Sulfur(vi) fluoride exchange as a key reaction for synthesizing biaryl sulfate core derivatives as potent hepatitis C virus NS5A inhibitors and their structure-activity relationship studies

Article abstract of DOI:10.1039/c8ra05471a

Extremely potent, new hepatitis C virus (HCV) nonstructural 5A (NS5A) featuring substituted biaryl sulfate core structures was designed and synthesized. Based on the previously reported novel HCV NS5A inhibitors featuring biaryl sulfate core structures which exhibit two-digit picomolar half-maximal effective concentration (EC₅₀) values against HCV genotype 1b and 2a, the new inhibitors equipped with the sulfate core structures containing diversely substituted aryl groups were explored. In this study, highly efficient, chemoselective coupling reactions between an arylsulfonyl fluoride and an aryl silyl ether, known as the sulfur(vi) fluoride exchange (SuFEx) reaction, were utilized. Among the inhibitors prepared based on the SuFEx chemistry, compounds 14, 15 and 29 exhibited two-digit picomolar EC₅₀ values against GT-1b and single digit or sub nanomolar activities against the HCV GT-2a strain. Nonsymmetrical inhibitors containing an imidazole and amide moieties on each side of the sulfate core structures were also synthesized. In addition, a biotinylated probe targeting NS5A protein was prepared for labeling using the same synthetic methodology.

Full text of DOI:10.1039/c8ra05471a

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PAPER
Sulfur(VI) fluoride exchange as a key reaction for
synthesizing biaryl sulfate core derivatives as
potent hepatitis C virus NS5A inhibitors and their
structure–activity relationship studies†
Cite this: RSC Adv., 2018, 8, 31803
a
b
c
d
e
Youngsu You, Hee Sun Kim, Jung Woo Park, Gyochang Keum, Sung Key Jang
a
and B. Moon Kim
*
Extremely potent, new hepatitis C virus (HCV) nonstructural 5A (NS5A) featuring substituted biaryl sulfate core
structures was designed and synthesized. Based on the previously reported novel HCV NS5A inhibitors
featuring biaryl sulfate core structures which exhibit two-digit picomolar half-maximal effective concentration
(EC50) values against HCV genotype 1b and 2a, the new inhibitors equipped with the sulfate core structures
containing diversely substituted aryl groups were explored. In this study, highly efficient, chemoselective
coupling reactions between an arylsulfonyl fluoride and an aryl silyl ether, known as the sulfur(VI) fluoride
exchange (SuFEx) reaction, were utilized. Among the inhibitors prepared based on the SuFEx chemistry,
compounds 14, 15 and 29 exhibited two-digit picomolar EC50 values against GT-1b and single digit or sub
nanomolar activities against the HCV GT-2a strain. Nonsymmetrical inhibitors containing an imidazole and
amide moieties on each side of the sulfate core structures were also synthesized. In addition, a biotinylated
probe targeting NS5A protein was prepared for labeling using the same synthetic methodology.
Received 26th June 2018
Accepted 28th August 2018
DOI: 10.1039/c8ra05471a
rsc.li/rsc-advances
Currently, six NS5A inhibitors are on the market for use with
ribavirin, NS3/NS4A protease inhibitors, or NS5B inhibi-
Introduction
7,30–32
Hepatitis C virus (HCV) is known to cause serious liver infec- tors.
In 2014, the US Food and Drug Administration (FDA)
tion, which may result in liver cirrhosis and eventually hepa- approved ledipasvir (GS-5885) with the NS5B inhibitor, sofos-
1
–7
33,34
tocellular carcinoma (HCC). Currently, more than 170 million buvir.
Ombitasvir (ABT-267) was also approved by the FDA in
people are infected with HCV worldwide, and approximately 3 combination with paritaprevir (NS3/4A protease inhibitor),
to 4 million people are newly infected by this insidious disease ritonavir (protease inhibitor), and dasabuvir (NS5B inhibitor)
8–16
35–37
yearly.
for HCV genotype 1 infections.
Daclatasvir (BMS-790052) in
In recent years, many pharmaceutical companies and labo- combination with sofosbuvir (2015), elbasvir (MK-8742) in
ratories worldwide have focused their studies on understanding combination with grazoprevir (NS3/4A protease inhibitor), and
the HCV life cycle and developing direct-acting agents (DAAs) as velpatasvir (GS-5816) along with sofosbuvir in 2016 are among
17–22
38–41
next-generation therapies.
One of the most notable HCV other FDA-approved HCV DAAs.
Recently, the FDA approved
target proteins is the nonstructural 5A (NS5A) protein, which is Vosevi® in 2017, which is a combination of sofosbuvir, velpa-
believed to be involved in viral replication and assembly of new tasvir, and voxilaprevir (NS3/4A inhibitor); a new pan-genotype
23–29
virions.
drug, Mavyret® (glecaprevir/pibrentasvir), was also approved
in the same year.
32,42,43
a
Daclatasvir, which was rst reported in 2010, exhibits
Department of Chemistry, College of Natural Sciences, Seoul National University,
Seoul 08826, South Korea. E-mail: kimbm@snu.ac.kr
b
extremely high antiviral activities against a few genetic variants,
44–48
Division of Integrative Biosciences and Biotechnology, Pohang University of Science and against genotype 1b with EC50 value of 9 pM.
Therefore,
numerous DAAs have been introduced based on the daclatasvir
structure, which has a symmetrical phenylimidazole core with
and Technology, Pohang 37673, South Korea
c
Supercomputing Modeling & Simulation Center, Division of Data Analysis, Korea
Institute of Science and Technology Information (KISTI), 245 Daehak-ro, Yuseong-gu,
49–53
proline–valine–carbamate motif.
Novel, excessively potent
Daejeon, 34141, South Korea
d
NS5A inhibitors have been introduced by many pharmaceutical
Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and
54–58
companies.
However, treatment with daclatasvir quickly
Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02455, South Korea
e
Department of Life Sciences, Pohang University of Science and Technology, Pohang resulted in the development of drug-resistance, such as muta-
3
†
1
7673, South Korea
tion on L31 and Y93 residues in NS5A protein and the antiviral
activities were reduced by up to 15 000 times.
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra05471a
59–63
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We recently reported the design, synthesis, and structure– SuFEx chemistry for the construction of symmetrical and
activity-relationship (SAR) studies of novel NS5A inhibitors nonsymmetrical biaryl sulfate-based HCV NS5A inhibitors,
64
based on a biaryl sulfate core structure (Fig. 1). These which exhibit extremely high inhibitory activities.
compounds showed high antiviral activities and good additive In 2014, Sharpless and coworkers introduced the SuFEx click
effects as a combination treatment with an NS5B inhibitor, reaction for chemoselectively linking two different phenol
6
4
74,75
sofosbuvir, without showing any cytotoxicity.
Encouraged by the initial results, we optimized the biaryl through the reaction of an aryl uorosulfate and an aryl silyl
sulfate core part and carried further SAR studies focused on the ether in the presence of catalytic amount of 1,8-
sulfate core structures. During this investigation, we discovered diazabicyclo(5.4.0)undec-7-ene (DBU).
derivatives.
The sulfate coupling reaction was accomplished
a
76–78
The synthesis of
VI
that some substituted biaryl sulfate core structures were not sulfate compounds from the S –F group proceeded with high
readily accessible using traditional coupling reactions with yields and at a fast rate because of the driving force that formed
74
sulfonyl diimidazole (SDI). For example, in the cross-coupling the strong Si–F and S–O bond. Furthermore, the resulting
reactions of o-uorophenol derivatives with SDI, only a mono- compounds exhibited considerably high stabilities against
meric aryl sulfonyl imidazole was obtained. Likewise, the hydrolysis, nucleophilic substitution, thermolysis, and reduc-
74,77–79
sulfonyl diimidazole coupling strategy would not be appropriate tion.
for selective construction of nonsymmetrical biaryl sulfate core only inert silyl uorides as by-products.
structures (Fig. 2). that this chemoselective SuFEx reaction could be used for the
Moreover, this useful coupling reaction produces
75,78,80,81
We envisioned
Although the rst-generation DAA HCV drugs have synthesis of novel HCV NS5A inhibitors containing biaryl
symmetrical structures similar to daclatasvir, many of the next- sulfates possessing various substitution at the o-position of the
generation DAAs developed to treat multi-genotype strains tend phenol derivatives or a nonsymmetric biaryl sulfate core
65–69
to possess nonsymmetric structures.
Since there have been structures.
reports of NS5A inhibitors with a high degree of variation at the
center core, we envisioned that nonsymmetrical biaryl sulfate
core structures with substituted aryl groups would be worth
Results and discussion
69–73
investigating and, thus, were included in our SAR studies.
We prepared various aminophenol derivatives (3a–d) as
For the synthesis of the nonsymmetrical biaryl sulfate core substrates for the SuFEx reaction (Scheme 1). The reduction of
structures, SuFEx chemistry appeared to be an extremely nitro groups to amines was carried out with commercially
74
attractive strategy. Here, we report the fruitful exploitation of available nitrophenols 1a–b using 10 wt% palladium on acti-
82
vated charcoal (Pd/C). The amide linked monomers 3a–d were
prepared from amino-phenol derivatives 2a–d and N-Boc-L-
proline using general amide coupling methods in the presence
83,84
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI).
The imidazole-linked monomers 7a–c were prepared from the
acetophenone derivatives following an established proce-
64,85
dure.
a-Bromination of acetophenone 4 was carried out in
the presence of copper(II) bromide to provide a-bromoaceto-
85,86
phenone 5 in an 81% yield.
Then, the resulting product 5
was treated with N-Boc-L-proline in the presence of DIPEA to
85
Fig. 1 Discovery of biaryl sulfate derivatives as potent HCV NS5A yield ester 6a. Likewise, commercially available a-bromoace-
inhibitors.
tophenone derivatives 5b and 5c were converted to the corre-
sponding esters 6b and 6c, respectively. Synthesis of the
imidazole derivatives 7a–c was accomplished by the reaction of
85
6a–c with ammonium acetate.
The introduction of a uorosulfonyl (fosyl) group was ach-
ieved by the reaction of the corresponding phenol derivatives
with sulfuryl uoride (SO F ) and 1.7 equiv. DIPEA in
2
2
74
dichloromethane (Table 1). Since SO F is a gas, the reaction
2
2
mixture was stirred rapidly (>1000 rpm) to enhance the liquid–
7
6,78
gas contact.
tored using thin layer chromatography (TLC). The structures of
The reaction was complete within 5 h, moni-
1
13
the fosylated products were conrmed using proton ( H), C,
1
9
and F nuclear magnetic resonance (NMR) spectroscopy;
1
9
especially, F NMR chemical shis of uorine at fosyl group
76,87
ranged from ca. 31 to 40 ppm.
These uorosulfate deriva-
tives exhibited good stability against hydrolysis and could be
7
7,78,80,88
stored on the shelf for months.
Compounds 8a–g were
Fig. 2 Biaryl sulfate core based nonstructural 5A (NS5A) inhibitors and
modification using sulfur(VI) fluoride exchange (SuFEx) chemistry.
obtained in good yields (>80%) as shown in Table 1.
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Scheme 1 Synthesis of intermediates; reagents and conditions: (a) Pd/C, H
2
, MeOH, room temperature (rt), 24 h, 99%; (b) EDCI, N-Boc-L-
proline, CH
2
Cl
2
, rt, 4 h, 59–96%; (c) CuBr
2
, EA/CHCl
3
, reflux, 8 h, 81%; (d) N-Boc-L-proline, N,N-diisopropylethylamine (DIPEA), acetonitrile, rt,
ꢀ
5
h, 72–91%; (e) NH
4
OAc, toluene, 95 C, 20 h, 45–52%.
a
Table 1 Preparation of fluorosulfate monomers
monomer containing an imidazole moiety (compounds 10g–
1
0j), 2.2–2.3 equiv. DBU was used for the SuFEx coupling
products.
We synthesized nonsymmetric or o-uoro substituted biaryl
sulfate based NS5A inhibitors using appropriate precursors
Tables 3 and 4). The tert-butyloxycarbonyl (Boc)-protecting
group for the proline moiety was deprotected with 50% (v/v)
(
64,90,91
TFA in CH Cl .
Volatiles were removed from the reaction
2
2
mixture under reduced pressure and the residue was directly
coupled with a capping group such as N-methyloxycarbonyl-
protected valine (val) or phenylglycine (phg) in the presence of
64
EDCI and HOBt. Finally, the biaryl sulfate based HCV NS5A
inhibitors were obtained aer purication of the crude prod-
64
ucts using silica gel chromatography.
To determine the antiviral activities of each compound, we
measured the EC50 of inhibitors for genotype-1b (GT-1b) and 2a
(
GT-2a) using the HCV replicon and HCV cell culture (HCVcc)
91
systems, respectively. We used the human hepatoma Huh
.5.1 cell line to investigate the EC50 of the compounds in the
HCV replicon systems for GT-1b, which encodes the bicistronic
7
9,91–93
NK5.1 gene and Gaussia luciferase (Gluc) reporter gene.
On the other hand, the inhibitory activities of GT-2a were
measured using the HCVcc system with JFH 5a-Rluc-ad34,
which is a derivative of JFH1 containing a Renilla luciferase
a
Yields of isolated products.
90,94,95
reporter and cell culture adaptive mutations.
The antiviral activities (EC ) of the tested compounds con-
5
0
We then constructed the sulfate core structures using aryl taining amide groups on both side of the sulfate core against
79
silyl ether counterparts through SuFEx chemistry (Table 2).
replicon (GT-1b) and HCVcc (GT-2a) are listed in Table 3. We
Silylation of phenol groups was achieved using the rst selected the compounds that exhibited <1 nM EC50 for GT-
standard method with tert-butyldimethylsilyl chloride 1b, and then measured the HCVcc EC50 for GT-2a. In the amide
8
9
(
TBDMSCl) and imidazole. The coupling reaction between linker series, the D-valine or D-phenylglycine derivatives were
an aryl fosylate and aryl silyl ether was carried out in the pres- chosen as only capping groups because these groups showed
64
ence of DBU in dimethyl formamide (DMF) according to the highest levels of antiviral activities in our previous study.
74,75
ꢀ
Sharpless' protocol.
Then, the mixture was stirred at 50 C
We rst carried out the SAR studies of inhibitors containing
for 12 h. The biaryl sulfate products (compounds 10a–10f) were a non-symmetric sulfate core. The inhibitor 11 (Table 3, entry 5)
formed in good to excellent yields in the presence of catalytic containing 3-aminophenyl (4-aminophenyl) sulfate derivative
74
amounts of DBU (20–30 mol%). However, in case of the equipped with a D-phenylglycine capping moiety showed 36-fold
0
coupling reactions involving the silyl ether or uorosulfate lower potency against the GT-1b than the symmetric p,p -
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6
4
Table 2 Preparation of biaryl sulfate core structures using sulfur(VI) showed activity at two-digit nanomolar levels in GT-1b.
a
fluoride exchange (SuFEx) chemistry
However, replacement of the methyl groups of BMK-21028 with
uorine (compound 13) improved the inhibitory activity against

GT-1b strain with 0.0204 nM EC50 (Table 3, entry 7). This result,
surprisingly, was a 1127-fold higher inhibitory activity than that
of BMK-21028 was, suggesting that the o-position in the biaryl
groups is extremely important for increasing the potency.
