Antiretroviral (HIV-1) activity of azulene derivatives

Article abstract of DOI:10.1016/j.bmc.2016.02.038

The antiretroviral activity of azulene derivatives was detected for the first time. A series of eighteen diversely substituted azulenes was synthesized and tested in vitro using HIV-1 based virus-like particles (VLPs) and infectious HIV-1 virus in U2OS and TZM-bl cell lines. Among the compounds tested, the 2-hydroxyazulenes demonstrated the most significant activity by inhibiting HIV-1 replication with IC₅₀ of 2-10 and 8-20 μM for the VLPs and the infectious virus, respectively. These results indicate that azulene derivatives may be potentially useful candidates for the development of antiretroviral agents.

Full text of DOI:10.1016/j.bmc.2016.02.038

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antiretroviral (HIV-1) activity of azulene derivatives
⇑
Julia Peet, Anastasia Selyutina, Aleksei Bredihhin
Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
a r t i c l e i n f o
a b s t r a c t
Article history:
The antiretroviral activity of azulene derivatives was detected for the first time. A series of eighteen
diversely substituted azulenes was synthesized and tested in vitro using HIV-1 based virus-like particles
(VLPs) and infectious HIV-1 virus in U2OS and TZM-bl cell lines. Among the compounds tested, the 2-
hydroxyazulenes demonstrated the most significant activity by inhibiting HIV-1 replication with IC50
Received 18 November 2015
Revised 17 February 2016
Accepted 27 February 2016
Available online xxxx
of 2–10 and 8–20 lM for the VLPs and the infectious virus, respectively. These results indicate that
azulene derivatives may be potentially useful candidates for the development of antiretroviral agents.
Keywords:
Azulene
Ó 2016 Elsevier Ltd. All rights reserved.
Antiviral
HIV
HIV-1 inhibition
1. Introduction
and heteroaryl groups to maximize the scope of this study. Polar
and hydrophilic compounds were preferred because of their supe-
The human immunodeficiency virus 1 (HIV-1)1 is a pandemic
virus, and the number of people infected with HIV-1 is constantly
increasing. In 2014, about 37 million people were living with HIV
and 1.2 million have died from HIV-associated diseases.2 Currently,
there are about 30 anti-HIV drugs of different classes available;
however, the search for new highly active and safe compounds
targeting the HIV remains a very important task, which could
potentially affect lives of millions of people.
Azulene derivatives have been shown to have anti-inflamma-
tory,3,4 antibacterial,5–7 anti-ulcer,8–10 anti-cancer11–14 and other
activities,15–18 but to the best of our knowledge, their antiviral
effect has not been reported before. However, strong antiviral
activity against the Herpes simplex virus was demonstrated by reis-
wigin diterpenes that have similar structures to azulene but with a
non-aromatic skeleton.19 In the current study, we screened azulene
and its derivatives for compounds with high anti-HIV activity.
rior solubility and bioavailability. To study the structure–activity
relationship (SAR) and possible mechanisms of action of the inves-
tigated compounds, we included well-studied and documented
compounds with biological activity such as guaiazulene (2) and
sodium gualenate (3) in the list of tested compounds.
The synthesis of azulene derivatives is often a complicated
task, and in some cases requires multistep procedures or even
the construction of an azulene core that already bears the desired
substituents. Recently, our research group has developed several
methods that significantly simplified the synthesis of azulene
derivatives, and most of the tested compounds in this study were
prepared according to the procedures described in our previous
publications.20,21 Thus, compounds 5,
6 and 12 were easily
obtained starting with azulene (1) (see Scheme 1), and com-
pounds 7, 10, 11, 17 and 18 were obtained using the Negishi
cross-coupling of bromoazulenes with corresponding organozinc
reagents.20 In addition, compounds 4 and 16 were synthesized
via azulenyl zinc intermediates.21 Compound 14 was prepared
according to the procedure described by Zhang22 and further
decarboxylated to produce 13.23 Compound 15 can be obtained
by decarboxylation of 14,24 however, we have obtained it more
conveniently by the direct boronation of azulene followed by oxi-
dation with hydrogen peroxide as was described by Kurotobi.25
Sodium gualenate (3) was easily prepared from guaiazulene (2)
and sulfuric acid; however, compound 9 was obtained only with
a low yield using the Buchwald–Hartwig amination. Compound 8
was prepared by the hydrolysis of ethyl 4-(3-chloroazulen-1-yl)
benzoate.
