Synthesis and SARs of indole-based α-amino acids as potent HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1039/c4ob01333f

A series of non-nucleoside reverse transcriptase inhibitors derived from indole-based α-amino acids were designed and synthesized. Their inhibitory activities were detected by a TZM-bl cell assay on HIV virus type HIV-1_IIIB. The comprehensive understanding of the SAR was obtained by utilizing the variation of the substituents of the indole-based α-amino acids. From the screened compounds, the novel inhibitors 19 and 29 were identified to be highly potent candidates with EC₅₀ values of 0.060 μM and 0.045 μM respectively (CC₅₀ values of 109.545 μM and 49.295 μM and SI values of 1825.8 and 1095.4). In most cases, the variation of substituents at different positions had a significant effect on the potency of activities. The results also indicate that the indole-based α-amino acids as efficient NNRTIs displayed comparable anti-HIV-1 activities to the reference drug NVP. We hope the identification of these indole-based amino acids as efficient NNRTIs of RT could stimulate researchers to develop more diversified anti-HIV drugs. This journal is

Full text of DOI:10.1039/c4ob01333f

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Synthesis and SARs of indole-based α-amino acids
as potent HIV-1 non-nucleoside reverse
transcriptase inhibitors†
Cite this: DOI: 10.1039/c4ob01333f
Xin Han,a Haoming Wu,b Wei Wang,a Chune Dong,a Po Tien,b Shuwen Wu*b and
Hai-Bing Zhou*a
A series of non-nucleoside reverse transcriptase inhibitors derived from indole-based α-amino acids were
designed and synthesized. Their inhibitory activities were detected by a TZM-bl cell assay on HIV virus type
HIV-1IIIB. The comprehensive understanding of the SAR was obtained by utilizing the variation of the substi-
tuents of the indole-based α-amino acids. From the screened compounds, the novel inhibitors 19 and 29
were identified to be highly potent candidates with EC50 values of 0.060 μM and 0.045 μM respectively
(CC50 values of 109.545 μM and 49.295 μM and SI values of 1825.8 and 1095.4). In most cases, the variation
of substituents at different positions had a significant effect on the potency of activities. The results also
indicate that the indole-based α-amino acids as efficient NNRTIs displayed comparable anti-HIV-1 activities
to the reference drug NVP. We hope the identification of these indole-based amino acids as efficient
NNRTIs of RT could stimulate researchers to develop more diversified anti-HIV drugs.
Received 30th June 2014,
Accepted 21st August 2014
DOI: 10.1039/c4ob01333f
www.rsc.org/obc
The acquired immunodeficiency syndrome (AIDS) is an incur-
able and potentially fatal disease with more than 30 million
people being affected worldwide. It is also estimated that more
than 25 million people died of AIDS. Therefore, current con-
ditions present a public health challenge on a global scale.1,2
Extensive research has been focused on this area and potential
drug targets have been identified. Since HIV-1 is the patho-
genic factor of AIDS, the design and synthesis of anti-HIV-1
reagents play an important role in the development of AIDS
drugs.3,4 So far, several types of anti-HIV-1 drugs have been
reported, for example AZT is the first anti-HIV-1 drug by FDA
as a nucleoside reverse transcriptase inhibitor (NRTI); non-
nucleoside reverse transcriptase inhibitors (NNRTIs), which
target the reverse transcriptase (RT) of HIV-1, represent a sig-
nificant progress in HIV treatment.5 Nevirapine (NVP) and
delavirdine (DLV), as the first-generation NNRTIs, have already
been approved for use in HIV-1 treatment; efavirenz (EFV),
etravirine and rilpivirine, as the new generation NNRTIs, have
also been applied in clinical treatment of HIV widely
(Scheme 1).6–9 Despite the major improvement achieved with
NNRTIs, drug resistance remains an unsolved problem; there-
fore, new bioactive compounds that can effectively inhibit
Scheme 1 Structures of HIV-1 reverse transcriptase inhibitors dis-
cussed in the text.
commonly observed NNRTI-associated resistant viruses are
highly desirable.
Currently, efforts on the development of novel small mole-
cules as reverse transcriptase inhibitors based on the indole
scaffold are greatly intensified. For example, in 2011, Alexandre
and co-workers reported a series of 3-aryl-phospho-indole (API,
7, Scheme 2) NNRTIs of HIV-1.10 These APIs showed similar
binding style in the palm domain of the p66 chain to the
known AIDS-drug EFV through at least three interactions with
K101, W229, F227, Y181, and Y188. Later, some new indolyl-
arylsulfone (IAS, 8, Scheme 2) derivatives were developed by
Silvestri and co-workers as highly potent NNRTIs and the
corresponding anti-HIV-1 activities lowered to approximately
2.0 nM in MT-4 cells.11–19 Then, Pelly and co-workers reported
aSchool of Pharmaceutical Sciences, State Key Laboratory of Virology,
Wuhan University, Wuhan, China, 430071
bCollege of Life Sciences, Wuhan University, Wuhan, China, 430072.
E-mail: zhouhb@whu.edu.cn, shuwenwu@hotmail.com
†Electronic supplementary information (ESI) available: NMR spectra and HPLC
profiles. See DOI: 10.1039/c4ob01333f
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catalyzed by Lewis acids. To the best of our knowledge, there is
no report on indole-based α-amino acid based small molecular
inhibitors against HIV-1.
Herein, we report the development of α-trifluoromethyl-
α-(heteroaryl)-glycine derivatives as potent HIV-1 NNRTIs. The
initial compounds were synthesized by Friedel–Crafts amino-
alkylation of indoles with imine catalyzed by Lewis acids.30
The anti-HIV activity was detected using a HIV-1IIIB/TZM-bl
indicator cell culture system. Overall, we anticipated that the
identified indole-based amino acid scaffold may aid in the
design of new NNRTIs and stimulate other researchers to
develop more diversified anti-HIV drugs.
Scheme 2 Indole-based NNRTIs.
novel cyclopropylindole (9, Scheme 2) compounds inhibiting
HIV-1 in 293 T cells and VLP (virus-like particles) with an EC50
of 65 nM.20 More recently, the Jiang group reported a novel
series of trifluoromethyl indole derivatives as potent HIV-1
NNRTIs in MT-2 cells (10, Scheme 2).21
Chemistry
The synthesis pathways are outlined in Scheme 4. All com-
pounds were prepared by the Friedel–Crafts reaction catalyzed
by Lewis acids in good to excellent yields (87–96%) (Table 1).
Firstly, the imines 13 and 14 were prepared from methyl or
ethyl trifluoropyruvates and PMP (p-methoxy-phenylamine) in
the presence of TsOH in benzene at reflux with 85% and 87%
yields, respectively. Compounds 15–40 were prepared in one
step from indoles and imines 13 catalyzed by 20 mol% of
Cu(CF3COO)2 in CH2Cl2 at 25 °C. The compounds 42–49 were
prepared by the Friedel–Crafts reaction of indoles and imine
14 in the presence of 1 equivalent of AlCl3 in CH2Cl2 followed
by deprotection of PMP in the presence of 3 equivalents of CAN.
