Discovery of naphtho[1,2-d]oxazole derivatives as potential anti-HCV agents through inducing heme oxygenase-1 expression

Article abstract of DOI:10.1016/j.ejmech.2017.12.006

A number of naphtho[1,2-d]oxazole derivatives were synthesized and evaluated for their anti-HCV virus activity. Among them, compound 18 was the most active, exhibited approximately 21-folds more active anti-HCV activity (IC₅₀ of 0.63 μM) than t

Full text of DOI:10.1016/j.ejmech.2017.12.006

Accepted Manuscript
Discovery of naphtho[1,2-d]oxazole derivatives as potential anti-HCV agents through
inducing heme oxygenase-1 expression
Chih-Hua Tseng, Chun-Kuang Lin, Yeh-Long Chen, Chin-Kai Tseng, Jar-Yu Lee, Jin-
Ching Lee
PII:
S0223-5234(17)31012-7
10.1016/j.ejmech.2017.12.006
EJMECH 9984
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 July 2017
Revised Date: 30 November 2017
Accepted Date: 2 December 2017
Please cite this article as: C.-H. Tseng, C.-K. Lin, Y.-L. Chen, C.-K. Tseng, J.-Y. Lee, J.-C. Lee,
Discovery of naphtho[1,2-d]oxazole derivatives as potential anti-HCV agents through inducing
heme oxygenase-1 expression, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.006.
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Graphical abstract

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Discovery of naphtho[1,2-d]oxazole derivatives as potential anti-HCV agents through
inducing heme oxygenase-1 expression
Chih-Hua Tsenga,b,c,d,e*, Chun-Kuang Linf,g, Yeh-Long Chen d,e,h, Chin-Kai Tsengi,j, Jar-Yu Leeh, and
Jin-Ching Leec,k,l*
aSchool of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807,
Taiwan
bDepartment of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical
University, Kaohsiung City 807, Taiwan
cResearch Center for Natural Products and Drug Development, Kaohsiung Medical University,
Kaohsiung City 807, Taiwan
dCenter for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung
City 807, Taiwan
eDepartment of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung City 807,
Taiwan
fDoctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung,
Taiwan
gDoctoral Degree Program in Marine Biotechnology, Academia Sinica, Taipei, Taiwan
hDepartment of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical
University, Kaohsiung City 807, Taiwan
iInstitute of Basic Medical Sciences, College of Medicine, National Cheng Kung University,
Tainan, Taiwan
jCenter of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung
University, Tainan, Taiwan
kDepartment of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung
City 807, Taiwan
lGraduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung, Taiwan
*Corresponding author. Tel.: +886 7 3121101 x2163; fax: +886 7 3125339.

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E-mail addresses: chihhua@kmu.edu.tw; jclee@kmu.edu.tw

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Abstract
A number of naphtho[1,2-d]oxazole derivatives were synthesized and evaluated for their
anti-HCV virus activity. Among them, compound 18 was the most active, exhibited approximately
21-folds more active anti-HCV activity (IC50 of 0.63 µM) than that of ribavirin (IC50 = 13.16 µM).
Compound 18 was less cytotoxic than ribavirin, and the selective index (SI) of 18 is approximately
28-folds higher than that of ribavirin (229.10 v.s. 8.08). By using heme oxygenase-1 (HO-1)
promoter-based assay and western blotting, compound 18 could induce HO-1 promoter activity,
and protein expression. The antiviral effect of compound 18 was attenuated by HO-1 specific
inhibitor SnPP treatment, which indicated that compound 18 suppressed HCV replication through
inducing HO-1 expression. We further found that compound 18 reduced bach1 expression resulting
in increasing the activity of Nrf-2 binding element. Moreover, the induction of HO-1 by compound
18 reduced HCV NS3/4A protease activity and induced the antiviral interferon responses.
Therefore, compound 18 can be considered as a supplemental antiviral agent or a lead compound
for further developing more effective agents against HCV replication.
Keywords: Naphtho[1,2-d]oxazole, Ribavirin, NS3/4A protease, Anti-HCV activity

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1. Introduction
The Hepatitis C virus (HCV) belongs to the family Flaviviridae and is closely related to the
West Nile virus (WNV), Yellow Fever virus (YFV), Japanese Encephalitis Virus (JEV), and
Dengue virus (DENV) which contains a single-positive strand RNA genome [1,2]. HCV causes
both acute and chronic infection, and eighty percent of HCV infection results in chronic liver
disease, such as cirrhosis and hepatocellular carcinoma (HCC) [3-6]. Annually, approximately 3
million people are newly infected by HCV, and 350,000 to 500,000 people die from HCV relative
liver disease [7]. Unfortunately, the development of HCV vaccine is impeded by viral genetic
heterogeneity leading to immune evasion and the lake of a relevant animal model. The standard of
care (SOC) for HCV patients is a combination treatment of pegylated interferon and synthetic
guanosine ribavirin, but the sustained viral response rate is dependent on the HCV genotype, being
successfully in 45-80%, and the severe adverse effect occurs commonly in a large number of
patients [8,9]. Directly acting antiviral agent (DAAs) have been intensively developed and licensed
in recent years, particularly targeting the viral NS3/4A protease, the NS5A phosphoprotein, and the
NS5B polymerase, but the limitations of DAAs are the high cost, drug resistance and heavy pill
load [10,11]. Discovering of therapeutic agents targeting host factors may help develop
pan-genotype and less side effect antiviral strategy against HCV [12]. Therefore, the development
of anti-HCV supplemental agents with higher effective and more affordable side effect profiles are
still required to be discovered.
The heme oxygenase (HO-1), a heme-degrading enzyme, exhibits anti-inflammatory and
antioxidant effect in vitro and in vivo [13]. Degradation of heme by HO-1 results in the production
of biliverdin, ferrous iron, and carbon monoxide (CO), which are regarded as a potential protectant
against liver injury [14]. HO-1 induction or overexpression has been demonstrated to reduce
several virus replication, including HCV [15], DENV [16], hepatitis B virus (HBV) [17], and
human immunodeficiency virus (HIV) [18]. In addition, the HO-1 product biliverdin, but not CO
and ferrous iron, interferes with HCV replication by increasing endogenous anti-viral interferon

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response and might directly inhibit the activity of HCV protease NS3/4A [19]. Consequently, the
development of HO-1-inducible agents may offer a therapeutic strategy for HCV patients by
simultaneously targeting both host and viral factors.
Previous study revealed that the most induction of HO-1 on the transcriptional level was
regulated by kelch-like ECH-associated protein 1 (keap1)/NF-E2-related factor 2 (Nrf2) signaling
pathway [20]. In the normal condition, keap1 protein is bound to the Nrf2 transcriptional factor,
which results in Nrf2 protein degradation [20]. Upon the oxidative stress, the Nrf2 is released from
keap1 and then translocated to the nucleus where it binds to antioxidant response element (ARE)
resulting in transcriptional activation of HO-1 and other antioxidant enzymes [21]. In the absence
of oxidative stress, the transcription factor BRCA1 associated C-terminal helicase 1 (Bach1), as a
transcription inhibitor, forms heterodimers with the leucine zipper subfamily of small Maf proteins
and binds to the Maf-recognition element (MARE) in the HO-1 promoter region [22]. The HO-1
expression is higher in the bach1-deficient mice compared with control mice, which indicated that
bach1 is a repressor of HO-1 gene [23].
Many efforts had been devoted to the discovery of novel anti-HCV agents for the past few
years [24-30]. In order to discover novel drug candidates, we have synthesized certain
anilinoquinoline, anilinobenzothiazole, and anilinocoumarin derivatives and evaluated for their
anti-HCV activities. Our results indicated that compound 1 inhibited the growth of HCV with an
EC50 value of 7.0 µM [31]. Compound 2 inhibited HCV RNA-dependent RNA polymerase (RdRp)
activity and HCV RNA replication (EC50 = 8 µM) [32]. Compound 3 exhibited strong anti-HCV
activity at protein and RNA levels at non-toxic concentrations, with an EC50 value of 12 µM [33].
We also have discovered that compounds 4 and 5 inhibit the DENV2 RNA expression in
Huh-7-DV-Fluc cells with potencies approximately equal to that of ribavirin [34]. In continuation
of our efforts to discover potential antiviral agents, we demonstrate herein the synthesis of
naphtho[1,2-d]oxazole derivatives (target compounds, Figure 1) and their evaluations related to the
inhibition of HCV/NS5B by inducing HO-1 expression.

