1-Phenyl-: N -(benzothiazol-2-yl)methanimine derivatives as Middle East respiratory syndrome coronavirus inhibitors

Article abstract of DOI:10.1039/d0ra08442e

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to human health, and currently there are no effective or specific therapies available to treat it. Herein a series of 1-phenyl-N-(benzothiazol-2-yl)methanimine derivatives with inhibitory activity against MERS-CoV are described. The compound 4f with a 50% inhibition concentration value of 0.09 μM is a promising inhibitor that warrants further evaluation, towards the development of potential anti-MERS-CoV drugs. This journal is

Full text of DOI:10.1039/d0ra08442e

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1-Phenyl-N-(benzothiazol-2-yl)methanimine
derivatives as Middle East respiratory syndrome
Cite this: RSC Adv., 2020, 10, 43299
coronavirus inhibitors†
Min-Qi Hu,‡a Heng Li,‡bc Ying Lin,a Ying Zhang,a Jie Tang,ad Jian-Ping Zuo,b
a
bc
bc
a
*
*
*
Li-Fang Yu,
Xian-Kun Tong,
Wei Tang
and Fan Yang
Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to human health, and
currently there are no effective or specific therapies available to treat it. Herein a series of 1-phenyl-N-
(benzothiazol-2-yl)methanimine derivatives with inhibitory activity against MERS-CoV are described. The
compound 4f with a 50% inhibition concentration value of 0.09 mM is a promising inhibitor that warrants
further evaluation, towards the development of potential anti-MERS-CoV drugs.
Received 3rd October 2020
Accepted 20th November 2020
DOI: 10.1039/d0ra08442e
rsc.li/rsc-advances
MERS-CoV infection. Therefore, the development of specic
antiviral drugs for the treatment of MERS-CoV is urgently
Introduction
needed.
Viral genomic studies indicate that MERS-CoV is an envel-
Middle East respiratory syndrome coronavirus (MERS-CoV) is
a novel zoonotic virus in the Middle East.1,2 It rst appeared in
Saudi Arabia in June 2012,3 then spread to the rest of the Middle
East and South Korea.4 MERS-CoV is primarily transmitted from
animals to humans,5 but it can reportedly spread from human
to human through close contact.6,7 It can cause severe respira-
tory disease, with symptoms of fever, coughing, and dyspnea,
and may contribute to severe gastrointestinal diseases and renal
failure.8 As at 03 June 2020, the World Health Organization had
reported 2494 laboratory-conrmed cases of MERS-CoV infec-
tion, including 858 conrmed deaths (an approximately 35%
fatality rate) in over 27 countries.
MERS-CoV poses a severe threat to human health, as illus-
trated by the recent global coronavirus disease-2019 (COVID-19)
pandemic caused by severe acute respiratory syndrome coro-
navirus 2. Although some reported therapeutics for COVID-19
have recently been reported,9–17 their curative effect is still very
limited and there can be side effects.18,19 Similarly, despite
ongoing global efforts to develop small-molecule inhibitors,20–26
peptide inhibitors,27–30 neutralizing antibodies,31–33 and
vaccines34–36 for the treatment and prevention of MERS-CoV,
there is no effective approved specic antiviral therapy for
oped virus with positive-sense single-stranded RNA that belongs
to lineage C in the genus Betacoronavirus of the family Coro-
naviridae under the Nidovirales order.3 It is the sixth corona-
virus known to infect humans, and the rst known lineage C
Betacoronavirus associated with human infection.37,38 The
MERS-CoV genome is approximately 30 kilobases in size and
contains more than 10 open reading frames (ORFs).39 The large
replicase ORF1a and ORF1b produce polyproteins PP1a and
PP1ab, which are subsequently cleaved into 15 or 16 nonstruc-
tural proteins.40–42 The region downstream of ORF1b encodes at
least four main structural proteins: spike (S), membrane (M),
envelope (E), and nucleocapsid (N).
The S protein serves as the critical surface-located trimeric
glycoprotein of MERS-CoV and induces entry into host cells,43
and it is composed of subunits S1 and S2. The receptor-binding
domain on the S1 subunit attaches to dipeptidyl peptidase 4,
which is a well-known target for diabetes.44–49 The conformation
of the S2 subunit then changes and a six-helix bundle fusion
core is formed, which enables the formation of the viral enve-
lope, facilitating tight host cell membrane binding for fusion
and subsequent host cell entry.50 Thus the S protein plays
a pivotal role in mediating the entry of the virus into target host
cells.
aShanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, School of Chemistry and Molecular Engineering, East China Normal
University, Shanghai, 200062, China. E-mail: fyang@chem.ecnu.edu.cn
According to previous reports, MERS-CoV pseudovirus
expressing S protein, which allows for single-cycle infection in
cells expressing dipeptidyl peptidase 4, can be used to rapidly
screen MERS-CoV entry inhibitors.51 In the current study, novel
small-molecule inhibitors of entry events of pseudovirus
expressing S protein were identied via high-throughput
screening technology using a cell-based assay.
bShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,
201203, China. E-mail: tangwei@simm.ac.cn; xktong@simm.ac.cn
cSchool of Pharmacy, University of Chinese Academy of Sciences, Beijing, 100049,
China
dShanghai Greenchem & Biotech Co. Ltd., Shanghai, 200062, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra08442e
‡ These authors contributed equally to this work.
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Fig. 1 High-throughput screening output. (A) Data from the 48 000-compound orthogonal cocktail library primary screening experiments were
plotted as inhibition of MERS-S pseudovirus infection in Huh-7 cells. Inhibition was calculated as 100% minus the percentage of luciferase
readout of each well against the control well with no drug, i.e., 100% ꢀ Luc (compound)/Luc (control) ꢁ 100%. (B) The structure of the hit
compound 1a. (C) Dose-dependent curve of inhibition activity and toxicity of compound 1a in a MERS-S pseudovirus assay. (D) Flow chart of the
high-throughput screening and validation procedure.
for anti-MERS-CoV activity (Fig. 1A). Compound library samples
Materials and methods
Screening was performed using an orthogonal cocktail library52
composed of 48 000 compounds that had never been screened
were orthogonally pooled as mixtures of 10 per well at 2 mg mLꢀ1
each, with duplicate representation for each compound
(Fig. 1D). This bidirectional orthogonal pooling strategy enables
greater screening efficiency and throughput when using large
Scheme 1 Reagents and conditions. (a) 4-Aminophenylboronic acid pinacol ester or 5-aminopyridine-3-boronic acid pinacol ester, Pd(dppf)
Cl2$CH2Cl2, K2CO3, DMF, 70 ꢂC, 16 h. (b) ArCHO, AcOH, MeOH, reflux, 2 h. (c) NaBH4, MeOH, room temperature, 1 h. (d) 2-Hydroxy-3-
methoxybenzoyl chloride, NaHCO3, H2O, Et2O, room temperature, 12 h.
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compound libraries. Using this approach, compound 1a was resonance spectrum of 1a revealed corresponding peaks in the
identied as a modest entry inhibitor (Fig. 1B) with a 50% range of d 114.2–166.1 ppm which were assigned to azomethine
inhibition concentration (IC50) of 0.73 mM (Fig. 1C). Using 1a as (–CH]N–) carbon and aryl carbons. A peak at d 55.2 ppm was
a hit compound, a series of 1-phenyl-N-(benzothiazol-2-yl) identical to the methoxy carbon. The 1H–1H COSY spectrum
methanimine derivatives were designed and synthesized, and demonstrated chemical shis of three protons on ring C at
structure–activity relationships and their inhibitory potencies d 6.87–7.06 ppm, and two protons near the nitrogen atom side
against MERS-S pseudovirus were investigated.
on ring B at d 7.36–7.41 ppm. Correlation between the azome-
thine proton and the two protons near the nitrogen atom side
1
on ring B was observed in the H–1H NOSEY correlation spec-
trum, indicating that the imine moiety of compound 1a is E-
geometry. The HRMS (ESI) spectrum of 1a depicted a signal at
m/z 383.0823, indicating a [M + Na]+ signal with the molecular
formula C21H16N2NaO2S, which is concordant with the struc-
ture of 1a. Characteristic spectra and data are shown in the
ESI.†
Results and discussion
Four major regions were partitioned for diversication; the
benzothiazole portion (blue), the linker region (pink), the imine
portion (yellow), and the phenyl portion (green) (Fig. 1B).
Compounds 1a–1g were synthesized rst as outlined in Scheme
1. Commercially available aryl bromides were used as the
starting material, and the corresponding intermediate amines
3a–3d were prepared using the Suzuki–Miyaura cross-coupling
reaction.53 Compounds 1a–1e were generated using the reac-
tion of an intermediate amine with the corresponding aromatic
aldehyde in the presence of AcOH as catalyst.54 Compound 1g
was generated by the reaction of 3a with 2-hydroxy-3-
methoxybenzoyl chloride. Compound 1f was generated by
a reduction reaction of 1a with NaBH4.55 Their IC50 values
against MERS-S pseudovirus and 50% cytotoxic concentration
(CC50) values are summarized in Table 1. The Schiff base
structure of hit compound 1a was conrmed by comprehensive
Compounds with benzoxazole (1b) or benzimidazole (1c)
motifs lost inhibitory activity, as did the compound without
Table 2 Inhibitory activity of 1h–1u against MERS-S pseudovirus
a
b
Compound
Ar
IC50
CC50
1h
0.57 ꢃ 0.11
0.52 ꢃ 0.03
>100
1
1i
>100
>100
>100
>100
spectral data. The H NMR spectrum depicted a sharp singlet
peak at d 8.67 parts per million (ppm) indicating the presence of
azomethine proton (–CH]N–). Phenolic-OH proton and 1j
methoxy protons exhibited singlet peaks at d 13.44 and
0.20 ꢃ 0.05
0.40 ꢃ 0.01
0.22 ꢃ 0.03
d 3.94 ppm respectively. A multiplet signal at d 6.87–8.14 ppm
1k
was assigned to the aromatic protons. The 13C nuclear magnetic
1l
Table 1 Inhibitory activity of 1a–1g against MERS-S pseudovirus
1m
0.34 ꢃ 0.24
>100
>100
1n
1o
1p
>10.00
0.14 ꢃ 0.06
>100
>100
>10.00
1q
1r
6.52 ꢃ 1.14
0.62 ꢃ 0.31
>100
>100
a
b
Compound
X
Y
IC50
CC50
1a
1b
1c
1d
1e
1f
S
O
CH
CH
CH
N
—
—
0.73 ꢃ 0.66
>10.0
>10.0
>100
>100
>100
>100
>100
>100
>100
>100
1s
1t
>10.00
>100
>100
8.80 ꢃ 0.01
NH
S
—
—
—
—
2.7 ꢃ 0.19
>10.0
1u
>10.00
>100
>100
>10.0
>10.0
2.0
1g
—
—
HRPc
HRPc
—
2.00
a
b
a
b
IC50: 50% inhibitory concentration (mM). CC50: 50% cytotoxic
IC50: 50% inhibitory concentration (mM). CC50: 50% cytotoxic
c
c
concentration (mM). HRP: peptide mimics of heptad repeat domain concentration (mM). HRP: peptide mimics of heptad repeat domain
in the MERS spike protein, which is responsible for conformational in the MERS spike protein, which is responsible for conformational
change during spike protein-mediated viral–host membrane fusion.
change during spike protein-mediated viral–host membrane fusion.
