Discovery of N-substituted oseltamivir derivatives as potent and selective inhibitors of H5N1 influenza neuraminidase

Article abstract of DOI:10.1021/jm500892k

To discover group-1-specific neuraminidase (NA) inhibitors that are especially involved in combating the H5N1 virus, two series of oseltamivir derivatives were designed and synthesized by targeting the 150-cavity. Among these, compound 20l was the most potent N1-selective inhibitor, with IC₅₀ values of 0.0019, 0.0038, and 0.0067 μM against NAs from three H5N1 viruses. These values are better than those of oseltamivir carboxylate. Compound 32 was another potent N1-selective inhibitor that exhibited a 12-fold increase in activity against the H274Y mutant relative to oseltamivir carboxylate. Molecular docking studies revealed that the 150-cavity was an auxiliary binding site that may contribute to the high selectivity of these compounds. The present work is a significant breakthrough in the discovery of potent group-1-specific neuraminidase inhibitors, which may be further investigated for the treatment of infection by the H5N1 virus.

Full text of DOI:10.1021/jm500892k

Subscriber access provided by Binghamton University | Libraries
Article
Discovery of N-substituted Oseltamivir Derivatives as Potent
and Selective Inhibitors of H5N1 Influenza Neuraminidase
Yuanchao Xie, Dongqing Xu, Bing Huang, Xiuli Ma, Wenbao
Qi, Fangyuan Shi, Xinyong Liu, Yingjie Zhang, and Wenfang Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500892k • Publication Date (Web): 25 Sep 2014
Downloaded from http://pubs.acs.org on September 27, 2014
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 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
Discovery of N-substituted Oseltamivir Derivatives as Potent and
Selective Inhibitors of H5N1 Influenza Neuraminidase
7
8
9
Yuanchao Xie,1 Dongqing Xu,1 Bing Huang,2 Xiuli Ma,2 Wenbao Qi,3 Fangyuan,
Shi,1 Xinyong Liu *,1, Yingjie Zhang *,1 and Wenfang Xu*,1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong
University, 44, West Culture Road, Jinan, Shandong 250012, P. R. China
2 Institute of poultry science, Shandong Academy of Agricultural Sciences, 1, Jiaoxiao
Road, Jinan, Shandong 250023, P. R. China
3 College of Veterinary Medicine, South China Agricultural University, 483, Wushan
Road, Tianhe District, Guangzhou 510642, P. R. China
ABSTRACT
To discover Group-1-specific neuraminidase (NA) inhibitors that are especially
involved in combating the H5N1 virus, two series of oseltamivir derivatives were
designed and synthesized by targeting the 150-cavity. Among these, compound 20l
was the most potent N1-selective inhibitor, with IC50 values of 0.0019 µM, 0.0038
µM and 0.0067 µM against NAs from three H5N1 viruses. These values are better
than those of oseltamivir carboxylate. Compound 32 was another potent N1-selective
inhibitor that exhibited a 12-fold increase in activity against the H274Y mutant
relative to oseltamivir carboxylate. Molecular docking studies revealed that the
150-cavity was an auxiliary binding site that may contribute to the high selectivity of
these compounds. The present work is a significant breakthrough in the discovery of
potent Group-1-specific neuraminidase inhibitors, which may be further investigated
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
for the treatment of infection by the H5N1 virus.
5
6
7
INTRODUCTION
8
9
Recently, increasing attention has been paid to the possibility of another global
flu pandemic. In 2009, a swine-origin H1N1 influenza virus rapidly spread throughout
the world, giving rise to a serious public panic.1 H7N9 avian virus is a lethal avian
influenza virus endemic to China that has evolved to infect humans since April 2013.2
It is known that the highly pathogenic avian influenza A (H5N1) virus, with a 60%
mortality, is a great threat to humans. Though there is no evidence of efficient
human-to-human transmission, some studies have showed that the H5N1 virus
exhibits an increased propensity for acquiring human receptor specificity.3
Neuraminidase (NA), which plays an important role in the viral life cycle, is a
good target for anti-influenza drug design. To date, three NA inhibitors have been
developed as effective treatments of influenza A and B infections: zanamivir (1),
oseltamivir (2) and peramivir (3, Figure 1). Of these, orally administered oseltamivir
is the first choice and has been widely used since approved in 1999, though recently,
resistant viral strains have seriously narrowed the drug’s clinical application.4, 5
Zanamivir and peramivir (which are only approved in Japan, Korea and China at
present) are mainly administered by inhalation from a nebulizer or intravenous routes,
which is very inconvenient for patients. In addition, viral strains that are resistant to
the two drugs are also constantly being reported.6, 7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
These emerging problems with the current NA inhibitors increase the demand for
the development of novel anti-influenza drugs. In 2006, one important discovery
ACS Paragon Plus Environment

Page 3 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
regarding the structure of the influenza virus NA provided a great opportunity for
inhibitor design. The X-ray crystallographic structures show that in Group-1 NAs (N1,
N4, N5, N8 subtypes), there is a large cavity termed the 150-cavity adjacent to the
active site that is not found in Group-2 NAs (N2, N3, N6, N7, N9 subtypes).8
Previously, NA inhibitors were mainly designed based on the crystal structures of N2
and N9, which are both Group-2 enzymes. This finding is very meaningful for
exploring Group-1-specific NA inhibitors, especially against H1N1 and H5N1
influenza virus. The H1N1 virus has caused at least three flu pandemics since the
early 20th century, leading to a tremendous loss of life. At present, this virus is
variable and still very dangerous to human health. The H5N1 influenza virus, which
sees occasional outbreaks in poultry and causes human death, is a significant global
pandemic threat. Thus, the development of high potent Group-1-specific NA
inhibitors may be of considerable value against H1N1 and H5N1 viral infection.
Enlightened by the new identified cavity of Group-1 NAs, researchers at first
tried to screen for potential inhibitors by using various computer-aided drug design
tools. A large number of compounds were proposed, but they were only predicted to
have high binding affinity for NA (mostly based on N1 from the H5N1 virus) without
being biologically evaluated in vivo or in vitro.9-14 Zanamivir and oseltamivir (OS)
are good leads to be optimized in the search for novel inhibitors.15-18 Based on their
structures, a few derivatives with selective NA inhibitory activities have been
discovered, such as compounds 419, 520 and 6.21 Compounds 4 and 5, designed with
the 150-cavity, are N1-selective inhibitors, but these compounds exhibit low
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
5
6
7
8
9
inhibitory activities. Compound 6 is derived from zanamivir and shows selectivity for
the NA of H5N1, but it is not equally potent against the NA of H1N1. In 2013, Lin
and co-workers discovered one acylguanidine-modified zanamivir analog (7), the
hydrophobic substituent (naphthylthiomethyl) of which was thought to interact with
the 150-cavity. However, this compound did not exhibit increased activity relative to
zanamivir or selectivity for the N1 enzyme.22 More recently, the group of Martin
designed several oseltamivir analogs bearing N-substituted guanidines, two of which
presented good activity against NAs from wild-type and oseltamivir-resistant strains.
Their findings also suggested that it was hard to access the 150-cavity by appending
substituents to the terminal nitrogen atoms of the exocyclic guanidine moiety.23
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Structures of the approved NA inhibitors (1)-(3) and compounds 4-7.
DESIGN OF OSELTAMIVIR DERIVATIVES
The main objective of our research was to discover potent Group-1 selective NA
inhibitors that act as anti-influenza agents. As shown in Figure 2 (A), the C5-NH2 of
OS carboxylate in the NA active center is very close to the 150-cavity. It is thus
feasible to take advantage of the cavity as an auxiliary binding site by modifying the
ACS Paragon Plus Environment

