European Journal of Medicinal Chemistry 75 (2014) 267e281
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Mini-review
A review of non-nucleoside anti-hepatitis B virus agents Fan Zhang*, Gang Wang School of Pharmacy, Liaoning Medical University, No. 40, Section 3, Songpo Road, Linghe District, Jinzhou 121001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2013 Received in revised form 12 January 2014 Accepted 17 January 2014 Available online xxx
Hepatitis B Virus is the most common cause of chronic liver disease worldwide. Currently approved agents of chronic HBV infection treatment include interferon and nucleoside analogues. However, the side effects of interferon and the viral resistance of nucleoside analogues make the current treatment far from satisfactory. Therefore, new drugs with novel structures and mechanisms are needed. Recently, a number of non-nucleoside HBV inhibitors have been obtained from natural sources or prepared by synthesis/semi-synthesis. Some of them exhibited potent anti-HBV activity with novel mechanisms. These compounds provide useful information for the medicinal chemist to develop novel non-nucleoside compounds as anti-HBV agents. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Hepatitis B virus Inhibitor Non-nucleoside Small-molecule inhibitors Natural products Synthetic compounds
1. Introduction Hepatitis B virus (HBV) infection is a major cause of viral hepatitis worldwide [1,2]. According to the World Health Organization (WHO) reports, more than 2 billion people have been infected with HBV at some time in their lives. Among them, about 350 million people remain infected chronically and become carriers of the virus [3e5]. Every year, HBV infection accounts 1 million people deaths from cirrhosis, liver failure, and hepatocellular carcinoma. Currently, seven drugs have been approved by FDA for the treatment of HBV infection: interferon alfa, pegylated interferon alfa2a, lamivudine, adefovir, entecavir, telbivudine, and tenofovir [6e8]. Interferon alfa and pegylated interferon alfa-2a are immunomodulatory agents, and their potential mechanism of action is the restitution of the host immune system [9,10]. However, the need for parenteral administration, the poor long-term response, and the high frequency of adverse side effects make interferon not ideal [11e 13]. The mechanism of action of nucleoside analogues is suggested to be through the interaction of their triphosphate derivatives, formed after cellular metabolic transformation, with HBV DNA polymerase or reverse transcriptase as substrates and/or inhibitors [14]. Lamivudine is the first nucleoside analogue approved to treat chronic HBV infection. However, HBV generates drug-resistance to lamivudine easily, and the rate of the resistance increases with the elongation of the therapeutic duration [15,16]. Genotypic resistance * Corresponding author. Tel.: þ86 416 4673440. E-mail address: [email protected] (F. Zhang). 0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2014.01.046
can be detected in 14%e32% after 1 year of lamivudine treatment and increases with the duration of treatment to 60%e70% after 5 years of treatment. Among adefovir-treated patients, the cumulative incidence of resistance over 5 year has been reported to be 29% in HBeAgnegative patients and 42% in HBeAg-positive patients [17,18]. As the nucleoside analogues inhibit HBV replication through their unique interaction with HBV DNA polymerase, emergence of HBV variants resistant to antiviral therapy is facing all the nucleoside analogues [19e21]. The development of multidrug-resistant has increased the risk of exacerbation of liver disease. The emergence of drug resistance during long-term therapy with nucleoside analogues is almost inevitable and represents a clinical challenge. It is therefore important to continue research to identify new anti-HBV targets and discover novel compounds with unique mode of action for controlling viral replication as well as preventing drug resistance and its complications in the long term [22e30]. This review will focus on the medicinal chemistry of nonnucleoside HBV inhibitors. In the last several years in particular, many non-nucleoside compounds obtained from natural sources or prepared by synthesis/semi-synthesis have been reported as novel HBV inhibitors. 2. Natural products 2.1. Wogonin and its analogues Wogonin (1, Fig. 1) isolated from Scutellaria baicalensis was identified as an effective anti-HBV agent [31,32]. At a dose of 20 mg/
268
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
OH HO
O
OH OH
O
O HO
HO
O
O
OH
HO OH
O
HO OH OH
O
OH
O
2
1
HO
O
OH OH
O OH
O
4
3 OH
Fig. 1. Structures of wogonin, baicalein, hyperin and quercetin.
mL, wogonin displayed an anti-hepatitis B surface antigen (antiHBsAg) activity with an inhibition percentage of 45.4 2.7%, furthermore, both the relaxed circular and the linear forms of HBV DNA were significantly reduced in the wogonin-treated group. Wogonin was also found to significantly reduce the secretion of hepatitis B e antigen (HBeAg) in human hepatoblastoma-derived liver Hep-G2 cells (HepG2.2.15 cells) with an IC50 value of 4 mg/ mL [33]. However, wogonin’s closely related analogue, baicalein (2), had minimal effects on HBsAg secretion in the assay. In wogonintreated ducks, plasma duck hepatitis B virus (DHBV) DNA levels were significantly reduced and remained lower than control level even at 10 days after end of the treatment. Wogonin also demonstrated anti-HBV activity in human HBV-transgenic mice, with persisted inhibition of HBsAg while there was a more rapid rebound of serum HBsAg level with lamivudine treatment. The polyphenolic extract from Geranium carolinianum L. was found to suppress the secretion of the HBV antigens and reduce HBV DNA level in vitro [34]. This extract also reduced the plasma and the liver DNA level in the DHBV infected ducks. Among isolated compounds from the extract, hyperin (3) showed significant inhibitory effect on both of HBsAg and HBeAg secretion, while quercetin (4) had no activity against HBeAg [35]. These results indicated that a galactose group at 3-position of quercetin enhanced the anti-HBV potency. 2.2. Helioxanthin and its analogues Helioxanthin (5, Fig. 2), an arylnaphthalene lignan lactone, was isolated from the root of Taiwania cryptomerioides [36]. Helioxanthin demonstrated an anti-HBV activity with an EC50 value of 1.0 mmol/L [37]. Modification of helioxanthin resulted in several derivatives with improved activity. Structure activity relationship (SAR) study revealed that the acid-hydrolysed derivative 6, the lactam derivative 7 and the cyclic hydrazide derivative 8 exhibited enhanced anti-HBV activity with EC50 values of 0.8, 0.08 and 0.03 mmol/L, respectively [37]. Introduction of halogen atoms at 5position of helioxanthin would reduce the antiviral activity. Modifications of the carboxylic acid and amide groups in compound 6
Artemisinin (20, Fig. 4) isolated from Artemisia annua had been widely used for antimalarial treatment [43]. Moreover, it was discovered to additionally use as anticancer, antiangiogenesis, antiviral, immunosuppressive, and antifungal agent. Recently, Romero and co-workers [44,45] revealed that artemisinin and its semi-synthetic derivative, artesunate (21), could induce strong inhibition of HBsAg secretion (IC50 values of 55 and 2.3 mmol/L, respectively) at concentrations at which host cell viability was not affected. When artesunate and lamivudine were administered together, synergic anti-HBV effects were also found in the assay. 2.4. Alisol analogues Six triterpenes, alisol A 24-acetate (22, Fig. 5), 25-anhydroalisol A (23), 13b,17b-epoxyalisol A (24), alisol B 23-acetate (25), alisol F
O O
O
O
O NH2 OH
O O
2.3. Artemisinin and its analogues
O
O
O
by dicarboxylic acid or diamide substituents, also resulted in a significant loss of activity. The replacement of a methylenedioxy group by two methoxy substituents in A ring exhibited more or less decreased activity. Further study revealed that the antiviral mechanism was completely different from nucleoside analogues [38,39]. The target of action was at the viral RNA transcription step and not at the HBV DNA polymerase. Compound 8 modified the interaction of hepatocyte nuclear factors (HNF) with HBV promoters, and down-regulated HNF expression in HBV-harbouring cells [40]. Janmanchi and co-workers [41,42] synthesized a series of helioxanthin analogues (Fig. 3) and their anti-HBV activity was evaluated. Modifications at the lactone rings and methylenedioxy unit of helioxanthin could modulate the antiviral activity. Among them, compound 13 was the most effective anti-HBV agent, which could suppress the secretion of viral surface antigen and e antigen in HepA2 cells with EC50 values of 0.06 and 0.14 mmol/L, respectively [41]. Compound 13 not only inhibited HBV DNA with wild-type and lamivudine-resistant strain but also suppressed HBV mRNA, core protein and viral promoters.
NH NH
NH O
O
O
O
O
A O O
5
O O
6
O
O O
7
Fig. 2. Structures of helioxanthin and its analogues.
O
8
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
Furthermore, epoxide group at 13(17)-position resulted in the decrease of suppressant property on the secretion of HBsAg and HBeAg. Among them, compound 31 showed high activities against secretion of HBsAg and HBeAg with IC50 values of 0.0048 and 0.011 mmol/L, respectively, and remarkable selective index values (SIHBsAg > 333 and SIHBeAg > 145, respectively) [49]. Anti-HBV activity of astragaloside IV (34, Fig. 7) isolated from Radix Astragali was reported by Wang and co-workers [50]. At dose of 200 mg/mL, astragaloside IV effectively suppressed secretion of HBsAg by 31.8% and HBeAg by 23.3% after 9 days of treatment. In Peking ducklings, it reduced DHBV levels in the serum with 64.0% inhibition and showed no significant toxicity at 120 mg/kg [50].
O R1
O
R2
O
O
O
O R2
O O
R1
12: 13: 14: 15: 16: 17: 18: 19:
9: R1 = R2 = CH2OH 10: R1 = CH2OAc, R2 = CH2OH 11: R1 = R2 = CH2 OAc
R1 = OCH3, R2 = OBn R1 = OCH3, R2 = OH R1 = OBn, R2 = OCH3 R1 = OH, R2 = OCH3 R1 = R2 = OBn R1 = R2 = OH R1 = R2 = OCH3 R1 = OCF3 , R2 = H
2.5. Matrine and its analogues Several matrine analogues (Fig. 8) isolated from the roots of Sophora flavescens and Sophora tonkinensis and semi-synthetic were investigated for their in vitro anti-HBV activity [51e53]. Compounds 37, 39, 41 and 42 exhibited a potential anti-HBV activity and low toxicity in HepG2.2.15 cells. SAR study revealed that the N / O group (40, 41) was not important element to keep the activity. Compounds with double bond in D ring, while without hydroxyl group, were more potent than other analogues. Introduction of a N / O group (41) or an a-hydroxyl group (43 and 44) into compound 42 (with unsaturated D ring) negatively influenced the antiHBV activity, especially the HBeAg inhibitory potency. As for compounds with saturated D ring, this influence was dramatically opposite. The presence of the N / O group (40) and a-substituted hydroxyl group (36) in matrine (35) markedly enhanced the potency. Moreover introduction of a proper substituent at the 13- and/ or 14-position(s) (37, 38, 39), especially electron-withdrawing groups, might enhance the activity. Matrinic acid (45) and oxymatrinic acid (46) with the D ring opening, still retained moderate anti-HBV activity. It was deduced that D ring might not be required for activity. Twenty-six Nsubstituted matrinic acid analogues were synthesized by Du and co-workers [54]. Among these analogues, compound 47 as host heat-stress cognate 70 (Hsc70) down-regulator exhibited a broadspectrum anti-HBV and anti-HCV effects with a mechanism different from that of nucleosides currently used in clinical patients [55]. SAR study showed that the carboxyl group at 11-position was required for activity and introduction of a substituent on nitrogen
Fig. 3. Structures of derivatives of helioxanthin synthesized by Janmanchi and coworkers.
