Synthesis of novel [3,2-b ]indole fused oleanolic acids as potential inhibitors of cell proliferation

Seven new indole-fused oleanolic acid derivatives were synthesized from oleanolic acid for their ability to inhibit cell proliferation in NRP.152 cells.


Results and Discussion
In the present paper we describe the synthesis of seven new indole-fused oleanolic acid derivatives, 3-9, for evaluation in the NRP.252 cell assay.Fused heterocyclic derivatives of steroids and alkaloids are well documented, 25 and biologically active indole-fused examples are of particular interest. 268][29][30] In contrast, only one research group has described the synthesis of indole-fused triterpenoids. 31,32Interestingly, a number of indole-fused diterpenes, such as the penitrems, are Penicillium fungal metabolites. 33Our syntheses of the target compounds 3-9 (Figure 1) are based on the Fischer indole synthesis, 34,35

Figure 1
As we have previously described, 16 sequential diazomethane treatment and Jones oxidation of oleanolic acid (1) furnished keto ester 10 in 94% yield (Scheme 1).Fischer indolization of 10 with phenylhydrazine in acetic acid gave the known 32 fused indole ester 11 in 90% yield.Cleavage of this hindered methyl ester with lithium iodide in DMF 36 afforded 3 in 54% yield.The corresponding C-3 ketone obtained from 1 also underwent Fischer indolization to give 3 in 61% yield, but a persistent yellow contaminant could not be removed from 3 by either crystallization or silica gel chromatography.

Scheme 1
Due to the susceptibility of the indole ring in 3 to side reactions, particularly oxidation, modifications to the C-ring were performed prior to indolization.Thus, as shown in Scheme 2, and as we have previously described, 20 the synthesis of 3,12-diketone 12 was accomplished via an acid mediated epoxide rearrangement that occurred upon treatment of 10 with m-CPBA.Fischer indolization of 12 (74%) followed by ester cleavage (59%) gave the desired fused indole 4. The highly hindered C-12 ketone in 12 remains unaffected under these Fischer indole reaction conditions. 37Likewise, as we have reported, 20 allylic oxidation of 10 gave the known C-12,13 enone 13 (45% yield), which, upon Fischer indolization (79% yield) and ester cleavage (55% yield), afforded fused indole 5.The indole ring substituted analogs 6-9 were synthesized by employing the appropriate substituted phenylhydrazine in the Fischer indolization (Scheme 3).Thus, treatment of 10 with 2-chlorophenylhydrazine 38 gave indole 14 that could be converted to indole 6 by ester cleavage.This indolization reaction was accompanied by 31% of uncyclized hydrazone.A sequence starting with 3-fluorophenylhydrazine yielded a mixture of indoles 15 and 16, which were separated by sequential column and preparative silica gel chromatography in a 2:1 ratio, respectively.Cleavage of the methyl esters afforded 7 and 8.

Scheme 3
Finally, the 5-methoxyindole derivative 9 was synthesized directly from 3-keto acid 17 by Fischer indolization in 62% yield.The known keto acid 17 was prepared from oleanolic acid (1) by Jones oxidation (95% yield) as previously described. 16Interestingly, the corresponding methyl ester analog that was prepared by indolization of 10 decomposed under the lithium iodide ester cleavage conditions.Unfortunately, attempts to effect indolization of C-12 ring C ketone derivatives were unsuccessful, presumably due to the hindered nature of this position.For example, we could not prepare the phenylhydrazone of ketones 18 and 19, or effect indolization of ketones 19 and 20 with 2-iodoaniline using the palladium-annulation method of Chen et al. 39  Oleanolic acid (1), indoles 3-9, and 10, 12, and 13 were screened in vitro for their ability to inhibit proliferation of premalignant, non-tumorigenic prostate cells.Of the compounds prepared in the present study, only 4 and 5 showed some activity (IC 50 <5 µM).All of the others were essentially inactive in this assay (>5 µM).For comparison, TGF-β has IC 50 = 0.000014 µΜ. 40herefore, in view of the disappointing activity in this assay of this series of fused-indole oleananes, we are not currently pursuing the study of additional examples of indole-fused triterpenoids.

Experimental Section
General Procedures.Flash column chromatography was done with Select Scientific silica gel (230-400 mesh). 1 H (300 MHz) and 13 C (75 MHz) NMR spectra were recorded on a Varian XL-300 spectrometer in CDCl 3 solvent; chemical shifts are reported with reference to the δ 7.27 signal of CHCl 3 ( 1 H NMR) and δ 77.23 signal of CDCl 3 ( 13 C NMR) as an internal standard.

General procedure for Fischer indolization
A mixture of ketone 10 (89.3 mg, 0.191 mmol), phenylhydrazine (0.02 mL, d = 1.1, 1.05 eq), and glacial acetic acid (2 mL) was heated at reflux under N 2 for 30 min.During this period the color changed from colorless to bright yellow.The reaction mixture was pipetted into distilled water (50 mL) and extracted with ether (4 x 20 mL).The combined ether extracts were washed with 5% aqueous NaOH (2 x 20 mL) and brine (2 x 20 mL), dried (Na 2 SO 4 ), and concentrated in vacuo to afford a yellow solid.Flash chromatography over silica gel and elution with hexaneethyl acetate afforded indole 11 (92.4 mg, 90%) as an amorphous pale yellow solid.The synthesis of indole 9 from ketone 17 was worked up by simply pouring into water, extracting with ethyl acetate, and processing in the usual way to give an amorphous product after flash chromatography.Indoles 9, 11, and 14-16 were all amorphous solids and were directly converted into the corresponding acids as described below.

General procedure for ester cleavage
A mixture of indole ester (0.09 mmol) and lithium iodide (0.45 mmol) in DMF (1.5 mL) under N 2 was heated at reflux for 15 h.The mixture was allowed to cool, treated with water (20 mL) and 10% aqueous hydrochloric acid (5 mL), and extracted with dichloromethane (3 x 20 mL).The organic layer was washed with water, dried (MgSO 4 ), and concentrated in vacuo to afford the crude acid.Purification was effected by preparative TLC (hexane/ethyl acetate, 4:1) to give 3-8 as amorphous solids, for which melting points could not be obtained.The amounts of compounds, which were needed for biological screening, were insufficient for crystallization.Spectra data of 3-9 are tabulated in Tables 1-3.
Scheme 4 and are depicted in Schemes 1-4.