Design, synthesis, and biological evaluation of novel trifluoromethyl indoles as potent HIV-1 NNRTIs with an improved drug resistance profile

Article abstract of DOI:10.1039/c3ob42186d

A novel series of trifluoromethyl indole derivatives have been designed, synthesized and evaluated for anti-HIV-1 activities in MT-2 cells. The hydrophobic constant, acute toxicity, carcinogenicity and mutagenicity were predicted. Trifluoromethyl indoles 10i and 10k showed extremely promising activities against WT HIV-1 with IC₅₀ values at the low nanomolar level, similar to efavirenz, better than nevirapine, and also possessed higher potency towards the drug-resistant mutant strain Y181C than nevirapine. Preliminary SAR and docking studies of detailed binding mode provided some insights for discovery of more potent NNRTIs. the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42186d

Organic &
Biomolecular Chemistry
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Design, synthesis, and biological evaluation of
novel trifluoromethyl indoles as potent HIV-1
NNRTIs with an improved drug resistance profile†
Cite this: Org. Biomol. Chem., 2014,
12, 3446
Hai-Xia Jiang,‡a Dao-Min Zhuang,‡b Ying Huang,§a Xing-Xin Cao,a Jian-Hua Yao,§a
Jing-Yun Li,b Jian-Yong Wang,c Chen Zhang*a and Biao Jiang*a,c
A novel series of trifluoromethyl indole derivatives have been designed, synthesized and evaluated
for anti-HIV-1 activities in MT-2 cells. The hydrophobic constant, acute toxicity, carcinogenicity and
mutagenicity were predicted. Trifluoromethyl indoles 10i and 10k showed extremely promising activities
against WT HIV-1 with IC50 values at the low nanomolar level, similar to efavirenz, better than nevirapine,
and also possessed higher potency towards the drug-resistant mutant strain Y181C than nevirapine.
Preliminary SAR and docking studies of detailed binding mode provided some insights for discovery of
more potent NNRTIs.
Received 5th November 2013,
Accepted 24th March 2014
DOI: 10.1039/c3ob42186d
www.rsc.org/obc
Introduction
Human immunodeficiency virus (HIV) reverse transcriptase
(RT) is responsible for producing the DNA copy of the viral
RNA genome that will be integrated into the human DNA. HIV
RT, together with protease and integrase, is one of three key
enzymes in the HIV life cycle and the primary target of many
antiviral drugs.1 Non-nucleoside reverse transcriptase inhibi-
tors (NNRTIs) target an allosteric binding pocket on HIV-1 RT
in a noncompetitive manner, causing distortion in the three-
dimensional structure of the enzyme to inhibit RT catalytic
function.2 Currently, five NNRTIs drugs have been approved by
the U.S. Food and Drug Administration (FDA) (Fig. 1). The
first-generation NNRTIs drugs nevirapine (NVP), efavirenz
(EFV) and delavirdine (DLV) are limited in their clinical use
Fig. 1 Current marketed NNRTIs.
due to the rapid emergence of drug resistance. In particular,
K103N and Y181C are the two mutations most frequently
observed in patients exposed to various NNRTIs.
Etravirine (TMC125) and rilpivirine (TMC278), which
belong to the diarylpyrimidine (DAPY) family, were approved
as second generation NNRTIs by the FDA in 2008 and 2011
respectively, and demonstrated activities against HIV-1 strains
that developed resistance to first generation NNRTIs. In
patients failing etravirine-containing regimens, Y181C was
shown to commonly occur.3–5 Furthermore, the pharmaco-
kinetic profiles of most DAPY derivatives are not satisfactory
due to their low water solubility.6–8 There are also many unde-
sired side effects to TMC125, such as toxic epidermal necroly-
sis and erythema multiforme, hypersensitivity reactions
characterized by rash, and sometimes even organ dysfunction,
including hepatic failure. Efforts to develop the next gen-
eration of NNRTIs9–11 based on novel scaffolds have focused
aCAS Key Laboratory of Synthetic Chemistry of Natural Substances,
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
345 Lingling Road, Shanghai 200032, China. E-mail: jiangb@mail.sioc.ac.cn,
zhangchen@mail.sioc.ac.cn; Fax: (+86) 021-6416-6128; Tel: (+86) 021-54925566
bDepartment of AIDS Research, State Key Laboratory of Pathogen and Biosecurity,
Beijing Institute of Microbiology and Epidemiology, Beijing, China.
E-mail: lijy@nic.bmi.ac.cn; Tel: (+86) 010-6694-8672
cShanghai Advanced Research Institute, Chinese Academy of Sciences,
99 Haike Road, Shanghai 201210, China
†Electronic supplementary information (ESI) available: Copies of 1H, 13C NMR,
MS, IR and HPLC spectra of target compounds 9a–9e and 10a–10q. See DOI:
10.1039/c3ob42186d
‡These authors contributed equally to this study.
§Laboratory of Computer Chemistry and Cheminformatics, Shanghai Institute
of Organic Chemistry, Chinese Academy of Sciences. These authors were respon-
sible for the predictions and docking calculations in this study.
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and that of 9d, 10e, 10l and 10m was at a low-toxic level (500 ≤
LD50 < 5000 mg kg−1 (low tox.)). The carcinogenicity (predicted
by CISOC-PSCT)21 of compounds 10e, 10l, 10i, 10k, 10j and
10m was N (probability of non carcinogenic is high), except for
TMC278 and compound 9d, which were P (probability of non
carcinogenic is low). AMES (predicted by CISOC-PSMT)22 of all
compounds, as listed in Table 1, was negative. For TMC278,
the prediction results almost corresponded to experimental
results.
Therefore, the preliminary prediction of some drug-like pro-
perties served as an encouragement for us to synthesize these
novel trifluoromethyl indoles.
Fig. 2 Structure of reference L-737, 126 (1) and IAS 2.
Chemistry
on the design of compounds with high potency, low toxicity,
improved resistant profiles, and better pharmacokinetic
profiles.
The synthesis of new trifluoromethyl indoles is depicted in
Scheme 1.
Variously substituted indoles were prepared with a view to
examining the SARs by typical Fischer indole synthesis. The
phenylhydrazine 3 reacted with methylpyruvate in methanol to
afford the phenylhydrazone 4, followed by Fischer cyclization
of phenylhydrazone 4 to give the aromatic indole 5 under
acidic conditions. The detailed mechanism for the Fischer
reaction23 of phenylhydrazine 3 and methylpyruvate is as
follows (Scheme 2): phenylhydrazone 4 is firstly formed from
the condensation reaction of commercially available methyl-
pyruvate and phenylhydrazine 3, followed by the isomerization
of phenylhydrazone 4 to result in the enamine A. After proto-
nation, enamine A is transformed into imine B through a
cyclic [3,3]-sigmatropic rearrangement. Then aminoacetal C is
produced by cyclization of the resulting imine B. Subsequently
imine D is obtained by aminoacetal C losing one molecule of
NH3 under acid catalysis, followed by protonation to deliver
the energetically favorable aromatic indole 5.
Aromatic indole 5 was converted into carbohydrazide 6 with
N2H4·H2O (85%) in ethanol, followed by diazotization reaction
with NaNO2 in acetic acid to produce azide 7. Subsequently,
the Curtius rearrangement of azide 7 yielded carbamate 8 in
ethanol. The detailed mechanism for the rearrangement24 is
as follows (Scheme 3): nitrene F is formed from substituted
azide 7 by loss of one molecule of nitrogen, followed by the
rearrangement of acyl nitrene F by migration of the indole
group to form the desired isocyanate G. The nucleophilic
addition reaction of the resulting isocyanate G and EtOH deli-
vers the desired ethyl carbamate 8. Subsequently, carbamate 8
reacts with trifluoroacetic anhydride in dry DMF or dry diethyl
ether at 0 °C to afford the desired compounds 9a–9e. Finally,
some of the most potent trifluoromethyl indole analogues,
10a–10l, were prepared in dry THF by reaction with various
The development of indolylarylsulfones (IASs) NNRTIs was
based on the Merck derivative L-737, 126 (1)12 as a reference
compound (Fig. 2). The potent activity of IAS 2 against the
NNRTI resistant mutants was correlated to the presence of a
3-(3,5-dimethylphenyl) sulfonyl moiety.13 The sulfone group14
is similar to the trifluoromethyl group in efavirenz allowing
the inhibitor to assume a butterfly-like conformation, which is
commonly found in many other NNRTIs. Inspired by the good
potency and bioavailability profiles15–17 of the trifluoromethyl
group, if the sulfonyl moiety of IASs could be replaced by a tri-
fluoromethyl group to produce a new potential NNRTI, it
would be possible to improve the activity against resistant
mutants and reduce toxicity.
