Article pubs.acs.org/jmc
Discovery of an Orally Active and Liver-Targeted Prodrug of 5‑Fluoro-2′-Deoxyuridine for the Treatment of Hepatocellular Carcinoma Youmei Peng,†,‡,# Wenquan Yu,†,# Ertong Li,† Jinfeng Kang,† Yafeng Wang,§ Qinghua Yang,§ Bingjie Liu,† Jingmin Zhang,§ Longyu Li,§ Jie Wu,† Jinhua Jiang,‡ Qingduan Wang,‡ and Junbiao Chang*,† †
College of Chemistry and Molecular Engineering and §School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, People’s Republic of China ‡ Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, People’s Republic of China S Supporting Information *
ABSTRACT: We report a series of novel O-(substituted benzyl) phosphoramidate prodrugs of 5-fluoro-2′-deoxyuridine for the treatment of hepatocellular carcinoma. Through structure optimization, the o-methylbenzyl analog (1t) was identified as an orally bioavailable and liver-targeted lead compound. This lead prodrug is well-tolerated at a dose up to 3 g/kg in Kuming mice via oral administration. An efficacy study demonstrated that it possesses good inhibitory effect (61.67% and 72.50%, respectively) on tumor growth in a mouse xenograft model. A metabolism study in Sprague−Dawley rats suggested that 1t can release the desired 5′-monophosphate in the liver with high liver-targeting index.
1. INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most lethal cancers and the third most common cause of cancer-related deaths globally with 626,000 new patients per year.1,2 The current therapeutic options for HCC include resection, transplantation, radiofrequency ablation, chemoembolization, and sorafenib (4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino] phenoxy]-N-methyl-pyridine-2-carboxamide).3 However, most HCC cases are diagnosed in their advanced stages, which strictly limits the treatment options.4 As a multikinase inhibitor, sorafenib is the only systemic therapy for advanced HCC, but its actual benefit is still relatively small.1 The difficulties associated with chemotherapy in HCC may derive from drug resistance mechanisms, such as rapid drug metabolism via primary and/or secondary metabolism pathways, removal of drugs from tumor cells by drug transport systems, and inactivation of oncolytic drugs by intracellular agents such as glutathione. These mechanisms limit the uptake and retention of the drug molecules in tumor cells and consequently decrease their overall therapeutic benefit.5 A major reason for the inefficacy of nucleoside antitumor drugs is that they cannot be efficiently phosphorylated by specific nucleoside kinases.5,6 On the other hand, nucleoside © XXXX American Chemical Society
phosphates, or nucleotides, cannot themselves be used as viable drugs due to their poor chemical stability along with high polarity that vitiates their cellular uptake.7,8 In order to improve the potency of nucleosides, numerous prodrugs have been designed by masking the monophosphates thus bypassing the first-step phosphorylation, which is rate-limiting. Previously, phosphate and phosphonate prodrugs containing cyclic 1,3propanyl esters have been reported. These can be activated via cytochrome P450-catalyzed oxidative cleavage reaction in the liver to form higher concentrations of the biologically active nucleoside triphosphate.6,9−11 However, prodrugs of this type will also produce a potentially toxic byproduct, aryl vinyl ketone, which may cause cytotoxicity and genetic toxicity.12,13 Aryloxy phosphoramidates, another type of prodrug, can partially avoid the dependence of the current drugs on active transport, saturate efflux transportion and nucleoside kinasemediated activation,14,15 and they are also resistant to metabolic deactivation.15 But once the carboxyl ester is hydrolyzed by esterases in blood and nontargeted tissues, the phenol group in the phosphate moiety is cleaved spontaneously by an Received: November 19, 2015
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DOI: 10.1021/acs.jmedchem.5b01807 J. Med. Chem. XXXX, XXX, XXX−XXX
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intramolecular nucleophilic attack,16,17 resulting in an alanyl phosphate metabolite, which is a very polar diacid, and will be poorly transported across cell membranes to the targeted tissues. In addition, studies have shown that although teratogenic effects have not been associated with exposure to phenol, high doses of phenol are fetotoxic.18 This report describes the development of novel phosphoramidate prodrugs of 5-fluoro-2′-deoxyuridine (FDUR) for the treatment of HCC by oral administration. FDUR itself is of particular value in the clinical treatment of liver cancer,19−21 but like most nucleosides, it is inactive and requires phosphorylation to the primary bioactive entity, the 5′-monophosphate (FdUMP).15 FdUMP acts as a potent suicide-type inhibitor of thymidylate synthase, a key enzyme in DNA synthesis, which prompts a potent toxic event in the cell.22 In order to reach the therapeutic goal and minimize systemic toxicities, FDUR in clinical practice was administered via hepatic arterial infusion (HAI) to provide direct drug delivery into the tumor feeding vessels.20,21 Unfortunately, many liver cancer patients cannot tolerate a catheter inserted into the hepatic artery and a pump implanted under the skin. In this paper, we report the design and synthesis of a series of novel phosphoramidate prodrugs of FDUR bearing O(substituted benzyl) moieties (Scheme 1). Through structure
and efficacy of compound 1t were also assessed in Kunming (KM) mice and in a mouse xenograft model.
