A Potent, Selective, and Orally Bioavailable HCV NS5A Inhibitor for Treatment of Hepatitis C Virus: (S)-1-((R)-2-(Cyclopropanecarboxamido)-2-phenylacetyl)-N-(4-phenylthiazol-2-yl)pyrrolidine-2-carboxamide

Article abstract of DOI:10.1021/acs.jmedchem.6b00962

Starting from the initial lead 4-phenylthiazole 18, a modest HCV inhibitor (EC₅₀ = 9440 nM), a series of structurally related thiazole derivatives has been identified as a novel chemical class of potent and selective HCV NS5A inhibitors. The introduction of a carboxamide group between the thiazole and pyrrolidine ring (42) of compound 18 resulted in a dramatic increase in activity (EC₅₀ = 0.92 nM). However, 42 showed only moderate pharmacokinetic properties and limited oral bioavalability of 18.7% in rats. Further optimization of the substituents at the 4-position of the thiazole ring and pyrrolidine nitrogen of the lead compound 42 led to the identification of compound 57, a highly potent and selective NS5A inhibitor of HCV (EC₅₀ = 4.6 nM), with greater therapeutic index (CC₅₀/EC₅₀ > 10000). Pharmacokinetic studies revealed that compound 57 had a superior oral exposure and desired bioavailability of 45% after oral administration in rats.

Full text of DOI:10.1021/acs.jmedchem.6b00962

Subscriber access provided by UNIV OF REGINA
Article
A Potent, Selective, and Orally Bioavailable HCV NS5A Inhibitor for
Treatment of Hepatitis C Virus: (S)-1- ((R)-2-(Cyclopropanecarboxamido)-2-
phenylacetyl)-N-(4-phenylthiazol-2-yl)pyrrolidine-2-carboxamide
Iou-Jiun Kang, Sheng-Ju Hsu, Hui-Yun Yang, Teng-Kuang Yeh, Chung-Chi Lee, Yen-Chun Lee,
Ya-Wen Tian, Jen-Shin Song, Tsu-An Hsu, Yu-Sheng Chao, Andrew Yueh, and Jyh-Haur Chern
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00962 • Publication Date (Web): 14 Dec 2016
Downloaded from http://pubs.acs.org on December 14, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
A Potent, Selective, and Orally Bioavailable HCV NS5A Inhibitor for
Treatment
(R)-2-(Cyclopropanecarboxamido)-2-phenylacetyl)-N-(4-phenylthiaz
ol-2-yl)pyrrolidine-2-carboxamide
of
Hepatitis
C
Virus:
(S)-1-
(
14
15
16
17
18
19
2
2
#
#
Iou-Jiun Kang, Sheng-Ju Hsu, Hui-Yun Yang, Teng-Kuang Yeh, Chung-Chi Lee,
2
23
24
Yen-Chun Lee, Ya-Wen Tian, Jen-Shin Song, Tsu-An Hsu, Yu-Sheng Chao, Andrew
25
2
27
*
*
Yueh, Jyh-Haur Chern
28
29
30
31
3
3
34
35
Institute of Biotechnology and Pharmaceutical Research, National Health Research
Institutes, Miaoli County 350, Taiwan, ROC
36
37
38
39
ˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍˍ
40
41
42
*
To whom correspondence should be addressed. Dr. Jyh-Haur Chern, E-mail:
4
4
45
46
47
48
49
50
51
jhchen@nhri.org.tw; Tel.: 886-37-246-166 ext. 35716; Fax: 886-37-586-456.
Corresponding address: Institute of Biotechnology and Pharmaceutical Research,
National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli
County 350, Taiwan, ROC
52
53
5
5
56
57
58
#Authors with equal contribution
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 114
1
2
3
4
5
6
7
8
9
ABSTRACT
1
1
Starting from the initial lead 4-phenylthiazole 18, a modest HCV inhibitor (EC50
=
12
13
14
9
440 nM), a series of structurally related thiazole derivatives has been identified as a
15
16
17
novel chemical class of potent and selective HCV NS5A inhibitors.
The
18
19
2
2
2
23
24
25
26
27
28
29
30
31
3
3
34
introduction of a carboxamide group between the thiazole and pyrrolidine ring (42) of
compound 18 resulted in a dramatic increase in activity (EC50 = 0.92 nM). However,
42 showed only moderate pharmacokinetic properties and limited oral bioavalability
of 18.7% in rats. Further optimization of the substituents at the 4-position of the
thiazole ring and pyrrolidine nitrogen of the lead compound 42 led to the
identification of compound 57, a highly potent and selective NS5A inhibitor of HCV
35
36
37
38
39
(EC50 = 4.6 nM), with greater therapeutic index (CC50/EC50 > 10,000).
40
41
42
Pharmacokinetic studies revealed that compound 57 had a superior oral exposure and
4
4
45
desired bioavailability of 45% after oral administration in rats.
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
1
1
Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) that was first
12
13
14
1
identified in 1989. HCV has at least six major genotypes, each containing multiple
15
16
17
2
subtypes, with genotype 1 being the most common worldwide. An estimated 170
18
19
20
million people globally have chronic hepatitis C (CHC) that eventually develops into
2
2
2
24
3
cirrhosis, hepatocellular carcinoma or liver failure. Genotype 1 has been historically
25
2
27
28
29
30
31
3
3
the most difficult to treat due to the fact that traditional dual therapy (pegylated
interferon plus ribavirin) produces relatively low sustained virological response (SVR)
rates (42% - 46%).4
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
In May 2011, the combination use of the first generation protease inhibitors telaprevir
and boceprevir (Figure 1) with pegylated interferon and ribavirin (triple therapy) was
approved by the Food and Drug Administration (FDA) for the treatment of HCV
genotype 1.5 Telaprevir- or boceprevir-based triple therapy is more effective than
traditional dual therapy and has an increased SVR rate, higher than 70%, in
genotype-1-infected patients.5c-e, 6 However, these triple therapies are limited by
treatment-related adverse effects and poor tolerability, especially in difficult-to-treat
5
5
56
57
58
59
60
patients, such as those with cirrhosis or advanced liver fibrosis.5c-e,
6a-e, 7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 114
1
2
3
4
5
6
7
8
9
1
1
12
13
Pharmaceutical developer Vertex Pharmaceuticals Inc. and Merck & Co. Inc. decide
to stop selling telaprevir and boceprevir on October 2014 and December 2015,
respectively, due to the alternative direct-acting antivirals (DAAs) with better efficacy
and tolerability, approved by FDA since 2013.8
14
15
16
17
18
19
20
Insert Figure 1
2
2
23
24
25
2
27
Recently, newly approved DAAs (Figure 2), such as the NS3/4A protease inhibitors
28
2
30
simeprevir and paritaprevir, the NS5A inhibitors ledipasvir,11 ombitasvir12 and
9
10
31
3
3
1
3
14
15
daclatasvir, the NS5B polymerase inhibitors sofosbuvir and dasabuvir have led to
34
35
36
dramatic progress in treating CHC. These emerging DAAs are being investigated in
37
3
39
interferon-free regimens consisting of one to three DAAs with or without ribavirin.16
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
However, the efficacy of monotherapy with single DAAs is always insufficient and
increases the risk of emerging resistant strains.17 Combination therapy with DAAs
exhibiting different mechanisms is often necessary to reduce drug resistance and
dose-related toxicity as well as to enhance the effectiveness of the antivirals.
Currently, the NS5A inhibitor ledipasvir is most commonly used in combination with
the NS5B polymerase inhibitor sofosbuvir for treatment in chronic hepatitis C
57
58
59
6
genotype 1 patients.
This drug has been tested and shown efficacy in
ACS Paragon Plus Environment