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
C5-NH2 of OS. It should also be considered that the NH2 group, which forms strong
hydrogen bonds with Glu119 and Asp151, is very important for the activity of the
molecule. In 2010, Pinto and colleagues synthesized a series of OS analogs bearing
substituted triazoles at the C-5 position.19 The activities of these compounds were
dramatically decreased, highlighting the importance of the basic group at the site.
Therefore, we planned to develop our compounds with two substituents at the C-5
NH2: one substituent would occupy the potential binding site and one basic group
would bind with Glu119 or Asp151. We thus designed two series of OS derivatives,
which were the substituted guanidines and the secondary amines (Figure 2). The
acylguanidine-modified zanamivir analogs reported by Lin et al. were also designed
to lock the 150-cavity of Group-1 NAs, but the result was unsatisfying.22 The various
substituents at the C-4 guanidine group were introduced by amide bonds, which
decreased the alkalinity of the guanidine group. In our study, all the derivatives were
designed by modifying the C-5 guanidino group and the amino group with alkyl
groups to increase the alkalinity and to reduce the polarity relative to the OS
carboxylate. In view of the importance of alkalinity, our design strategy is more
reasonable.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Comparison of the crystal structures of N1 (PDB 2HU0, A) and N2 (PDB
4GZQ, B) and the general structures of the designed compounds.
CHEMISTRY
Compound 9, with a guanidine group instead of NH2, was reported to have
comparable or even better activities relative to OS carboxylate.24 We synthesized this
compound as one of the positive controls. Another two OS derivatives, (11) and (14),
were prepared by converting the COOH groups of oseltamivir and compound
9 to
hydroxamic acid (CONHOH) to explore whether the CONHOH was good for the
activity, as shown in Scheme 1.
Commercial oseltamivir phosphate was the primary starting material. In Scheme
1, compound
9 was synthesized using a three-step route that included guanylation,
hydrolysis and N-Boc deprotection and was similar to the reported method.25 The
guanylation reaction was performed with N,N’-bis(tert-butoxycarbonyl)thiourea
(SC(NHBoc)2)/HgCl2. Treatment of compound
8 with a solution of NH2OK in
CH3OH gave intermediate 10. Compound 13 was synthesized by the same method
from Boc-protected oseltamivir 12. The target compounds 11 and 14 were all
prepared as hydrochlorides with 3 M HCl/EtOAc.
Scheme 1. Synthesis of compounds 9, 11 and 14a
ACS Paragon Plus Environment

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (a) SC(NHBoc)2, HgCl2, Et3N, DMF, rt; (b) NH2OK,
CH3OH, rt; (c) HCl/EtOAc, CH3OH, rt; (d) (Boc)2O, Et3N, CH2Cl2, rt.
In Scheme 2, oseltamivir phosphate was reacted with a range of different
aldehydes in the presence of NaBH3CN to afford the key intermediate 15. The
synthesis of compound 17 was achieved in three steps: guanylation, hydrolysis and
Boc deprotection. Compound 20 was also synthesized either in three steps (Boc
protection, hydrolysis and deprotection) from intermediate 15 as a trifluoroacetate salt
or by direct hydrolysis of intermediate 15 with NaOH.
Scheme 2. Synthesis of OS derivatives (17) and (20)a
aReagents and conditions: (a) RCHO, NaBH3CN, EtOH, rt; (b) SC(NHBoc)2, HgCl2,
Et3N, DMF, rt; (c) NaOH, CH3OH, H2O, 50 °C;(d) CF3COOH, CH2Cl2, rt; (e)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
(Boc)2O, Et3N, CH2Cl2, rt; (f) NaOH, CH3OH, H2O, 50 °C, 4 h, then CH3COOH.
5
6
7
Because the R2 groups of compounds 20a-m were all hydrophobic, we further
8
9
designed compound 23, which contained one NH2 at the end of the substituent. The
structure and its synthetic route are shown in Scheme 3. 3-(Boc-amino)propanal was a
key reagent that could be prepared from 3-aminopropanol through two steps: Boc
protection and PCC (pyridinium chlorochromate) oxidation. The following synthetic
procedure was the same as that in Scheme 2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of compounds 23a
aReagents and conditions: (a) NaBH3CN, EtOH, rt; (b) (Boc)2O, Et3N, CH2Cl2, rt; (c)
NaOH, CH3OH, H2O, 50 °C;(d) CF3COOH, CH2Cl2, rt.
Compounds 20h and 20l of the second series exhibited good NA inhibitory
activities (data shown in Table 2). Therefore, we continued to synthesize another five
derivatives, compounds 30-32. In Scheme 4, the two 2-alkoxybenzaldehydes were
prepared by reaction of 2-hydroxylbenzaldehyde with 2-(bromomethyl)furan and
3-bromoprop-1-ene and were used for the synthesis of compounds 30a and 30b. A
Suzuki reaction of the aryl bromides with boronic acid gave another three aldehydes
that were used in synthesizing compounds 31 and 32
Scheme 4. Synthesis of compounds 30
32a
.
-
ACS Paragon Plus Environment

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aReagents and conditions: (a) acetone, K2CO3, reflux; (b) THF, H2O, K2CO3
Pd(PPh3)4, reflux.
RESULTS AND DISCUSSION
Three N1 enzymes from influenza (H5N1) viral strains and two N2 enzymes
from influenza (H9N2) viral strains were the representatives of Group-1 and Group-2
NAs for the NA inhibition assay. In Table 1, OS carboxylate was found to be more
potent against N2 (IC50 = 0.0028
µ
M, H9N2-415) than N1 (IC50 = 0.017
µM,
H5N1-1220), which is consistent with reported data.26 Compound
9, by contrast,
which has a guanidine group, showed better inhibitory activity against N1 than
against N2. The two controls did not equally inhibit the two NA subtypes and showed
a certain degree of selectivity.
Compounds 11 and 14, which had a CONHOH group instead of a COOH group,
exhibited significantly decreased activities (> 40-fold) than the two controls. It could
be confirmed that CONHOH was not a good group for binding with the S1 pocket.
The S1 pocket in the NA active site contains three basic residues, Arg 118, Arg 292,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 47
1
2
3
4
5
6
7
8
9
and Arg 371, which can strongly interact with acidic groups, such as a COOH27 or
phosphoryl group.25 The CONHOH group, with an intramolecular hydrogen bond, is
weaker than COOH in acidity, which may have largely contributed to the poor
activities of the two compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The first series of OS derivatives (17a-l) were less potent than the two controls
but displayed a certain degree of selectivity for N1. The substituents (R1CH2-) at the
guanidines of these compounds were unfavorable for the activity and probably
affected the binding of the other structural parts to the corresponding pockets of the
active site. Compounds 17a and 17e were the best two compounds of this series with
IC50 values of 0.088-0.35
respectively. Generally, Compounds 17a
were better than the alkyl-containing compounds 17f-k.
µ
M (17a) and 0.15-0.47
µM (17e) against H5N1 and H9N2,
-e
, which both have aromatic groups at R1,
Table 1, NA inhibitory activities of compounds 11, 14 and 17a-l
NA inhibition assay, IC50 (µM)a
H5N1-1220b
> 0.1
H5N1-1206c
> 0.2
H5N1-QJd
> 0.2
H9N2-415e
0.20 0.015
0.11 0.01
0.21 0.015
2.60 0.18
H9N2-S2f
0.16 0.01
0.12 0.01
0.35 0.028
2.64 0.25
Compound
R1
11
14
> 0.1
> 0.2
0.18
17a
17b
0.20 0.015
1.17 0.11
0.088 0.007
3.02 0.21
0.19 0.013
> 3.0
ACS Paragon Plus Environment

