Design and biological evaluation of cinnamic and phenylpropionic amide derivatives as novel dual inhibitors of HIV-1 protease and reverse transcriptase

Article abstract of DOI:10.1016/j.ejmech.2021.113498

Upon the basis of both possible ligand-binding site interactions and the uniformity of key residues in active sites, a novel class of HIV-1 PR/RT dual inhibitors was designed and evaluated. Cinnamic acids or phenylpropionic acids with more flexible chain and smaller steric hindrance were introduced into the inhibitors, giving rise to significant improvement in HIV-1 RT inhibitory activity by one or two orders of magnitude, with comparable or even improved potency against PR at the same time, compared with coumarin anologues in our previous studies. Among these inhibitors, 38d displayed a 19-fold improvement in anti-PR activity with IC₅₀ value of 0.081 nM compared to the control DRV. In addition, inhibitor 38c exhibited an excellent anti-RT IC₅₀ value of 0.43 μM, only a 4.7-fold less potent activity than the control EFV. More significantly, the disparate ratio between HIV-1 PR and RT inhibition became more reasonable with ratio of 1: 10.4, just as 37b. Furthermore, the assays on HIV-1 late stage and early stage supported the rationality of designing dual inhibitors. The SAR data as well as molecular modeling studies provided new insight for further optimization of more potent HIV-1 PR/RT dual inhibitors.

Full text of DOI:10.1016/j.ejmech.2021.113498

European Journal of Medicinal Chemistry 220 (2021) 113498
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design and biological evaluation of cinnamic and phenylpropionic
amide derivatives as novel dual inhibitors of HIV-1 protease and
reverse transcriptase
,
,
Mei Zhu a 1, Qi Shan a 1, Ling Ma a, Jiajia Wen a, Biao Dong a, Guoning Zhang a,
Minghua Wang a, Juxian Wang a, Jinming Zhou b **, Shan Cen a ***, Yucheng Wang a
,
,
, *
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, 100050, China
b Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Department of Chemistry, Zhejiang Normal University, Jinhua, 321004,
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Upon the basis of both possible ligand-binding site interactions and the uniformity of key residues in
active sites, a novel class of HIV-1 PR/RT dual inhibitors was designed and evaluated. Cinnamic acids or
phenylpropionic acids with more flexible chain and smaller steric hindrance were introduced into the
inhibitors, giving rise to significant improvement in HIV-1 RT inhibitory activity by one or two orders of
magnitude, with comparable or even improved potency against PR at the same time, compared with
Received 21 November 2020
Received in revised form
15 April 2021
Accepted 17 April 2021
Available online 24 April 2021
coumarin anologues in our previous studies. Among these inhibitors, 38d displayed
improvement in anti-PR activity with IC50 value of 0.081 nM compared to the control DRV. In addi-
tion, inhibitor 38c exhibited an excellent anti-RT IC50 value of 0.43 M, only a 4.7-fold less potent activity
a 19-fold
Keywords:
HIV-1 PR/RT dual inhibitors
Cinnamic acids
Phenylpropionic acids
Disparate ratio
Molecular modeling
m
than the control EFV. More significantly, the disparate ratio between HIV-1 PR and RT inhibition became
more reasonable with ratio of 1: 10.4, just as 37b. Furthermore, the assays on HIV-1 late stage and early
stage supported the rationality of designing dual inhibitors. The SAR data as well as molecular modeling
studies provided new insight for further optimization of more potent HIV-1 PR/RT dual inhibitors.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
development of systematic biology and pharmacology in recent
years [7e17]. In fact, our attention is continuously focused on the
The application of highly active antiretroviral therapy (HAART)
since the mid-1990s has ushered a new era for HIV/AIDS and
reduced the mortality and morbidity rates among HIV infection
patients dramatically [1e3]. However, the complicated side effects,
increased virulence and continuous emergence of multidrug
resistant HIV-1 variants call for novel and potent HIV-1 inhibitors
imminently [4e6]. In consideration of the high resistance barrier
and low toxicity, multi-target drugs have gradually came into focus
on anti-HIV-1 drug development, and an increasing number of
research on HIV-1 dual inhibitors has been emerged, with the rapid
design of dual inhibitors of HIV-1 protease (PR) and reverse tran-
scriptase (RT), which constitute the core of HIV/AIDS chemotherapy
[18].
As is known, both HIV-1 PR and RT play key roles in virus
replication [19,20]. Among which, HIV-1 RT converts RNA as a
template into proviral DNA, while PR catalyzes the proteolytic
cleavage of polypeptide precursors into mature enzyme [21].
Furthermore, the aspartic acid (Asp) residues in the catalytically
active homodimer of HIV-1 PR form hydrogen bond interactions
with the central hydroxy group of inhibitors [22]. Intriguingly, Asp
residues of the catalytically active
b-sheet in p66 of RT will be
repositioned and deactivated, when non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs) are bounded to the allosteric pocket of
heterodimer RT [23,24]. Thus, the uniformity of key residues in
active sites of the two viral enzymes makes it reasonable to search
for common inhibitors. In addition, hydrogen bonds are indis-
pensable for enhancing backbone binding of PR and RT with
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: zhoujinming@zjnu.edu.cn (J. Zhou), shancen@imb.pumc.edu.
cn (S. Cen), wangyucheng@imb.pumc.edu.cn (Y. Wang).
1
These authors made equal contributions to this work.
https://doi.org/10.1016/j.ejmech.2021.113498
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
inhibitors [25e27]. In brief, it makes sense that dual inhibitors of
HIV-1 PR and RT are worth investing in, according to the designed
multifunctional ligands (DMLs).
In previous studies, we designed a series of coumarin de-
rivatives that were effective on HIV-1 PR and partially on RT [18].
Particularly, inhibitor 38d with a 3, 4-dihydroxyphenylpropiony
group as the P2 ligand and 6-methoxypyridine-4-sulfonamide as
the P20 ligand displayed a 19-fold improvement in antiviral activity
with IC50 value of 0.081 nM compared to DRV. Furthermore, in-
hibitors 35a, 35b, 38a, 38b and 38e containing
a 3, 4-
Taken compounds A and B for example, they exhibited IC50,
values of 11.1 and 155.9 nM, respectively, and IC50, RT values of 367.0
and 75.3 mM, respectively. Molecular modeling suggested that the
dihydroxyphenylacryloy group or 3, 4-dihydroxyphenylpropiony
group in P2 ligand showed appreciable antiviral IC50 values under
1 nM.
PR
steric hindrance might be the causation for suboptimal RT inhibi-
tory activity. In an effort to develop potent dual inhibitors of HIV-1
PR and RT, modifications have been done as follows: (1) Introduc-
tion of cinnamic acids or phenylpropionic acids with long linear
linker instead of coumarins to better fit into the spindly binding
pocket of RT; (2) Maintenance of aromatic rings in P2 ligand to keep
In general, as shown in Table 1 and 2 and Fig. 2, inhibitors with
phenylpropiony groups as the P2 ligands exhibited an improve-
ment in activity than those with phenylacryloy groups as the P2
ligands, presumably due to some degree of flexibility that adapted
to intra-protease better. And going forward, inhibitors with 3, 4-
dihydroxy substitution in P2 ligands showed better activity than
those with 3-methoxy-4-hydroxy substitution or tri-substitution in
P2 ligands. Namely, introduction of methoxy group resulted in a
significant loss of activity, which might be attributed to both steric
bulk and reduced van der Waals contacts, such as 37a, 36a vs 35a or
40a, 39a vs 38a. Indeed, it could also be verified via the molecular
modeling for inhibitors 38a, 38c and 38d in Fig. 6, which showed
important ligand-binding site interactions between hydroxy group
in P2 ligand and the S2 subsite of PR.
In order to investigate the effect of functionalized hydrophobic
P10 ligand on HIV-1 protease activity, n-propyl, cyclopropyl and
isopropyl were taken into consideration at the same time. However,
these new introduced structures resulted in obvious loss of inhib-
itory activity compared to an isobutyl P10 ligand, which might be
due to the steric hindrance or flexibility to some extent, or the
impaired hydrophobic interaction with the active site of PR.
Besides, for the aim of promoting van der Waals interactions in
the S20 subsite of HIV-1 PR, several electron-donating groups were
introduced into the P20 ligand. As a whole, inhibitors with a 4-
methoxy substituent exhibited superior activity than the corre-
sponding 4-amino and 4-methylthio substituents, except for 35d,
37d, 39a and 40a. The oxygen atom in 4-methoxy group might form
powerful hydrogen bond (O/HeN) with Asp300 or Asp290 in the
protease S20 subsite and other van der Waals interactions [34]. The
4-amino group could make weak hydrogen bonds involved directly
interactions (NeH/N) with the main chain amides or water-
mediated interactions (NH/H2O/HOOC) with the side chain ox-
ygen of Asp300 or Asp290 in S20 active subsite [35]. However, the
weaker electron-donating ability of sulfur atom might form feeble
hydrogen bond (S/HeN) with Asp300 or Asp290 in the protease S20
subsite.
p-p interactions with both PR and RT [28]; (3) Introduction of
hydroxy group or methoxy group to aromatic rings to strengthen
interactions with the active sites; (4) Maintenance of nonpeptide
HIV-1 protease inhibitor (PI) structural scaffold to keep excellent PR
inhibitory activity; (5) Introduction of aliphatic chains or rings in
P10 ligand to better adapt into both active cavities. Notably, the
results showed that the anti-RT activity of the new designed in-
hibitors enhanced significantly by one or two orders of magnitude,
with comparable or even improved potency against PR at the same
time. As exemplified by compound 38c, it exhibited dual inhibitory
activity with IC50, PR value of 2.02 nM and IC50, RT value of 0.43
respectively, which was close to the two positive controls (DRV
with IC50, PR value of 1.52 nM and EFV IC50, RT value of 0.091 M).
mM,
m
Furthermore, the disparate ratio between PR and RT inhibition
decreased sharply from 1: 15548 of compound A to 1: 10.4 of 37b
and seemed more reasonable as shown in Fig. 1.
2. Results and discussion
2.1. Chemistry
Strategies outlined in Scheme 1 are the synthesis of amine
compounds 21e28 prepared from the commercially available ma-
terial (2S, 3S)-1, 2-epoxy-3-(boc-amino)-4-phenylbutane (1),
similarly as reported in the literature [29]. The epoxide 1 was
reacted with isobutylamine (2), n-propylamine (3), cyclopropyl-
amine (4) or isoprppylamine (5) in acetonitrile at reflux for 6 h to
provide the corresponding amino alcohols (6e9) in yields of
83e90%. Treatment of the corresponding amino alcohols with p-
substitutedbenzenesulfonyl
chlorides
(10e12)
or
6-
methoxypyridine-3-sulfonyl chloride (13) in the presence of DIEA
and DMAP furnished sulfonamide derivatives (14e20), in excellent
yields (82%e91%), which were then exposed to trifluoroacetic acid
at 0e25 ꢀC for 3 h to remove the BOC-group and afforded the
corresponding amines in yields of 75e88% [30]. Catalytic hydro-
genation of 22 over 10% Pd/C in methanol effected reduction of the
nitro group to the corresponding diamine 23 in 94% yield [31].
Reaction of the amines with substituted cinnamic acids 29e31
or substituted phenylpropionic acids 32e34 (which were reduced
via cinnamic acids under the condition of hydrogen and 10% Pd/C),
in anhydrous DMF in the presence of EDCI/HOBt/DMAP at 0e25 ꢀC
for 2e3 h provided the corresponding target inhibitors 35a- 40g in
yields of 75e87%, as shown in Scheme 2 [31,32].
In addition, the types of aromatic rings in P20 ligand showed
remarkable effects on antiviral activity of inhibitors. In general,
inhibitors containing a pyridine ring showed more robust activity
than those with a benzene ring, such as 36d vs 36a, 38d vs 38a, 39d
vs 39a and 40d vs 40a, except for 35d and 37d. Taken 38d for
example, it displayed an almost 5-fold improved antiviral IC50 value
of 0.081 nM compared to 38a with IC50 value of 0.39 nM. The results
above indicated that the methoxypyridine P20 ligand exerted more
favorable interactions with the S20 subsite of protease than a 4-
methoxybenzene P20 ligand as shown in Fig. 6 [36].
2.3. Anti-RT activity assay
All the inhibitors were evaluated in an in vitro HIV-1 RT activity
assay as shown in Tables 1 and 2 [37]. Just as expected, inhibitors
with a linear linker in P2 ligand exhibited a significant enhance-
ment of RT activity compared with coumarin derivatives in our
previous studies [18]. Further confirmation could be acquired
through the molecular modeling of 38a, 38c and 38d in Fig. 7, in
which phenylacryloy groups or phenylpropiony groups with linear
linkers in P2 ligands fitted into the spindly binding pocket of RT
2.2. Anti-PR activity assay
We first evaluated the activity of inhibitors against HIV-1 wild-
type protease using the FRET-based protease activity assay in vitro
[33]. The results are shown in Tables 1 and 2, and darunavir (DRV) is
chosen as the control. Most of these inhibitors exhibited very
potent inhibitory effect with IC50 values in low nanomolar range.
2

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
Fig. 1. Design and modification of novel dual inhibitors of HIV-1 PR and RT. (A) Molecular modeling of compound A with HIV-1 RT. (B) Compound A and B. (C) Novel dual inhibitors
of HIV-1 PR and RT with rational ratio.
well.
Many inhibitors showed potency with RT IC50 values in low
micromolar range in the biochemical assays. In particularly, in-
hibitor 38c with a 3, 4-dihydroxyphenylpropiony group as the P2
ligand and 4-(methylthio) phenylsulfonamide as the P20 ligand
for the aim of rationalizing structure-activity relationships (SARs)
precisely. Inhibitors were distributed around two perpendicular
axes crossing the PR IC50 axis (X axis) at 30 nM and the RT IC50 axis
(Y axis) at 50 mM (bolded crosshair in the center of each graph). The
two axes spliced the graph into four quarters corresponding to PR/
RT dual inhibitors (lower left quarter), RT selective inhibitors (lower
right quarter), PR selective inhibitors (upper left quarter), and in-
hibitors of lower potency (upper right quarter).
As can be seen in Fig. 4, many of these inhibitors were distrib-
uted in the lower left quarter, suggesting of potency for both HIV-1
PR inhibitory activity and RT inhibitory activity, such as 35a, 35b,
35c, 35d, 35f, 36b, 38a, 38b, 38c, 38d, 38e, 38f, 38g, 39b, 39c, 39g.
The preliminary results of the new kind of HIV-1 PR/RT dual in-
hibitors were encouraging for further optimization to improve
antiviral activity.
exhibited an excellent antiviral IC50 value of 0.43
less potent than the control Efavirenz (EFV).
mM, only a 4.7-fold
In generally, just as appeared in HIV-1 PR inhibitory activity,
compounds with phenylpropiony groups as the P2 ligands showed
superior anti-RT activity than those with phenylacryloy groups as
the P2 ligands to a greater or lesser extent in Fig. 3. Among the
phenylpropionic amide analogues, inhibitors with the 4-(methyl-
thio) phenylsulfonamide or 6-methoxypyridine-4-sulfonamide P20
ligand exhibited optimal activity, such as 38c, 38d, 39c and 40d.
While, among cinnamic amide analogues, inhibitors with the 4-
aminophenylsulfonamide P20 ligand showed slight enhancement
of antiviral activity, such as 35b, 36b and 37b. However, there was
no discernible regularity in potency for inhibitors with different
aliphatic chains in P10 ligands.
2.5. HIV-1 infectivity assay on HIV-1 late stage and early stage
By extension, in order to further verify the targets of the dual
inhibitors, compounds 37b and 38c were carried out assays on the
late stage and early stage of HIV-1 life cycle, with DRV and EFV as
the controls [38]. As shown in Table 3 and Fig. 5, inhibitor 38c
2.4. Evaluation of biological activity
Both HIV-1 PR and RT antiviral IC50 values of inhibitors were
plotted against each other in the correlation plots as shown in Fig. 4,
exhibited antiviral EC50 value of 2.31
HIV-1 late stage at the concentration of 10
m
M and inhibition of 97% on
mM, as well as EC50 value
3

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
Scheme 1. Syntheses of compounds 21e28. Reagents and conditions: (i) R1NH2, CH3CN, 80 ꢀC, 6 h; (ii) Aryl sulfonyl chloride, DIEA, DMAP, THF, 0 ꢀC ~ r.t, 3e5 h; (iii) CF3COOH, DCM,
ꢀC ~ r.t, 3 h; (iv) H2 (gas), 50 psi, 10% Pd/C, CH3OH, r. t, 2 h.
0
Scheme 2. Syntheses of compounds 35a-40g. Reagents and conditions: (v) EDCI, HOBt, DMAP, anhydrous DMF, Argon, 0 ꢀC ~ r.t, 3 h; (vi) H2 (gas), 50 psi, 10% Pd/C, CH3OH, r. t,
2e3 h.
4

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
Table 1
Inhibitory activity of inhibitors 35aꢁ37 g.
Comp.
R1
R2
R3
R4
R5
X
PR IC50 (nM) a
RT IC50 (m
M) a
35a
35b
35c
35d
35e
35f
35g
36a
36b
36c
36d
36e
36f
36g
37a
37b
37c
37d
37e
37f
37g
DRV
EFV
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH2CH3
C3H5
OCH3
NH2
OH
OH
OH
OH
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
e
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
e
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
e
0.16 0.01
0.20 0.06
16.15 6.37
2.86 0.45
2.55 0.92
19.48 4.02
53.96 8.16
2.93 0.38
3.09 1.14
15.83 3.31
2.42 1.15
24.11 1.47
97.63 23.28
98.81 15.41
13.1 3.31
144.4 48.2
65.05 18.17
69.51 9.53
36.66 11.80
294.10 67.50
527.20 86.27
1.52 0.38
e
40.8 0.7
7.5 1.0
SCH 3
OCH3
OCH3
OCH3
OCH3
OCH3
NH2
SCH 3
OCH3
OCH3
OCH3
OCH3
OCH3
NH2
SCH 3
OCH3
OCH3
OCH3
OCH3
e
21.5 2.0
35.5 1.0
53.5 4.0
31.5 5.0
53.5 2.0
146 8.0
43.5 2.0
99.5 3.5
124.0 1.0
106.0 3.0
43.5 2.0
87.5 5.0
125.0 1.0
1.5 1.0
51.5 3.0
11.5 2.0
90.5 7.0
33.5 6.0
199.0 7.0
e
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH2CH3
C3H5
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH2CH3
C3H5
CH(CH3)2
e
e
e
e
e
e
e
0.091 0.008
a
All assays were conducted in triplicate, and the data shown represent mean values ( 1 standard deviation) derived from the results of three independent experiments.
Table 2
Inhibitory activity of inhibitors 38aꢁ40 g.
Comp.
R1
R2
R3
R4
R5
X
PR IC50 (nM) a
RT IC50 (m
M) a
38a
38b
38c
38d
38e
38f
38g
39a
39b
39c
39d
39e
39f
39g
40a
40b
40c
40d
40e
40f
40g
DRV
EFV
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH2CH3
C3H5
OCH3
NH2
OH
OH
OH
OH
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
e
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
e
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
e
0.39 0.14
0.56 0.03
2.02 0.50
0.081 0.017
0.54 0.08
4.04 1.22
1.47 0.66
1.10 0.53
0.58 0.23
7.73 2.99
0.16 0.05
1.70 0.90
32.47 14.17
9.56 6.81
104.1 20.3
68.91 17.09
89.81 28.4
42.20 3.604
86.44 32.42
160.8 74.87
70.11 49.95
1.52 0.38
e
3.5 2.0
9.5 2.0
0.43 0.100
2.5 0.5
SCH 3
OCH3
OCH3
OCH3
OCH3
OCH3
NH2
SCH 3
OCH3
OCH3
OCH3
OCH3
OCH3
NH2
SCH 3
OCH3
OCH3
OCH3
OCH3
e
7.5 1.0
45.5 2.0
20.5 2.0
206.0 9.0
31.5 5.0
3.5 0.0
241.0 8.0
234.0 5.0
285.0 4.0
40.5 1.0
73.0 4.0
113.0 12.0
60.7 4.0
2.97 0.14
6.5 1.0
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH2CH3
C3H5
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH2CH3
C3H5
9.0 2.0
299.0 10.0
e
CH(CH3)2
e
e
e
e
e
e
e
0.091 0.008
a
All assays were conducted in triplicate, and the data shown represent mean values ( 1 standard deviation) derived from the results of three independent experiments.
5

