Synthesis and in vitro antibacterial activity of novel fluoroalkyl-substituted pyrazolyl oxazolidinones

Article abstract of DOI:10.1039/c5ra11782h

A series of novel oxazolidinone derivatives bearing fluoroalkyl-substituted pyrazole as the C-ring structure were designed, synthesized and evaluated for their antibacterial activity against six Gram-positive bacterial pathogens. Most of the target compounds have good antibacterial activity. Especially, compounds 13f, 13i and 13l show excellent activity comparable to linezolid.

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Synthesis and in vitro antibacterial activity of novel
fluoroalkyl-substituted pyrazolyl oxazolidinones
Lili Yan,a Jingjing Wu,*a Heng Chen,a Shaowu Zhang,a Zhi Wang,a Hui Wangb and Fanhong Wu*a
Received 00th January 20xx,
Accepted 00th January 20xx
A series of novel oxazolidinone derivatives bearing fluoroalkyl-substituted pyrazole as the C-ring structure were designed,
synthesized and evaluated for their antibacterial activity against six Gram-positive bacterial pathogens. Most of the target
compounds have good antibacterial activity. Especially, compounds 13f, 13i and 13l show excellent activity comparable to
linezolid.
DOI: 10.1039/x0xx00000x
www.rsc.org/
with pyrazolo[1,5-a]pyridines as the C-ring substructure were
described by Hideyuki Suzuki et al.16 Youfu Luo’s group have
synthesized an oxazolidinone compound with 4-(pyridin-2-yl)-
1 Introduction
Common
multi-drug-resistant
Gram-positive
bacterial
1H-pyrrole, which has formed nanoassemblies without any
carrier and display antimicrobial activity on MRSA.17
pathogens such as methicillin-resistant Staphylococcus aureus
(MRSA) and Staphylococcus epidermidis (MRSE), vancomycin-
O
O
resistant
Enterococci
(VRE),
and
penicillin-resistant
N
N
N
O
N
N
O
H
O
C
N
B
N
A
N
Streptococcus pneumoniae (PRSP) have become serious health
threats.1-3 Oxazolidinones, as exemplified by linezolid, are a
new class of synthetic antibacterial agents that have high
activity against a broad range of Gram-positive Bacteria.4
Linezolid was developed by Pharmacia and Upjohn Company
and approved in 2000 for the treatment of serious infections
caused by the above-mentioned strains.5 However, bacterial
resistance to linezolid has been found recently in Gram-
positive bacteria Staphylococcus aureus and Enterococcus
faecium.6 Hence, numerous researches on the synthesis and
bioassay of various oxazolidinones have been developed.7-11
These studies have focused mainly on the modification of A-
ring (oxazolidinone), B-ring (phenyl), C-ring (morpholine), and
the C-5 side chain on the A-ring substructure of linezolid.
Among them, series of oxazolidinones containing nitrogen
heterocyclic structures as C-ring unit were reported and some
analogues have good activity comparable or superior to
linezolid against Gram-positive bacterial pathogens (Figure
1).12-13 Oxazolidinones containing a pyrroloaryl substituent
have been synthesized by J&J Pharmaceutical, and one
compound was approved to begin phase I clinical trial.14
Gyochang Keum has disclosed some oxazolidinones with 3-
azabicyclo[3.3.0]octanyl rings as C-ring exhibit potent in vitro
antibacterial activity.15 Potent oxazolidinone antibacterials
N
Me
O
ONa
ONa
P
Me
F
F
O
O
C-5 side chain
Linezolid
tedizolid
O
O
H
N
N
Me
NH
F
N
O
N
N
H
radezolid
Figure 1 Structure of linezolid and its analogs.
For decades, there is considerable interest in introducing
fluorine-containing groups into organic molecules since the
growing application of organic fluorine compounds in
pharmaceuticals.18,19 Many commercial drugs containing
trifluoromethyl and difluoromethyl groups have come to
market.19 However, literature survey showed that the
incorporation of these fluoroalkyl groups into oxazolidinones
has not been studies systematically. Consequently, we are
interested in developing fluoroalkyl-containing oxazolidinones
and expect these compounds might result in potent
antibacterial activity. According to the literature, pyrazole
cycle has been broadly used as a biologically active scaffold in
medicine science.20 Herein, we reported the synthesis of novel
oxazolidinone derivatives bearing fluoroalkyl-substituted
pyrazole as the C-ring structure and their antibacterial
activities against methicillin-resistant Staphylococcus aureus
(MRSA), methicillin-resistant Staphylococcus epidermidis
(MRSE), methicillin-sensitive Staphylococcus aureus (MSSA),
methicillin-sensitive Staphylococcus epidermidis (MSSE),
penicillin-resistant Streptococcus pneumoniae (PRSP) and
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Enterococcus faecium (EF). Three target compounds show compounds 10, which then underwent cyclization in the
good activity comparable to linezolid.
presence of carbonyl diimidazole (CDI) to form the 2-
oxazolidinones 11 Access to the fluoroalkyl-substituted
.
pyrazolyl oxazolidinones 13 were achieved via the N-Boc
deprotection and subsequent acetylation reaction. The
structures of these target compounds are shown in Table 1. All
2 Result and discussion
2.1 Chemistry
1
the H-NMR, 13C-NMR, 19F-NMR and Mass data of compounds
The synthetic routes to fluoroalkyl-substituted pyrazolyl
oxazolidinone analogues are outlined in Schemes 1-3. The key
13a-l were in accordance with the proposed molecular
intermediate
and described in Scheme 1.21 (S)-Epichlorohydrin
with benzaldehyde and aqueous ammonia in ethanol to form
compound after the addition of a solution of hydrochloric
acid. The reaction of compound and di-tert-butyldicarbonate
provided the key intermediate in high yield.
3
was prepared following literature procedures
structures.
1
reacted
Table 1 Structure of the target compounds 13a-l.
2
2
3
Scheme 1 Reagents and conditions: (a) PhCHO, NH3, CH2Cl2, 40 °C for 6 h, then 25 °C for
14 h; (b) HCl, H2O, toluene, 40-45 °C, 3.5 h; (c) (Boc)2O, Et3N, CH2Cl2, 25 °C, 12 h.
2.2 Antibacterial activity
The antibacterial activity of the target compounds 13a-l and
linezolid, known for comparison were evaluated against Gram-
positive bacterial strains: methicillin-resistant Staphylococcus
aureus (MRSA) (5 strains), methicillin-sensitive Staphylococcus
aureus (MSSA) (5 strains), methicillin-resistant Staphylococcus
epidermidis (MRSE) (5 strains), methicillin-sensitive
Staphylococcus epidermidis (MSSE) (5 strains), penicillin-
resistant Streptococcus pneumoniae (PRSP) (5 strains),
Enterococcus faecium (EF) (5 strains) and Staphylococcus
aureus ATCC43300 and ATCC29213, as quality-control strains,
using two-fold agar dilution method. The minimum inhibitory
concentrations (MIC) of these compounds against the above
bacteria are presented in Table 2.
Scheme 2 Reagents and conditions: (d) (RfCO)2O, Pyridine, CH2Cl2, 0 °C for 1 h, then 20
°C for 12 h; (e) EtOH, , reflux with stirring, 24h; (f) TFA, CH2Cl2, 25 °C , 12 h; (g) H2, Pd/C,
MeOH, 25 °C , 3 h.
The results revealed that most of the compounds exhibited
good to moderate antibacterial activity. Compounds 13f, 13i
and 13l appear to exhibit similar antibacterial activity in
comparison with linezolid, while compounds have 5-fluoroalkyl
Scheme 3 Reagents and conditions: (h) neat, 150 °C, 12 h; (i) CDI, Et3N, THF, 25 °C , 12
h; (j) TFA, CH2Cl2, 25 °C , 12 h; (k) Ac2O, Et3N, CH2Cl2, 25 °C , 12 h.
pyrazole moiety such as 13b
activity against all the tested bacterial strains with MIC values
greater than 8 μg mL-1. In addition, compounds 13a
13c 13e
, 13d, 13g, 13k showed poor
The synthesis of key intermediates
2. β-ethyloxyvinyl fluoroalkyl ketones
9
is depicted in Scheme
were prepared from
,
,
5
and 13h were 2-fold less active than linezolid. According to the
above result, it is found that compounds have 5-fluoroalkyl-4-
methyl pyrazole moiety showed higher MIC value against
Gram-positive bacteria than those have 5-fluoroalkyl-3-methyl
pyrazole and 5-fluoroalkyl pyrazole moiety. When 4-methyl-5-
trifluromethyl pyrazole was used as C-ring, compound with a
fluorine atom at the 3-position of phenyl ring 13f showed 2-4
fold potency advantages compared to compound with no
fluorine atom on phenyl ring 13c. Compounds with 5-
ethyl vinyl ether and its derivatives with fluoroalkylacetic
anhydride under base condition.22 Treatment of compounds
with p-nitrophenylhydrazines in ethanol gave exclusively the
5-hydroxy-4,5-dihydropyrazoles . Dehydration and reduction
of the nitro group afforded the corresponding pyrazoles 9 23
Scheme 3 indicated the synthetic route of the target
compounds 13 24
The preparation involved a coupling reaction
5
6
7
.
