A new series of HAPs as anti-HBV agents targeting at capsid assembly

Article abstract of DOI:10.1016/j.bmcl.2014.07.032

A series of novel Heteroaryldihydropyrimidines (HAPs) derivatives were designed and synthesized as potent inhibitors of HBV capsid assembly. These compounds were prepared from efforts to optimize an earlier series of HAPs, and compounds Mo1, Mo7, Mo8, Mo10, Mo12, and Mo13 demonstrated potent inhibition of HBV DNA replication at submicromolar range.

Full text of DOI:10.1016/j.bmcl.2014.07.032

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4247–4249
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new series of HAPs as anti-HBV agents targeting at capsid assembly
b,
a,b
Xiu-yan Yang a,b, Xiao-qian Xu a,b, Hua Guan c, LI-li Wang b, Qin Wu d, , Guo-ming Zhao , Song Li
⇑
⇑
a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
b Department of Medicinal Chemistry, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China
c Beijing Institute of Radiation Medicine, Beijing 100850, PR China
d The Liver Cirrhosis Diagnosis and Treatment Centre, The 302 Military Hospital of China, Beijing 100039, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel Heteroaryldihydropyrimidines (HAPs) derivatives were designed and synthesized as
potent inhibitors of HBV capsid assembly. These compounds were prepared from efforts to optimize
an earlier series of HAPs, and compounds Mo1, Mo7, Mo8, Mo10, Mo12, and Mo13 demonstrated potent
inhibition of HBV DNA replication at submicromolar range.
Received 25 February 2014
Revised 23 June 2014
Accepted 11 July 2014
Available online 30 July 2014
Ó 2014 Published by Elsevier Ltd.
Keywords:
Anti-HBV
Capsid
HAPs
SEC
Despite widely-adoption of an effective vaccine to prevent HBV,
chronic HBV infections remain a serious public health problem
worldwide. Over 350 million people are chronically infected or
exposed to the risk of developing severe complications including
cirrhosis and hepatocellular carcinoma. So far, very limited
immune modulators (IFN-a and pegIFN-a) and nucleoside/nucleo-
tide analogs (lamivudine, entecavir, telbivudine, adefovir and ten-
ofovir) have been approved by the FDA for treating chronic
hepatitis B (CHB). However, the use of interferon-a is limited due
to its low efficiency in Asian patients, high cost and serious side
effects, while nucleoside/nucleotide analogs targeting at the viral
DNA polymerase often result in drug-resistance virus after long-
term treatment.1,2 Therefore, there is a compelling demand for
developing safer and more effective anti-hepatitis B agents with
new structures and novel mechanisms of treatment.
Heteroaryldihydropyrimidines (HAPs) were reported as a highly
potent non-nucleoside inhibitors of HBV replication. And it took
effect by binding with core proteins and misdirecting the assembly
of the capsid in vitro.3 Moreover, the HAPs were also effective in
the transgenic mice model.4,5 Unfortunately, the reported HAPs
were hydrophobic due to their special chemical structures.6 The
poor water solubility significantly affected the drug candidate’s
pharmacokinetic/pharmacodynamic properties and eventually
limited their chance of being developed into pharmaceutical
products. Therefore, it is important to design and synthesize novel
HAPs analogs with both good water solubility and high antiviral
activities.
We herein report the approaches for the design, synthesis, char-
acterization and in vitro antiviral activities of new series of HAPs
with good water solubility. The preliminary structure-activity rela-
tionship (SAR) of HAPs has been evaluated for further discovery of
optimal compounds (Fig. 1). As indicated in our earlier report,7
2,4,6-trifluorophenyl substitution on the R1 is better for improving
the antiviral activity and 4-(2-Chloro-4-fluoro-phenyl) substitu-
tion on the R2 led to the strongest antiviral effect. So we reserve
both of them. The R5 group of the HAPs core faces a hydrophobic
pocket. Introduction of larger substitutions at this position is
expected to be tolerated or even enhance the binding affinity.8 This
observation prompted us to introduce a medium or long nitroge-
nous heterocyclic group on R5. Moreover, the obtained compounds
can be transformed into its corresponding water-soluble salt eas-
ily. The salt can be dissolved in buffer directly and no longer need
to add the auxiliary solvent such as DMSO. In addition, the solution
can be stored stably for a long time.
