Cyanohydrin as an anchoring group for potent and selective inhibitors of enterovirus 71 3C protease

Article abstract of DOI:10.1021/acs.jmedchem.5b01013

Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C^pro) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2′S,5S)-16 and (1R,2S,2′S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C^pro. Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.

Full text of DOI:10.1021/acs.jmedchem.5b01013

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Brief Article
Cyanohydrin as an Anchoring Group for Potent and
Selective Inhibitors of Enterovirus 71 3C Protease
Yangyang Zhai, Xiangshuai Zhao, Zhengjie Cui, Man Wang, Yaxin Wang, Linfeng Li, Qi
Sun, Xi Yang, Debin Zeng, Ying Liu, Yuna Sun, Zhiyong Lou, Luqing Shang, and Zheng Yin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01013 • Publication Date (Web): 16 Nov 2015
Downloaded from http://pubs.acs.org on November 23, 2015
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Journal of Medicinal Chemistry
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Cyanohydrin as an Anchoring Group for Potent and Selective Inhibi-
tors of Enterovirus 71 3C Protease
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†
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Yangyang Zhai, Xiangshuai Zhao, Zhengjie Cui, Man Wang, Yaxin Wang, Linfeng Li, Qi
§
†,||
†,||
†,||
#
‡
*,†,||
Sun, Xi Yang, Debin Zeng, Ying Liu, Yuna Sun, Zhiyong Lou, Luqing Shang,
and Zheng
*,†,||
Yin
†
College of Pharmacy and State Key Laboratory of ElementoꢀOrganic Chemistry, Nankai University, Tianjin 300071, China
|
|
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China
‡
Laboratory of structural Biological & Ministry of Education (MOE), and Laboratory of Protein Science, School of Medicine
and Life Sciences, Tsinghua University, Beijing 100084, China
§
College of Chemistry, and Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Central China Normal
University, Wuhan 430079, China
#
National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing 100101, China
Supporting Information Placeholder
pro
ABSTRACT: Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C ) inhibitors have been synthesized and assayed
for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S, 2S, 2’S, 5S)ꢀ16 and (1R, 2S,
2
’S, 5S)ꢀ16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result
pro
of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C . Cyanohydrin as an anchoring group with
high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.
INTRODUCTION
for cysteine protease inhibitors is the development of a highly
selective functional group that can interact nonꢀcovalently
with the cysteine residue. Cyanohydrin is scarcely reported as
Cysteine proteases are an important class of enzymes that
are heavily involved in various human physiological processꢀ
1
5
es. Functional abnormalities of cysteine proteases may lead to
an anchoring group of cysteine protease inhibitors, and cyaꢀ
pro
human diseases, for instance, inflammatory diseases, neuroꢀ
degenerative disorders, rheumatoid arthritis, and Alzheimer’s
disease, among others. Thus, cysteine proteases are an attracꢀ
tive class of targets for drug discovery. Many inhibitors have
been discovered and developed, for example, belnacasan as a
caspaseꢀ1 inhibitor for the treatment of interleukinꢀ1β convertꢀ
nohydrins as human EV71 3C inhibitors with high selectiviꢀ
ty are introduced here.
2
Human EV71, one of the major pathogens of hand, foot and
mouth disease, has caused several large outbreaks in infants
and children since 1997, mostly in the AsiaꢀPacific region.
Unfortunately, there are currently no antiviral drugs or vacꢀ
3
6
pro
ing enzymeꢀrelated diseases. Peptidomimetic approaches
cines clinically available. EV71 3C , a virusꢀencoded cysteꢀ
ine protease, plays a key role in the process of virus replicaꢀ
tion. The singleꢀstranded, positiveꢀsense RNA genome enꢀ
codes a large polyprotein precursor, which is subsequently
cleaved into four structural proteins (VP1ꢀVP4) and seven
have been widely adopted in drug discovery targeting cysteine
proteases. Based on the peptide substrate cleavage specificity
of the proteases, peptidomimetic inhibitors generally share a
common structure: 1) a modified peptide recognition sequence
for specific binding to the protease and 2) a Cꢀterminal elecꢀ
trophilic functional group for anchoring the activeꢀsite cysteꢀ
6
pro
nonꢀstructural proteins (2Aꢀ3D). 3C is indispensable for all
EV71 polyprotein cleavage processes, with the exception of
1
6
ine. Based on the characterization of the binding modes, the
the cleavages of VP1/2A and 3C/3D by 2A protease. Meanꢀ
pro
7
inhibitors can be broadly divided into the reversibles (e.g.,
aldehydes, nitriles, αꢀketo amides, etc.) and the irreversibles
while, 3C reportedly interferes with host cell function.
pro
Therefore, the essential role of 3C makes it an attractive
target for antiꢀEV71 drug discovery. In this brief article, we
introduce cyanohydrin as an anchoring group of cysteine proꢀ
tease inhibitors with high selectivity against human EV71
1
(e.g., α, βꢀunsaturated esters, disulfides, etc.). According to
the mechanism of the interaction, the activity of the inhibitors
is proportional to the electrophilicity of the reactive group.
However, the covalentꢀirreversible modifiers have seen limited
use as potential drug candidates because the strong reactive
electrophiles are generally accompanied by poor selectivity
pro
3C .
RESULTS AND DISCUSSION
pro
A literature survey of EV71 3C inhibitors revealed that, in
1
toward other proteases and potential toxicity caused by the
formation of protein adducts . Therefore, the important issue
addition to the good inhibition exhibited by rupintrivir (IC =
50
8 9
4
2.3 ± 0.5 µM) , derivative 1 (IC < 0.5 µM, EC50 = 0.096 ±
50
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.006 µM) with aldehyde as an electrophile (Figure 1) posꢀ
Scheme 1. Representative Synthetic Scheme of Aldehyde 6.
pro
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sessed even better activity. The crystal structure of EV71 3C
1
0
pro 8, 11
and the coꢀcrystal structure of rupintrivir/EV71 3C
revealed that, in the (S)ꢀγꢀlactam fragment, the carbonyl moieꢀ
ty forms a hydrogen bond with the imidazole segment of
His161 and the amide nitrogen donates a hydrogen bond to the
Thr142 backbone. Our research was initiated based on the
hypothesis that the replacement of the (S)ꢀγꢀlactam ring by a
(S)ꢀδꢀlactam ring could improve the potency against EV71
pro
3
C . As shown in Figure 2, the (S)ꢀδꢀlactam ring displayed
shorter hydrogen bond distances and more preferable orientaꢀ
tion than the (S)ꢀγꢀlactam ring when being docked into EV71
3
lactam ring was synthesized together with aldehyde 1 as a
reference (Scheme 2). The key intermediate 2 was prepared
similarly to literature, and the stereochemistry and high diaꢀ
stereoꢀselectivity were consistent with publications (see
Scheme 1, Figure 1 and Part Q in SI).
group of 2 with TFA followed by an amide bond formation
using EDCI as coupling reagent resulted in 3, which was subꢀ
sequently reacted with cinnamic acid to give 4 through a simiꢀ
lar procedure. Alcohol 5, obtained by the reduction of ester 4
Table 1. Enzyme inhibitory activity, antiviral activity and cytotoxicity of
pro
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1, 5, 6 and 8ꢀ16 as EV71 3C inhibitors
pro
C . To validate our hypothesis, aldehyde 6 with a (S)ꢀδꢀ
9, 12
Removal of the Boc
α, b
b
b
IC50
EC50
CC50
NO.