2. Results and discussion
2.1. Chemistry
The series of diversely substituted azulenes that was used in
this study is shown in Figure 1. We incorporated a variety of sub-
stituents including halogeno, carboxyethyl, hydroxy, phenyl, aryl
⇑
Corresponding author.
E-mail address: aleksei.bredihhin@ut.ee (A. Bredihhin).
http://dx.doi.org/10.1016/j.bmc.2016.02.038
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Peet, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.038

2
J. Peet et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Cl
N
Cl
Br
N
SO3Na
Cl
Cl
Cl
Br
Br
1
2
4
3
5
6
Cl
Cl
CO2Et
CO2Et
N
O
O
CO2H
N
CO 2Et
9
8
10
11
7
12
CO2Et
F
CN
N
CO2Et
OH
CO2Et
OH
OH
CO2Et
14
15
16
17
18
13
Figure 1. Structure of compounds investigated in this study.
Cl
h
b
5
Br
SO3Na
2
3
Br
Cl
Cl
a
i
c
6
Br
N
Br
6
d
9
CO2Et
1
O
e
R1
R2
R1
j
f
12
Br
OH
7,10,11,17,18
15
R1
I
R1
R1
R2
k
l
Cl
Cl
ZnCl
g
4,16
CO2Et
CO2Et
Cl
O
8
m
n
OH
OH
CO2Et
CO2H
CO2Et
14
13
Scheme 1. Synthesis of substituted azulenes. Reagents and conditions: (a) NCS, DCM, 0 °C to rt, 25 min; (b) NBS, DCM, 0 °C to rt, 25 min; (c) 2.0 equiv NBS, DCM, 0 °C to rt,
25 min; (d) AlCl3, ClCO2Et, rt,1 h; (e) pin2B2, [IrCl(cod)]2, bpy, cyclohexane, reflux, 14 h; (f) H2O2 (aq 30%), EtOH, 0 °C, 2 h; (g) NaOH (aq 10%), MeOH/1,2-dimethoxyethane,
reflux, 2 h; (h) H2SO4, Ac2O, 0 °C to rt, 4 h; (i) Pd2(dba)3, BINAP, tBuONa, morpholine, PhMe, 100 °C, 2 h; (j) Pd(dba)2, SPhos, RZnCl, THF, rt, 0.5–5 h; (k) (1) iPrMgClꢀLiCl, 20 min;
(2) ZnCl2, 15 min, THF, ꢁ30 °C; (l) Pd(dba)2, SPhos, RHal, THF, rt or 50 °C, 0.5–2 h; (m) EtONa, diethyl malonate, EtOH, 72 h, rt; (n) KOH (2 M in EtOH), reflux, 3 h.
2.2. Evaluation of antiretroviral activity
genome RNA, and thus Gluc activity in the infected cells correlates
with the efficiency of infection.26 To estimate the antiretroviral
activity, we have infected the U2OS cells with VLPs in the presence
of the test compounds. In the first screening, the compounds were
applied at their corresponding highest non-toxic concentrations
(Table 1) while 0.5% DMSO (used for dilution of compounds) and
nevirapine (an HIV-1 non-nucleoside reverse transcriptase inhibi-
tor) were used as the negative and positive controls, respectively.
Among the tested substances, guaiazulene (2) and compounds 5,
6, 10, 12, 13, 14 and 15 inhibited Gluc activity by 48% or more com-
pared to the negative control (Table 1). Guaiazulene (2) and com-
pounds 5, 6 and 12 showed moderate antiretroviral activity and
inhibited Gluc activity to 48%, 32%, 36% and 52%, respectively;
however, since these compounds were applied at a relatively high
First, the cytotoxicity of the compounds was tested using the
xCELLigence RTCA DP instrument and U2OS cell line. The studied
compounds were applied in several concentrations, and viability
of the cells was compared with viability of the cells, treated with
0.5% dimethyl sulphoxide (DMSO (served as a vehicle control)).