With a novel scaffold in mind, we simultaneously sought to
streamline the discovery process. The main goal is to minimize
the number of compounds that have to be synthesized and
assayed to obtain possible drug candidates. The hydrophobic
constant, acute toxicity, carcinogenic toxicity and mutagenic
toxicity of several of the designed compounds with representa-
tive structures were first predicted to exclude the compounds
that did not obey “Lipinski’s rule of five”18 or with potential
toxicity. Further molecular docking studies were performed in
order to validate whether the compounds can efficiently target
the nonnucleoside binding site (NNBS) of HIV-1 RT. Herein,
we report the design, synthesis, anti-HIV-1 evaluation and pre-
liminary structure–activity relationships (SARs) of a new series
of trifluoromethyl indoles.
Results and discussion
Prediction
The predictions of the hydrophobic constant (log P), acute toxi- Grignard reagents at 0 °C for 1–2 hours.
city, carcinogenicity and mutagenicity of some representative
Meanwhile, compounds 10m and 10n were produced in dry
and structurally diverse compounds are listed in Table 1. The DMSO by proline/AcK-catalyzed aldol reaction at 40 °C for 3–4
data listed in Table 1 show that the log P values (predicted by days. The detailed catalytic mechanism for the proline-cata-
CISOC-log P19) of compounds 9d, 10e, 10l, 10i, 10k, 10j and lyzed direct aldol reaction25 is as follows (Scheme 4): enamine
10m were in line with “Lipinski’s rule of five”.18 The acute toxi- I is firstly formed from the condensation reaction of commer-
city (predicted by CISOC-PSAT20) of compounds 10i, 10k and cially available proline and acetone promoted by AcK. Sub-
10j was at a non-toxic level (LD50 ≥ 5000 mg kg−1 (non tox.)), sequently, ketone 9c or 9e is added to the reaction mixture and
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Table 1 Prediction results of log P, acute, carcinogenic and mutagenic toxicity
Compd
Structure
Log P (exp./pred.)
3.80–5.47d/3.70
Acute tox.a (exp./pred.)
Car. tox.b (exp./pred.)
AMESc (exp./pred.)
TMC278
Oral, rat: LD50 = 980 mg/kge/3.46
P f/Pb
Ng/Nh
9d
Nullh/4.23
Nullh/3.35
Nullh/4.62
Nullh/5.02
Nullh/Pb
Nullh/Nb
Nullh/Nh
Nullh/Nh
10i
10k
10j
Nullh/4.80
Nullh/4.20
Nullh/5.08
Nullh/5.02
Nullh/Nb
Nullh/Nb
Nullh/Nh
Nullh/Nh
10e
Nullh/3.58
Nullh/4.97
Nullh/Nb
Nullh/Nh
10l
Nullh/3.93
Nullh/2.93
Nullh/4.88
Nullh/4.89
Nullh/Nb
Nullh/Nb
Nullh/Nh
Nullh/Nh
10m
a Rat, oral, less than 2, LD50 < 1 mg kg−1; equal or more than 2 and less than 3, 1 ≤ LD50 < 50 mg kg−1; equal or more than 3 and less than 4, 50
≤ LD50 < 500 mg kg−1; equal or more than 4 and less than 5, 500 ≤ LD50 < 5000 mg kg−1; equal or more than 5, LD50 ≥ 5000 mg kg−1 b Rat, oral,
P: probability of non carcinogenic is low; N: probability of non carcinogenic is high. c Salmonella typhimurium. d http://www.drugbank.ca/drugs/
DB08864.
e http://www.drugbank.ca/drugs/DB08864. f http://www.drugbank.ca/system/fda_labels/DB08864.pdf?1368312297. g http://www.
drugbank.ca/system/fda_labels/DB08864.pdf?1368312297. h P: positive; N: negative; Null: no experimental data.
.
is attacked by the enamine I to deliver the iminium product J. 24 hours. Since the stabilities of compounds 10o, 10p and 10q
Finally, iminium salt J is hydrolyzed to give aldol product 10m were too poor to meet the requirements for biological assays,
or 10n with the release of one molecule of proline, which we decided not to continue with our efforts to synthesize com-
allows the catalytic cycle to continue until the conversion is pounds with an aryl moiety as R2.
finished.
Following the same procedure as for compounds 10a–10l,
Biological activity
we also tried to synthesize compounds with a phenyl or
3,5-dimethylphenyl group as R2 (10o, 10p and 10q) (Fig. 3).
The in vitro Cell-Titer Glo assay method was used to evaluate
However, these compounds were very unstable and their
purities as detected by HPLC decreased by about 10% in
the 19 new trifluoromethyl indoles (9a–9e and 10a–10n) along
with two FDA-approved drugs, NVP and EFV, as reference com-
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Scheme 4
A
plausible mechanism for the proline-catalyzed direct
aldol reaction.
Scheme 1 Synthesis of compounds 9a–10n. Reagents and conditions:
(a) methylpyruvate, MeOH, reflux; (b)
4 N HCl–MeOH, reflux; (c)
N2H4·H2O (85%), EtOH, reflux; (d) NaNO2, AcOH, 1,4-dioxane, 0 °C; (e)
EtOH, reflux; (f) Trifluoroacetic anhydride, DMF or diethyl ether, 0 °C;
(g) R2MgCl/R2MgBr, THF, 0 °C; (h) Acetone, Proline/AcK (1/1), DMSO,
40 °C.
Fig. 3 Structure of 10o, 10p and 10q.
Table 2 Anti-HIV activities and cytotoxicity of compounds 9a–9e and
10a–10n in MT-2 cellsa
IC50 b (μM)
Compd
WT IIIB
Y181C
TC50 c (μM)
TId
>73.6
>1
>1
“A” type
9a
9b
9c
9d
9e
2.716
>200
57.531
57.068
200
200
142.758
200
162.289
200
>200
>200
67.5
>200
>200
>3.5
>1
“B” type
10a
10b
10c
10d
10e
10f
10g
10h
10m
10i
10j
10k
10l
10n
0.031
0.003
20.182
0.022
5.839
12.831
12.061
6.556
3.335
9.897
2.337
0.003
5.048
0.003
1.471
200
21.047
6.252
20.5
58.0
>200
54.9
>200
28.3
85.1
>200
35.5
23.3
33.2
24.0
54.3
>200
1.0
2672.6
>34.3
>4.3
>16.6
4.3
25.5
>20.2
15.2
6849.5
6.6
7051.8
36.9
NDe
15.760
38.425
10.169
18.152
13.343
29.875
13.901
8.010
Scheme 2 A plausible mechanism for the Fischer indole synthesis.
1.932
7.627
51.744
>1
NVP
EFV
>30
0.021
a Data represent the mean of at least three separate experiments.
b Compound concentration required to protect MT-2 cells against viral
cytopathogenicity by 50%. c Compound concentration that decreases
the uninfected MT-2 cell viability by 50%. d Selectivity index: TC50/IC50
(WT) ratio. e ND: not determined.
Scheme 3 A plausible mechanism for the Curtius rearrangement.
pounds. These compounds were assayed for their cytotoxicities where Tyr181 was replaced by Cys. The results, expressed as
and anti-HIV-1 activities in MT-2 cells infected with wild-type TC50 (50% cytotoxic concentration), IC50 (50% HIV-1 cytopro-
(WT) HIV-1 (strain IIIB), and Y181C mutant HIV-1 strain, tective concentration against HIV-1 induced cytopathic effect)
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10k, and the key residue Lys101, but the total scores of the
three compounds were almost identical. Their total scores
were higher than for TMC278, which corresponded to the IC50
values in Table 2. On the other hand, although the total score
of EFV was still higher than for compounds 10i and 10k, the
length of the hydrogen bond between EFV and LYS101 (O96)
was shorter than for LYS101 (O96) and 10i or 10k. Taken
together, these results suggest that it is reasonable for the IC50
values of 10i and 10k to be similar to EFV.
Furthermore, although 10e and 10l were located in the
active site, the IC50 value of 10l is almost 6-fold lower than 10e.
This result is probably due to the fact that the allyl group of
compound 10l can bind into the region formed by Tyr 181,
Tyr188, Phe227 and Trp229 while the methyl group of com-
pound 10e can’t.29 However, the extension of the allyl group of
compound 10l did not appear to fill the space between Tyr
181, Tyr188 and Trp229 any better than the n-butyl group of
compound 10k. In addition, the total scores of compounds
10e and 10l were higher than for compounds 10i and 10k,
which all fits with the IC50 values in Table 2.
The superimpositions of EFV and 10i, 10k, 10e and 10l are
shown in Fig. 5. In general, the superimpositions of the
benzene moiety of EFV and the corresponding benzene moi-
eties of compounds 10i, 10k and 10l were better than for EFV
and compound 10e.
The values for the calculation of the interactions between
the six compounds and their target, Y181C mutant HIV-1
RT (PDB code: 1JKH), are shown in Table 5 and Fig. 6.
According to the calculation results, the location of EFV in
the active site is also basically consistent with the crystal
structure.
