Discovery of phenylalanine derivatives as potent HIV-1 capsid inhibitors from click chemistry-based compound library

Article abstract of DOI:10.1016/j.ejmech.2018.09.029

The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of HIV-1 replication and is considered an important, clinically unexploited therapeutic target. As such, small drug-like molecules that inhibit this critical HIV-1 protein h

Full text of DOI:10.1016/j.ejmech.2018.09.029

Accepted Manuscript
Discovery of phenylalanine derivatives as potent HIV-1 capsid inhibitors from click
chemistry-based compound library
Gaochan Wu, Waleed A. Zalloum, Megan E. Meuser, Lanlan Jing, Dongwei Kang,
Chin Ho Chen, Ye Tian, Fangfang Zhang, Simon Cocklin, Kuo-Hsiung Lee, Xinyong
Liu, Peng Zhan
PII:
S0223-5234(18)30801-8
10.1016/j.ejmech.2018.09.029
EJMECH 10740
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 July 2018
Revised Date: 5 September 2018
Accepted Date: 10 September 2018
Please cite this article as: G. Wu, W.A. Zalloum, M.E. Meuser, L. Jing, D. Kang, C.H. Chen, Y. Tian, F.
Zhang, S. Cocklin, K.-H. Lee, X. Liu, P. Zhan, Discovery of phenylalanine derivatives as potent HIV-1
capsid inhibitors from click chemistry-based compound library, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.09.029.
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ACCEPTED MANUSCRIPT
EC = 4.33 ± 0.83 µM；
50
CC > 57.74 µM
50
Click Chemistry-based discovery
of potent HIV-1 CA inhibitor
SPR results of compound 13m

ACCEPTED MANUSCRIPT
Discovery of Phenylalanine Derivatives as Potent HIV-1 Capsid
Inhibitors from Click Chemistry-based Compound Library
a
b
c
a
a
Gaochan Wu , Waleed A. Zalloum , Megan E. Meuser , Lanlan Jing , Dongwei Kang ,
d
a
e
c,*
f,*
Chin Ho Chen , Ye Tian , Fangfang Zhang , Simon Cocklin , Kuo-Hsiung Lee ,
a,*
a,*
Xinyong Liu , Peng Zhan
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, 250012 Ji'nan, Shandong, PR China
b
Department of Pharmacy, Faculty of health science, American University of Madaba,
P.O Box 2882, Amman 11821, Jordan
c
Department of Biochemistry & Molecular Biology, Drexel University College of
Medicine, Philadelphia, Pennsylvania, USA
d
Duke University Medical Center, Box 2926, Surgical Oncology Research Facility,
Durham, NC 27710, USA
e
Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical
Sciences, Shandong University, Jinan, China
f
Natural Products Research Laboratories, Eshelman School of Pharmacy, University
of North Carolina, Chapel Hill, NC 27599, USA
*
Corresponding authors. E-mail address: sc349@drexel.edu (Cocklin S.);
khlee@unc.edu (Lee. K.H.); xinyongl@sdu.edu.cn (Liu X.Y.);
zhanpeng1982@sdu.edu.cn (Zhan P.)
Abstract: The HIV-1 capsid (CA) protein plays essential roles in both early and late
stages of HIV-1 replication and is considered an important, clinically unexploited
therapeutic target. As such, small drug-like molecules that inhibit this critical HIV-1
protein have become a priority for several groups. Therefore, in this study we explore
small molecule targeting of the CA protein, and in particular a very attractive
inter-protomer pocket. We report the design, parallel synthesis, and anti-HIV-1
activity evaluation of a series of novel phenylalanine derivatives as HIV-1 CA protein
1

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inhibitors synthesized via Cu(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition
(CuAAC) reaction. We demonstrate robust inhibitory activity over a range of
potencies against the HIV-1 NL4-3 reference strain. In particular, compound 13m
exhibited the greatest potency and lowest toxicity within this new series with an EC50
value of 4.33 µM and CC value of >57.74 µM (SI > 13.33). These values are very
50
similar to the lead compound PF-74 (EC = 5.95 µM, CC > 70.50 µM, SI > 11.85)
5
0
50
in our assay, despite significant structural difference. Furthermore, we demonstrate via
surface plasmon resonance (SPR) binding assays that 13m interacts robustly with
recombinant HIV-1 CA and exhibits antiviral activity in both the early and late stages
of HIV-1 replication. Overall, the novel parallel synthesis and structure-activity
relationships (SARs) identified within this study set the foundation for further rational
optimization and discovery of CA-targeting compounds with improved potency.
Keywords: HIV-1; Phenylalanine derivatives; HIV-1 capsid protein; CuAAC; Surface
plasmon resonance; Molecular dynamics simulation
1
. Introduction
Acquired immune deficiency syndrome (AIDS), primarily caused by human
immunodeficiency virus type 1 (HIV-1), remains a global problem. Although
considerable progress has been made with regards to improving the quality and length
of life of patients living with HIV with the application of combinatorial antiretroviral
therapy (cART) regimen, current therapies still suffer from adverse side effects and
the ever-present problems of natural and emergent drug resistance[1, 2]. Accordingly,
there is a continuing need for new anti-HIV drugs, especially those acting on novel
targets with new scaffolds and mechanisms of action.
The HIV-1 capsid (CA) protein plays crucial roles in both early (uncoating,
nuclear entry, reverse transcription, integration, etc.) and late stages (maturation and
assembly) of the HIV-1 life cycle [3, 4]. In the early stage, the fusion of the virus with
target cell membranes triggers the disassembly or uncoating of CA protein, which is
tightly associated with the reverse transcription and synthesis of viral cDNA [5, 6].
2

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Although viral uncoating is not completely understood, the correct spatiotemporal
disassembly of the capsid during the initial stage of infection is necessary for the
completion of reverse transcription and, hence, infectivity. Moreover, in the late stage
of HIV-1 replication, CA protein assembly and maturation are essential for the
formation and release of infectious viral particles. Extensive mutational analysis has
been performed on CA; analysis that has revealed an extreme genetic fragility, with
most changes to the capsid being detrimental for the above processes and the fitness
of the virus [7, 8]. Therefore, the HIV-1 CA protein has emerged as an attractive
antiviral target for the development of small-molecular inhibitors [9-12].
To date, a series of structurally diverse small-molecular compounds have been
identified as HIV-1 CA inhibitors [13-20]. Among them, PF-74 (Fig.1a) is the most
widely studied small molecule targeting the HIV-1 CA protein (Fig.1a). PF-74 was
developed by Pfizer, USA, and has been demonstrated to inhibit the replication of the
majority of isolates at low to sub-micromolar concentrations [11, 13, 21-23]. However,
the relatively low inhibitory activity and poor drug-like qualities, most critically
extremely poor metabolic stability [13] indicate the urgent needs for further structural
optimization of PF-74. Recently, the binding mode of PF-74 in the inter-protomer
binding pocket in the CA protein hexamer has been described by several groups in
crystallographic studies using native and disulphide stabilized constructs [24-26]. The
availability of these structures greatly facilitates the structure-based design of novel
compounds based upon the PF-74 scaffold. PF-74 consists of a phenylalanine core
(
red part, Fig.1a), an indole substituent (pink part, Fig.1a) and a linker between them
blue part, Fig.1a). The interprotomer pocket in which PF-74 binds is defined by four
(
α-helices (H3, H4, H5, and H7) in the NTD interface of one protomer; the
phenylalanine core forms interactions with Asn57, with Ile73, Ala105, Tyr130 and
Thr107 (Fig.1b). The methylindole ring of PF-74 is oriented towards the CTD
interface of the adjacent subunit within the same hexametric ring and interacts with
Arg173, Gln63, and Lys182 (Fig.1b) [24-26]. Of particular note, the plasticity of the
CTD-NTD interface provides additional space for further modification of the indole
3