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< Insert Figure 1 here >
2. Chemistry
Treatment of sodium 1,2-naphthoquinone-4-sulfonate (6) with 4-chloroaniline afforded
4-(4-chlorophenylamino)naphthalene-1,2-dione (8) in 74% yield. Preparation of compounds 7 and
9 were previously described [35]. The reaction of compounds 7 - 9 respectively with p-substituted
benzaldehyde gave their corresponding naphtho[1,2-d]oxazole derivatives 10 – 12 in 55–75% yield.
Methylation of compounds 10 – 12 with iodomethane afforded 13 - 15 as described in Scheme 1.
Under similar reaction conditions, compounds 7 – 9 were reacted with furaldehyde to give
their corresponding naphtho[1,2-d]oxazole derivatives 16 – 18 in 58–74% yield. Methylation of
compounds 16 – 18 with iodomethane afforded compounds 19 - 21 as described in Scheme 2.
< Insert Scheme 1,2 here >
3. Biological Results and Discussion
3.1. Anti-HCV activities and Cytotoxicities
The anti-HCV and cytotoxicities of naphtho[1,2-d]oxazole derivatives are summarized in
Table 1. Ava-5 cells were treated with compounds 10 – 21 or the positive ribavirin respectively at a
concentration of 5 or 20 µM for 3 days. HCV RNA levels were then determined by RT-qPCR
analysis. The cell cytotoxicity was determined by XTT assay in the Ava-5 cells treatment with 20
and 200 µM of pyrazolylquinoline derivatives for 3 days. Compounds which exhibited > 50%
inhibition of HCV at a concentration of 20 µM were considered as active. Results from Table 1
indicated N,2-diphenylnaphtho[1,2-d]oxazol-5-amine (10a) and most of its substituted phenyl
derivatives 10b, 10c, and 11 – 15 were inactive with the exception of compounds 11b and 12a
which exhibited 55% and 87% inhibition of HCV at a concentration of 20 µM.
2-(Furan-2-yl)-N-phenylnaphtho[1,2-d]oxazol-5-amine (16) and its 4-chlorophenyl derivatives 17
were inactive. However, its 4-methoxyphenyl derivative 18 demonstrated a significant anti-HCV

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activity, with a 95% inhibition of HCV. Further N-methylation of compound 18 reduced anti-HCV
activity, in which compound 21 was inactive. The concentration that inhibited 50% DENV
replication (IC50), the concentration that inhibited 50% cell growth (CC50), and the selective index
(SI : CC50/IC50) of compound 11b, 12a, and 18 were determined with ribavirin as a positive control.
Results from Table 2 indicated that compound 11b was less active than ribavirin while compound
12a was more active. Among them, compound 18 was the most active, exhibited approximately
21-folds more active anti-HCV activity (IC50 of 0.63 µM) than that of ribavirin (IC50 = 13.16 µM).
In addition, compound 18 was less cytotoxic than ribavirin. The selective index (SI) of 18 is
approximately 28-folds higher than that of ribavirin (229.10 v.s. 8.08).
< Insert Table 1,2 here >
3.2. Compound 18 reduced HCV protein synthesis and RNA replication
To further confirm the anti-HCV effect of compound 18, we treated compound 18 at indicated
concentrations in Ava5 cells for 3 days. The western blotting and RT-qPCR were performed to
determine the HCV replication, and the results showed that compound 18 dose-dependently
reduced HCV protein synthesis and RNA replication without cell cytotoxicity in HCV replicon
cells (Fig. 2A and 2B). Compound 18 exhibited an IC50 value of 0.82 µM in reducing HCV RNA
replication. We then further used the HCV JFH-1 infectious assay to confirm the inhibition effect of
compound 18 on HCV replication under the same condition described above, and the result
consistently showed that compound dose-dependently reduced HCV replication (Fig. 2c).
< Insert figure 2 here >
3.3. Compound 18 reduced HCV replication through inducing HO-1 expression
To determine whether compound 18 have impact on HO-1 gene expression in Ava5 cells, we
first analyzed the level of HO-1 gene transcription in 18-treated cells using HO-1 promoter-based
luciferase reporter assay. Ava5 cell were transfected with pHO-1-Luc, a luciferase gene driven by
HO-1 promoter, reporter plasmid and then were treated with compound 18 at indicated
concentrations for 3 days. The result indicated that compound 18 significant induced HO-1

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promoter activity in a concentration-dependent manner, compared with the DMSO-treated cells
(Fig. 3A). Next, we performed western blotting to determine the HO-1 protein expression in Ava5
cells. As expected, compound 18 dose-dependently induced HO-1 protein level in Ava5 cells (Fig.
3B). To confirm the elevated HO-1 levels by compound 18 contributed to the anti-HCV replication,
we used the specific inhibitor of HO-1 SnPP for evaluating the inhibition effect of compound 18 on
HCV replication. Ava5 cells were co-incubated with 1 µM compound 18 and increasing
concentration of SnPP (0-5 µM) for 3 days. As shown in Fig. 4A, SnPP attenuated the inhibition
effect of compound 18 on HCV replication (lane 2-4), compared with SnPP-untreated cells (lane 2).
Consistently, the RT-qPCR showed the similar result that SnPP restored the HCV RNA level under
the same conditions described above (Fig. 4B).
< Insert figure 3,4 here >
3.4. Anti-HCV activity of compound 18 is correlated with the down-regulation of bach-1 expression
To investigate the detailed mechanism of compound 18-induced HO-1 expression against
HCV replication, we analyzed whether Nrf2, one of major transcription factors for activation of
HO-1 expression, involved in anti-HCV activity. We treated compound 18 to Aca5 cells at
indicated concentration. Results of Western blotting revealed that compound 18 did not affect Nrf2
and Keap-1 expression but dose-dependently reduced bach-1 expression (Fig. 5A). Furthermore,
nuclear bach-1 protein level was also reduced in a dose-dependent manner in compound 18-treated
Ava5 cells (Fig. 5B). To confirm the induction effect of HO-1 by compound 18 through reducing
bach-1 expression leading to increasing Nrf2-mediated ARE activation for HO-1 expression, we
transfected p2xARE-Luc, a reporter plasmid with ARE-driven firefly luciferase, into Ava5 cells
and Huh-7 cells, followed by treatment of compound 18 at indicated concentrations. The result of
luciferase activity showed that compound 18 dose-dependently increased activity of Nrf2 binding
region, compared with Ava5 cells without compound 18 treatment (Fig. 5c).
< Insert figure 5 here >
3.5. Compound 18 reduced HCV NS3/4A protease activity and activated antiviral IFN response