Table 3 Inhibitory activity of compounds 10i and 10k against WT HIV-1
RTa
EC50 b (μM)
WT IIIB
Compd
10ib
133.33
18.59
0.08
10kb
EFVc
NVPc
0.4
a Data represent the mean of at least three separate experiments.
b Effective concentration (EC50, μM) required to inhibit the RT activity
of the indicated strain by 50%. c Ref. 14c, IC50 (μM).
and TI (selectivity index represented by the TC50/IC50 ratio)
values, are listed in Table 2.
As shown in Table 2, most trifluoromethyl indoles showed
moderate to potent inhibitory activities against WT HIV-1 with
IC50 values in the range of 57.068–0.003 μM. Among them,
compounds 10i and 10k displayed good anti-HIV-1 activities
against WT HIV-1 with an IC50 value of 0.003 μM, and com-
pound 10k showed the greatest selectivity (TI > 7079). Further-
more, compound 10k also showed potential activity against
the Y181C mutant virus with an IC50 value of 1.932 μM.
Based on the results of the antiviral assay (Table 2), some
important SAR information can be summarized as follows: (1)
“B” type trifluoromethyl indoles (10a–10n) were more potent
against both the WT HIV-1 and the Y181C resistant mutant
virus than “A” type (9f–9e); (2) substituents at the C5 position
of the benzene ring influenced the activity: Br > Cl; (3) indoles
with an NO2 substituent at the C7 position of the benzene ring
lost their activities against WT HIV-1 strain and their toxicity
increased (9e and 10n); (4) as far as the R2 group of “B” type
indoles was concerned, as for the R1 group at the same posi-
tion on the benzene ring, derivatives with R2 = n-butyl were
more potent than those with R2 = methyl, isopropyl, allyl or
acetonyl. Compounds 10b, 10k and 10g were superior to other
compounds against WT HIV-1.
The calculation of the interactions between ligands and
Y181C mutant HIV-1 RT showed that: (1) the total scores of
compounds 10i and 10k corresponded well to their biological
assays. (2) The length of the hydrogen bond between residue
Lys101 and EFV was shorter than for Lys101 and 10i or 10k.
Furthermore, in the active site, the superimposition of com-
pound 10k and EFV was better than for 10i and EFV. These
data were consistent with the biological assay results: the IC50
value of EFV was lower than those of 10k and 10i, and the IC50
value of 10k was lower than that of 10i. (3) Although the hydro-
gen bond lengths between Lys101 and 10i or 10k were identi-
HIV-1 RT inhibitory activities for compounds 10i and 10k
with the better cellular antiviral activities are listed in Table 3.
Molecular modeling
In order to investigate the binding mode of our new com- cal, the IC50 value of 10k was almost 10-fold lower than that of
pounds, interactions between the NNBS of HIV-1 RT and com- 10i. This result is probably due to the fact that the n-butyl
pounds 10e, 10l, 10i and 10k were calculated by FlexX.26 group of compound 10k could bind into the region formed by
Coordinates of the NNBS were taken from the crystal structure Cys181, Tyr188, Phe227, and Trp229.29 Its extension was seen
of the WT HIV-1 RT/TMC278 complex (PDB code: 2ZD1)27 and to fill the space between Cys181, Tyr188, and Trp229. In
Tyr181Cys mutant HIV-1 RT/EFV complex (PDB code: 1JKH).28
addition, the IC50 value of 10i for the Y181C mutant was 1000-
The values for the calculation of the interactions between fold higher than for the wild-type strain. The mutation of
the six compounds and target WT HIV-1 RT (PDB code: 2ZD1) Tyr181 to Cys181 also prevented the methyl group from enter-
are shown in Table 4 and Fig. 4. According to the calculation ing the region.
results, the location of TMC278 in the active site was basically
consistent with that in the crystal structure. In general, the Y181C mutant HIV-1 RT NNBS showed that the three
lower the total score of a compound, the higher its activity. compounds were bound outside of the active site. Although
The binding modes of compounds NVP, 10e and 10l in the
The docking results of ligands and WT HIV-1 RT showed there were hydrogen bonds between Lys101 and the three
that not only were there hydrogen bonds between EFV, 10i or compounds NVP, 10e and 10l, these hydrogen bonds were
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Table 4 Interactions between ligands and WT HIV-1 RT (2ZD1)
Compd
Structure
IC50 (μM)
Total score (kcal mol−1
)
H-bond (Å)
TMC278a (Cry.)
0.0004
No calculation
Lys101 (O96)–TMC278 (H40): 1.78
Lys101 (H92)–TMC278 (N2): 2.22
TMC278 (Cal.)
EFV
−34.27
−13.58
Lys101 (O96)–TMC278(H40): 1.62
Lys101 (H92)–TMC278(N2): 2.20
LYS101 (O96)–EFV(H25): 1.38
LYS101 (H92)–EFV(O5): 1.92
0.003
NVP
0.031
0.003
−16.17
−18.27
LYS103 (H154)–NVP(N17): 1.82
LYS103 (H156)–NVP(N3): 1.82
10i
Lys101 (O96)–10i (H27): 1.91
Lys101 (H92)–10i (O12): 1.58
Lys101 (H92)–10i (O13): 2.65
10k
10e
0.003
−18.27
−11.47
Lys101 (O96)–10k (H30): 2.14
Lys101 (H92)–10k (O12): 1.67
12.0614
Lys101 (O96)–10e (H27): 1.81
Lys101 (H92)–10e (O21): 2.23
10l
1.4712
−16.36
Lys101 (O96)–10l (H29): 2.16
Lys101 (H92)–10l (O21): 1.65
a Ref. 30.
Fig. 4 Interactions between HIV-1 RT WT (PDB: 2ZD1) and TMC278 (red), other compounds. (a) TMC278 (cry.); (b) TMC278 (red, cry.), blue one docking
result; (c) TMC278 (red, cry.), EFV (blue, docking result); (d) TMC278 (red, cry.), NVP (blue, docking result); (e) TMC278 (red, cry.), 10i (blue, docking result);
(f) TMC278 (red, cry.), 10k (blue, docking result); (g) TMC278 (red, cry.), 10e (blue, docking result); (h) TMC278 (red, cry.), 10l (blue, docking result).
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Fig. 5 Superimpositions of EFV and compounds 10i, 10k, 10e and 10l. (a) EFV (red), 10i (blue); (b) EFV (red), 10k (blue); (c) EFV (red), 10e (blue); (d)
EFV (red), 10l (blue).
different from those between Lys101 and EFV, 10i and 10k. unusual for most NNRTIs binding to the active site. Gener-
The main difference was in the particular atoms of Lys101 ally, the larger the difference in electronegativity between the
that were involved in hydrogen bonds to 10e and 10l. The H atom and the other atom (N, O, or F), the stronger the
total scores of 10e and 10l were also higher than for 10i and H-bond. This also inspired us to pay more attention to
10k, which was consistent with the IC50 values in Table 2.
special H–F hydrogen bonds for further fluorine-containing
Based on the molecular modelling investigations on the drugs design, which will also contribute to strengthen inter-
binding mode of ligands to the NNBS of Y181C mutant HIV-1 action of the ligand and target and improve biological
RT, together with the SAR studies as described above, we pos- activity.
tulated that selecting a suitable substituent (R2) to fit the
In summary, the calculation results of the interaction
pocket formed by Cys181, Tyr188, Phe227, and Trp229 would between the targets and our newly designed and synthesized
be beneficial towards enhancing the interaction between the compounds was consistent with the biological evaluation. For
inhibitors and Y181C mutant HIV-1 RT.
the Tyr181Cys mutant in the active site, two different mole-
Moreover, a hydrogen bond was formed between 10k cular models provided insights for further molecular design
(F25)–Cys181 (H214) in the mutant Y181C, which was and structural optimization.
Table 5 Interaction between ligands and Y181C mutant HIV-1 RT (1JKH)
Compd
Structure
IC50 (μM)
Total score
H-bond (Å)
EFV (cry.)
EFV (cal.)
