Design, Synthesis, and Evaluation of 3-((4-(t-Butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones as Neuraminidase Inhibitors

Article abstract of DOI:10.1002/cjoc.201500738

A series of novel 3-((4-(t-butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones (7a-7z) were designed, synthesized and evaluated for their ability of inhibiting neuraminidase (NA) of in?uenza H1N1 virus. Some compounds displayed moder

Full text of DOI:10.1002/cjoc.201500738

FULL PAPER
DOI: 10.1002/cjoc.201500738
Design, Synthesis, and Evaluation of 3-((4-(t-Butyl)-2-(2-
benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-
2(1H)-ones as Neuraminidase Inhibitors
Yilin Fang,a Mengwu Xiao,a Aixi Hu,*,a Jiao Ye,a Wenwen Lian,b and Ailin Liu*,b
a College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, China
b Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing
100050, China
A series of novel 3-((4-(t-butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones (7a－7z)
were designed, synthesized and evaluated for their ability of inhibiting neuraminidase (NA) of influenza H1N1 virus.
Some compounds displayed moderate influenza NA inhibitory activity. Compound 7l with the scaffold of
2-(2-(2-methoxybenzylidene)hydrazinyl)thiazole was the best one, exhibiting moderate NA inhibitory activity with
IC50 of 44.66 μmol/L. Structure-activity relationship showed that compounds with methoxy or hydroxy groups at
the ortho position, fluorine and nitro groups at the meta position and chlorine and bromine groups at the para posi-
tion of phenyl ring were more active. Docking study indicated that compound 7l has important interactions with
some key residues (including Asp151, Glu119, Arg292, Tyr406, and Asn347) and binds to 430-cavity adjacent to
NA active site.
Keywords
neuraminidase inhibitor, 3-((4-(t-butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-
2(1H)-ones, 2-(2-hydrazinyl)thiazole scaffold, synthesis, docking
Introduction
gence of viruses resistance during the treatment of in-
fluenza infections has been widely reported in recent
years.[10-13] With the emergence of drug-resistant
influenza strains[14-16] and in view of a highly pathogenic
flu pandemic, it is urgent to develop new antiviral strat-
egies to fight against potential human influenza pan-
demic.
The influenza virus, a viral respiratory pathogen re-
sponsible for frequent seasonal epidemics, can cause
grave threat to human health and economic problems.[1]
Despite advances in the understanding of molecular and
cellular aspects of influenza, the disease remains the
major cause of mortality and morbidity among patients
with respiratory diseases.[2] The viral functions of
influenza contain two major glycoproteins, hemaggluti-
nin and neuraminidase (NA),[3] and NA plays at least
two critical roles in the virus life cycle, including the
facilitation of virion progeny release and general mobil-
ity of the virus in the respiratory tract.[4] Hence, NA has
been considered as an important target for designing
agents against influenza viruses.
Currently, two major classes of anti-influenza medi-
cations have been approved by the Food and Drug Ad-
ministration (FDA) for the treatment of influenza (Fig-
ure 1): M2 protein ion channel inhibitors (amantadine
and rimantadine),[5] and NA inhibitors (Oseltamivir,
Zanamivir and Peramivir).[6-8] Zanamivir and Peramivir
are rarely used because of their low bioavailability and
rapid elimination analysis. Oseltamivir is the only orally
available drug currently in use.[9] However, the emer-
HO
O
NH2
NH2
O
NH2
HN
O
Amantadine
OH
Rimantadine
COOH
Oseltamivir
O
HO
H
O
HO
HO
NH
N
OH
HN
NH2
H
HN
NH2
NH
NH
O
O
Zanamivir
Peramivir
Figure 1 Chemical structures of clinically used anti-influenza
drugs approved by the FDA.
*
E-mail: axhu@hnu.edu.cn, liuailin@imm.ac.cn; Tel./fax: 0086-0731-88822286
Received October 17, 2015; accepted December 8, 2015; published online January 8, 2016.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201500738 or from the author.
Chin. J. Chem. 2016, 34, 403—411
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Fang et al.
FULL PAPER
2-Oxoquinoline is an important heterocyclic com-
pound, which shows a wide variety of pharmacological
properties. Many 2-oxoquinoline derivatives have been
reported to have antimicrobial,[17] anti-angiogenic,[18]
anticancer,[19] antioxidant,[20] antimalarial,[21] and an-
ti-inflammatory properties.[22,23] The thiazole nucleus is
a multifunctional group and used to modify pharma-
cophores and improve their biological and pharmacol-
ogical activities, such as anticancer,[24-26] antimalarial,[27]
antimicrobial,[28] and anti-inflammatory activities.[29]
Recently, thiazole derivatives were reported as NA in-
hibitors.[30] In our previous study,[31] 2-(2-hydrazinyl)-
thiazole derivatives were found to exhibit inhibitory
effects against the influenza A NA in vitro. Hence, it is
considered that simultaneous administration of 2-oxo-
quinoline and 2-(2-hydrazinyl)thiazole structures in a
single molecule, would lead to a rapid onset of NA in-
hibitory effect. So a series of novel 3-((4-(t-butyl)-2-(2-
benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-
2(1H)-ones 7a－7z (Figure 2) were designed as NA
inhibitors. The overall strategies for the synthesis of 7a
－7z were outlined in Scheme 1. The NA inhibitory
activity was evaluated in vitro.
and used directly without further depuration. Melting
points were determined using an X-4 electrothermal
digital melting point apparatus and are uncorrected. Nu-
clear magnetic resonance (NMR) was recorded on a
Varian INOVA-400 spectrometer in DMSO-d6 or
CDCl3, whereas tetramethylsilane (TMS) (δ 0) was used
as an internal standard for spectra recorded. Elemental
analysis was carried out on an Elementar Vario EL
CHNS Elemental Analyzer. Analyses for C, H and N
were within 0.4% of the theoretical values.
Synthesis of 2-chloro-3-formylquinoline (1) and 2-
oxo-3-formyquinoline (2)
Compounds 1 and 2 were synthesized according to
the literature.[32] The Vilsmeier cyclisation of acetani-
lide was carried out by adding POCl3 (17 mL) to the
substrate in DMF (5 mL) at 0－5 ℃, followed by heat-
ing at 90 ℃ for 16 h. After the reaction, the mixture
was poured into ice water and then filtered to offer pale
powder of compound 1. Yellow solid, yield 80.3%, m.p.
149－152 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 10.39
(s, 1H, O＝CH), 8.99 (s, 1H, C＝CH), 8.29 (d, J＝8.1
Hz, 1H), 8.07－7.96 (m, 2H), 7.77 (t, J＝7.4 Hz, 1H).
13C NMR (100 MHz, DMSO-d6) δ: 189.64, 149.26,
148.82, 141.64, 134.15, 130.49, 128.53, 128.05, 126.61.
Anal. calcd for C10H6ClNO: C 62.68, H 3.16, N 7.31;
found C 62.66, H 3.18, N 7.29.
Experimental
All chemicals and solvents were analytical reagents
Figure 2 Design of novel 3-((4-(t-butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones as NA inhibitors.