(
µM)
(µM)
(µM)
>100
1
ꢀꢀ
3.81 ± 0.19
3.07 ± 0.20
6
8
0.54 ± 0.02
>10
0.26 ± 0.07
>100
>100
>100
with NaBH , was finally oxidized to aldehyde 6 with
4
Dess−Martin periodinane. The biological activities (Table 1)
indicated that 6 (IC50 = 0.54 ± 0.02 µM, EC50 = 0.26 ± 0.07
µM) presented approximately 7ꢀ to 10ꢀfold better activities
than 1 (IC50 = 3.81 ± 0.19 µM, EC50 = 3.07 ± 0.20 µM) in both
the protease and cellular assays. The observed IC50 and EC50
values for 1 were different from the previously reported valꢀ
ues because of the use of different enzyme biochemical asꢀ
says and cellꢀbased EV71 assays . Additionally, the cellular
activity is more potent than that of enzymatic activity, which
is consistent with the observations in the published literatures
about 3C and 3Cꢀlike protease inhibitors.
efficacy and low toxicity of 6 (CC50 > 100 µM) directed our
attention to the (S)ꢀ ꢀlactam ring analogues.
1
1
9.49 ± 1.11
1.13 ± 0.10
4.59 ± 0.27
0.12 ± 0.05
>100
>100
9
9
>10
>10
>100
>100
>100
5
13
14
14
28.40 ± 1.33
10
12
>10
>100
>100
>100
8ꢀ9, 15
The enhanced
1.05 ± 0.09
0.15 ± 0.08
δ
1
3
1.62 ± 0.13
0.10 ± 0.06
>100
15
0.10 ± 0.02
0.28 ± 0.03
0.11 ± 0.05
0.056 ± 0.004
0.045 ± 0.008
0.048 ± 0.006
>100
>100
>100
>100
16
Figure 1. Structures of rupintrivir, 1 and 6.
(
1S, 2S,
0
0
.31 ± 0.03
.27 ± 0.05
2
’S, 5S)ꢀ16
1R, 2S,
2’S, 5S)ꢀ16
(
α
The pH condition of biochemical assay for the inhibitors was 7.0 except (1S,
S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16 was 6.0. Each data presents the
b
2
average results from three independent experiments, and error bars represent
SEMs (n=3).
Despite the significant improvement in biological activities,
6
as an aldehyde inhibitor may lead to various issues, such as
16
low selectivity, in further development. Therefore, we shiftꢀ
ed our efforts to replace aldehyde with other anchoring funcꢀ
tional groups. Nitriles are wellꢀestablished cysteine protease
17
inhibitors, and αꢀketo amides possess good inhibition to calꢀ
1
8
pain, which is a calciumꢀactivated cysteine protease. A series
of compounds were synthesized (Scheme 2). The intermediate
carboxylic acid was reacted with carbonyldiimidazole after the
saponification of ester 4 with aqueous lithium hydroxide, and
then the resulting imidazolide was quenched in situ with aqueꢀ
ous NH to provide amide 7. Amide 7 was converted to nitrile
3
Figure 2. Comparison between the docking models of 1 (yellow) and 6
19
pro
8 by (CF CO) Oꢀmediated dehydration. Reaction of aldeꢀ
(green) bound to EV71 3C
.
3 2
2
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pro
hyde 6 with KCN and NaHSO afforded the corresponding
potential mapped on the solventꢀaccessible surface of 3C .
3
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cyanohydrin 9. Cyanohydrin 9 was then converted to the
The most important interactions between (1R, 2S, 2’S, 5S)ꢀ9
and 3C are highlighted in this figure. Similar to the rupinꢀ
trivirꢀliganded protease complex, the overall structure of the
(1R, 2S, 2’S, 5S)ꢀ9ꢀbound form of EV71 3C adopts the typꢀ
ical chymotrypsinꢀlike fold. The S1 pocket is formed by
Thr142, His161, Gly163 and Gly164. The (S)ꢀδꢀlactam ring
engages in two intermolecular hydrogenꢀbonding interactions
with the sideꢀchain and the backbone of Thr142 as donors
with distances of 3.1 Å and 2.7 Å, respectively. Additionally,
the NH proton of His161 forms a hydrogen bond (1.9 Å) with
21
pro
methyl ester with hydrochloric acid gas and ethyl acetate.
8
Then the carboxylic acid, derived from the hydrolysis of the
intermediate methyl ester, coupled with amine to provide 10.
αꢀKeto amide 11 was the oxidative products of 10. 14 was
achieved in a twoꢀstep process via the reduction of the correꢀ
pro
2
2
sponding thionocarbonate ester derivative of 9. Ester 12 was
the methyl ester derivative of 9. The biological results showed
that, nitrile 8 surprisingly exhibited no inhibitory activities
(
IC50 > 10 µM and EC50 > 100 µM) (Table 1). In addition, the
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antiꢀEV71 activities of αꢀketo amide 11 (IC50 = 9.49 ± 1.11
µM and EC50 = 4.59 ± 0.27 µM) presented greater than 10ꢀ
fold activity loss compared to 6. The further study of nitriles
and αꢀketo amides was terminated due to the unsatisfactory
biological activities.
the lone pair supplied by the (S)ꢀδꢀlactam ring oxygen. The
larger S pocket is able to accommodate the more deeply sunk
2
P fragment. Arg39 is located at the back of the S pocket,
2
2
forming a πꢀπ stacking interaction with the P benzene ring.
