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Letters Discovery of S-Phase Arresting Agents Derived from Noscapine James T. Anderson,* Anthony E. Ting,* Sherry Boozer, Kurt R. Brunden, Joel Danzig, Tom Dent, John J. Harrington, Steven M. Murphy, Rob Perry, Amy Raber, Stephen E. Rundlett, Jianmin Wang, Nancy Wang, and Youssef L. Bennani Athersys, Inc., 3201 Carnegie Avenue, Cleveland, Ohio 44115-2634 Received July 20, 2004 Abstract: Analogues of the natural product noscapine were synthesized, and their potential as antitumor agents were examined. The discovery of a novel regio- and stereoselective O-demethylation led to the synthesis of several O-alkylated analogues that induced an unexpected S-phase arrest of mammalian cells. Compound 4a was the most potent analogue inhibiting cell proliferation at an EC50 of 1.9 µM.
The eukaryotic cell cycle is a series of tightly regulated events that result in the transmission of genetic material from one generation to the next. The fidelity of these processes is monitored by cell cycle checkpoints that induce cell cycle arrest when damage is detected. If the damage cannot be repaired, these checkpoints may also cause cells to undergo apoptosis. Many chemotherapeutic agents take advantage of these checkpoint controls to preferentially kill rapidly dividing cancer cells. However, there are several problems common to existing cancer chemotherapies that mainly result from a narrow therapeutic index leading to undesired side effects. Increased drug resistance in tumors is another problem.1 Thus, there is still a need for effective drugs with an improved safety profile. Noscapine 1 is a naturally occurring phthalideisoquinoline alkaloid obtained from opium. It has been used orally in humans as an antitussive agent and displays a low toxicity profile.2 Additionally, it has been known for over 60 years that noscapine acts as a weak anticancer agent.3 Recently, Joshi et al. have reported that noscapine causes cells to arrest in the G2/M phase of the cell cycle and can cause tumor regression in animal models when administered at 120 mg/kg (ip) daily for 3 weeks.4 Here, we report on efforts to discover potent noscapine derivatives that may be suitable for development as anticancer agents. Because of limited literature outlining procedures for the chemical modification of noscapine, we initially chose to probe the structure-activity relationship (SAR) at the 7-position of noscapine using the chemistry of Schmidhammer and Klotzer (Scheme 1).5 Thus, nos* To whom correspondence should be addressed. For J.T.A.: phone, 216-431-9900; fax, 216-361-9596; e-mail,
[email protected]. For A.E.T.: phone, 216-431-9900; fax, 216-361-9596; e-mail, ating@ athersys.com.
Figure 1. FACS analysis of HEK293 cells treated for 16 h with (A) 0.5% DMSO, (B) 50 µM noscapine, and (C) 50 µM 2.
Scheme 1. Benzyloxide Addition
capine was treated with sodium benzyloxide in benzyl alcohol and DMF and heated to 120 °C for 16 h to give a 1:1 mixture of diastereomers 2. This substitution typically proceeded in low yield with epimerization of the phthalide stereocenter and was limited to electronrich alkoxides. The mixture of diastereomers 2 showed an unexpected S-phase arrest on HEK293 cells that were analyzed for DNA content by flow cytometry (Figure 1 and Table 1). It was later revealed after HPLC purification that each of the diastereomers showed effects and potency similar to those of the mixture 2 in the cell cycle assay at 50 µM. Moreover, the mixture 2 was found to inhibit the growth of HEK293 cells with an EC50 of 15 µM. To determine whether the S-phase arrest observed in HEK293 cells treated with mixture 2 was due to the induction of apoptosis, the cells were examined for the presence of apoptotic nuclei following treatment with mixture 2 or buffer control. In contrast to HEK293 cells treated with the apoptotic agent etoposide (p < 0.05), no apoptosis was observed in cells incubated with mixture 2 (p > 0.5). This was consistent
10.1021/jm0494220 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/30/2005
Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2757
Table 1. Effects on Cell Cycle Distribution
Scheme 3. Phenol Alkylation
a 1:1 mixture of diastereomers. b Concentrations 2- to 10-fold lower did not show a difference in the FACS profile when compared to DMSO control.
Scheme 2. 7-O-Demethylation of Noscapine
with the fact that cells treated with mixture 2 displayed a similar morphology to DMSO-treated cells. The observation of an S-phase arrest was an unexpected result because noscapine has been reported to arrest cells in the G2/M-phase of the cell cycle. The fact that a change in size of the 7-alkoxy group gave such a dramatic result warranted further SAR exploration. We first examined the optimization of the substitution reaction to address the problem of epimerization, low yield, and general limitations of the SAR. A series of reaction conditions focusing around the metal counterion were investigated. Heating a DMF solution of 1 in the presence of potassium benzyloxide (BnOH, KHMDS) gave results similar to those from the original sodium salt conditions, and the lithium analogue (BnOH, nBuLi) resulted in no reaction after 3 h at 110 °C. However, the use of MeMgBr and BnOH in toluene-THF at 110 °C for 3-4-h did not result in benzyloxide substitution but instead resulted in the regioselective O-demethylation at the 7-position to give phenol 3 (Scheme 2).5,6 The regio- and stereoselective nature of the reaction was proven by X-ray crystallography.7 It is reasonable to hypothesize that the source of regioselectivity is due to the conjugative stabilization of the resultant phenoxide by the carbonyl group. However, it is evident that magnesium plays an important role in the outcome of the reaction. Ethanol and 2-propanol were investigated as replacements for benzyl alcohol in the demethylation; however, no reaction was observed at refluxing temperatures over a 24 h period. Thus, the nucleophilic and
high-boiling properties of benzyl alcohol may be advantageous, although other high-boiling alcohols were not investigated. The use of benzyl alcohol did not detract from the synthesis because it could be easily removed by acid-base extraction. We were gratified that under these novel O-demethylation conditions little to no epimerization occurred.8 This allowed for the alkylation of phenol 3 using alkyl halides to give products as pure stereoisomers (Scheme 3). Although the yields of alkylated products were generally low (
