Design, synthesis and neuraminidase inhibitory activity of N-(5-benzyl-4-(tert-butyl)thiazol-2-yl)benzamides

Article abstract of DOI:10.1007/s00044-015-1487-5

A series of N-(5-benzyl-4-(tert-butyl)thiazol-2-yl)benzamides were synthesized and the structures were characterized by ¹H NMR, MS and elemental analyses. The crystal structures of compounds F5 and F16 were determined by single-crystal X-ray diffraction. The neuraminidase inhibitory activities of compounds F1-F32 were evaluated in vitro at the concentration of 40 μg/mL. The results indicated that compounds F8, F26 and F32 exhibited most potent inhibitory activity against NA. Molecular docking was performed by LeDock to further explain the structure-activity relationship of compound F26. The docking modeling showed that compound F26 was in good combination with oseltamivir binding sites of NA and could be a potential NA inhibitor agent.

Full text of DOI:10.1007/s00044-015-1487-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1487-5
ORIGINAL RESEARCH
Design, synthesis and neuraminidase inhibitory activity of
N-(5-benzyl-4-(tert-butyl)thiazol-2-yl)benzamides
Zhilin Wu1 Junmei Peng1 Aixi Hu1 Jiao Ye1 Guoxi Li1
•
•
•
•
Received: 13 February 2015 / Accepted: 15 December 2015
Ó Springer Science+Business Media New York 2015
Abstract A series of N-(5-benzyl-4-(tert-butyl)thiazol-2-
yl)benzamides were synthesized and the structures were
characterized by 1H NMR, MS and elemental analyses. The
crystal structures of compounds F5 and F16 were deter-
mined by single-crystal X-ray diffraction. The neu-
raminidase inhibitory activities of compounds F1–F32
were evaluated in vitro at the concentration of 40 lg/mL.
The results indicated that compounds F8, F26 and F32
exhibited most potent inhibitory activity against NA.
Molecular docking was performed by LeDock to further
explain the structure–activity relationship of compound
F26. The docking modeling showed that compound F26
was in good combination with oseltamivir binding sites of
NA and could be a potential NA inhibitor agent.
(Hay et al., 2001). Up to now, 16 hemagglutinin antigen
(HA) subtypes (H1–H16) and 9 neuraminidase (NA) sub-
types (N1–N9) have been reported. Some of them, such as
H1N1, H2N2, H3N2, H5H1, and H7N7, have caused sev-
eral large-scale influenza pandemic in history, resulting in
more than 100 millions of deaths (Potter, 2001; Hilleman,
2002; Dawood et al., 2012). In March 2009, pandemic
influenza caused by H1N1 broke out in Mexico and spread
to the rest of the world rapidly (Donaldson et al., 2009).
Till July 2010, more than 214 countries and overseas ter-
ritories or communities had reported laboratory-confirmed
cases, including at least 18,398 deaths (WHO, 2010).
Recently, H7N9 (Zhu et al., 2013) and H10N8 (Wappes,
2013) were first found to be infectious to human and cause
death.
Keywords Benzamide Á Thiazole Á
NA inhibitory activity Á Crystal structure Á
Molecular docking
Currently, there are two classes of drugs to treat
influenza: M2 inhibitors and NA inhibitors (Ison, 2012).
Amantadine and rimantadine are two representatives of M2
inhibitors, which act specifically against influenza A by
blocking the ion channel of the M2 protein. But the pro-
tection they provided is very limited due to their narrow
spectrum of activity (De Clercq, 2001). NA inhibitors are
the most effective agents in treating both influenza A and
B. Zanamivir and oseltamivir are two NA inhibitors that
approved in most countries, while laninamivir and per-
amivir are accepted in more limited market (Baz et al.,
2007). Although zanamivir-resistant strains are rarely
reported, oseltamivir-resistant strains in H5N1 and H1N1
have emerged in some areas (Storms et al., 2012). Osel-
tamivir was reported to cause vomiting and nausea despite
its good oral activity (Sun et al., 2006). Zanamivir has very
low oral bioavailability compared to its excellent antiviral
activity when administered intranasally (Ryan et al., 1995;
Abed et al., 2008). Thus, it is an urgent task to develop new
and more effective antiviral drugs.
Introduction
Influenza, commonly known as ‘‘the flu,’’ is an infectious
disease caused by the influenza virus (type A, B and
C) (Stamboulian et al., 2000; Chang et al., 2007). The type
A viruses are the most virulent human pathogens among
the three influenza types and cause the most severe disease.
The influenza A virus can be subdivided into different
serotypes based on the antibody response to these viruses
& Aixi Hu
axhu@hnu.edu.cn
1
College of Chemistry and Chemical Engineering, Hunan
University, Changsha, Hunan Province, China
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Med Chem Res
Thiazole is an important heterocycle that shows wide
excellent pharmacological properties, and is the core
structure of many clinical preferred first-line drugs such as
antibiotics cefixime (Moghadam et al., 2010), anticar-
cinogen dasatinib (Teoh et al., 2011) and antiphlogistic
meloxicam (Ah et al., 2010). In recent years, thiazole
derivatives were also developed as influenza NA inhibitors
and showed good activity (Kessler et al., 2010; Liu et al.,
2011; Rossignal, 2012). From another aspect, the crystal
structure of NA inhibitor and the structure–activity rela-
tionship (Wang et al., 2001; Wang and Wade, 2001; Sun
et al., 2006; Vavricka et al., 2011) were studied. In our
previous studies, 1,3-thiazine derivatives 1 (Li et al.,
2013a) and 2 (Li et al., 2013b) were found to possess
potent NA inhibitory activity. For further modification and
optimization, thiazole ring was selected to replace thiazine
ring, and a series of N-(5-benzyl-4-(tert-butyl)thiazol-2-
yl)benzamides were synthesized (Fig. 1).
Results and discussion
Synthesis
Compounds F1–32 were prepared by the methodologies
outlined in Scheme 1. The synthesis of compounds C1–2
was carried out by hydrogenation catalyzed by newly
prepared Raney Ni. Compounds C3–4 were synthesized by
addition reaction of unsaturated ketones (B1–2) and nitro-
methane. Reacting thiourea with a-brominated ketones
D1–4 led to compounds E1–4. The acylation reactions of
2-aminothiazoles (E1–4) with benzoic acids (G1–10) were
conducted in the presence of dicyclohexyl carbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP) (Stocks et al.,
1995; Zhang et al., 2004) and generated 32 target com-
pounds (F1–32). The yields of compounds F with sub-
stituent Y = CH2NO2 were much lower than those with
substituent Y = H, because the nitro group reduced the
Fig. 1 Design of the target compounds
Scheme 1 Synthesis of thiazole derivatives F1-32. Reagents and
conditions: (a) pinacolone, NaOH, EtOH, ice-bath, 0.5 h, then 60 °C,
2 h; (b1) H2, Raney Ni, 3 MPa, EtOH, 80 °C, 2 h; (b2) MeNO2,
K2CO3, EtOH, reflux, 2–3 h; (c1) CuBr2, EtOH, reflux, 4 h; (c2) Br2,
CHCl3, reflux, 1 h; (d) (1) thiourea, EtOH reflux, 7.5 h (E1 and E2),
18 h (E3 and E4); (2) aqueous ammonia; (e) DMAP, DCC, CH2Cl2,
rt, 1.5–24 h
123

Med Chem Res
electron cloud density of the NH2 and increased the diffi-
culty of electrophilic substitution.
and 164.2°, 141.6°, 178.2°. From the crystal structure of
compound F16 (Fig. 2), we can see that the molecule is
made up of three planes including the 4-nitrophenyl ring
(Plane 1), thiazole ring (Plane 2) and 2,4-dichlorophenyl
ring (Plane 3). The dihedral angle between Plane 1 and
Plane 2 is 13.199°, while the one between Plane 2 and Plane
3 is 87.899°, almost perpendicular.
Crystal structure
Compounds F5 and F16 were determined by single-crystal
X-ray diffraction. The crystal data of compound F5 has been
deposited at the Cambridge Crystallographic Data Centre as
supplementary publication no. 950255 and published in our
previous article (Shen et al., 2014). The crystal data and
structure refinement for compounds F5 and F16 are shown
in Table 1. Different to the normal empirical formula of
compound F5, the empirical formula of compound F16 has
two extra H atoms and one extra O atom because each
molecule of the crystal is companied by a water molecule
(shown in Figs. 2, 3). The water molecule plays an impor-
tant role in maintaining stability of the crystal structure by
forming three intermolecular hydrogen bonds: O(4)–
H(4A)ÁÁÁN(1), N(2)–H(2)ÁÁÁO(4) and O(4)–H(4B)ÁÁÁO(1).