Page 5 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
treatment-naive and treatment experienced patients. However, the cost of the
fixed-dose combination (ledipasvir 90 mg/sofosbuvir 400 mg) has been a
controversial topic. It costs $1,125 per pill in the US, translating to $94,500 for a
1
1
12
13
14
1
2-week treatment course. Therefore, it was aimed to discover and develop a novel,
15
16
17
18
19
potent, and orally bioavailable HCV NS5A inhibitor with lower molecular weight and
cost.
20
2
2
23
24
25
2
27
Insert Figure 2
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
As shown in Figure 2, ledipasvir, ombitasvir, and daclatasvir are potent inhibitors of
HCV NS5A, which contain hetero-dimeric and homo-dimeric structures with large
molecular weights. In fact, the extremely high cost of this class of HCV NS5A
inhibitors may be due to their complicated structures and difficult synthesis. To
simplify the chemical structure and reduce the molecular weight of the HCV NS5A
inhibitor, daclatasvir, several heterocyclic compounds 6, 12, 18, and 22 (Figure 3)
have been designed, synthesized, and evaluated for their inhibitory activity against 1b
replicon assay in our anti-HCV screening program. Interestingly, the imidazole 6,
and thiazoles 18 and 22 were identified as the initial leads of HCV inhibitors.
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 114
1
2
3
4
5
6
7
8
9
Insert Figure 3
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
In addition to daclatasvir, ombitasvir is also a potent HCV NS5A inhibitor with a
specific carboxamide group between the phenyl and pyrrolidine ring (Figure 2).
This interesting structure prompts us to further investigate a series of new amide
analogs based on the scaffold of the thiazole compound 18. The introduction of a
carboxamide group between the thiazole and pyrrolidine ring (42) resulted in a
dramatic increase in activity (EC50 = 0.92 nM). Further optimization of the
substituents at the 4-position of the thiazole ring and pyrrolidine nitrogen of the lead
compound 42 led to the identification of compound 57 (Figure 4), a highly potent and
selective NS5A inhibitor of HCV (EC50 = 4.6 nM), with greater therapeutic index
34
35
36
37
38
39
(CC50/EC50 > 10,000).
40
41
42
4
4
45
Insert Figure 4
46
47
48
49
50
51
52
RESULTS AND DISCUSSION
53
5
5
56
5
58
Chemistry
59
6
The synthesis of the imidazole derivative 6 was carried out as shown in Scheme 1.
ACS Paragon Plus Environment

Page 7 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Coupling reaction of 2-aminoacetophenone hydrochloride 1 with N-Boc-L-proline in
the presence of EDC/HOBt·H O at room temperature gave the corresponding amide 2
2
1
1
in 40% yield. Reaction of 2 with ammonium acetate in the presence of acetic acid at
12
13
14
o
1
60 C led to the formation of the imidazole derivative 3 in 74% yield. Deprotection
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
of 3 with trifluoroacetic acid gave the pyrrolidine 4 in 82% yield, which was then
coupled with N-Boc-D-phenylglycine to give the protected pyrrolidine 5 in 67% yield.
Deprotection of 5 with trifluoroacetic acid gave the corresponding amine, which was
reacted with 4-morpholinecarbonyl chloride in the presence of triethylamine to give
the desired imidazole 6 in 31% overall yield.
31
3
3
34
35
36
Insert Scheme 1
37
38
39
40
41
42
4
4
45
46
47
48
The oxadiazole derivative 12 was prepared as outlined in Scheme 2. Reaction of the
benzonitrile 7 (Scheme 2) with hydroxylamine hydrochloride in the presence of
DIPEA gave the amidoxime 8 in 85% yield, which was then coupled with
49
50
51
N-Boc-L-proline in the presence of TBTU/HOBt·H O followed by thermal
2
52
53
5
5
56
57
58
59
60
cyclization of the O-acylated intermediate to give the oxadiazole intermediate 9 in
53% yield. Deprotection of 9 with trifluoroacetic acid gave the corresponding
pyrrolidine 10 in 90% yield, which was then coupled with N-Boc-D-phenylglycine in
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 114
1
2
3
4
5
6
7
8
9
1
1
12
13
the presence of EDC/HOBt·H O to give the protected pyrrolidine 11 in 89% yield.
2
Deprotection of 11 with trifluoroacetic acid gave the corresponding amine, which was
reacted with 4-morpholinecarbonyl chloride in the presence of triethylamine to give
the desired oxadiazole 12 in 77% overall yield.
14
15
16
17
18
19
20
Insert Scheme 2
2
2
23
24
25
2
27
The 4-phenylthiazole derivative 18 was prepared as outlined in Scheme 3. Reaction
28
2
30
of N-Boc-L-proline with (Boc)
O and (NH
)
2
CO in the presence of pyridine gave the
2
4
3
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
corresponding amide 13 in 95% yield, which was reacted with Lawesson’s reagent in
THF at 70 ℃ to give the thioamide 14 in 89% yield. Condensation of 14 with
phenacyl bromide at refluxing ethanol provided the thiazole intermediate 15 in 74%
yield. Similarly deprotection of 15 with trifluoroacetic acid gave the pyrrolidine 16
in 82% yield, which was then coupled with N-Boc-D-phenylglycine in the presence of
46
47
48
EDC/HOBt·H O to give the protected pyrrolidine 17 in 71% yield. Deprotection of
2
49
50
51
52
53
1
7 with trifluoroacetic acid gave the corresponding amine, which was reacted with
-morpholinecarbonyl chloride in the presence of triethylamine to give the desired
4
5
5
56
5
thiazole 18 in 58% overall yield.
58
59
60
ACS Paragon Plus Environment