.
between the compound
3 and intermediates 9 to give
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difluoromethyl-4-methyl pyrazole as C-ring have equal activity not (13i vs 13l).
no matter the fluorine atom substituted at the phenyl ring or
Table 2 Minimal-Inhibitory Concentrations (MICs) of Oxadiazolesa 13a-l.
Minimum inhibitory concentrations (MIC) (μg mL-1)
Compound
MRSA
2-4
MSSA
2-4
MRSE
4
MSSE
2-4
>16
2-4
8-16
2-4
1-2
16
PRSP
2-4
>16
2-4
8
EF
4
ATCC43300
ATCC29213
13a
13b
13c
4
>16
4
2
16
2
>16
2-4
>16
2-4
>16
4
>16
4
13d
13e
8-16
2-4
8-16
2-4
8-16
4
8
8
8
2-4
1-2
8-16
2-4
1-2
4
4
4
2
13f
0.5-2
8-16
2-4
0.5-1
8-16
2-4
1-2
8-16
4
1-2
8-16
4
2
0.5
8
13g
8
13h
13i
2-4
1-2
4-8
>16
1-2
1-2
4
2
1-2
1-2
1-2
4-8
>16
1-2
1-2
1-2
4
2
1
13j
4-8
4-8
8
4
13k
>16
1-2
>16
1-2
>16
1-2
1-2
>16
1-2
1-2
>16
2
>16
1
13l
Linezolid
1-2
1-2
2
1
2.3 Molecular docking
molecular docking result, along with the biological assay data,
suggests that compounds 13f and 13l prove to be potential
antibacterials.
To elucidate the antibacterial activity of the target compounds
at the molecular level, we explored the interaction mode
Next, we applied the molecular docking protocol to predict
the affinity of compounds 13b and 13k, which both show
lower antibacterial activity in the bioassay, to the Deinococcus
radiodurans linezolid-bound 50S ribosomal subunit crystal
structure. The scores of 13b and 13k are both lower than
linezolid (Table 3). It might due to that in both two compounds,
the trifluoromethyl group and methyl group in the pyrazole
ring occupy in meta position, which lead to a large steric space
and make the target compounds hard to fit into the binding
pocket of the subunit.
between the representative compounds 13f, 13l, linezolid and
the Deinococcus radiodurans linezolid-bound 50S ribosomal
subunit crystal structure (Protein Data Bank accession codes
3DLL) using Surflex-dock.25 The predicted binding affinity (total
scores) of docking studies was shown in Table 3. The scores of
linezolid, 13f and 13l are with values of 6.47, 7.92 and 8.29,
respectively. The docking result was shown in Figure 2. The
figure 1A depicted the docking result of 13f and linezolid. From
the figure 1A, we found the –CF3 group occupy more space
than the –H atom, which make the –CF3 group combine with
the residues of binding site very easy and result in high
antibacterial activity of 13f. The figure 1B showed the docking
result of 13l and linezolid, according to the figure, the –CHF2
group has a huger space compared to the –H atom. Therefore
compound 13l also showed high antibacterial activity
comparable to linezolid. The steric space of –CF3 is bigger than
–CHF2, and it is not conductive to the antibacterial activity.
Hence, the docking result showed that compound 13l exhibit
higher antibacterial activity than that of compound 13f. The
Table 3 Total-score of compounds 13f, 13l and linezolid
Compound
13f
Total-score
7.92
8.29
13l
linezolid
13b
6.47
4.66
13k
6.10
Figure 2 The docking results of linezolid, 13f and 13l.
of Gram-positive bacterial pathogens. Most of the target
compounds exhibited potent in vitro antibacterial activity.
3 Conclusion
Compounds have 5-fluoroalkyl-4-methyl pyrazole moiety 13f
,
In summary, we have synthesized a series of oxazolidinone-
type antibacterials bearing fluoroalkyl-substituted pyrazole as
the C-ring structure and evaluated their activity against a panel
13i and 13l showed high antibacterial activity comparable to
linezolid with minimum inhibitory concentration (MIC) values
of 1-2, 1-2, 1-2 μg mL-1. Molecular docking studies of linezolid
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and the representative compounds with most high activity 13f, 7a-l. Then, the compounds 7a-l was dissolved in CH2Cl2 and
13l and low activity 13b 13k, was performed and gave 6.47, added trifluoroacetic acid (3.42 g, 30 mmol) dropwise. The
,
7.92, 8.20, 4.66, 6.10 total-score respectively. The synthesis of mixture was stirred for 12 h at room temperature. The
more fluoroalkyl-containing oxazolidinone analogues is reaction was quenched with water and extracted with CH2Cl2
ongoing for structure−acꢀvity relaꢀonship studies.
(3 × 60 mL). The combined organic layers were washed with
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by flash column
chromatography using petroleum ether/ethyl acetate (8:1) as
eluent to give the pure products 8a-l in good yields.
4 Experimental
4.1 Materials and measurements
All reagents were obtained from commercial suppliers and
used without further purification. All NMR spectra were
recorded on a Bruker Avance 500 (resonance frequencies 500
MHz for 1H and 126 MHz for 13C) equipped with a 5 mm
inverse broadband probe head with z-gradients at 295.8K with
standard Bruker pulse programs. The samples were dissolved
in 0.6 ml CDCl3 (99.8% D.TMS). Chemical shifts were given in
values of δH and δC referenced to residual solvent signals (δH
7.26 for 1H, δC 77.0 for 13C in CDCl3). The 19F NMR spectra were
A mixture of compound
8 (10 mmol) and 5 % Pd/C (0.25 g)
in methanol (30 mL) was stirred under H2 for 3h at room
temperature. The reaction mixture was filtered through
diatomaceous earth, and the residue was washed with
methanol. The filter was concentrated under reduced
pressure. The crude liquid was purified by flash column
chromatography using petroleum ether/ethyl acetate (3:1) as
eluent to yield the pure products 9a-l
.
4.3.1 4-(5-(trifluoromethyl)-1 -pyrazol-1-yl)aniline (9a). Light
H
yellow liquid, yield: 90%. 1H NMR (500 MHz, CDCl3) δ (ppm)
7.64-7.62 (m, 1H, pyrazole-H), 7.23-7.21 (m, 2H, ArHs), 6.75-
6.73 (m, 2H, ArHs), 6.67-6.65 (m, 1H, pyrazole-H), 3.81 (br, 2H,
NH2); 13C NMR (126 MHz, CDCl3) δ (ppm) 147.7, 139.0, 132.7
obtained using
a 500 spectrometer (471 MHz) using
trifluorotoluene as external standard. Conventional pulse
sequences for NOESY, HMQC and HMBC with standard Bruker
pulse programs; 200-ms mixing time for NOESY; J in Hz. High
resolution mass spectra (HRMS) were recorded on a Bruker
solan X 70 FT-MS (samples was dissolved in CH3OH and the ion
source was ESI ), and the energy was 22.5eV at MS/MS.
Melting points are uncorrected.
2
1
(q, JC-F = 39.0 Hz), 129.8, 127.0, 120.0 (q, JC-F = 268.7 Hz),
114.7, 108.2; 19F NMR (471 MHz, CDCl3) δ (ppm) -62.3 (s, 3F).
4.3.2
4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)
aniline (9b). Light yellow liquid, yield: 91%. 1H NMR (500 MHz,
CDCl3) δ (ppm) 7.21-6.69 (m, 4H, ArHs), 6.54 (s, 1H, pyrazole-
H), 3.53 (br, 2H, NH2), 2.34 (s, 3H, CH3); 13C NMR (126 MHz,
4.2 Synthesis of key intermediate
hydroxypropyl)carbamate 3
(S)-tert-butyl (3-chloro-2-
2
CDCl3) δ (ppm) 148.3, 147.3, 133.1 (q, JC-F = 39.0 Hz), 130.0,
1
127. 4, 120.5 (q, JC-F = 268.9 Hz), 114.7, 107.8, 13.3; 19F NMR
The synthesis of (S)-tert-butyl (3-chloro-2-hydroxypropyl)
carbamate had been reported by William R. Perrault. We
prepared this compound completely according to the
literature procedure and obtained compound
in high yield.23
(471 MHz, CDCl3) δ (ppm) -62.7 (s, 3F).
3
4.3.3
4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-1-yl)
aniline (9c). Light yellow liquid, yield: 90%. 1H NMR (500 MHz,
CDCl3) δ (ppm) 7.45-7.42 (m, 1H, pyrazole-H), 7.18-7.16 (m, 2H,
ArHs), 6.65-6.62 (m, 2H, ArHs), 3.89 (br, 2H, NH2), 2.22 (s, 3H,
CH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 147.5, 140.6, 130.6,
3
4.3 General procedure for the preparation of 4-(5-(fluoroalkyl)-1
pyrazol-1-yl)anilines 9a-l
H-
2
1
129.2 (q, JC-F = 37.1 Hz), 127.3, 120.9 (q, JC-F = 269.6 Hz),
119.1, 114.6, 9.0; 19F NMR (471 MHz, CDCl3) δ (ppm) -62.7 (s,
3F).
