Development of a Synthesis Process for a Novel HCV NS5A Inhibitor, Emitasvir

Article abstract of DOI:10.1021/acs.oprd.0c00519

A new approach to the synthesis of Emitasvir (DAG181), a small molecule hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor, is described. Enantioselective enzymatic desymmetrization with hydrolases in the synthetical approach can significantly avoid the loss of chiral raw materials in the resolution process. The synthesis route is further optimized and scaled-up to establish a new process that is applied to the preparation of kilogram scales of target active pharmaceutical ingredients (APIs). Compared with our initial process, the overall yield of target API was dramatically improved from 17 to 40%.

Full text of DOI:10.1021/acs.oprd.0c00519

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Article
Development of a Synthesis Process for a Novel HCV NS5A Inhibitor,
Emitasvir
Hongming Xie, Haiwang Liu, Yingjun Zhang,* Enhuo Huang, Yahui Feng, Xuwen Xiang,
Qinghong Fang, Zhihong Peng, Wanrong Dong, and Delie An*
Cite This: Org. Process Res. Dev. 2021, 25, 838−848
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sı Supporting Information
ABSTRACT: A new approach to the synthesis of Emitasvir (DAG181), a small molecule hepatitis C virus (HCV) nonstructural
protein 5A (NS5A) inhibitor, is described. Enantioselective enzymatic desymmetrization with hydrolases in the synthetical approach
can significantly avoid the loss of chiral raw materials in the resolution process. The synthesis route is further optimized and scaled-
up to establish a new process that is applied to the preparation of kilogram scales of target active pharmaceutical ingredients (APIs).
Compared with our initial process, the overall yield of target API was dramatically improved from 17 to 40%.
KEYWORDS: hepatitis C virus inhibitor, Emitasvir, enantioselective synthesis, process development
1
. INTRODUCTION
shown in Scheme 1. Our initial synthesis plan for 6 includes
chemical resolution from diastereomers, which has poor atom
economy. At the same time, chiral impurities are difficult to
remove, resulting in only 38% yield and 96% enantiomeric
purity after recrystallization in acetone (Scheme 1). This
problem can be fully solved with enantioselective enzymatic
desymmetrization by hydrolases based on our previous
Worldwide, an estimated 71 million people were infected by
the hepatitis C virus (HCV). For more than half of people who
are infected with HCV, it turns into a long-term and chronic
disease. Numerous infected persons will later develop cirrhosis
or hepatocellular carcinoma. WHO estimated that 399 000
HCV infectors died in 2016 and most of them came from
1
,2
12
HCV-induced liver cirrhosis or hepatocellular carcinoma.
research, thereby increasing the chiral purity and avoiding
Unlike hepatitis A and B, currently, there is no effective vaccine
at least 50% loss of chiral raw materials in the resolution
to prevent hepatitis C infection. Therefore, the development of
process.
3
−5
antiviral drugs
plays an important role in preventing and
Due to the launch of Emitasvir, there is an urgent need for a
stable process route that can be mass-produced. Herein, we
reported our efforts to develop a novel process-scale synthesis
of Emitasvir. The new process was an improvement over the
original synthesis process, with increasing overall yield,
reducing reaction byproducts, and meeting regulatory guide-
lines at the same time.
controlling HCV infectious diseases.
HCV nonstructural protein 5A (NS5A) inhibitors that are
important components of anti-HCV drugs have attracted
considerable attention. They presented strong antiviral activity
and efficient antiviral response and are effective for a wide
range of HCV genotypes. In addition to marked HCV NS5A
6
−10
inhibitors,
several new NS5A inhibitors in the clinical stage
reported promising results.
11
2. RESULTS AND DISCUSSION
Emitasvir (Figure 1) is a new inhibitor of HCV NS5A,
which was developed for the treatment of genotype 1 hepatitis
C patients and has shown excellent antiviral properties in
clinical studies. In 2020, Emitasvir was approved by the
National Medical Products Administration (NMPA). Structur-
ally, Emitasvir has a bridgehead carbon chiral center, and the
key intermediate for preparing Emitasvir is compound 6, as
2.1. Retrosynthesis of Emitasvir. As shown in Scheme 2,
acylation of commercially available 3,6-dihydroxybenzonorbor-
nane 1 with acetic anhydride afforded a diester compound.
After enantioselective enzymatic desymmetrizations with
hydrolases, enantiopure 2 was obtained. Intermediate 2 was
converted into triflate compound 3, which was then coupled
with an imidazole moiety. With further hydrolysis, phenolic 4
was produced, followed by triflation to 5. DAG181 was finally
Received: November 30, 2020
Published: April 2, 2021
Figure 1. HCV NS5A inhibitor Emitasvir.
©
2021 American Chemical Society
https://doi.org/10.1021/acs.oprd.0c00519
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Scheme 1. Synthesis Routes for Key Intermediate 6
Scheme 2. Key Intermediates in the Retrosynthesis of Emitasvir (DAG181)
Scheme 3. Preparation of Intermediate 2
generated by another Suzuki coupling from 5 and amide
formation from 6.
absolute configuration we need is different from our previous
11
research. Therefore, the enzymatic desymmetrization reac-
tion was reoptimized here and diacetyl compound 2′ was
utilized as the starting material. The enzymatic desymmetriza-
tion reaction was initially tested with different hydrolases
(Table 1).
As shown in Table 1, basic protease showed promising
results, producing the target product in 84% yield and 90%
enantiomeric excess (ee) value. Because 2′ has poor solubility
2
.2. Process Development. 2.2.1. Enzymatic Desym-
metrization. First, the acetylation of 1 was prepared using
acetic anhydride and 4-dimethylaminopyridine (DMAP) in
methyl tert-butyl ether (MTBE) (Scheme 3). The acetic
anhydride was added slowly to the reaction mixture under 0−5
°C while stirring, and the reaction mixture was stirred at 30 °C.
For the next enzymatic desymmetrization reaction, the
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a
Table 1. Enzyme Screening for Enzymatic Desymmetrization of 2′
b
HPLC yield (%)
enzymes
AK
L3
L1
L5
P1
P4
2′
2a
2
1
ee value (%)
26.97
17.81
65.46
34.26
61.70
81.18
94.13
83.00
94.49
7.02
57.50
64.65
21.39
23.33
7.24
1.25
0.87
7.40
3.75
14.45
16.28
12.40
39.22
30.60
17.15
4.43
9.12
1.29
83.89
1.09
1.27
0.75
3.19
0.00
0.00
0.00
0.00
0.00
4.89
59.83
59.77
26.61
−25.40
−61.73
−86.41
−67.17
−10.41
48.81
4
35
M
PPL
basic protease
4.20
−90.46
a
Reaction conditions: 2′ (50 mg, 0.19 mmol, 1.0 equiv), enzyme (30 mg), isopropyl acetate (IPAc) (1 mL, 20 mL/g), and buffer (0.1 M, 1 mL, pH
b
=
7.0), Na CO (40 mg, 0.38 mmol, 2.0 equiv), 24 h at 20−25 °C. Determined by high-performance liquid chromatography (HPLC) analysis.