0
The C
2
symmetric 6,6 -diuoro-substituted phenol deriva-
tives containing the D-phenylglycine moieties 14 (Table 3, entry
8
1
) also showed increased inhibitory activities against both GT-
b and GT-2a, compared to BMK-21020, which has o-methyl
0
groups at 6- and 6 -positions of the biaryl groups (Table 3, entry
64
4
). Although the inhibitory activities against GT-1b were
increased, compound 14 showed low GT-2a inhibitory activity
single nanomolar level EC50) and compound 13 showed no
(
inhibitory activities at 10 nM.
Nonsymmetrically substituted diuorobiarylsulfate 15 (Table 3,
0
entry 9) with 2- and 2 -uoro-substitution exhibited 5-fold weaker
activity for GT-1b and 2-fold higher potency for GT-2a compared to
symmetric compound 13. The replacement of the D-phenylglycine
with D-valine moiety (compounds 16 and 17) showed lower
inhibitory activities except for compound 18, which had GT-1b
potency to single nanomolar range (Table 3, entries 10, 11, and
12, respectively). The fact that the SAR results were highly depen-
dent on the substitution and the sulfate linking the position of the
biaryl groups indicates that the inhibitory activity of various HCV
genotype is highly sensitive to the structural features of the core
moiety. These results indicate that ortho-substitution in a phenyl
group with uorine blocks the free rotation, which lowers the
antiviral activities against GT-1b. Nevertheless, replacing it with
hydrogen, which lacks steric hindrance relatively, enhanced the
inhibitory activities against the GT-2a gene.
As shown in Table 2, we designed the NS5A inhibitor series
consisting of imidazole linkers with the aim of enhancing the
96–98
antiviral activities and solubility.
In our previous report, the
imidazole linked biaryl sulfate inhibitors containing an L-valine
capping group (BMK-21025, Table 4, entry 1) showed good
64
activities against GT-1b and GT-2a. Thus, we introduced
nonsymmetric or substituted sulfate core structures in
imidazole-linked inhibitors to determine if it would inuence
the inhibitory activities and the results are summarized in Table
4
. Compound 19, containing o-uoro substituted biaryl sulfate
a
b
Yields of isolated products. The reactions were carried out with DBU with D-valine derivative, showed no inhibitory activity at 100 nM
(2.2–2.3 equiv.).
against replicon GT-1b (Table 4, entry 2). Compound 20 with an
N-methoxycarbonyl-L-valine capping group resulted in two-
substituted inhibitor BMK-21007 did, while the antiviral activ- digit nM EC50 on GT-1b (Table 4, entry 3). The replacement of
ities on GT-2a were reduced by >6-fold compared to those of the the L-valine moiety with D-phenylglycine moiety as in compound
0
symmetrically m,m -substituted biaryl sulfate inhibitor BMK- 21 dramatically enhanced the antiviral activity against GT-1b
64
2
1014 (Table 3, entries 1 and 2, respectively). The replacement (Table 4, entry 4). However, these results showed 25-fold lower
of the D-phenylglycine with D-valine moieties as shown in activity than that of inhibitors linked with amides such as
0
compound 12 induced no apparent inhibitory activity against compound 15. Introduction of m-, p -disubstituted biaryl sulfate
GT-1b at 100 nM concentration (Table 3, entry 6).
core as in compound 22 demonstrated that the potencies for
We next investigated the inhibitors containing o-uoro both GT-1b and GT-2a were better than that of compound 21
substituted biaryl groups, expecting increased potency based on with an o-uoro substituent (Table 4, entry 5). While the D-valine
the restricted rotational conformation around the sulfate core. derivative 23 showed no inhibition at 100 nM, the opposite
In a previous study, compound BMK-21028 (Table 3, entry 3), stereoisomeric L-valine derivative 24 showed improved potency
0
which was substituted with methyl at 2- and 2 -positions,
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a
Table 3 Synthesis and in vitro antiviral activities of inhibitors comprising an amide linked biaryl sulfate core
b
b
Entry
1
Compounds
Biaryl sulfate group
Capping group (R)
Replicon EC50 (GT-1b, nM)
HCVcc EC50 (GT-2a, nM)
BMK-21007
(R)-Phenyl
0.0100
0.0198
0.128
2
3
BMK-21014
BMK-21028
(R)-Phenyl
(R)-Phenyl
0.0199
d
23.0
—
4
BMK-21020
(R)-Phenyl
0.110
0.361
2.23
5
6
7
11
12
13
(R)-Phenyl
(R)-Isopropyl
(R)-Phenyl
0.129
c
—d
>100
c
0.0204
>10.0
8
9
14
15
16
17
(R)-Phenyl
0.0224
0.0241
1.22
(R)-Phenyl
0.627
c
d
1
0
1
(R)-Isopropyl
(R)-Isopropyl
>100
—
c
—d
—d
1
>100
c
1
2
18
(R)-Isopropyl
>1.00
a
b
EC50, half-maximal (50%) effective concentration; GT-1b, genotype-1b; GT-2a, genotype-2a. All experiments were performed three times except
c
d
for compounds that exhibited >1 nM EC50 for GT-1b. Experiment was performed once. Not determined.
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a
Table 4 Synthesis and in vitro antiviral activities of sulfate core-inhibitors equipped with imidazole moieties
b
b
Entry
1
Compounds
Biaryl sulfate group
Capping group (R)
Replicon EC50 (GT-1b) (nM)
HCVcc EC50 (GT-2a) (nM)
BMK-21025
(S)-Isopropyl
0.0323
0.882
c
—d
2
3
19
20
(R)-Isopropyl
(S)-Isopropyl
>100
c
—d
>10.0
4
5
6
21
22
23
(R)-Phenyl
(R)-Phenyl
(R)-Isopropyl
0.584
0.139
9.70
3.59
c
d
>100
—
c
7
24
(S)-Isopropyl
0.260
>10.0
a
b
EC50, half-maximal (50%) effective concentration; GT-1b, genotype-1b; GT-2a, genotype-2a. All experiments were performed three times except
c
d
for compounds that exhibited >1 nM EC50 for GT-1b. Experiment was performed once. Not determined.
for GT-1b (Table 4, entries 6 and 7, respectively). However, it proline-D-phenylglycine carbamate derivative in moderate yields
99
showed no marked antiviral activity against GT-2a.
as shown in Scheme 2. The nonsymmetric compound 29
Based on the SAR study, it can be concluded that compounds exhibited excellent inhibitory activity for GT-1b. However, it
with asymmetric biaryl sulfate core and D-phenylglycine capping exhibited nanomolar levels of antiviral activity against GT-2a,
derivatives (compounds 11 and 22) showed the best activities for which indicated no dramatic change in activity.
both genotypes. We proceeded to synthesize nonsymmetric
We also used SuFEx chemistry to prepare the biotin-tagged,
compounds, exploiting the chemoselectivity of SuFex chemistry. biaryl sulfate core based NS5A inhibitor as shown in Scheme
0
In our previous study, we reported that m,m -biaryl sulfate 3. Biotin binds specic proteins such as streptavidin and avidin
6
4
44
derivatives exhibit high inhibitory activities against NS5A.
with extremely high affinity. Because of this specicity, bio-
100,101
Especially, C -symmetric biaryl sulfate comprising amide (BMK- tinylation of NS5A inhibitors has been reported. The
1014) and imidazole (BMK-21025) linkers showed extremely introduction of biotin into one proline was readily carried out
2
2
high antiviral activities against two genotypes. Based on these using SuFex chemistry. Although the NS5A inhibitor containing
results, we synthesized compound 29 having a hybridized struc- the biaryl sulfate with biotin-tag 32 showed a slightly decreased
ture of the two compounds. Due to the orthogonality of SuFEx, we EC50 against genotype-1b (9.59 nM), it was 10 times higher than
were able to synthesize the biaryl sulfate compound containing the previously reported biotinylated tag molecules based on
63,102
imidazole-proline-L-valine carbamate derivative and amide- BMS compound (BMS-671 and BMS-690).
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To understand the observed antiviral activities (EC50) of inhib-
itors from a 3D structural perspective, three representative inhib-
itors, BMK-21007, BMK-21014, and 11, were docked in an NS5A
protein dimer model. The model was constructed using the NS5A
103
domain 1 crystal structure (3FQQ) and amino terminal alpha
104
helical NMR structure (1R7G).
Fig. 3 illustrates the 3D-
interaction models of BMK-21007, BMK-21014, and 11 with the
NS5A dimer. The docking model demonstrates that inhibitors
bound across the dimer interface with Thr95A, which forms
a hydrogen bond with the nonsymmetric biaryl sulfate core. The
interactions of inhibitors and the residues at the binding site are
detailed in two dimensional (2D) docking representation (ESI,
Fig. S4†). In the case of BMK-21007, the biaryl sulfate moiety can
engage in hydrophobic interaction with Gln54A and Gln54B,
whereas the methyl groups of the carbamate groups make p-alkyl
interaction with Pro58 and Tyr93 of the NS5A protein dimer
(Fig. 3A and S4A†). The amide linkers of BMK-21014 make
hydrogen bond with Gln54, Thr56, and Gln62, whereas both
methyl groups of the carbamates involve p-alkyl interaction with
Pro58 and Tyr93 (Fig. 3B and S4B†). In compound 11 (Fig. 3C), the
sulfate moiety appears to interact with Thr95 by hydrogen
bonding. Interestingly, Tyr93A has p-alkyl interaction with the
pyrrolidine ring and the methyl moiety of methyl carbamate,
whereas Tyr93B shows p-donor hydrogen bond interaction with
the phenyl group of D-phenylglycine. There are no interactions with
the residues of Pro58 (ESI, Fig. S4C†).
As shown in a superposed model (Fig. 3D, S4A and S4B†), the
symmetric inhibitors BMK-21007 and BMK-21014 pose reduced
steric hindrance of D-phenylglycine moiety compared to nonsym-
metrical inhibitor 11, where only one methyl group of the carba-
mates is involved in p-alkyl interaction with Tyr93A. Also, CDocker
interaction energies of BMK-21007, BMK-21014, and 11 are
Scheme 2 Synthesis of 3-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrro-
lidin-2-yl)-1H-imidazol-5-yl)phenyl(3-((S)-1-((R)-2-((methox-
ꢁ
1
ycarbonyl)amino)-2-phenylacetyl)pyrrolidine-2-carboxamido)phenyl) ꢁ58.41, ꢁ57.20, and ꢁ46.19 kcal mol , respectively, which
sulfate.
suggests that BMK-21007 and BMK-21014 might be better placed
in the binding mode compared to 11. In the case of inhibitor 22,
hydrogen bond interactions are found not only between the sulfate
and Gly54A but also between the imidazole and Thr56 (ESI,
Fig. 3 Docking of (A) BMK-21007, (B) BMK-21014, and (C) 11 to an
NS5A (3FQQ) dimer model. For clarity purpose, only residues having
interactions were shown. BMK-21007, BMK-21014, and 11 are shown in
stick and surrounding residues were shown in line. Various interactions
are shown in dashes. (For more clarity see the ESI, Fig. S4†). (D) Super-
Scheme 3 (a) TBDMSCl, Imidazole, CH
2
Cl
2
, rt, 4 h, 81%; (b) DBU, DMF,
posed model of inhibitors inside 3FQQ dimer model. BMK-21007, BMK-
1014, and 11 are shown blue, green and red, respectively.
ꢀ
5
0
2 2
C, 12 h, 75%; (c) TFA, CH Cl , rt, 30 m; (d) EDCI, HOBt, DIPEA,
2
Biotin, CH
2
Cl , rt, 16 h, 96%.
2
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Fig. S4D†). Both ends of the carbamate groups appear to show p- analyses were carried out on an Agilent HP1100 system (Santa
alkyl interaction with Tyr93A and Tyr93B, and especially Tyr93A is Clara, CA, USA), composed of an auto sampler, quaternary pump,
engaged in hydrogen bond interaction with the carbonyl moiety of photodiode array detector (DAD), and HP Chemstation soware.
one carbamate. Interestingly, one methyl group of the carbamate The separation was carried out on a poroshell 120 EC-C18
makes hydrophobic interaction with Pro58B, whereas the other column 50 ꢂ 4.6 mm, 2.7 mm with acetonitrile (A), 0.1% TFA
ꢁ1
side correlates with Pro97B. Therefore, the D-phenylglycine of in water (B), as a mobile phase at a ow rate of 1 mL min at
ꢀ
inhibitor 22 poses reduced steric hindrance compared to inhibitor 20 C. Method: 5% A and 95% B (0 min), 50% A and 50% B (15
11 due to the hydrogen bonding of the carbamate group (ESI, min), 95% A and 5% B (24 min), 95% A and 5% B (25 min), 5% A
Fig. S6†). CDocker interaction energy of inhibitor 22 is and 95% B (26 min), 5% A and 95% B (27 min). All materials were
ꢁ1
ꢁ
54.76 kcal mol , which bodes well with its EC50 activity.
purchased from a commercial supplier and used without further
purication unless otherwise noted. The H NMR-spectra of
1
intermediates and nal products exhibit a mixture of rotamers at
ambient temperature in DMSO-d .
6
Conclusions
In conclusion, using SuFEx chemistry we achieved chemoselective
coupling of aryl uorosulfates and aryl silyl ethers in the presence
of a catalytic amount of a base. Furthermore, this method was used
to synthesize new NS5A inhibitors containing various substituted
sulfate core structures with high yields. These compounds
exhibited high inhibitory activities against HCV viral proliferation
by binding NS5A protein. We easily synthesized inhibitors con-
taining nonsymmetric or o-uoro substituted biaryl sulfate core
structures using the SuFEx reaction in a “click chemistry” manner.
Furthermore, the monomeric halves of BMK-21014 and BMK-
64,90,91
General procedure for synthesis of capping groups
Na CO (2.6 mmol) was added to aq. NaOH (5 mL of 1 M/H O, 5
mmol) solution of an amino acid (5.00 mmol) and the resulting
solution was cooled with ice-water bath. Methyl chloroformate
5.40 mmol) was added dropwise, the cooling bath was removed
and the reaction mixture was stirred at ambient temperature for
2
3
2
(
3
.25 h. The reaction mixture was washed with ether (3 ꢂ 9 mL),
and the aqueous phase was cooled with ice-water bath and
acidied with concentrated HCl to a pH region of 1–2, and
extracted with CH Cl (3 ꢂ 9 mL). The organic phase was dried
21025, which showed excellent activity in our previous report, were
2
2
easily coupled using SuFEx chemistry to form nonsymmetric
biarylsulfate core-based inhibitor 29. Compounds 14, 15, and 29
had two-digit pM EC50 values against GT-1b and single digit or sub
nanomolar values for GT-2a. In addition, a sulfate core-containing
compound conjugated with biotin 32 was synthesized in
a straightforward manner and showed high activity compared to
that of reference biotinylated compounds. Based on these results,
SuFEx chemistry proved to be highly useful in constructing
symmetric and nonsymmetric sulfate core-containing NS5A
inhibitors, and could potentially be applied to the synthesis of
a wider range of compounds in medicinal chemistry.
(
MgSO ), ltered, and concentrated in vacuo to afford capping
4
groups as a white solid.
1
(
Methoxycarbonyl)-D-valine (Cap-1). Yield (760 mg, 87%); H
NMR (DMSO-d
6
, d ¼ 2.5 ppm, 300 MHz): d 12.54 (s, 1H), 7.32 (d,
1
H), 3.84 (t, 1H), 3.54 (s, 3H), 2.03 (m, 1H), 0.87 (d, 6H).