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To investigate whether HO-1-mediated reduction effect on HCV NS3/4A protease activity
involved in the anti-viral effect of compound 18, we performed a cell-based reporter assay by
co-transfecting pEG(DE4AB)SEAP reporeter plasmid and pNS3/4A, a NS3/4A protein expression
plasmid, into Huh-7 cells with compound 18 treatment at indicated concentration. The reporter
plasmid was illustrated in Figure 6A, and the secreted SEAP activity in the cell culture medium
displays NS3/4A protease activity. Results of SEAP activity indicated that compound 18
dose-dependently reduced NS3/4A protease activity (Fig. 6B). To further investigate whether the
induction effect of compound 18 on HO-1 was able to activate cellular anti-viral interferon (IFN)
response, we transfected a pISRE-luc reporter plasmid, an IFN-stimulated response element
(ISRE)-driven firefly luciferase plasmid, and then incubated with compound 18 at increasing
concentrations. As shown in Figure 7A, results of luciferase activity showed compound 18
significantly increased ISRE promoter activity in a dose-dependent manner. We further determined
the ISRE-mediated IFN expression in the compound 18-treated Ava5 cells by RT-qPCR. Similar
results indicated that compound 18 dose-dependently increased IFN-α-2, -α-5 and -α-17 mRNA
expression, compared with DMSO-treated Ava5 cells (Fig. 7B). The previous study revealed that
activation of type I interferon could facilitate the expression of IFN-stimulated genes (ISGs) which
are important regulators involved in immunomodulatory and antiviral responses against to viral
infection, including HCV and hepatitis B virus [36]. To confirm whether compound 18 stimulated
the expression of antiviral ISGs, including 2′-5′-oligoadenylate synthetase 1 (OAS1), OAS3, and
protein kinase PKR, were determined by RT-qPCR. As shown in Figure 7C to E, compound 18
dose-dependently increased ISG expression at 1.0 and 2.5 µM.
< Insert figure 6,7 here >
4. Conclusion
In the present study, we revealed that compound 18, 4-methoxyphenyl derivative of
2-(furan-2-yl)-N-phenylnaphtho[1,2-d]oxazol-5-amine, was the most effective anti-HCV agents
among the naphtho[1,2-d]oxazole derivatives and exhibited IC50 values of 0.63 µM, compared with

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the reference drug ribavirin (IC50 = 13 µM). The selective index (SI) of 18 is approximately
28-folds higher than that of ribavirin (229.10 v.s. 8.08) in Ava5 cells. We further determined the
anti-viral mechanism of compound 18, and our data exhibited that compound 18 strongly
up-regulated HO-1 promoter activity and protein expression (Figs. 2 and 3). The anti-viral activity
of compound 18 was attenuated by SnPP treatment (Fig. 4), which suggested that compound 18
reduced HCV replication through inducing HO-1 expression. The pathway of Nrf2-mediated HO-1
expression was investigated following exposure to compound 18, and we found that compound 18
did not affect the Nrf2 and Keap1 expression but significantly reduced transcriptional repressor
bach1 expression (Fig. 5). In addition, our results revealed that HO-1-mediated reduction effect on
HCV NS3/4A protease activity and enhancement effect on the antiviral IFN response involved in
antiviral activity of compound 18 (Figs. 6 and 7). Taken together, compound 18 reduced HCV
replication through reducing bach1 expression resulting in HO-1 induction (Fig. 8). In addition,
compound 18 is easy to be synthesized, which suggested that it can be a possible lead for further
development of novel drug candidates against HCV replication.
< Insert figure 8 here >
5. Experimental
5.1. General
Melting points were determined on an Electrothermal IA9100 melting point apparatus and are
13
uncorrected. Nuclear magnetic resonance (1H and C) spectra were recorded on a Varian Gemini
200 spectrometer or Varian-Unity-400 spectrometer. Chemical shifts were expressed in parts per
million (δ) with tetramethylsilane (TMS) as an internal standard. Thin-layer chromatography was
performed on silica gel 60 F-254 plates purchased from E. Merck and Co.. Mass spectra were
recorded on Bruker APEX II (ESI) mass spectrometer. The elemental analyses were performed in
the Instrument Center of National Science Council at National Cheng-Kung University and
National Taiwan University using Heraeus CHN-O Rapid EA, and all values are within ± 0.4% of
the theoretical compositions.

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5.2.1. 4-[(4-Chlorophenyl)amino]naphthalene-1,2-dione (8). Compound 8 was prepared from
1,2-naphthoquinone-4-sulfonic acid sodium salt (6) and 4-chloroaniline according to literature
o
conditions [35]. Yield 74% as a red solid. Mp. 275-276 C (Dec). 1H NMR (400 MHz, DMSO-d6):
5.91 (br s, 1H, 3-H), 7.21–7.38 (m, 2H, Ar-H), 7.54 (m, 2H, Ar-H), 7.73–7.77 (m, 1H, 7-H),
7.85–7.89 (m, 1H, 6-H), 8.06 (d, 1H, J = 7.6 Hz, 5-H), 8.31 (d, 1H, J = 7.2 Hz, 8-H), 9.80 (br s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6): 100.65, 103.72, 115.23, 121.99, 123.94, 127.59 (2C),
128.33, 129.52 (2C), 133.53, 134.47, 149.38, 154.19, 176.17, 180.93. ESIMS [M+H]+: 284. Anal.
calcd for C16H10ClNO2: C 67.74, H 3.55, N 4.94; found: C 67.65, H 3.42, N 5.05.
5.3. General procedure for the preparation of naphtho[1,2-d]oxazole compounds 10a-12c and
16-18.
To a suspension of 7, 8 or 9 (2.0 mmol) in acetic acid (15 mL) was added ammonium acetate (3.78
g, 49.0 mmol) and an appropriate benzaldehyde or furaldehyde (3.0 mmol). The reaction mixture
was heated at 150oC for 2.5 h (TLC monitoring). The solvent was removed in vacuo and the
residue suspended in H2O (20 mL). The crude product was purified by flash chromatography on
silica gel and recrystallized with MeOH to afford the naphtho[1,2-d]oxazole products.
5.3.1. N,2-Diphenylnaphtho[1,2-d]oxazol-5-amine (10a). Yield 62% as a yellow solid. Mp.
o
1
158.0-159.0 C. H NMR (400 MHz, CDCl3): 6.02 (br s, 1H, NH), 6.96-7.00 (m, 1H, Ar-H), 7.08
(d, 2H, J = 7.6 Hz, Ar-H), 7.29-7.33 (m, 2H, Ar-H), 7.48-7.71 (m, 6H, Ar-H), 8.12 (d, 1H, J = 8.8
Hz, Ar-H), 8.27 (m, 2H, Ar-H), 8.61 (d, 1H, J = 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
100.35, 118.09, 121.22, 122.57, 122.91, 125.25, 125.65, 127.00 (2C), 127.39, 127.54, 128.70 (2C),
128.87 (2C), 129.54 (2C), 130.72, 133.17, 137.99, 144.13, 148.35, 161.45. ESIMS [M+H]+: 337.
Anal. calcd for C23H16N2O·0.1 H2O: C 81.67, H 4.84, N 8.28; found: C 81.61, H 4.85, N 8.14.
5.3.2. 2-(4-Chlorophenyl)-N-phenylnaphtho[1,2-d]oxazol-5-amine (10b). Yield 71% as a yellow
o
solid. Mp. 199-200 C. 1H NMR (400 MHz, CDCl3): 6.01 (br s, 1H, NH), 6.80-7.02 (m, 1H, Ar-H),
7.08 (m, 2H, Ar-H), 7.30-7.35 (m, 2H, Ar-H), 7.46-7.58 (m, 4H, Ar-H), 7.67-7.71 (m, 1H, Ar-H),
8.11 (d, 1H, J = 8.4 Hz, Ar-H), 8.27 (m, 2H, Ar-H), 8.57 (d, 1H, J = 8.4 Hz, Ar-H). 13C NMR (100