Page 9 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Insert Scheme 3
1
1
12
13
14
15
16
17
The 5-phenylthiazole derivative 22 was prepared as outlined in Scheme 4. Reaction
of the amide 2 with Lawesson’s reagent at refluxing THF gave the thiazole
intermediate 19 in 81% yield. Similarly deprotection of 19 with trifluoroacetic acid
gave the pyrrolidine 20 in 88% yield, which was then coupled with
18
19
20
2
2
2
24
N-Boc-D-phenylglycine in the presence of EDC/HOBt·H O to give the protected
2
25
2
27
28
29
30
31
3
3
pyrrolidine 21 in 72% yield. Deprotection of 21 with trifluoroacetic acid gave the
corresponding amine, which was reacted with 4-morpholinecarbonyl chloride in the
presence of triethylamine to give the desired thiazole 22 in 56% overall yield.
34
35
36
37
38
39
Insert Scheme 4
40
41
42
4
4
45
To search for more potent HCV inhibitors, exploration of the substituents at the
46
47
48
4
-position of the thiazole ring and the pyrrolidine nitrogen of the amide analog of the
49
50
51
52
53
5
5
56
57
thiazole 18 was carried out. A series of new amide derivatives 42-51 were designed
and synthesized according to the procedures as shown in Scheme 5 beginning from
the commercially available 4-substituted-2-aminothiazole 23a-e. The coupling
reaction of 23a-e with N-Boc-L-proline in the presence of HATU/DIPEA in DMF at
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 114
1
2
3
4
5
6
7
8
9
o
5
0 C gave the corresponding amides 24a-e.
Deprotection of 24a-e with
trifluoroacetic acid at room temperature gave the pyrrolidine derivatives 25a-e, which
was then coupled with N-Boc protected glycine derivatives 26-31 in the presence of
1
1
12
13
14
EDC/HOBt·H O to give the corresponding pyrrolidines 32-41 in good yields
2
15
16
17
(76-93%). Deprotection of 32-41with trifluoroacetic acid gave the corresponding
18
19
20
2
2
23
24
25
26
amines, which was reacted with 4-morpholinecarbonyl chloride in the presence of
triethylamine to give the target compounds 42-51 in moderate to good yields
(41-89%).
27
28
29
30
31
3
3
Insert Scheme 5
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
The synthesis of amides 52-68, ureas 69-76, and thioureas 77-81 were performed
according to the procedures as shown in Scheme 6. Deprotection of the Boc
protected thiazole 32 with trifluoroacetic acid at room temperature gave the
corresponding amine, which was reacted with a variety of commercially available
acyl chlorides in the presence of triethylamine at room temperature to give the
corresponding amides 52-68 in moderate to good yields (21-90%). In addition, the
ureas 69-76 were prepared by reacting the amine intermediate with the corresponding
isocyanates, while the thioureas 77-81 were prepared by reacting the amine
ACS Paragon Plus Environment

