Synthesis and broad-spectrum antiviral activity of some novel benzo-heterocyclic amine compounds

Article abstract of DOI:10.3390/molecules19010925

A series of novel unsaturated five-membered benzo-heterocyclic amine derivatives were synthesized and assayed to determine their in vitro broad-spectrum antiviral activities. The biological results showed that most of our synthesized compounds exhibited potent broad-spectrum antiviral activity. Notably, compounds 3f (IC50 = 3.21-5.06 M) and 3g (IC50 = 0.71-34.87 M) showed potent activity towards both RNA viruses (influenza A, HCV and Cox B3 virus) and a DNA virus (HBV) at low micromolar concentrations. An SAR study showed that electron-withdrawing substituents located on the aromatic or heteroaromatic ring favored antiviral activity towards RNA viruses.

Full text of DOI:10.3390/molecules19010925

Molecules 2014, 19, 925-939; doi:10.3390/molecules19010925
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Broad-Spectrum Antiviral Activity of Some Novel
Benzo-Heterocyclic Amine Compounds
Da-Jun Zhang †, Wen-Fang Sun †, Zhao-Jin Zhong †, Rong-Mei Gao, Hong Yi, Yu-Huan Li,
Zong-Gen Peng * and Zhuo-Rong Li *
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: pumcpzg@126.com (Z.-G.P.);
l-z-r@263.net (Z.-R.L.); Tel.: +86-10-6301-0984 (Z.-G.P.); +86-10-6302-7185 (Z.-R.L.);
Fax: +86-10-6301-7302 (Z.-G.P. & Z.-R.L.).
Received: 28 November 2013; in revised form: 8 January 2014 / Accepted: 9 January 2014 /
Published: 15 January 2014
Abstract: A series of novel unsaturated five-membered benzo-heterocyclic amine
derivatives were synthesized and assayed to determine their in vitro broad-spectrum antiviral
activities. The biological results showed that most of our synthesized compounds exhibited
potent broad-spectrum antiviral activity. Notably, compounds 3f (IC50 = 3.21–5.06 μM) and
3g (IC50 = 0.71–34.87 μM) showed potent activity towards both RNA viruses (influenza A,
HCV and Cox B3 virus) and a DNA virus (HBV) at low micromolar concentrations. An
SAR study showed that electron-withdrawing substituents located on the aromatic or
heteroaromatic ring favored antiviral activity towards RNA viruses.
Keywords: unsaturated five-membered benzo-heterocyclic amines; broad-spectrum
antiviral activity; DNA and RNA virus; SAR
1. Introduction
Viral infections pose a threat to virtually every organism in every domain of life. People over
65 years of age account for 90% of seasonal influenza-associated deaths, and influenza viruses with
either high mortality or morbidity have heightened fears that the next influenza pandemic will occur

Molecules 2014, 19
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soon [1,2]. The hepatitis C virus (HCV) is a significant bloodborne human pathogen that affects an
estimated 3% of the world’s population, 80% of whom progress to a chronic infectious state [3,4].
Currently, the standard of care (SOC) is a combination of pegylated interferon and ribavirin, but this
regimen can cause severe side effects and poorly sustained viral response [5,6]. Although two protease
inhibitors (Victrelis (boceprevir) and Incivek (telaprevir)) were approved by the USA FDA in 2011,
both must be used in combination with ribavirin and pegylated interferon, and there is still an unmet
need for the treatment of HCV infections. Hepatitis B virus (HBV) is a hepatotropic noncytopathic
envelope virus that causes acute and chronic hepatitis. Although a vaccine has been developed, chronic
hepatitis B (CHB) caused by hepatitis virus infection is still one of the most serious human viral
infectious diseases worldwide. Worldwide more than 350 million people suffer from chronic HBV
infections, many of whom subsequently develop more severe liver diseases such as cirrhosis and
hepatocellular carcinoma (HCC) [7]. Coxsackie B viruses are single-strand RNA viruses, and infection
with Cox B can cause fever, headache, chest pain and other problems. Cardiac infection with Cox B3
can result in acute myocarditis that is spontaneously resolved or chronic myocarditis with prolonged
viral persistence [8]. Currently, there is no specific treatment or vaccine available for Coxsackie
virus infections.
Most current antiviral drugs, including those in development, are direct-acting antiviral (DAA)
molecules that specifically target viral proteins. These drugs are narrow in spectrum and are vulnerable
to the rapid emergence of viral resistance [9]. The emergence of drug-resistant viruses, especially
multidrug-resistant strains, represents a significant problem in current clinical practice that needs to be
addressed and should be considered a high priority for new avenues of research [10,11]. To fulfill all
of these requirements, novel classes of antivirals are needed [12]. Additionally, due to the high
mutation rates that are particularly prevalent in RNA viruses, the lifetime of specific antiviral
therapeutics is often severely limited. Broad spectrum antivirals would be one way of circumventing
this problem [13]. Consequently, it is highly desirable to generate these types of drugs, and scientists
are encouraged to research and develop novel broad spectrum antivirals.
Previously, we have reported a novel class of (5-oxazolyl)phenyl amines that act as potential
inosine monophosphate dehydrogenase (IMPDH) inhibitors and exhibit broad-spectrum antiviral
activities [14]. In continuance of our endeavor to search for novel antiviral agents with broad-spectrum
antiviral activity, we designed a novel class of unsaturated five-membered benzo-heterocyclic
derivatives using a ring transforming strategy that is based on our previously reported structures
(Figure 1).
Figure 1. The chemical structure of the designed target compounds.
Moreover, the secondary amine derivatives showed potent antiviral activity against CoxB3, CoxB6
and HCV in our previous study. Therefore, the secondary amine derivatives were primarily
synthesized in this work. Additionally, unsaturated five-membered benzo-heterocyclic scaffolds appear