To a stirred solution of pyridine (1.58 g, 20 mmol) and ethyl
vinyl ether derivatives (22 mmol) in CH2Cl2 (50 mL) was
dropwise added 2,2,2-trifluoroacetic anhydride or 2,2-
difluoroacetic anhydride (20 mmol) at -5°C. The reaction
mixture was stirred for 1 h at 0 °C, and then for another 12h at
20°C. After the completion of reaction, the mixture was
concentrated in vacuo, diluted with ethylene chloride (50 mL)
and poured into saturated sodium bicarbonate solution (50
ml). The two phases were separated and the organic layer was
washed with water, dried by anhydrous Na2SO4. The solvent
was removed in reduced pressure to provide oil products 4-
ethoxy-1-fluoroalkyl-but-3-en-2-ones 5a-f in high yields.
Compounds 5a-f were used for next step without further
purification.
4.3.4 3-fluoro-4-(5-(trifluoromethyl)-1H-pyrazol-1-yl)aniline
1
(9d). Light yellow liquid, yield: 89%. H NMR (500 MHz, CDCl3)
δ (ppm)7.73-7.70 (m, 1H, pyrazole-H), 7.14-7.11 (m, 1H, ArHs),
6.79-6.76 (m, 1H, pyrazole-H), 6.43-6.40 (m, 2H, ArHs), 4.01
1
(br, 2H, NH2); 13C NMR (126 MHz, CDCl3) δ (ppm) 158.7 (d, JC-F
3
= 250.8 Hz), 150.1 (d, JC-F = 10.6 Hz), 139.8, 134.2 (q,
2’
J
C-F
=
1’
39.0 Hz), 129.8, 119.8 (q,
2
J
= 264.6 Hz), 116.4, 110.0, 108.0,
C-F
101.8 (d, JC-F = 27.7 Hz); 19F NMR (471 MHz, CDCl3) δ (ppm) -
62.8 (s, 3F), -121.9 (s, 1F).
4.3.5 3-fluoro-4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-
1
1-yl)aniline (9e). Light yellow liquid, yield: 90%. H NMR (500
MHz, CDCl3) δ (ppm) 7.14-7.11 (m, 1H, ArH), 6.57-6.55 (m, 1H,
Compound
5 (15 mmol) and (4-nitrophenyl)hydrazine 6 (15
pyrazole-H), 6.44-6.39 (m, 2H, ArHs), 3.93 (br, 2H, NH2), 2.35(s,
1
mmol) in 50 mL ethanol was heated at reflux for 24 h, followed
by cooling and concentration in vacuo. The residue was
dissolved in EtOAc (100 mL), washed with water (3 × 100 mL)
and brine (100 mL). The organic layer was dried by anhydrous
Na2SO4 and concentrated in vacuo to afford the brown solid
3H, CH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 158.6 (d, JC-F
=
2’
250.9 Hz), 149.8 (d, 3JC-F = 10.7Hz), 149.1, 134.6 (q,
J
= 38.8
C-F
1’
Hz), 130.0, 119.7 (q,
2
J
C-F
= 269.6 Hz), 116.6, 110.0, 107.6,
101.8 (d, JC-F = 23.0 Hz), 13.4; 19F NMR (471 MHz, CDCl3) δ
(ppm) -62.7 (s, 3F), -121.9 (s, 1F).
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1
4.3.6 3-fluoro-4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol- MHz, CDCl3) δ (ppm) 155.2 (d, JC-F = 247.6 Hz), 147.6 (d, 3JC-F
=
1
1-yl)aniline (9f). Light yellow liquid, yield: 92%. H NMR (500 10.6 Hz), 145.1 (t,
2’
4
= 28.6 Hz), 131.4 (d, JC-F = 6.1Hz),
= 10.4 Hz), 116.8, 112.6 (t, 1’JC-F = 232.5k
J
C-F
3’
MHz, CDCl3) δ (ppm) 7.53-7.50 (m, 1H, pyrazole-H), 7.16-7.12 126.0, 119.0 (d,
J
C-F
(m, 1H, ArH), 6.57-6.53 (m, 1H, ArH), 6.47-6.44 (m, 1H, ArH), Hz), 110.9 102.3 (d, JC-F = 23.2 Hz), 8.0; 19F NMR (471 MHz,
3.82 (br, 2H, NH2), 2.36 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3) CDCl3) δ (ppm) -112.1 (s, 2F), -124.7 (s, 1F).
2
1
δ (ppm) 158.8 (d, JC-F = 250.8 Hz), 149.6 (d, JC-F = 10.8 Hz),
3
2’
1’
149.1, 141.4, 135.1 (q,
J
= 36.6 Hz), 130.0, 120.2 (q,
J
C-F
=
4.4 General procedure for the preparation of (
S)-tert-butyl (2-
C-F
264.5 Hz), 110.0, 107.6, 101.9 (d, 2JC-F = 22.9 Hz), 13.4;19F NMR hydroxy-3-((4-(5-(fluoroalkyl)-1
H
-pyrazol-1-yl)phenyl)amino)
(471 MHz, CDCl3) δ (ppm) -62.8 (s, 3F), -121.6 (s, 1F).
propyl)carbamates 10a-l
4.3.7 4-(5-(difluoromethyl)-1H-pyrazol-1-yl)aniline (9g).
1
Light yellow liquid, yield: 92%. H NMR (500 MHz, CDCl3) δ
The mixture of compound
9
(10 mmol) and compound
3 (3.14
g, 15 mmol) was heated to 150
°C and stirred overnight. After
(ppm)7.67-7.64 (m, 1H, pyrazole-H), 7.23-7.21 (m, 2H, ArHs),
6.75-6.70 (m, 2H, ArHs), 6.58 (t, 2JH-F = 49.5 Hz, 1H, CF2H), 6.48-
6.44 (m, 1H, pyrazole-H),3.88 (br, 2H, NH2); 13C NMR (126 MHz,
the completion of reaction, the residue was purified by flash
column chromatography using petroleum ether/ethyl acetate
(8:1) as eluent to give the pure products 10a-l
.
2
CDCl3) δ (ppm) 147.4, 139.5, 136.3 (t, JC-F = 30.1 Hz), 129.6,
1
4.4.1 (S)-tert-butyl (2-hydroxy-3-((4-(5-(trifluoromethyl)-
1H-pyrazol-1-yl)phenyl)amino)propyl) carbamate (10a). 1H
NMR (500 MHz, CDCl3) δ (ppm) 7.67 (br, 1H, NHCO), 7.20-7.19
(m, 1H, pyrazole-H), 7.20-7.17 (m, 2H, ArHs), 6.80-6.70 (m, 2H,
ArHs), 6.70-6.60 (m, 1H, pyrazole-H), 5.10-5.09 (m, 1H, Ar-NH)
4.01-4.00 (m, 1H, CH), 3.40-3.21 (m, 4H, 2CH2), 1.48 (s, 9H,
3CH3).
126.5, 115.0, 108.7 (t, JC-F = 235.0 Hz), 106.4; 19F NMR (471
MHz, CDCl3) δ (ppm) -110.1 (s, 2F).
4.3.8
4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)
aniline (9h). Light yellow liquid, yield: 89%. 1H NMR (500 MHz,
CDCl3) δ (ppm) 7.21-7.19 (m, 2H, ArHs), 6.69-6.67 (m, 2H,
2
ArHs), 6.52 (t, JH-F = 53.9 Hz, 1H, CF2H), 6.49-6.48 (m, 1H,
pyrazole-H), 3.77 (br, 2H, NH2), 2.35(s, 3H, CH3); 13C NMR (126
4.4.2
(S)-tert-butyl
(2-hydroxy-3-((4-(3-methyl-5-
propyl)
2
MHz, CDCl3) δ (ppm) 148.8, 147.1, 136.9 (t, JC-F = 29.9 Hz),
(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)
129.6, 126.5, 115.0, 108.7 (t, JC-F = 234.7 Hz), 105.9, 13.4; 19F
1
carbamate (10b). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.67 (br,
1H, NHCO), 7.21-7.15 (m, 2H, ArHs), 6.90-6.85 (m, 2H, ArHs),
6.84-6.80 (m, 1H, pyrazole-H) ,5.61-5.60 (m, 1H, Ar-NH), 4.00-
3.98 (m, 1H, CH), 3.40-3.21 (m, 4H, 2CH2), , 2.37 (s, 3H,
pyrazole-CH3), 1.50 (s, 9H, 3CH3).
NMR (471 MHz, CDCl3) δ (ppm) -110.1 (s, 2F).