Page 11 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
intermediate with the corresponding isothiocyanates in the presence of triethylamine.
Alternatively, the urea derivatives can also be successfully synthesized by the
treatment of the amine intermediate with CDI and primary amine or triphosgene in the
presence of triethylamine.
14
15
16
17
18
19
20
Insert Scheme 6
2
2
23
24
25
2
27
Biological Evaluation
28
2
30
31
3
In order to simplify the chemical structure of the HCV NS5A inhibitor, daclatasvir,
and find better therapeutic agents, several heterocyclic compounds 6, 12, 18, and 22
3
34
35
36
(Figure 3) have been designed, synthesized, and evaluated for their inhibitory activity
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
against 1b replicon assay in our anti-HCV screening program. As shown in Table 1,
the imidazole 6, and thiazoles 18 and 22 showed potent inhibitory activity against
HCV genotype 1b with an EC50 value of 98, 9440 and 2970 nM, respectively. All of
the above compounds (6, 18 and 22) exhibit low cytotoxicity (CC50 >50 μM),
indicating a good therapeutic window. Surprisingly, a complete loss in activity was
observed when A ring was replaced with a 1,2,4-oxadiazole ring (12). This result
suggests that the type of ring A is important for the activity of this class of inhibitors.
Compound 18 was selected for further optimization due to its convenient synthesis
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 114
1
2
3
4
5
6
7
8
9
with better yield and excellent thermal and chemical stability though compound 6 and
2
2 also show inhibitory activity.
1
1
12
13
14
Insert Table 1
15
16
17
18
19
20
As shown in Table 2, it is very interesting to note that the introduction of a
2
2
2
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
carboxamide group between the thiazole and pyrrolidine ring resulted in a dramatic
increase in inhibitory activity, compound 42 was found to be 10000-fold more potent
than 18 (18 vs 42, EC50 = 9440 vs 0.92 nM). The reason for this is probably that the
amide group of 42 can increase the strength of intermolecular interactions via H
bonding (since the secondary amide group has an H atom as H-bond donor and a
carbonyl group as the H-bond acceptor). With this result in hand, we turned our
investigation to the phenyl ring of 42 in order to assess the effect of relatively flexible
alkyl groups (Table 2). As shown in compound 43, removal of phenyl group at
position 4 of the thiazole ring of 42 resulted in drastic loss in activity. (EC50 from 0.92
nM of 42 → 9650 nM of 43). Furthermore, replacement of the phenyl group at
position 4 of the thiazole ring with three other types of alkyl groups, that is, methyl
(44), tert-butyl (45), and cyclohexyl (46) resulted in significant loss of activity. To
gain a deeper insight in the analysis of the activity-flexibility/rigidity relationship for
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 13 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
substituents at the 4-position of the thiazole ring (42-46), an interesting trend was
observed. It was found that analogues with more rigid substituents such as phenyl
1
1
(42) or cyclohexyl (46) at the 4-position of the thiazole ring displayed higher potency.
12
13
14
When the R group of 42 was changed from phenyl (42) to methyl (47), a more than
2
15
16
17
1
400 times decrease in activity was observed (42 vs 47, EC50 = 0.92 vs 1310 nM).
18
19
2
2
2
23
24
Further replacement of the methyl group (47) by the ethyl (48), n-propyl (49),
i-propyl (50) and tert-butyl (51) had no noticeable effect on activity. These above
25
2
27
results imply that a more rigid phenyl ring in both R
1
and R positions (42) should be
2
28
29
30
necessary for improving the inhibitory activity against HCV (42 vs 43-51).
31
3
3
34
35
36
Insert Table 2
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
The next step in our design was to investigate if conversion of the morpholine moiety
in 42 to different alkyl, alicyclic, aromatic or heterocyclic groups could provide
additional anti-HCV potency (Table 3). Replacement of the morpholino urea with a
variety of terminal amides (52-56 for methyl, ethyl, n-propyl, i-propyl and tert-butyl,
respectively), a double-digit decrease in activity was observed compared with 42 (42
vs 52-56, EC50 = 0.92 vs 15-86 nM). This decreased activity was also seen in
cyclopentyl (58) and cyclohexyl (59) analogues. (58, EC50 = 13 nM; 59, EC50 = 14
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 114
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
nM). Interestingly, cyclopropyl derivative 57 showed only a single-digit drop in
activity compared with 42 (42 vs 57, EC50 = 0.92 vs 4.6 nM). Replacing the
alicyclic cyclohexyl ring of 59 with an aromatic phenyl ring (60) showed no
significant change in activity (59 vs 60, EC50 = 14 vs 15 nM). Introduction of a
nitrogen atom into the phenyl ring of 60 yielded three positional isomers, 2'-pyridyl
(61), 3'-pyridyl (62) and 4'-pyridyl (63) derivatives. Interestingly, the 4′-pyridyl
derivative 63 was more active compared with the corresponding phenyl ring
derivative 60 (63 vs 60, EC50 = 3.8 vs 15 nM), although the other two isomers 61 and
2
23
24
25
26
27
28
29
30
6
2 showed no increase in activity (61, EC50 = 22 nM; 62, EC50 = 13 nM). A
31
3
3
34
35
comparison of activity data from pyridine substituted amides (61-63) suggested that
the nitrogen atom at the 4′-position of the terminal pyridine ring is optimal.
36
37
38
39
40
41
42
It would be of interest to study the effect of different nitrogen-containing alkyl
4
4
45
substituents (64-67) at the R position. Replacement of the isopropyl group (55)
3
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
with a dimethylamino group (64) gave a 3-fold enhancement in activity (55 vs 64,
EC50 = 36 vs 11 nM). Increasing the alkyl chain length of the dimethylamine from
methyl (64) to ethyl (65) resulted in equipotent anti-HCV activity (65, EC50 = 18 nM).
Interestingly, the replacement of the N,N-diethylamino group (65) by a bioisostere
such as pyrrolidine (66) or piperidine (67) ring enhanced the activity by 5.8-fold (65
ACS Paragon Plus Environment

Page 15 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
vs 66-67, EC50 = 18 vs 3.1 nM). Further modification to this moiety by placing
a N-methyl group at the 4-position of the piperidine ring of 67 generated compound
1
1
12
13
6
8 that was as active as 42. (68, EC50 = 0.85 nM). These findings suggest that
-membered heterocycles with two heteroatoms such as morpholine (42) and
6
14
15
16
17
piperazine (68) at the R3 position are preferred.
18
19
20
2
2
2
24
Insert Table 3
25
26
27
28
2
30
Structure activity relationship (SAR) studies upon changing the amide moiety of
31
3
3
5
7-63 to a urea group led to synthesis the urea derivatives 69-71 (Table 4).
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
Cyclopropyl (69) and cyclopentyl (70) urea compounds did not significantly change
the potency comparing to their amide counterpart 57 and 58 (69 vs 57 and 70 vs 58,
EC50 = 3.5 vs 4.6 and 13 vs 13 nM), while cyclohexyl urea 71 led to 4-fold increased
potency as compared to its corresponding amide 59 (71 vs 59, EC50 = 3.3 vs 14 nM).
Modification of the cyclohexyl ring (71) by increasing the ring size to a 7-membered
cycloheptyl ring (72) or replacement with a phenyl ring (73) was leading to slightly
increased activity compared with 71 (72, EC50 = 3.8 nM; 73, EC50 = 2.3 nM). The
3-pyridine substituted urea 75 was found to be 11- to 15-fold more active than its
58
59
6
corresponding 2-pyridyl (74) and 4-pyridyl (76) derivatives (75 vs 74 and 76, EC50 =
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 114
1
2
3
4
5
6
7
8
9
1
3
.9 vs 30 and 22 nM). It was interesting to observe that the phenyl (73) and
-pyridyl (75) substituted ureas exhibited a better inhibitory activity compared to their
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
corresponding amide analogues (60 and 62). Further bioisosteric replacement of the
urea oxygen with sulfur gave the corresponding thioureas 77-81 (Table 4).
Cyclopentyl substituted thiourea 78 was found to be more active than the oxygen
counterpart 70 (78 vs 70, EC50 = 5.2 vs 13 nM). The remaining cyclopropyl (77),
cyclohexyl (79), phenyl (80) and 3-pyridyl (81) substituted thioureas possessed
similar or reduced activity when compared to their corresponding urea analogues (69,
26
27
28
29
30
7
1, 73 and 75).
31
3
3
34
35
36
Insert Table 4
37
38
39
40
41
42
In vivo studies
4
4
45
46
47
48
49
50
51
Following in vitro studies, five compounds (42, 57, 60, 63 and 66) were selected for
further studies in vivo based on the in vitro results and availability. The in vivo rat
PK data for the compounds selected has been summarized in Table 5 and 6.
52
53
5
5
56
5
As can be seen from Table 5, compound 57 showed a favorable PK profile after a
58
59
6
single iv administration of 1 mg/kg. Low clearance (Cl = 27.1 ± 4.6 mL/min/kg)
ACS Paragon Plus Environment