2
The P phenylethylene group occupies the S pocket, with its
3
3
Scheme 2. Synthetic Scheme of 8ꢀ14.
phenyl ring extending vertically along the binding groove. The
cyanohydrin occupies the S ’ pocket and is in close proximity
1
to the protease catalytic center. Compared to α, βꢀunsaturated
ethyl ester in rupintrivir, one of striking differences is that the
cyanohydrin moiety of (1R, 2S, 2’S, 5S)ꢀ9 interacts with
Cys147. Neither –CN nor –OH form a hydrogen bond with the
thiol of Cys147. However, because of the electronꢀ
withdrawing profile of –OH and ꢀCN, the alphaꢀcarbon of
cyanohydrin exhibits electrophilicity to a certain extent, which
forms a nonꢀcovalent interaction (2.8 Å) with the thiol (Figure
3
B). This specific binding element is critical for the cyanohyꢀ
drin function. Furthermore, the –CN of cyanohydrin particiꢀ
pates in a bridge with a tightly bound water molecule in the S1’
pocket to stabilize the binding of the inhibitorꢀprotein complex,
and the nitrogen is within hydrogenꢀbonding distances of 2.5
Å and 2.3 Å to the backbones of Gln146 and Cys147, respecꢀ
tively. The ꢀOH of cyanohydrin interacts with His40 (2.3 Å)
as a hydrogen bond acceptor. Kinetic measurements were also
To our surprise, compared to aldehyde 6, cyanohydrin 9, the
synthetic precursor of αꢀketo amides, presented a relatively
weak enzyme inhibitory activity (IC50 = 1.13 ± 0.1 µM) but
greater than 2ꢀfold enhancement in cellular activity with an
EC50 of 0.12 ± 0.05 µM. To rapidly obtain more knowledge
regarding the cyanohydrin group, we focused on the modificaꢀ
tions of both –CN and –OH in the cyanohydrin group based on
the epimeric mixture of cyanohydrins 9. With the removal of –
CN or –OH, the inhibitors exhibited a dramatic loss in efficacy
pro
performed. The inhibition of EV71 3C by 9 was independꢀ
ent of incubation time and competitive, with a K of 0.77 ꢁM
i
(see Part C in SI). These observations were consistent with a
reversible, nonꢀcovalent mechanism of inhibition.
pro
against 3C , indicating that both –CN and –OH play critical
roles in activity. The virus replication in virusꢀinfected RD
cells was attenuated by the treatment of 14 with an EC50 of
2
8.40 ± 1.33 µM, whereas 5 provided no inhibition, even at
the highest concentration tested (EC50 >100 µM). It was hyꢀ
pothesized that the nitrile group and the alphaꢀcarbon in cyaꢀ
nohydrin contributed synergistically to the activity. Hence,
further studies on –CN/–OH performed. Following the reꢀ
placement of –CN with an amide group, αꢀhydroxy amide 10,
exhibited no activity against EV71 at the highest concentration
tested (IC50 > 10 µM and EC50 > 100 µM). Moreover, with the
esterification of –OH by a phenoxythiocarbonyl or acetyl
group, derivatised cyanohydrins 12 (IC50 = 1.05 ± 0.09 µM
and EC50 = 0.15 ± 0.08 µM) and 13 (IC50 = 1.62 ± 0.13 µM
and EC50 = 0.10 ± 0.06 µM) presented nearly identical efficaꢀ
pro
cy as 9 against 3C and EV71 viral replication. These findꢀ
ings suggested that the presence of the –CN group and the
electrophilicity of the alphaꢀcarbon play important roles in the
pro
process of cyanohydrin binding to EV71 3C .
In parallel to the structureꢀactivity relationship study, the Xꢀ
ray crystal structure study resulted in elucidation of the coꢀ
Figure 3. (A) Xꢀray Coꢀcrystal structure of (1R, 2S, 2’S, 5S)ꢀ9/EV71
pro
pro
pro
3C . (B) Partial enlargement for (1R, 2S, 2’S, 5S)ꢀ9/EV71 3C complex
crystal structure of (1R, 2S, 2’S, 5S)ꢀ9/EV71 3C , which was
at S1’ pocket.
determined at a 2.7 Å resolution during the crystallization
Further optimization of 9 led to the discovery of cyanohyꢀ
experiment of the epimeric mixture of cyanohydrins 9 with
drin 16, which contained the P paraꢀfluorine benzyl group and
pro
2
EV71 3C (Protein Data Bank accession code 5BPE). The
P3 5ꢀmethylisoxazole fragment (Scheme 3). The docking modꢀ
els of (1S, 2S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16 bound to
binding mode is presented in Figure 3 with the electrostatic
3
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pro
EV71 3C (Figure 4) indicated that the introduction of paraꢀ
fluoro benzyl influences the electron distribution of the S2
pocket, thus enhancing the interaction with positively charged
Arg39. Additionally, the P3ꢀisoxazole could form hydrogen
interactions with surrounding residues (Ser128, Leu127) of
cyanogenic glycosides can potentially degrade into hydrogen
30
1
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1
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cyanide in a process known as cyanogenesis, we focused
more attention on the toxicity of the cyanohydrin derivative as
pro
an EV71 3C inhibitor. Compared to a LD of 2.6 mg/kg for
50
31
KCN in mice via intravenous injection, the acute toxicokiꢀ
pro
3C . There is an intramolecular hydrogen bond formed beꢀ
netic behavior of (1R, 2S, 2’S, 5S)ꢀ16 represented tolerated
tween the atoms on the skeleton structure. Indeed, as shown in
toxicity with a LD of 96.5 mg/kg.
Scheme 3. Synthetic Scheme of 16.
50
Table 1, the epimeric mixture of cyanohydrins 16 exhibited
pro
better inhibition against EV71 3C (IC50 = 0.28 ± 0.03 µM)
2
3
than that of 9, which was consistent with 15 (IC50 = 0.10 ±
.02 µM) that had more than 5ꢀfold improved inhibitory activꢀ
0
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ity than that of 6. We next separated the epimeric mixture of
cyanohydrins 16 to (1S, 2S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ
1
6. The absolute configuration of the chiral carbon of cyanoꢀ
24
hydrin was determined by modified Mosher’s method and
further supported by H NMR and the calculated results of
1
energy minimization by MOE (see Part B in SI). Epimerizaꢀ
tion of the chiral carbon of cyanohydrin only occurred at pH >
6.0 in TrisꢀHCl buffer, while no epimerization was observed at
pH≦6.0 in buffer and other solvents (see Figure 20ꢀ23 in SI).