O
H O
OH
O
O O
H O
H
O
O
H
O O
O O
H
H 20
21
Fig. 4. Structures of artemisinin and artesunate.
(26), and alisol F 24-acetate (27), were isolated from the rhizome of Alisma orientalis. They exhibited inhibitory activity against HBsAg with IC50 values range from 0.6 to 15.4 mmol/L. Alisol F 24-acetate also displayed significant activity against HBeAg with an IC50 value of 5.1 mmol/L and a SI value of 18.5 [46]. Ester derivatives of alisol (Fig. 6) were synthesized by Zhang and co-workers [47e49]. SAR study revealed that acetylation of the free hydroxyl group at 25-position was important for potent anti-HBV activity of alisol A derivatives and small acyloxy substituents at 11, 23, 24-positions may be preferred for anti-HBV activity.
HO
OH
HO
HO
H
HO
24 OH
HO
AcO
O
OAc OH
HO O
H
H H
H
23
O
O 25
OH
H O
22
O
HO
H O
H
O
HO
H O
OH
OH
OAc HO
269
H O
H 26
Fig. 5. Structures of six triterpene derivatives.
H 27
OH
270
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O O
O
O O
O
O
O
O
O
O
O O O
O H
H O
O
H
H O
H
28
30
O
O O
O O
O
O
O O
O
H
H O
H
31
OEt
O
H O
O
O
OMe
O
H O
O
O
O
O
O
O
O
O
O
H
29
O
O
O
O
O
O
H
32
33
Fig. 6. Structures of ester derivatives of alisol.
Among the alkaloids isolated from the ethanol extract, acutumidine (48, Fig. 9) and its analogues (49e51) showed moderate activity against HBsAg secretion with IC50 values range from 0.45 to 2.02 mmol/L [56]. Moreover, antiviral activities of N-methyl substituted derivatives were less potent than the corresponding non-substituted derivatives.
OH O OH
HO
O
HO
O
2.7. Chrysophanol and its analogues
OH
OH
O
OH O
34
Several chrysophanol and its analogues (Fig. 10) were isolated from Rheum palmatum L. ethanol extract and evaluated for their anti-HBV activity [57]. Among them, chrysophanol (52), physcion (53), aloe-emodin (54) and 8-O-b-D-glucoside (55) showed similar inhibitory activities against HBV DNA, HBsAg and HBeAg with IC50 values about 300 mg/mL [57]. However, 8-O-b-D-glucoside exhibited less toxicity with SI value more than 42. This result revealed that a galactose group at 8-position of chrysophanol could reduce the toxicity of these analogues.
OH OH
Fig. 7. Structures of astragaloside IV.
atom at 12-position, especially substituted benzyl, might significantly improve the anti-HBV activity.
2.8. Dehydrocavidine and its analogues 2.6. Acutumidine and its analogues Dehydrocavidine (56, Fig. 11), dehydeoapocavidine (57) and dehydroisoapocavidine (58) were isolated from Corydalis saxicola [58e60]. They demonstrated anti-HBsAg effect with inhibition
The ethanol extract of Hypserpa nitida Miers exhibited inhibitory activity against the section of HBsAg and HBeAg in an assay [56].
R3 14 R2 15 D 13 H 16 N 12 17 11 H 7 5 4 8 6 H H N 3 9 R1 1 2 X 10 O
35 : R 1=R2 =R3 =H 36 : R 1=OH, R 2=R 3=H 37: R 1=R3 =H, R2 =OC2 H 5 38 : R 1=H, R2 =R3 =OCH 2 C6 H5 39: R 1=H, R2 =OCOCH2 Cl, R3 =OH 40: R 1=R2 =R3 =H, X=O
O N
N X
41: 42: 43: 44:
N
R2
H H
R1
H
11 7
H
5
COOH
4
H R1
R1 =R 2 =H, X=O R1 =R 2 =H R1 =OH, R2 =H R1 =H, R 2 =OH
Fig. 8. Structures of matrine and its analogues.
3
H
N
H
2 X 10 45: R1 =H 46: R 1 =H, X=O 47: R1 =CH 2PhOCH3 -p
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O
O
OH Cl
O
O
NH
O
O
N
O
O
O O 49
48
O
OH Cl
N
OH Cl
O
NH
O
O
271
OH Cl
O
N
O
O
O
O
50
51
Fig. 9. Structures of acutumidine and its analogues.
B infection in China [62,63]. An active component, protocatechuic aldehyde (61, Fig. 12), had been approved to be a strong inhibitor of HBV. As it could significantly inhibit the production of HBV DNA (IC50 ¼ 4.17 mg/mL), HBsAg (EC50 ¼ 3.94 mg/mL) and HBeAg (EC50 ¼ 2.46 mg/mL), while there might be at least two targets of protocatechuic aldehyde [64]. Protocatechuic aldehyde also reduced the level of DHBV DNA in DHBV-infected ducks at dosage of 50 mg/kg.
O
O R1
R2 HO OH O
OH
OH O
52: R1 = H, R2 = CH3 53: R1 = CH3O, R 2 = CH 3 54: R1 = H, R2 = CH3O
OH
H
O
O
OH
H 55
2.11. Caudatin and its analogues
Fig. 10. Structures of chrysophanol and its analogues.
percentages about 50%, and anti-HBeAg effect with inhibition percentages about 40% [59]. Within the concentration range from 16.5 to 250 mg/mL, the inhibitory effect of these compounds was dose-dependent. The known alkaloid dihydrochelerythrine (59) was also found to be a potent inhibitor of HBV. Dihydrochelerythrine could inhibit the secretions of both of HBsAg and HBeAg with IC50 values less than 0.05 mmol/L and a CC50 value of 0.16 mmol/ L [58]. 2.9. Triterpenoid saponin Zhao and co-workers isolated a triterpenoid saponin (60, Fig. 12) from Potentilla anserina L., which was frequently used as Traditional Chinese Medicine (TCM) in hepatitis B treatment. Compound 60 exhibited anti-HBV activity against secretion of HBsAg (IC50 ¼ 57.67 mg/mL, SI ¼ 4.10), HBeAg (IC50 ¼ 30.05 mg/mL, SI ¼ 7.86) and HBV DNA (IC50 ¼ 19.45 mg/mL, SI ¼ 12.14) in HepG2.2.15 cells [61]. The levels of DHBV DNA in the Peking ducklings treated with compound 60 (0.2 g/kg body wt) for 10 days were markedly reduced by 58.48%, and a long-term effect of inhibiting HBV was also found in the assay.
Caudatin (62, Fig. 13) from Cynanchum auriculatum (Bai-ShouWu in Chinese) as a prospective anti-HCC drug with the mechanisms of inhibiting cell proliferation and inducing cell apoptosis had been reported [65e68]. In the anti-HBV assay, caudatin was found to have activities against the secretion of HBsAg and HBV DNA replication with IC50 values of 142.67 mmol/L (SI ¼ 1.7) and 40.62 mmol/L (SI ¼ 6.0) in HepG2.2.15 cells, respectively [69]. A series of caudatin derivatives were synthesized by Wang and coworkers [69,70]. Most of the 3-O-substituted derivatives showed effective anti-HBV activity, especially inhibiting the HBV DNA replication with IC50 values range from 2.44 to 22.89 mmol/L [69]. However, the 3,8-O-di and 3,17-O-diacyl derivatives (63, 64) lost suppressant properties on the HBV and cytotoxicity, which suggested that the free hydroxyl groups were not only the necessary for the activity, but also the factor of leading the cytotoxicity. The oxidative derivative 66, expoxide 67 and reduction product 68 exhibited decreased anti-HBV activity compared to caudatin. Among these derivatives, compound 65 by attaching of the cinnamic acid to caudatin, displayed the most significant inhibitory effects on the secretion of HBsAg, HBeAg and HBV DNA replication with IC50 values of 5.52, 5.52, 2.44 mmol/L, respectively, and good safety (LD50 > 1250 mg/kg) [70]. Preliminary mechanism study proposed that compound 65 exerted antiviral effects via
2.10. Protocatechuic aldehyde Salvia miltiorrhiza was considered to be one of the most highly recommended and widely accepted medicines for treating hepatitis
R1 R2
O O
N
O
OH
O O
56 : R 1 = Me, R 2 = Me 57 : R 1 = Me, R 2 = H 58 : R 1 = H , R2 = Me
O
N
O
HO
H
O HO
O
HO HO OH O
O 59
Fig. 11. Structures of dehydrocavidine and its analogues.
OH 60
OH
HO
CHO
HO 61
Fig. 12. Structures of triterpenoid saponin and protocatechuic aldehyde.
272
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O
O
O O
O
O
O
OR 2
R1 O
OH O
O
O
OH
OH O
OH O
HO
67
68
O
63: R 1 = R2 = O
, R3 = H O
64: R 1 = R3 =
, R2 = H
N
O
H 3CO
65: R 1 =
HO
66
62: R 1 = R2 = R3 = H
OH
OH
OH
OH
OH
O
OH
OH
OR 3
O O
O
, R 2 = R3 = H
H 3CO OCH3 Fig. 13. Structures of caudatin and its analogues.
interfering HBV promoters and enhancers to influence HBV transcriptions.
presence of acetyl at 25-position was an important feature in conferring anti-HBV activity.
