Identification and Structure-Activity Relationships of Diarylhydrazides as Novel Potent and Selective Human Enterovirus Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01803

Enterovirus 71 (EV71) plays an important role in hand-foot-and-mouth disease. In this study, a series of diarylhydrazide analogues was synthesized, and the systematic exploration of SAR led to potent enterovirus inhibitors, of which compound 15 exhibits significant improvements in inhibition potency with an EC₅₀ value of 0.02 μM against EV71. It is very interesting that this class of diarylhydrazides exhibits activities against a series of human enteroviruses at the picomolar level, including EV71 and Coxsackieviruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5, and CVB6 (EC₅₀ as low as 0.5 nM). Compared with the reference antienterovirus drug 1 (enviroxime) and known inhibitor 5 (WIN 51711), the four highly selective compounds 15, 27, 41 and 47 inhibited EV71 replication with EC₅₀ values of 0.17-0.02 μM and SI values in a range of 978.4-12338. A preliminary mechanistic study indicated that VP1 might be the target site for this type of compound.

Full text of DOI:10.1021/acs.jmedchem.5b01803

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Article
Identification and Structure-Activity Relationships of Diarylhydrazides
as Novel Potent and Selective Human Enterovirus Inhibitors
Xin Han, Ningyuan Sun, Haoming Wu, Deyin Guo, Po Tien, Chune Dong, Shuwen Wu, and Hai-Bing Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01803 • Publication Date (Web): 17 Feb 2016
Downloaded from http://pubs.acs.org on February 21, 2016
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Identification and Structure-Activity Relationships of
Diarylhydrazides as Novel Potent and Selective Human
Enterovirus Inhibitors
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Xin Han,a Ningyuan Sun,b Haoming Wu,b Deyin Guo,b Po Tien,b Chune Dong,a
Shuwen Wu*b and HaiꢀBing Zhou*a
a State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis
and Drug Discovery (Wuhan University), Ministry of Education, Wuhan University
School of Pharmaceutical Sciences, Wuhan 430071, China
b College of Life Sciences, Wuhan University, Wuhan 430072, China
zhouhb@whu.edu.cn; shuwenwu@hotmail.com
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ABSTRACT
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Enterovirus 71 (EV71) plays an important role in handꢀfootꢀandꢀmouth disease. In
this study, a series of diarylhydrazide analogues was synthesized, and the systematic
exploration of SAR led to potent enterovirus inhibitors, of which compound 15
exhibits significant improvements in inhibition potency, with an EC50 value of 0.02
ꢁM against EV71. It is very interesting that this class of diarylhydrazides exhibits
activities against a series of human enteroviruses at the picomolar level, including
EV71 and Coxsackieviruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5 and CVB6
(EC50 as low as 0.5 nM). Compared with the reference antiꢀenterovirus drug 1
(enviroxime) and known inhibitor 5 (WIN 51711), the four highly selective
compounds 15, 27, 41 and 47 inhibited EV71 replication with EC50 values of
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0.17ꢀ0.02 ꢁM and SI values in a range of 978.4ꢀ12338. A preliminary mechanistic
study indicated that VP1 might be the target site for this type of compound.
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Introduction
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Handꢀfootꢀandꢀmouth disease (HFMD), which was first reported as an outbreak in
Taiwan, China, in 1998, is one of the most common infectious diseases among
children.1,2 In recent years, HFMD has become a prevalent disease in many countries
in the AsianꢀPacific region. Enterovirus 71 (EV71) and coxsackievirus 16 are the
most common etiologic agents of HFMD. EV71, first isolated in 1969 as a member of
the piconaviridae family, is also associated with some other serious diseases such as
mild childhood exanthema, herpangina, aseptic meningitis, encephalitis, and
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poliomyelitisꢀlike paralysis.3,4 Recently, there have been
a
number of
enterovirusꢀassociated HFMD breakouts, leading to high mortality and making the
disease a serious public health issue in China.5ꢀ8 Several approaches have been
attempted to combat enterovirus infections, but there are no effective antiviral drugs
against human enterovirus presently in clinical use.9ꢀ11 Therefore, the development of
novel antiꢀenteroviral agents for HFMD therapy is urgently needed. EV71 is a
singleꢀstranded RNA virus in the genus enterovirus, and its open reading frame (ORF)
includes three consecutive parts, P1, P2 and P3. The viral capsid has icosahedral
symmetry and is composed of 60 identical units, each consisting of four structural
proteins VP1ꢀVP4. These VPs (viral proteins) are important capsid proteomes that
form the enterovirus and contain significant numbers of cellꢀrecognition sites that
could be used to guide the antivirus drug discovery.12ꢀ14 To date, a number of
molecules have been reported to inhibit enterovirus, the earliest and the most widely
studied of which is enviroxime 1 (Figure 1).15ꢀ19 Vinylacetylene analogues of 1
(enviroxime) have also been utilized by the Spitzer group as poliovirus and coxsackie
A21 inhibitors in tissue culture.20,21 In 2003, 2ꢀaminoimidazo[1,2ꢀb] pyridazines were
reported to exhibit broad spectrum activity against human picornaviruses in a plaque
reduction assay and a cytopathic effect assay.22 More recently, Chern and coꢀworkers
developed pyrazolo[3,4ꢀd]pyrimidine (2, Figure 1) as an enterovirus inhibitor, in
particular, an antiꢀcoxsackievirus agent.23 In 2010, PérezꢀPérez and coꢀworkers
developed 9ꢀarylpurines (3, Figure 1) that elicit activity against a variety of
enteroviruses with EC50 values of 5ꢀ8 ꢁM.15,16 In 2013, MacLeod et al. identified
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imidazopyrazine derivatives as interesting inhibitors of EV71, with EC50 values as
low as 11 nM (4, Figure 1).24 Some natural products have also been found to have
potent enterovirus inhibitory activity.25ꢀ27 Although antiꢀHFMD drugs mainly include
siteꢀcombining blockers that contain scavenger receptor B2 (SCARB2), Pꢀselectin
glycoprotein ligandꢀ1 (PSGL1),28,29 3C protease inhibitors,1,30 and RNA
interference,31 because of their low bioactivities and bioavailabilities, as well as the
complex structures of the existing human enterovirus inhibitors, novel enterovirus
inhibitors with simplified scaffolds, higher bioactivities and bioavailabilities are
urgently needed from the point of view of new drug discovery.32ꢀ35 Hence, it is highly
desirable to develop more effective EV71 inhibitors for the treatment of HFMD and
enterovirusꢀrelated diseases. Some recent examples including WIN 51711 (5, Figure
1), a picornavirus inhibitor, were found to have a broad spectrum of activities against
enteroviruses by interfering with the capsidꢀreceptor binding site through inhibiting
the virus attachment to the cells and the uncoating of the viral RNA.36 In 2015,
itraconazole, a clinical drug for antifungal therapy, was found to be a broad spectrum
inhibitor of enteroviruses through acting on the novel targets oxyꢀsterolꢀbinding
protein (OSBP) and OSBPꢀrelated protein 4 (ORP4) by Van Kuppeveld and
coꢀworkers.37 Very recently, we have developed thiopheneꢀ2ꢀcarboxamides (6, Figure
1) as antiꢀEV71 agents, which showed low micromolar activity against EV71 (EC50 =
1.42 ꢁM) compared with the reference drug 1 (enviroxime).38
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N
N
N
Ph
CH3
N
N
N
N
NH2
S
N
N
N
N
SO2
N
N
HO
Br
N
3 9-arylpurine
2 pyrazolo[3,4-d]pyrimidines
1 Enviroxime
SO2Me
N
H
O
F
N
N
N
N
Br
O
N
S
O
HO
4 Imidazopyrazine
O
N
F
6 Thiophene-carboxamide
5 WIN 51711
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Figure 1. Structures of known enterovirus inhibitors
As a part of our longꢀterm interest in the development of antiviral agents,39ꢀ41 as well
as the further exploration of antiꢀEV71 drugs that might have superior efficacy and
fewer side effects than existing therapeutic agents, diarylhydrazide derivatives with
significant antiviral activity were identified based on the result of our previous work.
Two hit compounds 10 and 11 based on a 2,6ꢀdichlorosubstituted phenyl hydrazide
scaffold showed subꢀmicromolar potency against EV71, with EC50 values of 0.47 µM
and 0.94 µM, respectively, but suffer from poor selectivity indexes (Figure 2). The
subsequent chemical optimization led to the potential compound 15 with improved
bioactive properties and a higher selectivity index. It is worth noting that these
compounds are easy to synthesize. As far as we are aware, a hydrazide scaffold has
not been studied as an EV71 inhibitor, although certain hydrazide derivatives are
known to exhibit antibacterial, antimycotic, algicidal and anthelmintic activities
(Figure 2).42ꢀ44 Hence, an attempt has been made to explore the potential of these
newly discovered hydrazides as leads for antiꢀEV71 drug development.
