Synthesis and evaluation of Apogossypol atropisomers as potential Bcl-xL antagonists
Introduction
Programmed cell death plays an essential role in normal tissue homeostasis, ensuring a proper balance of cell production and cell loss. Defects in the regulation of programmed cell death promote tumorgenesis, and also contribute significantly to chemoresistance [1], [2]. The Bcl-2 (B-cell lymphocyte/leukemia-2) family of proteins is central for the regulation of apoptosis [3], [4], [5]. Overexpression of anti-apoptotic Bcl-2-family proteins, such as Bcl-2, Bcl-XL, Mcl-1, Bfl-1, Bcl-W and Bcl-B, occurs in many human cancers and leukemia’s and therefore these proteins are very attractive targets for the development of novel anti-cancer agents [6], [7], [8], [9]. Bcl-2 family proteins also have pro-apoptotic members such as Bak, Bax, Bad, Bim or Bid. Anti-apoptotic and pro-apoptotic Bcl-2 family proteins dimerize and negate each other’s function [10]. Structural studies have elucidated a hydrophobic crevice on the surface of anti-apoptotic proteins that binds the BH3 dimerization domain of proapoptotic family members. Thus, molecules that mimic proapoptotic BH3 domain will displace anti-apoptotic proteins hence inducing apoptosis.
We and others have reported that the natural product (+/−) Gossypol is a potent inhibitor of Bcl-2, Bcl-XL and Mcl-1, functioning as a BH3 mimic [11], [12], [13]. Naturally, Gossypol is composed of the (+) and (−) enantiomers (atropisomers) due to hindered rotation along the single CC bond between the two substituted naphthalene groups. Given that (+/−) Gossypol has toxicity problems likely due to two reactive aldehyde groups, we selected (+/−) Apogossypol, a derivative that lacks two aldehyde groups, but that retains most of anti-Bcl-2 properties in vitro and against cancer cells [14]. Recently, we further compared the efficacy and toxicity in mice of (+/−) Gossypol and Apogossypol. Our preclinical in vivo data supports the hypothesis that (+/−) Apogossypol has superior efficacy and markedly reduced toxicity compared to (+/−) Gossypol [15]. We also evaluated the single-dose pharmacokinetic characteristics of (+/−) Apogossypol in mice, revealing superior blood concentrations over time compared to (+/−) Gossypol, due to slower clearance of the compound [16]. These observations indicate that further development of (+/−) Apogossypol for cancer therapy is warranted.
Here we first prepared the individual isomers of Apogossypol and we further investigated their activity in vitro and in cell, given that similar studies with Gossypol revealed a marked differential activity for the isolated enantiomers [17]. Indeed (−) Gossypol and not its natural racemic mixture, is currently under clinical evaluation [18]. The synthesis of (+) Apogossypol has been reported by Seshadri and Sharma However, no details on optical rotation or HPLC separation were provided [19]. The synthesis of (−) Apogossypol has not been reported. In this current work we focused our attention on preparing and evaluating activities of (+) and (−) atropisomers of Apogossypol (Fig. 1).