A general synthetic procedure is illustrated in Scheme 1. The
appropriate commercially available nitrile 1 was first treated with
hydroxylamine hydrochloride to afford N0-hydroxybenzimidamide
2, and followed by hydrogenolysis in the presence of 5% Pd/C to
give benzamidine acetate 3. Coupling of 3 with ethyl 4-chloroace-
toacetate and 2-chloro-4-fluorobenzaldehyde via a Biginelli reac-
tion afforded 4. Thereafter 4 reacted with various amines to
⇑
Corresponding authors. Tel.: +86 010 66874615; fax: +86 010 68211656.
E-mail addresses: wuqin6@126.com (Q. Wu), zhaogm@bmi.ac.cn (G. Zhao).
http://dx.doi.org/10.1016/j.bmcl.2014.07.032
0960-894X/Ó 2014 Published by Elsevier Ltd.

4248
X. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4247–4249
Mo1, R5=piperazinyl-2-one, R6=F
Mo2, R5=morpholinyl, R6=F
F
O
R2
R3
Mo3, R5=thiomorpholinyl, R6=F
O
N
Mo4, R5=pyrrolidinyl, R6=F
O
Cl
Mo5, R5=piperidinyl, R6=F
R4
N
H
R1
O
N
F
R6
Mo6, R5=4-methylpiperidinyl, R6=F
Mo7, R5=1-methylpiperazinyl, R6=F
Mo8, R5=1-ethylpiperazinyl, R6=F
Mo9, R5=1-propylpiperazinyl, R6=F
Mo10, R5= morpholinyl, R6=morpholinyl
Mo11, R5=ethyl carboxylate-1-piperazinyl, R6=F
Mo12, R5= 2,2'-dihydroxy-diethylamino, R6=F
Mo13, R5=morpholinyl hydrochloride, R6=F
R1 = Ar, heteroAr
N
H
R2 = Ar
R5
R3, R4= CH3,C2H5
F
HAPs
Figure 1. Design of the new derivates Mo1–13.
(IC50 = 6.66
l
mol), bearing a diethanolamino group, was more
R6
OH
N
+
R6
potent inhibitor of HBV replication than 3TC (14.17
the opening diethanolamino group was replaced by rigidified mor-
pholinyl to give compounds Mo2, Mo10, Mo13 (the IC50 values
10.57 lmol, 4.35 lmol, 2.35 lmol, respectively), their anti-HBV
lmol). When
R6
NH2
NH2
O
CN
F
ii,iii
i
O-
NH2
F
F
F
F
F
3
2
1
activities were reserved or even improved. Replacing the O atom
of morpholinyl with S,C,N resulted in the compounds Mo1, Mo2–
9. The thiomorpholinyl derivate Mo3 lost anti-HBV activity. The
piperidinyl derivates Mo5 and Mo6 showed good efficacy against
HBV DNA replication (IC50 = 10.24, 9.56
pyrrolidinyl derivates Mo4 showed none anti-HBV activity. In the
five ‘piperazinyl’ derivates, Mo1 (IC50 = 5.74 mol), Mo7
(IC50 = 7.55 mol) and Mo8 (IC50 = 6.71 mol) also showed high
inhibition of HBV replication, however, When chain length at
piperazine N1 more than two carbon atoms, such as Mo9 and
Mo11, both of them gave none antiviral activity. It illuminated that
the anti-HBV activity largely depends on the size, length and char-
acter of R5 substituent.
To gain deeper insights into the mechanisms of our compounds,
Mo13 was investigated to examine the effect on preformed Hepa-
titis B virus (HBV) capsid and on HBV capsid assembly by Size
exclusion chromatography (SEC).11,12 Recovered protein was
assigned to the void (aberrant capsid induced by Mo13, 8.5 min),
capsid (9.5 min) and dimer (12.5 min) based on the HPLC
chromatogram.