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Figure 2. Identification of diarylhydrazide 15 as a potent and highly selective EV71
inhibitor from the systematic SAR study of the diarylhydrazide analogues. EC50:
effective concentration (ꢁM) for 50% inhibition of EV71, as evaluated by an
MTSꢀbased CPE reduction assay in Vero cells; CC50: cytotoxic concentration (ꢁM) to
induce 50% death of noninfected cells, as evaluated by an MTSꢀbased CPE reduction
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assay in Vero cells; SI: selectivity index calculated as CC50/EC50 ratio; EV71:
Enterovirus 71; CVB1: Coxsackie virus B1; CVB2: Coxsackie virus B2.
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RESULTS AND DISCUSSION
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Chemical Synthesis
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The general synthetic route for the preparation of all of the designed hydrazides 10ꢀ59
is illustrated in Schemes 1 and 2. The key intermediate arylhydrazine hydrochloride 8
was prepared through the simple SnCl2 reduction of the corresponding aryldiazenes 7
derived from various anilines (Schemes 1). According to the reported method, the
arylhydrazine hydrochloride 8 coupling with acyl chloride 9 in the presence of
triethylamine in CH2Cl2 at room temperature afforded the final products 10ꢀ59 in
75ꢀ95% yields (Scheme 2).45
Scheme 1. Synthesis of arylhydrazine hydrochlorides. Reagents and reaction
conditions: (a) NaNO2 (1.1 equiv), H2O, HCl (3 equiv), ꢀ5 °C ꢀ rt; (b) SnCl2 (2 equiv),
H2O, HCl, rt, 12 h.
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Scheme 2. Synthesis of the target compounds 10ꢀ59. Reagents and reaction
conditions: (a) triethylamine (2.6 equiv), CH2Cl2, rt, 2 h.
Structure-Activity Relationship Analysis
The activities of all new hydrazide compounds were tested against diversified
enterovirus serotypes in a neutralization test. In addition, the references
antiꢀenterovirus drug 1 (enviroxime) and known inhibitor 5 (WIN 51711) were tested
for comparison. The results of this extensive biological evaluation are shown in Table
1. As a global observation, it is noteworthy that the two phenyl rings A and B
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displayed distinct SARs found in most of the antiꢀenterovirus agents. Initially,
compounds 10 and 11 showed good activities against EV71 (EC50 = 0.47 ꢁM and 0.94
ꢁM, respectively) compared to 1 (enviroxime) (EC50 = 0.15 ꢁM) and 5 (WIN 51711)
(EC50 = 19.48 ꢁM) (entries 1ꢀ4). Based on the structures of 10 and 11, in the
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subsequent
chemical
optimization,
diverse
substituents
including
electronꢀwithdrawing and electronꢀdonating groups on the acyl phenyl ring A were
screened in the presence of the same 2,6ꢀdichloro on the hydrazine phenyl ring B. The
results revealed that electronꢀdonating groups are favorable substituents in the control
of the bioactivity, and the corresponding hydrazides exhibited potent antiꢀenterovirus
activity (entries 3ꢀ9), with the best one (15) possessing a N(Me)2 at the paraꢀposition
of the acyl phenyl ring A, showing the lowest EC50 of 0.02 ꢁM (entry 8). On the other
hand, the unsubstituted or chloroꢀsubstituted hydrazide on the acyl phenyl ring A and
some other heterocyclic rings, e.g., pyridine (20) and thiophene (21), showed no
activity against EV71 (entries 11ꢀ14). Other substrates, for example, transferring the
OMe from the paraꢀ to orthoꢀposition, adding a chlorine atom onto the hydrazine
phenyl ring B, or exchanging the position of rings A and B, also showed no activities
against EV71 (entries 9, 10, 15 and 16). In the presence of the tꢀBu on phenyl ring A,
phenyl ring B with different groups, e.g., 2,6ꢀOMe2, 2,6ꢀEt2, 2,6ꢀPr2, 2,6ꢀMe2, 2,6ꢀBr2,
2,6ꢀF2 or 2ꢀClꢀ6ꢀMe, has an inhibitory activity that decreases with the increase in the
side chain by a large margin (entries 17ꢀ20, 26, 34 and 43). This observation could be
verified by computer modeling (Figure 7), which indicated that there is not enough
space between the 2,6ꢀdisubstituted phenyl ring B and the acting site in the protease to
accommodate a bulky substituent. Moreover, upon introducing 2,6ꢀdimethyl groups
instead of 2,6ꢀdichloro groups on phenyl ring B, reduced inhibitory activities were
obtained, except for with a tꢀBu group at the paraꢀposition of phenyl ring A (entries
20ꢀ25). On the other hand, the replacement of phenyl ring B with unsymmetric
2ꢀClꢀ6ꢀMe groups at the same position led to comparable activity to the corresponding
2,6ꢀdimethyl substituted ring (entries 26ꢀ32). There was no obvious change in activity
upon altering the 4ꢀOMe to 4ꢀSMe in the presence of the same phenyl ring B (entries
29 and 33). The 2,6ꢀdibromo substituted ring B usually led to increased antiꢀEV71
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activity; most notably, compounds 41 and 49 gave EC50 values of 0.08 ꢁM and 0.86
ꢁM, respectively (entries 34ꢀ42). Interestingly, the 2,6ꢀdifluoro substituted ring B
exhibited a severely reduced activity compared with the corresponding 2,6ꢀdichloro
and 2,6ꢀdibromo analogues (entries 43ꢀ47). Moreover, analogues 55ꢀ59 with
electronꢀwithdrawing substituents, i.e., 4ꢀF (55), 4ꢀBr (56), 4ꢀCN (57), 4ꢀNO2 (58),
3ꢀCl (59), on phenyl ring A show no activity against EV71. One can conclude that the
installation of an electronꢀwithdrawing group on phenyl ring A usually leads to no
activity. Among all of the novel antiꢀEV71 hydrazide agents, there were three
compounds (15, 41 and 47) having a better inhibitory activity than the reference
antiꢀEV71 agent 1 (enviroxime) (entries 1, 8, 34 vs 40) and eight compounds
possessing comparable activities to that of 1 (entries 3, 6, 7, 20, 25, 30, 32 and 38).
These diarylhydrazide analogues were further tested using another cell line, RD cells.
All of the compounds, except for 12 and 39, which show slightly better inhibitory
activities, showed a smaller inferior inhibitory activity for EV71 in RD cells than
those of Vero cells (Table 1). The most potent compound 15 was the most remarkable,
with an EC50 value of 0.03 ꢁM in RD cells (entry 8). Most of the compounds had no
or little cellular toxicity and high selectivity. These biological results demonstrate that
this series of hydrazides consists of potent and versatile antiꢀEV71 agents with a high
selectivity index and potential for pharmaceutical use.