The highest non-toxic concentration of the compound was deter-
mined as the concentration that reduced cell viability by less than
20% (Table 1).
The first screening for antiretroviral activity was performed
using HIV-1-based virus-like particles (VLPs). This assay is fast, safe
and sensitive, and therefore, allowed the convenient preliminary
evaluation of the antiretroviral activity of test compounds. VLP
particles encode the Gaussia luciferase (Gluc) reporter gene in their
concentrations (75 lM), we have assumed that their IC50 values
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J. Peet et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
additional one or two ethoxycarbonyl groups. In the same time,
compound 12, which has only one ethoxycarbonyl group and no
Toxicity and antiretroviral activity of tested compounds
OH group, showed much lower antiretroviral activity (IC50
>
Compound Highest non-toxic
concentration
Toxicity
Antiretroviral activity:
normalized Gluc
activitya
assay: cell
75 M). That led us to the conclusion, that the presence of the
l
(lM)
viabilitya
OH group in the second position is the key structural element
DMSO
1
2
3
4
5
6
7
8
0.5%
150
75
500
0.5
75
75
75
50
5
100
104
106
91
80
101
96
88
92
80
100
76
48
154
141
32
36
94
103
98
required for demonstrating a high antiretroviral activity.
Because compounds 13, 14 and 15 showed a positive effect in
the first screening for anti-HIV activity (performed using HIV-
1-based VLPs), we have further tested their ability to inhibit
infectious HIV-1 virus replication using TZM-bl cells (Table 2).
The toxicity of the compounds in the experiments with TZM-bl
cells was comparable with their toxicity against U2OS cells (CC50
of 13, 14 and 15 was 260 30, 65 15, and 60
7 lM, respectively,
9
Table 2). Surprisingly, compound 13 was not active against HIV-1
virus (data not shown). In contrast, compounds 14 and 15 were
10
11
12
13
14
15
16
17
18
18.75
150
75
150
50
5
50
150
150
80
83
85
89
80
80
86
80
36
70
52
6.4
3.5
51
80
63
active with of IC50 of 20 7 and 8.5
3 lM, respectively, and their
SI values were 3 and 7, respectively (Table 2).
We have supposed, that antiretroviral activity of compounds 13,
14, and 15 can be explained by their inhibition of an important
HIV-1 enzyme—reverse transcriptase. In order to test this hypoth-
esis we tested compounds 13, 14, and 15 for their ability to inhibit
reverse transcriptase activity in the cell-free assay. These com-
80
NT
124
0.01
Nevirapine 50
a
Percentage of negative control (DMSO); NT—not tested.
pounds were tested at the high concentration (500
compound 14 inhibited the reaction. IC50 (14) in the cell-free assay
was 104.2 M which indicates that compound 14 acts as a
lM) and only
4
l
reverse transcriptase inhibitor.
would be well above physiological concentrations and, therefore,
they were excluded from further analysis. Interestingly, compound
10 showed slightly better results than compounds 2, 5, 6 and 12
did; however, its activity was also considered too low for further
analysis.
The most prominent inhibitory effect was demonstrated by
compounds 13, 14 and 15, which all bear a hydroxyl (OH) group
at position 2. Compound 15 inhibited Gluc activity only to 51%,
which was similar to the effect exhibited by guaiazulene (2) and
compound 12; however, this occurred at a concentration that
There are two types of reverse transcriptase inhibitors—nucle-
oside (nucleotide) and non-nucleoside. Compounds from the first
group mimic natural substrates of reverse transcriptase (nucleo-
tides). We assume that compound 14 is a non-nucleoside reverse
transcriptase inhibitor because it does not resemble nucleoside
or nucleotide structure. Non-nucleoside inhibitors can have very
diverse chemical structures, but all of them bind reverse transcrip-
tase allosterically which leads to the change in the enzyme confor-
mation and inhibition of its activity.