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
17c
17d
17e
1.44 0.15
0.55 0.032
0.15 0.018
1.56 0.20
0.25 0.014
0.27 0.029
3.20 0.24
0.55 0.03
0.43 0.050
2.37 0.21
0.49 0.044
0.47 0.055
2.16 0.18
0.36 0.038
0.37 0.045
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17f
17g
17h
17i
0.67 0.075
0.23 0.033
0.78 0.075
1.14 0.20
1.14 0.18
0.39 0.041
3.62 0.39
> 3.0
3.90 0.42
0.45 0.053
> 3.0
> 5.0
> 5.0
> 5.0
> 5.0
> 5.0
> 5.0
> 3.0
1.48 0.22
> 5.0
1.23 0.18
4.88 0.55
> 5.0
17j
0.37 0.045
0.95 0.12
2.54 0.32
> 3.0
> 3.0
17k
17l
> 3.0
> 5.0
0.79 0.087
0.017 0.001
0.0069 0.001
1.78 0.24
0.013 0.002
0.0023
> 3.0
> 5.0
> 5.0
OS-C
9
0.0090
0.0026
0.0028
0.0064
0.0031
0.011 0.001
aConcentration required to reduce NA activity to 50% of control NA activity (IC50). Values were the mean of three
experiments. The standard derivations omitted for conciseness were
<
20% of the mean.
bA/chicken/china/1220/2012(H5N1).
cA/duck/china/1206/2012(H5N1).
dA/duck/china/QJ/01(H5N1).
eA/chicken/china/415/2013(H9N2). fA/chicken/shandong/S2/02(H9N2).
As shown in Table 2, the second series of OS derivatives (20a-m) exhibited
selective inhibition against NAs of H5N1 influenza virus and were much more potent
than the first series. Compound 20l was the best compound among the second series,
with IC50 values of 0.0019
µ
M, 0.0038
µ
M and 0.0067
µM against the three types of
H5N1 NAs and IC50 values of 1.20
µ
M and 0.58
µ
M against the two types of H9N2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
5
6
7
8
9
NAs. Obviously, compound 20l displayed a high degree of selectivity for N1 over N2.
For the inhibitory activity against the NA of the H5N1-1220 strains, Compound 20l
was approximately 8-fold more potent than the OS carboxylate and 3-fold more
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
potent than compound
9.
Several other compounds in the series, such as compounds 20d
,
20e, 20h and 20i,
showed comparable or even better inhibitory activities relative to the OS carboxylate
against N1. The presence of Cl at the ortho-position of the phenyl group (compound
20h) enhanced the activity 2- to 10-fold, while the CH3O, OH, or F at the position
(compounds 20f, 20g and 20j) decreased the activity. Compounds 20a-c, with alkyl
groups at the R2 position, were also found to be good N1 selective inhibitors but were
not as potent as the control. Compound 20a bears a 3-pentyl group and showed the
best activity against N2 (IC50 = 0.30 µM and 0.070 µM for H9N2-515 and H9N2-S2,
respectively) within the series. Compound 20k has a double bond in the R2 group and
was approximately 2-fold more active than compound 20m, which had a single bond
at the same position. This suggested that rigid R2 groups were favorable for NA
inhibitory activity. To some extent, this is consistent with the structure-activity
relationships (SARs) of most compounds in series 1 and series 2.
Table 2, NA inhibitory activities of compounds 20a-ma
NA inhibition assay, IC50 (µM)
ACS Paragon Plus Environment

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound
20a
R2
H5N1-1220
H5N1-1206
H5N1-QJ
H9N2-415
H9N2-S2
0.027 0.003
0.043 0.004
0.056 0.007
0.083 0.01
0.053 0.010
0.019 0.004
0.117 0.020
0.054 0.004
0.076 0.008
0.099 0.012
0.054 0.008
0.080 0.013
> 0.20
0.30 0.035
2.28 0.35
> 5.0
0.070 0.009
0.63 0.078
1.75 0.23
20b
0.019 0.002
0.078 0.009
0.015 0.003
0.0071
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
20c
20d
0.74 0.11
2.44 0.38
1.64 0.31
0.099 0.016
0.42 0.08
0.25 0.041
20e
20f
0.031 0.006
0.021 0.004
0.0036
20g
20h
20i
> 5.0
> 5.0
0.063 0.009
0.014 0.002
0.038 0.005
0.090 0.010
0.194 0.034
0.029 0.004
0.056 0.007
0.108 0.018
0.98 0.08
1.15 0.15
2.72 0.31
0.043 0.007
0.042 0.007
0.65 0.01
0.013 0.003
0.010 0.002
20j
20k
20l
0.014 0.003
0.0019
0.048 0.009
0.0038
0.106 0.016
0.0067
4.02 0.54
1.20 0.23
> 5.0
3.23 0.52
0.58 0.11
> 5.0
20m
0.032 0.006
0.081 0.011
0.190 0.021
aValues were the mean of three independent experiments. The standard derivations omitted for conciseness were <
20% of the mean.
The NA inhibition results of the other six OS derivatives are listed in Table 3.
Compound 23 bears one NH2 and exhibited decreased activity against N1 in
comparison to compound 20b, suggesting that basic groups in the R2 substituent are
not favorable for the activity. Combining the SAR results described above, it can be
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
6
7
8
9
concluded that the potential binding region of the R2 group of the second series was
probably hydrophobic. Compounds 30a and 30b have large substituents at the
ortho-position of the phenyl group and were less potent against N1 than compounds
20f or 20g. Among the other three compounds, compound 32 was highly selective and
was the most potent against the three types of H5N1 NAs, with IC50 values of 0.0021
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
µ
M, 0.0043 µM and 0.014 µM. Compounds 31a and 31b with substituents (Cl and Ph)
at the biphenyl group were less potent than compound 20l with respect to the N1
inhibitory activity. However, for the N2 inhibitory activity, compound 31b was much
more potent (10 to 100-fold) than compounds 31a and 20l, indicating that the Ph at
the biphenyl group probably formed some special interactions with the N2 enzyme.
Table 3, NA inhibitory activities of compounds 23 and 30-32a
NA inhibition assay, IC50 (µM)
Compound
23
R2
H5N1-1220
> 0.10
H5N1-1206
> 0.20
H5N1-QJ
> 0.20
H9N2-415
H9N2-S2
0.95 0.08
1.53 0.13
4.70 0.58
2.02 0.35
1.05 0.17
0.72 0.08
1.91 0.17
0.34 0.042
30a
> 0.20
> 0.20
0.059 0.01
0.090 0.012
0.011 0.002
30b
> 0.20
> 0.20
31a
0.027 0.005
0.044 0.005
31b
0.043 0.006
0.039 0.005
0.058 0.006 0.032 0.004
0.038 0.004
ACS Paragon Plus Environment