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
Fig. 2. Anti-HIV-1 PR activity of inhibitors with phenylacryloy groups vesus phenylpropiony groups. (A) Inhibitors with phenylacryloy groups. (B) Inhibitors with phenylpropiony
groups.
Fig. 3. Anti-HIV-1 RT activity of inhibitors with phenylacryloy groups vesus phenylpropiony groups. (A) Inhibitors with phenylacryloy groups s. (B) Inhibitors with phenylpropiony
groups.
of 8.32
And 37b showed antiviral EC50 value of 37.35
30% on HIV-1 late stage, and EC50 value of 68.02
m
M and inhibition of 68% on HIV-1 early stage, respectively.
M and inhibition of
M and inhibition
interactions between the P2 ligand and the S2 subsite of PR, which
verified the importance of hydroxy group that formed additional
ligand-binding site interactions. In addition, hydrogen bonding
interaction was also observed between the hydroxy group in the
scaffold and residue Asp25’. And one of the sulfonamide oxygens
formed hydrogen bonding interaction with amide NHs of Ile500
located in the flaps. Moreover, favorable van der Waals interactions
between P20 ligand and the S20 subsite made great contributions to
the potent antiviral activity. Moreover, inhibitor 38c also fitted into
the protease cave well, but with a slightly lower negative binding
score of ꢁ12.18 kcal/mol compared to 38a, which implied weaker
binding with the enzyme and was in accordance with its PR
inhibitory activity [40]. As shown in Fig. 6 (C), inhibitor 38d fitted
into the active cavity of the protease and formed several strong
hydrogen-bonding contacts with PR. What’s particularly important
was that the nitrogen atom of pyridine in P20 moiety formed direct
hydrogen bond interaction with the backbone NH of Asp300, which
was in line with its high anti-PR potency.
m
m
of 14% on HIV-1 early stage. Both of the two inhibitors exhibited
small difference of inhibition on HIV-1 late stage and early stage.
However, the controls DRV (HIV-1 PR inhibitor) and EFV (HIV-1 RT
inhibitor) showed sharp disparity on the late stage and early stage
of HIV-1 life cycle. For instance, DRV exhibited inhibition of 99% on
HIV-1 late stage and 2% on early stage. EFV exhibited inhibition of
15% on HIV-1 late stage and 85% on early stage, respectively. The
results above supported the rationality of designing dual inhibitors.
2.6. Molecular modeling studies
For the aim of investigating possible hydrogen bonding in-
teractions and hydrophobic contacts, molecular modeling work for
inhibitors 38a, 38c and 38d were performed using the molecular
modeling software MOE (Molecular Operating Environment)
(version 2009.06). Both HIV-1 PR crystal structure (PDB-ID: 4mc9)
and RT crystal structure (PDB-ID: 2yng) were obtained from the
protein data bank [39].
As shown in Fig. 6, inhibitor 38a adapted into HIV-1 PR binding
site nicely with a binding score of ꢁ13.54 kcal/mol. Furthermore,
there formed hydrogen bonding as well as other van der Waals
In Fig. 7 (A), inhibitor 38a with a flexible chain in P2 ligand could
fit into the narrow pocket (non-nucleoside inhibitor binding
pocket, NNIBP) of HIV-1 reverse transcriptase with a binding score
of ꢁ8.10 kcal/mol. One of the sulfonamide oxygens formed a strong
hydrogen bond with residue Lys103, which formed p-p interaction
with the aromatic ring in P1 ligand at the same time. The benzene
6

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
Fig. 4. Scatter plot for the inhibition of PR and RT enzymes. (A) HIV-1 PR/RT dual inhibitors with different substitutions. (B) Inhibitors were categorized according to their P2 ligands.
(C) Inhibitors were categorized according to their P10 ligands. (D) Inhibitors were categorized according to their P20 ligands. Inhibitors with IC50 values against PR above 60 nM or
IC50 values against RT above 100 mM had been arbitrary positioned at the 60 nM or 100 mM value, respectively.
Table 3
Antiviral Activity of 37b and 38c on HIV-1 late stage and early stage.
a
b
Compd.
Activity on HIV-1 late stage
EC50 M)
Activity on HIV-1 early stage a
EC50 M) Inhibition (%) (10 mM)
Ratio
(
m
Inhibition (%) (10
mM)
(m
37b
38c
DRV
EFV
37.35 0.42
2.31 0.03
30
97
99
15
68.02 0.21
8.32 0.28
14
68
2
2.14
1.43
49.50
5.67
e
e
e
e
85
a
All assays were conducted in quintuplicate.
Ratio is defined by inhibition on HIV-1 late stage/inhibition on HIV-1 early stage, or inhibition on HIV-1 early stage/inhibition on HIV-1 late stage.
b
ring in P2 ligand kept
p
-
p
interaction with Tyr188 of RT [28]. As
sulfur atom in P20 ligand interacted with the backbone of Val106 by
a hydrogen bond, which played an important role in enhancing
shown in Fig. 7 (B), inhibitor 38c fitted into the RT active cavity well,
with a binding score of ꢁ9.56 kcal/mol. The aromatic ring of P20
anti-RT activity [41]. Likewise,
p-p interaction, hydrogen bonding
ligand formed
Pro236. Similarly,
p
-
p
p
interactions with both residues Val106 and
p interaction was also observed between the
or hydrophobic contacts were also observed in the structural
analysis of 38d modeled into the active subsite of HIV-1 reverse
transcriptase in Fig. 7 (C).
-
aromatic ring in P2 ligand and Lys223 residue of RT. Moreover, the
7

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
respectively. Thus, the molecular dynamic results indicated that
38c and 38d would bind to the dual targets (PR and RT).
3. Conclusion
In conclusion, we have designed a novel class of HIV-1 PR/RT
dual inhibitors on the basis of both possible ligand-binding site
interactions and the uniformity of the key residues in active sites.
Compared with coumarin derivatives in our previous studies,
introduction of phenylpropiony groups or phenylacryloy groups as
P2 ligands made notable enhancement in HIV-1 RT inhibitory ac-
tivity with one or two orders of magnitude, with comparable or
even improved potency against PR at the same time. More impor-
tantly, the disparate ratio between PR and RT inhibition decreased
sharply and seemed more reasonable with ratio of 1: 10.4, as
example of 37b, which gave encouragement for further optimiza-
tion to improve both PR and RT inhibitory potency. Besides, the
inconspicuous disparity in the assays of inhibitors 37b and 38c on
the late stage and early stage of HIV-1 life cycle supported the ra-
tionality of designing dual inhibitors as shown in Fig. 5.
In addition, the PR antiviral activity of inhibitors with a
methoxypyridine P20 ligand showed improved antiviral IC50 values
than those with a 4-methoxybenzene as the P20 ligand. For
instance, inhibitor 38d with a 3, 4-dihydroxyphenylpropiony group
as the P2 ligand and a 6-methoxypyridine-4-sulfonamide as the P20
ligand displayed an almost 5-fold improved PR antiviral IC50 value
of 0.081 nM compared to 38a with a benzene in the P20 ligand.
Moreover, to a certain extent, inhibitors with phenylpropiony P2
ligands exhibited superior anti-RT activity than those with phe-
nylacryloy groups as the P2 ligands. Taken 38c with a 3, 4-
dihydroxyphenylpropiony group as the P2 ligand for example, it
Fig. 5. Inhibition of compounds 37b, 38c, DRV and EFV on HIV-1 late stage and early
stage.
2.7. Molecular dynamic simulation studies
In order to confirm the druggability nature of the ligands and
their binding mechanism, we run 10 ns MD simulations (molecular
dynamic simulations) for the complex 38c/PR, 38c/RT, 38 d/PR, and
38d/RT as shown in Fig. 8. The RMSD analysis (<2.5 Å) showed that
the binding mode for all four complexes during the simulation
changed little. Thus, it indicated that the docked initial binding
modes could represent the interactions between the inhibitors and
the active sites of HIV-1 PR or RT, and validated the rationality of
designing dual inhibitors.
Also, on the basis of the MD simulation trajectory, the binding
energies for the 38c/PR, 38c/RT, 38 d/PR, and 38d/RT complexes
were evaluated using the MMPBSA method, and the results are
listed in Table 4. As results, both 38c and 38d showed good binding
exhibited an excellent antiviral IC50 value of 0.43 mM. In compari-
son, inhibitor 35c with a 3, 4-dihydroxyphenylacrylol P2 ligand
displayed a nearly 50-fold decrement in RT activity with IC50 value
affinity with the protease with the calculated
D
G
(ꢁ11.59
and ꢁ13.88 kcal/mol). While the predicted binding free energy
of 21.5
mM.
between 38c、38d and RT was ꢁ8.37 and ꢁ8.75 kcal/mol,
According to the molecular modeling studies, both hydroxy
Fig. 6. Molecular modeling for inhibitors 38a, 38c and 38d within HIV-1 PR model. (A) Ligplot interaction of 38a. (B) Ligplot interaction of 38c. (C) Ligplot interaction of 38d. Ligand
exposures were represented as purple spheres and hydrogen bonding was depicted as blue or green arrows.
Fig. 7. Molecular modeling for inhibitors 38a, 38c and 38d within HIV-1 RT model. (A) Ligplot interaction of 38a. (B) Ligplot interaction of 38c. (C) Ligplot interaction of 38d. Ligand
exposures were represented as purple spheres, hydrogen bonding was depicted as blue or green arrows, and
p-p interactions were depicted as green line.
8

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
Fig. 8. Molecular dynamic simulations for the complex 38c/PR, 38c/RT, 38 d/PR, and 38d/RT. (A) Molecular dynamic simulations for the complex 38c/PR. (B) Molecular dynamic
simulations for the complex 38c/RT. (A) Molecular dynamic simulations for the complex 38 d/PR. (B) Molecular dynamic simulations for the complex 38d/RT.
DMSO‑d6: 1H,
d
¼ 2.49 ppm, 13C,
d
¼ 39.5 ppm), and coupling
Table 4
The predicted binding free energy between 38c、38d and HIV-1 PR and RT using
MMPBSA (kcal/mol).
constants were reported in Hz. All the target compounds were
characterized by 1H and 13C NMRs and HRMS spectra.
Complexes
DGpolar
DGnopolar
D
Gmm/pbsa
-TS
DGBind
38c/PR
38 d/PR
38c/RT
38d/RT
59.95
58.32
62.61
63.74
ꢁ100.20
ꢁ100.62
ꢁ96.06
ꢁ97.21
ꢁ40.25
ꢁ42.3
28.66
28.42
25.08
25.72
ꢁ11.59
ꢁ13.88
ꢁ8.37
4.1.1. Tert-butyl ((2S, 3R)-3-hydroxy-4-(isobutylamino)-1-
phenylbutan-2-yl)carbamate (6)
ꢁ33.45
ꢁ34.47
To a stirred solution of (2S, 3S)-1, 2-epoxy-3-(boc-amino)-4-
phenylbutane (1, 20.0 g, 75.94 mmol) in acetonitrile (82 mL) at
25 ꢀC was added isobutylamine (2, 19.0 mL, 189.46 mmol). The
resulting mixture was heated at reflux for 6 h. After this period, the
reaction mixture was concentrated under reduced pressure and the
residue was purified by column chromatography on silica gel (10%
CH3OH in CH2Cl2 as the eluent) to provide the corresponding amine
(21.2 g, 83%) as white amorphous solid: 1H NMR (500 MHz, CD3OD)
ꢁ8.75
group and aromatic ring in P2 ligands were indispensable for
additional hydrogen bondings and other van der Waals interactions
in both PR and RT inhibitory potency, which was consistent with
our anticipation. Moreover, the flexible chain in P2 ligand resulted
in a significant enhancement of dual antiviral activity. In addition,
substitutions in P20 ligand played an important role in antiviral
potency. We would put focus on improving anti-RT activity in our
next study, and the benzene ring in P1 ligand might be an appro-
priate modification site inspired by molecular modeling studies in
Fig. 7 (A).
d
7.29e7.16 (m, 5H, H-20, H-30, H-40, H-50, H-60), 3.71e3.59 (m, 2H,
H-2, H-3), 3.16e3.08 (m, 1H, H-1a), 2.83e2.74 (m, 1H, H-4a),
2.68e2.56 (m, 2H, H-1b, H-4b), 2.54e2.48 (m, 1H, H-100a),
2.46e2.39 (m, 1H, H-100b), 1.88e1.77 (m, 1H, H-200), 1.32 (s, 9H, Boc-
CH3), 0.96 (d, J ¼ 6.4 Hz, 6H, 300a-CH3, 300b-CH3); 13C NMR (126 MHz,
CDCl3)
d 155.9, 137.9, 129.5, 128.4, 126.3, 79.3, 70.6, 58.0, 54.2, 51.4,
36.7, 28.4, 28.3, 20.5; LC-MS (ESI) [MþH]þ m/z 337.2.
4. Experimental section
4.1.2. Tert-butyl ((2S, 3R)-3-hydroxy-1-phenyl-4-(propylamino)
butan-2-yl)carbamate (7)
4.1. Chemistry
The compound was obtained from n-propylamine (3) in 90%
All experiments requiring anhydrous conditions were con-
ducted in flame-dried glassware fitted with rubber septa under a
positive pressure of dry argon, unless otherwise noted. The solvent
dichloromethane was distilled under an argon atmosphere from
calcium hydride. All reactions were monitored by the thin-layer
chromatography (TLC) on silica gel plates (GF-254) and visualized
with the UV light. Flash column chromatography was performed on
yield as white powder as described for 6: 1H NMR (500 MHz, CDCl3)
d
7.30e7.27 (m, 2H, H-30, H-50), 7.23e7.22 (m, 3H, H-20, H-40, H-60),
4.65 (s, 1H, 2-NH), 3.80 (s, 1H, H-3), 3.46 (s, 1H, H-2), 2.98 (d,
J ¼ 13.5 Hz, 1H, H-1a), 2.86 (s, 1H, H-4a), 2.69 (s, 2H, H-1b, H-4b),
2.57 (s, 2H, H-100a, H-100b), 1.50e1.48 (m, 2H, H-200a, H-200b), 1.36 (s,
9H, Boc-CH3), 0.93 (t, J ¼ 6.0 Hz, 3H, 300-CH3); 13C NMR (101 MHz,
CDCl3) d 155.9, 137.9, 129.5, 128.34, 126.3, 79.4, 70.8, 54.2, 51.7, 51.3,
a CombiFlash®Rf 200 system employing silica gel (50e75
mm,
36.6, 28.3, 23.2, 11.7; LC-MS (ESI, M þ Hþ) m/z 323.3.
Qingdao Haiyang Chemical Co. Ltd). High resolution mass spectra
were obtained on an Autospee Ultima-TOF spectrometer. 1H NMR
and 13C NMR spectra were recorded in CDCl3, CD3OD or DMSO‑d6
on a Bruker AVANCE III 400 MHz, 500 MHz or 600 MHz spec-
trometer (Bruker Inc) with tetramethylsilane (TMS) as the internal
4.1.3. Tert-butyl ((2S, 3R)-4-(cyclopropylamino)-3-hydroxy-1-
phenylbutan-2-yl)carbamate (8)
The compound was obtained from cyclopropylamine (4) in 86%
reference. The chemical shifts were given in
d
(ppm) referenced to
yield as white powder as described for 6: 1H NMR (500 MHz, CDCl3)
the respective solvent peak (CDCl3: 1H,
d
¼ 7.26 ppm, 13C,
d
7.22e7.16 (m, 5H, H-20, H-30, H-40, H-50, H-60), 3.71 (s, 1H, H-3),
d
¼ 77.16 ppm; CD3OD: 1H,
d
¼ 3.31 ppm, 13C,
d
¼ 49.00 ppm;
3.41 (s, 1H, H-2), 2.91 (d, J ¼ 13.5 Hz, 1H, H-1a), 2.78e2.72 (m, 3H,
9

