Discovery of a Silicon-Containing Pan-Genotype Hepatitis C Virus NS5A Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.0c00082

We describe a study leading to the discovery of compound 11, a pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with excellent potency, metabolic stability, and pharmacokinetics (PK). Compound 11 incorporating a 4-silapiperidine group was discovered by further optimizing our previous lead with a triethylsilyl moiety. It displayed great potency against genotype 1 subtype a (GT1a), -1b, -2a, -3a, -4a, -5a, and -6a with an EC50 range of 0.33-17 pM and improved potency against the resistance-associated variant GT1a-M28T. Pharmacokinetics (PK) study indicated that compound 11 has reasonable PK exposures with a high liver distribution in preclinical animal species (mouse, rat, and dog). The results of a 14 day repeat-dose toxicity study identified the safety of compound 11.

Full text of DOI:10.1021/acs.jmedchem.0c00082

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Article
Discovery of A Silicon-containing Pan-
genotype Hepatitis C Virus NS5A Inhibitor
Baomin Liu, Kuo Gai, Hui Qin, Jie Wang, Xushi Liu, Yuan Cao, Qin Lu, Dandan Lu, Deyang
Chen, Hengqiao. Shen, wei Song, Jia Mei, Xiaojin Wang, Hongjiang Xu, and Yinsheng Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00082 • Publication Date (Web): 15 Apr 2020
Downloaded from pubs.acs.org on April 16, 2020
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Discovery of A Silicon-containing Pan-genotype Hepatitis C Virus NS5A Inhibitor
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Baomin Liu, Kuo Gai, Hui Qin, Jie Wang, Xushi Liu, Yuan Cao, Qin Lu, Dandan Lu, Deyang
Chen, Hengqiao Shen, Wei Song, Jia Mei, Xiaojin Wang, Hongjiang Xu, Yinsheng Zhang*
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Jiangsu Key Laboratory of Targeted Antiviral Research, Chia Tai Tianqing Pharmaceutical
Group Co., LTD, 1099 Fuying Road, Jiangning District, Nanjing 211122, Jiangsu Province,
China
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ABSTRACT: We describe the study leading to the discovery of compound 11, a pan-genotypic
HCV NS5A inhibitor with excellent potency, metabolic stability, and pharmacokinetics.
Compound 11 incorporating a 4-silapiperidine group was discovered by further optimizing our
previous lead with a triethylsilyl moiety. It displayed great potency against GT1a, -1b, -2a, -3a,
-4a, -5a and -6a with an EC50 range of 0.33~17 pM and improved potency against resistance-
associate variant GT1a_ M28T. Pharmacokinetics (PK) study indicated that compound 11 has
reasonable PK exposures with a high liver distribution in preclinical animal species (mouse, rat
and dog). The results of a 14-day repeat-dose toxicity study identified safety of compound 11.
INTRODUCTION
The World Health Organization (WHO) estimates that about 71 million people worldwide
are infected with hepatitis C virus (HCV).1 HCV infection often leads to severe liver diseases
including cirrhosis, hepatocellular carcinoma and other complications.2 Traditional therapy for
HCV infection relies upon a combination of pegylated interferon-α and ribavirin, a poorly
tolerated regimen with relative low sustained virologic response (SVR) rates (40-65%).3 In
recent years, a significant improvement in treating HCV has been made due to the development
of direct-acting antiviral agents (DAAs) with higher rates of SVR and less side effects, such as
6
the NS3/4A protease inhibitors simeprevir,4 paritaprevir,5 grazoprevir and glecaprevir,7 the
NS5B polymerase inhibitors sofosbuvir 8 and dasabuvir,9 the NS5A inhibitors daclatasvir,10
ombitasvir,11 ledipasvir,12 elbasvir, 13 velpatasvir8 and pibrentasvir 14 (Figure 1). Administration
of a combination of all oral DAAs has become the current treatment paradigm for HCV with
improved efficacy, shorter treatment duration and less adverse effects. Several DAA-based
regimens have been approved for the treatment of pan-genotypic HCV infection with SVR rates
over 95% only requiring 12 or 8 weeks of therapy, such as sofosbuvir/velpatasvir,8,15
grazoprevir/elbasvir 16 and glecaprevir/pibrentasvir.17
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Hepatitis C virus nonstructural protein 5A (NS5A) has been treated as an attractive viral
target for HCV therapy. NS5A has no known enzymatic activity, but plays an essential role in
HCV RNA replication and virus assembly.18 The mechanism of HCV NS5A inhibitors remains
unclear. In the absence of enzymatic activity for NS5A, the identification of inhibitors has been
driven by cell-based replicon activities. The NS5A inhibitors have become the most commonly
used DAAs combined with an NS5B polymerase inhibitor and/or an NS3/4A protease inhibitor
for treating patients who have HCV. First generation NS5A inhibitors demonstrated high SVR
rates in patient with HCV genotype 1 subtype a (GT1a) while exhibited weak activity against
several other genotypes. In addition, most of NS5A inhibitors approved for market showed a
low resistance barrier to GT1a.19 Therefore, many research groups have been working toward
the discovery of more potent HCV NS5A inhibitors against a wider variety of genotypes as well
as NS5A resistance mutations particularly in the prevalent GT1a.
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Incorporation of a silicon atom into drug molecules provides an innovative avenue and a
source of chemical diversity in drug discovery.20 Silicon has a larger covalent radius than carbon,
and also a larger carbon-silicon bond length compared to the corresponding carbon-carbon bond.
Silicon-containing analogs are more lipophilic than their carbon counterparts. These unique
physicochemical properties of silicon render it ideal for medicinal applications that no other
element can effectively achieve.21 Our previous research efforts have identified a pan-genotype
22
HCV NS5A inhibitor 1 through a “silicon-carbon switch” strategy (Figure 2). Despite its
potency against HCV genotypes 1a, 1b, 2a, 3a, 4a, and 5a in single picomolar range, 1 showed
relatively low activity against 6a with sub-nM activity. In a continued effort to develop pan-
genotype HCV NS5A inhibitors, we explored the impact of structural modifications to 1 (Figure
2), with the goal of improving the potency against GT6a and maintaining the good potency
against the other genotypes. Herein, we disclose a novel series of silicon-containing NS5A
inhibitors and identification of compound 11 with pan-genotypic potency and ideal PK profiles.
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N
N
N
O
H
O
NH
O
N
2HCl
O
N
N
HN
NH
N
N
O
O
8
9
HN
O
HN
O
O
NH
NH
O
O
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O
O
O
Ombitasvir
Daclatasvir
NH
HN
N
F
F
N
H
N
N
N
O
N
N
N
O
N
H
N
O
O
N
O
O
HN
HN
NH
O
O
NH
O
O
O
O
O
Ledipasvir
Elbasvir
F
O
O
NH
O
N
O
N
F
F
O
H
N
N
N
F
F
HN
N
O
HN
NH
O
N
NH
N
N
O
N
N
Velpatasvir
O
O
O
O
O
O
NH
HN
Pibrentasvir
O
O
Figure 1. Chemical structure of HCV NS5A inhibitors approved by FDA.
Figure 2. Chemical structure of compound 1 and structural modifications strategy for novel
NS5A inhibitor design.
RESULTS AND DISCUSSION
In our previous work, compound 1 was discovered as a silicon-containing pan-genotype
HCV NS5A inhibitor with considerably less potency against GT6a than GT1~5 22. We sought
to explore the impact of silicon-containing groups as “X groups”, and the “cap” groups as “Y
groups”, with a goal of improving the potency against GT6a (Figure 2). Replacing the Moc-
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Val-“end-capping” group of compound 1 with the Moc-Thr(OMe)-capping group led to
compound 3 and modifying the 4-position of the central N-phenyl ring with silyl groups yielded
compound 2 and 4 (Table 1). These compounds were tested in GT1a, GT1b, GT3a and GT6a
sub-genomic replicon assays. In vitro biological data for those compounds are demonstrated in
Table 1. As indicated in the Table 1, all the compounds provided picomolar activity against the
genotypes GT1a, -1b, -3a, and new compounds 2, 3 & 4 delivered an obvious improvement in
performance against GT6a (80~274 pM vs. 817 pM). However, those compounds still exhibited
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Table 1. In Vitro Activity of Compounds 1-6 against HCV GT1a, GT1b, GT3a and
GT6a in the Replicon Assay
R1
H
H
N
N
(S) N (S)
N
N
H
H
O
O
N
O
O
N
O
O
Y
O
O
Y
Replicon EC50 (pM) a
Compound
R1
Y
GT1a
11.0
GT1b
2.80
GT3a
5.80
GT6a
817
1b
SiEt3
2
3
4
SiMe3
12.0
2.30
7.90
1.60
1.00
1.40
7.00
2.00
3.00
247
121
80.0
H3CO
H3CO
H3CO
SiEt3
SiMe2t-Bu
a
b
Mean of triplicate well values. EC50 stands for 50% effective concentration. In vitro
biological data for compound 1 are slightly different from previous report and the differences
might result from experimental error.
relatively weaker potencies against GT6a than other genotypes. Compared with compound 1,
Moc-Thr(OMe)-cap analogue 3 showed potency improvement across the genotypes tablulated.