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Scheme 2 Reagents and conditions. (a) 4-Aminophenylboronic acid pinacol ester, Pd(dppf)Cl2$CH2Cl2, K2CO3, DMF, 70 ꢂC, 16 h. (b) Thiophene-
3-carbaldehyde, AcOH, MeOH, reflux, 2 h. (c) For 4o: 4-aminophenol, t-BuONa, K2CO3, DMF, room temperature, 5 h; for 4p: 4-amino-
benzenethiol, K2CO3, MeCN, reflux, 3 h.
Table 3 Inhibitory activity of 4a–4p against MERS-S pseudovirus
a
b
Compound
R
IC50
CC50
4a
4b
4c
4d
4e
—
—
—
—
7-F
6-F
5-F
4-F
6-OEt
6-Me
6-NHMe
6-NMe2
6-COOH
6-CONHMe
—
>10.00
>10.00
>10.00
>10.00
1.67 ꢃ 0.30
0.09 ꢃ 0.01
1.00 ꢃ 0.07
0.53 ꢃ 0.02
>10.00
>10.00
>10.00
>10.00
>10.00
>10.00
>10.00
>10.00
2.00
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
HRPc
—
—
a
IC50: 50% inhibitory concentration (mM). b CC50: 50% cytotoxic concentration (mM). c HRP: peptide mimics of heptad repeat domain in the MERS
spike protein, which is responsible for conformational change during spike protein-mediated viral–host membrane fusion.
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a phenyl group B (1e) in the linker region. Modest inhibitory suggest that compound 4f has potential for the subsequent
activity was evident when the phenyl group B was replaced with development of novel anti-MERS-CoV agents.
a pyridyl group (1d). When the imine portion was reduced or
replaced with amide (1f and 1g), inhibitory activity was mark-
edly reduced. Modication was then focused on the phenyl ring
Conclusion
C of the compound, and derivatives 1h–1m were designed and
synthesized in a similar way as 1a. Their IC50 values on MERS-S
pseudovirus and CC50 values are summarized in Table 2.
All the derivatives demonstrated inhibitory activities with
IC50 values ranging from 0.20 mM to 0.57 mM, and no cell
toxicity. Either changing the position of the hydroxyl group and
methoxy group or the absence of these groups increased the
activity slightly, the compounds containing 3-OH were more
potent. Changing the phenyl ring C to a pyridine ring did not
elicit signicant changes in inhibitory activity.
Given the results of 1h–1m, further structural extensions
were investigated in the phenyl ring C area. A series of ve-
member aromatic heterocycles were introduced to replace the
phenyl ring C. Derivatives 1o and 1r, substituted by a 3-thienyl
group, exhibited marked inhibitory activity with respective IC50
values of 0.14 mM and 0.62 mM. The introduction of a nitro
group on the thiophene motif caused a slight reduction in
inhibitory activity. Compound 1q exhibited modest inhibitory
capacity, indicating that the substitution mode inuenced
activity. None of the compounds in this series exhibited any
cytotoxicity.
Based on the above results, while retaining the 3-thienyl
group at ring C position a structure–activity relationship
investigation was conducted in which structural extensions at
the benzothiazole part of this compound were assessed. As
shown in Scheme 2, commercially available aryl bromides were
used as starting materials, and the corresponding intermediate
amines 3f–3u were prepared using Suzuki–Miyaura cross-
coupling or a nucleophilic substitution reaction. The nal
compounds 4a–4p were readily generated via a similar method
that has been previously outlined in Scheme 1. Their IC50 values
against MERS-S pseudovirus and their CC50 values are
summarized in Table 3.
Replacement of a benzothiazole unit (4a–4d) abolished the
inhibitory activity (Table 3), further conrming that the ben-
zothiazole motif played a pivotal role in inhibitory capacity. An
atom of uorine—the most electronegative of the halogens—
was then introduced at different benzothiazole positions, and
the derivatives obtained (4e–4h) exhibited diverse inhibitory
capacity. Introduction of a uorine atom at the 6-position of the
phenyl ring A (4f) increased inhibition activity, with an IC50
value of 0.09 mM, and introduction of a uorine atom at another
In summary, a series of new 1-phenyl-N-(benzothiazol-2-yl)
methanimine derivatives was developed as MERS-CoV entry
inhibitors via modication of a hit compound, which was ob-
tained by screening an orthogonal cocktail library composed of
48 000 compounds. All of the compounds were assessed for
their capacity to inhibit the entry of pseudovirus expressing
MERS-CoV S protein. Structure–activity relationships indicated
that benzothiazole was the best option for region A of the
structure, and the presence of a phenyl ring B and an imine
portion favored the retention of inhibitory activity against
MERS-CoV. The introduction of a 6-member aromatic ring or
a 3-thiophene ring at ring C position also favored inhibitory
activity. Remarkably, compound 4f, with an IC50 value of 0.09
mM, was more potent than the initial hit compound. In addi-
tion, none of these types of structures exhibited cytotoxicity. The
present study demonstrates an approach for the structural
modication of hit compounds, and a novel skeleton for new
anti-MERS-CoV drug research. Further research on this series of
derivatives is ongoing.
Experimental
Chemistry
Commercial reagents and solvents were purchased commercial
suppliers and used without further purication. All non-
aqueous reactions were under a nitrogen atmosphere and all
non-aqueous reaction vessels were oven-dried. Flash column
chromatography was performed using Qingdao Ocean silica gel
(200–300) with the indicated eluents. All nal compounds were
characterized by their NMR and HRMS spectra, unless stated
otherwise. 1H NMR spectra were recorded at a spectrometer
frequency of 400 MHz, and 13C NMR spectra at 100 MHz on
Bruker Avance 400. Chemical shis are reported in d (ppm)
using signals of tetramethylsilane (TMS) as the internal stan-
dard. High-resolution mass spectra (HRMS) were measured on
a Bruker ESI-TOF high-resolution mass spectrometer. Melting
points (mp) were uncorrected and were recorded on a Buchi B-
54 melting point apparatus.
General procedure for the synthesis of compounds 3a–3d and
3f–3s
position resulted in modest inhibitory potency. The introduc- 2a–2q (1 mmol), the corresponding pinacol borate (1.2 mmol)
tion of other groups such as alkoxy, alkyl, amino, and carboxyl and Pd(dppf)Cl2$CH2Cl2 (0.05 mmol) were suspended in DMF
groups (4i–4n) reduced inhibitory activity. The reduction in (6 mL) in the presence of 2 M K2CO3 (2 mL) and stirred at 70 ꢂC
inhibitory capacity is most likely due to the steric effect of the overnight under nitrogen. The reaction mixture was diluted
bulky group. To increase the exibility of the compounds, with H2O (40 mL) and organics were extracted with EtOAc (3ꢁ
heteroatom was introduced between the benzothiazole group 30 mL). The combined organic layers were washed with brine
and phenyl ring B to obtain 4o and 4p, but unfortunately (30 mL), dried over MgSO4, ltered and concentrated in vacuo to
inhibitory activity was abolished. The CC50 values of these afford crude mixture. The crude mixture was then puried by
compounds were greater than 100 mM, indicating no toxicity or ash column chromatography in a mixture of petroleum ether
at least lower toxicity to uninfected cells. The results of the study and ethyl acetate to yield the pure products 3a–3d and 3f–3s.
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4-(Benzo[d]thiazol-2-yl)aniline (3a). Yellow solid; yield
4-(6-Fluorobenzo[d]thiazol-2-yl)aniline (3k). White solid;
60%.1H NMR (400 MHz, DMSO-d6) d 8.01 (d, J ¼ 7.9 Hz, 1H, yield 71%. 1H NMR (400 MHz, chloroform-d) d 7.94–7.90 (m, 1H,
ArH), 7.89 (d, J ¼ 8.1 Hz, 1H, ArH), 7.76 (d, J ¼ 8.4 Hz, 2H, ArH), ArH), 7.85 (d, J ¼ 7.8 Hz, 2H, ArH), 7.52 (d, J ¼ 8.1 Hz, 1H, ArH),
7.48–7.42 (m, 1H, ArH), 7.37–7.31 (m, 1H, ArH), 6.67 (d, J ¼ 7.20–7.14 (m, 1H, ArH), 6.73 (d, J ¼ 8.9 Hz, 2H, ArH), 4.02 (s, 2H,
8.4 Hz, 2H, ArH), 5.88 (s, 2H, NH2). 13C NMR (100 MHz, chlo- NH2). 13C NMR (100 MHz, chloroform-d) d 167.2 (d, J ¼ 3.2 Hz),
roform-d) d 168.5, 154.3, 149.2, 134.7, 129.2, 126.1, 124.4, 124.0, 159.0 (d, J ¼ 244.5 Hz), 149.8 (d, J ¼ 1.8 Hz), 148.2, 134.5 (d, J ¼
122.6, 121.4, 114.7.
11.1 Hz), 128.0 (2C), 122.6, 122.2 (d, J ¼ 9.2 Hz), 113.8 (2C), 113.5
4-(Benzo[d]oxazol-2-yl)aniline (3b). Light orange solid; yield (d, J ¼ 24.5 Hz), 106.7 (d, J ¼ 26.7 Hz).