In order to obtain the enzyme inhibitory activity of the pure
isomer (1S, 2S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16, the pH
condition of biochemical assay was adjusted to 6.0, in which
there should be no cyanohydrin epimerization (see Figure 24
in SI) and little impact was observed with the assay. Interestꢀ
ingly, (1S, 2S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16 exhibited
similar activities in protease and cellular assays. The interacꢀ
tions of (1S, 2S, 2’S, 5S)ꢀ16/(1R, 2S, 2’S, 5S)ꢀ16 docked to
Figure 4. Comparison between the docking models of (1S, 2S, 2’S, 5S)ꢀ
16 (purple) and (1R, 2S, 2’S, 5S)ꢀ16 (green) bound to EV71 3C
pro
.
pro
EV71 3C could help to explain their unsubstantial difference
in IC values (0.31 ± 0.03 µM vs 0.27 ± 0.05 µM). As illusꢀ
50
trated in Figure 4, in addition to the nonꢀcovalent bond beꢀ
tween the alphaꢀcarbon and the thiol in the S ’ pocket, (1S, 2S,
1
2
’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16 have almost the same
interactive distances between the nitrogen of –CN and His40.
Moreover, the corresponding distances between –OH and
Gly145/Cys147 are 2.0 Å and 4.2 Å in the (1S, 2S, 2’S, 5S)ꢀ
16 model, respectively, compared to 2.2 Å and 3.1 Å in the
(1R, 2S, 2’S, 5S)ꢀ16 model. However, although aldehyde 15
presented a strong enzyme inhibitory potency compared to the
corresponding cyanohydrin 16, improved antiviral activity of
Figure 5. 15, (1S, 2S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16 (100 µM)
protease inhibitory activities of HNE, hTrypsin, hChymotrypsin, PPE,
pro
pro
AChE, BChE, EV71 3C and SARS CoV M . Vertical bars represent
the standard deviation of each data point (n=3).
Selectivity is one of the general issues for protease inhibiꢀ
tors, particularly those with reactive electrophiles such as alꢀ
dehyde. We profiled selected inhibitors containing either cyaꢀ
nohydrin or aldehyde while keeping the rest of the structure
constant against a list of proteases available to our research
group. The inhibition ratios of 15, (1S, 2S, 2’S, 5S)ꢀ16 and
1
6 was observed in the cellular assay. The calculated Log P
value of 16 (0.54) is 1.5ꢀfold higher than that of aldehyde 15
0.34). This could be the main factor that the cyanohydrin 16
(
could have better cellular penetration compared with the aldeꢀ
hyde 15 (EC50 = 0.11 ± 0.05 µM). Further, aldehyde as a reacꢀ
tive functional group could react with various nuleophiles in
the cell. Therefore, the aldehyde may be consumed by nucleoꢀ
phile in the cell that results in less exposure. In addition, the
epimeric mixture of cyanohydrins 16 (EC50 = 0.056 ± 0.004
µM), (1S, 2S, 2’S, 5S)ꢀ16 (EC50 = 0.045 ± 0.008 µM) and (1R,
2S, 2’S, 5S)ꢀ16 (EC50 = 0.048 ± 0.006 µM) exhibited equipoꢀ
tent antiviral activities in the cellꢀbased assay. Compared with
the reported inhibitor 1 with an aldehyde group (EC50 = 3.07 ±
0.20 µM), cyanohydrin derivatives (1S, 2S, 2’S, 5S)ꢀ16/(1R,
(1R, 2S, 2’S, 5S)ꢀ16 were determined against serine protease
(human neutrophil elastase (HNE), human trypsin (hTrypsin),
human chymotrypsin (hChymotrypsin), porcine pancreatic
elastase (PPE), acetylcholine esterase (AChE) and butyrylchoꢀ
linesterase (BChE)) and cysteine protease (EV71 3C and
pro
Severe Acute Respiratory Syndrome Coronaviruses main proꢀ
pro 32
tease (SARS CoV M )). As indicated by the results summaꢀ
rized in Figure 5, in contrast to the low selectivity of 15, (1S,
2
S, 2’S, 5S)ꢀ16 and (1R, 2S, 2’S, 5S)ꢀ16 both exhibited signifꢀ
2
S, 2’S, 5S)ꢀ16 exhibited more than 80ꢀfold higher antiviral
pro pro
icantly high selectivities for EV71 3C and SARS CoV M
activities. Additionally, favorable CC50 values (CC50 > 100
µM) were observed for all of the inhibitors in the in vitro cytoꢀ
toxicity assay.
over other proteases. The selectivities of ((1S, 2S, 2’S, 5S)ꢀ16
and (1R, 2S, 2’S, 5S)ꢀ16 are presumably related to the speciꢀ
ficity of the cyanohydrin. Therefore, cyanohydrin potentially
represents a good choice as an anchoring group for cysteine
protease inhibitors because of its high selectivity and good
inhibitory activity.
Cyanohydrin derivatives are present in a number of natural
2
5
products,
s6uch
as
mandelonitrile,
cyanohydrin
2
phosphonate, and αꢀketoglutarate cyanohydrin (αꢀKgCN) in
animals. More importantly, cyanohydrin derivatives have
27
CONCLUSIONS
been used as prescription drugs for treating asthma and proved
In conclusion, cyanohydrin derivatives as potent inhibitors
28, 29
to possess high potential in cancer chemotherapy
. Because
pro
of EV71 3C have been described. The coꢀcrystal structure of
4
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Page 5 of 7
Journal of Medicinal Chemistry
pro
cyanohydrin inhibitor and 3C was resolved, and the interacꢀ
(m, 1H), 3.35 (d, J = 11.7 Hz, 1H), 3.31 – 3.17 (m, 3H), 2.23 (dt, J = 13.6,
6.8 Hz, 1H), 2.17 – 2.06 (m, 1H), 1.98 (s, 2H), 1.91 – 1.83 (m, 1H), 1.69
pro
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tions between the cyanohydrin group and 3C were revealed.
Based on the data analysis of the coꢀcrystal structure, a numꢀ
ber of potent EV71 3C inhibitors were discovered, and the
antiviral efficacies of the inhibitors were significantly imꢀ
proved. Importantly, cyanohydrin as an anchoring group with
high selectivity and excellent inhibitory activity provides a
useful choice for cysteine protease inhibitors.
1
3
(
dd, J = 20.0, 11.4 Hz, 1H), 1.59 – 1.45 (m, 1H); C NMR (100 MHz,
CDCl ) δ: 200.15, 174.90, 171.91, 165.58, 141.57, 136.42, 134.69, 129.82,
29.70, 129.58, 129.53, 128.82, 128.60, 128.57, 127.88, 127.01, 120.31,
57.41, 54.27, 42.31, 38.82, 37.49, 30.79, 27.53, 21.30. HRMS (ESMS):
pro
3
1
+
C
26
H
29
N
3
NaO
Preparation of Amide 7. To a solution of 4 (2.0 g, 4.12 mmol) in a soluꢀ
tion of MeOH: H O (60 mL, 25:3 v/v) was added LiOH (0.15 g, 6.18
4
(M+Na) , calcd. 470.2050, found 470.2052.