2
3
a
Table 2. Results of Cosolvent Screening for Enzymatic Desymmetrization of 2′
b
HPLC yield
cosolvent
2′
2a
2
1
ee value
cyclohexane
diisopropyl ether
MTBE
DCM
n-butanol
isopropyl acetate
ethyl acetate (EA)
DMF
DMSO
methanol
64.05%
1.47%
4.46%
99.49%
7.03%
19.37%
34.94%
0.93%
1.97%
N/A
1.83%
2.03%
3.88%
N/A
11.15%
4.32%
6.73%
31.86%
N/A
31.37%
85.50%
85.27%
N/A
76.23%
70.84%
54.49%
34.51%
52.89%
40.52%
1.68%
11.00%
6.39%
N/A
5.60%
3.61%
2.19%
17.31%
37.46%
13.84%
−88.98%
−95.36%
−91.30%
N/A
−74.48%
−88.50%
−78.01%
−3.99%
−100%
19.82%
−34.31%
a
Reaction conditions: 2′ (50 mg, 0.19 mmol, 1.0 equiv), basic protease (30 mg), cosolvent (1 mL, 20 mL/g), buffer (0.1 M, 1 mL, pH 7.0),
b
Na CO (40 mg, 0.38 mmol, 2.0 equiv), 24 h at 20−25 °C. Determined by HPLC analysis.
2
3
in aqueous solutions, different organic cosolvents were then
evaluated. MTBE and diisopropyl ether (DIPE) were found as
good candidates, providing as high as 95% ee value and 85%
yield (Table 2). Kinetic data (Figure S1) indicated that the
enzyme-specific activity for this enzymatic desymmetrization
reaction is 20.1 U/g, which is not high. Therefore, 60% (g/g)
enzyme was needed here. When the enzymatic desymmetriza-
tion reaction was produced on a gram scale, MTBE showed
better solubility for 2′ than DIPE. Therefore, MTBE was
employed as a cosolvent in the next gram-scale optimization.
Further optimization indicated that pH 7 (Table S1) and 20−
2.2.2. Triflation. In the next step, 2 was treated with Tf O,
resulting in formation of triflate ester 3 (Scheme 4). As a key
2
Scheme 4. Formation of Intermediate 3
intermediate, compound 3 was optimized with a variety of
reaction parameters. Effects of parameters, including the
2
5 °C (Table S2) were optimal conditions for this enzymatic
reaction. Finally, enzyme loading (Table S3) was screened.
The results indicated that the reaction achieved higher
conversion and less byproduct with 60% enzyme (g/g). A
kilogram scale further verified the feasibility of these optimal
conditions in commercial production (see Section 4).
solvent and base type, base amount, Tf O amount,
2
concentration, and temperature, were investigated here for
large-scale production. A number of different solvents/bases
were examined (as shown in Table 3). Combinations such as
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a
Table 3. Solvent and Base Screening
of 4 and 4a (Scheme 5). For the first step, intermediate 4a was
synthesized from Suzuki coupling of 3 with 9. Optimization
was conducted with several parameters including the base, Pd
catalyst, solvent, and temperature. As shown in Table 4, it was
discovered that a weak base such as KOAc provided low
conversion for both 9 and 3. For stronger bases such as
K CO , K PO , Na CO , and t-BuONa used (Table 4, entries
2
3
3
4
2
3
b
b
b
entry
solvent/base
3
(%) 3a (%)
2 (%)
2
−4, and 6), a large amount of 9 still remained, although
1
2
3
4
5
6
7
8
dichloromethane/pyridine
EtOAc/pyridine
91.99
87.37
2.80
2.15
2.01
2.11
2.33
2.33
2.21
2.42
0.36
3.56
0.08
0.04
0.27
0.24
0.18
0.42
excessive 3 was fully consumed. Mild bases such as KHCO
3
and NaHCO (Table 4, entries 9 and 10) that provide higher
3
dichloromethane/Et N
91.40
93.22
92.07
92.70
92.19
92.12
3
conversion for 9 are better candidates. It can be attributed to
the fact that 3 is easily hydrolyzed at strong base conditions,
and hydrolyzed product 3b has been experimentally confirmed
dichloromethane/DIPEA
EtOAc/Et N
3
EtOAc/DIPEA
EtOAc/DIPEA/DMAP (0.1 equiv)
IPAc/DIPEA
(
Scheme 6) to be a bad substrate for Suzuki coupling with 9.
To further explain the obviously different yields obtained
between K CO and NaHCO , several control experiments
2
3
3
a
Reaction conditions: compound 2 (1.0 g, 4.6 mmol, 1.0 equiv), base
2.0 equiv), solvent (10 mL), Tf O (1.7 g, 6.0 mmol, 1.3 equiv), −10
have been performed, as shown in Table 5. We here set 3 as 1
equiv to trace the effect of 3b on the reaction yield. First,
kinetic data (Figure S2) indicated that the amount of
hydrolyzed product 3b was continuously increased, albeit
coupling was faster than hydrolysis. Second, hydrolyzed
product 3b could not react with 9 (Scheme 6), which means
that the lower yield in stronger base conditions is partially
attributed to more hydrolyzed product 3b. At last, an obvious
decrease in the effective yield (4 plus 4a) from 87 to 59% has
been observed when extra 10 mol % 3b was added in standard
conditions (Table 5, entries 1 and 3). Therefore, hydrolyzed
product 3b itself is not a good substrate for Suzuki coupling,
and at the same time, it inhibits the coupling to some extent,
which explains the lower yield under the stronger base.
(
2
b
°C, 5 h. Determined by HPLC analysis.
dichloromethane (DCM)/N,N-diisopropylethylamine
DIPEA) and EtOAc/DIPEA (Table 3, entries 4 and 6)
could afford the desired triflate ester 3 in a higher yield and less
byproduct 3a. Considering EtOAc is more process-friendly
than DCM, EtOAc/DIPEA was chosen. The base and Tf O
amount were further investigated. DIPEA (2.0 equiv) provided
the highest conversion and yield compared with 1.5 equiv
Table S4). For Tf O amount screening, the starting material 2
was not fully converted with 1.1 equiv of Tf O. When the
amount was increased to 1.2 equiv, a higher conversion was
achieved. However, for 1.3 equiv of Tf O, the amount of
(
2
(
2
2
2
Compared with KHCO , NaHCO as the base showed
3
3
byproduct 3a increased and the yield of product 3 decreased.
