Design, Synthesis And Evaluation Of Trehalose-based Compounds As Anti-invasive Agents

A characteristic feature of cancer cells is their ability to dissemination into distant, often specific tissues. Cancer cells invade beyond the constraints of the normal tissue from which they originate; this invasion permits them to enter into the circulation from where they can reach distant organs and eventually form secondary tumors, called metastasis. Metastasizing is a complex, multi-step biochemical process, which includes the detachment of cancer cells from primary tumor, migration, adhesion and invasion of cancer cells into the blood or lymphatic vessels, extravasation out of the vessel, and finally interaction with the target tissue and growth out to micrometastasis and macrometastasis/secondary tumors. Metastasis is responsible for 90% of cancer patient deaths, and it remains a major hurdle in cancer treatment. Anticancer drug development strategies are generally aimed at direct inhibition of cancer cell growth. Most current chemotherapy treatments are antiproliferatives, which seek to retard growth of the primary tumor or even reduce the existing tumor burden. However, many tumor cells are unaffected by this type of chemotherapy. Indeed, some cells are able to stay dormant for months, or even years, and to grow later into new tumors. Furthermore, migrating cells are known to show a decreased proliferation rate and are thus less sensitive to standard chemotherapy. As invasion is a measure of tumor cell activity and crucial at all the different steps of the metastasis process, and closely related to the final outcome of cancer, treatments directly targeting metastatic diseases by specifically inhibiting the invasive behavior of tumor cells are extremely attractive. Such approaches are expected to extend the patient's lifespan, even though they might not directly inhibit late-stage tumor proliferation. The development of anti-invasive and anti-metastasis drugs is a major challenge in current cancer research.Brartemicin is a trehalose-based inhibitor of tumor cell invasion produced by the actinomycete of the genus Nonomuraea. In order to explore the preliminary structure-activity relationship and obtain more potent inhibitors, a series of Brartemicin analogs were synthesized through the Mitsunobu coupling of the secondary hydroxyls benzyl protected a,a-D-trehalose with benzoic acid derivatives, followed by modification of functional groups and deprotection. These compounds were evaluated for their inhibitory activity against invasion of murine colon 26-L5 carcinoma cells in vitro. Among the synthetic analogs tested, three compounds exhibited better anti-invasive activity than natural Brartemicin. Compound 6,6'-bis(2,3-dimethoxybenzoyl)-a,a-D-trehalose is 2.6-fold more potent against murine colon 26-L5 carcinoma cells in vitro. Therefore, it is considered to be a promising lead molecule for the antimetastasis.In this dissertation, Brartemicin was selected as lead compound. Structure modification was selectively performed on 6,6'-OH of Brartemicin. Two series of ester and amide analogs were designed and synthesized, and their structures were comfirmed by 1H-NMR,13C-NMR, MS.This dissertation consists of five parts shown in below:Part one:Background of the studyIn this part, the definition of tumor invasion and metastasis, the biological process of tumor invasion and metastasis, and the possible targets were outlined. The anti-invasive and anti-metastastic agents were introduced and reviewed, including their structure, biological activity, mechanism and others.Part two:Design of the target compoundsBased on the structure of Brartemicin,6-and 6'-positions are rationalized as the major structural modification sites:(1) according to the principle of the molecular similarity in drug design, a series of 6,6'-diesterated trehalose analogs were designed. The electrical properties, hydrogen bond donating and accepting abilities, and steric factors are considered for the substituents on the aromatic ring; (2) considering that amides are often used to replace esters in drug design and they are more stable than the ester analogs in vivo, a series of 6,6'-diaminated trehalose analogs were designed. To find more potent analogs and systematic study the structure-activity relationships, drug-like molecular fragments and privileged structures are applied to design the structurally diverse derivatives.Part three:Synthetic routes design and discussionsThree synthetic routes are designed for the synthesis of the 6,6'-ester and 6,6'-amide series of trehalose analogs. The synthesis of intermediates 2,2',3,3',4,4'-hexabenzyl-a,a-D-trehalose and 6,6'-diamino-2,2,3,3',4,4'-hexabenzoyl-6,6'-dideoxy-a,a-D-trehalose dihydrochloride were improved, and the steric effects on the acyl migration on the later were discussed.Part four:Synthetic experimentsA total 75 compounds were prepared that includes 68 novel ones (34 intermediates,34 target compounds). Their structures were determined by 1H-NMR,13C-NMR and ESI-MS, etc.Part five:Biological evaluations and discussionsBosed on the reconstituted basement membrane invasion assay method, the synthesized compounds were evaluated against colon 26-L5 for their anti-tumor cell invasion activity. In the 6,6'-ester series, compounds JYL-017 and JYL-041 exhibited strong anti-invasive activities. Analog JYL-017 was 2.6-fold more potent than natural Brartemicin, while JYL-041 was slight better than it. Analogs JYL-016, JYL-020, JYL-023 and JYL-024 showed good activity, with an IC50 of 1.64,1.72, 1.60,1.64μM, while compounds JYL-022 and JYL-037 exhibited weak activity (IC5o> 1.71μM). In the 6,6'-amide series,2-OH and 2-OMe analgs exhibited strong activity, and compound JYL-068 was slight better than Brartemicin. 2,3-dimethoxylated analog JYL-064 and 2,6-difluorinated analog JYL-065 exhibited weak acitivity (IC5o>1.61μM). The structure-activity-relatioships were briefly discussed. All compounds showed no cytotoxicity even at the concentration of 16μM by MTT method.In the preliminary anti-invasive mechanism studied, all the compounds did not show the inhibitory activity against matrix metalloproteinases MMP-2 and MMP-9. This indicates that these matrix metalloproteinases are not the targets of these active trehalose analogs. In addition, these compounds did noe affect the migration of the tumor cells.Part six:Summary and outlooksIn conclusion, total 34 novel trehalose analogs were design and synthesized. In the reconstituted basement membrane invasion assay,3 compounds exhibited better anti-invasive activity than natural Brartemicin. Especially, compound 6,6'-bis(2,3-dimethoxybenzoyl)-a,a-D-trehalose (JYL-017) is 2.6-fold more potent against murine colon 26-L5 carcinoma cells in vitro. Therefore, it is considered to be a promising lead molecule for the antimetastasis.The structure-activity-relatioships were discussed and the anti-invasive mechanisms were studied. However, the anti-invasive mechanism and the targets for these active trehalose analogs were still unclear. Further studies are needed to elucidate these questions.