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MHz, CDCl3): 99.79, 118.28, 121.41, 122.53 (2C), 122.83, 125.34, 125.52, 126.04, 126.92, 127.48,
128.16 (2C), 129.18 (2C), 129.56 (2C), 132.99, 136.75, 138.32, 143.89, 148.45, 160.39. ESIMS
[M+H]+: 371. Anal. calcd for C23H15ClN2O: C 74.49, H 4.08, N 7.55; found: C 74.14, H 3.93, N
7.57.
5.3.3. 2-(4-methoxyphenyl)-N-phenylnaphtho[1,2-d]oxazol-5-amine (10c). Yield 68% as a yellow
o
solid. Mp. 184-185 C. 1H NMR (400 MHz, CDCl3): 3.89 (s, 3H, OCH3), 6.97-7.07 (m, 5H, Ar-H),
7.28-7.32 (m, 2H, Ar-H), 7.48.-7.52 (m, 2H, Ar-H), 7.64-7.68 (m, 1H, Ar-H), 8.10 (d, 1H, I = 8.8
Hz, Ar-H), 8.23 (m, 2H, Ar-H), 8.61 (m, 1H, Ar-H). 13C NMR (100 MHz, CDCl3): 55.44, 100.54,
114.37 (2C), 117.90, 119.71, 121.07, 122.66 (2C), 122.98, 125.22, 125.72, 127.33, 128.92 (2C),
129.51 (2C), 131.99, 132.46, 137.62, 144.26, 147.96, 161.59, 161.95. ESIMS [M+H]+: 367. Anal.
calcd for C24H18N2O2: C 78.67, H 4.95, N 7.65; found: C 78.93, H 4.92, N 7.72.
5.3.4. N-(4-Chlorophenyl)-2-phenylnaphtho[1,2-d]oxazol-5-amine (11a). Yield 63% as a yellow
o
solid. Mp.: 174-175 C. 1H NMR (400 MHz, CDCl3): 6.01 (br s, 1H, NH), 6.96 (d, 2H, J = 8.8 Hz,
Ar-H), 7.22-7.26 (m, 2H, Ar-H), 7.51-7.55 (m, 5H, Ar-H), 7.66-7.70 (m, 1H, Ar-H), 8.07 (d, 1H, J
= 8.4 Hz, Ar-H), 8.27 (m, 2H, Ar-H), 8.61 (d, 1H, J = 8.4 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
101.32, 118.85, 122.65 (2C), 122.97, 125.42, 125.65, 125.84, 127.07 (2C), 127.33, 127.51, 128.92
(2C), 129.14, 129.45 (2C), 130.91, 133.49, 137.40, 143.07, 148.09, 161.66. ESIMS [M+H]+: 371.
Anal. calcd for C23H15ClN2O: C 74.49, H 4.08, N 7.55; found: C 74.62, H 3.95, N 7.49.
5.3.5. N,2-Bis(4-chlorophenyl)naphtho[1,2-d]oxazol-5-amine (11b). Yield 56% as a yellow solid.
o
1
Mp.: 206-207 C. H NMR (400 MHz, CDCl3): 6.01 (br s, 1H, NH), 6.96-6.99 (m, 2H, Ar-H),
7.23-7.27 (m, 2H, Ar-H), 7.48-7.57 (m, 4H, Ar-H), 7.68-7.72 (m, 1H, Ar-H), 8.08 (d, 1H, J = 8.4
Hz, Ar-H), 8.18-8.22 (m, 2H, Ar-H), 8.59 (d, 1H, J = 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3):
100.95, 119.03, 122.60 (2C), 122.91, 125.52, 125.78, 125.86, 125.97, 126.97, 127.61, 128.24 (2C),
129.23 (2C), 129.48 (2C), 133.61, 136.92, 137.65, 142.90, 148.24, 160.68. ESIMS [M+H]+: 405.
Anal. calcd for C23H14Cl2N2O: C 68.16, H 3.48, N 6.91; found: C 68.13, H 3.43, N 7.31.

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5.3.6. N-(4-Chlorophenyl)-2-(4-methoxyphenyl)naphtho[1,2-d]oxazol-5-amine (11c). Yield 62% as
o
a red solid. Mp.: 191-192 C. 1H NMR (400 MHz, CDCl3): 3.89 (s, 3H, OCH3), 6.01 (br s, 1H, NH),
6.91-6.94 (m, 2H, Ar-H), 7.00-7.04 (m, 2H, Ar-H), 7.20-7.25 (m, 2H, Ar-H), 7.47-7.51 (m, 2H,
Ar-H), 7.63-7.67 (m, 1H, Ar-H), 8.04 (d, 1H, J = 8.4 Hz, Ar-H), 8.19-8.22 (m, 2H, Ar-H), 8.58 (d,
1H, J = 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3): 55.43, 101.82, 114.36 (2C), 118.53, 119.79,
122.73 (2C), 122.94, 125.30, 125.95, 127.34 , 128.88 (2C), 129.39 (2C), 129.79, 132.45, 133.44,
134.96, 136.80, 143.33, 147.73, 161.93. ESIMS [M+H]+: 401. Anal. calcd for C24H17ClN2O2: C
71.91, H 4.27, N 6.99; found: C 71.55, H 4.24, N 7.26.
5.3.7. N-(4-Methoxyphenyl)-2-phenylnaphtho[1,2-d]oxazol-5-amine (12a). Yield 55% as a yellow
o
1
solid. Mp.: 210-211 C. H NMR (400 MHz, CDCl3): 3.84 (s, 3H, OCH3), 6.01 (br s, 1H, NH),
6.93-6.95 (m, 2H, Ar-H), 7.10-7.14 (m, 2H, Ar-H), 7.47-7.58 (m, 5H, Ar-H), 7.66-7.70 (m, 1H,
Ar-H), 8.09 (d, 1H, J = 8.4 Hz, Ar-H), 8.24-8.28 (m, 2H, Ar-H), 8.61-8.62 (m, 1H, Ar-H). 13C
NMR (100 MHz, CDCl3): 55.62, 95.98, 115.00, 117.66, 121.82 (2C), 122.87, 123.04, 123.98,
125.04 , 126.91 (2C), 127.36, 128.86 (2C), 130.61 (2C), 131.09, 135.99, 140.78, 143.08, 148.82,
155.76, 160.69. ESIMS [M+H]+: 367. Anal. calcd for C24H18N2O2: C 78.67, H 4.95, N 7.65 found:
C 78.34, H 5.25, N 7.27.
5.3.8. 2-(4-Chlorophenyl)-N-(4-methoxyphenyl)naphtho[1,2-d]oxazol-5-amine (12b). Yield 69% as
o
a yellow solid. Mp.: 190-191 C. 1H NMR (400 MHz, CDCl3): 3.84 (s, 3H, OCH3), 6.00 (br s, 1H,
NH), 6.92-6.96 (m, 2H, Ar-H), 7.13-7.15 (m, 2H, Ar-H), 7.44-7.56 (m, 4H, Ar-H), 7.66-7.70 (m,
1H, Ar-H), 8.08 (d, 1H, J = 8.4 Hz, Ar-H), 8.13-8.15 (m, 2H, Ar-H), 8.55 (d, 1H, J = 8.4 Hz, Ar-H).
13C NMR (100 MHz, CDCl3): 55.61, 95.63, 114.97 (2C), 121.79, 122.85, 122.97 (2C), 123.85,
125.04, 126.20, 126.92 , 127.39, 127.97 (2C), 128.39, 129.12 (2C), 129.23, 131.45, 136.43, 148.96,
155.78, 159.72. ESIMS [M+H]+: 401. Anal. calcd for C24H17ClN2O2: C 71.91, H 4.27, N 6.99;
found: C 71.54, H 4.04, N 6.91.
5.3.9. N,2-Bis(4-methoxyphenyl)naphtho[1,2-d]oxazol-5-amine (12c). Yield 75% as a yellow solid.
o
Mp.: 186-187 C. 1H NMR (400 MHz, CDCl3): 3.83 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.91-6.93