Page 15 of 47
Journal of Medicinal Chemistry
1
2
3
4
32
0.0021
0.0043
0.014 0.002
3.52 0.53
0.21 0.037
5
6
7
8
aValues were the mean of three independent experiments. The standard derivations omitted for conciseness were <
9
20% of the mean.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The H274Y mutant NA exhibits high-level resistance to oseltamivir, which is
presently a great concern. In Table 4, oseltamivir carboxylate was found to be quite
insensitive to this mutant enzyme, with an IC50 value of 2.1
µM, which is
approximately 1000-fold lower than the IC50 against the wild type enzyme. Among
these oseltamivir derivatives, compound 32 displayed obvious increased activity (IC50
= 0.16
µM) relative to the OS carboxylate towards the H274Y mutant and was
deemed a suitable leading compound for further structural optimizations.
Table 4, NA (H274Y, H5N1) inhibitory activities of selected compoundsa
Compound
OS-Cb
17a
17e
20l
31a
32
IC50 (
µ
M),
1.0 0.1 1.78 0.1 1.16 0.1 1.91 0.2 0.16 0.02 2.1 0.2
H5N1(H274Y)
aValues were the mean of three independent experiments. bThe positive control, oseltamivir carboxylate.
The binding models of compounds 20l and 32 with the N1 enzyme were
analyzed using the computer software programs SYBYL and MOE. As shown in
Figure 3 A, the two compounds perfectly bound with the enzyme. For example, the
biphenylmethyl group of compound 20l occupied the 150-cavity while the other
structural part of the compound interacted with the active site in a manner similar to
the binding pattern of oseltamivir carboxylate. This result was in good agreement with
the purpose of our design. In the previous section, we hypothesized that the binding
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
7
8
9
region of the biphenyl group was hydrophobic. In Figure 3 B, it was clear that most of
the residues around the group were hydrophobic, such as Trp 438, Val 116 and Gly
147. The side chain of Gln 136, Arg 128 and Arg 152 also made certain contributions
to the hydrophobicity of the cavity. Among these residues, Val 116 seemed to be the
most important because it was directly facing the biphenyl groups. Compound 20l
exhibited poor activities against N2, which can be easily explained by the enzyme’s
structure. Because of the closed 150-loop, the active site of N2 is relatively smaller
than that of N1 (Figure 2 B), and would not well accommodate compound 20l or other
compounds bearing a large group at C-5 position. Their structures could be distorted
in the active site, resulting in unfavorable interactions with the corresponding pockets.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. The docking results of compound 20l (light blue) and 32 (green) with
N1(PDB 2HU0, A) compared with the binding mode of OS carboxylate and the key
residues that may form potential interactions with compound 20l (B).
It is interesting to observe that compound 20l was more potent than 32 against
the wild type H5N1 NAs, but about six-fold less potent against the H274Y mutant.
The molecular basis of oseltamivir resistance caused by the mutation H274Y has been
ACS Paragon Plus Environment

Page 17 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
elucidated by Collins et al.28 In the NA active site, the replacement of histidine with a
bulkier tyrosine residue could disrupt the hydrophobic pocket formed by the
methylene of Glu 276, thus preventing oseltamivir from making hydrophobic contact
with the pocket. With respect to the two compounds, the only difference between
them was the C-4 substituent on the phenyl ring linked to the C-5 NH2 of oseltamivir
carboxylate. Compound 20l had a higher activity against the wild type N1 enzymes
than compound 32 and almost completely occupied the 150-cavity due to the
biphenylmethyl group. The 4-thiophen-2-ylphenyl group of compound 32 at the same
position was relatively small. In the case of binding with wild type H5N1 NAs,
compound 32 could not bind tightly like 20l and thus presented lower activities.
However, in the case of binding with the H274Y mutant enzyme, the
4-thiophen-2-ylphenyl group of compound 32 was more prone to moving, thereby
adjusting the molecule to the changes that were caused by the mutation. Further
explanation would be better assisted by the crystal structures of the enzyme-inhibitor
complexes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, we compared the amino acid sequences of two NAs (H5N1-1220 and
H5N1-1206) with the protein (2HU0) that was used for the docking analysis. We
found that the NA of H5N1-1220 showed a high degree (> 97%) of similarity to
2HU0. Only 8 of the 368 residues were different between these two proteins, and not
included in the active site or the 150-loop region. The NA of H5N1-1206, with 12
different residues, showed a slightly lower degree of similarity to 2HU0 (96.7%, data
shown in the supporting information). The amino acid sequence of the two proteins
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
made a great contribution to the credibility of the docking analysis.
5
6
7
CONCLUSION
8
9
Because the 150-cavity was first reported, many studies have been conducted to
explore N1 selective NA inhibitors but no outstanding progress has been made. In this
manuscript, we designed and synthesized two series of oseltamivir derivatives with
modifications at the C-5 NH2 position. The secondary amine derivatives were found
to be much better than the N-substituted guanidines. To the best of our knowledge,
this work contributes the most selective N1 inhibitors, such as compound 20l, which
possesses IC50 values of 0.0019 µM, 0.0038 µM and 0.0067 µM against the three
types of H5N1. Moreover, compound 32 exhibited increased activity against the
H274Y mutant. Collectively, we provide one rational design strategy for the discovery
of Group-1-specific NAs inhibitors. Considering the interesting anti-mutant potential
of compound 32, we hope that detailed SARs studies and further derivatization of
compounds 32 and 20l based on this strategy might lead to more potent compounds
against drug-resistant NAs.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
Materials
Used
for
Biological
Experiments.
Influenza
virus
(A/chicken/china/1220/2012,
A/duck/china/1206/2012,
A/duck/china/QJ/01.
A/chicken/china/415/2013 and A/chicken/shandong/S2/02) were kindly provided by
the College of Veterinary Medicine, South China Agricultural University, P.R. China
and handled under biosafety level 3 (BSL-3) conditions. The H5N1 NA (H274Y) was
obtained from Sino Biological Inc. The substrate used in the enzyme inhibition assay,
ACS Paragon Plus Environment

