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J. Med. Chem. 2010, 53, 660–668 DOI: 10.1021/jm901237x
Design, Synthesis, and Antihepatitis B Virus Activities of Novel 2-Pyridone Derivatives Zhiliang Lv, Chunquan Sheng,* Tiantian Wang, Yikai Zhang, Jia Liu, Jilu Feng, Hailing Sun, Hanyu Zhong, Chunjuan Niu, and Ke Li* Department of Medicinal Chemistry, College of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China Received August 18, 2009
A series of novel 2-pyridone derivatives were synthesized and evaluated for their antihepatitis B virus (HBV) activity and cytotoxicity in vitro. Moderate to good activity against HBV DNA replication was observed in these 2-pyridone analogues. The most active compounds were 5d and 6l, with good inhibitory activity against HBV DNA replication (IC50 = 0.206 and 0.12 μM, respectively) and remarkable high selectivity (selectivity indexes of >532 and 467, respectively). A pharmacophore model of the synthesized compounds was proposed by the GASP program. The pharmacophore model consists of three hydrophobic points, four HBA points, and one HBD point. The 2-pyridone derivatives represent a novel class of HBV inhibitors, which are worth further optimization. Introduction a
Hepatitis B virus (HBV ) is a cause of chronic liver disease worldwide, affecting more than 5% of the world’s population.1 HBV can cause both acute and chronic infections. Chronic HBV infection leads to liver damage, cirrhosis, and liver cancer with high mortality. There are approximately 350-400 million who are chronically infected, resulting in 0.5-1.2 million deaths annually.2,3 HBV infection represents a serious global medical, social, and economic burden. Important viral-encoded genes include the envelope protein, hepatitis B surface antigen (HBsAg), the primary serum marker of viral infection; a structural nucleocapsid, hepatitis B core antigen; and a soluble nucleocapsid protein, hepatitis B e antigen (HBeAg), which combined with serum levels of HBV DNA are serum markers of active viral replication.2 The replication of HBV shares unusually similar features with retroviruses, and eradication of HBV infection turns out to be difficult because stable, covalently closed circular DNA (cccDNA), which is relatively resistant to antiviral action and immune clearance, becomes established in hepatocyte nuclei and integrated into the host genome.2,4 Seven drugs have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of HBV infection either as monotherapy or in combination. These drugs are categorized as interferons (interferon-R and pegylated interferon R-2a) and nucleotide analogues (lamivudine, telbivudine,5 entecavir,6 adefovir and tenofovir3,7) (Figure 1). Until now, the major therapeutic option for HBV carriers was R-interferon. Unfortunately, its use is limited because the success rate is low, the therapy is expensive, and serious side effects (such as *To whom correspondence should be addressed. For C.S.: phone/ fax, 86-21-81871233; e-mail,
[email protected]. For K.L.: phone/ fax, 86-21-81871237; e-mail,
[email protected]. a Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; cccDNA, covalently closed circular DNA; SARs, structure-activity relationships; SI, selectivity index; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; vdW, van der Waals.
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Published on Web 12/15/2009
influenza-like symptoms, depression, and insomnia) are observed.8,9 Lamivudine has been used for the treatment of HBV infection for a few years. It is orally effective and well tolerated.10 However, the emergence of resistance to lamivudine has been reported.11 Adefovir dipivoxil was approved by the U.S. Food and Drug Administration in 2002 for the treatment of chronic hepatitis B infection. Adefovir dipivoxil can reduce serum HBV DNA levels significantly and can lead to seroconversion in 20-27% of the patients treated for 12 weeks at a daily dose of 30 mg or greater.12 However, adefovir dipivoxil therapy is limited by dose-related side effects such as nephrotoxicity, lactic acidosis, and severe hepatomegaly with steatosis.13 More recently, telbivudine and entecavir were marked with pharmacological profile. However, their chemical structures are similar to those of lamivudine and adefovir dipivoxil. They may act by the same biological mechanism and encounter the same problems as lamivudine and adefovir dipivoxil. Therefore, there is a tremendous clinical need to develop novel classes of antiviral agents for the treatment of HBV infection. In the screening of an in-house library for anti-HBV activity, we identified 5-(2-hydroxy-4-methoxybenzoyl)-1-phenylpyridin-2(1H )-one (5a) as a modest inhibitor of HBV, with an IC50 value of 20 μM in inhibiting HBV DNA replication and low cytotoxicity (CC50=179 μM) in vitro. Because the chemical scaffold of compound 5a differs from those of all reported antiHBV agents, we were interested in synthesizing its derivatives and analyzing their structure-activity relationships (SARs). In the present investigation, we designed and synthesized a new series of 5-(2-hydroxy-4-methoxybenzoyl)pyridin-2-one derivatives with compound 5a as the starting point. The antiHBV activities of the compounds were investigated, and a pharmacophore model was built to guide further structural optimization. Chemistry The synthetic route of compound 4 was depicted in Scheme 1. 3-Iodo-7-methyloxy-4H-chromone (3) was synthesized by a reported procedure.14 Coupling of compound 3 r 2009 American Chemical Society
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 661
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Figure 1. Chemical structures of the anti-HBV agents and lead compound 5a.