Molecules 2014, 19
927
to be privileged structures, which were widely distributed among bioactive compounds and successful
drugs [15–18]. Consequently, a novel class of unsaturated five-membered benzo-heterocycle-
substituted amines were synthesized and assayed for their broad spectrum antiviral activity in vitro,
and their structure-activity relationships (SARs) were also investigated.
Scheme 1. Synthetic route to the target compounds 1a–4b.
Reagents and conditions: (a) 10% Pd/C, H2(50 psi), EtOH, rt; and (b) various substituted aldehydes, EtOH, rt
or reflux, 8 h, then NaBH4, rt, 3 h.
2. Results and Discussion
2.1. Chemistry
As depicted in Scheme 1, a series of novel benzo-heterocyclic amine derivatives containing the
substituted or non-substituted aromatic/heteroaromatic rings were synthesized. The commercially
available compounds 1–4 were chosen as starting materials and were reduced at 50 psi hydrogen over
Pd/C (10%) to produce the intermediate compounds 1M–4M which have been reported in the
literature [19–22]. The target compounds were obtained via reductive amination with a yield of
50%–65%. Specifically, compounds 1M–4M were condensed with commercially available substituted
aldehydes at room temperature or under reflux in ethanol to produce the intermediate Schiff bases,

Molecules 2014, 19
928
which were directly reduced without further purification in the presence of NaBH4 to generate the
target compounds. In the case of compounds 4a and 4b, which were insoluble in methylene chloride,
ethyl acetate was used to extract the target products after workup. The chemical structures of the
synthesized target compounds were all confirmed by 1H-NMR, 13C-NMR and HRMS.
2.2. Biological Evaluation
The synthesized target compounds were assayed for their broad spectrum antiviral activity towards
influenza A, HBV, HCV and Cox B3 virus in vitro. Specifically, the activity against influenza A and
Cox B3 was determined by the cytopathic effect (CPE) inhibitory assay, and oseltamivir and ribavirin
(RBV) were used as positive controls, respectively. The inhibitory activities towards HBV and HCV
were tested by real time PCR, and lamivudine (3TC) and teleprevir were used as positive controls,
respectively. The results are summarized in Table 1.
In general, the majority of our synthesized compounds showed definitive broad spectrum activity
against the viruses tested especially compounds 3f and 3g, which exhibited potent activity towards the
entire virus panel tested. IC50 values ranged from 0.71 μM to 34.87 μM. Additionally, most of our
synthesized compounds showed potent inhibitory activity towards Influenza A, HCV and Cox B3 virus
simultaneously, including compounds 2c,e,g, 3d–g, 3i and 4a. However, only 3 compounds (1a, 3f and
3g) showed inhibitory activity toward HBV. Based on these results, we concluded that our synthesized
compounds were more sensitive to RNA viruses (Influenza A, HCV and Cox B3) when compared to a
DNA virus (HBV).
2.2.1. Anti-influenza Virus Activity
The majority of the synthesized compounds exhibited potent antiviral activity towards influenza
A/hanfang/359/95 virus. This was particularly true for compounds 1d, 2c,e,g, 3b–f,h,i and 4a because
the IC50 values were superior or comparable to the positive control drugs (RBV and oseltamivir).
Notably, compound 3d showed both potent inhibitory activity against influenza A (IC50 = 3.36 μM)
and the highest SI value (80.8) among the entire panel of synthesized compounds.
With regard to the structure-activity relationships, the activity of compounds 4a,b, and 3e,g were
superior to their indole and benzoxazole scaffold counterparts; we concluded that the benzimidazole
and benzothiazole moieties favored anti-influenza activity. Replacement of the hydrogen at the 4
position of the thiophene ring (2d and 3h) with a chloro group (2e and 3i) increased the compounds’
anti-influenza activity. The derivatives 2b and 3d containing a strong electron withdrawing group
(NO2) substituted at the para-position of the benzene ring also showed potent inhibitory activity.
Migration of the NO2 group from the para-position (3d) to the meta-position (3c) slightly decreased
the activity. Based on these results, we concluded that an electron withdrawing substituent on the
aromatic or heteroaromatic ring favored anti-influenza A activity.

Molecules 2014, 19
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Table 1. The antiviral activity and cytotoxicity of the compounds.
Influenza A/hanfang/359/95
Cox B3
HCV
HBV
NO.
TC50 (μM) IC50 (μM)
SI
-
-
-
2.2
-
7.0
11.5
11.6
8.0
-
TC50 (μM) IC50 (μM) SI TC50 (μM) IC50 (μM)
SI
11.3
NT
16.8
NT
TC50 (μM) IC50 (μM)
SI
5.6
-
-
-
-
-
-
NT
-
NT
NT
-
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
4a
4b
RBV
3.35
2.86
4.52
>1.60
>1.65
>0.66
11.84
>10.60
41.26
11.59
34.43
10.95
>98.32
9.04
>10.13
10.02
12.47
3.36
8.60
4.15
8.34
8.61
4.79
2.86
1.93
1.69
3.52
31.34
1.24
9.29
3.55
56.73
8.98
10.13
5.66
7.23
1.82
1.16
3.21
5.03
10.43
2.53
52.98
-
1.7
3.0
13.0
39.1
15.7
8.2
83.5
10.8
8.2
15.88
NT
46.78
NT
1.40
NT
2.77
8.02
3.21
16.67
28.87
28.87
16.67
28.87
NT
1.44
>1.85
>5.56
>16.67
>16.67
>5.56
>16.67
NT
>11.11
NT
NT
>16.67
>33.33
NT
>5.56
>5.56
5.06
0.71
NT
>5.56
>16.67
NT
NT
>50
NT
NT
25.15
66.10
55.13
257.52
103.36
100.35
29.08
295.10
80.80
21.08
67.23
112.45
181.3
20.05
12.48
65.57
65.03
13.18
826.44
2.09
26.50
64.04
288.23
133.16
434.24
87.30
425.44
56.04
21.08
55.95
45.01
272.01
24.10
21.66
94.60
78.14
27.42
826.44
1.73
NT
89.63
>200
105.44
136.53
45.78
NT
27.57
108.82
126.59
NT
142.86
43.58
54.99
138.38
153.72
38.24
>200
NT
32.95
>66.67
10.48
>66.67
17.72
NT
14.18
48.99
38.01
NT
22.50
9.75
5.04
34.87
47.64
9.81
>66.67
NT
2.7
<3.0
10.1
<2.0
2.6
NT
1.9
2.2
3.3
NT
6.3
4.5
10.9
4.5
3.2
3.9
<3.0
NT
14.3
>8.2
NT
19.25
NT
NT
5.2
9.0
2.1
6.2
-
28.87
57.74
NT
9.62
9.62
28.87
21.48
NT
9.62
28.87
NT
NT
>50
5.6
3.6
80.8
2.8
5.2
3.6
11.9
8.0
3.9
-
3.0
20.5
544.0
212.5
NT
NT
11.9
15.6
99.5
17.2
3.9
13.0
6.2
5.2
15.6
-
4.0
5.2
3.9
-
NT
-
-
5.7
30.3
NT
-
26.24
6.58
3.42
213.80
>0.41
4.93
-
NT
NT
-
NT
NT
>54.1
NT
14.84
440.02
4765.44
4030.42
NT
25.75
254.07
8205.90
NT
6.38
48.89
2120.4
NT
NT
NT
79.04
>200
NT
NT
NT
5.51
24.37
NT
NT
NT
21.43
8.76
18.97
NT
NT
NT
>100
NT
Oseltamivir
3TC
Telaprevir
NT
NT
NT
NT
NT
2920.0
NT
NT
1.85
NT
NT
NT
32.12
0.011
TC50, 50% cytotoxic concentration; IC50, 50% inhibition concentration; SI, the selectivity index. NT means not tested.