2.12. Hemslecin A and its analogues
3. Synthetic compounds
Hemslecin A (69, Fig. 14), a cucurbitane-type terpene, widely present in this genus had been used to cure inflammatory diseases in clinical practice [71]. Hemslecin A and its derivatives were evaluated for their anti-HBV activity in HepG2.2.15 cells [72]. Compound 69 showed moderate activity against HBV DNA replication (IC50 ¼ 11.4 mmol/L), with obvious toxicity (CC50 ¼ 66.2 mmol/L), which led to a low SI value (SI ¼ 5.8) [72]. Most of the derivatives displayed enhanced anti-HBV activity, of which compounds 70e75 exhibited not only significant activity inhibiting HBV DNA replication with IC50 values of 2.8e11.6 mmol/ L, but also high SI values. SAR study indicated that (1) mono- or di-acylation of hydroxyl group(s) at 2-position and/or 3-position resulted in enhancement of antiviral activity. (2) The acylation of hydroxyls at 2-, 3- and 16-position decreased cytotoxicity. (3) Epoxide of double bond at 5(6)-position decreased the cytotoxicity and maintained activity against HBV DNA replication. (4) The
3.1. 2,2-Bisheterocycles
HO O H
R1 O
H
O
OR 3
HO OAc
R2 O
HO HO
69: 70: 71: 72: 73:
R1 = R1 = R1 = R1 = R1 =
A library of subunits of leucamide A (76, Fig. 15), a bioactive cydicheptapeptide [73], were screened in several in vitro assays to determine their antiviral activity. Among them, compound 77 showed moderate activity against HBV DNA with an IC50 value of 76.4 mmol/L [74]. In order to investigate their SAR, many 2,2bisheterocycle compounds were synthesized and evaluated their anti-HBV activity. SAR study indicated that a bulky aliphatic group at 5-positon of the heterocycle B ring had an advantageous effect on the activity against HBV DNA replication. The inhibitory activity of acid analogue 78 (IC50 ¼ 60 mmol/L) substantially diminished compared with its ester analogue 79 (IC50 ¼ 2.4 mmol/L) and 80 (IC50 ¼ 1.1 mmol/L) [74]. The A ring could be replaced with the thiophene ring (81) or the benzo[b]thiophene ring (82). The most promising compound 83 showed significant potency
R2 = R 3 = H R 3 = H, R2 = AcO R2 = AcO, R 3 = H CH 3 CH 2CO, R2 = R3 = H R2 = R 3 = C 2H 5 OCH 2 CO
O H
H
O
OH
HO O H
OAc HO HO
O
74
O O
Fig. 14. Structures of hemslecin A and its analogues.
OH
H
OH 75
Cl
O OAc
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O
O N S
HN
O
O
N
N S A
NH O
HN
O
O N H
O
S
S
N
N
S O
N
78: R = H 79: R = Me 80: R = Et
S O
N
O
82
R
O
77
S
O
S
76
81
O B N
N
O
N
S
273
O
S
S
N
N 83
O O
Fig. 15. Structures of leucamide A and 2,2-bisheterocycle tandems.
(IC50 ¼ 0.14 mmol/L) against HBV DNA replication and was about 500-fold more potent than the lead compound 76 (IC50 ¼ 76.4 mmol/L), and even more potent than lamivudine (IC50 ¼ 0.26 mmol/L) [74]. These compounds were inactive at 200 mg/mL in HBV polymerase assays, indicating that the action mechanism did not involve inhibition of the DNA polymerase. 3.2. Acrylamide derivatives In a screening in the HepAD38 cellular assay, a series of phenylpropenamide derivatives (84e91, Fig. 16) displayed potent antiHBV activity with IC50 values less than 4 mmol/L and no general toxicity [75,76]. Among them, the E isomers showed weak activity or no activity against HBV. SAR study revealed that vinyl bromides were slightly more active and less toxic than the corresponding vinyl chlorides. In general, substitution at 4-position of A ring had little effect on the activity. Substitution at 4-position of B ring resulted in compounds with improved inhibitory activity. Unsubstituted piperidine and pyrrolidine amides were preferred. Additionally, AT-61 (84) and AT-130 (91) also showed antiviral activity against the YVDD lamivudine resistant variant in vitro [77]. AT-61 was highly selective, inhibiting only the replication of human
R1
A
A
R1 O
O N O
Cl HN
B
N O
Br HN R2
84 : R 1 = H, R 2 = H 85 : R 1 = 4-F, R2 = H 86: R 1 = H, R 2 = 4-F
B 87: R 1 = 88: R 1 = 89: R 1 = 90: R 1 = 91: R 1 =
R2 4-Me, R 2 = H H, R 2 = 4-F H, R 2 = 4-Cl H, R 2 = 4-NO2 2-MeO, R 2 = 4-NO2
Fig. 16. Structures of phenylpropenamides.
HBV but not the replication of other viruses. Mechanism of phenylpropenamide derivatives was initially considered to inhibit HBV replication by interfering with the packaging of pregenomic RNA into the immature core particles not the HBV polymerase [78,79]. Subsequent studies revealed an assembly effector mechanism underlying the apparent blocking of RNA packaging [80]. Katen and co-workers had determined that AT-130 bound to a hydrophobic pocket located at the contact between B and C-dimers of the HBV capsid protein and induced subtle tertiary and quaternary structure changes that resulted in morphologically normal capsides without the reverse transcriptase pregenomic RNA complex [81]. The bioisoteric replacement of the phenyl in phenylpropenamide with 1,2,3-thiadiazolyl (Fig. 17) resulted in a new class of acrylamide analogues, which possessed the similar SAR with the phenylpropenamides. Compounds 92e94 that replaced A or B ring by 1,2,3-thiadiazole still had anti-HBV DNA activity with IC50 values of 10.4, 3.59 and 9.00 mg/mL, respectively [82]. However, when A and B rings were simultaneously replaced by 1,2,3-thiadiazole (95), the anti-HBV activity was eliminated and the cytotoxicity was higher than that of monosubstituted analogues. 3.3. Imino sugars Imino sugars (Fig. 18), N-butyl-deoxy-nojirimycin (N-Bu-DNJ, 96) and N-nonyl-deoxy-nojirimycin (NN-DNJ, 97) had been shown to be effective against HBV in tissue culture and in the woodchuck model in several reports [83e87]. In order to understand the SAR of this series, Mehta and co-workers synthesized many imino sugar derivatives. These derivatives could be divided into two functional groups: an imino sugar head group and an alkyl tail. SAR study indicated that the head group played a critical role in antiviral activity. Variation of the sugar head group was allowed to a large extent maintained antiviral activity. As for the alkyl tail, the side chain length appeared to be critical, with antiviral activity decreasing sharply with side chains of fewer than eight carbons. Interruption of the side chain with oxygenation (N-7-oxa-decylDGJ, 100) also reduced antiviral activity. NN-DGJ (98) was also effective against the lamivudine resistant virus. Moreover, NN-DGJ did not inhibit DNA polymerase or envelop antigen production [88]. It either prevented the maturation of HBV nucleocapsids or destabilized the formed nucleocapsids [89].
274
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
R
N N S
A O
Br
N HN
N N S
O N
Br
O
HN
Br
N
O
HN
S N N 92: R = 2-Cl 93 : R = 4-Cl
O
O
O
S N N 95
94
Fig. 17. Structures of 1,2,3-thiadiazole substituted acrylamides.
HO
(CH2)3CH 3 N
HO
(CH2)8CH 3 N
HO
OH
HO
HO
OH
(CH2)8CH 3 N
(CH2)8CH 3 N
HO
HO
OH
OH
(CH2)6O(CH 2) 2CH3 N
HO HO
OH
OH
OH
OH
OH
OH
96
97
98
99
100
Fig. 18. Structures of imino sugars.
3.4. 2,5-Pyridinedicarboxylic acid derivatives
3.5. Triazoles
Lee and co-workers [90] synthesized a series of pyridinedicarboxylic acid derivatives and evaluated their anti-HBV activity. Some compounds (101e111, Fig. 19) showed potent inhibitory effect against HBV polymerase (TC50 values less than 10 mg/L) and low cytotoxicity (CC50 values more than 100 mmol/L), which indicated that these 2,5-pyridinedicarboxylic acids were excellent anti-HBV agents [90]. SAR study showed that isopropyl, tert-butyl and 3-imidazolylpropyl groups were preferred for excellent activity. The amino substituted by the isobutyl group resulted in compounds 103 and 109 with improved activity. This might be the only class of non-nucleoside HBV polymerase inhibitors until now.
As triazoles possessed significant antifungal and antibacterial properties, El-Barbary and co-workers [91] synthesized several new derivatives and evaluated their anti-HBV activity. Compounds 112, 113 and 114 (Fig. 20) showed a moderate activity against HBV with IC50 values of 35, 63 and 40 mmol/L, respectively, and CC50 values more than 100 mmol/L [91]. An oxadiazole compound 115 was identified as a HBV inhibitor with an EC50 value of 1.63 mmol/L [92]. It inhibited the expression of HBsAg and HBeAg in a concentration-dependent manner with no cytotoxic effect. The transcription of HBV was not affected by compound 115. It was likely that compound 115
H2 N
O HO
N H
N
N
R
N
N
HN
O N H
N N O
O 101
102: R = HOCH 2CH2 103: R = i-Pr
HN
O R
N H
N
104: R = 105: R = 106 : R = 107 : R =
HN
O N
N
N
N
N
O HOCH 2CH 2 HOCH 2C(CH 3) 2 2-pyridylmethyl 3-imidazolypropyl
R
N H
N N 108: 109: 110: 111:
N
N O R = CH 3OCH 2CH 2 R = i-Pr R = 3-cyclopropyl R = 3-imidazolypropyl
Fig. 19. Structures of 2,5-pyridinedicarboxylic acids.
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
N
N
N N
N S O AcO NH 2
Cl
NH2
N
N
S
OAc 112
275
OAc
OAc
N N S NH 2
Cl
114
113 O O
N O O S
N N
N
N
HS
O
N
S
O
NH
N
Cl
S
N N
117
116
115
Fig. 20. Structures of 1,2,4-triazoles, 1,3,4-oxadiazoles and 1,2,3-thiadiazoleacetamides.
interaction between the core protein and PreS region of the surface protein [94]. Among them, compound KKJ00626 (117) inhibited the production of HBV particles from transiently HBV-producing HuH7 cells with an IC50 value of 0.12 mmol/L [94].
affected events downstream of transcription in the viral replication process. In the investigation of 1,2,3-thiadiazole derivatives as antiviral agents, compound 116 was identified as a HBV inhibitor [93]. At 0.6 mmol/mL, compound 116 was found to reduce the secretion of HBsAg and HBeAg by 88.6% and 15.5%, respectively. In addition, compound 116 also inhibited HBV DNA with an IC50 value of 400 mmol/L and the possible mode of anti-HBV activity was inhibition of virus replication [93]. In a modified enzyme-linked immunosorbent assay, a chemical library consisting of 5600 compounds was screened for their
3.6. Benzimidazoles In a random screening, compound 118 (Fig. 21) was identified as an inhibitor of HBV with an IC50 value of 14.2 mmol/L against HBV DNA [95]. SAR study revealed that substituents, such as methyl, isopropyl, methoxy and nitro, were tolerated at the O
O N
N N S O O O 2N
O
Cl
N
Cl
N S O O
N O
Cl
N
Cl
N
N
Cl
O
R 124: 125: 126: 127: 128: 129:
N S O O
O
N N N
O
123
R
O H N
NH 2
N O S O
R=H R = 2-F R = 4-F R = 2,6-diF R = 2-Cl R = 4-Me
N
120
122 N
N H
Cl
N
N
121
O
N
O
O N
Cl
119
118
Cl
O
O
N
N NH 2
N O S O
130 : R = H 131: R = F
Fig. 21. Structures of benzimidazoles.