F
F
O
Cl
O
Cl
lmol, respectively), while
v
iv
O
N
F
R6
R6
O
N
F
l
N
N
l
l
Cl
R5
F
F
Mo1-13
4
Scheme 1. 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) 2-chloro-4-fluorobenzal-dehyde,ethyl 4-chloroacetoacetate,
NaOAc, EtOH, reflux, 20 h; (v) K2CO3, DMF, rt, 24 h.
produce Mo1–13. All the new compounds were characterized by
1H NMR, 13C NMR and MS spectrums.9
Inhibition activities for HBV replication of the compounds
Mo1–13 were determined in the HepG2.2.15 cells, which constitu-
tively produces HBV genomes, and secretes virus-like particles.10
Lamivudine (3TC) was used as positive control. To ascertain the
cytotoxic effects of the tested compounds, the cell viability was
determined after the cells exposed to the compounds for 48 h
(Table 1).
A comparison of the IC50 values of the derivatives Mo1–13
clearly indicated that the size and polarity of R5 substituent had
obvious effect on its antiviral activities (Table 1). Mo12
By SEC, we had observed the effect of compounds Mo13 on
Cp149 (Fig. 2). There was no change of the capsid morphology
detected at low concentration of Mo13 (Cp149:Mo13 = 4:1 or
2:1). At higher concentration (Cp149:Mo13 = 1:2 or 1:4), the
increasing continuous spectrum of void and the decreasing dimers
were observed. The results suggested that Mo13 can break the
equilibrium and change the product of HBV core protein
Table 1
In vitro anti-HBV evaluation of compounds Mo1–Mo13
Compds
Cytotoxicity (
TC50
l
mol)
TC0
4.7
4.83
4.68
Anti-HBV activity
mol) SI
5.74 5.72
IC50 (l
Mo1
Mo2
Mo3
Mo4
Mo5
Mo6
Mo7
Mo8
Mo9
Mo10
Mo11
Mo12
Mo13
3TC
32.85
33.75
32.73
77.29
25.21
32.98
98.74
41.3
31.25
55.25
29.6
54.02
31.48
5.75
10.57
3.19
14.96
14.56
4.65
14.1
13.78
4.45
12.81
4.22
13.98
4.51
10.24
9.56
7.55
6.71
2.46
13.08
6.16
4.35
12.72
6.66
2.35
8.11
13.37
0.82
14.17 1.43
Figure 2. SEC showed the effect of Mo13 on capsid assembly.

X. Yang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4247–4249
4249
ArH), 7.258–7.235 (2H, m, ArH), 6.000 (1H, s, CH), 3.955–3.919 (2H, m,
J = 7.2 Hz, CH2), 3.834 (2H, br, J = 14.0 Hz, CH2), 2.509–2.500 (4H, m), 1.726 (4H,
br), 1.065–1.030 (3H, t, J = 7.0 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d 165.7,
163.4, 163.2, 163.1, 161.5, 160.6, 159.0, 148.4, 140.9, 135.7, 132.7, 116.9, 115.5,
108.0, 104.8, 101.7, 61.1, 54.1, 52.1, 51.3, 22.9, 13.7. MS (EI) M+ 495.1.
Mo5: 1H NMR (400 MHz, DMSO-d6) d 9.490(1H, br, NH), 7.275–7.272 (3H, m,
ArH), 7.252–7.246 (2H, m, ArH), 6.007(1H, s, CH), 3.958–3.922 (2H, q, J = 7.3 Hz,
CH2), 3.758–3.601 (2H, dd, J = 4.8 Hz, J = 33.0 Hz, CH2), 2.508–2.499 (4H, br),
1.524–1.397 (4H, br), 1.397–1.385 (2H, br) 1.070–1.034 (3H, t, J = 7.0 Hz, CH3).
13C NMR (400 MHz, DMSO-d6) d 165.3, 163.2, 162.8, 161.4, 160.3, 158.9, 148.2,
139.8, 136.5, 133.2, 116.7, 115.4, 107.9, 104.3, 101.2, 61.1, 53.6, 52.1, 49.5, 25.3,
23.7, 14.2. MS (EI) M+ 509.0.
self-assembly. By structural biology, we established a screening
system for anti-HBV compounds that target on nucleocapsid. SEC
would be a much better method to discover the strong antiviral
compounds because of its objectivity, convenience and precision.