Methoxycarbonyl)-L-valine (Cap-2). Yield (5.00 g, 86%); H
NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz): d 12.51 (br s, 1H), 7.32
1
(
6
(
d, 1H), 3.84 (t, 1H), 3.54 (s, 3H), 2.03 (m, 1H), 0.88 (d, 6H).
(
R)-2-((Methoxycarbonyl)amino)-2-phenylacetic acid (Cap-3).
1
Yield (1.40 g, 67%); H NMR (DMSO-d , d ¼ 2.5 ppm, 500 MHz):
d 12.79 (br s, 1H), 7.96 (d, 1H), 7.40–7.29 (m, 5H), 5.13 (d, 1H),
.55 (s, 3H).
6
3
Experimental
General methods and material
General procedure for synthesis of aminophenol
1
13
19
The H, C and F NMR-spectra were measured with a Varian/ A stirred mixture of nitro phenol (12.7 mmol) in MeOH (50 mL)
Oxford As-500 (500 MHz), an Agilent 400-MR DD2 Magnetic at room temperature was treated with 10 wt% palladium on
Resonance System (400 MHz), or Bruker/Avance DPX-300 (300 charcoal (100 mg). Hydrogen gas was passed through the
MHz) spectrophotometer. Chemical shis were measured as part mixture for 24 h, and then the palladium on charcoal was
per million (d values) from Tetramethylsilane (TMS) as an removed by ltration through Celite. The ltrate was concen-
internal standard at probe temperature in CD
DMSO-D6 for neutral compounds. Coupling constants are
provided in Hz, with the following spectral pattern (CD OD, d ¼ 3.31 ppm, 400 MHz): d 6.69–6.65 (t, 1H), 6.34–6.25
3
OD or CDCl
3
or trated in vacuo, and was obtained as solid product.
1
3-Amino-2-uorophenol (2a). Yield (1.65 g, 99%); H NMR
3
1
3
designations: s, singlet; d, doublet; t, triplet; q, quartet; quint, (m, 2H); C NMR (CD
quintet; m, multiplet; br, broad; app, apparent. Reactions were 143.90, 141.59, 124.71, 109.49, 107.95; F NMR (CD
conducted, puried, and analyzed according to methods widely MHz): d ꢁ161.96; HRMS (ESI) m/z: anal. calcd for [M + H]
3
OD, d ¼ 49.00 ppm, 100 MHz): d 146.13,
19
3
OD, 376
+
practiced in the eld, while taking necessary precautions in the
exclusion of moisture and/or oxygen where appropriate. Evapo-
C
6
H
7
FNO: 128.0506; found 128.0482.
5-Amino-2-uorophenol (2b). Yield (809 mg, 99%); H NMR
OD, d ¼ 3.31 ppm, 400 MHz): d 6.78–6.73, (t, 1H), 6.34–6.31
1
ration of solvents was performed at reduced pressure using (CD
a rotary evaporator. TLC was performed using silica gel 60F254 (q, 1H), 6.17–6.13 (m, 1H); C NMR (CD OD, d ¼ 49.00 ppm,
3
1
3
3
1
9
coated on an aluminium sheet (E. Merck, Art.5554). High reso- 100 MHz): d 147.94, 145.65, 145.15, 116.60, 107.67, 106.22;
lution mass spectra (HRMS) were recorded on a ThermoFinnigan NMR (CD
OD, 376 MHz): d ꢁ152.91; HRMS (ESI) m/z: anal.
LCQ™ Classic, Quadrupole Ion-Trap Mass Spectrometer. HPLC calcd for [M + H] C
F
3
+
6
7
H FNO: 128.0506; found 128.0508.
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1
3
General procedure for synthesis of amide linked intermediate
3
7.19–7.13 (m, 1H), 5.63 (s, 1H), 4.38 (s, 2H); C NMR (CDCl ,
d ¼ 77.16 ppm, 100 MHz): d 190.18, 155.97, 153.50, 144.29,
A mixture of amino phenol (12.6 mmol), N-Boc-L-proline (3.25 g,
1
9
1
31.19, 122.62, 118.44, 116.35, 30.63; F NMR (CDCl , 376
3
1
5.1 mmol), and EDCI (2.11 g, 16.4 mmol) in CH
was stirred at room temperature for 4 h. The mixture was then
poured in 1 N aq. HCl solution and Extracted with CH Cl . The
2 2
Cl (30 mL)
+
MHz): ꢁ130.70; HRMS (ESI) m/z: anal. calcd for [M + Na] C -
8
6 2
H BrFNaO : 254.9427; found 254.9428.
2
2
organic layer dried over magnesium sulfate, and then was
puried by the column chromatography (n-hexane/ethyl
acetate). The residue was obtained as solid product.
General procedure for synthesis of keto ester derivatives
2-Bromo-acetophenone (0.416 mmol) and N-Boc-L-proline
tert-Butyl
pyrrolidine-1-carboxylate (3a). Yield (3.79 g, 93%); H NMR propylethylamine (0.541 mmol) was added dropwise to the
DMSO-d
, d ¼ 2.5 ppm, 500 MHz): d 9.84 (s, 1H), 9.63 (s, 1H), 7.32– mixture and was stirred at room temperature for 5 h. The
.21 (m, 1H), 6.90–6.89 (d, 1H), 6.71–6.70 (d, 1H), 4.42–4.33 (m, 1H), mixture was then poured in 1 N aq. HCl solution and extracted
.40 (s, 1H), 3.36–3.32 (m, 1H), 2.20–2.12 (m, 1H), 1.87–1.78 (m, 3H), with CH Cl . The organic layer dried over magnesium sulfate,
(S)-2-((2-uoro-3-hydroxyphenyl)carbamoyl)- (0.499 mmol) in acetonitrile (10 mL) put into the ask. Diiso-
1
(
6
7
3
2
2
13
1.40–1.31 (app br s, 9H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 and then was puried by the column chromatography (n-
MHz): d 171.90, 153.34, 145.34, 142.79, 127.08, 123.46, 114.31, hexane/ethyl acetate). The residue was obtained as white solid.
19
113.46, 78.66, 60.03, 46.67, 31.23, 28.02, 23.43; F NMR (DMSO-d6,
1-(tert-Butyl) 2-(2-(4-uoro-3-hydroxyphenyl)-2-oxoethyl) (S)-
1
3
76 MHz): d ꢁ147.91, ꢁ149.01; HRMS (ESI) m/z: anal. calcd for [M + pyrrolidine-1,2-dicarboxylate (6a). Yield (110 mg, 72%); H NMR
+
Na] C H FN NaO : 347.1378; found 347.1379.
(DMSO-d , d ¼ 2.5 ppm, 400 MHz): d 10.43 (s, 1H), 7.55–7.48 (m,
16
21
2
4
6
tert-Butyl
pyrrolidine-1-carboxylate (3b). Yield (748 mg, 59%);
NMR (DMSO-d
(S)-2-((4-uoro-3-hydroxyphenyl)carbamoyl) 2H), 7.34–7.29 (m, 1H), 5.56–5.36 (m, 2H), 4.35–4.39/4.08–4.03
1
H
(2 m, 0.86H + 0.28H), 3.41–3.34 (m, 1H), 3.28–3.16 (m, 1H),
1
3
6
, d ¼ 2.5 ppm, 400 MHz): d 9.83 (s, 2H), 7.38– 2.32–2.09 (m, 1H), 1.92–1.76 (m, 3H), 1.36 (app br s, 9H);
C
7
.36 (d, 1H), 7.05–7.00 (t, 1H), 6.95–6.91 (m, 1H), 4.23–4.13 NMR (DMSO-d , d ¼ 39.52 ppm, 100 MHz): d 191.37, 172.33,
6
(
m, 1H), 3.44–3.38 (m, 1H), 3.35–3.32 (m, 1H), 2.23–2.12 (m, 155.97, 153.00, 145.44, 130.79, 120.36, 116.90, 116.55, 79.06,
1
3
19
1
H), 1.91–1.75 (m, 3H), 1.39–1.28 (app br s, 9H); C NMR 66.41, 58.56, 46.22, 30.47, 29.55, 27.96, 23.88, 23.12; F NMR
(
1
6
DMSO-d , d ¼ 39.52 ppm, 100 MHz): d 171.19, 153.11, (DMSO-d , 376 MHz): d ꢁ127.51; HRMS (ESI) m/z: anal. calcd for
6
6
+
48.05, 146.15, 144.59, 135.46, 115.49, 109.99, 109.09, 78.40, [M + Na] C H FNNaO : 390.1323; found 390.1323.
18 22 6
1
9
0.30, 46.49, 30.92, 30.09, 28.09, 27.89, 23.87, 23.29; F NMR
1-(tert-Butyl) 2-(2-(4-hydroxyphenyl)-2-oxoethyl) (S)-pyrrolidine-
1
(
DMSO-d
6
, 376 MHz): d ꢁ141.97; HRMS (ESI) m/z: anal. calcd 1,2-dicarboxylate (6b). Yield (1.20 g, 74%); H NMR (DMSO-d ,
6
+
for [M + Na] C16
H
21FN
2
NaO
4
: 347.1378; found 347.1379.
d ¼ 2.5 ppm, 400 MHz): d 10.49 (s, 1H), 7.86–7.84 (d, 2H), 5.52–5.32
tert-Butyl (S)-2-((4-hydroxyphenyl)carbamoyl)pyrrolidine-1- (m, 2H), 4.34–4.29 (m, 1H), 3.40–3.37 (m, 1H), 3.32–3.28 (m, 1H),
1
carboxylate (3c). Yield (5.36 g, 87%). H NMR (DMSO-d
6
, d ¼ 2.33–2.22 (m, 1H), 2.18–2.12 (m, 1H), 1.91–1.84 (m, 2H), 1.39–1.36
13
2
6
3
.5 ppm, 400 MHz): d 9.68 (s, 1H), 9.16 (s, 1H), 7.37–7.34 (d, 2H), (app br s, 9H); C NMR (DMSO-d , d ¼ 39.52 ppm, 100 MHz):
6
.70–6.67 (d, 2H), 4.22–4.12 (m, 1H), 3.43–3.37 (m, 1H), 3.35– d 190.47, 172.31, 162.68, 152.95, 130.41, 125.35, 115.44, 78.94,
.29 (m, 1H), 2.19–2.12 (m, 1H), 1.91–1.74 (m, 3H), 1.39–1.28 66.11, 58.55, 46.17, 30.43, 27.93, 23.05; HRMS (ESI) m/z: anal. calcd
1
3
+
(app br s, 9H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): for [M + Na] C H NNaO : 372.1418; found 372.1419.
18
23
6
d 170.76, 153.28, 130.70, 121.02, 115.02, 78.38, 60.27, 46.56,
3
1-(tert-Butyl) 2-(2-(3-hydroxyphenyl)-2-oxoethyl) (S)-pyrrolidine-
1
1.03, 30.23, 27.97, 23.96, 23.39; HRMS (ESI) m/z: anal. calcd for 1,2-dicarboxylate (6c). Yield (1.47 g, 91%); H NMR (DMSO-d ,
6
+
[M + Na] C16
H
22
N
2
NaO
4
: 329.1472; found 329.1472.
d ¼ 2.5 ppm, 400 MHz): d 9.88 (s, 1H), 7.45–7.43 (d, 1H), 7.37–7.33
tert-Butyl (S)-2-((3-hydroxyphenyl)carbamoyl)pyrrolidine-1- (m, 2H), 7.10–7.08 (d, 1H), 5.58–5.38 (m, 2H), 4.37–4.31 (m, 1H),
1
carboxylate (3d). Yield (5.41 g, 96%); H NMR (DMSO-d , d ¼ 3.41–3.31 (m, 2H), 2.34–2.10 (m, 2H), 1.92–1.82 (m, 2H), 1.40–1.37
6
1
3
2
7
1
1
3
1
.5 ppm, 400 MHz): d 9.83 (s, 1H), 9.36 (s, 1H), 7.18 (s, 1H), 7.08– (app br s, 9H); C NMR (DMSO-d , d ¼ 39.52 ppm, 100 MHz):
6
.04 (t, 1H), 6.96–6.94 (d, 1H), 6.45–6.43 (d, 1H), 4.24–4.15 (m, d 192.42, 172.30, 157.84, 153.00, 135.18, 130.04, 121.13, 118.74,
H), 3.37 (m, 1H), 3.33–3.32 (m, 1H), 2.17–2.08 (m, 1H), 1.86– 113.90, 79.00, 66.55, 58.59, 46.21, 30.48, 29.56, 28.11, 27.94, 23.87,
1
3
+
.75 (m, 3H), 1.39–1.27 (app br s, 9H); C NMR (DMSO-d
6
, d ¼ 23.12; HRMS (ESI) m/z: anal. calcd for [M + Na] C H NNaO :
18
23
6
9.52 ppm, 100 MHz): d 171.40, 157.63, 153.23, 140.15, 129.28, 372.1418; found 372.1417.
10.38, 110.11, 106.55, 78.49, 60.40, 46.60, 31.06, 27.98, 23.44;
+
HRMS (ESI) m/z: anal. calcd for [M + Na]
29.1472; found 329.1474.
-Bromo-1-(4-uoro-3-hydroxyphenyl)ethan-1-one (5a).
16 22 2 4
C H N NaO :
General procedure for synthesis of imidazole linked
intermediate
3
2
A
A mixture of keto ester (0.554 mmol) and ammonium acetate
mixture of 4-uoro-3-hydroxyacetophenone (100 mg, 0.649
mmol), copper(II) bromide (174 mg, 0.779 mmol) in ethyl
acetate (5 mL)/chloroform (5 mL) was heated and reuxed for
(
8.30 mmol) were suspended in toluene (10 mL). The reaction
ꢀ
mixture was heated to 90 C and stirred for 20 h. The mixture
2
was then poured in H O and extracted with ethyl acetate. The
8
h. The mixture was then ltered to remove the copper(I)
organic layer dried over magnesium sulfate and evaporated in
vacuo. Aer evaporation, the residue was puried by the column
chromatography (n-hexane/ethyl acetate). Then product was
obtained as yellow solid.
bromide. The ltrate was concentrated in vacuo, and was ob-
tained as yellow solid (123 mg, 81%); H NMR (CDCl
7
1
3
, d ¼
.26 ppm, 400 MHz): d 7.67–7.64 (m, 1H), 7.56–7.51 (m, 1H),
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tert-Butyl (S)-2-(5-(4-uoro-3-hydroxyphenyl)-1H-imidazol-2- 141.01, 131.00, 119.33, 115.30, 111.07, 78.61, 60.48, 46.57,
1
19
yl)pyrrolidine-1-carboxylate (7a). Yield (101 mg, 52%); H NMR 30.91, 30.11, 27.87, 23.98, 23.39; F NMR (CD
DMSO-d
OD, 376 MHz):
3
+
(
6
, d ¼ 2.5 ppm, 400 MHz): d 12.06/11.83–11.76 (s/d, 38.49; HRMS (ESI) m/z: anal. calcd for [M + Na] C16
.16H + 0.76H), 9.70 (s, 1H), 7.35 (s, 2H), 7.10–7.01 (m, 2H), NaO S: 411.0997; found 411.0998.