N O 132
NH 2 N O S O
276
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
R2 HO
R2 N
R1
N H
HO
N
SR1 N H 137: R 1 = CH2 CH3 , R 2 = 2-methylimidazol-1-yl 138 : R 1 = CH(CH 3 )2 , R 2 = imidazol-1-yl 139 : R 1 = CH(CH 3) 2, R2 = 2-methylimidazol-1-yl
S
133: R 1 = H, R 2 = imidazol-1-yl 134: R 1 = 4-Me, R2 = imidazol-1-yl 135: R 1 = H, R 2 = 2-methylimidazol-1-yl 136 : R 1 = 4-Me, R2 = 2-methylimidazol-1-yl
Fig. 22. Structures of 1H-benzimidazol-5-ols.
A group of 1-methyl-1H-benzimidazol-5-ol analogues (Fig. 22) with different Mannich side chains at 4-position of the benzimidazole core was synthesized and evaluated their anti-HBV activity in HepG2.2.15 cells [98,99]. Among them, imidazolyl derivatives (133e139) showed more potent inhibition of HBV DNA replication or the secretion of HBeAg than lamivudine with IC50 values between 7.8 and 44.5 mmol/L [98,99]. Besides, 2-methylimidazol-1-yl analogues (135, 136, 137, 139) had less cytotoxicity than corresponding imidazol-1-yl analogues (133, 134, 138). Converting sulfur into sulfinyl and sulfonyl groups, the cellular toxicity was obviously reduced, whereas a complete loss in anti-HBV DNA activity was also observed.
benzenesulfonyl group at N-1 position of the benzimidazole core. Removal of the N-1 substituent resulted in a tremendous loss of activity, while introducing 4-methylbenzoyl to N-1 position of the benzimidazole core would decrease the cytotoxicity. Replacement of the phthalimide group with maleimide yielded in compound 120 with improved activity. The alkyl linker was important for potent antiviral activity, and the methylene and propylene analogues were more toxic. Contrarily, replacement of sulfonyl group with alkyl substituents at N-1 position could dramatically increase antiviral activity and selectivity. The most promising compounds 121 and 122 showed potent anti-HBV activity (IC50 values of 0.9 and 0.7 mmol/L, respectively) and high selectivity (SI more than 111 and 714, respectively) [95]. In order to improve the water-solubility and oral bioavailability, eighteen novel derivatives of compound 121 were synthesized and evaluated for their anti-HBV activity and cytotoxicity in the HepG2.2.15 cell line [96]. Among them, compound 123 with IC50 < 0.41 mmol/L and SI > 81.2, was the most promising compound, nearly three times higher selectivity than lamivudine [96]. Introduction of large substituents at 5- or 6-position of the benzoimidazole ring resulted in a new series of benzimidazole derivatives (124e132) with anti-HBV activity [97]. SAR study showed that the substituent on the benzoyl ring was important at 6-position. The halide substituted analogues 125e128 demonstrated more active than the methyl substituted analogue 129. However, 5-position substituted derivative 130 without a substituent on the benzoyl ring was found to be more potent than the fluoro substituted derivative 131. The best compounds were 127 and 132 with IC50 values both of 0.70 mmol/L [97].
3.7. Indoles Arbidol (140, Fig. 23), licensed in Russia for treatment of influenza A and B infections, was reported to possess inhibitory activity of several viruses such as the respiratory syncytial virus, parainfluenza virus, rhinovirus and hepatitis C virus [100e105]. Recent studies extended its inhibitory activity to HBV with IC50 values of 22.85 mg/mL against HBV DNA and 47.45 mg/mL against HBsAg [106,107]. Its derivatives 141e145 also exhibited significant antiHBV activity with IC50 values less than 60 mg/mL [108e110]. SAR study revealed that introduction of fluoro or chloro group on the phenyl ring could improve anti-HBV activity. Oxidation of sulfide into sulfinyl resulted in compounds with increased activity. Mannich functionalities did not show significant distinctness of antiHBV activity effects. Bromo group at 6-position had little influence on activity and cytotoxicity. Arbidol was found to inhibit
N N
O
O N
O
HO
O
N
O
HO N
Br
S
N
S
F
N
Br
O 140
141
142 N
O
N
O
HO Br
F N
S
O
O
O 143
O
HO N
S O
O N
O
HO
F
Br
O
N
O
O
HO
S OO
144 Fig. 23. Structures of ethyl 5-hydroxy-1H-indole-3-carboxylates.
N
145
S OO
F
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
R Cl
Cl
R Cl
N H
OH
O
N H 149: R = 150 : R = 151: R =
146: R = H 147: R = F 148: R = Cl
Cl
277
O
nO N H 152: n = 153: n = 154 : n = 155 : n = 156: n =
O H F Cl
O 2, R 2, R 2, R 1, R 3, R
R =F = OCH 3 = CF3 = OCH 3 = OCH 3
R O
R Cl
Cl
NH 2 N H
N O N H
O
O
159: R = H 160: R = F
157: R = H 158: R = F
Fig. 24. Structures of 4-aryl-6-chloro-quinolin-2-ones and benzodiazepine-2-ones.
R
O
O
OH
N N H
S
NO2
161: R = H 162: R = Ac
O
N N H
S
R
OH
O
O
Cl
S
N H
163: R = Br 164 : R = Cl
OH
N
N N H
S
Cl
166
165
Fig. 25. Structures of nitazoxanide, tizoxanide, RM-4832, RM-4848, RM-4850 and RM-4852.
membrane fusion during influenza virus or HCV infection and suppress these viruses entry into target cells [101,111,112]. However, its model of action on HBV had not been disclosed until now. 3.8. Quinolin-2-ones and benzodiazepine-2-ones Cheng and co-workers [113,114] synthesized several 4-arylquinolin-2-one and 5-aryl-1,4-benzodizaepin-2-one derivatives. Compounds 146e160 (Fig. 24) inhibited the secretion of HBsAg with IC50 values range from 0.01 to 2.84 mmol/L. SAR study exhibited that compounds 146e148 were less active than corresponding hydroxyl ethyl derivatives 149e151, which revealed that introduction of hydroxyethyl to 3-position of 4-aryl-6-chloroquinoline-2-one was an important feature in conferring enhanced anti-HBV activity. In addition, ester derivatives 152e155 displayed optimal profile inhibiting not only HBsAg and HBeAg secretion but also HBV DNA replication. 3-Amino derivatives 157 and 158 exhibited increased cytotoxicity and activity. Preliminary mechanism study suggested that compound 153 mainly promoted the transcription activity of HBV enhancers ENⅠ and ENⅡ to exert antiHBV action. The ring-expansion derivatives 159 and 160 were also active against HBsAg secretion with IC50 values of 67 and 56 mmol/L, respectively [114]. 3.9. Thiazolides Nitazoxanide (161, Fig. 25), a drug with anti-infective activity against anaerobic bacteria, protozoa and viruses, was currently in
phase II clinical development for treating chronic hepatitis C [115e 118]. Recently, Korba and co-workers [119] had proved that nitazoxanide and its analogues tizoxanide (162), RM-4832 (163), RM4848 (164), RM-4850 (165) and RM-4852 (166) were potent inhibitors of HBV with EC50 values range from 0.12 to 1.2 mmol/L. Nitazoxanide and RM4850 were also effective against several HBV lamivudine-resistant and one adefovir dipovoxil-resistant constructs in transient transfection assays in HuH7 cells. 3.10. Quinolines Liu and co-workers [120,121] had reported several quinoline derivatives (Fig. 26) with potent anti-HBV activity. In 7hydroxyquinolines, compounds 167 (IC50 ¼ 3.5 mmol/L, SI ¼ 37.9) and 168 (IC50 ¼ 2.6 mmol/L, SI ¼ 61.7) were more potent than the positive control lamivudine (IC50 ¼ 343.2 mmol/L, SI ¼ 7.0) against
O O HO
N
R O S
O
R
O
N N
O
HO O F
N
S O F
167 : R = H 168: R = F
169: R = pyrrolidin-1-yl 170: R = 1H-imidazol-1-yl
Fig. 26. Structures of ethyl 6(7)-hydroxyquinoline-3-carboxylates.
278
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
F N
R1
Cl
176: R 1 = O
, R 2 = OH F
R2 Cl
171: R 1 = 172: R 1 = 173: R 1 = 174: R 1 = 175: R 1 =
177: R 1 =
OMs, R 2 = OMs Cl, R 2 = OAc OCH 3 , R 2 = OH Cl, R 2 = OH N3 , R 2 = N 3
N
, R2 = OTBDPS
178: R 1 = HN N
HN N
N
N
N
, R2 = OTBDPS
N
179: R 1 =
, R2 = OH
Fig. 27. Structures of 4-aryl-6-chloro-quinolines.