At the same time, non-racemic trifluoromethylated indoles
(TFMIs) represent another type of indole-based derivative;
however, there has been limited information regarding the
enantioselective discrimination of reverse transcriptase (RT) of
HIV-1 with these non-racemic indole derivatives. In 2013, our
group developed enantioselective and efficient inhibitors of
reverse transcriptase (RT) of HIV-1 based on TFMIs by a TZM-
bl cell assay (11, Scheme 2).22,23 The inhibitory activity of the
two enantiomers and racemic ones was compared. TZM-b1
cells exhibited strong enantioselective discrimination for the
R-indole derivatives. In order to further understand the struc-
ture–activity relationship (SAR) of the indole-based inhibitors
of reverse transcriptase and find more efficient NNRTIs of this
type, in this work we have developed an indole-based α-amino
acid library by means of changing the –OH group of TFMIs to
an amine group (Scheme 3). The conception of this project is
largely based on the following facts: (1) amino acids (α-amino
acids) are among the most basic components of living things,
many of which, including unnatural amino acids, are drug
candidates or building blocks for peptide drugs;24–29 (2) on the
other hand, indole-based α-amino acids could be readily
prepared by the Friedel–Crafts reaction of indoles and imines
Structure–activity relationship (SAR)
studies
All compounds were assayed for their anti-HIV activity using a
HIV-1IIIB/TZM-bl indicator cell culture system as previously
described.22,31 The in vitro anti-HIV-1 activity and cytotoxicity
Scheme 3 The comparison of –OH with –NH2 on the anti-HIV activity.
Scheme 4 Synthetic routes to target compounds.
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Table 1 Anti-HIV activity of α-trifluoromethyl-α-(heteroaryl)-glycine derivatives 15–49 in TZM-bl cells
Entry
Compounds
R1
R2
R3
Yield (%)
EC50a (µM)
CC50b (µM)
SIc
1
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
NVP
H
H
5-F
5-Cl
5-Br
5-NO2
5-CN
5-CHO
5-COOMe
5-Me
5-OMe
5-OH
6-F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1-Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
92
96
93
90
91
89
94
91
91
90
92
90
89
87
91
89
92
93
91
94
90
89
88
89
90
93
92
91
94
90
91
90
92
91
89
—
2.445
7.416
0.434
1.715
0.060
1.057
2.120
1.909
0.813
2.398
2.055
5.955
0.342
0.574
0.045
0.785
1.606
8.659
46.473
NAe
69.869
285.320
>131.475
>124.729
>109.545
78.491
28.6
38.5
>302.9
>72.7
>1825.8
74.3
42.4
66.7
>142.9
>55.6
>61.5
13.3
>384.4
>217.3
1095.4
61.5
40.0
8.1
2d
3
4
5
6
7
8
9
89.957
>127.283
>116.183
>133.321
>126.472
79.405
>131.475
>124.729
49.295
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27f
28
29
30
31
32
33
34
35
36
6-Cl
6-Br
6-NO2
6-CN
6-Me
4-COOMe
4-OH
4-Me
2-Me
7-Me
H
H
H
H
H
48.302
64.255
69.936
>116.183
>132.341
56.616
>133.321
>133.321
>133.321
>112.239
>112.239
40.778
>2.5
—
—
NA
NA
—
4.991
10.990
9.821
NA
8.410
3.139
NA
4.239
1.481
NA
>26.7
>12.1
>11.4
—
4.8
30.4
—
>30.8
>76.9
—
1-Isopentyl
1-Pentyl
Et
Et
Et
H
H
H
H
H
H
H
H
H
—
Me
Me
Me
Me
Me
Me
Me
Me
—
95.510
5-F
5-Cl
103.424
130.434
113.921
>139.736
>139.725
34.935
5-Br
2-Me
5-Me
7-Me
5-OMe
—
NA
5.590
NA
—
6.2
132.314
150.208
—
0.034
4417.9
a EC50: the effective concentration (μM) for 50% inhibition of HIV-1 (HIV-1IIIB strain) as evaluated with the luciferase activity in TZM-bl cells.
b CC50: the cytotoxic concentration (μM) to induce 50% death of non-infected cells, as evaluated with the MTT method in TZM-bl cells. c SI: the
selectivity index calculated as the CC50/EC50 ratio. d NH2 was replaced by OH, ref. 22. e NA: no activity. f NH2 was protected by PMP.
associated with the corresponding compounds are described determining the anti-HIV-1 activity. Overall, the compounds
in Table 1. As shown in Table 1, most of the indole-based with an electron-withdrawing group on the phenyl ring exhibi-
α-amino acids showed moderate to potent inhibitory ted excellent inhibition (entries 3–9). The same trend was
activity against HIV-1IIIB with EC50 values in the range of observed when the substitution occurred at the 6-position of
46.473–0.045 µM (entries 1–35). Among them, the target com- the phenyl ring (entries 13–17). Exhilaratingly, the compounds
pounds 19 and 29 displayed excellent anti-HIV-1 activities with an electron-donating group on the same position pro-
against HIV-1IIIB with EC50 values of 0.060 and 0.045 µM and vided superior inhibitory activity to the corresponding indole-
good selectivity indices (SI, CC50/EC50) of 1825.8 and 1095.4, alkyl trifluoropyruvate derivatives (entries 10–12). Other impor-
respectively (entries 5 and 15). The indole-based α-amino acid tant SAR information is as follows: (1) installation of the ethyl
15 exhibited better anti-HIV-1 activity than 16, in which the group at R3 induced more robust inhibition than their methyl
amine group was replaced by OH, giving a moderate EC50 of analogues (entries 1 vs. 28, 3 vs. 29, 4 vs. 30, 4 vs. 31, 10 vs. 33,
2.445 µM in TZM-bl cells (entries 1 and 2). A decreased 11 vs. 35, 23 vs. 34); (2) when an electron-withdrawing group is
potency was observed when the NH2 group was protected by present at the C-5 position of the phenyl ring, instead of the
PMP (entries 1 vs. 27). Further study revealed that the substitu- C-6 position, the potency of the resulting compound dimin-
ent on the phenyl ring of indole was an important factor in ished (entries 3 vs. 13, 4 vs. 14, 5 vs. 15, 6 vs. 16, 7 vs. 17); (3)
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having a substituent group at the 4-position or the 2-position Rac-15 and 19 exhibited better anti-HIV-1 activity than the
resulted in an overall decrease in potency (entries 9 vs. 19, 10 corresponding OH analogues 16 and 50. The results indicated
vs. 21, 12 vs. 20); (4) N-protected indoles also gave moderate that the NH2 group could enhance the inhibitory activities
but reduced anti-HIV-1 activities which demonstrated the sig- against HIV-1IIIB
.