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
H-1b, H-4a, H-4b), 2.07 (s, 1H, H-100), 1.29 (s, 9H, Boc-CH3), 0.39 (s,
2H, H-200a, H-300a), 0.28 (s, 2H, H-200b, H-300b); 13C NMR (126 MHz,
137.8, 134.1, 129.6, 128.5, 127.7, 126.4, 125.4, 79.7, 72.7, 58.6, 54.7,
53.7, 35.5, 29.7, 28.3, 27.2, 20.1, 19.9, 14.8; LC-MS (ESI, M þ Hþ) m/z
523.3.
CDCl3)
d 156.0, 137.9, 129.6, 128.4, 126.4, 79.5, 70.7, 54.0, 51.4, 36.6,
30.6, 28.3, 6.81, 6.26; LC-MS (ESI, M þ Hþ) m/z 321.3.
4.1.8. Tert-butyl ((2S, 3R)-3-hydroxy-4-((N-isobutyl-6-
methoxypyridine)-3-sulfonamido)-1-phenylbutan-2-yl)carbamate
(17)
4.1.4. Tert-butyl ((2S, 3R)-3-hydroxy-4-(isopropylamino)-1-
phenylbutan-2-yl)carbamate (9)
The compound was obtained from n-propylamine (3) in 85%
The compound was obtained by 6 coupling with 6-
methoxypyridine-3-sulfonyl chloride (13) in the presence of DIEA
yield as white powder as described for 6: 1H NMR (500 MHz, CDCl3)
d
7.29e7.27 (m, 2H, H-30, H-50), 7.24e7.22 (m, 3H, H-20, H-40, H-60),
and DMAP procedure in 80% yield (white amorphous solid) as
4.59 (s, 1H, 3-OH), 3.79 (s, 1H, H-3), 3.41 (s, 1H, H-2), 3.00 (d,
J ¼ 13.5 Hz, 1H, H-1a), 2.86 (s, 1H, H-4a), 2.76 (s, 1H, H-100),
2.69e2.67 (m, 2H, H-1b, H-4b), 1.36 (s, 9H, Boc-CH3), 1.06 (s, 3H,
described for 14: 1H NMR (500 MHz, CDCl3) 8.59 (s, 1H, H-6000),
d
7.87 (d, J ¼ 8.0 Hz, 1H, H-2000), 7.30e7.24 (m, 5H, H-20, H-30, H-40, H-
50, H-60), 6.81 (d, J ¼ 8.0 Hz, 1H, H-5000), 4.65 (s, 1H, H-3), 4.00 (s, 3H,
4000-OCH3), 3.85e3.76 (m, 3H, H-2, H-4a, H-4b), 3.01e2.90 (m, 4H,
H-1a, H-1b, H-100a, H-100b), 1.87 (s, 1H, H-200), 1.35 (s, 9H, Boc-CH3),
200a-CH3), 1.05 (s, 3H, 200b eCH3); 13C NMR (101 MHz, CDCl3)
d 155.9,
137.9, 129.6, 128.4, 126.3, 79.4, 71.0, 54.0, 49.0, 48.7, 36.6, 28.3, 23.3,
22.9; LC-MS (ESI, M þ Hþ) m/z 323.3.
0.89 (s, 6H, 300a-CH3, 300b-CH3); 13C NMR (101 MHz, CDCl3)
d 166.4,
156.2, 147.4, 137.7, 137.3, 129.5, 128.5, 128.0, 126.5, 111.5, 79.9, 72.7,
58.3, 54.9, 54.3, 53.4, 35.5, 28.2, 27.1, 20.1, 19.9; LC-MS (ESI, M þ Hþ)
m/z 509.3.
4.1.5. Tert-butyl ((2S, 3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)carbamate (14)
To a stirred solution of 6 (5.0 g, 14.86 mmol) in tetrahydrofuran
(40 mL) at 0 ꢀC was added DIEA (3.68 mL, 16.34 mmol) and DMAP
(0.18 g, 1.49 mmol) in batches, followed by a mixture of 4-
methoxybenzenesulfonyl chloride (10, 3.37 g, 16.34 mmol) and
tetrahydrofuran (15 mL). The resulting mixture was stirred at 0 ꢀC
for 0.5 h and at 25 ꢀC for another 3e5 h. The mixture was then
concentrated under reduced pressure and extracted with ethyl
acetate and dried over anhydrous Na2SO4. Removal of solvent,
followed by column chromatography over silica gel (20% EtOAc in
n-hexane as the eluent), yielded compound 14 (6.14 g, 82%) as
4.1.9. Tert-butyl ((2S, 3R)-3-hydroxy-4-((4-methoxy-N-
propylphenyl)sulfonamido)-1-phenylbutan-2-yl)carbamate (18)
The compound was obtained by
7 coupling with 4-
methoxybenzenesulfonyl chloride (10) in the presence of DIEA
and DMAP procedure in 80% yield (white amorphous solid) as
described for 14: 1H NMR (500 MHz, CDCl3)
d
7.72 (d, J ¼ 7.5 Hz, 2H,
H-2000, H-6000), 7.30e7.22 (m, 5H, H-20, H-30, H-40, H-50, H-60), 6.97 (d,
J ¼ 7.5 Hz, 2H, H-3000, H-5000), 3.87 (s, 3H, 4000-OCH3), 3.80 (s, 2H, H-2,
H-3), 3.12e3.10 (m, 3H, H-4a, H-4b, H-1a), 3.04e2.99 (m, 2H, H-1b,
H-100a), 2.94 (s, 1H, H-100b), 1.55e1.51 (m, 2H, H-200a, H-200b), 1.35 (s,
9H, Boc-CH3), 0.85 (d, J ¼ 6.0 Hz, 3H, 300-CH3); 13C NMR (101 MHz,
white amorphous solid: 1H NMR (400 MHz, CDCl3)
d 7.72 (d,
J ¼ 6.9 Hz, 2H, H-2000, H-6000), 7.33e7.17 (m, 5H, H-20, H-30, H-40, H-50,
H-60), 6.98 (d, J ¼ 6.9 Hz, 2H, H-3000, H-5000), 3.87 (s, 3H, 4000-OCH3),
3.84e3.74 (m, 2H, H-2, H-3), 3.13e2.80 (m, 6H, H-1a, H-1b, H-4a, H-
4b, H-100a, H-100b), 1.92e1.80 (m, 1H, H-200), 1.35 (s, 9H, Boc-CH3),
0.89 (dd, J ¼ 14.4, 5.2 Hz, 6H, 300a-CH3, 300b-CH3); 13C NMR
CDCl3)
d 163.0, 156.2, 137.9, 130.4, 129.6, 129.4, 128.5, 126.5, 114.4,
79.8, 72.7, 55.7, 54.7, 52.6, 52.3, 35.4, 29.7, 28.3, 22.0, 11.2; LC-MS
(ESI, M þ Hþ) m/z 493.3.
(101 MHz, CDCl3)
d
163.0, 156.0, 137.9, 130.0, 129.6, 129.5, 128.5,
4.1.10. Tert-butyl ((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
126.4, 114.3, 79.7, 72.8, 58.6, 55.6, 54.7, 53.7, 35.5, 28.3, 27.2, 20.1,
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)carbamate (19)
The compound was obtained by 8 coupling with 4-
methoxybenzenesulfonyl chloride (10) in the presence of DIEA
19.9; LC-MS (ESI, M þ Hþ) m/z 507.0.
4.1.6. Tert-butyl ((2S, 3R)-3-hydroxy-4-((N-isobutyl-4-nitrophenyl)
sulfonamido)-1-phenylbutan-2-yl)carbamate (15)
and DMAP procedure in 80% yield (white amorphous solid) as
described for 14: 1H NMR (500 MHz, CD3OD)
d
7.80 (d, J ¼ 8.5 Hz,
The compound was obtained by
6
coupling with 4-
2H, H-2000, H-6000), 7.25e7.23 (m, 4H, H-20, H-30, H-50, H-60), 7.17e7.12
(m, 1H, H-40), 7.09 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 3.92e3.89 (m, 1H,
H-3), 3.87 (s, 3H, 4000-OCH3), 3.68e3.64 (m, 1H, H-2), 3.40 (dd,
J ¼ 14.5, 3.0 Hz, 1H, H-1a), 3.14 (dd, J ¼ 13.5, 3.0 Hz, 1H, H-1b), 3.03
(dd, J ¼ 14.0, 9.0 Hz, 1H, H-4a), 2.57 (dd, J ¼ 13.5, 11.0 Hz, 1H, H-4b),
2.04e1.97 (m, 1H, H-100), 1.29 (s, 9H, Boc-CH3), 1.02e0.89 (m, 2H, H-
200a, H-300a), 0.77e0.70 (m, 1H, H-200b), 0.68e0.61 (m, 1H, H-300b);
nitrobenzenesulfonyl chloride (11) in the presence of DIEA and
DMAP procedure in 91% yield (white amorphous solid) as described
for 14: 1H NMR (400 MHz, CDCl3)
d
8.35 (d, J ¼ 8.8 Hz, 2H, H-3000, H-
5000), 7.98 (d, J ¼ 8.8 Hz, 2H, H-2000, H-6000), 7.35e7.31 (m, 2H, H-30, H-
50), 7.29e7.22 (m, 3H, H-20, H-40, H-60), 3.85e3.76 (m, 2H, H-2, H-3),
3.25e3.20 (m, 2H, H-4a, H-4b), 3.03e2.96 (m, 3H, H-1a, H-100a, H-
100b), 2.94e2.87 (m, 1H, H-1b), 1.95e1.86 (m, 1H, H-200), 1.38 (s, 9H,
Boc-CH3), 0.90 (dd, J ¼ 6.4, 4.8 Hz, 6H, 300a-CH3, 300b-CH3); 13C NMR
13C NMR (101 MHz, CD3OD)
d 164.8, 157.9, 140.4, 131.1, 130.5, 130.4,
129.1, 127.0, 115.3, 79.9, 73.5, 56.6, 56.3, 56.2, 37.1, 33.3, 28.7, 8.84,
(101 MHz, CDCl3)
d
156.4, 150.0, 145.0, 137.5, 129.4, 128.6, 128.5,
7.56; LC-MS (ESI, M þ Hþ) m/z 491.4.
126.7, 124.3, 80.2, 72.2, 57.5, 55.2, 52.5, 35.6, 28.2, 26.9, 20.0, 19.8;
LC-MS (ESI, M þ Hþ) m/z 521.9.
4.1.11. Tert-butyl ((2S, 3R)-3-hydroxy-4-((N-isopropyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)carbamate (20)
4.1.7. Tert-butyl ((2S, 3R)-3-hydroxy-4-((N-isobutyl-4-(methylthio)
phenyl)sulfonamido)-1-phenylbutan-2-yl)carbamate (16)
The compound was obtained by 9 coupling with 4-
methoxybenzenesulfonyl chloride (10) in the presence of DIEA
The compound was obtained by 6 coupling with 4-(methylthio)
benzenesulfonyl chloride (12) in the presence of DIEA and DMAP
procedure in 85% yield (white amorphous solid) as described for
and DMAP procedure in 85% yield (white amorphous solid) as
described for 14: 1H NMR (500 MHz, CDCl3)
d
7.74 (d, J ¼ 7.5 Hz, 2H,
H-2000, H-6000), 7.30e7.22 (m, 5H, H-20, H-30, H-40, H-50, H-60), 6.96 (d,
J ¼ 7.5 Hz, 2H, H-3000, H-5000), 4.10e4.05 (m, 1H, H-3), 3.87 (s, 3H, 4000-
OCH3), 3.78 (s, 2H, H-2, H-4a), 3.20 (d, J ¼ 15.5 Hz, 1H, H-4b), 3.07
(dd, J ¼ 15.0, 9.5 Hz, 2H, H-1a, H-1b), 2.94 (s, 1H, H-100), 1.35 (s, 9H,
Boc-CH3), 1.03 (d, J ¼ 5.0 Hz, 3H, 200a-CH3), 0.90 (s, 3H, 200b eCH3);
14: 1H NMR (500 MHz, CDCl3)
d
7.65 (d, J ¼ 7.0 Hz, 2H, H-2000, H-6000),
7.30e7.24 (m, 7H, H-20, H-30, H-40, H-50, H-60, H-3000, H-5000),
3.81e3.75 (m, 2H, H-2, H-3), 3.13e2.92 (m, 5H, H-1a, H-4a, H-4b, H-
100a, H-100b), 2.83 (s, 1H, H-1b), 2.52 (s, 3H, 4000-SCH3), 1.85 (s, 1H, H-
200), 1.34 (s, 9H, Boc-CH3), 0.90 (d, J ¼ 4.5 Hz, 3H, 300a-CH3), 0.87 (d,
13C NMR (101 MHz, CDCl3)
d 162.9, 155.9, 137.8, 131.4, 129.6, 129.3,
J ¼ 4.5 Hz, 3H, 300b-CH3); 13C NMR (101 MHz, CDCl3)
d
156.1, 145.9,
128.4, 126.4, 114.3, 79.5, 74.3, 55.6, 54.4, 50.2, 46.7, 35.7, 29.7, 28.3,
10

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
21.7, 19.8; LC-MS (ESI, M þ Hþ) m/z 493.3.
H-40, H-50, H-60, H-3000, H-5000), 3.75 (s,1H, H-3), 3.30e3.21 (m, 2H, H-
2, H-4a), 3.14 (d, J ¼ 4.0 Hz, 1H, H-4b), 3.03e2.99 (m, 1H, H-100a),
2.96 (d, J ¼ 13.5 Hz, 1H, H-100b), 2.90e2.86 (m, 1H, H-1a), 2.52e2.48
(m, 4H, H-1b, 4000-SCH3), 1.93e1.86 (m, 1H, H-200), 0.92 (d, J ¼ 5.0 Hz,
3H, 300a-CH3), 0.89 (d, J ¼ 5.0 Hz, 3H, 300b-CH3); 13C NMR (101 MHz,
4.1.12. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-
4-methoxybenzenesulfonamide (21)
A solution of 14 (5.0 g, 9.87 mmol) in a mixture of 10 mL tri-
fluoroacetic acid and 10 mL CH2Cl2 was stirred for 3 h at 25 ꢀC. After
this period, the reaction mixture was concentrated under reduced
pressure and the residue was redissolved in 10 mL CH2Cl2. Satu-
rated aqueous sodium bicarbonate were added dropwise to
neutralize the superfluous acid. The mixture was then extracted
with CH2Cl2 and dried over anhydrous Na2SO4. Removal of solvent
under reduced pressure, followed by column chromatography over
silica gel (10% CH3OH in CH2Cl2 as the eluent), yielded compound
21 (3.13 g, 78%) as white amorphous solid: 1H NMR (400 MHz,
CDCl3)
d 145.8, 138.7, 134.4, 129.3, 128.7, 127.7, 126.5, 125.5, 73.0,
58.5, 55.7, 52.6, 39.0, 27.2, 20.2, 19.9, 14.8; LC-MS (ESI) [M þ H]þ m/z
423.2.
4.1.16. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-
6-methoxypyridine-3-sulfonamide (25)
The compound was obtained by 17, which was exposed to tri-
fluoroacetic acid to remove the BOC-group in 77% yield (white
amorphous solid) as described for 21: 1H NMR (500 MHz, CDCl3)
CDCl3)
d
7.67 (d, J ¼ 8.8 Hz, 2H, H-2000, H-6000), 7.29e7.20 (m, 5H, H-20,
d
8.63 (s, 1H, H-6000), 7.93 (d, J ¼ 8.5 Hz, 1H, H-2000), 7.31e7.30 (m, 2H,
H-30, H-40, H-50, H-60), 6.95 (d, J ¼ 8.8 Hz, 2H, H-3000, H-5000),
4.22e4.15 (m, 1H, H-3), 3.85 (s, 3H, 4000-OCH3), 3.81e3.78 (m, 1H, H-
2), 3.23e3.18 (m, 1H, H-4a), 3.13e3.00 (m, 3H, H-4b, H-100a, H-100b),
2.81e2.70 (m, 2H, H-1a, H-1b), 1.69e1.59 (m, 1H, H-200), 0.75 (d,
J ¼ 2.8 Hz, 3H, 300a-CH3), 0.73 (d, J ¼ 2.8 Hz, 3H, 300b-CH3); 13C NMR
H-30, H-50), 7.26e7.21 (m, 3H, H-20, H-40, H-60), 6.83 (d, J ¼ 8.5 Hz,
1H, H-5000), 4.01 (s, 3H, 4000-OCH3), 3.76 (s, 1H, H-3), 3.32e3.28 (m,
2H, H-2, H-4a), 3.13 (d, J ¼ 4.0 Hz, 1H, H-4b), 3.03e2.91 (m, 3H, H-
1a, H-100a, H-100b), 2.50 (t, J ¼ 11.5 Hz, 1H, H-1b), 1.92 (t, J ¼ 5.5 Hz,
1H, H-200), 0.92 (d, J ¼ 6.5 Hz, 3H, 300a-CH3), 0.90 (d, J ¼ 6.5 Hz, 3H,
(101 MHz, CD3OD)
d
164.5, 140.2, 132.0, 130.6, 130.4, 129.6, 127.5,
300b-CH3); 13C NMR (101 MHz, CDCl3)
d 166.1, 147.1, 138.3, 137.1,
115.3, 73.7, 58.9, 57.0, 56.2, 53.1, 39.1, 28.1, 20.5, 20.4; LC-MS (ESI,
128.9, 128.3, 128.0, 126.2 111.1, 72.5, 57.8, 55.3, 53.9, 52.0, 38.7, 26.7,
M þ Hþ) m/z 407.3.
19.8, 19.6; LC-MS (ESI) [M þ H]þ m/z 408.3.
4.1.13. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-
4-nitrobenzenesulfonamide (22)
The compound was obtained by 15 which was exposed to tri-
fluoroacetic acid to remove the BOC-group in 83% yield (white
amorphous solid) as described for 21: 1H NMR (400 MHz, DMSO‑d6)
4.1.17. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-
N-propylbenzenesulfonamide (26)
Compound 18 was exposed to trifluoroacetic acid at 0e25 ꢀC for
3 h to remove the BOC-group and afforded the corresponding
amine 26 in yield of 88% as white amorphous solid as described for
d
8.38 (d, J ¼ 8.0 Hz, 2H, H-3000, H-5000), 8.06 (d, J ¼ 8.0 Hz, 2H, H-2000,
21: 1H NMR (500 MHz, CDCl3)
d
7.76 (d, J ¼ 7.5 Hz, 2H, H-2000, H-6000),
H-6000), 7.38e7.27 (m, 5H, H-20, H-30, H-40, H-50, H-60), 5.64 (s, 1H, 3-
OH), 3.91 (s, 1H, H-3), 3.49e3.37 (m, 2H, H-2, H-4a), 3.07 (dd,
J ¼ 13.6, 8.4 Hz, 2H, H-4b, H-100a), 2.99 (dd, J ¼ 14.0, 6.4 Hz, 1H, H-
100b), 2.87 (dd, J ¼ 14.0, 6.4 Hz, 1H, H-1a), 2.83e2.72 (m, 1H, H-1b),
1.93e1.84 (m, 1H, H-200), 0.82 (d, J ¼ 6.4 Hz, 3H, 300a-CH3), 0.76 (d,
7.30e7.22 (m, 5H, H-20, H-30, H-40, H-50, H-60), 6.98 (d, J ¼ 7.5 Hz, 2H,
H-3000, H-5000), 3.87 (s, 3H, 4000-OCH3), 3.81 (s, 1H, H-3), 3.27e3.20 (m,
3H, H-2, H-4a, H-4b), 3.15e3.05 (m, 2H, H-1a, H-1b), 2.99 (d,
J ¼ 13.5 Hz, 1H, H-100a), 2.60e2.51 (m, 1H, H-100b), 1.55 (d, J ¼ 6.0 Hz,
2H, H-200a, H-200b), 0.85 (t, J ¼ 6.5 Hz, 3H, 300-CH3); 13C NMR
J ¼ 6.4 Hz, 3H, 300b-CH3); 13C NMR (101 MHz, CD3OD)
d
151.4, 147.0,
(101 MHz, CDCl3) d 162.9, 138.4, 130.6, 129.4, 129.3, 128.7, 126.6,
140.0, 130.4, 129.9, 129.7, 127.5, 125.3, 72.8, 57.8, 57.2, 51.9, 39.3,
114.3, 72.4, 55.6, 55.5, 52.1, 51.4, 38.2, 21.9, 11.2; LC-MS (ESI) [M þ
27.8, 20.3; LC-MS (ESI, M þ Hþ) m/z 422.3.
H]þ m/z 393.3.
4.1.14. 4-Amino-N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-
N-isobutylbenzenesulfonamide (23)
4.1.18. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-
cyclopropyl-4-methoxybenzenesulfonamide (27)
To a solution of compound 22 (1.26 g, 3.0 mmol) in 10 mL CH3OH
was added 10% Pd/C (0.13 g). The mixture was stirred at 25 ꢀC under
a 50 psi hydrogen pressure for 2 h. The reaction mixture was
filtered over Celite and washed with CH3OH. Removal of solvent
under reduced pressure, followed by column chromatography on
silica gel (10% CH3OH in CH2Cl2 as the eluent) afforded the corre-
sponding aromatic amine (1.10 g, 94%) as white amorphous solid:
Compound 19 was exposed to trifluoroacetic acid at 25 ꢀC for 3 h
to remove the BOC-group and afforded the corresponding amine 27
in yield of 75% as white amorphous solid as described for 21: 1H
NMR (500 MHz, CD3OD)
d
7.80 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000),
7.32e7.20 (m, 5H, H-20, H-30, H-40, H-50, H-60), 7.10 (d, J ¼ 8.5 Hz, 2H,
H-3000, H-5000), 3.96e3.93 (m, 1H, H-3), 3.88 (s, 3H, 4000-OCH3), 3.43
(dd, J ¼ 14.0, 4.0 Hz, 1H, H-2), 3.16e3.09 (m, 2H, H-1a, H-1b), 3.02
(dd, J ¼ 13.5, 4.5 Hz, 1H, H-4a), 2.56 (dd, J ¼ 13.5, 9.5 Hz, 1H, H-4b),
2.04e1.93 (m, 1H, H-100), 0.97e0.84 (m, 2H, H-200a, H-300a),
0.74e0.67 (m, 1H, H-200b), 0.67e0.58 (m, 1H, H-300b); 13C NMR
1H NMR (500 MHz, CD3OD)
d
7.52 (d, J ¼ 8.6 Hz, 2H, H-2000, H-6000),
7.35e7.29 (m, 4H, H-20, H-30, H-50, H-60), 7.26e7.23 (m, 1H, H-40),
6.73 (d, J ¼ 8.6 Hz, 2H, H-3000, H-5000), 3.83e3.80 (m, 1H, H-3), 3.37
(dd, J ¼ 14.9, 3.8 Hz, 1H, H-2), 3.15 (dt, J ¼ 8.8, 4.3 Hz, 1H, H-4a),
3.08e3.00 (m, 2H, H-4b, H-100a), 2.95 (dd, J ¼ 13.6, 7.8 Hz, 1H, H-
100b), 2.86 (dd, J ¼ 13.6, 7.2 Hz, 1H, H-1a), 2.58 (dd, J ¼ 13.5, 9.4 Hz,
1H, H-1b), 2.00e1.95 (m 1H, H-200), 0.91 (d, J ¼ 6.6 Hz, 3H, 300a-CH3),
(101 MHz, CD3OD)
d 164.8, 140.2, 131.1, 130.6, 130.5, 129.7, 127.5,
115.3, 72.9, 57.0, 56.2, 55.2, 38.7, 33.1, 8.58, 7.53; LC-MS (ESI) [M þ
H]þ m/z 391.3.
0.88 (d, J ¼ 6.6 Hz, 3H, 300b-CH3); 13C NMR (151 MHz, CDCl3)
d
150.5,
4.1.19. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-
isopropyl-4-methoxybenzenesulfonamide (28)
138.7, 130.9, 129.5, 129.3, 128.6, 126.5, 114.1, 73.0, 58.7, 55.7, 52.6,
39.0, 27.2, 20.2, 19.9; LC-MS (ESI) [M þ H]þ m/z 392.5.
Compound 20 was exposed to trifluoroacetic acid at 25 ꢀC for 3 h
to remove the BOC-group and afforded the corresponding amine 28
in yield of 85% as white amorphous solid as described for 21: 1H
4.1.15. N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-
4-(methylthio)benzenesulfonamide (24)
NMR (500 MHz, CDCl3)
d
7.78 (d, J ¼ 7.0 Hz, 2H, H-2000, H-6000),
The compound was obtained by 16, which was exposed to tri-
fluoroacetic acid to remove the BOC-group in 81% yield (white
amorphous solid) as described for 21: 1H NMR (500 MHz, CDCl3)
7.30e7.21 (m, 5H, H-20, H-30, H-40, H-50, H-60), 6.97 (d, J ¼ 7.0 Hz, 2H,
H-3000, H-5000), 4.08 (t, J ¼ 5.5 Hz, 1H, H-3), 3.88 (s, 1H, H-2), 3.86 (s,
3H, 4000-OCH3), 3.36 (d, J ¼ 15.0 Hz, 1H, H-4a), 3.24e3.16 (m, 2H, H-
1a, H-4b), 3.05 (d, J ¼ 13.5 Hz, 1H, H-1b), 2.60 (t, J ¼ 11.5 Hz, 1H, H-
d
7.70 (d, J ¼ 7.5 Hz, 2H, H-2000, H-6000), 7.30e7.21 (m, 7H, H-20, H-30,
11