Scheme 1 Synthesis of 3-((4-(t-butyl)-2-(2-benzyli-denehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
O
CHO
Cl
CHO
O
NHCOCH3
(a)
O
(d)
(b)
t-BuCOCH3
Bu-t
N
(c)
N
H
N
O
H
1
3
2
O
NHCSNH2
N
Bu-t
Bu-t
R
(e)
6a 6z
Br
N
O
N
O
H
H
(f)
4
5
a: R = 2-OH; b: R = 5-Cl-2-OH; c: R = 5-Br-2-OH; d: R = 5-NO2-2-OH;
e: R = 3,5-diCl-2-OH; f: R = 3,5-diBr-2-OH; g: R = 3-Br-5-NO2-2-OH;
h: R = 3,5-diNO2-2-OH; i: R = 2-CH3; j: R = 3-CH3; k: R = 4-CH3;
l: R = 2-OCH3; m: R = 3-OCH3; n: R = 4-OCH3; o: R = 2-F; p: R = 3-F
q:R = 4-F; r: R = 2-Cl; s: R = 3-Cl; t: R = 4-Cl; u: R = 2-Br; v: R = 3-Br;
w: R = 4-Br; x: R = 2-NO2; y: R = 3-NO2; z: R = 4-NO2
t-Bu
R
N
N
HN
.
HBr
N
S
H
O
7a 7z
Reagents and conditions: (a) DMF, POCl3, 90 ℃, 16 h; (b) 70% AcOH, 95 ℃, 6 h; (c) NaOH, EtOH, reflux 2 h; (d) Raney Ni, EtOH, 90 ℃,
8 h; (e) CuBr2, CHCl3-EtOAc, 80 ℃, 4 h; (f) EtOH, reflux
404
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Chin. J. Chem. 2016, 34, 403—411

3-((4-(t-Butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones
Then compound 1 (10 mmol) was treated with 70%
acetic acid aqueous solution (200 mL) at 90 ℃ for 6 h
and the solution was cooled down to room temperature
to offer needle crystals of compound 2. Yellow solid,
NMR (400 MHz, CDCl3) δ: 7.89 (s, 1H), 7.86 (d, J＝8.5
Hz, 1H), 7.69 (d, J＝7.1 Hz, 1H), 7.62－7.57 (m, 1H),
7.40－7.36 (m, 1H), 5.57 (dd, J＝9.3, 6.8 Hz, 1H),
3.58－3.52 (m, 2H, CH2), 1.31 (s, 9H, 3×CH3); 13C
NMR (100 MHz, CDCl3) δ: 205.85, 161.56, 141.98,
128.14, 124.41, 122.68, 122.43, 120.67, 119.45, 115.39,
39.05, 25.83, 21.73, 21.16. Anal. calcd for C16H18BrNO2:
C 57.16, H 5.40, N 4.17; found C 57.12, H 5.42, N 4.15.
1
yield 81.0%, m.p. 304－306 ℃. H NMR (400 MHz,
DMSO-d6) δ: 12.19 (s, 1H, NH), 10.25 (s, 1H, O＝CH),
8.49 (s, 1H, C＝CH), 7.91 (d, J＝7.8 Hz, 1H), 7.66 (t,
J＝7.8 Hz, 1H), 7.37 (d, J＝8.3 Hz, 1H), 7.25 (t, J＝7.6
Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 190.05,
161.76, 142.69, 141.43, 133.97, 131.21, 125.87, 122.94,
118.42, 115.69. Anal. calcd for C10H7NO: C 69.36, H
4.07, N 8.09; found C 69.34, H 4.11, N 8.07.
General procedure for the synthesis of (E)-2-benzy-
lidenehydrazinecarbothioamide (6a－6z)
Compounds 6a－6z were synthesized according to
the literature.[33] Aldehyde (0.01 mmol) was added to a
stirring mixture of thiosemicarbazide (2 mmol) and
EtOH (20 mL). The resulting mixture was heated at
80 ℃ and stirred for 2 h, and the reaction mixture was
cooled down to room temperature to offer needle crys-
tals of compound 6.
Synthesis of (E)-3-(4,4-dimethyl-3-oxopent-1-en-1-
yl)quinolin-2(1H)-one (3)
The mixture of compound 2 (1 mmol), ethanol (20
mL), pinacolone (1.2 mmol) and 0.5 g NaOH was
blended together and reacted at 80 ℃ for 2 h. After
cooling down to room temperature, powders or crystals
of compound 3 were obtained. Orange solid, yield
87.2%, m.p. 209－211 ℃. 1H NMR (400 MHz, CDCl3)
δ: 8.07－7.97 (m, 2H), 7.71 (d, J＝15.5 Hz, 1H), 7.61 (d,
J＝7.7 Hz, 1H), 7.53 (t, J＝8.1 Hz, 1H), 7.37 (d, J＝8.1
Hz, 1H), 7.23 (d, J＝7.4 Hz, 1H), 1.27 (s, 9H, 3×CH3);
13C NMR (100 MHz, CDCl3) δ: 200.36, 158.61, 136.74,
133.09, 126.45, 123.34, 121.27, 119.51, 117.78, 115.10,
111.37, 38.36, 21.40. Anal. calcd for C16H17NO2: C
75.27, H 6.71, N 5.49; found C 75.23, H 6.72, N 5.47.
General procedure for the synthesis of 3-((4-(t-bu-
tyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)-
quinolin-2(1H)-ones (7a－7z)
Compound 5 (1 mmol) was added into a refluxing
solution of an appropriate compound 6 (1.2 mmol) in
EtOH (15 mL). The mixture was kept at this tempera-
ture for more than 4 h. After completion of the reaction
as indicated by TLC, the mixture was cooled to room
temperature, then filtered to get the crude products. The
crude products were recrystallized from hot DMSO and
95% alcohol, then the mixture was cooled to offer
compounds 7a－7z.
(E)-3-((4-(t-Butyl)-2-(2-(2-hydroxybenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7a) Yellow solid, yield 64.6%, m.p. 257－259 ℃. 1H
NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H, NH),
10.25 (br, 1H, NH), 8.40 (s, 1H, N＝CH), 7.67 (d, J＝
8.7 Hz, 2H), 7.55 (d, J＝7.7 Hz, 1H), 7.48 (t, J＝7.3 Hz,
1H), 7.33 (d, J＝8.2 Hz, 1H), 7.21 (t, J＝7.0 Hz, 1H),
7.16 (t, J＝7.6 Hz, 1H), 6.88 (d, J＝8.2 Hz, 1H), 6.84 (t,
J＝7.5 Hz, 1H), 4.02 (s, 2H, CH2), 1.35 (s, 9H, 3×CH3);
13C NMR (100 MHz, DMSO-d6) δ: 165.97, 165.17,
163.63, 147.53, 143.76, 141.50, 140.29, 138.60, 137.06,
134.38, 132.27, 129.53, 127.73, 124.82, 124.58, 122.39,
121.70, 120.42, 66.13, 52.99, 35.47, 30.52, 21.52. Anal.
calcd for C24H24N4O2S: C 66.64, H 5.59, N 12.95; found
C 66.68, H 5.57, N 12.92.