.80–4.73 (d, 1H), 3.52 (s, 1H), 2.32–2.15 (m, 1H), 1.97–1.76 (m,
tert-Butyl (S)-2-((2-uoro-3-((uorosulfonyl)oxy)phenyl)-
H21FN -
2
0
4
4
3
1
2
6
1
3
H), 1.39–1.15 (app br s, 9H); C NMR (DMSO-d , d ¼ carbamoyl)pyrrolidine-1-carboxylate (8c). Yield (364 mg,
6
1
9.52 ppm, 100 MHz): d 153.39, 150.76, 144.58, 138.79, 131.95, 83%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 10.18 (s,
15.97, 115.18, 113.60, 111.31, 78.60, 58.75, 55.20, 46.28, 33.32, 1H), 8.04–7.95 (d, 1H), 7.54 (s, 1H), 7.39–7.35 (t, 1H), 4.45–
1
9
7.88, 23.88, 23.14; F NMR (DMSO-d
140.30; HRMS (ESI) m/z: anal. calcd for [M + H] C18
48.1718; found 348.1719.
tert-Butyl
(S)-2-(5-(4-hydroxyphenyl)-1H-imidazol-2-yl)- 146.52, 144.00, 136.75, 128.31, 124.97, 118.45, 78.61, 59.87,
pyrrolidine-1-carboxylate (7b). Yield (887 mg, 47%);
6
, 376 MHz): d ꢁ138.76, 4.36 (m, 1H), 3.42 (s, 1H), 3.37–3.33 (m, 1H), 2.23–2.16 (m,
+
13
ꢁ
H
23FN
3
O
3
:
1H), 1.92–1.80 (s, 3H), 1.40–1.31 (app s, 9H); C NMR
(DMSO-d
, d ¼ 39.52 ppm, 100 MHz): d 172.20, 153.08,
3
6
1
19
H
6
46.55, 31.04, 29.94, 27.86, 23.94, 23.30; F NMR (DMSO-d ,
NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz): d 11.76 (br s, 1H), 376 MHz): d 39.69, ꢁ139.30, ꢁ140.16; HRMS (ESI) m/z: anal.
6
+
9
4
1
.48 (br s, 1H), 7.53 (s, 2H), 7.20 (s, 1H), 6.77–6.75 (d, 2H), calcd for [M + Na]
.83–4.76 (d, 1H), 3.54–3.35 (d, 2H), 2.20–1.81 (m, 4H), 1.39– 429.0902.
.17 (app br s, 9H); C NMR (DMSO-d
MHz): d 155.93, 153.82, 153.47, 150.09, 125.54, 115.30, 78.23, carbamoyl)pyrrolidine-1-carboxylate (8d). Yield (260 mg,
5.26, 46.34, 33.41, 31.88, 27.93, 23.85, 23.16; HRMS (ESI) m/ 90%); H NMR (DMSO-d
16 20 2 2 6
C H F N NaO S: 429.0902; found
1
3
6
, d ¼ 39.52 ppm, 100
tert-Butyl (S)-2-((4-uoro-3-((uorosulfonyl)oxy)phenyl)-
1
5
6
, d ¼ 2.5 ppm, 400 MHz): d 10.42 (s,
+
z: anal. calcd for [M + Na] C18
H
23
N
3
NaO
3
: 352.1632; found 1H), 8.14–8.12 (d, 1H), 7.64–7.56 (m, 2H), 4.24–4.15 (m, 1H),
3.41 (s, 1H), 3.31 (s, 1H), 2.22–2.17 (m, 1H), 1.93–1.78 (m,
(S)-2-(5-(3-hydroxyphenyl)-1H-imidazol-2-yl)- 3H), 1.39–1.25 (app s, 9H);
3
52.1634.
tert-Butyl
1
3
C NMR (DMSO-d
6
, d ¼
1
pyrrolidine-1-carboxylate (7c). Yield (456 mg, 45%);
H
39.52 ppm, 100 MHz): d 172.14, 153.63, 153.05, 149.36,
NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz): d 11.88 (br s, 1H), 146.90, 136.55, 135.74, 135.60, 120.93, 118.14, 113.36, 78.65,
6
1
9
9
1
.41 (br s, 1H), 7.34–7.11 (m, 4H), 6.60 (s, 1H), 4.85–4.77 (d, 60.46, 46.59, 30.92, 30.11, 27.90, 24.00, 23.40; F NMR
H), 3.55 (s, 1H), 3.35 (s, 1H), 2.22–2.14 (m, 1H), 1.99–1.82 (DMSO-d
, 376 MHz): d 39.41, ꢁ136.14; HRMS (ESI) m/z: anal.
6
1
3
+
(
m, 3H), 1.39–1.16 (app br s, 9H); C NMR (DMSO-d
6
, d ¼ calcd for [M + Na]
C H F N NaO S: 429.0902; found
16 20 2 2 6
3
1
2
9.52 ppm, 100 MHz): d 157.66, 153.51, 129.43, 115.25, 429.0900.
13.14, 111.25, 78.32, 55.33, 48.72, 46.42, 33.49, 31.93, 27.96,
3.93, 23.25; HRMS (ESI) m/z: anal. calcd for [M + Na]
tert-Butyl (S)-2-(5-(3-((uorosulfonyl)oxy)phenyl)-1H-imidazol-
2-yl)pyrrolidine-1-carboxylate (8e). Yield (49.8 mg, 80%); H NMR
+
1
C18H23N NaO : 352.1632; found 352.1634.
(DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 8.31–8.25 (d, 2H), 8.14–8.10
3
3
(t, 1H), 7.74–7.70 (t, 1H), 7.63–7.61 (d, 1H), 5.17–5.13 (t, 1H), 3.66–
3
3
.61 (m, 1H), 3.44–3.37 (m, 1H), 2.43–2.34 (m, 1H), 2.11–1.90 (m,
General procedure for synthesis of fosylated intermediate
13
H), 1.38–1.16 (app s, 9H); C NMR (DMSO-d , d ¼ 39.52 ppm,
6
A mixture of phenol derivative (0.800 mmol) and N,N-diisopro- 100 MHz): d 153.82, 152.56, 150.85, 150.11, 131.82, 130.54,
pylethylamine (1.20 mmol) in CH
sulfuryl uoride at room temperature for 5 h. The mixture was 23.45, 17.96, 16.69, 12.20. F NMR (DMSO-d , 376 MHz): 38.53;
3 5
then poured in 1 N aq. HCl solution and extracted with CH Cl . HRMS (ESI) m/z: anal. calcd for [M + H] C18H23FN O S: 412.1337;
2 2
Cl
(5 mL) was stirred under 125.80, 121.20, 117.86, 116.57, 79.34, 53.31, 46.54, 32.94, 27.73,
1
9
6
+
2
2
The organic layer dried over magnesium sulfate, ltered, and found 412.1339.
concentrated in vacuo. Without any purication, the residue was
tert-Butyl
(S)-2-(5-(4-uoro-3-((uorosulfonyl)oxy)phenyl)-
obtained as an oil.
1H-imidazol-2-yl)pyrrolidine-1-carboxylate (8f). Yield (59.6 mg,
1
tert-Butyl (S)-2-((4-((uorosulfonyl)oxy)phenyl)carbamoyl)- 92%); H NMR (DMSO-d
, d ¼ 2.5 ppm, 400 MHz): d 12.07–12.00
pyrrolidine-1-carboxylate (8a). Yield (278 mg, 90%); H NMR (d, 1H), 8.03–8.01 (d, 1H), 7.91–7.87 (m, 1H), 7.66–7.64 (m, 1H),
DMSO-d
6
1
(
2
1
6
, d ¼ 2.5 ppm, 400 MHz): d 10.31 (s, 1H), 7.82–7.80 (d, 7.58–7.54 (t, 1H), 4.82–4.77 (d, 1H), 3.53 (s, 1H), 3.34–3.29 (m,
13
H), 7.53–7.51 (d, 2H), 4.28–4.21 (m, 1H), 3.42 (s, 2H), 2.21 (s, 1H), 2.22 (s, 1H), 1.96–1.85 (m, 3H), 1.39–1.15 (app s, 9H);
C
1
3
H), 1.89–1.79 (m, 3H), 1.38–1.27 (app s, 9H); C NMR (DMSO- NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): d 153.36, 151.81,
d , d ¼ 39.52 ppm, 100 MHz): d 171.97, 153.13, 144.74, 139.67, 151.06, 149.33, 136.79, 133.63, 125.85, 118.19, 113.46, 78.26,
6
1
9
1
2
21.53, 120.75, 78.57, 60.46, 46.58, 31.01, 30.16, 27.92, 23.99, 55.23, 46.31, 33.29, 31.84, 27.86, 23.83, 23.10; F NMR (DMSO-
1
9
3.39; F NMR (CD
3
OD, 376 MHz): 31.23; HRMS (ESI) m/z: anal.
d
6
, 376 MHz): d 39.85, ꢁ134.19; HRMS (ESI) m/z: anal. calcd for
+
+
calcd for [M + Na] C16
H21FN
2
NaO
6
S: 411.0997; found 411.1001. [M + H] C18
H F N O S: 430.1243; found 430.1243.
22 2 3 5
tert-Butyl (S)-2-((3-((uorosulfonyl)oxy)phenyl)carbamoyl)-
tert-Butyl (S)-2-(5-(4-((uorosulfonyl)oxy)phenyl)-1H-imidazol-
1
1
pyrrolidine-1-carboxylate (8b). Yield (1.66 g, 87%); H NMR 2-yl)pyrrolidine-1-carboxylate (8g). Yield (100 mg, 80%); H NMR
DMSO-d
(
6
, d ¼ 2.5 ppm, 400 MHz): d 10.43–10.41 (d, 1H), 7.98 (s, (DMSO-d
H), 7.62–7.59 (t, 1H), 7.55–7.50 (m, 1H), 7.26–7.24 (d, 1H), 2H), 7.68–7.64 (d, 1H), 7.57–7.56 (d, 2H), 4.88–4.79 (m, 1H), 3.56–
.27–4.17 (m, 1H), 3.46–3.40 (m, 1H), 3.37–3.31 (m, 1H), 2.24– 3.53 (t, 1H), 3.39–3.35 (m, 1H), 2.28–2.17 (m, 1H), 1.99–1.85 (m,
6
, d ¼ 2.5 ppm, 500 MHz): d 12.50 (s, 1H), 7.93–7.91 (d,
1
4
2
1
3
13
.16 (m, 1H), 1.94–1.77 (m, 3H), 1.39–1.25 (app s, 9H); C NMR 3H), 1.39–1.15 (app s, 9H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm,
(
DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): d 172.23, 153.06, 149.63, 126 MHz): d 153.22, 151.07, 147.88, 126.11, 121.25, 78.35, 54.90,
31812 | RSC Adv., 2018, 8, 31803–31821
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1
9
4
6.32, 33.23, 31.81, 28.15, 27.83, 23.82, 23.12; F NMR (DMSO- ꢁ4.52; HRMS (ESI) m/z: anal. calcd for [M + H]+ C24
38 3 3
H N O Si:
+
d6, 376 MHz): 38.27; HRMS (ESI) m/z: anal. calcd for [M + H]
444.2677; found 444.2679.
C H FN O S: 412.1337; found 412.1340.
tert-Butyl (S)-2-(5-(3-((tert-butyldimethylsilyl)oxy)-4-uoro-
1
8
23
3 5
phenyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (9e). Yield
1
(
638 mg, 69%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz):
General procedure for silyl protection
d 12.12–11.81 (m, 1H), 7.43 (s, 1H), 7.35–7.31 (m, 2H), 7.23–7.10
A mixture of phenol derivative (2.29 mmol), tert-butyldime- (m, 1H), 4.83–4.76 (m, 1H), 3.53 (s, 1H), 2.21–2.10 (m, 1H), 1.98–
thylsilyl chloride (3.44 mmol), imidazole (6.87 mmol) in THF 1.84 (m, 3H), 1.39 (s, 4H), 1.15 (s, 9H), 0.97 (s, 9H), 0.18 (s, 6H);
1
3
(
10 mL) was stirred at room temperature for 4 h. The mixture
was then poured in H O and extracted with CH Cl
. The organic 142.40, 138.33, 132.30, 118.02, 117.76, 116.26, 116.07, 111.83,
layer dried over magnesium sulfate, and then was puried by 78.15, 55.30, 46.25, 33.25, 31.81, 27.84, 25.42, 23.73, 23.06,
C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): d 153.10, 150.70,
2
2
2
1
9
the column chromatography (n-hexane/ethyl acetate). The 18.01, ꢁ4.82; F NMR (DMSO-d , 376 MHz): d ꢁ134.91,
6
+
residue was obtained as sticky oil.
ꢁ136.21; HRMS (ESI) m/z: anal. calcd for [M + H] C24
Si: 462.2583; found 462.2585.
tert-Butyl (S)-2-((4-((tert-butyldimethylsilyl)oxy)phenyl)-
3
H37FN -
tert-Butyl (S)-2-((3-((tert-butyldimethylsilyl)oxy)phenyl)-
O
3
carbamoyl)pyrrolidine-1-carboxylate (9a). Yield (759 mg,
1
9
1
4
2
9
5%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 9.93 (s, carbamoyl)pyrrolidine-1-carboxylate (9f). Yield (63.0 mg,
1
H), 7.35–7.24 (d, 1H), 7.16–7.09 (m, 2H), 6.52–6.51 (d, 1H), 92%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 500 MHz): d 9.83–9.81
.23–4.13 (m, 1H), 3.44–3.38 (m, 1H), 3.32–3.30 (m, 1H), 2.21– (d, 1H), 7.47–7.44 (m, 2H), 6.79–6.78 (d, 2H), 4.22–4.13 (m,
.12 (m, 1H), 1.90–1.75 (m, 3H), 1.39–1.26 (app s, 9H), 0.93 (s, 1H), 3.43–3.40 (m, 1H), 3.32–3.30 (m, 1H), 2.20–2.13 (m, 1H),
1
3
H), 0.17 (s, 6H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 1.91–1.75 (m, 3H), 1.39–1.27 (app s, 9H), 0.94 (s, 9H), 0.16 (s,
13
MHz): d 171.50, 155.27, 153.11, 140.22, 129.48, 114.63, 6H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz): d 170.98,
1
1
H
12.27, 110.84, 78.44, 60.40, 46.56, 30.95, 27.89, 25.55, 23.43, 153.16, 150.73, 132.92, 120.77, 119.73, 78.40, 60.29, 46.55,
+
7.95, ꢁ4.53; HRMS (ESI) m/z: anal. calcd for [M + Na] C22- 31.02, 27.95, 25.58, 23.96, 23.37, 17.93; HRMS (ESI) m/z: anal.