Page 17 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
associated with a relatively low volume of distribution at steady state (Vss = 1.32 ±
0
.04 L/kg), moderate half-life (t1/2 = 1.3 ± 0.3 h), and high AUC (635 ± 116 ng/mL×h)
was seen with 57 iv administration. Compound 63 showed highest clearance (Cl =
2.2 ± 8.9 mL/min/kg) and lowest AUC (295.6 ± 33 ng/mL×h). Compound 60
displayed highest Vss (4.2 ± 0.9 L/kg), longest t1/2 (2.3 ± 0.3 h), moderate CL (44.8 ±
.2 mL/min/kg) and moderate AUC (367 ± 27 ng/mL×h).
6
3
2
2
23
24
25
2
27
Insert Table 5
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
As shown in Table 6, Compound 57 exhibited rapid absorption (Tmax = 0.8 ± 0.4 h),
high Cmax (247.3 ± 93.6 ng/mL) and AUC (1436 ± 293 ng/mL×h) as well as
relatively high bioavailability (F = 45%) in rats after oral administration of 5 mg/kg.
Compound 42 has similar Cmax (247.3 ± 93.6 ng/mL) and Tmax (0.8 ± 0.3 h) but
lower AUC (404 ± 76 ng/mL×h) and bioavailability (F = 18.7%). Lower
bioavailability was also reported for other inhibitors, including 60 (F = 31%), 63 (F =
48
49
50
51
1
0.1%), and 66 (20.4%). These results indicate that compound 57 had a superior PK
52
53
5
5
56
profile compared to the other compounds evaluated, characterized by significantly
higher oral bioavailability and AUC.
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 114
1
2
3
4
5
6
7
8
9
Insert Table 6
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
Furthermore, drug resistance studies have been carried out to elucidate how
compound 57 works in the HCV RNA replication. The resistance profile showed
that the N terminus of NS5A is the region responsible for the 57-mediated inhibition
of HCV1b replicon activity. It may be possible that 57 exert inhibitory activity by
directly binding to NS5A due to it produce very similar resistance data to daclatasvir,
a NS5A inhibitor, in the drug resistance mutations within the N terminus of NS5A.18
Of course, there is still a need to clarify the binding site and molecular mechanism of
action of this class of inhibitors. Nevertheless, the findings from this study provide
very useful information to develop novel anti-HCV agents.
34
35
36
37
38
39
40
41
42
CONCLUSION
4
4
45
46
47
48
In
this
paper,
we
described
the
discovery
of
49
50
51
(S)-1-((R)-2-(cyclopropanecarboxamido)-2-phenylacetyl)-N-(4-phenylthiazol-2-yl)pyr
52
53
5
5
56
57
58
59
60
rolidine-2-carboxamide (57) as a potent, selective, and orally bioavailable HCV
NS5A inhibitor for the treatment of HCV infection. A medicinal chemistry program
based on the scaffold of 4-phenylthiazole 18 has led to the identification of 57, a
ACS Paragon Plus Environment

Page 19 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
potent HCV NS5A inhibitor with EC50 value of 4.6 nM, which showed high
therapeutic index (CC50/EC50 > 10,000). In addition, compound 57 displayed
promising pharmacokinetic properties in rats following oral administration.
Previously drug resistance data suggested that 57 is likely to inhibit HCV replication
by directly binding to HCV NS5A, and it is currently under preclinical development
for the treatment of HCV infection. Further SAR and mechanistic studies are still in
progress and will be reported elsewhere.
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 114
1
2
3
4
5
6
7
8
9
EXPERIMENTAL SECTION
1
1
General Methods
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
All commercial chemicals and solvents are reagent grade and were used without
further treatment unless otherwise noted. 1H NMR spectra were obtained with a
Varian Mercury-300 or a Varian Mercury-400 spectrometer. Chemical shifts were
recorded in parts per million (ppm, ) and were reported relative to the solvent peak
or TMS. Coupling constants (J) are reported in hertz (Hz). Splitting patterns are
described by using the following abbreviations: s, singlet; d, doublet; t, triplet; q,
quartet; br, broad; m, multiplet. LC/MS data were measured on an Agilent
MSD-1100 ESI-MS/MS System. All tested compounds were detected at UV 254nm
unless otherwise stated. Column chromatography was performed with silica gel
37
38
39
40
41
42
(Merck Kieselgel 60, 230-400 mesh). Reactions were monitored by TLC using
4
4
45
Merck 60 F254 silica gel glass backed plates and visualized under ultraviolet
46
47
48
irradiation (254 nm and 360 nm) or by spraying with phosphomolybdic acid reagent
49
50
51
o
(
Aldrich) followed by heating at 80 C. Melting points were determined on an
52
53
5
5
56
57
58
59
60
Electrothermal IA9000 Series Digital Melting Point Apparatus. Purity of the final
compounds was determined on a Hitachi 2000 series HPLC system with a reverse
phase C18 column (Agilent ZORBAX Eclipse XDB-C18 5 m, 4.6 mm x 150 mm),
ACS Paragon Plus Environment