Anti-NA activity assay
Compounds F1–32 were preliminarily evaluated for their
NA inhibitory activity in vitro at the concentration of
40 lg/mL. The results showed that most of the compounds
displayed good inhibitory activity against NA (Table 2).
Among all the tested compounds, F8, F26 and F32 had the
best inhibitory activities with inhibition rate of 45.56, 59.89
and 49.63 %, respectively.
The interaction between NA active sites and substituent
groups is mainly an electrostatic interaction which depends
on the relative structure and position of the functional
˚
The distances and angles are respectively 2.05, 2.42, 2.01 A
Table 1 Crystal data and structure refinement for compounds F5 and F16
Compound
F5
F16
Empirical formula
Formula weight
Temperature
C21H19ClF2N2OS
420.89
C21H21Cl2N3O4S
482.37
173(2) K
173(2) K
˚
0.71073 A
˚
0.71073 A
Wavelength
Crystal system
Space group
Triclinic
P-1
Triclinic
P-1
˚
˚
Unit cell dimensions
a = 12.6479(13) A, a = 110.227(2)°
a = 7.7746(9) A, a = 109.031(2)°
˚
˚
b = 13.1204(13) A, b = 106.763(2)°
b = 11.1100(13) A, b = 100.859(2)°
˚
˚
c = 14.1341(15) A, c = 99.676(2)°
2011.5(4) A
c = 14.2061(17) A, c = 101.269(2)°
1094.8(2) A
3
˚
3
˚
Volume
Z
4
2
Density (calculated)
Absorption coefficient
F(000)
1.390 Mg/m3
0.326 mm-1
1.463 Mg/m3
0.426 mm-1
872
0.43 9 0.41 9 0.26 mm3
500
0.43 9 0.41 9 0.26 mm3
Crystal size
Theta range for data collection
Index ranges
1.83–26.00°
-15 B h B 15, -16 B k B 16, -17 B l B 17
15,736
2.02–26.00°
-9 B h B 9, -13 B k B 13, -17 B l B 17
8578
Reflections collected
Independent reflections
Completeness to theta = 26.00°
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I [ 2sigma(I)]
R indices (all data)
Largest diff. peak and hole
7779 [R(int) = 0.0186]
98.2 %
4249 [R(int) = 0.0165]
98.3 %
Semi-empirical from equivalents
0.9201 and 0.8726
Full-matrix least-squares on F2
7779/0/517
Semi-empirical from equivalents
0.8973 and 0.8381
Full-matrix least-squares on F2
4249/0/286
1.023
1.061
R1 = 0.0365, wR2 = 0.0880
R1 = 0.0507, wR2 = 0.0978
-3
R1 = 0.0363, wR2 = 0.0940
R1 = 0.0428, wR2 = 0.0982
-3
˚
˚
0.374 and -0.218 e.A
0.377 and -0.361 e.A
123

Med Chem Res
away from the active sites. Tert-butyl and acylamino were
hydrophobic groups which could enhance the bioavailability
of the compounds. Tert-butyl could also be accommodated
in the hydrophobic pocket of SA cavity and enhance the
combining capacity. The result indicated that the structure of
compound F26 could bind to the active sites of NA quite
well and could be used as a lead compound for further
design of NA inhibitors (Fig. 4).
Molecular dock
To further understand the structure–activity relationship
(SAR) of the target compounds, molecular docking was per-
formed by LeDock (http://lephar.com/) with the simulations
of compounds F8, F26 and F32 into the active sites of pH1N1
N1 NA (p09N1) comparing the binding mode of p09N1-
oseltamivir (PDBcode:3TI6). Thedockingresult showedthat
compound F26 was nicely accommodated in SA cavity
(Fig. 5a). To make a judgment of the accuracy of the docking
parameters, oseltamivir (acid form) was redocked to p09N1.
The docking modeling coincided with the original ligand in
the protein very well (Fig. 5b), which proved that the docking
parameters were appropriate. The interactions between com-
poundF26andactivesitesofp09N1wereanalyzedbyLigPlot
(Laskowski and Swindells, 2011) and shown in Fig. 6.
Fig. 2 Crystal structure of compound F16
groups (Hu and Wang, 2004). That is why the tested com-
pounds with different substituents or with the same sub-
stituents but at different positions had absolutely different
inhibitory activities. For substituent Z, 4-NO2 was the most
effective group in improving the inhibitory activity of the
compounds. Almost all the compounds with 4-NO2 (F8, F16
and F26) had higher inhibition rate compared with those
with the same group X and Y but different group Z. The only
exception was compound F32 which had two nitro groups at
the 3- and 5-position and displayed a good activity similar to
compound F26. On the other hand, the replacement of –H
with –CH2NO2 at benzyl position could also enhance the
activity of the compounds. The reason to explain why those
nitro-substituted compounds showed the best activity might
be that the nitro group could provide oxygen atoms as
hydrogen bond receptors from the key amino acid residues
(Arg118, Arg292 and Arg371). It was further verified by
molecular docking performed by LeDock. As for the sub-
stituent group Y on the other benzene ring, there was not
much difference between the inhibition rate of compounds
with 4-Cl and 2,4-diCl, because this benzene ring was far
The binding energy for the best binding mode of com-
pounds F8, F26 and F32 were respectively -7.51, -8.91,
and -9.43 kcal/mol, while the one of redocked oseltamivir
was -6.57 kcal/mol. The comparison of compound F26
and oseltamivir binding to p09N1 showed that although the
molecule of compound F26 was much bigger than oselta-
mivir, the key active groups were quite coincident and
played an analogous role in binding the ligands to the
active cavity. For substituent Y, the nitro group of –CH2
NO2, similar to the carboxyl of oseltamivir, played a sig-
nificant role in the conformation with the key amino acid
Fig. 3 Hydrogen bond diagram
of compound F16 (H atoms
bonded to C atoms have been
omitted)
123

Med Chem Res
Table 2 Inhibitory activity of compounds F1-32 against NA in vitro (40 lg/mL)
Compd
X
Y
Z
inhibition
rate/ %
Compd
X
Y
Z
inhibition
rate/ %
F1
4-Cl
H
2-CH3O
\10
F18
2,4-
diCl
H
2-CH3;3-
NO2
12.94
F2
F3
F4
F5
F6
F7
F8
F9
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
H
H
H
H
H
H
H
H
2-Cl
\10
F19
F20
F21
F22
F23
F24
F25
F26
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
2-CH3O
2-Cl
\10
4-Cl
\10
19.03
43.19
\10
2,6-diCl
2,6-diF
2-CF3
\10
4-Cl
15.19
\10
2,6-diCl
2,6-diF
2-CF3
16.35
29.89
43.66
59.89
3,5-diCF3
4-NO2
10.06
45.56
33.54
3,5-diCF3
4-NO2
3,5-
diNO2
F10
F11
4-Cl
2,4-
H
H
2-Cl
4-Cl
17.93
16.68
F27
F28
4-Cl
4-Cl
CH2NO2
CH2NO2
3,5-diNO2
42.94
16.18
2-CH3;3-
NO2
diCl
2,4-
diCl
2,4-
diCl
2,4-
diCl
2,4-
diCl
2,4-
diCl
2,4-
diCl
F12
F13
F14
F15
F16
F17
a
H
H
H
H
H
H
2,6-diCl
2,6-diF
2-CF3
\10
F29
2,4-
CH2NO2
CH2NO2
CH2NO2
CH2NO2
2,6-diF
\10
diCl
2,4-
diCl
2,4-
diCl
2,4-
diCl
\10
F30
2-CF3
11.55
37.10
49.63
72.6
\10
F31
4-NO2
3,5-diCF3
4-NO2
13.86
36.94
25.70
F32
3,5-diNO2
Oseltamivira
3,5-
diNO2
The concentration of oseltamivir was 40 ng/mL
Fig. 4 Structure–activity
relationship of compound F
residues surrounded which were stabilized by four hydro-
gen bonds. Two of them were formed between the two
oxygen atoms of the nitro group and the guanidyl of
Arg371. The other two hydrogen bonds were formed by the
two oxygen atoms with the guanidyl of Arg292 and
Arg118, respectively. In addition to these hydrogen bonds,
hydrophobic interaction was also found in the docking
modeling (Fig. 6), which mainly took shape between the
tert-butyl of compound F26 and the hydrophobic chain of
Glu276 and Glu277. Besides, some weak hydrophobic
interactions were found to exist between compound F26
and the hydrophobic chains of the amino acid residues
surrounded such as Arg224, Ser179, Trp178 and Ser246.