In summary, we have described the successful structural mod-
ification of HAPs to Mo1–13, a novel class of HBV capsid assembly
inhibitor with good water solubility. These newly developed deriv-
atives demonstrated excellent activities against HBV replication
in vitro. We also confirmed that Mo13 can induce Cp149 assembly
appropriately to aberrant capsid by SEC. The results indicated that
the design of new HBV capsid assembly inhibitor by optimizing the
size and character of R5 substituent is a feasible and promising
strategy and SEC is a nice method to screening them conveniently.
Mo6: 1H NMR (400 MHz, DMSO-d6) d 9.486 (1H, br, NH), 7.475–7.424 (3H, m,
ArH), 7.300–7.286 (2H, m, ArH), 6.007 (1H, s, CH), 4.036–3.963 (2H, q,
J = 7.2 Hz, CH2),3.740–3.660 (2H, dd, J = 14.0 Hz, CH2), 2.860–1.626 (4H, br),
1.340–1.192 (4H, br), 1.052–1.034 (3H, t, J = 7.0 Hz, CH3), 0.902–0.886 (4H, m).
13C NMR (400 MHz, DMSO-d6) d 165.9, 163.4, 163.1, 161.6, 159.1, 147.8, 139.2,
135.9, 132.6, 116.8, 115.5, 108.2, 104.1, 101.5, 61.4, 52.8, 51.9, 45.2, 32.2, 30.1,
19.6, 13.9. MS (EI) M+ 523.1.
Acknowledgments
Mo7: 1H NMR (400 MHz, DMSO-d6) d 9.523 (1H, br, NH), 7.410 (2H, m, ArH),
7.276 (3H, m, ArH), 6.008 (1H, s, CH), 3.963–3.945 (2H, q, J = 7.0 Hz, CH2),
3.740–3.628 (2H, dd, J = 14.01 Hz, CH2), 2.513–2.495 (8H, br), 2.155 (3H, s,
CH3), 1.056 (3H, t, J = 7.0 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d 166.7,
164.1, 163.7, 162.0, 161.2, 159.6, 148.9, 141.2, 135.4, 133.4, 133.2, 117.6, 116.1,
108.7, 104.0, 102.3, 61.7,53.6, 52.1, 51.0, 49.4, 49.1, 42.2, 14.2. MS (EI) M+
524.2.
We gratefully acknowledge financial support from the National
Nature Science Foundation of China (Grant No. 21172263).
Supplementary data
Mo8: 1H NMR (400 MHz, DMSO-d6) d 9.488 (1H, br, NH), 7.263 (2H, m, ArH),
7.240 (3H, m, ArH), 6.011 (1H, s, CH), 3.965–3.948 (2H, q, J = 7.0 Hz, CH2),
3.733–3.667 (2H, dd, J = 14.01 Hz, CH2), 2.506–2.492 (10H, br), 1.057 (3H, t,
J = 7.0 Hz, CH3), 0.984 (3H, t, J = 7.2 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d
166.4, 164.1, 163.7, 162.0, 161.1, 159.5, 149.0, 143.1, 135.7, 133.3, 133.1, 117.6,
116.1, 108.2, 104.5, 102.3, 61.7, 55.3, 52.6, 52.1, 49.2, 45.1, 14.2, 12.9. MS (EI)
M+ 538.0.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.07.
032. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Mo9: 1H NMR (400 MHz, DMSO-d6) d 9.489 (1H, br, NH), 7.263 (2H, m, ArH),
7.240 (3H, m, ArH), 6.008 (1H, s, CH), 3.964–3.946 (2H, q, J = 7.0 Hz, CH2),
3.731–3.663 (2H, dd, J = 14.01 Hz, CH2), 2.505–2.496 (8H, br), 2.214 (2H, t,
J = 7.5 Hz, CH2), 1.447–1.392 (2H, q, J = 7.5 Hz, CH2), 1.057 (3H, t, J = 7.0 Hz,
CH3), 0.843 (3H, t, J = 7.2 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d 166.3,
164.0, 163.8, 163.6, 162.0, 161.1, 159.5, 149.0, 143.1, 135.9, 133.2, 133.0, 117.6,
116.1, 107.5, 104.9, 102.3, 61.4, 57.1, 54.4, 52.1, 49.7, 49.3, 49.0, 17.1, 14.3,11.5.