ACCEPTED MANUSCRIPT
(m, 2H, Ar-H), 6.99-7.18 (m, 5H, Ar-H), 7.52 (br s, 1H, N-H), 7.64-7.67 (m, 2H, Ar-H), 8.08 (d,
1H, J = 8.8 Hz, Ar-H), 8.20-8.21 (m, 2H, Ar-H), 8.61 (m, 1H, Ar-H). 13C NMR (100 MHz, CDCl3):
55.42, 55.60, 96.20, 114.34 (2C), 114.97 (2C), 119.65, 121.90 (2C), 122.56, 123.06, 123.97, 124.97,
127.29, 128.77 (2C), 130.49, 131.09, 136.09, 140.41, 148.44, 155.57, 160.86, 161.82. ESIMS
[M+H]+: 397. Anal. calcd for C25H20N2O3: C 75.74, H 5.08, N 7.07; found: C 75.37, H 5.08, N
7.19.
5.3.10. 2-(Furan-2-yl)-N-phenylnaphtho[1,2-d]oxazol-5-amine (16). Yield 63% as a yellow solid.
o
1
Mp.: 125-126 C. H NMR (400 MHz, CDCl3): 6.61 (dd, J = 3.2, 1.6 Hz, 1H, Fu-4-H), 6.97-7.00
(m, 1H, Ar-H), 7.07-7.09 (m, 2H, Ar-H), 7.22 (d, 1H, J = 3.2 Hz, Fu-3-H), 7.29-7.33 (m, 2H, Ar-H),
7.51-7.55 (m, 2H, Ar-H and N-H), 7.65-7.69 (m, 2H, Ar-H), 8.10 (d, 1H, J = 8.4 Hz, Ar-H), 8.60 (d,
1H, J = 8.4 Hz, Ar-H). 13C NMR (100 MHz, CDCl3): 99.67, 112.18, 112.85, 118.27, 121.41,
122.48 (2C), 122.96, 125.39, 125.57, 126.76, 127.51, 129.53 (2C), 132.32, 138.38, 143.86, 145.08,
147.81, 150.28, 153.76. ESIMS [M+H]+: 327. Anal. calcd for C21H14N2O2: C 77.29, H 4.32, N 8.58;
found: C 77.12, H 4.21, N 8.45.
5.3.10. N-(4-Chlorophenyl)-2-(furan-2-yl)naphtho[1,2-d]oxazol-5-amine (17). Yield 74% as a
o
yellow solid. Mp.: 172-173 C. 1H NMR (400 MHz, CDCl3): 6.10 (br s, 1H, NH), 6.62 (dd, J = 3.2,
1.6 Hz, 1H, Fu-4-H), 6.94-6.98 (m, 2H, Ar-H), 7.22-7.26 (m, 3H, Ar-H), 7.53-7.57 (m, 2H, Ar-H),
7.67-7.71 (m, 2H, Ar-H), 8.07 (dd, 1H, J = 8.4, 0.8 Hz, Ar-H), 8.61 (d, 1H, J = 8.0 Hz, Ar-H). 13C
NMR (100 MHz, CDCl3): 100.91, 112.20, 112.94, 118.97,121.36, 122.57, 123.01 (2C), 125.55,
125.85, 126.87, 127.61, 129.46 (2C), 133.14, 137.65, 142.88, 145.16, 147.61, 151.06, 154.08.
ESIMS [M+H]+: 361. Anal. calcd for C21H13ClN2O2·0.6 H2O: C 67.87, H 3.85, N 7.54; found: C
67.56, H 3.75, N 7.54.
5.3.11. 2-(Furan-2-yl)-N-(4-methoxyphenyl)naphtho[1,2-d]oxazol-5-amine (18). Yield 58% as a
o
1
yellow solid. Mp.: 175-176 C. H NMR (400 MHz, CDCl3): 3.83 (s, 3H, OCH3), 6.01 (br s, 1H,
NH), 6.59 (dd, J = 3.2, 1.6 Hz, 1H, Fu-4-H), 6.92 (d, 2H, J = 8.4 Hz, Ar-H), 7.11-7.20 (m, 3H,
13
Ar-H), 7.52-7.70 (m, 3H, Ar-H), 8.08 (d, 1H, J = 8.8 Hz, Ar-H), 8.59 (m, 1H, Ar-H). C NMR

ACCEPTED MANUSCRIPT
(100 MHz, CDCl3): 55.61, 95.54, 112.09 (2C), 112.31, 114.96, 117.77, 120.81, 121.74 (2C),
123.01, 123.91, 125.12, 127.42, 130.79, 135.44, 140.99, 144.85, 148.19, 153.12, 155.81. ESIMS
[M+H]+: 357. Anal. calcd for C22H16N2O3: C 74.15, H 4.53, N 7.86; found: C 74.22, H 4.38, N
7.77.
5.4. General procedure for preparation of N-methyl-N,2-diphenylnaphtho[1,2-d]oxazole
derivatives: 13a-15c and 19-21.
To a stirred solution of naphtho[1,2-d]oxazole derivatives 10a-12c or 16-18 (1.0 mmol) in dry
DMF (20 mL) was added NaH (60% in oil, 0.50 g) at 0oC and methyliodide (3 mmol) was added at
rt for 10 min (TLC monitoring). The organic phase was washed with brine, dried over MgSO4,
filtered and concentrated under vacuo. Crude product was purified by flash chromatography on
silica gel (MeOH/CH2Cl2 1/50) and crystallized from MeOH.
5.4.1. N-Methyl-N,2-diphenylnaphtho[1,2-d]oxazol-5-amine (13a). Yield 78% as a yellow solid.
o
1
Mp. 124-125 C. H NMR (400 MHz, CDCl3): 3.44 (s, 3H, NCH3), 6.65-6.68 (m, 2H, Ar-H),
6.74-6.79 (m, 1H, Ar-H), 7.15-7.21 (m, 2H, Ar-H), 7.47-7.57 (m, 4H, Ar-H), 7.66-7.70 (m, 2H,
Ar-H), 7.98 (d, 1H, J = 8.4 Hz, 9-H), 8.30-8.33 (m, 2H, Ar-H), 8.64-8.67 (m, 1H, 6-H). 13C NMR
(100 MHz, CDCl3): 40.54, 110.74, 113.84 (2C), 117.69, 122.83, 124.84, 125.84, 127.28 (2C),
127.32, 127.39, 127.42, 128.92 (2C), 129.00 (2C), 129.14, 131.10, 136.35, 143.80, 148.02, 150.08,
162.74. ESIMS [M+H]+: 351. Anal. calcd for C24H18N2O: C 82.26, H 5.18, N 7.99; found: C 81.96,
H 5.38, N 7.72.
5.4.2. 2-(4-Chlorophenyl)-N-methyl-N-phenylnaphtho[1,2-d]oxazol-5-amine (13b). Yield 83% as a
o
yellow solid. Mp: 150-151 C. 1H NMR (400 MHz, CDCl3): 3.45 (s, 3H, NCH3), 6.66-6.68 (m, 2H,
Ar-H), 6.75-6.79 (m, 1H, Ar-H), 7.16-7.20 (m, 2H, Ar-H), 7.48-7.54 (m, 3H, Ar-H), 7.65 (s, 1H,
4-H), 7.67-7.71 (m, 1H, 7-H), 7.99 (d, 1H, J = 8.4 Hz, 9-H), 8.23-8.26 (m, 2H, Ar-H), 8.62 (d, 1H,
J = 8.0 Hz, 6-H). 13C NMR (100 MHz, CDCl3): 40.56, 110.64, 113.92 (2C), 117.81, 122.78, 124.90,
125.92, 125.96, 127.29, 127.51, 128.51 (2C), 129.02 (2C), 129.20, 129.28 (2C), 136.27, 137.27,