0.021
No calculation
−22.99
Lys101 (H56)–EFV (O5): 2.72
Lys101 (H56)–EFV (O5): 1.99
Lys101 (O60)–EFV (H25): 1.77
NVP
>30
−13.81
Lys101 (H74)–NVP (N3): 1.91
Lys101 (H76)–NVP (N17): 1.90
10i
13.9
−11.35
−12.58
−10.95
Lys101 (H56)–10i (O12): 2.06
Lys101 (H56)–10i (O13): 2.71
Lys101 (O60)–10i (H27): 1.89
10k
10e
1.93
Lys101 (H56)–10k (O12): 2.06
Lys101 (H56)–10k (O13): 2.71
Lys101 (O60)–10k (H30): 1.89
Cys181 (H214)–10k (F25): 2.60
38.4248
Lys101 (H76)–10e (O22):1.83
Lys103 (H118)–10e (O21): 1.91
10l
7.6286
−8.80
Lys101 (H76)–10l (O22):1.78
Lys103 (H118)–10l (O21): 1.78
Lys103 (H118)–10l (O14): 2.47
Lys103 (H119)–10l (O14): 2.01
3452 | Org. Biomol. Chem., 2014, 12, 3446–3458
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Fig. 6 Interaction between the Y181C mutant HIV-1 RT (PDB: 1JKH), EFV (red) and other compounds. (a) EFV (cry.); (b) EFV (red, cry.; blue, docking
result); (c) EFV (red, cry.), NVP (blue, docking result); (d) EFV (red, cry.), 10i (blue, docking result); (e) EFV (red, cry.), 10k (blue, docking result); (f) EFV
(red, cry.), 10e (blue, docking result); (g) EFV (red, cry.), 10l (blue, docking result).
purified with silica gel column to give the product methyl
2-(2-phenylhydrazono) propanoate 4 in a yield of 80–95%.
General procedure for preparation of target compounds 5. A
Experimental
Chemistry
All commercially available reagents and solvents were used solution of hydrazone 4 (1 mmol) in MeOH (20 mL) with cat.
without further purification. Reactions were monitored by H2SO4 or 4 N HCl–MeOH was refluxed under Ar. After
thin-layer chromatography (TLC) and column chromatography 2.5 hours, the resulting reaction solution was poured into
was carried out on silica gel H (10 40 mm). IR spectra were crushed ice and extracted with DCM (2 × 100 mL). The organic
recorded on a FT-IR spectrometer and only major peaks are layer was washed with sat. NaHCO3 (2 × 10 mL), water and
reported. NMR spectra were recorded on a Bruker DRX 300 or then brine, separated, dried with MgSO4, filtered and evapor-
400 MHz spectrometer. Mass spectra were obtained using an ated under reduced pressure to give the product 5 in a yield of
Agilent 592N spectrometer and a Shimadzu LCMS-2010EV. The 65–85%.
purity of compounds was determined by analytical HPLC
General procedure for preparation of target compounds
using a Kromasil 5u 100A C18 column (4.6 × 150 mm, Nacalai 6. To a solution of methyl 1H-indole-2-carboxylate 5 (5 mmol)
Tesque, Inc., Kyoto, Japan) at a flow rate of 1.0 mL min−1 on a in EtOH (10 mL) was added hydrazine hydrate (4 ml, 85%) and
Shimadzu SPD20A LC-20AT (Shimadzu Corp., Ltd, Kyoto, the reaction solution was stirred at reflux for 5 hours. After
Japan). Gradient conditions: (1) solvent A (acetonitrile) and cooling to room temperature, the solid was collected by fil-
solvent B (water): 0–20.00 min, 10/90 (A/B)–100/0 (A/B) (linear tration, washed with cold EtOH and dried to give the product 6
gradient); 20.00–30.00 min, 100% A. Eluting products were in a yield of 85–95%.
detected by UV at 254 nm; (2) solvent A (acetonitrile) and
solvent B (water): 0–20.00 min, 80/20 (A/B). Eluting products 7. To a solution of 1H-indole-2-carbohydrazide 6 (10 mmol) in
were detected by UV at 214 nm. dioxane (10 mL) was added AcOH (10 mL) at 0 °C with stirring.
General procedure for preparation of target compounds
Statistical analysis was performed using GraphPad Prism Then NaNO2 (800 mg) in H2O (2 mL) was added dropwise at
5.0 (GraphPad Software, Inc., San Diego, CA). Data were pro- 0 °C and the reaction mixture was stirred at the same tempera-
cessed using non-linear regression and a standard sigmoidal ture for 0.5–1 hour. The solids were collected by filtration,
dose response model to obtain IC50 values. Dose–response washed well with ice water and dried to give the azide product
data are presented as means standard errors from three inde- 7 in a yield of 80–95%.
pendent experiments, with triple wells for each concentration.
General procedure for preparation of target compounds 8. A
solution of azide 7 (1 mmol) in dry EtOH (20 mL) was refluxed
for 1–5 hours. After cooling to room temperature, the solids
were removed by filtration, and the filtrate was concentrated
under reduced pressure to give the carbamate product 8 in a
yield of 85–90%.
General procedure for preparation of target compounds 9a–
9e. To a solution of carbamate 8 (1 mmol) in dry DMF (1 mL)
or dry diethyl ether (10 ml) was added dropwise TFAA
General procedure for preparation of target compounds 9a–9e
General procedure for preparation of target compounds
4. To a solution of phenylhydrazine hydrochloride 3 (5 mmol)
in MeOH (20 mL) was added methylpyruvate (1.4–1.5 mmol)
and the reaction was stirred at reflux until the starting material
was consumed completely (monitored by TLC). The solvents
were removed under reduced pressure and the residue was
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(2.5 mmol) at 0 °C with stirring. After completion (monitored s, NH), 10.75 (br s, NH), 7.82 (s, 1H), 7.62 (d, J = 8.6 Hz, 1H),
by TLC), water was added and the solution was extracted with 7.39 (dd, J = 8.6, 1.7 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 1.38 (t, J =
EtOAc (2 × 100 mL). The organic layer was separated, dried 7.1 Hz, 3H); 13C NMR (101 MHz, in acetone-d6): δ 173.33 (q, J =
with Na2SO4, filtered and concentrated to give the residue. The 36 Hz), 152.78, 150.01, 132.60, 126.00, 123.75, 121.54 (q, J =
residue was purified with silica gel column to give the 6 Hz), 117.4 (q, J = 286 Hz), 116.13, 114.54, 94.32, 63.10, 13.55;
trifluoroacetyl carbamate products 9a–9e in a yield of 75–90%.
Ethyl 5-chloro-3-(2,2,2-trifluoroacetyl)-1H-indol-2-ylcarbamate
MS (EI) m/z: 380(99.36), 378(100) [M]+, 354, 334, 309, 299, 283,
265, 237, 226, 201, 183, 158, 129, 102, 75, 64, 51, 43; IR (cm−1):
3370.4, 3279.7, 2991.2, 1735.1, 1639.1, 1566.9, 1529.5, 1471.3,
1431.9, 1377.3, 1332.1, 1271.6, 1255.8, 1214.7, 1193.4, 1182.3,
1144.2, 1126.9, 1072.8, 1061.6, 1013.7, 950.5, 862.5, 814.9,
775.4, 767.7, 742.8, 706.2, 688.3, 641.8, 585.8, 532.9, 456.3,
419.7. HPLC purity: 99% (254 nm), tR: 20.95 min; 98%
(214 nm), tR: 5.76 min.
(9a). Yield 85%; 1H NMR (400 MHz, in acetone-d6):
δ
7.67–7.65 (m, 2H), 7.24 (dd, J = 8.6, 1.8 Hz, 1H), 4.37 (q, J = 7.1
1
Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H); H NMR (300 MHz, in DMSO-
d6): δ 1.30–1.34 (m, 3H), 4.29–4.34 (m, 2H), 7.30 (d, J = 8.1 Hz,
1H), 7.53 (s, 1H), 7.65 (d, J = 8.7 Hz, 1H), 10.70 (s, 1 H), 12.80
(m, 1 H). 13C NMR (101 MHz, in acetone-d6): δ 173.31 (q, J = 36
Hz), 152.75, 150.12, 132.28, 128.48, 123.22, 118.57 (q, J = 6 Hz),
Ethyl 7-nitro-3-(2,2,2-trifluoroacetyl)-1H-indol-2-ylcarbamate
117.37 (q, J = 286 Hz), 114.14, 94.46, 63.09, 13.55; MS (EI) m/z: (9e). Yield 75%; 1H NMR (300 MHz, CDCl3): δ 12.26 (br s, NH),
334[M]+, 288, 265, 256, 237, 226, 219, 203, 193, 178, 165, 158, 10.67 (br s, NH), 8.17 (br d, J = 8.5 Hz, 1H), 8.14 (br d, J = 7.9
149, 137, 129, 125, 109, 97, 91, 77, 69, 57, 51, 43, 41; IR (cm−1): Hz, 1H), 7.40 (t, J = 8.2 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.42 (t,
3373.7, 3276.2, 2992.4, 2914.3, 1736.4, 1639.1, 1568.2, 1531.4, J = 7.1 Hz, 3H); 13C NMR (101 MHz, in acetone-d6): δ 174.15 (q,
1476.7, 1454.1, 1435.2, 1377.8, 1330.5, 1276.6, 1256.1, 1215.7, J = 38 Hz), 153.33, 150.47, 133.38, 126.36 (q, J = 5 Hz), 125.15,
1194.8, 1183.2, 1145.2, 1125.5, 1083.7, 1062.8, 1014.9, 954.5, 123.27, 119.10, 117.06 (q, J = 286 Hz), 94.56, 63.77, 13.52; MS
863.8, 817.9, 775.6, 768.1, 750.2, 714.4, 690.9, 638.4, 537.3, (EI) m/z: 345 [M]+, 329, 300, 284, 276, 256, 248, 232, 226, 214,
456.7, 435.7. HPLC purity: 99% (254 nm), tR: 21.29 min; 99% 204, 184, 158, 129, 102, 90, 75, 69, 51, 43; IR (cm−1): 3359.2,
(214 nm), tR: 6.10 min.