MS (EI) M+ 552.0.
References and notes
1. Janssen, H. L.; Van-Zonneveld, M.; Senturk, H.; Zeuzem, S.; Akarca, U. S.;
Cakaloglu, Y.; Simon, C.; So, T. M.; Gerken, G.; de Man, R. A. Lancet 2005, 365,
123.
2. Zoulim, F.; Poynard, T.; Degos, F.; Slama, A.; El Hasnaoui, A.; Blin, P.; Mercier, F.;
Deny, P.; Landais, P.; Parvaz, P. J. Viral Hepat. 2006, 13, 278.
3. Hacker, H. J.; Deres, K.; Mildenberger, M.; Schröder, C. H. Biochem. Pharmacol.
2003, 66, 2273.
4. Deres, K.; Schroder, C. H.; Paessens, A.; Goldmann, S.; Hacker, H. J.; Weber, O.;
Krämer, T.; Niewöhner, U.; Pleiss, U.; Stoltefuss, J. Science 2003, 299, 893.
5. Bourne, C. R.; Finn, M. G.; Zlotnick, A. J. Virol. 2006, 80, 11055.
6. Stray, S. J.; Zlotnick, A. J. Mol. Recognit. 2006, 19, 542.
Mo10: 1H NMR (400 MHz, DMSO-d6) d 9.254(1H, br, NH), 7.444–7.320 (3H, m,
ArH), 6.901–6.755 (2H, m, ArH), 5.993 (1H, s, CH), 3.957–3.940 (2H, q,
J = 7.0 Hz, CH2) 3.771–3.755 (2H, dd, J1 = 15.4 Hz, J2 = 22.6 Hz, CH2), 3.600 (4H,
br), 3.402–3.333 (4H, m), 2.851–2.829 (4H, t, J = 4.4 Hz), 2.508–2.499 (4H, m),
1.041(3H, t, J = 7.0 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d 166.0, 164.7,
162.6, 162.2, 160.1, 153.8, 147.8, 145.8, 138.7, 133.2, 130.9, 117.1, 115.1, 114.0,
102.7, 98.0, 96.8, 66.7, 66.4, 59.9, 56.2, 56.0, 53.9, 51.8, 14.5. MS (EI) M+ 578.0.
Mo11: 1H NMR (400 MHz, DMSO-d6) d 9.581(1H, br, NH), 7.452–7.415 (1H, q,
ArH), 7.408–7.379 (1H, dd, ArH), 7.280–7.215 (3H, m, ArH), 6.015 (1H, s, CH),
4.064–4.012 (2H, q, J = 6.8 Hz, CH2), 3.981–3.928 (2H, q, J = 7.6 Hz, CH2), 3.815–
3.672 (2H, dd, CH2), 3.400–3.391 (4H, t, CH2), 2.506–2.447 (4H, t, CH2), 1.181
(3H, t, J = 6.8 Hz, CH3), 1.050 (3H, t, J = 7.6 Hz, CH3). 13C NMR (400 MHz, DMSO-
d6) d 165.9, 164.3, 162.6, 161.9, 160.1, 159.5, 155.2, 146.9, 142.5, 139.3, 132,7,
131.1, 116.9, 115.3, 110.7, 101.5, 98.2, 61.3, 60.0, 56.4, 55.4, 52.9, 43.7, 15.1,
14.4. MS (EI) M+ 583.0.
7. Zhao, G. M.; Wang, X. K.; Ju, X. J.; Guan, H.; Zhong, W.; Wang, L. L.; Xiao, J. H.;
Yang, X. H.; Li, S. Scientia Sinica Vitae 2011, 41, 971.
8. Goldmann, S.; Stoltefuss, J.; Niewohner, U.; Schlemmer, K. H.; Keldenich, J.;
Paessens, A.; Weber, O.; Deres, K. PCT patent WO01/68641, 2001.