nificance of NH in heterocycles for blocking the reverse tran- Lastly, in order to ascertain whether the compounds were
scriptase (entries 24, 25 and 26); (5) the cytotoxicity (CC50 also inhibitory in primary T-lymphocyte cells using virus
values) study showed that indole-based α-amino acids 19 and strains that belong to different clades of HIV-1,31 compounds
29 had good SI values of >1825.8 and 1095.4, respectively 17 and 29 were included in this study (Table 3). The com-
(entries 5 and 15).
pounds showed moderate inhibitory activity against virus
In order to demonstrate whether the chirality and the members that belonged to clade A, clade B, clade C, and group
amino group have a significant influence on the anti-HIV-1 O (EC50 in the nanomolar or lower micromolar range). The
activity or not, firstly the chiral indole-based α-amino acids clade viruses (92US657 and 93IN101), in contrast to HIV-1IIIB
,
were synthesized by the Mitsunobu reaction of indole-alkyl tri- were R-tropic, the clade viruses (BCF02 and 92UG029) were
fluoropyruvate derivatives as shown in Scheme 5. With this X-tropic, while the clade virus (93TH051) was X/R tropic. These
method, R-15, S-15, R-19 and S-19 were obtained in moderate data indicated that this class of compounds possesses a broad
yields but with the retained enantiopurities. In the analysis of spectrum of anti-HIV-1 activity.
different stereoisomers for the anti-HIV activity using a
To further understand the structure–binding relationships,
HIV-1IIIB/TZM-bl indicator cell culture system, the results indi- the binding mode of R-19 was studied by docking experiments
cated that the R-enantiomer showed much better anti-HIV into the non-nucleoside binding site (NNBS) of RT. The crystal
activity than the corresponding S-enantiomer (Table 2). In the structure of WT RT NNBS with the indolylarylsulfone scaffold
cases of compounds R-15 and R-19, the anti-HIV activities (PDB 2rf2) was used as a model in the docking experiments
lowered to 1.293 μM and 0.047 μM, respectively, revealing that due to the similar structure of indolylarylsulfone to those of
the chirality had a very significant effect on the activity of the indole-based α-amino acids.32 AutoDock Vina33 was chosen to
ligands. Secondly, in Table 2, one can see that all the R- and study the binding modes of R-19 and the result showed good
binding score (−8.5 kcal mol−1), in which R-19 took the
similar position to that of the indolylarylsulfone in the protein
complex structure, and a H-bond was formed between the
indole NH and the carbonyl oxygen of Lys101. In the proposed
structure of the R-19-protein, both trifluoromethyl and ethyl
groups fitted well in the pocket (Scheme 6A). The NH2 group
seems to have weak interactions with the WT RT NNBS similar
to the OH group in our previous work.22 The possible binding
modes provided by AutoDock Vina revealed that this series of
indole-based α-amino acids could be embedded in the pocket
formed by Y181, Y188, F227, W229, etc. The superimposition
of R-19 and the previous compound 11 in the same active site
is shown in Scheme 6B which gave a highly coincident confor-
mation in RT.
Scheme 5 Mitsunobu reaction for preparation of chiral indole-based
α-amino acids.
Table 2 The chiral analysis of OH and NH2 in indole-based trifluoropyruvate derivatives for anti-HIV-1 activity
Config.
R
Rac
S
R
Rac
S
EC50a (μM)
CC50b (µM)
SIc
X = OH
X = NH2
X = OH
X = NH2
X = OH
X = NH2
2.994
1.293
278.522
115.284
93.0
7.416
2.445
285.320
69.869
38.5
79.379
NAd
306.46
115.284
3.9
0.093
0.047
34.140
90.375
367.1
2.895
0.060
50.326
>109.545
17.4
3.004
0.438
46.563
90.375
15.5
89.2
28.6
—
1922.9
>1825.8
206.3
a EC50: the effective concentration (μM) for 50% inhibition of HIV-1 (HIV-1IIIB strain) as evaluated with the luciferase activity in TZM-bl cells.
b CC50: the cytotoxic concentration (μM) to induce 50% death of non-infected cells, as evaluated with the MTT method in TZM-bl cells. c SI: the
selectivity index calculated as the CC50/EC50 ratio. d NA: no activity.
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Table 3 Inhibitory activity of compounds 17 and 29 against different
primary HIV-1 strains in peripheral blood mononuclear cells (PBMC)
EC50a (μM)
Clade A
(92UG029)
Clade B
(92US657)
Clade C
(93IN101)
Group O
(BCF02)
Entry
Cmpds
Scheme 7 Schematic representation of the mechanism of DNA
polymerization (black) and a proposed molecular mechanism of indole
α-amino acids (IAA, red).
1
2
17
29
0.779
0.075
1.190
0.117
1.450
0.101
1.075
0.175
a EC50
: 50% effective concentration or compound concentration
required to inhibit HIV-1 p24 production in virus-infected PBMC. Data
are the mean of two independent experiments.
to other polymerases;34–36 it completes three structurally dis-
tinct steps in a clockwise cycle. However, binding of an IAA to
RT may cause several conformational changes. IAA shifts the
primer end from the P site to the P′ site. This P′-complex is
catalytically incompetent as the primer terminus moves out
from the polymerase active site. The thumb subdomain is
locked at a hyper-extended position, interactions between DNA
and the polymerase domain are reduced, the dNTP-binding
site is distorted and the RNase H active site is repositioned
with respect to the polymerase active site.
In conclusion, we have synthesized a series of indole
α-amino acids bearing various substituents with low nano-
molar concentrations and weak cytotoxicity as novel NNRTIs
in the HIV-1IIIB/TZM-bl indicator cell culture system compared
with the reference drug NVP. Of all these results, higher inhibi-
tory activities against HIV-1IIIB were obtained compared with
our previous indole-alkyl trifluoropyruvate derivatives. A com-
prehensive understanding of SAR of indole α-amino acids is
reported for the first time. Detailed SAR discussions indicated
that variation of substituents at different positions had a sig-
nificant effect on their potency. At the same time, the activity
difference of the stereoisomers was also investigated in this
study. It was found that changing the R-isomer to the S-isomer
greatly reduced the potency and even induced inactivity. Fur-
thermore, this work fully expanded the scope of indole deriva-
tives as anti-HIV agents. We hope the indole based amino
acids identified herein can stimulate researchers to develop
more diversified and effective anti-HIV drugs. The preparation
and in-depth pharmacological evaluation of the chiral –NH2
bearing compounds with good anti-HIV activity are underway
in our laboratory and will be reported in due course.