Therefore, 1.2 equiv is the optimal amount (Table S5).
Subsequently, the influence of temperature on the yield was
evaluated. Higher yield and less byproduct 3a were obtained
higher conversion and yield. Other ligands such as Sphos and
Xphos both produced lower conversion for 3 and 9 (Table 4,
entries 11 and 12). The reaction in anhydrous DME or pure
water only afforded a small amount of target product (Table 4,
entries 13 and 14). Therefore, Pd(PPh ) and NaHCO were
when Tf O was added at −10 °C (Table S6).
2
3
4
3
2
.2.3. Suzuki Coupling to 4. Intermediate 9 was synthesized
finally chosen for further optimization. The amount of
from the coupling reaction of bis(pinacolato)diboron (B Pin )
NaHCO needed was investigated from 1.0 to 2.5 equiv.
2
2
3
with commercially available precursor 8, and 8 was
Results indicated that 1.5 equiv of NaHCO was enough to get
3
1
3
commercially produced by the reported method. The
kilogram scale is explained in detail in Section 4. The reaction
of 3 and 9 in the presence of Pd(PPh ) and a base afforded
as high as 91% effective yield (4 plus 4a, Table S7). The ratio
of DME to water was then screened from 2:1 to 4:1, and 2:1
was the optimal ratio as it provided a high effective yield with
less 9 remained (Table S8). The volume of the solvent was set
as 10 mL/g based on the screening results. When the reaction
temperature was decreased to 60 or 70 °C, the reaction
became slower, and more starting material 9 was left (Table
S9). Therefore, 80 °C was chosen as the optimal temperature.
3
4
not only the coupling product 4a but also the corresponding
hydrolysis product 4, which was detected in the crude reaction
mixture. As hydrolysis product 4 is the actual target precursor
for the next Suzuki coupling, one-pot synthesis of product 4
was developed here by adding K CO to the reaction mixture
2
3
Scheme 5. Preparation of Intermediate 4
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a
Table 4. Catalyst and Base Screening
b
b
b
b
entry
catalyst
Pd(PPh3)4
base
KF
4a (%)
4
(%)
9
(%)
3
(%)
1
2
3
4
5
6
7
8
9
85.43
26.09
10.53
35.87
66.60
10.55
13.66
41.77
51.28
62.28
26.07
0.87
0.67
3.31
29.54
34.5
30.13
16.13
36.95
20.10
28.57
0
1.87
0
0
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(dppf)Cl2
PdCl (PPh )
Na CO3
8.40
2.38
2.38
0.37
0.55
32.26
2.84
35.03
23.29
2.20
0.14
0
2
K PO4
3
K CO3
0
2
KOAc
t-BuONa
15.33
0
0
0
0.13
0
12.51
10.06
22.46
21.56
Cs CO3
2
K CO3
2
3
2
2
Pd(PPh3)4
Pd(PPh3)4
NaHCO3
KHCO3
NaHCO3
NaHCO3
NaHCO3
NaHCO3
10
11
12
13
14
1.72
c
Pd(OAc) /SPhos
38.28
47.76
69.25
60.48
2
c
Pd(OAc) /XPhos
2
d
e
Pd(PPh3)4
Pd(PPh3)4
1.70
15.33
0.20
a
Reaction conditions: 9 (1.0 g, 2.3 mmol, 1.0 equiv), 3 (0.88 g, 2.5 mmol, 1.1 equiv), Pd catalyst (4 mol %), base (2.5 equiv), dimethyl ether
b
c
d
(DME) (15 mL), water (5 mL), 80 °C, 10 h. Determined by HPLC analysis. Pd(OAc) (4 mol %) and a ligand (10 mol %). Anhydrous DME
2
e
as the solvent. Pure water as the solvent.
Scheme 6. No Reaction between Hydrolyzed Products 3b and 9
a
Table 5. Control Experiments for Suzuki Coupling
b
b
b
b
b
entry
conditions
4a (%)
4
(%)
9
(%)
3
(%)
3b (%)
1
2
3
NaHCO3
K CO
84.90
55.04
56.52
2.51
2.97
2.21
6.20
31.03
23.90
3.34
0.24
8.06
1.05
3.58
5.29
2
3
NaHCO with extra 10 mol % 3b
3
a
Reaction conditions: 9 (1.0 g, 2.3 mmol, 1.0 equiv), 3 (0.88 g, 2.5 mmol, 1.1 equiv), Pd(PPh ) (105 mg, 4 mol %), base (2.5 equiv), DME (15
3
4
b
mL), water (5 mL), 80 °C, 3 h. Determined by HPLC analysis.
The Pd catalyst amount could be decreased to 0.03 equiv
without lowering the reaction conversion (Table S10). Final
conditions utilized gave 4 and 4a totally in 89% HPLC assay
yield.
1.5 equiv of K CO . Compared with 2.5 equiv of K CO , the
2 3 2 3
result for 2.0 equiv of K CO was almost the same. Therefore,
2
3
2.0 equiv of K CO was utilized. Finally, 60 °C was set as the
2
3
reaction temperature after considering the boiling point of
methanol and the reaction rate.
After the Suzuki coupling reaction was finished from the
HPLC result, K CO was directly added to the reaction system
to facilitate the overall transformation from 4a to 4 in a one-
2.2.4. Selective Triflation. As the important precursor for
the next step of Suzuki coupling, compound 5 was obtained
from the triflation of 4. The transformation was initially
accomplished using trifluoromethanesulfonic anhydride
2
3
pot model. The amount of K CO was screened, as shown in
2
3
Table 6. It was discovered that 4a was not fully consumed with
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a
Table 6. Screening of the Base Amount
b
b
entry
K CO (equiv)
4
(%)
4a (%)
2
3
1
2
3
1.5
2.0
2.5
83.15
85.67
85.38
2.18
0
0
a
Reaction conditions: compound 9 (1.0 g, 2.3 mmol, 1 equiv), 3 (0.88 g, 2.5 mmol, 1.1 equiv), Pd(PPh ) (75 mg, 0.03 equiv), NaHCO (0.286 g,
3
4
3
3
.4 mmol, 1.5 equiv), DME/H O (2/1, 10 mL) at 80 °C for 5 h, then add methanol (10 mL/g) and K CO (0.63 g, 4.6 mmol, 2.0 equiv), and stir
2
2
3
b
at 60 °C for 10 h. Determined by HPLC analysis.
a
Table 7. Screen of Triflating Reagents and Solvents/Bases
b
b
b
b
entry
triflating reagents (equiv)
solvent/base
DCM/pyridine
DCM/DIPEA
T (°C)
4
(%)
5
(%)
5a (%)
5b (%)
1
2
3
4
5
6
7
8
9
Tf O(1.5)
2
0
0
0
−20
−20
0
0
0
0
0
0
0
60
0
0
33.29
53.50
53.09
9.27
2.95
11.80
5.46
67.93
71.01
29.37
28.89
32.39
52.25
5.48
37.86
4.01
12.11
4.05
1.74
1.94
1.42
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
14.97
27.85
29.52
20.04
34.59
n.d.
n.d.
n.d.
n.d.
n.d.