Particularly, the compound 3 was approximately 7-fold more potent than compound 1 against
GT6a, suggesting that the incorporation of O-methylthreonine might be beneficial for GT6a
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inhibition. Of the three new compounds, compound 4 which has a SiMe2t-Bu group at the 4-
position of the central N-phenyl ring showed the best activity against GT6a. It was
approximately 10-fold more potent than compound 1 against GT6a. While the SiMe3-
substituted analogue 2 showed the least potency improvement in GT6a compared with 3 and 4.
There appears to be a general trend that bulky substituents at the 4-position of the central N-
phenyl ring correlate with an improved ability to suppress GT6a.
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Improvement in potencies against GT6a was attainable by modifying “X group” and “Y
group”, while retaining potent activities against GT1a, GT1b and GT3a. Our primary results
gave us confidence that greater potencies with comparable activities across the various HCV
genotypes could be achieved by exploring modifications of “X group” and/or “Y group”. We
next followed the trend mentioned above to introduce more bulky sila-groups to the 4-position
of the central N-phenyl ring with the aim to obtain potent pan-genotype inhibitors. Analogues
with a 4-silapiperidine group at the 4-position of the central N-phenyl ring were synthesized
and evaluated in our research. In order to avoid the possible undesired PK properties, a fluorine
atom was introduced to 3-position of the central N-phenyl ring. As demonstrated in Table 2,
the compounds were tested as either the pure 2S, 5S or 2R, 5R diastereomers. The valine capped
compounds 5-8 showed high potent activities against GT1a and GT1b, with replicon potencies
in the 0.4-0.9 pM range of EC50. A comparison of the 2S, 5S trans-pyrrolidine compound 5 with
the 2R, 5R isomer 6 revealed that the stereochemistry at carbons 2 and 5 was unrelated with
their potencies against GT1a and GT1b. Difference was also not observed between the 2S, 5S
trans-pyrrolidine compound 7 and its 2R, 5R counterpart 8 in GT1a and GT1b inhibition. The
trend was not in line with what we and others reported that a stereochemical dependence for
potencies of compounds against GT1a.22,23 However, the antiviral activities of the valine capped
compounds 5-8 against GT3a and GT6a revealed a stereochemical dependence. The 2S, 5S
isomer 5 was approximately 5.8- and 4.6-fold more potent than the 2R, 5R isomer 6 against
GT3a and GT6a, respectively. Likewise, the 2S, 5S isomer 7 was more active against GT3a
(3.4-fold) and GT6a (5.3-fold) than the 2R, 5R isomer 8. Comparison of compound 5 and 7
revealed that introduction of additional fluorine atom to the 5-position of the central N-phenyl
ring of the valine capped compounds barely affected the potencies against GT1a, GT1b and
GT3a, but led to 2-fold improved potency against GT6a.
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Testing the O-methylthreonine capped compounds also demonstrated a stereochemical
dependence for their replicon potencies. The 2S, 5S isomer 9 was 1.9- to 5.5-fold more potent
against genotypes tested than the corresponding 2R, 5R isomer 10. Activity comparison of
9
Table 2. In Vitro Activity of Compounds 5-12 Against HCV GT1a, GT1b, GT3a and
GT6a in the Replicon Assay
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Si
N
R2
F
H
H
N
N
N
N
N
H
H
O
O
2
5
O
N
N
O
O
O
O
Y
Y
O
Replicon EC50 (pM) a
Compounds
Configuration
R2
Y
GT1a
0.800
GT1b
0.400
GT3a
8.80
GT6a
182
5
2S,5S
H
H
F
6
7
8
9
2R,5R
2S,5S
2R,5R
2S,5S
0.800
0.900
0.900
12.0
0.500
0.400
0.400
2.90
51.0
11.0
37.0
6.00
831
88.0
463
242
F
H
H3CO
H3CO
H3CO
H3CO
10
11
12
2R,5R
2S,5S
2R,5R
H
F
F
65.0
1.20
12.0
16.0
1.70
1.70
14.0
3.00
4.80
454
17.0
114
a Mean of triplicate well values. All experiments were performed at least twice. EC50 stands for
50% effective concentration.
compound 11 with 12 revealed a similar trend of increased potency for the 2S, 5S isomer against
genotypes tested vs the corresponding 2R, 5R isomer, with the exception of that the potencies
of the two isomers agaist GT1b remained unchanged. Compound 12 (2R, 5R isomer) was
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significantly less active against GT1a (10-fold) and GT6a (6.7-fold) than the compound 11 (2S,
5S isomer), with similar activity against GT3a. The compound 11 exhibited significantly
improved activity against GT6a (14.2-fold) vs the compound 9, indicating that introduction of
additional fluorine atom to the 5-position of the central N-phenyl ring contributed to the
improvement of potency against GT6a.
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As a result of its desirable potency shown in Table 2, compound 11 was selected for further
in vitro virologic evaluation. As shown in Table 3, 11 demonstrated picomolar potency across
GT1~GT6, with replicon potencies in the 0.33-3.0 pM range for GT1-GT5 and 17 pM for GT6a
. To determine the in vitro resistance profile of compound 11, replicon resistance selection was
conducted with 11 in HCV replicons containing NS5A from GT1a or GT1b. As indicated in
Table 4, the greatest resistance emerged with the Q30E, Y93H, Y93C, and Y93N variants in
genotype 1a. GT1a_Q30E, GT1a_Y93H, GT1a_Y93C , and GT1a_Y93N conferred 175.3-
to >833-fold resistance to 11. Examination of the data reveals that 11 demonstrated a similar
potency against resistant variants in GT1a when compared to the known NS5A inhibitor
ombitasbir, although there are some differences in fold-resistance and variants selected. One
particular exception is found for GT1a_ M28T, which was susceptible to 11 (1.9-fold) but
showed high fold resistance for Ombitasbir.23 Variants of genotype 1b, where tested, showed
lower fold resisitance to 11 when compared to variants of genotype 1a, especially for Y93H
(>833 vs 12.2) and L31V (>23 vs 2.3). GT1b_L31W and GT1b_ L31V conferred 11.4- and 2.3-
fold resistance, respectively. GT1b_ P32L remained fully susceptible to inhibition by 11.
Table 3. Antiviral Activity of 11 in HCV Replicon Cell Lines Containing NS5A from
GT1-6
Replicon EC50 (pM) a
GT1a
1.2
GT1b
1.7
GT2a
0.38
GT3a
3.0
GT4a
0.33
GT5a
1.6
GT6a
17
a Mean of triplicate well values. All experiments were performed at least twice. EC50 stands for
50% effective concentration.
Table 4. Activity of 11 against HCV Genotype 1a and 1b NS5A Resistance-associated
Variants in vitro
HCV genotype
variant
M28T
EC50 (pM) a
2.37
Fold-resistance b
1.90
1a
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L31M
L31V
Q30K
Q30E
Y93H
Y93C
Y93N
12.2
27.0
9.90
23.0
30.5
175
37.5
216
>1000
>250
>250
>833
>203
>203
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1b
Y93H
L31W
L31V
P32L
20.4
19.1
3.81
1.64
12.2
11.4
2.30
1.00
a As determined in transient-transfection assays.
b Fold-resistance is relative to the value of the respective wild-type replicon.
Given its in vitro pan-genotypic potency, we turned our attention to pharmacokinetic (PK)
profiles of compound 11. In vitro metabolic stability in mouse, rat and human microsomes
(MLM, RLM and HLM, respectively) for compound 11 and ombitasvir was shown in Table 5.
The rat and human microsomal stability of compound 11 was similar to ombitasvir.
Table 5. In Vitro Metabolic Stability Data
compound
MLM (% remaining a)
RLM (% remaining a)
HLM (% remaining a)
11
60.4
63.5
76.3
73.5
80.9
82.5
Ombitasvir
a Percentage of parent compound remaining after 60 min of incubation.
Pharmacokinetic characterization of compound 11 in mouse was summarized in Table 6.
Its T1/2 (half-life) and AUC (area under curve) were similar to ombitasvir. A low oral
bioavailability (7.2%) of compound 11 was observed, with similarity to the reported oral
bioavailability of pibrentasvir (7.9%),24 an approved pan-genotype HCV NS5A inhibitor. Dog
PK profile of compound 11 was also investigated. As shown in Figure 3 and Table 7, compound
11 demonstrated a longer T1/2 in dog than in mouse (21.2 vs 12.2). The AUC was also acceptable
considering its picomolar level potency against GT1~6 and the similar AUCs with ombitasvir.