Page 19 of 47
Journal of Medicinal Chemistry
1
2
3
4
2’-(4-methylumbelliferyl)-α-D-acetylneuraminic
acid
sodium
salt
hydrate
5
6
(4-MU-NANA) (Sigma, M8639), was purchased from Sigma.
7
8
9
NA Enzyme Inhibitory Assay. The NA inhibition assay was performed
according to the standard method.29 Influenza virus suspensions (H5N1-1220,
H5N1-1206, H5N1-QJ, H9N2-415, H9N2-S2) obtained from the allantoic fluid of
embryonated chicken eggs were inactivated and used for biological evaluation. The
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
substrate, 2’-(4-methylumbelliferyl)-α-D-acetylneuraminic acid sodium salt hydrate
(4-MU-NANA) (Sigma, M8639), was cleaved by NA to yield a quantifiable
fluorescent product. The tested compounds were dissolved in DMSO and diluted to
the
2-(N-morpholino)-ethanesulfonic acid, 4 mM CaCl2, pH 6.5). In a 96-well plate, 10
L of the diluted virus supernatant, 70 L of MES buffer and 10 L of compounds at
corresponding
concentrations
in
MES
buffer
(32.5
mM
µ
µ
µ
different concentrations were added successively and incubated for 5 min at 37 °C.
The reaction was started by the addition of the substrate. After incubation for 30 to 60
min, the reaction was terminated by adding 150 µL 0.2 M glycine-NaOH (pH 10.2) in
water. Fluorescence was recorded (excitation at 360 nm and emission at 450 nm) and
substrate blanks were subtracted from the sample readings. The 50% inhibitory
concentration (IC50) was calculated by plotting the percent of inhibition of NA activity
versus the inhibitor concentration.
General Information on Synthesis. Oseltamivir phosphate was provided by
Shandong Qidu Pharmaceutical Co., Ltd. The other commercially available chemicals
and reagents were purchased from Aladdin, TCI, J&K, ENERGY CHEMICAL and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 47
1
2
3
4
5
6
Sinopharm Chemical Reagent Co., Ltd with purities of at least 97%. All the tested
compounds were found to be > 95% pure by HPLC analysis, which was performed on
7
8
9
a Shimadzu HPLC instrument using a C18 column (5 ꢀm，4.6 mm × 250 mm) with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
two solvent systems (either methanol/water (0.1% CF3COOH in methanol) with a
gradient elution of 55% to 90% methanol over 30 minutes at 1.0 mL/min, or
acetonitrile/buffer (0.2 M KH2PO4, pH = 6) with a gradient elution of 10% to 80%
1
acetonitrile over 25 minutes at 1.0 mL/min). The H-NMR spectra were determined
using either a Brucker Avance 300 spectrometer or the 600 model with TMS as an
internal standard. The solvents for NMR were DMSO-d6 (δ 2.5 for 1H) and CD3OD
1
3.3 for H). ESI-MS was determined using an API 4000 LC/MS spectrometer
(δ
(Applied Biosystems, USA). HRMS analysis was performed using an Agilent 6520
Q-TOF LC/MS spectrometer (Agilent, Germany). All reactions were monitored by
thin-layer chromatography (TLC) on 25.4 × 76.2 mm silica gel plates (GF-254). The
silica gel used for column chromatography was 200-300 mesh. Melting points were
determined using an electrothermal melting point apparatus and were uncorrected.
Synthetic Methods. Synthesis of compound 9. Compound 9 was synthesized
according to the reported method.25
(3
R
,4
R,5
S)-4-acetamido-5-guanidino-3-(pentan-3-yloxy)cyclohex-1-enecarbo
1
xylic acid trifluoroacetate salt (9). White solid (yield 52%), mp 140 °C. H NMR
(D2O, 300 MHz):
Hz), 3.72-3.82 (m, 1H), 3.44-3.52 (m, 1H), 2.80 (dd, 1H,
(m, 1H), 1.98 (s, 3H), 1.32-1.58 (m, 4H), 0.83 (t, 3H, = 7.5 Hz), 0.78 (t, 3H,
δ
6.79 (s, 1H), 4.29 (d, 1H,
J
= 8.7 Hz), 3.87 (dd, 1H,
= 17.4, 5.1 Hz), 2.29-2.41
= 7.5
J = 10.8, 8.7
J
J
J
ACS Paragon Plus Environment

Page 21 of 47
Journal of Medicinal Chemistry
1
2
3
4
Hz). 13C NMR (D2O, 75 MHz):
δ
174.82, 169.41, 156.91, 138.33, 128.76, 84.34,
5
6
7
75.47, 54.91, 50.65, 29.93, 25.67, 25.30, 22.07, 8.61, 8.57. HRMS calcd for
8
9
C
15
H
27
N4
O4
[M
+
H]+: 327.2032, found:
m/z 327.2025.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of compounds 11 and 14. To a solution of compound 8 (0.55 g, 1.0
mmol) in 5.0 mL of anhydrous methanol, a solution of NH2OK (0.14 g, 6.0 mmol) in
3.5 mL of anhydrous methanol was added. The mixture was stirred for 0.5 h and then
concentrated under vacuum. The solution was acidified with 1 M HCl to pH 5-6 and
extracted with EtOAc (3 × 10 mL). The organic layers were dried over MgSO4 and
evaporated under vacuum. The crude product was purified by column
chromatography to give compound 10, which was dissolved in 20.0 mL HCl/EtOAc.
The mixture was stirred at room temperature overnight. The precipitate was filtered
and then dried to give compound 11 as a white solid. Compound 14 was prepared
using the same method from intermediate 12.
(3
-1-enecarboxamide hydrochloride (11). White solid (yield 59%), mp 142-143°C. 1H
NMR (D2O, 300 MHz): 6.27 (s, 1H), 4.33 (d, 1H, = 8.4 Hz), 3.69-3.87 (m, 2H),
3.40-3.47 (m, 1H), 2.66 (dd, 1H, = 17.1, 5.1 Hz), 2.30−2.39 (m, 1H), 1.93 (s, 3H),
1.28-1.51 (m, 4H), 0.73-0.82 (m, 6H). 13C NMR (D2O, 75 MHz):
174.74, 156.81,
132.69, 84.46, 75.38, 54.93, 50.51, 29.89, 25.59, 25.20, 21.98, 8.52, 8.45. HRMS
calcd for C15 [M 342.2134.
H]+: 342.2141, found:
(3 ,4 ,5
R,4R,5S)-4-acetamido-5-guanidino-N-hydroxy-3-(pentan-3-yloxy)cyclohex
δ
J
J
δ
H
28
N5
O4
+
m/z
R
R
S)-4-acetamido-5-amino-N-hydroxy-3-(pentan-3-yloxy)cyclohex-1-e
1
necarboxamide hydrochloride (14). White solid (yield 49%), mp 136-138°C. H
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
NMR (D2O, 300 MHz):
δ
6.34 (s, 1H), 4.26 (d, 1H,
J
= 9.0 Hz), 3.98-4.05 (m, 1H),
= 17.1, 5.1 Hz), 2.46-2.56 (m,
3.54-3.62 (m, 1H), 3.44-.53 (m, 1H), 2.81 (dd, 1H,
J
7
8
9
1H), 2.03 (s, 3H), 1.35-1.58 (m, 4H), 0.76-0.85 (m, 6H). 13C NMR (D2O, 75 MHz):
175.27, 166.63, 132.83, 128.22, 84.47, 75.05, 52.65, 49.07, 28.23, 25.46, 25.06, 22.42,
8.52, 8.47. HRMS calcd for C14 [M 300.1915.
H]+: 300.1923, found:
δ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
26N
3
O
4
+
m/z
The general procedure for the synthesis of compounds 17a-l. To a solution of
oseltamivir phosphate (0.82 g, 2.0 mmol) and aldehyde (2.4 mmol, 1.2 eq) in 25 mL
ethanol, NaBH3CN (0.25 g, 4.0 mmol, 2 eq) was slowly added. The reaction was
stirred at room temperature for 6 h and then concentrated. To the residue, 20 mL
saturated NaHCO3 solution was added and the mixture was extracted with EtOAc.
The combined extracts were dried over anhydrous MgSO4 and concentrated to give
the crude product, intermediate 15. Compound 15l (containing a biphenyl group at R2),
1
white solid (84%), H NMR (CDCl3, 300 MHz):
δ
7.53-7.60 (m, 4H), 7.30-7.46 (m,
= 7.2 Hz), 4.18-4.26 (m, 3H), 3.93-3.98 (m, 1H),
3.72-3.82 (m, 2H), 3.33-3.41 (m, 1H), 3.15-3.24 (m, 1H), 2.80 (dd, 1H, = 17.7, 5.1
Hz), 2.22-2.35 (m, 1H), 2.01 (s, 3H), 1.46-1.56 (m, 4H), 1.30 (t, 3H, = 7.2 Hz), 0.90
(t, 6H, 170.65, 166.52, 140.92, 139.95,
= 7.5 Hz). 13C NMR (CDCl3, 75 MHz):
5H), 6.80 (s, 1H), 5.50 (d, 1H,
J
J
J
J
δ
139.29, 137.19, 129.38, 128.75, 128.57, 127.35, 127.17, 127.03, 81.77, 74.53, 60.88,
55.81, 53.60, 50.25, 30.41, 26.15, 25.77, 23.76, 14.24, 9.52, 9.42. HRMS calcd for
C
29
H
39
N2
O4
[M
+
H]+: 479.2910, found:
m/z 479.2904.
Without further purification, intermediate 15 (1.0 mmol), Et3N (0.29 ml, 2.0
mmol, 2 eq) and SC(NHBoc)2 (0.33 g, 1.2 mmol, 1.2 eq) were dissolved in 15 ml
ACS Paragon Plus Environment