1
65%. H NMR (400 MHz, DMSO-d6) d 7.86 (d, J ¼ 8.3 Hz, 2H,
4-(5-Fluorobenzo[d]thiazol-2-yl)aniline (3l). White solid;
ArH), 7.70–7.63 (m, 2H, ArH), 7.36–7.27 (m, 2H, ArH), 6.70 (d, J yield 59%. 1H NMR (400 MHz, DMSO-d6) d 8.01 (dd, J ¼ 8.8,
¼ 8.4 Hz, 2H, ArH), 5.96 (s, 2H, NH2). 13C NMR (100 MHz, 5.4 Hz, 1H, ArH), 7.76 (d, J ¼ 8.5 Hz, 2H, ArH), 7.72 (dd, J ¼ 10.0,
DMSO-d6) d 163.6, 152.5, 149.8, 142.1, 128.9 (2C), 124.3, 124.0, 2.6 Hz, 1H, ArH), 7.25–7.17 (m, 1H, ArH), 6.68 (d, J ¼ 8.5 Hz, 2H,
118.7, 113.5 (2C), 112.7, 110.2.
ArH), 5.98 (s, 2H, NH2). 13C NMR (100 MHz, DMSO-d6) d 170.9,
4-(1H-Benzo[d]imidazol-2-yl)aniline (3c). Light orange solid; 161.3 (d, J ¼ 240.0 Hz), 154.8 (d, J ¼ 12.3 Hz), 152.4, 129.4 (d, J ¼
1
yield 70%. H NMR (400 MHz, DMSO-d6) d 12.43 (s, 1H, imid- 2.0 Hz), 128.8 (2C), 122.9 (d, J ¼ 9.9 Hz), 119.8, 113.5 (2C), 112.3
azole), 7.87 (d, J ¼ 8.2 Hz, 2H, ArH), 7.49 (s, 2H, ArH), 7.14–7.10 (d, J ¼ 24.7 Hz), 107.8 (d, J ¼ 23.6 Hz).
(m, 2H, ArH), 6.69 (d, J ¼ 8.2 Hz, 2H, ArH), 5.58 (s, 2H, NH2). 13
C
4-(4-Fluorobenzo[d]thiazol-2-yl)aniline (3m). White solid;
NMR (100 MHz, DMSO-d6) d 152.6, 150.5, 127.7 (2C), 121.2, yield 61%. 1H NMR (400 MHz, DMSO-d6) d 7.85 (dd, J ¼ 7.6,
117.3, 113.5 (2C).
1.4 Hz, 1H, ArH), 7.77 (d, J ¼ 8.6 Hz, 2H, ArH), 7.37–7.28 (m, 2H,
6-(Benzo[d]thiazol-2-yl)pyridin-3-amine (3d). Yellow solid; ArH), 6.67 (d, J ¼ 8.7 Hz, 2H, ArH), 5.99 (s, 2H, NH2). 13C NMR
yield 70%. This intermediate is used directly in the next step (100 MHz, DMSO-d6) d 169.0, 154.3 (d, J ¼ 252.5 Hz), 152.5,
without further purication.
142.2 (d, J ¼ 13.2 Hz), 136.5 (d, J ¼ 3.9 Hz), 129.0 (2C), 125.1 (d, J
4-(Thiazolo[4,5-b]pyridin-2-yl)aniline (3f). Yellow solid; yield ¼ 7.1 Hz), 119.5, 118.0 (d, J ¼ 4.0 Hz), 113.5 (2C), 111.9 (d, J ¼
24%. 1H NMR (400 MHz, chloroform-d) d 8.66 (d, J ¼ 3.6 Hz, 1H, 17.7 Hz).
ArH), 8.17 (d, J ¼ 7.9 Hz, 1H, ArH), 8.00 (d, J ¼ 8.4 Hz, 2H, ArH),
4-(6-Ethoxybenzo[d]thiazol-2-yl)aniline (3n). Brown solid;
7.25–7.21 (m, 1H, ArH), 6.74 (d, J ¼ 8.6 Hz, 2H, ArH), 4.09 (s, 2H, yield 38%. 1H NMR (400 MHz, chloroform-d) d 7.88–7.82 (m, 3H,
NH2).
ArH), 7.31 (s, 1H, ArH), 7.04 (d, J ¼ 8.9 Hz, 1H, ArH), 6.73 (d, J ¼
4-(4,5,6,7-Tetrahydrobenzo[d]thiazol-2-yl)aniline (3g). Light 8.1 Hz, 2H, ArH), 4.09 (q, J ¼ 6.8 Hz, 2H, CH2), 3.96 (s, 2H, NH2),
yellow solid; yield 62%. 1H NMR (400 MHz, chloroform-d) d 7.68 1.45 (t, J ¼ 6.9 Hz, 3H, CH3). 13C NMR (100 MHz, chloroform-d)
(d, J ¼ 8.6 Hz, 2H, ArH), 6.67 (d, J ¼ 8.5 Hz, 2H, ArH), 3.84 (s, 2H, d 166.0, 156.6, 148.8, 148.7, 135.8, 128.8 (2C), 124.2, 122.9,
NH2), 2.83–2.75 (m, 4H, CH2), 1.91–1.81 (m, 4H, CH2). 13C NMR 115.5, 114.8 (2C), 105.0, 64.1, 14.9.
(100 MHz, chloroform-d) d 164.2, 149.7, 146.8, 126.6 (2C), 126.3,
123.9, 113.8 (2C), 25.9, 22.6, 22.4, 22.1.
4-(6-Methylbenzo[d]thiazol-2-yl)aniline (3o). Yellow solid;
yield 60%. 1H NMR (400 MHz, chloroform-d) d 7.90–7.85 (m, 3H,
4-(Benzo[d]imidazo[2,1-b]thiazol-2-yl)aniline (3h). Light ArH), 7.64 (s, 1H, ArH), 7.26–7.22 (m, 1H, ArH), 6.73 (d, J ¼
yellow solid; yield 45%. 1H NMR (400 MHz, chloroform-d) d 7.82 8.5 Hz, 2H, ArH), 3.97 (s, 2H, NH2), 2.48 (s, 3H, CH3). 13C NMR
(s, 1H, ArH), 7.70–7.65 (m, 3H, ArH), 7.57 (d, J ¼ 8.0 Hz, 1H, (100 MHz, chloroform-d) d 166.1, 151.2, 142.9, 134.2, 134.0,
ArH), 7.46–7.40 (m, 1H, ArH), 7.34–7.28 (m, 1H, ArH), 6.74 (d, J 128.1, 127.8, 127.6, 126.3, 121.3, 118.9, 21.1.
¼ 8.4 Hz, 2H, ArH), 3.74 (s, 2H, NH2). 13C NMR (100 MHz,
2-(4-Aminophenyl)-N-methylbenzo[d]thiazol-6-amine (3p).
chloroform-d) d 147.1, 146.7, 145.0, 131.3, 129.2, 125.4 (2C), Orange solid; yield 70%. 1H NMR (400 MHz, chloroform-d)
125.1, 123.6, 123.5, 123.3, 114.3 (2C), 111.4, 104.2.
d 7.82 (d, J ¼ 8.5 Hz, 2H, ArH), 7.77 (d, J ¼ 8.7 Hz, 1H, ArH), 6.98
4-(Imidazo[1,2-a]pyridin-2-yl)aniline (3i). Yellow solid; yield (s, 1H, ArH), 6.77–6.70 (m, 3H, ArH), 3.91 (s, 2H, NH2), 2.90 (s,
56%. 1H NMR (400 MHz, chloroform-d) d 8.07 (d, J ¼ 6.7 Hz, 1H, 3H, CH3). 13C NMR (100 MHz, chloroform-d) d 162.7, 147.4,
ArH), 7.76 (d, J ¼ 8.5 Hz, 2H, ArH), 7.73 (s, 1H, ArH), 7.59 (d, J ¼ 146.0, 145.8, 135.7, 127.5 (2C), 123.6, 121.8, 113.8 (2C), 113.0,
9.1 Hz, 1H, ArH), 7.15–7.09 (m, 1H, ArH), 6.77–6.70 (m, 3H, 100.8, 30.0.
ArH), 3.75 (s, 2H, NH2). 13C NMR (100 MHz, chloroform-d)
2-(4-Aminophenyl)-N,N-dimethylbenzo[d]thiazol-6-amine
d 145.4, 145.3, 144.5, 126.2 (2C), 124.3, 123.3, 123.2, 116.2, 114.2 (3q). Orange solid; yield 72%. 1H NMR (400 MHz, chloroform-d)
(2C), 111.0, 105.7.
d 7.85–7.81 (m, 3H, ArH), 7.10 (s, 1H, ArH), 6.93 (dd, J ¼ 9.0,
4-(7-Fluorobenzo[d]thiazol-2-yl)aniline (3j). Yellow solid; 2.5 Hz, 1H, ArH), 6.72 (d, J ¼ 8.6 Hz, 2H, ArH), 3.92 (s, 2H, NH2),
1
yield 69%. H NMR (400 MHz, DMSO-d6) d 7.80–7.73 (m, 3H, 3.02 (s, 6H, CH3). 13C NMR (100 MHz, chloroform-d) d 163.9,
ArH), 7.50–7.42 (m, 1H, ArH), 7.24–7.18 (m, 1H, ArH), 6.69 (d, J 148.5, 148.5, 146.2, 136.6, 128.6 (2C), 124.6, 122.5, 114.8 (2C),
¼ 8.3 Hz, 2H, ArH), 6.04 (s, 2H, NH2). 13C NMR (100 MHz, 113.2, 103.3, 41.2.
DMSO-d6) d 169.0 (d, J ¼ 1.5 Hz), 156.8 (d, J ¼ 2.5 Hz), 156.3 (d, J
2-(4-Aminophenyl)benzo[d]thiazole-6-carboxylic acid (3r).
1
¼ 246.1 Hz), 152.6, 129.1 (2C), 127.5 (d, J ¼ 7.5 Hz), 120.1 (d, J ¼ Yellow solid; yield 60%. H NMR (400 MHz, DMSO-d6) d 12.99
16.2 Hz), 119.2, 118.0 (d, J ¼ 3.3 Hz), 113.6 (2C), 109.9 (d, J ¼ (br, 1H, COOH), 8.62 (s, 1H, ArH), 8.00 (d, J ¼ 8.5 Hz, 1H, ArH),
18.7 Hz).
7.93 (d, J ¼ 8.6 Hz, 1H, ArH), 7.79 (d, J ¼ 8.3 Hz, 2H, ArH), 6.67
(d, J ¼ 8.3 Hz, 2H, ArH), 6.04 (s, 2H, NH2). 13C NMR (100 MHz,
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DMSO-d6) d 171.7, 167.0, 156.8, 152.7, 133.8, 129.1 (2C), 127.3,
126.3, 123.8, 121.3, 119.6, 113.5 (2C).