ACCEPTED MANUSCRIPT
substituent and linker of PF-74, as exemplified by compounds I and II with improved
potency (Fig.1). Interestingly, in these compounds, the methoxy moiety was
frequently observed in the aniline substituent of phenylalanine core (Fig.1) [27, 28].
Fig.1 The design of novel phenylalanine derivatives as HIV-1 CA inhibitors. (a) Structure of
PF-74; (b) The binding mode of PF-74 in the binding site of CA protein hexamer. Red dashed
lines indicate H-bond interactions.
Given the attractiveness of the CA protein as a target, and the wealth of structural
and mechanistic information available, we chose PF-74 to focus our optimization
efforts upon. Specifically, in this study, we chose to explore modification of
methylindole moiety as this region has been demonstrated to be quite tolerant to
changes [13]. However, unlike previous studies, we demonstrate the successful
implementation of a parallel synthesis strategy using the facile Cu(I)-catalyzed
alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction (Fig.1) [29-32]. This
strategy, installs a 1,2,3-triazole unit as a linker in the newly designed compounds,
further diversifying the compounds and, most likely, improving solubility (Fig. 1). We
4

ACCEPTED MANUSCRIPT
chose not to modulate the phenylalanine core region of PF-74 and its reported
analogues but chose to include the methoxy-bearing aniline substituent present within
the high potency analogues (PF-74).
Therefore, in this novel study, we report the parallel synthesis and antiviral
activity of 20 newly designed phenylalanine derivatives. Furthermore, we
demonstrate via surface plasmon resonance (SPR) binding assays that representative
compounds from this study interact robustly with recombinant HIV-1 CA and exhibit
antiviral activity in both the early and late stages of HIV-1 replication. Overall, the
novel parallel synthesis and structure-activity relationships (SARs) identified within
this study set the foundation for further rational optimization and discovery of
CA-targeting compounds with improved potency.
2
. Results and discussion
2
.1 Chemistry
The straightforward synthetic route towards the target compounds was depicted
in Scheme 1. (Tert-butoxycarbonyl)-L-phenylalanine (1) was selected as the starting
material. It was reacted with 4-methoxy-N-methylaniline, followed by removing the
Boc protection to produce the free amine (3). Then, 3 was treated with propiolic acid
to yield the key intermediate 4. Meanwhile, the substituted benzyl bromide (5) or
chloride (9) were converted to azide 6 or 10 by nucleophilic substitution with NaN3,
respectively. Further oxidation of 10 in the presence of 3-chloroperbenzoic acid
generated compounds 11 and 12. Substituted phenylboric acids 7 were reacted with
NaN by the catalytic CuSO .5H O to provide the corresponding azides 8. Finally, the
3
4
2
target products (13) were obtained by CuAAC reaction between the azide substituents
6, 8, 11 or 12) and key intermediate 4. The newly synthesized compounds were
characterized by MS and NMR spectra.
(
5

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Scheme 1. The synthetic route of phenylalanine derivatives (13a-13t). Reagents and
Conditions. (i) 4-methoxy-N-methylaniline, PyBop, DIEA, CH Cl , r.t., 8-9h; (ii):
2
2
CF COOH; CH Cl , r.t., 6-7h; (iii) propiolic acid, HATU, DIEA, CH Cl , r.t., 12h;
3
2
2
2
2
.
(
iv): NaN , DMF, r.t., 12-13h; (v): NaN , CuSO 5H O, CH OH; (vi):
3 3 4 2 3
3
1
3
-Chloroperbenzoic acid, CH Cl , 0℃,1h; (vii): 3-chloroperbenzoic acid, CH Cl ,
2 2 2 2
.
4
5h at r.t.; (viii): CuSO 5H O, L-ascorbic acid sodium salt, THF/H O (V:V = 1:1),
2
2
0-60℃, 4-6h.
2
.2 Anti-HIV activity evaluation in MT-4 cells
Having demonstrated the utility of our new parallel synthesis scheme, we next
sought to test the newly synthesized compounds for in vitro anti-HIV-1 activity in a
multicycle assay using fully infectious HIV-1 NL virus and MT-4 target cells. PF-74
4
-3
was included as an in-line control to allow for direct comparison with the new
compounds. Finally, the toxicity of PF-74 and the new compounds towards the MT-4
cells was also assessed. Table 1 shows the anti-HIV potency (EC , as measured by a
5
0
luciferase gene expression assay [33]), cytotoxicity (CC ) as well as selectivity index
5
0
(
SI, the ratio of CC /EC ) for each of the compounds and control.
50 50
6

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Table 1. Anti-HIV-1 activity and cytotoxicity of the novel phenylalanine derivatives
in MT-4 cells infected with the HIV-1 NL4-3 virus.
a
b
Compounds
R
EC50 (µM)
CC50 (µM)
SI
1
3a
8.00 ± 2.37
> 20.68
> 2.58
c
c
d
1
3b
NA
NA
ND
c
c
d
1
3c
NA
NA
ND
d
1
3d
>
19.84
> 19.84
ND
c
c
d
1
3e
NA
NA
ND
7

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d
1
3f
>
>
19.84
21.53
> 19.84
ND
ND
d
1
3g
CN
> 21.53
> 21.53
1
3h
9
.11 ± 2.88
.98 ± 3.10
> 2.36
> 2.40
> 1.91
1
3i
8
> 21.53
> 19.43
CN
1
3j
1
0.16 ± 2.22
c
c
d
1
3k
NA
NA
ND
NO2
d
1
3l
>
19.43
> 19.43
> 57.74
ND
1
3m
4
.33 ± 0.83
> 13.33
8

ACCEPTED MANUSCRIPT
d
1
3n
>
19.25
> 19.25
ND
1
3o
1
4.93 ± 3.94
> 20.02
> 19.47
> 1.34
> 2.82
1
3p
3q
6
.91 ± 2.43
c
c
d
1
NA
NA
ND
1
3r
6
.65 ± 1.47
> 19.32
> 18.74
> 2.91
c
d
1
3s
NA
ND
OH
O
O
1
3t
H C
3
10.45 ± 3.08
5.95 ± 1.32
> 16.56
> 70.50
> 1.59
N
O
N
H
-
> 11.85
PF-74
a
EC : the concentration of the compound required to achieve 50% protection of MT-4 cells
50
against HIV-1-induced cytopathic effect, determined in at least triplicate against HIV-1 in MT-4
cells; values are the mean ± SD of at least two parallel tests.
b
CC : the concentration of the compound required to reduce the viability of uninfected cells by
5
0
9