Molecules 2014, 19
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However, in the case of compound 3e, introduction of an electron-donating methoxyl group at the
ortho-position of the benzene ring (3f) also increased antiviral activity, and this effect may not be due to
its electron-donating property, but rather to the hydrogen bond-accepting properties of the methoxyl
group. A complete loss in activity of compound 3k containing a bulky anthracene group may be due to
steric hindrance.
2.2.2. Anti Cox B3 Virus Activity
As shown in Table 1, all of the synthesized compounds except 3k showed potent inhibitory activity
towards Cox B3 at low micromolar concentrations, especially compounds 1a–d, 2a,c,e, 3d–f and 3i,
and the IC50 values were less than 5.0 μM. Notably, compounds 3d and 2c exhibited relatively high SI
values (99.5 and 83.5, respectively). Based on the activity of compounds 1b, 2c, 3e and 4a, we
concluded that only the benzimidazole slightly disfavored anti-Cox B3 activity, and there was no
significant difference among the other three scaffolds. Similar to the anti-influenza activity SAR, the
derivatives containing electron-withdrawing groups (2e, 3d and 3i) showed relatively more potent
activity. Substitution of an electron-donating methoxyl group (3f) for the hydrogen atom at the
ortho- position of the benzene ring (3e) disfavored antiviral activity. Migration of the NO2 group from
the para position (3d) to the meta-position (3c) also decreased its activity. Compound 3k showed no
inhibitory activity, likely due to high steric hindrance.
2.2.3. Anti HCV Activity
As shown in Table 1, compounds 1a,c, 3e,f,i and 4a showed relatively potent anti-HCV activity
with IC50 values of less than 10.0 μM. The IC50 values of all of the synthesized compounds were
inferior to the positive control telaprevir, an NS3/4A protease inhibitor. However, the synthesized
compounds exhibited less cytotoxicity when compared to telaprevir. Based on the activity of
compounds 1c, 3g and 4b, the indole scaffold appeared to be the optimal fragment for anti-HCV
activity. Given that compounds 2e and 3i, which contain chloro groups at the thiophene ring, were
more potent that their non-substituted counterparts, we concluded that the electron-withdrawing
substituents were favorable for anti-HCV activity, a result that is consistent with the SAR of both anti-
influenza A and anti-Cox B3 activities. The most potent of the synthesized compounds was also the
compound containing a chloro group at the para-position of the benzene ring (1a). The derivatives
containing a styryl substituent (2c, 3e, 3f and 4a) also showed potent anti-HCV activity at low
micromolar concentrations.
2.2.4. Anti-HBV Activity
HBV was less sensitive to the synthesized compounds compared to influenza A, Cox B3 and HCV
virus, and only compounds 1a, 3f and 3g showed definitive activity towards HBV. Among these,
compounds 1a and 3g were more potent than the positive control 3TC, especially compound 3g which
exhibited an IC50 value of less than 1.0 μM (0.7 μM). Some simple SARs can be determined based on
the biological results. Although compound 3f showed potent inhibitory activity towards HBV,
compound 3e which lacks a methoxyl group at the ortho-position of the benzene ring did not exhibit

Molecules 2014, 19
931
any activity. Hence, the methoxyl group was indispensable for the anti-HBV activity of compound 3f.
Additionally, substitution of the indole (1c) or benzimidazole (4b) scaffold for the benzothiazole (3g)
fragment led to a complete loss of antiviral activity.
3. Experimental
3.1. General Information
All solvents and reagents were of chemical or analytical grade. The progress of the reactions was
1
13
monitored by TLC using solvent systems of different polarities. H-NMR and C-NMR spectra were
recorded with a BrukerBioSpin GmbH (Rheinstetten, Germany) spectrometer at 400 or 500 MHz.
Chemical shifts are reported in parts per million relative to a tetramethylsilane internal standard. Melting
points were determined with an X6 microscope melting point apparatus and were not corrected. All mass
spectra (MS, HRMS) were recorded on a LTQ-Orbitrap linear ion trap high-resolution mass
spectrometer (ThermoFisher Scientific, San Jose, CA, USA).
3.2. Chemistry
3.2.1. General procedure for the synthesis of 1M–4M
A mixture of compounds 1–4 (6.13 mmol) in ethanol (30 mL) was treated with 10% Pd/C (20 wt. %
of 1–4) and subjected overnight to 50 psi H2 (g) in a Parr hydrogenation apparatus. The reaction
mixture was filtered and concentrated in vacuo. Pure products 1M–4M were obtained via flash
chromatography by eluting with a gradient of 30%–40% EtOAc/hexanes.
1H-Indol-6-ylamine (1M). Gray solid, mp: 67–68 °C; 90% yield; 1H-NMR (400 MHz, CDCl3) δ (ppm):
7.87 (1H, brs), 7.40 (1H, d, J = 8.4 Hz), 7.00 (1H, m), 6.67(1H,s), 6.57 (1H, dd, J1 = 8.4 Hz, J2 = 2.0 Hz),
6.42 (1H, m).
Benzooxazol-6-ylamine (2M). Yellow solid, mp: 86–87 °C; 86% yield; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 8.32 (1H, s), 7.39 (1H, d, J = 8.8 Hz), 6.78 (1H, d, J = 2.0 Hz), 6.64 (1H, dd, J1 = 8.8 Hz,
J2 = 2.0 Hz), 5.36 (1H, br).
Benzothiazol-6-ylamine (3M). Yellow solid, mp: 88–89 °C; 88% yield; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 8.88 (1H, s), 7.71 (1H, d, J = 8.4 Hz), 7.12 (1H, d, J = 2.0 Hz), 6.81 (1H, dd, J1 = 8.4 Hz,
J2 = 2.0 Hz), 5.40 (2H, br).
1
3H-Benzoimidazol-5-ylamine (4M). Brown solid, mp: 121–122 °C; 85% yield; H-NMR (400 MHz,
DMSO-d6) δ (ppm): 7.85 (1H, s), 7.24 (1H, d, J = 8.4 Hz), 6.66 (1H, s), 6.50 (1H, dd, J1 = 8.4 Hz,
J2 = 2.0 Hz), 4.80 (2H, br).
3.2.2. General procedure for the synthesis of 1a–1e, 2a–2g, 3a–3f
A mixture of the appropriate amine (1M–3M, 2 mmol) and the appropriate aldehyde (2.2 mmol) in
ethanol (10 mL) was stirred at room temperature for 8 h and NaBH4 (2.2 mmol) was subsequently