+
36
N
2
NaO
4
Si: 443.2337; found 443.2337.
calcd for [M + Na]
22 36 2 4
C H N NaO Si: 443.2337; found
tert-Butyl (S)-2-((3-((tert-butyldimethylsilyl)oxy)-4-uoro- 443.2340.
phenyl)carbamoyl)pyrrolidine-1-carboxylate (9b). Yield
tert-Butyl
, d ¼ 2.5 ppm, 400 MHz): 1H-imidazol-2-yl)pyrrolidine-1-carboxylate (9g). Yield (545 mg,
(S)-2-(5-(3-((tert-butyldimethylsilyl)oxy)phenyl)-
1
(
318 mg, 73%); H NMR (DMSO-d
6
1
d 9.95 (s, 1H), 7.51–7.38 (q, 1H), 7.09 (s, 2H), 4.22–4.12 (m, 81%); H NMR (DMSO-d
, d ¼ 2.5 ppm, 500 MHz): d 11.88–11.82
H), 3.42–3.39 (m, 1H), 3.35–3.30 (m, 1H), 2.21–2.12 (m, 1H), (app s, 1H), 7.44–7.18 (m, 4H), 6.63 (s, 1H), 4.83–4.77 (d, 1H), 3.54
.87–1.75 (m, 3H), 1.39–1.25 (app br s, 9H), 0.95 (s, 9H), 0.17 (s, 1H), 3.36 (s, 1H), 2.22–2.14 (d, 1H), 2.07 (s, 1H), 1.97–1.83 (m,
6
1
1
(
1
3
13
s, 6H); C NMR (DMSO-d , d ¼ 39.52 ppm, 100 MHz): 2H), 1.39–1.16 (app s, 9H), 0.96 (s, 9H), 0.19 (s, 6H); C NMR
6
d 171.38, 153.08, 150.51, 148.14, 142.08, 135.72, 115.92, (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz): d 155.26, 153.39, 139.10,
1
ꢁ
13.02, 78.41, 60.40, 46.55, 30.94, 27.85, 25.34, 23.42, 17.95, 129.35, 117.52, 115.55, 112.01, 78.10, 55.27, 46.26, 33.27, 31.79,
1
9
4.89; F NMR (DMSO-d
m/z: anal. calcd for [M + Na]
6
, 376 MHz): d ꢁ138.76; HRMS (ESI) 30.63, 27.84, 25.57, 23.74, 23.08, 17.93; HRMS (ESI) m/z: anal.
+ +
22 2 4 38 3 3
C H35FN NaO
Si: 461.2242; calcd for [M + H] C24H N O Si: 444.2677; found 444.2681.
found 461.2242.
tert-Butyl (S)-2-((3-((tert-butyldimethylsilyl)oxy)-2-uoro-
phenyl)carbamoyl)pyrrolidine-1-carboxylate (9c). Yield
General procedure for SuFEx reaction
1
(
294 mg, 73%); H NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz): A mixture of silylated monomer (0.408 mmol), fosylated
6
d 9.71 (s, 1H), 7.54–7.38 (m, 1H), 7.02–6.98 (t, 1H), 6.78–6.74 monomer (0.272 mmol) and DBU (0.05 mmol) in DMF (10 mL)
ꢀ
(
t, 1H), 4.40–4.33 (m, 1H), 3.43–3.38 (m, 1H), 2.24–2.08 (m, was stirred at 50 C for 12 h. The mixture was then poured in 1 N
1
0
H), 1.91–1.77 (m, 3H), 1.40–1.30 (app br s, 9H), 0.96 (s, 9H), aq. HCl solution and extracted with CH
2
Cl
2
. The organic layer
1
3
.17 (s, 6H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): dried over magnesium sulfate, and then was puried by the
d 171.82, 153.14, 142.85, 127.23, 123.61, 123.56, 117.48, column chromatography (n-hexane/ethyl acetate). The residue
1
ꢁ
16.90, 78.51, 59.85, 46.54, 31.05, 27.88, 25.38, 23.32, 18.00, was obtained as solid.
1
9
0
4.84; F NMR (DMSO-d , 376 MHz): d ꢁ143.28, ꢁ144.70;
Di-tert-butyl 2,2 -((((sulfonylbis(oxy))bis(4,1-phenylene))-bis(aza-
6
+
0
HRMS (ESI) m/z: anal. calcd for [M + Na] C
H
FN NaO Si: nediyl))bis(carbonyl))(2S,2 S)-bis(pyrrolidine-1-carboxylate)
(10a).
2
2
35
2
4
1
4
61.2242; found 461.2242.
tert-Butyl
Yield (76.1 mg, 90%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz):
(S)-2-(5-(4-((tert-butyldimethylsilyl)oxy)phenyl)- d 10.28 (s, 1H), 7.72–7.70 (d, 4H), 7.39–7.37 (d, 4H), 4.23–4.16 (m,
1
6
1
H-imidazol-2-yl)pyrrolidine-1-carboxylate (9d). Yield (269 mg, 2H), 3.39–3.32 (m, 4H), 2.21–2.16 (t, 2H), 1.86–1.78 (m, 6H), 1.38–
1
13
0%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 500 MHz): d 11.77 (s, 1.25 (app br s, 18H); C NMR (DMSO-d , d¼ 39.52 ppm, 100
6
H), 7.60–7.59 (d, 2H), 7.30 (s, 1H), 6.81–6.80 (d, 2H), 4.83–4.75 MHz): d 172.2, 153.5, 145.2, 138.8, 121.9, 120.9, 79.0, 63.0, 60.7,
+
(m, 1H), 3.56–3.50 (m, 1H), 3.37–3.34 (m, 1H), 2.23–2.12 (m, 46.8, 31.2, 28.2, 23.7; HRMS (ESI) m/z: anal. calcd for [M + Na]
1
0
H), 2.03–1.83 (m, 3H), 1.39–1.16 (app br s, 9H), 0.95 (s, 9H), C H N NaO S: 697.2514; found 697.2515.
32 42 4 10
1
3
0
.18 (s, 6H); C NMR (DMSO-d , d ¼ 39.52 ppm, 126 MHz):
Di-tert-butyl 2,2 -((((sulfonylbis(oxy))bis(3,1-phenylene))-bis(aza-
6
0
d 153.70, 153.37, 139.36, 128.82, 125.44, 119.76, 78.12, 55.20, nediyl))bis(carbonyl))(2S,2 S)-bis(pyrrolidine-1-carboxylate)
4
(10b).
1
6.25, 33.30, 31.76, 27.88, 25.57, 23.79, 23.05, 17.94, ꢁ3.21, Yield (1.84 g, 69%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz):
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1
d 10.32 (s, 1H), 8.21–7.77 (m, 3H), 7.59–7.57 (d, 2H), 7.47 (s, 1H), Yield (32.2 mg, 69%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz):
7
.40–7.22 (m, 1H), 7.12–7.08 (t, 1H), 4.24–4.18 (d, 2H), 3.41 (br s, 4H), d 11.99 (s, 2H), 7.88–7.85 (d, 3H), 7.53 (s, 2H), 7.39–7.38 (d, 3H),
.20 (s, 2H), 1.88–1.80 (m, 6H), 1.39–1.24 (app br s, 18H); C NMR 7.30–7.27 (d, 1H), 6.91–6.87 (d, 1H), 4.84–4.77 (d, 2H), 4.03–3.98 (t,
13
2
(
1
DMSO-d , d¼ 39.52 ppm, 100 MHz): d 172.0, 153.1, 149.9, 140.8, 1H), 3.53 (s, 2H), 3.29–3.25 (m, 1H), 2.22–2.14 (m, 2H), 2.05–1.93
6
13
30.6, 118.4, 115.3, 111.4, 78.6, 60.5, 46.6, 30.9, 27.9, 23.4; HRMS (m, 2H), 1.85–1.71 (m, 4H), 1.39–1.15 (app br s, 18H); C NMR
+
(ESI) m/z: anal. calcd for [M + Na] C32
H
42
N
4
NaO10S: 697.2514; found (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): d 174.6, 153.4, 147.9, 125.7,
6
97.2514.
tert-Butyl
121.1, 78.3, 59.6, 55.2, 46.4, 33.3, 31.0, 30.0, 28.0, 23.9, 23.2; HRMS
+
45 6 8
(S)-2-((3-(((4-((S)-1-(tert-butoxycarbonyl) (ESI) m/z: anal. calcd for [M + Na] C36H N O S: 721.3014; found
pyrrolidine-2-carboxamido)phenoxy)-sulfonyl)oxy)phenyl)ca- 721.3014.
0
rbamoyl)pyrrolidine-1-carboxylate (10c). Yield (170 mg,
Di-tert-butyl 2,2 -(((sulfonylbis(oxy))bis(3,1-phenylene))bis-(1H-
1
0
9
1
3%); H NMR (DMSO-d , d ¼ 2.5 ppm, 500 MHz): d 10.34– imidazole-5,2-diyl))(2S,2 S)-bis(pyrrolidine-1-carboxylate)
(10h).
6
1
0.23 (d, 2H), 7.96–7.88 (m, 1H), 7.75–7.73 (d, 2H), 7.58–7.55 Yield (54.4 mg, 67%); H NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz):
6
(
t, 1H), 7.49–7.37 (m, 3H), 7.11–7.10 (d, 1H), 4.26–4.16 (m, d 12.04–11.97 (br d, 2H), 8.21–7.76 (t, 4H), 7.64–7.61 (d, 1H), 7.52–
2
1
H), 3.45–3.40 (m, 2H), 3.36–3.31 (m, 2H), 2.23–2.15 (m, 2H), 7.44 (q, 2H), 7.37–7.20 (m, 2H), 6.93–6.74 (q, 1H), 4.83–4.77 (d, 2H),
1
3
.93–1.77 (m, 6H), 1.39–1.24 (app br s, 18H); C NMR 3.53 (s, 2H), 3.39–3.36 (m, 2H), 2.22–2.15 (m, 2H), 1.98–1.79 (m,
1
3
(
DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz): d 171.80, 153.08, 6H), 1.38–1.14 (app br s, 18H); C NMR (DMSO-d , d ¼ 39.52 ppm,
6
1
1
2
49.91, 144.95, 144.91, 140.75, 138.75, 130.59, 121.56, 120.58, 100 MHz): d 153.4, 150.4, 138.1, 131.3, 130.5, 123.5, 119.2, 117.8,
18.31, 115.45, 111.39, 78.54, 60.41, 46.58, 30.95, 30.14, 116.4, 116.2, 78.2, 55.2, 46.3, 33.3, 27.9, 23.1; HRMS (ESI) m/z: anal.
+
+
7.90, 23.99, 23.40; HRMS (ESI) m/z: anal. calcd for [M + Na]
calcd for [M + Na] C H N O S: 721.3014; found 721.3014.
36 45 6 8
C32H42N NaO S: 697.2514; found 697.2515.
tert-Butyl (S)-2-(5-(3-(((4-(2-((S)-1-(tert-butoxycarbonyl)-pyrrolidin-
4
10
0
Di-tert-butyl 2,2 -((((sulfonylbis(oxy))bis(4-uoro-3,1-phenyl- 2-yl)-1H-imidazol-5-yl)phenoxy)sulfonyl)oxy)-phenyl)-1H-imidazol-2-
0
1
ene))bis(azanediyl))bis(carbonyl))(2S,2 S)-bis(pyrrolidine-1-carb-
yl)pyrrolidine-1-carboxylate (10i). Yield (721 mg, 52%); H NMR
, d ¼ 2.5 ppm, 400 MHz): d 8.25–8.04 (m, 6H), 7.66–7.51 (d,
1
oxylate) (10d). Yield (217 mg, 78%); H NMR (DMSO-d
6
, d ¼ (DMSO-d
6
2
2
3
.5 ppm, 400 MHz): d 10.33 (s, 2H), 8.11–8.08 (t, 2H), 7.60–7.58 (d, 4H), 5.12 (s, 2H), 3.63 (s, 2H), 3.41 (s, 2H), 2.38 (s, 2H), 2.07–1.91 (m,
H), 7.53–7.48 (t, 2H), 4.23–4.13 (m, 2H), 3.42–3.40 (m, 2H), 3.35– 6H), 1.38–1.15 (app br s, 18H); C NMR (DMSO-d
13
6
, v ¼ 39.52 ppm,
.32 (m, 2H), 2.21–2.18 (m, 2H), 1.91–1.78 (m, 6H), 1.38–1.23 (app 100 MHz): d 153.71, 152.45, 150.67, 150.56, 150.22, 149.56, 131.39,
1
3
br s, 18H); C NMR (DMSO-d , d ¼ 39.52 ppm, 100 MHz): 127.37, 124.78, 122.07, 117.89, 116.19, 115.71, 79.19, 52.97, 46.45,
6
d 171.92, 153.60, 153.04, 149.87, 147.41, 136.27, 119.94, 117.90, 32.89, 31.98, 27.70, 23.92, 23.38; HRMS (ESI) m/z: anal. calcd for [M +
1
9
+
113.55, 78.60, 60.45, 46.57, 30.92, 30.11, 27.86, 23.99, 23.40;
F
45 6 8
H] C36H N O S: 721.3014; found 721.3016.
0
NMR (DMSO-d
calcd for [M + Na] C32
6
, 376 MHz): d ꢁ135.44; HRMS (ESI) m/z: anal.
Di-tert-butyl 2,2 -(((sulfonylbis(oxy))bis(4-uoro-3,1-phe-
+
0
H
40
F
2
N
4
NaO10S: 733.2325; found 733.2324. nylene))bis(1H-imidazole-5,2-diyl))(2S,2 S)-bis(pyrrolidine-1-
0
1
Di-tert-butyl 2,2 -((((sulfonylbis(oxy))bis(2-uoro-3,1-phenyl- carboxylate) (10j). Yield (305 mg, 63%); H NMR (DMSO-d
ene))bis(azanediyl))bis(carbonyl))(2S,2 S)-bis(pyrrolidine-1-carb-
6
, v
0
¼ 2.5 ppm, 400 MHz): d 12.01 (s, 2H), 7.92–7.91 (d, 2H), 7.82
1
oxylate) (10e). Yield (231 mg, 98%); H NMR (DMSO-d , d ¼ (s, 2H), 7.58 (s, 2H), 7.52–7.47 (t, 2H), 4.82–4.76 (d, 2H), 3.52
6
2
1
3
1
.5 ppm, 400 MHz): d 10.12 (s, 2H), 8.00–7.97 (t, 1H), 7.90–7.87 (t, (s, 2H), 3.37–3.17 (m, 2H), 2.22–2.13 (m, 2H), 1.95–1.84 (t,
1
3
H), 7.43–7.40 (t, 2H), 7.35–7.31 (t, 2H), 4.43–4.34 (m, 2H), 3.44– 2H), 1.37–1.14 (app br s, 18H); C NMR (DMSO-d , d ¼
6
.39 (m, 2H), 3.30 (m, 2H), 2.32–2.11 (m, 2H), 1.91–1.76 (m, 6H), 39.52 ppm, 100 MHz): d 153.35, 152.22, 150.24, 136.98,
1
3
.40–1.29 (br s, 18H); C NMR (DMSO-d
6
, v ¼ 39.52 ppm, 100 136.87, 133.21, 125.00, 118.39, 117.68, 113.11, 78.24, 55.22,
19
6
MHz): d 172.17, 153.07, 137.06, 128.08, 124.67, 124.04, 118.49, 46.29, 33.23, 31.78, 27.83, 23.77, 23.07; F NMR (DMSO-d ,
1
9
+
7
(
8.61, 59.83, 46.55, 31.04, 29.98, 27.90, 23.95, 23.31; F NMR 376 MHz): d ꢁ133.22; HRMS (ESI) m/z: anal. calcd for [M + H]
DMSO-d S: 757.2826; found 757.2828.
, 376 MHz): d ꢁ138.71, ꢁ139.67; HRMS (ESI) m/z: anal.
calcd for [M + Na] C H F N NaO S: 733.2325; found 733.2321.