(E)-3-((4-(t-Butyl)-2-(2-(5-chloro-2-hydroxyben-
zylidene)hydrazinyl)thiazol-5-yl)methyl)quinolin-
2(1H)-one (7b) Beige solid, yield 50.0%, m.p. 275－
278 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H,
NH), 10.41 (br, 1H, NH), 8.21 (s, 1H, N＝CH), 7.67 (d,
J＝7.2 Hz, 1H), 7.57 (s, 1H), 7.52 (s, 1H), 7.47 (t, J＝
7.2 Hz, 1H), 7.31 (d, J＝8.4 Hz, 1H), 7.21 (dd, J＝2.8,
2.4 Hz, 1H), 7.15 (t, J＝7.2 Hz, 1H), 6.88 (d, J＝8.8 Hz,
1H), 3.99 (s, 2H, CH2), 1.32 (s, 9H, 3×CH3); 13C NMR
(100 MHz, DMSO-d6) δ: 166.49, 165.87, 162.11,
143.86, 143.06, 141.63, 140.31, 137.98, 137.12, 134.96,
132.79, 132.21, 129.89, 128.36, 127.76, 127.03, 124.31,
Synthesis of 3-(4,4-dimethyl-3-oxopentyl)quinolin-
2 (1H)-one (4)
Compound 3 (0.1 mol), Raney Ni 1.28 g and EtOH
(120 mL) were added into autoclave and the mixture
was stirred at 80 ℃ under 2.5 MPa H2 for 3 h. After the
reaction was completed, the mixture was reduced to
normal pressure, and cooled down to room temperature
to offer needle crystals of compound 4. Yellow solid,
1
yield 90.0%, m.p. 183－185 ℃. H NMR (400 MHz,
CDCl3) δ: 11.53 (s, 1H, NH), 7.74 (s, 1H), 7.54 (d, J＝
7.2 Hz, 1H), 7.46 (t, J＝7.6 Hz, 1H), 7.34 (d, J＝8.1 Hz,
1H), 7.20 (t, J＝7.5 Hz, 1H), 2.95 (s, 4H, 2×CH2), 1.13
(s, 9H, 3×CH3); 13C NMR (100 MHz, CDCl3) δ:
210.47, 159.31, 133.25, 132.65, 127.84, 124.66, 122.36,
117.64, 115.32, 110.64, 39.26, 30.33, 21.45, 20.86. Anal.
calcd for C16H19NO2: C 74.68, H 7.44, N 5.44; found C
74.65, H 7.42, N 5.46.
Synthesis of 3-(2-bromo-4,4-dimethyl-3-oxopentyl)-
quinolin-2(1H)-one (5)
To a solution of compound 4 (0.02 mol) in a mixture
of CHCl3 (15 mL) and EtOAc (15 mL), CuBr2 (0.04 mol)
was added in portions during a period of 0.5 h at 80 ℃,
the reaction mixture was allowed to reflux for 4 h. The
solution was washed with HCl (aq) until the blue faded.
The aqueous phase was then made neutral with water,
and the product was extracted with EtOAc (10 mL×3).
The combined organic phase was dried over Na2SO4 and
concentrated under reduced pressure to offer compound
1
5. White solid, yield 69.1%, m.p. 190－193 ℃. H
Chin. J. Chem. 2016, 34, 403—411
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Fang et al.
FULL PAPER
123.67, 120.04, 40.64, 35.53, 32.27. Anal. calcd for
C24H23ClN4O2S: C 61.73, H 4.96, N 12.00; found C
61.68, H 4.93, N 12.02.
(E)-3-((2-(2-(3-Bromo-2-hydroxy-5-nitrobenzyli-
dene)hydrazinyl)-4-(t-butyl)thiazol-5-yl)methyl)-
quinolin-2(1H)-one (7g) Red solid, yield 61.5%, m.p.
1
(E)-3-((2-(2-(5-Bromo-2-hydroxybenzylidene)-
hydrazinyl)-4-(t-butyl)thiazol-5-yl)methyl)quinolin-
2(1H)-one (7c) Yellow solid, yield 67.7%, m.p. 259－
261 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 11.91 (s, 1H,
NH), 10.42 (br, 1H, NH), 8.19 (s, 1H, N＝CH), 7.67 (d,
J＝10.8 Hz, 2H), 7.56 (s, 1H), 7.56 (t, J＝7.6 Hz, 1H),
7.33－7.30 (m, 2H), 7.15 (t, J＝7.2 Hz, 1H), 6.83 (d,
J＝8.4 Hz, 1H), 3.99 (s, 2H, CH2), 1.31 (s, 9H, 3×CH3);
13C NMR (100 MHz, DMSO-d6) δ: 167.44, 166.03,
164.59, 143.81, 141.49, 137.71, 137.10, 135.48, 134.58,
134.32, 132.41, 132.26, 129.42, 127.69, 126.90, 124.55,
124.25, 120.39, 119.94, 35.52, 31.05, 23.72. Anal. calcd
for C24H23BrN4O2S: C 56.36, H 4.53, N 1095; found C
56.17, H 4.51, N 10.93.
267－270 ℃. H NMR (400 MHz, DMSO-d6) δ: 11.91
(s, 1H, NH), 8.48 (d, J＝2.5 Hz, 1H), 8.34 (d, J＝2.7 Hz,
1H), 7.69 (d, J＝7.9 Hz, 1H), 7.62 (s, 1H), 7.47 (t, J＝
7.6 Hz, 1H), 7.32 (d, J＝8.2 Hz, 1H), 7.15 (t, J＝7.6 Hz,
1H), 3.98 (s, 2H, CH2), 1.34 (s, 9H, 3×CH3); 13C NMR
(100 MHz, DMSO-d6) δ: 161.80, 140.56, 138.16,
136.39, 130.03, 127.92, 127.87, 122.13, 119.55, 115.14,
56.34, 30.30, 18.86. Anal. calcd for C24H22BrN5O4S: C
51.80, H 3.99, N 12.59; found C 51.61, H 3.96, N 12.57.
(E)-3-((4-(t-Butyl)-2-(2-(2-hydroxy-3,5-dinitroben
zylidene)hydrazinyl)thiazol-5-yl)methyl)quinolin-
2(1H)-one (7h) Red solid, yield 43.3%, m.p. 259－
262 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 11.91 (s, 1H,
NH), 11.64 (s, 1H, OH), 9.05 (s, 1H), 8.68－8.62 (m,
1H), 8.46 (d, J＝9.1 Hz, 2H), 7.67 (d, J＝7.7 Hz, 1H),
7.62 (s, 1H, C＝CH), 7.47 (t, J＝7.7 Hz, 1H), 7.32 (d,
J＝8.2 Hz, 1H), 7.15 (t, J＝7.5 Hz, 1H), 4.00 (s, 2H,
CH2), 1.34 (s, 9H, 3×CH3); 13C NMR (100 MHz,
CDCl3) δ: 178.43, 161.75, 157.58, 157.35, 138.09,
137.31, 137.24, 137.13, 136.40, 132.75, 129.99, 127.87,
126.09, 125.83, 122.28, 122.10, 121.35, 119.41, 115.10,
35.79, 30.34, 27.53. Anal. calcd for C24H22N6O6S: C
55.17, H 4.24, N 16.08; found C 55.21, H 4.26, N 16.04.
(E)-3-((4-(t-Butyl)-2-(2-(2-methylbenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(E)-3-((4-(t-Butyl)-2-(2-(2-hydroxy-5-nitrobenzyl-
idene)hydrazinyl)thiazol-5-yl)methyl) quinolin-
2(1H)-one (7d) Orange solid, yield 72.2%, m.p.