Table 6. Pharmacokinetics of Compound 11 in Mouse a
iv
po
compound
dose
T1/2
(h)
Vc
(L/kg)
AUClast
CLp
dose
T1/2
(h)
Tmax
(h)
Cmax
(ng/mL)
AUClast
F (%)
(mg/kg)
(ng·h/mL)
(L/h/kg)
(mg/kg)
(ng·h/mL)
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2
-
12.2
1.56
-
17863
-
0.1
-
10
10
8.0
7.7
7.3
3.0
454
583
6446
6480
7.2%
-
b
ombitasvirc
-
aFormulation: po 2:2:96 (v/v) Alcohol : Tween 80 : Glucose injection, iv: diluted po formulation for 2.5
times with 10% glucose injection. b Not tested. c The data was disclosed in our previous report.22
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Figure 3. PK study of compound 11 in dog (PO 5 mpk).
Table 7. Dog PK Profile of Compound 11 after Oral Administrationa
dose (mpk)
T1/2 (h)
Tmax (h)
Cmax
(ng/mL)
395
AUC0-48h
(ng·h/mL)
8020
AUC0-∞
(ng·h/mL)
10104
MRT (h)
17.5
5
21.2
5.3
aFormulation: Phosal 53 MCT / PEG-400 / Poloxamer 124 / Cremophor RH40 (40/20/20/20,
by weight).
In order to learn more metabolic properties, the liver distribution of both 11 and
pibrentasvir were evaluated in mouse and rats. The concentrations of 11 and pibrentasvir in
plasma and liver after oral administration are summarized in Table 8. At each time point, the
concentration of 11 in mouse and rat plasma was lower than that of pibrentasvir, but the
situation in the liver was just the opposite. The maximum concentration of 11 and pibrentasvir
occurred at 3 h in plasma and liver. The liver concentration of 11 was much higher than its
plasma concentration at the same time point, suggesting 11 was a NS5A inhibitor with high
liver distribution. The mean plasma concentration ratios (LPRs) of 11 at 3 h in mouse and rat
were 24.6 and 5.2, respectively, suggesting that 11 can distribute into liver easily. By contrast,
LPRs of pibrentasvir were much lower in mouse and rat, with LPRs of 0.39 and 1.1 at 3 h in
mouse and rat, respectively, indicating that pibrentasvir did not possess liver targeting effect.
Table 8. Liver Distribution of 11 and Pibrentasvir in Mouse and Rat (mean ± SD, n = 3)a
11
Pibrentasvir
Liver
species
Time (h)
Plasma
Liver
LPRb
Plasma
LPRb
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(ng/ml)
(ng/g)
(ng/ml)
(ng/g)
0.5
3
160 ± 57.5
308 ± 57.6
58.6 ± 6.8
436 ± 126
1585 ± 131
625 ± 147
8.8
414 ± 97.9
1541 ± 171
718 ± 114
94 ± 42
193 ± 67
69.6 ±13
0.43
0.39
0.11
mouse
24.6
17.9
24
9
10
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0.5
3
40.9 ± 16.2
45.2 ± 7.2
16.4 ± 6.4
359 ± 107
1111 ± 202
293 ± 39.1
2.7
5.2
103 ± 17.0
96.4 ± 40.2
30.1 ± 5.85
155 ± 23.2
102 ± 50.6
18.9 ± 4.71
1.5
1.1
rat
8
10.7
0.63
a Dosing at 10 mg/kg. b Mean liver concentration / Mean plasma concentration ratio (LPR).
The in vitro cell toxicity of compound 11 was assessed by using Huh7 cells. CC50 value of
compound 11 was determined to be > 50 μM, indicating no cell toxicity was observed in the
assay.
To evaluate the safety of compound 11, a 14-day repeated oral dose toxicity study was
conducted and the results were available in supporting information. Mice were administered 11
once daily by gavage at a high dose of 300 mg/kg. All animals were observed daily and detailed
clinical signs untill they were killed after the 14th adminstration. As demonstrated in Table 9,
there was no statistically significant difference in body weight between the control and 11. In
addition, no abnormality was observed on the organs. Also, hematology data showed no
statistically significant difference between the control and 11 (Table 10). Blood chemistry data
indicated a statistically significant increase of cholesterol in 11 (P < 0.01) (Table 11), with no
abnormality in other liver-related indicators. In summary, animal studies identified no toxicity
with 11.
CHEMISTRY
As shown in Scheme 1, compounds 2-4 (Table 1) were synthesized starting from silicon-
containing di-aniline compounds 13a, 13b and 13c, which were prepared according to previous
methods and purified as single 2S, 5S isomers.22 The amide side chains were attached to the
central pyrrodine core by peptide-coupling compounds 13a, 13b and 13c with N-
(methoxycarbonyl)-O-methyl-L-threonyl-L-proline 14.
Scheme 1a. Preparation of Compound 2-4
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8
9
R
N
R
N
OH
H
H
N
N
N
O
N
O
O
N
O
H2N
NH2
+
O
O
O
O
O
O
NH
OMe
O
NH
OMe
HN
O
2: R = SiMe3
3: R = SiEt3
4: R = SiMe2t-Bu
14
13a : R = SiMe3
13b : R = SiEt3
13c : R = SiMe2t-Bu
10
11
12
13
14
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17
18
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52
53
54
55
56
57
58
59
60
OMe
a Reagents and conditions: EDCI, HOBt, NMM, DMF, 60 °C, 2 h, 29-47% yield.
Compounds 5-8 (Table 2) were obtained using the preparation illustrated in Scheme 2. 4,4-
Dimethyl-1,4-azasilinane hydrochloride 15, 3,4-difluoronitrobenzene 16a and 3,4,5-
trifluoronitrobenzene 16b were purchased, while (1R,4R)-1,4-bis(4-nitrophenyl)butane-1,4-
diyl dimethanesulfonate and (1S,4S)-1,4-bis(4-nitrophenyl)butane-1,4-diyl dimethanesulfonate
were prepared based on known procedures.22 16a and 16b was treated with 15 under alkaline
condition to give nitro compounds 17a and 17b, respectively, which were in turn reduced by
hydrogen under Pd/C to the corresponding aniline compounds 18a and 18b. The aniline
compounds were reacted with S, S-dimesylate or R, R-dimesylate to give R, R-pyrrolidine
derivatives (19b, 19d) or S, S-pyrrolidine derivatives (19a, 19c), respectively. Reduction of the
nitro groups by catalytic hydrogenation gave di-aniline compounds (20a, 20b, 20c and 20d).
Hydrogenation with platinum (IV) oxide was the best condition to minimize stereochemical
isomerization. A (methoxycarbonyl)-L-valyl-L-proline moiety was finally introduced to give
5~8. As demonstrated in Scheme 3, compound 9~12 were also synthesized in a similar method
by switching the valine and O-methylthreonine “caps” in the preparation.
Scheme 2a. Preparation of Compound 5-8
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9
Si
N
Si
N
F
R
F
R
F
R
F
Si
i
iii
ii
+
N
H
.HCl
10
11
12
13
14
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16
17
18
19
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22
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NO2
NO2
17a : R = H
17b : R = F
NH2
18a R = H
18b R = F
15
16a : R = H
16b : R = F
OH
Si
Si
N
N
O
O
N
O
NH
R
F
R
F
21
iv
OMe
O2N
NO2
H2N
NH2
v
N
5
N
2
5
2
19a : R = H, 2S/5S
19b : R = H, 2R/5R
19c : R = F, 2S/5S
19d : R = F, 2R/5R
20a : R = H, 2S/5S
20b : R = H, 2R/5R
20c : R = F, 2S/5S
20d : R = F, 2R/5R
Si
N
R
F
H
H
N
N
N
N
N
O
O
2
5
O
O
5 : R = H, 2S/5S
6 : R = H, 2R/5R
7 : R = F, 2S/5S
8 : R = F, 2R/5R
O
NH
OMe
HN
O
OMe
a Reagents and conditions: (i) triethylamine, DMF, r.t, 12 h; (ii) H2, 10% Pd/C, THF, r.t, 12 h;
(iii) (1R,4R)-1,4-bis(4-nitrophenyl)butane-1,4-diyl dimethanesulfonate (19r) or (1S,4S)-1,4-
bis(4-nitrophenyl)butane-1,4-diyl dimethanesulfonate (19s), DMF, 60 °C, 24 h; (iv) H2,
platinum dioxide, THF, r.t, 3 h; (v) EDCI, HOBt, N-methylmorpholine, DMF, 55 °C, 2 h.
Scheme 3a. Preparation of Compound 9-12
Si
N
Si
N
OH
R
F
R
F
O
N
O
H
H
+
O
N
N
H2N
NH2
N
N
O
NH
OMe
O
N
N
O
O
O
2
5
2
5
O
O
14
20a : R = H, 2S/5S
20b : R = H, 2R/5R
20c : R = F, 2S/5S
20d : R = F, 2R/5R
9 : R = H, 2S/5S
10 : R = H, 2R/5R
11 : R = F, 2S/5S
12 : R = F, 2R/5R
O
NH
OMe
HN
O
OMe
a Reagents and conditions: EDCI, HOBt, N-methylmorpholine, DMF, 60 °C, 2 h.