6
36 43 2 6 8
C H F N O
+
3
2
40
2
4
10
tert-Butyl (S)-2-((3-(((5-((S)-1-(tert-butoxycarbonyl)pyrrole-dine-
2
-carboxamido)-2-uorophenoxy)sulfonyl)oxy)-2-uoro-phenyl)car-
1
General procedure for synthesis of inhibitors
bamoyl)pyrrolidine-1-carboxylate (10f). Yield (112 mg, 58%); H
NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 10.33 (s, 1H), 10.11 (s, A mixture of precursor (0.151 mmol) in CF CO H (3 mL)/CH Cl
3
2
2
2
1H), 8.12–7.90 (m, 2H), 7.61 (s, 1H), 7.54–7.49 (t, 1H), 7.40–7.31 (m, (3 mL) was stirred at room temperature for 30 m. The volatile
2H), 4.43–4.36 (m, 1H), 4.23–4.13 (m, 1H), 3.40 (s, 2H), 3.30 (s, 2H), component was removed in vacuo, 1-ethyl-3-(3-
2.21 (s, 2H), 1.88–1.79 (m, 6H), 1.39–1.23 (app br s, 18H); C NMR dimethylaminopropyl)carbodiimide (0.392 mmol), hydroxy-
13
(
1
DMSO-d , d ¼ 39.52 ppm, 100 MHz): d 171.95, 153.61, 153.04, benzotriazole hydrate (0.392 mmol), capping group (0.362
6
49.87, 147.41, 137.09, 136.21, 128.05, 124.70, 124.01, 119.90, mmol) were added in batches over 4 min to a solution of N,N-
1
17.92, 113.55, 78.61, 60.45, 46.56, 30.92, 30.12, 27.88, 23.99, 23.39; diisopropylethylamine (0.754 mmol) in CH Cl (10 mL) and the
2
2
19
F NMR (DMSO-d
6
, 376 MHz): d ꢁ135.48, ꢁ138.91, ꢁ139.77; reaction mixture stirred at room temperature for overnight. The
+
HRMS (ESI) m/z: anal. calcd for [M + Na] C32
H
40
F
2
N
4
NaO10S: mixture was then poured in 1 N aq. HCl solution and extracted
with CH Cl . The organic layer dried over magnesium sulfate,
733.2325; found 733.2325.
2
2
0
Di-tert-butyl 2,2 -(((sulfonylbis(oxy))bis(4,1-phenylene))bis-(1H- and then was puried by the column chromatography (CH
2
Cl
2
/
0
imidazole-5,2-diyl))(2S,2 S)-bis(pyrrolidine-1-carboxylate)
(10g). MeOH). The residue was obtained as solid.
31814 | RSC Adv., 2018, 8, 31803–31821
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-((S)-1-((R)-2-((Methoxycarbonyl)amino)-2-phenylacetyl)-
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3
d ¼ 39.52 ppm, 100 MHz): d 170.71, 168.39, 156.07, 149.70,
pyrrolidine-2-carboxamido)phenyl (4-((S)-1-((R)-2-((methoxy- 147.73, 137.20, 136.26, 128.59, 128.03, 119.96, 117.92, 113.47,
1
9
carbonyl)amino)-2-phenylacetyl)pyrrolidine-2-carboxamido)- 60.69, 56.67, 51.63, 46.96, 29.21, 24.31; F NMR (DMSO-d ,
6
1
phenyl) sulfate (11). Yield (106.7 mg, 83%); H NMR (DMSO- 376 MHz): d ꢁ135.20; HRMS (ESI) m/z: anal. calcd for [M +
+
d
6
, d ¼ 2.5 ppm, 400 MHz): d 10.63–10.31/10.27/10.17 (m/s/s, Na] C42
42 2 6
H F N NaO12S: 915.2442; found 915.2443.
2-Fluoro-3-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phe-
0
7
5
4
3
1
.87H + 0.5H + 0.57H), 7.91–7.89 (d, 1H), 7.80–7.72 (m, 3H),
.68–7.56 (m, 2H), 7.50–7.26 (m, 12H), 7.17–7.05 (m, 2H), nylacetyl)pyrrolidine-2-carboxamido)phenyl (2-uoro-5-((S)-
.53–5.42/5.37–5.34/4.94–4.90 (m/t/t, 1.61H + 0.19H + 0.18H), 1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)-pyrrolid-
.61–4.39 (m, 2H), 3.86–3.81 (qui, 1H), 3.73–3.66 (m, 1H), ine-2-carboxamido)phenyl) sulfate (15). Yield (112 mg, 58%);
1
.54 (s, 6H), 3.22–3.17 (q, 1H), 3.13–3.09 (t, 1H), 2.32–2.14 (m,
H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 10.46/10.27/
1
3
H), 2.07–1.76 (m, 8H), 1.23/0.85 (2 s, 0.30H + 0.10H);
, d ¼ 39.52 ppm, 100 MHz): d 170.59, 168.40, (m, 2H), 7.74–7.64 (m, 3H), 7.55–7.50 (t, 1H), 7.39–7.21 (m,
56.09, 149.89, 145.03, 140.74, 138.70, 137.21, 130.65, 128.62, 11H), 7.09–7.04 (d, 2H), 5.51–5.20/5.10–5.08/4.86–4.84 (m/d/
C
10.21/10.11 (4 s, 0.57H+0.53H + 0.32H + 0.36H), 8.11–7.80
NMR (DMSO-d
6
1
1
5
+
28.06, 121.61, 120.65, 118.31, 115.50, 111.35, 60.71, 56.71, d, 1.96H + 0.06H + 0.08H), 4.68/4.57–4.55/4.46–4.43/4.36–
1.65, 46.98, 29.25, 24.30; HRMS (ESI) m/z: anal. calcd for [M 4.34 (4 m, 0.28H + 0.40H + 0.46H + 0.62H), 3.81–3.67 (m, 2H),
+
Na] C42H44N NaO12S: 879.2630; found 879.2631.
3.52 (s, 6H), 3.17–3.08 (m, 2H), 2.27–2.16 (m, 1H), 1.99–1.80
(m, 7H), 1.23/0.85 (2 s, 0.31H + 0.10H); C NMR (DMSO-d ,
6
6
1
3
3
-((S)-1-((Methoxycarbonyl)-D-valyl)pyrrolidine-2-carboxamido)
phenyl
(4-((S)-1-((methoxycarbonyl)-D-valyl)-pyrrolidine-2- d ¼ 39.52 ppm, 100 MHz): d 170.92, 168.38, 156.30, 156.05,
carboxamido)phenyl) sulfate (12). Yield (46.7 mg, 71%); H NMR 149.85, 147.40, 137.16, 136.77, 136.25, 136.08, 128.58, 128.35,
DMSO-d
1
(
6
, d ¼ 2.5 ppm, 400 MHz): d 10.64/10.54/10.13/10.02 (4 s, 128.01, 124.81, 123.48, 123.06, 119.72, 117.96, 117.78, 113.31,
.2H+0.2H + 0.86H + 0.82H), 7.87 (s, 1H), 7.76–7.73 (d, 2H), 7.61– 60.67, 56.65, 51.54, 46.94, 29.18, 24.30; F NMR (DMSO-d
.59 (d, 2H), 7.51–7.36 (m, 5H), 7.14–7.08 (m, 1H), 4.43–4.39 (q, 376 MHz): d ꢁ73.82, ꢁ135.52, ꢁ139.50, ꢁ139.97; HRMS (ESI)
1
9
0
7
2
6
,
+
42 2 6
H), 4.12–4.07 (t, 2H), 3.79 (s, 2H), 3.64–3.58 (q, 2H), 3.55–3.53 m/z: anal. calcd for [M + Na] C42H F N NaO12S: 915.2442;
(
8
app s, 6H), 3.48–3.45 (m, 1H), 2.16–2.08 (m, 2H), 2.04–1.92 (m, found 915.2441.
1
3
H), 0.89–0.86 (t, 10H), 0.80–0.77 (t, 1H), 0.72–0.68 (m, 1H);
, v ¼ 39.52 ppm, 100 MHz): d 171.01, 170.71, 2-carboxamido)phenyl) sulfate (16). Yield (77.7 mg, 80%); H
70.24, 156.92, 149.85, 144.94, 140.71, 138.67, 130.61, 121.57, NMR (DMSO-d
C
Bis(2-uoro-5-((S)-1-((methoxycarbonyl)-D-valyl)pyrrolidine-
1
NMR (DMSO-d
6
1
1
2
6
, d ¼ 2.5 ppm, 500 MHz): d 10.63/10.15 (2 s,
20.50, 118.19, 115.42, 111.22, 60.32, 60.27, 58.01, 51.56, 47.11, 0.35H+1.62H), 8.04 (s, 2H), 7.63–7.61 (d, 2H), 7.52–7.48 (t, 2H),
9.76, 29.41, 29.37, 24.39, 24.35, 19.09, 18.32; HRMS (ESI) m/z: 7.36–7.35 (d, 2H), 4.38–4.36 (d, 2H), 4.11–4.07 (t, 2H), 3.78–
+
anal. calcd for [M + Na] C H N NaO S: 811.2943; found 3.74 (m, 2H), 3.63–3.57 (m, 2H), 3.53 (s, 6H), 2.15–2.08 (m, 2H),
36
48
6
12
8
11.2913.
Bis(2-uoro-3-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-
phenylacetyl)pyrrolidine-2-carboxamido)phenyl) sulfate (13). d 170.87, 170.22, 156.93, 149.64, 147.66, 136.25, 120.42,
2.08–1.84 (m, 8H), 0.88–0.86 (t, 10H), 0.77–0.76 (d, 1H), 0.67–
1
3
0.67 (d, 1H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz):
1
Yield (41.0 mg, 49%); H NMR (DMSO-d
MHz): d 10.21 (s, 1H), 10.10 (s, 1H), 8.02–7.97 (q, 2H), 7.72– 29.95, 24.44, 19.09, 18.29; F NMR (DMSO-d
6
, v ¼ 2.5 ppm, 400 119.35, 117.33, 113.56, 113.09, 60.63, 59.92, 57.96, 51.42,
1
9
, 376 MHz):
6
+
7
7
.66 (q, 2H), 7.41–7.40 (m, 6H), 7.37–7.29 (m, 7H), 7.27–7.25/ d ꢁ135.35; HRMS (ESI) m/z: anal. calcd for [M + Na] C36
.12–7.10 (2 d, 0.48H + 0.35H), 5.51–5.45 (q, 2H), 4.69–4.67
NaO12S: 847.2755; found 847.2752.
2-Fluoro-3-((S)-1-((methoxycarbonyl)-D-valyl)pyrrolidine-2-
.53 (s, 6H), 3.20–3.14 (q, 1H), 3.10–3.08 (d, 1H), 2.18–2.16 (d, carboxamido)phenyl (2-uoro-5-((S)-1-((methoxycarbonyl)-D-
H -
46
2 6
F N
(
m, 1H), 4.57–4.55 (m, 1H), 3.81 (s, 1H), 3.71–3.65 (m, 1H),
3
1
1
3
H), 2.03–1.78 (m, 7H), 1.23/0.85 (2 s, 2.02H + 0.62H);
, d ¼ 39.52 ppm, 126 MHz): d 170.92, 168.45, (40.3 mg, 39%); H NMR (DMSO-d
68.25, 156.33, 155.97, 136.74, 128.60, 128.38, 128.35, 127.99, d 10.64/10.45/10.16/9.97 (4 s, 0.16H + 0.22H + 0.81H + 0.71H),
24.76, 123.15, 118.26, 118.03, 60.26, 56.65, 51.55, 46.85, 8.08–8.07 (d, 1H), 7.99–7.82 (m, 1H), 7.64–7.60 (m, 1H), 7.53–
C
valyl)pyrrolidine-2-carboxamido)phenyl) sulfate (17). Yield
1
NMR (DMSO-d
6
6
, d ¼ 2.5 ppm, 400 MHz):
1
1
2
1
9
9.14, 24.69, 24.27; F NMR (DMSO-d
6
, 376 MHz): d ꢁ74.67, 7.48 (t, 1H), 7.44–7.28 (m, 4H), 5.17–5.15/5.03–5.01/4.60–4.58
+
ꢁ
139.84, ꢁ139.84; HRMS (ESI) m/z: anal. calcd for [M + Na]
(3 d, 0.14H + 0.16H + 0.77H), 4.38–4.37 (d, 1H), 4.12–4.07 (t,
2H), 3.87–3.71 (m, 2H), 3.64–3.58 (m, 1H), 3.53–3.50 (app s,
6H), 3.46–3.45 (m, 1H) 2.21–2.09 (m, 2H), 1.99–1.81 (m, 8H),
C
H
F N NaO S: 915.2442; found 915.2445.
4
2
42
2
6
12
Bis(2-uoro-5-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-
1
3
phenylacetyl)pyrrolidine-2-carboxamido)phenyl) sulfate (14). 1.23 (s, 0.32H), 0.89–0.66 (m, 12H); C NMR (DMSO-d
6
, d ¼
, d ¼ 2.5 ppm, 400 39.52 ppm, 100 MHz): d 170.90, 170.22, 156.84, 149.90,
MHz): d 10.64/10.47/10.32/10.28 (4 s, 0.06H + 0.3H + 0.33H + 147.44, 146.40, 143.89, 137.12, 136.06, 128.18, 124.71, 123.31,
1
Yield (63.4 mg, 52%); H NMR (DMSO-d
6
1
7
.19H), 8.13–8.01 (m, 2H), 7.87–7.72 (m, 2H), 7.65 (s, 2H), 119.81, 117.89, 113.39, 60.28, 58.00, 51.54, 47.09, 29.75,
1
9
.55–7.50 (t, 2H), 7.42–7.29 (m, 8H), 7.25–7.23/7.06–7.03 (2 24.38, 19.07, 18.30; F NMR (DMSO-d
, 376 MHz): d ꢁ134.98,
6
m, 0.52H + 0.74H) 7.06–7.04 (t, 1H), 5.52–5.47/5.35–5.33/ ꢁ135.41, ꢁ138.11, ꢁ139.92; HRMS (ESI) m/z: anal. calcd for
+
4
3
(
.87–4.85 (m/d/d, 1.56H + 0.26H + 0.20H), 4.48–4.35 (m, 2H), [M + Na] C36
46 2 6
H F N NaO12S: 847.2755; found 847.2754.
.84–3.81 (m, 2H), 3.53 (s, 6H), 3.20–3.09 (m, 2H), 2.04–1.77
Bis(2-uoro-3-((S)-1-((methoxycarbonyl)-D-valyl)pyrrolidine-2-
m, 8H), 1.23/0.85 (2 s, 0.35H + 0.07H); C NMR (DMSO-d6, carboxamido)phenyl) sulfate (18). Yield (97.9 mg, 74%); H NMR
1
3
1
6
(DMSO-d , v ¼ 2.5 ppm, 400 MHz): d 10.45/10.14/9.97 (3 s, 0.33H
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+
4
2
0.20H + 1.44H), 7.98–7.81 (m, 2H), 7.48–7.29 (m, 6H), 5.17–5.15/ 3.71 (m, 1H), 3.54–3.51 (app br s, 6H), 3.46–3.38 (d, 1H), 3.17–
.60–4.58 (2 d, 0.27H + 1.48H), 4.12–4.07 (t, 1H), 3.87–3.79 (m, 3.15 (d, 1H), 2.37–2.22 (m, 2H), 2.08–1.89 (m, 6H), 1.23 (s,
1
3
H), 3.66–3.60 (m, 4H), 3.50 (s, 6H), 2.25–2.11 (m, 2H), 1.98–1.85 0.20H); C NMR (DMSO-d , d ¼ 39.52 ppm, 126 MHz): d 168.99,
6
1
3
(m, 8H), 1.23 (s, 0.15H), 0.89–0.71 (m, 12H); C NMR (DMSO-d6, 156.34, 155.97, 150.33, 149.40, 136.86, 131.50, 128.69, 128.62,
d ¼ 39.52 ppm, 100 MHz): d 170.53, 156.87, 146.52, 144.01, 128.31, 128.12, 127.79, 127.44, 127.23, 124.83, 122.11, 122.06,
1
4
37.14, 128.15, 124.70, 123.42, 118.19, 59.91, 58.04, 51.45, 48.64, 117.94, 115.90, 57.00, 56.73, 53.48, 51.62, 47.01, 30.88, 24.14;
1
9
+
6
7.13, 29.75, 29.16, 24.37, 19.08, 18.32; F NMR (DMSO-d , 376 HRMS (ESI) m/z: anal. calcd for [M + H]
46 47 8
C H N O10S:
MHz): d ꢁ78.40, ꢁ142.54, ꢁ144.44; HRMS (ESI) m/z: anal. calcd 903.3130; found 903.3130.