2. CHEMISTRY Prodrug 1 was synthesized as outlined in Scheme 2. Treatment of a benzyl alcohol (2) with phosphoryl oxychloride (POCl3) in the presence of triethylamine gave the phosphorodichloride (3), which is further treated with benzylamine to provide the chlorophosphoramidate (4). Finally, the reaction of 4 with FDUR (5) in the presence of N-methylimidazole (NMI) afforded the desired prodrug 1a−r. The analog 1s was synthesized by using L-alanine isopropyl ester hydrochloride in place of benzylamine (Scheme 3). To synthesize the RP-enantiomer (1t) of the racemic prodrug 1s, (SP)-O-perfluorophenyl phosphoramidate 7 was prepared by the procedure used for the synthesis of 1s. Treatment of phosphorochloridate 4s with pentafluorophenol gave diastereomers 6 with the SP-enantiomer as the major isomer. The enantiomer 7 can be obtained through recrystallization from a mixture of diisopropyl ether and petroleum ether (Scheme 3). Selective protection of the 5′- and 3′-hydroxy groups in the nucleoside 5 with DMTrCl and TBSCl, respectively, gave intermediate 9, which was treated with TFA to provide the 3′TBS protected nucleoside (10). Reaction of compound 10 with intermediate 7 in the presence of t-butyl magnesium chloride, followed by recrystallization in a mixture of ethanol and water afforded prodrug 1t (Scheme 4) as the pure RP-enantiomer (confirmed by X-ray, see the Supporting Information).
Scheme 1. Lead Optimization of the O-(Substituted Benzyl) Phosphoramidate Prodrugs of FDUR
3. RESULTS AND DISCUSSION 3.1. In Vitro Evaluation and Lead Optimization. First, we performed the optimization of the O-benzyl moiety in the newly designed prodrug structure. In view of the poor stability of the amino acid ester moiety in rodents,24 to blood circulating esterases, we initially synthesized the prodrugs (1a−r) containing a benzylamine group for optimization. These Nbenzyl phosphoramidate prodrugs are all quite stable in rat plasma (t1/2 > 24 h), the single exception being the O-(omethoxy)benzyl analog (1e) for which t1/2 = 3.4 ± 0.5 h. The activation rate in vitro of the prodrug analogs (1a−r) in rat liver S9 were determined by measurement of the formation of FdUMP, the primary bioactive entity, upon incubation for 0, 10, 20, 40, or 60 min with 50 μM of the tested compounds. As shown in Table 1, the unsubstituted O-benzyl analog (1a) showed a moderate activation rate for the formation of FdUMP. In most cases, incorporation of substituents in the benzene ring of the O-benzyl group resulted in higher rate of activation. Generally, compounds bearing electron-donating groups showed better activation rates than those with electronwithdrawing groups, and ortho-substituted analogs possess slightly better activation rates than the meta- and para-
optimization, the O-(o-methylbenzyl) analog (1t) was identified as an orally bioavailable lead compound which targets the liver.23 Metabolism studies in Sprague−Dawley rats demonstrated that this phosphoramidate prodrug can release the desired FdUMP in the liver with a high liver-targeting index and thus minimize extra hepatic exposure. The acute toxicity
Scheme 2. Synthesis of O-(Substituted Benzyl) Phosphoramidate Prodrugs (1)a−c
a
POCl3, Et3N, CH2Cl2, −78 °C. bBnNH2, Et3N, DCM, −78 °C to rt. cFDUR (5), NMI, CH2CI2, rt, overnight. B
DOI: 10.1021/acs.jmedchem.5b01807 J. Med. Chem. XXXX, XXX, XXX−XXX
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Scheme 3. Synthesis of O-(o-Methylbenzyl) Phosphoramidate Prodrug (1s) and (SP)-O-Perfluorophenyl Phosphoramidate (7)a−e
POCl3, Et3N, CH2Cl2, −78 °C. bL-alanine isopropyl ester hydrochloride, Et3N, DCM, −78 °C to rt. c5, Et3N, CH2Cl2, rt. dpentafluorophenol, Et3N, CH2Cl2, rt. erecrystallization in a mixture of diisopropyl ether and petroleum ether.