HBV DNA [120]. The imidazolyl group could be replaced with the 2methylimidazolyl group with little effect on anti-HBV activity. Electron-withdrawing fluoro atom on the phenyl at 2-postion was superior to these with electron-donating methoxy, methyl or no substitution. In 6-hydroxyquinolines, compounds 169 and 170 inhibited the secretion of both HBsAg and HBeAg. They also demonstrated potent inhibition of HBV DNA replication with IC50 values of 10.7 and 4.7 mmol/L, respectively [121]. SAR study revealed that fluoride could enhance the activity while oxidation of the sulfide into the sulfinyl group had little influence on both of anti-HBV activity and cytotoxicity. As for Mannich groups, pyrrolidinyl, piperidyl and imidazolyl groups were preferred at 5-position while morpholinyl and 4-methyl piperazinyl groups were not. Another series of 4-aryl-6-chloro-quinoline derivatives (Fig. 27) were synthesized and assayed for their anti-HBV activity and cytotoxicities in HepG2.2.15 cells with tenofovir as positive control by Guo and co-workers [122]. Nine compounds 171e179 displayed significant inhibition against HBV DNA replication with IC50 values range from 4.4 to 9.8 mmol/L. Of them, compounds 175, 177 and 178 had low cytotoxicities, resulting in high SI values. The preliminary SAR study indicated that TBDPS substitution at 3-position is an important feature in the conferring relatively low cytotoxicity, but for 2-N-substituted analogues TBDPS substitution caused the loss of suppressant property on the secretion of HBsAg and HBeAg. A novel 6H-benzothiopyrano[4,3-b]quinoline skeleton (Fig. 28) was found to be an inhibitor of HBV by Jia [123,124]. Seven
compounds 180e186 showed potent anti-HBV activity against HBV DNA with IC50 values
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Mini-review
A review of non-nucleoside anti-hepatitis B virus agents Fan Zhang*, Gang Wang School of Pharmacy, Liaoning Medical University, No. 40, Section 3, Songpo Road, Linghe District, Jinzhou 121001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 August 2013 Received in revised form 12 January 2014 Accepted 17 January 2014 Available online xxx
Hepatitis B Virus is the most common cause of chronic liver disease worldwide. Currently approved agents of chronic HBV infection treatment include interferon and nucleoside analogues. However, the side effects of interferon and the viral resistance of nucleoside analogues make the current treatment far from satisfactory. Therefore, new drugs with novel structures and mechanisms are needed. Recently, a number of non-nucleoside HBV inhibitors have been obtained from natural sources or prepared by synthesis/semi-synthesis. Some of them exhibited potent anti-HBV activity with novel mechanisms. These compounds provide useful information for the medicinal chemist to develop novel non-nucleoside compounds as anti-HBV agents. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Hepatitis B virus Inhibitor Non-nucleoside Small-molecule inhibitors Natural products Synthetic compounds
1. Introduction Hepatitis B virus (HBV) infection is a major cause of viral hepatitis worldwide [1,2]. According to the World Health Organization (WHO) reports, more than 2 billion people have been infected with HBV at some time in their lives. Among them, about 350 million people remain infected chronically and become carriers of the virus [3e5]. Every year, HBV infection accounts 1 million people deaths from cirrhosis, liver failure, and hepatocellular carcinoma. Currently, seven drugs have been approved by FDA for the treatment of HBV infection: interferon alfa, pegylated interferon alfa2a, lamivudine, adefovir, entecavir, telbivudine, and tenofovir [6e8]. Interferon alfa and pegylated interferon alfa-2a are immunomodulatory agents, and their potential mechanism of action is the restitution of the host immune system [9,10]. However, the need for parenteral administration, the poor long-term response, and the high frequency of adverse side effects make interferon not ideal [11e 13]. The mechanism of action of nucleoside analogues is suggested to be through the interaction of their triphosphate derivatives, formed after cellular metabolic transformation, with HBV DNA polymerase or reverse transcriptase as substrates and/or inhibitors [14]. Lamivudine is the first nucleoside analogue approved to treat chronic HBV infection. However, HBV generates drug-resistance to lamivudine easily, and the rate of the resistance increases with the elongation of the therapeutic duration [15,16]. Genotypic resistance * Corresponding author. Tel.: þ86 416 4673440. E-mail address: [email protected] (F. Zhang). 0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2014.01.046
can be detected in 14%e32% after 1 year of lamivudine treatment and increases with the duration of treatment to 60%e70% after 5 years of treatment. Among adefovir-treated patients, the cumulative incidence of resistance over 5 year has been reported to be 29% in HBeAgnegative patients and 42% in HBeAg-positive patients [17,18]. As the nucleoside analogues inhibit HBV replication through their unique interaction with HBV DNA polymerase, emergence of HBV variants resistant to antiviral therapy is facing all the nucleoside analogues [19e21]. The development of multidrug-resistant has increased the risk of exacerbation of liver disease. The emergence of drug resistance during long-term therapy with nucleoside analogues is almost inevitable and represents a clinical challenge. It is therefore important to continue research to identify new anti-HBV targets and discover novel compounds with unique mode of action for controlling viral replication as well as preventing drug resistance and its complications in the long term [22e30]. This review will focus on the medicinal chemistry of nonnucleoside HBV inhibitors. In the last several years in particular, many non-nucleoside compounds obtained from natural sources or prepared by synthesis/semi-synthesis have been reported as novel HBV inhibitors. 2. Natural products 2.1. Wogonin and its analogues Wogonin (1, Fig. 1) isolated from Scutellaria baicalensis was identified as an effective anti-HBV agent [31,32]. At a dose of 20 mg/
268
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
OH HO
O
OH OH
O
O HO
HO
O
O
OH
HO OH
O
HO OH OH
O
OH
O
2
1
HO
O
OH OH
O OH
O
4
3 OH
Fig. 1. Structures of wogonin, baicalein, hyperin and quercetin.
mL, wogonin displayed an anti-hepatitis B surface antigen (antiHBsAg) activity with an inhibition percentage of 45.4 2.7%, furthermore, both the relaxed circular and the linear forms of HBV DNA were significantly reduced in the wogonin-treated group. Wogonin was also found to significantly reduce the secretion of hepatitis B e antigen (HBeAg) in human hepatoblastoma-derived liver Hep-G2 cells (HepG2.2.15 cells) with an IC50 value of 4 mg/ mL [33]. However, wogonin’s closely related analogue, baicalein (2), had minimal effects on HBsAg secretion in the assay. In wogonintreated ducks, plasma duck hepatitis B virus (DHBV) DNA levels were significantly reduced and remained lower than control level even at 10 days after end of the treatment. Wogonin also demonstrated anti-HBV activity in human HBV-transgenic mice, with persisted inhibition of HBsAg while there was a more rapid rebound of serum HBsAg level with lamivudine treatment. The polyphenolic extract from Geranium carolinianum L. was found to suppress the secretion of the HBV antigens and reduce HBV DNA level in vitro [34]. This extract also reduced the plasma and the liver DNA level in the DHBV infected ducks. Among isolated compounds from the extract, hyperin (3) showed significant inhibitory effect on both of HBsAg and HBeAg secretion, while quercetin (4) had no activity against HBeAg [35]. These results indicated that a galactose group at 3-position of quercetin enhanced the anti-HBV potency. 2.2. Helioxanthin and its analogues Helioxanthin (5, Fig. 2), an arylnaphthalene lignan lactone, was isolated from the root of Taiwania cryptomerioides [36]. Helioxanthin demonstrated an anti-HBV activity with an EC50 value of 1.0 mmol/L [37]. Modification of helioxanthin resulted in several derivatives with improved activity. Structure activity relationship (SAR) study revealed that the acid-hydrolysed derivative 6, the lactam derivative 7 and the cyclic hydrazide derivative 8 exhibited enhanced anti-HBV activity with EC50 values of 0.8, 0.08 and 0.03 mmol/L, respectively [37]. Introduction of halogen atoms at 5position of helioxanthin would reduce the antiviral activity. Modifications of the carboxylic acid and amide groups in compound 6
Artemisinin (20, Fig. 4) isolated from Artemisia annua had been widely used for antimalarial treatment [43]. Moreover, it was discovered to additionally use as anticancer, antiangiogenesis, antiviral, immunosuppressive, and antifungal agent. Recently, Romero and co-workers [44,45] revealed that artemisinin and its semi-synthetic derivative, artesunate (21), could induce strong inhibition of HBsAg secretion (IC50 values of 55 and 2.3 mmol/L, respectively) at concentrations at which host cell viability was not affected. When artesunate and lamivudine were administered together, synergic anti-HBV effects were also found in the assay. 2.4. Alisol analogues Six triterpenes, alisol A 24-acetate (22, Fig. 5), 25-anhydroalisol A (23), 13b,17b-epoxyalisol A (24), alisol B 23-acetate (25), alisol F
O O
O
O
O NH2 OH
O O
2.3. Artemisinin and its analogues
O
O
O
by dicarboxylic acid or diamide substituents, also resulted in a significant loss of activity. The replacement of a methylenedioxy group by two methoxy substituents in A ring exhibited more or less decreased activity. Further study revealed that the antiviral mechanism was completely different from nucleoside analogues [38,39]. The target of action was at the viral RNA transcription step and not at the HBV DNA polymerase. Compound 8 modified the interaction of hepatocyte nuclear factors (HNF) with HBV promoters, and down-regulated HNF expression in HBV-harbouring cells [40]. Janmanchi and co-workers [41,42] synthesized a series of helioxanthin analogues (Fig. 3) and their anti-HBV activity was evaluated. Modifications at the lactone rings and methylenedioxy unit of helioxanthin could modulate the antiviral activity. Among them, compound 13 was the most effective anti-HBV agent, which could suppress the secretion of viral surface antigen and e antigen in HepA2 cells with EC50 values of 0.06 and 0.14 mmol/L, respectively [41]. Compound 13 not only inhibited HBV DNA with wild-type and lamivudine-resistant strain but also suppressed HBV mRNA, core protein and viral promoters.
NH NH
NH O
O
O
O
O
A O O
5
O O
6
O
O O
7
Fig. 2. Structures of helioxanthin and its analogues.
O
8
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
Furthermore, epoxide group at 13(17)-position resulted in the decrease of suppressant property on the secretion of HBsAg and HBeAg. Among them, compound 31 showed high activities against secretion of HBsAg and HBeAg with IC50 values of 0.0048 and 0.011 mmol/L, respectively, and remarkable selective index values (SIHBsAg > 333 and SIHBeAg > 145, respectively) [49]. Anti-HBV activity of astragaloside IV (34, Fig. 7) isolated from Radix Astragali was reported by Wang and co-workers [50]. At dose of 200 mg/mL, astragaloside IV effectively suppressed secretion of HBsAg by 31.8% and HBeAg by 23.3% after 9 days of treatment. In Peking ducklings, it reduced DHBV levels in the serum with 64.0% inhibition and showed no significant toxicity at 120 mg/kg [50].
O R1
O
R2
O
O
O
O R2
O O
R1
12: 13: 14: 15: 16: 17: 18: 19:
9: R1 = R2 = CH2OH 10: R1 = CH2OAc, R2 = CH2OH 11: R1 = R2 = CH2 OAc
R1 = OCH3, R2 = OBn R1 = OCH3, R2 = OH R1 = OBn, R2 = OCH3 R1 = OH, R2 = OCH3 R1 = R2 = OBn R1 = R2 = OH R1 = R2 = OCH3 R1 = OCF3 , R2 = H
2.5. Matrine and its analogues Several matrine analogues (Fig. 8) isolated from the roots of Sophora flavescens and Sophora tonkinensis and semi-synthetic were investigated for their in vitro anti-HBV activity [51e53]. Compounds 37, 39, 41 and 42 exhibited a potential anti-HBV activity and low toxicity in HepG2.2.15 cells. SAR study revealed that the N / O group (40, 41) was not important element to keep the activity. Compounds with double bond in D ring, while without hydroxyl group, were more potent than other analogues. Introduction of a N / O group (41) or an a-hydroxyl group (43 and 44) into compound 42 (with unsaturated D ring) negatively influenced the antiHBV activity, especially the HBeAg inhibitory potency. As for compounds with saturated D ring, this influence was dramatically opposite. The presence of the N / O group (40) and a-substituted hydroxyl group (36) in matrine (35) markedly enhanced the potency. Moreover introduction of a proper substituent at the 13- and/ or 14-position(s) (37, 38, 39), especially electron-withdrawing groups, might enhance the activity. Matrinic acid (45) and oxymatrinic acid (46) with the D ring opening, still retained moderate anti-HBV activity. It was deduced that D ring might not be required for activity. Twenty-six Nsubstituted matrinic acid analogues were synthesized by Du and co-workers [54]. Among these analogues, compound 47 as host heat-stress cognate 70 (Hsc70) down-regulator exhibited a broadspectrum anti-HBV and anti-HCV effects with a mechanism different from that of nucleosides currently used in clinical patients [55]. SAR study showed that the carboxyl group at 11-position was required for activity and introduction of a substituent on nitrogen
Fig. 3. Structures of derivatives of helioxanthin synthesized by Janmanchi and coworkers.