ACCEPTED MANUSCRIPT
5
0%, determined in at least triplicate against HIV-1 in MT-4 cells; values were averaged from at
least four independent experiments.
c
NA: No anti-HIV-1 activity or cytotoxicity at the test concentration.
d
ND: Not determined.
As shown in the Table 1, it can be observed that some of newly synthesized
compounds exhibited from moderate to excellent activity against HIV-1 NL4-3 virus
with EC values ranging from 14.93 µM to 4.33 µM and SI values between 1.34 and
50
1
6
3.33. Among them, 13m (EC = 4.33 ± 0.83 µM, CC > 57.74 µM), 13p (EC =
50 50 50
.91 ± 2.43 µM, CC >19.47 µM) and 13r (EC = 6.65 ± 1.47 µM, CC > 19.32 µM)
5
0
50
50
turned out to be the most potent HIV-1 inhibitors, which were equipotent with PF-74
EC = 5.95 ± 1.32 µM, CC > 70.50 µM). In particular, 13m also exhibited an
(
5
0
50
equivalent SI value (13.33) comparable to PF-74 (SI: 11.85).
The exploratory SARs analysis was derived from the results. Firstly, our
attention was focused on the SAR of the substituted benzyls (13a-13n). When making
a comparison between 13m and 13n with a terminal β- and α-substituted naphthalene
group in their structures, respectively, 13m is far more active than 13n in inhibiting
HIV-1 NL , indicating that β-substituted naphthalene might form a better interaction
4
-3
with the CTD of CA protein. Similarly, variation of the position of identical
substituent in the benzyl (from the ortho-position to the para-position) resulted in
different inhibitory activity (13a (2-Me) > 13b (3-Me) and 13c (4-Me); 13i (4-CN) ≈
1
3h (3-CN) > 13g (2-CN); 13j (2-NO ) > 13k (3-NO ) and 13l (4-NO )), with the
2 2 2
exception of compounds that were endowed with chlorinated benzyl (13d/13e/13f).
These results indicated that benzyl with different positions of the same substituent
would have a great influence on the inhibitory activity against HIV-1 NL4-3 virus. In
addition, it was noteworthy that different substituents at the same position
demonstrated significant effects on the activity results, as suggested by comparison of
1
3a/13d/13g/13j, 13b/13e/13h/13k and 13c/13f/13i/13l.
Next, we turned our attention to the SAR of synthesized compounds with
substituted benzenes (13o/13p). Detailed comparison of the activities of 13o with 13p
1
0

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suggested that one more flexible substituent at the benzene (13p) might strengthen the
interaction with the CTD binding site compared to substituent bearing with rigid
construction (13o). Further optimization of phenylalanine derivatives was focused on
incorporating a group including S atom or its oxidation groups to the linker.
Comparing the anti-HIV-1 (NL ) activity between 13q, 13r and 13s reveals that had
4
-3
certain influence on the antiviral activity (Table 1). Among compounds above, 13r
exhibited the best inhibitory activity with the EC value of 6.65 ± 1.47 µM, which is
5
0
comparable to the reference agent PF-74.
Finally, the commercially available zidovudine unit was introduced as one CTD
substituent, resulting in the compound 13t with moderate activity toward HIV-1 NL4-3
(
EC = 10.45 ± 3.08 µM).
5
0
On the whole, preliminary SAR studies of these newly designed compounds
revealed that the anti-HIV-1(NL ) activities of phenylalanine derivatives were not
4
-3
only sensitive to types of CTD substituents but also affected by different linkage
positions. The biological evaluation results and the SAR analysis described above will
be beneficial to further design of CA protein inhibitors targeting the conformationally
dynamic CTD-NTD interface.
2
.3 Binding to HIV-1 CA protein
We next sought to demonstrate that the newly synthesized and designed
compounds retained target specificity to the HIV-1 CA protein. We chose to perform
this analysis on three of our highest potency compounds, 13m, 13p, and 13r. The
successful use of surface plasmon resonance (SPR) to quantify the binding
interactions of CA-targeting small molecules has been previously reported [13-16].
Therefore, we chose this methodology for our studies.
Moreover, to ascertain whether or not there was any oligomeric preference of the
compounds, we chose three different available CA protein constructs; the CA NTD,
monomeric CA, and hexameric CA. Again, PF-74 was utilized as the reference
compound. Figure 2 shows the results of this analysis.
1
1

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(A)
(B)
(C)
1
2

ACCEPTED MANUSCRIPT
(D)
Fig. 2 SPR results of compounds 13m (B), 13p (C) and 13r (D) binding to three kinds of CA
proteins (NTD, monomer, and hexamer) respectively with PF-74 as the reference (A).
1
3

ACCEPTED MANUSCRIPT
As depicted in Fig. 2A, compounds 13m, 13p, 13r, and PF-74 all bind to the
three different CA protein constructs (NTD, monomer and hexamer). PF-74 interacts
the tightest overall, with equilibrium dissociation constant (K ) values for the NTD,
D
monomer, and hexamer, of 3.6 µM, 2.7 µM and 64.3 nM, respectively. These values
are consistent with the previous affinity studies reported in the literature [13, 24, 26]
and demonstrate that PF-74 has a preference for binding to the hexameric form of the
CA protein. This result, again, is consistent with the structural information regarding
the binding mode of PF-74 in which the molecule makes contact with two adjacent
protomers in the CA hexamer. In contrast to the oligomeric preference shown by
PF-74, compounds 13m, 13p, and 13r, display comparable affinities to all of the CA
protein constructs used, with the exception of 13r that appears to prefer the
monomeric form of CA. Moreover, compounds 13m, 13p, and 13r have a markedly
different kinetic signatures to PF-74, having rapid on- and off-rates, compared to the
rapid on- but relatively slow off-rate of the parental compound. Taken together, this
analysis demonstrates that the novel 1,2,3-triazole-containing phenylalanine
derivatives retain the target specificity of the parental PF-74 and can be classified as
HIV-1 CA inhibitors. Moreover, the use of SPR to determine not only an affinity but a
kinetic signature for the compounds may provide a rapid method of screening for
higher potency analogues. A recent study has demonstrated that the antiviral potency
of a small series of HIV-1 entry inhibitors can be best correlated with the off-rate
parameter of their interaction with their protein target [34]. As such, next-generation
analogues in this series could be prioritized via a rapid off-rate screening strategy.
2
.5 Determination of the action stage of 13m
The observation that all of the new compounds interact with both monomeric and
hexameric forms of the HIV-1 CA protein has implications for the stage at which they
exert there antiviral effect. PF-74 binds to both CA monomer and hexamer and
inhibits HIV-1 replication in the early, infective stages, and the late, assembly stages
of the virus lifecycle [11]. We, therefore, sought to see if this dual-stage mode of
action was true for our new compounds and chose to focus on 13m as the most potent
1
4