Materials and methods
Chemistry
All reagents and solvents needed in the experiment were of
analytical reagents grade and could be used directly with-
out further purification. Melting points were measured on
123

Med Chem Res
Fig. 5 Molecular docking modeling of compound F26 with p09N1. a Comparison of compound F26 (green) and oseltamivir (magenta) binding
to p09N1. b Comparison of redocked oseltamivir (yellow) and original ligand (magenta) (Color figure online)
Fig. 6 Interactions between compound F26 and the oseltamivir binding site. H-bonds are displayed as green dashed lines. Hydrophobic
interactions are indicated as red opposite arcs (Color figure online)
123

Med Chem Res
(CDCl3, 400 MHz), d:1.11 (s, 9H, 3 9 CH3), 4.53–5.30
(m, 4H, 2 9 CH, CH2), 7.23–7.41 (m, 3H, C6H3).
an X-4 electrothermal digital melting point apparatus and
1
uncorrected. H NMR spectra were recorded on a Bruker
advanced instrument with TMS as internal standard at
400 MHz with chemical shifts (d) expressed in ppm. EI-
MS spectra were recorded on a Finnigan LCQ advantage
mass spectrometer. Elemental analyses were performed on
a Vario EL III (Germany) instrument.
General procedures for the synthesis of compounds E3–E4
A mixture of 5.0 mmol compound D3 or D4, 0.5 mmol
thiourea and 50 mL ethanol was stirred for 18 h under
reflux. The mixture was concentrated under reduced pres-
sure to remove some of the solvent, neutralized by aqueous
ammonia and then moved into a refrigerator to separate out
light yellow solid overnight.
The compounds in series B, C, D and E were synthe-
sized according to reported methods (Hu and Cao, 2011;
Shen et al., 2012; Gresser et al., 2011) (Scheme 1).
General procedures for the synthesis of compounds C3–C4
4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thiazol-
1
2-amine (E3) Yield 82 %, m.p.: 148–151 °C. H NMR
A mixture of 15 mmol compound B, 75 mmol nitro-
methane and 1.5 mmol potassium carbonate in 50 mL
ethanol was stirred for 3 h under reflux. When the mixture
was cooled to room temperature, 50 mL water was added.
The mixture was moved into a refrigerator to separate out
white solid overnight.
(DMSO-d6, 400 MHz), d: 1.30 (s, 9H, 3 9 CH3),
4.96–5.02 (m, 1H, CH), 5.26–5.41 (m, 2H, CH2), 6.94 (br,
2H, NH2), 7.42 (s, 4H, C6H4).
4-(Tert-butyl)-5-(1-(2,4-dichlorophenyl)-2-nitroethyl)thia-
zol-2-amine (E4) Yield 65 %, m.p.: 165–167 °C. 1H
NMR (CDCl3, 400 MHz), d: 1.29 (s, 9H, 3 9 CH3), 4.55
(dd, J = 13.6 Hz, J = 9.6 Hz, 1H, CH2NO2), 4.86 (dd,
J = 13.6 Hz, J = 9.6 Hz, 1H, CH2NO2), 5.13 (br, 2H,
NH2), 5.88 (t, J = 9.6 Hz, 1H, CH), 7.25 (dd, J = 8.4 Hz,
J = 2.0 Hz, 1H, C6H3 5-H), 7.34 (d, J = 8.4 Hz, 1H, C6H3
6-H), 7.45 (d, J = 2.0 Hz, 1H, C6H3 3-H).
5-(4-Chlorophenyl)-2,2-dimethyl-6-nitrohexan-3-one (C3)
Yield 91 %, m.p.: 68–70 °C. 1H NMR (CDCl3, 400 MHz),
MHz), d: 1.08 (s, 9H, 3 9 CH3), 2.89 (dd, J = 17.6 Hz,
J = 7.2 Hz, 1H, CH2), 2.98 (dd, J = 17.6 Hz, J = 7.2 Hz,
1H, CH2), 4.02–4.05 (m, 1H, CH), 4.60 (dd, J = 12.0 Hz,
J = 7.2 Hz, 1H, O2NCH2), 4.68 (dd, J = 12.0 Hz,
J = 7.2 Hz, 1H, O2NCH2), 7.17 (d, J = 8.0 Hz, 2H, C6H4
2,6-H), 7.30 (d, J = 8.0 Hz, 2H, C6H4 3,5-H).
General procedures for the synthesis of compounds F1–32
5-(2,4-Dichlorophenyl)-2,2-dimethyl-6-nitrohexan-3-one (C4)
Yield 88 %, m.p.: 84–86 °C. 1H NMR (CDCl3,
400 MHz), d: 1.12 (s, 9H, 3 9 CH3), 3.03–3.06 (m, 2H,
CH2), 4.42–4.43 (m, 1H, CH), 4.75 (d, J = 6.0 Hz, 2H,
CH2NO2), 7.15 (d, J = 8.4 Hz, 1H, C6H3 6-H), 7.22 (dd,
J = 8.4 Hz, J = 2.0 Hz, 1H, C6H3 5-H), 7.42 (d,
J = 2.0 Hz, 1H, C6H3 3-H).
A mixture of 2-aminothiazole (E1–4, 1.5 mmol), benzoic
acid (G1–10, 1.6 mmol) and DMAP (1.5 mmol) in
dichloromethane (40 mL) was stirred at room temperature
for 0.5 h, and then dicyclohexylcarbodiimide (DCC,
1.6 mmol) was added. The reaction mixture was kept
stirring at room temperature and detected by thin-layer
chromatography (Vpetroleum ether:Vethyl acetate = 5:1). When
the reaction was completed, the mixture was filtered to
remove the undissolved N, N0-dicyclohexylurea (DCU).
The filtrate was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure. The crude products
were purified by column chromatography on silica gel
using a gradient of petroleum ether and ethyl acetate as
eluent to give the desired products (F1–32).
General procedures for the synthesis of compounds D3–D4
A mixture of 10 mmol compounds C3 or C4, 3 drops of
acetone and 15 mL chloroform was heated to reflux, fol-
lowed by the addition of a solution of 12 mmol bromine in
5 mL chloroform. The mixture was stirred for another 1 h,
concentrated under reduced pressure to remove the solvent
and recrystallized in ethanol to afford compound D3 or D4
as yellow solid.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-2-methoxy-
benzamide (F1) Yield 70 %, m.p.: 142–143 °C. 1H NMR
(CDCl3, 400 MHz), d: 1.38 (s, 9H, 3 9 CH3), 4.10 (s, 3H,
OCH3), 4.23 (s, 2H, CH2), 7.05 (d, J = 8.0 Hz, 1H, CH3
OC6H4 3-H), 7.10–7.14 (m, 3H, C6H4Cl 2,6-H, CH3OC6H4
5-H), 7.26 (d, J = 8.4 Hz, 2H, C6H4Cl 3,5-H), 7.10–7.56
(m, 1H, CH3OC6H4 4-H), 8.25 (dd, J = 8.0 Hz,
J = 2.0 Hz, 1H, CH3OC6H4 6-H), 10.82 (bs, 1H, NHCO).
ESI–MS (m/z): 413.1[M-1]. Anal. Calad. (%): C, 63.68; H,
4-Bromo-5-(4-chlorophenyl)-2,2-dimethyl-6-nitrohexan-3-
one (D4) Yield 86 %, m.p.: 117–119 °C. 1H NMR
(CDCl3, 400 MHz), d: 0.94 (s, 9H, 3 9 CH3), 4.22–5.22
(m, 4H, 2 9 CH, CH2), 7.15 (d, J = 8.4 Hz, 2H, C6H4 2,6-
H), 7.29 (d, J = 8.4 Hz, 2H, C6H4 3,5-H).
4-Bromo-5-(2,4-dichlorophenyl)-2,2-dimethyl-6-nitrohexan-
3-one (D4) Yield 60 %, m.p.: 129–131 °C. 1H NMR
123

Med Chem Res
5.59; N, 6.75; S, 7.73. Found (%): C, 63.67; H, 5.58; N,
6.76; S, 7.75.