Experimental section
Materials
Scheme 6 (A) Molecular orientations of R-19 and H-bonding between Unless otherwise noted, reagents and materials were obtained
the indole NH moiety of R-19 and lysine-101 by the AutoDock Vina
docking result with good score; (B) the superimposition between R-19
(red, bearing NH2) with 11 (green, bearing OH) on the same active site in
the RT.
from commercial suppliers and were used without further
purification. THF and CH2Cl2 were dried over Na and CaCl2
and distilled prior to use, respectively. Reactions were moni-
tored by thin layer chromatography (TLC) and column chromato-
graphy purification was performed using 230–400 mesh silica
The possible mechanism of action by this novel class of
indole α-amino acids (IAAs) is proposed as shown in
Scheme 7. The mechanism of polymerization by RT is similar
gel. NMR spectra were measured on Bruker DRX and DMX
1
spectrometers at 400 MHz for H spectra and at 100 MHz for
13C spectra and calibrated from the residual solvent signal.
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The imines 13 and 14,37 1-isopentylindole and 1-pentylindole,38 tr = 13.958 min, the major enantiomer at tr = 20.863 min; 88%
were prepared according to the literature procedures. All final ee; [α]2D0 = +12.6 (c = 0.56, CHCl3). LR-ESI: [M]+ 286.
products were characterized by 1H NMR, 13C NMR and MS ana-
Ethyl 3,3,3-trifluoro-2-hydroxy-2-(1H-indol-3-yl)-propanoate
lyses. Except for the known compounds,39,40 all new compounds 16.22 99% yield. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.89
were also characterized and confirmed by HRMS.
(d, J = 8.1 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H), 7.34 (d, J = 8.1 Hz,
1H), 7.28–7.10 (m, 3H), 4.65–4.27 (m, 2H), 1.33 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 169.42, 136.35, 125.13,
124.99, 124.44, 122.70, 121.15, 121.14, 120.53, 111.39, 108.65,
General procedure for the synthesis of indole-based α-amino
acid compounds
(i) Indole-based α-amino acid ethyl esters. With Cu- 64.25, 13.93. HRMS (ESI) calcd for C13H12F3NO3 [M + Na]+
(CF3CO2)2 (0.2 equiv.) as the Lewis acid, the Friedel–Crafts 310.0667, found, 310.0647.
reaction of indole (0.2 mmol, 23.4 mg) with ethyl 3,3,3-tri-
fluoro-2-((4-methoxyphenyl)imino)propanoate (0.2
mmol, propanoate 17. 93% yield. 1H NMR (400 MHz, CDCl3) δ 8.33
55 mg, 1 equiv.) progressed in CH2Cl2 at room temperature (s, 1H), 7.58 (dt, J = 12.2, 6.1 Hz, 1H), 7.50 (d, J = 2.6 Hz, 1H),
Ethyl
2-amino-3,3,3-trifluoro-2-(5-fluoro-1H-indol-3-yl)-
giving full conversion and excellent yield.
7.28–7.25 (m, 1H), 6.97 (td, J = 9.0, 2.5 Hz, 1H), 4.46–4.37 (m,
(ii) Indole-based α-amino acid methyl esters. With AlCl3 2H), 1.36 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
1
(1 equiv.) as the Lewis acid, the Friedel–Crafts reaction of δ 169.18, 158.12 (d, JC–F = 235.33 Hz), 132.88, 126.10, 125.62,
indole (0.2 mmol, 23.4 mg) with methyl 3,3,3-trifluoro-2- 119.49, 113.92, 111.99 (d, JC–F = 10.10 Hz), 111.31 (d, JC–F
2
3
=
((4-methoxyphenyl)-imino)propanoate (0.2 mmol, 52.2 mg, 27.27 Hz), 106.33, 64.39, 55.38, 13.92, 13.77. HRMS (ESI) calcd
1 equiv.) progressed in CH2Cl2 at room temperature giving for C13H12F4N2O2 [M + Na]+ 327.0733, found, 327.0738.
almost full conversion and excellent yield. Then, the PMP
Ethyl
2-amino-2-(5-chloro-1H-indol-3-yl)-3,3,3-trifluoro-
group could be removed in the presence of 3 eq. CAN (Ceric propanoate 18. 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.36
Ammonium Nitrate) in the mixture solvents of CH3CN and (s, 1H), 7.92 (s, 1H), 7.50 (d, J = 2.6 Hz, 1H), 7.26 (d, J = 4.6 Hz,
H2O at 0 °C within 30 min.41,42
1H), 7.18 (dd, J = 8.7, 1.9 Hz, 1H), 4.47–4.39 (m, 2H), 1.37 (t,
J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 169.10, 134.74,
126.36, 126.19, 125.77, 123.13, 120.99, 112.29, 108.56, 72.39,
Synthesis of the chiral indole-based α-amino acid R-15
1.17 mL H2SO4 (98%) was added to a mixture of NaN3 (2.8 g), 64.46, 13.92. LR-ESI: [M]+ 320.
H2O (2.8 mL) and 16.7 mL benzene dropwise at 0 °C. After Ethyl
2-amino-2-(5-bromo-1H-indol-3-yl)-3,3,3-trifluoro-
40 min, the mixture solvent was filtered and dried with Na2SO4 propanoate 19. 91% yield. 1H NMR (400 MHz, CDCl3) δ 8.34
for use in the next step. The freshly prepared HN3 in a solution (s, 1H), 8.00 (s, 1H), 7.35 (s, 1H), 7.18 (d, J = 10.3 Hz, 1H), 7.11
of benzene was added to a mixture of S-16 (86.2 mg, (d, J = 8.6 Hz, 1H), 4.40–4.27 (m, 3H), 1.28 (t, J = 7.1 Hz, 3H).
0.3 mmoL), PPh3 (94.4 mg, 0.36 mmol) and 5 mL THF at once, 13C NMR (100 MHz, CDCl3) δ 169.09, 135.02, 127.65, 126.78,
and then 1 mL THF containing DIAD (0.36 mmol) was added 125.65, 125.29, 123.94, 113.94, 112.79, 108.33, 64.52, 62.69,
dropwise slowly at 0 °C. After 4 h at 50 °C, 1 mL THF solution 13.92. The enantiomeric excess (ee) was determined by HPLC
of PPh3 (84.7 mg, 0.3 mmol) was added to the mixture and with a Chiralpak OD-H column (90 : 10 hexane–2-propanol,
then the temperature was reduced to 40 °C for 40 min; to this 1.0 mL min−1, 220 nm); Rac- 11.545 min/15.367 min; R- the
0.2 mL H2O was added and the reaction mixture was kept at minor enantiomer at tr = 10.968 min, the major enantiomer at
40 °C for 3 h. Then, the solvent was removed under vacuum, tr = 14.863 min; −87% ee; [α]2D0 = −8.27 (c = 0.7, CHCl3); S- the
and the residue was dissolved in a mixture of CH2Cl2 and major enantiomer at tr = 12.125 min, the minor enantiomer at
CF3CO2H; the organic phase was retained, and the aqueous tr = 16.253 min; 67% ee; [α]2D0 = +14.77 (c = 0.85, CHCl3);
phase was removed under vacuum to give a salt. The salt was LR-ESI: [M]+ 364.
neutralized with NEt3 and extracted with ether, the ether was
removed giving the target compound R-15 with 90% ee and propanoate 20. 89% yield. H NMR (400 MHz, CDCl3) δ 10.58
Ethyl
2-amino-3,3,3-trifluoro-2-(5-nitro-1H-indol-3-yl)-
1
45% yield.