ACCEPTED MANUSCRIPT
representative of our derivatives. To achieve this, we utilized the modular nature of
the single round infection assay, in which singly-round infective HIV-1 particles are
generated recombinantly in HEK293T cells, and then used to infect U87.CD4.CCR5
cells: this allows for the separation of early and late stages of the replication cycle
[
35].
Effects on post-integration and assembly events were identified by producing
Env pseudotyped viruses in the absence or presence of various concentrations of 13m.
HIV-1B41 Env pseudotyped virus (which encodes for firefly luciferase as a reporter
gene) were diluted ten-fold and used to infect U87.CD4.CCR5 target cells.
Compound-induced late-stage effects were demonstrated by a decrease in infectivity
in the target cells, compared to the control virus that was generated without the
compound present. Compound-induced early-stage effects were observed by using
virus produced in the absence of compound to infect U87.CD4.CCR5 target cells in
the absence or presence of various concentrations of 13m. As can be seen in Table 2,
1
3m displays effects in both the post-integration, assembly and pre-integration,
infective stages of the HIV-1 life-cycle. The control compound PF-74 also exhibits
this dual-stage inhibition profile. These preliminary mechanism-of-action studies set
the stage for more rigorous studies with these and higher potency compounds.
Table 2. Early and late stage anti-HIV-1 activity and cytotoxicity of 13m and PF-74
using HIV-1 Env-pseudotyped virus.
Therapeutic
Compounds
PF-74
IC Early Stage
IC Late Stage
CC50
(µM)
Index
5
0
50
(
µM)
(µM)
(CC /IC )
5
0
50
0.1 ± 0.02
7.0 ± 0.8
8.3 ± 4.3
73 ± 15
54.8 ± 2
17.3
2.8
13m
31 ± 11
IC : the concentration of the compound required to achieve 50% infection of HIV-1
50
Env-pseudotyped virus in U87.CD4.CCR5 target cells. SD of 3 parallel tests are indicated.
1
5

ACCEPTED MANUSCRIPT
CC : the concentration of the compound required to reduce the viability of treated cells by 50%.
5
0
SD of 3 parallel tests are indicated.
Therapeutic Index: defined as the CC /IC . The IC used was the average of the early and late
5
0
50
50
stage IC values.
50
2
.5. Molecular Dynamics (MD) simulation
To better understand the activity results and find the potential binding modes of
this series of molecules, the representative compound 13m was analyzed by MD
simulation to find its binding to the active site of CA HIV-1 monomer. Figure 3A
shows the root mean square deviation (RMSD) of the protein amino acid residues for
all heavy atoms during the 1µs MD simulation. It is clear from the figure that the
protein structure has deviated from the starting X-ray structure 5HGL. Furthermore,
the figure shows that the protein structure could exist in different conformational
ensembles due to the different RMSD values. To further investigate the deviation of
the protein from the starting X-ray structure, the root mean squares fluctuation
(RMSF) of residues was calculated and plotted in Figure 3B. This figure shows that
most amino acid residues have deviated from the starting structure, which also
indicates the presence of the protein in other conformational forms. The presence of
the protein structure in different conformational forms could be accompanied with
different binding modes of 13m. Accordingly, RMSD of 13m heavy atoms was
calculated in reference to the first frame of the MD simulation and plotted in Figure
3
C. Indeed, it is obvious from the figure that 13m is present in different structural
clusters, which potentially indicate binding to the active site in different modes.
A
1
6

ACCEPTED MANUSCRIPT
B
C
Fig. 3 A) RMSD (all heavy atoms) of amino acid residues of CA HIV-1 monomer in reference to
the first frame of the 1µs MD simulation. B) RMSF of the backbone Cα atoms for residues of CA
HIV-1 monomer during the 1µs MD simulation. C) RMSD (all heavy atoms) of the bound 13m
during the 1µs of the MD simulation based on the first frame of the simulation
.
According to the RMSD and RMSF results, the entire trajectory has been
clustered based on 13m (no fit), after alignment of protein residues, to see the binding
modes of 13m in the active site. The clustering procedure yielded nine different
structural clusters with two most populated. Figure 4 shows representative structures
of the two most populated clusters with their corresponding expanded views for 13m
binding to the active site. According to the clustering results, it is clear that CA HIV-1
monomer exists in two different conformational ensembles with RMSD between the
representative structures of 6.4 Ǻ. This high RMSD value indicates that they are
totally different. Inspection of the expanded views of the first and second clusters in
the figure shows that in the most populated cluster 13m binds in similar way to PF-74
structure, where the core scaffold is oriented to the inside of the active site and the
substituent is oriented to the outside of the active site. On the other hand, in the
second most populated cluster, the core of 13m is oriented to the outside of the active
1
7

ACCEPTED MANUSCRIPT
site and the substituent is oriented to the inside of the active site, which is in contrast
to the binding of PF-74. According to these results, the binding of 13m in two
different binding modes could potentially induce the two different conformations of
CA HIV-1 monomer. Furthermore, binding of 13m in two different binding modes
could enhance its activity due to the increased chance of binding to the active site.
Fig. 4 Representative structures of the first (A) and second (C) conformational clusters and their
corresponding expanded views of 13m binding to the first (B) and second clusters (D). Protein
part of the representative structures was illustrated as surface to show the binding site and
conformations.
Interaction of 13m with CA HIV-1 monomer fixed it to the open conformation as
shown by Jacques et. al in 5HGL X-ray structure[21]. Figures 5 A and C show that
GLN50 (in both clusters) is kept far from PRO1, HIS12, THR48 and ASN51 triad,
which is important to form hydrogen bond to induce the close conformation. This
1
8

ACCEPTED MANUSCRIPT
could fix the hexamer to one conformation and disables its function. In the first
conformational cluster 13m binds in similar mode of PF-74 but with different amino
acid residues. Where, the phenyl ring of the core region could form hydrophobic
interaction with LYS70 similar to PF-74. Furthermore, 13m forms hydrophobic
interactions with MET66 and LEU56. It also forms aliphatic hydrogen boning with
ASN74 through its methoxy group in the core region, and hydrogen bond with ASN57
at the substituent region. 13m in the other binding mode, which is different from the
binding mode of PF-74, forms hydrophobic interaction with LYS70 through its
substituent region in contrast to the first binding mode. Also, it forms hydrophobic
interactions with MET66 and ALA64 through its core region. Interaction of 13m with
CA HIV-1 monomer in different binding modes involves more amino acids, which
increases the possibility of inhibiting this protein and could accounts for its high
inhibitory activity.
Fig. 5 Illustration of the open form conformation of CA HIV-1 monomer in first (A) and second
1
9