2
mmol) at RT. After 1.5 h, the pH of the reaction mixture was adjusted to
3.0. The mixture was extracted with EtOAc (30 mL×2), and the organic
phase was dried over Na SO and concentrated to afford a white solid
2 4
crude product (1.77 g). A solution of the crude product (1.77 g) and 1, 1ꢀ
carbonyldiimidazole (1.0 g, 6.18 mmol) in 30 mL of anhydrous THF was
stirred at RT for 15 min. To the reaction mixture was added 1.6 mL of
EXPERIMENTAL SECTION
General. All reagents were purchased from commercial suppliers and
used as received. NMR spectra were recorded on a Bruker AVANCEꢀ400
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
400 MHz) (Bruker, Karlsruhe, Germany) NMR spectrometer. Molecular
mass was determined by ESI mass spectrometry using a Shimadzu
LCMSꢀ2020 (Shimadzu, Kyoto, Japan). Optical rotations were measured
with an Insmark IPꢀ120 Automatic Polarimeter (Insmark, Shanghai, Chiꢀ
NH
RT for 2 h. The reaction mixture was partitioned between EtOAc and H
The organic phase was dried over Na SO and concentrated, and the resiꢀ
3
(aq., 25%, 41.2 mmol), and the resulting suspension was stirred at
2
O.
D
na). Measurements were collected at 15 °C in DCM at 589 nm. [α] valꢀ
2
4
o
ues are given in units of ( × mL)/(g × dm). HRMS were recorded on a
highꢀresolution ESIꢀFTICR mass spectrometer (Varian 7.0 T, Varian,
USA). All tested compounds exhibited purities of > 95% as analyzed by
HPLC (Dionex UltiMate 3000, Germany).
General Procedure for the Synthesis of Aldehydes (1, 6 and 15).
Preparation of Ester 3. To a solution of 2 (2.0 g, 6.7 mmol) in anhydrous
due was purified by column chromatography (DCM: MeOH, 20:1 v/v) to
1
afford the pure product as a white solid 7 (1.54 g, 81%). H NMR (400
MHz, MeODꢀd
4
) δ: 7.54 (s, 2H), 7.30 (m, 9H), 6.69 (d, J = 15.8 Hz, 1H),
.79 – 4.69 (m, 1H), 4.41 (dd, J = 11.7, 3.6 Hz, 1H), 3.29 – 3.16 (m, 3H),
.07 (dd, J = 13.7, 8.5 Hz, 1H), 2.28 (m, 2H), 2.04 – 1.93 (m, 1H), 1.90 –
4
3
1
13
4
.77 (m, 2H), 1.70 (m, 1H), 1.51 (m, 1H); C NMR (100 MHz, MeODꢀd )
DCM (30 mL) was added CF
Then, the reaction mixture was stirred at RT for 2 h and then concentrated.
To a solution of the residue obtained in DCM (40 mL) was added Et
3
COOH (5.0 mL, 67 mmol) slowly at 0 °C.
δ: 175.42, 175.33, 172.68, 167.28, 140.96, 136.96, 134.80, 129.56, 128.99,
1
3
4
28.57, 128.24, 127.51, 126.57, 121.23, 119.99, 55.44, 51.13, 41.54,
3
N
+
7.41, 37.05, 33.53, 25.84, 20.68. LCꢀMS (ESI) (m/z): 463.2 (M+H) ,
dropꢀwise to adjust the pH to 7.0 at 0 °C, and then BocꢀLꢀPheꢀOH (1.8 g,
6.7 mmol), EDCI (1.53 g, 8.0 mmol) and HOBt (1.08 g, 8.0 mmol) were
+
85.2 (M+Na) .
3
Preparation of Nitrile 8. To a solution of 7 (1.77 g, 3.83 mmol) and Et N
sequentially added. After 20 min, Et
dropꢀwise. Then, the reaction mixture was stirred at RT for 3 h, followed
by washing with H O (50 mL×2), saturated citric acid solution (50 mL×
), saturated NaHCO solution (50 mL×2) and brine (50 mL×2). The
organic phase was dried over Na SO and concentrated, and the residue
3
N (3.8 mL, 26.8 mmol) was added
(1.33 mL, 9.58 mmol) in 100 mL of anhydrous THF, trifluoroacetic acid
anhydride (TFAA) (0.97 g, 4.60 mmol) was added dropꢀwise at ꢀ5 °C. The
reaction mixture was stirred at 0 °C for 2 h and then partitioned between
EtOAc and H O. The organic phase was dried over Na SO and concenꢀ
2 2 4
trated, and the residue was purified by column chromatography (DCM:
2
2
3
2
4
was purified by column chromatography (EtOAc: petroleum ether, 3: 1
MeOH, 35:1 v/v) to afford the pure product as a white solid 8 (1.51 g,
15
1
v/v) to afford the pure product as a light yellow oil 3 (2.6 g, 5.83 mmol,
89%). [α]
D
6
= ꢀ9.0 (c=0.16, DCM); H NMR (400 MHz, DMSOꢀd ) δ:
1
8
7
7%). H NMR (400 MHz, CDCl
3
) δ: 7.68 (d, J = 6.4 Hz, 1H), 7.29 (t, J =
7.55 (d, J = 7.0 Hz, 3H), 7.46 – 7.37 (m, 4H), 7.28 (d, J = 4.2 Hz, 4H),
6.70 (d, J = 15.8 Hz, 1H), 5.04 (dd, J = 15.6, 7.9 Hz, 1H), 4.59 (m, 1H),
3.06 (m, 3H), 2.87 (dd, J = 13.6, 9.5 Hz, 1H), 2.29 – 2.16 (m, 2H), 1.78
.2 Hz, 2H), 7.22 (d, J = 7.3 Hz, 3H), 5.18 (d, J = 7.5 Hz, 1H), 4.54 (dd, J
= 12.3, 4.7 Hz, 1H), 3.71 (s, 3H), 3.31 (d, J = 2.5 Hz, 2H), 3.16 (dd, J =
13.8, 5.5 Hz, 1H), 3.04 (dd, J = 12.7, 6.9 Hz, 1H), 2.40 – 2.30 (m, 1H),
13
(m, 3H), 1.65 – 1.51 (m, 1H), 1.40 (m, 1H); C NMR (100 MHz, DMSOꢀ
) δ: 172.48, 171.75, 165.33, 139.58, 137.93, 135.23, 130.02, 129.59,
129.42, 128.64, 128.01, 126.91, 122.19, 119.96, 116.99, 54.67, 41.55,
2
.30 – 2.20 (m, 1H), 2.15 – 2.07 (m, 1H), 1.95 – 1.82 (m, 2H), 1.80 – 1.67
d
6
13
(
m, 1H), 1.54 (q, J = 12.9 Hz, 1H), 1.40 (s, 9H); C NMR (100 MHz,
CDCl ) δ: 174.60, 172.24, 171.85, 170.79, 155.34, 136.68, 129.55, 128.41,
126.73, 79.82, 55.24, 52.34, 50.52, 42.41, 38.60, 37.84, 33.71, 28.27,
3
39.08, 37.88, 37.34, 34.48, 26.55, 21.63. HRMS (ESMS): C26
(M+Na) , calcd.467.2054, found 467.2060.