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CONCLUSIONS
6
7
8
9
In summary, we have disclosed several novel silicon-containing N-phenylpyrrolidines as
HCV NS5A inhibitors. By incorporating a 4-silapiperidine group with a O-methylthreonine
“cap” we identified compound 11 as a HCV NS5A inhibitor with significantly improved
potency against the GT 6a (EC50 = 17 pM). This lead also showed an excellent in vitro profile
in many other genotypes with an EC50 range of 0.33-3.0 pM against GT 1a, 1b, 2a, 3a, 4a and
5a. An improved potency against variant GT1a_ M28T was also observed for compound 11
when compared to ombitasvir. Compound 11 showed a good stability profile in in mouse, rat
10
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and human microsomes, and furter in vivo studies revealed that 11 also demonstrated an
acceptable rat and dog PK profile in combination with high liver distribution in both mouse
and rat. Results of the cytotoxicity test and the 14-day repeat-dose toxicity study identified the
good safety profile of compound 11. All the encouraging results suggested that compound 11
is a promising silicon-containing pan-genotype HCV NS5A inhibitor.
EXPERIMENTAL SECTION
Replicon Assays. Detailed replicon biological assays were conducted according to our previous
report.22 The methods to measure the effects of individul amino acid variants on the activity of
an inhibitor in HCV replicon cell culture assays were described previously. Briefly, the
resistance-associated variants in NS5A were each introduced into GT 1a-H77, GT 1b-Con1, or
chimeric replicons. In transient assays, the replicon containing the variant was transfected via
electroporation into a Huh-7 derived cell line. The inhibitory effect of compounds on HCV
replication was determined by measuring decrease in luciferase signal. The percent inhibition
of HCV replicon replication was calculated for each compound concentration and the EC50
value was calculated using nonlinear regression curve fitting to the 4-parameter logistic
equation in the GraphPad Prism software.
Pharmacokinetic Profiles. In vitro PK assessment was performed using pooled mouse, rat and
human liver microsomes obtained from BD Gentest. A typical standard reaction mixture 300
mL consisted of the pooled liver microsomes 0.5 mg/mL, 1 mM NADPH, 5 mM MgCl2, 100
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mM potassium phosphate buffer (pH 7.4) and 1 μM of test compounds. After a 5 min pre-
incubation at 37 °C, the reactions were initiated by addition of NADPH and incubation
proceeded for 60 min at 37 °C in a shaking metal bath. The reaction was stopped by transferring
60 mL aliquots to the tubes on ice and adding 120 mL amounts of ice-cold acetonitrile
containing internal standards. Concentration of the test compounds was measured by UPLC-
MS/MS.
10
11
12
13
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19
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In vivo PK assessment: Compound 11 was dosed in overnight fasted mice, rats or dogs via
iv and po administration. Unless otherwise noted, compound 11 was formulated in 2:2:96 (v/v)
Alcohol : Tween 80 : Glucose injection for single 10 mg/kg po dosing in mouse. And po
formulation was diluted by Glucose injection for 2.5 times as iv formulation for single 2 mg/kg
iv dosing in mouse. Compound 11 was formulated in Phosal 53 MCT / PEG-400 / Poloxamer
124 / Cremophor RH40 (40/20/20/20, by weight) for single 5 mg/kg po dosing in dog. The
concentration of compounds in the plasma and liver was measured by the ultra-performance
liquid chromatography-mass spectrometry analysis (UPLC-MS/MS).
Toxicity study. Cytotoxicity test was performed as previous method.22 Repeated-dose toxicity
study was carried out for 14 days. After acclimation, animals were randomly allocated to two
groups, each containing five males and five females, and orally administered compound 11 at
the dose of 300mg/kg or the vehicle alone once daily by gavage. Mice were killed after the 14th
administration. Animals in all groups were observed daily and detailed clinical signs evident.
Their body weights and food intake were monitored on day 1 of administration and thereafter
twice per week. Body weights were used for calculation of the volume of the test chemical to
be administered. Hematology, blood biochemistry and serum hormone measurements and
histopathology was performed according to previous methods.
Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and
used without further purification. 1 H NMR spectra (500 MHz) were collected on a Bruker-500
FT NMR spectrometer with tetramethylsilane (TMS) as internal standard. Mass spectra (HRMS)
were recorded on an AB SCIEX 3200 Q TRAP Mass Spectrometer. High resolution mass
spectra (HRMS) were recorded on an AB SCIEX Triplet TOF 4600 Mass Spectrometer. All
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final compounds were purified to > 96% purity as determined by HPLC analyse. Reverse phase
HPLC purity determinations were performed on an Agilent 1260 HPLC system. Compound 14,
15, 16a, 16b and 21 were purchased from commercial resources. Compounds 13a, 13b and
13c, (1S,4S)-1,4-bis(4-nitrophenyl)butane-1,4-diyl dimethanesulfonate, (1R,4R)-1,4-bis(4-
nitrophenyl)butane-1,4-diyl dimethanesulfonate and compound 1 were prepared according to
the reported methods.22 Ombitasvir and pibrentasvir were synthesized according to the reported
methods with 99% purity.23,24
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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Dimethyl
((2S,2'S,3R,3'R)-((2S,2'S)-(((((2S,5S)-1-(4-
(trimethylsilyl)phenyl)pyrrolidine-2,5-diyl)bis(4,1-
phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-
oxobutane-1,2-diyl))dicarbamate (2). A mixture of 13a (0.54 g, 1.35 mmol), N-
(methoxycarbonyl)-O-methyl-L-threonyl-L-proline (1.16 g, 4.04 mmol), EDCI (0.77 g, 4.0
mmol), HOBt (0.55 g, 4.04 mmol) and N-methylmorpholine (0.41 g, 4.04mmol) in DMF (10
mL) was stirred at 60 °C for 2 h. The mixture was diluted with ethyl acetate (50 mL) and washed
with water, brine and dried over sodium sulfate. The drying agent was filtered off, and the
solvent was concentrated in vacuo. The residue was purified by column chromatography on
silica gel using ethyl acetate to give compound 2 (0.36 g, 47%). 1H NMR (500 MHz, CDCl3) δ
8.87 (s, 2 H), 7.44-7.42 (m, 4 H), 7.13-7.11 (m, 4 H), 6.99-6.96 (m, 2 H), 6.33-6.31 (m, 2 H),
5.73-5.71 (m, 2 H), 5.14-5.13 (m, 2 H), 4.83-4.81 (m, 2 H), 4.69-4.67 (m, 2 H), 3.79-3.77 (m,
2 H), 3.75-3.72 (m, 4 H), 3.75-3.72 (s, 6 H), 3.34 (s, 6 H), 2.50-2.45 (m, 4 H), 2.06-2.01 (m, 8
H), 1.20 (d, J =6 Hz, 6 H ), 0.16 (s, 6 H), 0.08 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 170.2,
169.0, 156.8, 144.7, 139.5, 136.6, 128.6, 126.6, 120.0, 114.0, 62.7, 60.8, 60.4, 57.4, 55.5, 52.4,
48.0, 32.3, 27.6, 24.9, 14.7, 1.92, 1.32; MS (ESI) m/z 964.5 (M+Na)+; HPLC: 98.2% purity;
HRMS (ESI) m/z calcd for [M+H]+ C49H67N7O10Si: 942.4797, found 942.4854.
Dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-(((((2S,5S)-1-(4-(triethylsilyl)phenyl)pyrrolidine-
2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-
methoxy-1-oxobutane-1,2-diyl))dicarbamate (3). A mixture of 13b (0.60g, 1.35 mmol), N-
(methoxycarbonyl)-O-methyl-L-threonyl-L-proline (1.23 g, 4.05 mmol), EDCI (0.63 g, 4.05
mmol), HOBt (0.54 g, 4.05 mmol) and N-methylmorpholine (0.68 g, 6.75 mmol) in DMF (10
mL) was stirred at 60 °C for 4 h. The mixture was poured into water (50 mL), extracted with
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dichlormethane (50 mL× 3), and the combined organic layers were dried with Mg2SO4. The
drying agent was filtered off, and the solvent was concentrated in vacuo. The residue was
purified on C18 column (DAC 100*250mm,YMC Triart Prep C18-S), eluting with a mixture
9
1
of 82% acetonitril and 18% water to give compound 3 (0.43g, 32.4%). H NMR (DMSO-d6,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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41
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500 MHz) δ 9.93(s, 2H), 7.51 (d, J = 8.5 Hz, 4 H), 7.29 (d, J = 8.0 Hz, 2 H), 7.15 (d, J = 8.5
Hz, 4 H), 7.03 (d, J = 8.5 Hz, 2 H), 6.28(d, J = 8.5 Hz, 2 H), 5.19 (d, J = 6.5 Hz, 2 H), 4.43 (dd,
J = 5.0 Hz, 8.0 Hz, 2 H), 4.27 (d, J = 7.5 Hz, 2 H), 3.68 (m, 2 H), 3.54 (m, 2 H), 3.49 (s, 6 H),
3.47 (m, 2 H), 3.26 (s, 6 H), 2.46 (m, 2 H), 2.07 (m, 2 H), 1.99 (d, J = 6.5 Hz, 2 H),1.90 (m, 2
H), 1.88 (t, J = 5.0 Hz, 2 H), 1.63 (d, J = 5.5 Hz, 2 H), 1.15 (d, J = 6.0 Hz, 6 H), 0.83 (t, J = 8.0
Hz, 9 H), 0.60 (q, J =8.0 Hz, 6 H); 13C NMR (125 MHz, DMSO-d6) δ 170.6, 157.1, 145.7,
138.1, 134.8, 126.7, 119.6, 113.8, 76.8, 62.3, 60.6, 57.4, 56.6, 51.9, 47.8, 32.4, 29.9, 25.1, 15.9,
7.9, 3.5, 1.6; MS (ESI) m/z: 1006.5 [M + Na]+ ; HPLC: 99.9% purity; HRMS (ESI) m/z calcd
for [M+H]+ C52H73N7O10Si: 984.5266, found 984.5347.
