Design, Synthesis And Biological Evaluation Of Rarα/Hdac-based Dual-target Anticancer Drugs

1. Research backgroundLeukemia, the malignant disease of hematopoietic system, has long been a threat to human life and health. With the development of the process of industrialization and environmental degradation, the incidence of leukemia, especially childhood leukemia incidence rate is rising.The pathogenesis of leukemia includes chromosomal translocations and hematopoiesis-related oncogenes or antioncogenes mutations. Signaling pathways affect the processs of leukemia. But due to the differences in pathogenesis of different types of leukemia, the cure methods become complexity and variability.Retinoids, the general term of more than4,000natural and synthetic compounds, include a variety of compounds which have enter the clinic and the first-line therapeutic agents of tumor treatment, such as all-trans retinoic acid (ATRA) and Tamibarotene(Am80). Am80is a selective RARa agonist developed by Japanese company Nippon Shinyaku in June2005(trade name:Amnolake).Histone deacetylase (HDAC), involved in the regulation of gene transcription with leukemia and other malignancies, is becoming the new target of cancer therapy: the discovery of the recruitment of HDAC enzyme by nuclear receptors in cancer provide a rationale for inhibition of HDAC activity to release transcriptional repression. Histone deacetylase inhibitors (HDACIs) is divided into six categories. HDACIs are amenable to the design of multi-targeted small molecules because their inherent chemical flexibility and synergy with various anticancer agents. HDACIs can release transcriptional repression caused by the specific oncogene product, and the leukemia treatment mechanisms include:causing cell arrest, differentiation and/or apoptosis of tumor cells, particularly those patients who were highly resistant to ATRA. The advantages of the histone deacetylase inhibitor is better targeted, accordingly toxicity smaller, the disadvantage is the low bioavailability and fast metabolism. Several HDACIs are now undergoing clinical trials and SAHA is the first FAD-approved pan-HDACi to enter the clinic.2. Rational drug design of target compoundsCurrently identified HDACIs shared the following structural character:Cap group, linker and ZBG (zinc binding group). Based on the3D structures of known HDACIs and binding modes of these compounds in complex with HDACs, according to the Principles of Hybridization and Prodrug, we designed and synthesized a series of novel HDAC inhibitors. The retinoic acid receptor RARα agonist Tamibarotene (AM80) was selected as a hydrophobic fragment of the new compounds; glycolic acid, glycine,6-aminocaproicacid and other connection chains of varying lengths as linkers; different hydroximic acid and carboxyl structure as Zn2+binding group. We hope that these new compounds play the role of combined treatment:at first the compounds could maintain the utility structure of HDACIs and have the activity of it, then the Am80can be disposed to play the activity of the Am80in vivo.3. Synthesis of designed target compoundsThe important intermediate AM80was synthesized using2,5-Dimethyl-2,5-hexanediol as starting material through a reaction sequence including halogenation reaction, Friedel-Crafts alkylation, nitration, catalytic hydrogenation, acalytion, condensation and hydrolysis. The condensation of Am80with o-phenylenediamine, hydroxylamine hydrochloride, hydroxy acetic acid and glycin led to all the target compounds. The structures of target compounds and important intermediate were identified by'H-NMR, ESI-MS spectra4. Biological evaluation of the synthesized target compoundsPreliminary activity assay was carried out in vitro. Three kinds of enzymes were selected, which are HDAC, APN and Gelatinase. The results showed that most target compounds exhibited highly selective inhibition against HDAC as compared with APN and MMP-2. The compounds10b and12b were as potent as the positive control SAHA against HDAC. Compounds10b and12b were also assayed for their anti-proliferation activity towards ES-2, HCT116, MDA-MB-231, K562, HL-60, NB4and PC-3cell lines. MTT method was employed. The result showed that10b and12b exhibited higher antiproliferation activity against ES-2, PC-3, K562and HL-60cell lines than positive drug SAHA, and as compared with the AM80,10b have stronger antiproliferative activity against HL-60cell line and12b have stonger antiproliferative activity against NB4cell line.5. Pharmacokinetic studyThis study was performed to describe the compounds (10b,12b) concentration-time courses administrated either orally or intravenously to rats, to select an optimized administration scheme.12b and10b which have interesting bioactivity were administered (oral and i.v.) to rats to investigate whether it is actually metabolized to tamibarotene(AM80). The results show that after i.v. administration,10b kept the utility at first then disposed AM80after5minutes. Comparison of tamibarotene concentration profile after i.v. administration of10b, the MRT prolonged from4.44h to18.09h, t1/2from2.22h to3.88h, Tmax from0.08h to4h. These results indicate10b is possibly useful as a prodrug of AM80. Compared with iv administration,10b and12b all had low bioavailability after oral administration.6. Conclusion and prospectA series of novel HDAC inhibitors based on the scaffold of AM80were designed and synthesized. Preliminary enzyme activity assay and anti-proliferation assays of seven cell lines showed10b and12b possess potential inhibitory activity. Pharmacokinetic study showed that10b and12b, especially10b disposed AM80after i.v. administration and play the role of synergistic effects. These compounds are promising lead compounds for developing new generation of HDAC inhibitors as antitumor agents.