greater cytotoxicity in a human hepatoma cell line (SMMC-7721) than in a breast cancer cell line (MCF7), with the exception of H1-H4. Compounds E2 and G4 showed better inhibition of the cancer cells than CPT. Fluorescent accumulation of E2 was detected in the SMMC-7721 cells and E2 entered this cell line more effectively than it entered MCF-7 cells. The lactone stability of the analogues tested in rat blood serum generally increased, indicating a more stable lactone character than CPT. In vivo, the use of an ester bond to connect a bile acid with CPT (G4) produced the best anti-tumour activity. Taken together, these findings indicated that introducing a bile acid group at the 20-hydroxyl group of CPT affected drug activity more than introduction at the 10-hydroxyl group. The results of this study suggested that CPT-bile acid analogues are promising candidates for anti-cancer therapy.