ACCEPTED MANUSCRIPT
144.13, 148.09, 150.04, 161.73. ESIMS [M+H]+: 385. Anal. calcd for C24H17ClN2O·0.4 H2O: C
73.52, H 4.58, N 7.15; found: C 73.24, H 4.30, N 7.01.
5.4.3. 2-(4-Methoxyphenyl)-N-methyl-N-phenylnaphtho[1,2-d]oxazol-5-amine (13c). Yield 81% as
o
1
a yellow solid. Mp. 129-130 C. H NMR (400 MHz, CDCl3): 3.44 (s, 3H, NCH3), 3.90 (s, 3H,
OCH3), 6.64-6.67 (m, 2H, Ar-H), 6.74-6.78 (m, 1H, Ar-H), 7.03-7.07 (m, 2H, Ar-H), 7.15-7.20 (m,
2H, Ar-H), 7.46-7.50 (m, 1H, 8-H), 7.64-7.69 (m, 2H, 4- and 7-H), 7.97 (d, 1H, J = 8.4 Hz, 9-H),
8.24-8.27 (m, 2H, Ar-H), 8.62-8.64 (m, 1H, 6-H). 13C NMR (100 MHz, CDCl3): 40.51, 55.45,
110.77, 113.73 (2C), 114.37 (2C), 117.57, 120.06, 122.82, 124.79, 125.71, 127.17, 127.23, 128.98
(2C), 129.05 (2C), 129.09, 136.48, 143.20, 147.80, 150.12, 162.02, 162.95. ESIMS [M+H]+: 381.
Anal. calcd for C25H20N2O2·0.6 H2O: C 76.75, H 5.46, N 7.16; found: C 76.67, H 5.15, N 7.37.
5.4.4. N-(4-Chlorophenyl)-N-methyl-2-phenylnaphtho[1,2-d]oxazol-5-amine (14a). Yield 75% as
o
1
a white solid. Mp. 159-160 C. H NMR (400 MHz, CDCl3): 3.43 (s, 3H, NCH3), 6.56-6.58 (m,
2H, Ar-H), 7.10-7.12 (m, 2H, Ar-H), 7.49-7.57 (m, 4H, Ar-H), 7.65 (s, 1H, 4-H), 7.67-7.71 (m, 1H,
7-H), 7.92 (d, 1H, J = 8.8 Hz, 9-H), 8.32 (m, 2H, Ar-H), 8.65 (d, 1H, J = 8.0 Hz, 6-H). 13C NMR
(100 MHz, CDCl3): 40.67, 110.81, 114.85 (2C), 122.60, 122.95, 124.59, 125.31, 126.00, 127.33
(2C), 127.33, 127.51, 128.82 (2C), 128.87, 128.95 (2C), 131.20, 136.61, 143.17, 147.91, 148.67,
162.91. ESIMS [M+H]+: 385. Anal. calcd for C24H17ClN2O·0.4 H2O: C 73.52, H 4.58, N 7.15;
found: C 73.35, H 4.47, N 7.03.
5.4.5. N,2-bis(4-Chlorophenyl)-N-methylnaphtho[1,2-d]oxazol-5-amine (14b). Yield 75% as a
o
yellow solid. Mp. 213-214 C. 1H NMR (400 MHz, CDCl3): 3.43 (s, 3H, NCH3), 6.55-6.59 (m, 2H,
Ar-H), 7.09-7.13 (m, 2H, Ar-H), 7.49-7.53 (m, 3H, Ar-H), 7.63 (s, 1H, 4-H), 7.68-7.71 (m, 1H,
13
7-H), 7.92 (d, 1H, J = 8.4 Hz, 9-H), 8.24-8.26 (m, 2H, Ar-H), 8.63 (d, 1H, J = 8.4 Hz, 6-H). C
NMR (100 MHz, CDCl3): 40.69, 110.69, 114.91 (2C), 122.69, 122.89, 124.64, 125.82, 126.11,
127.32, 127.63, 128.53 (2C), 128.84 (2C), 128.92, 129.30 (2C), 136.52, 137.37, 143.48, 147.97,
148.63, 161.90. ESIMS [M+H]+: 419. Anal. calcd for C24H16Cl2N2O·0.5 H2O: C 67.59, H 3.97, N
6.57; found: C 67.34, H 3.75, N 6.84.

ACCEPTED MANUSCRIPT
5.4.6. N-(4-Chlorophenyl)-2-(4-methoxyphenyl)-N-methylnaphtho[1,2-d]oxazol-5-amine (14c).
o
1
Yield 76% as a yellow solid. Mp. 172-173 C. H NMR (400 MHz, CDCl3): 3.42 (s, 3H, NCH3),
3.91 (s, 3H, OCH3), 6.54-6.58 (m, 2H, Ar-H), 7.04-7.13 (m, 4H, Ar-H), 7.47-7.51 (m, 1H, 8-H),
7.63 (s, 1H, 4-H), 7.66-7.70 (m, 1H, 7-H), 7.90 (d, 1H, J = 8.4 Hz, 9-H), 8.25-8.28 (m, 2H, Ar-H),
13
8.64-8.66 (m, 1H, 6-H). C NMR (100 MHz, CDCl3): 40.65, 55.47, 110.76, 114.43 (2C), 114.78
(2C), 119.85, 122.51, 122.98, 124.53, 125.90, 127.14, 127.38, 128.81 (2C), 128.84, 129.14 (2C),
136.52, 142.65, 147.67, 148.72, 162.16, 163.11. ESIMS [M+H]+: 415. Anal. calcd for
C25H19ClN2O2: C 72.37, H 4.62, N 6.75; found: C 72.38, H 4.76, N 6.87.
5.4.7. N-(4-Methoxyphenyl)-N-methyl-2-phenylnaphtho[1,2-d]oxazol-5-amine (15a). Yield 71% as
1
a yellow solid. Mp. 120-121 oC. H NMR (400 MHz, CDCl3): 3.41 (s, 3H, NCH3), 3.75 (s, 3H,
OCH3), 6.67-6.71 (m, 2H, Ar-H), 6.76-6.79 (m, 2H, Ar-H), 7.46-7.57 (m, 4H, Ar-H), 7.60 (s, 1H,
4-H), 7.64-7.68 (m, 1H, 7-H), 8.05-8.07 (m, 1H, 9-H), 8.30-8.32 (m, 2H, Ar-H), 8.62-8.64 (m, 1H,
6-H). 13C NMR (100 MHz, CDCl3): 41.47, 55.68, 109.21, 114.54 (2C), 116.48 (2C), 122.74,
123.04, 123.98, 125.08, 125.58, 127.21 (2C), 127.27, 127.48, 128.91 (2C), 130.99, 135.92, 144.87,
145.16, 148.14, 152.65, 162.42. ESIMS [M+H]+: 381. Anal. calcd for C25H20N2O2: C 78.93, H 5.30,
N 7.36; found: C 78.83, H 5.24, N 7.23.
5.4.8. 2-(4-Chlorophenyl)-N-(4-methoxyphenyl)-N-methylnaphtho[1,2-d]oxazol-5-amine (15b).
Yield 78% as a yellow solid. Mp. 137-1338 oC. 1H NMR (400 MHz, CDCl3): 3.41 (s, 3H, NCH3),
3.75 (s, 3H, OCH3), 6.68-6.78 (m, 4H, Ar-H), 7.46-7.53 (m, 3H, Ar-H), 7.58 (s, 1H, 4-H),
7.65-7.69 (m, 1H, 7-H), 8.06 (d, 1H, J = 8.4 Hz, 9-H), 8.22-8.26 (m, 2H, Ar-H), 8.59 (d, 1H, J =
8.4 Hz, 6-H). 13C NMR (100 MHz, CDCl3): 41.52, 55.68, 109.01, 114.55 (2C), 116.63 (2C),
122.66, 125.15, 125.68, 126.01, 127.37, 128.42 (2C), 128.93, 129.25 (2C), 130.25, 135.57, 137.11,
144.84, 145.48, 148.22, 152.75, 161.41. ESIMS [M+H]+: 415. Anal. calcd for C25H19ClN2O2·0.5
H2O: C 70.84, H 4.76, N 6.61; found: C 70.53, H 4.62, N 6.72.
5.4.9. N,2-Bis(4-methoxyphenyl)-N-methylnaphtho[1,2-d]oxazol-5-amine (15c). Yield 75% as a
o
1
yellow solid. Mp. 119-120 C. H NMR (400 MHz, CDCl3): 3.40 (s, 3H, NCH3), 3.74 (s, 3H,