(E)-2-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)-6-
methoxyphenol (1a). Orange solid; yield 63%. Mp 175–176 C.
ꢂ
1H NMR (400 MHz, chloroform-d) d 13.44 (s, 1H, OH), 8.67 (s,
1H, –CH]N–), 8.14 (d, J ¼ 8.2 Hz, 2H, ArH), 8.07 (d, J ¼ 8.2 Hz,
1H, ArH), 7.90 (d, J ¼ 7.9 Hz, 1H, ArH), 7.52–7.48 (m, 1H, ArH),
7.41–7.36 (m, 3H, ArH), 7.06–6.99 (m, 2H, ArH), 6.93–6.87 (m,
1H, ArH), 3.94 (s, 3H, CH3). 13C NMR (100 MHz, chloroform-d)
d 166.1, 162.3, 153.1, 150.5, 149.3, 147.5, 134.0, 131.2, 127.7,
125.4, 124.2, 123.0, 122.2, 120.8, 120.6, 118.0, 117.7, 114.2, 55.2.
HRMS (ESI) m/z: calculated for C21H16N2NaO2S [M + Na]+:
383.0825, found: 383.0823.
2-(4-Aminophenyl)-N-methylbenzo[d]thiazole-6-
1
carboxamide (3s). Light yellow solid; yield 47%. H NMR (400
MHz, DMSO-d6) d 8.51 (q, J ¼ 4.2 Hz, 1H, –(C]O)NH–), 8.48 (s,
1H, ArH), 7.95–7.88 (m, 2H, ArH), 7.79 (d, J ¼ 8.4 Hz, 2H, ArH),
6.68 (d, J ¼ 8.6 Hz, 2H, ArH), 5.99 (s, 2H, NH2), 2.82 (d, J ¼
4.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d 170.4, 166.1,
155.6, 152.5, 133.6, 130.4, 129.0 (2C), 125.3, 121.1, 121.1, 119.7,
113.5 (2C), 26.4.
(E)-2-(((4-(Benzo[d]oxazol-2-yl)phenyl)imino)methyl)-6-
methoxyphenol (1b). Orange solid; yield 55%. Mp 218–219 C.
Synthesis of 4-(benzo[d]thiazol-2-yloxy)aniline (3t)
ꢂ
4-Aminophenol (2 mmol) was dissolved in DMF (5 mL) followed
by the addition of t-BuONa (2.4 mmol) and stirred at room
temperature for 1 h. Then 2a (2 mmol) and K2CO3 (2.4 mmol)
were added and stirred for another 4 h. The reaction mixture
was diluted with H2O (40 mL) and organics were extracted with
EtOAc (3ꢁ 30 mL). The combined organic layers were washed
with brine (30 mL), dried over MgSO4, ltered and concentrated
in vacuo to afford crude mixture. The crude mixture was then
puried by ash column chromatography in a mixture of
petroleum ether and ethyl acetate to yield the pure product 3t.
1H NMR (400 MHz, chloroform-d) d 13.35 (s, 1H, OH), 8.69 (s,
1H, –CH]N–), 8.31 (d, J ¼ 8.3 Hz, 2H, ArH), 7.80–7.76 (m, 1H,
ArH), 7.61–7.57 (m, 1H, ArH), 7.42 (d, J ¼ 8.2 Hz, 2H, ArH), 7.38–
7.34 (m, 2H, ArH), 7.06–7.01 (m, 2H, ArH), 6.94–6.88 (m, 1H,
ArH), 3.95 (s, 3H, CH3). 13C NMR (100 MHz, chloroform-d)
d 162.7, 161.5, 150.6, 149.9, 149.8, 147.5, 141.2, 127.9 (2C),
124.6, 124.2, 123.7, 123.0, 120.8 (2C), 119.0, 118.0, 117.8, 114.3,
109.6, 55.2. HRMS (ESI) m/z: calculated for C21H16N2NaO3 [M +
Na]+: 367.1053, found: 367.1041.
(E)-2-(((4-(1H-Benzo[d]imidazol-2-yl)phenyl)imino)methyl)-
1
Yellow oil, yield 80%. H NMR (400 MHz, chloroform-d) d 7.73
1
6-methoxyphenol (1c). Red solid; yield 36%. Mp > 220 ꢂC. H
(d, J ¼ 8.1 Hz, 1H, ArH), 7.63 (d, J ¼ 8.0 Hz, 1H, ArH), 7.40–7.34
(m, 1H, ArH), 7.25–7.21 (m, 1H, ArH), 7.12 (d, J ¼ 8.7 Hz, 2H,
ArH), 6.71 (d, J ¼ 8.7 Hz, 2H, ArH), 3.74 (s, 2H, NH2). 13C NMR
(100 MHz, chloroform-d) d 173.6, 149.4, 147.2, 145.0, 132.2,
126.2, 123.8, 121.8 (2C), 121.6, 121.3, 115.9 (2C).
NMR (400 MHz, DMSO-d6) d 13.10 (s, 1H, OH), 12.94 (s, 1H,
imidazole), 9.06 (s, 1H, –CH]N–), 8.27 (d, J ¼ 8.2 Hz, 2H, ArH),
7.68 (d, J ¼ 7.5 Hz, 1H, ArH), 7.60 (d, J ¼ 8.2 Hz, 2H, ArH), 7.54
(d, J ¼ 7.5 Hz, 1H, ArH), 7.28 (d, J ¼ 7.5 Hz, 1H, ArH), 7.23–7.20
(m, 2H, ArH), 7.15 (d, J ¼ 7.7 Hz, 1H, ArH), 6.95–6.91 (m, 1H,
ArH), 3.84 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d 163.9,
150.7, 150.6, 149.0, 147.9, 143.9, 135.0, 128.6, 127.6 (2C), 123.9,
122.6, 122.0 (2C), 121.7, 119.3, 118.8, 118.7, 115.8, 111.3, 55.9.
HRMS (ESI) m/z: calculated for C21H17N3NaO2 [M + Na]+:
366.1213, found: 366.1198.
Synthesis of 4-(benzo[d]thiazol-2-ylthio)aniline (3u)
4-Aminobenzenethiol (2 mmol) and 2a (2 mmol) was dissolved
in MeCN (5 mL) followed by the addition of K2CO3 (2.4 mmol)
and stirred at reux for 3 h. Upon completion, the reaction was
allowed to cold to room temperature. The reaction mixture was
diluted with H2O (40 mL) and organics were extracted with
EtOAc (3ꢁ 30 mL). The combined organic layers were washed
with brine (30 mL), dried over MgSO4, ltered and concentrated
in vacuo to afford crude mixture. The crude mixture was then
puried by ash column chromatography in a mixture of
petroleum ether and ethyl acetate to yield the pure product 3u.
White solid; yield 50%. 1H NMR (400 MHz, chloroform-d) d 7.84
(d, J ¼ 8.2 Hz, 1H, ArH), 7.62 (d, J ¼ 8.0 Hz, 1H, ArH), 7.50 (d, J ¼
8.4 Hz, 2H, ArH), 7.41–7.35 (m, 1H, ArH), 7.25–7.19 (m, 1H,
ArH), 6.74 (d, J ¼ 8.4 Hz, 2H, ArH), 4.02 (s, 2H, NH2). 13C NMR
(100 MHz, chloroform-d) d 172.4, 153.3, 148.0, 136.6 (2C), 134.4,
125.0, 122.8, 120.6, 119.7, 115.6, 114.9 (2C).
(E)-2-(((6-(Benzo[d]thiazol-2-yl)pyridin-3-yl)imino)methyl)-6-
1
ꢂ
methoxyphenol (1d). Yellow solid; yield 64%. Mp > 220 C. H
NMR (400 MHz, chloroform-d) d 13.00 (s, 1H, OH), 8.71 (s, 1H,
–CH]N–), 8.61 (d, J ¼ 2.4 Hz, 1H, ArH), 8.43 (d, J ¼ 8.4 Hz, 1H,
ArH), 8.09 (d, J ¼ 8.2 Hz, 1H, ArH), 7.96 (d, J ¼ 8.0 Hz, 1H, ArH),
7.74 (dd, J ¼ 8.4, 2.5 Hz, 1H, ArH), 7.54–7.49 (m, 1H, ArH), 7.44–
7.41 (m, 1H, ArH), 7.09–7.03 (m, 2H, ArH), 6.96–6.90 (m, 1H,
ArH), 3.95 (s, 3H, CH3). 13C NMR (100 MHz, chloroform-d)
d 167.5, 163.8, 153.3, 150.4, 148.7, 147.5, 144.6, 142.3, 135.2,
127.6, 125.3, 124.7, 123.2, 122.5, 121.0, 120.3, 118.1, 117.9,
114.7, 55.2. HRMS (ESI) m/z: calculated for C20H15N3NaO2S [M +
Na]+: 384.0777, found: 384.0786.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-phenylmethanimine
(1h). Yellow solid; yield 22%. Mp 200–202 ꢂC. 1H NMR (400
MHz, chloroform-d) d 8.51 (s, 1H, –CH]N–), 8.14 (d, J ¼ 8.5 Hz,
2H, ArH), 8.08 (d, J ¼ 8.2 Hz, 1H, ArH), 7.96–7.93 (m, 2H, ArH),
7.91 (d, J ¼ 8.0 Hz, 1H, ArH), 7.52–7.47 (m, 4H, ArH), 7.41–7.36
(m, 1H, ArH), 7.32 (d, J ¼ 8.4 Hz, 2H, ArH). 13C NMR (100 MHz,
chloroform-d) d 166.6, 160.1, 153.4, 153.2, 134.9, 134.0, 130.8,
130.2, 128.0 (2C), 127.8 (2C), 127.6 (2C), 125.3, 124.1, 122.1,
120.6, 120.5 (2C). HRMS (ESI) m/z: calculated for C20H14N2NaS
[M + Na]+: 337.0770, found: 337.0752.
General procedure for the synthesis of compounds 1a–1d, 1h–
1u and 4a–4p
The catalytic amount of acetic acid was added to a solution of
3a–3d, 3f–3u (0.44 mmol) and the corresponding aromatic
aldehyde (0.53 mmol) in MeOH (4 mL), the mixture was stirred
at reux for 2 h. The resulting precipitate was isolated by
suction ltration and washed with MeOH (8 mL) to give the pure
products 1a–1d, 1h–1u and 4a–4p.