Molecules 2014, 19
932
added to the solution. After 3 h, the reaction was completed. Water (5 mL) was then added to the
solution and neutralized with aqueous 10% HCl. CH2Cl2 (40 mL) was added to the solution, and the
organic layer was separated, washed with saturated aqueous sodium chloride and subsequently dried
over anhydrous Na2SO4. The solvent was removed under a vacuum. The residue was purified by
chromatography on silica gel (EtOAc/n-hexane = 1/3) to generate the corresponding compounds.
Characterization data for the title compounds:
1
(4-Chlorobenzyl)-(1H-indol-6-yl)-amine (1a). White solid, mp: 135–137 °C, 50% yield; H-NMR
(DMSO-d6, 500 MHz) δ (ppm): 10.42 (1H, s), 7.39–7.31 (4H, m), 7.20–7.18 (1H, m), 6.93–6.92 (1H,
m), 6.47–6.45 (1H, m), 6.35 (1H, s), 6.15 (1H, s), 5.93 (1H, t, J = 6.0 Hz), 4.26 (2H, d, J = 6.0 Hz).
13C-NMR (125 MHz, DMSO-d6) δ (ppm): 144.6, 140.4, 137.8, 130.4, 129.4 (2C), 128.6 (2C),
122.2, 120.7, 119.9, 109.7, 101.3, 93.3, 47.1. HR-MS (ESI+) m/z: 257.0844 [M+H]+, calcd for
+
C15H14ClN2 , 257.0845.
1
(E)-(1H-Indol-6-yl)-(3-phenylallyl)-amine (1b). White solid, mp: 130–132 °C, 53% yield; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 10.47 (1H, s), 7.40–7.39 (2H, m),7.32–7.29 (2H, m), 7.22–7.19 (2H,
m), 6.96–6.95 (1H, m), 6.61 (1H, d, J = 16.0 Hz), 6.51 (1H, s), 6.47 (1H, dd, J1 = 8.5 Hz, J2 = 2.0 Hz),
13
6.43–6.41 (1H, m), 6.17 (1H, s), 5.52 (1H, t, J = 5.5 Hz), 3.86–3.84 (2H, m). C-NMR (125 MHz,
DMSO-d6) δ (ppm): 145.0, 138.0, 137.4, 130.2, 129.3, 129.2 (2C), 127.7, 126.5 (2C), 122.2, 120.6,
+
119.9, 109.7, 101.3, 93.2, 46.3. HR-MS (ESI+) m/z: 249.1390 [M+H]+, calcd for C17H17N2 , 249.1392.
1
(1H-Indol-6-yl)-(5-methylfuran-2-ylmethyl)-amine (1c). White solid, mp: 100–102 °C, 50% yield; H-
NMR (400 MHz, DMSO-d6) δ (ppm): 10.49 (1H, s), 7.19 (1H, d, J = 8.4 Hz), 6.96–6.95 (1H, m), 6.52
(1H, s), 6.46 (1H, dd, J1 = 8.4 Hz, J2 = 2.0 Hz), 6.17–6.16 (1H, m), 6.12 (1H, d, J = 2.8 Hz), 5.95 (1H,
13
d, J = 2.0 Hz), 5.63 (1H, t, J = 6.0 Hz), 4.15 (2H, d, J = 6.0 Hz), 2.22 (3H, s). C-NMR (125 MHz
DMSO-d6) δ (ppm): 152.4, 150.7, 144.6, 137.9, 122.3, 120.6, 120.0, 109.6, 107.9, 106.7, 101.3, 93.3,
41.5, 13.8. HR-MS(ESI+) m/z: 227.1185 [M+H]+, calcd for C14H15N2O+, 227.1184.
(1H-Indol-6-yl)-(5-methylthiophen-2-ylmethyl)-amine (1d). White solid, mp: 89–91 °C, 50% yield; 1H-
NMR (500 MHz, DMSO-d6) δ (ppm): 10.47 (1H, s), 7.19 (1H, d, J = 8.5 Hz),6.96–6.95 (1H, m), 6.79
(1H, d, J = 3.0 Hz), 6.60 (1H, d, J = 2.0 Hz), 6.51 (1H, s), 6.47 (1H, dd, J1 = 8.5 Hz, J2 = 2.0 Hz), 6.17
(1H, s), 5.78 (1H, t, J = 6.0 Hz), 4.33 (2H, d, J = 6.0 Hz), 2.35 (3H, s). 13C-NMR (125 MHz
DMSO-d6) δ (ppm): 144.5, 143.1, 137.9, 137.8, 125.2, 124.6, 122.3, 120.6, 120.1, 109.8, 101.3, 93.6,
43.6, 15.5. HR-MS(ESI+) m/z: 243.0954 [M+H]+, calcd for C14H15N2S+, 243.0956.
4-(Benzooxazol-6-ylaminomethyl)-2-bromo-6-methoxyphenol (2a). White solid, mp: 129–131 °C, 53%
yield; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.27 (1H, s), 8.33 (1H, s), 7.42 (1H, d, J = 8.4 Hz), 7.08
(1H, s), 7.01 (1H, s), 6.76 (1H, d, J = 2.0 Hz), 6.72 (1H, dd, J1 = 8.4 Hz, J2 = 2.0 Hz), 6.51 (1H, t,
J = 6.0 Hz), 4.20 (2H, d, J = 6.0 Hz), 3.80 (3H, s).13C-NMR (125 MHz DMSO-d6) δ (ppm): 151.6,
151.4, 148.9, 148.2, 142.9, 132.2, 130.5, 123.2, 120.3, 112.4, 111.1, 109.7, 92.9, 56.6, 46.6.
HR-MS(ESI+) m/z: 349.0178 [M+H]+, calcd for C15H14N2O3Br+, 349.0188.