ACCEPTED MANUSCRIPT
OCH3), 3.90 (s, 3H, OCH3), 6.66-6.69 (m, 2H, Ar-H), 6.75-6.78 (m, 2H, Ar-H), 7.03-7.06 (m, 2H,
Ar-H), 7.44-7.49 (m, 1H, 8-H), 7.58 (s, 1H, 4-H), 7.63-7.67 (m, 1H, 7-H), 8.04 (d, 1H, J = 8.4 Hz,
9-H), 8.23-8.27 (m, 2H, Ar-H), 8.61-8.63 (m, 1H, 6-H). 13C NMR (100 MHz, CDCl3): 41.38, 55.43,
55.68, 109.29, 114.36 (2C), 114.53 (2C), 116.26, 120.00, 122.75, 125.00, 125.48, 127.03, 127.14,
128.87, 128.99 (2C), 135.60, 144.59, 144.90, 147.86, 152.54, 161.98, 162.62. ESIMS [M+H]+: 411.
Anal. calcd for C26H22N2O3: C 76.08, H 5.40, N 6.82; found: C 76.04, H 5.39, N 6.78.
5.4.10. 2-(Furan-2-yl)-N-methyl-N-phenylnaphtho[1,2-d]oxazol-5-amine (19). Yield 71% as a gray
solid. Mp. 126-127 oC. 1H NMR (400 MHz, CDCl3): 3.44 (s, 3H, NCH3), 6.63-6.68 (m, 3H, Ar-H),
6.75-6.79 (m, 1H, Ar-H), 7.16-7.20 (m, 2H, Ar-H), 7.27 (dd, 1H, J = 3.6, 0.8 Hz, Fu-3-H),
7.48-7.52 (m, 1H, 8r-H), 7.63 (s, 1H, 4-H), 7.63-7.70 (m, 2H, Ar-H), 8.05 (d, 1H, J = 8.4 Hz, 9-H),
8.64-8.67 (m, 1H, 6-H). 13C-NMR (100 MHz, CDCl3): 40.54, 110.52, 112.26, 113.50, 113.90 (2C),
117.79, 122.88, 124.85, 126.01, 127.17, 127.52, 129.01 (2C), 129.25, 135.81, 142.78, 144.10,
145.43, 147.44, 155.00, 155.04. ESIMS [M+H]+: 341. Anal. calcd for C22H16N2O2: C 77.63, H 4.74,
N 8.23; found: C 77.58, H 4.81, N 7.99.
5.4.11. N-(4-Chlorophenyl)-2-(furan-2-yl)-N-methylnaphtho[1,2-d]oxazol-5-amine (20). Yield 79%
as a yellow solid. Mp. 200-201 oC. 1H NMR (400 MHz, CDCl3): 3.42 (s, 3H, NCH3), 6.54-6.58 (m,
2H, Ar-H), 6.64 (dd, 1H, J = 3.6, 2.0 Hz, Fu-4-H), 7.09-7.13 (m, 2H, Ar-H), 7.29 (dd, 1H, J = 3.6,
0.8 Hz, Fu-3-H), 7.49-7.53 (m, 1H, 8-H), 7.62 (s, 1H, 4-H), 7.67-7.71 (m, 2H, Ar-H), 7.91 (d, 1H, J
= 8.4 Hz, 9-H), 8.66 (d, 1H, J = 8.0 Hz, 6-H). 13C-NMR (100 MHz, CDCl3): 40.68, 110.57, 112.29,
113.64, 114.90 (2C), 122.70, 123.00, 124.61, 126.17, 127.21, 127.64, 128.84 (2C), 128.90, 136.10,
142.73, 143.47, 145.51, 147.33, 148.60, 155.19. ESIMS [M+H]+: 375. Anal. calcd for
C22H15ClN2O2·0.3 H2O: C 69.49, H 4.14, N 7.37; found: C 69.28, H 3.92, N 7.27.
5.4.12. 2-(Furan-2-yl)-N-(4-methoxyphenyl)-N-methylnaphtho[1,2-d]oxazol-5-amine (21). Yield
76% as a yellow solid. Mp. 146-147 oC. 1H NMR (400 MHz, CDCl3): 3.40 (s, 3H, NCH3), 3.75 (s,
3H, OCH3), 6.63 (dd, 1H, J = 3.6, 1.6 Hz, Fu-4-H), 6.66-6.79 (m, 4H, Ar-H), 7.26 (dd, 1H, J = 3.6,
0.8 Hz, Fu-3-H), 7.46-7.50 (m, 1H, 8-H), 7.57 (s, 1H, 4-H), 7.64-7.67 (m, 1H, 7-H), 7.68 (dd, 1H, J

ACCEPTED MANUSCRIPT
= 1.6, 0.8 Hz, Fu-5-H), 8.05 (d, 1H, J = 8.8 Hz, 9-H), 8.62-8.64 (m, 1H, 6-H). 13C NMR (100 MHz,
CDCl3): 41.51, 55.67, 108.89, 112.22, 113.25, 114.53 (2C), 116.61 (2C), 122.78, 125.10, 125.73,
127.10, 127.39, 128.96, 135.08, 142.86, 144.78, 145.31, 145.44, 147.57, 152.73, 154.74. ESIMS
[M+H]+: 371. Anal. calcd for C23H18N2O3: C 74.58, H 4.90, N 7.56; found: C 74.49, H 4.85, N
7.51.
5.5. Cytotoxicity and antiviral activity assays
5.5.1. Compounds
Compounds were dissolved in DMSO at 10 mM and then diluted in culture medium.
5.5.2. Cells culture and reagents
Ava5 cells [37], human hepatoma cells (Huh-7) harbaoring HCV replicon genome, were cultured in
DMEM culture medium with 10% heat-inactivated fetal bovine serum, 1% antibiotic–antimycotic,
1% non-essential amino acids and 1 mg·mL−1 G418 and . Huh-7 cells were maintained in DMEM
with 10% heat-inactivated fetal bovine serum, 1% antibiotic-antimycotic, and 1% non-essential
amino acids and were incubated at 37 °C with a 5% CO2 supplement. An HO-1-specific inhibitor [tin
protoporphyrin IX dichloride (SnPP)] was purchased from Sigma (St. Louis, MO, USA).
5.5.3. Cytotoxicity assays
For cytotoxicity tests, run in parallel with antiviral assays, plates at an initial density of (5 x 103
cells/well) were treated with or without serial dilutions of test compounds. Cell viability was
determined after 72 h at 37 oC in a humidified CO2 (5%) atmosphere by the (2,3-bis [2-methyloxy-
4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) (XTT) method [38].
5.5.4. Tansfection and luciferase activity assay
Ava5 cells were transfected with the HO-1 promoter-driven luciferase plasmid, pHO-1-Luc, using
the T-proTM transfection reagent (Ji-Feng Biotechnology Co., Ltd., Taipei, Taiwan) according to the
manufacturer's instructions. The transfected cells were treated with compound 18 at various
concentrations for 3 days. Each transfection complex contains 0.1 µg pCMV-SEAP, a secreted
alkaline phosphatase (SEAP) expression vector, serving as a transfection control for normalization