4.3.9
4-(5-(difluoromethyl)-4-methyl-1H-pyrazol-1-yl)
1
aniline (9i). Light yellow liquid, yield: 89%. H NMR (500 MHz,
CDCl3) δ (ppm) 7.47-7.46 (m, 1H, pyrazole-H), 7.18-7.16 (m, 2H,
2
ArHs), 6.67-6.66 (m, 2H, ArHs), 6.57 (t, JH-F = 53.1 Hz, 1H,
4.4.3
(S)-tert-butyl
(2-hydroxy-3-((4-(4-methyl-5-
propyl)
CF2H), 3.92 (br, 2H, NH2), 2.26 (s, 3H, CH3); 13C NMR (126 MHz,
(trifluoromethyl)-1H-pyrazol-1-yl)phenyl)amino)
2
CDCl3) δ (ppm) 147.3, 140.7, 132.4 (t, JC-F = 29.1 Hz), 129.8,
carbamate (10c). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.67 (br,
1H, NHCO), 7.43-7.40 (m, 1H, pyrazole-H), 7.21-7.15 (m, 2H,
ArHs), 6.92-6.83 (m, 2H, ArHs) , 6.00-5.98 (m, 1H, Ar-NH), 4.00-
3.99 (m, 1H, CH), 3.39-3.18 (m, 4H), 2.26 (s, 3H, pyrazole-CH3),
1.49 (s, 9H, 3CH3).
126.6, 117.8, 114.9, 109.7 (t, JC-F = 233.5 Hz), 8.5; 19F NMR
1
(471 MHz, CDCl3) δ (ppm) -111.4 (s, 2F).
4.3.10
4-(5-(difluoromethyl)-1H-pyrazol-1-yl)-3-fluoro
aniline (9j). Light yellow liquid, yield: 90%. 1H NMR (500 MHz,
CDCl3) δ (ppm) 7.72-7.71 (m,1H, pyrazole-H), 7.17-7.16 (m, 1H,
ArH), 6.57 (t, 2JH-F = 53.6 Hz, 1H, CF2H), 6.46-6.44 (m, 2H, ArHs),
4.4.4 (S)-tert-butyl (3-((3-fluoro-4-(5-(trifluoromethyl)-1H-
pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl)
carbamate
6.43-6.40 (m, 1H, pyrazole-H),4.05 (br, 2H, NH2); 13C NMR (126
(10d). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69 (br, 1H, NHCO),
7.44-7.41 (m, 1H, pyrazole-H), 7.40-7.37 (m, 1H, ArH), 6.88-
6.82 (m, 2H, ArHs), 6.77-6.75 (m, 1H, pyrazole-H), 5.12-5.10
(m, 1H, Ar-NH), 4.04-4.02 (m, 1H, CH), 3.37-3.18 (m, 4H, 2CH2),
1.49 (s, 9H, 3CH3).
1
MHz, CDCl3) δ (ppm) 157.8 (d, JC-F = 249.4 Hz), 149.9 (d, 3JC-F
=
=
10.7 Hz), 140.2, 137.9 (t, 2’JC-F = 29.6 Hz), 129.6, 116.3 (d, 3’JC-F
13.0 Hz), 110.5, 108.5 (t, 1’JC-F = 237.0 Hz), 106.4, 101.8 (d, 2JC-F
= 22.9 Hz); 19F NMR (471 MHz, CDCl3) δ (ppm) -112.4 (s, 2F), -
122.7 (s, 1F).
4.4.5 (S)-tert-butyl (3-((3-fluoro-4-(3-methyl-5-(trifluoro-
4.3.11 4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1-yl)-3-
fluoroaniline (9k). Light yellow liquid, yield: 88%. 1H NMR (500
MHz, CDCl3) δ (ppm) 7.12-7.11 (m, 1H, ArH), 6.51 (t, 2JH-F = 54.3
Hz, 1H, CF2H), 6.40-6.39 (m, 2H, ArHs), 6.38-6.37 (m, 1H,
pyrazole-H),4.17 (br, 2H, NH2), 2.33 (s, 3H, CH3); 13C NMR (126
methyl)-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxy
propyl)
carbamate (10e). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.72 (br,
1H, NHCO), 7.33-7.30 (m, 1H, ArH), 6.64-6.60 (m, 2H, ArHs),
6.23-6.20 (m, 1H, pyrazole-H), 4.03-4.01 (m, 1H, CH), 3.39-3.18
(m, 4H, 2CH2), 2.38 (s, 3H, pyrazole-CH3), 1.51 (s, 9H, 3CH3).
4.4.6 (S)-tert-butyl (3-((3-fluoro-4-(4-methyl-5-(trifluoro-
methyl)-1H-pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl) car-
bamate (10f). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.70 (br, 1H,
NHCO), 7.44-7.41 (m, 1H, pyrazole-H), 6.64-6.60 (m, 3H, ArHs),
4.01-3.99 (m, 1H, CH), 3.37-3.14 (m, 4H), 2.27 (s, 3H, pyrazole-
CH3), 1.50 (s, 9H, 3CH3).
1
MHz, CDCl3) δ (ppm) 157.8 (d, JC-F = 249.4 Hz), 158.9, 156.9,
149.7, 138.4 (t, 2’JC-F = 29.4 Hz), 116.3 (d, 1JC-F = 13.6 Hz), 110.4,
1’
2
107.7 (t,
J
= 236.6 Hz), 107.0, 101.8(d, JC-F = 22.9Hz), 13.4;
C-F
19F NMR(471 MHz, CDCl3) δ (ppm) -112.4 (s, 2F), -122.7 (s, 1F).
4.3.12 4-(5-(difluoromethyl)-4-methyl-1H-pyrazol-1-yl)-3-
1
fluoroaniline (9l). Light yellow liquid, yield: 92%. H NMR (500
MHz, CDCl3) δ (ppm) 7.58-7.55 (m, 1H, pyrazole-H), 7.53-7.48
2
(m, 1H, ArH),6.77 (t, JH-F = 54.3 Hz, 1H, CF2H), 6.48-6.45 (m,
2H, ArHs), 3.97 (br, 2H, NH2), 2.25 (s,3H, CH3); 13C NMR (126
4.4.7 (S)-tert-butyl (3-((4-(5-(difluoromethyl)-1H-pyrazol-1-
yl)phenyl)amino)-2-hydroxypropyl)carbam-ate (10g). 1H NMR
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(500 MHz, CDCl3) δ (ppm) 7.70 (br, 1H, NHCO), 7.54-7.52 (m,
4.5.1 (S)-tert-butyl ((2-oxo-3-(4-(5-(trifluoromethyl)-1H-
1H, pyrazole-H), 7.50-7.48 (m, 2H, ArHs), 6.77-6.68 (m, 2H, pyrazol-1-yl)phenyl)oxazolidin-5-yl)methyl)car-bamate (11a).
ArHs), 6.64-6.62 (m, 1H, pyrazole-H), 6.43 (t, 2JH-F = 53.4 Hz, 1H, 1H NMR (500 MHz, CDCl3) δ (ppm) 7.75-7.74 (m, 1H, pyrazole-
CF2H), 4.00-3.99 (m, 1H, CH), 3.40-3.25 (m, 4H, 2CH2), 1.51 (s, H),7.73-7.71 (m, 2H, ArHs), 7.56-7.53 (m, 2H, ArHs), 6.79-6.77
9H, 3CH3).
4.4.8 (S)-tert-butyl (3-((4-(5-(difluoromethyl)-3-methyl-1H- CH), 4.10-4.09 (m, 1H, NCH2-Ha), 3.89-3.86 (m, 1H, NCH2-Hb),
pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl) carbamate 3.73-3.70 (m, 2H, CH2), 1.43 (s, 9H, 3CH3).
(10h). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.70 (br, 1H), 7.40-
4.5.2 (S)-tert-butyl ((3-(4-(3-methyl-5-(trifluoromethyl)-1H-
(m, 1H, pyrazole-H), 6.19-6.18 (m, 1H, NH), 4.85-4.84 (m, 1H,
7.32 (m, 2H, ArHs), 6.65-6.63 (m, 2H, ArHs), 6.62-6.60 (m, 1H, pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl) carbamate
2
pyrazole-H), 6.31 (t, JH-F = 53.8 Hz, 1H, CF2H), 4.00-3.99 (m, (11b). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.71-7.59 (m, 4H,
1H, CH), 3.40-3.25 (m, 4H, 2CH2), 2.33 (s, 3H, pyrazole-CH3), ArHs), 6.70 (s, 1H, pyrazole-H), 6.52-6.43 (m, 1H, NH), 4.93-
1.48 (s, 9H, 3CH3).
4.4.9 (S)-tert-butyl (3-((4-(5-(difluoromethyl)-4-methyl-1H- NCH2-Hb), 3.62-3.59 (m, 2H, CH2), 1.97 (s, 3H, pyrazole-CH3),
pyrazol-1-yl)phenyl)amino)-2-hydroxypropyl) carbamate 1.47 (s, 9H, 3CH3).