261－263 ℃. 1H NMR (400 MHz, DMSO-d6) δ: 11.92
(s, 1H, NH), 11.70 (s, 1H, OH), 8.42 (d, J＝2.9 Hz, 1H),
8.29 (s, 1H, N＝CH), 8.07 (dd, J＝9.1, 2.9 Hz, 1H),
7.67 (d, J＝7.5 Hz, 1H), 7.59 (s, 1H), 7.47 (t, J＝7.7 Hz,
1H), 7.32 (d, J＝8.2 Hz, 1H), 7.15 (t, J＝8.0 Hz, 1H),
7.06 (d, J＝9.1 Hz, 1H), 4.01 (s, 2H, CH2), 1.33 (s, 9H,
3×CH3); 13C NMR (100 MHz, DMSO-d6) δ: 166.68,
166.58, 145.19, 143.00, 141.36, 137.85, 134.88, 132.75,
130.88, 127.00, 126.57, 126.26, 124.33, 121.84, 120.01,
40.80, 35.45, 32.29. Anal. calcd for C24H23N5O4S: C
60.36, H 4.85, N 14.67; found C 60.19, H 4.83, N 14.65.
(E)-3-((4-(t-Butyl)-2-(2-(3,5-dichloro-2-hydroxy-
benzylidene)hydrazinyl)thiazol-5-yl)methyl) quino-
lin-2(1H)-one (7e) Red solid, yield 62.0%, m.p.
1
(7i) Beige solid, yield 48.0%, m.p. 225－228 ℃. H
NMR (400 MHz, CDCl3) δ: 13.02 (s, 1H, NH), 12.91 (s,
1H, OH), 11.60 (s, 1H, NH), 8.41 (s, 1H, N＝CH),
7.68－7.63 (m, 2H), 7.60 (d, J＝8.6 Hz, 1H), 7.56 (d,
J＝7.3 Hz, 1H), 7.42 (d, J＝8.1 Hz, 1H), 7.30 (d, J＝7.6
Hz, 2H), 7.16 (dd, J＝15.1, 7.5 Hz, 2H), 4.19 (s, 2H,
CH2), 2.43 (s, 3H, CH3), 1.56 (s, 9H, 3×CH3); 13C
NMR (100 MHz, CDCl3) δ: 166.54, 162.89, 149.55,
144.05, 138.33, 138.05, 137.69, 131.19, 131.01, 130.87,
130.38, 129.91, 127.82, 127.57, 126.24, 123.20, 119.56,
116.43, 115.55, 35.20, 30.28, 27.71, 19.91. Anal. calcd
for C25H26N4OS: C 69.74, H 6.09, N 13.01; found C
69.56, H 6.07, N 12.97.
(E)-3-((4-(t-Butyl)-2-(2-(3-methylbenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7j) Yellow solid, yield 42.2%, m.p. 221－224 ℃. 1H
NMR (400 MHz, CDCl3) δ: 13.03 (s, 1H, NH), 12.76 (s,
1H, OH), 10.98 (s, 1H, NH), 8.16 (s, 1H, N＝CH),
7.63－7.53 (m, 3H), 7.50 (d, J＝7.9 Hz, 2H), 7.35 (d,
J＝8.2 Hz, 1H), 7.29 (d, J＝7.6 Hz, 1H), 7.16 (d, J＝7.8
Hz, 2H), 4.17 (s, 2H, CH2), 2.36 (s, 3H, CH3), 1.55 (s,
9H, 3×CH3); 13C NMR (100 MHz, CDCl3) δ: 166.25,
163.13, 150.67, 143.77, 141.96, 138.44, 137.83, 130.88,
129.84, 129.58, 129.52, 127.79, 127.71, 123.19, 119.56,
116.49, 115.70, 35.33, 30.24, 27.75, 21.58. Anal. calcd
for C25H26N4OS: C 69.74, H 6.09, N 13.01; found C
69.54, H 6.08, N 12.99.
1
245－247 ℃. H NMR (400 MHz, CDCl3) δ: 11.07 (s,
1H, NH), 8.31 (s, 1H, N＝CH), 7.66 (s, 1H), 7.60－7.55
(m, 2H), 7.41 (d, J＝2.4 Hz, 1H), 7.34 (d, J＝8.5 Hz,
1H), 7.29 (d, J＝7.6 Hz, 1H), 7.20 (d, J＝2.4 Hz, 1H),
4.19 (s, 2H, CH2), 1.58 (s, 9H, 3×CH3); 13C NMR (100
MHz, DMSO-d6) δ: 161.81, 151.19, 138.11, 136.34,
132.90, 130.00, 129.13, 127.90, 123.60, 122.12, 121.82,
119.46, 115.13, 35.86, 30.28, 27.58. Anal. calcd for
C24H22Cl2N4O2S: C 57.49, H 4.42, N 11.17; found C
57.30, H 4.39, N 11.15.
(E)-3-((4-(t-Butyl)-2-(2-(3,5-dibromo-2-hydroxy-
benzylidene)hydrazinyl)thiazol-5-yl)methyl) quino-
lin-2(1H)-one (7f) Yellow solid, yield 46.2%, m.p.
1
258－260 ℃. H NMR (400 MHz, DMSO-d6) δ: 11.92
(s, 1H, NH), 8.25 (s, 1H, N＝CH), 7.71 (s, 1H), 7.70 (d,
J＝2.4 Hz, 1H), 7.65 (d, J＝8.4 Hz, 1H), 7.60 (s, 1H),
7.46 (t, J＝7.7 Hz, 1H), 7.32 (d, J＝8.2 Hz, 1H), 7.15 (t,
J＝7.5 Hz, 1H), 4.05 (s, 2H, CH2), 1.33 (s, 9H, 3×CH3);
13C NMR (100 MHz, DMSO-d6) δ: 161.81, 152.72,
138.12, 136.34, 134.47, 132.84, 130.81, 130.01, 127.91,
122.77, 122.13, 119.46, 118.60, 115.14, 111.45, 111.13,
110.91, 35.82, 30.00, 27.46. Anal. calcd for
C24H22Br2N4O2S: C 48.83, H 3.76, N 9.49; found C
48.63, H 3.74, N 9.46.
(E)-3-((4-(t-Butyl)-2-(2-(4-methylbenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7k) Yellow solid, yield 38.3%, m.p. 176－178 ℃.
406
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© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, 34, 403—411

3-((4-(t-Butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones
1H NMR (400 MHz, CDCl3) δ: 13.04 (s, 1H, NH), 12.79
(s, 1H, OH), 11.58 (s, 1H, NH), 8.16 (s, 1H, N＝CH),
7.65 (s, 1H), 7.63－7.56 (m, 2H), 7.40 (d, J＝8.1 Hz,
1H), 7.33－7.27 (m, 2H), 7.19－7.14 (m, 2H), 6.96 (dd,
J＝8.6, 2.2 Hz, 1H), 4.18 (s, 2H, CH2), 3.78 (s, 3H,
CH3), 1.56 (s, 9H, 3×CH3); 13C NMR (100 MHz,
CDCl3) δ: 166.54, 162.89, 149.55, 144.05, 138.33,
138.05, 137.69, 131.19, 131.01, 130.87, 130.46, 130.38,
127.82, 127.57, 126.24, 123.20, 119.56, 116.43, 115.55,
35.39, 30.28, 27.64, 19.97. Anal. calcd for C25H26N4OS:
C 69.74, H 6.09, N 13.01; found C 69.56, H 6.06, N
12.97.