O
H O
OH
O
O O
H O
H
O
O
H
O O
O O
H
H 20
21
Fig. 4. Structures of artemisinin and artesunate.
(26), and alisol F 24-acetate (27), were isolated from the rhizome of Alisma orientalis. They exhibited inhibitory activity against HBsAg with IC50 values range from 0.6 to 15.4 mmol/L. Alisol F 24-acetate also displayed significant activity against HBeAg with an IC50 value of 5.1 mmol/L and a SI value of 18.5 [46]. Ester derivatives of alisol (Fig. 6) were synthesized by Zhang and co-workers [47e49]. SAR study revealed that acetylation of the free hydroxyl group at 25-position was important for potent anti-HBV activity of alisol A derivatives and small acyloxy substituents at 11, 23, 24-positions may be preferred for anti-HBV activity.
HO
OH
HO
HO
H
HO
24 OH
HO
AcO
O
OAc OH
HO O
H
H H
H
23
O
O 25
OH
H O
22
O
HO
H O
H
O
HO
H O
OH
OH
OAc HO
269
H O
H 26
Fig. 5. Structures of six triterpene derivatives.
H 27
OH
270
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O O
O
O O
O
O
O
O
O
O
O O O
O H
H O
O
H
H O
H
28
30
O
O O
O O
O
O
O O
O
H
H O
H
31
OEt
O
H O
O
O
OMe
O
H O
O
O
O
O
O
O
O
O
O
H
29
O
O
O
O
O
O
H
32
33
Fig. 6. Structures of ester derivatives of alisol.
Among the alkaloids isolated from the ethanol extract, acutumidine (48, Fig. 9) and its analogues (49e51) showed moderate activity against HBsAg secretion with IC50 values range from 0.45 to 2.02 mmol/L [56]. Moreover, antiviral activities of N-methyl substituted derivatives were less potent than the corresponding non-substituted derivatives.
OH O OH
HO
O
HO
O
2.7. Chrysophanol and its analogues
OH
OH
O
OH O
34
Several chrysophanol and its analogues (Fig. 10) were isolated from Rheum palmatum L. ethanol extract and evaluated for their anti-HBV activity [57]. Among them, chrysophanol (52), physcion (53), aloe-emodin (54) and 8-O-b-D-glucoside (55) showed similar inhibitory activities against HBV DNA, HBsAg and HBeAg with IC50 values about 300 mg/mL [57]. However, 8-O-b-D-glucoside exhibited less toxicity with SI value more than 42. This result revealed that a galactose group at 8-position of chrysophanol could reduce the toxicity of these analogues.
OH OH
Fig. 7. Structures of astragaloside IV.
atom at 12-position, especially substituted benzyl, might significantly improve the anti-HBV activity.
2.8. Dehydrocavidine and its analogues 2.6. Acutumidine and its analogues Dehydrocavidine (56, Fig. 11), dehydeoapocavidine (57) and dehydroisoapocavidine (58) were isolated from Corydalis saxicola [58e60]. They demonstrated anti-HBsAg effect with inhibition
The ethanol extract of Hypserpa nitida Miers exhibited inhibitory activity against the section of HBsAg and HBeAg in an assay [56].
R3 14 R2 15 D 13 H 16 N 12 17 11 H 7 5 4 8 6 H H N 3 9 R1 1 2 X 10 O
35 : R 1=R2 =R3 =H 36 : R 1=OH, R 2=R 3=H 37: R 1=R3 =H, R2 =OC2 H 5 38 : R 1=H, R2 =R3 =OCH 2 C6 H5 39: R 1=H, R2 =OCOCH2 Cl, R3 =OH 40: R 1=R2 =R3 =H, X=O
O N
N X
41: 42: 43: 44:
N
R2
H H
R1
H
11 7
H
5
COOH
4
H R1
R1 =R 2 =H, X=O R1 =R 2 =H R1 =OH, R2 =H R1 =H, R 2 =OH
Fig. 8. Structures of matrine and its analogues.
3
H
N
H
2 X 10 45: R1 =H 46: R 1 =H, X=O 47: R1 =CH 2PhOCH3 -p
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O
O
OH Cl
O
O
NH
O
O
N
O
O
O O 49
48
O
OH Cl
N
OH Cl
O
NH
O
O
271
OH Cl
O
N
O
O
O
O
50
51
Fig. 9. Structures of acutumidine and its analogues.
B infection in China [62,63]. An active component, protocatechuic aldehyde (61, Fig. 12), had been approved to be a strong inhibitor of HBV. As it could significantly inhibit the production of HBV DNA (IC50 ¼ 4.17 mg/mL), HBsAg (EC50 ¼ 3.94 mg/mL) and HBeAg (EC50 ¼ 2.46 mg/mL), while there might be at least two targets of protocatechuic aldehyde [64]. Protocatechuic aldehyde also reduced the level of DHBV DNA in DHBV-infected ducks at dosage of 50 mg/kg.
O
O R1
R2 HO OH O
OH
OH O
52: R1 = H, R2 = CH3 53: R1 = CH3O, R 2 = CH 3 54: R1 = H, R2 = CH3O
OH
H
O
O
OH
H 55
2.11. Caudatin and its analogues
Fig. 10. Structures of chrysophanol and its analogues.
percentages about 50%, and anti-HBeAg effect with inhibition percentages about 40% [59]. Within the concentration range from 16.5 to 250 mg/mL, the inhibitory effect of these compounds was dose-dependent. The known alkaloid dihydrochelerythrine (59) was also found to be a potent inhibitor of HBV. Dihydrochelerythrine could inhibit the secretions of both of HBsAg and HBeAg with IC50 values less than 0.05 mmol/L and a CC50 value of 0.16 mmol/ L [58]. 2.9. Triterpenoid saponin Zhao and co-workers isolated a triterpenoid saponin (60, Fig. 12) from Potentilla anserina L., which was frequently used as Traditional Chinese Medicine (TCM) in hepatitis B treatment. Compound 60 exhibited anti-HBV activity against secretion of HBsAg (IC50 ¼ 57.67 mg/mL, SI ¼ 4.10), HBeAg (IC50 ¼ 30.05 mg/mL, SI ¼ 7.86) and HBV DNA (IC50 ¼ 19.45 mg/mL, SI ¼ 12.14) in HepG2.2.15 cells [61]. The levels of DHBV DNA in the Peking ducklings treated with compound 60 (0.2 g/kg body wt) for 10 days were markedly reduced by 58.48%, and a long-term effect of inhibiting HBV was also found in the assay.
Caudatin (62, Fig. 13) from Cynanchum auriculatum (Bai-ShouWu in Chinese) as a prospective anti-HCC drug with the mechanisms of inhibiting cell proliferation and inducing cell apoptosis had been reported [65e68]. In the anti-HBV assay, caudatin was found to have activities against the secretion of HBsAg and HBV DNA replication with IC50 values of 142.67 mmol/L (SI ¼ 1.7) and 40.62 mmol/L (SI ¼ 6.0) in HepG2.2.15 cells, respectively [69]. A series of caudatin derivatives were synthesized by Wang and coworkers [69,70]. Most of the 3-O-substituted derivatives showed effective anti-HBV activity, especially inhibiting the HBV DNA replication with IC50 values range from 2.44 to 22.89 mmol/L [69]. However, the 3,8-O-di and 3,17-O-diacyl derivatives (63, 64) lost suppressant properties on the HBV and cytotoxicity, which suggested that the free hydroxyl groups were not only the necessary for the activity, but also the factor of leading the cytotoxicity. The oxidative derivative 66, expoxide 67 and reduction product 68 exhibited decreased anti-HBV activity compared to caudatin. Among these derivatives, compound 65 by attaching of the cinnamic acid to caudatin, displayed the most significant inhibitory effects on the secretion of HBsAg, HBeAg and HBV DNA replication with IC50 values of 5.52, 5.52, 2.44 mmol/L, respectively, and good safety (LD50 > 1250 mg/kg) [70]. Preliminary mechanism study proposed that compound 65 exerted antiviral effects via
2.10. Protocatechuic aldehyde Salvia miltiorrhiza was considered to be one of the most highly recommended and widely accepted medicines for treating hepatitis
R1 R2
O O
N
O
OH
O O
56 : R 1 = Me, R 2 = Me 57 : R 1 = Me, R 2 = H 58 : R 1 = H , R2 = Me
O
N
O
HO
H
O HO
O
HO HO OH O
O 59
Fig. 11. Structures of dehydrocavidine and its analogues.
OH 60
OH
HO
CHO
HO 61
Fig. 12. Structures of triterpenoid saponin and protocatechuic aldehyde.
272
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O
O
O O
O
O
O
OR 2
R1 O
OH O
O
O
OH
OH O
OH O
HO
67
68
O
63: R 1 = R2 = O
, R3 = H O
64: R 1 = R3 =
, R2 = H
N
O
H 3CO
65: R 1 =
HO
66
62: R 1 = R2 = R3 = H
OH
OH
OH
OH
OH
O
OH
OH
OR 3
O O
O
, R 2 = R3 = H
H 3CO OCH3 Fig. 13. Structures of caudatin and its analogues.
interfering HBV promoters and enhancers to influence HBV transcriptions.
presence of acetyl at 25-position was an important feature in conferring anti-HBV activity.