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luciferase activity. The luciferase activity assay was performed using the Bright-Glo Luciferase
assay system (Promega) according to the manufacturer’s instructions.
5.5.5. Immunoblot analysis
Ava5 cells were seeded in 24-well plates at a density of 5 × 104 cells per well for over-night and
treated with indicated reagent at proper concentrations for 3 days. Cells were washed twice with
cold PBS and lysed by RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet
P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25
µg/mL aprotinin, and 25 µg/mL leupeptin) and stored at −20 °C. The protein concentration was
determined by the Bradford method. 10 µg protein were separated by 10% SDS-PAGE and
transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat dried milk and
incubated with anti-NS5B, anti-GAPDH and anti-HO-1 antibodies, followed by 30 min with
secondary antibodies in 5% milk containing Tris-buffered saline (TBS) and 0.5% Tween. The
blotting signal was developed using an ECL detection kit (PerkinElmer, CT, USA) and was counted
by the software Quantity One (Bio-Rad, CA, USA) [39].
5.5.6. Real-time quantitative PCR (RT-qPCR) assay
Total cellular RNA were extracted by a Total RNA Miniprep Purification kit (GMbiolab, Taiwan)
according to the manufacturer’s instructions and were transcribed to cDNA by M-MLV reverse
transcriptase (Promega, USA) with HCV 3’UTR (5’-acttgatctgcagagaggcc-3’ ) or oligo dT primer.
The mRNA expression of target gene were determined by quantitative real-time RT-PCR with
specific primers as previous study described [15]. The CT value of each sample was determined by
the ABI Step One Real-Time PCR-System. The PCR primers were as follows: GAPDH,
5’-gtcttcaccaccatggagaa-3’ (forward), and 5’-atggcatggactgtggtcat-3’ (reverse); NS5B,
5’-ggaaaccaagctgcccatca-3’ (forward), and 5’-cctccacggatagaagttta-3’ (reverse). Each relative
mRNA levels were obtained by normalization of the endogenous cellular gapdh gene.
5.5.7. Preparation of HCVcc stock and viral infection assay

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Huh-7 cells stably expressing pEF/JFH1-Rz/N plasmid were cultured to produce cell
culture-drived infectious HCV (HCVcc), and the condition medium were collected to harvest HCV
particles [40]. Fivity percent cell culture infective dose of harvested vrial particle was determined
by immunofluorescence assay with the specific anti-Core antibodies (Abcam). For the antiviral
activity assay, the Huh-7 cells were seeded into a 24 well plate with the density of 5 × 104 cells per
well and infected with HCVcc at a multiplicity of infection of 0.1 for 6 hr. The infected cells were
treated with various concentrations of compound 18 for 3 days. The RT-qPCR was used to analyze
the anti-HCV activity of compound 18.
5.5.8. Statistical analysis
All data were presented as mean ± SD of three independent experiments with triplicate sample.
Statistical analysis was calculated by Student's t-test, and the significant difference was considered
if *P<0.05.
Acknowledgment. Financial support of this work by the Minister of Science and Technology of the
Republic of China (Most 106-2320-B-037-015, MOST 105-2320-B-037-007, MOST
105-2320-B-037-011, MOST 103-2320-B-037-011-MY3) and Kaohsiung Medical University
(KMU-TP104E16, KMU-TP104E42, KMU-TP104H07, KMU-TP104H08, KMU-TP105E02,
KMU-TP105E16, KMU-TP105E33, KMU-TP105H07, KMU-TP105H08, 105KMUOR02) are
gratefully acknowledged. We also thank Center for Research Resources and Development at
Kaohsiung Medical University for the instrumentation and equipment support.
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Figure captions:
Figure 1. Structures
methylbenzothiazole (2),
of
3’-nitrophenylaminoquinoline
(1),
2-(4-nitroanilino)-6-
3-(3’,4’,5’-trimethoxyanilin-1’-yl)methylaminocoumarin
(3),
2-(hydroxyphenylmethyl)-3-(4-methoxyphenyl)quinoline
(4),
2-(4-hydroxybenzoyl)-3-(4-
hydroxyphenyl)quinoline (5), and target compounds.
Figure 2. Compound 18 dose-dependently reduced HCV replication. Compound 18 reduced
HCV (A) protein synthesis and (B) RNA replication in Ava5 cells. Ava5 cells were treated with
compound 18 at indicated concentrations for 3 days. Cell lysates and cellular mRNA were
subjected to Western blotting and RT-qPCR. Relative HCV RNA were normalized by cellular
gapdh mRNA levels. Cell variability was determined in compound 18-treated Ava5 cells at
indicated concentrations for 3 days. (C) Compound 18 inhibited HCV JFH-1 replication in Huh7.5
cells in a dose-dependently manner. Huh7.5 cells were infected with HCV JFH-1 at an MOI of 1,
and the infected cells were treated with compound 18 for 3 days. RT-qPCR was performed to
determined relative HCV RNA level. Data was presented as the mean ± SD of three independent
experiments; *P < 0.05.
Figure 3. Compound 18 induced HO-1 expression in Ava5 cells. Compound 18 induced (A)
HO-1 promoter activity and (B) HO-1 protein expression. Ava5 cells were transfected with
pHO-1-Luc at 0.5 µg, and the transfected cells were treated with compound 18 for 3 days. The cell
lysate was subjected to luciferase activity assay. Ava5 cells were treated with compound 18 at
indicated concentrations, and the cell lysate was subjected to western blotting with anti-HO-1 and
anti-GAPDH antibodies. Data was presented as the mean ± SD of three independent experiments;
*P < 0.05.
Figure 4. Compound 18 reduced HCV replication through inducing HO-1 expression. (A and
B) The reduction effect of compound 18 on HCV replication was attenuated by SnPP treatment.
Ava5 cells were co-incubated with compound 18 at 1 µM and increasing concentrations of SnPP
from 0-5 µM for 3 days. Cell lysate and cellular mRNA were subjected to Western blotting and

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RT-qPCR. Relative HO-1 mRNA were normalized by cellular gapdh mRNA levels. Data was
presented as the mean ± SD of three independent experiments.
Figure 5. Compound 18 induced HO-1 expression through reduceing bach1 expression. (A)
Compound 18 dose-dependently reduced bach1 expression. Ava5 cells were treated with compound
18 at indicated concentrations for 3 days. Cell lysate was subjected to western blotting with
anti-Nrf2, anti-Keap-1, anti-bach1, and anti-GAPDH antibodies. (B) Compound 18 reduced the
translocation of bach1 into nuclear. Ava5 cells were treated with compound 18 at indicated
concentrations for 3 days. Nuclear fraction was subjected to western blotting with anti-bach1,
anti-Nrf2, and anti-GAPDH antibodies. (C) Compound 18 induced the Nrf2- mediated ARE
transactivation. Huh-7 and Ava5 cells were transfected with p2xARE-Luc, and the
transfected-Ava5 cells were treated with compound 18 for 3 days. Cell lysate was subjected to
luciferase activity assay. Data was presented as the mean ± SD of three independent experiments;
*P < 0.05.
Figure 6. Compound 18 reduced HCV NS3/4A protease activity. (A) The schematic diagram of a
reporter vector indicating NS3/4A protease activity. The reporter vector included the HCV NS3/4A
cleavage site flanked by egfp and seap genes, named as pEG(DE∆4AB)SEAP. (B) Compound 18
reduced NS3/4A protease activity in a concentration-dependent manner. Huh-7 cells were
co-transfected with pEG(DE∆4AB)SEAP and the NS3/4A expression vector, and the cells were
treated with compound 18 for 3 days. Cell lysate was subjected to SEAP activity assay, and the fold
change of NS3/4A activity was present the relative to the compound 18-untreated control (defined
as 1). Data was presented as the mean ± SD of three independent experiments; *P < 0.05.
Figure 7. Compound 18 induced antiviral IFN responses. (A) Compound 18 induced the activity of
ISRE. Ava5 cells were transfected with pISRE-Luc, and the cells were treated with compound 18 at
indicated concentrations for 3 days. The cell lysate was subjected to luciferase activity assay. (B)
Compund 18 elevated IFN-α-2, IFN-α-5, and IFN-α-17 expression. (C) Compound 18 induced
IFN-mediated antiviral gene expression. Ava5 cells were treated with compound 18 at indicated