Tf O(1.5)
2
Tf O(1.5)
2
DCM/Et N
11.82
64.94
56.71
84.77
92.97
3.49
23.19
66.88
67.79
64.33
30.35
89.94
98.80
3
Tf O(1.5)
2
DCM/pyridine
DCM/pyridine
DCM/DIPEA
Tf O(2.0)
2
Tf NPh(1.2)
2
Tf NPh(1.2)
2
DCM/Et N
3
Tf NPh(1.2)
2
DCM/pyridine
EtOAc/DIPEA
Tf NPh(1.2)
2
10
11
12
13
14
15
Tf NPh(1.2)
2
EtOAc/Et N
3
Tf NPh(1.2)
2
IPAc/Et N
n.d.
n.d.
n.d.
n.d.
3
Tf NPh(1.2)
2
THF/Et N
3
Tf NPh(1.2)
2
THF/K CO3
2
Tf NPh(1.2)
2
EtOAc/Et N/DMAP(0.1)
3
Tf NPh(1.2)
2
DCM/Et N/DMAP(0.05)
0.20
n.d.
3
a
b
Reaction conditions: compound 4 (1.0 g, 1 equiv), base (2.0 equiv), solvent (10 mL), triflating reagents, 5 h. Determined by HPLC analysis.
(
Tf O) as a triflating reagent. However, Tf O produced two
bases, we fortunately observed around 93% yield with
triethylamine (TEA) as a base. For other solvents such as
EtOAc, IPAc, or tetrahydrofuran (THF), reaction yields were
not satisfying (entries 9−14). Finally, the reaction yield was
promoted to 98.8% when 0.05 equiv of DMAP was added to
2
2
obvious byproducts, i.e., 5a and 5b, because triflation also
happened on the imidazole ring. Other base reagents (Table 7,
entries 2 and 3) were investigated to affect the selectivity but
gave no improvement. Decreasing the reaction temperature
(
entry 4) or increasing the amount of Tf O (entry 5) could
DCM/Et N (entry 15). Other parameters, such as the amount
2
3
increase the conversion but gave no obvious improvement to
the selectivity. Fortunately, sulfonylating reagents such as
perfluorobutylsulfonylfluoride (C F SO F) and N-phenyl-bis-
of Tf NPh and Et N, concentration, and temperature, were
2
3
also investigated. The final optimal conditions included 1.2
equiv of Tf NPh, 2 equiv of Et N, DCM (15 mL/g), and 5 ± 5
4
9
2
2
3
(
trifluoromethane-sulfonimide) (Tf NPh) were found as better
°C, as shown in detail in Section 4.
2
reagents, which can eliminate the formation of imidazole-
sulfonylated byproduct 5a and over-sulfonylated byproduct 5b
(
2.2.5. Suzuki Coupling and Amide Coupling. Compound
11, the precursor for second Suzuki coupling, was synthesized
from the coupling of bis(pinacolato)diboron (B Pin ) with
entry 6). Compared with C F SO F, Tf NPh provided better
4 9 2 2
2
2
1414
results. Thus, optimizing the process using Tf NPh as a
commercially available compound 10.
The kilogram scale
2
triflating reagent was carried out. After screening different
is shown in Section 4. The optimal condition for Suzuki
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a
Table 8. Screening of De-boc Reagents from 6 to 7
b
b
entry
reagents
solvent
CH Cl
EtOAc
MeOH
MeOH
EtOAc
time (h)
7
(%)
6
(%)
mono-boc byproducts 7a/7b (%)
1
2
3
4
5
TMSCl/MeOH
HCl
HCl(aq)
10
10
22
5
99.74
99.80
57.51
93.74
41.61
0
0
0/0
0/0
19.58/16.88
0/0
2
2
6.22
0
49.48
SOCl /MeOH
2
TMSCl/MeOH
17
3.80/4.99
a
b
Reaction conditions: 6 (1.0 g, 1.35 mmol, 1.0 equiv), de-boc reagents (15 equiv), solvent (12 mL), 25 °C. Determined by HPLC analysis.
Scheme 7. Preparation of DAG181
coupling between 5 and 11 was similar to the reaction in
reaction mixture was washed with water once and 5% citric
acid aqueous solution three times. Solid DAG181 was then
obtained in 90% yield and >99% purity after evaporating the
organic solvent (Scheme 7).
Section 2.2.3, with the only change being using base K CO
2
3
instead of NaHCO3.
The de-boc step was screened with several conditions
(
Table 8) such as HCl−EtOAc, SOCl /MeOH, and
2
Based on all optimizations described above, we finished the
synthesis of DAG181 at the kilogram scale (see Section 4).
Starting with compound 1, we were able to afford the target
compound DAG181 in 40% overall yield and >99% HPLC
area purity without any purification by column chromatog-
raphy (Scheme 8). This new robust process shows significant
improvement in yield compared with the previous route (17%,
see Scheme 1). Both impurities and byproducts in each step of
the new process could be easily removed via washing or
filtration.
trimethylsilyl chloride (TMSCl)/MeOH. Both HCl−EtOAc
and TMSCl/MeOH afforded high yields. However, for HCl−
EtOAc, storage and transportation are the main concerns. For
entries 3 and 5, boc groups were not fully removed and mono-
boc byproducts were introduced. Therefore, boc-protection
groups were finally removed by an excess amount of
trimethylsilyl chloride (TMSCl, 2.0 g/g) in a mixture of
methanol and dichloromethane to give 7. The final step in the
synthesis of DAG181 involved 1-ethyl-3-(3-dimethylamino-
propyl)-carbodiimide (EDCI)/Oxyma-mediated amide cou-
pling of 12 with 7. When the reaction was finished, the
8
44
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Org. Process Res. Dev. 2021, 25, 838−848

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pubs.acs.org/OPRD
Article
Scheme 8. Synthesis Route for DAG181
3
. CONCLUSIONS
ybenzonorbornene (1.00 kg, 1 equiv) and DMAP (0.035 kg,
.05 equiv) was treated with MTBE (13 L) and triethylamine
1.74 L, 2.2 equiv). After the reaction mixture was cooled
down to 0−5 °C, acetic anhydride (1.18 L, 2.2 equiv) was
added slowly while stirring at a controlled temperature
between 0 and 20 °C. Then, the mixture was warmed to 30
0
(
In summary, a robust and novel synthesis approach to
Emitasvir was developed with high purity and reproducibility.