Page 23 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
DMF, followed by the addition of HgCl2 (0.41 g, 1.5 mmol, 1.5 eq). The reaction
mixture was stirred at room temperature for 3 h and was subsequently diluted with
EtOAc and filtered through a pad of Celite. The filtrate was evaporated under reduced
pressure to give a colorless oil, which was dissolved in 15 ml CH3OH and 5 ml H2O.
NaOH (0.24 g, 6.0 mmol, 6 eq) was added and the solution was stirred at room
temperature for 1 h. After adjusting the pH to 5-6 with 1 M HCl, the mixture was
extracted with EtOAc. The combined extracts were dried and concentrated.
Chromatographic purification afforded compound 16.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A solution of compound 16 (0.20 mmol) and TFA (1.0 mL, 13.0 mmol) in
CH2Cl2 (1.0 mL) was stirred at room temperature for 24 h. The solvent was
evaporated and dried under vacuum. The residue was triturated in ether to give a
white solid, which was collected by filtration. The solid was dried to give compounds
17a-l.
(3
)cylohex-1-enecarboxylic acid trifluoroacetate salt (17a). White solid (63%), mp
128-132 °C. 1H NMR (CD3OD, 300 MHz):
7.38 (dd, 1H, = 5.1, 1.2 Hz), 7.07 (dd,
1H, = 3.6, 1.2 Hz), 7.00 (dd, 1H, = 5.1, 3.6 Hz), 6.84 (s, 1H), 4.8 (s, 2H),
4.25-4.32 (m, 2H), 4.13-4.20 (m, 1H), 3.39-3.47 (m, 1H), 2.85 (dd, 1H, = 17.4, 5.1
Hz), 2.60-2.71 (m, 1H), 2.01 (s, 3H), 1.49-1.58 (m, 4H), 0.95 (t, 3H, = 7.2 Hz), 0.91
(t, 3H, 171.80, 166.92, 157.73, 138.36,
= 7.2 Hz). 13C NMR (CD3OD, 75 MHz):
136.92, 128.25, 126.02, 125.36, 124.49, 81.90, 74.99, 55.93, 52.14, 42.22, 27.89,
25.37, 24.85, 21.07, 7.93, 7.67. HRMS calcd for C20 S [M
H]+: 423.2066,
R,4R,5S)-4-acetamido-3-(pentan-3-yloxy)-5-(1-(thiophen-2-ylmethyl)guanidino
δ
J
J
J
J
J
J
δ
H31
N
4
O4
+
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 47
1
2
3
4
5
6
7
8
9
found:
(3
-enecarboxylic acid trifluoroacetate salt (17b). White solid (36%), mp 122-124°C.
1H NMR (DMSO-d6, 300 MHz):
12.56 (br, 1H), 8.01 (d, 1H, = 8.7 Hz), 7.51 (br,
4H), 7.37 (t, 2H, = 7.5 Hz), 7.26 (t, 1H, = 7.5 Hz),7.14 (d, 1H, = 7.5 Hz), 6.62 (s,
1H), 4.49-4.64 (m, 2H), 4.12-4.26 (m, 2H), 3.98-4.11 (m, 1H), 3.35-3.43 (m, 1H),
2.51-2.63 (m, 1H), 2.30-2.40 (m, 1H), 1.84 (s, 3H), 1.40-1.50 (m, 4H), 0.85 (t, 3H,
= 7.2 Hz), 0.80 (t, 3H, 169.37, 166.94,
= 7.2 Hz). 13C NMR (DMSO-d6, 75 MHz):
157.88, 137.33, 136.94, 128.88, 128.40, 126.87, 125.76, 81.16, 75.16, 56.00, 51.64,
45.86, 27.97, 25.72, 25.10, 22.76, 9.40, 8.86. HRMS calcd for C22 [M
H]+:
417.2502, found: 417.2548.
(3 ,4 ,5 )-4-acetamido-5-(1-(4-isopropylbenzyl)guanidino)-3-(pentan-3-ylo
xy)cyclohex-1-enecarboxylic acid trifluoroacetate salt (17c). White solid (62%),
mp 127-130°C. 1H NMR (DMSO-d6, 300 MHz):
12.62 (br, 1H), 7.99 (d, 1H, = 8.1
Hz), 7.46 (br, 4H), 7.24 (d, 2H, = 8.1 Hz), 7.05 (d, 2H, = 8.1 Hz), 6.62 (s, 1H),
m
/
z
423.2065.
R
,4
R
,5 )-4-acetamido-5-(1-benzylguanidino)-3-(pentan-3-yloxy)cyclohex-1
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
δ
J
J
J
J
J
J
δ
H
33
N4
O4
+
m/z
R
R S
δ
J
J
J
4.44-4.59 (m, 2H), 4.11-4.25 (m, 2H), 3.98-4.10 (m, 1H), 3.35-3.43 (m, 1H),
2.80-2.94 (m, 1H), 2.56-2.67 (m, 1H), 2.30-2.42 (m, 1H), 1.84 (s, 3H), 1.39-1.50 (m,
4H), 1.19 (d, 6H,
NMR (DMSO-d6, 75 MHz):
126.33, 125.71, 81.17, 75.15, 55.98, 51.71, 45.66, 33.01, 27.96, 25.71, 25.09, 23.87,
J
= 6.9 Hz), 0.85 (t, 3H,
J = 7.2 Hz), 0.80 (t, 3H, J
= 7.2 Hz). 13C
δ
169.35, 166.97, 157.80, 146.98, 137.35, 134.19, 128.90,
23.81, 22.76, 9.40, 8.85. HRMS calcd for C25
459.2966.
H39N
4
O4
[M m/z
H]+: 459.2971, found:
+
ACS Paragon Plus Environment