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
100), 1.04 (d, J ¼ 4.5 Hz, 3H, 200a-CH3), 1.01 (d, J ¼ 4.5 Hz, 3H, 200b-
1H, H-4b), 3.10 (dd, J ¼ 13.5, 9.0 Hz, 1H, isobutyl-CH2a), 2.85 (dd,
J ¼ 13.5, 6.0 Hz, 1H, isobutyl-CH2b), 2.76 (dd, J ¼ 13.5, 6.0 Hz, 1H, H-
1a), 2.68 (dd, J ¼ 13.5,11.5 Hz,1H, H-1b), 2.05e1.99 (m,1H, isobutyl-
CH), 0.94 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.86 (d, J ¼ 6.5 Hz,
CH3); 13C NMR (101 MHz, CDCl3)
d 162.9, 138.6, 131.5, 129.4, 128.7,
126.7, 114.4, 73.9, 56.0, 55.6, 50.3, 45.7, 38.4, 21.4, 20.5; LC-MS (ESI)
[M þ H]þ m/z 393.5.
3H, one of isobutyl-CH3); 13C NMR (126 MHz, CD3OD)
d 168.4,164.3,
4.1.20. 3-(3, 4-Dihydroxyphenyl)propanoic acid (32)
148.4, 146.3, 142.0, 139.6, 130.9, 130.2, 129.8, 128.8, 127.7, 126.7,
121.6, 117.7, 116.0, 114.8, 114.6, 74.4, 59.0, 55.5, 55.0, 54.0, 36.4, 27.5,
20.0; HRMS (ESI) m/z calcd. for C30H35N2O7S ([M ꢁ H]-): 567.2159,
found 567.2175.
To a solution of (E)-3-(3, 4-dihydroxyphenyl)acrylic acid (29,
0.50 g, 2.78 mmol) in CH3OH (4 mL) was added 10% Pd/C (0.05 g).
The mixture was stirred at 25 ꢀC under a 50 psi hydrogen pressure
for 3 h. The reaction mixture was filtered over Celite and washed
with CH3OH. Removal of solvent under reduced pressure, followed
by column chromatography on silica gel (12% CH3OH in CH2Cl2 as
the eluent) afforded the corresponding aromatic amine (0.48 g,
95%) as pale yellow amorphous solid: 1H NMR (500 MHz, CD3OD)
4.1.24. (E)-N-((2S, 3R)-4-((4-amino-N-isobutylphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(3,4-
dihydroxyphenyl)acrylamide (35b)
The compound was obtained by 29 which was coupled with 4-
d
6.67e6.64 (m, 2H, H-20, H-50), 6.52 (d, J ¼ 7.0 Hz, 1H, H-60), 2.75 (t,
amino-N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-iso-
J ¼ 7.5 Hz, 2H, H-3a, H-3b), 2.52 (t, J ¼ 7.5 Hz, 2H, H-2a, H-2b); 13
C
butylbenzenesulfonamide (23) through EDCI/HOBt/DMAP coupling
procedure in 75% yield (white amorphous solid) as described for
NMR (101 MHz, CD3OD)
d
177.0, 146.2, 144.6, 133.8, 120.5, 116.4,
116.3, 37.2, 31.5; LC-MS (ESI) [M ꢁ H]- m/z 181.4.
35a: 1H NMR (500 MHz, CD3OD)
d
7.45 (d, J ¼ 8.5 Hz, 2H, H-2000, H-
6000), 7.29e7.21 (m, 5H, H-30, four of 1-phenyl-H), 7.13 (t, J ¼ 7.0 Hz,
1H, one of 1-phenyl-H), 6.99 (s, 1H, H-200), 6.88 (d, J ¼ 8.0 Hz, 1H, H-
500), 6.76 (d, J ¼ 8.0 Hz, 1H, H-600), 6.62 (d, J ¼ 8.5 Hz, 2H, H-3000, H-
5000), 6.32 (d, J ¼ 16.0 Hz, 1H, H-20), 4.14 (t, J ¼ 6.5 Hz, 1H, H-3), 3.90
(t, J ¼ 6.5 Hz, 1H, H-2), 3.32 (d, J ¼ 8.5 Hz, 1H, H-4a), 3.24 (brd,
J ¼ 12.0 Hz, 1H, H-4b), 3.01 (dd, J ¼ 13.5, 8.5 Hz, 1H, isobutyl-CH2a),
2.88 (dd, J ¼ 15.0, 8.5 Hz, 1H, isobutyl-CH2b), 2.77e2.67 (m, 2H, H-
1a, H-1b), 2.02e1.95 (m, 1H, isobutyl-CH), 0.92 (d, J ¼ 6.5 Hz, 3H,
one of isobutyl-CH3), 0.86 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3);
4.1.21. 3-(4-Hydroxy-3-methoxyphenyl)propanoic acid (33)
The compound was obtained by (E)-3-(4-hydroxy-3-
methoxyphenyl)acrylic acid (30) via reduction reaction under the
condition of hydrogen and 10% Pd/C in 92% yield (white amorphous
solid) as described for 32: 1H NMR (500 MHz, CD3OD)
d 6.78 (s, 1H,
H-20), 6.70 (d, J ¼ 8.0 Hz, 1H, H-50), 6.64 (d, J ¼ 8.0 Hz, 1H, H-60), 3.81
(s, 3H, 30-OCH3), 2.82 (t, J ¼ 7.5 Hz, 2H, H-3a, H-3b), 2.55 (t,
J ¼ 7.5 Hz, 2H, H-2a, H-2b); 13C NMR (101 MHz, CD3OD)
d 177.0,
148.8, 145.8, 133.7, 121.6, 116.1, 113.0, 56.3, 37.1, 31.7; LC-MS (ESI)
13C NMR (126 MHz, CD3OD)
d 169.0, 154.1, 148.9, 146.8, 142.5, 140.1,
[M ꢁ H]- m/z 195.4.
130.5,130.3, 129.2, 128.2, 127.2, 125.7, 122.2,118.2, 116.5, 115.1, 114.5,
74.7, 59.4, 55.5, 54.5, 36.8, 28.1, 20.6, 20.5; HRMS (ESI) m/z calcd. for
4.1.22. 3-(4-Hydroxy-3, 5-dimethoxyphenyl)propanoic acid (34)
The compound was obtained by (E)-3-(4-hydroxy-3, 5-
dimethoxyphenyl)acrylic acid (31) via reduction reaction under
the condition of hydrogen and 10% Pd/C in 94% yield (pale yellow
amorphous solid) as described for 32: 1H NMR (500 MHz, CD3OD)
C
29H34N3O6S ([M ꢁ H]-): 552.2163, found 552.2191.
4.1.25. (E)-3-(3, 4-dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isobutyl-4-(methylthio)phenyl)sulfonamido)-1-phenylbutan-2-yl)
acrylamide (35c)
d
6.49 (s, 2H, H-20, H-60), 3.81 (s, 6H, 30-OCH3, 50-OCH3), 2.82 (t,
The title compound was obtained by 29 which was coupled with
J ¼ 7.5 Hz, 2H, H-3a, H-3b), 2.57 (t, J ¼ 7.5 Hz, 2H, H-2a, H-2b); 13
C
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
NMR (101 MHz, CD3OD)
d
176.9, 149.2, 134.8, 133.0, 106.5, 56.7, 37.1,
(methylthio)benzenesulfonamide (24) through EDCI/HOBt/DMAP
coupling procedure in 76% yield (white amorphous solid) as
32.1; LC-MS (ESI) [M ꢁ H]- m/z 225.4.
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.64 (d, J ¼ 7.0 Hz,
4.1.23. (E)-3-(3, 4-dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)
acrylamide (35a)
2H, H-2000, H-6000), 7.28e7.24 (m, 7H, four of 1-phenyl-H, H-30, H-3000,
H-5000), 7.14 (s, 1H, one of 1-phenyl-H), 7.01 (s, 1H, H-200), 6.91 (d,
J ¼ 7.5 Hz, 1H, H-500), 6.77 (d, J ¼ 7.0 Hz, 1H, H-600), 6.34 (d,
J ¼ 15.5 Hz, 1H, H-20), 4.08 (s, 1H, H-3), 3.90 (s, 1H, H-2), 3.35 (s, 1H,
H-4a), 3.25 (d, J ¼ 14.0 Hz, 1H, H-4b), 3.14e3.10 (m, 1H, isobutyl-
CH2a), 2.85 (dd, J ¼ 13.0, 10.0 Hz, 1H, isobutyl-CH2b), 2.77 (d,
J ¼ 13.5 Hz, 1H, H-1a), 2.67 (t, J ¼ 12.0 Hz, 1H, H-1b), 2.34 (s, 3H, 4000-
SCH3), 2.03 (s, 1H, isobutyl-CH), 0.95 (d, J ¼ 4.5 Hz, 3H, one of
isobutyl-CH3), 0.86 (d, J ¼ 4.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDCI, 0.29 g, 1.5 mmol) and 1-hydroxybenzotriazole (HOBt,
0.15 g, 1.1 mmol) were sequentially added in batches to a stirred
solution of (E)-3-(3, 4-dihydroxyphenyl)acrylic acid (29, 0.18 g,
1.0 mmol) and N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-
isobutyl-4- methoxybenzenesulfonamide (21, 0.43 g, 1.05 mmol) in
3 mL dry DMF at 0 ꢀC under an argon atmosphere. The reaction
mixture was stirred for 10 min at 0 ꢀC and additional 1 h at 25 ꢀC. 4-
Dimethylaminopyridine (DMAP, 0.024 g, 0.20 mmol) was added
and the reaction mixture was stirred for another 2 h at 25 ꢀC. The
solvent was removed under reduced pressure. Water (6 mL) was
added to the residue and extracted with CH2Cl2 (3 ꢂ 6 mL). The
combined organic layers were dried over Na2SO4. Removal of sol-
vent under reduced pressure, followed by column chromatography
over silica gel column. Elution with 5% CH3OH in CH2Cl2 gave
compound 35a (0.40 g, 78%) as white amorphous solid: 1H NMR
(101 MHz, CD3OD)
d 168.9, 148.9, 147.4, 146.8, 142.6, 140.1, 135.3,
130.3, 129.3, 129.0, 128.2, 127.2, 126.3, 122.2, 118.1, 116.6, 115.1, 74.9,
59.4, 55.5, 54.4, 37.1, 28.0, 20.5, 14.4; HRMS (ESI) m/z calcd. for
C
30H35N2O6S2 ([M ꢁ H]-): 583.1937, found 583.1914.
4.1.26. (E)-3-(3, 4-dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isobutyl-6-methoxypyridine)-3-sulfonamido)-1-phenylbutan-2-yl)
acrylamide (35d)
The title compound was obtained by 29 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-6-
(500 MHz, CD3OD)
d
7.69 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000), 7.28e7.21
methoxypyridine-3-sulfonamide (25) through EDCI/HOBt/DMAP
(m, 5H, H-30, four of 1-phenyl-H), 7.14 (t, J ¼ 7.0 Hz, 1H, one of 1-
phenyl-H), 7.01 (s, 1H, H-200), 6.94e6.90 (m, 3H, H-500, H-3000, H-
5000), 6.77 (d, J ¼ 8.0 Hz, 1H, H-600), 6.34 (d, J ¼ 15.0 Hz, 1H, H-20), 4.10
(dd, J ¼ 12.5, 5.0 Hz,1H, H-3), 3.91 (t, J ¼ 7.0 Hz,1H, H-2), 3.70 (s, 3H,
4000-OCH3), 3.33 (d, J ¼ 14.0 Hz, 1H, H-4a), 3.26 (dd, J ¼ 14.0, 2.6 Hz,
coupling procedure in 82% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
8.55 (s, 1H, H-6000),
7.97 (d, J ¼ 8.5 Hz, 1H, H-2000), 7.28e7.24 (m, 5H, H-30, four of 1-
phenyl-H), 7.14 (s, 1H, one of 1-phenyl-H), 6.98 (s, 1H, H-200), 6.88
(d, J ¼ 8.0 Hz, 1H, H-500), 6.82 (d, J ¼ 8.5 Hz, 1H, H-5000), 6.75 (d,
12

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
J ¼ 7.5 Hz, 1H H-600), 6.30 (d, J ¼ 15.5 Hz, 1H, H-20), 4.10 (s, 1H, H-3),
3.89 (s, 3H, 4000-OCH3), 3.86 (s, 1H, H-2), 3.37 (d, J ¼ 15.5 Hz, 1H, H-
4a), 3.21 (d, J ¼ 14.0 Hz,1H, H-4b), 3.15e3.11 (m, 1H, isobutyl-CH2a),
3.04e3.00 (m, 1H, isobutyl-CH2b), 2.86 (dd, J ¼ 13.0, 3.5 Hz, 1H, H-
1a), 2.68 (t, J ¼ 12.0 Hz, 1H, H-1b), 2.02 (s, 1H, isobutyl-CH), 0.93 (d,
J ¼ 5.0 Hz, 3H, one of isobutyl-CH3), 0.87 (d, J ¼ 5.0 Hz, 3H, one of
7.17e7.13 (m, 2H, one of 1-phenyl-H, H-30), 6.96 (brs, 3H, H-3000, H-
5000, H-200), 6.84 (d, J ¼ 7.0 Hz, 1H, H-500), 6.74 (d, J ¼ 7.0 Hz, 1H, H-600),
6.38 (d, J ¼ 15.5 Hz, 1H, H-20), 5.16 (s, 1H, 3-OH), 4.06e3.97 (m, 2H,
H-3, H-2), 3.78 (s, 1H, isopropyl-CH), 3.71 (s, 3H, 4000-OCH3),
3.24e3.16 (m, 2H, H-4a, H-4b), 2.77 (dd, J ¼ 14.5, 7.5 Hz, 1H, H-1a),
2.61 (t, J ¼ 12.5 Hz,1H, H-1b),1.13 (d, J ¼ 4.0 Hz, 3H, one of isopropyl
-CH3), 0.80 (d, J ¼ 4.0 Hz, 3H, one of isopropyl -CH3); 13C NMR
isobutyl-CH3); 13C NMR (101 MHz, CD3OD)
d 169.1, 167.8, 148.9,
148.5, 146.7, 142.6, 140.0, 139.0, 130.3, 130.1, 129.3, 128.2, 127.2,
122.2, 118.0, 116.4, 115.1, 112.3, 74.2, 58.5, 55.6, 54.7, 53.8, 36.9, 27.9,
205, 20.4; HRMS (ESI) m/z calcd. for C29H34N3O7S ([M ꢁ H]-):
568.2117, found 568.2094.
(101 MHz, DMSO‑d6) d 165.6, 162.7, 147.8, 146.0, 140.1, 139.6, 131.8,
129.6, 128.4, 126.8, 126.2, 120.9, 118.9, 116.3, 114.7, 114.2, 74.1, 55.9,
53.8, 50.1, 47.2, 35.6, 22.7, 19.6; HRMS (ESI) m/z calcd. for
C
29H33N2O7S ([M ꢁ H]-): 553.2008, found 553.2015.
4.1.27. (E)-3-(3, 4-dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((4-
methoxy-N-propylphenyl)sulfonamido)-1-phenylbutan-2-yl)
acrylamide (35e)
4.1.30. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (36a)
The title compound was obtained by 29 which was coupled with
The title compound was obtained by (E)-3-(4-hydroxy-3-
methoxyphenyl)acrylic acid (30) which was coupled with N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
methoxybenzenesulfonamide (21) through EDCI/HOBt/DMAP
coupling procedure in 87% yield (white amorphous solid) as
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-N-
propylbenzenesulfonamide (26) through EDCI/HOBt/DMAP
coupling procedure in 80% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.70 (d, J ¼ 7.5 Hz,
2H, H-2000, H-6000), 7.28e7.23 (m, 5H, H-30, four of 1-phenyl-H), 7.14
(s, 1H, one of 1-phenyl-H), 7.00 (s, 1H, H-200), 6.95 (d, J ¼ 7.5 Hz, 2H,
H-3000, H-5000), 6.89 (d, J ¼ 7.5 Hz, 1H, H-500), 6.76 (d, J ¼ 7.0 Hz, 1H, H-
600), 6.33 (d, J ¼ 15.5 Hz, 1H, H-20), 4.14 (s, 1H, H-3), 3.87 (d,
J ¼ 8.0 Hz, 1H, H-2), 3.72 (s, 3H, 4000-OCH3), 3.36 (d, J ¼ 14.5 Hz, 2H,
H-4a, H-4b), 3.24 (d, J ¼ 15.0 Hz, 1H, propyl-1-CH2a), 3.00 (d,
J ¼ 5.5 Hz, 1H, propyl-1-CH2b), 2.90 (dd, J ¼ 13.5, 9.0 Hz, 1H, H-1a),
2.69 (t, J ¼ 12.0 Hz, 1H, H-1b), 1.61e1.54 (m, 2H, propyl-2-CH2a,
propyl-2-CH2b), 0.86 (t, J ¼ 6.0 Hz, 3H, propyl-CH3); 13C NMR
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.69 (d, J ¼ 8.5 Hz,
2H, H-2000, H-6000), 7.32 (d, J ¼ 16.0 Hz, 1H, H-30), 7.24e7.21 (m, 4H,
four of 1-phenyl-H), 7.15e7.11 (m, 2H, one of 1-phenyl-H, H-200),
7.03 (d, J ¼ 8.0 Hz, 1H, H-500), 6.94 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000),
6.80 (d, J ¼ 8.0 Hz, 1H, H-600), 6.39 (d, J ¼ 16.0 Hz, 1H, H-20), 4.11 (dd,
J ¼ 12.0, 5.5 Hz,1H, H-3), 3.90 (d, J ¼ 7.5 Hz, 1H, H-2), 3.87 (s, 3H, 300-
OCH3), 3.72 (s, 3H, 4000-OCH3), 3.34 (d, J ¼ 16.0 Hz, 1H, H-4a),
3.26e3.23 (m, 1H, H-4b), 3.09 (dd, J ¼ 13.5, 9.0 Hz, 1H, isobutyl-
CH2a), 2.86 (dd, J ¼ 15.0, 8.5 Hz, 1H, isobutyl-CH2b), 2.77 (dd,
J ¼ 13.5, 6.0 Hz, 1H, H-1a), 2.68 (dd, J ¼ 13.5, 11.5 Hz, 1H, H-1b),
2.03e2.00 (m, 1H, isobutyl-CH), 0.94 (d, J ¼ 6.5 Hz, 3H, one of
isobutyl-CH3), 0.86 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3b); 13C
(101 MHz, CD3OD)
d 169.0, 164.5, 149.0, 146.8, 142.6, 140.2, 131.8,
130.6, 130.3, 129.3, 128.2, 127.3, 122.2, 118.2, 116.6, 115.4, 115.1, 74.7,
56.1, 55.4, 53.5, 53.2, 36.8, 22.7, 11.5; HRMS (ESI) m/z calcd. for
C
29H33N2O7S ([M ꢁ H]-): 553.2008, found 553.2002.
NMR (126 MHz, CD3OD) d 168.8, 164.5, 150.0, 149.4, 142.4, 140.1,
131.5, 130.7, 130.3, 129.3, 128.2, 127.2, 123.2, 118.5, 116.6, 115.3, 111.6,
74.8, 59.3, 56.4, 56.1, 55.5, 54.4, 37.0, 28.0, 20.5; HRMS (ESI) m/z
calcd. for C31H37N2O7S ([M ꢁ H]-): 581.2316, found 581.2337.
4.1.28. (E)-N-((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(3, 4-
dihydroxyphenyl)acrylamide (35f)
The title compound was obtained by 29 which was coupled with
N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-cyclopropyl-4-
methoxybenzenesulfonamide (27) through EDCI/HOBt/DMAP
coupling procedure in 75% yield (white amorphous solid) as
4.1.31. (E)-N-((2S, 3R)-4-((4-amino-N-isobutylphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (36b)
The title compound was obtained by 30 which was coupled with
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.74 (d, J ¼ 7.5 Hz,
4-amino-N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-iso-
2H, H-2000, H-6000), 7.29e7.23 (m, 5H, H-30, four of 1-phenyl-H), 7.14
(s, 1H, one of 1-phenyl-H), 7.00 (s, 1H, H-200), 6.97 (d, J ¼ 7.5 Hz, 2H,
H-3000, H-5000), 6.91 (d, J ¼ 7.5 Hz, 1H, H-500), 6.77 (d, J ¼ 7.0 Hz, 1H, H-
600), 6.35 (d, J ¼ 15.5 Hz, 1H, H-20), 4.14 (s, 1H, H-3), 3.99 (s, 1H, H-2),
3.73 (s, 3H, 4000-OCH3), 3.37 (d, J ¼ 20.0 Hz, 2H, H-4a, H-4b), 3.26 (s,
1H, cyclopropyl-CH), 2.97e2.92 (m, 1H, H-1a), 2.69 (t, J ¼ 12.0 Hz,
1H, H-1b), 1.03 (s, 1H, cyclopropyl-CH2a), 0.96 (s, 1H, cyclopropyl-
CH2a’), 0.77 (s, 1H, cyclopropyl-CH2b), 0.62 (s, 1H, cyclopropyl-
butylbenzenesulfonamide (23) through EDCI/HOBt/DMAP coupling
procedure in 80% yield (white amorphous solid) as described for
35a: 1H NMR (500 MHz, CD3OD)
d
7.46 (d, J ¼ 8.5 Hz, 2H, H-2000, H-
6000), 7.34 (d, J ¼ 16.0 Hz, 1H, H-30), 7.27e7.20 (m, 4H, four of 1-
phenyl-H), 7.13 (t, J ¼ 6.5 Hz, 1H, one of 1-phenyl-H), 7.07 (s, 1H,
H-200), 7.00 (d, J ¼ 8.0 Hz, 1H, H-500), 6.79 (d, J ¼ 8.0 Hz, 1H, H-6000),
6.63 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.39 (d, J ¼ 16.0 Hz, 1H, H-2 ),
4.20e4.13 (m, 1H, H-3), 3.92e3.89 (m, 1H, H-2), 3.84 (s, 3H, 300-
OCH3), 3.38 (d, J ¼ 13.5 Hz, 1H, H-4a), 3.24 (d, J ¼ 13.5 Hz, 1H, H-4b),
3.01 (dd, J ¼ 13.5, 8.5 Hz, 1H, isobutyl-CH2a), 2.90 (dd, J ¼ 15.0,
8.5 Hz, 1H, isobutyl-CH2b), 2.78e2.68 (m, 2H, H-1a, H-1b),
2.03e1.97 (m, 1H, isobutyl-CH), 0.92 (d, J ¼ 6.5 Hz, 3H, one of iso-
butyl-CH3), 0.86 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
CH2b’); 13C NMR (101 MHz, CD3OD)
d 168.9, 164.7, 148.9, 146.8,
142.5,140.2,131.2, 130.3,129.7, 129.3,128.2,127.2,122.1,118.2,116.5,
115.3 115.1, 74.0, 56.7, 56.1, 55.2, 36.7, 33.8, 9.38, 7.37; HRMS (ESI) m/
z calcd. for C29H31N2O7S ([M ꢁ H]-): 551.1852, found 551.1852.
4.1.29. (E)-3-(3, 4-dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isopropyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)
acrylamide (35g)
(126 MHz, CD3OD) d 168.4, 153.7, 149.4, 148.8, 141.9, 139.7, 130.1,
129.9, 128.8, 127.8, 126.8, 125.3, 122.8, 118.2, 116.1, 114.0, 111.1, 74.3,
59.0, 56.0, 55.1, 54.1, 36.4, 27.7, 20.2, 20.1; HRMS (ESI) m/z calcd. for
The title compound was obtained by 29 which was coupled with
C
30H36N3O6S ([M ꢁ H]-): 566.2319, found 566.2300.
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isopropyl-4-
methoxybenzenesulfonamide (28) through EDCI/HOBt/DMAP
4.1.32. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-4-(methylthio)phenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (36c)
coupling procedure in 79% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d
9.34 (s, 1H, 30-
OH), 9.13 (s, 1H, 40-OH), 8.04 (d, J ¼ 8.0 Hz, 1H, 2-NH), 7.70 (d,
The title compound was obtained by 30 which was coupled with
J ¼ 7.6 Hz, 2H, H-2000, H-6000), 7.26e7.23 (m, 4H, four of 1-phenyl-H),
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
13