Scheme 1. Synthesis of the Key Intermediate 4a
Reagents and conditions: (a) ether, Na, ethyl formate, 0 °C, 8 h, yield 95%; (b) CH3COOH, conc HCl, 100 °C, 30 min, yield 98%; (c) piperidine, methanol, reflux, 3 h, yield 99%; (d) dichloromethane, I2, room temp, overnight, yield 97%; (e) PdCl2(Ph3P)2, CuI, K2CO3, THF/H2O, 100 °C, 10 h, yield 80%. a
with methyl acrylate by a Heck reaction15,16 afforded the key intermediate 4. In order to establish a robust synthetic procedure of compound 4, the reaction conditions were optimized. Various solvents, bases, metal catalysts, and temperatures were investigated. The mixed solvent (THF/H2O = 9:1) was found to be the best choice with K2CO3 as the base and PdCl2(Ph3P)2 as the metal catalyst. The optimal reaction temperature and time were 100 °C and 10 h, respectively. In the presence of triethylamine, target compounds 5a-s were obtained by reacting intermediate 4 with various substituted phenylamines and alkylamines (molar ratio of 1: 1) via three sequencing steps in one pot. First, the ester group was ammonolyzed by amines. Second, the chromone ring was opened by the attack of the amine group. Last, the 2-pyrinone ring was formed by a rearrangement process. When intermediate
4 was reacted with excess benzylamines and alkylamines, compounds 6a-o were obtained. However, reacting compound 4 with excess phenylamines could not afford corresponding Schiff bases. In order to determine the configuration of the Schiff base, the crystal structure of compound 6c was determined. Figure 2 shows the ORTEP drawing of compound 6c, indicating that the Schiff base is in cis-configuration. Results and Discussion In Vitro Virological Examination and Preliminary Structure-Activity Relationships (SARs). The potential anti-HBV activity and cytotoxicity of the synthesized 2-pyridones were tested in HepG2 2.2.15 cells, and adefovir was used as a reference antiviral drug. The results are summarized in Table 1. The anti-HBV activity of each compound was
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expressed as the concentration of compound that achieved 50% inhibition (IC50) of HBsAg, HBeAg, and HBV DNA replication. The cytotoxicity of each compound was expressed as the concentration of compound required to kill 50% (CC50) of the HepG2 2.2.15 cells. The selectivity index (SI), an important pharmaceutical parameter that estimates possible future clinical development, was determined as the
Figure 2. ORTEP drawing of compound 6c.
ratio of CC50 value to IC50 value. The bioactivity of each compound was evaluated by the combination of its IC50 values and SI. Compounds 5a-l have different patterns of substitution on the phenyl ring attached to the N atom of 2-pyridones. As shown in Table 1, these compounds generally exhibited moderate to good anti-HBV potency. Various substitutions on the phenyl ring are important for the antiviral activity. 4-Ethoxyl (compound 5d) and 3-methyl (compound 5f) analogues showed excellent anti-HBV activity with their IC50 values against HBV DNA replication of 0.206 and 0.8 μM, respectively, which were more potent than reference drug adefovir. They also showed higher inhibitory activity against HBsAg and HBeAg than adefovir. Moreover, compounds 5d and 5f were almost nontoxic against HepG2 2.2.15 cells, which resulted in their high SI values. When the 3-methyl group of compound 5f was moved to position 2 (compound 5g) and position 4 (compound 5e), their anti-HBV activity was decreased by 6- to 14-fold. 2,5-2CH3 derivative (compound 5h) also showed good inhibitory activity against HBV DNA replication (IC50 = 0.5 μM). However, it was very toxic with a CC50 value of 0.5 μM (SI = 1). If the 4-methyl group of compound 5e was replaced by a chlorine atom (compound 5i), the inhibitory activity against HBV DNA replication was decreased slightly (IC50 = 6.2 μM).
Table 1. In Vitro Anti-HBV Activity and Cytotoxicity of the Target Compounds HBsAg
HBeAg
DNA replication
compd
CC50 (μM)a
IC50 (μM)b
SIc
IC50 (μM)b
SIc
IC50 (μM)b
SIc
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o adefovir
179 346 >113.9 >109.6 >119.4 159 >119.4 0.5 164 >117.9 177 133 15 131 67 79 696.1 232 244 102 51 8 10 10 201.4 32 20 387 164 120 56 98 >121.9 20 540
176 >113.9 >113.9 >109.6 >119.4 29 18 13 61 >117.9 >109.3 >110.2 99 >139.3 >132.7 46 846.2 276 77 40 72 13 >86.9 13 496.5 36 >117.8 201 123 >121.9 111 29 >121.9 98 305
1.02 d d d d 5.48 >6.63 0.04 2.69 d 86.9 40 >402.8 2 >117.8 71 10 >121.9 56 5 54 40 286
2.36 1.56 >2.3 >1.99 >59.7 530 >1194 0.01 102.5 d 10.85 1.0 26.45 >1.2 d 11.88 0.15 3.93 2.34 3.95 4.09 2.3