(E)-3-((4-(t-Butyl)-2-(2-(2-methoxybenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7l) Orange solid, yield 49.9%, m.p. 239－241 ℃. 1H
NMR (400 MHz, CDCl3) δ: 12.86 (s, 1H, NH), 11.67 (s,
1H, NH), 8.57 (s, 1H, N＝CH), 7.72 (dd, J＝7.7, 1.4 Hz,
1H), 7.65 (s, 1H), 7.58 (dd, J＝14.2, 7.5 Hz, 2H), 7.41
(d, J＝8.2 Hz, 1H), 7.36 (t, J＝8.7 Hz, 1H), 7.30 (d, J＝
7.5 Hz, 1H), 6.90－6.82 (m, 2H), 4.18 (s, 2H, CH2),
3.85 (s, 3H, OCH3), 1.57 (s, 9H, 3×CH3); 13C NMR
(100 MHz, CDCl3) δ: 161.40, 158.25, 153.60, 141.77,
138.89, 133.50, 132.88, 127.81, 125.93, 124.95, 122.86,
121.46, 118.26, 115.81, 115.70, 114.64, 111.47, 110.74,
106.23, 50.56, 30.43, 25.36, 22.78. Anal. calcd for
C25H26N4O2S: C 67.24, H 5.87, N 12.55; found C 67.05,
H 5.85, N 12.53.
(E)-3-((4-(t-Butyl)-2-(2-(3-methoxybenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7m) Beige solid, yield 42.3%, m.p. 209－212 ℃. 1H
NMR (400 MHz, CDCl3) δ: 13.13 (s, 1H, NH), 12.81 (s,
1H, OH), 11.24 (s, 1H, NH), 8.16 (s, 1H, N＝CH), 7.65
(s, 1H), 7.63－7.56 (m, 2H), 7.40 (d, J＝8.1 Hz, 1H),
7.33－7.27 (m, 2H), 7.19－7.14 (m, 2H), 6.96 (dd, J＝
8.6, 2.2 Hz, 1H), 4.18 (s, 2H, CH2), 3.78 (s, 3H, OCH3),
1.56 (s, 9H, 3×CH3); 13C NMR (100 MHz, DMSO-d6)
δ: 189.28, 174.53, 161.19, 161.10, 159.85, 138.90,
135.68, 135.31, 132.19, 130.41, 129.69, 127.36, 124.31,
122.86, 121.25, 119.40, 117.88, 115.50, 114.06, 55.60,
30.65. Anal. calcd for C25H26N4O2S: C 67.24, H 5.87, N
12.55; found C 67.36, H 5.89, N 12.52.
(E)-3-((4-(t-Butyl)-2-(2-(4-methoxybenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7n) Yellow solid, yield 52.0%, m.p. 178－181 ℃.
1H NMR (400 MHz, CDCl3) δ: 12.98 (s, 1H, NH), 12.69
(s, 1H, OH), 11.60 (s, 1H, NH), 8.14 (s, 1H, N＝CH),
7.65 (s, 1H), 7.63－7.56 (m, 2H), 7.54 (d, J＝8.8 Hz,
2H), 7.42 (d, J＝8.2 Hz, 1H), 7.31 (d, J＝7.6 Hz, 1H),
6.86 (d, J＝8.7 Hz, 2H), 4.18 (s, 2H, CH2), 3.82 (s, 3H,
OCH3), 1.55 (s, 9H, 3×CH3); 13C NMR (100 MHz,
DMSO-d6) δ: 173.35, 162.54, 161.22, 160.78, 138.84,
138.66, 135.84, 132.09, 130.95, 129.66, 127.50, 126.60,
123.65, 122.80, 119.42, 115.50, 114.85, 55.79, 38.51,
30.64, 27.48. Anal. calcd for C25H26N4O2S: C 67.24, H
5.87, N 12.55; found C 67.07, H 5.84, N 12.51.
NMR (400 MHz, CDCl3) δ: 13.18 (s, 1H, NH), 13.05 (s,
1H, OH), 10.63 (s, 1H, NH), 8.43 (s, 1H, N＝CH), 7.79
(t, J＝7.6 Hz, 1H), 7.68－7.57 (m, 3H), 7.43－7.31 (m,
3H), 7.14－7.08 (m, 2H), 4.18 (s, 2H, CH2), 1.55 (s, 9H,
3×CH3); 13C NMR (100 MHz, DMSO-d6) δ: 189.20,
175.34, 162.87, 161.08, 153.49, 138.88, 135.53, 134.20,
132.17, 129.66, 128.25, 127.24, 125.34, 124.68, 122.75,
119.28, 116.62, 116.41, 115.44, 38.56, 30.59, 29.72.
Anal. calcd for C24H23FN4O2S: C 66.34, H 5.34, N
12.89; found C 66.15, H 5.31, N 12.87.
(E)-3-((4-(t-Butyl)-2-(2-(3-fluorobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7p) Yellow solid, yield 44.2%, m.p. 248－251 ℃.
1H NMR (400 MHz, CDCl3) δ: 13.27 (s, 1H, NH), 12.91
(s, 1H, OH), 10.39 (s, 1H, NH), 8.17 (s, 1H, N＝CH),
7.65－7.54 (m, 3H), 7.37－7.29 (m, 5H), 7.12 (d, J＝
8.5 Hz, 1H), 4.18 (s, 2H, CH2), 1.56 (s, 9H, 3×CH3);
13C NMR (100 MHz, DMSO-d6) δ: 163.78, 161.69,
161.37, 138.12, 136.71, 132.48, 131.19, 131.11, 130.02,
127.85, 123.11, 122.10, 119.37, 116.72, 116.63, 115.11,
112.88, 112.65, 35.76, 30.34, 27.25. Anal. calcd for
C24H23FN4O2S: C 66.34, H 5.34, N 12.89; found C
66.15, H 5.31, N 12.86.
(E)-3-((4-(t-Butyl)-2-(2-(4-fluorobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(7q) Beige solid, yield 68.6%, m.p. 266－269 ℃. H
NMR (400 MHz, DMSO-d6) δ: 11.91 (s, 1H, NH), 7.97
(s, 1H, N＝CH), 7.67－7.59 (m, 4H), 7.47 (t, J＝7.6 Hz,
1H), 7.32 (d, J＝8.0 Hz, 1H), 7.22 (t, J＝8.8 Hz, 1H),
7.15 (t, J＝7.2 Hz, 1H), 4.00 (s, 2H, CH2), 1.32 (s, 9H, 3
×CH3); 13C NMR (100 MHz, DMSO-d6) δ: 163.14,
161.13, 159.98, 138.86, 135.64, 132.16, 131.43, 131.35,
130.57, 129.66, 127.34, 124.26, 122.77, 119.34, 116.54,
116.32, 115.47, 38.53, 30.46. Anal. calcd for C24H23F-
N4O2S: C 66.34, H 5.34, N 12.89; found C 66.14, H
5.32, N 12.87.