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
(methylthio)benzenesulfonamide (24) through EDCI/HOBt/DMAP
HRMS (ESI) m/z calcd. for C30H35N2O7S ([M ꢁ H]-): 567.2165, found
coupling procedure in 85% yield (white amorphous solid) as
567.2161.
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d 7.98 (d,
J ¼ 9.0 Hz, 1H, 400-OH), 7.67 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000), 7.33 (d,
J ¼ 8.5 Hz, 2H, H-3000, H-5000), 7.27e7.24 (m, 5H, H-30, four of 1-
phenyl-H), 7.17 (dd, J ¼ 8.5, 4.0 Hz, 1H, one of 1-phenyl-H), 7.14
(s, 1H, H-200), 7.02 (d, J ¼ 8.0 Hz, 1H, H-500), 6.83 (d, J ¼ 8.0 Hz, 1H, H-
600), 6.48 (d, J ¼ 16.0 Hz, 1H, H-20), 5.19 (d, J ¼ 6.0 Hz, 1H, 3-OH),
4.03e3.98 (m,1H, H-3), 3.84 (s, 3H, 300-OCH3), 3.73 (d, J ¼ 4.0 Hz,1H,
H-2), 3.33 (s, 1H, H-4a), 3.13 (d, J ¼ 12.0 Hz, 1H, H-4b), 3.08 (dd,
J ¼ 13.5, 9.0 Hz, 1H, isobutyl-CH2a), 2.79 (dd, J ¼ 15.0, 8.5 Hz, 1H,
isobutyl-CH2b), 2.73 (dd, J ¼ 13.5, 6.0 Hz, 1H, H-1a), 2.62 (dd,
J ¼ 13.5, 11.5 Hz, 1H, H-1b), 2.44 (s, 3H, 4000-SCH3), 2.04e1.99 (m, 1H,
isobutyl-CH), 0.90 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.82 (d,
J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR (151 MHz, DMSO‑d6)
4.1.35. (E)-N-((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (36f)
The title compound was obtained by 30 which was coupled with
N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-cyclopropyl-4-
methoxybenzenesulfonamide (27) through EDCI/HOBt/DMAP
coupling procedure in 79% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.79 (d, J ¼ 8.5 Hz,
2H, H-2000, H-6000), 7.37 (d, J ¼ 16.0 Hz, 1H, H-30), 7.32 (d, J ¼ 7.5 Hz,
2H, two of 1-phenyl-H), 7.27 (t, J ¼ 7.5 Hz, 2H, two of 1-phenyl-H),
7.18 (t, J ¼ 7.5 Hz, 1H, one of 1-phenyl-H), 7.15 (s, 1H, H-200), 7.07 (d,
J ¼ 8.0 Hz, 1H, H-500), 7.02 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.84 (d,
J ¼ 8.0 Hz, 1H, H-600), 6.46 (d, J ¼ 16.0 Hz, 1H, H-20), 4.23e4.19 (m,
1H, H-3), 4.06e4.03 (m, 1H, H-2), 3.92 (s, 3H, 300-OCH3), 3.79 (s, 3H,
4000-OCH3), 3.44 (dd, J ¼ 14.5, 3.5 Hz, 1H, H-4a), 3.32 (dd, J ¼ 14.5,
3.0 Hz, 1H, H-4b), 3.02 (s, 1H, cyclopropyl-CH), 3.01 (dd, J ¼ 14.5,
8.5 Hz,1H, H-1a), 2.74 (dd, J ¼ 14.0,11.0 Hz,1H, H-1b),1.08e1.05 (m,
1H, cyclopropyl-CH2a), 1.01e0.94 (m, 1H, cyclopropyl-CH2a’),
0.83e0.78 (m, 1H, cyclopropyl-CH2b), 0.69e0.64 (m, 1H, cyclo-
d
165.6, 148.8, 148.3, 145.2, 140.0, 139.6, 134.8, 129.5, 128.5, 128.1,
126.8, 126.3, 125.6, 122.0, 119.3, 116.2, 111.2, 73.1, 57.5, 56.0, 53.9,
53.1, 35.6, 26.6, 20.4, 14.2; HRMS (ESI) m/z calcd. for C31H37N2O6S2
([M ꢁ H]-): 597.2093, found 597.2091.
4.1.33. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-6-methoxypyridine)-3-sulfonamido)-1-
phenylbutan-2-yl)acrylamide (36d)
propyl-CH2b’); 13C NMR (151 MHz, DMSO‑d6)
d 165.0, 162.4, 148.2,
The title compound was obtained by 30 which was coupled with
147.7, 139.5, 138.9, 129.6, 128.9, 128.4, 127.8, 126.3, 125.6, 121.4,
118.8, 115.6, 114.2, 110.6, 71.3, 55.4, 54.8, 52.9, 34.4, 32.0, 8.0, 6.1;
HRMS (ESI) m/z calcd. for C30H33N2O7S ([M ꢁ H]-): 565.2008, found
565.1999.
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-6-
methoxypyridine-3-sulfonamide (25) through EDCI/HOBt/DMAP
coupling procedure in 85% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
8.60 (d, J ¼ 2.0 Hz,
1H, H-6000), 8.02 (dd, J ¼ 9.0, 2.0 Hz, 1H, H-2000), 7.37 (d, J ¼ 15.5 Hz,
1H, H-30), 7.32e7.26 (m, 4H, four of 1-phenyl-H), 7.19 (t, J ¼ 6.5 Hz,
1H, one of 1-phenyl-H), 7.14 (s, 1H, H-200), 7.06 (d, J ¼ 8.0 Hz, 1H, H-
500), 6.87 (d, J ¼ 8.5 Hz, 1H, H-5000), 6.83 (d, J ¼ 8.0 Hz, 1H, H-600), 6.41
(d, J ¼ 15.5 Hz, 1H, H-20), 4.17e4.13 (m, 1H, H-3), 3.94 (s, 3H, 300-
OCH3), 3.92 (s, 3H, 4000-OCH3), 3.90 (d, J ¼ 8.0 Hz, 1H, H-2), 3.42 (dd,
J ¼ 15.0, 2.0 Hz, 1H, H-4a), 3.26 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-4b), 3.18
(dd, J ¼ 13.5, 8.5 Hz, 1H, isobutyl-CH2a), 3.07 (dd, J ¼ 15.0, 9.0 Hz,
1H, isobutyl-CH2b), 2.92 (dd, J ¼ 13.5, 6.5 Hz, 1H, H-1a), 2.73 (dd,
J ¼ 14.0, 11.0 Hz, 1H, H-1b), 2.13e2.05 (m, 1H, isobutyl-CH), 0.98 (d,
J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.91 (d, J ¼ 6.5 Hz, 3H, one of
4.1.36. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isopropyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (36g)
The title compound was obtained by 30 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isopropyl-4-
methoxybenzenesulfonamide (28) through EDCI/HOBt/DMAP
coupling procedure in 79% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.77 (d, J ¼ 8.5 Hz,
2H, H-2000, H-6000), 7.39 (d, J ¼ 16.0 Hz, 1H, H-30), 7.33 (d, J ¼ 7.5 Hz,
2H, two of 1-phenyl-H), 7.28 (t, J ¼ 7.5 Hz, 2H, two of 1-phenyl-H),
7.19 (d, J ¼ 7.0 Hz, 1H, one of 1-phenyl-H), 7.16 (s, 1H, H-200), 7.08 (d,
J ¼ 8.0 Hz, 1H, H-500), 6.97 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.84 (d,
J ¼ 8.0 Hz, 1H, H-600), 6.48 (d, J ¼ 16.0 Hz, 1H, H-20), 4.24e4.20 (m,
1H, H-3), 4.15e4.09 (m, 1H, H-2), 4.01 (t, J ¼ 7.0 Hz, 1H, isopropyl-
CH), 3.91 (s, 3H, 300-OCH3), 3.76 (s, 3H, 4000-OCH3), 3.39 (d, J ¼ 3.0 Hz,
1H, H-4a), 3.31 (d, J ¼ 2.0 Hz, 1H, H-4b), 3.02e2.96 (m, 1H, H-1a),
2.75 (dd, J ¼ 14.0, 11.0 Hz, 1H, H-1b), 1.20 (d, J ¼ 6.5 Hz, 3H, one of
isobutyl-CH3); 13C NMR (151 MHz, DMSO‑d6)
d 165.5, 165.1, 148.2,
147.7, 146.7, 139.3, 139.0, 138.0, 128.9, 127.9, 126.3, 125.7, 121.5, 118.7,
115.6, 111.0, 110.6, 71.8, 56.0, 55.4, 53.9, 53.4, 51.9, 35.0, 25.9, 19.8;
HRMS (ESI) m/z calcd. for C30H36N3O7S ([M ꢁ H]-): 582.2274, found
582.2262.
4.1.34. (E)-3-(4-hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((4-methoxy-N-propylphenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (36e)
isopropyl -CH3), 0.94 (d, J ¼ 6.5 Hz, 3H, one of isopropyl -CH3); 13
C
NMR (151 MHz, CD3OD)
d 168.9, 164.5, 150.1, 149.4, 142.5, 140.3,
132.7, 130.6, 130.4, 129.3, 128.2, 127.2, 123.3, 118.6, 116.6, 115.4, 111.7,
75.7, 56.5, 56.1, 55.5, 51.7, 48.3, 37.2, 22.5, 20.1; HRMS (ESI) m/z
calcd. for C30H35N2O7S ([M ꢁ H]-): 567.2165, found 567.2160.
The title compound was obtained by 30 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-N-
propylbenzenesulfonamide (26) through EDCI/HOBt/DMAP
coupling procedure in 82% yield (white amorphous solid) as
4.1.37. (E)-3-(4-hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (37a)
The title compound was obtained by (E)-3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylic acid (31) which was coupled with N-
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.76 (d, J ¼ 9.0 Hz,
2H, H-2000, H-6000), 7.37 (d, J ¼ 16.0 Hz, 1H, H-30), 7.32e7.26 (m, 4H,
four of 1-phenyl-H), 7.19 (t, J ¼ 7.0 Hz, 1H, one of 1-phenyl-H), 7.15
(s, 1H, H-200), 7.07 (d, J ¼ 8.0 Hz, 1H, H-500), 7.01 (d, J ¼ 9.0 Hz, 2H, H-
3000, H-5000), 6.84 (d, J ¼ 8.0 Hz, 1H, H-600), 6.44 (d, J ¼ 16.0 Hz, 1H, H-
20), 4.22e4.18 (m, 1H, H-3), 3.94e3.89 (m, 4H, H-2, 300-OCH3), 3.79
(s, 3H, 4000-OCH3), 3.43 (dd, J ¼ 15.0, 3.0 Hz, 1H, H-4a), 3.32e3.27 (m,
2H, H-4b, propyl-1-CH2a), 3.08e3.03 (m, 1H, propyl-1-CH2b), 2.97
(dd, J ¼ 15.0, 8.5 Hz, 1H, H-1a), 2.75 (dd, J ¼ 14.0, 11.0 Hz, 1H, H-1b),
1.70e1.58 (m, 2H, propyl-2-CH2a, propyl-2-CH2b), 0.90 (t, J ¼ 7.5 Hz,
((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
methoxybenzenesulfonamide (21) through EDCI/HOBt/DMAP
coupling procedure in 83% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d
8.80 (s, 1H, 400-
OH), 7.93 (d, J ¼ 8.5 Hz, 1H, 2-NH), 7.69 (d, J ¼ 7.5 Hz, 2H, H-2000, H-
6000), 7.32e7.24 (m, 5H, H-30, four of 1-phenyl-H), 7.14 (s, 1H, one of
1-phenyl-H), 7.00 (d, J ¼ 7.5 Hz, 2H, H-3000, H-5000), 6.85 (s, 2H, H-200,
H-600), 6.49 (d, J ¼ 15.5 Hz, 1H, H-20), 5.15 (d, J ¼ 5.0 Hz, 1H, 3-OH),
4.01 (d, J ¼ 4.5 Hz, 1H, H-3), 3.80 (s, 6H, 300-OCH3, 500-OCH3), 3.73 (s,
3H, propyl-CH3); 13C NMR (151 MHz, DMSO‑d6)
d 165.1, 162.3, 148.3,
147.8, 139.5, 139.0, 130.6, 129.2, 129.1, 128.0, 126.3, 125.8, 121.6,
118.9, 115.7, 114.3, 110.7, 72.4, 55.5, 53.3, 51.6, 50.9, 34.8, 21.0, 11.1;
14

