Synthesis of amide-spacered dimers of ursolic and oleanolic acid

Transdermal therapeutic systems can release drug substances slowly and in a controlled manner from a drug depot. To provide a slow-release form of ursolic and oleanolic acid amide-spacered dimers were synthesized from the parent acids and diamines of variable chain lengths. These dimers were assayed in sulforhodamine B (SRB) assays for their cytotoxicity since as a pre-requisite for their use in slow-release forms these substances must not exert any unfavorable side-effects such as cytotoxicity. As a result of long term incubation up to 96 hours, none of these compounds showed any significant cytotoxicity in sulforhodamine B assays.


Introduction
In recent years, the search for biologically active compounds has increasingly turned to secondary natural substances. A newly awakened interest consists in the isolation, synthesis and biological investigation of dimeric compounds. Such dimers consist of two identical monomeric basic structures, which are connected to each other by a suitable linker. Some dimers seem to hold increased biological activity as compared to the corresponding monomers [1][2][3][4][5] . The formation of dimers, trimers, etc… is a common phenomenon in nature to regulate, for example, the activity of proteins 6, especially of enzymes. The synthesis and biological investigation, however, of dimeric structures derived from triterpenes have been fairly studied [7][8][9][10] . The chemistry of triterpenoic dimers started with Dolby's and Debono's dimerization 11 of citronellal. Since then several dimers have been described 7 . Of special interest are 3-amino-oleanolic acid derived dimers holding an adipinic acid-derived spacer 12 showing some activity for the HIV-1 protease; oleanolic acid derivatives 13,14 were inhibitors of the glycogen phosphorylase.
The number of dimers obtained with an incorporation of the C-28 carboxyl group remains limited probably due to its steric hindrance 15 also found for lupane derived dimers 16 . Only a few dimers holding an amide spacer 13,14,16 have been described so far. These compounds were tested as enzyme inhibitors or antivirals butby and largeno cytotoxic data have been provided for these compounds.
To surpass some problems usually associated with low solubility and to develop an application providing a slow-release of the drug, trans-dermal applications have been suggested and used quite successfully for different kinds of drugs 17 . Thereby, the transdermal application involves the application of ointments, creams or gels to the skin, and the active ingredients of which are absorbed by the skin. This allows them to enter the bloodstream and from there to their actual site of action. Transdermal patches, also known as "transdermal therapeutic systems" (TTS), are a frequently used method of transdermal application. They can release drug substances slowly and in a controlled manner from a drug depot. As a prerequisite for their use, these substances must not exert any unfavorable side-effects, such as cytotoxicity.
We became interested in the syntheses of dimeric OA and UA amides linked together by an alkyl chain of variable length. OA and UA were acetylated and 3-O-acetylated 1 [24][25][26] and 2 27,28 were obtained (Scheme 1). The reaction of 1 with oxalyl chloride followed by the addition of 0.5 equivalents of ethylene-diamine gave dimeric 3.
All of the dimers were screened for their cytotoxic activity in sulforhodamine assays (SRB), but none of these compounds showed any significant cytotoxicity (EC50 > 30 M cut-off) when tested with several human tumor cell lines (A375, HT29, SW1736, MCF7, A2780, FaDu, A549) as well with nonmalignant mouse fibroblasts (NIH 3T3). No cytotoxic effect was observed even upon applying a prolonged incubation time of 96 hours. Presently, the skin penetration and the stability of these dimers as well as they're in vivo degradation is studied in more detail in our laboratories.

Conclusion
A slow-release form of ursolic and oleanolic acid, amide-spacered dimers were synthesized from the parent acids and diamines of variable chain lengths. These dimers were assayed in SRB assays; even upon long term incubation up to 96 hours, none of these compounds showed any significant cytotoxicity in sulforhodamine B assays.