(E)-3-((4-(t-Butyl)-2-(2-(2-chlorobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(7r) Beige solid, yield 72.7%, m.p. 250－253 ℃. H
NMR (400 MHz, CDCl3) δ: 13.10 (s, 1H, NH), 11.37 (s,
1H, NH), 8.56 (s, 1H, N＝CH), 7.81 (d, J＝7.8 Hz, 1H),
7.66 (s, 1H), 7.58 (dd, J＝16.4, 7.8 Hz, 2H), 7.42－7.36
(m, 2H), 7.34－7.28 (m, 2H), 7.18 (t, J＝7.6 Hz, 1H),
4.18 (s, 2H, CH2), 1.55 (s, 9H, 3×CH3); 13C NMR (100
MHz, DMSO-d6) δ: 189.85, 175.78, 161.16, 156.87,
139.10, 135.58, 134.76, 133.51, 132.30, 130.97, 130.53,
129.79, 128.84, 128.13, 127.28, 124.98, 122.84, 119.37,
115.53, 38.66, 30.67. Anal. calcd for C24H23ClN4O2S: C
63.92, H 5.14, N 12.42; found C 63.73, H 5.12, N 12.40.
(E)-3-((4-(t-Butyl)-2-(2-(3-chlorobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(7s) Beige solid, yield 53.0%, m.p. 241－243 ℃. H
NMR (400 MHz, CDCl3) δ: 13.24 (s, 1H, NH), 12.85 (s,
1H, OH), 11.16 (s, 1H, NH), 8.14 (s, 1H, N＝CH), 7.65
(s, 1H), 7.61 (d, J＝4.8 Hz, 1H), 7.59－7.53 (m, 2H),
7.43 (d, J＝7.7 Hz, 1H), 7.37 (d, J＝7.8 Hz, 2H),
7.33－7.27 (m, 2H), 4.19 (s, 2H, CH2), 1.57 (s, 9H, 3×
CH3); 13C NMR (100 MHz, DMSO-d6) δ: 189.58,
(E)-3-((4-(t-Butyl)-2-(2-(2-fluorobenzylidene)hy-
drazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one (7o)
White solid, yield 65.1%, m.p. 207－210.5 ℃. 1H
Chin. J. Chem. 2016, 34, 403—411
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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407

Fang et al.
FULL PAPER
175.20, 161.16, 159.87, 139.02, 138.95, 136.06, 134.06,
132.23, 131.63, 131.18, 128.44, 127.35, 127.29, 124.71,
122.84, 119.40, 115.52, 38.61, 30.67. Anal. calcd for
C24H23ClN4O2S: C 63.92, H 5.14, N 12.42; found C
63.75, H 5.11, N 12.41.
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7x) Yellow solid, yield 59.2%, m.p. 189－192 ℃. 1H
NMR (400 MHz, DMSO-d6) δ: 13.18 (s, 1H, NH),
13.05 (s, 1H, OH), 10.61 (s, 1H, NH), 8.43 (s, 1H, N＝
CH), 7.79 (t, J＝7.6 Hz, 1H), 7.68－7.57 (m, 3H), 7.34
(dd, J＝15.8, 7.7 Hz, 2H), 7.10 (dd, J＝17.2, 9.3 Hz,
2H), 4.18 (s, 2H, CH2), 1.55 (s, 9H, 3×CH3); 13C NMR
(100 MHz, DMSO-d6) δ: 165.86, 162.15, 147.87,
145.64, 143.88, 143.82, 139.63, 139.24, 138.95, 137.28,
135.82, 135.41, 134.53, 132.99, 130.10, 129.87, 127.75,
124.20, 120.43, 61.20, 35.54, 23.71. Anal. calcd for
C24H23N5O3S: C 62.46, H 5.02, N 15.17; found C 62.37,
H 4.98, N 15.19.
(E)-3-((4-(t-Butyl)-2-(2-(3-nitrobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7y) Yellow solid, yield 62.9%, m.p. 242－245 ℃. 1H
NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H, NH),
10.63 (s, 1H, NH), 8.38 (s, 1H), 8.15 (d, J＝5.7 Hz, 1H),
8.10 (s, 1H), 8.01 (d, J＝8.0 Hz, 1H), 7.70－7.64 (m,
1H), 7.58 (s, 1H), 7.47 (t, J＝7.7 Hz, 1H), 7.32 (d, J＝
8.4 Hz, 1H), 7.15 (t, J＝7.5 Hz, 1H), 4.02 (s, 2H, CH2),
1.33 (s, 1H, 9H, 3×CH3); 13C NMR (100 MHz,
DMSO-d6) δ: 161.61, 148.56, 138.11, 137.11, 136.84,
136.35, 135.28, 134.77, 132.45, 132.40, 130.65, 129.96,
123.30, 121.25, 120.25, 117.24, 113.77, 36.05, 30.84,
27.43. Anal. calcd for C24H23N5O3S: C 62.46, H 5.02, N
15.17; found C 62.58, H 5.04, N 15.14.
(E)-3-((4-(t-Butyl)-2-(2-(4-nitrobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7z) Orange solid, yield 81.3%, m.p. 237－240 ℃. 1H
NMR (400 MHz, DMSO-d6) δ: 11.90 (s, 1H, NH), 8.22
(d, J＝8.6 Hz, 2H), 8.13－8.08 (m, 1H), 8.04 (s, 1H),
7.80 (d, J＝8.7 Hz, 2H), 7.60 (s, 1H), 7.47 (t, J＝7.7 Hz,
1H), 7.32 (d, J＝8.2 Hz, 1H), 7.15 (t, J＝7.5 Hz, 1H),
4.02 (s, 2H, CH2), 1.33 (s, 9H, 3×CH3); 13C NMR (100
MHz, DMSO-d6) δ: 165.36, 161.83, 147.13, 141.39,
138.14, 136.36, 133.29, 130.01, 127.89, 127.00, 124.42,
122.15, 119.47, 115.15, 35.89, 30.83, 27.20. Anal. calcd
for C24H23N5O3S: C 62.46, H 5.02, N 15.17; found C
62.25, H 5.00, N 15.14.
(E)-3-((4-(t-Butyl)-2-(2-(4-chlorobenzylidene)-
hydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-one
(7t) Yellow solid, yield 62.6%, m.p. 254－257 ℃. 1H
NMR (400 MHz, DMSO-d6) δ: 11.91 (s, 1H, NH), 7.98
(s, 1H, N＝CH), 7.66 (d, J＝7.9 Hz, 1H), 7.61－7.58
(m, 3H), 7.49－7.43 (m, 3H), 7.32 (d, J＝8.3 Hz, 1H),
7.15 (t, J＝7.5 Hz, 1H), 4.01 (s, 2H, CH2), 1.33 (s, 9H, 3
×CH3); 13C NMR (100 MHz, DMSO-d6) δ: 167.24,
165.85, 164.95, 143.81, 141.49, 137.71, 135.48, 134.82,
134.58, 134.32, 132.41, 129.42, 127.69, 126.90, 124.51,
124.25, 120.39, 119.94, 35.52, 31.15, 23.63. Anal. calcd
for C24H23ClN4O2S: C 63.92, H 5.14, N 12.42; found C
63.72, H 5.11, N 12.39.