We successfully demonstrated the chemistry and process on a
kilogram scale. In contrast to the original procedure, which had
at least 50% loss of chiral raw materials in the resolution
process, the new scalable route dramatically improved the
overall yield from 17 to 40% with enzymatic desymmetrization.
For the synthesis of key intermediate 5, triflating reagent
°
C and maintained at this temperature for 30 min. When the
HPLC result indicated that 3′,6′-dihydroxybenzonorbornene
was less than 1%, water (3.0 L) was added with quickly stirred
(
≤40 °C). The organic phase was separated while still hot and
then washed with water. The separated organic phase was
treated with Na CO (0.82 kg, 2.0 equiv) and 0.1 M NaH PO
4
Tf NPh was found as a better reagent, which could eliminate
2
the formation of imidazole triflate byproduct. Impurities in all
steps of the new synthesis approach could be easily removed
via washing and filtration without any purification by column
chromatography. Several kilograms of active pharmaceutical
ingredients have been manufactured by this process to date,
and the final product has been obtained with high purity of
above 99%.
2
3
2
buffer (6 L, pH = 7). The solution was charged with basic
protease (0.60 kg), and the reaction mixture was stirred at 20−
25 °C for 24 h until the amount of the starting material was
<5.00% as determined by HPLC. The reaction mixture was
filtered through a Celite bed, and the filter cake was washed
with MTBE (1.5 L). After separation, the combined organic
layer was washed with water (3.0 L) and concentrated under
vacuum at 45 °C. Crude product 2 was added to cyclohexane
4
. EXPERIMENTAL SECTION
.1. Materials and Instrumentation. Commercially
4
(
10.0 L), and the mixture was warmed to 70−75 °C and
available materials purchased from Alfa Aesar or Kelong were
used as received. Unless otherwise indicated, all reagents and
solvents were used as commercially available without further
purification. Compounds 8 and 10 were purchased from Aneo
stirred until the solid dissolved completely. The mixture was
cooled to 10−20 °C and stirred at this temperature for 2 h for
crystallization. After filtration, the filter cake was dried at 40−
13,14
5
0 °C for 12 h to afford compound 2 as an off-white solid
Chem-Tech, which were synthesized by reported methods.
(
1.02 kg, 82% yield, 98% purity, 97.82% ee). Liquid
The enzymes were purchased from NovoCata Biotechnology
Co., Ltd. (AK, 435, M, PPL, basic protease) or Amano
Enzyme, Inc. (P1, L1, L3, L5, P4). NMR spectra were
measured on a Bruker Avance 400 or 600 spectrometer in the
solvents indicated. The enantiomeric purity of compound 2
was monitored on an Agilent 1260 chromatograph with a
Daicel Chiralpak IA column (4.6 mm × 250 mm) using 90:10
hexane/ethanol as the eluent at a flow rate of 0.8 mL/min and
UV detection at 210 nm. High-resolution mass spectrometry
+
chromatography−mass spectrometry (LC−MS) [M + H]
calcd, 219.1; found, 219.2. H NMR (400 MHz, CDCl ) δ
1
3
6
1
.60 (d, J = 8.6 Hz, 1H), 6.45 (d, J = 8.6 Hz, 1H), 5.32 (s,
H), 3.52 (s, 1H), 3.31 (s, 1H), 2.30 (s, 3H), 1.88−1.82 (m,
2H), 1.76−1.69 (m, 1H), 1.51−1.45 (m, 1H), 1.29−1.16 (m,
2H). C NMR (101 MHz, CDCl ) δ 170.31, 146.82, 140.85,
1
3
3
137.69, 134.33, 119.52, 114.08, 48.98, 41.00, 39.77, 26.29,
25.88, 20.88.
(
HRMS) was performed on a Fourier transform ion cyclotron
resonance mass spectrometer.
.1.1. (1R,4S)-8-Hydroxy-1,2,3,4-tetrahydro-1,4-methano-
naphthalen-5-yl Acetate (2). A mixture of 3′,6′-dihydrox-
4.1.2. (1R,4S)-8-(((Trifluoromethyl)sulfonyl)oxy)-1,2,3,4-
tetrahydro-1,4-methano-naphthalen-5-yl Acetate (3). Com-
pound 2 (1.00 kg, 1 equiv) and DIPEA (1.60 L, 2 equiv) were
4
dissolved in EtOAc (15 L). Tf O was added slowly, and the
2
8
45
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Article
reaction mixture was maintained at −10 °C. The reaction
mixture was then stirred for 5 h. When HPLC analysis
subsequently indicated that <0.40% of 2 remained, water (4 L)
was added. The organic phase was collected and washed with
water (3 L) again. The organic solvent was then distilled off
with ethanol (3 L) for 5 h at 50 °C. The temperature was
decreased down to 0 °C, and white solid compound 5 (1.23
kg) was obtained by centrifugation in 96% yield and 99.5%
HPLC purity. High-resolution mass spectrometry (HRMS) [M
+
+ H] : calcd for C H F N O S: 604.2093; found: 604.2099.
3
0
33
3
3
5
1
under reduced pressure, affording oil compound 3 (1.50 kg) in
H NMR (400 MHz, CDCl ) δ 7.88 (d, J = 7.9 Hz, 1H), 7.54
3
+
9
3
3% yield and 94.1% HPLC purity. LC−MS [M + H] calcd,
(d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.32−7.26 (m,
1H), 7.25−7.18 (m, 1H), 7.04 (d, J = 8.5 Hz, 1H), 5.00 (d, J =
5.3 Hz, 1H), 3.72 (s, 1H), 3.63 (s, 1H), 3.44 (s, 2H), 2.19 (s,
2H), 2.09−1.88 (m, 4H), 1.88−1.77 (m, 1H), 1.63−1.57 (m,
1
51.1; found, 351.1; H NMR (400 MHz, CDCl ) δ 6.96 (d, J
3
=
8.9 Hz, 1H), 6.84 (d, J = 8.9 Hz, 1H), 3.65 (d, J = 1.8 Hz,
1
2
2
1
4
H), 3.40 (d, J = 1.7 Hz, 1H), 2.32 (s, 3H), 2.01−1.87 (m,
1
3
H), 1.86−1.79 (m, 1H), 1.61−1.56 (m, 1H), 1.39−1.29 (m,
1H), 1.57−1.45 (m, 9H), 1.46−1.38 (m, 2H). C NMR (151
1
3
H). C NMR (101 MHz, CDCl ) δ 168.78, 143.30, 142.68,
42.03, 140.37, 120.86, 119.32, 118.73 (q, J = 322.2 Hz),
9.33, 41.49, 41.38, 25.92, 25.40, 20.76.