(10i). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.71 (br, 1H, NHCO),
4.5.3 (S)-tert-butyl ((3-(4-(4-methyl-5-(trifluoromethyl)-1H-
4.90 (m, 1H, CH), 4.09-4.07 (m, 1H, NCH2-Ha), 3.76-3.69 (m, 1H,
7.15-7.13 (m, 1H, pyrazole-H), 7.10-7.02 (m, 2H, ArHs), 6.67- pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl) carbamate
2
6.60 (m, 2H, ArHs), 6.44 (t, JH-F = 52.8 Hz, 1H, CF2H), 4.02-4.01 (11c). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69-7.65 (m, 1H,
(m, 1H, CH), 3.45-3.30 (m, 4H, 2CH2), 2.23 (s, 3H, pyrazole- pyrazole-H), 7.64-7.60 (m, 1H, ArH), 7.32-7.30 (m, 3H, ArHs),
CH3), 1.49 (s, 9H, 3CH3).
4.4.10 (S)-tert-butyl (3-((4-(5-(difluoromethyl)-1H-pyrazol- NCH2-Ha), 3.77-3.75 (m, 1H, NCH2-Hb), 3.60-3.57 (m, 2H, CH2),
1-yl)-3-fluorophenyl)amino)-2-hydroxypropyl) carbamate 1.99 (s, 3H, pyrazole-CH3), 1.47 (s, 9H, 3CH3).
(10j). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.73 (br, 1H, NHCO),
4.5.4 (S)-tert-butyl ((3-(3-fluoro-4-(5-(trifluoromethyl)-1H-
6.86-6.83 (m, 1H, NH), 4.72-4.70 (m, 1H, CH), 4.09-4.01 (m, 1H,
7.50-7.48 (m, 1H, pyrazole-H), 7.40-7.28 (m, 1H, ArH), 6.72- pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate
2
6.65 (m, 2H, ArHs), 6.62-6.60 (m, 1H, pyrazole-H), 6.42 (t, JH-F (11d). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69-7.67 (m, 1H,
= 54.4 Hz, 1H, CF2H), 4.02-4.00 (m, 1H, CH), 3.44-3.32 (m, 4H, pyrazole-H), 7.65-7.63 (m, 1H, ArH), 7.34-7.30 (m, 2H, ArHs),
2CH2), 1.50 (s, 9H, 3CH3).
6.87-6.86 (m, 1H, pyrazole-H), 6.85-6.84 (m, 1H, NH), 4.74-4.72
4.4.11 (S)-tert-butyl (3-((4-(5-(difluoromethyl)-3-methyl- (m, 1H, CH), 4.09-4.05 (m, 1H, NCH2-Ha), 3.84-3.81 (m, 1H,
1H-pyrazol-1-yl)-3-fluorophenyl)amino)-2-hydr-oxypropyl)
carba- mate (10k). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69 (br,
NCH2-Hb), 3.70-3.65 (m, 2H, CH2), 1.45 (s, 9H, 3CH3).
4.5.5 (S)-tert-butyl ((3-(3-fluoro-4-(3-methyl-5-
1H, NHCO), 7.24-7.16 (m, 2H, ArHs), 6.72-6.64 (m, 1H, ArH), (trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin- 5-
6.62-6.60 (m, 1H, pyrazole-H), 6.44 (t, 2JH-F = 54.4 Hz, 1H, CF2H), yl)methyl)carbamate (11e). 1H NMR (500 MHz, CDCl3) δ (ppm)
4.00-3.99 (m, 1H, CH), 3.39-3.30 (m, 4H, 2CH2), 2.30 (s, 3H, 7.74-7.71 (m, 1H, ArH), 7.54-7.50 (m, 1H, ArH), 7.40-7.37 (m,
pyrazole-CH3), 1.53 (s, 9H, 3CH3).
4.4.12 (S)-tert-butyl (3-((4-(5-(difluoromethyl)-4-methyl- 4.78-4.75 (m, 1H, CH), 4.09-4.06 (m, 1H, NCH2-Ha), 3.86-3.84
1H-pyrazol-1-yl)-3-fluorophenyl)amino)-2-hydroxypropyl) (m, 1H, NCH2-Hb), 3.77-3.68 (m, 2H, CH2), 1.98 (s, 3H, pyrazole-
carba- mate (10l). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.67 (br, CH3), 1.49 (s, 9H, 3CH3).
1H, NHCO), 7.19-7.16 (m, 1H, pyrazole-H), 7.12-7.08 (m, 1H, 4.5.6 (S)-tert-butyl
1H, ArH), 6.76-6.74 (m, 1H, pyrazole-H), 6.34-6.31 (m, 1H, NH),
((3-(3-fluoro-4-(4-methyl-5-
ArH), 6.80-6.55 (m, 2H, ArHs), 6.64 (t, 2JH-F = 54.2 Hz, 1H, CF2H), (trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin- 5-
4.00-3.99 (m, 1H, CH), 3.39-3.30 (m, 4H, 2CH2), 2.20 (s, 3H, yl)methyl)carbamate (11f). 1H NMR (500 MHz, CDCl3) δ (ppm)
pyrazole-CH3), 1.47 (s, 9H, 3CH3).
7.71-7.68 (m, 1H, pyrazole-H), 7.50-7.48 (m, 1H, ArH), 7.42-
7.39 (m, 1H, ArH), 6.78-6.76 (m, 1H, ArH), 5.94-5.92 (m, 1H,
4.5 General procedure for the preparation of (
S
)-tert-butyl ((2-oxo- NH), 4.24-4.22 (m, 1H, CH), 4.02-4.00 (m, 1H, NCH2-Ha), 3.79-
3-(4-(5-(fluoroalkyl)-1
H
-pyrazol-1-yl)phenyl)oxazolidin-5-yl) meth-
3.76 (m, 1H, NCH2-Hb), 3.68-3.65 (m, 2H, CH2), 1.97 (s, 3H,
pyrazole-CH3), 1.47 (s, 9H, 3CH3).
yl)carbamates 11a-l
4.5.7 (S)-tert-butyl ((3-(4-(5-(difluoromethyl)-1H-pyrazol-1-
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)carbamate (11g). 1H
NMR (500 MHz, CDCl3) δ (ppm) 7.69-7.67 (m, 1H, pyrazole-H),
7.66-7.60 (m, 2H, ArHs), 7.59-7.56 (m, 2H, ArHs), 6.68-6.66 (m,
1H, pyrazole-H), 6.58 (t, 2JH-F = 55.7 Hz, 1H, CF2H), 6.49-6.45 (m,
1H, NH), 4.83-4.80 (m, 1H, CH), 4.04-4.01 (m, 1H, NCH2-Ha),
3.86-3.84 (m, 1H, NCH2-Hb), 3.67-3.64 (m, 2H, CH2), 1.43 (s, 9H,
3CH3).
Triethylamine (1.62 g, 16 mmol) and carbonyl diimidazole (CDI)
(2.59 g, 16 mmol) was added dropwise to a solution of
compound 10 (8 mmol) in THF (50 mL) at room temperature.
The mixture was stirred for 14 h. After the completion of
reaction, the reaction was quenched with water and extracted
with EtOAc (3 × 50 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The residue was
purified by flash column chromatography using petroleum
ether/ethyl acetate (4:1) as eluent to give the pure products
4.5.8 (S)-tert-butyl ((3-(4-(5-(difluoromethyl)-3-methyl-1H-
pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl) carbamate
(11h). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.70-7.68 (m, 2H,
ArHs), 7.51-7.48 (m, 2H, ArHs), 6.86-6.83 (m, 1H, pyrazole-H),
11a-l
.
2
6.50 (t, JH-F = 54.9 Hz, 1H, CF2H), 6.41-6.39 (m, 1H, NH), 4.77-
6 | J. Name., 2012, 00, 1-3
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4.74 (m, 1H, CH), 4.09-4.06 (m, 1H, NCH2-Ha), 3.78-3.75 (m, 1H,
4.6.1 (S)-N-((2-oxo-3-(4-(5-(trifluoromethyl)-1H-pyrazol-1-
(13a). Light
C. 1H NMR (500
NCH2-Hb), 3.63-3.61 (m, 2H, CH2), 1.97 (s, 3H, pyrazole-CH3), yl)phenyl)oxazolidin-5-yl)methyl)acetamide
1.50 (s, 9H, 3CH3). yellow solid, yiled: 85%, m.p: 145.2-147.8
°
4.5.9 (S)-tert-butyl ((3-(4-(5-(difluoromethyl)-3-methyl-1H- MHz, CDCl3) δ (ppm) 7.72-7.71 (m, 1H, pyrazole-H), 7.70-7.68
pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl) carbamate (m, 2H, ArHs), 7.53-7.51 (m, 2H, ArHs), 6.83-6.82 (m, 1H,
(11i). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69-7.67 (m, 1H, pyrazole-H), 6.21-6.20 (m, 1H, NH), 4.84-4.83 (m, 1H, CH),
pyrazole-H), 7.58-7.54 (m, 3H, ArHs), 7.10-7.07 (m, 1H, ArH), 4.16-4.12 (m, 1H, NCH2-Ha), 3.90-3.87 (m, 1H, NCH2-Hb), 3.76-
2
6.70 (t, JH-F = 53.7 Hz, 1H, CF2H), 6.43-6.41 (m, 1H, NH), 4.71- 3.61 (m, 2H, CH2), 2.05 (s, 3H, COCH3); 13C NMR (126 MHz,
4.68 (m, 1H, CH), 4.11-4.07 (m, 1H, NCH2-Ha), 3.76-3.73 (m, 1H, CDCl3) δ (ppm) 171.2, 154.3, 139.7, 138.8, 135.0, 132.6, 126.5,
1
NCH2-Hb), 3.54-3.50 (m, 2H, CH2), 1.93 (s, 3H, pyrazole-CH3), 119.9 (q, JC-F = 268.3 Hz), 118.3, 109.0, 72.1, 47.5, 41.9, 23.1;
1.46 (s, 9H, 3CH3).
