Design, Synthesis, And Anti-tumor Activity Of Limonene Analogues

Farnesyl transferase inhibitors (FTIs) are one of the most well-studied oncogene-targeted therapy because Ras is a key protein in tumorigenesis and is found mutated in approximately 30% of all human cancers. The relation between Ras and tumor was introduced briefly, and the effect mechanism and development of FTIs were given in this thesis.Monoterpene d-limonene was reported to have preventive and therapeutic effects against a variety of tumors in animal models. One of the probable mechanisms may be its ability to inhibit farnesyl transferase. This agent has been put into Phase I clinical trials and the preliminary results indicated that it was well tolerated in cancer patients. However, higher concentrations of d-limonene are needed to inhibit the growth of tumor cells in vitro. In Crowell's study, some hydroxy-containing metabolites of limonene were observed with more potential abilities to inhibit tumor cell growth than d-limonene. According to him, the low polarity of d-limonene structure might limit its ability to transverse cellular membranes and resulted in the less antitumor activity.Based on this observation, we designed a new group of limonene analogues containing polar groups in the structure without breaking unsaturated double bond and ring structure. The structural modification of l-carvone was focused on the 10-carbon and the carbonyl moiety. Benzoates and the hydrophilic amines were linked to the terpenoid moiety in order to enhance polarity and to alter hydrophility. In this study, l-carvone was chosen as the starting reagent and sixty-one novel compounds were synthesized via chlorination, nucleophilic substitution and reduction. Among these derivatives, forty-one compounds contain carvone skeleton, eleven compounds contain carveol skeleton and nine compounds contain limonene skeleton. The structures of novel compounds were confirmed by MS, ¹H-NMR and IR spectra, and the characteristics of ¹H NMR spectra were summarized.To assess the anti-proliferation effect of the target compounds, experiments with 'MTT assay' were performed in human prostate LNCaP and Dul45 cancer cells. The results demonstrate that the IC_50s(50% inhibitive concentrations) of major compounds were lower than l-carvone, l-carveol and l-limonene, and IC_50sof compounds A11,B8,B9,B14,B15 and C11 was lower than 20μM. The results showed that the antitumor activities of l-limonene derivatives were more potent than that of l-carvone and l-carveol derivatives with same substitutes. Some structure-activity relationships (SAR) were concluded.To investigate the potential mechanism of these compounds, compounds A8, B6, C7, D6 and E6, l-limonene and l-carvone were selected for this purpose and the famesyl transferase inhibition was investigated by measuring the HDJ2 unfarnesyl form using western blot analyses. However, no one of these compounds increased the unfarnesyl form of HDJ2. To further seek for the potent targets, the protein and phosphorylated forms of both p38 and ERK were investigated. The results suggested that phosphorylated ERK and p21^wafl protein level was significantly increased. This result indicated that these derivatives might inhibit cell growth through activation of ERK which leads to upregulation of p21^wafl protein and cell cycle arrest. The mechanism of these compounds which activate ERK is worthy of farther study.