Dimethyl
((2S,2'S,3R,3'R)-((2S,2'S)-(((((2S,5S)-1-(4-(tert-butyldimethylsilyl)phenyl)
pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-
diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamate (4). A mixture of 13c (0.50g, 1.1
mmol), N-(methoxycarbonyl)-O-methyl-L-threonyl-L-proline (0.98 g, 3.3 mmol), EDCI (0.65
g, 3.3 mmol), HOBt (0.46 g, 3.3 mmol) and N-methylmorpholine (0.34 g, 3.3mmol) in DMF
(10 mL) was stirred at 60 °C for 2 h. The mixture was diluted with ethyl acetate (50 mL) and
washed with water, brine and dried over sodium sulfate. The drying agent was filtered off, and
the solvent was concentrated in vacuo. The residue was purified by column chromatography on
silica gel using ethyl acetate and C18 column (DAC 100*250mm,YMC Triart Prep C18-S),
eluting with a mixture of 82% acetonitril and 18% water to give compound 4 (0.32 g, 29%) as
a white solid. 1H NMR (500 MHz, CDCl3) δ 8.90 (s, 2 H), 7.43 (d, J = 8.0 Hz, 4 H), 7.12 – 7.09
(m, 6 H), 6.29 (d, J = 8.0 Hz, 2 H), 5.72(d, J = 7.5 Hz, 2H), 5.12 (d, J = 5.0 Hz, 2 H), 4.81 (s,
2 H), 4.67 (s, 2 H), 3.78-3.68 (m, 10 H), 3.40-3.33 (m, 6 H), 2.49-2.44 (m, 4 H), 2.09-2.03 (m,
8 H), 1.74 (d, J = 4.5 Hz, 2 H), 1.20 (d, J = 4.0 Hz, 6 H), 0.78 (s, 9 H), 0.13 (s, 3 H), 0.11 (s, 3
H); 13C NMR (125 MHz, CDCl3) δ 170.3, 169.0, 156.8, 145.2, 139.7, 136.6, 135.0, 126.5, 121.9,
120.0, 113.3, 62.6, 60.8, 57.4, 55.5, 52.4, 48.0, 32.2, 27.5, 26.6, 24.9, 17.1, 14.7, 14.2, -6.1; MS
+
(ESI) m/z 1006.5 (M+Na) ; HPLC: 98.4% purity; HRMS (ESI) m/z calcd for [M+H]+
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C52H73N7O10Si: 984.5266, found 984.5347.
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1-(2-fluoro-4-nitrophenyl)-4,4-dimethyl-1,4-azasilinane (17a). To a suspension of 4,4-
dimethyl-1,4-azasilinane hydrochloride (15, 19.3g, 105.8mmol) in DMF (200mL) was added
triethylamine (48.2 g, 349.1 mmol) dropwise at room temperature and stirred for 10 min. Then，
3,4-difluoronitrobenzene (16.8 g, 105.8 mmol) was added dropwise at 0 °C, and the mixture
was stirred at room temperature overnight. The reaction solution was poured into water (1L)
and stirred for 10 min. The solid was collected by filtration and washed with water to give
compound 17a as a yellow solid (28.1g, 99.0%). 1H NMR (500 MHz, DMSO-d6) δ 7.91-7.95
(m, 2 H), 7.10 (d, J = 9.5 Hz, 1 H), 3.71 (t, J = 6.3 Hz, 4 H), 0.86 (t, J = 6.3 Hz, 4 H), 0.10 (s,
6 H). 13C NMR (125 MHz, DMSO-d6): δ 151.5, 144.3, 137.2, 122.1, 117.0, 113.3, 49.1, 13.7, -
2.4; MS (ESI) m/z 269.1 [M + H]+.
9
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4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-fluoroaniline (18a). To a solution of 1-(2-fluoro-
4-nitrophenyl)-4,4-dimethyl-1,4-azasilinane (17a, 28.0 g, 104.3 mmol) in THF (200 mL) was
added 10% palladium on carbon (5.0 g). The reaction container was flushed with N2, and the
mixture was stirred under 1 atm H2 overnight. The mixture was filtered through Celite, and the
filtrate was concentrated under vacuum to give a crude product that was purified by stirring in
petroleum ether. The title compound was obtained as a white solid (22.1 g, 88.9%). 1H NMR
(500 MHz, DMSO-d6) δ 6.75-6.79 (m, 1H), 6.25-6.31 (m, 2H), 4.90 (s, 2H), 3.04-3.06 (m, 4H),
0.79-0.81(m, 4H), 0.08 (s, 6H); 13C NMR (125 MHz, DMSO-d6) δ 155.8, 145.5, 131.8, 122.5,
109.9, 102.3, 52.2, 15.1, -2.4; MS (ESI) m/z 239.1 [M + H]+.
1-(4-((2S,5S)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2-fluorophenyl)-4,4-dimethyl-
1,4-azasilinane (19a). The mixture of (1R,4R)-1,4-bis(4-nitrophenyl)butane-1,4-diyl
dimethanesulfonate (1.5 g, 3.1 mmol) and 4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-fluoroaniline
(18a, 7.32 g, 31 mmol) in DMF (15 mL) was stirred at 60 °C for 24 h. The reaction solution
was poured into 2 N aq HCl (100 mL) and stirred for 10min. The solid was collected by
filtration and dissolved in dichloromethane (50 mL). The mixture was washed with water and
dried over sodium sulfate, filtered, and concentrated under vacuum to give a crude product that
was purified by column chromatography on silica gel using a solvent gradient of 10% ethyl
acetate in petrol to give 19a as a yellow solid (1.2 g, 75%). 1H NMR (500 MHz, CDCl3) δ 8.24
(s, 4 H), 7.36 (d, J = 8.0 Hz, 4 H), 6.71 (s, 1 H), 6.00 (m, 2 H), 5.29 (s, 2 H), 3.11 (s, 2 H), 2.63
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(s, 2 H), 1.96 (s, 2 H), 1.58 (m, 2 H), 0.85 (m, 4 H), 0.07 (s, 6 H); 13C NMR (125 MHz, CDCl3)
δ 144.7, 126.8, 124.4, 63.4, 32.1, 27.1, 11.1, -3.0; MS (ESI) m/z: 535.1 [M + H]+.
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4,4'-((2S,5S)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-fluorophenyl)pyrrolidine-2,5-
diyl)dianiline (20a). To a solution of 19a (1.67 g, 3.13 mmol) in THF (20 mL) was added
platinum dioxide (0.7 g, 3.13 mmol). The reaction container was flushed with N2, and the
mixture was stirred under 1 atm H2 for 3 h. The mixture was filtered through Celite, and the
filtrate was concentrated under vacuum to give a crude product that was purified by column
chromatography on silica gel using a solvent gradient of 0-20% ethyl acetate in petrol to give
20a as a white solid (1.2 g, 81%). 1H NMR (500 MHz , CDCl3): δ 6.95 (d, J = 8.0 Hz, 4 H),
6.64 (d, J = 8.5 Hz, 4 H), 6.06 (m, 3 H), 4.98 (d, J = 7.0 Hz, 2 H), 3.12 (s, 2 H), 2.50 (s, 2 H),
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1.75 (d, J = 5.0 Hz, 2 H), 1.26 (m, 2 H), 0.88 (m, 4 H), 0.12 (s, 6 H); C NMR (125 MHz,
CDCl3) δ 159.7, 147.6, 137.3, 131.4, 126.9, 120.7, 115.5, 114.4, 108.1, 107.9, 62.6, 52.1, 50.9,
32.9, 15.1, 14.5, -2.4, -2.5; MS (ESI) m/z 475.1 [M + H]+.
Dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-(((((2S,5S)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-
3-fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))
bis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-diyl))dicarbamate (9). The
mixture of 20a (0.53 g, 1.12 mmol), N-(methoxycarbonyl)-O-methyl-L-threonyl-L-proline
(0.97 g, 3.35 mmol), EDCI (0.64 g, 3.35 mmol), HOBt (0.45 g, 3.35 mmol) and N-
methylmorpholine (0.68 g, 6.75 mmol) in DMF (10 mL) was stirred at 55 °C for 12 h. The
mixture was poured into water (50 mL). The solid was collected by filtration and dissolved in
dichloromethane (50 mL). The mixture was washed with brine and dried over sodium sulfate,
filtered, and concentrated under vacuum to give a crude product that was purified by column
chromatography on silica gel using a solvent gradient of 0–66% ethyl acetate in petrol to give
9 as a white solid (0.5 g, 44%). 1H NMR (500 MHz , CDCl3) δ 9.93 (s, 2 H), 7.50 (d, J = 8.5
Hz, 4 H), 7.29 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.5 Hz, 4 H), 6.71 (m, 1 H), 5.96 (m, 2 H), 5.13
(d, J = 6.0 Hz, 4H), 4.44 (m, 2 H), 4.28 (m, 2 H), 3.82 (m, 2 H), 3.65 (m, 2 H), 3.54 (s, 6 H),
3.48 (m, 2 H), 3.26 (s, 6 H), 2.98 (m, 4 H), 2.45 (m, 2 H), 2.16 (m, 2 H), 2.09 (m, 2 H), 1.89
(m, 4 H), 1.62 (d, J = 5.5 Hz, 2 H), 1.14 (d, J = 6.0 Hz, 6 H), 0.73 (t, J = 5.5 Hz, 2 H), 0.04 (s,
6 H); 13C NMR (125 MHz, CDCl3) δ 170.6, 169.2, 157.1, 139.1, 138.1, 126.7, 119.7, 76.8, 62.6,
60.7, 57.4, 56.6, 52.0, 47.8, 32.6, 29.2, 25.1, 22.6, 16.0, 15.0, 14.4, -2.5; MS (ESI) m/z
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1015.7[M + H]+; HPLC: 96.0% purity; HRMS (ESI) m/z calcd for [M+H]+ C52H71FN8O10Si:
1015.5124, found1015.5170.
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1-(2,6-difluoro-4-nitrophenyl)-4,4-dimethyl-1,4-azasilinane (17b). To a suspension of
4,4-dimethyl-1,4-azasilinane hydrochloride (20.6g, 124.4 mmo) in DMF (200mL) was added
triethylamine (51.6g, 3373.0mmol) dropwise at room temperature and stirred for 10 min. Then
3,4,5-trifluoronitrobenzene (20.0 g, 113.0 mmol) was added dropwise at 0 °C and the mixture
was stirred at room temperature overnight. The reaction solution was poured into water (1L)
and stirred for 10min. The solid was collected by filtration and washed with water to give
compound 17b as a yellow solid (31.2 g, 96.4%). 1H NMR (500 MHz, DMSO-d6) δ 7.93 (dd,
J = 10.0 Hz, 1.0 Hz, 2 H), 3.49-3.51(m, 4H), 0.85-0.88(m, 4 H), 0.12 (s, 6 H); 13C NMR (125
MHz, DMSO-d6) δ 155.8, 153.9, 136.6, 109.4, 50.8, 15.1, -2.6; MS (ESI) m/z 287.1 [M + H]+.
4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-difluoroaniline (18b). To a solution of 1-(2,6-
difluoro-4-nitrophenyl)-4,4-dimethyl-1,4-azasilinane (31.0 g, 108.3 mmol) in THF (200 mL)
was added 10% palladium on carbon (5.0 g). The reaction container was flushed with N2, and
the mixture was stirred under 1 atm H2 overnight. The mixture was filtered through Celite, and
the filtrate was concentrated under vacuum to give a crude product that was purified by stirring
in petroleum ether. The title compound was obtained as a white solid (23.4 g, 84.3%). 1H NMR
(500 MHz, DMSO-d6): δ 6.10-6.12 (m, 2 H), 5.34 (s, 2 H), 3.10-3.13 (m, 4 H), 0.77-0.79 (m, 4
H), 0.08 (s, 6 H); 13C NMR (125 MHz, DMSO-d) δ 161.2, 159.3, 118.9, 97.3, 52.4, 15.7, -2.4;
MS (ESI) m/z 257.1 [M + H]+.
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1-(4-((2R,5R)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2-fluorophenyl)-4,4-dimethyl-
1,4-azasilinane (19b). The mixture of (1S,4S)-1,4-bis(4-nitrophenyl)butane-1,4-diyl
dimethanesulfonate (2.5 g, 5.1 mmol) and 4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-fluoroaniline
(9.8 g, 40.9 mmol) in DMF (20 mL) was stirred at 60 °C for 24 h. The reaction solution was
poured into 2 N aq HCl (100 mL) and stirred for 10min. The solid was collected by filtration
and dissolved in dichloromethane (50 mL). The mixture was washed with water and dried over
sodium sulfate, filtered, and concentrated under vacuum to give a crude product that was
purified by column chromatography on silica gel using a solvent gradient of 10% ethyl acetate
in petrol to give 19b as a brown solid (1.5 g, 53.6%). 1H NMR (500 MHz , CDCl3) δ 8.21(d, J
= 5.5 Hz, 4 H), 7.37 (d, J = 6.5 Hz, 4 H), 6.74 (s, 1 H), 5.99 (m, 2 H), 5.28 (s, 2 H), 3.11 (s, 2
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H), 2.59 (s, 2 H), 1.89 (s, 2 H), 1.58 (s, 2 H), 0.85 (s, 4 H), 0.07 (s, 6 H); 13 C NMR (125 MHz,
CDCl3) δ 147.4, 127.0, 124.1, 121.8, 109.1, 62.9, 51.9, 32.2, 14.8, -3.1; MS (ESI) m/z: 535.1
[M + H]+.
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4,4'-((2R,5R)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-fluorophenyl)pyrrolidine-2,5-
diyl)dianiline (20b). To a solution of 19b (1.5 g, 2.72 mmol) in THF (20 mL) was added
platinum dioxide (0.3 g, 1.36 mmol). The reaction container was flushed with N2, and the
mixture was stirred under 1 atm H2 for 3 h. The mixture was filtered through Celite, and the
filtrate was concentrated under vacuum to give a crude product that was purified by by column
chromatography on silica gel using a solvent gradient of 0-20% ethyl acetate in petrol to give
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20b as a white solid (0.6 g, 46.5%). H NMR (500 MHz, CDCl3) δ 6.95(d, J = 8.0 Hz, 4 H),
6.64(d, J = 8.0 Hz, 4 H), 6.05 (m, 3 H), 4.98 (s, 2 H), 3.11 (s, 4 H), 2.49 (s, 2 H), 1.74 (s, 2 H),
0.85 (m, 4 H), 0.09 (s, 6 H); 13C NMR (125 MHz, CDCl3) δ 144.9, 127.0, 115.3, 102.2, 62.9,
32.6, 29.7, -3.0; MS (ESI) m/z: 475.1 [M + H]+.
Dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-(((((2R,5R)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-
yl)-3-fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))
bis(azanediyl))bis(carbonyl))bis (pyrrolidine-2,1-diyl))bis(3-methoxy-1-oxobutane-1,2-
diyl))dicarbamate (10). The mixture of 20b (0.55 g, 1.2 mmol), N-(methoxycarbonyl)-O-
methyl-L-threonyl-L-proline (1.0 g, 3.5 mmol), EDCI (0.67 g, 3.5 mmol), HOBt (0.5 g, 3.5
mmol) and N-methylmorpholine (0.71 g, 7 mmol) in DMF (10 mL) was stirred at 55 °C for 12
h. The mixture was poured into water (50 mL). The solid was collected by filtration and
dissolved in dichloromethane (50 mL). The mixture was washed with brine and dried over
sodium sulfate, filtered, and concentrated under vacuum to give a crude product that was
purified by column chromatography on silica gel using a solvent gradient of 0–66% ethyl
acetate in petrol to give 10 as a white solid (0.55 g, 46.6%). 1H NMR (500 MHz, DMSO-d6) δ
9.94 (s, 2 H), 7.51 (d, J = 8.5 Hz, 4 H), 7.29 (d, J = 7.5 Hz, 2 H), 7.13 (d, J = 8.5 Hz, 4 H), 6.70
(m, 1 H), 5.96 (m, 2 H), 5.13 (d, J = 6.0 Hz, 2 H), 4.44 (dd, J = 5.0 Hz, 8.0 Hz, 2 H), 4.28 (t, J
= 7.5 Hz, 2 H), 3.69 (m, 2 H), 3.54 (m, 2 H), 3.49 (s, 6 H), 3.47 (d, J = 6.5 Hz, 2 H), 3.25 (s, 6
H), 2.98 (m, 4 H), 2.44 (m, 2 H), 2.15 (m, 2 H), 2.00 (d, J = 6.0 Hz, 2 H), 1.89 (m, 4 H), 1.62
(d, J = 5.5 Hz, 2 H), 1.13 (d, J = 6.5 Hz, 6 H), 0.74 (t, J = 6.0 Hz, 4 H), 0.04 (s, 6 H); 13C NMR
(125 MHz, DMSO-d6) δ 170.6, 169.2, 157.2, 155.3, 141.7, 139.1, 138.1, 131.1, 126.7, 122.1,
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119.6, 109.5, 101.9, 76.8, 62.5, 60.7, 57.5, 56.6, 52.0, 47.8, 32.6, 29.9, 25.1, 16.0, 15.0, 1.6, -
2.5; MS (ESI) m/z: 1037.50 [M + Na]+; HPLC: 97.9% purity; HRMS (ESI)m/z calcd for
[M+H]+ C52H71FN8O10Si: 1015.5124, found 1015.5170.