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7.92–7.90 (m, 2H, ArH), 7.54–7.46 (m, 2H, ArH), 7.40–7.37 (m,
1H, ArH), 7.27 (d, J ¼ 8.3 Hz, 2H, ArH), 6.97 (s, 1H, ArH). 13C
NMR (100 MHz, chloroform-d) d 166.6, 153.5, 153.2, 151.7,
146.2, 143.5, 134.0, 130.1, 127.5 (2C), 125.3, 124.9, 124.1, 122.0,
120.6, 120.4 (2C), 106.8. HRMS (ESI) m/z: calculated for
(E)-2-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)phenol
(1i). Light yellow solid; yield 60%. Mp 205–207 ꢂC. 1H NMR (400
MHz, DMSO-d6) d 12.81 (s, 1H, OH), 9.06 (s, 1H, –CH]N–),
8.19–8.17 (m, 3H, ArH), 8.07 (d, J ¼ 8.1 Hz, 1H, ArH), 7.71 (d, J ¼
7.5 Hz, 1H, ArH), 7.60 (d, J ¼ 8.4 Hz, 2H, ArH), 7.58–7.54 (m, 1H,
ArH), 7.47–7.44 (m, 2H, ArH), 7.03–6.99 (m, 2H, ArH). 13C NMR
(100 MHz, DMSO-d6) d 166.6, 164.3, 160.3, 153.6, 150.7, 134.5,
133.7, 132.6, 131.1, 128.4 (2C), 126.7, 125.5, 122.8, 122.4 (2C),
122.3, 119.3, 119.3, 116.7. HRMS (ESI) m/z: calculated for
C
18H13N2OS [M + H]+: 305.0743, found: 305.0755.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-yl)
methanimine (1o). Light yellow solid; yield 58%. Mp 162–
1
ꢂ
164 C. H NMR (400 MHz, chloroform-d) d 8.51 (s, 1H, –CH]
N–), 8.12 (d, J ¼ 8.2 Hz, 2H, ArH), 8.07 (d, J ¼ 8.1 Hz, 1H, ArH),
7.90 (d, J ¼ 7.9 Hz, 1H, ArH), 7.85 (s, 1H, ArH), 7.71 (d, J ¼
5.0 Hz, 1H, ArH), 7.51–7.47 (m, 1H, ArH), 7.41–7.36 (m, 2H,
ArH), 7.29 (d, J ¼ 8.2 Hz, 2H, ArH). 13C NMR (100 MHz, chlo-
roform-d) d 167.6, 155.0, 154.5, 154.3, 140.6, 135.1, 131.2, 130.9,
128.6 (2C), 126.9, 126.3, 126.0, 125.1, 123.1, 121.6, 121.5 (2C).
HRMS (ESI) m/z: calculated for C18H13N2S2 [M + H]+: 321.0515,
found: 321.0513.
C
20H15N2OS [M + H]+: 331.0900, found: 331.0902.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(3-methoxyphenyl)
methanimine (1j). Light yellow solid; yield 45%. Mp 118–119 ꢂC.
1H NMR (400 MHz, DMSO-d6) d 8.67 (s, 1H, –CH]N–), 8.15–8.13
(m, 3H, ArH), 8.06 (d, J ¼ 8.1 Hz, 1H, ArH), 7.57–7.54 (m, 3H,
ArH), 7.48–7.42 (m, 4H, ArH), 7.14 (d, J ¼ 8.2 Hz, 1H, ArH), 3.84
(s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d 166.8, 161.8,
159.5, 153.8, 153.6, 137.2, 134.4, 130.4, 130.0, 128.3 (2C), 126.6,
125.4, 122.7, 122.3, 122.0 (2C), 121.9, 118.1, 112.7, 55.2. HRMS
(ESI) m/z: calculated for C21H17N2NaOS [M + H]+: 345.1056,
found: 345.1071.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(1H-pyrrol-3-yl)
methanimine (1p). White solid; yield 55%. Mp > 220 ꢂC. 1H
NMR (400 MHz, DMSO-d6) d 11.41 (s, 1H, pyrrole), 8.48 (s, 1H,
–CH]N–), 8.13 (d, J ¼ 8.0 Hz, 1H, ArH), 8.08 (d, J ¼ 8.1 Hz, 2H,
ArH), 8.04 (d, J ¼ 8.1 Hz, 1H, ArH), 7.56–7.52 (m, 1H, ArH), 7.47–
7.43 (m, 2H, ArH), 7.30 (d, J ¼ 8.1 Hz, 2H, ArH), 6.91 (s, 1H, ArH),
6.59 (s, 1H, ArH). 13C NMR (100 MHz, DMSO-d6) d 167.1, 156.4,
155.6, 153.6, 134.3, 129.0, 128.2 (2C), 126.6, 125.3, 125.3, 123.0,
122.6, 122.2, 121.7 (2C), 120.5, 106.2. HRMS (ESI) m/z: calcu-
lated for C18H14N3S [M + H]+: 304.0903, found: 304.0894.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiophen-2-yl)
(E)-3-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)
benzene-1,2-diol (1k). Red solid; yield 26%. Mp > 220 ꢂC. 1H
NMR (400 MHz, DMSO-d6) d 9.01 (s, 1H, –CH]N–), 8.19–8.13
(m, 3H, ArH), 8.07 (d, J ¼ 8.1 Hz, 1H, ArH), 7.59 (d, J ¼ 8.2 Hz,
2H, ArH), 7.57–7.54 (m, 1H, ArH), 7.49–7.45 (m, 1H, ArH), 7.15
(d, J ¼ 6.9 Hz, 1H, ArH), 6.99 (d, J ¼ 6.8 Hz, 1H, ArH), 6.84–6.80
(m,1H, ArH). 13C NMR (100 MHz, DMSO-d6) d 166.6, 164.7,
153.6, 150.5, 149.4, 145.6, 134.5, 131.1, 128.4 (2C), 126.7, 125.5,
122.9, 122.8, 122.4 (2C), 122.3, 119.4, 119.3, 118.9. HRMS (ESI)
m/z: calculated for C20H14N2NaO2S [M + Na]+: 369.0668, found:
369.0654.
methanimine (1q). Light green solid; yield 57%. Mp 184–185 ꢂC.
1H NMR (400 MHz, chloroform-d) d 8.62 (s, 1H, –CH]N–), 8.12
(d, J ¼ 8.1 Hz, 2H, ArH), 8.07 (d, J ¼ 8.2 Hz, 1H, ArH), 7.90 (d, J ¼
8.0 Hz, 1H, ArH), 7.59–7.46 (m, 3H, ArH), 7.41–7.35 (m, 1H,
(E)-5-(((4-(Benzo[d]thiazol-2-yl)phenyl)imino)methyl)-2-
methoxyphenol (1l). Yellow solid; yield 54%. Mp 214–215 ꢂC. 1H
NMR (400 MHz, DMSO-d6) d 9.40 (br, 1H, OH), 8.52 (s, 1H,
–CH]N–), 8.14–8.12 (m, 3H, ArH), 8.05 (d, J ¼ 8.1 Hz, 1H, ArH),
7.57–7.52 (m, 1H, ArH), 7.47–7.44 (m, 2H, ArH), 7.39–7.35 (m,
3H, ArH), 7.06 (d, J ¼ 8.3 Hz, 1H, ArH), 3.85 (s, 3H, CH3). 13C
NMR (100 MHz, DMSO-d6) d 166.9, 161.3, 154.3, 153.6, 151.3,
146.8, 134.4, 129.9, 128.9, 128.2 (2C), 126.6, 125.4, 122.9, 122.7,
122.3, 121.9 (2C), 113.7, 111.6, 55.6. HRMS (ESI) m/z: calculated
for C21H17N2O2S [M + H]+: 361.1005, found: 361.0986.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(pyridin-3-yl)
ArH), 7.32 (d, J ¼ 8.2 Hz, 2H, ArH), 7.18–7.14 (m, 1H, ArH). 13
C
NMR (100 MHz, chloroform-d) d 167.6, 154.2, 153.7, 153.7,
142.6, 135.0, 133.0, 131.3, 131.1, 128.6 (2C), 127.9, 126.4, 125.1,
123.1, 121.7 (2C), 121.6. HRMS (ESI) m/z: calculated for
C
18H12N2NaS2 [M + Na]+: 343.0334, found: 343.0333.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(5-nitrothiophen-3-
ꢂ
yl)methanimine (1r). Yellow solid; yield 74%. Mp 213–215 C.
1H NMR (400 MHz, DMSO-d6) d 8.70 (s, 1H, –CH]N–), 8.60 (s,
1H, ArH), 8.42 (s, 1H, ArH), 8.16–8.14 (m, 3H, ArH), 8.06 (d, J ¼
8.2 Hz, 1H, ArH), 7.57–7.53 (m, 1H, ArH), 7.48–7.43 (m, 3H,
ArH). 13C NMR (100 MHz, DMSO-d6) d 166.7, 155.4, 153.6, 153.1,
152.1, 138.7, 138.1, 134.4, 130.8, 128.3 (2C), 127.2, 126.7, 125.5,
122.8, 122.3, 122.1 (2C). HRMS (ESI) m/z: calculated for
methanimine (1m). Yellow solid; yield 17%. Mp 151–153 ꢂC. 1H
NMR (400 MHz, chloroform-d) d 9.05 (s, 1H, –CH]N–), 8.73 (d, J
¼ 4.7 Hz, 1H, ArH), 8.55 (s, 1H, ArH), 8.32 (d, J ¼ 8.0 Hz, 1H,
ArH), 8.15 (d, J ¼ 8.1 Hz, 2H, ArH), 8.08 (d, J ¼ 8.2 Hz, 1H, ArH),
7.91 (d, J ¼ 8.0 Hz, 1H, ArH), 7.52–7.48 (m, 1H, ArH), 7.46–7.42
(m, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.33 (d, J ¼ 8.1 Hz, 2H,
ArH). 13C NMR (100 MHz, chloroform-d) d 167.3, 158.0, 154.2,
153.6, 152.3, 151.1, 135.1, 135.0, 131.8, 131.6, 128.6 (2C), 126.4,
125.2, 123.9, 123.2, 121.6, 121.5 (2C). HRMS (ESI) m/z: calcu-
lated for C19H14N3S [M + H]+: 316.0903, found: 316.0893.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(furan-3-yl)
C
18H12N3O2S2 [M + H]+: 366.0365, found: 366.0360.