H
28
N
4
NaO
3
+
+
2
1.49. LCꢀMS (ESI) (m/z): 448.2 (M+H) .
General Procedure for the Synthesis of Cyanohydrins (9 and 16).
Preparation of Cyanohydrin 9. To a solution of 6 (500 mg, 1.1 mmol) in
DCM (20 mL) was added saturated NaHSO (149 mg, 1.4 mmol) solution.
3
Preparation of Alcohol 5. Ester 4 was prepared through an analogous
procedure to 3 utilizing cinnamic acid (86% yield). To a solution of 4 (1.0
g, 2.2 mmol) in anhydrous MeOH (30.0 mL) was added NaBH
22 mmol) slowly at 0 °C. Then, the reaction mixture was stirred at RT for
h. Saturated NH Cl solution (20 mL) was added to quench the reaction.
4
(0.84 g,
The mixture was stirred at RT for 0.5 h, and then an aqueous solution of
KCN (86 mg, 1.3 mmol) was added. The mixture was stirred at RT for 12
h. Then, the organic phase was collected, and the aqueous layer was exꢀ
tracted with DCM (30 mL×3). The combined organic phase was washed
with brine (30 mL×2), dried over Na SO , and concentrated, and the
2
4
Subsequently, volatiles were evaporated, and DCM (40 mL×2) was
added to extract the aqueous residue. The organic phase was washed with
2
4
brine (30 mL×2), dried over Na
2
SO
4
, and concentrated, and the residue
residue was purified by column chromatography (DCM: MeOH, 33:1 v/v)
was purified by column chromatography (DCM: MeOH, 30:1 v/v) to
to afford the pure product as a white solid 9 (480 mg, 1.01 mmol, 92%).
1
5
1
afford the pure product as a white solid 5 (0.81 g, 1.8 mmol, 82%). [α]
D
ꢀ
3
H NMR (400 MHz, CDCl ) δ: 7.54 (d, J = 15.6 Hz, 1H), 7.49 – 7.38 (m,
1
1
5.8 (c=0.15 g/100mL, DCM); H NMR (400 MHz, MeODꢀd
4
) δ: 7.52 (t,
2H), 7.36 – 7.15 (m, 8H), 6.43 (d, J = 4.7 Hz, 1H), 4.96 (dd, J = 13.9, 6.8
Hz, 1H), 4.57 – 4.46 (m, 1H), 4.23 (dd, J = 12.6, 9.2 Hz, 1H), 3.29 – 3.05
(m, 4H), 2.24 (m, 2H), 2.03 – 1.90 (m, 1H), 1.86 – 1.70 (m, 2H), 1.65 (dd,
J = 10.7 Hz, 3H), 7.44 – 7.19 (m, 8H), 6.68 (d, J = 15.8 Hz, 1H), 4.74 (d,
J = 5.8 Hz, 1H), 4.00 (d, J = 2.7 Hz, 1H), 3.48 – 3.39 (m, 1H), 3.34 (s,
1H), 3.27 – 3.13 (m, 3H), 3.04 (dd, J = 13.6, 8.4 Hz, 1H), 2.33 (dd, J =
11.6, 7.2 Hz, 1H), 2.06 (t, J = 12.5 Hz, 2H), 1.79 (s, 1H), 1.75 – 1.59 (m,
13
J = 12.8, 5.3 Hz, 1H), 1.44 (dd, J = 22.0, 11.3 Hz, 1H); C NMR (100
MHz, CDCl ) δ: 175.42, 172.86, 166.07, 141.67, 136.36, 134.62, 129.83,
129.48, 128.80, 128.58, 127.92, 126.99, 120.23, 118.72, 64.30, 54.57,
3
1
3
2
1
1
4
H), 1.48 (dd, J = 21.2, 11.0 Hz, 1H); C NMR (100 MHz, MeODꢀd ) δ:
77.45, 173.75, 168.34, 142.22, 138.46, 136.26, 130.94, 130.45, 129.99,
29.54, 128.92, 127.87, 121.57, 65.50, 56.71, 43.02, 39.11, 38.61, 33.89,
51.25, 42.25, 38.47, 31.50, 26.99, 21.15. HRMS (ESMS): C27
(M+Na) , calcd. 497.2159, found 497.2158.
H
30
N
4
NaO
4
+
+
27.16, 22.09. HRMS (ESMS): C26
H
32
N
3
O
4
(M+H) , calcd. 450.2387,
Cyanohydrin 16 (94% yield) was purified by column chromatography
found 450.2384.