Molecules 2014, 19
933
1
Benzooxazol-6-yl-(4-nitrobenzyl)-amine (2b). Brown solid, mp: 154–156 °C, 65% yield; H-NMR
(400 MHz, DMSO-d6) δ (ppm): 8.33 (1H, s), 8.19 (2H, d, J = 8.8 Hz), 7.64 (2H, d, J = 8.8 Hz),
7.45–7.43 (1H, d, J = 8.4 Hz), 6.80 (1H, t, J = 6.0 Hz), 6.73–6.72 (2H, m), 4.49 (2H, d, J = 6.0 Hz).
13C-NMR (125 MHz DMSO-d6) δ (ppm): 151.6, 151.5, 149.0, 147.9, 147.0, 130.7, 128.7 (2C), 124.0
+
(2C), 120.5, 112.3, 93.0, 46.6. HR-MS (ESI+) m/z: 270.0879 [M+H]+, calcd for C14H12N3O3 , 270.0879.
1
(E)-Benzooxazol-6-yl-(3-phenylallyl)-amine (2c). Light green solid, mp: 100–103 °C, 55% yield; H-
NMR (400 MHz, DMSO-d6) δ (ppm): 8.34 (1H, s), 7.45–7.40 (3H, m), 7.33–7.29 (2H,m), 7.24–7.20
(1H,m), 6.82 (1H, d, J = 2.0 Hz), 6.74 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.63 (1H, d, J = 16.0 Hz),
13
6.38 (1H, m), 6.27(1H, t, J = 5.6 Hz), 3.91–3.87 (2H, m). C-NMR (125 MHz DMSO-d6) δ (ppm):
151.7, 151.3, 148.5, 137.2, 130.8, 129.1 (2C), 128.9, 128.1, 127.8, 126.6 (2C), 120.2, 112.3, 92.7,
45.7. HR-MS (ESI+) m/z: 251.1176 [M+H]+, calcd for C16H15N2O+, 251.1184.
Benzooxazol-6-yl-thiophen-2-ylmethylamine (2d). Light green solid, mp: 62–64 °C, 62% yield;
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.35 (1H, s), 7.43 (1H, d, J = 8.8 Hz), 7.37 (1H, d,
J = 5.2 Hz), 7.09 (1H, d, J = 3.2 Hz), 6.98–6.95 (1H, m), 6.85 (1H, d, J = 2.0 Hz), 6.75 (1H, dd,
13
J1 = 8.8 Hz, J2 = 2.0 Hz), 6.59 (1H, t, J = 6.0 Hz), 4.50 (2H, d, J = 6.0 Hz). C-NMR (125 MHz
DMSO-d6): δ (ppm): 151.6, 151.5, 148.0, 144.2, 130.6, 127.3, 125.6, 125.1, 120.3, 112.5, 93.1, 42.7.
HR-MS (ESI+) m/z: 231.0582 [M+H]+, calcd for C12H11N2OS+, 231.0592.
Benzooxazol-6-yl-(5-chlorothiophen-2-ylmethyl)-amine (2e). Light green solid, mp: 100–102 °C, 58%
yield; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.36 (1H, s), 7.45 (1H, d, J = 8.8 Hz), 6.97–6.95 (2H,
m), 6.86 (1H, d, J = 2.0 Hz), 6.73 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.63 (1H, t, J = 6.0 Hz), 4.45 (2H,
J = 6.0 Hz). 13C-NMR (125 MHz DMSO-d6) δ (ppm): 151.7, 151.5, 147.7, 144.2, 130.9, 126.9, 126.7,
125.3, 120.1, 112.6, 93.3, 42.9. HR-MS (ESI+) m/z: 265.0210 [M+H]+, calcd for C12H10N2OSCl+,
265.0202.
1
Benzooxazol-6-yl-pyrimidin-5-ylmethyl-amine (2f). Gray solid, mp: 175–177 °C, 51% yield; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 9.06 (1H, s), 8.81 (2H, s), 8.35 (1H, s), 7.45 (1H, d, J = 8.5 Hz), 6.85
(1H, d, J = 1.5 Hz), 6.74 (1H, dd, J1 = 8.5 Hz, J2 = 1.5 Hz), 6.61 (1H, t, J = 6.0 Hz), 4.38 (2H, d,
13
J = 6.0 Hz). C-NMR (125 MHz DMSO-d6) δ (ppm): 157.6, 156.8 (2C), 156.0, 151.7, 151.6, 133.5,
130.8, 120.5, 112.5, 93.2, 42.6. HR-MS (ESI+) m/z: 227.0934 [M+H]+, calcd for C12H11N4O+, 227.0933.
1
Benzooxazol-6-yl-quinolin-8-ylmethylamine (2g). Gray solid, mp: 98–100 °C, 63% yield; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 8.99–8.98 (1H, m), 8.39(1H, d, J = 8.5 Hz), 8.29 (1H, s), 7.87 (1H, d,
J = 8.0 Hz), 7.73 (1H, d, J = 7.0 Hz), 7.60–7.57 (1H, m), 7.56–7.52 (1H, m), 7.42–7.40 (1H, m),
6.75–6.74 (2H, m), 6.62 (1H, t, J = 6.0 Hz), 4.95 (2H, d, J = 6.0 Hz). 13C-NMR (125 MHz DMSO-d6)
δ (ppm): 151.7, 151.3, 150.2, 148.6, 146.4, 137.4, 137.0, 130.4, 128.4, 127.9, 127.4, 126.8, 121.9,
120.3, 112.2, 92.7, 42.4. HR-MS (ESI+) m/z: 276.1125 [M+H]+, calcd for C17H14N3O+, 276.1137.
4-(Benzothiazol-6-ylaminomethyl)-2-bromo-6-methoxyphenol (3a). White solid, mp: 193–195 °C, 54%
yield; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.27 (1H, s), 8.89 (1H, s), 7.54 (1H,d, J = 8.4 Hz), 7.10
(1H, d, J = 2.4 Hz), 7.08 (1H, s), 7.01 (1H, d, J = 2.4 Hz), 6.89 (1H, dd, J1 = 8.4 Hz, J2 = 2.4 Hz), 6.53