C6H4 2,6-H), 7.29 (d, J = 8.0 Hz, 2H, C6H4 3,5-H), 8.05
(s, 1H, C6H3 4-H), 8.47 (s, 2H, C6H3 2,6-H), 9.76 (bs, 1H,
NHCO). ESI–MS (m/z): 519.1[M-1]. Anal. Calad. (%): C,
53.03; H, 3.68; N, 5.38; S, 6.16. Found (%): C, 52.97; H,
3.72; N, 5.33; S, 6.23.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-2-chloroben-
zamide (F2) Yield 66 %, m.p.: 105–107 °C. 1H NMR
(CDCl3, 400 MHz), d: 1.34 (s, 9H, 3 9 CH3), 4.24 (s, 2H,
CH2), 7.15 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.27 (d,
J = 8.4 Hz, 2H, C6H4 3,5-H), 7.35–7.48 (m, 3H, C6H4
4,5,6-H), 7.79 (d, J = 8.8 Hz, 1H, C6H4 3-H), 9.47 (bs,
1H, NHCO). ESI–MS (m/z): 417.1[M-1]. Anal. Calad.
(%): C, 60.14; H, 4.81; N, 6.68; S, 7.65. Found (%): C,
60.03; H, 4.87; N, 6.62; S, 7.76.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-4-nitroben-
zamide (F8) Yield 67 %, m.p.: 184–185 °C. 1H NMR
(CDCl3, 400 MHz), d: 1.37 (s, 9H, 3 9 CH3), 4.24 (s, 2H,
CH2), 7.15 (d, J = 8.4 Hz, 2H, C6H4Cl 2,6-H), 7.28 (d,
J = 8.4 Hz, 2H, C6H4Cl 3,5-H), 8.13 (d, J = 8.8 Hz, 2H,
C6H4NO2 2,6-H), 8.34 (d, J = 8.8 Hz, 2H, C6H4NO2 3,5-
H), 9.75 (bs, 1H, NHCO). ESI–MS (m/z): 428.1[M-1].
Anal. Calad. (%): C, 58.67; H, 4.69; N, 9.77; S, 7.46.
Found (%): C, 58.70; H, 4.72; N, 9.75; S, 7.43.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-4-chloroben-
zamide (F3) Yield 80 %, m.p.: 115–117 °C. 1H NMR
(CDCl3, 400 MHz), d: 1.36 (s, 9H, 3 9 CH3), 4.24 (s, 2H,
CH2), 7.14 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 7.27 (d,
J = 8.0 Hz, 2H, C6H4 3,5-H), 7.48 (d, J = 8.8 Hz, 2H,
COC6H4 3,5-H), 7.88 (d, J = 8.8 Hz, 2H, COC6H4 2,6-H),
9.49 (bs, 1H, NHCO). ESI–MS (m/z): 417.1[M-1]. Anal.
Calad. (%): C, 60.14; H, 4.81; N, 6.68; S, 7.65. Found (%):
C, 60.09; H, 4.83; N, 6.61; S, 7.75.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-3,5-dini-
trobenzamide (F9) Yield 69 %, m.p.: 93–95 °C. 1H
NMR (CDCl3, 400 MHz), d: 1.47 (s, 9H, 3 9 CH3), 4.20
(s, 2H, CH2), 7.16 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.31
(d, J = 8.4 Hz, 2H, C6H4 3,5-H), 9.15 (d, J = 2.0 Hz, 1H,
C6H3 4-H), 9.31 (d, J = 2.0 Hz, 2H, C6H3 2,6-H), 10.22
(bs, 1H, NHCO). ESI–MS (m/z): 473.1[M-1]. Anal. Calad.
(%): C, 53.11; H, 4.03; N, 11.80; S, 6.75. Found (%): C,
53.08; H, 4.09; N, 11.72; S, 6.80.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-2,6-
dichlorobenzamide (F4) Yield 72 %, m.p.: 173–176 °C.
1H NMR (CDCl3, 400 MHz), d: 1.33 (s, 9H, 3 9 CH3),
4.23 (s, 2H, CH2), 7.16 (d, J = 8.4 Hz, 2H, C6H4 2,6-H),
7.29 (d, J = 8.4 Hz, 2H, C6H4 3,5-H), 7.32–7.37 (m, 3H,
C6H3), 9.13 (bs, 1H, NHCO). ESI–MS (m/z): 451.0[M-1].
Anal. Calad. (%): C, 55.58; H, 4.22; N, 6.17; S, 7.07.
Found (%): C, 55.52; H, 4.29; N, 6.11; S, 7.12.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-2-methyl-
3-nitrobenzamide (F10) Yield 68 %, m.p.: 157–158 °C.
1H NMR (CDCl3, 400 MHz), d: 1.37 (s, 9H, 3 9 CH3),
2.59 (s, 3H, CH3), 4.25 (s, 2H, CH2), 7.16 (d, J = 8.4 Hz,
2H, C6H4 2,6-H), 7.29 (d, J = 8.4 Hz, 2H, C6H4 3,5-H),
7.45 (t, J = 8.0 Hz, 1H, C6H3 5-H), 7.72 (d, J = 8.0 Hz,
1H, C6H3 6-H), 7.93 (d, J = 8.0 Hz, 1H, C6H3 4-H), 9.06
(bs, 1H, NHCO). ESI–MS (m/z): 442.1[M-1]. Anal. Calad.
(%): C, 59.52; H, 4.99; N, 9.47; S, 7.22. Found (%): C,
59.45; H, 5.08; N, 9.40; S, 7.27.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-2,6-diflu-
1
orobenzamide (F5) Yield 84 %, m.p.: 130–132 °C. H
NMR (CDCl3, 400 MHz), d: 1.35 (s, 9H, 3 9 CH3), 4.24
(s, 2H, CH2), 7.02–7.03 (m, 2H, C6H3 3,5-H), 7.15 (d,
J = 8.4 Hz, 2H, C6H4 2,6-H), 7.28 (d, J = 8.4 Hz, 2H,
C6H4 3,5-H), 7.43–7.51 (m, 1H, C6H3 4-H), 9.22 (bs, 1H,
NHCO). ESI–MS (m/z): 419.1[M-1]. Anal. Calad. (%): C,
59.92; H, 4.55; N, 6.66; S, 7.62. Found (%): C, 59.90; H,
4.52; N, 6.64; S, 7.61.
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-2-
chlorobenzamide (F11) Yield 78 %, m.p.: 148–150 °C.
1H NMR (CDCl3, 400 MHz), d: 1.34 (s, 9H, 3 9 CH3),
4.30 (s, 2H, CH2), 7.08 (d, J = 8.4 Hz, 1H, C6H3 6-H),
7.18 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, C6H3 5-H),
7.39–7.81 (m, 5H, ClC6H3 3-H, C6H4), 9.50 (bs, 1H,
NHCO). ESI–MS (m/z): 451.0[M-1]. Anal. Calad. (%): C,
55.58; H, 4.22; N, 6.17; S, 7.07. Found (%): C, 55.61; H,
4.27; N, 6.15; S, 7.05.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-2-(triflu-
oromethyl)benzamide
(F6) Yield
71 %,
m.p.:
1
125–127 °C. H NMR (CDCl3, 400 MHz), d: 1.34 (s, 9H,
3 9 CH3), 4.23 (s, 2H, CH2), 7.16 (d, J = 8.4 Hz, 2H,
C6H4Cl 2,6-H), 7.28 (d, J = 8.4 Hz, 2H, C6H4Cl 3,5-H),
7.63–7.64 (m, 3H, C6H4CF3 3,4,5-H), 7.77–7.79 (m, 1H,
C6H4CF3 6-H), 8.91(bs, 1H, NHCO). ESI–MS (m/z):
450.9[M-1]. Anal. Calad. (%): C, 58.34; H, 4.45; N, 6.19;
S, 7.08. Found (%): C, 58.23; H, 4.51; N, 6.13; S, 7.19.
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-4-
chlorobenzamide (F12) Yield 75 %, m.p.: 137–140 °C.
1H NMR (CDCl3, 400 MHz), d: 1.36 (s, 9H, 3 9 CH3),
4.29 (s, 2H, CH2), 7.06 (d, J = 8.4 Hz, 1H, C6H3 6-H),
7.18 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H, C6H3 5-H), 7.41 (d,
J = 2.4 Hz, 1H, C6H3 3-H), 7.49 (d, J = 8.8 Hz, 2H, C6H4
3,5-H), 7.89 (d,, J = 8.8 Hz, 2H, C6H4 2,6-H), 9.57 (bs,
1H, NHCO). ESI–MS (m/z): 451.0[M-1]. Anal. Calad.