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Table 1. Antiviral Evaluation of the Hydrazides against EV71a
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EC50 (ꢁM)b
CC50 (ꢁM)c
SId
Entry Cmpd
R1
R2
Vero
Cells
RD
Cells
Vero
Cells
RD
Cells
Vero
Cells
RD
Cells
1
2
1
Enviroxime
WIN 51711
4ꢀtꢀBu
0.15 ± 0.06
19.48 ± 6.57
0.47 ± 0.10
0.94 ± 0.27
1.28 ± 0.48
0.33 ± 0.07
0.42 ± 0.08
0.02 ± 0.00
0.68 ± 0.13
>100
0.24 ± 0.07
15.32 ± 4.64
0.85 ± 0.24
1.68 ± 0.53
0.96 ± 0.34
0.37 ± 0.10
0.44 ± 0.15
0.03 ± 0.01
1.36 ± 0.37
>100
>223.21
>223.21
>1488.1
7.6
>949.8
10.9
5
147.77 ± 38.84 167.39 ± 47.84
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 6ꢀCl2
2, 4, 6ꢀCl3
4ꢀOEt
30.84 ± 7.59
>258.74
>271.04
>246.01
>247.51
>246.76
>257.07
NDe
36.72 ± 9.3
>258.74
>271.04
>246.01
>247.51
>246.76
>257.07
ND
65.6
43.2
4
4ꢀEt
4ꢀMe
4ꢀOEt
4ꢀnꢀPr
4ꢀN(Me)2
4ꢀOMe
2ꢀOMe
H
>275.3
>211.8
>745.5
>589.3
>154
5
>281.3
>664.9
>562.5
6
7
8
>12338 >8225.3
9
>378
--f
--
>189
ꢀꢀ
10
11
12g
13
14
15
16
>100
>100
ND
ND
ꢀꢀ
4ꢀCl
>100
>100
ND
ND
--
ꢀꢀ
ꢀꢀ
>100
>100
ND
ND
--
ꢀꢀ
ꢀꢀ
>100
>100
ND
ND
--
ꢀꢀ
4ꢀOEt
2,6ꢀCl2
>100
>100
ND
ND
--
ꢀꢀ
>100
>100
ND
ND
--
ꢀꢀ
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4ꢀtꢀBu
4ꢀtꢀBu
4ꢀtꢀBu
4ꢀtꢀBu
4ꢀEt
2, 6ꢀOMe2 10.41 ± 3.78
27.39 ± 8.52
>100
>243.60
>243.60
ND
>23.4
--
>8.9
ꢀꢀ
2, 6ꢀEt2
2, 6ꢀPr2
>100
ND
ND
>100
>100
ND
--
ꢀꢀ
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24
25
26
27
28
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31
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33
34
35
36
37
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39
40
41
42
43
44
45
46
47
48
49
2, 6ꢀMe2
2, 6ꢀMe2
2, 6ꢀMe2
2, 6ꢀMe2
2, 6ꢀMe2
2, 6ꢀMe2
2ꢀClꢀ6ꢀMe
2ꢀClꢀ6ꢀMe
0.17 ± 0.04
1.57 ± 0.43
5.31 ± 1.35
12.32 ± 4.14
0.95 ± 0.44
0.29 ± 0.08
0.79 ± 0.20
6.37 ± 1.46
0.42 ± 0.15
2.75 ± 0.87
13.56 ± 3.27
28.97 ± 7.64
2.64 ± 0.85
0.69 ± 0.21
0.97 ± 0.37
10.52 ± 2.59
26.87 ± 8.56
15.38 ± 4.34
0.82 ± 0.30
2.16 ± 0.43
0.16 ± 0.07
15.43 ± 4.26
0.29 ± 0.07
1.58 ± 0.73
24.65 ± 8.53
2.63 ± 0.84
0.53 ± 0.24
4.62 ± 1.57
0.07 ± 0.03
2.43 ± 0.72
>100
166.33 ± 31.81 188.49 ± 50.27
135.27 ± 36.63 164.31 ± 42.37
978.4
86.2
454.2
59.7
4ꢀMe
>314.56
>295.94
>314.56
>295.94
>59.2
>24
>23.2
>10.2
>107.2
>410.9
65.1
4ꢀOMe
4ꢀnꢀPr
4ꢀN(Me)2
4ꢀtꢀBu
4ꢀEt
>283.31
>283.31
>298.2
>973.5
62.3
>282.32
>282.32
49.24 ± 13.16
81.74 ± 17.87
>291.18
63.23 ± 20.48
105.42 ± 27.53
>291.18
12.8
10.0
4ꢀMe
2ꢀClꢀ6ꢀMe 12.96 ± 4.62
>22.5
>47.9
>673.1
87.2
>10.8
>17.9
>319.3
52.4
4ꢀOMe
4ꢀOEt
4ꢀnꢀPr
2ꢀClꢀ6ꢀMe
2ꢀClꢀ6ꢀMe
2ꢀClꢀ6ꢀMe
5.74 ± 1.82
0.39 ± 0.07
0.92 ± 0.19
0.28 ± 0.08
6.13 ± 2.17
0.08 ± 0.03
0.93 ± 0.61
10.81 ± 6.17
3.32 ± 0.71
0.37 ± 0.10
3.54 ± 0.92
0.03 ± 0.01
0.86 ± 0.44
54.24 ± 16.42
>275.16
>275.16
>262.49
>262.49
80.25 ± 22.85
92.83 ± 25.05
113.28 ± 31.38
106.43 ± 28.72
4ꢀN(Me)2 2ꢀClꢀ6ꢀMe
331.5
17.4
682.3
8.7
4ꢀSMe
4ꢀtꢀBu
4ꢀEt
2ꢀClꢀ6ꢀMe
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
2, 6ꢀBr2
106.58 ± 27.44 134.42 ± 31.58
>187.73
>200.96
>187.73
>200.96
2346.6
>216.1
13.5
>640.7
>127.0
6.6
4ꢀMe
146.33 ± 27.08 163.27 ± 30.61
4ꢀOMe
4ꢀOEt
4ꢀnꢀPr
4ꢀN(Me)2
H
>199.97
>193.19
>199.97
>193.19
>194.12
>193.65
>216.19
ND
60.2
>76.1
>361.8
>42.0
>2934.2
>89.1
ꢀꢀ
>522.1
>54.8
>6455
>251.4
2
>194.12
>193.65
>216.19
2ꢀOMe
109.48 ± 17.05
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4ꢀtꢀBu
4ꢀOMe
4ꢀOEt
2, 6ꢀF2
2, 6ꢀF2
2, 6ꢀF2
2, 6ꢀF2
2, 6ꢀF2
2.19 ± 0.81
74.03 ± 24.22
66.72 ± 25.39
27.38 ± 5.80
4.27 ± 0.92
>100
149.84 ± 28.69 183.84 ± 26.35
68.4
3.9
43.0
ꢀꢀ
>287.51
>273.71
>275.57
>274.64
ND
ND
>100
4.1
ꢀꢀ
10
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15
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49
4ꢀnꢀPr
51.44 ± 8.53
>275.57
>274.64
10.1
15.3
>5.4
>11.7
4ꢀN(Me)2
17.95 ± 12.98 23.46 ± 11.47
a All data are mean values ± standard deviation for at least three independent experiments; EC50: effective concentration (ꢁM) for 50%
inhibition of EV71, as evaluated by an MTSꢀbased CPE reduction assay in corresponding cells; c CC50: cytotoxic concentration (ꢁM) to induce
50% death of noninfected cells, as evaluated by an MTSꢀbased CPE reduction assay in corresponding cells; d SI: selectivity index calculated as
CC50/EC50 ratio; e ND: Not determined; f Not calculated because the EC50 was too high; g Other electronꢀwithdrawing substituents, e.g., 4ꢀF (55),
4ꢀBr (56), 4ꢀCN (57), 4ꢀNO2 (58), and 3ꢀCl (59), were also found to have no activities.
b
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An analysis of the antiꢀEV71 activities of alternate substituents at the 4ꢀposition of
phenyl ring A with a 2,6ꢀCl2 substituted phenyl ring B is presented in Figure 3A. The
results reveal that the derivatives with substituents N(Me)2 (15), OEt (13), tꢀBu (10)
and nꢀPr (14) have better antiꢀEV71 activities compared to others, with N(Me)2 being
the best in the series. However, with no substituent or the electronꢀwithdrawing group
Cl at the 4ꢀposition of phenyl ring A, there is almost no activity, with EC50 values
greater than 100 ꢁM. N(Me)2 is also a favorable active moiety in the determination of
antiꢀEV71 activity in the presence of an 2,6ꢀCl2, 2,6ꢀMe2, 2ꢀClꢀ6ꢀMe or 2,6ꢀBr2
substituted phenyl ring B, although not the 2,6ꢀF2 substituted ring (Figure 3B). These
results verify that aryl phenylhydrazides that have electronꢀdonating groups at the
4ꢀposition of phenyl ring A and disubstituted groups on phenyl ring B usually have
excellent inhibitory activities for EV71.
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A
B
Figure 3. A) and B) Graphical exhibit of the effect of substituents of phenyl rings A
and B on the antiꢀEV71 activity of the compounds in Vero cells.
The four most potent antiꢀEV71 agents, compounds 15, 27, 41 and 47, were further
selected for evaluation against a panel of other human enteroviruses, including
Coxsackie viruses B1 (CVB1), CVB2, CVB3, CVB4, CVB5 and CVB6. We were
pleased to find that these compounds exhibited inspiring activity against a variety of
human enteroviruses. The results are illustrated in Table 2, showing that all of the
compounds exhibited significant activity against Coxsackievirus, with EC50 values
ranging from lower micromolar to nanomolar concentrations. Compounds 15 and 47
proved to be the two most potent antiꢀenterovirus agents against CVB2, with EC50
values as low as picomolar, 0.0009 ꢁM and 0.0005 ꢁM, respectively. In terms of
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CVB1, antiviral activity in the low nanomolar range was also found in the presence of
compounds 15, 27 and 47 (EC50 = 0.0013ꢀ0.005 ꢁM). Although these compounds
showed weaker activities against CVB3, CVB4, CVB5 and CVB6 than against CVB1
and CVB2, the EC50 values were still in a range of low micromolar to nanomolar
concentrations (EC50 = 6.52ꢀ0.02 ꢁM). It is noteworthy that for all enteroviruses tested,
compound 47 showed much better antiviral activity than reference drug 1
(enviroxime), while compound 15 showed superior activity against CVB1–CVB4
than 1 (enviroxime).