Molecules 2014, 19
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(1H, d, J = 6.0 Hz), 4.22 (2H, d, J = 6.0 Hz), 3.80 (3H, s).13C-NMR (125 MHz DMSO-d6) δ (ppm):
149.9, 148.9, 147.5, 145.4, 143.0, 135.9, 132.2, 123.5, 123.2, 115.0, 111.1, 109.7, 102.0, 56.6, 46.5.
HR-MS (ESI+) m/z: 364.9953 [M+H]+, calcd for C15H14N2O2SBr+, 364.9959.
2-(Benzothiazol-6-ylaminomethyl)-6-methoxyphenol (3b). Pale yellow solid, mp: 206–208 °C, 59%
1
yield; H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.87 (1H, s), 8.73 (1H, s), 7.72 (1H, d, J = 8.8 Hz),
7.05 (1H, d, J = 2.0 Hz), 6.88 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.85 (1H, s), 6.83 (1H, s), 6.72–6.68
13
(1H, m), 6.38 (1H, t, J = 6.0 Hz), 4.26 (2H, d, J = 6.0 Hz), 3.80 (3H, s). C-NMR (125 MHz
DMSO-d6) δ (ppm): 149.6, 147.9, 147.8, 145.2, 144.4, 135.9, 126.4, 123.5, 120.7, 119.1, 114.8, 110.9,
101.5, 56.3, 41.9. HR-MS (ESI+) m/z: 287.0851 [M+H]+, calcd for C15H15N2O2S+, 287.0854.
1
Benzothiazol-6-yl-(3-nitrobenzyl)-amine (3c). Yellow solid, mp: 126–128 °C, 54% yield; H-NMR
(500 MHz, DMSO-d6) δ (ppm): 8.89 (1H, s), 8.24 (1H, s), 8.09 (1H, dd, J1 = 8.0 Hz, J2 = 1.5 Hz), 7.84
(1H, d, J = 8.0 Hz), 7.74 (1H, d, J = 9.0 Hz), 7.64–7.61 (1H, m), 7.11 (1H, d, J = 2.5 Hz), 6.90 (1H,
dd, J1 = 9.0 Hz, J2 = 2.5 Hz), 6.80 (1H, t, J = 6.0 Hz), 4.49 (2H, d, J = 6.0 Hz). 13C-NMR (125 MHz
DMSO-d6) δ (ppm): 150.2, 148.4, 147.1, 145.6, 143.2, 136.0, 134.4, 130.3, 123.7, 122.3, 122.2, 115.0,
102.1, 46.2. HR-MS (ESI+) m/z: 286.0661 [M+H]+, calcd for C14H12N3O2S+, 286.0650.
1
Benzothiazol-6-yl-(4-nitrobenzyl)-amine (3d). Brown solid, mp: 157–159 °C, 57% yield; H-NMR
(400 MHz, DMSO-d6) δ (ppm): 8.90 (1H, s), 8.20 (2H, d, J = 8.4 Hz), 7.75 (1H, d, J = 8.8 Hz), 7.64
(2H, d, J = 8.4 Hz), 7.07 (1H, d, J = 2.4 Hz), 6.89 (1H, dd, J1 = 8.8 Hz, J2 = 2.4 Hz), 6.84–6.80 (1H, t,
13
J = 6.4 Hz), 4.50 (2H, d, J = 6.4 Hz). C-NMR (125 MHz DMSO-d6) δ (ppm): 150.2, 149.0, 147.2,
147.0, 145.5, 136.0, 128.7 (2C), 124.0 (2C), 123.7, 114.9, 102.0, 46.5. HR-MS (ESI+) m/z: 286.0652
[M+H]+, calcd for C14H12N3O2S+, 286.0650.
1
(E)-benzothiazol-6-yl-(3-phenyl-allyl)-amine (3e). Brown solid, mp: 99–101 °C, 57% yield; H-NMR
(400 MHz, DMSO-d6) δ (ppm): 8.89 (1H, s), 7.75 (1H, d, J = 8.8 Hz), 7.41 (2H, d, J = 7.6 Hz),
7.33–7.29 (2H,m), 7.24–7.20 (1H, m), 7.16 (1H, d, J = 2.4 Hz), 6.90 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz),
6.63 (1H, d, J = 16.0 Hz), 6.43–6.36 (1H, m), 6.30 (1H, t, J = 5.2 Hz), 2.50 (2H, t, J = 5.2 Hz).
13C-NMR (125 MHz DMSO-d6) δ (ppm): 149.8, 147.7, 145.3, 137.2, 136.0, 130.8, 129.1(2C), 128.1,
127.8, 126.6(2C), 123.5, 115.0, 101.7, 45.5. HR-MS (ESI+) m/z: 267.0956 [M+H]+, calcd for
C16H15N2S+, 267.0956.
(E)-benzothiazol-6-yl-[3-(2-methoxy-phenyl)-allyl]-amine (3f). Pale yellow solid, mp: 107–109 °C,
60% yield; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.89 (1H, s), 7.75 (1H, d, J = 8.8 Hz), 7.46 (1H,
dd, J1 = 7.6 Hz, J2 = 1.6 Hz), 7.24–7.20 (1H, m), 7.15 (1H, d, J = 2.4 Hz), 6.98 (1H, d, J = 8.8 Hz),
6.92–6.95 (3H, m), 6.38–6.28 (2H, m), 3.92–3.90 (2H, m), 3.77 (3H, s). 13C-NMR (125 MHz DMSO-d6)
δ (ppm): 156.7, 149.7, 147.8, 145.2, 136.0, 129.1, 128.5, 127.0, 125.7, 125.6, 123.5, 121.0, 115.0,
111.8, 101.7, 55.8, 46.0. HR-MS (ESI+) m/z: 297.1060 [M+H]+, calcd for C17H17N2OS+, 297.1062.
Benzothiazol-6-yl-(5-methylfuran-2-ylmethyl)-amine (3g). Pale yellow solid, mp: 52–54 °C, 63% yield;
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.90 (1H, s), 7.74 (1H, d, J = 8.8 Hz), 7.20 (1H, d, J = 2.0 Hz),
6.91 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.41 (1H, t, J = 6.0 Hz), 6.21 (1H, d, J = 2.8 Hz), 5.98–5.97