4.5.10 (S)-tert-butyl ((3-(4-(5-(difluoromethyl)-1H-pyrazol- for C16H15F3N4O3Na [M+Na]+: 391.0994, found: 391.0999
1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)methyl)carbamate 4.6.2 (S)-N-((3-(4-(3-methyl-5-(trifluoromethyl)-1H-pyrazol-
(11j). 1H NMR (500 MHz, CDCl3) δ (ppm) 7.71-7.70 (m, 1H, 1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide
(13b).
pyrazole-H), 7.69-7.68 (m, 1H, ArH), 7.53-7.35 (m, 2H, ArHs), Light yellow solid, yiled: 83%, m.p: 148.4-150.2
C. 1H NMR
19F NMR (471 MHz, CDCl3) δ (ppm) -62.8 (s, 3F). HRMS calcd.
°
6.77-6.75 (m, 1H, pyrazole-H), 6.68 (t, 2JH-F = 54.6 Hz, 1H, CF2H), (500 MHz, CDCl3) δ (ppm) 7.66-7.48 (m, 4H, ArHs), 6.61 (s, 1H,
6.55-6.53 (m, 1H, NH), 4.76-4.72 (m, 1H, CH), 4.09-4.07 (m, 1H, pyrazole-H), 6.41-6.32 (m, 1H, NH), 4.83-4.82 (m, 1H, CH),
NCH2-Ha), 3.78-3.74 (m, 1H, NCH2-Hb), 3.61-3.59 (m, 2H, CH2), 4.14-4.10 (m, 1H, NCH2-Ha), 3.88-3.85 (m, 1H, NCH2-Hb), 3.71-
1.48 (s, 9H, 3CH3).
3.63 (m, 2H, CH2), 2.37 (s, 3H, pyrazole-CH3), 2.04 (s, 3H,
4.5.11 (S)-tert-butyl ((3-(4-(5-(difluoromethyl)-3-methyl- COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 171.2, 154.3,
2
1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl)
149.1, 138.5, 135.1, 133.1 (q, JC-F = 37.8 Hz), 126.1, 119.8 (q,
methyl) carbamate (11k). 1H NMR (500 MHz, CDCl3) δ (ppm) 1JC-F = 268.9 Hz), 118.2, 108.7, 72.1, 47.2, 41.9, 23.0, 13.3; 19F
7.69-7.37 (m, 3H, ArHs), 6.75-6.73 (m, 1H, pyrazole-H), 6.65 (t, NMR (471 MHz, CDCl3) δ (ppm) -62.8 (s, 3F). HRMS calcd. for
2JH-F = 54.8 Hz, 1H, CF2H), 6.57-6.53 (m, 1H, NH), 4.89-4.85 (m, C34H34F6N8O6Na [2M+Na]+: 787.2403, found: 787.2412.
1H, CH), 4.11-4.09 (m, 1H, NCH2-Ha), 3.78-3.76 (m, 1H, NCH2-
Hb), 3.59-3.55 (m, 2H, CH2), 1.97 (s, 3H, pyrazole-CH3), 1.51 (s, 1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide
9H, 3CH3). Off-white solid, yiled: 82%, m.p: 179.4-180.9
C. 1H NMR (500
4.5.12 (S)-tert-butyl ((3-(4-(5-(difluoromethyl)-4-methyl- MHz, CDCl3) δ (ppm) 7.66-7.64 (m, 1H, pyrazole-H), 7.63-7.62
1H-pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl) (m, 1H, ArH), 7.44-7.42 (m, 3H, ArHs), 6.75-6.73 (m, 1H, NH),
4.6.3 (S)-N-((3-(4-(4-methyl-5-(trifluoromethyl)-1H-pyrazol-
(13c).
°
methyl) carbamate (11l). 1H NMR (500 MHz, CDCl3) δ (ppm) 4.81-4.80 (m, 1H, CH), 4.12-4.08 (m, 1H, NCH2-Ha), 3.88-3.85
7.77-7.75 (m, 1H, pyrazole-H), 7.74-7.70 (m, 2H, ArHs), 7.30- (m, 1H, NCH2-Hb), 3.69-3.60 (m, 2H, CH2), 2.26 (s, 3H, pyrazole-
7.29 (m, 1H, ArH), 6.97-6.95 (m, 1H, NH), 6.83 (t, 2JH-F = 53.6 Hz, CH3), 2.02 (s, 3H, COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm)
2
1H, CF2H), 4.90-4.88 (m, 1H, CH), 4.06-4.03 (m, 1H, NCH2-Ha), 171.3, 154.4, 141.4, 138.6, 135.6, 129.2 (q, JC-F = 37.6 Hz),
3.76-3.73 (m, 1H, NCH2-Hb), 3.65-3.62 (m, 2H, CH2), 1.96 (s, 3H, 126.7, 120.7 (q, 1JC-F = 269.4 Hz), 119.9, 118.2, 72.1, 47.5, 41.9,
pyrazole-CH3), 1.49 (s, 9H, 3CH3).
23.0, 9.0; 19F NMR (471 MHz, CDCl3) δ (ppm) -62.8 (s, 3F).
HRMS calcd. for C17H17F3N4O3Na[M+Na]+: 405.1150, found:
4.6 General procedure for the preparation of (
S
)-
N
-((2-oxo-3-(4-(5- 405.1149.
4.6.4 (S)-N-((3-(3-fluoro-4-(5-(trifluoromethyl)-1H-pyrazol-
1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13d).
C. H NMR (500
(fluoroalkyl)-1H-pyrazol-1-yl)phenyl)oxazolidin-5-yl)methyl) acet-
amides 13a-l
1
Off-white solid, yiled: 80%, m.p: 165.1-167.3
°
To a solution of compound 11 (6 mmol) in CH2Cl2 (50 mL) was
added dropwise trifluoroacetic acid (1.37g, 12 mmol). The
mixture was stirred for 12h at room temperature. The solvent
was removed in reduced pressure to provide the crude
products 12a-l, which were used for next step without further
purification.
MHz, CDCl3) δ (ppm) 7.74-7.71 (m, 1H, pyrazole-H), 7.70-7.67
(m, 1H, ArH), 7.44-7.30 (m, 2H, ArHs), 6.88-6.86 (m, 1H,
pyrazole-H), 6.84-6.82 (m, 1H, NH), 4.84-4.82 (m, 1H, CH),
4.13-4.09 (m, 1H, NCH2-Ha), 3.88-3.85 (m, 1H, NCH2-Hb), 3.68-
3.63 (m, 2H, CH2), 2.01 (s, 3H, COCH3); 13C NMR (126 MHz,
1
CDCl3) δ (ppm) 171.5, 158.9 (d, JC-F = 253.3 Hz), 154.2, 141.0
(d, 3JC-F = 10.6 Hz), 140.3, 134.1 (q, 2’JC-F = 38.8 Hz), 129.7, 122.1
Acetic anhydride (1.23g, 12 mmol) was added dropwise to a
mixture of compound 12 (6 mmol) in CH2Cl2 (50 mL) and
triethylamine (1.82 g, 18 mmol). The resulting solution was
stirred for 12h at room temperature. After the completion of
reaction, the reaction was quenched with water and extracted
with CH2Cl2 (3 × 50 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The residue was
purified by flash column chromatography using ethyl acetate
3’’
1’
(d,
J
C-F
= 13.2 Hz), 119.6 (q,
J
C-F
= 268.8 Hz), 113.0, 108.5,
106.3 (d, JC-F = 25.4 Hz), 72.3, 47.4, 41.8, 22.9; 19F NMR (471
MHz, CDCl3) δ (ppm) -62.8 (s, 3F), -118.8 (s, 1F). HRMS calcd.
for C16H14F4N4O3Na[M+Na]+: 409.0900, found: 409.0913.