9. Mo1: 1H NMR (400 MHz, DMSO-d6) d 9.73(1H, br, NH), 7.82 (1H, br, NH), 7.43–
7.42 (2H, m, ArH), 7.29–7.25 (3H, m, ArH), 6.01 (1H, s, CH), 3.98–3.93 (2H, q,
J = 7.3 Hz, CH2), 3.84–3.71 (2H, dd, CH2), 3.18 (2H, m, CH2), 3.06–3.05 (2H, q,
CH2), 2.51–2.50 (2H, m, CH3), 1.050 (3H, t, J = 7.3 Hz, CH3). 13C NMR (400 MHz,
DMSO-d6) d 168.0, 165.9, 164.3, 162.6, 161.9, 159.4, 146.7, 142.6, 139.4, 132.7,
131.1, 116.9, 115.4, 110.7, 101.4, 98.4, 60.1, 57.3, 56.3, 54.1, 49.0, 14.6. MS
(FAB) M+ 525.0.
Mo12: 1H NMR (400 MHz, DMSO-d6) d 9.644 (1H, br, NH), 7.486–7.449 (1H, q,
ArH), 7.396–7.367 (1H, dd, ArH), 7.251–7.220 (3H, m, ArH), 6.007(1H, s, CH),
4.533–4.508 (2H, t, J = 5.2 Hz, OH), 4.021–4.006 (2H, d, CH2), 3.946–3.928 (2H,
q, J = 7.2 Hz, CH2), 3.454–3.451 (4H, m, CH2), 2.646–2.632 (4H, t, J = 5.6 Hz,
CH2), 1.043 (3H, t, J = 7.2 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d 165.9,
164.3, 162.5, 161.9, 161.8, 160.0, 159.3, 150.2, 142.3, 139.7, 132.6, 131.3, 116.8,
115.4, 110.6, 101.5, 95.3, 59.8, 59.2, 57.4, 56.4, 54.2, 14.5. MS (EI) M+ 530.0.
10. Korba, B. E.; Gerin, J. L. Antiviral Res. 1992, 19, 55.
Mo2: 1H NMR (400 MHz, DMSO-d6) d 9.629(1H, br, NH), 7.415–7.413 (2H, m,
ArH), 7.278–7.259 (3H, m, ArH), 6.011 (1H, s, CH), 3.966–3.948 (2H, q,
J = 7.0 Hz, CH2) 3.787–3.751 (2H, dd, J = 14.01 Hz, CH2), 3.614–3.608 (4H, br),
2.504 (4H, br), 1.056 (3H, t, J = 7.0 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d
165.9, 164.4, 162.6, 162.0, 160.1, 159.5, 146.8, 142.7, 139.3, 132.7, 131.2, 116.9,
115.4, 110.5, 101.5, 98.5, 66.5, 60.0, 56.2, 55.9, 53.7, 14.4. MS (EI) M+ 511.1.
Mo3: 1H NMR (400 MHz, DMSO-d6) d 9.471 (1H, br, NH), 7.405 (2H, m, ArH),
7.242 (3H, m, ArH), 6.009 (1H, s, CH), 3.964–3.946 (2H, q, J = 7.0 Hz, CH2),
3.804–3.690 (2H, dd, J = 14.01 Hz, CH2), 2.716 (4H, br), 2.653 (4H, br), 1.053
(3H, t, J = 7.0 Hz, CH3). 13C NMR (400 MHz, DMSO-d6) d 166.3, 163.9, 163.6,
162.1, 161.1, 159.6, 148.8, 140.2, 136.1, 133.2, 133.1, 117.4, 116.0, 109.7, 105.4,
102.2, 61.6, 54.8, 54.5, 51.8, 24.3, 14.2. MS (EI) M+ 527.0.
11. Stray, S. J.; Bourne, C. R.; Punna, S.; Lewis, W. G.; Finn, M. G.; Zlotnick, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 8138.
12. SEC general procedure: Capsid assembly was initiated by mixing Cp149 with
test compounds in 2ꢀ buffer incubation 24 h, respectively. Assembly reactions
were examined on a Superose column (Biosep-SEC-S3000) mounted on HPLC
system equipped with an auto injection module. The column was equilibrated
with 100 mM HEPES pH 7.5, 300 mM NaCl at 0.6 ml/min.
Mo4: 1H NMR (400 MHz, DMSO-d6) d 9.662(1H, br, NH), 7.418–7.278 (3H, m,