3282.4, 3086.4, 3031.7, 3018.2, 2997.1, 1728.1, 1645.1, 1575.1,
1530.2, 1517.2, 1471.3, 1461.3, 1434.3, 1376.4, 1338.9, 1309.1,
1271.7, 1238.9, 1221.8, 1187.8, 1152.0, 997.3, 951.2, 891.5,
860.9, 806.2, 776.4, 738.9, 705.5, 683.8, 653.1, 562.1, 477.0,
418.3. HPLC purity: 98% (254 nm), tR: 21.00 min; 97%
(214 nm), tR: 5.57 min.
Ethyl 6-chloro-3-(2,2,2-trifluoroacetyl)-1H-indol-2-ylcarbamate
(9b). Yield 87%; 1H NMR (400 MHz, in acetone-d6): δ 11.88 (br
s, NH), 10.80 (br s, NH), 7.73 (s, 1H), 7.72 (d, J = 10.3 Hz, 1H),
7.30 (d, J = 7.4 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.39 (t,
J = 7.1 Hz, 3H); 13C NMR (101 MHz, in acetone-d6): δ 173.46 (q,
J = 36 Hz), 152.84, 150.19, 134.39, 128.35, 123.34, 120.71,
120.29 (q, J = 5 Hz), 117.42 (q, J = 285 Hz), 112.75, 94.62, 63.10,
13.55; MS (EI) m/z: 334[M]+, 314, 288, 261, 233, 219, 201, 193,
165, 137, 129, 110, 102, 87, 75, 64, 58, 51, 43; IR (cm−1):
3359.7, 3252.8, 3072.3, 2989.2, 2910.7, 1741.3, 1637.8, 1581.1,
1563.3, 1530.9, 1489.9, 1477.0, 1454.4, 1378.9, 1353.2, 1327.5,
1301.1, 1275.7, 1254.8, 1196.6, 1172.9, 1151.1, 1133.4, 1116.7,
1065.0, 1016.2, 972.6, 943.8, 906.5, 886.2, 811.9, 772.8, 743.6,
706.7, 692.1, 641.1, 596.3, 577.7, 527.1, 490.4, 407.7. HPLC
purity: 100% (254 nm), tR: 21.10 min; 100% (214 nm), tR:
5.65 min.
Ethyl 5,7-dichloro-3-(2,2,2-trifluoroacetyl)-1H-indol-2-ylcarba-
mate (9c). Yield 82%; 1H NMR (400 MHz, in acetone-d6): δ 7.66
(d, J = 1.2 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 4.45 (q, J = 7.1 Hz,
2H), 1.41 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, in acetone-d6):
δ 173.82 (q, J = 33 Hz), 153.36, 150.36, 128.87, 128.74, 124.32,
122.58, 117.78 (q, J = 6 Hz), 117.63, 117.06 (q, J = 286 Hz),
95.08, 63.64, 13.51; MS (EI) m/z: 368 [M]+, 322, 299, 271, 255,
227, 220, 199, 192, 171, 163, 136, 127, 109, 100, 95, 86, 76, 69,
62, 52, 43; IR (cm−1): 3365.4, 3262.0, 3121.5, 3075.4, 2994.7,
2947.1, 1734.5, 1647.1, 1619.8, 1576.0, 1528.1, 1462.2, 1446.2,
1421.6, 1371.0, 1350.7, 1332.2, 1279.1, 1231.1, 1218.3, 1197.6,
1179.5, 1143.4, 1096.8, 1071.0, 1024.0, 995.9, 954.3, 893.3,
871.0, 860.0, 769.1, 754.5, 715.8, 698.5, 633.9, 586.2, 560.1,
459.2, 441.2. HPLC purity: 99% (254 nm), tR: 23.63 min; 99%
(214 nm), tR: 12.83 min.
General procedure for preparation of target compounds
10a–10l and 10o–10q
To a solution of trifluoroacetyl carbamate 9a–9d (1 mmol) in
dry THF (10 mL) Grignard reagents (2–4 mmol) were added
dropwise at 0 °C under Ar. After stirring at 0 °C for 1–2 hours,
the resulting reaction mixture was quenched with sat. NH4Cl
and extracted with EtOAc (2 × 100 mL). The organic layer was
separated, washed with brine, dried with Na2SO4, filtered and
concentrated under reduced pressure to give the residue. The
residue was purified with silica gel chromatography to give the
products 10a–10l in a yield of 52–85%.
Ethyl
5-chloro-3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)-1H-
indol-2-ylcarbamate (10a). Yield 83%; 1H NMR (300 MHz, in
acetone-d6): δ 10.86 (br s, 1H), 9.15 (br s, 1H), 7.43 (d, J = 8.4
Hz, 1H), 7.35 (s, 1H), 6.91 (d, J = 8.6 Hz, 1H), 6.40 (br s, 1H),
4.11 (q, J = 7.1 Hz, 2H), 1.91 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H); 13
C
NMR (101 MHz, in acetone-d6): δ 153.04, 135.85, 131.26,
126.71 (q, J = 286.0 Hz), 126.20, 125.21, 119.79, 117.68, 112.69,
92.54, 74.81 (q, J = 30.7 Hz), 61.63, 22.42, 13.77; LR-ESI: [M −
H]+ 349.0; IR (cm−1): 3425.6, 3364.3, 2999.0, 2981.4, 1697.6,
1635.9, 1586.7, 1489.6, 1473.6, 1440.9, 1381.5, 1361.1, 1298.6,
1257.1, 1235.6, 1204.2, 1178.5, 1161.5, 1134.2, 1095.2, 1074.7,
1038.5, 924.0, 909.1, 862.6, 835.9, 796.2, 763.1, 740.0, 705.1,
687.4, 649.7, 606.5, 585.6, 566.3, 529.6, 438.6, 412.1; HPLC
purity: 97% (254 nm), tR: 18.76 min; 96% (214 nm), tR:
3.42 min.
Ethyl 5-bromo-3-(2,2,2-trifluoroacetyl)-1H-indol-2-ylcarbamate
(9d). Yield 80%; 1H NMR (400 MHz, in acetone-d6): δ 11.87 (br
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Ethyl
5-chloro-3-(1,1,1-trifluoro-2-hydroxyhexan-2-yl)-1H- 3406.0, 3358.7, 3004.0, 2983.8, 1693.2, 1636.5, 1587.6, 1503.4,
indol-2-ylcarbamate (10b). Yield 81%; 1H NMR (400 MHz, in 1485.7, 1473.6, 1441.2, 1386.5, 1362.9, 1293.7, 1250.2, 1209.3,
acetone-d6): δ 10.97 (br s, 1H), 9.37 (br s, 1H), 7.55 (d, J = 8.6 1185.9, 1163.5, 1095.7, 1044.9, 1020.8, 929.3, 908.7, 873.2,
Hz, 1H), 7.45 (s, 1H), 7.04 (dd, J = 8.6, 1.7 Hz, 1H), 6.30 (br s, 842.8, 809.1, 764.1, 743.0, 706.2, 692.0, 650.7, 623.1, 608.0,
1H), 4.24 (q, J = 7.1 Hz, 2H), 2.65 (td, J = 13.9, 4.5 Hz, 1H), 2.05 584.5, 557.7, 443.0; HPLC purity: 96% (254 nm), tR: 21.61 min;
(m, 1H), 1.54 (m, 1H), 1.36 (dd, J = 14.6, 7.2 Hz, 1H), 1.29 (t, J = 97% (214 nm), tR: 6.81 min.