(s, 1H), 8.97 (s, 1H), 8.10 (d, J = 9.1 Hz, 1H), 7.72 (s, 1H),
Ethyl
2-amino-3,3,3-trifluoro-2-(1H-indol-3-yl)-propanoate 7.50–7.44 (m, 1H), 5.16 (s, 1H), 4.52–4.38 (m, 2H), 2.44 (s, 1H),
15. The compound was prepared according to the general pro- 1.40 (td, J = 7.1, 1.3 Hz, 3H). 13C NMR (100 MHz, CDCl3)
1
cedure in 92% yield. H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), δ 168.50, 141.95, 139.72, 128.19, 124.52, 118.81, 117.53,
7.88 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 111.69, 110.69, 67.01, 64.13, 13.73. HRMS (ESI) calcd for
7.24–7.19 (m, 1H), 7.18–7.11 (m, 1H), 4.45–4.34 (m, 2H), 1.31 C13H12F3N3O4 [M + Na]+ 354.0678, found, 354.0681.
(t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 168.37, 135.28,
Ethyl
2-amino-2-(5-cyano-1H-indol-3-yl)-3,3,3-trifluoro-
124.03, 123.94, 123.41, 121.63, 121.09, 120.00, 119.46, 110.38, propanoate 21. 94% yield. 1H NMR (400 MHz, CDCl3) δ 9.08
107.46, 72.52 (q), 63.21, 12.85. The enantiomeric excess (ee) (s, 1H), 8.36 (s, 1H), 7.64 (d, J = 2.7 Hz, 1H), 7.49–7.40 (m, 2H),
was determined by HPLC with a Chiralpak OD-H column 4.55 (d, J = 16.2 Hz, 1H), 4.51–4.36 (m, 2H), 1.38 (t, J = 7.1 Hz,
(90 : 10 hexane–2-propanol, 1.0 mL min−1, 220 nm); Rac- 3H). 13C NMR (100 MHz, CDCl3) δ 168.76, 138.17, 127.56,
15.015 min/21.832 min; R- the major enantiomer at tr
=
126.90, 125.35, 125.11, 124.69, 112.54, 109.47, 103.54, 72.98,
13.677 min, the minor enantiomer at tr = 21.258 min; −90% 64.66, 13.94. HRMS (ESI) calcd for C14H12F3N3O2 [M + Na]+
ee; [α]2D0 = −10.27 (c = 1.0, CHCl3); S- the minor enantiomer at 334.0779, found, 334.0783.
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Ethyl
2-amino-3,3,3-trifluoro-2-(5-formyl-1H-indol-3-yl)- (s, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 7.41 (d, J = 2.5 Hz,
propanoate 22. 91% yield, 1H NMR (400 MHz, acetone-d6) δ 1H), 7.26–7.23 (m, 1H), 4.45–4.36 (m, 2H), 1.33 (t, J = 7.1 Hz,
11.03 (s, 1H), 10.05 (s, 1H), 8.55 (s, 1H), 7.80–7.70 (m, 2H), 3H). 13C NMR (100 MHz, CDCl3) δ 169.15, 137.14, 125.03,
7.63 (d, J = 8.5 Hz, 1H), 4.46–4.36 (m, 2H), 1.32 (t, J = 7.1 Hz, 124.84, 124.07, 123.90, 122.54, 116.33, 114.30, 108.87, 76.12,
3H). 13C NMR (100 MHz, acetone-d6) δ 192.72, 168.90, 140.97, 64.44, 13.94. HRMS (ESI) calcd for C13H12BrF3N2O2 [M + Na]+
131.01, 128.11, 127.94, 127.52, 126.32, 126.29, 122.62, 113.38, 386.9932, found, 386.9935.
113.33, 77.84, 63.90, 14.25. HRMS (ESI) calcd for
C14H13F3N2O3 [M + H]+ 315.0957, found, 315.0961.
Ethyl
2-amino-3,3,3-trifluoro-2-(6-nitro-1H-indol-3-yl)-
propanoate 30. 89% yield. 1H NMR (400 MHz, acetone-d6)
Methyl 3-(2-amino-3-ethoxy-1,1,1-trifluoro-3-oxopropan-2-yl)- δ 11.24 (s, 1H), 8.98 (d, J = 1.8 Hz, 1H), 8.09 (dd, J = 9.0, 2.3 Hz,
1H-indole-5-carboxylate 23. 91% yield. 1H NMR (400 MHz, 1H), 7.87 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 9.0 Hz, 1H), 4.45–4.38
acetone-d6) δ 10.90 (s, 1H), 8.75 (s, 1H), 7.85 (dd, J = 8.6, (m, 2H), 1.35 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, acetone-
1.6 Hz, 1H), 7.72 (d, J = 2.3 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), d6) δ 168.70, 142.79, 140.78, 129.75, 126.41, 125.73, 123.57,
6.24 (s, 1H), 4.44–4.35 (m, 2H), 3.88 (s, 3H), 1.33 (t, J = 7.1 Hz, 119.26, 118.07, 113.13, 112.10, 77.79, 64.06, 14.20. HRMS (ESI)
3H). 13C NMR (100 MHz, acetone-d6) δ 169.03, 168.20, 140.33, calcd for C13H12F3N3O4 [M + Na]+ 354.0678, found, 354.0681.
127.73, 126.54, 125.98, 125.07, 123.90, 122.84, 112.43, 64.30,
63.84, 51.98, 14.21. LR-ESI: [M]+ 344.
Ethyl
2-amino-2-(6-cyano-1H-indol-3-yl)-3,3,3-trifluoro-
propanoate 31. 92% yield. 1H NMR (400 MHz, CDCl3) δ 9.14
Ethyl
2-amino-3,3,3-trifluoro-2-(5-methyl-1H-indol-3-yl)- (s, 1H), 8.36 (s, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.44 (dt, J = 8.5,
propanoate 24. 90% yield. 1H NMR (400 MHz, acetone-d6) 4.8 Hz, 2H), 4.59 (s, 1H), 4.50–4.37 (m, 2H), 1.37 (t, J = 7.1 Hz,
δ 10.40 (s, 1H), 7.69 (s, 1H), 7.51 (d, J = 2.5 Hz, 1H), 7.32 (d, J = 8.3 3H). 13C NMR (100 MHz, CDCl3) δ 167.73, 137.18, 126.49,
Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 5.98 (s, 1H), 4.40–4.29 (m, 2H), 125.91, 124.26, 124.09, 123.67, 120.83, 119.65, 111.56, 108.34,
2.39 (s, 3H), 1.29 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, acetone- 102.37, 69.48, 63.61, 12.89. HRMS (ESI) calcd for
d6) δ 169.39, 136.16, 129.26, 126.72, 126.69, 125.81, 124.38, 123.88, C14H12F3N3O2 [M + Na]+ 334.0779, found, 334.0783.