ACCEPTED MANUSCRIPT
(C) clusters. Binding interactions of 13m to the first (B) and second clusters (D).
3
. Conclusions
In brief, taking the most studied HIV CA inhibitor PF-74 as lead compound, we
designed and expeditiously synthesized a series of 1,2,3-triazole-containing
phenylalanine derivatives via CuAAC reaction. Among them, 13m exhibited the best
anti-HIV-1 activity (EC = 4.33 µM, SI > 13.33), being similar to the lead PF-74
5
0
(
EC = 5.95 µM, SI > 11.85). Direct binding studies using SPR demonstrated that
50
this series of phenylalanine derivatives interact with the HIV-1 CA protein,
irrespective of its oligomeric status. In-line with this observation, we have
demonstrated the compound 13m inhibits the replication of HIV-1 in both the early
and late stages. Finally, through molecular dynamics simulation, we can conclude that
1
3m potentially has two different binding modes to HIV-1 CA monomer, which has
implication for the precise manner of CA protein inhibition in each of the discrete
stages of replication. We envisioned that the conformationally dynamic CTD-NTD
interface still have ample space for further modification to form potential interaction
with nearby hotpot residues. Ongoing studies regarding the further development of
these novel capsid inhibitors from our lab will be reported in due course.
4
. Experimental section
4
.1. Chemistry
All melting points of the compounds were determined on a micro melting point
1
13
apparatus and were uncorrected. H-NMR and C-NMR spectra were obtained via a
Bruker Avance-400 NMR-spectrometer in DMSO or CDCl using TMS as internal
3
reference. Chemical shifts were expressed in ꢀ units (ppm) and J values were
presented in hertz (Hz). Related mass spectra dates were determined by a LC
Autosampler Device: Standard G1313A instrument. TLC was performed on Silica Gel
GF254 for TLC and spots were visualized by irradiation with UV light (ꢁ = 254 nm).
Meanwhile, flash column chromatography was performed on column packed with
Silica Gel 60 (200-300 mesh). Rotary evaporator under reduced pressure condition
was used to concentrate the reaction solutions. Solvents were of reagent grade and
2
0

ACCEPTED MANUSCRIPT
purified with standard methods when necessary.
.1.1
4
Tert-butyl(S)-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)carba
mate (2)
A solution of (tert-butoxycarbonyl)-L-phenylalanine (1, 8.75 mmol, 2.3 g) in 15 mL
dichloromethane was added PyBop (10.9 mmol, 5.7 g) at 0℃, and the mixture stirred
for 0.5
h. Subsequently,
DIEA (21.87 mmol, 3.61
mL)
and
4
-methoxy-N-methylaniline (7.29 mmol, 1.0 g) were added to the mixture and then
stirred at room temperature for another 8-9 h (monitored by TLC). The resulting
mixture was evaporated under reduced pressure and the residue was initially washed
by 1N HCl and extracted with ethyl acetate (3 × 20 mL). Then, the combined organic
layer was washed with saturated sodium bicarbonate (3 × 50 mL), dried over
anhydrous Na SO , filtered, and concentrated under reduced pressure to afford
2
4
1
corresponding crude intermediate 2 as yellow oil with a yield of 94%. H NMR (400
MHz, DMSO-d ) δ 7.21 (d, J = 8.4 Hz, 3H), 7.15 (d, J = 7.1 Hz, 2H), 7.02 (d, J = 8.4
6
Hz, 2H), 6.85 – 6.75 (m, 2H), 4.15 (q, J = 5.4 Hz, 1H), 3.80 (s, 3H), 3.12 (s, 3H),
1
3
2
1
3
4
.81–2.54 (m, 2H), 1.30 (s, 9H). C NMR (100 MHz, DMSO) δ 172.21, 158.98,
55.74, 138.53, 136.13, 129.28, 128.47, 126.70, 115.21, 78.33, 55.94, 53.53, 37.86,
+
7.10, 28.65. ESI-MS: m/z 385.4 (M + 1) , C H N O (384.2).
22
28
2
4
.1.2 (S)-2-amino-N-(4-methoxyphenyl)-N-methyl-3-phenylpropanamide (3)
Trifluoroacetate (34.2 mmol, 5.0 eq) was added dropwise to intermediate 2 (2.63 g,
.84 mmol, 1.0 eq) in 30 mL dichloromethane and stirred at room temperature for 6-7
6
h. Then, the resulting mixture solution was alkalized to pH = 9 with saturated sodium
bicarbonate solution, and then extracted with dichloromethane (3 × 20 mL). The
combined organic layer was dried over anhydrous Na SO , filtered, and concentrated
2
4
under reduced pressure to yield the crude product, which was purified by flash
1
column chromatography to afford compound 3 as yellow oil with the yield of 84%. H
NMR (400 MHz, DMSO-d ) δ 7.34–7.15 (m, 3H), 6.90 (m, 6H), 3.77 (s, 3H), 3.35 (t,
6
J = 6.9 Hz, 1H), 3.06 (s, 3H), 2.76 (dd, J = 12.9, 6.8 Hz, 1H), 2.46 (dd, J = 12.9, 7.1
2
1

ACCEPTED MANUSCRIPT
1
3
Hz, 1H). C NMR (100 MHz, DMSO) δ 174.96, 158.76, 139.02, 136.36, 129.51,
1
1
4
28.93, 128.46, 126.54, 115.03, 55.83, 53.38, 42.25, 37.45. ESI-MS: m/z 285.3 (M +
+
) , C H N O (284.1).
17
20
2
2
.1.3
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)propiolamide
(4)
Propiolic acid (4.22 mmol, 0.3 g) and HATU (5.28 mmol, 2.0 g) were mixed in
dichloromethane and stirred in an ice bath for 1 h. Then, the intermediate 3 (6.33
mmol, 1.80 g) and DIEA (7.03 mmol, 1.16 mL) were added to the above solution
slowly at 0℃. The reaction system was then stirred at room temperature for additional
1
2 h. The solvent was removed under reduced pressure and then 1N HCl (30 mL) was
added, extracted with ethyl acetate (3 × 30 mL). The combined organic layer was
washed with saturated sodium bicarbonate (3 × 50 mL). The resulting organic layer
was washed with saturated salt water, dried over anhydrous Na SO , filtered, and
2
4
concentrated under reduced pressure to afford corresponding crude product, which
was purified by flash column chromatography to yield compound 4 as yellow oil with
1
the yield of 38%. H NMR (400 MHz, DMSO-d ) δ 9.16 (d, J = 7.8 Hz, 1H), 7.17 (td,
6
J = 7.0, 4.5 Hz, 5H), 7.00 (d, J = 8.9 Hz, 2H), 6.85 – 6.82 (m, 2H), 4.45 (ddd, J = 9.7,
1
3
7
.7, 4.5 Hz, 1H), 3.79 (s, 3H), 3.12 (s, 3H), 2.91 – 2.71 (m, 2H), 2.69 (s, 1H). C
NMR (100 MHz, DMSO) δ 170.90, 159.10, 151.91, 137.93, 135.87, 129.19, 128.63,
+
1
26.96, 115.17, 78.37, 76.80, 55.91, 52.53, 37.85, 36.86. ESI-MS: m/z 337.6 (M + 1) ,
C H N O (336.1).
20
20
2
3
4
4
.1.4 General procedure for the synthesis of azide substituents
.1.4.1 General procedure for the synthesis of azide substituents (6 and 10)
Benzyl bromide (5, 1 g, 1 eq) and sodium azide (NaN , 1.5 eq) were dissolved in 10
3
mL N, N-dimethylformamide (DMF). The mixture was added 20 mL water after
stirred at room temperature for 12-13 h, and then extracted by ethyl acetate (3 × 20
mL). The organic phase was combined, dried over anhydrous Na SO , filtered, and
2
4
concentrated to yield the crude azide substituent 6, which was used for the next step
without further purification. The abovementioned method was applied to the
1
substituent (azidomethyl)(phenyl)sulfane (10, C H N S). H NMR (400 MHz,
7
7
3
2
2