2
4.6.5
1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)
amide (13e). Off-white solid, yiled: 87%, m.p: 153.5-155.2
1H NMR (500 MHz, CDCl3) δ (ppm) 7.71-7.68 (m, 1H, ArH),
7.45-7.42 (m, 1H, ArH), 7.33-7.30 (m, 1H, ArH), 6.64-6.60 (m,
1H, pyrazole-H), 6.23-6.20 (m, 1H, NH), 4.85-4.84 (m, 1H, CH),
(S)-N-((3-(3-fluoro-4-(3-methyl-5-(trifluoromethyl)-
acet-
C.
°
as eluent to give the pure target products 13a-l
.
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J. Name., 2013, 00, 1-3 | 7
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Journal Name
4.13-4.10 (m, 1H, NCH2-Ha), 3.88-3.84 (m, 1H, NCH2-Hb), 3.76- 13C NMR (126 MHz, CDCl3) δ (ppm) 171.5, 154.5, 141.4, 138.3,
2
3.65 (m, 2H, CH2), 2.38 (s, 3H, pyrazole-CH3), 2.05 (s, 3H, 135.1, 132.2 (t, JC-F = 28.2 Hz), 125.9, 118.9, 118.7, 109.2 (t,
COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 171.2, 158.0 (d, 1JC- 1JC-F = 235.5 Hz), 72.2, 47.5, 41.9, 22.9, 8.4; 19F NMR (471 MHz,
3
F = 253.4 Hz), 153.9, 149.8, 140.7 (d, JC-F = 10.5 Hz), 134.6 (q, CDCl3) δ (ppm) -111.0 (s, 2F). HRMS calcd. for C34H36F4N8O6Na
2’
J
C-F
= 39.1 Hz), 129.8, 122.4 (d, 3’JC-F = 12.5 Hz), 119.5 (q, 1’JC-F
=
[2M+Na]+: 751.2592, found: 751.2590.
2
269.6 Hz), 112.8, 108.2, 106.3 (d, JC-F = 25.6 Hz), 72.1, 47.4,
41.9, 23.1, 13.4; 19F NMR (471 MHz, CDCl3) δ (ppm) -62.8 (s, fluorophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13j).
3F), -118.6 (s, 1F). HRMS calcd. for C17H16F4N4O3Na [M+Na]+: Light yellow solid, yiled: 79%, m.p: 149.5-152.2
423.1056, found: 423.1055. (500 MHz, CDCl3) δ (ppm) 7.74-7.73 (m, 1H, pyrazole-H), 7.72-
4.6.6 (S)-N-((3-(3-fluoro-4-(4-methyl-5-(trifluoromethyl)- 7.70 (m, 1H, ArH), 7.50-7.28 (m, 2H, ArHs), 6.72-6.71 (m, 1H,
4.6.10 (S)-N-((3-(4-(5-(difluoromethyl)-1H-pyrazol-1-yl)-3-
°
C. 1H NMR
2
1H-pyrazol-1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)
acet- pyrazole-H), 6.70-6.68 (m, 1H, NH), 6.59 (t, JH-F = 54.4 Hz, 1H,
amide (13f). Off-white solid, yiled: 83%, m.p: 160.3-162.2
°
C. CF2H), 4.86-4.82 (m, 1H, CH), 4.13-4.11 (m, 1H, NCH2-Ha), 3.89-
1H NMR (500 MHz, CDCl3) δ (ppm) 7.69-7.66 (m, 1H, pyrazole- 3.85 (m, 1H, NCH2-Hb), 3.70-3.66 (m, 2H, CH2), 2.03 (s, 3H,
H), 7.65-7.28 (m, 3H, ArHs), 6.65-6.61 (m, 1H, NH), 4.85-4.83 COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 171.4, 156.8 (d, 1JC-
3
= 251.5 Hz), 154.1, 140.8, 140.4 (d, JC-F = 10.5 Hz), 137.8 (t,
(m, 1H, CH), 4.13-4.09 (m, 1H, NCH2-Ha), 3.88-3.84 (m, 1H,
NCH2-Hb), 3.68-3.65 (m, 2H, CH2), 2.36 (s, 3H, pyrazole-CH3),
2.03 (s, 3H, COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 171.4,
F
2’
J
J
= 27.8 Hz), 129.3, 122.4 (d, 3’JC-F = 12.9 Hz), 113.4, 108.5 (t,
C-F
1’
2
= 237.0 Hz), 107.3, 106.4 (d, JC-F = 25.4 Hz), 72.2, 47.4,
C-F
1
3
158.0 (d, JC-F = 253.1 Hz), 154.1, 149.8, 142.0, 140.7 (d, JC-F
2’
=
41.8, 23.0; 19F NMR (471 MHz, CDCl3) δ (ppm) -112.6 (s, 2F), -
3’
11.6 Hz), 134.6 (q,
1’
J
= 38.8 Hz), 129.7 (d,
J
= 11.4 Hz), 119.9 (s, 1F). HRMS calcd. for C16H15F3N4O3Na[M+Na]+:
C-F
C-F
2
120.5 (q,
J
C-F
= 269.2 Hz), 112.9, 108.2, 106.3 (d, JC-F = 25.6 391.0994, found: 391.1010.
Hz), 72.2, 47.4, 41.9, 23.0, 13.4; 19F NMR (471 MHz, CDCl3) δ
(ppm) -62.8 (s, 3F), -118.6 (s, 1F). HRMS calcd. for pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl) methyl)
C17H16F4N4O3Na[M+Na]+: 423.1056, found: 423.1055.
acetamide (13k). Light yellow solid, yiled: 81%, m.p: 151.3-
4.6.7
4.6.11
(S)-N-((3-(4-(5-(difluoromethyl)-3-methyl-1H-
1
(S)-N-((3-(4-(5-(difluoromethyl)-1H-pyrazol-1- 152.7 °C. H NMR (500 MHz, CDCl3) δ (ppm) 7.64-7.27 (m, 3H,
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13g). Light ArHs), 7.26-7.24 (m, 1H, pyrazole-H), 6.54 (t, 2JH-F = 54.4 Hz, 1H,
1
C. H NMR (500 CF2H), 6.47-6.42 (m, 1H, NH), 4.80-4.76 (m, 1H, CH), 4.08-4.05
yellow solid, yiled: 82%, m.p: 154.4-154.9 °
MHz, CDCl3) δ (ppm) 7.73-7.71 (m, 1H, pyrazole-H), 7.70-7.68 (m, 1H, NCH2-Ha), 3.83-3.78 (m, 1H, NCH2-Hb), 3.62-3.57 (m,
(m, 2H, ArHs), 7.55-7.51 (m, 2H, ArHs), 6.77-6.72 (m, 1H, 2H, CH2), 2.29 (s, 3H, pyrazole-CH3), 1.96 (s, 3H, COCH3); 13C
1
pyrazole-H), 6.64 (t, 2JH-F = 53.4 Hz, 1H, CF2H), 6.59-6.55 (m, 1H, NMR (126 MHz, CDCl3) δ (ppm) 172.1, 156.8 (d, JC-F = 251.0
3
NH), 4.86-4.80 (m, 1H, CH), 4.14-4.10 (m, 1H, NCH2-Ha), 3.90- Hz), 154.3, 150.2, 140.2 (d, JC-F = 9.9 Hz), 138.3 (t,
2’
J
= 28.9
C-F
3’
1’
3.86 (m, 1H, NCH2-Hb), 3.70-3.67 (m, 2H, CH2), 2.04 (s, 3H, Hz), 129.3, 122.4 (d,
J
= 12.6 Hz), 113.4, 108.5 (t,
2
J
C-F
=
C-F
COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 171.3, 154.4, 236.6 Hz), 107.0, 106.3 (d, JC-F = 26.0 Hz), 72.2, 47.4, 41.8,
2
140.2, 138.4, 136.3 (t, JC-F = 29.7 Hz), 134.9, 125.8, 118.7, 22.7, 13.3; 19F NMR (471 MHz, CDCl3) δ (ppm) -112.7 (s, 2F), -
108.5 (t, 1JC-F = 236.1 Hz), 107.6, 72.2, 47.6, 42.0, 23.1; 19F NMR 120.1 (s, 1F). HRMS calcd. for C17H17F3N4O3Na[M+Na]+:
(471 MHz, CDCl3) δ (ppm) -109.9 - -109.8 (m, 2F). HRMS calcd. 405.1150, found: 405.1166.
for C16H16F2N4O3Na[M+Na]+: 373.1088, found: 373.1098.
