2,4-Diaryl-4,6,7,8-tetrahydroquinazolin-5(1H)-one derivatives as anti-HBV agents targeting at capsid assembly

Article abstract of DOI:10.1016/j.bmcl.2009.10.119

A series of novel 2,4-diaryl-4,6,7,8-tetrahydroquinazolin-5(1H)-one derivatives were designed and synthesized as potent inhibitors of HBV capsid assembly. These compounds arose from efforts to rigidify an earlier series of heteroaryldihydropyrimidines (HA

Full text of DOI:10.1016/j.bmcl.2009.10.119

Bioorganic & Medicinal Chemistry Letters 20 (2010) 299–301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,4-Diaryl-4,6,7,8-tetrahydroquinazolin-5(1H)-one derivatives as anti-HBV
agents targeting at capsid assembly
Xuejun Zhu a,b, , Guoming Zhao b, , Xiaoping Zhou a, Xiaoqian Xu b,c, Guangqiang Xia b, Zhibing Zheng b,
b,
Lili Wang b, Xiaohong Yang a, , Song Li
*
*
a School of Pharmacy, Jilin University, Changchun 130021, PR China
b Department of Medicinal Chemistry, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China
c School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2009
Revised 29 September 2009
Accepted 27 October 2009
Available online 30 October 2009
A series of novel 2,4-diaryl-4,6,7,8-tetrahydroquinazolin-5(1H)-one derivatives were designed and syn-
thesized as potent inhibitors of HBV capsid assembly. These compounds arose from efforts to rigidify
an earlier series of heteroaryldihydropyrimidines (HAPs), and compounds 12, 13, 20, 24, 30 and 32
showed potent inhibition of HBV capsid assembly, especially 24 with IC50 value at sub-micromolar range.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
2,4-Diaryl-4,6,7,8-tetrahydroquinazolin-
5(1H)-one
Hepatitis B virus (HBV)
Nucleocapsid assembly
Inhibitor
More than 350 million people around the world are chronically
infected with the hepatitis B virus (HBV), and estimated one mil-
lion patients die each year due to the long-term complications of
liver cirrhosis or hepatocellular carcinomas. So far, six antiviral
target against various genotypes of HBV resistant mutants.7 Finally,
only a few small molecular agents that inhibit the interaction
between core proteins or misdirect the assembly of capsid have
been reported so far3,8,9 (Fig. 1). Heteroaryldihydropyrimidines
(HAPs), first developed by Bayer investigators,3,9 were shown to
bind the HBV core proteins and misdirect the assembly of the cap-
sid in vitro, rather than affect the protein synthesis.3 Moreover, the
HAP compounds were also effective in the transgenic mice
agents have been approved for treating chronic hepatitis
B
(CHB): two immune modulators (IFN- and pegIFN- ), and four
a
a
polymerase inhibitors (lamivudine, entecavir, telbivudine and ade-
fovir).1 However, there are several disadvantages of current treat-
ments,2 such as the emergence of resistant mutants, poor
tolerability, and the inefficiency of eradicating HBV from CHB
patients. Therefore, development of more effective therapeutic
agents for HBV infection is still highly necessary.
Aside from the HBV polymerase, various potential new targets
have recently been investigated to prevent HBV replication, such
as the virus nucleocapsid assembly and the host cell ER glucosi-
dases.3,4 Some features involved in the capsid assembly make the
disruption of this process an attractive target for antivirus discov-
ery. First, encapsidation of HBV pregenomic RNA is a very impor-
tant process in HBV life cycle. The assembly disruption will affect
the virus replication.4c,5 Furthermore, the encapsidation is an evo-
lutionary constraint process.6 Compared to the polymerase, the
genetic stability makes capsid assembly process a better drug
* Corresponding authors. Tel.: +86 431 85619660.
E-mail address: xjzh1980@hotmail.com (X. Yang).
They contributed equally to this Letter.