ACCEPTED MANUSCRIPT
DMSO-d ) δ 7.52–7.47 (m, 2H), 7.42–7.35 (m, 2H), 7.33 – 7.27 (m, 1H), 4.87 (s,
6
2
H).
4
.1.4.2 General procedure for the synthesis of azide substituents (8)
Different substituted phenylboronic acid compounds (7, 1 g, 1 eq), sodium azide
.
(
NaN , 1.5 eq) and copper sulfate pentahydrate (CuSO 5H O, 0.1 eq) were mixed in
3
4
2
the 10 mL methanol and then stirred at room temperature for 8-9 h. Subsequently, the
reaction solvent was removed under reduced pressure and 20 mL of water was added.
Aqueous solution was extracted by ethyl acetate (3 × 20 mL), the combined phase
was dried over anhydrous Na SO , filtered, and concentrated to afford the crude azide
2
4
substituents 8 which was used for the next step directly.
.1.4.3 General procedure for the synthesis of azide substituents (11 and 12)
Under ice cooling, after a solution of 3-chloroperbenzoic acid (mCPBA, 580 mg, 75%,
.26 mmol) in CH Cl (1.0 mL) was added dropwise to a solution of the intermediate
4
1
2
2
(
azidomethyl)(phenyl)sulfane (10, 168 µL, 1.17 mmol) in dry CH Cl (10 mL), the
2 2
mixture was stirred at room temperature for 15 h. Then saturated sodium bicarbonate
solution was poured into the reaction mixture and extracted with ethyl acetate (3 × 50
mL). The combined organic phase was dried over anhydrous sodium sulphate and
then evaporated. The residue was purified using flash chromatography with
n-hexane/ethylacetate to afford azide substituent ((azidomethyl)sulfinyl)benzene (12,
1
C H N O S). H NMR (400 MHz, Chloroform-d) δ 8.00 – 7.94 (m, 2H), 7.77 – 7.70
7
7
3
2
(
m, 1H), 7.63 (dd, J = 8.4, 7.1 Hz, 2H), 4.31 (s, 2H, CH2).
This method was also applied to
(azidomethyl)sulfinyl)benzene (11, C H N OS). Compared to the synthesis of azide
synthesize
substituent
(
7
7
3
substituent ((azidomethyl)sulfinyl)benzene (12), the only difference of
((azidomethyl)sulfinyl)benzene (11) synthesis is that the reaction would be finished
1
after reacting with mCPBA at 0℃ merely for 1 h. H NMR (400 MHz, Chloroform-d)
δ 7.64–7.56 (m, 2H), 7.52–7.35 (m, 3H), 4.21 (d, J = 12.0 Hz, 1H), 3.97 (d, J = 11.9
Hz, 1H)
4
.1.5 General procedure for the synthesis of target compounds (13a-13t)
2
3

ACCEPTED MANUSCRIPT
The key intermediate 4 (1.0 eq), azide substituents (6, 8, 10, 11 and 12, 1.1 eq),
ascorbic acid sodium (0.6 eq) and CuSO .5H O (0.3 eq) were dissolved in the solution
4
2
of tetrahydrofuran/water (v:v = 1:1). The resulting mixture was stirred at 30-60℃ for
-6 h. Then the reaction mixture was extracted with ethyl acetate (3 × 10 mL), and the
4
combined organic phase was washed with saturated salt water (3 × 10 mL), dried over
anhydrous MgSO , filtered, and concentrated under reduced pressure to give the
4
corresponding crude target product, which was purified by flash column
chromatography to afford product 13a-13t. Yield: 30%-80%.
4
.1.5.1
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(2-methyl
benzyl)-1H-1,2,3-triazole-4-carboxamide (13a). White solid, yield: 70%. mp: 60-62℃.
1
H NMR (400 MHz, DMSO-d ) δ 8.49 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.31 – 7.12
6
(m, 8H), 7.08 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.92 – 6.82 (m, 2H), 5.66
(s, 2H), 4.66 (q, J = 7.4 Hz, 1H), 3.80 (s, 3H), 3.12 (s, 3H), 2.81 (m, 2H), 2.30 (s, 3H).
1
3
C NMR (100 MHz, DMSO-d ) δ 171.28, 159.56, 159.07, 142.56, 137.98, 136.76,
6
1
1
5
35.93, 134.20, 130.94, 129.35, 129.24, 129.12, 128.93, 128.60, 127.04, 126.89,
+
26.79, 115.19, 55.92, 51.76, 51.68, 37.88, 37.27, 19.09. ESI-MS: m/z 484.5 (M + 1) ,
+
06.4 (M + 23) , C H N O (483.2).
2
8
29
5
3
4
.1.5.2
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(3-methyl
benzyl)-1H-1,2,3-triazole-4-carboxamide (13b). Yellow solid, yield: 30%. mp:
1
5
7
4
2
1
1
8-60℃. H NMR (400 MHz, DMSO-d ) δ 8.59 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H),
6
.23 – 7.12 (m, 8H), 7.01 (d, J = 8.2 Hz, 3H), 6.88 (d, J = 6.8 Hz, 2H), 5.60 (s, 2H),
.67 (q, J = 6.0 Hz, 1H), 3.81 (s, 3H), 3.12 (d, J = 4.9 Hz, 3H), 2.89 (d, J = 7.1 Hz,
1
3
H), 2.29 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 171.28, 159.56, 159.08, 142.57,
6
37.97, 136.77, 135.93, 134.19, 130.94, 129.35, 129.23, 129.13, 128.93, 128.60,
27.03, 126.89, 126.79, 115.19, 55.91, 51.75, 51.69, 37.88, 37.31, 19.09. ESI-MS:
+
+
m/z 484.5 (M + 1) , 506.4 (M + 23) , C H N O (483.2).
2
8
29
5
3
4
.1.5.3
S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(4-methyl
benzyl)-1H-1,2,3-triazole-4-carboxamide (13c). Yellow solid, yield: 55%. mp:
(
2
4

