Design, Synthesis And Biological Evaluation Of Novel Retinoid Derivatives As Multitarget Anticancer Agents

1. Research backgroundLeukemia, which seriously threatens human health, is a kind of common malignant disease of hematopoietic system and an epidemiological survey showed that this disease had constituted the leading cause of death due to cancer in children and persons under the age of 39 years. The pathogenesis of leukemia, which is dependent on the type of leukemia, is a complicated multi-stage process involved in a variety of factors. Non-random chromosomal translocations, hematopoiesis-related oncogenes or antioncogenes mutations are the important pathogenesises of human leukemia, as well as disorders of hematopoietic transcription factors or cytokine network can promote the development of leukemia. With the rapid development of modern molecular biology and correlated science, the molecular mechanisms of leukemogenesis will be further elucidated.Retinoids, as the representative drugs for the induction differentiation therapy of tumors, have been successfully used in the treatment of leukemia, which can serve as the ligands of retinoic acid receptors (RAR) or retinoid X receptors (RXR) in vivo to exert the antitumor effects, such as inducing tumor cell differentiation, promoting tumor cell apoptosis, and inhibiting tumor cell proliferation. Currently, retinoids have become one of the most common drugs for the treatment of leukemia, for example, the effective response rate to ATRA in the treatment of APL is of up to 70-80%. However, retinoid resistance, which can lead to "'Leukemia Treatment Failure", frequently occurred in the clinical applications of the existing retinoids. Therefore, it is crucial to develop novel retinoid derivatives to overcome drug resistance.Histone deacetylases (HDAC) are a class of key enzymes implicated in regulation of gene transcription, and histone deacetylase inhibitors (HDACI) have been demonstrated to be fairly effective for the treatment of several types of leukemias and some other types of cancers. HDACI can exert its anti-leukemic effects through a variety of mechanisms, such as blocking cell cycle progression, promoting cell differentiation, inducing cell apoptosis, inhibiting angiogenesis, inducing tumor immunity, and so on.Nitric oxide (NO) is an key cellular messenger molecule in vivo, which is involved in many physiological processes, including vascular relaxation, neurotransmission, immune responses, and etc.. A variety of studies have found that NO played an important role in the tumoricidal activity of the human immune system, and it is a cytotoxic or apoptosis-inducing agent against tumor cells under appropriate conditions of concentration, as well as could prevent cancer cells from metastasis and effectively overcome tumor cell resistance to conventional therapeutics. NO donors, as a source of exogenous NO. constitute a powerful way to supply NO for diseases therapy, and some of them displayed remarkable antileukemic activity, which might be expected to be lead compounds to develop a new type of antileukemic agents.Multi-target drug design, namely that the drugs are designed as single molecules to modulate multiple physiological targets simultaneously, directly or indirectly through metabolites, has become an important strategy in the field of drug design and discovery. Through the synergic multi-target actions, multi-target drugs might provide a significantly enhanced efficacy, reduced side effects or decreased drug resistance during the treatment of complex diseases, which are involved in multiple pathological pathways, such as cancers, cardiovascular diseases, and neurodegenerative diseases. Generally, multi-target drugs might be obtained by conjugated pharmacophore approach, namely that two distinct pharmacophores (or drugs) are joined into one molecule by a cleavable or non-cleavable spacer. Another way is merged pharmacophore approach that two distinct pharmacophores (or drugs) are overlapped by taking advantage of structural commonalities to form a simpler molecule with multi-target actions.2. Rational drug design of target compoundsConventional multi-target therapy, such as combination chemotherapy, is an important basis for the development of multi-target drug design. A series of studies have shown that the combined treatment with RAR ligands and RXR ligands can result in a synergistic effect, which can bring out more potent proliferation-inhibiting activity, apoptosis-inducing activity and differentiation-inducing activity on tumor cells, as well as the combined treatment with RAR agonists (such as ATRA) and HDAC inhibitors (such as butyrate, valproic acid, SAHA, FK228, MS-275, and TSA) can afford remarkably improved efficacy, reduced side effects and particularly decreased retinoid resistance against acute leukemia (such as APL). In addition, the combination of retinoids (such as ATRA) with some other antitumor drugs, such as As2O3 and cisplatin, can also produce a synergistic effect to improve the efficacy against leukemia or solid tumors.In our work, on the basis of an extensive literature review, we have designed four series of 52 novel retinoid derivatives as multi-target agents through the pharmacophore combination approach under the guidance of the multitarget-directed drug design strategy, in which tamibarotene (AM80). a RARa selective agonist, was used as a basic model drug to be coupled with N,N-dimethyl ethanolamine, RXR ligands, HDAC inhibitors, or furoxans as NO-donors through various spacers, such as diols, diamines, ethanolamine, and diverse amino acids. Among these new multi-target compounds, we hope to find out more potent anti-leukemic agents with better efficacy and lower toxicity.3. Synthesis of the designed target compoundsIn the present study, four series of 52 novel target compounds,4 model drugs and 72 key intermediates were synthesized, and then all of the target compounds, the model drugs and some key intermediates with novel structures were characterized by 1H-NMR, ESI-MS, HR-MS and IR spectra.According to relevant literature, four model drugs, including tamibarotene (AM80), bexarotene(LGD1069),4-(3,5,5,8,8-pentamethy1-5,6,7,8-tetrahydro-naphthalene-2-carbonyl) benzoic acid (LG) and valproic acid (VPA), were synthesized and then improved synthesis processes for them were also developed. AM80-DMEA derivatives were prepared