Figure 1. Representative HBV capsid assembly inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.119

300
X. Zhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 299–301
Scheme 1. Reagents and conditions: (i) 0.05 equiv CH3ONa, anhydrous MeOH, 0 °C,
Figure 2. Design of 4,6,7,8-tetrahydroquinazolin-5(1H)-one derivatives.
overnight; (ii) ammonium chloride, ambient temperature, 24 h; (iii) substituted
cyclohexane-1,3-dione, substituted benzaldehyde, 1.5 equiv NaOAc, EtOH, reflux,
20 h.
model.10 In 2006, the cocrystal structure of HBV capsid-HAP was
solved by Bourne and coworkers.11 HAP1 binds to a hydrophobic
groove located at the interface between the capsid protein
subunits.12 However, due to the low resolution at 5 Å and only
the ‘apo’ structure was released, it was difficult to perform the
structure-based design of new capsid assembly inhibitors by using
this crystal structure.
We reported herein the synthesis, characterization and in vitro
antiviral activities of 2-aryl-4,6,7,8-tetrahydroquinazolin-5(1H)-
one derivatives, a novel class of HBV capsid assembly inhibitor.
The derivatives were designed by rigidifying an earlier series of
HAPs (Fig. 2).
A general synthetic procedure is illustrated in Scheme 1. In
most cases the appropriate commercially available nitrile 7 was
converted to the carboxyamidine 8 via treatment with sodium
methoxide in anhydrous methanol at room temperature, followed
by reaction with ammonium chloride.13 These carboxyamidines
were then converted to the 2,4-diaryl-4,6,7,8-tetrahydroquinazo-
lin-5(1H)-ones 9–32 by reacting with cyclohexane-1,3-dione and
benzaldehyde under the Biginelli condition.14
Scheme 2. Reagents and conditions: (i) hydroxylamine hydrochloride, K2CO3,
DMSO, ambient temperature, overnight; (ii) Ac2O, AcOH, rt, 30 min; (iii) 5% Pd/C,
H2, 2 atm, 2.5 h; (iv) substituted cyclohexane-1,3-dione, 2-chloro-4-fluorobenzal-
dehyde, 1.5 equiv NaOAc, EtOH, reflux, 20 h.
Compounds 36 and 37 were prepared according to the meth-
od described in Scheme 2. 2,4,6-Trifluorobenzonitrile 33 was
first treated with hydroxylamine hydrochloride to afford 2,4,
6-trifluoro-N0-hydroxybenzimidamide 34, and followed by hydro-
genation in the presence of 5% Pd/C to give 2,4,6-trifluorobenzimi-
damide acetate 35.15 Coupling of 35 with cyclohexane-1,3-dione
and 2-chloro-4-fluorobenzaldehyde afforded compounds 36 and
37. All the new compounds described were characterized by 1H
NMR, MS and HRMS spectrums.16
Inhibition activities for HBV replication of the final compounds
9–32 and 36–37 were determined as described previously17 in the
HepG2.2.15 cells, which constitutively produces HBV genomes,
Table 1
In vitro anti-HBV evaluation of compounds 9–32, and 36–37
R1
R2
R3
R4
R5
DNA replication IC50
(lM)
Cytotoxicity IC50 (lM)
T.I.b
a
a
Compds
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyrazin-2-yl
Pyrazin-2-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-3-yl
Furan-2-yl
3-Fluoropyridin-2-yl
3-Fluoropyridin-2-yl
Pyrazin-2-yl
Pyrazin-2-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-4-yl
3,5-Difluoropyridin-2-yl
Thiazol-2-yl
Thiazol-2-yl
2,4,6-Trifluorophenyl
2,4,6-Trifluorophenyl
H
Cl
H
Cl
Cl
Cl
Cl
Cl
H
H
H
F
F
F
H
F
H
F
H
F
F
F
F
H
F
H
F
H
F
F
F
F
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
H
CH3
H
6.60
na
na
4.30
2.30
na
25.10
na
na
na
na
1.10
na
571.31
589.20
515.62
619.50
777.21
1470.40
82.80
1124.62
280.50
368.90
717.0
1369.42
371.10
773.41
622.94
623.80
67.32
145.10
173.23
142.39
134.80
41.17
86.56
—
—
144.07
337.92
—
9.25
—
—
—
—
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
1244.93
—
54.46
—
14.20
na
0.44
26.20
25.30
22.40
16.47
4.92
1.05
8.75
3.12
na
1417.73
2.56
5.73
7.74
8.60
27.28
39.21
5.73
19.16
—
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
47.68
56.99
122.60
5.60
F
F
F
36
37
CH3
CH3
na
—
Bay41-4109
Lamivudine
0.78
0.22
567
727
a
Values are means of three experiments, na = not active at the maximum nontoxic concentration.