ACCEPTED MANUSCRIPT
1
6
3-65℃. H NMR (400 MHz, DMSO-d ) δ 8.56 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H),
6
7
.36 – 7.08 (m, 9H), 7.00 (d, J = 8.4 Hz, 2H), 6.92 – 6.83 (m, 2H), 5.58 (s, 2H), 4.66
(q, J = 7.3 Hz, 1H), 3.80 (s, 3H), 3.12 (s, 3H), 2.88 (d, J = 7.1 Hz, 2H), 2.28 (s, 3H).
1
3
C NMR (100 MHz, DMSO-d ) δ 171.27, 159.58, 159.07, 142.67, 138.12, 137.98,
6
1
5
35.92, 133.09, 129.80, 129.34, 129.24, 128.60, 128.47, 126.90, 115.19, 55.91, 53.34,
+
+
1.73, 37.88, 37.27, 21.17. ESI-MS: m/z 484.5 (M + 1) , 506.4 (M + 23) ,
C H N O (483.2).
28
29
5
3
4
.1.5.4
(S)-1-(2-chlorobenzyl)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropa
n-2-yl)-1H-1,2,3-triazole-4-carboxamide (13d). Yellow solid, yield: 60%. mp:
1
1
7
7
3
1
1
3
62-164℃. H NMR (400 MHz, DMSO-d ) δ 8.56 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),
6
.54 (dd, J = 7.6, 1.7 Hz, 1H), 7.40 (pd, J = 7.5, 1.7 Hz, 2H), 7.29 – 7.11 (m, 6H),
.01 (d, J = 8.5 Hz, 2H), 6.94 – 6.75 (m, 2H), 5.76 (s, 2H), 4.67 (q, J = 7.3 Hz, 1H),
13
.80 (s, 3H), 3.12 (s, 3H), 2.89 (d, J = 7.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ
6
71.28, 159.52, 159.08, 142.50, 137.99, 135.93, 133.30, 133.12, 131.07, 130.87,
30.15, 129.35, 129.24, 128.60, 128.26, 127.45, 126.89, 115.19, 55.92, 51.78, 51.41,
+
+
7.88, 37.25. ESI-MS: m/z 504.3 (M + 1) , 526.4 (M + 23) , C H ClN O (503.1).
2
7
26
5
3
4
.1.5.5
(S)-1-(3-chlorobenzyl)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropa
n-2-yl)-1H-1,2,3-triazole-4-carboxamide (13e). White solid, yield: 65%. mp:
1
2
7
8
3
1
1
3
4
88-290℃. H NMR (400 MHz, DMSO-d ) δ 8.64 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H),
6
.42 (dd, J = 6.0, 2.9 Hz, 3H), 7.37 – 7.25 (m, 1H), 7.23 – 7.10 (m, 5H), 7.01 (d, J =
.4 Hz, 2H), 6.93 – 6.80 (m, 2H), 5.67 (s, 2H), 4.67 (q, J = 7.3 Hz, 1H), 3.80 (s, 3H),
1
3
.12 (s, 3H), 2.89 (d, J = 7.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.26,
6
59.54, 159.07, 142.77, 138.44, 138.00, 135.92, 133.78, 131.24, 129.35, 129.25,
28.75, 128.60, 128.37, 127.27, 127.18, 126.89, 115.19, 55.92, 52.75, 51.78, 37.88,
+
+
7.24. ESI-MS: m/z 504.3 (M + 1) , 526.3 (M + 23) , C H ClN O (503.1).
27
26
5
3
.1.5.6
S)-1-(4-chlorobenzyl)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropa
(
n-2-yl)-1H-1,2,3-triazole-4-carboxamide (13f). White solid, yield: 50%. mp:
1
1
72-174℃. H NMR (400 MHz, DMSO-d ) δ 8.61 (s, 1H), 8.34 (d, J = 8.1 Hz, 1H),
6
2
5

ACCEPTED MANUSCRIPT
7
2
3
1
1
.51 – 7.42 (m, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.26 – 7.09 (m, 5H), 7.01 (d, J = 8.8 Hz,
H), 6.93 – 6.81 (m, 2H), 5.65 (s, 2H), 4.67 (q, J = 7.3 Hz, 1H), 3.80 (s, 3H), 3.12 (s,
1
3
H), 2.89 (d, J = 7.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.26, 159.54,
6
59.08, 142.75, 137.98, 135.93, 135.08, 133.47, 130.40, 129.34, 129.28, 129.24,
28.60, 127.16, 126.89, 115.20, 55.92, 52.73, 51.75, 37.88, 37.26. ESI-MS: m/z 504.3
+
+
(
M + 1) , 526.3 (M + 23) , C H ClN O (503.1).
27 26 5 3
4
.1.5.7
(S)-1-(2-cyanobenzyl)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan
-2-yl)-1H-1,2,3-triazole-4-carboxamide (13g). Light yellow solid, yield: 58%. mp:
1
1
7
7
2
68-170℃. H NMR (400 MHz, DMSO-d ) δ 8.62 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H),
6
.93 (dd, J = 7.7, 1.3 Hz, 1H), 7.77 – 7.70 (m, 1H), 7.61 – 7.55 (m, 1H), 7.38 (d, J =
.8 Hz, 1H), 7.17 (dt, J = 13.7, 7.5 Hz, 5H), 7.01 (d, J = 8.5 Hz, 2H), 6.91 – 6.84 (m,
H), 5.87 (s, 2H), 4.67 (q, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.13 (s, 3H), 2.90 (d, J = 6.9
1
3
Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.25, 159.47, 159.08, 142.63, 138.84,
6
1
37.99, 135.92, 134.36, 133.91, 129.94, 129.79, 129.35, 129.25, 128.60, 127.65,
26.89, 117.38, 115.20, 111.66, 55.93, 51.79, 51.76, 37.89, 37.24. ESI-MS: m/z 495.4
1
+
+
+
(
M + 1) , 512.5 (M + 18) , 517.4 (M + 23) , C H N O (494.2).
28 26 6 3
4
.1.5.8
(S)-1-(3-cyanobenzyl)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan
-2-yl)-1H-1,2,3-triazole-4-carboxamide (13h). White solid, yield: 62%. mp:
1
1
7
7
3
1
1
5
68-170℃. H NMR (400 MHz, DMSO-d ) δ 8.65 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H),
6
.90 – 7.81 (m, 2H), 7.62 (dt, J = 15.3, 7.8 Hz, 2H), 7.17 (dt, J = 13.8, 7.6 Hz, 5H),
.01 (d, J = 8.5 Hz, 2H), 6.93 – 6.82 (m, 2H), 5.72 (s, 2H), 4.67 (q, J = 7.3 Hz, 1H),
13
.81 (s, 3H), 3.12 (s, 3H), 2.89 (d, J = 7.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ
6
71.24, 159.50, 159.08, 142.82, 137.97, 137.57, 135.93, 133.45, 132.59, 132.25,
30.60, 129.35, 129.23, 128.60, 127.37, 126.89, 118.85, 115.20, 112.22, 55.92, 52.60,
+
+
1.73, 37.88, 37.31. ESI-MS: m/z 495.4 (M + 1) , 517.5 (M + 23) , C H N O
3
28
26
6
(494.2).
4
.1.5.9
(S)-1-(4-cyanobenzyl)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan
2
6