N-(4-(Tert-butyl)-5-(4-chlorobenzyl)thiazol-2-yl)-3,5-bis(tri-
fluoromethyl)benzamide
1
(F7) Yield
58–61 °C. H NMR (CDCl3, 400 MHz), d: 1.39 (s, 9H,
3 9 CH3), 4.22 (s, 2H, CH2), 7.15 (d, J = 8.0 Hz, 2H,
73 %,
m.p.:
123

Med Chem Res
(%): C, 55.58; H, 4.22; N, 6.17; S, 7.07. Found (%): C,
55.60; H, 4.28; N, 6.12; S, 7.02.
1H NMR (CDCl3, 400 MHz), d: 1.34 (s, 9H, 3 9 CH3),
2.60 (s, 3H, CH3), 4.31 (s, 2H, CH2), 7.08 (d, J = 8.0 Hz,
1H, 2,4-diClC6H3 6-H), 7.21 (dd, J = 8.0 Hz, J = 2.0 Hz,
1H, 2,4-diClC6H3 5-H), 7.43–7.94 (m, 4H, 2,4-diClC6H3
3-H, C6H3), 9.13 (bs, 1H, NHCO). ESI–MS (m/z):
476.1[M-1]. Anal. Calad. (%): C, 55.23; H, 4.42; N, 8.78;
S, 6.70. Found (%): C, 55.20; H, 4.46; N, 8.75; S, 6.72.
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-2,6-
difluorobenzamide (F13) Yield 78 %, m.p.: 133–135 °C.
1H NMR (CDCl3, 400 MHz), d: 1.32 (s, 9H, 3 9 CH3),
4.29 (s, 2H, CH2), 7.00–7.51 (m, 6H, 2 9 C6H3), 9.40 (bs,
1H, NHCO). ESI–MS (m/z): 453.0[M-1]. Anal. Calad.
(%): C, 55.39; H, 3.98; N, 6.15; S, 7.04. Found (%): C,
55.44; H, 4.02; N, 6.13; S, 7.02.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-2-methoxybenzamide (F19) Yield 53 %, m.p.:
1
73–76 °C. H NMR (CDCl3, 400 MHz), d: 1.43 (s, 9H,
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-2-
(trifluoromethyl)benzamide (F14) Yield 73 %, m.p.:
3 9 CH3), 4.11 (s, 3H, OCH3), 4.84 (dd, J = 13.2 Hz,
J = 8.0 Hz, 1H, CH2NO2), 4.97 (dd, J = 13.2 Hz,
J = 8.0 Hz, 1H, CH2NO2), 5.63 (t, J = 8.0 Hz, 1H, CH),
7.06 (d, J = 8.0 Hz, 1H, C6H4OCH3 3-H), 7.13 (t,
J = 8.0 Hz, 1H, C6H4OCH3 5-H), 7.32 (s, 4H, C6H4Cl),
7.56 (t, J = 8.0 Hz, 1H, C6H4OCH3 4-H), 8.26 (d,
J = 8.0 Hz, 1H, C6H4OCH3 6-H), 10.91 (bs, 1H, NHCO).
ESI–MS (m/z): 472.1[M-1]. Anal. Calad. (%): C, 58.28; H,
5.10; N, 8.87; S, 6.77. Found (%): C, 58.25; H, 5.12; N,
8.85; S, 6.74.
1
130–133 °C. H NMR (CDCl3, 400 MHz), d: 1.32 (s, 9H,
3 9 CH3), 4.29 (s, 2H, CH2), 7.09 (d, J = 8.4 Hz, 1H,
C6H3 6-H), 7.21 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, C6H3
5-H), 7.42 (d, J = 2.0 Hz, 1H, C6H3 3-H), 7.61–7.80 (m,
4H, C6H4), 9.24 (bs, 1H, NHCO). ESI–MS (m/z): 485.1[M-
1]. Anal. Calad. (%): C, 54.22; H, 3.93; N, 5.75; S, 6.58.
Found (%): C, 54.25; H, 3.95; N, 5.72; S, 6.54.
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-3,5-
bis(trifluoromethyl)benzamide (F15) Yield 78 %, m.p.:
1
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-2-chlorobenzamide (F20) Yield 55 %, m.p.:
110–111 °C. H NMR (CDCl3, 400 MHz), d: 1.42 (s, 9H,
3 9 CH3), 4.29 (s, 2H, CH2), 7.08 (d, J = 8.4 Hz, 1H, 2,4-
diClC6H3 6-H), 7.21 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H, 2,4-
diClC6H3 5-H), 7.43 (d, J = 2.4 Hz, 1H, 2,4-diClC6H3
3-H), 8.07 (s, 1H, C6H3 4-H), 8.48 (s, 2H, C6H3 2,6-H),
9.85 (bs, 1H, NHCO). ESI–MS (m/z): 553.0[M-1]. Anal.
Calad. (%): C, 49.74; H, 3.27; N, 5.04; S, 5.77. Found (%):
C, 49.77; H, 3.29; N, 5.05; S, 5.75.
1
95–98 °C. H NMR (CDCl3, 400 MHz), d: 1.40 (s, 9H,
3 9 CH3), 4.84 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2
NO2), 4.97 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2NO2),
5.64 (t, J = 8.0 Hz, 1H, CH), 7.30–7.35 (m, 4H, C6H4Cl),
7.40–7.42 (m, 1H, NHCOC6H4Cl 6-H), 7.47–7.49 (m, 2H,
NHCOC6H4Cl 4,5-H), 7.82 (d, J = 7.6 Hz, 1H,
NHCOC6H4Cl 3-H), 9.51 (bs, 1H, NHCO). ESI–MS (m/z):
476.1[M-1]. Anal. Calad. (%): C, 55.23; H, 4.42; N, 8.78;
S, 6.70. Found (%): C, 55.16; H, 4.49; N, 8.71; S, 6.77.
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-4-ni-
1
trobenzamide (F16) Yield 73 %, m.p.: 100–101 °C. H
NMR (CDCl3, 400 MHz), d: 1.38 (s, 9H, 3 9 CH3), 4.30
(s, 2H, CH2), 7.07 (d, J = 8.0 Hz, 1H, C6H3 6-H), 7.20
(dd, J = 8.0 Hz, J = 2.0 Hz, 1H, C6H3 5-H), 7.42 (d,
J = 2.0 Hz, 1H, C6H3 3-H), 8.15 (d, J = 8.8 Hz, 2H, C6H4
2,6-H), 8.37 (d, J = 8.8 Hz, 2H, C6H4 3,5-H), 9.86 (bs,
1H, NHCO). ESI–MS (m/z): 462.1[M-1]. Anal. Calad.
(%): C, 54.32; H, 4.12; N, 9.05; S, 6.91. Found (%): C,
54.33; H, 4.15; N, 9.02; S, 6.88.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-4-chlorobenzamide (F21) Yield 51 %, m.p.:
1
115–119 °C. H NMR (CDCl3, 400 MHz), d: 1.44 (s, 9H,
3 9 CH3), 4.85 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2
NO2), 4.97 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2NO2),
5.65 (t, J = 8.0 Hz, 1H, CH), 7.29 (d, J = 8.4 Hz, 2H,
C6H4Cl 2,6-H), 7.34 (d, J = 8.4 Hz, 2H, C6H4Cl 3,5-H),
7.50 (d, J = 8.0 Hz, 2H, NHCOC6H4Cl 3,5-H), 7.94 (d,
J = 8.0 Hz, 2H, NHCOC6H4Cl 2,6-H), 9.70 (bs, 1H,
NHCO). ESI–MS (m/z): 476.1[M-1]. Anal. Calad. (%): C,
55.23; H, 4.42; N, 8.78; S, 6.70. Found (%): C, 55.21; H,
4.45; N, 8.75; S, 6.66.
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-3,5-
dinitrobenzamide (F17) Yield 68 %, m.p.: 165–167 °C.
1H NMR (CDCl3, 400 MHz), d: 1.46 (s, 9H, 3 9 CH3),
4.26 (s, 2H, CH2), 7.12 (d, J = 8.4 Hz, 1H, 2,4-diClC6H3
6-H), 7.24 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, 2,4-diClC6H3
5-H), 7.44 (d, J = 2.0 Hz, 1H, 2,4-diClC6H3 3-H), 9.16 (t,
J = 2.4 Hz, 1H, C6H3 4-H), 9.30 (d, J = 2.4 Hz, 2H, C6H3
2,6-H), 10.15 (bs, 1H, NHCO). ESI–MS (m/z): 507.0[M-
1]. Anal. Calad. (%): C, 49.52; H, 3.56; N, 11.00; S, 6.30.