4.6.12
(S)-N-((3-(4-(5-(difluoromethyl)-4-methyl-1H-
4.6.8 (S)-N-((3-(4-(5-(difluoromethyl)-3-methyl-1H-pyrazol-1- pyrazol-1-yl)-3-fluorophenyl)-2-oxooxazolidin-5-yl) methyl)
yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13h). Off- acetamide (13l). Light yellow solid, yiled: 80%, m.p: 163.2-
C. 1H NMR (500 165.9
1
C. H NMR (500 MHz, CDCl3) δ (ppm) 7.79-7.77 (m, 1H,
white solid, yiled: 78%, m.p: 148.0-149.3
°
MHz, CDCl3) δ (ppm) 7.64-7.63 (m, 2H, ArHs), 7.49-7.47 (m, 2H, pyrazole-H), 7.76-7.64 (m, 2H, ArHs), 7.21-7.19 (m, 1H, ArH),
°
2
ArHs), 6.75-6.72 (m, 1H, pyrazole-H), 6.57 (t, 2JH-F = 53.8 Hz, 1H, 6.84-6.83 (m, 1H, NH), 6.74 (t, JH-F = 54.2 Hz, 1H, CF2H), 4.86-
CF2H), 6.53-6.51 (m, 1H, NH), 4.83-4.80 (m, 1H, CH), 4.11-4.08 4.82 (m, 1H, CH), 4.10-4.06 (m, 1H, NCH2-Ha), 3.86-3.83 (m, 1H,
(m, 1H, NCH2-Ha), 3.88-3.85 (m, 1H, NCH2-Hb), 3.68-3.64 (m, NCH2-Hb), 3.70-3.68 (m, 2H, CH2), 2.24 (s, 3H, pyrazole-CH3),
2H, CH2), 2.35 (s, 3H, pyrazole-CH3), 2.02 (s, 3H, COCH3); 13C 2.03 (s, 3H, COCH3); 13C NMR (126 MHz, CDCl3) δ (ppm) 171.5,
NMR (126 MHz, CDCl3) δ (ppm) 171.3, 154.4, 149.6, 138.1, 154.3, 153.4 (d, 1JC-F = 248.4 Hz), 145.9 (t, 2’JC-F = 29.2 Hz), 137.7
2
1
136.7 (t, JC-F = 29.2 Hz), 135.0, 125.6, 118.7, 108.4 (t, JC-F
=
(d, 3JC-F = 10.1 Hz), 131.2 (d, 4JC-F = 9.7 Hz), 124.4, 123.8 (d, 3’JC-F
235.5 Hz), 107.3, 72.1, 47.5, 41.9, 23.0, 13.4; 19F NMR (471 = 9.8 Hz), 118.6, 113.7, 112.3 (t, 1’JC-F = 237.0 Hz), 106.8 (d, 2JC-F
MHz, CDCl3) δ (ppm) -109.9 - -109.8 (m, 2F). HRMS calcd. for = 26.5 Hz), 72.2, 47.4, 41.8, 23.0, 8.0; 19F NMR (471 MHz,
C17H18F2N4O3Na[M+Na]+: 387.1245, found: 387.1249.
CDCl3) δ (ppm) -112.6 (s, 2F), -122.4 (s, 1F). HRMS calcd. for
4.6.9 (S)-N-((3-(4-(5-(difluoromethyl)-4-methyl-1H-pyrazol- C17H17F3N4O3Na[M+Na]+: 405.1150, found: 405.1167.
1-yl)phenyl)-2-oxooxazolidin-5-yl)methyl)acetamide (13i).
C. H NMR (500 4.7 Antibacterial activity assay
1
Off-white solid, yiled: 83%, m.p: 156.3-158.6
°
MHz, CDCl3) δ (ppm) 7.63-7.62 (m, 1H, pyrazole-H), 7.62-7.61
The antibacterial activities of the target compounds were
tested against six Gram-positive bacterial strains using linezolid
as a positive control. The strains were isolated and identified
by Jiangsu Province People's Hospital and kept in this
(m, 1H, ArH), 7.48-7.43 (m, 3H, ArHs), 7.06-7.02 (m, 1H, NH),
2
6.59 (t, JH-F = 52.8 Hz, 1H, CF2H), 4.80-4.76 (m, 1H, CH), 4.09-
4.05 (m, 1H, NCH2-Ha), 3.85-3.83 (m, 1H, NCH2-Hb), 3.64-3.60
(m, 2H, CH2), 2.23 (s, 3H, pyrazole-CH3), 1.98 (s, 3H, COCH3);
8 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
7
8
9
Q. Xin, H. Fan, B. Guo, H. He, S. Gao, H. Wang, Y. Huang and
Y. Yang, J. Med. Chem., 2011, 54, 7493−7502.
laboratory at -80 °C. In vitro activities of the compounds were
tested in nutrient broth (NB) for bacteria by two-fold serial
agar dilution method using the method recommended by
National Committee for Clinical Laboratory Standards (NCCLS).
Compounds were dissolved in 50% water in DMSO to prepare
a stock solution that had a concentration of 320 μg/mL. Serial
2-fold dilutions were prepared from the stock solution with
M. F. Gordeev and Z. Y. Yuan, J. Med. Chem., 2014, 57
4487−4497.
,
B. Guo, H. Fan, W. Chu, H. Wang, Y. Huang, X. Chen and Y.
Yang, J. Med. Chem., 2013, 56, 2642−2650.
10 E. Spink, D. Ding, Z. Peng, M. A. Boudreau, E. Leemans, E.
Lastochkin, W. Song, K. Lichtenwalter, P. I. O’Daniel, S. A.
Testero, H. Pi, V. A. Schroeder, W. R. Wolter, N. T. Antunes,
M. A. Suckow, S. Vakulenko, M. Chang and S. Mobashery, J.
Med. Chem., 2015, 58, 1380−1389.
11 T. Yang, J. Chen, Y. Fu, K. Chen, J. He, W. Ye, Z. Sang and Y.
Luo, Org. Process Res. Dev., 2014, 18, 511−519.
12 P. Philippe, D. A. Carisa, F. Edward, M. Purvi and D. Anita, J.
Am. Med. Assoc., 2013, 309, 559−569.
sterile water and then 10-fold diluted with Mueller
(MH) agar medium to provide concentration ranges of
16 0.0312 μg/mL. The tested organisms were grown in MH
broth medium (containing 5% sheep serum) at 35 C for 8 h
−Hinton
−
°
and were adjusted to the turbidity of the 0.5 McFarland
standard to a concentration of 108 CFU ml-1. The bacterial
suspensions were inoculated onto the drug-supplemented MH
13 K. Michalska, I. Karpiuk, M. Krol and S. Tyski, Bioorg. Med.
Chem., 2013, 21, 577−591.
agar plates with a multipoint inoculator and incubated at 35 °C
14 S. D. Paget, B. D. Foleno, C. M. Boggs, R. M. Goldschmidt, D.
J. Hlasta, M. A. Weidner-Wells, H. M. Werblood, E. Wira, K.
for 16-20 h. The minimum inhibitory concentrations (MIC)
values were determined at the end of the incubation period.
The lowest concentration of the compound that prevented
visible growth was considered as MIC.
Bush and M. J. Macielag, Bioorg. Med. Chem. Lett., 2003, 13
4173−4177.
,
15 D. Bhattarai, S. H. Lee, S. H. Seo, G. Nam, S. B. Kang, A. N.
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4.8 Molecule docking
Energy minimization: The conformations of compounds 13b
,
13f 13l 13k and linezolid were optimized using the minimize
,
,
17 C. Gong, T. Yang, X. Yang, Y. Liu, W. Ang, J. Tang, W. Pi, L.
Xiong, Y. Chang, W. Ye, Z. Wang, Y. Luo, X. Zhao and Y. Wei,
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18 S. Purser, P. R. Moore, S. Swallow and V. Gouverneur, Chem.
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21 W. R. Perrault, B. A. Pearlman, D. B. Godrej, A. Jeganathan, K.
Yamagata, J. J. Chen, C. V. Lu, P. M. Herrinton, R. C.
Gadwood, L. Chan, M. A. Lyster, M. T. Maloney, J. A.
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22 X. Fang, Y. Chen, D. He, X. Yang and F. Wu, J. Fluorine Chem.,
2008, 129, 1167−1172.
module of SYBYL-X 2.1.26 The force field was calculated with
tripos field and the atom charges were also calculated using
Gasteiger-Huckel method.27
Docking calculations: The docking studies of compounds 13b
,
13f 13l 13k and linezolid were performed by the surflex-dock
,
,
method.25 The three compounds were docked into the
Deinococcus radiodurans 50S ribosomal subunit crystal
structure (PDB ID: 3DLL) by a posteriori scoring function in
Surflex-dock. Before the docking program was performed, the
ligand of the crystal structure was used to build the protomol,
which is the representation of a ligand making every potential
interaction with the binding pocket. In this paper, the
protomol of SYBYL was built by automatic docking and other
setups were default setting up. The binding affinity was
characterized by the total-scores and expressed in -lgKd unit.
23 S. P. Singh, D. Kumar, B. G. Jones and M. D. Threadgill, J.
Fluorine Chem., 1999, 94, 199−203.
24 A. Greco, R. De Marco, S. Tani, D. Giacomini, P. Galletti, A.
Tolomelli, E. Juaristi and L. Gentilucci, Eur. J. Org. Chem.,
2014, 7614–7620.
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Acknowledgements
We are grateful for financial supports from the National
Natural Science Foundation of China (Grant No. 21472126,
21172148, 21302128).
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