ACCEPTED MANUSCRIPT
-2-yl)-1H-1,2,3-triazole-4-carboxamide (13i). White solid, yield: 45%. mp:
1
1
7
6
3
1
1
90-192℃. H NMR (400 MHz, DMSO-d ) δ 8.65 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H),
6
.91 – 7.81 (m, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.17 (dt, J = 13.7, 7.6 Hz, 5H), 7.07 –
.96 (m, 2H), 6.94 – 6.82 (m, 2H), 5.77 (s, 2H), 4.68 (q, J = 7.2 Hz, 1H), 3.81 (s, 3H),
1
3
.13 (s, 3H), 2.90 (d, J = 7.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.26,
6
59.53, 159.08, 142.82, 141.51, 138.01, 135.92, 133.25, 129.34, 129.25, 129.20,
28.61, 127.54, 126.89, 118.97, 115.20, 111.51, 55.92, 52.90, 51.80, 37.88, 37.20.
+
+
ESI-MS: m/z 495.4 (M + 1) , 517.5 (M + 23) , C H N O (494.2).
2
8
26
6
3
4
.1.5.10
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(2-nitrobe
nzyl)-1H-1,2,3-triazole-4-carboxamide (13j). Yellow solid, yield: 60%. mp: 63-65℃.
1
H NMR (400 MHz, DMSO-d ) δ 8.57 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.16 (d, J =
6
8
.0 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.18 (dt, J = 13.2, 6.8
Hz, 5H), 7.09 (d, J = 7.7 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 6.94 – 6.83 (m, 2H), 6.02
(s, 2H), 4.68 (q, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.13 (s, 3H), 2.91 (d, J = 6.9 Hz, 2H).
1
3
C NMR (100 MHz, DMSO-d ) δ 171.28, 159.51, 159.09, 148.01, 142.64, 138.00,
6
1
1
35.94, 134.93, 130.82, 130.69, 130.26, 129.35, 129.25, 128.61, 127.89, 126.90,
+
25.63, 115.20, 55.92, 51.80, 50.80, 37.88, 37.25. ESI-MS: m/z 515.5 (M + 1) , 537.4
+
(
M + 23) , C H N O (514.2).
2
7
26
6
5
4
.1.5.11
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(3-nitrobe
nzyl)-1H-1,2,3-triazole-4-carboxamide (13k). Light yellow solid, yield: 67%. mp:
1
6
8
7
2
1-63℃. H NMR (400 MHz, DMSO-d ) δ 8.69 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H),
6
.25 (t, J = 2.0 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.69 (t, J =
.9 Hz, 1H), 7.18 (dd, J = 18.4, 7.9 Hz, 5H), 7.01 (d, J = 8.4 Hz, 2H), 6.92 – 6.83 (m,
H), 5.83 (s, 2H), 4.67 (q, J = 7.2 Hz, 1H), 3.80 (s, 3H), 3.12 (s, 3H), 2.89 (d, J = 6.9
1
3
Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.24, 159.50, 159.08, 148.37, 142.83,
6
1
1
1
38.11, 137.98, 135.93, 135.23, 130.94, 129.34, 129.24, 128.59, 127.44, 126.89,
23.73, 123.38, 115.20, 55.92, 52.51, 51.77, 37.88, 37.25. ESI-MS: m/z 515.5 (M +
+
+
) , 537.4 (M + 23) , C H N O (514.2).
2
7
26
6
5
4
.1.5.12
2
7

ACCEPTED MANUSCRIPT
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(4-nitrobe
nzyl)-1H-1,2,3-triazole-4-carboxamide (13l). Light yellow solid, yield: 57%. mp:
1
6
5-67℃. H NMR (400 MHz, DMSO-d ) δ 8.67 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),
6
8
.29 – 8.20 (m, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.17 (dt, J = 13.8, 7.7 Hz, 5H), 7.01 (d,
J = 8.8 Hz, 2H), 6.93 – 6.84 (m, 2H), 5.84 (s, 2H), 4.68 (q, J = 7.2 Hz, 1H), 3.81 (s,
1
3
3
1
1
H), 3.13 (s, 3H), 2.90 (d, J = 7.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.25,
6
59.51, 159.09, 147.77, 143.44, 142.85, 137.99, 135.93, 129.53, 129.34, 129.24,
28.61, 127.60, 126.89, 124.42, 115.21, 55.93, 52.64, 51.78, 37.88, 37.25. ESI-MS:
+
m/z 515.4 (M + 1) , C H N O (514.2).
27
26
6
5
4
.1.5.13
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(naphthal
en-2-ylmethyl)-1H-1,2,3-triazole-4-carboxamide (13m). White solid, yield: 65%. mp:
1
7
2-74℃. H NMR (400 MHz, DMSO-d ) δ 8.66 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H),
6
7
.93 (dd, J = 8.9, 4.9 Hz, 3H), 7.87 (s, 1H), 7.54 (dd, J = 6.5, 3.1 Hz, 2H), 7.49 – 7.41
(m, 1H), 7.24 – 7.12 (m, 5H), 7.00 (d, J = 8.4 Hz, 2H), 6.94 – 6.82 (m, 2H), 5.82 (s,
1
3
2
H), 4.67 (q, J = 7.4 Hz, 1H), 3.80 (s, 3H), 3.12 (s, 3H), 2.88 (d, J = 7.0 Hz, 2H). C
NMR (100 MHz, DMSO-d ) δ 171.26, 159.57, 159.08, 142.74, 137.97, 135.93,
6
1
33.57, 133.21, 133.01, 129.35, 129.23, 129.05, 128.60, 128.31, 128.09, 127.46,
27.19, 127.07, 126.98, 126.88, 126.11, 115.20, 55.91, 53.73, 51.73, 37.88, 37.31.
1
+
+
ESI-MS: m/z 520.4 (M + 1) , 542.4 (M + 23) , C H N O (519.2).
3
1
29
5
3
4
.1.5.14
(S)-N-(1-((4-methoxyphenyl)(methyl)amino)-1-oxo-3-phenylpropan-2-yl)-1-(naphthal
en-1-ylmethyl)-1H-1,2,3-triazole-4-carboxamide (13n). White solid, yield: 68%. mp:
1
7
8
3
6
8-80℃. H NMR (400 MHz, DMSO-d ) δ 8.54 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H),
6
.16 (d, J = 8.1 Hz, 1H), 7.99 (t, J = 8.5 Hz, 2H), 7.57 (ddt, J = 20.7, 14.9, 7.3 Hz,
H), 7.43 (d, J = 7.1 Hz, 1H), 7.24 – 7.09 (m, 5H), 6.99 (d, J = 8.4 Hz, 2H), 6.91 –
.80 (m, 2H), 6.15 (s, 2H), 4.64 (q, J = 7.6 Hz, 1H), 3.79 (s, 3H), 3.10 (s, 3H), 2.87 (d,
1
3
J = 5.3 Hz, 2H). C NMR (100 MHz, DMSO-d ) δ 171.25, 159.50, 159.06, 142.56,
6
1
1
3
4
37.95, 135.91, 133.84, 131.54, 130.99, 129.67, 129.33, 129.21, 128.58, 127.85,
27.35, 127.12, 126.88, 126.69, 126.05, 123.59, 115.17, 55.91, 51.72, 51.54, 37.86,
+
+
7.29. ESI-MS: m/z 520.4 (M + 1) , 542.4 (M + 23) , C H N O (519.2).
31
29
5
3
.1.5.15
2
8