The Synthesis of Glycyrrhetinic Acid Derivatives Containing A Nitrogen Heterocycle and Their Antiproliferative Effects in Human Leukemia Cells

Fifteen novel glycyrrhetinic acid derivatives containing a nitrogen heterocycle at C-30 and with different A-ring substituents were designed and synthesized. All of these derivatives have improved antiproliferative effects against human HL-60 leukemia cells. Compounds with a cyano-enone functionality on the A-ring exhibit greater growth inhibitory effects, compared to those with a 2-hydroxymethylene-3-keto, an isoxazole, or a 2-cyano-3-keto group. N-(2-cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b) was found to be two-fold more potent than methyl 2-cyano-3,11-dioxooleana-1,12- dien-30-oate (CDODO-Me-11).

To determine the importance of introducing a cyano-enone at the A-ring and methyl esterification at C30, glycyrrhetinic acid derivatives containing a nitrogen heterocycle at C-30 and with some additional modifications of the A-ring were designed and synthesized. The antiproliferative abilities of these compounds were tested in human HL-60 leukemia cells and compared with that of GA, methyl 2-cyano-3,11-dioxo-oleana-1,12-dien-30-oate (CDODO-Me-11) and methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-30-oate (CDODO-Me-12), which have been found to be potent antileukemia agents [4]. The synthesis of these compounds and their abilities to inhibit growth of HL-60 cells are presented in this communication.

Chemistry
Four groups of GA derivatives with a different A-ring structure and a replacement at the C-30 carboxylic group with a nitrogen heterocycle -piperidine, 4-piperidyl piperidine, 4-methyl piperazine, or piperazine -were synthesized starting from GA (Scheme 1). Compounds 1-3 were obtained using a routine method [5,9,10]. A benzyl group instead of a methyl group was used to protect the C-30 carboxylic acid as halogenolysis of the methyl ester with LiI in DMF gave an 11-oxo-13(18)-en by-product in approximately 20% yield [8].
The benzyl group of compound 3 was removed under mild conditions to give the key intermediate 4 in 76% yield, which was used as the precursor for generating the 2-hydroxymethylene-3-keto compounds 5a-5c and the isoxazole 6. Addition of oxalyl chloride to compound 4 in chloroform gave the corresponding acyl chloride. Amides 5a-5c were prepared in 50%-72% yield by condensation between the acyl chloride and the corresponding nitrogen heterocycle.
Using the same procedures employed for obtaining compounds 5a-5c, the isoxazole amides 7a-7d were obtained from isoxazole 6, which was prepared in 85% yield by refluxing compound 4 with hydroxylamine hydrochloride in glacial acetic acid. Under basic conditions compounds 7a-7d were isomerized to the corresponding 2-cyano-3-keto compounds 8a-8d, that were then dehydrogenated using dichloro  in toluene to give the cyanoenone amides 9a-9d in fair to moderate yields.

HL-60 cell growth inhibition activity
The abilities of compounds 5a-5c, 7a-7d, 8a-8d and 9a-9d to inhibit growth of HL-60 cells were determined and compared to that of GA, CDODO-Me-11 and CDODO-Me-12 ( Table 1). All of these new synthetic compounds had GI 50 values in the 0.5-20 μM range, which were much lower than that of GA (>40 μM). The activity order among these compounds was cyano-enone amides 9 > 2-hydroxymethylene-3-keto compounds 5 > isoxazoles 7 and 2-cyano-3-keto compounds 8. Since compounds 8a-8d have decreased activities comparing to that of compounds 9a-9d, it suggests that a double bond between C-1 and C-2 is required for maintaining high activity in these cyano-enone amides. Compounds 9a, 9c and 9d have activities similar to that of CDODO-Me-11. The antiproliferative activity of N- (2-cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b) is two-fold more than that of CDODO-Me-11, indicating that the methyl ester could be replaced to further improve the antiproliferative activities of these compounds. Although 9b has lower activity than that of CDODO-Me-12, which has a 12-oxo-9(11)-en structure in the C-ring, the simple synthesis of 9b compared to that of CDODO-Me-12 makes this compound potentially more useful. The antitumor effects of compound 9b in additional cell lines are worthy of further study.

General
GA (purity over 98%) was purchased from Shanghai Haokang Chemicals Co. Ltd., China. Other reagents were bought from commercial suppliers in analytic grade and used without further purification, unless noted otherwise. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker ARX-300 instrument with tetramethylsilane as an internal standard. Infra-red (IR) spectra were recorded on a Bruker IR-27G spectrometer. Mass spectra (MS) were determined on a Finnigan MAT/USA spectrometer (LC-MS). HRMS spectra were obtained on a Bruker micrOTOF-Q in an ESI mode. The melting points were determined on an electrically heated X4 digital visual melting point apparatus and are uncorrected. TLC plates (Alugram silica gel G/UV254) were purchased from Macherey-Nagel GmbH & Co.

General procedure for the preparation of 2-hydroxymethylene-30-amides 5a-5c
To a solution of 4 (2.34 g, 4 mmol) in chloroform (10 mL), oxalyl chloride (2 mL) was added. The mixture was stirred at r.t. for 1 hr and excess oxalyl chloride was removed by co-evaporation with hexane (three times). The obtained solid was dissolved in chloroform (30 mL) and an appropriate nitrogen heterocycle (6 mmol) was added. The mixture was stirred at r.t. for 5 min and washed three times with 5% aqueous HCl solution. The organic layer was dried over anhydrous MgSO 4 . After MgSO 4 was removed by filtration, the filtrate was concentrated in vacuum and the residue was purified on a silica gel column with chloroform-methanol (v/v) = 50:1 to give a white solid. The Rf values were determined using TLC plates with chloroform-methanol (v/v) = 10:1.

Cell growth inhibition assay
All compounds were dissolved in DMSO. A stock solution of 20 mmol/L of each compound was prepared in DMSO and stored in aliquots at -20 ºC. A working solution was diluted with ethanol and fresh medium before assaying. The final concentration of ethanol in the medium was less than 1% and the final concentration of DMSO was less than 0.1%. Cells were seeded at a density of 4 × 10 4 cells/mL in 24 well plates with various concentrations of the tested compounds and incubated for 3 days. Total cell number in each group was determined using a hemocytometer. The cell growth inhibitory ability was expressed as the ratio of the cell number in the groups treated with the compounds to that of cells treated with DMSO and/or ethanol. The concentration (GI 50 ) which inhibited half of cell growth was calculated.

Conclusions
Fifteen novel glycyrrhetinic acid derivatives containing a nitrogen heterocycle at C-30 and with different substituents on the A-ring were designed and synthesized. The antiproliferative effects of these compounds were determined in HL-60 cells. The results reveal that: 1) introduction of the cyano-enone moiety into the A-ring of GA significantly improves the antiproliferative activities in leukemia cells and 2) N- (2-cyano-3,11-dioxoolean-1,12-dien-30-yl)-4-piperidyl piperidine (9b) is the most active compound among these novel GA derivatives with a 11-oxo-12-en structure.