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1-(4-((2S,5S)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2,6-difluorophenyl)-4,4-
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dimethyl-1,4-azasilinane (19c). The mixture of (1R,4R)-1,4-bis(4-nitrophenyl)butane-1,4-diyl
dimethanesulfonate (1.5 g, 3.0 mmol) and 4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-
difluoroaniline (7.88 g, 30.71 mmol) in DMF (10 mL) was stirred at 60 °C for 24 h. The reaction
solution was poured into 2 N aq HCl (200 mL) and stirred for 10min. The solid was collected
by filtration and dissolved in dichloromethane (50 mL). The mixture was washed with water
and dried over sodium sulfate, filtered, and concentrated under vacuum to give a crude product
that was purified by column chromatography on silica gel using a solvent gradient of 10% ethyl
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acetate in petrol to give 19c as a yellow solid (1.2 g, 70.6%). H NMR (500 MHz, CDCl3) δ
8.24 (d, J = 8.5 Hz, 4 H), 7.37 (d, J = 8.5 Hz, 4 H), 5.78 (d, J = 11.5 Hz, 2 H), 5.25 (d, J = 6.5
Hz, 2 H), 3.29 (s, 4 H), 2.60 (s, 2 H), 2.06 (s, 2 H), 0.97 (s, 4 H), 0.10 (s, 6 H); 13C
NMR(125MHz, CDCl3): δ 149.7, 147.4, 126.8, 124.3, 97.9, 97.7, 63.0, 53.1, 32.1, 14.6, -3.2;
MS (ESI) m/z: 553.2 [M+H]+.
4,4'-((2S,5S)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-difluorophenyl)pyrrolidine-
2,5-diyl)dianiline (20c). To a solution of 19c (2.1 g, 3.8 mmol) in THF (20 mL) was added
platinum dioxide (0.43 g, 1.9 mmol). The reaction container was flushed with N2, and the
mixture was stirred under 1 atm H2 for 3 h. The mixture was filtered through Celite, and the
filtrate was concentrated under vacuum to give a crude product that was purified by by column
chromatography on silica gel using a solvent gradient of 0-20% ethyl acetate in petrol to give
20c as a white solid (0.72 g, 40%). 1H NMR (500 MHz , CDCl3) δ 6.94 (d, J = 8.0 Hz, 4 H),
6.65 (d, J = 8.5 Hz, 4H), 5.82 (d, J = 12.0 Hz, 2 H), 4.95 (d, J = 5.5 Hz,2 H), 3.17 (s, 4 H), 2.49
(s, 2 H), 1.73(d, J = 5.5 Hz, 2 H), 0.82 (s,4 H), 0.07 (s, 6 H); 13C NMR (125 MHz , CDCl3) δ
160.8, 158.9, 145.0, 142.6, 133.2, 126.9, 115.4, 97.3, 63.0, 52.3, 32.6, 15.5, 14.1, -3.0; MS (ESI)
m/z : 493.1 [M + H]+.
Dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-(((((2S,5S)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-
3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-
phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-
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oxobutane-1,2-diyl))dicarbamate (11). The mixture of 20c (0.50 g, 1.02 mmol), N-
(methoxycarbonyl)-O-methyl-L-threonyl-L-proline (0.9 g, 3.05 mmol), EDCI (0.58 g, 3.05
mmol), HOBt (0.42 g, 3.05 mmol) and N-methylmorpholine (0.62 g, 6.1 mmol) in DMF (10
mL) was stirred at 55 °C for 12 h. The mixture was poured into water (50 mL). The solid was
collected by filtration and dissolved in dichloromethane (50 mL). The mixture was washed with
brine and dried over sodium sulfate, filtered, and concentrated under vacuum to give a crude
product that was purified by column chromatography on silica gel using a solvent gradient of
0–66% ethyl acetate in petrol to give 11 as a white solid (0.6 g, 57.1%). 1H NMR (500 MHz ,
DMSO-d6) δ 9.95 (s, 2 H), 7.52 (d, J = 8.5 Hz, 4 H), 7.29 (d, J = 8.0 Hz, 2 H), 7.14 (d, J = 8.5
Hz, 4 H), 5.81 (d, J = 12.5 Hz, 2 H), 5.15 (d, J = 6.0 Hz, 2 H), 4.44 (dd, J = 4.5 Hz, 8.0 Hz, 2
H ), 4.28 (d, J = 8.0 Hz, 2 H), 3.83 (m, 2 H), 3.68 (d, J = 7.0 Hz, 2 H), 3.54 (s, 6 H), 3.49 (t, J
= 6.5 Hz, 2 H), 3.23 (s, 6 H), 3.04 (t, J = 5.5 Hz, 4 H), 2.45 (m, 2 H), 2.16 (m, 2 H), 2.00 (d, J
= 6.0 Hz, 2 H), 1.90 (m, 4 H), 1.62 (d, J = 5.5 Hz, 2 H), 1.15 (d, J = 6.0 Hz, 6 H), 0.71(t, J =
6.0 Hz, 4 H), 0.04(s, 6 H); 13C NMR (125 MHz, DMSO-d6) δ 170.6, 169.2, 160.7, 158.8, 157.1,
142.8, 138.5, 126.7, 119.7, 118.7, 97.7, 76.8, 62.6, 60.7, 57.4, 56.6, 52.3, 47.8, 32.5, 29.9, 25.1,
16.0; MS (ESI) m/z: 1055.3 [M + Na]+; HPLC: 98.2% purity; HRMS (ESI) m/z calcd for
[M+H]+ C52H70F2N8O10Si: 1033.5030, found 1033.5068.
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1-(4-((2R,5R)-2,5-bis(4-nitrophenyl)pyrrolidin-1-yl)-2,6-difluorophenyl)-4,4-
dimethyl-1,4-azasilinane (19d). The mixture of (1S,4S)-1,4-bis(4-nitrophenyl)butane-1,4-diyl
dimethanesulfonate (2.0 g, 4.09 mmol) and 4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-
difluoroaniline (8.4 g, 32.75 mmol) in DMF (20 mL) was stirred at 60 °C for 24 h. The reaction
solution was poured into 2 N aq HCl (50 mL) and stirred for 10min. The mixture was partitioned
between water and ethyl acetate (20 mL × 2). The combined organic layers were dried over
sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by
column chromatography on silica gel using a solvent gradient of 10% ethyl acetate in petrol to
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give 19d as a yellow solid (2.0 g, 88%). H NMR (500 MHz, CDCl3) δ 8.31-8.24 (m, 4 H),
7.60-7.58 (m, 2 H), 7.38-7.36 (m, 4 H), 5.76 (d, J = 11 Hz, 2 H), 3.30-3.20 (m, 4 H), 2.70-2.60
(m, 3 H), 2.03-2.02 (m, 1 H), 0.92-0.90 (m, 4 H), 0.09 (s, 6 H); MS (ESI) m/z 553.2 [M+H]+.
4,4'-((2R,5R)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-difluorophenyl)pyrrolidine-
2,5-diyl)dianiline (20d). To a solution of 19d (2.0 g, 3.62 mmol) in THF (20 mL) was added
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platinum dioxide (0.82 g, 3.62 mmol). The reaction container was flushed with N2, and the
mixture was stirred under 1 atm H2 for 3 h. The mixture was filtered through Celite, and the
filtrate was concentrated under vacuum to give a crude product that was purified by column
chromatography on silica gel using a solvent gradient of 0-20% ethyl acetate in petrol to give
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20c as a white solid (0.95 g, 53%). H NMR (500 MHz, CDCl3) δ 6.95 (d, J = 8 Hz, 4 H),
6.66 (d, J = 8 Hz, 4 H), 5.82 (d, J = 11.5 Hz, 2 H), 4.95 (d, J = 6 Hz, 2 H), 3.17-3.15 (m, 4 H),
2.51-2.47 (m, 2 H), 1.74-1.72 (m, 2 H), 0.83-0.81 (m, 4 H), 0.08 (s, 6 H); MS(ESI) m/z 493.2
[M+H]+.