121.52, 112.18, 64.30, 63.53, 21.80, 14.28. LR-ESI: [M]+ 300.
Ethyl
Ethyl
2-amino-3,3,3-trifluoro-2-(6-methyl-1H-indol-3-yl)-
2-amino-3,3,3-trifluoro-2-(5-methoxy-1H-indol-3-yl)- propanoate 32. 93% yield. 1H NMR (400 MHz, CDCl3) δ 8.12
propanoate 25. 92% yield. 1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.65 (s, 1H), 7.28 (d, J = 2.3 Hz, 1H), 7.16 (d, J = 8.3 Hz,
(s, 1H), 7.36 (dd, J = 5.3, 2.3 Hz, 2H), 7.20 (d, J = 8.9 Hz, 1H), 1H), 7.03 (dd, J = 8.3, 0.9 Hz, 1H), 4.46–4.39 (m, 2H), 2.43 (s,
6.87 (dd, J = 8.8, 2.4 Hz, 1H), 4.47–4.31 (m, 2H), 3.84 (s, 3H), 3H), 1.32 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
1.33 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 169.39,
δ
169.49, 134.65, 129.83, 125.34, 125.04, 124.48,
154.44, 131.43, 125.66, 124.96, 122.20, 113.16, 112.11, 108.10, 124.34, 120.55, 111.09, 107.92, 67.10, 64.23, 21.63, 13.88.
102.70, 64.23, 63.02, 55.82, 13.99. LR-ESI: [M]+ 316. HRMS (ESI) calcd for C14H15F3N2O2 [M + Na]+ 323.0983,
Ethyl
2-amino-3,3,3-trifluoro-2-(5-hydroxy-1H-indol-3-yl)- found, 323.0985.
propanoate 26. 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.29
Methyl 3-(2-amino-3-ethoxy-1,1,1-trifluoro-3-oxopropan-2-yl)-
(s, 1H), 7.38 (s, 1H), 7.29 (s, 1H), 7.19–7.13 (m, 1H), 6.78 (d, J = 1H-indole-4-carboxylate 33. 91% yield. 1H NMR (400 MHz,
8.7 Hz, 1H), 4.70 (s, 1H), 4.39–4.33 (m, 2H), 1.37 (d, J = 7.1 Hz, acetone-d6) δ 10.90 (s, 1H), 8.76 (s, 1H), 7.86 (dd, J = 8.6, 1.5
3H). 13C NMR (100 MHz, CDCl3) δ 169.33, 149.84, 131.53, Hz, 1H), 7.73 (d, J = 2.5 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H),
125.83, 125.19, 120.05, 112.64, 112.08, 105.49, 70.03, 64.62, 4.53–4.31 (m, 2H), 3.89 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H).
13.76. HRMS (ESI) calcd for C13H13F3N2O3 [M + H]+ 303.0957, 13C NMR (100 MHz, acetone-d6) δ 169.01, 168.81, 168.23,
found, 303.0961.
Ethyl 2-amino-3,3,3-trifluoro-2-(6-fluoro-1H-indol-3-yl)- 112.45, 112.40, 79.03, 63.86, 52.01, 14.22. HRMS (ESI) calcd for
propanoate 27. 89% yield. 1H NMR (400 MHz, acetone-d6) δ C15H15F3N2O4 [M + Na]+ 345.1062, found, 345.1066.
140.18, 127.73, 127.58, 126.54, 125.95, 125.07, 123.92, 122.84,
10.64 (s, 1H), 7.93–7.86 (m, 1H), 7.59 (s, 1H), 7.19 (d, J = 9.8
Ethyl
2-amino-3,3,3-trifluoro-2-(4-hydroxy-1H-indol-3-yl)-
Hz, 1H), 6.95–6.86 (m, 1H), 4.37 (m, J = 10.8, 7.4, 3.7 Hz, 2H), propanoate 34. 94% yield. 1H NMR (400 MHz, CDCl3) δ 9.13
1.29 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, acetone-d6) (s, 1H), 8.36 (s, 1H), 7.63 (d, J = 2.6 Hz, 1H), 7.44 (dt, J = 8.5,
1
δ 169.09, 160.54 (d, JC–F = 237.35 Hz), 137.69, 126.59, 126.35, 4.9 Hz, 2H), 4.58 (s, 1H), 4.44 (m, J = 25.1, 10.7, 7.1, 3.6 Hz,
2
123.22, 123.12, 109.81, 109.04 (d, JC–F = 24.24 Hz), 98.35 (d, 2H), 1.37 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
3JC–F = 25.25 Hz), 78.14, 63.67, 14.26. HRMS (ESI) calcd for δ 168.76, 138.25, 127.49, 126.99, 125.25, 125.14, 120.71,
C13H12F4N2O2 [M + Na]+ 327.0733, found, 327.0736.
112.63, 109.32, 103.34, 67.04, 64.62, 13.91. HRMS (ESI) calcd
2-amino-2-(6-chloro-1H-indol-3-yl)-3,3,3-trifluoro- for C13H13F3N2O3 [M + Na]+ 303.0957, found, 303.0953.
propanoate 28. 87% yield. 1H NMR (400 MHz, acetone-d6) δ
Ethyl 2-amino-3,3,3-trifluoro-2-(4-methyl-1H-indol-3-yl)-
Ethyl
10.75 (s, 1H), 7.95 (d, J = 1.3 Hz, 1H), 7.66 (d, J = 2.5 Hz, 1H), 7.47 propanoate 35. 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.22
(d, J = 8.7 Hz, 1H), 7.15 (dd, J = 8.7, 2.1 Hz, 1H), 4.38 (qd, J = 7.1, (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 2.6 Hz, 1H), 7.06 (d,
4.0 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, acetone- J = 7.9 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 4.44–4.31 (m, 2H), 2.42
d6) δ 168.98, 136.22, 127.59, 127.52, 125.86, 123.72, 122.91, (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
121.38, 113.98, 109.44, 77.86, 63.78, 14.24. LR-ESI: [M]+ 320.
Ethyl 2-amino-2-(6-bromo-1H-indol-3-yl)-3,3,3-trifluoro- 120.58, 118.79, 109.13, 64.23, 62.21, 16.46, 13.92. HRMS (ESI)
propanoate 29. 91% yield. 1H NMR (400 MHz, CDCl3) δ 8.30 calcd for C14H15F3N2O2 [M + Na]+ 323.0983, found, 323.0986.