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13
14
15
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27
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Table 2. Antiviral Evaluation of Compounds 15, 27, 41, 47 and 1 (enviroxime)
against a Selected Panel of Enterovirusesa
Virus
EC50 (ꢁM)b
(strain)
1
15
27
41
47
(enviroxime)
0.75 ± 0.20
0.15 ± 0.03
2.48 ± 0.65
> 186
CVB1
CVB2
CVB3
CVB4
CVB5
0.0013 ± 0.0003
0.0009 ± 0.0004
0.95 ± 0.23
0.0016 ± 0.0005
0.0016 ± 0.0006
0.09 ± 0.03
2.61 ± 0.99
1.57 ± 0.60
0.87 ± 0.35
6.52 ± 1.70
0.98 ± 0.48
0.92 ± 0.44
0.005 ± 0.002
0.0005 ± 0.0004
0.03 ± 0.01
2.57 ± 1.07
5.62 ± 1.79
0.34 ± 0.10
0.12 ± 0.04
0.10 ± 0.02
2.27 ± 0.73
3.19 ± 0.92
0.06 ± 0.02
CVB6
0.94 ± 0.47
0.56 ± 0.21
0.02 ± 0.01
a
All data are mean values ± standard deviation for at least three independent
b
experiments; EC50: effective concentration (ꢁM) for 50% inhibition of EV71, as
evaluated by an MTSꢀbased CPE reduction assay in Vero cells.
Proposed Targets of Hydrazides Acting on the Enterovirus
Based on the enterovirus 71 genome structure, in the single long open reading frame
(ORF), there are three or four consecutive parts P1, P2, P3, and P1’.14 The enterovirus
entering into the host cell leads to the translation of polyprotein. The synthesized
polyprotein could be hydrolyzed into 4 types of structural protein, i.e., VP1, VP2,
VP3, and VP4 at P1, and 7 types of nonꢀstructural protein, i.e., 2Aꢀ2C at P2 and
3Aꢀ3D at P3, in the presence of its own 3C protease. These VPs are important capsid
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proteomes that form the enterovirus and contain a significant number of
cellꢀrecognition sites. As such, if there is a small molecule that can enter into the
hydrophobic pocket of the VP, it should be an effective antiꢀenterovirus inhibitor.
Preliminary mechanistic studies indicated that our compounds inhibit the virus
replication at the early stages, referring to the attachment or uncoating of the virion,
by testing compound 15 and the two reference agents 5 (WIN 51711) and 1
(enviroxime) through a timeꢀcourse experiment (Figure 4). Similar to 5 (WIN 51711),
a capsidꢀbinding inhibitor that is different from 1 (enviroxime), a 3A protease
inhibitor, it is likely that compound 15 acts on the capsid to prevent the uncoating of
the virion.11,36,46
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15
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59
60
Figure 4. Time course experiment. Compound 15 (c = 0.05 ꢁg/mL), 1 (enviroxime)
(c = 0.53 ꢁg/mL) and 5 (WIN 51711) (c = 20 ꢁg/mL) were added to the RD cells after
being infected by 0.01 MOI EV71 at the corresponding time.
Fluorescenceꢀbased thermal stability assays were also conducted to verify the
above proposed mechanism.36,47 The result indicated that when the RNA genome in
EV71 virions is incubated with 5 ꢁg/mL compound 15, 2 °C higher temperatures are
needed to achieve the equal staining of the genome compared to native virions
without compound 15 (Figure 5). A similar phenomenon occurred when the positive
control 5 (WIN 51711) instead of compound 15 was utilized in the same system
(Figure 5). Thus, the possible mechanism of inhibition of these ligands is the
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stabilizing effect of compound 15 on the EV71 capsid, thereby preventing the
5
6
uncoating of the virion.
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59
60
Figure 5. Impact of hydrazide on EV71 virion stability. SYBR green fluorescent
assays to measure stability of EV71 particles. EV71 virions were mixed with SYBR
green dyes I and II and heated to the indicated temperatures. The fluorescent signal
increases as the dye binds to the RNA that is released from thermally destabilized
particles. Red line, EV71 virions; Green line, EV71 with 5
ꢁ
g/mL compound 15;
Yellow line, EV71 with 50
without virus.
ꢁg/mL compound 5 (WIN 51711); Purple line, control
To exclude the possibility that the antiviral activity was due to toxic side effects,
we tested the effect of compound 15 on virus production over a range of
concentrations during a single replication cycle.37 For comparison, we chose the
antiꢀEV71 agent 5 (WIN 51711) as a positive control in the virus titer assay (pink line,
Figure 6). The results demonstrate that compound 15 is active against EV71 in a
single replication cycle without apparent toxicity (green line, Figure 6).
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Figure 6. Virus titers in supernatants. RD cells were treated with compound 5 (WIN
51711) (pink line) or 15 (green line) and infected with EV71. The virus titers in the
supernatants were determined at 72 hpi.
These observations are consistent with the molecular modeling result of compound 15
with EV71 (enterovirus 71) using the crystal complex of the latter with 5 (WIN 51711)
as a template, with the aid of the software of AutoDock Vina. It is believed that 5
(WIN 51711) is an active enterovirus and rhinovirus inhibitor targeting the viral
capsid protein VP1 to interfere with or block the combination of the virus and host
cell.36,49,50 A significant VP1 pocket (blue area) can be found in the coꢀcrystal
structure of the native EV71 virion and 5 (WIN 51711) (PDB: 3ZFF), as shown in
Figure 7A. Similar to the orientation of 5 (WIN 51711) in the native EV71 virion, the
typical hydrazide compound 15 could also bind into the hydrophobic pocket of
subunit VP1 (Figure 7B). The crystal complex of EV71 with 5 clearly shows that the
latter fits into the VP1 pocket as a slender shape with one aromatic ring at each end
(Figure 7C). Moreover, a hydrogen bond formed between the NH of 5 and the residue
ASP 112. Similarly, we found that compound 15 could enter into the hydrophobic
VP1 pocket that was formed by a series of necessary residues, e.g., Ile 111, Asp 112,
Ile 113, Phe 131, Phe 135, Val 192, Met 195 and Tyr 201, as an active pocket between
the ligand and macromolecule (Figure 7D). However, a similar hydrogen bond was
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not found between 15 and ASP 112 because the aromatic ring A of compound 15
binds farther away from the Trp 203 ꢀ Ile 113 ꢀ Asp 112 cave due to its shorter length
than 5, although this does not affect the inhibitory activity against enteroviruses. In
addition to the hydrophobic interaction, several π–π interactions between the two
aromatic rings A and B and the residues Tyr 201, Phe 135 and Phe 155 were also
found to be significant contributors to the enhanced binding force and higher
inhibitory activity of 15.
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A
B
D
C
Figure 7. Model of diarylhydrazide 15 bound into the VP1 pocket of EV71 virion and
comparisons with 5 (WIN 51711). (A) The coꢀcrystal structure of EV71 and 5 (PDB:
3ZFF) is shown as an overview type with capsid protein subunits VP1 (blue), VP2
(red), VP3 (green), and VP4 (yellow) in a cartoon representation. (B) Computer
modeling of the complex of 15 with EV71 at the same VP1 pocket (PDB: 3ZFF) for 5.
(C) VP1 is shown in cartoon representation in blue with side chains of residues
forming the hydrophobic pocket shown as sticks and 5 (WIN 51711) binding deep
into the pocket. (D) Compound 15 in the same hydrophobic VP1 pocket formed by
side chains of residues in cartoon representation.
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Conclusions
5
6
7
8
In summary, we have synthesized a series of diarylhydrazide analogues and evaluated
their biological activities against EV71. These compounds possess high antiꢀEV71
activity and induce a very low cytotoxic effect in Vero cells and RD cells. The SAR
results indicate that the electronꢀdonating groups at the 4ꢀposition of phenyl ring A
and 2,6ꢀdisubstituted phenyl ring B play key roles for their inhibitory activity, which
is supported by the molecular modeling of the complex structure of the EV71 protease
with 15. Among all of the hydrazide compounds studied, 15 is the best one, with an
EC50 value of 0.02 ꢁM against EV71. Compounds 15, 27, 41 and 47 also exhibited
excellent inhibitory activity for other human enteroviruses, including CVB1, CVB2,
CVB3, CVB4, CVB5, and CVB6 (EC50 = 0.0005ꢀ6.52 ꢁM). Therefore, this series of
diarylhydrazide compounds could be considered as a novel class of enterovirus
inhibitors with a simple chemical structure. A preliminary mechanistic study of
hydrazides with enterovirus 71 indicated that the attachment site of VP1 might be the
target of this type of compound. Further SAR studies and inꢀdepth mechanistic
research of hydrazides with enteroviruses are currently underway in our group and
will be reported in due course.