Dimethyl ((2S,2'S,3R,3'R)-((2S,2'S)-(((((2R,5R)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-
yl)-3,5-difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-
phenylene))bis(azanediyl))bis(carbonyl))
bis(pyrrolidine-2,1-diyl))bis(3-methoxy-1-
oxobutane-1,2-diyl))dicarbamate (12). The mixture of 20d (0.40 g, 0.81 mmol), N-
(methoxycarbonyl)-O-methyl-L-threonyl-L-proline (0.70 g, 2.44 mmol), EDCI (0.47 g, 2.44
mmol), HOBt (0.33g, 3.44 mmol) and N-methylmorpholine (0.41 g, 4.05mmol) in DMF (10
mL) was stirred at 60 °C for 1 h. The mixture was poured into water (30 mL). The mixture was
partitioned between water and ethyl acetate (20 mL × 2). The combined organic layers were
dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was
purified by column chromatography on silica gel using a solvent gradient of 66% ethyl acetate
in petrol to give 12 as a white solid (0.32g, 38%). 1H NMR (500 MHz, DMSO-d6) δ 9.94 (s, 2
H), 7.51 (d, J = 8.5 Hz, 4 H), 7.29 (d, J = 7.5 Hz, 2 H), 7.13 (d, J = 8.5 Hz, 4 H), 6.70 (m, 1 H),
5.96 (m, 2 H), 5.13 (d, J = 6.0 Hz, 2 H), 4.44 (dd, J = 5.0 Hz, 8.0 Hz, 2 H), 4.28 (t, J = 7.5 Hz,
2 H), .3.69 (m, 2 H), 3.54 (m, 2 H), 3.49 (s, 6 H), 3.47 (d, J = 6.5 Hz, 2 H), 3.25 (s, 6 H), 2.98
(m, 4 H), 2.44 (m, 2 H), 2.15 (m, 2 H), 2.00 (d, J = 6.0 Hz, 2 H), 1.89 (m, 4 H), 1.62 (d, J = 5.5
Hz, 2 H), 1.13 (d, J = 6.5 Hz, 6 H), 0.73 (t, J = 6.0 Hz, 4 H), 0.04 (s, 6 H); 13C NMR (125 MHz,
DMSO-d6) δ 170.6, 169.2, 157.2, 155.3, 141.7, 139.1, 138.1, 131.1, 126.7, 122.1, 119.6, 109.5,
101.9, 76.8, 62.5, 60.6, 57.5, 56.6, 52.0, 47.8, 32.6, 29.9, 25.1, 16.0, 15.0, 1.6, -2.5; MS (ESI)
m/z: 1055.50 [M + Na]+; HPLC: 96.73% purity; HRMS (ESI) m/z calcd for [M+H]+
C52H70F2N8O10Si: 1033.5030, found 1033.5134.
Dimethyl
((2S,2'S)-((2S,2'S)-(((((2S,5S)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-
fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis
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(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (5). Prepared from
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6
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20a and (methoxycarbonyl)-L-valyl-L-proline (21) in the same method as 9. H NMR (500
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8
9
MHz , DMSO-d6) δ 9.97 (s, 2 H), 7.50 (d, J = 8.5 Hz, 4 H), 7.28 (d, J = 8.5 Hz, 2 H), 7.13 (d,
J = 8.5 Hz, 4 H), 6.71-6.72 (m, 1 H), 5.95-6.05 (m, 2 H), 5.12 (d, J = 6.5 Hz, 2 H), 4.44 -4.43
(m, 2 H), 4.04-4.10 (m, 2 H), 3.82-3.75 (m, 2 H), 3.65-3.65 (m, 2 H), 3.53 (s, 6 H), 3.41-3.31
(m, 2 H), 2.97-2.98 (m, 4 H), 2.44-2.48 (m, 2 H), 2.16-2.14 (m, 2 H), 2.09-1.87 (m, 8 H), 1.62
(d, J = 5.5 Hz, 2 H), 0.95-0.86 (m, 12 H), 0.73 (t, J = 6.3 Hz, 2 H), 0.04 (s, 6 H); HPLC: 99.8%
purity; HRMS (ESI) m/z: calcd for [M+H]+ C52H71FN8O8Si: 983.5148; found 983.5158.
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Dimethyl
((2S,2'S)-((2S,2'S)-(((((2R,5R)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3-
fluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (6). Prepared from
1
20b and (methoxycarbonyl)-L-valyl-L-proline (21) in the same method as 10. H NMR
(DMSO-d6, 500 MHz) δ 9.98 (s, 2 H), 7.51 (d, J = 8.0 Hz, 4 H), 7.27 (d, J = 8.5 Hz, 2 H), 7.13
(d, J = 8.0 Hz, 4 H), 5.89-6.09 (m, 3 H), 5.15-5.21 (m, 2 H), 4.44 (dd, J = 5.0 Hz, 8.0 Hz, 2 H),
4.04 (t, J = 8.5 Hz, 2 H), 3.75-3.85 (m, 2 H), 3.63-3.69 (m, 2 H), 3.63-3.58 (m, 3 H), 3.53 (s, 6
H), 3.10-2.95 (m, 1 H), 2.51-2.44 (m, 2 H), 2.15-1.86 (m, 10 H), 1.65-1.55 (m, 2 H), 0.94-0.86
(m, 16 H), 0.06 (s, 6 H); HPLC: 99.7% purity; HRMS (ESI) m/z: calcd for [M+H]+
C52H71FN8O8Si: 983.5148; found 983.5143.
Dimethyl ((2S,2'S)-((2S,2'S)-(((((2S,5S)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-
difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis
(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (7). Prepared from
1
20c and (methoxycarbonyl)-L-valyl-L-proline (21) in the same method as 11. H NMR (500
MHz , DMSO-d6) δ 9.98 (s, 2 H), 7.52 (d, J = 8.5 Hz, 4 H), 7.28 (d, J = 8.5 Hz, 2 H), 7.13 (d,
J = 8.5 Hz, 4 H), 5.95-6.05 (m, 2 H), 5.15 (d, J = 6.5 Hz, 2 H), 4.45 -4.43 (m, 2 H), 4.11-4.07
(m, 2 H), 3.82-3.75 (m, 2 H), 3.65-3.63 (m, 2 H), 3.53 (s, 6 H), 3.41-3.31 (m, 2 H), 2.97-2.98
(m, 4 H), 2.44-2.48 (m, 2 H), 2.16-2.14 (m, 2 H), 2.09-1.87 (m, 8 H), 1.62 (d, J = 5.5 Hz, 2 H),
0.95-0.86 (m, 12 H), 0.73 (t, J = 6.3 Hz, 2 H), 0.04 (s, 6 H); HPLC: 99.6% purity; HRMS (ESI)
m/z: calcd for [M+H]+ C52H70F2N8O8Si:1001.5054; found 1001.5092.
Dimethyl ((2S,2'S)-((2S,2'S)-(((((2R,5R)-1-(4-(4,4-dimethyl-1,4-azasilinan-1-yl)-3,5-
difluorophenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis
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(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-1,2-diyl))dicarbamate (8). Prepared from
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20d and (methoxycarbonyl)-L-valyl-L-proline (21) in the same method as 12. H NMR (500
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9
MHz , DMSO-d6) δ 9.99 (s, 2 H), 7.53 (d, J = 8.5 Hz, 4 H), 7.28 (d, J = 8.5 Hz, 2 H), 7.13 (d,
J = 8.5 Hz, 4 H), 5.80 (d, J = 7.0 Hz, 2 H), 5.15 (d, J = 6.5 Hz, 2 H), 4.45-4.43 (m, 2 H), 4.04
(t, J = 8.5 Hz, 2 H), 3.82-3.75 (m, 2 H), 3.65-3.63 (m, 2 H), 3.53 (s, 6 H), 3.12-3.02 (m, 6 H),
2.97-2.98 (m, 4 H), 2.20-1.87 (m, 14 H), 1.62 (d, J = 5.5 Hz, 2 H), 0.94-0.84 (m, 12 H), 0.73
(t, J = 6.3 Hz, 2 H), 0.04 (s, 6 H); HPLC: 99.4% purity; HRMS (ESI) m/z: calcd for [M+H]+
C52H70F2N8O8Si:1001.5054; found 1001.5083.
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ASSOCIATED CONTENT
The Supporting Information is avilable free of charge on the ACS Publications website at DOI:
XXXXXX
1
13
Molecular Formula Strings, HPLC spectrum of targeted compounds, H and C NMR spectra
for compound 11, Body and organ weights, Hematology data for mouse orally treated with 11 for
14 days, blood chemistry data for mouse orally treated with 11 for 14 days.
AUTHOR INFORMATION
Corresponding Author
*Email: zys@cttq.com;
ORCID
Yinsheng Zhang: http://orcid.org/0000-0001-5390-671X
ACKNOWLEDGMENTS
This study was partially supported by the Natural Science Foundation of Jiangsu Province
(Grants No BK20170454)，China Postdoctoral Science Foundation (Grants No 2019M651989)
and Department of science and technology of Jiangsu Province (Sukyyz [2019] No. 1627).
ABBREVIATIONS USED
GT, genotype; PK, pharmacokinetics; AUC, area under the curve; CLp, systemic clearance;
EDCI,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride;
HOBt,
1-
hydroxybenzotriazole; NMM, N-methylmorpholine; DMF, N,N-dimethylformamide; THF,
tetrahydrofuran.
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TABLE OF CONTENTS GRAPHIC
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80x64mm (300 x 300 DPI)
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