Molecules 2014, 19
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13
(1H, J = 2.8 Hz), 4.23 (2H, d, J = 6.0 Hz), 2.22 (3H, s). C-NMR (125 MHz DMSO-d6) δ (ppm):
151.4, 151.1, 149.6, 147.3, 145.4, 135.0, 123.5, 115.0, 108.2, 106.7, 101.9, 40.5, 13.8. HR-MS (ESI+)
m/z: 245.0742 [M+H]+, calcd for C13H13N2OS+, 245.0749.
Benzothiazol-6-yl-thiophen-2-ylmethylamine (3h). Light brown solid, mp: 148–150 °C, 61% yield;
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.91 (1H, s), 7.75 (1H, d, J = 8.8 Hz), 7.37 (1H, d, J = 4.8
Hz), 7.19 (1H, d, J = 2.0 Hz), 7.09 (1H, d, J = 3.2 Hz), 6.98–6.95 (1H, m), 6.91 (1H, dd, J1 = 8.8 Hz,
13
J2 = 2.0 Hz), 6.62 (1H, t, J = 6.0 Hz), 4.52 (2H, d, J = 6.0 Hz). C-NMR (125 MHz DMSO-d6) δ
(ppm): 150.1, 147.2, 145.6, 144.2, 135.9, 127.3, 125.6, 125.2, 123.5, 115.1, 102.2, 42.5. HR-MS
+
(ESI+) m/z: 247.0362 [M+H]+, calcd for C12H11N2S2 , 247.0364.
Benzothiazol-6-yl-(5-chlorothiophen-2-ylmethyl)-amine (3i). Brown solid, mp: 104–106 °C, 62%
1
yield; H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.93 (1H, s), 7.76 (1H, d, J = 8.8 Hz), 7.19 (1H, d,
J = 2.0 Hz), 6.97–6.95 (2H, m), 6.90 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.67 (1H, t, J = 6.0 Hz),
13
4.46 (2H, d, J = 6.0 Hz). C-NMR (125 MHz DMSO-d6) δ (ppm): 150.4, 146.9, 145.7, 144.2, 135.9,
126.9, 126.7, 125.3, 123.6, 115.1, 102.4, 42.8. HR-MS (ESI+) m/z: 280.9980 [M+H]+, calcd for
C12H10N2S2Cl+, 280.9974.
Benzothiazol-6-yl-pyrimidin-5-ylmethyl-amine (3j). Pale yellow solid, mp: 127–129 °C, 51% yield;
1H-NMR (500 MHz, DMSO-d6) δ (ppm): 9.06 (1H, s), 8.91 (1H, s), 8.81 (2H, s), 7.75 (1H, d,
J = 9.0 Hz), 7.19 (1H, d, J = 2.5 Hz), 6.90 (1H, dd, J1 = 9.0 Hz, J2 = 2.5 Hz), 6.64 (1H, t, J = 6.0 Hz),
13
4.39 (2H, d, J = 6.0 Hz). C-NMR (125 MHz DMSO-d6) δ (ppm): 157.6, 156.8 (2C), 150.4, 147.0,
145.7, 136.0, 133.5, 123.7, 115.1, 102.2, 42.5. HR-MS (ESI+) m/z: 243.0710 [M+H]+, calcd for
C12H11N4S+, 243.0704.
Anthracen-9-ylmethyl-benzothiazol-6-yl-amine (3k). Yellow solid, mp: 174–176 °C, 64% yield;
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.96 (1H, s), 8.66 (1H, s), 8.30 (2H,d, J = 8.4 Hz), 8.15–8.13
(2H, m), 7.79 (1H, d, J = 8.8 Hz), 7.60–7.52 (5H, m), 7.01 (1H, dd, J1 = 8.8 Hz, J2 = 2.0 Hz), 6.32 (1H,
t, J = 4.0 Hz), 5.14 (2H, d, J = 4.0 Hz). 13C-NMR (125 MHz DMSO-d6) δ (ppm): 149.8, 148.2, 145.4,
136.1, 131.6 (2C), 130.7 (2C), 130.2, 129.4 (2C), 127.8, 126.8 (2C), 125.7 (2C), 125.0 (2C), 123.5,
115.1, 101.4, 40.6. HR-MS (ESI+) m/z: 341.1104 [M+H]+, calcd for C22H17N2S+, 341.1112.
Preparation of (E)-(3H-Benzoimidazol-5-yl)-(3-phenyl-allyl)-amine (4a). A mixture of compound 4M
(2 mmol) and trans-3-phenylpropenal (2.2 mmol) in ethanol (15 mL) was stirred at room temperature
for 8 h, and NaBH4 (2.2 mmol) was then added to the solution which was mixed overnight. The solvent
concentration was halved by diluting it with water and was extracted by ethyl acetate (3 × 15 mL). The
combined organic layers were dried over Na2SO4 and evaporated. The residue was purified by flash
chromatography on silica using a CH2Cl2/MeOH gradient and the compound 4a was generated in the
1
form of a yellow solid, mp: 140–142 °C, 52% yield; H-NMR (400 MHz, DMSO-d6) δ (ppm): 7.90
(1H, s), 7.45–7.29 (6H,m), 7.23–7.20 (1H, m), 6.65–6.61 (3H, m), 6.42 (1H, t, J = 5.2 Hz), 3.87 (2H,
d, J = 5.2 Hz). 13C-NMR (125 MHz DMSO-d6) δ (ppm): 145.7, 139.9, 137.3, 130.4, 129.4, 129.1 (2C),
129.0, 128.0, 127.7, 126.5 (2C), 117.5, 111.5, 94.7, 46.3. HR-MS (ESI+) m/z: 250.1341 [M+H]+, calcd
+
for C16H16N3 , 250.1344.