9
10
11
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13
14
15
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47
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60
Experimental Section
General Methods. Unless otherwise noted, reagents and materials were obtained
from commercial suppliers and were used without further purification. The TEA and
CH2Cl2 were dried over CaCl2 and distilled prior to use, respectively. Reactions were
monitored by thinꢀlayer chromatography (TLC) and column chromatography
purifications performed using a 230–400 mesh silica gel. NMR spectra were
1
measured on a Bruker Biospin AV400 instrument at 400 MHz for H NMR spectra,
100 MHz for 13C NMR spectra, and 375 MHz for 19F NMR spectra and calibrated
from the residual solvent signal. Highꢀresolution mass spectra were obtained from the
Shanghai Mass Spectrometry Center. Melting points were determined by an Xꢀ4
Beijing Tech melting point apparatus and were uncorrected. All compounds assayed
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were >95% pure, as determined by an HPLC analysis conducted on an Agilent
Technologies 1220 Infinity system with UV detection at 254 nm using both a
normalꢀphase column and a reversedꢀphase column. The normalꢀphase HPLC was
recorded on a Thermo Scientific Hypersil Silica column using hexane/ethyl acetate
(80:20) as the eluent, and the reversedꢀphase HPLC was recorded on an
Agilent ZORBAX SBꢀC18 column using MeOH/H2O (90:10) as the eluent (see
Supporting Information for details).
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Representative
procedure
for
synthesis
of
4-(tert-butyl)-N'-(2,6-
dichlorophenyl)-benzohydrazide 10: To a solution of (2,6ꢀdichlorophenyl)hydrazine
hydrochloride (55.5 mg, 0.26 mmol) in 3 mL of CH2Cl2 was added 52.6 mg (2.6 eq.)
of Et3N. The mixture was stirred at room temperature for 0.5 h and then
4ꢀ(tertꢀbutyl)benzoyl chloride (39.3 mg, 0.2 mmol) was added at 0 °C. After the
reaction was completed, the solvents were removed, and the residue was purified by
flash chromatography on silica gel, eluting with petroleum ether/ethyl acetate (8:1) to
yield
62.1
mg
(92%
as
a
white
solid)
of
1
4-(tert-Butyl)-N'-(2,6-dichlorophenyl)benzohydrazide 10: mp 150ꢀ151 °C; H NMR
(400 MHz, Acetoneꢀd6) δ 9.86 (d, J = 2.4 Hz, 1H), 7.91 – 7.87 (m, 2H), 7.52 (d, J =
8.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 3.2 Hz, 1H), 6.96 (t, J = 8.0 Hz,
1H), 1.33 (s, 9H). 13C NMR (100 MHz, Acetone-d6) δ 167.00, 155.90, 142.98, 130.86,
130.40, 129.94, 128.26, 126.23, 125.60, 123.86, 35.50, 31.42. HRMS (ESI) calcd for
C17H18Cl2N2NaO [M + Na]+ 359.0694, found 359.0687.
N'-(2,6-Dichlorophenyl)-4-ethylbenzohydrazide 11: yield 93% as a white solid; mp
1
113ꢀ114 °C; H NMR (400 MHz, Acetoneꢀd6) δ 9.86 (d, J = 1.8 Hz, 1H), 7.87 (d, J =
8.2 Hz, 2H), 7.32 (dd, J = 8.0, 3.2 Hz, 4H), 7.17 (d, J = 3.0 Hz, 1H), 6.96 (t, J = 8.0
Hz, 1H), 2.71 – 2.65 (m, 2H), 1.22 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 167.08, 149.25, 142.98, 131.12, 129.94, 128.78, 128.53, 125.61, 123.87,
29.33, 15.80. HRMS (ESI) calcd for C15H14Cl2N2NaO [M + Na]+ 331.0381, found
331.0368.
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N'-(2,6-Dichlorophenyl)-4-methylbenzohydrazide 12: yield 91% as a white solid; mp
140ꢀ141 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.86 (s, 1H), 7.84 (d, J = 8.2 Hz, 2H),
7.30 (dd, J = 16.4, 8.0 Hz, 4H), 7.16 (d, J = 2.8 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H), 2.37
(s, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.10, 143.00, 142.97, 130.87, 129.94,
129.93, 128.42, 125.61, 123.91, 123.87, 21.45. HRMS calcd for C14H12Cl2N2NaO [M
+ Na]+ 317.0224, found 317.0215.
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N'-(2,6-Dichlorophenyl)-4-ethoxybenzohydrazide 13: yield 94% as a white solid; mp
154ꢀ155 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.77 (s, 1H), 7.93 – 7.89 (m, 2H),
7.33 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 3.2 Hz, 1H), 7.01 – 6.95 (m, 3H), 4.10 (t, J = 7.0
Hz, 2H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 166.71, 162.83,
143.09, 130.22, 129.93, 125.59, 123.81, 114.95, 64.33, 14.98. HRMS (ESI) calcd for
C15H15Cl2N2O2 [M + H]+ 325.0511, found 325.0504.
N'-(2,6-Dichlorophenyl)-4-propylbenzohydrazide 14: yield 95% as a white solid; mp
125ꢀ126 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.86 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H),
7.31 (t, J = 8.6 Hz, 4H), 7.16 (d, J = 3.0 Hz, 1H), 6.96 (t, J = 8.0 Hz, 1H), 2.69 – 2.59
(m, 2H), 1.69 – 1.59 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 167.10, 147.66, 142.98, 131.15, 129.94, 129.37, 128.44, 125.62, 123.87,
38.40, 25.10, 14.03. HRMS (ESI) calcd for C16H16Cl2N2NaO [M + Na]+ 345.0537,
found 345.0531.
N'-(2,6-Dichlorophenyl)-4-(dimethylamino)benzohydrazide 15: yield 83% as a white
solid; mp 156ꢀ157 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.56 (s, 1H), 7.84 – 7.80 (m,
2H), 7.31 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 3.2 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H), 6.76 –
6.72 (m, 2H), 3.02 (s, 6H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.14, 153.78, 143.39,
129.89, 129.75, 125.57, 123.66, 120.09, 111.79, 40.14. HRMS (ESI) calcd for
C15H15Cl2N3NaO [M + Na]+ 346.0490, found 346.0482.
N'-(2,6-Dichlorophenyl)-4-methoxybenzohydrazide 16: yield 88% as a white solid; mp
148ꢀ150 °C; 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 4.6 Hz, 1H), 7.76 (d, J = 8.8
Hz, 2H), 7.28 – 7.25 (m, 2H), 7.05 (d, J = 4.8 Hz, 1H), 6.92 (m, 3H), 3.84 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 166.19, 162.75, 141.35, 129.03, 128.95, 126.11, 124.23,
124.04, 114.03, 55.44. HRMS (ESI) calcd for C14H12Cl2N2NaO2 [M + Na]+ 347.0330,
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found 347.0332.
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N'-(2,6-Dichlorophenyl)-2-methoxybenzohydrazide 17: yield 88% as a white solid; mp
128ꢀ129 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 10.08 (d, J = 4.8 Hz, 1H), 8.04 (dd, J
= 7.8, 1.8 Hz, 1H), 7.51 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H), 7.40 (d, J = 5.2 Hz, 1H), 7.34
(d, J = 8.0 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 7.08 – 7.04 (m, 1H), 6.99 (t, J = 8.0 Hz,
1H), 4.06 (s, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 164.97, 158.75, 143.00, 134.31,
132.56, 129.91, 126.20, 124.66, 121.86, 120.68, 112.86, 56.72. HRMS (ESI) calcd for
C14H12Cl2N2NaO2 [M + Na]+ 347.0330, found 347.0334.
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N'-(2,6-Dichlorophenyl)benzohydrazide 18: yield 90% as a white solid; mp
139ꢀ140 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.92 (s, 1H), 7.94 (dd, J = 5.2, 3.2 Hz,
2H), 7.56 (ddd, J = 6.6, 3.8, 1.2 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.34 (d, J = 8.0 Hz, 2H),
7.19 (d, J = 2.8 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H). 13C NMR (100 MHz, Acetoneꢀd6) δ
167.11, 142.91, 133.73, 132.59, 129.95, 129.33, 128.38, 125.63, 123.91. HRMS (ESI)
calcd for C13H10Cl2N2NaO [M + Na]+ 303.0068, found 303.0056.