(E)-3-((2-(2-(2-Bromobenzylidene)hydrazinyl)-4-
(t-butyl)thiazol-5-yl)methyl)quinolin-2(1H)-one (7u)
Yellow solid, yield 48.1%, m.p. 168－171 ℃. 1H NMR
(400 MHz, CDCl3) δ: 11.16 (s, 1H, NH), 8.22 (s, 1H, N
＝CH), 7.94 (dd, J＝7.9, 1.5 Hz, 1H), 7.55－7.50 (m,
3H), 7.47 (d, J＝7.3 Hz, 1H), 7.33 (d, J＝8.1 Hz, 1H),
7.25－7.13 (m, 3H), 4.19 (s, 2H, CH2), 1.39 (s, 9H, 3×
CH3); 13C NMR (100 MHz, DMSO-d6) δ: 189.83,
175.78, 161.14, 159.16, 139.06, 138.97, 135.56, 133.75,
133.67, 132.41, 132.27, 129.75, 129.18, 128.60, 127.26,
125.13, 124.96, 122.81, 119.35, 115.52, 38.65, 30.67.
Anal. calcd for C24H23BrN4OS: C 58.18, H 4.68, N
11.31; found C 58.00, H 4.66, N 11.29.
(E)-3-((2-(2-(3-Bromobenzylidene)hydrazinyl)-4-
(t-butyl)thiazol-5-yl)methyl)quinolin-2(1H)-one (7v)
Yellow solid, yield 55.0%, m.p. 181－183 ℃. 1H NMR
(400 MHz, DMSO-d6) δ: 11.90 (s, 1H, NH), 7.95 (s, 1H,
N＝CH), 7.75 (s, 1H), 7.66 (d, J＝7.4 Hz, 1H), 7.57 (d,
J＝4.3 Hz, 2H), 7.51 (d, J＝7.1 Hz, 1H), 7.46 (t, J＝7.7
Hz, 1H), 7.36－7.31 (m, 2H), 7.14 (t, J＝7.5 Hz, 1H),
4.01 (s, 2H, CH2), 1.32 (s, 9H, 3×CH3); 13C NMR (100
MHz, DMSO-d6) δ: 164.58, 161.82, 138.13, 137.05,
136.53, 132.99, 132.09, 131.28, 130.03, 128.67, 127.90,
125.69, 122.46, 122.15, 119.47, 116.35, 115.15, 35.95,
30.62, 27.48. Anal. calcd for C24H23BrN4OS: C 58.18, H
4.68, N 11.31; found C 57.98, H 4.67, N 11.27.
(E)-3-((2-(2-(4-Bromobenzylidene)hydrazinyl)-4-
(t-butyl)thiazol-5-yl)methyl)quinolin-2(1H)-one (7w)
Yellow solid, yield 45.1%, m.p. 264－267 ℃. 1H NMR
(400 MHz, CDCl3) δ: 13.21 (s, 1H, NH), 12.81 (s, 1H,
OH), 11.09 (s, 1H, NH), 8.14 (s, 1H, N＝CH), 7.67 (s,
1H), 7.59 (dd, J＝18.4, 7.7 Hz, 2H), 7.50－7.44 (m, 4H),
7.39 (d, J＝8.3 Hz, 1H), 7.31 (t, J＝7.3 Hz, 1H), 4.19 (s,
2H, CH2), 1.56 (s, 9H, 3×CH3); 13C NMR (100 MHz,
CDCl3) δ: 166.54, 162.89, 149.55, 144.05, 138.33,
138.05, 131.19, 131.01, 130.87, 130.38, 127.82, 127.57,
126.24, 123.20, 119.56, 116.43, 115.55, 35.39, 30.28,
27.64. Anal. calcd for C24H23BrN4OS: C 58.18, H 4.68,
N 11.31; found C 58.01, H 4.65, N 11.28.
NA inhibition assay
The NA inhibition assay was conducted using NA
isolated from a subtype of the influenza A/PR/8/34
(H1N1) virus. The substrate used in the enzyme inhibi-
tion assay, 2'-(4-methylumbelliferyl)-α-D-acetylneura-
minic acid sodium salt hydrate (MUNANA), was pur-
chased from Sigma. The NA inhibition assay was per-
formed according to the standard method.[34] The NA
substrate (MUNANA) was cleaved by NA to generate a
quantifiable fluorescent product.[35] The reaction mixture
was dissolved in DMSO consisting of the test compound,
a viral suspension (as the source of NAs), and MUN-
ANA (32.5 mmol/L) in MES buffer containing 4
mmol/L CaCl2 (pH 6.5). After incubation for 60 min at
37 ℃ in a black 96-well plate, then the reaction was
terminated by adding 150 μL of 34 mmol/L NaOH. The
resulting fluorescence was quantified at an excitation
(E)-3-((4-(t-Butyl)-2-(2-(2-nitrobenzylidene)-
408
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© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, 34, 403—411

3-((4-(t-Butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones
wavelength of 360 nm and an emission wavelength of
450 nm.[36] The 50% inhibitory concentration (IC50) was
calculated by plotting the percent of inhibition of NA
activity versus the inhibitor concentration.
using an NA activity assay. The inhibition rates of the
NA inhibitory activity of target compounds 7a－7z and
Oseltamivir (as a positive control) were shown in Table
1. As shown in Table 1, all target compounds showed
inhibition effects on influenza H1N1 virus NAs (inhibi-
tion rates 20.03%－54.83%), while the inhibition rate of
the positive control Oseltamivir was 72.6%, at the con-
centration of 40 μg/mL and 4 ng/mL, respectively.
Compounds 7a, 7c, 7e, 7l, 7p and 7w had moderate
NA inhibitory activity, their IC50 values were calculated
and shown in Table 2. As shown in Table 2, the pre-
liminary influenza NA inhibition assay indicated that
compound 7l had mild inhibitory activity (with IC50
value of 44.66 μmol/L), other compounds 7a, 7c, 7e, 7p
and 7w had less inhibitory activity relative to compound
7l (with IC50 values of 62.56, 55.90, 78.41, 69.32 and
49.08 μmol/L, respectively).
Docking study
In order to determine the interaction between
3-((4-(t-butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-
yl)methyl)-quinolin-2(1H)-ones possessing 2-(2-hydra-
zinyl)thiazole scaffold and the NA active site, the most
potent compound 7l was docked into the active sites of
NA (PDB entry: 3TI6) using LeDock (http://www.
lephar.com/) with default parameters. The binding
pocket was set as [Xmin＝37.4, Xmax＝19.8, Ymin＝4.0,
Y
max＝21.5, Zmin＝12.0, Zmax＝32.2] around the active
site. The results were analyzed and visualized using
PyMOL (http://pymol.sourceforge.net/).