(DCM: MeOH, 33:1 v/v) to afford the two pure products as white solids
2
8
Preparation of Aldehyde 6. To a solution of 5 (2.1 g, 4.7 mmol) in anhyꢀ
drous DCM (30.0 mL) was added DessꢀMartin reagent (3.0 g, 7.1 mmol)
slowly at 0 °C. Then, the reaction mixture was stirred at RT for 1 h. A
(1S, 2S, 2’S, 5S)ꢀ16. 44% yield. [α]
D
= ꢀ160.04 (c=0.031 g/100mL,
1
DCM); H NMR (400 MHz, CDCl
3
) δ: 8.60 (d, J = 6.8 Hz, 1H), 7.60 (d, J
= 8.1 Hz, 1H), 7.21 (dd, J = 7.7, 5.7 Hz, 2H), 6.96 (t, J = 8.5 Hz, 2H),
6.37 (s, 1H), 4.92 (q, J = 14.0, 7.1 Hz, 1H), 4.54 (d, J = 2.9 Hz, 1H), 4.20
(m, 1H), 3.30 (m, 2H), 3.23 (dd, J = 14.0, 5.8 Hz, 1H), 3.13 (dd, J = 14.0,
solution of NaHCO
0 min, DCM (30.0 mL×2) was added to extract the mixture. The organꢀ
ic phase was washed with brine (30 mL×2), dried over Na SO , and
concentrated, and the residue was purified by column chromatography
3 2 2 3
and Na S O was added to quench the reaction. After
1
7
4
.3 Hz, 1H), 2.46 (s, 3H), 2.28 (tt, J = 10.4, 5.1 Hz, 2H), 2.02 (dd, J = 8.4,
.0 Hz, 1H), 1.87 (dd, J = 9.5, 4.1 Hz, 1H), 1.79 – 1.62 (m, 2H), 1.54 (dd,
2
4
13
3
J = 22.2, 11.0 Hz, 1H); C NMR (100 MHz, CDCl ) δ: 175.38, 172.32,
(
DCM: MeOH, 35:1 v/v) to afford the pure product as a white solid 6
1
5
1
171.38, 161.97 (d, J C−F = 245.2 Hz), 159.18, 158.14, 131.81 (d, J C−F = 3.0
Hz), 130.99 (d, J C−F = 7.9 Hz), 118.26, 115.46 (d, J C−F = 21.2 Hz), 101.38,
(1.70 g, 3.8 mmol, 81%). [α]
D
= ꢀ2.4 (c=0.21 g/100mL, MeOH); H
3
NMR (400 MHz, CDCl ) δ: 9.22 (s, 1H), 7.48 (d, J = 4.4 Hz, 2H), 7.42 –
6
5.06, 54.46, 52.22, 42.29, 37.89, 37.52, 31.31, 27.26, 21.27, 12.31.
7
.17 (m, 9H), 6.47 (d, J = 15.6 Hz, 1H), 5.12 – 4.89 (m, 1H), 4.33 – 4.20
5
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Journal of Medicinal Chemistry
Page 6 of 7
+
HRMS (ESMS):
4
g/100mL, DCM); H NMR (400 MHz, CDCl
7
6
C
23
H
26FN
5
NaO
5
(M+Na) , calcd.494.1810, found
3H), 6.39 (d, J = 15.6 Hz, 1H), 5.01 (dd, J = 14.8, 6.9 Hz, 1H), 3.94 (m,
1H), 3.18 (s, 2H), 3.05 (m, 2H), 2.48 (dd, J = 16.6, 4.0 Hz, 1H), 2.23 –
2
8
94.1815. (1R, 2S, 2’S, 5S)ꢀ16. 50% yield. [α]
D
= ꢀ41.88 (c=0.028
) δ: 8.38 (d, J = 7.4 Hz, 1H),
.61 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 7.2 Hz, 2H), 6.96 (t, J = 8.2 Hz, 2H),
.35 (s, 1H), 4.93 (q, J = 14.1, 7.0 Hz, 1H), 4.57 (d, J = 4.3 Hz, 1H), 4.24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
2.12 (m, 2H), 2.12 – 2.04 (m, 1H), 1.95 – 1.82 (m, 1H), 1.80 – 1.67 (m,
13
1H), 1.66 – 1.47 (m, 2H), 1.36 (m, 1H); C NMR (100 MHz, CDCl
3
) δ:
174.65, 171.82, 165.52, 141.45, 136.50, 134.69, 129.83, 129.53, 128.85,
(m, 1H), 3.36 – 3.17 (m, 3H), 3.12 (dd, J = 14.0, 7.5 Hz, 1H), 3.29 (m,
H), 3.22 (dd, J = 13.7, 7.8 Hz, 1H), 3.12 (dd, J = 14.0, 7.5 Hz, 1H), 2.46
s, 3H), 2.36 – 2.14 (m, 2H), 2.10 – 1.97 (m, 1H), 1.77 (m, 3H), 1.51 (dd,
128.53, 127.87, 126.99, 120.45, 117.40, 54.07, 44.83, 42.19, 39.15, 37.94,
+
2
(
34.87, 27.13, 23.51, 21.52. HRMS (ESMS): C27
H
30
N
4
NaO
3
(M+Na) ,
calcd. 481.2210, found 481.2214.
Xꢀray Coꢀcrystal structure of EV71 3C Complexed with (1R, 2S,
2’S, 5S)ꢀ9. The inhibitor (1R, 2S, 2’S, 5S)ꢀ9 was mixed with EV71 3C
at a molar ratio of 5:1, and the optimal crystallization of EV71 3C was
13
pro
J = 21.8, 10.7 Hz, 1H); C NMR (100 MHz, CDCl
71.32, 161.96 (d, J C−F = 245.0 Hz), 159.10, 158.18, 131.86 (d, J C−F = 3.1
Hz), 131.01 (d, J C−F = 8.0 Hz), 118.62, 115.45 (d, J C−F = 21.3 Hz), 101.38,
4.19, 54.51, 51.25, 42.31, 37.72, 37.64, 31.43, 26.95, 21.19, 12.30.
3
) δ: 175.56, 172.00,
pro
1
pro
6
achieved by mixing 1.0 µL of protein with 1.0 µL of buffer containing
100 mM TrisꢀHCl (pH 7.7), 200 mM sodium citrate and 20% polyethꢀ
ylene glycol 3350 in a hangingꢀdrop vapor diffusion system at 22 °C.
Crystals appeared and reached their final size within 5 days. The resulting
crystals were soaked in reservoir solution containing 15% ethylene glycol
as the cryoꢀprotectant and flashꢀfrozen in liquid nitrogen. The native data
+
HRMS (ESMS):
94.1812.