MHz, CDCl , mixture of two tautomers) δ 156.62, 156.54,
3
3
149.95, 149.55, 149.14, 142.31, 142.14, 140.38, 140.24, 139.55,
137.77, 137.11, 135.31, 134.69, 134.06, 131.31, 129.31, 129.22,
128.82, 127.75, 125.09, 124.24, 123.94, 121.98, 119.85, 118.45,
118.29, 117.72, 115.60, 112.34, 80.54, 80.35, 54.24, 54.10,
49.41, 47.37, 43.00, 41.19, 28.52, 28.17, 28.04, 26.64, 25.96,
24.89.
4
.1.3. (S)-tert-Butyl 2-(5-(4-((1S,4R)-8-Hydroxy-1,2,3,4-tet-
rahydro-1,4-methanonaphthalen -5-yl)phenyl)-1H-imida-
zol-2-yl)pyrrolidine-1-carboxylate (4). To a stirred solution
of 9 (1.00 kg, 1 equiv), compound 3 (0.88 kg, 1.1 equiv) and
NaHCO (0.29 kg, 1.5 equiv) in DME/H O (2/1, 10 L total)
3
2
4.1.5. (S)-tert-Butyl 2-(5-(4-((1S,4R)-8-(2-((S)-1-(tert-
Butoxycarbonyl)pyrrolidin-2-yl)-1H-benzo[d]imidazol-5-yl)-
1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl)phenyl)-
1H-imidazol-2-yl)pyrrolidine-1-carboxylate (6). To a stirred
solution of 5 (1.00 kg, 1 equiv), 11 (0.72 kg, 1.05 equiv) and
K CO (0.46 kg, 2 equiv) in DME/H O (2/1, total 10 L) were
were added Pd(PPh ) (0.079 kg, 0.03 equiv) under N . After
3
4
2
addition, the temperature was increased to 80 °C, and the
reaction mass was stirred at that temperature for 10 h. When
HPLC analysis subsequently indicated that <0.3% of 9
remained, the temperature was decreased down to 60 °C.
K CO (0.54 kg, 2 equiv) and MeOH (8 L) were added, and
2
3
2
2
3
added Pd(PPh ) (0.057 kg, 0.03 equiv) under N . After
3 4
2
the reaction mixture was stirred for 10 h. When HPLC analysis
indicated that <0.1% of 4a remained, the temperature was
decreased down to 25 ± 5 °C. After filtration from Celite, the
organic solvent was distilled off under reduced pressure,
affording a solid crude product. After the crude solid was
collected by filtration, water (10 L) was added and the mixture
was triturated for 5 h at 25 ± 5 °C. The solid was collected by
filtration, and DCM (40 L) was subsequently added, followed
by stirring for 1 h at 40 °C. Then, n-heptane (10 L) was added,
and the reaction mixture was triturated for 4 h at 40 °C,
followed by stirring for 1 h at −10 °C. A gray solid was
obtained after centrifugation. This purification step was
repeated one to two times until HPLC analysis indicated the
purity of 4 ≥97%, affording white solid compound 4 (0.92 kg)
in 86% yield and 98.1% HPLC purity. High-resolution mass
addition, the temperature was increased to 80 °C. The reaction
mixture was stirred for 5 h at that temperature. When HPLC
analysis subsequently indicated that <0.3% of 5 remained, the
temperature was decreased down to 50 ± 5 °C. The organic
solvent was distilled off under reduced pressure, followed by
adding DCM (15 L) and water (3 L) while stirring. The
organic phase was collected and washed with water (3 L). After
removing the organic solvent, the residue was treated with
acetone (6.5 L). The formed suspension was triturated for 4 h
at a reflux temperature. The suspension was then cooled down
to 0 ± 5 °C, and solid was obtained by centrifugation. The
solid was further washed with acetone (1 L) and dried by
vacuum. White solid compound 6 (1.17 kg) was obtained in
+
95% yield and 99.2% HPLC purity. LC−MS [M + H] calcd,
1
741.4; found, 741.4. H NMR (600 MHz, CDCl ) δ 7.94−7.60
3
+
spectrometry (HRMS) [M + H] : calcd for C H N O :
(m, 4H), 7.58−7.49 (m, 2H), 7.40 (d, J = 8.1 Hz, 1H), 7.32−
7.20 (m, 3H), 5.24−5.16 (m, 1H), 5.07−4.99 (m, 1H), 3.70−
3.61 (m, 2H), 3.48 (s, 2H), 3.13−2.88 (m, 2H), 2.34−2.12
(m, 4H), 2.12−1.92 (m, 4H), 1.79−1.58 (m, 2H), 1.60−1.17
2
9
34
3
3
1
4
7
1
4
2
1
72.2600; found: 472.2605. H NMR (400 MHz, MeOD) δ
.71 (d, J = 7.7 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.30 (s,
H), 6.98 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H), 5.09−
.94 (m, 1H), 3.74−3.62 (m, 2H), 3.57−3.43 (m, 2H), 2.45−
.23 (m, 1H), 2.12−2.01 (m, 2H), 1.96−1.91 (m, 2H), 1.70−
1
3
(m, 22H). C NMR (151 MHz, CDCl ) δ 156.57, 156.50,
3
155.44, 154.51, 149.46, 146.45, 146.19, 139.23, 135.41, 134.47,
133.24, 129.12, 126.64, 125.99, 125.40, 124.63, 123.90, 80.73,
80.49, 54.72, 54.15, 49.10, 47.41, 47.37, 42.60, 42.51, 28.52,
28.42, 27.13, 27.07, 24.96, 24.89.