δ 169.47, 135.96, 125.03, 124.68, 124.18, 123.18, 120.73,
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Ethyl
2-amino-3,3,3-trifluoro-2-(2-methyl-1H-indol-3-yl)-
Methyl
2-amino-3,3,3-trifluoro-2-(5-fluoro-1H-indol-3-yl)-
propanoate 36. 89% yield. 1H NMR (400 MHz, CDCl3) δ 7.99 propanoate 43. 94% yield. 1H NMR (400 MHz, CDCl3) δ 8.40
(s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.27–7.24 (m, 1H), 7.11 (m, J = (s, 1H), 7.55 (dd, J = 10.2, 2.1 Hz, 1H), 7.49 (d, J = 2.5 Hz, 1H),
14.9, 7.1, 1.1 Hz, 2H), 4.47–4.31 (m, 2H), 3.98 (s, 1H), 2.52 (s, 7.31–7.23 (m, 1H), 6.98 (td, J = 8.9, 2.5 Hz, 1H), 4.39 (s, 1H),
3H), 1.34 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) 3.96 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 169.72, 158.17 (d,
δ 169.35, 135.25, 134.61, 126.81, 125.34, 121.65, 120.53, 1JC–F = 236.34 Hz), 136.12, 132.84, 126.03, 125.61, 125.51,
2
3
120.25, 110.31, 103.93, 63.60, 61.66, 14.25, 13.92. HRMS (ESI) 124.81, 112.03 (d, JC–F = 9.09 Hz), 111.38 (d, JC–F = 27.27 Hz),
calcd for C14H15F3N2O2 [M + Na]+ 323.0983, found, 323.0985.
108.71, 106.13, 63.60, 54.57. HRMS (ESI) calcd for
Ethyl
2-amino-3,3,3-trifluoro-2-(7-methyl-1H-indol-3-yl)- C12H10F4N2O2 [M + Na]+ 313.0576, found, 313.0573.
propanoate 37. 88% yield. 1H NMR (400 MHz, CDCl3) δ 8.23
Methyl 2-amino-2-(5-chloro-1H-indol-3-yl)-3,3,3-trifluoro-
(s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), propanoate 44. 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.39
7.11–7.01 (m, 2H), 4.45–4.34 (m, 2H), 2.46 (s, 3H), 1.33 (t, J = (s, 1H), 7.86 (d, J = 1.2 Hz, 1H), 7.43 (d, J = 2.6 Hz, 1H), 7.24 (d,
7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 169.44, 135.95, J = 8.6 Hz, 1H), 7.16 (dd, J = 8.7, 1.9 Hz, 1H), 4.42 (s, 1H), 3.95
124.99, 124.69, 124.10, 123.19, 120.73, 120.46, 118.89, 109.27, (s, 3H). 13C NMR (100 MHz, CDCl3) δ 169.66, 134.65, 126.44,
64.19, 62.13, 16.50, 13.93. LR-ESI: [M]+ 300.
Ethyl
2-amino-3,3,3-trifluoro-2-(1-methyl-1H-indol-3-yl)- 54.65. HRMS (ESI) calcd for C12H10ClF3N2O2 [M + Na]+
propanoate 38. 89% yield. 1H NMR (400 MHz, CDCl3) δ 7.88 329.0281, found, 329.0278.
(d, J = 8.1 Hz, 1H), 7.32 (d, J = 10.6 Hz, 2H), 7.27 (s, 1H), 7.15 Methyl
126.17, 125.71, 123.20, 121.91, 120.56, 112.41, 108.18, 63.97,
2-amino-2-(5-bromo-1H-indol-3-yl)-3,3,3-trifluoro-
(t, J = 7.5 Hz, 1H), 4.49–4.42 (m, 1H), 4.37–4.31 (m, 1H), 3.78 propanoate 45. 91% yield. 1H NMR (400 MHz, CDCl3) δ 8.40
(s, 3H), 1.34 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) (s, 1H), 8.04 (s, 1H), 7.43 (d, J = 2.6 Hz, 1H), 7.30 (dd, J = 8.7,
δ 169.47, 137.24, 128.84, 125.70, 124.99, 122.22, 121.28, 120.12, 1.8 Hz, 1H), 7.24–7.22 (m, 1H), 4.40 (s, 1H), 3.95 (s, 3H). 13C
109.52, 106.86, 68.11, 64.16, 33.04, 13.95. LR-ESI: [M]+ 300.
Ethyl
NMR (100 MHz, CDCl3) δ 169.62, 134.93, 126.84, 125.78,
2-amino-3,3,3-trifluoro-2-(1-isopentyl-1H-indol-3-yl)- 125.53, 125.39, 123.70, 123.56, 114.08, 112.79, 64.15, 54.65.
propanoate 39. 90% yield. 1H NMR (400 MHz, CDCl3) HRMS (ESI) calcd for C12H10BrF3N2O2 [M + Na]+ 372.9775,
δ 7.34–7.29 (m, 2H), 7.15 (d, J = 7.7 Hz, 1H), 7.09 (s, 1H), 6.94 found, 372.9772.
(t, J = 7.6 Hz, 1H), 4.26–4.05 (m, 4H), 1.70 (d, J = 7.2 Hz, 2H),
Methyl
2-amino-3,3,3-trifluoro-2-(2-methyl-1H-indol-3-yl)-
1.61–1.55 (m, 1H), 1.08 (dd, J = 34.7, 27.6 Hz, 3H), 0.96–0.91 propanoate 46. 90% yield. 1H NMR (400 MHz, CDCl3) δ 8.01
(m, 6H). 13C NMR (100 MHz, CDCl3) δ 168.69, 136.19, 129.24, (s, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.26–7.24 (m, 1H), 7.17–7.08
126.92, 121.94, 121.37, 119.14, 109.39, 108.00, 65.78, 61.98, (m, 2H), 3.92 (s, 1H), 3.91 (s, 3H), 2.49 (s, 3H). 13C NMR
44.77, 38.93, 25.75, 22.49, 22.39, 13.91. HRMS (ESI) calcd for (100 MHz, CDCl3) δ 169.79, 135.22, 134.59, 126.78, 122.46,
C18H24F3N2O2 [M + H]+ 357.1790, found, 357.1793.