2.12. Hemslecin A and its analogues
3. Synthetic compounds
Hemslecin A (69, Fig. 14), a cucurbitane-type terpene, widely present in this genus had been used to cure inflammatory diseases in clinical practice [71]. Hemslecin A and its derivatives were evaluated for their anti-HBV activity in HepG2.2.15 cells [72]. Compound 69 showed moderate activity against HBV DNA replication (IC50 ¼ 11.4 mmol/L), with obvious toxicity (CC50 ¼ 66.2 mmol/L), which led to a low SI value (SI ¼ 5.8) [72]. Most of the derivatives displayed enhanced anti-HBV activity, of which compounds 70e75 exhibited not only significant activity inhibiting HBV DNA replication with IC50 values of 2.8e11.6 mmol/ L, but also high SI values. SAR study indicated that (1) mono- or di-acylation of hydroxyl group(s) at 2-position and/or 3-position resulted in enhancement of antiviral activity. (2) The acylation of hydroxyls at 2-, 3- and 16-position decreased cytotoxicity. (3) Epoxide of double bond at 5(6)-position decreased the cytotoxicity and maintained activity against HBV DNA replication. (4) The
3.1. 2,2-Bisheterocycles
HO O H
R1 O
H
O
OR 3
HO OAc
R2 O
HO HO
69: 70: 71: 72: 73:
R1 = R1 = R1 = R1 = R1 =
A library of subunits of leucamide A (76, Fig. 15), a bioactive cydicheptapeptide [73], were screened in several in vitro assays to determine their antiviral activity. Among them, compound 77 showed moderate activity against HBV DNA with an IC50 value of 76.4 mmol/L [74]. In order to investigate their SAR, many 2,2bisheterocycle compounds were synthesized and evaluated their anti-HBV activity. SAR study indicated that a bulky aliphatic group at 5-positon of the heterocycle B ring had an advantageous effect on the activity against HBV DNA replication. The inhibitory activity of acid analogue 78 (IC50 ¼ 60 mmol/L) substantially diminished compared with its ester analogue 79 (IC50 ¼ 2.4 mmol/L) and 80 (IC50 ¼ 1.1 mmol/L) [74]. The A ring could be replaced with the thiophene ring (81) or the benzo[b]thiophene ring (82). The most promising compound 83 showed significant potency
R2 = R 3 = H R 3 = H, R2 = AcO R2 = AcO, R 3 = H CH 3 CH 2CO, R2 = R3 = H R2 = R 3 = C 2H 5 OCH 2 CO
O H
H
O
OH
HO O H
OAc HO HO
O
74
O O
Fig. 14. Structures of hemslecin A and its analogues.
OH
H
OH 75
Cl
O OAc
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
O
O N S
HN
O
O
N
N S A
NH O
HN
O
O N H
O
S
S
N
N
S O
N
78: R = H 79: R = Me 80: R = Et
S O
N
O
82
R
O
77
S
O
S
76
81
O B N
N
O
N
S
273
O
S
S
N
N 83
O O
Fig. 15. Structures of leucamide A and 2,2-bisheterocycle tandems.
(IC50 ¼ 0.14 mmol/L) against HBV DNA replication and was about 500-fold more potent than the lead compound 76 (IC50 ¼ 76.4 mmol/L), and even more potent than lamivudine (IC50 ¼ 0.26 mmol/L) [74]. These compounds were inactive at 200 mg/mL in HBV polymerase assays, indicating that the action mechanism did not involve inhibition of the DNA polymerase. 3.2. Acrylamide derivatives In a screening in the HepAD38 cellular assay, a series of phenylpropenamide derivatives (84e91, Fig. 16) displayed potent antiHBV activity with IC50 values less than 4 mmol/L and no general toxicity [75,76]. Among them, the E isomers showed weak activity or no activity against HBV. SAR study revealed that vinyl bromides were slightly more active and less toxic than the corresponding vinyl chlorides. In general, substitution at 4-position of A ring had little effect on the activity. Substitution at 4-position of B ring resulted in compounds with improved inhibitory activity. Unsubstituted piperidine and pyrrolidine amides were preferred. Additionally, AT-61 (84) and AT-130 (91) also showed antiviral activity against the YVDD lamivudine resistant variant in vitro [77]. AT-61 was highly selective, inhibiting only the replication of human
R1
A
A
R1 O
O N O
Cl HN
B
N O
Br HN R2
84 : R 1 = H, R 2 = H 85 : R 1 = 4-F, R2 = H 86: R 1 = H, R 2 = 4-F
B 87: R 1 = 88: R 1 = 89: R 1 = 90: R 1 = 91: R 1 =
R2 4-Me, R 2 = H H, R 2 = 4-F H, R 2 = 4-Cl H, R 2 = 4-NO2 2-MeO, R 2 = 4-NO2
Fig. 16. Structures of phenylpropenamides.
HBV but not the replication of other viruses. Mechanism of phenylpropenamide derivatives was initially considered to inhibit HBV replication by interfering with the packaging of pregenomic RNA into the immature core particles not the HBV polymerase [78,79]. Subsequent studies revealed an assembly effector mechanism underlying the apparent blocking of RNA packaging [80]. Katen and co-workers had determined that AT-130 bound to a hydrophobic pocket located at the contact between B and C-dimers of the HBV capsid protein and induced subtle tertiary and quaternary structure changes that resulted in morphologically normal capsides without the reverse transcriptase pregenomic RNA complex [81]. The bioisoteric replacement of the phenyl in phenylpropenamide with 1,2,3-thiadiazolyl (Fig. 17) resulted in a new class of acrylamide analogues, which possessed the similar SAR with the phenylpropenamides. Compounds 92e94 that replaced A or B ring by 1,2,3-thiadiazole still had anti-HBV DNA activity with IC50 values of 10.4, 3.59 and 9.00 mg/mL, respectively [82]. However, when A and B rings were simultaneously replaced by 1,2,3-thiadiazole (95), the anti-HBV activity was eliminated and the cytotoxicity was higher than that of monosubstituted analogues. 3.3. Imino sugars Imino sugars (Fig. 18), N-butyl-deoxy-nojirimycin (N-Bu-DNJ, 96) and N-nonyl-deoxy-nojirimycin (NN-DNJ, 97) had been shown to be effective against HBV in tissue culture and in the woodchuck model in several reports [83e87]. In order to understand the SAR of this series, Mehta and co-workers synthesized many imino sugar derivatives. These derivatives could be divided into two functional groups: an imino sugar head group and an alkyl tail. SAR study indicated that the head group played a critical role in antiviral activity. Variation of the sugar head group was allowed to a large extent maintained antiviral activity. As for the alkyl tail, the side chain length appeared to be critical, with antiviral activity decreasing sharply with side chains of fewer than eight carbons. Interruption of the side chain with oxygenation (N-7-oxa-decylDGJ, 100) also reduced antiviral activity. NN-DGJ (98) was also effective against the lamivudine resistant virus. Moreover, NN-DGJ did not inhibit DNA polymerase or envelop antigen production [88]. It either prevented the maturation of HBV nucleocapsids or destabilized the formed nucleocapsids [89].
274
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
R
N N S
A O
Br
N HN
N N S
O N
Br
O
HN
Br
N
O
HN
S N N 92: R = 2-Cl 93 : R = 4-Cl
O
O
O
S N N 95
94
Fig. 17. Structures of 1,2,3-thiadiazole substituted acrylamides.
HO
(CH2)3CH 3 N
HO
(CH2)8CH 3 N
HO
OH
HO
HO
OH
(CH2)8CH 3 N
(CH2)8CH 3 N
HO
HO
OH
OH
(CH2)6O(CH 2) 2CH3 N
HO HO
OH
OH
OH
OH
OH
OH
96
97
98
99
100
Fig. 18. Structures of imino sugars.
3.4. 2,5-Pyridinedicarboxylic acid derivatives
3.5. Triazoles
Lee and co-workers [90] synthesized a series of pyridinedicarboxylic acid derivatives and evaluated their anti-HBV activity. Some compounds (101e111, Fig. 19) showed potent inhibitory effect against HBV polymerase (TC50 values less than 10 mg/L) and low cytotoxicity (CC50 values more than 100 mmol/L), which indicated that these 2,5-pyridinedicarboxylic acids were excellent anti-HBV agents [90]. SAR study showed that isopropyl, tert-butyl and 3-imidazolylpropyl groups were preferred for excellent activity. The amino substituted by the isobutyl group resulted in compounds 103 and 109 with improved activity. This might be the only class of non-nucleoside HBV polymerase inhibitors until now.
As triazoles possessed significant antifungal and antibacterial properties, El-Barbary and co-workers [91] synthesized several new derivatives and evaluated their anti-HBV activity. Compounds 112, 113 and 114 (Fig. 20) showed a moderate activity against HBV with IC50 values of 35, 63 and 40 mmol/L, respectively, and CC50 values more than 100 mmol/L [91]. An oxadiazole compound 115 was identified as a HBV inhibitor with an EC50 value of 1.63 mmol/L [92]. It inhibited the expression of HBsAg and HBeAg in a concentration-dependent manner with no cytotoxic effect. The transcription of HBV was not affected by compound 115. It was likely that compound 115
H2 N
O HO
N H
N
N
R
N
N
HN
O N H
N N O
O 101
102: R = HOCH 2CH2 103: R = i-Pr
HN
O R
N H
N
104: R = 105: R = 106 : R = 107 : R =
HN
O N
N
N
N
N
O HOCH 2CH 2 HOCH 2C(CH 3) 2 2-pyridylmethyl 3-imidazolypropyl
R
N H
N N 108: 109: 110: 111:
N
N O R = CH 3OCH 2CH 2 R = i-Pr R = 3-cyclopropyl R = 3-imidazolypropyl
Fig. 19. Structures of 2,5-pyridinedicarboxylic acids.
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
N
N
N N
N S O AcO NH 2
Cl
NH2
N
N
S
OAc 112
275
OAc
OAc
N N S NH 2
Cl
114
113 O O
N O O S
N N
N
N
HS
O
N
S
O
NH
N
Cl
S
N N
117
116
115
Fig. 20. Structures of 1,2,4-triazoles, 1,3,4-oxadiazoles and 1,2,3-thiadiazoleacetamides.
interaction between the core protein and PreS region of the surface protein [94]. Among them, compound KKJ00626 (117) inhibited the production of HBV particles from transiently HBV-producing HuH7 cells with an IC50 value of 0.12 mmol/L [94].
affected events downstream of transcription in the viral replication process. In the investigation of 1,2,3-thiadiazole derivatives as antiviral agents, compound 116 was identified as a HBV inhibitor [93]. At 0.6 mmol/mL, compound 116 was found to reduce the secretion of HBsAg and HBeAg by 88.6% and 15.5%, respectively. In addition, compound 116 also inhibited HBV DNA with an IC50 value of 400 mmol/L and the possible mode of anti-HBV activity was inhibition of virus replication [93]. In a modified enzyme-linked immunosorbent assay, a chemical library consisting of 5600 compounds was screened for their
3.6. Benzimidazoles In a random screening, compound 118 (Fig. 21) was identified as an inhibitor of HBV with an IC50 value of 14.2 mmol/L against HBV DNA [95]. SAR study revealed that substituents, such as methyl, isopropyl, methoxy and nitro, were tolerated at the O
O N
N N S O O O 2N
O
Cl
N
Cl
N S O O
N O
Cl
N
Cl
N
N
Cl
O
R 124: 125: 126: 127: 128: 129:
N S O O
O
N N N
O
123
R
O H N
NH 2
N O S O
R=H R = 2-F R = 4-F R = 2,6-diF R = 2-Cl R = 4-Me
N
120
122 N
N H
Cl
N
N
121
O
N
O
O N
Cl
119
118
Cl
O
O
N
N NH 2
N O S O
130 : R = H 131: R = F
Fig. 21. Structures of benzimidazoles.