Page 21 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
o
operating at 25 C. Mobile phase A was acetonitrile. Mobile phase B was 10 mM
NH OAc aqueous solution containing 0.1% formic acid. The gradient system started
4
1
1
12
13
14
15
16
17
from A/B (10%/90%) at 0 min to A/B (90%/10%) at 45 min. The flow rate of the
mobile phase was 0.5 mL/min, and the injection volume of the sample was 5 μL.
Peaks were detected at 254 nm. The purity of all tested compounds is >95% purity.
18
19
20
2
2
2
24
A.1. Preparation of Compound 6, 12, 18 and 22
25
2
27
(S)-tert-butyl 2-(2-oxo-2-phenylethylcarbamoyl)pyrrolidine-1-carboxylate (2)
28
2
30
To a solution of Boc-L-proline (5.64 g, 2.62 mmol) in CH
Cl (30 mL) at room
2
2
31
3
3
temperature, 1-hydroxybenzotriazole monohydrate (HOBt·H O, 4 g, 2.62 mmol) and
2
34
35
36
ethyl-(N’,N’-dimethylamino)propylcarbodiimide hydrochloride (EDC, 5 g, 2.62
37
38
39
mmol) was added and then stirred for 30 min. To the above reaction mixture,
40
41
42
2
-aminoacetophenone hydrochloride
1
(3 g, 1.74 mmol) and N,
4
4
45
46
47
N-diisopropylethylamine (DIPEA, 3.4 mL, 2.62 mmol) was added in one portion
respectively, and then stirred at room temperature for 18 hours. The solution was
48
49
50
51
washed by 10% citric acid(aq.) and NaHCO3(aq.)
.
The result mixture was then
52
53
5
5
56
extracted with CH
yellow liquid.
2
Cl
2
, dried over MgSO , filtered and concentrated to give viscous
4
5
The liquid was purified with column chromatography
58
59
6
1
(Hexane:EtOAc = 2:1) to yield white solid (2.19 g, 40%): mp 96-97 ℃; H NMR
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 114
1
2
3
4
5
6
7
8
9
(
300MHz, CDCl
3
) δ 1.48 (br s, 9H), 1.85-1.99 (m, 2H), 2.02-2.18 (m, 2H), 3.42 (br s,
0
.4H), 3.53 (br s, 1.6H), 4.31 (br s, 0.5H), 4.40 (br s, 0.5H), 4.08-4.85 (m, 2H), 7.05
1
1
(br s, 1H), 7.47-7.65 (m, 3H), 7.97 (d, J = 3.7 Hz, 2H); LC/MS(ESI) m/z: 233.2
12
13
14
[
M-Boc + H]+.
15
16
17
18
19
2
2
2
23
24
25
26
27
28
29
30
(S)-tert-butyl 2-(5-phenyl-1H-imidazol-2-yl)pyrrolidine-1-carboxylate (3)
To a solution of compound 2 (2.19 g, 6.6 mmol) and ammonium acetate (12.7 g) in
xylenes (80 mL), acetic acid was added and then stirred at refluxed temperature (~160
℃) for 3 hours. After cooling to room temperature, the reaction mixture was
31
3
3
extracted with EtOAc/H
2
O. The organic layer was dried over MgSO , filtered and
4
34
35
36
concentrated in vacuo. The residue was purified with column chromatography
37
3
39
1
(
Hexane:EtOAc = 2:1) to provide compound 3 (1.52 g, 73.5%): mp 63-65 ℃; H
40
41
42
NMR (300MHz, CDCl ) δ 1.47 (br s, 9H), 1.93-1.99 (m, 2H), 2.07-2.15 (m, 2H), 2.97
3
4
4
45
(br s, 1H), 3.36-3.40 (m, 1H), 4.95-4.97 (m, 1H), 7.19-7.23 (m, 2H), 7.34 (t, J = 7.5
46
47
48
+
+
Hz, 3H), 7.63 (br s, 2H); LC/MS(ESI) m/z: 314.1 [M + H] , 336.1 [M + Na] .
49
50
51
52
53
5
(S)-5-phenyl-2-(pyrrolidin-2-yl)-1H-imidazole (4)
5
56
57
To a solution of compound 3 (1.52 g, 4.85 mmol) in CH
2
Cl (15 mL) at 0 ℃,
2
58
59
6
trifluoroacetic acid (7.5 mL) was added. Then, the reaction was stirred at room
ACS Paragon Plus Environment

Page 23 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
temperature for 1 hour.
Basification of the solution by NaHCO3(sat.) was
accomplished until pH value was about 8. The solution was extracted with CH
2
Cl .
2
1
1
The combined organic layers were dried by MgSO and concentrated in vacuo. The
4
12
13
14
crude product was used as starting material for next step without further purification.
15
16
17
1
Compound 4 (0.845 g, 82%): mp 196-198 ℃; H NMR (300MHz, CDCl
3
) δ
18
19
20
1
.84-1.98 (m, 2H), 2.21-2.28 (m, 2H), 3.02-3.14 (m, 2H), 4.48 (t, J = 7.9 Hz, 1H),
2
2
2
24
7.21 (d, J = 4.2 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.61 (d, J = 3.6 Hz, 2H);
25
2
27
+
+
LC/MS(ESI) m/z: 214.1 [M + H] , 236.1 [M + Na] .
28
29
30
31
3
3
tert-butyl
34
35
36
(R)-2-oxo-1-phenyl-2-((S)-2-(5-phenyl-1H-imidazol-2-yl)pyrrolidin-1-yl)ethylcarb
37
38
39
amate (5)
40
41
42
To a solution of N-Boc-D-phenylglycine (1.09 g, 4.36 mmol) in CH
2
Cl (25 mL) at
2
4
4
45
room temperature, HOBt·H O (0.73 g, 4.75 mmol) was added in one portion and then
2
46
47
48
49
50
51
52
53
5
the mixture was stirred for 10 min. To the above reaction mixture were added EDC
0.91 g, 4.75 mmol) and compound 4 (0.84 g, 3.96 mmol) respectively, and then
stirred for 18 hours at room temperature. The solution was washed by 10% citric
(
5
56
5
acid(aq.) and NaHCO3(aq.). The result mixture was then extracted with CH
2
Cl , dried
2
58
59
6
over MgSO , filtered and concentrated to give viscous yellow liquid. The liquid was
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 114
1
2
3
4
5
6
7
8
9
purified with column chromatography (Hexane:EtOAc = 2:1) to yield brown gel (1.18
1
g, 67%): mp 99-100 ℃; H NMR (400MHz, CDCl
3
) δ 1.21 (s, 9H), 1.87-2.19 (m, 4H),
1
1
2
.89 (br s, 1H), 3.19-3.25 (m, 1H), 3.60-3.79 (m, 1H), 5.31 (d, J = 4.0 Hz, 1H), 5.34
12
13
14
(d, J = 3.4 Hz, 1H), 5.66 (br s, 1H), 7.20-7.25 (m, 3H), 7.27-7.45 (m, 6H), 7.68 (br s,
15
16
17
18
19
+
+
2
9
H); LC/MS(APCI) m/z: 447.3 [M + H] , 347.3 [M-Boc + H] ; HPLC t = 43.75 min,
R
6.8%.
20
2
2
23
24
25
2
27
28
29
N-((R)-2-oxo-1-phenyl-2-((S)-2-(5-phenyl-1H-imidazol-2-yl)pyrrolidin-1-yl)ethyl)
morpholine-4-carboxamide (6)
30
31
3
3
To a solution of compound 5 (0.36 g, 0.81 mmol) in CH
2
Cl (10 mL) at 0℃,
2
34
35
36
37
38
trifluoroacetic acid (5 mL) was added. Then, the reaction was stirred at room
temperature for 1 hour. Basification of the solution by NaHCO3(sat.) was
39
40
41
42
accomplished until pH value was about 8. The solution was extracted with CH
2
Cl .
2
4
4
45
The combined organic layers were dried by MgSO and concentrated in vacuo to
4
46
47
48
afford product as starting material for next step without further purification. To the
49
50
51
above crude product in CH
2
Cl (10 mL) at ice bath, morpholine-4-carbonyl chloride
2
52
53
5
(
0.18 mL, 1.62 mmol) and Et N (0.23 mL, 1.62 mmol) were added respectively and
3
5
56
5
58
59
then stirred for 10 min. The result mixture was concentrated under reduced pressure
and then extracted with EtOAc. The organic layer was washed with brine, dried
60
ACS Paragon Plus Environment