7.1 Hz, 3H), 1.13 (m, 1H), 0.84 (t, J = 7.4 Hz, 3H); 13C NMR
Ethyl
5,7-dichloro-3-(1,1,1-trifluoro-2-hydroxy-3-methylbu-
(101 MHz, in acetone-d6): δ 153.04, 137.06, 131.23, 126.79 (q, tan-2-yl)-1H-indol-2-ylcarbamate (10f). Yield 75%; 1H NMR
J = 286.8 Hz), 126.07, 125.32, 119.83, 117.27 (d, J = 2.0 Hz), (400 MHz, in acetone-d6): δ 10.75 (br s, 1H), 9.47 (br s, 1H),
112.68, 89.92, 78.24 (q, J = 29.4 Hz), 61.61, 32.92, 24.57, 22.32, 7.53 (s, 1H), 7.19 (s, 1H), 6.02 (br s, 1H), 4.31 (q, J = 7.1 Hz,
13.78, 13.27; LR-ESI: [M − H]− 391.1; IR (cm−1): 3422.0, 3352.6, 2H), 3.01 (dt, J = 13.7, 6.8 Hz, 1H), 1.35 (t, J = 7.1 Hz, 3H), 1.27
2961.3, 2935.8, 2872.1, 1694.1, 1636.3, 1587.4, 1488.8, 1440.5, (dd, J = 6.8, 1.4 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H); 13C NMR
1385.0, 1363.1, 1281.8, 1256.2, 1234.6, 1212.3, 1183.1, 1152.4, (101 MHz, in acetone-d6): δ 153.65, 137.51, 127.86, 126.62,
1080.5, 1059.8, 1014.0, 983.3, 959.1, 933.4, 863.1, 796.8, 788.6, 126.58 (q, J = 287 Hz), 125.60, 119.29, 117.27, 116.17, 93.63,
766.2, 737.5, 716.6, 688.4, 652.2, 594.4, 532.1; HPLC purity: 81.15 (q, J = 28 Hz), 62.13, 31.72, 16.91, 15.13, 13.71; LR-ESI:
96% (254 nm), tR: 20.69 min; 99% (214 nm), tR: 4.97 min.
Ethyl 6-chloro-3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)-1H- 2937.5, 1704.6, 1690.5, 1638.4, 1587.2, 1559.7, 1491.2, 1473.2,
[M − H]− 411.2; IR (cm−1): 3455.1, 3414.7, 3352.9, 2981.5,
indol-2-ylcarbamate (10c). Yield 82%; 1H NMR (400 MHz, in 1447.7, 1388.1, 1374.9, 1308.0, 1283.6, 1253.9, 1209.8, 1186.3,
acetone-d6): δ 10.94 (br s, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.51 (d, 1151.7, 1113.5, 1091.2, 1070.6, 1017.6, 982.7, 889.5, 873.6,
J = 8.6 Hz, 1H), 7.05 (dd, J = 8.7, 2.0 Hz, 1H), 4.26 (q, J = 834.9, 808.2, 766.2, 740.2, 717.0, 652.1, 591.1, 543.8, 444.0;
7.1 Hz, 2H), 2.05 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H); 13C NMR HPLC purity: 95% (254 nm), tR: 23.23 min; 95% (214 nm), tR:
(101 MHz, in acetone-d6): δ 153.08, 135.20, 133.30, 126.73 (q, 10.66 min.
J = 284 Hz), 125.06, 123.80, 120.13, 119.59, 111.23, 92.75, 74.88
Ethyl 5,7-dichloro-3-(1,1,1-trifluoro-2-hydroxyhexan-2-yl)-1H-
(q, J = 30 Hz), 61.61, 22.47, 13.78; LR-ESI: [M + H]+ 351.0; indol-2-ylcarbamate (10g). Yield 83%; 1H NMR (400 MHz, in
IR (cm−1): 3442.5, 3370.4, 2993.1, 2960.3, 2926.9, 2871.6, acetone-d6): δ 10.75 (br s, 1H), 9.43 (br s, 1H), 7.49 (s, 1H), 7.18
2854.3, 1692.6, 1636.1, 1599.2, 1565.7, 1488.4, 1475.6, 1437.8, (d, J = 1.6 Hz, 1H), 6.35 (br s, 1H), 4.31 (q, J = 7.1 Hz, 2H), 2.66
1381.1, 1363.1, 1291.6, 1262.3, 1233.9, 1204.3, 1179.3, 1162.9, (td, J = 14.4, 4.5 Hz, 1H), 2.16–2.03 (m, 1H), 1.75–1.50 (m, 1H),
1131.4, 1162.9, 1090.7, 1069.7, 1041.0, 943.1, 927.9, 887.2, 1.47–1.26 (m, 5H), 1.22–1.08 (m, 1H), 0.86 (t, J = 7.4 Hz, 3H);
836.6, 798.8, 762.6, 732.4, 710.6, 643.2, 609.0, 587.7, 525.7, 13C NMR (101 MHz, in acetone-d6): δ 153.65, 138.00, 127.82,
457.5, 428.8; HPLC purity: 95% (254 nm), tR: 18.55 min; 96% 127.07, 126.60 (q, J = 285 Hz), 125.77, 119.30, 116.81, 116.22,
(214 nm), tR: 3.23 min.
91.72, 78.18 (q, J = 30 Hz), 62.15, 32.84, 24.54, 22.30, 13.73,
Ethyl 6-chloro-3-(1,1,1-trifluoro-2-hydroxypent-4-en-2-yl)-1H- 13.25; LR-ESI: [M − H]− 425.2; IR (cm−1): 3403.2, 3370.5,
indol-2-ylcarbamate (10d). Yield 85%; 1H NMR (400 MHz, in 2959.0, 2932.3, 2871.3, 1690.1, 1638.9, 1590.0, 1560.3, 1490.0,
acetone-d6): δ 10.97 (br s, 1H), 9.30 (br s, 1H), 7.64 (d, J = 1473.2, 1445.2, 1387.4, 1362.0, 1283.5, 1254.2, 1220.1, 1205.0,
1.5 Hz, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.07 (dd, J = 8.7, 1.9 Hz, 1182.3, 1155.2, 1087.8, 1068.5, 975.4, 936.8, 873.6, 844.7,
1H), 6.35 (br s, 1H), 5.67 (ddt, J = 17.1, 10.2, 6.9 Hz, 1H), 5.20 809.5, 767.7, 717.7, 691.2, 652.1, 635.4, 588.9, 525.4, 442.4;
(d, J = 17.2 Hz, 1H), 5.01 (d, J = 10.2 Hz, 1H), 4.26 (q, J = HPLC purity: 98% (254 nm), tR: 23.92 min; 99% (214 nm), tR:
7.1 Hz, 2H), 3.51 (dd, J = 15.0, 6.5 Hz, 1H), 2.88 (dd, J = 15.1, 13.84 min.
7.2 Hz, 1H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, in
Ethyl 5,7-dichloro-3-(1,1,1-trifluoro-2-hydroxypent-4-en-2-yl)-
1
acetone-d6): δ 153.08, 136.40, 133.31, 130.80, 124.31 (q, J = 284 1H-indol-2-ylcarbamate (10h). Yield 82%; H NMR (300 MHz,
Hz), 120.23, 119.61, 119.16, 111.25, 90.04, 77.70 (q, J = 29 Hz), CDCl3): δ 10.62 (br s, 1H), 8.91 (br s, 1H), 7.32 (s, 1H), 7.12 (d,
61.64, 37.78, 13.80; LR-ESI: [M − H]− 375.2; IR (cm−1): 3418.8, J = 1.3 Hz, 1H), 5.64 (dt, J = 16.3, 8.3 Hz, 1H), 5.33 (d, J = 11.2
3364.9, 2981.2, 2933.3, 1704.2, 1635.7, 1592.7, 1567.3, 1487.7, Hz, 1H), 5.29 (d, J = 4.5 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 3.31
1441.5, 1383.9, 1300.6, 1276.6, 1259.8, 1233.9, 1206.0, 1186.0, (dd, J = 14.6, 6.2 Hz, 1H), 2.86 (dd, J = 14.6, 8.4 Hz, 2H), 1.35 (t,
1172.6, 1150.2, 1132.1, 1067.2, 1020.5, 996.4, 956.6, 931.9, J = 7.1 Hz, 3H); 13C NMR (101 MHz, in acetone-d6): δ 153.65,
899.4, 884.7, 855.8, 802.3, 765.9, 727.6, 670.6, 589.0, 523.3; 137.94, 130.46, 127.82, 126.92, 126.33 (q, J = 285 Hz), 125.76,
HPLC purity: 97% (254 nm), tR: 19.51 min; 96% (214 nm), 119.44, 119.33, 117.19, 116.17, 91.69, 77.53 (q, J = 30 Hz),
tR: 3.74 min.
Ethyl 5,7-dichloro-3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)- 3351.0, 3095.3, 2996.1, 2930.0, 1863.5, 1686.1, 1639.0, 1589.3,
62.18, 37.60, 13.71; LR-ESI: [M − H]− 409.2; IR (cm−1): 3414.6,
1
1H-indol-2-ylcarbamate (10e). Yield 83%; H NMR (400 MHz, 1559.1, 1489.8, 1472.7, 1445.9, 1420.1, 1389.8, 1340.9, 1260.6,
in acetone-d6): δ 10.71 (br s, 1H), 9.33 (br s, 1H), 7.52 (s, 1H), 1210.0, 1184.5, 1167.4, 1152.6, 1091.3, 1075.6, 1201.4, 991.2,
7.18 (d, J = 1.4 Hz, 1H), 6.59 (br s, 1H), 4.31 (q, J = 7.1 Hz, 2H), 940.2, 940.6, 928.3, 872.0, 838.2, 771.1, 743.5, 723.5, 654.0,
2.07 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, in 620.4, 590.0, 538.8, 440.1; HPLC purity: 96% (254 nm), tR:
acetone-d6): δ 153.65, 136.76, 127.82, 127.20, 126.54 (q, J = 284 23.39 min; 98% (214 nm), tR: 8.25 min.