4-Chloro-N'-(2,6-dichlorophenyl)benzohydrazide 19: yield 92% as a white solid; mp
161ꢀ162 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 10.02 (s, 1H), 7.97 – 7.94 (m, 2H),
7.55 – 7.51 (m, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 2.8 Hz, 1H), 6.98 (t, J = 8.0
Hz, 1H). 13C NMR (100 MHz, Acetoneꢀd6) δ 166.23, 142.70, 138.21, 132.41, 130.21,
129.97, 129.53, 125.62, 124.00. HRMS (ESI) calcd for C13H9Cl3N2NaO [M + Na]+
336.9678, found 336.9674.
N'-(2,6-Dichlorophenyl)picolinohydrazide 20: yield 75% as a white solid; mp
143ꢀ144 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 10.09 (s, 1H), 8.66 (d, J = 4.8 Hz,
1H), 8.06 (d, J = 7.6 Hz, 1H), 8.00 (td, J = 7.6, 1.6 Hz, 1H), 7.62 (m, 1H), 7.36 (d, J =
8.0 Hz, 2H), 7.23 (d, J = 4.0 Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 163.66, 149.98, 149.59, 142.58, 138.60, 129.92, 127.82, 125.99, 124.40,
123.00. HRMS (ESI) calcd for C12H8Cl2N3O [M ꢀ H]ꢀ 280.0044, found 280.0040.
N'-(2,6-Dichlorophenyl)thiophene-2-carbohydrazide 21: yield 85% as a white solid;
mp 155ꢀ156 °C; 1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.59 (d, J = 3.0 Hz, 1H),
7.52 (dd, J = 5.0, 0.8 Hz, 1H), 7.28 – 7.25 (m, 2H), 7.09 (dd, J = 4.8, 3.8 Hz, 1H),
6.99 – 6.89 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 161.38, 140.94, 135.40, 130.95,
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129.19, 129.17, 128.96, 127.85, 126.13, 124.27. HRMS (ESI) calcd for
C11H8Cl2N2NaOS [M + Na]+ 308.9632, found 308.9626.
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4-Ethoxy-N'-(2,4,6-trichlorophenyl)benzohydrazide 22: yield 90% as a white solid;
mp 160ꢀ162 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.70 (s, 1H), 7.76 (d, J = 8.6 Hz,
2H), 7.26 (s, 2H), 7.03 (s, 1H), 6.84 (d, J = 8.4 Hz, 2H), 4.01 – 3.95 (m, 2H), 1.24 (t,
J = 6.9 Hz, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 166.75, 162.88, 142.36, 132.51,
130.25, 129.43, 127.09, 126.13, 125.40, 114.98, 64.34, 15.00. HRMS (ESI) calcd for
C15H13Cl3N2NaO2 [M + Na]+ 380.9940, found 380.9944.
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2,6-Dichloro-N'-(4-ethoxyphenyl)benzohydrazide 23: yield 88% as a white solid; mp
163ꢀ164 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.58 (s, 1H), 7.71 (s, 2H), 7.52 – 7.44
(m, 4H), 6.94 (d, J = 9.0 Hz, 2H), 4.05 (d, J = 7.0 Hz, 2H), 1.36 (d, J = 7.0 Hz, 3H).
13C NMR (100 MHz, Acetoneꢀd6) δ 162.56, 156.75, 137.77, 132.80, 131.88, 129.02,
128.39, 122.10, 122.01, 115.44, 115.00, 64.20, 15.16. HRMS (ESI) calcd for
C15H14Cl2N2NaO2 [M + Na]+ 347.0330, found 347.0325.
4-(tert-Butyl)-N'-(2,6-dimethoxyphenyl)benzohydrazide 24: yield 87% as a white solid;
mp 147ꢀ148 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.50 (d, J = 6.9 Hz, 1H), 7.82 (d,
J = 8.6 Hz, 2H), 7.52 – 7.47 (m, 2H), 7.31 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.4 Hz,
1H), 6.66 (d, J = 8.4 Hz, 2H), 3.90 (s, 6H), 1.33 (s, 9H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 155.38, 151.63, 131.51, 127.86, 127.12, 126.15, 125.93, 123.04, 105.97,
56.69, 35.44, 31.54, 31.45. HRMS (ESI) calcd for C19H24N2NaO3 [M + Na]+ 351.1685,
found 351.1689.
4-(tert-Butyl)-N'-(2,6-diethylphenyl)benzohydrazide 25: yield 84% as a white solid;
mp 152ꢀ154 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 8.99 (s, 1H), 8.01 (d, J = 8.4 Hz,
2H), 7.57 (d, J = 8.4 Hz, 2H), 7.27 – 7.10 (m, 4H), 2.66 – 2.61 (m, 4H), 1.37 (s, 9H),
1.15 (t, J = 7.6 Hz, 6H). 13C NMR (100 MHz, Acetoneꢀd6) δ 166.59, 155.49, 143.15,
133.10, 128.31, 128.24, 127.00, 126.23, 35.48, 31.48, 25.67, 15.00. HRMS (ESI)
calcd for C21H28N2NaO [M + Na]+ 347.2099, found 347.2093.
4-(tert-Butyl)-N'-(2,6-diisopropylphenyl)benzohydrazide 26: yield 83% as a white
solid; mp 169ꢀ171 °C; 1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 8.4 Hz, 2H), 7.80 (d,
J = 8.4 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.43 (s, 1H), 7.32 (d, J = 7.4 Hz, 1H), 7.22
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(d, J = 7.7 Hz, 2H), 3.15 (m, 2H), 1.36 (s, 9H), 1.21 (s, 6H), 1.19 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ 166.81, 155.36, 146.47, 131.63, 131.32, 128.43, 127.12, 126.80,
125.75, 125.65, 123.53, 35.01, 31.21, 28.90, 23.68, 22.72. HRMS (ESI) calcd for
C23H32N2NaO [M + Na]+ 347.2099, found 347.2093.
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4-(tert-Butyl)-N'-(2,6-dimethylphenyl)benzohydrazide 27: yield 92% as a white solid;
mp 155ꢀ156 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.54 (d, J = 2.4 Hz, 1H), 7.85 –
7.81 (m, 2H), 7.51 – 7.46 (m, 2H), 6.93 (d, J = 7.4 Hz, 2H), 6.79 (d, J = 7.4 Hz, 1H),
6.50 (d, J = 3.2 Hz, 1H), 2.48 (s, 6H), 1.31 (s, 9H). 13C NMR (100 MHz, Acetoneꢀd6)
δ 167.64, 155.65, 146.10, 131.32, 129.64, 129.03, 128.11, 126.17, 123.10, 35.46,
31.44, 19.06. HRMS (ESI) calcd for C19H24N2NaO [M + Na]+ 319.1786, found
319.1776.
N'-(2,6-Dimethylphenyl)-4-ethylbenzohydrazide 28: yield 91% as a white solid; mp
127ꢀ128 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.54 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H),
7.30 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 7.5 Hz, 2H), 6.78 (t, J = 7.6 Hz, 1H), 6.50 (d, J =
3.2 Hz, 1H), 2.70 – 2.65 (m, 2H), 2.48 (s, 6H), 1.21 (t, J = 7.6 Hz, 3H). 13C NMR
(100 MHz, Acetoneꢀd6) δ 167.66, 149.00, 146.11, 131.57, 129.62, 129.03, 128.73,
128.35, 123.09, 19.02, 15.79. HRMS (ESI) calcd for C17H20N2NaO [M + Na]+
291.1473, found 291.1469.
N'-(2,6-Dimethylphenyl)-4-methylbenzohydrazide 29: yield 93% as a white solid; mp
139ꢀ142 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.54 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H),
7.27 (d, J = 8.0 Hz, 2H), 6.94 (d, J = 7.4 Hz, 2H), 6.78 (t, J = 7.6 Hz, 1H), 6.49 (d, J =
3.2 Hz, 1H), 2.48 (s, 6H), 2.36 (s, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.64,
146.11, 142.73, 131.31, 129.88, 129.62, 129.03, 128.23, 123.08, 21.39, 19.01. HRMS
(ESI) calcd for C16H18N2NaO [M + Na]+ 277.1317, found 277.1308.
N'-(2,6-Dimethylphenyl)-4-methoxybenzohydrazide 30: yield 90% as a white solid;
mp 180ꢀ181 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.48 (s, 1H), 7.90 – 7.86 (m, 2H),
7.00 – 6.97 (m, 2H), 6.93 (d, J = 7.4 Hz, 2H), 6.77 (t, J = 7.6 Hz, 1H), 6.48 (d, J = 3.2
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Hz, 1H), 3.85 (s, 3H), 2.48 (s, 6H). C NMR (100 MHz, Acetoneꢀd6) δ 167.31,
163.32, 146.22, 130.03, 129.60, 128.97, 126.23, 123.02, 114.47, 55.78, 19.01. HRMS
(ESI) calcd for C16H18N2NaO2 [M + Na]+ 293.1266, found 293.1262.