by the esterification reactions of the RARa agonist AM80 as the starting material with DMEA as a choline precursor, followed by salification reactions of the tertiary amine structure of DMEA moieties with various inorganic acids or organic acids. AM80-HDACI derivatives were synthesized by the acylation reactions of the acyl chloride compounds of HDAC inhibitors, butyric acid or valproic acid as the original material with a single hydroxyl group or amino group from various diols, diamines or ethanolamine, followed by condensation reactions with AM80. AM80-RXR-agonist derivatives were obtained by the condensation reactions of the RXR agonist bexarotene or LG as the source material with a single hydroxyl group or amino group from various diols, diamines or ethanolamine, followed by condensation reactions with AM80. AM80-NO-donor derivatives were prepared by multi-step reactions from benzaldehyde as the starting material. First,3-(hydroxymethyl)-4-phenyl-1,2,5-oxadiazole-2-oxide, as a furoxan NO-donor, was obtained by Claisen-Schmidt condensation reaction, NaBH4 reduction, and nitrosation reaction from benzaldehyde, followed by chlorination, etherification, hydrolysis, and esterification reactions to produce a variety of key intermediates, which were interacted with AM80, AM80-diol derivatives, AM80-diphenol derivatives, AM80-diamine derivatives or AM80-ethanolamine derivatives to give the expected target compounds.4. Biological evaluation of the synthesized target compoundsIn vitro pharmacodynamic screening of 52 target compounds was conducted in human leukemic HL-60, NB4 and K562 cell lines, and In vivo preliminary pharmacodynamic evaluation of the representative target compounds 21 d and 51 p was also investigated in non-obese diabetic-severe combined immunodeficiency (NOD-SC1D) mice. Furthermore, a preliminary phannacokinetic study for the representative compound 21 d was carried out in Wistar rats. In addition, a variety of experiments were performed to evaluate the metabolic characteristics of the representative target compounds, including in vitro NO release experiments of AM80-NO donor derivatives, in vitro metabolism experiments of the representative target compounds 31q,41 p and 53f in rat liver microsomes incubation system, and so on.The cell proliferation assay showed that most of the AM80-DMEA derivatives exhibited higher antiproliferative activity against HL-60, NB4 and K562 cell lines than the control AM80. Especially, compounds 21d,21 e and 21f, which exhibited about 40,60 and 50 fold respectively stronger antiproliferative activity against HL-60 cells than AM80, showed good potential to develop into drugs. Within the AM80-HDACI derivatives, compound 31s exhibited significantly stronger antiproliferative activity against HL-60, NB4 and K.562 cell lines than the control AM80, compound 31 n exhibited higher antiproliferative activity against HL-60 and K562 cells than AM80, and compounds 31m and 31u displayed more potent antiproliferative activity against K562 cells than AM80. However, most of the AM80-RXR agonist derivatives showed less antiproliferative activity against HL-60, NB4 and K562 cell lines than the positive drug AM80, and only compound 41m exhibited slightly stronger antiproliferative activity against HL-60 and K562 cell lines than AM80. All of the AM80-NO-donor derivatives exhibited higher antiproliferative activity against the acute myeloid leukemic HL-60 and NB4 cell lines than against the chronic myeloid leukemic K562 cell lines, and six of the target compounds (51v,52p,52s,52t,53f and 53g) displayed significantly stronger antiproliferative effects against HL-60 and NB4 cells than the positive drug AM80, while four compounds (51v,52p,52t and 53t) exhibited more potent antiproliferative activity against K562 cells than AM80. In particular, compound 52p exhibited the best antiproliferative activities against HL-60, NB4 and K562 cells with the IC50 of 0.16μM,0.19μM and 7.12μM respectively, which were respectively 56 times,25 times and 6 times stronger than that of AM80. Altogether, the target compounds with excellent biological activity accounted for over 50% of the total number of AM80-NO-donor derivatives, and it was suggested that NO-donor retinoic acid receptor agonist hybrids as multi-target agents might bring out more potent antileukemic activity.The results from pharmacodynamic studies in NOD-SC1D mice showed that the representative target compounds 21d and 51 p exhibited similar antileukemic effects with the control drug AM80, while compound 21d, compared to AM80, exhibited better water solubility and lower skin irritation, which could be made into injection to afford the improved bioavailability and could be expected to conduct in-depth development as a me-too drug. Furthermore, the results from pharmacokinetic study in Wistar rats showed that the representative target compound 21 d was more easily absorbed than AM80 under oral administration, as well as it could be completely metabolized in the body to release the parent drugs AM80 and DMEA to exert a synergistic efficacy under injection administration.The results from in vitro metabolism studies in rat liver microsomes showed that all of the representative target compounds 31q,41 p and 53f had undergone a significant metabolic degradation in rat liver microsomes incubation system and their major metabolites were AM80. so it was suggested that hydrolysis was the major metabolic reactions for three representative target compounds. Nitric oxide release test in vitro showed that all of the AM80-NO-donor derivatives could release a significant level of nitric oxide in PBS solution containing 3.1mmol·L-1 L-cys, particularly, compounds 52p-52t, which containing various amino acids as spacers, generally have a relatively large percentage of NO released.5. ConclusionIn conclusion, we have designed and synthesized four series of novel antileukemic multi-target agents by pharmacophore combination approach under the guidance of the multitarget-directed drug design strategy, and have also conducted a preliminary biological activity evaluation and SAR analysis of these target compounds. More than ten of the target compounds exhibited more potent antiproliferative activity against human leukemic cell lines than the control AM80. These findings might provide a new insight for the development of novel antileukemic drugs with multi-target actions.