C
23
H26FN
5
NaO
5
(M+Na) , calcd.494.1810, found
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Preparation of αꢀHydroxy amide 10. 9 (379 mg, 0.80 mmol) was disꢀ
solved in a solution of 4 N HCl/EtOAc (4 mL) and MeOH (4 mL), and the
mixture was refluxed for 16 h under a N2 atmosphere. After concentrating,
the residue was dissolved in DCM, washed with brine (30 mL×2), dried
pro
set of EV71 3C in complex with inhibitor was collected to 2.7 Å resoluꢀ
over Na
2
SO
4
and concentrated. The obtained residue was dissolved in
tion at beamline BL17U of the Shanghai Synchrotron Radiation Facility
(SSRF) with a wavelength of 0.9789 Å. The data were processed and
MeOH: H
2
O (150 mL, 25:3 v/v) at RT, and then LiOH (28.8 mg, 1.2
33
mmol) was added. After 1.5 h, the pH of the reaction mixture was adjustꢀ
ed to 3.0. The mixture was extracted with EtOAc (100 mL×3), and the
organic phase was dried over Na SO and concentrated to afford the pure
2 4
scaled using the HKL2000 package. The space group was identified as
C2, and one molecule was found per asymmetric unit. The complex strucꢀ
34
ture was determined using PHENIX with the crystal structure of EV71
pro
3
C
(PDB code: 3OSY) as the initial searching model. Manual model
product as a white solid intermediate, which was engaged in an amide
bond formation with propanꢀ1ꢀamine (56.74 mg, 0.96 mmol) to afford a
35
construction and refinement were performed using COOT and PHENIX
following rigid body refinement, energy minimization, and individual Bꢀ
factor refinement. The quality of the final refined model was verified
1
white solid 10 (353 mg, 0.68 mmol, 85% yield). H NMR (400 MHz,
CDCl
3
) δ: 7.54 (d, J = 15.7 Hz, 1H), 7.44 – 7.38 (m, 2H), 7.30 (dd, J = 5.1,
36
using the program PROCHECK . Data collection and the final model
1
1
1
.4 Hz, 3H), 7.20 – 7.06 (m, 3H), 6.91 (t, J = 8.6 Hz, 2H), 6.53 (d, J =
5.7 Hz, 1H), 5.44 (s, 1H), 4.95 (dd, J = 14.0, 6.4 Hz, 1H), 4.36 – 4.26 (m,
H), 3.16 (m, 4H), 2.46 – 2.18 (m, 1H), 2.10 – 1.98 (m, 1H), 1.98 – 1.86
statistics are summarized in Table S1 of the SI.
(m, 1H), 1.71 (dd, J = 8.7, 4.0 Hz, 1H), 1.64 – 1.32 (m, 6H), 1.26 (s, 1H),
ASSOCIATED CONTENT
1
3
0
1
1
3
3
.88 (t, J = 7.4 Hz, 3H); C NMR (100 MHz, CDCl ) δ: 175.71, 172.22,
Supporting Information.
71.35, 166.03, 141.40, 134.64, 132.54, 131.06, 130.98, 129.82, 128.81,
27.88, 120.45, 115.34, 115.13, 74.20, 54.66, 53.46, 52.16, 42.31, 40.78,
8.11, 37.40, 30.75, 29.71, 27.21, 22.80, 21.14, 11.40. HRMS (ESMS):
Detailed synthetic procedures, compound characterization, crysꢀ
tallographic data, and biochemical and antiviral assays. This maꢀ
terial is available free of charge via the Internet at
http://pubs.acs.org.
+
C
30
H
38
N
3
O
5
(M+H) , calcd. 520.2806, found 520.2810.
15
D
1
αꢀKeto Amide 11. [α]
MHz, CDCl ) δ: 7.58 (d, J = 15.6 Hz, 1H), 7.46 (dd, J = 6.3, 2.7 Hz, 2H),
.39 – 7.31 (m, 3H), 7.19 (dd, J = 8.3, 5.5 Hz, 2H), 7.05 (t, J = 5.9 Hz,
1H), 6.94 (t, J = 8.6 Hz, 2H), 6.39 (d, J = 15.6 Hz, 1H), 5.29 – 5.22 (m,
H), 5.03 (dd, J = 14.0, 6.1 Hz, 1H), 3.31 – 3.24 (m, 3H), 3.14 (t, J = 6.5
Hz, 1H), 2.29 (m, 1H), 2.17 – 1.97 (m, 3H), 1.93 – 1.83 (m, 1H), 1.79 –
.68 (m, 1H), 1.64 – 1.50 (m, 3H), 1.25 (s, 2H), 0.94 (t, J = 7.4 Hz, 3H);
= ꢀ40.8 (c=0.08 g/100mL, DCM); H NMR (400
3
7
AUTHOR INFORMATION
Corresponding Author
1
1
zheng_yin@nankai.edu.cn
shanglq@nankai.edu.cn
1
3
C NMR (100 MHz, CDCl
141.54, 134.65, 132.14, 131.24, 131.16, 129.83, 128.83, 127.88, 120.24,
3.81, 53.57, 42.35, 38.57, 37.95, 32.62, 22.51, 21.59, 11.37. HRMS
3
) δ: 195.03, 175.00, 171.03, 165.43, 159.49,
Notes
5
+
(ESMS): C30
H
35
N
3
NaO
5
(M+Na) , calcd. 540.2469, found 540.2461.
The authors declare no competing financial interests.
General Procedure for the Synthesis of Derivatised cyanohydrins (12
and 13). Preparation of Derivatised cyanohydrin 12. To a solution of 9
(237 mg, 0.5 mmol) in DCM (30 mL) was added DMAP (488.68 mg, 4.0
ACKNOWLEDGMENTS
mmol) and phenyl chlorothionoformate (345.26 mg, 2.0 mmol) under a N
2
We thank the staff of SSRF BL17U1 for their support in collectꢀ
ing the diffraction data. This work was supported by the National
Basic Research Program of China (973 program, Grant Nos.
atmosphere at 0 °C. The reaction mixture was stirred at RT for 2 h, poured
into water and extracted with DCM. The organic phase was washed with
H O, dried over Na SO , and concentrated, and the residue was purified
2 2 4
2
013CB911104, 2013CB911100), the National Natural Science
by column chromatography (DCM: MeOH, 50:1 v/v) to afford the pure
1
product as a yellow solid 12 (289.8 mg, 0.48 mmol, 95%). H NMR (400
Foundation of China (Grant Nos. 21202087, 81322023,
31170678, 31370733), the Tianjin Science and Technology Proꢀ
gram (Grant Nos. 13JCYBJC24300, 13JCQNJC13100), and the
3
MHz, CDCl ) δ: 7.59 (d, J = 15.6 Hz, 1H), 7.44 (dd, J = 10.9, 4.3 Hz, 4H),
7
.34 (dd, J = 8.1, 5.5 Hz, 5H), 7.31 – 7.28 (m, 3H), 7.13 (d, J = 7.8 Hz,
2H), 7.00 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 15.7 Hz, 1H), 5.77 (d, J = 4.7
Hz, 1H), 5.18 (dd, J = 14.3, 6.7 Hz, 1H), 4.56 – 4.42 (m, 1H), 3.23 (tt, J =
"111" Project of the Ministry of Education of China (Project No.
B06005).
1
2
3.7, 6.7 Hz, 4H), 2.60 (td, J = 13.8, 3.2 Hz, 1H), 2.34 (t, J = 13.6 Hz, 1H),
.10 – 1.97 (m, 1H), 1.93 – 1.77 (m, 2H), 1.77 – 1.62 (m, 1H), 1.59 – 1.45
1
3
REFERENCES
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