Found (%): C, 49.50; H, 3.58; N, 11.02; S, 6.27.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-2,6-dichlorobenzamide (F22) Yield 61 %,
m.p.: 101–104 °C. 1H NMR (CDCl3, 400 MHz), d: 1.41 (s,
9H, 3 9 CH3), 4.84 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H,
CH2NO2), 4.99 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2
NO2), 5.64 (t, J = 8.0 Hz, 1H, CH), 7.26–7.38 (m, 7H,
C6H4, C6H3), 9.06 (bs, 1H, NHCO). ESI–MS (m/z):
N-(4-(Tert-butyl)-5-(2,4-dichlorobenzyl)thiazol-2-yl)-2-methyl-
3-nitrobenzamide (F18) Yield 74 %, m.p.: 181–182 °C.
123

Med Chem Res
509.9[M-1]. Anal. Calad. (%): C, 51.52; H, 3.93; N, 8.19;
S, 6.25. Found (%): C, 51.55; H, 3.95; N, 8.14; S, 6.21.
CH2NO2), 4.98 (dd, J = 13.6 Hz, J = 8.0 Hz, 1H, CH2
NO2), 5.60 (t, J = 8.0 Hz, 1H, CH), 7.30 (d, J = 8.4 Hz,
2H, C6H4 2,6-H), 7.38 (d, J = 8.4 Hz, 2H, C6H4 3,5-H),
9.22 (s, 1H, C6H3 4-H), 9.29 (s, 2H, C6H3 2,6-H), 10.00
(bs, 1H, NHCO). ESI–MS (m/z): 531.9[M-1]. Anal. Calad.
(%): C, 49.49; H, 3.78; N, 13.12; S, 6.01. Found (%): C,
49.47; H, 3.75; N, 13.09; S, 6.09.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-2,6-difluorobenzamide (F23) Yield 60 %, m.p.:
1
88–91 °C. H NMR (CDCl3, 400 MHz), d: 1.40 (s, 9H,
3 9 CH3), 4.83 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2
NO2), 4.97 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2NO2),
5.64 (t, J = 8.0 Hz, 1H, CH), 7.04 (t, J = 8.4 Hz, 2H,
C6H3 3,5-H), 7.30 (d, J = 8.8 Hz, 2H, C6H4 2,6-H), 7.34
(d, J = 8.8 Hz, 2H, C6H4 3,5-H), 7.48–7.54 (m, 1H, C6H3
4-H), 9.44 (bs, 1H, NHCO). ESI–MS (m/z): 478.1[M-1].
Anal. Calad. (%): C, 55.06; H, 4.20; N, 8.76; S, 6.68.
Found (%): C, 55.02; H, 4.24; N, 8.72; S, 6.64.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-2-methyl-3-nitrobenzamide (F28) Yield 57 %,
1
m.p.: 84–87 °C. H NMR (CDCl3, 400 MHz), d: 1.42 (s,
9H, 3 9 CH3), 2.61(s, 3H, CH3), 4.85 (dd, J = 13.2 Hz,
J = 8.0 Hz, 1H, CH2NO2), 4.98 (dd, J = 13.2 Hz,
J = 8.0 Hz, 1H, CH2NO2), 5.66 (t, J = 8.0 Hz, 1H, CH),
7.31 (d, J = 8.8 Hz, 2H, C6H4 2,6-H), 7.36 (d, J = 8.8 Hz,
2H, C6H4 3,5-H), 7.47 (t, J = 8.0 Hz, 1H, C6H3 5-H), 7.74
(d, J = 8.0 Hz, 1H, C6H3 6-H), 7.95 (d, J = 8.0 Hz, 1H,
C6H3 4-H), 9.11 (bs, 1H, NHCO). ESI–MS (m/z):
501.0[M-1]. Anal. Calad. (%): C, 54.92; H, 4.61; N, 11.14;
S, 6.38. Found (%): C, 54.90; H, 4.64; N, 11.12; S, 6.35.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-2-(trifluoromethyl)benzamide
(F24) Yield
1
46 %, m.p.: 105–108 °C. H NMR (CDCl3, 400 MHz), d:
1.32 (s, 9H, 3 9 CH3), 4.97 (dd, J = 13.6 Hz, J = 8.0 Hz,
1H, CH2NO2), 5.22 (dd, J = 13.6 Hz, J = 8.0 Hz, 1H,
CH2NO2), 5.64(t, J = 8.0 Hz, 1H, CH), 7.40–7.45 (m, 5H,
C6H4Cl, C6H4CF3 5-H), 7.55–7.62 (m, 3H, C6H4CF3 3,4,6-
H), 9.12 (bs, 1H, NHCO). ESI–MS (m/z): 509.9[M-1].
Anal. Calad. (%): C, 53.96; H, 4.13; N, 8.21; S, 6.26.
Found (%): C, 53.98; H, 4.15; N, 8.18; S, 6.22.
N-(4-(Tert-butyl)-5-(1-(2,4-dichlorophenyl)-2-nitroethyl)
thiazol-2-yl)-2,6-difluorobenzamide (F29) Yield 38 %,
1
m.p.: 92–94 °C. H NMR (CDCl3, 400 MHz), d: 1.34 (s,
9H, 3 9 CH3), 4.74 (dd, J = 14.0 Hz, J = 9.2 Hz, 1H,
CH2NO2), 4.93 (dd, J = 14.0 Hz, J = 6.4 Hz, 1H, CH2
NO2), 5.64 (q, J = 9.2 Hz, J = 6.4 Hz, 1H, CH),
7.25–7.28 (m, 1H, C6H3 5-H), 7.38 (d, J = 8.8 Hz, 1H,
C6H3 6-H), 7.46 (d, J = 2.0 Hz, 1H, C6H3 3-H), 7.03–7.07
(m, 2H, NHCOC6H3 3, 5-H), 7.49–7.53 (m, 1H,
NHCOC6H3 4-H), 9.33 (bs, 1H, NHCO). ESI–MS (m/z):
511.9 [M-1]. Anal. Calad. (%): C, 51.37; H, 3.72; N, 8.17;
S, 6.23. Found (%): C, 51.28; H, 3.76; N, 8.15; S, 6.30.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-3,5-bis(trifluoromethyl)benzamide (F25) Yield
1
41 %, m.p.: 94–97 °C. H NMR (CDCl3, 400 MHz), d:
1.45 (s, 9H, 3 9 CH3), 4.86 (dd, J = 13.2 Hz, J = 8.0 Hz,
1H, CH2NO2), 4.98 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H,
CH2NO2), 5.64 (t, J = 8.0 Hz, 1H, CH), 7.30 (d,
J = 8.8 Hz, 2H, C6H4 2,6-H), 7.36 (d, J = 8.8 Hz, 2H,
C6H4 3,5-H), 8.10 (s, 1H, C6H3 4-H), 8.46 (s, 2H, C6H3
2,6-H), 9.66 (bs, 1H, NHCO). ESI–MS (m/z): 578.1[M-1].
Anal. Calad. (%):C, 49.70; H, 3.48; N, 7.25; S, 5.53. Found
(%): C, 49.72; H, 3.49; N, 7.22; S, 5.50.
N-(4-(Tert-butyl)-5-(1-(2,4-dichlorophenyl)-2-nitroethyl)
thiazol-2-yl)-2-(trifluoromethyl)benzamide (F30) Yield
1
44 %, m.p.: 91–93 °C. H NMR (CDCl3, 400 MHz), d:
1.36 (s, 9H, 3 9 CH3), 4.75 (dd, J = 14.0 Hz, J = 9.2 Hz,
1H, CH2NO2), 4.94 (dd, J = 14.0 Hz, J = 6.4 Hz, 1H,
CH2NO2), 5.60 (q, J = 9.2 Hz, J = 6.4 Hz, 1H, CH), 7.29
(dd, J = 8.4 Hz, J = 2.0 Hz, 1H, C6H3 5-H), 7.39 (d,
J = 8.4 Hz, 1H, C6H3 6-H), 7.48 (d, J = 2.0 Hz, 1H, C6H3
3-H), 7.65–7.70 (m, 4H, C6H4), 9.18 (bs, 1H, NHCO).
ESI–MS (m/z): 544.1 [M-1]. Anal. Calad. (%): C, 50.56;
H, 3.69; N, 7.69; S, 5.87. Found (%): C, 50.45; H, 3.72; N,
7.71; S, 5.93.