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiazol-4-yl)
methanimine (1s). Golden solid; yield 69%. Mp 173–174 ꢂC. 1H
NMR (400 MHz, chloroform-d) d 8.93 (s, 1H, ArH), 8.72 (s, 1H,
–CH]N–), 8.17–8.10 (m, 3H, ArH), 8.07 (d, J ¼ 8.2 Hz, 1H, ArH),
7.91 (d, J ¼ 8.0 Hz, 1H, ArH), 7.53–7.47 (m, 1H, ArH), 7.42–7.36
(m, 3H, ArH). 13C NMR (100 MHz, chloroform-d) d 167.5, 154.5,
154.2, 154.1, 153.7, 153.6, 135.1, 131.7, 128.6 (2C), 126.4, 125.2,
123.2, 122.3, 121.7 (2C), 121.6. HRMS (ESI) m/z: calculated for
methanimine (1n). Light yellow solid; yield 60%. Mp 170–
17H11N3NaS2 [M + Na]+: 344.0287, found: 344.0275.
1
ꢂ
C
172 C. H NMR (400 MHz, chloroform-d) d 8.44 (s, 1H, –CH]
N–), 8.12 (d, J ¼ 8.3 Hz, 2H, ArH), 8.07 (d, J ¼ 8.1 Hz, 1H, ArH),
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiazol-5-yl)
methanimine (1t). Yellow solid; yield 72%. Mp 180–182 C. H
1
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NMR (400 MHz, chloroform-d) d 8.95 (s, 1H, ArH), 8.71 (s, 1H, 144.5, 139.8, 130.5, 129.2, 125.8 (2C), 125.7, 124.9, 124.5, 123.6,
–CH]N–), 8.29 (s, 1H, ArH), 8.13 (d, J ¼ 8.2 Hz, 2H, ArH), 8.07 120.3 (2C), 116.5, 111.4, 106.9. HRMS (ESI) m/z: calculated for
(d, J ¼ 8.2 Hz, 1H, ArH), 7.91 (d, J ¼ 8.0 Hz, 1H, ArH), 7.53–7.47
(m, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.32 (d, J ¼ 8.2 Hz, 2H,
C
18H14N3S [M + H]+: 304.0903, found: 304.0897.
(E)-N-(4-(7-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-
ArH). 13C NMR (100 MHz, chloroform-d) d 167.3, 157.0, 154.2, yl)methanimine (4e). Light yellow solid; yield 38%. Mp 166–
1
ꢂ
153.0, 151.3, 147.8, 138.3, 135.0, 131.9, 128.6 (2C), 126.4, 125.2, 167 C. H NMR (400 MHz, chloroform-d) d 8.50 (s, 1H, –CH]
123.2, 121.7, 121.6 (3C). HRMS (ESI) m/z: calculated for N–), 8.12 (d, J ¼ 8.5 Hz, 2H, ArH), 7.88–7.85 (m, 2H, ArH), 7.71
C
17H12N3S2 [M + H]+: 322.0467, found: 322.0471.
(d, J ¼ 5.1 Hz, 1H, ArH), 7.48–7.40 (m, 2H, ArH), 7.30 (d, J ¼
(E)-N-(4-(Benzo[d]thiazol-2-yl)phenyl)-1-(thiazol-2-yl)
8.5 Hz, 2H, ArH), 7.13–7.09 (m, 1H, ArH). 13C NMR (100 MHz,
methanimine (1u). Light green solid; yield 64%. Mp 193–195 ꢂC. chloroform-d) d 167.6 (d, J ¼ 1.7 Hz), 156.1 (d, J ¼ 250.1 Hz),
1H NMR (400 MHz, chloroform-d) d 8.75 (s, 1H, –CH]N–), 8.15 156.0 (d, J ¼ 2.5 Hz), 154.2, 153.8, 139.5, 130.2, 129.5, 127.7 (2C),
(d, J ¼ 8.1 Hz, 2H, ArH), 8.08 (d, J ¼ 8.2 Hz, 1H, ArH), 8.03 (d, J ¼ 126.2 (d, J ¼ 7.3 Hz), 125.9, 124.9, 121.1 (d, J ¼ 16.5 Hz), 120.5
3.1 Hz, 1H, ArH), 7.91 (d, J ¼ 8.0 Hz, 1H, ArH), 7.55 (d, J ¼ (2C), 117.9 (d, J ¼ 3.5 Hz), 109.5 (d, J ¼ 18.7 Hz). HRMS (ESI) m/z:
3.0 Hz, 1H, ArH), 7.53–7.47 (m, 1H, ArH), 7.42–7.36 (m, 3H, calculated for C18H12FN2S2 [M + H]+: 339.0420, found: 339.0418.
ArH). 13C NMR (100 MHz, chloroform-d) d 167.2, 166.9, 154.2,
(E)-N-(4-(6-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-
153.8, 152.1, 144.8, 135.1, 132.5, 128.7 (2C), 126.4, 125.3, 123.2, yl)methanimine (4f). Light yellow solid; yield 52%. Mp 187–
1
ꢂ
123.0, 121.9 (2C), 121.7. HRMS (ESI) m/z: calculated for 189 C. H NMR (400 MHz, chloroform-d) d 8.50 (s, 1H, –CH]
C
17H11N3NaS2 [M + Na]+: 344.0287, found: 344.0271.
(E)-N-(4-(Thiazolo[4,5-b]pyridin-2-yl)phenyl)-1-(thiophen-3-
N–), 8.08 (d, J ¼ 8.3 Hz, 2H, ArH), 8.00 (dd, J ¼ 8.8, 4.8 Hz, 1H,
ArH), 7.86 (s, 1H, ArH), 7.70 (d, J ¼ 4.9 Hz, 1H, ArH), 7.60–7.56
yl)methanimine (4a). Light yellow solid; yield 32%. Mp 176– (m, 1H, ArH), 7.43–7.39 (m, 1H, ArH), 7.29 (d, J ¼ 8.3 Hz, 2H,
178 C. H NMR (400 MHz, DMSO-d6) d 8.72–8.68 (m, 2H, ArH ArH), 7.25–7.19 (m, 1H, ArH). 13C NMR (100 MHz, chloroform-d)
1
ꢂ
and –CH]N–), 8.65 (d, J ¼ 8.0 Hz, 1H, ArH), 8.27 (s, 1H, ArH), d 166.3 (d, J ¼ 3.3 Hz), 159.4 (d, J ¼ 245.7 Hz), 154.1, 153.5, 149.8
8.20 (d, J ¼ 8.1 Hz, 2H, ArH), 7.73–7.69 (m, 2H, ArH), 7.50–7.45 (d, J ¼ 1.8 Hz), 139.5, 135.0 (d, J ¼ 11.1 Hz), 130.1, 129.8, 127.4
(m, 4.7 Hz, 1H, ArH), 7.42 (d, J ¼ 8.1 Hz, 2H, ArH). 13C NMR (100 (2C), 125.9, 124.9, 122.9 (d, J ¼ 9.2 Hz), 120.5 (2C), 113.9 (d, J ¼
MHz, DMSO-d6) d 170.1, 164.2, 156.5, 154.9, 148.4, 140.3, 133.1, 24.6 Hz), 106.8 (d, J ¼ 26.8 Hz). HRMS (ESI) m/z: calculated for
131.8, 129.7, 128.4 (2C), 128.3, 128.0, 125.4, 122.0 (2C), 120.3.
HRMS (ESI) m/z: calculated for C17H11N3NaS2 [M + Na]+:
344.0287, found: 344.0289.
C
18H12FN2S2 [M + H]+: 339.0420, found: 339.0431.
(E)-N-(4-(5-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-
yl)methanimine (4g). Khaki solid; yield 51%. Mp 183–185 ꢂC. 1H
NMR (400 MHz, chloroform-d) d 8.49 (s, 1H, –CH]N–), 8.09 (d, J
(E)-N-(4-(4,5,6,7-Tetrahydrobenzo[d]thiazol-2-yl)phenyl)-1-
(thiophen-3-yl) methanimine (4b). Khaki solid; yield 60%. Mp ¼ 8.5 Hz, 2H, ArH), 7.85 (dd, J ¼ 3.0, 1.2 Hz, 1H, ArH), 7.81 (dd, J
150–151 ꢂC. 1H NMR (400 MHz, chloroform-d) d 8.49 (s, 1H, ¼ 8.8, 5.1 Hz, 1H, ArH), 7.73 (dd, J ¼ 9.6, 2.5 Hz, 1H, ArH), 7.70
–CH]N–), 7.91 (d, J ¼ 8.2 Hz, 2H, ArH), 7.82 (s, 1H, ArH), 7.69 (d, J ¼ 5.1 Hz, 1H, ArH), 7.40 (dd, J ¼ 5.1, 2.9 Hz, 1H, ArH), 7.28
(d, J ¼ 5.1 Hz, 1H, ArH), 7.41–7.37 (m, 1H, ArH), 7.22 (d, J ¼ (d, J ¼ 8.5 Hz, 2H, ArH), 7.17–7.13 (m, 1H, ArH). 13C NMR (100
8.2 Hz, 2H, ArH), 2.87–2.79 (m, 4H, CH2), 1.94–1.86 (m, 4H, MHz, chloroform-d) d 169.0, 160.9 (d, J ¼ 243.2 Hz), 154.1, 154.1
CH2). 13C NMR (100 MHz, chloroform-d) d 163.1, 153.5, 151.9, (d, J ¼ 12.1 Hz), 153.6, 139.5, 130.1, 129.8, 129.3 (d, J ¼ 1.9 Hz),
150.4, 139.7, 130.8, 129.6, 128.0, 126.1 (2C), 125.8, 124.9, 120.3 127.5 (2C), 125.9, 124.9, 121.2 (d, J ¼ 9.9 Hz), 120.5 (2C), 112.7
(2C), 25.9, 22.7, 22.4, 22.0. HRMS (ESI) m/z: calculated for (d, J ¼ 25.0 Hz), 108.2 (d, J ¼ 23.6 Hz). HRMS (ESI) m/z: calcu-
C
18H17N2S2+[M + H]+: 325.0828, found: 325.0814.
lated for C18H12FN2S2 [M + H]+: 339.0420, found: 339.0423.