Section snippets
Preparation of Apogossypol enantiomers
Racemic (+/−) gossypol acetic acid (5.0 g, Yixin Pharmaceutical Co.) was dissolved in 120 ml of diethyl ether and washed with water (2 × 100 ml) to remove acetic acid [20]. The ether layer was dried over MgSO4 followed by removal of the solvent under vacuum to give gossypol as yellow brown solid. l-Phenylalanine methyl ester hydrochloride (13.8 g, Sigma–Aldrich) was dissolved into 200 ml of CH2Cl2 and then washed with saturated NaHCO3 solution (2 × 50 ml) to remove hydrochloride. The CH2Cl2 layer was
Resolution of Apogossypol enantiomers
The optical rotation (α) of (+) and (−) Gossypol, expressed as +348° and −352°, respectively, and 1H NMR data (600 MHz, CDCl3: δ1.55 (d, 12H, J = 5.5 Hz); 2.16 (s, 6H); 3.94 (m, 2H); 5.80 (s, 2H); 6.44 (s, 2H); 7.79 (s, 2H); 11.15 (s, 2H); 15.19 (s, 2H)) are in excellent agreement with published physical data for (+) and (−) gossypol [25]. The optical rotation of compounds 3a and 3b are −92.5° and +95.0°, respectively. In comparison, optical rotation of racemic Apogossypol is +2.0°. Since there are
Conclusions
Both (−) and (+) Apogossypol display similar in vitro binding and displacement properties and cellular activity as racemic Apogossypol. In comparison, Gossypol enantiomers have remarkable stereo-selective cytotoxic difference in several tumor cell lines. Due to the presence of two aldehyde groups, Gossypol enantiomers are more anisotropic in shape than Apogossypol enantiomers. In fact, the optical rotation difference between (+) and (−) Gossypol enantiomers are 700°. In comparison, the optical
Conflicts of interest
The licensing rights for Apogossypol have been transferred to Coronado Biosciences, San Diego. Drs. Reed and Pellecchia are consultants and shareholders for Coronado Biosciences, hence declare a possible conflict of interest.
Acknowledgements
We thank NIH (Grant No. U01 AI061139) and Coronado Biosciences (CSRA #08–02) for financial support and the NCI-RAID program for technical and scientific support.
References (26)
- et al.
Apoptosis: a link between cancer genetics and chemotherapy
Cell
(2002) - et al.
Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells
Biochem. Pharmacol.
(2003) - et al.
Rational design and real time in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-xL
Chem. Biol.
(2004) - et al.
Bcl-2 antagonist ApoGossypol (NSC736630) displays single-agent activity in Bcl-2 transgenic mice and has superior efficacy with less toxicity compared with Gossypol (NSC19048)
Blood
(2008) - et al.
Quantitative determination of apogossypol, a pro-apoptotic analog of gossypol, in mouse plasma using LC/MS/MS
J. Pharm. Biomed. Anal.
(2006) - et al.
Stereo-specific cytotoxic effects of gossypol enantiomers and gossypolone in tumor cell lines
Cancer Lett.
(1999) - et al.
Development and validation of a genetic algorithm for flexible docking
J. Mol. Biol.
(1997) Dysregulation of apoptosis in cancer
J. Clin. Oncol.
(1999)Apoptosis-based therapies
Nat. Rev. Drug Discov.
(2002)Molecular biology of chronic lymphocytic leukemia: implications for therapy
Semin. Hematol.
(1998)
The Bcl-2 protein family: arbiters of cell survival
Science
BCL-2 family members and the mitochondria in apoptosis
Genes Dev.
Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells
Proc. Natl. Acad. Sci. USA
Cited by (27)
Single and dual target inhibitors based on Bcl-2: Promising anti-tumor agents for cancer therapy
2020, European Journal of Medicinal ChemistryCitation Excerpt :Several side effects may be related to the two reactive aldehyde groups. In order to reduce the side effects of levo enantiomer of gossypol, two reactive aldehyde groups were removed to generate new higher potent inhibitors, semi-synthetic derivatives, apogossypol and apogossypolone [49] (Fig. 4). Subsequently, a potent derivative of apogossypol, BI-97C1 (Sabutoclax), was found.
Targeting apoptosis pathways in lung cancer
2013, Cancer LettersCitation Excerpt :Gossypol, one of the first known BH3 mimetics, is a natural polyphenol derived from cotton plants that was found to bind the BH3 pocket of BCL-2, BCL-XL and MLC-1 to cause inhibition of their activities [46]. Molecular modeling and structure-based analyses has led to the development of an improved variant, named apogossypol, which also inhibits BLF-1 [47]. Further improvements have been recently reported leading to derivatives with EC50 concentrations in the nanomolar range in NSCLC cells [48].
Sabutoclax, a Mcl-1 antagonist, inhibits tumorigenesis in transgenic mouse and human xenograft models of prostate cancer
2012, Neoplasia (United States)Synthesis of benzyl substituted naphthalenes from benzylidene tetralones
2012, Tetrahedron LettersSynthesis of hemigossypol and its derivatives
2010, Tetrahedron Letters