121.71, 120.43, 120.10, 110.40, 103.89, 77.37, 77.05, 76.73,
Ethyl
2-amino-3,3,3-trifluoro-2-(1-pentyl-1H-indol-3-yl)- 64.59, 53.74, 14.05. LR-ESI: [M]+ 286.
propanoate 40. 93% yield. 1H NMR (400 MHz, CDCl3) δ 7.31
Methyl
2-amino-3,3,3-trifluoro-2-(5-methyl-1H-indol-3-yl)-
(d, J = 7.5 Hz, 2H), 7.15 (t, J = 7.7 Hz, 1H), 7.10 (s, 1H), 6.95 (d, propanoate 47. 92% yield. 1H NMR (400 MHz, CDCl3) δ 8.19
J = 7.3 Hz, 1H), 4.26–4.02 (m, 4H), 1.84–1.78 (m, 2H), 1.33–1.26 (s, 1H), 7.62 (s, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.24 (d, J = 7.5 Hz,
(m, 4H), 1.17 (dt, J = 14.2, 5.2 Hz, 3H), 0.88 (d, J = 6.7 Hz, 3H). 1H), 7.05 (d, J = 8.3 Hz, 1H), 4.34 (s, 1H), 3.93 (s, 3H), 2.45 (s,
13C NMR (100 MHz, CDCl3) δ 168.72, 136.26, 129.40, 126.95, 3H). 13C NMR (100 MHz, CDCl3) δ 170.01, 134.58, 130.02,
121.96, 121.38, 119.16, 109.47, 107.92, 69.45 (q), 61.99, 46.54, 125.38, 124.46, 124.30, 120.36, 111.03, 107.96, 77.36, 77.04,
29.84, 29.04, 22.33, 14.00, 13.93. HRMS (ESI) calcd for 76.72, 63.40, 54.46, 21.65. HRMS (ESI) calcd for C13H13F3N2O2
C18H24F3N2O2 [M + H]+ 357.1790, found, 357.1792.
[M + Na]+ 309.0827, found, 309.0824.
Methyl 2-amino-3,3,3-trifluoro-2-(7-methyl-1H-indol-3-yl)-
Ethyl 3,3,3-trifluoro-2-(1H-indol-3-yl)-2-((4-methoxyphenyl)-
1
amino)-propanoate 41. 92% yield. H NMR (400 MHz, CDCl3) propanoate 48. 91% yield. 1H NMR (400 MHz, CDCl3) δ 8.22
δ 8.27 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.33 (d, J = 8.2 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.08 (t,
Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.52 (q, J J = 7.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 3.91 (s, 3H), 2.44 (s,
= 9.1 Hz, 4H), 5.01 (s, 1H), 4.31–4.14 (m, 2H), 3.62 (s, 3H), 1.11 3H). 13C NMR (100 MHz, CDCl3) δ 170.02, 135.91, 124.94,
(t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 168.46, 153.74, 124.65, 124.08, 123.26, 120.89, 120.59, 118.53, 109.07, 65.43,
137.55, 135.84, 125.62, 124.96, 122.62, 120.58, 120.45, 119.57, 54.45, 16.48. HRMS (ESI) calcd for C13H13F3N2O2 [M + Na]+
113.83, 111.15, 108.05, 68.42, 63.22, 55.36, 13.77. HRMS (ESI) 309.0827, found, 309.0823.
calcd for C20H19F3N2O3 [M + H]+ 393.1426, found, 393.1421.
Methyl 2-amino-3,3,3-trifluoro-2-(5-methoxy-1H-indol-3-yl)-
Methyl 2-amino-3,3,3-trifluoro-2-(1H-indol-3-yl)-propanoate propanoate 49. 89% yield. 1H NMR (400 MHz, CDCl3) δ 8.24
1
42. 91% yield. H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.84 (s, 1H), 7.39 (d, J = 2.3 Hz, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.24 (d,
(d, J = 8.0 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 8.1 Hz, J = 8.9 Hz, 1H), 6.89 (dd, J = 8.9, 2.4 Hz, 1H), 4.35 (s, 1H), 3.94
1H), 7.21 (dd, J = 11.7, 4.6 Hz, 1H), 7.18–7.12 (m, 1H), 4.40 (s, (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 169.91,
1H), 3.91 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 169.98, 136.31, 154.59, 131.37, 125.68, 124.83, 122.10, 113.24, 112.06, 108.12,
125.08, 124.93, 124.42, 122.76, 120.83, 120.67, 111.47, 108.45, 102.59, 64.13, 55.83, 54.46. HRMS (ESI) calcd for
64.42, 54.49. LR-ESI: [M]+ 272.
C13H13F3N2O3 [M + Na]+ 325.0776, found, 325.0779.
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Paper
Anti-HIV-1 activity assays
D. Wernic, P. Schroeder, M. Amad, P. Edwards,
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The inhibitory activity of the compounds against the labora-
tory-adapted HIV-1IIIB strain was examined using the HIV-1IIIB
/
TZM-bl indicator cell culture system as previously
described.31,43 Briefly, 1.5 × 103 TZM-bl cells were seeded in a
volume of 300 L into each well of a 48-well plate and incubated
overnight at 37 °C. The cells were then infected with HIV-1IIIB
at a multiplicity of infection (M.O.I.) of 0.1 and incubated in
the presence or absence of tested compounds for two days at
37 °C. After that they were lysed in lysis buffer, and the lucifer-
ase substrate was added to the cell lysates as recommended by
the manufacturer’s protocol (Promega). The luciferase activi-
ties were quantified using a luciferase reporter assay system
(Promega, Madison, WI). The percentage of the compound
inhibitory activity was calculated using the following formula:
[1 − (E − N)/(P − N)] × 100%, where N represents the negative
control, P represents the positive control and E corresponds to
the experimental group. The EC50 values (effective concen-
tration for 50% inhibition) were calculated using the computer
program Calcusyn. The inhibitory activity of the experimental
compounds in primary HIV-1 replication was determined as
previously described. Peripheral blood mononuclear cells
(PBMCs) were isolated from the blood of healthy donors by
standard density gradient centrifugation using a Histopaque-
1077 (Sigma). PBMCs were cultured in RPMI 1640 medium
containing 15% FBS, 1% penicillin–streptomycin and IL-2
(20 units mL−1). Three-day PHA-stimulated (5 µg mL−1) and
polybrene-treated (2 µg mL−1) cells were infected with the
corresponding primary HIV-1 strain at a M.O.I. of 0.01 in the
presence or absence of the tested compounds. The super-
natants were collected on day 4 post-infection and tested for
p24 antigen expression by ELISA using the RETRO-TEK HIV-1
p24 antigen ELISA kit (USA). The absorbance of the samples at
450 nm (A450) was read using a Tecan GENios microplate
reader (Tecan, Switzerland), and the EC50 values were calcu-
lated as described above.
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Acknowledgements
We are grateful to the NSFC (81172935, 81373255), the Key
Project of Ministry of Education (313040), Hubei Province’s
Outstanding Medical Academic Leader Program, the Scientific
and Technological Innovative Research Team of Wuhan
(2013070204020048), the National Mega Project on Major Drug
Development (2011ZX09401-302) and the Fundamental
Research Funds for the Central Universities (2014306020201,
2042014kf0204) for support of this research.
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