1
3
.60 (m, 1H), 1.53−1.39 (m, 4H), 1.25 (s, 9H). C NMR
(
151 MHz, MeOD) δ 156.53, 156.10, 152.51, 151.95, 150.73,
1
8
2
48.65, 140.93, 134.46, 129.78, 128.07, 127.85, 125.70, 114.75,
1.21, 57.14, 49.66, 47.82, 43.98, 40.87, 34.96, 28.76, 28.50,
8.00, 27.44, 24.70.
4.1.6. 2-((S)-Pyrrolidin-2-yl)-5-((1R,4S)-8-(4-(2-((S)-pyrroli-
din-2-yl)-1H-imidazol-5-yl)phenyl)-1,2,3,4-tetrahydro-1,4-
methanonaphthalen-5-yl)-1H-benzo[d]imidazole (7). To a
stirred solution of 6 (1.00 kg, 1 equiv) in DCM/MeOH (12 L/
1 L) was added trimethylsilyl chloride (TMSCl, 2.34 L) at 0 ±
5 °C. After addition, the temperature was increased to 25 ± 5
°C. The reaction mixture was stirred for 16 h at that
temperature. When HPLC analysis subsequently indicated that
<0.1% of 6 remained, the organic solvent was distilled off
under reduced pressure at 50 ± 5 °C. The residue was treated
with methanol (8 L), and the suspension was triturated for 8 h
at reflux temperature. After cooling down to 25 ± 5 °C, the
solid was collected by centrifugation and washed with
methanol (1 L). White solid compound 7 (0.88 kg) was
4
.1.4. (S)-tert-Butyl 2-(5-(4-((1S,4R)-8-(((Trifluoromethyl)-
sulfonyl)oxy)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-
yl)phenyl)-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (5).
To a stirred solution of 4 (1.00 kg, 1 equiv), DMAP (0.015
kg, 0.05 equiv) and Et N (0.59 L, 2 equiv) in DCM (15 L)
3
were added PhNTf (0.91 kg, 1.2 equiv) at 0 °C. After
2
addition, the reaction mixture was stirred for 10 h at 0 °C.
When HPLC analysis subsequently indicated that <0.5% of 4
remained, the stirring was stopped and the organic solvent was
distilled off under reduced pressure. Water (5 L) was then
added, and the reaction mixture was triturated for 5 h at 30 °C.
After centrifugation, the collected solid was further triturated
8
46
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pubs.acs.org/OPRD
Article
obtained under vacuum in 95% yield and 99.5% HPLC purity.
LC−MS [M − 4HCl + H] calcd, 541.3; found, 541.4. H
NMR (600 MHz, dimethyl sulfoxide (DMSO)) δ 10.72 (s,
at 25 ± 5 °C until HPLC analysis indicated that <0.2% of 7
remained. The reaction mixture was washed with water (5 L)
twice and 5% citric acid aqueous solution (5 L) three times.
The organic solvent was then distilled off under reduced
pressure at 40 ± 5 °C, followed by drying for 8 h at 50 ± 5 °C
under vacuum, affording white solid DAG181 (1.12 kg) with
90% yield and 99.3% HPLC purity. High-resolution mass
+
1
1
8
1
7
1
3
2
1
H), 10.52 (s, 1H), 10.25 (s, 1H), 9.95 (s, 1H), 8.30 (s, 1H),
.11 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 8.4 Hz, 1H), 7.79 (s,
H), 7.64 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.5 Hz, 1H), 7.43−
.20 (m, 2H), 5.17 (t, J = 7.7 Hz, 1H), 5.12 (t, J = 7.6 Hz,
H), 3.55 (s, 1H), 3.52 (s, 2H), 3.48−3.43 (m, 1H), 3.43−
.34 (m, 2H), 2.63−2.52 (m, 3H), 2.48−2.38 (m, 1H), 2.28−
.15 (m, 2H), 2.12−2.03 (m, 3H), 2.03−1.94 (m, 1H), 1.71−
+
spectrometry (HRMS) [M + H] : calcd for C H N O :
4
9
59
8
6
1
855.4558; found: 855.4527. H NMR (400 MHz, MeOD) δ
7.70 (d, J = 7.6 Hz, 2H), 7.63−7.53 (m, 2H), 7.50−7.41 (m,
2H), 7.39−7.27 (m, 2H), 7.22−7.14 (m, 2H), 5.32−5.25 (m,
1H), 5.19 (t, J = 6.4 Hz, 1H), 4.31−4.21 (m, 2H), 4.10−3.97
(m, 2H), 3.96−3.84 (m, 2H), 3.66 (s, 6H), 3.59−3.53 (m,
2H), 2.44−2.19 (m, 6H), 2.12−2.02 (m, 6H), 1.71−1.61 (m,
1
3
.63 (m, 1H), 1.52−1.47 (m, 1H), 1.47−1.37 (m, 2H).
C
NMR (151 MHz, DMSO) δ 149.31, 146.19, 146.06, 141.82,
1
1
5
2
40.53, 136.22, 135.34, 134.11, 133.60, 132.51, 129.07, 126.53,
26.08, 125.96, 125.77, 125.22, 116.34, 114.97, 114.25, 54.14,
2.17, 48.83, 45.57, 45.41, 42.14, 42.05, 29.76, 29.47, 26.60,
4.14, 23.76.
1
3
1H), 1.51−1.42 (m, 3H), 0.98−0.87 (m, 12H). C NMR
(151 MHz, MeOD) δ 173.50, 173.36, 159.47, 157.58, 151.05,
147.43, 147.24, 140.44, 136.82, 136.75, 136.72, 135.72, 135.69,
134.50, 129.99, 127.64, 127.06, 126.05, 125.89, 125.79, 124.77,
59.82, 59.73, 56.99, 56.40, 52.74, 49.94, 43.90, 43.87, 32.43,
32.31, 31.58, 31.47, 28.07, 28.02, 26.19, 26.05, 19.87, 19.82,
18.44, 18.30.
4
.1.7. (S)-tert-Butyl 2-(5-(4-(4,4,5,5-Tetramethyl-1,3,2-di-
oxaborolan-2-yl)phenyl)-1H-imidazol-2-yl)pyrrolidine-1-car-
boxylate (9). To a stirred solution of 8 (1.00 kg, 1 equiv) in
DME (6 L) were added B Pin (0.68 kg, 1.05 equiv), KOAc
2
2
(
0.62 kg, 2.5 equiv), and Pd(PPh ) Cl (0.027 kg, 0.015 equiv)
3 2 2
under N . After addition, the temperature was increased to 90
2
°
C, and the reaction mixture was stirred at 90 °C for 5 h.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.0c00519.