+
for [M + Na] C36
H
46
F
2
N
6
NaO12S: 847.2755; found 847.2757.
3-(2-((S)-1-((Methoxycarbonyl)-D-valyl)pyrrolidin-2-yl)-1H-
imidazol-5-yl)phenyl (4-(2-((S)-1-((methoxycarbonyl)-D-valyl)-
Bis(2-uoro-5-(2-((S)-1-((methoxycarbonyl)-D-valyl)pyrrolidin-
2
-yl)-1H-imidazol-5-yl)phenyl) sulfate (19). Yield (34.5 mg, 52%); pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl) sulfate (23). Yield
1
1
H NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz): d 8.08–8.06 (d, 2H), (107 mg, 86%); H NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz):
6
6
8
7
1
.01 (s, 2H), 7.92–7.91 (d, 2H), 7.80 (s, 1H), 7.73–7.68 (t, 1H), 7.59– d 8.13–8.08 (d, 2H), 7.95–7.87 (m, 4H), 7.78–7.63 (m, 3H),
.46 (m, 1H), 7.25–7.23 (d, 1H), 5.69–5.67/5.14–5.12 (2 d, 0.35H + 7.54–7.48 (m, 1H), 7.30–7.23 (m, 2H), 5.80–5.74/5.19–5.10 (d
.32H), 4.17–4.13 (t, 2H), 3.87–3.86 (d, 2H), 3.68–3.62 (q, 2H), 3.53
ꢁ
1
m
, 0.25H + 1.73H), 4.18–4.07 (m, 2H), 3.88–3.78 (m, 2H),
(
(
s, 6H), 2.32 (s, 2H), 2.08–1.67 (m, 8H), 1.23 (s, 0.47H), 0.90–0.29 3.68–3.66 (d, 2H), 3.53 (s, 6H), 2.36 (s, 2H), 2.08–1.96 (m, 8H),
1
3
13
m, 12H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): 1.23 (s, 0.39H), 0.90–0.68 (m, 12H), 0.33 (s, 1H); C NMR
d 170.71, 156.88, 149.66, 137.07, 136.94, 126.54, 120.06, 118.57, (DMSO-d , d ¼ 39.52 ppm, 100 MHz): d 170.80, 158.49,
6
1
1
15.78, 57.87, 53.48, 51.58, 46.95, 30.88, 29.69, 24.28, 19.25, 158.15, 156.87, 150.32, 149.57, 131.43, 127.33, 124.70, 122.08,
1
9
8.57, 18.41, 17.93; F NMR (DMSO-d , 376 MHz): d ꢁ74.27; 120.79, 117.78, 116.21, 115.85, 57.84, 53.19, 51.59, 47.00,
6
+
HRMS (ESI) m/z: anal. calcd for [M + H]
8
40 49 2 8
C H F N O10S: 30.89, 29.70, 24.34, 19.28, 17.89; HRMS (ESI) m/z: anal. calcd
+
71.3255; found 871.3256.
Bis(2-uoro-5-(2-((S)-1-((methoxycarbonyl)-L-valyl)pyrrolidin-
-yl)-1H-imidazol-5-yl)phenyl) sulfate (20). Yield (90.1 mg, 48%); imidazol-5-yl)phenyl (4-(2-((S)-1-((methoxycarbonyl)-L-valyl)-
for [M + H] C40
51 8
H N O10S: 835.3443; found 835.3446.
3-(2-((S)-1-((Methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-
2
1
H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 8.06–8.05 (d, 2H), pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl) sulfate (24). Yield
1
7
5
.99 (s, 2H), 7.90–7.88 (d, 2H), 7.71–7.67 (t, 2H), 7.30–7.28 (d, 2H), (110 mg, 54%); H NMR (DMSO-d , d ¼ 2.5 ppm, 400 MHz):
6
.45/5.11–5.08 (m/t, 0.13H + 1.99H), 4.10–4.06 (t, 2H), 3.86–3.76 d 12.37–12.39/12.16–12.13/11.92–11.86 (3 d, 0.14H+0.18H+1.51H),
(
1
d
1
5
m, 4H), 3.53 (s, 6H), 2.32–2.28 (t, 2H), 2.14–2.10 (m, 2H), 2.07– 7.85–7.83 (d, 2H), 7.76–7.73 (m, 2H), 7.61 (s, 1H), 7.54 (s, 1H), 7.48–
1
3
.94 (m, 6H), 1.23 (s, 0.46H), 0.81–0.75 (q, 12H); C NMR (DMSO- 7.44 (t, 1H), 7.39–7.36 (d, 2H), 7.31–7.26 (t, 2H), 7.19–7.16 (q, 1H),
, d ¼ 39.52 ppm, 100 MHz): d 170.95, 158.36, 158.03, 156.88, 5.22/5.08–5.03 (2 m, 0.14H+1.86H), 4.11–4.00 (m, 2H), 3.78 (s, 2H),
49.87, 137.07, 136.93, 126.37, 119.84, 118.72, 118.55, 57.93, 3.53 (s, 6H), 3.17–3.16 (d, 1H), 2.13 (s, 4H), 1.94–1.87 (m, 6H), 0.89–
6
1
9
13
3.34, 51.52, 47.05, 31.00, 29.30, 24.58, 19.13, 17.99; F NMR 0.80 (m, 12H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz):
(DMSO-d
6
, 376 MHz): d ꢁ78.32, ꢁ138.05; HRMS (ESI) m/z: anal. d 170.35, 156.77, 150.39, 149.67, 147.82, 137.76, 135.00, 130.43,
+
calcd for [M + H] C H F N O S: 871.3255; found 871.3254.
125.64, 123.38, 121.04, 117.89, 116.19, 113.71, 113.19, 57.99, 54.18,
4
0
49 2 8 10
Bis(2-uoro-5-(2-((S)-1-((R)-2-((methoxycarbonyl)amino)-2- 51.44, 48.61, 46.79, 30.96, 30.87, 29.84, 24.26, 18.51; HRMS (ESI) m/
+
phenylacetyl)pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl) sulfate z: anal. calcd for [M + H] C H N O S: 835.3443; found 835.3445.
40 51 8 10
1
(
4
21). Yield (34.5 mg, 73%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm,
Methyl ((R)-2-((S)-2-((3-hydroxyphenyl)carbamoyl)pyrrolidin-
00 MHz): d 8.11–8.03 (m, 4H), 7.98–7.93 (m, 2H), 7.79–7.71 1-yl)-2-oxo-1-phenylethyl)carbamate (25). Yield (839 mg, 79%);
1
(
(
m, 2H), 7.68–7.64 (t, 2H), 7.39–7.34 (m, 6H), 7.29 (s, 2H), 6.99
H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): d 10.00/9.86/9.73
s, 2H), 5.65–5.63/5.50–5.49/5.40–5.38 (3 d, 0.51H + 1.37H + (3 s, 0.20H + 0.14H + 0.72H), 9.37 (s, 1H), 7.98–7.97/7.71–7.69/
0
6
1
3
1
1
.49H), 5.17–5.16 (d, 2H), 3.93–3.82 (m, 2H), 3.53–3.51 (app s, 7.55–7.52 (d/d/m, 0.34H + 0.86H + 0.41H), 7.42–7.30 (m, 4H),
H), 3.18–3.12 (q, 2H), 2.22–2.15 (m, 2H), 2.02–2.00 (d, 4H), 7.20/7.15–7.13 (s/t, 0.70H+0.39H), 7.09–7.05 (t, 1H), 7.00–6.85/
1
3
.87 (s, 2H), 1.23 (s, 0.72H);
C NMR (DMSO-d , d ¼ 6.87–6.85 (2 d, 0.73H + 0.19H), 6.47–6.45 (d, 1H), 5.51–5.49/
6
9.52 ppm, 100 MHz): d 168.88, 155.95, 151.56, 149.53, 5.33–5.31/4.92–4.89 (3 d, 0.74H + 0.21H + 0.19H), 4.39–4.37 (t,
37.11, 136.95, 128.67, 128.27, 128.11, 127.78, 126.70, 120.27, 1H), 3.81 (s, 1H), 3.54 (s, 3H), 3.21–3.15 (q, 1H), 2.33–1.77 (m,
1
3
18.82, 118.63, 115.87, 56.99, 53.80, 51.61, 46.94, 30.91, 24.10; 4H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz): d 170.09,
1
9
F NMR (DMSO-d
ESI) m/z: anal. calcd for [M + H] C46
found 939.2939.
-(2-((S)-1-((R)-2-((Methoxycarbonyl)amino)-2-phenyl-acetyl)
pyrrolidin-2-yl)-1H-imidazol-5-yl)phenyl (4-(2-((S)-1-((R)-2-
(methoxycarbonyl)amino)-2-phenylacetyl)pyrrolidin-2-yl)-1H-
6
, 376 MHz): d ꢁ78.97, ꢁ133.12; HRMS 168.34, 157.61, 156.07, 140.03, 137.20, 129.31, 128.61, 128.04,
+
(
45 2 8
H F N O10S: 939.2942; 110.42, 110.04, 106.48, 60.69, 56.71, 51.64, 46.95, 29.34, 24.25;
+
HRMS (ESI) m/z: anal. calcd for [M + Na]
21 23 3 5
C H N NaO :
3
420.1530; found 420.1531.
Methyl ((S)-1-((S)-2-(5-(3-hydroxyphenyl)-1H-imidazol-2-yl)
(
pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)carbamate (26). Yield
1
1
imidazol-5-yl)phenyl) sulfate (22). Yield (53.3 mg, 35%);
NMR (DMSO-d
H
(455 mg, 64%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 500 MHz):
6
, d ¼ 2.5 ppm, 400 MHz): d 8.19–8.06 (m, 2H), d 11.67 (app br s, 1H), 9.44 (app br s, 1H), 7.36–7.25 (m, 2H),
7
7
1
.98–7.96 (d, 2H), 7.91–7.83 (m, 2H), 7.73–7.64 (m, 5H), 7.39– 7.17–6.86 (m, 3H), 6.56 (s, 1H), 5.24–5.23/5.08–5.07 (2 d,
.25 (m, 10H), 7.03 (s, 1H), 5.53–5.49/5.45–5.43/5.40–5.38 (t/d/d, 0.13H+0.81H), 4.08–4.05 (t, 1H), 3.80–3.76 (d, 2H), 3.54–3.44
.51H + 0.25H + 0.18H), 5.18 (m, 2H), 3.91–3.85 (d, 1H), 3.80– (app s, 3H), 2.27–2.08 (m, 2H), 2.01–1.86 (m, 3H), 0.89–0.84 (q,
31816 | RSC Adv., 2018, 8, 31803–31821
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1
3
6
1
5
H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz): d 170.43, yl-1-oxobutan-2-yl)carbamate (102 mg, 0.213 mmol) and DBU
ꢀ
57.61, 156.85, 129.33, 115.06, 113.06, 111.10, 58.05, 57.54, (34.5 mL, 0.230 mmol) in DMF (10 mL) was stirred at 50 C for
5.19, 54.25, 51.47, 46.87, 30.92, 29.88, 24.31, 19.04, 18.53; 12 h. The mixture was then poured in water and extracted with
HRMS (ESI) m/z: anal. calcd for [M + H] C H N O : 387.2027; CH Cl . The organic layer dried over magnesium sulfate, and
+
2
0
27
4
4
2
2
found 387.2029.
-((S)-1-((R)-2-((Methoxycarbonyl)amino)-2-phenylacetyl)-
pyrrolidine-2-carboxamido)phenyl sulfurouoridate (27). A yield (97.1 mg, 65%); H NMR (DMSO-d
mixture of methyl ((R)-2-((S)-2-((3-hydroxyphenyl)carbamoyl)- d 11.95 (s, 1H), 10.46/10.28 (2 s, 0.56H + 0.31H), 7.92–7.88 (d, 1H),
pyrrolidin-1-yl)-2-oxo-1-phenylethyl)carbamate (276 mg, 7.82–7.72 (m, 2H), 7.66–7.63 (m, 1H), 7.61–7.58 (t, 1H), 7.49–7.46
then crude product was puried by the column chromatography
3
(n-hexane/ethyl acetate). The residue was obtained as white solid;
1
6
, d ¼ 2.5 ppm, 500 MHz):
0
.695 mmol) and N,N-diisopropylethylamine (363 mL, 2.09 (t, 1H), 7.41–7.40 (d, 2H), 7.38–7.35 (t, 1H), 7.33–7.25 (m, 3H),
mmol) in CH Cl (10 mL) was stirred under sulfuryl uoride 7.22–7.20 (d, 1H), 7.12–7.08 (t, 1H), 7.03–6.63 (m, 1H), 5.51–5.46/
2
2
at room temperature for 5 h. The mixture was then poured in 5.32–5.31/5.22–5.20 (m/d/d, 0.76H + 0.06H + 0.11H), 5.07–5.04/
N aq. HCl solution and extracted with CH Cl . The organic 4.90–4.88 (m/d, 0.80H + 0.05H), 4.49–4.37 (m, 1H), 4.06–4.03 (t,
1
2
2
layer dried over magnesium sulfate, ltered, and concen- 1H), 3.79–3.65 (m, 2H), 3.53 (s, 6H), 3.45–3.39 (m, 1H), 3.23–3.08
trated in vacuo. Without any purication, the residue was (m, 1H), 2.19–2.08 (m, 2H), 1.95–1.94 (d, 2H), 1.92–1.85 (m, 2H),
1
13
obtained as a white oil; yield (255 mg, 76%); H NMR (DMSO- 1.65–1.55 (m, 3H), 1.23 (s, 0.81H) 0.87–0.81 (m, 6H); C NMR
, d ¼ 2.5 ppm, 400 MHz): d 10.58/10.51/10.41/10.34 (4 s, (DMSO-d , d ¼ 39.52 ppm, 126 MHz): d 170.76, 170.34, 168.34,
.02H + 0.23H + 0.11H + 0.53H), 7.99–7.86 (m, 1H), 7.74–7.66 168.15, 156.77, 156.29, 150.38, 149.89, 140.83, 136.78, 130.62,
m, 1H), 7.63–7.59 (t, 1H), 7.56–7.52 (t, 1H), 7.42–7.41 (d, 2H), 130.49, 128.56, 128.33, 128.00, 127.75, 123.42, 118.12, 116.18,
d
6
6
0
(
7
6
5
1
1
.38–7.35 (t, 2H), 7.34–7.32 (m, 1H), 7.29–7.24/7.06–7.05/ 115.30, 111.13, 60.59, 57.96, 56.60, 54.15, 51.41, 46.79, 30.96,
.72–6.70 (m/d/d, 0.88H + 0.28H + 0.06H), 5.52–5.47/5.35– 29.83, 29.20, 24.69, 24.19, 18.83, 18.48; HRMS (ESI) m/z: anal.