a
Scheme 4. Synthesis of the (SP)-O-(o-Methylbenzyl) Phosphoramidate Prodrug 1ta−e
a
DMTrCl, Py., 0 °C. bTBDMSCl, imidazole, DCM, rt. cTFA, DCM, rt. d7, t-BuMgCl/THF, 0 °C to rt. eTBAF/THF, rt.
toxicity study of the prodrug (1t) was conducted in KM mice. A single oral dose of the test compound at 2 and 3 g/kg was administered to the mice, and this was followed by observation for 15 days. During the study period, all mice remained alive, and no significant side effects were observed at either dose. Only negligible weight loss occurred at 1−5 days posttreatment (Figure 1). All the animals were sacrificed on day 15, and no damage to the heart, liver, spleen, lung or kidney was observed. These results demonstrated that orally administered prodrug 1t was well-tolerated in KM mice at dosage of up to 3 g/kg in the course of treatment and during the post-treatment period. 3.3. Assessment of Efficacy in KM Mice. For the assessment of the antitumor activity in vivo, a mouse xenograft model established with H22 cells in KM mice was employed. The tumor-bearing mice were dosed daily by oral gavage with prodrug 1t at 65, 130, or 260 mg/kg or sorafenib as the positive control (60 mg/kg) for 8 days (for detailed dose optimization see Section 5.7). The tumor weight was measured on day 8 and is given in Table 3. Compared to the control groups, the tested compound (1t) exhibited good inhibitory effect on the tumor growth. In particular, prodrug 1t at the dose of 130 or 260 mg/ kg significantly reduced tumor size with inhibitory rates of 61.67% and 72.50%, respectively, which are equally or more
substituted analogs. Cytostatic evaluation in vitro of these prodrugs with the SMMC7721 cell line identified the omethylbenzyl (1b) and o-methoxybenzyl analogs (1e) with potent inhibitory activities. Due to the lower stability of the omethoxybenzyl group (1e) in plasma, the o-methylbenzyl substituent (1b) was selected as the optimal structure design. Replacement of the N-benzyl group in compound 1b with the L-alanine isopropyl ester moiety led to the phosphoramidate 1s. Although replacement of the N-benzyl group with the Lalanine isopropyl ester moiety decreased the in vitro activity, study of the tissue distribution indicated that the amino acid ester analog possesses a much higher liver-targeting index (see Section 3.4). Consistent with the previous data,24 both the racemic prodrug 1s and its RP-enantiomer 1t have a short halflife ( 24 h, Table 2). Further evaluation demonstrated that prodrug 1t exhibited equally good inhibitory activity with the liver cancer cell line (SMMC7721) and slightly lower toxicity on a normal liver cell line (LO2) than its racemic form 1s. Besides, considering its superiority in synthesis and purification (see Section 5.1), we focused our further development efforts on this RP-phosphoramidate prodrug (1t). 3.2. Assessment of Acute Toxicity in KM Mice. Prior to the investigation of the in vivo anti-HCC efficacy, an acute C
DOI: 10.1021/acs.jmedchem.5b01807 J. Med. Chem. XXXX, XXX, XXX−XXX
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Table 1. Stability, Activation, and Cytostatic Activity of Prodrugs 1a−ra
a
Data are the means (±SD) of triplicate samples.
Table 2. Stability and Cytostatic Activity of Racemic Prodrug (1s) and Its RP-Enantiomer (1t)a
a
Data are the means (±SD) of two independent experiments, with each one performed in triplicates.
assess their liver-targeting indexes, compared to those of FDUR. Intragastric gavage (i.g.) administration of 52.17 mg/kg (0.096 mmol/kg) of 1t and 49.87 mg/kg (0.096 mmol/kg) of 1b produced 543 ng/g·h and