Molecules 2014, 19
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(3H-Benzoimidazol-5-yl)-(5-methyl-furan-2-ylmethyl)-amine (4b). Compound 4b was synthesized
using a method similar to that of 4a and was isolated as a brown solid. mp: 75–77 °C; 55% yield;
1H-NMR (400 MHz; DMSO-d6) δ (ppm): 8.07 (1H; s); 7.35 (1H; d; J = 8.8 Hz); 6.71–6.69 (2H, m);
6.18 (1H, d; J = 2.8 Hz); 6.00–5.99 (1H, m); 4.22 (2H; s); 2.25(3H; s). 13C-NMR (125 MHz DMSO-d6) δ
(ppm): 152.0; 150.9; 146.8; 139.8; 136.9; 131.5; 117.0; 112.1; 108.1; 106.7; 94.8; 41.3; 13.7. HR-MS
(ESI+) m/z: 228.1135 [M+H]+; calcd for C13H14N3O+; 228.1137.
3.3. Antiviral Assays
Madin-Darby Canine Kidney (MDCK) cells, African green monkey kidney cells (Vero) and Coxsackie
viruses (Cox B3 Nancy strain) were all purchased from ATCC. 2.2.15 cells, an HBV-transfected human
HepG2 cell line [23], were kindly provided by Y.C. Cheng (Yale Medical School, New Haven, CT,
USA). Huh7.5 human liver cells (kindly provided by Vertex Pharmaceuticals, Boston, MA) were
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA)
supplemented with 10% inactivated fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin
(Invitrogen). Influenza A strains were all obtained from the Institute of Virology, Chinese Academy of
Preventive Medicine. The plasmid pFL-J6/JFH/JC1 containing the full-length chimeric HCV cDNA
was kindly provided by Vertex Pharmaceuticals (Boston, MA, USA).
3.3.1. Cytotoxicity Assay
The cytotoxicity of the compounds in MDCK, Vero and HepG2.2.15 cells was monitored via
cytopathic effect (CPE) assays. The MDCK and Vero cells (2.5 × 104 /well) were plated in a 96-well
plate. Following a 24 h hold period, the monolayer of cells was incubated in the presence of various
concentrations of the test compounds. Following 48 h of culture at 37 °C under a 5% CO2 atmosphere
in an incubator, the cells were monitored by CPE. The TC50 value was calculated using Reed &
Muench analysis.
The HepG2.2.15 cells (2.5 × 104 /well) were plated in a 96-well plate, and after a 48 h incubation,
the supernatant was replaced by fresh culture medium containing the test compounds. Cells were
cultured for 3 days at 37 °C in a 5% CO2 atmosphere in an incubator, and the medium was
replaced with fresh medium containing the test compounds for an additional 3 days. The cells were
subsequently monitored by CPE and the TC50 value was calculated using Reed and Muench analysis.
The Huh7.5 cells (1 × 104 cells/well) were plated in 96-well plates. Six hours later the culture media
was replaced with fresh medium containing the test compounds at various concentrations. Cytotoxicity
was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
after 96 h. The 50% cytotoxicity concentration (TC50) was calculated with Reed and Muench methods.
3.3.2. Anti-Influenza Assays
Confluent MDCK cells grown in 96-well microplates were infected with influenza A at a median
tissue culture infective dose TCID50 of 100. After 2 h of viral adsorption at 37 °C, the monolayers were
washed with PBS and incubated at 37 °C in the maintenance medium with or without different
concentrations of test compounds. Viral cytopathic effect (CPE) was measured when the viral control

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group reached a value of 4 and the antiviral activities of the synthesized compounds were determined
by Reed and Muench analyses.
3.3.3. Anti-HBV Assays
The anti-HBV activities of test compounds and a positive control drug were tested in HepG2.2.15
cells. The HepG2.2.15 cells were cultured in 96-well plates and treated with the test compounds at 37 °C
for 3 days. The medium was removed, and fresh medium containing the test compounds was added for
an additional 3 days. The cells were harvested and intracellular DNA was extracted. The inhibition of
viral DNA replication in treated cells versus untreated cells was determined by real time PCR.
3.3.4. Anti-HCV Assays
Huh7.5 cells were seeded at a density of 3 × 104 cells/cm2. After 24 h of incubation, the cells were
infected with an HCV viral stock (approximately 45 IU per cell) and simultaneously treated with
Telaprevir, the test compounds, or the solvent control. The culture medium was removed 96 h after
inoculation, and the total intracellular RNA and total intracellular proteins were extracted with a
Qiagen Kit according to the manufacturer’s instructions. Intracellular HCV RNA was quantified using
a one-step real time RT-PCR kit (Invitrogen). Cytotoxicity was tested by the MTT assay, and IC50
values were calculated by the Reed and Muench methods.
3.3.5. Anti-Coxsackie B3 Activity Assays
Confluent Vero cells grown in 96-well plates were infected with a median tissue culture infective
dose of 100 (100TCID50) Cox B3 viruses. After 1 h of viral adsorption at 37 °C, the monolayers were
washed with phosphate buffered saline (PBS) and incubated at 37 °C in the maintenance medium
(MEM plus 2% fetal bovine serum (FBS)) with or without different concentrations of test compounds.
Viral cytopathic effect (CPE) was measured when the viral control group reached a level of 4 and the
antiviral activity of test compounds was determined by the Reed and Muench analyses.
4. Conclusions
A total of 24 novel benzo-heterocyclic amine derivatives were designed, synthesized and screened
for their antiviral activities towards influenza A, HBV, HCV and Cox B3 in this study. In general, the
target compounds were more effective against RNA viruses (influenza A, HCV and Cox B3) than the
DNA virus (HBV), and the majority of the synthesized compounds simultaneously exhibited potent
activity against the influenza A, HCV and Cox B3 viruses. Additionally, a similar trend was observed
among the SARs of the synthesized compounds towards the RNA virus. Different substituents on the
aromatic ring had certain effects on the activity. For example, an electron-withdrawing substituent on
the aromatic or heteroaromatic ring favored antiviral activity. Notably, compounds 3f and 3g showed
potent antiviral activity towards the entire virus panel tested at low micromolar concentrations,
especially compound 3f which was active against the entire virus panel tested with an IC50 value of
approximately 5.0 μM. The detailed structure optimization of compound 3f and the study of the
mechanisms of action of these compounds are ongoing in our laboratory.

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Acknowledgments
This work was supported by the National Natural Science Foundation of China (81273439) and the
National Mega-project for Innovative Drugs (2012ZX09301002-001-024-02).
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the entire panel of target compounds are available from the authors.
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