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
4H, H-2, 4000-OCH3), 3.32 (d, J ¼ 12.5 Hz, 1H, H-4a), 3.11 (d,
J ¼ 13.5 Hz, 1H, H-4b), 3.06e3.01 (m, 1H, isobutyl-CH2a), 2.77e2.73
(m, 1H, isobutyl-CH2b), 2.70e2.64 (m, 1H, H-1a), 2.60 (t, J ¼ 12.0 Hz,
1H, H-1b), 1.99 (s, 1H, isobutyl-CH), 0.87 (d, J ¼ 4.5 Hz, 3H, one of
isobutyl-CH3), 0.80 (d, J ¼ 4.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
J ¼ 9.0 Hz, 1H, H-2000), 7.27e7.24 (m, 5H, H-30, four of 1-phenyl-H),
7.17 (d, J ¼ 4.0 Hz, 1H, one of 1-phenyl-H), 6.96 (d, J ¼ 8.5 Hz, 1H, H-
5000), 6.85 (s, 2H, H-200, H-600), 6.47 (d, J ¼ 16.0 Hz, 1H, H-20), 5.17 (d,
J ¼ 6.5 Hz,1H, 3-OH), 4.04e4.02 (m, 1H, H-3), 3.92 (s, 3H, 4000-OCH3),
3.82 (s, 6H, 300-OCH3, 500-OCH3), 3.69 (d, J ¼ 7.0 Hz, 1H, H-2), 3.39 (s,
1H, H-4a), 3.14e3.06 (m, 2H, H-4b, isobutyl-CH2a), 2.97 (dd,
J ¼ 15.0, 8.5 Hz, 1H, isobutyl-CH2b), 2.83 (dd, J ¼ 13.5, 6.0 Hz, 1H, H-
1a), 2.65e2.60 (m, 1H, H-1b), 2.03e1.99 (m, 1H, isobutyl-CH), 0.89
(d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.83 (d, J ¼ 6.5 Hz, 3H, one of
(101 MHz, DMSO‑d6)
d 165.5, 162.7, 148.6, 140.0, 139.8, 137.8, 130.7,
129.8, 129.5, 128.5, 126.2, 125.7, 119.7, 114.7, 105.6, 73.1, 57.6, 56.4,
55.9, 53.8, 53.2, 36.2, 35.5, 26.7, 20.5; HRMS (ESI) m/z calcd. for
C
32H39N2O8S ([M ꢁ H]-): 611.2422, found 611.2398.
isobutyl-CH3); 3C NMR (151 MHz, DMSO‑d6)
d 166.1, 165.6, 148.5,
4.1.38. (E)-N-((2S, 3R)-4-((4-amino-N-isobutylphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylamide (37b)
147.3, 139.9, 138.5, 137.8, 129.5, 128.5, 126.3, 125.7, 119.7, 111.5, 105.7,
72.4, 56.5, 56.4, 54.5, 53.9, 52.4, 35.5, 26.5, 20.4, 20.3; HRMS (ESI)
m/z calcd. for C31H38N3O8S ([M ꢁ H]-): 612.2380, found 612.2379.
The title compound was obtained by 31 which was coupled with
4-amino-N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-iso-
4.1.41. (E)-3-(4-hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((4-methoxy-N-propylphenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (37e)
butylbenzenesulfonamide (23) through EDCI/HOBt/DMAP coupling
procedure in 75% yield (white amorphous solid) as described for
35a: 1H NMR (500 MHz, CD3OD)
d
7.46 (d, J ¼ 8.5 Hz, 2H, H-2000, H-
The title compound was obtained by 31 which was coupled with
6000), 7.32 (d, J ¼ 15.5 Hz, 1H, H-30), 7.26e7.19 (m, 4H, four of 1-
phenyl-H), 7.11 (t, J ¼ 7.0 Hz, 1H, one of 1-phenyl-H), 6.78 (s, 2H,
H-200, H-600), 6.62 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.41 (d, J ¼ 15.5 Hz,
1H, H-20), 4.19 (m, 1H, H-3), 3.92 (m, 1H, H-2), 3.81 (s, 6H, 300-OCH3,
500-OCH3), 3.38 (d, J ¼ 12.0 Hz, 1H, H-4a), 3.24 (d, J ¼ 12.0 Hz, 1H, H-
4b), 3.00 (dd, J ¼ 13.5, 8.5 Hz, 1H, isobutyl-CH2a), 2.90 (dd, J ¼ 15.0,
8.5 Hz, 1H, isobutyl-CH2b), 2.78e2.68 (m, 2H, H-1a, H-1b),
2.03e1.97 (m, 1H, isobutyl-CH), 0.91 (d, J ¼ 6.5 Hz, 3H, one of iso-
butyl-CH3), 0.85 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-N-
propylbenzenesulfonamide (26) through EDCI/HOBt/DMAP
coupling procedure in 84% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d 7.96 (d,
J ¼ 9.0 Hz, 1H, 400-OH), 7.73 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000),
7.28e7.25 (m, 5H, H-30, four of 1-phenyl-H), 7.21e7.15 (m,1H, one of
1-phenyl-H), 7.06 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.87 (s, 2H, H-200,
H-600), 6.51 (d, J ¼ 16.0 Hz, 1H, H-20), 5.23 (d, J ¼ 6.5 Hz, 1H, 3-OH),
4.07 (d, J ¼ 8.0 Hz, 1H, H-3), 3.82 (s, 6H, 300-OCH3, 500-OCH3), 3.79 (s,
3H, 4000-OCH3), 3.73 (brs, 1H, H-2), 3.38 (brs, 1H, H-4a), 3.27e3.21
(m, 1H, H-4b), 3.12 (d, J ¼ 12.0 Hz, 1H, propyl-1-CH2a), 2.95 (dt,
J ¼ 14.0, 7.0 Hz, 1H, propyl-1-CH2b), 2.83 (dd, J ¼ 14.5, 8.0 Hz, 1H, H-
1a), 2.64 (dd, J ¼ 13.5, 11.5 Hz, 1H, H-1b), 1.56e1.52 (m, 2H, propyl-
2-CH2a, propyl-2-CH2b), 0.81 (t, J ¼ 7.5 Hz, 3H, propyl-CH3); 3C NMR
(151 MHz, CD3OD)
129.8, 128.8, 126.7, 126.6, 125.2, 118.4, 114.0, 105.9, 74.2, 58.8, 56.3,
d 168.2, 153.6, 148.9, 142.0, 139.6, 138.4, 130.0,
54.9, 53.9, 36.3, 27.6, 20.1, 20.0; HRMS (ESI) m/z calcd. for
C
31H38N3O7S ([M ꢁ H]-): 596.2425, found 596.2426.
4.1.39. (E)-3-(4-hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-4-(methylthio)phenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (37c)
(151 MHz, DMSO‑d6) d 165.0, 162.2, 148.1, 139.5, 139.3, 137.3, 130.6,
129.2, 129.0, 128.0, 125.8, 125.2, 119.3, 114.3, 105.2, 72.4, 55.9, 55.5,
53.2, 51.6, 50.9, 34.7, 21.0, 11.1; HRMS (ESI) m/z calcd. for
The title compound was obtained by 31 which was coupled with
C
30H35N2O7S ([M ꢁ H]-): 567.2165, found 567.2175.
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
(methylthio)benzenesulfonamide (24) through EDCI/HOBt/DMAP
4.1.42. (E)-N-((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3, 5-
dimethoxyphenyl)acrylamide (37f)
coupling procedure in 85% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d 7.97 (d,
J ¼ 9.0 Hz, 1H, 400-OH), 7.67 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000), 7.33 (d,
J ¼ 8.5 Hz, 2H, H-3000, H-5000), 7.28e7.25 (m, 5H, H-30, four of 1-
phenyl-H), 7.18e7.16 (m, 1H, one of 1-phenyl-H), 6.87 (s, 2H, H-
200, H-600), 6.51 (d, J ¼ 16.0 Hz, 1H, H-20), 5.20 (d, J ¼ 4.5 Hz, 1H, 3-
OH), 4.03e4.01 (m, 1H, H-3), 3.82 (s, 6H, 300-OCH3, 500-OCH3), 3.73
(brs, 1H, H-2), 3.13 (d, J ¼ 12.0 Hz, 1H, H-4a), 3.10e3.05 (m, 2H, H-
4b, isobutyl-CH2a), 2.79 (dd, J ¼ 15.0, 8.5 Hz, 1H, isobutyl-CH2b),
2.73 (dd, J ¼ 14.0, 5.5 Hz,1H, H-1a), 2.62 (dd, J ¼ 13.5, 11.5 Hz, 1H, H-
1b), 2.44 (s, 3H, 4000-SCH3), 2.05e1.99 (m, 1H, isobutyl-CH), 0.90 (d,
J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.82 (d, J ¼ 6.5 Hz, 3H, one of
The title compound was obtained by 31 which was coupled with
N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-cyclopropyl-4-
methoxybenzenesulfonamide (27) through EDCI/HOBt/DMAP
coupling procedure in 78% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d 7.99 (d,
J ¼ 9.0 Hz, 1H, 400-OH), 7.75 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000),
7.30e7.26 (m, 5H, H-30, four of 1-phenyl-H), 7.18e7.14 (m,1H, one of
1-phenyl-H), 7.08 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.87 (s, 2H, H-200,
H-600), 6.53 (d, J ¼ 16.0 Hz,1H, H-20), 5.24 (s,1H, 3-OH), 4.06 (brs,1H,
H-3), 3.87 (brs, 1H, H-2), 3.82 (s, 6H, 300-OCH3, 500-OCH3), 3.79 (s, 3H,
4000-OCH3), 3.40 (d, J ¼ 4.0 Hz, 1H, H-4a), 3.14 (d, J ¼ 12.5 Hz, 1H, H-
4b), 2.83 (dd, J ¼ 14.0, 8.0 Hz, 1H, H-1a), 2.67e2.62 (m, 1H, H-1b),
1.97 (brs, 1H, cyclopropyl-CH), 1.03e0.97 (m, 2H, cyclopropyl-CH2a,
cyclopropyl-CH2a’), 0.76 (d, J ¼ 7.0 Hz, 1H, cyclopropyl-CH2b), 0.59
(d, J ¼ 7.0 Hz, 1H, cyclopropyl-CH2b’); 13C NMR (151 MHz, DMSO‑d6)
isobutyl-CH3); 13C NMR (151 MHz, DMSO‑d6)
d 165.5, 148.6, 145.2,
140.0, 139.9, 137.9, 134.8, 129.5, 128.5, 128.1, 126.3, 125.7, 125.6,
119.7, 105.7, 73.1, 57.5, 56.4, 53.9, 53.1, 35.6, 26.6, 20.4, 14.2; HRMS
(ESI) m/z calcd. for C32H39N2O7S2 ([M ꢁ H]-): 627.2199, found
627.2200.
d
165.0, 162.5, 148.1, 139.6, 139.3, 137.3, 129.7, 129.0, 128.6, 127.9,
4.1.40. (E)-3-(4-hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-6-methoxypyridine)-3-sulfonamido)-1-
phenylbutan-2-yl)acrylamide (37d)
125.7,125.2,119.3,114.3,105.2, 71.4, 55.9, 55.5, 54.8, 53.0, 34.5, 32.0,
8.07, 6.15; HRMS (ESI) m/z calcd. for C31H35N2O8S ([M ꢁ H]-):
595.2114, found 595.2103.
The title compound was obtained by 31 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-6-
4.1.43. (E)-3-(4-hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isopropyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)acrylamide (37g)
methoxypyridine-3-sulfonamide (25) through EDCI/HOBt/DMAP
coupling procedure in 82% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d
8.81 (s, 1H, 400-
The title compound was obtained by 31 which was coupled with
OH), 8.60 (s, 1H, H-6000), 8.06 (d, J ¼ 8.5 Hz, 1H, 2-NH), 7.94 (d,
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isopropyl-4-
15

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
methoxybenzenesulfonamide (28) through EDCI/HOBt/DMAP
4.1.46. 3-(3, 4-Dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isobutyl-4-(methylthio)phenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (38c)
coupling procedure in 79% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d 8.01 (d,
J ¼ 9.0 Hz,1H, 400-OH), 7.74 (d, J ¼ 8.5 Hz, 2H, H-2000, H-6000), 7.29e7.25
(m, 5H, H-30, four of 1-phenyl-H), 7.17 (t, J ¼ 7.0 Hz, 1H, one of 1-
phenyl-H), 7.01 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.88 (s, 2H, H-200,
H-600), 6.55 (d, J ¼ 16.0 Hz, 1H, H-20), 5.19 (d, J ¼ 6.0 Hz, 1H, 3-OH),
4.10 (d, J ¼ 8.5 Hz, 1H, H-3), 3.98 (dt, J ¼ 13.0, 6.5 Hz, 1H, H-2), 3.82
(s, 7H, isopropyl-CH, 300-OCH3, 500-OCH3), 3.75 (s, 3H, 4000-OCH3),
3.28 (dd, J ¼ 15.0, 2.5 Hz, 1H, H-4a), 3.20 (d, J ¼ 12.5 Hz, 1H, H-4b),
2.83 (dd, J ¼ 15.0, 8.0 Hz, 1H, H-1a), 2.66 (dd, J ¼ 13.5, 11.5 Hz,1H, H-
1b), 1.15 (d, J ¼ 6.5 Hz, 3H, one of isopropyl-CH3), 0.84 (d, J ¼ 6.5 Hz,
The title compound was obtained by 32 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
(methylthio)benzenesulfonamide (24) through EDCI/HOBt/DMAP
coupling procedure in 78% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.73 (d, J ¼ 8.5 Hz,
2H, H-2000, H-6000), 7.41 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 7.27 (t,
J ¼ 7.5 Hz, 2H, two of 1-phenyl-H), 7.23e7.18 (m, 3H, three of 1-
phenyl-H), 6.63 (d, J ¼ 8.0 Hz, 1H, H-500), 6.61 (s, 1H, H-200), 6.44
(d, J ¼ 7.0 Hz, 1H, H-600), 4.06e4.02 (m, 1H, H-3), 3.80e3.77 (m, 1H,
H-2), 3.32 (d, J ¼ 2.5 Hz, 1H, H-4a), 3.16 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-
4b), 3.05 (dd, J ¼ 13.5, 8.0 Hz, 1H, isobutyl-CH2a), 2.99 (dd, J ¼ 15.0,
8.5 Hz, 1H, isobutyl-CH2b), 2.92 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-1a),
2.67e2.62 (m, 1H, H-1b), 2.58 (dd, J ¼ 13.5, 6.5 Hz, 1H, H-30a), 2.54
(s, 3H, 4000-SCH3), 2.51 (d, J ¼ 8.5 Hz, 1H, H-30b), 2.34e2.28 (m, 2H,
H-20), 2.04e1.99 (m, 1H, isobutyl-CH), 0.94 (d, J ¼ 6.5 Hz, 3H, one of
isobutyl-CH3), 0.90 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
3H, one of isopropyl-CH3); 13C NMR (151 MHz, DMSO‑d6)
d 165.0,
162.2,148.1, 139.6,139.3,137.3, 131.5, 129.1, 129.0,127.9, 125.7,125.2,
119.3, 114.2, 105.2, 73.4, 55.9, 55.4, 53.2, 49.6, 46.7, 35.0, 22.1, 19.1;
HRMS (ESI) m/z calcd. for C31H37N2O8S ([M ꢁ H]-): 597.2271, found
597.2298.
4.1.44. 3-(3, 4-Dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (38a)
The title compound was obtained by 3-(3, 4-dihydroxyphenyl)
propanoic acid (32) which was coupled with N-((2R, 3S)-3-amino-
(151 MHz, CD3OD) d 175.3, 147.4, 146.2, 144.6, 140.0, 136.2, 133.8,
130.4 129.3, 128.9, 127.3, 126.5, 120.5, 116.4, 74.0, 58.9, 55.4, 54.1,
39.4, 36.7, 32.4, 28.1, 20.6, 20.5, 14.7; HRMS (ESI) m/z calcd. for
C
30H37N2O6S2 ([M ꢁ H]-): 585.2093, found 585.2079.
2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
ybenzenesulfonamide (21) through EDCI/HOBt/DMAP coupling
procedure in 77% yield (white amorphous solid) as described for
methox-
4.1.47. 3-(3, 4-Dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isobutyl-6-methoxypyridine)-3-sulfonamido)-1-phenylbutan-2-yl)
propanamide (38d)
35a: 1H NMR (500 MHz, CD3OD)
d
7.78 (d, J ¼ 8.5 Hz, 2H, H-2000, H-
The title compound was obtained by 32 which was coupled with
6000), 7.28 (t, J ¼ 7.5 Hz, 2H, two of 1-phenyl-H), 7.24e7.19 (m, 3H,
three of 1-phenyl-H), 7.10 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 6.63 (d,
J ¼ 8.0 Hz, 1H, H-500), 6.61 (s, 1H, H-200), 6.44 (d, J ¼ 8.0 Hz, 1H, H-600),
4.07e4.03 (m,1H, H-3), 3.89 (s, 3H, 4000-OCH3), 3.79 (t, J ¼ 6.0 Hz,1H,
H-2), 3.31 (d, J ¼ 2.5 Hz, 1H, H-4a), 3.17 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-
4b), 3.04 (dd, J ¼ 13.5, 8.0 Hz, 1H, isobutyl-CH2a), 2.97 (dd, J ¼ 15.0,
8.5 Hz, 1H, isobutyl-CH2b), 2.90 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-1a), 2.65
(dd, J ¼ 13.5, 11.0 Hz, 1H, H-1b), 2.60e2.50 (m, 2H, H-30), 2.36e2.25
(m, 2H, H-20), 2.05e1.97 (m,1H, isobutyl-CH), 0.94 (d, J ¼ 6.5 Hz, 3H,
one of isobutyl-CH3), 0.90 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3);
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-6-
methoxypyridine-3-sulfonamide (25) through EDCI/HOBt/DMAP
coupling procedure in 80% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
8.57 (d, J ¼ 2.0 Hz,
1H, H-6000), 7.99 (dd, J ¼ 9.0, 2.0 Hz, 1H, H-2000), 7.23 (t, J ¼ 7.5 Hz, 2H,
two of 1-phenyl-H), 7.18e7.14 (m, 3H, three of 1-phenyl-H), 6.90 (d,
J ¼ 9.0 Hz,1H, H-5000), 6.59 (d, J ¼ 8.0 Hz,1H, H-500), 6.57 (s,1H, H-200),
6.40 (d, J ¼ 8.0 Hz, 1H, H-600), 3.99 (d, J ¼ 11.0 Hz, 1H, H-3), 3.96 (s,
3H, 4000-OCH3), 3.75e3.72 (m, 1H, H-2), 3.27 (d, J ¼ 2.0 Hz, 1H, H-4a),
3.12 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-4b), 3.07e3.00 (m, 2H, isobutyl-
CH2a, isobutyl-CH2b), 2.89 (dd, J ¼ 13.5, 6.5 Hz, 1H, H-1a),
2.64e2.53 (m, 2H, H-30), 2.49 (dt, J ¼ 14.1, 7.8 Hz, 1H, H-1b),
2.52e2.46 (m, 1H, H-30b), 2.33e2.21 (m, 2H, H-20), 2.02e1.95 (m,
1H, isobutyl-CH), 0.91 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.86
(d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR (151 MHz, CD3OD)
13C NMR (151 MHz, CD3OD)
d 175.4, 164.7, 146.4, 144.8, 140.2, 133.9,
132.2, 130.8, 130.6, 129.4, 127.4, 120.6, 116.6, 116.5, 115.6, 74.2, 59.2,
56.4, 55.5, 54.3, 39.5, 36.8, 32.5, 28.3, 20.7, 20.6; HRMS (ESI) m/z
calcd. for C30H37N2O7S ([M ꢁ H]-): 569.2321, found 569.2316.
d
175.3, 167.9, 148.5, 146.2, 144.6, 140.0, 139.1, 133.8, 130.5, 130.4,
4.1.45. N-((2S, 3R)-4-((4-amino-N-isobutylphenyl)sulfonamido)-3-
hydroxy-1-phenylbutan-2-yl)-3-(3, 4-dihydroxyphenyl)
propanamide (38b)
129.3, 127.3, 120.5, 116.5, 116.4, 112.5, 73.6, 58.4, 55.4, 54.8, 53.6,
39.4, 36.9, 32.3, 28.0, 20.5; HRMS (ESI) m/z calcd. for C29H36N3O7S
([M ꢁ H]-): 570.2274, found 570.2288.
The title compound was obtained by 32 which was coupled with
4-amino-N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-iso-
4.1.48. 3-(3, 4-Dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((4-
methoxy-N-propylphenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (38e)
butylbenzenesulfonamide (23) through EDCI/HOBt/DMAP coupling
procedure in 75% yield (white amorphous solid) as described for
35a: 1H NMR (500 MHz, CD3OD)
d
7.49 (d, J ¼ 7.0 Hz, 2H, H-2000, H-
The title compound was obtained by 32 which was coupled with
6000), 7.25e7.18 (m, 5H, 1-phenyl-H), 6.71 (d, J ¼ 7.0 Hz, 2H, H-3000, H-
5000), 6.62 (d, J ¼ 7.5 Hz, 1H, H-500), 6.59 (s, 1H, H-200), 6.43 (d,
J ¼ 7.0 Hz, 1H, H-600), 4.04 (s, 1H, H-3), 3.78 (s, 1H, H-2), 3.29e3.24
(m, 1H, H-4a), 3.16 (d, J ¼ 13.0 Hz, 1H, H-4b), 2.94 (dd, J ¼ 18.5,
9.5 Hz, 1H, isobutyl-CH2a), 2.89e2.85 (m, 1H, isobutyl-CH2b),
2.84e2.80 (m,1H, H-1a), 2.65e2.60 (m,1H, H-1b), 2.54 (dd, J ¼ 18.5,
8.0 Hz, 2H, H-30), 2.29 (d, J ¼ 6.0 Hz, 2H, H-20), 1.97 (s, 1H, isobutyl-
CH), 0.92 (d, J ¼ 4.5 Hz, 3H, one of isobutyl-CH3), 0.88 (d, J ¼ 4.5 Hz,
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-N-
propylbenzenesulfonamide (26) through EDCI/HOBt/DMAP
coupling procedure in 80% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD) 7.97 (s, 1H, 400-OH),
d
7.73 (d, J ¼ 7.5 Hz, 2H, H-2000, H-6000), 7.23e7.16 (m, 5H, 1-phenyl-H),
7.05 (d, J ¼ 7.5 Hz, 2H, H-3000, H-5000), 6.60e6.57 (m, 2H, H-500, H-200),
6.40 (d, J ¼ 7.5 Hz, 1H, H-600), 4.03 (brs, 1H, H-3), 3.84 (s, 3H, 4000-
OCH3), 3.73 (brs, 1H, H-2), 3.22e3.11 (m, 3H, H-4a, H-4b, propyl-1-
CH2a), 3.06e3.00 (m, 1H, propyl-1-CH2b), 2.94 (d, J ¼ 10.0 Hz, 1H,
H-1a), 2.64e2.59 (m, 1H, H-1b), 2.53 (dd, J ¼ 16.5, 7.5 Hz, 2H, H-30),
2.32e2.26 (m, 2H, H-20), 1.56e1.50 (m, 2H, propyl-2-CH2a, propyl-
2-CH2b), 0.84 (s, 3H, propyl-CH3); 13C NMR (101 MHz, CD3OD)
3H, one of isobutyl-CH3); 13C NMR (101 MHz, CD3OD)
d 175.2,154.3,
146.2, 144.6, 140.0, 133.8, 130.5, 130.4, 129.2, 127.2, 125.8, 120.4,
116.4, 116.3, 114.4, 74.2, 59.4, 55.3, 54.4, 39.4, 36.7, 32.3, 28.2, 20.6,
20.5; HRMS (ESI) m/z calcd. for C29H36N3O6S ([M ꢁ H]-): 554.2325,
found 554.2317.
d
175.2, 164.5, 146.2, 144.6, 140.0, 133.7, 132.2, 130.5, 130.4, 129.2,
16