Hz), 125.63, 119.24, 117.21, 116.22, 94.40, 74.70 (q, J = 31 Hz),
Ethyl
5-bromo-3-(1,1,1-trifluoro-2-hydroxypropan-2-yl)-1H-
62.16, 22.25, 13.73; LR-ESI: [M − H]− 383.2; IR (cm−1): 3473.4, indol-2-ylcarbamate (10i). Yield 85%; 1H NMR (400 MHz, in
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acetone-d6): δ 10.84 (br s, 1H), 9.15 (br s, 1H), 7.50 (s, 1H), 7.37 1019.3, 995.8, 962.3, 931.9, 912.7, 882.1, 856.8, 800.8, 193.3,
(d, J = 8.5 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 6.37 (br s, 1H), 4.10 766.3, 728.6, 702.1, 671.1, 590.1, 522.2, 475.1; HPLC purity:
(q, J = 7.1 Hz, 2H), 1.90 (s, 3H), 1.15 (t, J = 7.1 Hz, 3H); 13C 95% (254 nm), tR: 19.55 min; 97% (214 nm), tR: 3.77 min.
NMR (101 MHz, in acetone-d6): δ 153.08, 135.73, 131.56,
Ethyl 5-bromo-3-(2,2,2-trifluoro-1-hydroxy-1-phenylethyl)-1H-
126.87, 126.72 (q, J = 287 Hz), 122.50, 120.66, 113.18, 112.89, indol-2-ylcarbamate (10o). Yield 63%; 1H NMR (300 MHz, in
92.47, 74.82 (q, J = 30 Hz), 61.66, 22.45, 13.78; LR-ESI: acetone-d6): δ 11.07 (br s, 1H), 9.27 (br s, 1H), 7.64 (m, 2H),
[M − H]− 393.0; IR (cm−1): 3424.4, 3364.7, 2978.8, 1697.5, 7.45 (m, 4H), 7.04 (dd, J = 8.6, 1.9 Hz, 1H), 6.45 (br s, 1H), 4.27
1635.9, 1591.6, 1488.2, 1472.6, 1439.9, 1383.0, 1359.5, 1297.0, (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz,
1257.3, 1235.4, 1204.3, 1177.9, 1159.3, 1135.4, 1095.1, 1064.3, in acetone-d6): δ 153.05, 138.85, 136.41, 131.29, 128.99, 128.45
1039.5, 861.8, 834.9, 793.9, 763.2, 733.1, 704.0, 674.5. 647.8. (2C), 127.66 (q, J = 2.0 Hz) (2C), 127.39, 126.49 (q, J = 287.0
605.6, 584.4, 533.6, 437.0; HPLC purity: 95% (254 nm), Hz), 122.36, 120.41, 112.92, 112.58, 92.64, 78.90 (q, J = 30.0
tR: 21.01 min; 95% (214 nm), tR: 3.30 min.
Hz), 61.75, 13.78; LR-ESI: [M − H]− 455.0; IR (cm−1): 3386.2,
Ethyl 5-bromo-3-(1,1,1-trifluoro-2-hydroxy-3-methylbutan-2- 3063.8, 2983.0, 2934.7, 1718.1, 1633.9, 1586.2, 1485.5, 1449.7,
yl)-1H-indol-2-ylcarbamate (10j). Yield 78%; 1H NMR 1388.1, 1358.0, 1230.4, 1254.1, 1203.6, 1164.5, 1078.1, 1002.2,
(400 MHz, in acetone-d6): δ 11.00 (br s, 1H), 9.45 (br s, 1H), 959.0, 935.0, 882.9, 859.2, 795.2, 765.2, 732.3, 709.0, 698.7,
7.65 (s, 1H), 7.53 (d, J = 8.6 Hz, 1H), 7.18 (dd, J = 8.6, 1.8 Hz, 681.3, 668.7, 653.5, 626.1, 590.7, 549.4; HPLC purity: 91%
1H), 5.98 (br s, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.02 (dt, J = 13.7, (254 nm), tR: 19.99 min.
6.9 Hz, 1H), 1.31 (dd, J = 9.5, 4.7 Hz, 3H), 1.26 (dd, J = 6.8, 1.5
Ethyl 5-chloro-3-(2,2,2-trifluoro-1-hydroxy-1-phenylethyl)-1H-
Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H); 13C NMR (101 MHz, in indol-2-ylcarbamate (10p). Yield 58%; 1H NMR (400 MHz, in
acetone-d6): δ 153.06, 136.43, 131.58, 126.76 (q, J = 287 Hz), acetone-d6): δ 11.03 (br s, 1H), 9.29 (br s, 1H), 7.68 (m, 2H),
126.26, 122.53, 120.73, 113.15, 112.85, 91.72, 81.23 (q, J = 7.50 (d, J = 8.6 Hz, 1H), 7.47–7.41 (m, 3H), 6.97 (s, 1H), 6.93
28 Hz), 61.61, 31.67, 16.98, 15.17, 13.77; LR-ESI: [M − H]− (dt, J = 8.6, 1.7 Hz, 1H), 6.36 (br s, 1H), 4.28 (q, J = 7.1 Hz, 2H),
421.3; IR (cm−1): 3409.4, 2980.0, 1709.8, 1632.7, 1585.6, 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, in acetone-d6): δ
1485.4, 1433.6, 1384.9, 1358.9, 1255.1, 1230.5, 1207.7, 1185.5, 153.12, 138.91, 136.61, 131.03, 129.00, 128.47 (2C), 127.69 (q,
1148.5, 1111.6, 1071.6, 1033.0, 982.2, 887.0, 864.3, 796.2, J = 2.0 Hz) (2C), 126.82, 126.55 (q, J = 287.0 Hz), 124.95,
766.5, 708.5, 681.5, 648.8, 589.6, 541.2; HPLC purity: 96% 119.76, 117.43, 112.47, 92.82, 78.97 (q, J = 30.0 Hz), 61.78,
(254 nm), tR: 20.49 min; 95% (214 nm), tR: 4.80 min.
Ethyl
13.83; LR-ESI: [M − H]− 411.0; IR (cm−1): 3391.6, 2933.4,
5-bromo-3-(1,1,1-trifluoro-2-hydroxyhexan-2-yl)-1H- 1699.5, 1635.3, 1588.7, 1487.9, 1450.4, 1385.8, 1361.0, 1230.0,
indol-2-ylcarbamate (10k). Yield 75%; 1H NMR (400 MHz, in 1254.6, 1204.2, 1165.3, 1082.4, 1002.6, 964.2, 935.2, 883.0,
acetone-d6): δ 10.85 (br s, 1H), 9.22 (br s, 1H), 7.46 (s, 1H), 7.37 860.5, 796.6, 765.4, 737.4, 714.4, 698.6, 668.6, 653.8, 626.9,
(d, J = 8.5 Hz, 1H), 7.03 (dd, J = 8.5, 1.8 Hz, 1H), 6.13 (br s, 1H), 594.6, 549.8; HPLC purity: 92% (254 nm), tR: 19.69 min.
4.10 (q, J = 7.1 Hz, 2H), 2.57–2.44 (m, 1H), 1.97–1.85 (m, 1H),
Ethyl
5-chloro-3-(1-(3,5-dimethylphenyl)-2,2,2-trifluoro-1-
1.49–1.33 (m, 1H), 1.29–1.19 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H), hydroxyethyl)-1H-indol-2-ylcarbamate (10q). Yield 52%; 1H
1.00 (ddd, J = 17.1, 10.1, 5.4 Hz, 1H), 0.68 (t, J = 8 Hz, 3H); 13C NMR (300 MHz, in acetone-d6): δ 10.89 (br s, 1H), 9.12 (br s,
NMR (101 MHz, in acetone-d6): δ 153.08, 136.95, 131.53, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.14 (s, 2H), 6.96 (s, 1H), 6.79 (d,
126.79 (q, J = 288 Hz), 126.73, 122.53, 120.29, 113.17, 112.97, J = 8.5 Hz, 1H), 6.67 (br s, 1H), 6.31 (s, 1H), 4.15 (q, J = 7.5 Hz,
89.83, 78.27 (q, J = 30 Hz), 61.64, 32.95, 24.55, 22.31, 13.78, 2H), 2.17 (s, 6H), 1.19 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz,
13.24; LR-ESI: [M − H]− 435.1; IR (cm−1): 3421.4, 3357.1, in acetone-d6): δ 153.09, 138.87, 137.82 (2C), 136.44, 131.01,
2961.2, 2935.3, 2867.0, 1692.6, 1634.8, 1585.2, 1487.1, 1444.6, 130.36, 126.86, 126.55 (q, J = 287.2 Hz), 125.28 (q, J = 2.0 Hz)
1385.9, 1362.0, 1281.8, 1257.0, 1235.7, 1213.4, 1183.3, 1157.3, (2C), 124.86, 119.73, 117.60, 112.42, 92.92, 78.94 (d, J = 29.7
1099.7, 1072.5, 1007.4, 983.1, 952.1, 932.4, 862.1, 794.6, 766.5, Hz), 61.73, 20.58 (2C), 13.81; LR-ESI: [M − H]− 439.2; IR
714.0, 678.9, 649.4, 591.2, 560.7, 528.9, 421.7; HPLC purity: (cm−1): 3515.2, 3422.5, 3399.3, 3381.4, 2982.1, 2918.6, 1725.6,
96% (254 nm), tR: 21.04 min; 97% (214 nm), tR: 5.50 min.