Structure-activity relationship (SAR)
Results and Discussion
From the above results, some interesting structure-
activity relationships could be concluded. Table 2 also
revealed that the substituents of R had great influence on
the inhibition activity. For the scaffold of substituted
salicylaldehyde, non-substituted group was better than
substituted salicylaldehyde group, such as compounds
7a, 7c and 7e with IC50 of 62.56, 55.90 and 78.41
μmol/L, respectively. For the scaffold of substituted
benzaldehyde, electron donor substituents of R seemed
to decrease the activity, except compound 7l (with IC50
of 44.66 μmol/L), other compounds had low inhibitory
activity. Moreover, electron withdrawing substituents of
R had important effects on the inhibition activity and
halogen groups were better than nitro groups, such as
compounds 7p and 7w with IC50 of 69.32 and 49.08
μmol/L respectively. Also, as shown in Table 1, we
found that most compounds with chlorine and bromine
Compounds 7a－7z were accomplished using the
general method outlined in Scheme 1. Starting materials,
2-chloro-3-formylquinoline (1) and 2-oxo-3-formyl-
quinoline (2) were prepared according to the reported
procedures.[32] Compound 3 was synthesized by the al-
dol condensation reaction of pinacolone and 2-oxo-3-
formylquinoline (2) under basic conditions, and then
compound 4 was prepared by the reduction of com-
pound 3 with H2 in EtOH. Compound 5 was synthesized
by the reaction of compound 4 with CuBr2 in CHCl3 and
EtOAc. Compounds 6a－6z were prepared by thio-
semicarbazide and different substituted benzaldehyde or
substituted salicylaldehyde in EtOH. Target compounds
7a－7z were obtained in a simple one-pot cyclization
reaction of compounds 6a－6z and compound 5.
In our study, compounds 7a－7z were evaluated
Table 1 The inhibitory activities of the 3-((4-(t-butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones 7a－7z and
reference compound Oseltamivir against NA in vitro
Inhibition rate/%
(40 μg/mL)
Inhibition rate/%
(40 μg/mL)
Compd.
R
Compd.
R
7a
2-OH
48.57
39.81
47.63
35.29
47.12
40.20
33.09
44.86
20.03
34.52
37.66
54.83
34.04
72.6a
7n
7o
7p
7q
7r
7s
4-OCH3
2-F
36.22
45.24
47.56
41.41
37.35
31.53
41.87
21.29
25.56
43.36
39.50
45.16
35.17
7b
5-Cl-2-OH
5-Br-2-OH
5-NO2-2-OH
3,5-diCl-2-OH
3,5-diBr-2-OH
3-Br-5-NO2-2-OH
3,5-diNO2-2-OH
2-CH3
7c
3-F
7d
4-F
7e
2-Cl
7f
3-Cl
7g
7t
4-Cl
7h
7u
7v
7w
7x
7y
7z
2-Br
3-Br
4-Br
2-NO2
3-NO2
4-NO2
7i
7j
3-CH3
7k
4-CH3
7l
2-OCH3
7m
3-OCH3
Oseltamivir
a Oseltamivir at the concentration of 4 ng/mL.
Chin. J. Chem. 2016, 34, 403—411
© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
409

Fang et al.
FULL PAPER
Table 2 IC50 values of the NA inhibitory activity of compounds
7a, 7c, 7e, 7l, 7p, and 7w
binding modes. Comparison of the interaction models of
7l and Oseltamivir with A/H1N1-NA is shown in Fig-
ures 3A－3D. The predicted binding mode of 7l indi-
cates a binding interface largely non-overlapping with
that of Oseltamivir (Figure 3D). What’s more, differing
from the binding pocket of Oseltamivir, sialic acid
binding cavity (SA cavity), compound 7l targets both
SA cavity and adjacent hydrophobic cavity (430-cavity),
which is a promising dual-site mode in NA inhibitor
design (Figure 3C). As shown in Figure 3A, 3C, the
amide group of the quinoline ring formed hydrogen
bonds with Arg292 and Asn347. The thiazole ring
formed a hydrogen bond with Asp151, and the t-butyl
group may accommodate a small hydrophobic pocket
formed by the hydrocarbon chains of Ile149 and Pro431.
In addition, a C＝N…HO hydrogen bonding interac-
tion between the －NH－N＝C group and the phenolic
hydroxyl group of Tyr406 was formed, the －NH－
group could interact with Glu119 residue by hydrogen
bond for stabilizing the interaction. And the methoxy
oxygen of benzene ring formed hydrogen bonds with
Arg152. Also, the 2-(1H)-quinoline ring could binds to
the 430-cavity formed by Pro431, Lys432, Arg371 and
Asn347. All in all, compound 7l forms 7 hydrogen
bonds with side chains of key residues of Asp151,
Glu119, Arg292, Tyr406, and Asn347. Therefore, com-
pound 7l is most likely to be capable of inhibiting the
catalytic action of NA by a tight binding in the
Compound
R
IC50/(μmol•L1)
7a
2-OH
62.56
7c
5-Br-2-OH
3,5-diCl-2-OH
2-OCH3
3-F
55.90
7e
78.41
7l
44.66
7p
69.32
7w
4-Br
49.08
Oseltamivir
0.00159
groups at para positions of benzene ring had better in-
hibitory activity than those with chlorine and bromine
groups at ortho and meta position. For the substituted
fluorine and nitro, it could be easy found that fluorine
groups had important effect on the inhibition activity
than nitro groups. And fluorine or nitro substituent at the
meta position of benzene ring would increase the activ-
ity. Therefore, in the present study, compound 7l exhib-
ited mild inhibitory activity, and it could be used as a
good representative of compounds 7a－7z for further
studies.
Molecular docking
In this study, the most potent compound 7l was
docked into the active site of NA to understand their
Figure 3 Comparison of the interaction models of 7l and Oseltamivir with A/H1N1-NA (3TI6). (A-B) Binding models of 7l (A) and
Oseltamivir (B) with A/H1N1-NA. 7l and Oseltamivir are shown in yellow sticks. The H-bond interactions are depicted as blue dashed
lines. (C) Diagram of neuraminidase active site with 7l. (D) Detailed comparison of the interactions of 7l (in cyan stick) and Oseltamivir
(in green thin stick) with A/H1N1-NA by structural superposition.
410
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© 2016 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2016, 34, 403—411

3-((4-(t-Butyl)-2-(2-benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-2(1H)-ones
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active site through the multiple hydrogen bond and hy-
drophobic interactions. As shown in Figure 3B, Osel-
tamivir forms 9 solid hydrogen bonds with side chains
of key residues of Asp151, Arg152, Glu119, Arg292,
Arg371 and Arg118. Thus, Comparing to Oseltamivir,
compounds 7l could not fit very well into the catalytic
site of the NA, which may lead to mild NA inhibitory
activity. Although compound 7l exhibited mild inhibi-
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binding modes, making it fascinating to further explore
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Conclusions
In this paper, a series of novel 3-((4-(t-butyl)-2-(2-
benzylidenehydrazinyl)thiazol-5-yl)methyl)quinolin-
2(1H)-ones were synthesized and evaluated for their
ability to inhibit NA of influenza H1N1 virus. Among
them, compound 7l (IC50＝44.66 μmol/L) was the best
compound with mild NA inhibitory activity. The dock-
ing analysis indicated that it has important interactions
with some key residues (including Asp151, Glu119,
Arg292, Tyr406, and Asn347) in the NA active site and
2-(1H)-quinoline ring could binds to the 430-cavity ad-
jacent to the active site. Compound 7l showed moderate
potent NA inhibitory activity, which can provide attrac-
tive structural bases to explore novel NA inhibitors.
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Acknowledgement
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This work was supported by the National Natural
Science Foundation of China (No. 21442014).
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