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N'-(2,6-Dimethylphenyl)-4-propylbenzohydrazide 31: yield 93% as a white solid; mp
117ꢀ118 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.54 (s, 1H), 7.82 (d, J = 8.0 Hz, 2H),
7.28 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 7.4 Hz, 2H), 6.78 (t, J = 7.6 Hz, 1H), 6.50 (d, J =
3.2 Hz, 1H), 2.64 – 2.59 (m, 2H), 2.48 (s, 6H), 1.63 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
13C NMR (100 MHz, Acetoneꢀd6) δ 167.70, 147.40, 146.11, 131.59, 129.63, 129.33,
129.04, 128.27, 123.10, 38.38, 25.11, 19.04, 14.03. HRMS (ESI) calcd for
C18H22N2NaO [M + Na]+ 305.1630, found 305.1619.
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4-(Dimethylamino)-N'-(2,6-dimethylphenyl)benzohydrazide 32: yield 81% as a white
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solid; mp 140ꢀ141 °C; H NMR (400 MHz, Acetoneꢀd6) δ 9.29 (s, 1H), 7.79 (d, J =
8.8 Hz, 2H), 6.92 (d, J = 7.4 Hz, 2H), 6.74 (m, 3H), 6.46 (s, 1H), 2.99 (s, 6H), 2.48 (s,
6H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.89, 153.64, 146.54, 132.14, 129.60,
128.82, 122.85, 120.58, 117.85, 111.80, 111.60, 40.15, 19.08. HRMS (ESI) calcd for
C17H21N3NaO [M + Na]+ 306.1582, found 306.1572.
4-(tert-Butyl)-N'-(2-chloro-6-methylphenyl)benzohydrazide 33: yield 93% as a white
solid; mp 112ꢀ113 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.69 (s, 1H), 7.88 – 7.84 (m,
2H), 7.52 – 7.49 (m, 2H), 7.18 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.86 (m,
2H), 2.50 (s, 3H), 1.32 (s, 9H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.31, 155.82,
144.24, 130.99, 130.88, 130.77, 128.43, 128.35, 128.17, 126.23, 124.87, 123.55,
123.52, 35.49, 31.48, 31.44, 19.47. HRMS (ESI) calcd for C18H21ClN2NaO [M + Na]+
339.1240, found 339.1233.
N'-(2-Chloro-6-methylphenyl)-4-ethylbenzohydrazide 34: yield 91% as a white solid;
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mp 140ꢀ141 °C; H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 4.4 Hz, 1H), 7.68 (d, J =
8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 7.3 Hz,
1H), 6.89 (t, J = 7.8 Hz, 1H), 6.65 (d, J = 4.6 Hz, 1H), 2.68 (q, J = 7.6 Hz, 2H), 2.52
(s, 3H), 1.24 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.50, 148.57,
142.09, 130.13, 129.54, 129.28, 127.94, 127.63, 126.87, 125.28, 123.74, 28.52, 18.25,
14.94. HRMS (ESI) calcd for C16H17ClN2NaO [M + Na]+ 311.0927, found 311.0931.
N'-(2-Chloro-6-methylphenyl)-4-methylbenzohydrazide 35: yield 90% as a white solid;
mp 125ꢀ126 °C; 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 8.0 Hz, 2H), 7.62 (s, 1H),
7.30 (d, J = 7.8 Hz, 3H), 7.18 (m, 2H), 2.44 (s, 3H), 2.33 (s, 3H). 13C NMR (100 MHz,
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CDCl3) δ 165.71, 142.66, 138.17, 132.79, 131.17, 129.46, 129.39, 127.78, 127.46,
127.01, 21.57, 19.15. HRMS (ESI) calcd for C15H15ClN2NaO [M + Na]+ 297.0771,
found 297.0766.
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N'-(2-Chloro-6-methylphenyl)-4-methoxybenzohydrazide 36: yield 92% as a white
solid; mp 145ꢀ147 °C; 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 4.2 Hz, 1H), 7.74 (d,
J = 8.8 Hz, 2H), 7.21 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.96 – 6.89 (m,
3H), 6.66 (d, J = 4.8 Hz, 1H), 3.85 (s, 3H), 2.55 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 162.68, 142.49, 130.44, 129.82, 128.92, 128.01, 125.70, 124.41, 124.13, 114.03,
55.46, 18.53. HRMS (ESI) calcd for C15H15ClN2NaO2 [M + Na]+ 313.0720, found
313.0713.
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N'-(2-Chloro-6-methylphenyl)-4-ethoxybenzohydrazide 37: yield 94% as a white solid;
mp 133ꢀ134 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.61 (s, 1H), 7.89 (d, J = 8.8 Hz,
2H), 7.18 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 7.4 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 6.85
(t, J = 7.6 Hz, 2H), 4.13 – 4.07 (m, 2H), 2.50 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H). 13C
NMR (100 MHz, Acetoneꢀd6) δ 167.06, 162.77, 144.43, 130.87, 130.74, 130.11,
128.33, 125.77, 124.84, 123.46, 114.95, 64.32, 19.48, 15.00. HRMS (ESI) calcd for
C16H17ClN2NaO2 [M + Na]+ 327.0876, found 327.0871.
N'-(2-Chloro-6-methylphenyl)-4-propylbenzohydrazide 38: yield 92% as a white solid;
mp 126ꢀ128 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.67 (s, 1H), 7.84 (d, J = 8.2 Hz,
2H), 7.30 (d, J = 8.2 Hz, 2H), 7.19 (t, J = 6.4 Hz, 1H), 7.06 (d, J = 7.4 Hz, 1H), 6.86
(m, 2H), 2.65 (d, J = 7.4 Hz, 2H), 2.51 (s, 3H), 1.67 – 1.61 (m, 2H), 0.92 (t, J = 7.3
Hz, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.42, 147.58, 144.26, 131.31, 130.88,
130.79, 130.56, 129.38, 128.34, 128.32, 124.89, 123.56, 123.53, 38.39, 25.10, 19.47,
14.02. HRMS (ESI) calcd for C17H19ClN2NaO [M + Na]+ 325.1084, found 325.1073.
N'-(2-Chloro-6-methylphenyl)-4-(dimethylamino)benzohydrazide 39: yield 78% as a
white solid; mp 133ꢀ134 °C; 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 4.4 Hz, 1H),
7.69 – 7.65 (m, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.88 (t, J = 7.8
Hz, 1H), 6.69 – 6.64 (m, 3H), 3.02 (s, 6H), 2.55 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 166.86, 152.82, 142.83, 130.37, 129.79, 128.58, 127.93, 125.60, 123.84, 118.64,
111.12, 40.07, 18.60. HRMS (ESI) calcd for C16H18ClN3NaO [M + Na]+ 326.1036,
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N'-(2-Chloro-6-methylphenyl)-4-(methylthio)benzohydrazide 40: yield 88% as a white
solid; mp 125ꢀ127 °C; 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.0 Hz, 2H), 7.60 (s,
1H), 7.31 (dd, J = 8.0, 4.8 Hz, 3H), 7.18 (m, 2H), 2.54 (s, 3H), 2.33 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 169.62, 144.38, 138.14, 132.70, 131.15, 129.98, 129.41, 127.84,
127.04, 125.49, 19.15, 15.03. HRMS (ESI) calcd for C15H15ClN2NaOS [M + Na]+
329.0491, found 329.0485.
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4-(tert-Butyl)-N'-(2,6-dibromophenyl)benzohydrazide 41: yield 93% as a white solid;
mp 168ꢀ169 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.83 (s, 1H), 7.91 (d, J = 8.4 Hz,
2H), 7.54 (dd, J = 11.6, 8.4 Hz, 4H), 7.05 (d, J = 2.8 Hz, 1H), 6.83 (t, J = 8.0 Hz, 1H),
1.34 (s, 9H). 13C NMR (100 MHz, Acetoneꢀd6) δ 166.90, 155.91, 144.70, 133.96,
130.83, 128.32, 126.22, 125.05, 114.82, 110.90, 35.50, 31.41. HRMS (ESI) calcd for
C17H18Br2N2NaO [M + Na]+ 446.9684, found 446.9689.