Page 25 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
over MgSO
4
, filtered and concentrated to yield crude product. The residue was
purified with column chromatography (Acetone:Hexane = 1:5) to afford the final
1
1
1
viscous solid 6 (0.113 g, 31%): H NMR (300MHz, CDCl
3
) δ 1.83-2.15 (m, 4H),
12
13
14
2
.61-2.72 (m, 1H), 3.21-3.38 (m, 5H), 3.41-3.69 (m, 4H), 3.83-3.89 (m, 1H), 5.35 (d,
15
16
17
J = 3.0 Hz, 1H), 5.46 (s, 1H), 7.16-7.23 (m, 2H), 7.24-7.49 (m, 8H), 7.67 (d, J = 3.7
18
19
20
1
3
Hz, 2H); C NMR (CDCl , 75MHz) δ 24.35, 30.01, 43.93, 46.70, 55.72, 56.76, 66.27,
3
2
2
2
24
124.85, 126.59, 127.72, 128.27, 128.51, 128.92, 129.27, 135.21, 148.25, 157.79,
25
2
27
+
170.68; LC/MS(APCI) m/z: 460.2 [M + H] ; HRMS (m/z): calcd for C26
H
30
N
5
3
O [M
28
2
30
+
+ H] 460.2349, found 460.2346; HPLC t
R
= 18.60 min, 96.8%.
31
3
3
34
35
36
N'-hydroxybenzimidamide (8)
37
38
39
40
41
42
4
4
45
To a solution of benzonitrile 7 (3 g, 29 mmol) in ethanol (50 mL) at room temperature,
hydroxylamine hydrochloride (2.02 g, 29 mmol) and DIPEA (5.1 mL, 29 mmol) were
added respectively. Then the reaction mixture was stirred at 90 ℃ for 5 hours.
After cooling to room temperature and removing the solvent, the result colorless
46
47
48
49
50
51
viscous liquid was extracted with EtOAc/H O. The organic layer was dried over
2
52
53
5
MgSO , filtered and concentrated in vacuo. The crude compound was washed with
4
5
56
5
n-Hexane and filtered to give white solid 8 (3.36 g, 85%) for next step without further
58
59
6
1
purification: mp 182-185 ℃; H NMR (400MHz, DMSO-d
6
) δ 7.56-7.59 (m, 2H),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 114
1
2
3
4
5
6
7
8
9
7
.67-7.76 (m, 3H), 9.09 (br s, 2H), 11.36 (br s, 1H); LC/MS(ESI) m/z: 137.1 [M +
H]+.
1
1
12
13
14
(S)-tert-butyl 2-(3-phenyl-1,2,4-oxadiazol-5-yl)pyrrolidine-1-carboxylate (9)
15
16
17
To a solution of Boc-L-proline (2.15g, 10 mmol) in DMF (18 mL),
18
19
20
O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU, 3.21
2
2
2
24
g, 10 mmol), HOBt·H O (0.31g, 2 mmol) and DIPEA (8.8 mL, 50 mmol) was added
2
25
2
27
28
29
30
31
3
3
respectively. The reaction mixture was stirred at room temperature for 5 min. To
the above reaction mixture, compound 8 (1.36 g, 10 mmol) was added, and then
stirred at room temperature for 1 hour, and at 110 ℃ for 2.5 hours. After cooling to
34
35
36
room temperature, the mixture was extracted with EtOAc/H
2
O, dried over MgSO ,
4
37
38
39
then filtered and concentrated to give crude yellow liquid. The crude compound was
40
41
42
purified with column chromatography (EtOAc:n-Hexane = 1:10) to provide
4
4
45
1
compound 9 (1.67 g, 53%): mp 109-110 ℃; H NMR (400MHz, CDCl
3
) δ 1.30 (s,
46
47
48
49
50
51
52
53
5
7
H), 1.47 (s, 2H), 1.99-2.20 (m, 3H), 2.36-2.44 (m, 1H), 3.50-3.60 (m, 1H), 3.68-3.74
m, 1H), 5.06-5.09 (m, 0.7H), 5.20 (br s, 0.3H), 7.49 (d, J = 3.6 Hz, 3H), 8.07 (d, J =
.8 Hz, 2H); LC/MS(ESI) m/z: 338.1 [M + Na]+.
(
3
5
56
57
58
59
6
(S)-3-phenyl-5-(pyrrolidin-2-yl)-1,2,4-oxadiazole (10)
ACS Paragon Plus Environment