(E)-N-(4-(Benzo[d]imidazo[2,1-b]thiazol-2-yl)phenyl)-1-
(E)-N-(4-(4-Fluorobenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-3-
ꢂ
(thiophen-3-yl) methanimine (4c). Khaki solid; yield 38%. Mp yl)methanimine (4h). Yellow solid; yield 36%. Mp 153–154 C.
162–163 ꢂC. 1H NMR (400 MHz, chloroform-d) d 8.54 (s, 1H, 1H NMR (400 MHz, chloroform-d) d 8.50 (s, 1H, –CH]N–), 8.15
–CH]N–), 7.98 (s, 1H, ArH), 7.90 (d, J ¼ 8.1 Hz, 2H, ArH), 7.81 (d, J ¼ 8.6 Hz, 2H, ArH), 7.86 (dd, J ¼ 2.9, 1.1 Hz, 1H, ArH), 7.70
(s, 1H, ArH), 7.73–7.68 (m, 2H, ArH), 7.62 (d, J ¼ 8.1 Hz, 1H, (dd, J ¼ 5.1, 0.9 Hz, 1H, ArH), 7.66 (dd, J ¼ 8.0, 0.8 Hz, 1H, ArH),
ArH), 7.49–7.43 (m, 1H, ArH), 7.40–7.32 (m, 2H, ArH), 7.28 (d, J 7.41 (dd, J ¼ 5.0, 2.6 Hz, 1H, ArH), 7.36–7.29 (m, 1H, ArH), 7.28
¼ 8.1 Hz, 2H, ArH). 13C NMR (100 MHz, chloroform-d) d 153.0, (d, J ¼ 8.6 Hz, 2H, ArH), 7.19 (ddd, J ¼ 10.4, 8.1, 0.8 Hz, 1H,
150.1, 147.1, 146.4, 139.9, 131.1, 130.6, 129.3, 129.2, 125.7, ArH). 13C NMR (100 MHz, chloroform-d) d 167.1, 154.8 (d, J ¼
125.2, 125.0 (2C), 124.9, 123.8, 123.4, 120.3 (2C), 111.6, 105.6. 257.2 Hz), 154.2, 153.7, 142.0 (d, J ¼ 13.4 Hz), 139.5, 136.6 (d, J ¼
HRMS (ESI) m/z: calculated for C20H14N3S2 [M + H]+: 360.0624, 3.6 Hz), 130.1, 129.6, 127.8 (2C), 125.9, 124.9, 124.7 (d, J ¼ 7.1
found: 360.0609.
Hz), 120.5 (2C), 116.3 (d, J ¼ 4.3 Hz), 111.0 (d, J ¼ 18.0 Hz).
(E)-N-(4-(Imidazo[1,2-a]pyridin-2-yl)phenyl)-1-(thiophen-3-
HRMS (ESI) m/z: calculated for C18H12FN2S2 [M + H]+: 339.0420,
yl)methanimine (4d). Light yellow solid; yield 34%. Mp 203– found: 339.0417.
1
ꢂ
205 C. H NMR (400 MHz, chloroform-d) d 8.53 (s, 1H, –CH]
(E)-N-(4-(6-Ethoxybenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-
N–), 8.10 (d, J ¼ 6.8 Hz, 1H, ArH), 7.98 (d, J ¼ 8.5 Hz, 2H, ArH), 3-yl)methanimine (4i). Yellow solid; yield 32%. Mp 181–183 ꢂC.
7.85 (s, 1H, ArH), 7.80 (s, 1H, ArH), 7.70 (d, J ¼ 5.1 Hz, 1H, ArH), 1H NMR (400 MHz, chloroform-d) d 8.51 (s, 1H, –CH]N–), 8.06
7.62 (d, J ¼ 9.1 Hz, 1H, ArH), 7.40–7.36 (m, 1H, ArH), 7.28 (d, J ¼ (d, J ¼ 8.2 Hz, 2H, ArH), 7.93 (d, J ¼ 8.9 Hz, 1H, ArH), 7.85 (s, 1H,
8.3 Hz, 2H, ArH), 7.19–7.13 (m, 1H, ArH), 6.79–6.74 (m, 1H, ArH), 7.70 (d, J ¼ 4.8 Hz, 1H, ArH), 7.41 (s, 1H, ArH), 7.34 (s, 1H,
ArH). 13C NMR (100 MHz, chloroform-d) d 153.1, 150.6, 144.7, ArH), 7.29 (d, J ¼ 8.2 Hz, 2H, ArH), 7.08 (d, J ¼ 9.4 Hz, 1H, ArH),
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4.11 (q, J ¼ 6.9 Hz, 2H, CH2), 1.47 (t, J ¼ 6.9 Hz, 3H, CH3). 13C 125.4, 122.2, 121.9 (2C), 121.6, 26.4. HRMS (ESI) m/z: calculated
NMR (100 MHz, chloroform-d) d 164.0, 156.0, 153.9, 152.9, for C20H15N3NaOS2 [M + Na]+: 400.0549, found: 400.0565.
147.7, 139.6, 135.3, 130.3, 129.9, 127.2 (2C), 125.9, 124.9, 122.5,
(E)-N-(4-(Benzo[d]thiazol-2-yloxy)phenyl)-1-(thiophen-3-yl)
120.4 (2C), 115.0, 103.9, 63.1, 13.8. HRMS (ESI) m/z: calculated methanimine (4o). White solid; yield 18%. Mp 95–97 ꢂC. 1H
for C20H16N2NaOS2 [M + Na]+: 387.0596, found: 387.0613.
NMR (400 MHz, DMSO-d6) d 8.66 (s, 1H, –CH]N–), 8.21 (s, 1H,
ArH), 7.94 (d, J ¼ 8.0 Hz, 1H, ArH), 7.73–7.67 (m, 2H, ArH), 7.66–
(E)-N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-1-(thiophen-
3-yl)methanimine (4j). Golden solid; yield 37%. Mp 191–192 ꢂC. 7.61 (m, 1H, ArH), 7.49 (d, J ¼ 8.4 Hz, 2H, ArH), 7.46–7.41 (m,
1H NMR (400 MHz, chloroform-d) d 8.50 (s, 1H, –CH]N–), 8.10 1H, ArH), 7.38–7.30 (m, 3H, ArH). 13C NMR (100 MHz, DMSO-d6)
(d, J ¼ 8.3 Hz, 2H, ArH), 7.94 (d, J ¼ 8.3 Hz, 1H, ArH), 7.85 (s, 1H, d 171.9, 155.8, 152.1, 149.9, 148.6, 140.4, 132.4, 131.8, 127.8,
ArH), 7.72–7.68 (m, 2H, ArH), 7.43–7.38 (m, 1H, ArH), 7.32–7.27 126.5, 125.4, 124.2, 122.4 (2C), 122.2, 121.7 (2C), 121.1. HRMS
(m, 3H, ArH), 2.50 (s, 3H, CH3). 13C NMR (100 MHz, chloroform- (ESI) m/z: calculated for C18H13N2OS2 [M + H]+: 337.0464, found:
d) d 166.6, 155.0, 154.2, 152.3, 140.6, 135.3, 135.2, 131.3, 131.0, 337.0474.
128.4 (2C), 127.9, 126.9, 125.9, 122.6, 121.5 (2C), 121.4, 21.6.
(E)-N-(4-(Benzo[d]thiazol-2-ylthio)phenyl)-1-(thiophen-3-yl)
1
HRMS (ESI) m/z: calculated for C19H15N2S2 [M + H]+: 335.0671, methanimine (4p). White solid; yield 20%. Mp 109–111 C. H
ꢂ
found: 335.0678.
NMR (400 MHz, DMSO-d6) d 8.66 (s, 1H, –CH]N–), 8.27 (s, 1H,
ArH), 7.94 (d, J ¼ 8.0 Hz, 1H, ArH), 7.87–7.80 (m, 3H, ArH), 7.72–
(E)-N-Methyl-2-(4-((thiophen-3-ylmethylene)amino)phenyl)
benzo[d]thiazol-6-amine (4k). Yellow solid; yield 59%. Mp 200– 7.68 (m, 1H, ArH), 7.67–7.63 (m, 1H, ArH), 7.48–7.43 (m, 1H,
1
201 ꢂC. H NMR (400 MHz, DMSO-d6) d 8.67 (s, 1H, –CH]N–), ArH), 7.41–7.31 (m, 3H, ArH). 13C NMR (100 MHz, DMSO-d6)
8.23 (s, 1H, ArH), 7.99 (d, J ¼ 8.2 Hz, 2H, ArH), 7.72 (d, J ¼ d 169.7, 156.8, 154.0, 153.5, 140.2, 136.6 (2C), 134.8, 133.1,
8.8 Hz, 1H, ArH), 7.70–7.68 (m, 1H, ArH), 7.65–7.64 (m, 1H, 127.9, 126.4, 125.4, 125.1, 124.4, 122.7 (2C), 121.7, 121.3. HRMS
ArH), 7.35 (d, J ¼ 8.2 Hz, 2H, ArH), 7.04 (s, 1H, ArH), 6.81 (d, J ¼ (ESI) m/z: calculated for C18H13N2S3 [M + H]+: 353.0235, found:
8.8 Hz, 1H, ArH), 6.11 (q, J ¼ 5.0 Hz, 1H, NH), 2.75 (d, J ¼ 4.9 Hz, 353.0232.
3H, CH3). 13C NMR (100 MHz, DMSO-d6) d 160.0, 155.7, 152.9,
148.4, 145.0, 140.4, 136.7, 132.6, 131.0, 127.8, 127.4 (2C), 125.4,
Synthesis of (E)-2-((benzo[d]thiazol-2-ylimino)methyl)-6-
methoxyphenol (1e)
122.9, 121.8 (2C), 114.3, 100.3, 29.9. HRMS (ESI) m/z: calculated
for C19H16N3S2 [M + H]+: 350.0780, found: 350.0757.