In vitro therapeutic index (IC50 cytotoxicity/IC50 complement inhibition).
b

X. Zhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 299–301
301
and secretes virus-like particles.18 Bay41-4109 and Lamivudine
References and notes
were used as positive control. To ascertain the cytotoxic effects
of all the tested compounds, the cell viability was determined after
the cells had been exposed to the compounds for 48 h17 (Table 1).
Based on the SAR results of HAPs, the effects of substitutions on
the 4-phenyl rings were first examined. 4-(2-Chloro-4-fluoro-
1. Ferir, G.; Kaptein, S.; Neyts, J. Rev. Med. Virol. 2008, 18, 19.
2. (a) Ghany, M.; Liang, T. J. Gastroenterology 2007, 132, 1574; (b) Shaw, T.;
Bartholomeusz, A. J. Hepatol. 2006, 44, 593; (c) Clercq, D. E. Exp. Opin. Emerg.
Drugs 2008, 13, 393.
3. (a) Deres, K.; Schroder, C. H. Science 2003, 299, 893; (b) Hacker, H. J.; Deres, K.
Biochem. Pharmacol. 2003, 66, 2273; (c) Stray, S. J.; Zlotnick, A. J. Mol. Rec. 2006,
19, 542.
phenyl) substitution (9, IC50 = 6.6 lM) led to the best antiviral
effect. Single substitution on the 4-phenyl ring such as 2-chloro
(10) or 4-fluoro (11) attenuated the activity. The activities were
susceptible to alterations on 4-phenyl rings. It was consistent with
literatures reported by Bayer investigators.9
4. (a) Block, T. M.; Lu, X.; Mehta, A. S.; Ferir, G. Nat. Med. 1998, 4, 610; (b) Block, T.
M.; Jordan, R. Antiviral. Chem. Chemother. 2001, 12, 317; (c) Wen, Y.-M.; Lin, X.;
Ma, Z.-M. Curr. Drug Targets 2003, 3, 241.
5. Le Pogam, S.; Yuan, T-T.; Sahu, G. K. J. Virol. 2000, 74, 9099.
6. Chain, B.; Myers, R.; Neyts, J. BMC Microb. 2005, 5, 33.
7. Choi, I.-G.; Yu, Y. G. Infect. Disorders—Drug Targets 2007, 7, 251.
8. (a) Zlotnick, A.; Ceres, P.; Singh, S. J. Virol. 2002, 76, 4848; (b) King, R. W.;
Ladner, S. K.; Miller, T. J. Antimicrob. Agents Chemother. 1998, 42, 3179.
9. (a) Stoltefuss, J.; Goldmann, S. WO Patent 9,954,326, 1999.; (b) Goldmann, S.;
Kramer, T. WO Patent 0,058,302, 2000.; (c) Goldmann, S.; Stoltefuss, J. DE
Patent 10,012,824A1, 2001.; (d) Goldmann, S.; Stoltefuss, J.; U. Niewohner. DE
Patent 10,013,126A1, 2001.; (e) Goldmann, S.; Stoltefuss, J. U.S. Patent
6,503,913B1, 2003.