Page 27 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To a solution of compound 9 (1 g, 3.2 mmol) in CH
2
Cl (10 mL) at 0 ℃,
2
trifluoroacetic acid (5 mL) was added. Then, the reaction was stirred at room
temperature for 1 hour. Basification of the solution by NaHCO3(sat.) was
1
1
12
13
14
accomplished until pH value was about 8. The solution was extracted with CH
2
Cl .
2
15
16
17
The combined organic layers were dried by Na
2
SO and concentrated in vacuo. The
4
18
19
20
crude product was used as starting material for next step without further purification.
2
2
2
24
1
Viscous solid compound 10 (0.61 g, 90%): H NMR (400MHz, CDCl
3
) δ 1.87-2.02
25
2
27
(m, 2H), 2.10-2.20 (m, 2H), 2.28-2.37 (m, 1H), 3.06-3.12 (m, 1H), 3.19-3.25 (m, 1H),
28
2
30
4.55 (dd, J = 5.6, 8.4 Hz, 1H), 7.44-7.52 (m, 3H), 8.07-8.10 (m, 2H); LC/MS(ESI)
31
3
3
+
+
m/z: 216.1 [M+H] , 238.1 [M + Na] .
34
35
36
37
38
39
tert-butyl
40
41
42
(R)-2-oxo-1-phenyl-2-((S)-2-(3-phenyl-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)ethylc
4
4
45
arbamate (11)
46
47
48
To a solution of N-Boc-D-phenylglycine (0.58 g, 2.3 mmol) in CH
2
Cl (10 mL) at
2
49
50
51
room temperature, HOBt·H O (0.43g, 2.8 mmol) was added in one portion; the
2
52
53
5
5
56
57
58
59
60
mixture was then stirred for 10 min. To the above reaction mixture were added EDC
(0.53 g, 2.8 mmol) and compound 10 (0.5g, 2.3 mmol) respectively, and then stirred
for 18 hours at room temperature. The solution was washed by 10% citric acid(aq.)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 114
1
2
3
4
5
6
7
8
9
and NaHCO3(aq.). The result mixture was then extracted with CH
2
Cl , dried over
2
Na
2
SO , filtered and concentrated to give viscous yellow liquid. The liquid was
4
1
1
purified with column chromatography (Hexane:EtOAc = 2:1) to yield white solid
12
13
14
1
(
0.93 g, 89%): mp 75-77 ℃; H NMR (300MHz, CDCl
3
) δ 1.37 (s, 4.5H), 1.41 (s,
15
16
17
18
19
4
1
.5H), 1.94-2.40 (m, 4H), 3.18-3.39 (m, 1H), 3.83-3.95 (m, 1H), 5.34 (d, J = 2.2 Hz,
H), 5.46-5.58 (m, 1H), 6.01 (d, J = 3.6 Hz, 1H), 7.30-7.52 (m, 8H), 7.78-8.10 (m,
20
2
2
2
24
+
+
2H); LC/MS(ESI) m/z: 349.1 [M-Boc + H] , 471.2 [M + Na] .
25
26
27
28
29
30
N-((R)-2-oxo-1-phenyl-2-((S)-2-(3-phenyl-1,2,4-oxadiazol-5-yl)pyrrolidin-1-yl)eth
31
3
3
yl)morpholine-4-carboxamide (12)
34
35
36
To a solution of compound 11 (0.5 g, 1.1 mmol) in CH
2
Cl (10 mL) at 0℃,
2
37
38
39
40
41
trifluoroacetic acid (5 mL) was added. Then, the reaction was stirred at room
temperature for 1 hour. Basification of the solution by NaHCO3(sat.) was
42
4
4
45
accomplished until pH value was about 8. The solution was extracted with CH
2
Cl .
2
46
47
48
The combined organic layers were dried by Na
2
SO and concentrated in vacuo to
4
49
50
51
afford crude product as starting material for next step without further purification.
52
53
5
To the above crude product in CH
2
Cl (10 mL) at ice bath, morpholine-4-carbonyl
2
5
56
5
chloride (0.16 mL, 1.3 mmol) and Et N (0.18 mL, 1.3 mmol) were added respectively
3
58
59
6
and then stirred for 10 min. The result mixture was concentrated under reduced
ACS Paragon Plus Environment

Page 29 of 114
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
pressure and then extracted with EtOAc. The organic layer was washed with brine,
dried over Na
2
SO , filtered and concentrated to yield crude product. The residue
4
1
1
was purified with column chromatography (Hexane:EtOAc = 4:1, and then
12
13
14
1
Hexane:EtOAc = 2:1) to afford the final product 12 (0.39 g, 76.6%): mp 89-92 ℃; H
15
16
17
NMR (300MHz, CDCl ) δ 1.93-2.45 (m, 4H), 3.14-3.27 (m, 1H), 3.31-3.48 (m, 4H),
3
18
19
20
3
.59-3.67 (m, 4H), 3.80-3.94 (m, 1H), 5.34 (dd, J = 3.0, 7.8 Hz, 1H), 5.62-5.72 (m,
2
2
23
24
1H), 6.12 (d, J = 3.2 Hz, 1H), 7.30-7.53 (m, 8H), 8.05-8.11 (m, 2H); 13C NMR
25
2
27
(CDCl , 75MHz) δ 24.41, 30.51, 43.72, 46.70, 53.99, 56.74, 66.33, 126.59, 127.52,
3
28
2
30
128.11, 128.30, 128.75, 129.01, 131.20, 137.65, 156.28, 168.39, 169.61, 179.06;
31
3
3
+
+
LC/MS(ESI) m/z: 462.2 [M + H] , 484.2 [M + Na] ; HRMS (m/z): calcd for
34
3
36
+
C
25
H
28
N
5
O
4
[M + H] 462.2141, found 462.2136; HPLC t
R
= 29.47 min, 98.4%.
37
38
39
40
41
42
(S)-tert-butyl 2-carbamoylpyrrolidine-1-carboxylate (13)
4
4
45
46
47
To a solution of compound Boc-L-proline (5 g, 23 mmol) in 1,4-dioxane (90 mL) at
room temperature, pyridine (1.16 mL, 13.9 mmol), ammonium carbonate (2.9 g, 30.2
48
49
50
51
mmol) and (Boc) O (6.59 g, 30.2 mmol) was added and then stirred for 18 hours.
2
52
53
5
After removed the solvent, the mixture was extracted with 20% citric acid/brine.
5
56
5
The organic layer was dried over MgSO , filtered and concentrated. The crude
4
58
59
6
compound was washed with n-Hexane and filtered to give white solid 13 (4.7 g, 95%)
ACS Paragon Plus Environment