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
127.2, 120.4,116.4, 116.3,115.4, 73.9, 56.2, 55.2, 53.2, 52.8, 39.3, 36.5,
32.3, 22.7, 11.5; HRMS (ESI) m/z calcd. for C29H35N2O7S ([M ꢁ H]-):
555.2165, found 555.2185.
isobutyl-CH), 0.93 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.89 (d,
J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR (151 MHz, CD3OD)
d
175.3, 164.7, 149.0, 146.0, 140.1, 133.8, 132.2, 130.8, 130.5, 129.4,
127.4, 121.8, 116.3, 115.5, 113.1, 74.2, 59.1, 56.5, 56.3, 55.5, 54.2, 39.4,
36.8, 32.6, 28.3, 20.7, 20.6; HRMS (ESI) m/z calcd. for C31H39N2O7S
([M ꢁ H]-): 583.2478, found 583.2488.
4.1.49. N-((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(3, 4-
dihydroxyphenyl)propanamide (38f)
The title compound was obtained by 32 which was coupled with
N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-cyclopropyl-4-
methoxybenzenesulfonamide (27) through EDCI/HOBt/DMAP
coupling procedure in 75% yield (white amorphous solid) as
4.1.52. N-((2S, 3R)-4-((4-amino-N-isobutylphenyl)sulfonamido)-3-
hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3-methoxyphenyl)
propanamide (39b)
The title compound was obtained by 33 which was coupled with
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.97 (s, 2H, 300-OH,
4-amino-N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-iso-
400-OH), 7.77 (d, J ¼ 7.5 Hz, 2H, H-2000, H-6000), 7.23e7.15 (m, 5H, 1-
butylbenzenesulfonamide (23) through EDCI/HOBt/DMAP coupling
procedure in 75% yield (white amorphous solid) as described for
phenyl-H), 7.08 (d,
J
¼
7.5 Hz, 2H, H-3000, H-5000), 6.58 (d,
J ¼ 9.0 Hz, 2H, H-500, H-200), 6.40 (d, J ¼ 7.0 Hz, 1H, H-600), 4.05 (s, 1H,
H-3), 3.88 (s, 1H, H-2), 3.85 (s, 3H, 4000-OCH3), 3.34e3.31 (m, 1H, H-
4a), 3.16 (d, J ¼ 13.5 Hz, 1H, H-4b), 3.01 (d, J ¼ 11.5 Hz, 1H, H-1a),
2.65e2.50 (m, 3H, H-1b, H-30), 2.28 (d, J ¼ 8.5 Hz, 2H, H-20), 1.98 (d,
J ¼ 4.5 Hz, 1H, cyclopropyl-CH), 0.93 (s, 2H, cyclopropyl-CH2a,
cyclopropyl-CH2a’), 0.69 (s, 1H, cyclopropyl-CH2b), 0.62 (s, 1H,
35a: 1H NMR (500 MHz, CD3OD)
d
7.47 (d, J ¼ 8.5 Hz, 2H, H-2000, H-
6000), 7.23e7.13 (m, 5H, 1-phenyl-H), 6.69 (d, J ¼ 7.0 Hz, 3H, H-3000, H-
5000, H-500), 6.62 (d, J ¼ 8.0 Hz,1H, H-600), 6.51 (d, J ¼ 8.0 Hz,1H, H-200),
4.02e4.01 (m, 1H, H-3), 3.77 (s, 3H, 300-OCH3), 3.75e3.73 (m, 1H, H-
2), 3.25e3.22 (m, 1H, H-4a), 3.15e3.12 (m, 1H, H-4b), 2.93e2.86 (m,
2H, isobutyl-CH2a, isobutyl-CH2b), 2.79 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-
1a), 2.66e2.54 (m, 3H, H-1b, H-30), 2.35e2.25 (m, 2H, H-20),
1.96e1.90 (m, 1H, isobutyl-CH), 0.89 (d, J ¼ 6.5 Hz, 3H, one of iso-
butyl-CH3), 0.85 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
cyclopropyl-CH2b’); 13C NMR (101 MHz, CD3OD)
d 173.7, 163.4,
163.3,144.8,143.1,138.7,132.3,129.7,129.0,127.8,125.8,119.0,115.0,
114.9, 113.9, 71.7, 54.8, 53.7, 37.9, 35.5, 35.0, 31.7, 30.9, 30.2, 7.24,
6.14; HRMS (ESI) m/z calcd. for C29H33N2O7S ([M ꢁ H]-): 553.2008,
found 553.2025.
(126 MHz, CD3OD)
d 174.1, 153.3, 147.8, 144.8, 139.0, 132.7, 129.5,
129.4, 128.2 126.2, 124.8, 120.6, 115.1, 113.5, 112.0, 73.2, 58.3, 55.3,
54.3, 53.4, 38.3, 35.7, 31.4, 27.2, 19.6, 19.5; HRMS (ESI) m/z calcd. for
4.1.50. 3-(3, 4-Dihydroxyphenyl)-N-((2S, 3R)-3-hydroxy-4-((N-
isopropyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (38g)
C
30H38N3O6S ([M ꢁ H]-): 568.2476, found 568.2470.
4.1.53. 3-(4-Hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-hydroxy-4-
((N-isobutyl-4-(methylthio)phenyl)sulfonamido)-1-phenylbutan-2-
yl)propanamide (39c)
The title compound was obtained by 32 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isopropyl-4-
methoxybenzenesulfonamide (28) through EDCI/HOBt/DMAP
The title compound was obtained by 33 which was coupled with
coupling procedure in 83% yield (white amorphous solid) as
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.97 (s, 1H, 400-OH),
(methylthio)benzenesulfonamide (24) through EDCI/HOBt/DMAP
7.76 (d, J ¼ 7.5 Hz, 2H, H-2000, H-6000), 7.22e7.16 (m, 5H, 1-phenyl-H),
7.04 (d, J ¼ 7.5 Hz, 2H, H-3000, H-5000), 6.62 (d, J ¼ 7.0 Hz, 1H, H-500),
6.58 (s, 1H, H-200), 6.42 (d, J ¼ 7.5 Hz, 1H, H-600), 4.08 (s, 1H, H-3),
4.03e3.95 (m, 1H, H-2), 3.84 (s, 4H, isopropyl-CH, 4000-OCH3),
3.21e3.17 (m, 2H, H-4a, H-4b), 2.97e2.94 (m, 1H, H-1a), 2.64 (t,
J ¼ 12.0 Hz, 1H, H-1b), 2.56e2.50 (m, 2H, H-30), 2.35e2.23 (m, 2H,
H-20), 1.09 (d, J ¼ 4.5 Hz, 3H, one of isopropyl-CH3), 0.93 (d,
J ¼ 4.5 Hz, 3H, one of isopropyl-CH3); 13C NMR (101 MHz, CD3OD)
coupling procedure in 78% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.73 (d, J ¼ 8.5 Hz,
2H, H-2000, H-6000), 7.41 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 7.26 (t,
J ¼ 7.5 Hz, 2H, two of 1-phenyl-H), 7.22e7.18 (m, 3H, three of 1-
phenyl-H), 6.73 (s, 1H, H-500), 6.65 (d, J ¼ 8.0 Hz, 1H, H-600), 6.55
(d, J ¼ 8.0 Hz, 1H, H-200), 4.06e4.02 (m, 1H, H-3), 3.82 (s, 3H, 300-
OCH3), 3.80e3.77 (m, 1H, H-2), 3.31 (d, J ¼ 2.5 Hz, 1H, H-4a), 3.16
(dd, J ¼ 14.0, 3.5 Hz, 1H, H-4b), 3.06e2.96 (m, 2H, isobutyl-CH2a,
isobutyl-CH2b), 2.91 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-1a), 2.68e2.60 (m,
3H, H-1b, H-30), 2.54 (s, 3H, 4000-SCH3), 2.37e2.30 (m, 2H, H-20),
2.04e1.98 (m, 1H, isobutyl-CH), 0.94 (d, J ¼ 6.5 Hz, 3H, one of iso-
butyl-CH3), 0.90 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
d
175.3, 164.6, 146.3, 144.7, 140.2, 133.8, 133.1, 130.6, 130.5, 129.3,
127.3, 120.5, 116.5, 116.4, 115.5, 75.1, 56.3, 55.4, 51.5, 48.0, 39.5, 36.8,
32.5, 22.2, 20.4; HRMS (ESI) m/z calcd. for C29H35N2O7S ([M ꢁ H]-):
555.2165, found 555.2172.
(151 MHz, CD3OD)
d 175.2, 148.9, 147.4, 145.9, 140.0, 136.2, 133.7,
4.1.51. 3-(4-Hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-hydroxy-4-
((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (39a)
The title compound was obtained by 3-(4-hydroxy-3-
methoxyphenyl)propanoic acid (33) which was coupled with N-
130.4, 129.3, 128.9, 127.3, 126.5, 121.7, 116.2, 113.0, 74.0, 58.9, 56.4,
55.4, 54.1, 39.3, 36.7, 32.5, 28.1, 20.5, 14.7; HRMS (ESI) m/z calcd. for
C
31H39N2O6S2 ([M ꢁ H]-): 599.2250, found 599.2280.
4.1.54. 3-(4-Hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-hydroxy-4-
((N-isobutyl-6-methoxypyridine)-3-sulfonamido)-1-phenylbutan-
2-yl)propanamide (39d)
((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
methoxybenzenesulfonamide (21) through EDCI/HOBt/DMAP
coupling procedure in 77% yield (white amorphous solid) as
The title compound was obtained by 33 which was coupled with
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.78 (d, J ¼ 9.0 Hz,
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-6-
2H, H-2000, H-6000), 7.26 (t, J ¼ 7.5 Hz, 2H, two of 1-phenyl-H),
7.23e7.18 (m, 3H, three of 1-phenyl-H), 7.10 (d, J ¼ 9.0 Hz, 2H, H-3000,
H-5000), 6.73 (s, 1H, H-500), 6.65 (d, J ¼ 8.0 Hz, 1H, H-600), 6.55 (d,
J ¼ 8.0 Hz, 1H, H-200), 4.07e4.03 (m, 1H, H-3), 3.89 (s, 3H, 4000-OCH3),
3.83 (s, 3H, 300-OCH3), 3.80e3.77 (m, 1H, H-2), 3.32 (dd, J ¼ 15.0,
3.0 Hz, 1H, H-4a), 3.17 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-4b), 3.02 (dd,
J ¼ 13.5, 8.0 Hz, 1H, isobutyl-CH2a), 2.97 (dd, J ¼ 15.0, 9.0 Hz, 1H,
isobutyl-CH2b), 2.89 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-1a), 2.70e2.58 (m,
3H, H-1b, H-30), 2.39e2.29 (m, 2H, H-20), 2.04e1.96 (m, 1H,
methoxypyridine-3-sulfonamide (25) through EDCI/HOBt/DMAP
coupling procedure in 80% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
8.57 (s, 1H, H-6000),
8.00 (d, J ¼ 8.5 Hz, 1H, H-2000), 7.26e7.12 (m, 5H, 1-phenyl-H), 6.91
(d, J ¼ 8.5 Hz, 1H, H-5000), 6.69 (s, 1H, H-500), 6.61 (d, J ¼ 7.0 Hz, 1H, H-
600), 6.52 (d, J ¼ 7.0 Hz, 1H, H-200), 3.97 (s, 4H, H-3, 4000-OCH3), 3.78 (s,
3H, 300-OCH3), 3.73 (s, 1H, H-2), 3.26 (s, 1H, H-4a), 3.12 (d,
J ¼ 13.5 Hz, 1H, H-4b), 3.06e2.99 (m, 2H, isobutyl-CH2a, isobutyl-
CH2b), 2.89 (dd, J ¼ 12.5, 6.0 Hz, 1H, H-1a), 2.63e2.57 (m, 3H, H-1b,
17

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
H-30), 2.36e2.28 (m, 2H, H-20), 2.03e1.91 (m, 1H, isobutyl-CH), 0.90
(d, J ¼ 5.0 Hz, 3H, one of isobutyl-CH3), 0.86 (d, J ¼ 5.0 Hz, 3H, one of
13C NMR (101 MHz, CD3OD)
d 175.1, 164.5, 148.8, 145.8, 140.1, 133.7,
133.0, 130.5, 130.4, 129.2, 127.2, 121.6, 116.1, 115.4, 112.9, 75.1, 56.3,
56.2, 55.3, 51.4, 47.9, 39.3, 36.7, 32.5, 22.1, 20.3; HRMS (ESI) m/z
calcd. for C30H37N2O7S ([M ꢁ H]-): 569.2321, found 569.2348.
isobutyl-CH3); 13C NMR (101 MHz, CD3OD)
d 175.2, 167.8, 148.8,
148.4,145.8,139.9,139.0,133.7,130.4,130.3,129.3,127.3,121.6,116.1,
112.9, 112.4, 73.6, 58.3, 56.3, 55.4, 54.8, 53.6, 39.3, 36.8, 32.4, 27.9,
20.4; HRMS (ESI) m/z calcd. for C30H38N3O7S ([M ꢁ H]-): 584.2430,
found 584.2443.
4.1.58. 3-(4-Hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)propanamide (40a)
4.1.55. 3-(4-Hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-hydroxy-4-
((4-methoxy-N-propylphenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (39e)
The title compound was obtained by 3-(4-hydroxy-3,5-
dimethoxyphenyl)propanoic acid (34) which was coupled with N-
((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
The title compound was obtained by 33 which was coupled with
methoxybenzenesulfonamide (21) through EDCI/HOBt/DMAP
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-N-
coupling procedure in 80% yield (white amorphous solid) as
propylbenzenesulfonamide (26) through EDCI/HOBt/DMAP
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.77 (d, J ¼ 8.5 Hz,
coupling procedure in 80% yield (white amorphous solid) as
2H, H-2000, H-6000), 7.27e7.24 (m, 2H, two of 1-phenyl-H), 7.21e7.18
(m, 3H, three of 1-phenyl-H), 7.09 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000),
6.44 (s, 2H, H-200, H-600), 4.08e4.03 (m, 1H, H-3), 3.88 (s, 3H, 4000-
OCH3), 3.82 (s, 7H, 300-OCH3, 500-OCH3, H-2), 3.38 (d, J ¼ 2.5 Hz, 1H,
H-4a), 3.15 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-4b), 3.05 (dd, J ¼ 13.5, 8.0 Hz,
1H, isobutyl-CH2a), 2.96 (dd, J ¼ 15.0, 8.5 Hz, 1H, isobutyl-CH2b),
2.89 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-1a), 2.71e2.59 (m, 3H, H-1b, H-30),
2.42e2.31 (m, 2H, H-20), 2.04e1.96 (m, 1H, isobutyl-CH), 0.93 (d,
J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.88 (d, J ¼ 6.5 Hz, 3H, one of
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.73 (d, J ¼ 7.0 Hz,
2H, H-2000, H-6000), 7.22e7.15 (m, 5H, 1-phenyl-H), 7.05 (d, J ¼ 7.0 Hz,
2H, H-3000, H-5000), 6.69 (s, 1H, H-500), 6.61 (d, J ¼ 7.0 Hz, 1H, H-600),
6.51 (d, J ¼ 7.0 Hz, 1H, H-200), 4.04 (s, 1H, H-3), 3.84 (s, 3H, 4000-OCH3),
3.78 (s, 3H, 300-OCH3), 3.73 (s, 1H, H-2), 3.28 (s, 1H, H-4a), 3.14e3.11
(m, 2H, H-4b, propyl-1-CH2a), 3.05 (d, J ¼ 6.0 Hz, 1H, propyl-1-
CH2b), 2.95 (dd, J ¼ 13.5, 9.5 Hz, 1H, H-1a), 2.69e2.53 (m, 3H, H-
1b, H-30), 2.31 (d, J ¼ 8.0 Hz, 2H, H-20), 1.57e1.51 (m, 2H, propyl-2-
CH2a, propyl-2-CH2b), 0.83 (s, 3H, propyl-CH3); 13C NMR (101 MHz,
isobutyl-CH3); 13C NMR (151 MHz, CD3OD)
d 175.1, 164.5, 149.1,
CD3OD)
d
175.1, 164.5, 148.8, 145.8, 140.0, 133.7, 132.3, 130.5, 130.4,
139.9, 134.9, 133.0, 132.0, 130.6, 130.3, 129.2, 127.2, 115.3, 106.5, 74.1,
58.9, 56.7, 56.2, 55.3, 54.0, 39.3, 36.6, 32.9, 28.1, 20.5, 20.4; HRMS
(ESI) m/z calcd. for C32H41N2O8S ([M ꢁ H]-): 613.2584, found
613.2585.
129.2,127.2,121.6,116.1,115.4,112.9, 73.9, 56.3, 56.2, 55.2, 53.2, 52.7,
39.3, 36.5, 32.5, 22.7, 11.5; HRMS (ESI) m/z calcd. for C30H37N2O7S
([M ꢁ H]-): 569.2321, found 569.2333.
4.1.56. N-((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3-
methoxyphenyl)propanamide (39f)
4.1.59. N-((2S, 3R)-4-((4-amino-N-isobutylphenyl)sulfonamido)-3-
hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3, 5-dimethoxyphenyl)
propanamide (40b)
The title compound was obtained by 33 which was coupled with
N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-cyclopropyl-4-
methoxybenzenesulfonamide (27) through EDCI/HOBt/DMAP
coupling procedure in 75% yield (white amorphous solid) as
The title compound was obtained by 34 which was coupled with
4-amino-N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-iso-
butylbenzenesulfonamide (23) through EDCI/HOBt/DMAP coupling
procedure in 81% yield (white amorphous solid) as described for
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.78 (d, J ¼ 7.0 Hz,
35a: 1H NMR (500 MHz, CD3OD)
d
7.46 (d, J ¼ 7.5 Hz, 2H, H-2000, H-
2H, H-2000, H-6000), 7.20e7.15 (m, 5H, 1-phenyl-H), 7.08 (d, J ¼ 7.5 Hz,
2H, H-3000, H-5000), 6.69 (s, 1H, H-500), 6.60 (d, J ¼ 7.0 Hz, 1H, H-600),
6.51 (d, J ¼ 7.5 Hz, 1H, H-200), 4.05 (s, 1H, H-3), 3.88e3.82 (m, 5H, H-
2, H-4a, 4000-OCH3), 3.78 (s, 3H, 300-OCH3), 3.16 (d, J ¼ 13.5 Hz, 1H, H-
4b), 3.00 (dd, J ¼ 12.0, 9.5 Hz, 1H, H-1a), 2.61e2.59 (m, 3H, H-1b, H-
30), 2.39e2.24 (m, 2H, H-20),1.98 (s,1H, cyclopropyl-CH), 0.91 (s, 2H,
cyclopropyl-CH2a, cyclopropyl-CH2a’), 0.68 (s, 1H, cyclopropyl-
CH2b), 0.61 (s, 1H, cyclopropyl-CH2b’); 13C NMR (101 MHz, CD3OD)
6000), 7.20e7.16 (m, 5H, 1-phenyl-H), 6.68 (d, J ¼ 7.5 Hz, 2H, H-3000, H-
5000), 6.40 (s, 2H, H-200, H-600), 4.02 (s, 1H, H-3), 3.77 (s, 7H, 300-OCH3,
500-OCH3, H-2), 3.27 (s, 1H, H-4a), 3.12 (d, J ¼ 14.0 Hz, 1H, H-4b),
2.95e2.85 (m, 2H, isobutyl-CH2a, isobutyl-CH2b), 2.79 (dd, J ¼ 12.0,
7.0 Hz, 1H, H-1a), 2.63e2.53 (m, 3H, H-1b, H-30), 2.32 (d, J ¼ 6.0 Hz,
2H, H-20), 1.98e1.82 (m, 1H, isobutyl-CH), 0.88 (d, J ¼ 4.5 Hz, 3H,
one of isobutyl-CH3), 0.85 (d, J ¼ 4.5 Hz, 3H, one of isobutyl-CH3);
13C NMR (101 MHz, CD3OD)
d 175.1, 154.4, 149.2, 140.1, 134.9, 133.1,
d
175.0, 164.7, 148.8, 145.8, 140.1, 133.6, 131.1, 130.5, 130.4, 129.2,
130.5, 130.4, 129.3, 127.3, 125.9, 114.5, 106.5, 74.4, 59.4, 56.7, 55.4,
127.2, 121.6, 116.1, 115.3, 112.9, 73.1, 56.3, 56.2, 55.1, 39.2, 36.4, 33.1,
32.5, 8.64, 7.55; HRMS (ESI) m/z calcd. for C30H35N2O7S ([M ꢁ H]-):
567.2165, found 567.2164.
54.4, 39.4, 36.7, 33.0, 28.3, 20.7, 20.6; HRMS (ESI) m/z calcd. for
C
31H40N3O7S ([M ꢁ H]-): 598.2587, found 598.2590.
4.1.60. 3-(4-Hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-4-(methylthio)phenyl)sulfonamido)-1-
phenylbutan-2-yl)propanamide (40c)
4.1.57. 3-(4-Hydroxy-3-methoxyphenyl)-N-((2S, 3R)-3-hydroxy-4-
((N-isopropyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)
propanamide (39g)
The title compound was obtained by 34 which was coupled with
The title compound was obtained by 33 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-4-
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isopropyl-4-
(methylthio)benzenesulfonamide (24) through EDCI/HOBt/DMAP
methoxybenzenesulfonamide (28) through EDCI/HOBt/DMAP
coupling procedure in 83% yield (white amorphous solid) as
coupling procedure in 78% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.73 (d, J ¼ 8.5 Hz,
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.76 (d, J ¼ 7.5 Hz,
2H, H-2000, H-6000), 7.41 (d, J ¼ 8.5 Hz, 2H, H-3000, H-5000), 7.27e7.24 (m,
2H, two of 1-phenyl-H), 7.21e7.18 (m, 3H, three of 1-phenyl-H),
6.45 (s, 2H, H-200, H-600), 4.07e4.03 (m, 1H, H-3), 3.82 (s, 6H, 300-
OCH3, 500-OCH3), 3.79 (d, J ¼ 6.5 Hz, 1H, H-2), 3.39 (d, J ¼ 2.5 Hz, 1H,
H-4a), 3.15 (dd, J ¼ 14.0, 3.5 Hz, 1H, H-4b), 3.07 (dd, J ¼ 13.5, 8.0 Hz,
1H, isobutyl-CH2a), 2.99 (dd, J ¼ 15.0, 8.5 Hz, 1H, isobutyl-CH2b),
2.92 (dd, J ¼ 13.5, 7.0 Hz, 1H, H-1a), 2.70e2.61 (m, 3H, H-1b, H-30),
2.54 (s, 3H, 4000-SCH3), 2.42e2.31 (m, 2H, H-20), 2.05e1.97 (m, 1H,
2H, H-2000, H-6000), 7.21e7.16 (m, 5H, 1-phenyl-H), 7.04 (d, J ¼ 7.5 Hz,
2H, H-3000, H-5000), 6.70 (s, 1H, H-500), 6.65 (d, J ¼ 7.5 Hz, 1H, H-600),
6.53 (d, J ¼ 7.5 Hz, 1H, H-200), 4.09 (s, 1H, H-3), 4.02e3.94 (m, 1H, H-
2), 3.84 (s, 4H, isopropyl-CH, 4000-OCH3), 3.78 (s, 3H, 300-OCH3), 3.20
(d, J ¼ 13.5 Hz, 1H, H-4a), 2.97e2.94 (m, 2H, H-4b, H-1a), 2.70e2.55
(m, 3H, H-1b, H-30), 2.38e2.33 (m, 2H, H-20), 1.08 (d, J ¼ 5.0 Hz, 3H,
one of isopropyl-CH3), 0.93 (d, J ¼ 5.0 Hz, 3H, one of isopropyl-CH3);
18