Page 25 of 47
Journal of Medicinal Chemistry
1
2
3
4
(3
R
,4
R,5
S)-4-acetamido-5-(1-(2-methoxybenzyl)guanidino)-3-(pentan-3-ylox
5
6
7
y)cyclohex-1-enecarboxylic acid trifluoroacetate salt (17d). White solid (54%), mp
8
9
1
126-130°C. H NMR (DMSO-d6, 300 MHz):
δ
12.62 (br, 1H), 8.00 (d, 1H,
J = 8.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hz), 7.58 and 7.37 (br, 4H), 7.27 (dt, 1H,
J = 8.4, 1.5 Hz), 6.89-7.03 (m, 3H), 6.60 (s,
1H), 4.36-4.51 (m, 2H), 4.14-4.26 (m, 2H), 3.95-4.08 (m, 1H), 3.82 (s, 3H), 3.35-3.43
(m, 1H), 2.49-2.58 (m, 1H, partially merged in DMSO), 2.03-2.16 (m, 1H), 1.82 (s,
3H), 1.33-1.51 (m, 4H), 0.84 (t, 3H,
(DMSO-d6, 75 MHz): 169.40, 166.86, 157.94, 156.22, 137.30, 128.75, 128.12,
124.95, 124.16, 120.05, 110.58, 81.22, 75.02, 55.54, 55.22, 51.57, 42.24, 27.66, 25.73,
J = 7.2 Hz), 0.80 (t, 3H, J
= 7.2 Hz). 13C NMR
δ
25.15, 22.69, 9.37, 8.90. HRMS calcd for C23
447.2602.
H35N
4
O5
[M m/z
H]+: 447.2607, found:
+
(3R,4R,5S)-5-(1-([1,1'-biphenyl]-4-ylmethyl)guanidino)-4-acetamido-3-(pent
an-3-yloxy)cyclohex-1-enecarboxylic acid trifluoroacetate salt (17e). White solid
1
(58%), mp 124-128°C. H NMR (CD3OD, 300 MHz):
δ 7.60-7.68 (m, 4H), 7.41-7.47
(m, 2H), 7.30-7.37 (m, 3H), 6.85 (s, 1H), 4.61-4.86 (m, 2H), 4.31-4.51 (m, 2H),
4.16-4.23 (m, 1H), 3.41-3.49 (m, 1H), 2.84 (dd, 1H, = 17.4, 5.1 Hz), 2.47-2.57 (m,
1H), 2.02 (s, 3H), 1.46-1.61 (m, 4H), 0.95 (t, 3H, = 7.5 Hz), 0.92 (t, 3H, = 7.5 Hz).
13C NMR (CD3OD, 75 MHz):
171.82, 166.97, 157.86, 139.95, 139.85, 137.02,
134.16, 128.13, 128.01, 126.61, 126.50, 126.00, 125.58, 81.94, 74.88, 55.68, 52.06,
27.54, 25.37, 24.88, 21.09, 7.93, 7.71. HRMS calcd for C28 [M
H]+:
493.2815, found: 493.2807.
(3 ,4 ,5
J
J
J
δ
H
37
N
4
O4
+
m/z
R
R
S)-4-acetamido-3-(pentan-3-yloxy)-5-(1-propylguanidino)cyclohex-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
1-enecarboxylic acid trifluoroacetate salt (17f). White solid (49%), mp 128-132°C.
5
6
7
1H NMR (DMSO-d6, 300 MHz):
δ 12.69 (br, 1H), 7.89 (d, 1H, J = 7.8 Hz), 7.32 (br,
8
9
4H), 6.62 (s, 1H), 4.14-4.20 (m, 1H), 3.91-4.00 (m, 2H), 3.32-3.40 (m, 1H), 3.06-3.20
(m, 2H), 2.51-2.56 (m, 2H, partially emerged in DMSO), 1.79 (s, 3H), 1.52-1.68 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1H), 1.23-1.50 (m, 5H), 0.75-0.87 (m, 9H). 13C NMR (DMSO-d6, 75 MHz):
166.96, 156.99, 137.09, 129.03, 80.96, 75.21, 55.50, 51.52, 44.33, 28.01, 25.64, 25.02,
22.66, 20.91, 10.62, 9.36, 8.75. HRMS calcd for C18 [M
H]+: 369.2502,
found: 369.2515.
(3 ,5 )-4-acetamido-5-(1-isobutylguanidino)-3-(pentan-3-yloxy)cyclohex
-1-enecarboxylic acid trifluoroacetate salt (17g). White solid (43%), mp 140-143°C.
1H NMR (DMSO-d6, 300 MHz):
12.69 (br, 1H), 7.93 (d, 1H, = 8.4 Hz), 7.40 (br,
4H), 6.61 (s, 1H), 4.12-4.21 (m, 1H), 3.92-4.18 (m, 1H), 3.82-3.90 (m, 1H), 3.31-3.40
(m, 1H), 3.22 (dd, 1H, = 15.3, 6.0 Hz), 2.81-2.90 (m, 1H), 2.51-2.60 (m, 2H),
1.90-2.00 (m, 1H), 1.79 (s, 3H), 1.32-1.49 (m, 4H), 0.75-0.87 (m, 12H). 13C NMR
(DMSO-d6, 75 MHz): 169.20, 167.09, 158.16, 136.99, 129.17, 80.91, 75.12, 55.50,
52.22, 48.87, 28.62, 25.66, 25.02, 22.78, 19.66, 19.55, 9.44, 8.77. HRMS calcd for
δ 169.12,
H33
N4
O
4
+
m/z
R,4R S
δ
J
J
δ
C
19
H
35
N
4
O
4
[M
,5
+
H]+: 383.2658, found:
m/z 383.2655.
(3
R
,4
R
S
)-4-acetamido-3-(pentan-3-yloxy)-5-(1-pentylguanidino)cyclohex-1
-enecarboxylic acid trifluoroacetate salt (17h). White solid (38%), mp 124-127°C.
1H NMR (CD3OD, 300 MHz):
6.83 (s, 1H), 4.27 (d, 1H, = 6.9 Hz), 4.11-4.20 (m,
1H), 4.02-4.09 (m, 1H), 3.37-3.45 (m, 1H), 3.21-3.28 (m, 2H), 2.75 (dd, 1H, = 17.1,
δ
J
J
5.1 Hz), 2.56-2.65 (m, 1H), 1.96 (s, 3H), 1.70-1.85 (m, 1H), 1.49-1.59 (m, 5H),
ACS Paragon Plus Environment