10. Weber, O.; Schlemmer, K. H.; Hartmann, E. Antiviral. Res. 2002, 54, 69.
11. Bourne, C.; Finn, M. G.; Zlotnick, A. J. Virol. 2006, 80, 11055.
12. Bourne, C.; Lee, S.; Venkataiah, B. J. Virol. 2008, 82, 10262.
13. Slee, H. D.; Chen, Y.; Zhang, X. J. Med. Chem. 2008, 51, 1719.
14. Kappe, C. O. Tetrahedron 1993, 49, 6937.
Compound12wasidentifiedasapotentanti-HBVagent. Replace-
ment of the 2-pyridin-2-yl substitution in 12 by pyrazin-2-yl, pyri-
din-3-yl, furan-2-yl, 3-fluoropyridin-2-yl, pyridin-4-yl, thiazol-2-yl
and 2,4,6-trifluorophenyl resulted in compounds 13, 15, 19, 20, 28,
31 and 36. Compounds 13 (IC50 = 2.3
showed increased activities against HBV replication, while com-
pounds 28 (IC50 = 16.47 M) and 31 (IC50 = 8.75 M) showed
lM) and 20 (IC50 = 1.1 lM)
l
l
decreased activities, and compounds 15, 19 and 36 lost their activi-
ties. At the same time, 4-(2-chlorophenyl) (14, 16, 20), 4-(4-fluoro-
phenyl) (17) and 4-phenyl (18) analogs were prepared. Compared
to their parent compounds, all these substitutions attenuated the
activities. This result further confirmed that the antiviral activity
has little tolerance to the changes of 4-(2-chloro-4-fluorophenyl).12
As revealed by the crystal structure, the 6-methyl group of the
HAP-1 core faces a hydrophobic tunnel.12 Installation of longer
hydrophobic substitutions at this position is expected to be toler-
ated or even enhance the binding affinity. This observation
prompted us to synthesize the 7,7-dimethyl substituted com-
pounds 21–27, 29–30, 32 and 37. Among this new series, com-
15. Judkins, B. D.; Allen, D. G.; Cook, T. A. Synth. Commun. 1996, 26, 4351.
16. Selected data for compound 9: 1H NMR (400 MHz, CDCl3) d 2.04–2.05 (2H, m,
CH2); 2.34–2.76 (4H, m, CH2); 5.79–5.97 (1H, BR, CH); 7.21–7.30 (3H, m, ArH);
7.39–7.43 (3H, m, ArH); 7.83 (1H, m, ArH); 8.34–8.42 (1H, m, ArH); 8.54–8.55
(1H, m, ArH); 8.78 (1H, s, NH); MS(EI) 303.2(M+); HRMS (m/z) calcd for
C19H17N3O: 1372; found 303.1370.
Compound 12: 1H NMR (400 MHz, CDCl3) d 2.13–2.15 (2H, m, CH2); 2.45 (2H,
s, CH2); 2.65–2.85 (2H, m, CH2); 6.14–6.22 (1H, m, CH); 6.90–9.92 (1H, m,
ArH); 7.16–7.26 (1H, m, ArH); 8.19–8.54 (2H, m, ArH); 8.77 (1H,s,NH);
MS(EI) 355.2 (M+); HRMS (m/z) calcd for C19H15N3OFCl: 355.0888; found
373.0889.
Compound 13: 1H NMR (400 MHz, CDCl3) d 2.14–2.19 (2H, m, CH2); 2.47
(2H, s, CH2); 2.72–2.87 (2H, m, CH2); 6.14–6.23 (1H, m, CH); 6.91–6.95 (1H,
m, ArH); 7.13–7.26 (2H, m, ArH); 8.21–8.52 (2H, m, ArH); 8.71 (1H, m, ArH);
9.58(1H, s, NH); MS (EI) 356.2 (M+); HRMS (m/z) calcd for C18H14N4OFCl:
356.0840; found 356.0839.