(E)-N,N-Dimethyl-2-(4-((thiophen-3-ylmethylene)amino)
The catalytic amount of acetic acid was added to a solution of 3e
phenyl)benzo[d]thiazol-6-amine (4l). Orange solid; yield 60%. (0.5 mmol) and 2-hydroxy-3-methoxybenzaldehyde (0.6 mmol)
Mp 177–178 ꢂC. 1H NMR (400 MHz, DMSO-d6) d 8.68 (s, 1H, in MeOH (4 mL), the mixture was stirred at reux for 2 h. The
–CH]N–), 8.24 (s, 1H, ArH), 8.02 (d, J ¼ 8.6 Hz, 2H, ArH), 7.83 resulting precipitate was isolated by suction ltration and
(d, J ¼ 9.1 Hz, 1H, ArH), 7.71–7.69 (m, 1H, ArH), 7.65–7.64 (m, washed with MeOH to give the pure product 1e. Orange solid;
1
ꢂ
1H, ArH), 7.36 (d, J ¼ 8.5 Hz, 2H, ArH), 7.32 (s, 1H, ArH), 7.01 (d, yield 32%. Mp 170–172 C. H NMR (400 MHz, chloroform-d)
J ¼ 9.0 Hz, 1H, ArH), 3.00 (s, 6H, CH3). 13C NMR (100 MHz, d 12.44 (s, 1H, OH), 9.26 (s, 1H, –CH]N–), 7.96 (d, J ¼ 8.2 Hz,
DMSO-d6) d 161.1, 155.8, 153.1, 148.7, 145.3, 140.4, 136.6, 132.7, 1H, ArH), 7.83 (d, J ¼ 8.0 Hz, 1H, ArH), 7.51–7.45 (m, 1H, ArH),
130.8, 127.9, 127.5 (2C), 125.4, 122.8, 1218 (2C), 113.3, 103.1, 7.39–7.33 (m, 1H, ArH), 7.13 (d, J ¼ 7.9 Hz, 1H, ArH), 7.06 (d, J ¼
40.5. HRMS (ESI) m/z: calculated for C20H18N3S2+[M + H]+: 8.0 Hz, 1H, ArH), 6.95–6.89 (m, 1H, ArH), 3.93 (s, 3H, –CH3). 13
C
364.0937, found: 364.0948.
NMR (100 MHz, chloroform-d) d 167.9, 166.4, 151.1, 150.5,
147.5, 133.7, 125.7, 124.3, 124.2, 122.0, 120.7, 118.3, 117.4,
(E)-2-(4-((Thiophen-3-ylmethylene)amino)phenyl)benzo[d]
thiazole-6-carboxylic acid (4m). Yellow solid; yield 86%. Mp: 115.8, 55.3. HRMS (ESI) m/z: calculated for C15H12N2NaO2S [M +
>220 ꢂC. 1H NMR (400 MHz, DMSO-d6) d 13.16 (br, 1H, COOH), Na]+: 307.0512, found: 307.0509.
8.77 (s, 1H, –CH]N–), 8.68 (s, 1H, ArH), 8.26 (s, 1H, ArH), 8.15
(d, J ¼ 8.1 Hz, 2H, ArH), 8.12–8.06 (m, 2H, ArH), 7.71–7.69 (m,
Synthesis of 2-(((4-(benzo[d]thiazol-2-yl)phenyl)amino)
methyl)-6-methoxy-phenol (1f)
1H, ArH), 7.66–7.64 (m, 1H, ArH), 7.40 (d, J ¼ 8.1 Hz, 2H, ArH).
13C NMR (100 MHz, DMSO-d6) d 170.4, 166.9, 156.4, 156.4,
154.6, 140.3, 134.5, 133.1, 129.8, 128.6 (2C), 127.9, 127.5, 127.5, NaBH4 (0.84 mmol) was added to a suspension of 1a (0.42
125.4, 124.3, 122.4, 121.9 (2C). HRMS (ESI) m/z: calculated for mmol) in MeOH (6 mL) and stirred at room temperature for 1 h.
C
19H13N2O2S2 [M + H]+: 365.0413, found: 365.0428.
Upon completion, MeOH was removed under reduced pressure.
(E)-N-Methyl-2-(4-((thiophen-3-ylmethylene)amino)phenyl)
The crude mixture was then puried by ash column chroma-
benzo[d]thiazole-6- carboxamide (4n). Yellow solid; yield 72%. tography in a mixture of petroleum ether and ethyl acetate to
Mp: >220 ꢂC. 1H NMR (400 MHz, DMSO-d6) d 8.69 (s, 1H, –CH] yield the pure product 1f. Light yellow solid; yield 66%. Mp 187–
N–), 8.61–8.55 (m, 2H, ArH and –(C]O)NH–), 8.26 (s, 1H, ArH), 188 ꢂC. 1H NMR (400 MHz, DMSO-d6) d 8.79 (s, 1H, OH), 8.00 (d,
8.15 (d, J ¼ 8.5 Hz, 2H, ArH), 8.09 (d, J ¼ 8.5 Hz, 1H, ArH), 7.98 J ¼ 7.4 Hz, 1H, ArH), 7.89 (d, J ¼ 8.1 Hz, 1H, ArH), 7.78 (d, J ¼
(d, J ¼ 8.5 Hz, 1H, ArH), 7.72–7.68 (m, 1H, ArH), 7.66–7.65 (m, 8.4 Hz, 2H, ArH), 7.46–7.43 (m, 1H, ArH), 7.35–7.31 (m, 1H,
1H, ArH), 7.40 (d, J ¼ 8.5 Hz, 2H, ArH), 2.84 (d, J ¼ 4.4 Hz, 3H, ArH), 6.88–6.84 (m, 2H, ArH and NH), 6.82–6.80 (m, 1H, ArH),
CH3). 13C NMR (100 MHz, DMSO-d6) d 169.2, 166.0, 156.4, 155.2, 6.73–6.68 (m, 3H, ArH), 4.30 (d, J ¼ 5.9 Hz, 2H, CH2), 3.80 (s, 3H,
154.5, 140.3, 134.4, 133.0, 131.5, 129.9, 128.5 (2C), 127.9, 125.6, CH3). 13C NMR (100 MHz, DMSO-d6) d 168.0, 153.9, 151.6, 147.3,
143.8, 133.7, 128.6, 126.2, 125.5, 124.3, 121.8, 121.7, 120.0,
43308 | RSC Adv., 2020, 10, 43299–43311
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120.0, 118.6, 111.9, 110.4, 55.8, 40.8. HRMS (ESI) m/z: calculated with an automatic liquid handler (FX from Beckman–Coulter).
for C21H19N2O2S [M + H]+: 363.1162, found: 363.1190.
Finally, the pseudotyped virus, thawed and diluted immediately
prior to use in cell culture medium, was added under a BSL-2
hood using Hydra (Thermo Fisher Scientic). The nal
concentration of DMSO in all wells was maintained at 2%. The
Synthesis of N-(4-(benzo[d]thiazol-2-yl)phenyl)-2-hydroxy-3-
methoxybenzamide (1g)
ꢂ
plates were incubated at 37 C in a humidied CO2 incubator
2-Hydroxy-3-methoxybenzoyl chloride (0.53 mmol) was dis-
solved in Et2O (0.5 mL) and then added dropwise to a stirring
solution of 3a (0.44 mmol) and NaHCO3 (0.88 mmol) in Et2O (1
mL) and H2O (0.5 mL) at 0 ꢂC. Then the reaction was allowed to
warm to room temperature and stirred for 12 h. The resulting
precipitate was isolated by suction ltration and washed with
H2O (2 mL), 2 N HCl (2 mL) and H2O (2 mL) sequentially to yield
the pure product 1g. White solid; yield 14%. Mp 215–216 ꢂC. 1H
NMR (400 MHz, DMSO-d6) d 11.33 (s, 1H, OH), 10.60 (s, 1H,
–(C]O)NH–), 8.13–8.09 (m, 3H, ArH), 8.04 (d, J ¼ 8.2 Hz, 1H,
ArH), 7.93 (d, J ¼ 8.4 Hz, 2H, ArH), 7.53 (d, J ¼ 7.8 Hz, 2H, ArH),
7.47–7.42 (m, 1H, ArH), 7.18 (d, J ¼ 8.0 Hz, 1H, ArH), 6.96–6.90
(m, 1H, ArH), 3.84 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
d 167.0, 166.8, 153.6, 148.4, 148.3, 141.1, 134.3, 128.4, 127.9
(2C), 126.6, 125.3, 122.6, 122.2, 120.9 (2C), 120.1, 118.6, 118.0,
115.5, 56.0. HRMS (ESI) m/z: calculated for C21H16N2NaO3S [M +
Na]+: 399.0774, found: 399.0774.
for 48 h. Bright-Glo substrate (Promega) was added directly to
each well and cell lysis was allowed to proceed in the dark for
5 min.
Cytotoxic effect of compounds was assayed by MTT method
which evaluated the reduction product of MTT by cellular
succinate dehydrogenase as an indicator of cell liability.
Toxicity was calculated as 100% minus percentage of OD@570
readout each well against the control well with no drug, 100% ꢀ
OD@570 (compound)/OD@570 (control) ꢁ 100%.
Luciferase activity was measured using the Envision micro-
plate luminometer (PerkinElmer). Inhibition was calculated as
100% minus percentage of luciferase readout each well against
the control well with no drug, 100% ꢀ Luc (compound)/Luc
(control) ꢁ 100%.
Measurement of IC50 and CC50
For determining CC50 and IC50, compounds were serially
diluted and assayed as described for the HTS screening, CC50
and IC50 were determined by regression analysis using 4
parameter regression.
Plasmid and pseudovirus production
To produce MERS spike protein pseudotyped HIV virions, we
constructed
a MERS spike protein (MERS-S) expression
plasmid. The codon-optimized gene of full-length S protein of
MERS-CoV (Genebank: AFS88936), with replacement of the N-
terminal signal peptide (aa 1–17) with CD5 signal sequence,
were synthesized (Inovogen, China) and insertion into
pcDNA3.1 vector, veried by sequencing. A HIV backbone vector
pNL4-3. Luc.R-E- was used for pseudovirus package. 293T cells
were co-transfected with MERS spike protein expression
plasmid plus pNL4.3LucR-E- plasmid using X-tremeGENE DNA
HP Transfection Reagent (Roche) according the manufacturer's
instructions.29 At 72 h post-transfection, supernatants were
harvested and ltered through 0.45 mm lter, the ltered
supernatant was further concentrated 10 folds by ultracentri-
fugation with 100 kDa cut-off, aliquoted and stored at ꢀ80 ꢂC as
stock vial. The p24 level of pseudovirus stock solution was
determined by ELISA kit according to the manual (ab218268,
Abcam).
Conflicts of interest
There are no conicts of interest to declare.
Acknowledgements
This work was supported by the National Key Research and
Development Program of China (2018YFC1200604). The
pNL4.3.Luc.R-.E- and MERS-S expression plasmid was kindly
gied by Dr Shibo Jiang and Dr Lu Lu from Fudan University.
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