Page 27 of 47
Journal of Medicinal Chemistry
1
2
3
4
1.28-1.47 (m, 4H), 0.87-0.97 (m, 9H). 13C NMR (CD3OD, 75 MHz):
δ 171.59, 167.06,
5
6
7
8
9
157.20, 137.06, 128.21, 81.88, 75.00, 55.43, 52.08, 43.31, 27.86, 27.62, 27.03, 25.35,
24.83, 21.55. 20.99, 12.38, 7.91, 7.65. HRMS calcd for C20 [M
H]+:
397.2815, found: 397.2808.
(3 ,4 ,5 )-4-acetamido-5-(1-butylguanidino)-3-(pentan-3-yloxy)cyclohex-1-
enecarboxylic acid trifluoroacetate salt (17i). White solid (41%), mp 116-118°C. 1H
NMR (CD3OD, 300 MHz): 6.83 (s, 1H), 4.27 (d, 1H, = 6.9 Hz), 4.11-4.18 (m, 1H),
4.02-4.10 (m, 1H), 3.33-3.45 (m, 1H), 3.22-3.30 (m, 2H), 2.75 (dd, 1H, = 17.1, 5.1
Hz), 2.57-2.67 (m, 1H), 1.97 (s, 3H), 1.70-1.80 (m, 1H), 1.45-1.60 (m, 5H), 1.31-1.43
(m, 2H), 0.89-1.01 (m, 9H). 13C NMR (CD3OD, 75 MHz):
171.60, 167.06, 157.19,
137.05, 128.22, 81.87, 75.00, 55.43, 52.10, 29.40, 27.61, 25.35, 24.83, 20.99, 18.95,
H
37
N
4
O
4
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
m/z
R
R S
δ
J
J
δ
12.21, 7.92, 7.65. HRMS calcd for C19
383.2652.
H35
N
4O
4
[M
+
H]+: 383.2658, found:
m/z
(3R,4R,5S)-4-acetamido-5-(1-isopentylguanidino)-3-(pentan-3-yloxy)cyclohe
x-1-enecarboxylic acid trifluoroacetate salt (17j). White solid (33%), mp
1
117-119°C. H NMR (CD3OD, 300 MHz):
δ
6.83 (s, 1H), 4.26 (d, 1H,
J
= 6.9 Hz),
4.03-4.19 (m, 2H), 3.38-3.45 (m, 1H), 3.21-3.33 (m, 2H), 2.75 (dd, 1H,
J = 17.1, 5.1
Hz), 2.53-2.63 (m, 1H), 1.97 (s, 3H), 1.61-1.81 (m, 2H), 1.45-1.59 (m, 4H), 1.20-1.33
(m, 1H), 0.87-1.00 (m, 12H). 13C NMR (CD3OD, 75 MHz):
171.64, 167.04, 157.19,
137.10, 128.17, 81.94, 75.03, 55.40, 52.05, 42.35, 35.99, 27.59, 25.61, 25.35, 24.83,
21.20, 20.98, 20.51, 7.92, 7.66. HRMS calcd for C20 [M
H]+: 397.2815,
found: 397.2808.
δ
H37
N4
O
4
+
m/z
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
(3
R
,4
R,5
S)-4-acetamido-5-(1-(2-ethylbutyl)guanidino)-3-(pentan-3-yloxy)cyc
5
6
7
lohex-1-enecarboxylic acid trifluoroacetate salt (17k). White solid (68%), mp
8
9
1
147-148°C. H NMR (CD3OD, 300 MHz):
δ 6.82 (s, 1H), 4.22-4.38 (m, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.11-4.18 (m, 1H), 3.98-4.08 (m, 1H), 3.36-3.45 (m, 1H), 3.26-3.30 (m, 1H), 3.10 (dd,
1H, = 15.9, 9.3 Hz), 2.81 (dd, 1H, = 17.1, 4.8 Hz), 2.55-2.65 (m, 1H), 1.98 (s, 3H),
1.73-1.83 (m, 1H), 1.47-1.59 (m, 4H), 1.30-1.44 (m, 4H), 0.87-0.98 (m, 12H). 13C
NMR (CD3OD, 75 MHz): 171.66, 167.07, 158.56, 136.92, 128.24, 81.83, 75.27,
56.95, 52.51, 38.09, 28.22, 25.31, 24.78, 22.45, 22.15, 21.10, 9.65, 8.76, 7.93, 7.61.
HRMS calcd for C21 [M 411.2966.
H]+: 411.2971, found:
(3 ,4 ,5 )-4-acetamido-3-(pentan-3-yloxy)-5-(1-(3-phenylpropyl)guanidino)
J
J
δ
H
39N
4
O
4
+
m/z
R
R S
cyclohex-1-enecarboxylic acid trifluoroacetate salt (17l). White solid (44%), mp
1
75-78°C. H NMR (CD3OD, 300 MHz):
δ 7.15-7.32 (m, 5H), 6.79 (s, 1H), 4.24 (d,
1H,
(m, 2H, partially merged in H2O), 2.53-2.77 (m, 3H), 2.38-2.47 (m, 1H), 2.05-2.12 (m,
1H), 1.96 (s, 3H), 1.67-1.79 (m, 1H), 1.46-1.58 (m, 4H), 0.93 (t, 3H, = 7.5 Hz), 0.89
(t, 3H, 171.58, 166.98, 157.22, 140.34,
= 7.5 Hz). 13C NMR (CD3OD, 75 MHz):
136.97, 128.13, 127.69, 127.54, 125.30, 81.91, 74.93, 55.45, 52.00, 42.80, 31.65,
29.14, 27.39, 25.34, 24.83, 20.99, 7.90, 7.66. HRMS calcd for C24 [M
H]+:
445.2815, found: 445.2810.
J = 8.1 Hz), 4.08-4.18 (m, 1H), 3.93-4.00 (m, 1H), 3.34-3.44 (m, 1H), 3.21-3.33
J
J
δ
H
37
N4
O4
+
m/z
The general procedure for the synthesis of compounds 20a-m. Method A: To a
solution of intermediate 15 (1.0 mmol) and Et3N (0.29 ml, 2 eq) in 20 ml THF was
added (Boc)2O ( 0.22 g, 1.0 mmol) in one portion. The reaction mixture was stirred
ACS Paragon Plus Environment

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
for 10 h at room temperature and then concentrated. The residue was then hydrolyzed
with NaOH, followed by chromatographic purification to give compound 19, which
was treated with TFA to give the target compounds according to the previous method.
Method B: To a solution of compound 15 (1.0 mmol) in 15 mL CH3OH and 5 mL
H2O, NaOH (0.24 g, 6 mmol, 6 eq) was added. The reaction mixture was stirred at
50 °C for 4 h. After the reaction was complete, the pH was adjusted to 5-6 with acetic
acid. The solvent was evaporated under vacuum and the obtained residue was purified
by column chromatography with CH2Cl2 and CH3OH to give the target compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3
R
,4
R,5
S)-4-acetamido-5-((2-ethylbutyl)amino)-3-(pentan-3-yloxy)cyclohex
1
-1-enecarboxylic acid (20a). White solid (32%), mp 138 °C. H NMR (CD3OD, 300
MHz):
δ 6.89 (s, 1H), 4.29 (d, 1H, J = 7.2 Hz), 4.17 (dd, 1H, J = 10.8 Hz, 8.1 Hz),
3.61-3.72 (m, 1H), 3.44-3.52 (m, 1H), 3.02-3.10 (m, 2H), 2.96 (dd, 1H,
J
= 17.4 Hz,
13
5.4 Hz), 2.57-2.66 (m, 1H), 2.07 (s, 3H), 1.34-1.70 (m, 9H), 0.89-0.99 (m, 12H). C
NMR (CD3OD, 75 MHz):
50.62, 37.41, 25.24, 25.14, 24.75, 22.17, 22.04, 21.43, 8.66, 8.62, 7.92, 7.64. HRMS
calcd for C20 [M 369.2749.
H]+: 369.2753, found:
(3 ,4 ,5 )-4-acetamido-5-(butylamino)-3-(pentan-3-yloxy)cyclohex-1-eneca
rboxylic acid acetate salt (20b). White solid (21%), mp 85-86°C. 1H NMR
(DMSO-d6, 300 MHz): 7.91 (d, 1H, = 8.7 Hz), 6.57 (s, 1H), 4.02 (d, 1H, = 8.1
δ 172.83, 166.65, 136.49, 126.90, 81.87, 74.06, 54.99,
H
37
N2
O4
+
m/z
R
R S
δ
J
J
Hz), 3.61-3.72 (m, 1H), 3.30-3.38 (m, 1H), 2.80-2.89 (m, 1H), 2.60-2.69 (m, 2H),
2.49-2.58 (m, 1H, partially emerged in DMSO), 2.00-2.12 (m, 1H), 1.90 (s, 3H), 1.84
(s, 3H), 1.23-1.45 (m, 8H), 0.76-0.89 (m, 9H). 13C NMR (DMSO-d6, 75 MHz):
δ
ACS Paragon Plus Environment