■
When HPLC analysis subsequently indicated that <0.1% of 8
remained, the organic solvent was distilled off under reduced
pressure. The formed residue was dissolved with EA (6 L),
followed by washing with water (3 L) and saturated NaCl
solution (3 L). The organic phase was collected, and the
organic solvent was then removed under reduced pressure. The
formed residue was triturated with n-heptane (4 L). The solid
was then collected by filtration. Dry compound 9 (1.03 kg)
sı
*
Results of partial condition screening for compounds 2,
1
13
3
, and 4 and H and C NMR spectra of key
intermediates (PDF)
1
was obtained with 92% yield and 98.4% HPLC purity. H
AUTHOR INFORMATION
Corresponding Authors
■
NMR (400 MHz, CDCl ) δ 7.85−7.33 (m, 4H), 7.25 (s, 1H),
3
5
2
1
.05−4.84 (m, 1H), 3.61−3.27 (m, 2H), 3.10−2.77 (m, 1H),
.31−2.03 (m, 2H), 2.05−1.80 (m, 1H), 1.48 (s, 9H), 1.34 (s,
2H).
Yingjun Zhang − The State Key Laboratory of Anti-Infective
Drug Development (No. 2015DQ780357), Sunshine Lake
Pharma Co. Ltd., Dongguan 523871, P. R. China;
Email: Zhangyingjun@hec.cn
Delie An − State Key Laboratory of Chemo/Biosensing and
Chemometrics, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, P. R.
China; Email: deliean@hnu.edu.cn
4
.1.8. (S)-tert-Butyl 2-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxa-
borolan-2-yl)-1H-benzo[d]imidazol-2-yl)pyrrolidine-1-car-
boxylate (11). To a stirred solution of 10 (1.00 kg, 1 equiv) in
DME (6.7 L) were added B Pin (0.73 kg, 1.05 equiv), KOAc
2
2
(
0.67 kg, 2.5 equiv), and Pd(dppf)Cl (0.040 kg, 0.02 equiv)
2
under N . After addition, the temperature was increased to 90
2
°
C, and the reaction mixture was stirred at 90 °C for 6 h.
Authors
When HPLC analysis subsequently indicated that <0.1% of 10
remained, the organic solvent was distilled off under reduced
pressure. The formed residue was dissolved with EA (5.4 L),
followed by washing with water (2.1 L) and saturated NaCl
solution (2.1 L). After filtration from Celite, the organic
solvent was distilled off under reduced pressure, affording a
solid crude product. The solid was triturated with n-heptane
Hongming Xie − State Key Laboratory of Chemo/Biosensing
and Chemometrics, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, P. R.
China
Haiwang Liu − The State Key Laboratory of Anti-Infective
Drug Development (No. 2015DQ780357), Sunshine Lake
Pharma Co. Ltd., Dongguan 523871, P. R. China;
orcid.org/0000-0002-5024-9715
Enhuo Huang − The State Key Laboratory of Anti-Infective
Drug Development (No. 2015DQ780357), Sunshine Lake
Pharma Co. Ltd., Dongguan 523871, P. R. China
Yahui Feng − HEC Research and Development Center, HEC
Pharm Group, Dongguan 523871, P. R. China
Xuwen Xiang − HEC Research and Development Center, HEC
Pharm Group, Dongguan 523871, P. R. China
Qinghong Fang − The State Key Laboratory of Anti-Infective
Drug Development (No. 2015DQ780357), Sunshine Lake
Pharma Co. Ltd., Dongguan 523871, P. R. China
Zhihong Peng − State Key Laboratory of Chemo/Biosensing
and Chemometrics, College of Chemistry and Chemical
(
(
2.1 L) and was then collected by filtration. Dry compound 11
0.96 kg) was obtained in 85% yield and 98.6% HPLC purity.
1
H NMR (400 MHz, CDCl ) δ 8.25−7.81 (m, 1H), 7.75−7.30
3
(
m, 2H), 5.18−4.98 (m, 1H), 3.66−3.28 (m, 2H), 3.11−2.92
m, 1H), 2.26−2.06 (m, 2H), 2.06−1.87 (m, 1H), 1.48 (s,
H), 1.34 (s, 12H).
(
9
4
.1.9. DAG181. To a stirred solution of 7 (1.00 kg, 1 equiv),
1
2 (0.52 kg, 2.05 equiv) in DCM (18 L) was added
triethylamine (TEA, 1.02 L, 5.0 equiv) slowly at 0 ± 5 °C.
EDCI (0.70 kg, 2.5 equiv) and DCM solution (2 L) of Oxyma
(
5
0.10 kg, 0.5 equiv) were then added dropwise over 1 h at 0 ±
°C. After addition, the reaction mixture was stirred for 1 h at
that temperature. Then, the mixture was continuously stirred
8
47
https://doi.org/10.1021/acs.oprd.0c00519
Org. Process Res. Dev. 2021, 25, 838−848

Organic Process Research & Development
pubs.acs.org/OPRD
Article
Engineering, Hunan University, Changsha 410082, P. R.
China
Wanrong Dong − State Key Laboratory of Chemo/Biosensing
and Chemometrics, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, P. R.
China; orcid.org/0000-0002-6541-2853
HCV NS5A: Mechanism of Resistance to an HCV Antiviral Agent.
PLoS One 2015, 10, No. e0122844.
(8) (a) Lawitz, E. J.; Dvory-Sobol, H.; Doehle, B. P.; Worth, A. S.;
McNally, J.; Brainard, D. M.; Link, J. O.; Miller, M. D.; Mo, H.
Clinical Resistance to Velpatasvir (GS-5816), a Novel Pan-Genotypic
Inhibitor of the Hepatitis C Virus NS5A Protein. Antimicrob. Agents
Chemother. 2016, 60, 5368−5378. (b) Younossi, Z. M.; Stepanova,
M.; Feld, J.; Zeuzem, S.; Jacobson, I.; Agarwal, K.; Hezode, C.; Nader,
F.; Henry, L.; Hunt, S. Sofosbuvir/velpatasvir Improves Patient-
reported Outcomes in HCV Patients: Results from ASTRAL-1
Placebo-controlled Trial. J. Hepatol. 2016, 65, 33−39.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.0c00519
Notes
(9) (a) Coburn, C. A.; Meinke, P. T.; Chang, W.; Fandozzi, C. M.;
The authors declare no competing financial interest.
Graham, D. J.; Hu, B.; Huang, Q.; Kargman, S.; Kozlowski, J.; Liu, R.;
McCauley, J. A.; Nomeir, A. A.; Soll, R. M.; Vacca, J. P.; Wang, D.;
Wu, H.; Zhong, B.; Olsen, D. B.; Ludmerer, S. W. Discovery of MK-
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