N O 132
NH 2 N O S O
276
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
R2 HO
R2 N
R1
N H
HO
N
SR1 N H 137: R 1 = CH2 CH3 , R 2 = 2-methylimidazol-1-yl 138 : R 1 = CH(CH 3 )2 , R 2 = imidazol-1-yl 139 : R 1 = CH(CH 3) 2, R2 = 2-methylimidazol-1-yl
S
133: R 1 = H, R 2 = imidazol-1-yl 134: R 1 = 4-Me, R2 = imidazol-1-yl 135: R 1 = H, R 2 = 2-methylimidazol-1-yl 136 : R 1 = 4-Me, R2 = 2-methylimidazol-1-yl
Fig. 22. Structures of 1H-benzimidazol-5-ols.
A group of 1-methyl-1H-benzimidazol-5-ol analogues (Fig. 22) with different Mannich side chains at 4-position of the benzimidazole core was synthesized and evaluated their anti-HBV activity in HepG2.2.15 cells [98,99]. Among them, imidazolyl derivatives (133e139) showed more potent inhibition of HBV DNA replication or the secretion of HBeAg than lamivudine with IC50 values between 7.8 and 44.5 mmol/L [98,99]. Besides, 2-methylimidazol-1-yl analogues (135, 136, 137, 139) had less cytotoxicity than corresponding imidazol-1-yl analogues (133, 134, 138). Converting sulfur into sulfinyl and sulfonyl groups, the cellular toxicity was obviously reduced, whereas a complete loss in anti-HBV DNA activity was also observed.
benzenesulfonyl group at N-1 position of the benzimidazole core. Removal of the N-1 substituent resulted in a tremendous loss of activity, while introducing 4-methylbenzoyl to N-1 position of the benzimidazole core would decrease the cytotoxicity. Replacement of the phthalimide group with maleimide yielded in compound 120 with improved activity. The alkyl linker was important for potent antiviral activity, and the methylene and propylene analogues were more toxic. Contrarily, replacement of sulfonyl group with alkyl substituents at N-1 position could dramatically increase antiviral activity and selectivity. The most promising compounds 121 and 122 showed potent anti-HBV activity (IC50 values of 0.9 and 0.7 mmol/L, respectively) and high selectivity (SI more than 111 and 714, respectively) [95]. In order to improve the water-solubility and oral bioavailability, eighteen novel derivatives of compound 121 were synthesized and evaluated for their anti-HBV activity and cytotoxicity in the HepG2.2.15 cell line [96]. Among them, compound 123 with IC50 < 0.41 mmol/L and SI > 81.2, was the most promising compound, nearly three times higher selectivity than lamivudine [96]. Introduction of large substituents at 5- or 6-position of the benzoimidazole ring resulted in a new series of benzimidazole derivatives (124e132) with anti-HBV activity [97]. SAR study showed that the substituent on the benzoyl ring was important at 6-position. The halide substituted analogues 125e128 demonstrated more active than the methyl substituted analogue 129. However, 5-position substituted derivative 130 without a substituent on the benzoyl ring was found to be more potent than the fluoro substituted derivative 131. The best compounds were 127 and 132 with IC50 values both of 0.70 mmol/L [97].
3.7. Indoles Arbidol (140, Fig. 23), licensed in Russia for treatment of influenza A and B infections, was reported to possess inhibitory activity of several viruses such as the respiratory syncytial virus, parainfluenza virus, rhinovirus and hepatitis C virus [100e105]. Recent studies extended its inhibitory activity to HBV with IC50 values of 22.85 mg/mL against HBV DNA and 47.45 mg/mL against HBsAg [106,107]. Its derivatives 141e145 also exhibited significant antiHBV activity with IC50 values less than 60 mg/mL [108e110]. SAR study revealed that introduction of fluoro or chloro group on the phenyl ring could improve anti-HBV activity. Oxidation of sulfide into sulfinyl resulted in compounds with increased activity. Mannich functionalities did not show significant distinctness of antiHBV activity effects. Bromo group at 6-position had little influence on activity and cytotoxicity. Arbidol was found to inhibit
N N
O
O N
O
HO
O
N
O
HO N
Br
S
N
S
F
N
Br
O 140
141
142 N
O
N
O
HO Br
F N
S
O
O
O 143
O
HO N
S O
O N
O
HO
F
Br
O
N
O
O
HO
S OO
144 Fig. 23. Structures of ethyl 5-hydroxy-1H-indole-3-carboxylates.
N
145
S OO
F
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
R Cl
Cl
R Cl
N H
OH
O
N H 149: R = 150 : R = 151: R =
146: R = H 147: R = F 148: R = Cl
Cl
277
O
nO N H 152: n = 153: n = 154 : n = 155 : n = 156: n =
O H F Cl
O 2, R 2, R 2, R 1, R 3, R
R =F = OCH 3 = CF3 = OCH 3 = OCH 3
R O
R Cl
Cl
NH 2 N H
N O N H
O
O
159: R = H 160: R = F
157: R = H 158: R = F
Fig. 24. Structures of 4-aryl-6-chloro-quinolin-2-ones and benzodiazepine-2-ones.
R
O
O
OH
N N H
S
NO2
161: R = H 162: R = Ac
O
N N H
S
R
OH
O
O
Cl
S
N H
163: R = Br 164 : R = Cl
OH
N
N N H
S
Cl
166
165
Fig. 25. Structures of nitazoxanide, tizoxanide, RM-4832, RM-4848, RM-4850 and RM-4852.
membrane fusion during influenza virus or HCV infection and suppress these viruses entry into target cells [101,111,112]. However, its model of action on HBV had not been disclosed until now. 3.8. Quinolin-2-ones and benzodiazepine-2-ones Cheng and co-workers [113,114] synthesized several 4-arylquinolin-2-one and 5-aryl-1,4-benzodizaepin-2-one derivatives. Compounds 146e160 (Fig. 24) inhibited the secretion of HBsAg with IC50 values range from 0.01 to 2.84 mmol/L. SAR study exhibited that compounds 146e148 were less active than corresponding hydroxyl ethyl derivatives 149e151, which revealed that introduction of hydroxyethyl to 3-position of 4-aryl-6-chloroquinoline-2-one was an important feature in conferring enhanced anti-HBV activity. In addition, ester derivatives 152e155 displayed optimal profile inhibiting not only HBsAg and HBeAg secretion but also HBV DNA replication. 3-Amino derivatives 157 and 158 exhibited increased cytotoxicity and activity. Preliminary mechanism study suggested that compound 153 mainly promoted the transcription activity of HBV enhancers ENⅠ and ENⅡ to exert antiHBV action. The ring-expansion derivatives 159 and 160 were also active against HBsAg secretion with IC50 values of 67 and 56 mmol/L, respectively [114]. 3.9. Thiazolides Nitazoxanide (161, Fig. 25), a drug with anti-infective activity against anaerobic bacteria, protozoa and viruses, was currently in
phase II clinical development for treating chronic hepatitis C [115e 118]. Recently, Korba and co-workers [119] had proved that nitazoxanide and its analogues tizoxanide (162), RM-4832 (163), RM4848 (164), RM-4850 (165) and RM-4852 (166) were potent inhibitors of HBV with EC50 values range from 0.12 to 1.2 mmol/L. Nitazoxanide and RM4850 were also effective against several HBV lamivudine-resistant and one adefovir dipovoxil-resistant constructs in transient transfection assays in HuH7 cells. 3.10. Quinolines Liu and co-workers [120,121] had reported several quinoline derivatives (Fig. 26) with potent anti-HBV activity. In 7hydroxyquinolines, compounds 167 (IC50 ¼ 3.5 mmol/L, SI ¼ 37.9) and 168 (IC50 ¼ 2.6 mmol/L, SI ¼ 61.7) were more potent than the positive control lamivudine (IC50 ¼ 343.2 mmol/L, SI ¼ 7.0) against
O O HO
N
R O S
O
R
O
N N
O
HO O F
N
S O F
167 : R = H 168: R = F
169: R = pyrrolidin-1-yl 170: R = 1H-imidazol-1-yl
Fig. 26. Structures of ethyl 6(7)-hydroxyquinoline-3-carboxylates.
278
F. Zhang, G. Wang / European Journal of Medicinal Chemistry 75 (2014) 267e281
F N
R1
Cl
176: R 1 = O
, R 2 = OH F
R2 Cl
171: R 1 = 172: R 1 = 173: R 1 = 174: R 1 = 175: R 1 =
177: R 1 =
OMs, R 2 = OMs Cl, R 2 = OAc OCH 3 , R 2 = OH Cl, R 2 = OH N3 , R 2 = N 3
N
, R2 = OTBDPS
178: R 1 = HN N
HN N
N
N
N
, R2 = OTBDPS
N
179: R 1 =
, R2 = OH
Fig. 27. Structures of 4-aryl-6-chloro-quinolines.
HBV DNA [120]. The imidazolyl group could be replaced with the 2methylimidazolyl group with little effect on anti-HBV activity. Electron-withdrawing fluoro atom on the phenyl at 2-postion was superior to these with electron-donating methoxy, methyl or no substitution. In 6-hydroxyquinolines, compounds 169 and 170 inhibited the secretion of both HBsAg and HBeAg. They also demonstrated potent inhibition of HBV DNA replication with IC50 values of 10.7 and 4.7 mmol/L, respectively [121]. SAR study revealed that fluoride could enhance the activity while oxidation of the sulfide into the sulfinyl group had little influence on both of anti-HBV activity and cytotoxicity. As for Mannich groups, pyrrolidinyl, piperidyl and imidazolyl groups were preferred at 5-position while morpholinyl and 4-methyl piperazinyl groups were not. Another series of 4-aryl-6-chloro-quinoline derivatives (Fig. 27) were synthesized and assayed for their anti-HBV activity and cytotoxicities in HepG2.2.15 cells with tenofovir as positive control by Guo and co-workers [122]. Nine compounds 171e179 displayed significant inhibition against HBV DNA replication with IC50 values range from 4.4 to 9.8 mmol/L. Of them, compounds 175, 177 and 178 had low cytotoxicities, resulting in high SI values. The preliminary SAR study indicated that TBDPS substitution at 3-position is an important feature in the conferring relatively low cytotoxicity, but for 2-N-substituted analogues TBDPS substitution caused the loss of suppressant property on the secretion of HBsAg and HBeAg. A novel 6H-benzothiopyrano[4,3-b]quinoline skeleton (Fig. 28) was found to be an inhibitor of HBV by Jia [123,124]. Seven
compounds 180e186 showed potent anti-HBV activity against HBV DNA with IC50 values