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
isobutyl-CH), 0.93 (d, J ¼ 6.5 Hz, 3H, one of isobutyl-CH3), 0.89 (d,
J ¼ 6.5 Hz, 3H, one of isobutyl-CH3); 13C NMR (151 MHz, CD3OD)
149.1, 140.1, 134.9, 132.9, 131.1, 130.6, 130.4, 129.2, 127.2, 115.3, 106.5,
73.2, 56.7, 56.2, 56.1, 55.1, 39.2, 36.4, 33.1, 33.0, 8.67, 7.57; HRMS
(ESI) m/z calcd. for C31H37N2O8S ([M ꢁ H]-): 597.2271, found
597.2267.
d
175.2, 149.3, 147.5, 140.0, 136.3, 135.0, 133.0, 130.4, 129.3, 128.9,
127.3, 126.6, 106.6, 74.1, 58.9, 56.8, 55.5, 54.0, 39.4, 36.7, 33.0, 28.1,
20.6, 20.5, 14.7; HRMS (ESI) m/z calcd. for C32H41N2O7S2 ([M ꢁ H]-):
629.2355, found 629.2358.
4.1.64. 3-(4-Hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isopropyl-4-methoxyphenyl)sulfonamido)-1-
phenylbutan-2-yl)propanamide (40g)
4.1.61. 3-(4-Hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((N-isobutyl-6-methoxypyridine)-3-sulfonamido)-1-
phenylbutan-2-yl)propanamide (40d)
The title compound was obtained by 34 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isopropyl-4-
The title compound was obtained by 34 which was coupled with
methoxybenzenesulfonamide (28) through EDCI/HOBt/DMAP
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl-6-
coupling procedure in 83% yield (white amorphous solid) as
methoxypyridine-3-sulfonamide (25) through EDCI/HOBt/DMAP
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.75 (d, J ¼ 7.5 Hz,
coupling procedure in 80% yield (white amorphous solid) as
2H, H-2000, H-6000), 7.20e7.15 (m, 5H, 1-phenyl-H), 7.04 (d, J ¼ 7.5 Hz,
2H, H-3000, H-5000), 6.41 (s, 2H, H-200, H-600), 4.08 (s, 1H, H-3),
4.03e3.93 (m, 1H, H-2), 3.84 (s, 4H, isopropyl-CH, 4000-OCH3), 3.78
(s, 6H, 300-OCH3, 500-OCH3), 3.33 (s, 1H, H-4a), 3.18 (d, J ¼ 13.5 Hz, 1H,
H-4b), 2.96 (dd, J ¼ 14.5, 8.0 Hz, 1H, H-1a), 2.67e2.57 (m, 3H, H-1b,
H-30), 2.41e2.33 (m, 2H, H-20), 1.08 (d, J ¼ 5.5 Hz, 3H, one of iso-
propyl-CH3), 0.92 (d, J ¼ 5.5 Hz, 3H, one of isopropyl-CH3); 13C NMR
described for 35a: 1H NMR (500 MHz, DMSO‑d6)
d 8.58 (s, 1H, H-
6000), 8.05 (d, J ¼ 8.0 Hz, 1H, H-2000), 7.77 (d, J ¼ 8.0 Hz, 1H, 400-OH),
7.22e7.15 (m, 5H,1-phenyl-H), 6.98 (d, J ¼ 8.0 Hz,1H, H-5000), 6.39 (s,
2H, H-200, H-600), 3.92 (s, 3H, 4000-OCH3), 3.84 (d, J ¼ 5.5 Hz, 1H, H-3),
3.70 (s, 6H, 300-OCH3, 500-OCH3), 3.59 (s,1H, H-2), 3.31 (d, J ¼ 14.5 Hz,
1H, H-4a), 3.09e3.04 (m, 1H, H-4b), 3.00 (d, J ¼ 13.5 Hz, 1H, iso-
butyl-CH2a), 2.93 (dd, J ¼ 12.5, 10.0 Hz, 1H, isobutyl-CH2b), 2.80 (d,
J ¼ 13.0 Hz, 1H, H-1a), 2.54e2.50 (m, 3H, H-1b, H-30), 2.23e2.20 (m,
2H, H-20), 1.95 (s, 1H, isobutyl-CH), 0.85 (d, J ¼ 3.5 Hz, 3H, one of
isobutyl-CH3), 0.80 (d, J ¼ 3.5 Hz, 3H, one of isobutyl-CH3); 13C NMR
(101 MHz, CD3OD)
d 175.1, 164.5, 149.1, 140.1, 134.9, 133.1, 132.9,
130.5, 130.4, 129.2, 127.2, 115.4, 106.5, 75.1, 56.7, 56.2, 55.4, 51.4,
48.0, 39.3, 36.7, 32.9, 22.1, 20.3; HRMS (ESI) m/z calcd. for
C
31H39N2O8S ([M ꢁ H]-): 599.2427, found 599.2434.
(101 MHz, DMSO‑d6)
d 171.1, 165.5, 147.7, 146.7, 139.2, 137.9, 133.5,
131.2, 129.1, 129.0, 127.8, 125.6, 111.0, 105.3, 71.5, 55.8, 53.9, 53.3,
4.2. Anti-HIV-1 PR activity assay
51.7, 37.6, 35.0, 31.3, 25.9, 19.8, 19.7; HRMS (ESI) m/z calcd. for
C
31H40N3O8S ([M ꢁ H]-): 614.2536, found 614.2533.
The inhibitory effect of all newly designed inhibitors were tested
using the method of fluorescence resonance energy transfer (FRET).
Peptide (Arg-Glu (EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(-
DABCYL)-Arg) purchased from AnaSpec was chosen as the sub-
strate. The energy transfer donor (EDANS) and acceptor (DABCYL)
dyes were labeled at two ends of the peptide to perform FRET.
Excitation and emission wavelengths were set at 340 nm and
490 nm. Inhibitors were dissolved in DMSO and diluted to appro-
priate concentrations. HIV-1 PR was cloned and heterologously
expressed in Escherichia coli and purified. The FRET assay reaction
buffer contained 0.1 M sodium acetate, 1 M sodium chloride, 1 mM
ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT),
2% DMSO and 1 mg/mL bovine serum albumin (BSA) with an
adjusted pH 4.7. The experiment was carried out in 96-well plates.
HIV-1 PR and the inhibitor were mixed and incubated for
20e30 min at room temperature and then the substrate was added.
Each reaction was recorded for about 10 min.
4.1.62. 3-(4-Hydroxy-3, 5-dimethoxyphenyl)-N-((2S, 3R)-3-
hydroxy-4-((4-methoxy-N-propylphenyl)sulfonamido)-1-
phenylbutan-2-yl)propanamide (40e)
The title compound was obtained by 34 which was coupled with
N-((2R,
3S)-3-amino-2-hydroxy-4-phenylbutyl)-4-methoxy-N-
propylbenzenesulfonamide (26) through EDCI/HOBt/DMAP
coupling procedure in 80% yield (white amorphous solid) as
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.73 (d, J ¼ 7.0 Hz,
2H, H-2000, H-6000), 7.21e7.17 (m, 5H, 1-phenyl-H), 7.06 (d, J ¼ 7.0 Hz,
2H, H-3000, H-5000), 6.41 (s, 2H, H-200, H-600), 4.03 (s, 1H, H-3), 3.85 (s,
3H, 4000-OCH3), 3.77 (s, 6H, 300-OCH3, 500-OCH3), 3.74 (s, 1H, H-2), 3.35
(s, 1H, H-4a), 3.17 (d, J ¼ 6.0 Hz, 1H, H-4b), 3.11 (d, J ¼ 14.0 Hz, 1H,
propyl-1-CH2a), 3.05 (d, J ¼ 6.0 Hz, 1H, propyl-1-CH2b), 2.94 (dd,
J ¼ 13.0, 9.5 Hz, 1H, H-1a), 2.69e2.60 (m, 3H, H-1b, H-30), 2.33 (d,
J ¼ 8.0 Hz, 2H, H-20), 1.53 (s, 2H, propyl-2-CH2a, propyl-2-CH2b),
0.83 (s, 3H, propyl-CH3); 13C NMR (101 MHz, CD3OD)
d 175.1, 164.6,
149.2, 140.0, 133.0, 132.3, 130.5, 130.4,129.2, 127.2, 115.4, 106.5, 74.0,
56.7, 56.2, 55.2, 53.1, 52.7, 39.3, 36.5, 32.9, 22.7,11.5; HRMS (ESI) m/z
calcd. for C31H39N2O8S ([M ꢁ H]-): 599.2427, found 599.2442.
4.3. Anti-HIV-1 RT activity assay
The activity of HIV-1 reverse transcriptase (RT) assay in vitro was
exploited on a novel one-step RT-PCR assay [37]. HIV-1 RT was
derived from clone HIVNL4-3 and belonged to Group B of HIV-1,
which was a gift from Ying Guo (Institute of Materia Medica,
PUMC). HIV-1 RT were used in the first step of Real-time PCR re-
action for converting RNA to DNA. Real-time PCR reaction was
performed using a one-step RT-PCR Kit (Takara, RR066A) according
to the manual from the manufacturer. Each reaction mixture had a
4.1.63. N-((2S, 3R)-4-((N-cyclopropyl-4-methoxyphenyl)
sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-3-(4-hydroxy-3, 5-
dimethoxyphenyl)propanamide (40f)
The title compound was obtained by 34 which was coupled with
N-((2R, 3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-cyclopropyl-4-
methoxybenzenesulfonamide (27) through EDCI/HOBt/DMAP
coupling procedure in 75% yield (white amorphous solid) as
total volume of 20
mRNA 20 ng (template), GAPDH primers 10
pounds 1 L at different concentrations, Ex Taq HS (polymerase)
mL, comprising 2X reaction buffer 10
mL, random
M (primer), com-
described for 35a: 1H NMR (500 MHz, CD3OD)
d
7.77 (d, J ¼ 7.5 Hz,
m
2H, H-2000, H-6000), 7.21e7.15 (m, 5H, 1-phenyl-H), 7.08 (d, J ¼ 7.5 Hz,
2H, H-3000, H-5000), 6.41 (s, 2H, H-200, H-600), 4.04 (s, 1H, H-3), 3.86 (s,
4H, H-2, 4000-OCH3), 3.77 (s, 6H, 300-OCH3, 500-OCH3), 3.35e3.31 (m,
1H, H-4a), 3.14 (d, J ¼ 14.0 Hz, 1H, H-4b), 2.99 (dd, J ¼ 13.0, 9.0 Hz,
1H, H-1a), 2.70e2.54 (m, 3H, H-1b, H-30), 2.38e2.32 (m, 2H, H-20),
1.99 (s, 1H, cyclopropyl-CH), 0.90 (d, J ¼ 3.0 Hz, 2H, cyclopropyl-
CH2a, cyclopropyl-CH2a’), 0.68 (s, 1H, cyclopropyl-CH2b), 0.61 (s,
m
and HIV-1 RT. The amount of RT per reaction was approximately
5 ng, which was equivalent to 100 mU. All the components were
added under an ice-cold condition. Primer sequence was as fol-
lowed: GAPDH-forward: GAAGGTGAAGGTCGGAGT; GAPDH-
reverse: GAAGATGGTGATGGGATTTC. We used
a conventional
real-time PCR machine (Agilent Mx3000P) to measure the fluo-
1H, cyclopropyl-CH2b’); 13C NMR (101 MHz, CD3OD)
d
175.0, 164.8,
rescence. QRT-PCR cycles consisting of 42 ꢀC for 5 min, 95 ꢀC for 10 s
19

M. Zhu, Q. Shan, L. Ma et al.
European Journal of Medicinal Chemistry 220 (2021) 113498
and 60 ꢀC for 60 s for 40 rounds were used. The results were
normalized using the DMSO group levels and calculated by the
2ꢁ△△Ct comparative method.
ID: 4mc9), the highest score is ꢁ11.75 kcal/mol, the value of RMSD
between the pose with highest score and the structure in crystal
complex is 1.92 Å. As for RT (PDB-ID: 2yng), the value of RMSD
between the pose with highest score (ꢁ12.25 kcal/mol) and the
structure in crystal complex is 2.00 Å. Thus, the docking results for
both PR and RT are consistent to the crystal structures, and the
docking protocol is suitable for the systems.
4.4. Infectivity assay on HIV-1 late stage
The inhibitory effect of compounds on HIV-1 infectivity were
determined using a single-round HIV-1 infectivity assay [38]. 293T
cells were co-transfected with either plasmid pNL4-3-E-R- (pHIV-
Notes
1
NL4-3) or DRV-resistant pNL4-3-E-R- variants (pHIV-1DRRVS) and
pHCMV-G (VSV-G) to produce VSV-G pseudotyped HIV-1. In-
hibitors dissolved in dimethylsulfoxide and diluted to appropriate
concentrations. Then they were added into culture medium for 5 h
of post-transfection. After incubating for 48 h at 37 ꢀC, pseudotyped
The authors declare no competing financial interest.
Declaration of competing interest
viruses in 10 mL of supernatant were used to infect SupT1 cells for
48 h, followed by measuring luciferase activity of newly infected
cells using Centro LB960 (Berthold).
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.5. Infectivity assay on HIV-1 early stage
Acknowledgments
To assess the effect of the compounds on HIV-1 infectivity, ex-
periments were carried out using VSVG-pseudotyped HIV-1. SupT1
cells (1 ꢂ 105/mL) were infected with VSVG-pseudotyped HIV-
1(NL4-3) in 96-well plates, and then the compounds were added at
This work was supported by National Science & Technology
Major Project “Key New Drug Creation and Manufacturing Pro-
gram”, China (2019ZX09201001-003-007 and 2019ZX09721001-
004-006), Natural Science Foundation of Zhejiang Province
(LY21B020006), CAMS Innovation Fund for Medical Sciences
(CIFMS 2016-I2M-1e011 and 2018-I2M-3e004) and The National
Mega-Project for Infectious Disease (2018ZX10301408).
the concentration of 10 mM. Equal volumes of DMSO were added
into the culture medium, in order to keep constant final DMSO
concentration as 1% (v/v). After 48 h, SupT1 cells were lysed and
firefly luciferase activities were determined using a firefly Lucif-
erase Assay System (Promega).
Appendix A. Supplementary data
4.6. Molecular modeling
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113498.
The molecular modeling work for inhibitors 38a, 38c and 38d
were performed in the molecular modeling software Molecular
Operating Environment (MOE) [42]. The HIV-1 protease crystal
structure (PDB-ID: 4mc9) and reverse transcriptase crystal struc-
ture (PDB-ID: 2yng) were both obtained from protein data bank
website (www.pdb.org). In general, the docking was performed
through “DOCK” module in MOE using the alpha triangle place-
ment method. Refinement of the docked poses was carried out
using the Forcefield refinement scheme and scored using both the
affinity dG and london dG scoring system.
References
[1] J.S.G. Montaner, V.D. Lima, R. Barrios, B. Yip, E. Wood, T. Kerr, K. Shannon,
P.R. Harrigan, R.S. Hogg, P. Daly, P. Kendall, Association of highly active anti-
retroviral therapy coverage, population viral load, and yearly new HIV di-
agnoses in British Columbia, Canada: a population-based study, Lancet 376
(2010) 532e539.
[2] P. Braitstein, M.W.G. Brinkhof, F. Dabis, M. Schechter, A. Boulle, P. Miotti,
R. Wood, C. Laurent, E. Sprinz, C. Seyler, D.R. Bangsberg, E. Balestre,
J.A.C. Sterne, M. May, M. Egger, Mortality of HIV-1-infected patients in the first
year of antiretroviral therapy: comparison between low-income and high-
income countries, Lancet 367 (2006) 817e824.
Before docking, preparation procedures were needed for both
receptors and inhibitors. The proteins were examined for missing
atoms, bonds and contacts, meanwhile ionization states and
hydrogen positions were assigned with the “Protonate-3D” tool in
the MOE suite. The water molecules farther than 4.5 Å from the
ligand or protein were also deleted. Then the structures were
subjected to energy minimization with Amber99 force field until
the root mean square (RMS) gradient fell below 0.1 cal molꢁ1 Åꢁ1, to
minimize contacts among hydrogen atoms. After the protein
preparation step, the coordinates of the binding site were deter-
mined by MOE Site Finder module based on the co-crystallized
ligand and saved as dummy atoms. The ligand structures with
stereochemistry were generated by Chemdraw 18.0. Then the
hydrogen atoms were added and protonation state (pH 7.0) was
assigned. All ligand structures were energy minimized with
MMFF94x force field. After optimization, the ligands were posi-
tioned into binding site (dummy atoms) of receptors using Dock
module in MOE software. The triangle matcher and induced fit
were applied as the placement and refinement method, with
London dG as initial scoring function and GBVI/WSA dG as the final
scoring function. Before the molecular docking for our compounds,
we used the ligand that complexed with the proteins (both HIV-1
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