1639.0, 1595.3, 1486.4, 1474.4, 1456.9, 1379.6, 1361.4, 1288.1,
Ethyl 5-bromo-3-(1,1,1-trifluoro-2-hydroxypent-4-en-2-yl)-1H- 1251.3, 1207.2, 1229.4, 1182.6, 1146.7, 1087.3, 1011.4, 974.4,
indol-2-ylcarbamate (10l). Yield 75%; 1H NMR (400 MHz, in 945.5, 919.6, 887.7, 855.0, 790.7, 760.3, 747.9, 726.4, 717.9,
acetone-d6): δ 11.02 (br s, 1H), 9.34 (br s, 1H), 7.67 (s, 1H), 7.53 685.6, 632.4, 594.2, 553.7, 525.4, 434.9; HPLC purity: 91%
(d, J = 8.6 Hz, 1H), 7.19 (dd, J = 8.6, 1.7 Hz, 1H), 6.42 (br s, 1H), (254 nm), tR: 20.78 min.
5.75–5.60 (m, 1H), 5.22 (dd, J = 17.1, 1.2 Hz, 1H), 5.04 (d, J =
10.2 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 3.51 (dd, J = 15.1, 6.4 Hz,
1H), 2.89 (dd, J = 15.2, 7.3 Hz, 1H), 1.32 (t, J = 7.1 Hz, 3H); 13C
General procedure for preparation of target compounds 10m
and 10n
NMR (101 MHz, in acetone-d6): δ 153.08, 136.92, 131.57,
130.77, 126.57, 126.52 (q, J = 286 Hz), 122.59, 120.61, 119.24, To a solution of trifluoroacetyl carbamate 9c and 9e (1 mmol)
113.17, 113.00, 89.72, 77.63 (q, J = 30 Hz), 61.68, 37.73, 13.79; and Proline/AcK (1/1, 0.3 mmol/0.3 mmol) in dry DMSO
LR-ESI: [M − H]− 419.2; IR (cm−1): 3407.1, 3367.6, 2983.2, (4 mL) was added acetone (1 ml) at 40 °C under Ar. After stir-
2933.3, 1703.0, 1633.6, 1582.8, 1491.2, 1443.5, 1361.0, 1296.9, ring at 40 °C for 3–4 days, the solvent was evaporated under
1275.5, 1258.2, 1235.3, 1211.7, 1182.4, 1145.1, 1094.5, 1072.4, reduced pressure and the residue was purified with silica gel
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chromatography to give the desired products 10 m and 10n in diluted inhibitors for 3 days. Virus yields were quantified
a yield of 50–60%. using TZM-bl as a reporter cell line. Briefly, MT-2 cells (1.5 ×
Ethyl 5,7-dichloro-3-(1,1,1-trifluoro-2-hydroxy-4-oxopentan-2- 104 per well) were infected with an HIV-1 IIIB in 100 μL of
yl)-1H-indol-2-ylcarbamate (10m). Yield 60%; 1H NMR RPMI 1640 medium containing 10% FBS in the presence or
(400 MHz, in acetone-d6): δ 10.78 (br s, 1H), 9.29 (br s, 1H), absence of a test compound at graded concentrations for 3
7.60 (s, 1H), 7.19 (d, J = 1.5 Hz, 1H), 7.00 (br s, 1H), 4.31 (q, J = days. Then 10 μL culture supernatants were transfer to the
7.1 Hz, 2H), 3.89 (d, J = 17.4 Hz, 1H), 3.54 (d, J = 17.4 Hz, 1H), same positions of a new 384-well plate containing TZM-bl cells
2.22 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, in (1.5 × 104 per well). 24 hours after infection, 20 μL of Bright-
acetone-d6): δ 206.61, 153.70, 137.34, 127.78, 126.78, 125.77, Glo Luciferase Assay reagent (Promega, Madison, WI) were
125.74 (q, J = 285 Hz), 119.43(2C), 117.11, 116.25, 92.59, 76.13 added to the wells for measurement of luminescence with a
(q, J = 30 Hz), 62.20, 44.24, 30.54, 13.71; LR-ESI: [M − H]− Victor 2 luminometer. The effective concentrations for 50%
425.2; IR (cm−1): 3380.7, 3349.8, 3246.0, 3072.2, 2997.5, inhibition (EC50) were calculated using GraphPad Prism 5.0.
2910.9, 1725.6, 1701.6, 1636.4, 1584.0, 1556.5, 1498.9, 1484.1,
HIV-1 infection assay using TZM-bl as a reporter cell line.
1469.6, 1433.1, 1388.6, 1361.4, 1330.4, 1268.1, 1236.0, 1171.7, Inhibition of HIV-1 infection was measured as reduction in
1151.1, 1090.5, 1079.3, 1040.1, 981.1, 922.6, 897.2, 873.3, luciferase gene expression after a single round of virus infec-
848.7, 767.2, 739.1, 726.0, 689.8, 611.3, 570.6, 530.6, 442.8, tion of TZM-bl cells as described previously. Briefly, 200
409.5; HPLC purity: 98% (254 nm), tR: 20.87 min; 99% TCID50 of virus with a resistant mutation at 181 (where Tyr
(214 nm), tR: 5.36 min.
was replaced by Cys) was used to infect TZM-bl cells in the
Ethyl 7-nitro-3-(1,1,1-trifluoro-2-hydroxy-4-oxopentan-2-yl)- presence of various concentrations of compounds. 48 hours
1
1H-indol-2-ylcarbamate (10n). Yield 60%; H NMR (400 MHz, after infection, 20 μL of Bright-Glo Luciferase Assay reagent
in acetone-d6): δ 11.94 (br s, 1H), 9.37 (br s, 1H), 8.08 (d, J = 8.0 (Promega, Madison, WI) were added to the wells for measure-
Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.03 ment of luminescence with a Victor 2 luminometer. The 50%
(br s, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.94 (d, J = 17.3 Hz, 1H), inhibitory concentration (IC50) was defined as the concen-
3.56 (d, J = 17.3 Hz, 1H), 2.21 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H); tration that caused a 50% reduction of luciferase activity (Rela-
13C NMR (101 MHz, in acetone-d6): δ 206.35, 153.56, 137.52, tive Light Units) compared to virus control wells.
132.54, 128.36, 126.17, 125.74 (q, J = 285 Hz), 125.70, 120.02,
116.67, 91.96, 76.23 (q, J = 31 Hz), 62.28, 44.55, 30.55, 13.74;
LR-ESI: [M − H]− 402.2, [M + Na]+ 426.3, [M + H]+ 404.3; IR
Acknowledgements
(cm−1): 3403.9, 3387.8, 2995.5, 2910.2, 1724.8, 1643.3, 1593.8,
1520.4, 1568.8, 1499.4, 1459.6, 1429.0, 1415.6, 1364.2, 1312.5, This work was financially supported by Important National
1343.0, 1234.4, 1201.7, 1168.1, 1150.4, 1114.7, 1084.5, 1039.2, Science & Technology Specific Projects (no. 2009ZX09501-007),
1021.2, 977.1, 917.9, 891.1, 871.3, 846.6, 800.2, 767.2, 734.6, National Natural Scientific Foundation of China (Key Program,
700.3, 634.9, 621.1, 565.7, 529.1, 487.5, 468.2; HPLC purity: no. 20832007), National Program on Key Basic Research
98% (254 nm), tR: 18.01 min; 97% (214 nm), tR: 2.90 min.
Project (973 Program, no. 2010CB126103) and National
Natural Science Foundation of China (General Program, no.
21072216).
Anti-HIV activity assay
The anti-HIV activity and cytotoxicity of compounds 9a–9e and
10a–10n were evaluated against wild-type HIV-1 strain IIIB and
resistant mutant strain Y181C HIV-1 in MT-2 cell cultures.
Notes and references
In vitro cytotoxicity assay in MT-2 cells
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3458 | Org. Biomol. Chem., 2014, 12, 3446–3458
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