N'-(2,6-Dibromophenyl)-4-ethylbenzohydrazide 42: yield 94% as a white solid; mp
146ꢀ148 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.82 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H),
7.55 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 2.8 Hz, 1H), 6.83 (t, J =
8.0 Hz, 1H), 2.72 – 2.66 (m, 2H), 1.22 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 166.99, 149.26, 144.69, 133.97, 131.08, 128.78, 128.59, 125.07, 114.84,
29.31, 15.79. HRMS (ESI) calcd for C15H14Br2N2NaO [M + Na]+ 418.9371, found
418.9365.
N'-(2,6-Dibromophenyl)-4-methylbenzohydrazide 43: yield 91% as a white solid; mp
123ꢀ124 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.82 (s, 1H), 7.87 (d, J = 8.2 Hz, 2H),
7.56 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 2.8 Hz, 1H), 6.83 (t, J =
8.0 Hz, 1H), 2.38 (s, 3H). 13C NMR (100 MHz, Acetoneꢀd6) δ 166.95, 144.70, 143.01,
133.96, 130.84, 129.91, 128.47, 125.06, 114.83, 21.43. HRMS (ESI) calcd for
C14H12Br2N2NaO [M + Na]+ 404.9214, found 404.9218.
N'-(2,6-Dibromophenyl)-4-methoxybenzohydrazide 44: yield 93% as a white solid; mp
1
150ꢀ151 °C; H NMR (400 MHz, Acetoneꢀd6) δ 10.18 (s, 1H), 8.05 (dd, J = 7.8, 1.8
Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.55 – 7.49 (m, 1H), 7.30 (d, J = 5.2 Hz, 1H), 7.21
(d, J = 8.4 Hz, 1H), 7.10 – 7.05 (m, 1H), 6.88 (t, J = 8.0 Hz, 1H), 4.10 (s, 3H). 13C
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NMR (100 MHz, Acetoneꢀd6) δ 164.84, 158.77, 144.88, 134.36, 133.89, 132.59,
125.94, 121.87, 120.56, 115.74, 112.88, 56.78. HRMS (ESI) calcd for
C14H12Br2N2NaO2 [M + Na]+ 420.9163, found 420.9159.
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N'-(2,6-Dibromophenyl)-4-ethoxybenzohydrazide 45: yield 95% as a white solid; mp
161ꢀ163 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.74 (s, 1H), 7.95 – 7.91 (m, 2H),
7.55 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 2.8 Hz, 1H), 7.00 – 6.97 (m, 2H), 6.82 (t, J = 8.0
Hz, 1H), 4.11 (q, J = 7.0 Hz, 2H), 1.38 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 166.63, 162.84, 144.80, 133.95, 130.30, 125.57, 125.01, 114.95, 114.82,
64.33, 15.00. HRMS (ESI) calcd for C15H14Br2N2NaO2 [M + Na]+ 434.9320, found
434.9325.
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N'-(2,6-Dibromophenyl)-4-propylbenzohydrazide 46: yield 92% as a white solid; mp
117ꢀ118 °C; 1H NMR (400 MHz, Acetoneꢀd6) δ 9.83 (s, 1H), 7.89 (d, J = 8.2 Hz, 2H),
7.55 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 2.8 Hz, 1H), 6.83 (t, J =
8.0 Hz, 1H), 2.66 – 2.61 (m, 2H), 1.65 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, Acetoneꢀd6) δ 167.01, 147.67, 144.70, 133.96, 131.11, 129.38, 128.51,
125.08, 114.86, 38.41, 25.10, 14.04. HRMS (ESI) calcd for C16H16Br2N2NaO [M +
Na]+ 432.9527, found 432.9524.
N'-(2,6-Dibromophenyl)-4-(dimethylamino)benzohydrazide 47: yield 81% as a white
solid; mp 156ꢀ158 °C; 1H NMR (400 MHz, Acetoneꢀd6)δ 9.53 (s, 1H), 7.87 – 7.82 (m,
2H), 7.54 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 3.0 Hz, 1H), 6.81 (t, J = 8.0 Hz, 1H), 6.73
(d, J = 9.0 Hz, 2H), 3.02 (s, 6H). 13C NMR (100 MHz, Acetoneꢀd6) δ 167.04, 153.78,
145.10, 133.92, 129.82, 124.86, 120.04, 114.79, 111.79, 40.14. HRMS (ESI) calcd for
C15H15Br2N3NaO [M + Na]+ 433.9480, found 433.9486.
N'-(2,6-Dibromophenyl)benzohydrazide 48: yield 90% as a white solid; mp
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142ꢀ143 °C; H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 4.4 Hz, 1H), 7.83 (d, J = 7.2
Hz, 2H), 7.49 (m, 5H), 7.01 (d, J = 4.8 Hz, 1H), 6.80 (t, J = 8.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3) δ 166.43, 143.05, 132.92, 132.29, 132.01, 128.83, 127.22, 125.38,
115.69. HRMS (ESI) calcd for C13H10Br2N2NaO [M + Na]+ 390.9058, found
390.9063.
N'-(2,6-Dibromophenyl)-2-methoxybenzohydrazide 49: yield 88% as a white solid; mp
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147ꢀ148 °C; H NMR (400 MHz, Acetoneꢀd6) δ 10.18 (s, 1H), 8.05 (dd, J = 7.8, 1.8
Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 5.1 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H),
7.10 – 7.05 (m, 1H), 6.88 (t, J = 8.0 Hz, 1H), 4.10 (s, 3H). 13C NMR (100 MHz,
Acetoneꢀd6) δ 164.84, 158.77, 144.88, 134.36, 133.89, 132.59, 125.94, 121.87, 120.56,
115.74, 112.88, 56.78. HRMS (ESI) calcd for C14H12Br2N2NaO2 [M + Na]+ 420.9163,
found 420.9158.
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4-(tert-Butyl)-N'-(2,6-difluorophenyl)benzohydrazide 50: yield 94% as a white solid;
mp 112ꢀ113 °C; 1H NMR (400 MHz, Acetone-d6) δ 9.90 (d, J = 3.0 Hz, 1H), 7.88 (d, J
= 8.6 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 3.2 Hz, 1H), 6.95 – 6.89 (m, 3H),
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1.33 (s, 9H). 13C NMR (100 MHz, Acetone-d6) δ 167.51, 154.72 (d, JCꢀF = 241.00
Hz), 154.67 (d, 2JCꢀF = 245.00 Hz), 131.02, 128.15, 127.44 (t, 3JCꢀF = 13.00 Hz; 4JCꢀF
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=
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12.00 Hz), 126.23, 121.73 (t, JCꢀF = 9.00 Hz; JCꢀF = 10.00 Hz), 112.48 (d, JCꢀF
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16.00 Hz), 112.41 (d, JCꢀF = 16.00 Hz), 35.49, 31.44. 19F NMR (375 MHz,
Acetone-d6) δ ꢀ129.06 (m, 2F), ꢀ164.90 (C6F6). HRMS (ESI) calcd for
C17H18F2N2NaO [M + Na]+ 327.1285, found 327.1289.
N'-(2,6-Difluorophenyl)-4-methoxybenzohydrazide 51: yield 93% as a white solid; mp
117ꢀ118 °C; 1H NMR (400 MHz, Acetone-d6) δ 9.86 (d, J = 4.2 Hz, 1H), 8.02 (dd, J =
7.8, 1.8 Hz, 1H), 7.51 (td, J = 7.2, 2.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.06 (td, J =
7.8, 0.8 Hz, 1H), 6.94 (m, 3H), 4.04 (s, 3H). 13C NMR (100 MHz, Acetone-d6) δ
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132.43, 127.32 (t, JCꢀF = 13.00 Hz; JCꢀF = 13.00 Hz), 122.22 (t, JCꢀF = 10.00 Hz;
6JCꢀF = 9.00 Hz), 121.79, 121.03, 112.84, 112.51 (d, 7JCꢀF = 16.00 Hz), 112.44 (d, 8JCꢀF
= 16.00 Hz), 56.62. 19F NMR (375 MHz, Acetone-d6) δ ꢀ129.04 (s, 2F), ꢀ164.90
(C6F6). HRMS (ESI) calcd for C14H12F2N2NaO2 [M + Na]+ 301.0765, found
301.0760.
N'-(2,6-Difluorophenyl)-4-ethoxybenzohydrazide 52: yield 95% as a white solid; mp
125ꢀ126 °C; 1H NMR (400 MHz, Acetone-d6) δ 9.80 (s, 1H), 7.89 (d, J = 8.8 Hz, 2H),
6.98 (d, J = 8.8 Hz, 3H), 6.96 – 6.86 (m, 3H), 4.11 (q, J = 6.8 Hz, 2H), 1.38 (t, J = 7.0
Hz, 3H). 13C NMR (100 MHz, Acetone-d6) δ 167.20, 162.82, 154.74 (d, 1JCꢀF = 241.00
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