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-4-nitrobenzamide (F26) Yield 59 %, m.p.:
1
131–133 °C. H NMR (CDCl3, 400 MHz), d: 1.44 (s, 9H,
3 9 CH3), 4.86 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2
NO2), 4.98 (dd, J = 13.2 Hz, J = 8.0 Hz, 1H, CH2NO2),
5.65 (t, J = 8.0 Hz, 1H, CH), 7.30 (d, J = 8.8 Hz, 2H,
C6H4Cl 2,6-H), 7.35 (d, J = 8.8 Hz, 2H, C6H4Cl 3,5-H),
8.16 (d, J = 8.8 Hz, 2H, C6H4NO2 2,6-H), 8.39 (d,
J = 8.8 Hz, 2H, C6H4NO2 3,5-H), 9.55 (bs, 1H, NHCO).
ESI–MS (m/z): 487.1[M-1]. Anal. Calad. (%): C, 54.04; H,
4.33; N, 11.46; S, 6.56. Found (%): C, 54.02; H, 4.35; N,
11.42; S, 6.53.
N-(4-(Tert-butyl)-5-(1-(2,4-dichlorophenyl)-2-nitroethyl)
thiazol-2-yl)-4-nitrobenzamide (F31) Yield 33 %, m.p.:
1
141–143 °C. H NMR (CDCl3, 400 MHz), d: 1.35(s, 9H,
3 9 CH3), 4.75 (dd, J = 13.6 Hz, J = 9.6 Hz, 1H, CH2
NO2), 4.94 (dd, J = 13.6 Hz, J = 6.0 Hz, 1H, CH2NO2),
6.02 (q, J = 9.6 Hz, J = 6.0 Hz, 1H, CH), 7.26 (dd,
J = 8.8 Hz, J = 2.0 Hz, 1H, C6H3 5-H), 7.36 (d,
J = 8.8 Hz, 1H, C6H3 6-H), 7.48 (d, J = 2.0 Hz, 1H, C6H3
N-(4-(Tert-butyl)-5-(1-(4-chlorophenyl)-2-nitroethyl)thia-
zol-2-yl)-3,5-dinitrobenzamide (F27) Yield 55 %, m.p.:
1
160–163 °C. H NMR (CDCl3, 400 MHz), d: 1.50 (s, 9H,
3 9 CH3), 4.88 (dd, J = 13.6 Hz, J = 8.0 Hz, 1H,
123

Med Chem Res
3-H), 8.15 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 8.38 (d,
J = 8.4 Hz, 2H, C6H4 3,5-H), 9.84 (bs, 1H, NHCO). ESI–
MS (m/z): 520.9 [M-1]. Anal. Calad. (%): C, 50.49; H,
3.85; N, 10.70; S, 6.13. Found (%): C, 50.43; H, 3.88; N,
10.65; S, 6.16.
suspended in MES buffer (pH = 6.5). The reaction was
started by adding the substrate MUNANA and then incu-
bated at 37 °C for 40 min. After incubation, the reaction
was terminated by adding sodium hydroxide solution. The
plate was read within 20 min under the parameters of
excitation wavelength at 360 nm and emission wavelength
at 450 nm (Liu et al., 2008). The inhibition rates of the
tested compounds were worked out according to the
decrement of the fluorescence intensity.
N-(4-(Tert-butyl)-5-(1-(2,4-dichlorophenyl)-2-nitroethyl)
thiazol-2-yl)-3,5-dinitrobenzamide (F32) Yield 35 %,
m.p.: 138–140 °C. 1H NMR (CDCl3, 400 MHz), d: 1.42 (s,
9H, 3 9 CH3), 4.78 (dd, J = 14.0 Hz, J = 10.0 Hz, 1H,
CH2NO2), 4.93 (dd, J = 14.0 Hz, J = 6.0 Hz, 1H, CH2
NO2), 5.60 (q, J = 10.0 Hz, J = 6.0 Hz, 1H, CH), 7.29
(dd, J = 8.4 Hz, J = 2.4 Hz, 1H, C6H3 5-H), 7.38 (d,
J = 8.4 Hz, 1H, C6H3 6-H), 7.50 (d, J = 2.4 Hz, 1H, C6H3
3-H), 9.21 (t, J = 2.4 Hz, 1H, NHCOC6H3 4-H), 9.27 (d,
J = 2.4 Hz, 2H, NHCOC6H3 2, 6-H), 9.97 (bs, 1H,
NHCO). ESI–MS (m/z): 566.1 [M-1]. Anal. Calad. (%): C,
46.49; H, 3.37; N, 12.32; S, 5.64. Found (%): C, 46.41; H,
3.40; N, 12.27; S, 5.74.
Molecular docking
Molecular docking of compounds F8, F26, F32 and osel-
tamivir to p09N1 was performed by LeDock with default
parameters. Before docking, the conformational energy of
ligand was minimized by MM2 force field. The dimensions
of the binding box were set as [Xmin = -37.4, Xmax
-19.8, Ymin = 4.0, Ymax = 21.5, Zmin = 12.0, Zmax
=
=
32.2] around the active site. The number of binding poses
of each ligand was 20.
X-ray crystal structural determination
The crystals of the compounds F5 and F16 suitable for
X-ray structure determination were grown in ethanol by
slow evaporation for about 15 days at room temperature.
X-ray intensity data were measured at 173(2) K on a
Bruker AXS SMART 1000 CCD diffractometer equipped
with a graphite monochromatic Mo Ka radiation (k =
Conclusion
A series of N-(5-benzyl-4-(tert-butyl)thiazol-2-yl)benza-
mides were synthesized and evaluated for their NA inhi-
bitory activity in vitro. Among the synthesized compounds,
nitro-substituted compounds (F8, F26 and F32) showed
good NA inhibitory activity with inhibition ratio 45.56,
59.89 and 49.63 %. The structure–activity relationship
(SAR) of compound F26 was discussed through molecular
docking performed by LeDock and the result indicated that
compound F26 was in good combination with the oselta-
mivir binding sites which could be further modified to
exhibit better potency.
˚
0.71073 A). Corrections for incident and diffracted beam
absorption effects were applied using SADABS (Sheldrick,
2004). The structures were solved by direct methods with
SHELXS-97 and expanded by difference Fourier tech-
niques. The non-hydrogen atoms were refined anisotropi-
cally, and hydrogen atoms were added according to
theoretical models. The structures were refined by full-
matrix least-squares techniques on F2 with SHELXL-97.
Acknowledgments The authors would like to acknowledge and
thank Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College for the NA inhibitory
activities test.
Determination of NA inhibitory activity
All the target compounds were tested for their NA inhibi-
tory activity in vitro and oseltamivir was used as standard
drug for comparison (Zhang et al., 2008; Liu et al., 2005).
The influenza virus (H1N1) donated by Chinese Center for
Disease Control and Prevention provided us with the
source of NA. 20-(4-Methylumbelliferyl)-a-D-acetyl neu-
raminic acid (MUNANA) is the substrate of NA and the
cleavage of MUNANA by NA could produce a fluorescent
product, which would emit a fluorescence of 450 nm under
the irradiation of excitation wavelength of 360 nm. The
intensity of fluorescence reflects the activity of NA sensi-
tively (Mitrasinovic, 2009).
References
´
Abed Y, Nehme B, Baz M, Boivin G (2008) Activity of the
neuraminidase inhibitor A-315675 against oseltamivir–resistant
influenza neuraminidases of N1 and N2 subtypes. Antiviral Res
77:163–166
Ah YC, Choi JK, Choi YK, Ki HM, Bae JH (2010) A novel
transdermal patch incorporating meloxicam: in vitro and in vivo
characterization. Int J Pharm 385:12–19
Baz M, Abed Y, Boivin G (2007) Characterization of drug–resistant
recombinant influenza A/H1N1 viruses selected in vitro with
peramivir and zanamivir. Antiviral Res 74:159–162
In the enzyme reaction system, a certain concentration
of the target compounds with the NA enzyme was
Chang SH, Zhang JJ, Liao XY, Zhu XX, Wang DH, Zhu J, Feng T,
Zhu BL, Gao GF, Wang J, Yang HM, Yu J, Wang J (2007)
123

Med Chem Res
Influenza Virus Database (IVDB): an integrated information
resource and analysis platform for influenza virus research.