pounds 24 (IC50 = 0.44
inhibition of HBV replication, which was comparable to Bay41-
4109. Although compounds 29 (IC50 = 4.92 M) and 32
(IC50 = 3.12 M) just showed moderate inhibition of HBV replica-
lM) and 30 (IC50 = 1.05 lM) showed good
l
Compound 20: 1H NMR (400 MHz, CDCl3) d 2.11–2.17 (2H, m, CH2); 2.39–
2.50 (3H, s, CH3); 2.64–2.78 (2H, m, CH2); 6.18 (1H, s, CH); 6.89–6.93 (1H,
m, ArH); 7.12–7.14 (1H, m, ArH); 7.24–7.28(1H, m, Ar); 7.42- 7.46 (1H, m,
ArH); 8.39–8.40 (1H, m, ArH); MS(EI)373.1 (M+); HRMS (m/z) calcd for
C19H14N3OClF2: 373.0793; found 373.0792.
l
tion, they were more potent than the 7-nonsubstituted compounds
28 and 31. It indicated that 7,7-dimethyl substitution was favor-
able for the antiviral activity.
Compound 24: 1H NMR (400 MHz, CDCl3) d 1.15 (3H, s, CH3); 1.16 (3H, s,
CH3); 2.31(2H, s, CH2); 2.53–2.69(2H, m, CH2); 6.07(1H, s, CH); 6.94- 6.96
(1H, m, ArH); 7.14–7.17 (1H, m, ArH); 7.23–7.26 (1H, m, ArH); 7.34- 7.38
(1H, m, ArH); 8.07–8.09 (1H, m, ArH); 8.69–8.71 (1H, m, ArH); 8.89(1H, m,
ArH); MS (EI) 383.1 (M+); HRMS (m/z) calcd for C21H19N3OFCl: 383.1201;
found 383.1198.
In summary, we have described the successful structural mod-
ification of HAPs to 4,6,7,8-tetrahydroquinazolin-5(1H)-ones, a
novel class of HBV capsid assembly inhibitor. These newly devel-
oped derivatives demonstrated excellent in vitro activities against
HBV replication. The results indicated that the design of new HBV
capsid assembly inhibitor by rigidifying structures of HAPs is a
feasible and promising strategy. The active compounds 24 and 30
could be used as the lead compounds for further modification.
Compound 30: 1H NMR (400 MHz, CDCl3) d 8.30–8.29 (m, 1H, ArH); 8.05 (s,
1H, NH); 7.32–7.29 (t, 1H, J1 = 2.4 Hz, J2 = 8.0 Hz, ArH); 7.29–7.26 (m, 1H,
ArH); 7.14- 7.11 (d, 1H, J1 = 2.4 Hz, J2 = 8.0 Hz, ArH); 6.94–6.90 (t, 1H,
J1 = 2.4 Hz, J2 = 8.0 Hz, ArH); 6.16 (s, 1H, CH); 2.70–2.53 (m, 2H, CH2); 2.30
(m, 4H, CH2); 1.16 (s, 3H, CH3); 1.15 (s, 3H, CH3); HRMS (m/z) calcd for
C21H17ClF3N3O: 419.1012; found 419.1010(M+).
Compound 32: 1H NMR (400 MHz, CDCl3) d 7.95–7.85 (m, 1H, ArH); 7.64–
7.47 (m, 1H, ArH); 7.34–7.24 (m, 1H, ArH); 7.13–7.11 (m, 1H, ArH); 6.11 (s,
1H, CH); 2.70–2.52 (m, 2H, CH2); 2.43–2.26 (m, 2H, CH2); 1.15 (s, 6H, CH3);
HRMS (m/z) calcd for C19H17ClFN3OS: 389.0765; found 389.0765(M+).
17. Korba, B. E.; Gerin, J. L. Antiviral. Res. 1992, 19, 55.
Acknowledgments
This work was supported by the National High Technology R&D Pro-
gram of China (2006AA020605) and National S&T Major Project
(2009ZX09103-26). We thank Dr. Shi Chang for providing Bay41-4109.
18. (a) Sells, M. A.; Chen, M. L.; Acs, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1005; (b)
Sells, M. A.; Zelent, A. Z.; Shvartsman, M. J. Virol. 1988, 62, 2836.