+
.33/4.90–4.88 (m/d/d, 0.88H + 0.08H + 0.08H), 4.50–4.38 (m, calcd for [M + H] C41
48 7
H N O11S: 846.3127; found 846.3129.
H), 3.83–3.62 (m, 1H), 3.54 (s, 3H), 3.20–3.10 (m, 1H), 2.29–
Methyl ((R)-2-((S)-2-((3-((tert-butyldimethylsilyl)oxy)-phe-nyl)
1
3
.78 (m, 4H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 100 MHz): carbamoyl)pyrrolidin-1-yl)-2-oxo-1-phenylethyl)-carbamate (30).
d 170.95, 168.40, 156.06, 149.63, 140.92, 137.15, 130.98, A mixture of methyl ((R)-2-((S)-2-((3-hydroxyphenyl)carbamoyl)
1
5
3
28.58, 128.39, 128.02, 119.34, 115.34, 111.06, 60.69, 56.66, pyrrolidin-1-yl)-2-oxo-1-phenyl-ethyl)carbamate (51.1 mg, 0.129
1
9
1.59, 46.94, 29.17, 24.28; F NMR (DMSO-d , 376 MHz): mmol), tert-butyldimethylsilyl chloride (29.1 mg, 0.193 mmol),
6
+
8.64; HRMS (ESI) m/z: anal. calcd for [M + Na] C21H22FN3- imidazole (26.3 mg, 0.0263 mmol) in CH Cl (10 mL) was stirred
2
2
NaO
7
S: 502.1055; found 502.1054.
at room temperature for 4 h. The mixture was then poured in
O and extracted with CH Cl . The organic layer dried over
magnesium sulfate, and then was puried by the column chro-
Methyl ((S)-1-((S)-2-(5-(3-((tert-butyldimethylsilyl)oxy)-phe-
nyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl)
H
2
2
2
carbamate (28). A mixture of methyl ((S)-1-((S)-2-(5-(3-hydrox- matography (n-hexane/ethyl acetate). The residue was obtained
1
yphenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-methyl-1-oxobutan- as sticky white oil; yield (53.5 mg, 81%); H NMR (DMSO-d
2
6
, d ¼
-yl)carbamate (158 mg, 0.409 mmol), tert-butyldimethylsilyl 2.5 ppm, 500 MHz): d 10.08/9.82 (2 s, 0.18H + 0.81H), 7.97–7.96/
chloride (123 mg, 0.817 mmol), imidazole (139 mg, 2.04 mmol) 7.71–7.69 (2 d, 0.13H + 0.81H), 7.41–7.31 (m, 4H), 7.26 (s, 1H),
in CH Cl (10 mL) was stirred at room temperature for 4 h. The 7.17–6.97 (m, 2H), 6.54 (s, 1H), 5.50–5.48/5.32–5.30/4.90–4.88 (3
2
2
2 2 2
mixture was then poured in H O and extracted with CH Cl . The d, 0.84H+0.14H + 0.13H), 4.37 (s, 1H), 3.81 (s, 1H), 3.53 (s, 3H),
organic layer dried over magnesium sulfate, and then was 3.18–3.16 (d, 1H), 1.98–1.77 (m, 4H), 0.96–0.95 (app s, 9H), 0.20–
1
3
puried by the column chromatography (n-hexane/ethyl 0.19 (app s, 6H); C NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz):
acetate). The residue was obtained as sticky white oil; yield d 170.21, 168.29, 156.01, 155.28, 140.18, 137.15, 129.46, 128.56,
1
(
187 mg, 91%); H NMR (DMSO-d
6
, d ¼ 2.5 ppm, 400 MHz): 128.00, 127.70, 114.58, 112.27, 110.74, 60.67, 56.66, 51.58, 46.90,
d 12.12/12.03/11.77 (3 s, 0.16H + 0.09H + 0.68H), 7.44–7.06 (m, 29.26, 25.54, 24.22, 17.89, ꢁ4.50; HRMS (ESI) m/z: anal. calcd for
+
5
H), 6.69–6.60 (m, 1H), 5.06–5.05 (d, 1H), 4.08–4.04 (t, 1H), 3.79 [M + Na] C H N NaO Si: 534.2395; found 534.2396.
27 37 3 5
(
(
s, 2H), 3.53 (s, 3H), 2.16–2.07 (m, 2H), 1.96–1.89 (m, 3H), 0.96
tert-Butyl (S)-2-((3-(((3-((S)-1-((R)-2-((methoxycarbonyl)-amino)-2-
phenylacetyl)pyrrolidine-2-carboxamido)phenoxy)-sulfonyl)oxy)phe-
1
3
s, 9H), 0.92–0.90 (d, 2H), 0.88–0.82 (q, 4H), 0.18 (s, 6H);
C
NMR (DMSO-d
6
, d ¼ 39.52 ppm, 126 MHz): d 170.30, 156.78, nyl)carbamoyl)pyrrolidine-1-carboxylate (31). A mixture of methyl
55.24, 149.15, 138.83, 136.71, 129.36, 117.49, 117.08, 115.60, ((R)-2-((S)-2-((3-((tert-butyldimethylsilyl)-oxy)phenyl)carbamoyl)p-
12.41, 57.96, 54.21, 51.42, 46.80, 31.01, 29.84, 25.61, 24.13, yrrolidin-1-yl)-2-oxo-1-phenylethyl)-carbamate (602 mg, 1.18
1
1
1
+
9.01, 18.56, 17.96; HRMS (ESI) m/z: anal. calcd For [M + H]
mmol), tert-butyl (S)-2-((3-((uorosulfonyl)oxy)phenyl)carbamoyl)
C H N O Si: 501.2892; found 501.2894.
pyrrolidine-1-carboxylate (914 mg, 0.235 mmol) and DBU (35.2
2
6
41 4 4
ꢀ
3-(2-((S)-1-((Methoxycarbonyl)-L-valyl)pyrrolidin-2-yl)-1H-imida- mL, 0.235 mmol) in DMF (10 mL) was stirred at 50 C for 12 h. The
zol-5-yl)phenyl
phenylacetyl)pyrrolidine-2-carboxamido)phenyl) sulfate (29). A with CH
mixture of 3-((S)-1-((R)-2-((methoxycarbonyl)-amino)-2- and then was puried by the column chromatography (n-hexane/
phenylacetyl)pyrrolidine-2-carboxamido)phenyl sulfurouoridat- ethyl acetate). The residue was obtained as white solid; yield
(3-((S)-1-((R)-2-((methoxycarbonyl)-amino)-2- mixture was then poured in 1 N aq. HCl solution and extracted
2
Cl . The organic layer dried over magnesium sulfate,
2
1
e (88.9 mg, 0.178 mmol), methyl ((S)-1-((S)-2-(5-(3-((tert-butyldi- (676 mg, 75%); H NMR (DMSO-d
methylsilyl)oxy)phenyl)-1H-imidazol-2-yl)pyrrolidin-1-yl)-3-meth-
6
, d ¼ 2.5 ppm, 500 MHz):
d 10.44–10.25 (m, 2H), 7.94–7.90 (t, 2H), 7.73–7.71 (d, 1H), 7.66–
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7
3
0
.57 (m, 2H), 7.49–7.45 (m, 2H), 7.42–7.40 (d, 2H), 7.38–7.24 (m, and 10% heat-inactivated fetal bovine serum (DFBS; Hyclone) at
ꢀ
H), 7.14–7.04 (m, 2H), 5.52–5.47/5.35–5.33/4.91–4.89 (m/d/d, 37 C in a humidied 6.0% CO incubator.
2
.83H + 0.10H + 0.09H), 4.51–4.38 (m, 1H), 4.27–4.25/4.20–4.17
(
3
d/q, 0.35H + 0.64H), 4.14–4.10 (q, 1H), 3.82 (s, 1H), 3.53 (s, 3H),
Virus production
m, 1H), 2.05–1.78 (m, 7H), 1.39–1.24 (app s, 9H); C NMR In vitro transcribed RNA of JFH5a-Rluc-ad34, which is a deriva-
DMSO-d
, d ¼ 39.52 ppm, 126 MHz): d 172.03, 170.78, 168.35, tive of JFH1 with adaptive mutations in the E2 and p7 regions,
53.06, 149.85, 140.74, 130.66, 128.57, 128.34, 128.00, 118.40, was transfected into Huh7.5.1 cells by electroporation. JFH5a-
.46–3.42 (m, 1H), 3.37–3.34 (m, 1H) 3.19–3.17 (d, 3H), 2.24–2.13
1
3
(
(
1
1
2
6
15.34, 111.40, 78.60, 60.46, 56.64, 51.61, 46.56, 30.91, 29.19, Rluc-ad34 virus contains a reporter Renilla luciferase (Rluc)
+
92
7.89, 24.27, 23.39; HRMS (ESI) m/z: anal. calcd for [M + Na]
for convenient virus proliferation assay. HCV-containing cell
C H N NaO S: 788.2572; found 788.2575.
culture media were collected 3–5 days aer electroporation and
ltered through a 0.45 mM pore size lter.
3
7
43
5
11
3-((S)-1-((R)-2-((Methoxycarbonyl)amino)-2-phenylacetyl)-pyrro-
lidine-2-carboxamido)phenyl (3-((S)-1-(5-((3aS,4S,6aR)-2-oxohex-
ahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl)-pyrrolidine-2-ca-
rboxamido)phenyl) sulfate (32). A mixture of tert-butyl (S)-2-((3-
Antiviral activity test with infectious HCV particles
(
((3-((S)-1-((R)-2-((methoxycarbonyl)amino)-2-phenylacetyl)
Huh 7.5.1 cells were inoculated with a JFH5a-Rluc-ad34 virus stock
pyrrolidine-2-carboxamido)phenoxy)sulfonyl)-oxy)phenyl)
and cultivated for 3 h. At 3 h aer the virus inoculation, HCV
carbamoyl)pyrrolidine-1-carboxylate (426 mg, 0.557 mmol) infected Huh7.5.1 cells were cultured with media containing seri-
in CF CO H (5 mL)/CH Cl (5 mL) was stirred at room ally diluted compounds. At 3 days aer the chemical treatment, the
3
2
2
2
temperature for 30 m. The volatile component was removed cells were harvested and their luciferase activities were measured
in vacuo, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide using a Renilla luciferase assay system (Promega) according to the
(
(
139 mg, 0.724 mmol), hydroxybenzotriazole hydrate manufacturer's direction. Finally, the luciferase activities were
97.8 mg, 0.724 mmol), 5-[(3aS,4S,6aR)-2-oxohexahydro-1H- normalized to those obtained from mock-treated cells.
thieno[3,4-d]imidazol-4-yl]pentanoic acid (163 mg, 0.668
mmol) were added in batches over 4 min to a solution of
N,N-diisopropylethylamine (485 mL, 2.78 mmol) in CH Cl
Cell line and cell culture
2
2
(
10 mL) and the reaction mixture stirred at room tempera- Huh 7.5.1 cells containing a bicistronic HCV replicon NK5.1-
ture for overnight. The mixture was then poured in 1 N aq. Gluc cell line (genotype 1b) were employed for the interroga-
64
2 2
HCl solution and extracted with CH Cl . The organic layer tion of the anti-HCV activities of the compounds. The rst
dried over magnesium sulfate, and then was puried by the open reading frame (ORF) of the replicon contains the Gaussia
column chromatography (n-hexane/EA). The residue was luciferase gene fused with foot-and-mouth disease virus (FMDV)
1
obtained as white solid; yield (479 mg, 96%); H NMR 2a gene and the neomycin phosphotransferase gene, and the
(
(
DMSO-d
4 s, 0.38H + 0.53H + 0.89H + 0.31H), 7.91–7.84 (m, 2H), replicon-containing cells were cultivated under the same
.75–7.58 (m, 3H), 7.50–7.44 (m, 2H), 7.41–7.29 (m, 5H), conditions as described above in presence of an additional
6
, d ¼ 2.5 ppm, 500 MHz): d 10.52/10.45/10.36/10.27 second ORF contains HCV nonstructural genes NS3-5. The
7
7
5
0
4
2
2
1
1
ꢁ
1
.23–7.04 (m, 2H), 6.44 (s, 1H), 6.39–6.34 (t, 1H), 5.50–5.45/ antibiotic G418 (0.5 mg mL , Calbiochem).
.32–5.30/5.21–5.19/5.09/4.88–4.87 (q/d/d/s/d, 0.84H
+
.07H + 0.05H + 0.03H + 0.10H), 4.54–4.25 (m, 4H), 4.13–
.07 (m, 1H), 3.81–3.56 (m, 3H), 3.53 (s, 3H), 3.20–3.00 (m,
Antiviral activity assay test with HCV replicon
H), 2.82–2.74 (m, 1H), 2.58–2.56 (d, 1H), 2.33–2.26 (m, 2H), Huh7.5.1 cells containing HCV replicon (NK5.1-Gluc) were
.17–2.12 (m, 1H), 2.17–1.96 (m, 2H), 1.91–1.81 (m, 5H), planted on a 12-well plate. At 16 h aer cultivation, the replicon
.64–1.58 (m, 1H), 1.51–1.44 (m, 3H), 1.40–1.33 (m, 2H), containing NK/R2AN-containing cells were incubated with
1
3
.23 (s, 1H); C NMR (DMSO-d , d ¼ 39.52 ppm, 126 MHz): media containing serially diluted compounds for 3 days. Aer
6
d 171.41, 170.81, 168.35, 162.71, 156.31, 149.87, 140.88, the chemical treatment, the cell culture media were collected
1
1
5
36.79, 130.62, 128.57, 128.33, 128.00, 118.22, 115.23, and luciferase activities were measured through the use of the
13.71, 111.17, 61.05, 60.61, 60.01, 59.19, 56.61, 55.47, Renilla luciferase assay system (Promega) according to manu-
1.53, 46.93, 33.35, 29.47, 29.20, 28.20, 24.46, 24.24; HRMS facturer's direction, and then normalized to those obtained
+
(
ESI) m/z: anal. calcd for [M + Na]
C
42
H
49
N
7
NaO11
S
2
:
from mock-treated cells.
9
15.0012; found 914.2826.
Molecular docking
The X-ray structure of 3FQQ and NMR structure of 1R7G were
retrieved from the protein data bank (PDB) and prepared via
homology modeling with Discovery Studio Client. The default
Measurement of the anti-HCV activities of compounds using
HCVcc
Cell line and cell culture. Huh 7.5.1 cells were grown in Dul- ligand settings were used to prepare all tautomers, and further
becco's Modied Eagle's medium (DMEM; Gibco) containing energy minimized. The inhibitors were then docked into each of
ꢁ
1
ꢁ1
antibiotics (100 U mL penicillin, 10 mg mL streptomycin) the NS5A dimer models using the CDOCKER.
31818 | RSC Adv., 2018, 8, 31803–31821
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1
1
5 T. I. Ng, P. Krishnan, T. Pilot-Matias, W. Kati, G. Schnell,
J. Beyer, T. Reisch, L. Lu, T. Dekhtyar, M. Irvin,
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H. Shen, W. Song, Y. Zhang, X. Wang, H. Xu and Y. Zhang,
Eur. J. Med. Chem., 2018, 148, 95–105.
Conflicts of interest
There are no conicts of interest to declare regarding this study.
Acknowledgements
We gratefully acknowledge the nancial support of the Bio R&D
Program
(No.
2012M3A9A9054974
and
No.
2
017M3A9F6029755) through the National Research Founda-
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