Nucleic Acids Res 35:376–380
against methicillin and cefixime resistant staphylococcus aureus
strains. Asian Pac J Trop Med 3:262–265
Potter CW (2001)
91:572–579
A history of influenza. J Appl Microbiol
Dawood FS, Iuliano AD, Reed C, Meltzer MI, Shay DK, Cheng PY,
Bandaranayake D, Breiman RF, Brooks WA, Buchy P, Feikin
DR, Fowler KB, Gordon A, Hien NT, Horby P, Huang QS, Katz
MA, Krishnan A, Lal R, Montgomery JM, Mølbak K, Pebody R,
Presanis AM, Razuri H, Steens A, Tinoco YO, Wallinga J, Yu
HJ, Vong S, Bresee J, Widdowson MA (2012) Estimated global
mortality associated with the first 12 months of 2009 pandemic
influenza A H1N1 virus circulation: a modelling study. Lancet
Infect Dis 12:687–695
De Clercq E (2001) Antiviral drugs: current state of the art. J Clin
Virol 22:73–89
Donaldson LJ, Rutter PD, Ellis BM, Greaves FEC, Mytton OT,
Pebody RG, Yardley IE (2009) Mortality from pandemic
A/H1N1 2009 influenza in England: public health surveillance
study. Brit Med J 339:5213–5220
Gresser R, Hartmann H, Wrackmeyer M, Leo K, Riede M (2011)
Synthesis of thiophene–substituted aza–BODIPYs and their
optical and electrochemical properties. Tetrahedron 67:7148–
7155
Hay AJ, Gregory V, Douglas AR, Lin YP (2001) The evolution of
human influenza viruses. Phil Trans R Soc Lond B 356:1861–
1870
Hilleman MR (2002) Realities and enigmas of human viral
influenza: pathogenesis, epidemiology and control. Vaccine
20:3068–3087
Hu AX, Cao G (2011) Hydrogen–bonded helices in 2–aminothiazole
derivatives: generation of chiral crystals from small achiral
molecules. Tetrahedron-Asymmetry 22:1332–1336
Hu J, Wang EH (2004) Studies on structure–activity relationship of
influenza neuraminidase inhibitors. Progress in Pharmaceutical
Sciences 28:204–208
Ison MG (2012) Clinical use of approved influenza antivirals: therapy
and prophylaxis. Influenza Other Respir Viruses 7(Suppl.
1):7–13
Kessler U, Ranadheera C, Joubert M, Giethlen B, Garrido F (2010)
Small molecule inhibitors of influenza A and B virus and
respiratory syncytial virus replication. Pat WO2010128163A2
Laskowski RA, Swindells MB (2011) LigPlot?: multiple ligand–
protein interaction diagrams for drug discovery. J Chem Inf
Model 51:2778–2786
Li W, Xia L, Hu AX, Liu AL, Peng JM, Tan WQ (2013a) Design and
synthesis of 4-Alkyl-2-amino-(acetamino)-6-aryl-1,3-thiazine
derivatives as influenza neuraminidase inhibitors. Arch Pharm
Chem Life Sci 346:635–644
Li W, Hu AX, Liu AL, Peng JM, Xia L, Tan WQ (2013b) Synthesis
and biological activities of 4-alkyl-6-aryl-1,3-thiazines. Chin J
Org Chem 33:1478–1482
Rossignal JF (2012) Use of thiazolide compounds for the prevention
and treatment of viral diseases, cancer and diseases caused by
intracellular infections. Pat WO2012158699A1
Ryan DM, Ticehurst J, Dempsey MH (1995) GG167 (4-guanidino-
2,4-dideoxy-2,3-dehydro-N-acetyl-neuraminic acid) is a potent
inhibitor of influenza virus in ferrets. Antimicrob Agents
Chemother 39:2583–2584
¨
Sheldrick GM (2004) SADABS. University of Gottingen, Germany,
Program for the absorption correction
Shen F, Hu AX, Luo XF, Ye J, Ou XM (2012) Synthesis
and Bioactivity of (E)-2-Benzylidenimino-4-(7-methoxy-2,2-
dimethyl-2,3-dihydrobenzofuran-5-yl) thiazole. Chin
Chem 32:388–392
J Org
Shen K, Peng JM, Li W, Yan XW, Hu AX (2014) Synthesis,
crystal structure and antitumor activity of N-(4-tert-butyl-5-(4-
chlorobenzyl)thiazol-2-yl)-2,6-difluorobenzamide. Chin J Struct
Chem 33:855–858
Stamboulian D, Bonvehi PE, Nacinovich FM, Cox N (2000)
Influenza. Infect Dis Clin North Am 14:141–166
Stocks MJ, Harrison RP, Teague SJ (1995) Macrocyclic ring closures
employing the intramolecular Heck Reaction. Tetrahedron Lett
36:6555–6558
Storms AD, Gubareva LV, Su S, Wheeling JT, Okomo-Adhiambo M,
Pan CY, Reisdorf E, St George K, Myers R, Wotton JT,
Robinson S, Leader B, Thompson M, Shannon M, Klimov A,
Fry AM (2012) Oseltamivir–resistant pandemic (H1N1) (2009)
virus infections, United States, 2010–11. Emerg Infect Dis
18:308–311
Sun C, Zhang XD, Huang H, Zhou P (2006) Synthesis and evaluation
of a new series of substituted acyl(thio)urea and thiadiazolo
[2,3–a] pyrimidine derivatives as potent inhibitors of influenza
virus neuraminidase. Bioorg Med Chem 14:8574–8581
Teoh D, Ayeni TA, Rubatt JM, Adams DJ, Grace L, Starr MD, Barry
WT, Berchuck A, Murphy SK, Secord AA (2011) Dasatinib
(BMS-35482) has synergistic activity with paclitaxel and
carboplatin in ovarian cancer cells. Gynecol Oncol 121:187–192
Vavricka CJ, Li Q, Wu Y, Qi JX, Wang MY, Liu Y, Gao F, Liu J,
Feng EG, He JH, Wang JF, Liu H, Jiang HL, Gao GF (2011)
Structural and functional analysis of laninamivir and its
octanoate prodrug reveals group specific mechanisms for
influenza NA inhibition. PLoS Pathog 7:e1002249
Wang T, Wade RC (2001) Comparative binding energy (COMBINE)
analysis of influenza neuraminidase–inhibitor complexes. J Med
Chem 44:961–971
Wang GT, Chen Y, Wang S, Gentles R, Sowin T, Kate W, Muchmore
S, Giranda V, Stewart K, Sham H, Kempf D, Laver WG (2001)
Design, synthesis, and structural analysis of influenza neu-
raminidase inhibitors containing pyrrolidine cores. J Med Chem
44:1192–1201
Liu AL, Cao HP, Du GH (2005) Drug screening for influenza
neuraminidase inhibitors. Sci China Life Sci 48:1–5
Liu AL, Wang HD, Lee SM, Wang YT, Du GH (2008) Structure–
activity relationship of flavonoids as influenza virus neu-
raminidase inhibitors and their in vitro anti–viral activities.
Bioorg Med Chem 16:7141–7147
Wappes J (2013) China reports first human case of H10N8 avian flu.
http://www.cidrap.umn.edu/news–perspective/ 2013/12/china–
reports–first–human–case–h10n8–avian–flu
Liu Y, Lei Z, Gong JZ, Fang H, Liu AL, Du GH, Xu WF (2011)
Design, synthesis, and biological activity of thiazole derivatives
as novel influenza neuraminidase inhibitors. J Enzyme Inhib
Med Chem 26:506–513
Mitrasinovic PM (2009) On the structure–based design of novel
inhibitors of H5N1 influenza A virus neuraminidase (NA).
Biophys Chem 140:35–38
WHO (2010) Pandemic (H1N1) 2009—update 111. http://www.who.
int/csr/don/2010_07_30/en/
Zhang AF, Okrasa L, Pakula T, Schlu¨ter AD (2004) Homologous
series of dendronized poly methacrylates with a methyleneoxy-
carbonyl spacer between the backbone and dendritic side chain:
synthesis, characterization, and some bulk properties. J Am
Chem Soc 126:6658–6666
Moghadam MS, Maleki S, Darabpour E, Motamedi H, Nejad SMS
(2010) Antibacterial activity of eight Iranian plant extracts
Zhang J, Wang Q, Fang H, Xu WF, Liu AL, Du GH (2008) Design,
synthesis, inhibitory activity, and SAR studies of hydrophobic
123

Med Chem Res
p-aminosalicylic acid derivatives as neuraminidase inhibitors.
Bioorgan Med Chem 16:3839–3847
Zhu H, Wang D, Kelvin DJ, Li L, Zheng Z, Yoon SW, Wong SS,
Farooqui A, Wang J, Banner D, Chen R, Zheng R, Zhou J, Zhang
Y, Hong W, Dong W, Cai Q, Roehrl MHA, Huang SSH, Kelvin
AA, Yao T, Zhou B, Chen X, Leung GM, Poon LLM, Webster
RG, Webby RJ, Peiris JSM, Guan Y, Shu Y (2013) Infectivity,
transmission, and pathology of human-isolated H7N9 influenza
virus in ferrets and pigs. Science 341:183–186
123