Despite we have found that compound 14 is a reverse transcrip-
tase inhibitor, the mechanism of action of compound 15 is still
unclear. It may directly target some HIV-1 enzyme or function,
but it can also act as host-targeting inhibitor. This suggests that
compound 15 may inhibit certain cellular enzyme or processes,
which is of immense importance for inhibiting viral replication.
Host-targeting inhibitors are a very promising group of drugs
because resistant forms of the virus cannot be easily developed.
was 15-fold lower (5 vs 75
shown by compounds 13 and 14, which suppressed Gluc activity to
lM). The most promising results were
6.4% and 3.5% (at 150 and 50
selected for further studies.
The CC50 and IC50 values for compounds 13, 14 and 15 (Table 2)
were determined using U2OS cells and HIV-1-based VLPs. The most
significant activity, as shown in Table 2, was demonstrated by
lM, respectively) and, therefore, were
compound 15 with IC50 = 2.2 0.5
toxicity (CC50 = 26.8 M) its selectivity index (SI, ratio of CC50
IC50) was 12. Compound 14 was less toxic (CC50 = 72.6 10
but also less active (IC50 = 4.4
lM, but due to its relatively high
However,
a serious drawback of host-targeting inhibitors is
7
l
/
their toxicity to the host organism and for this reason, a careful
optimization of such compounds is required.
l
M)
1
l
M) compared to 15 (Table 2).
Therefore, the selectivity index (SI = 16) of 14 was not significantly
different from that of compound 15. Compound 13 showed moder-
3. Conclusions
ate anti-HIV activity with IC50 = 10.1
1
l
M
while its
In summary, a series of azulene derivatives were synthesized,
and their antiretroviral activity was tested using a two-stage pro-
cess. The first screening was performed using HIV-1-based VLPs,
and three compounds (13, 14 and 15) showed the highest activity
in this analysis.
CC50 = 344.2 40 M, which resulted in the highest selectivity
l
index (SI = 34.1, Table 2).
The investigated compounds (13, 14 and 15) have several sim-
ilar structural features including the presence of an OH group at
position 2 in all of them while compounds 13 and 14 have an
Table 2
Inhibition of HIV-1 VLP and infectious HIV-1 by azulenes
Compounds
MW
First screen (VLP)
CC50 M (U2OS)
Test with HIV-1
,
l
IC50
,
lM
SI
CC50
,
l
M (TZM-bl)
IC50
,
l
M
SI
13
14
15
216.23
288.30
144.16
344.2 40
72.2 10
10.1
4.4
2.2 0.5
1
1
34.1
16.4
12.2
260 30
65 15
NA
20
8.5
—
3
7
7
3
26.8
7
60
7
Abbreviations used: MW—molecular weight; CC50—half-maximal cytotoxic concentration; IC50—half-maximal inhibitory concentration; SI—selectivity index; VLP—virus-like
particles; NA—not available.
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J. Peet et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
The compounds, that were selected based on their performance
(d, J = 7.5 Hz, 1H), 7.81–7.77 (m, 2H), 7.60 (t, J = 7.6 Hz, 1H),
7.37–7.29 (m, 2H). 13C NMR (100 MHz, CDCl3): d = 140.6, 136.5,
135.5, 134.9, 134.3, 134.0, 133.9, 144.1, 130.1, 128.9, 127.6,
125.0, 124.0, 115.0. HRMS (ESI): m/z [M+H]+ calcd for C17H11ClO2:
283.0520; found: 283.0514.
in the first screen, were further tested for their activity against the
infectious HIV-1 virus. One compound (13) was unable to inhibit
HIV-1 infection, whereas the other two were active, with
compound 15 being the most active (IC50 = 8.5
Additionally, we have found that compound 14 can act as a non-
nucleoside reverse transcriptase inhibitor with IC50 = 104.2 M.
3 lM and SI = 7).
4
l
4.1.3. 4-(3-Chloroazulen-1-yl) morpholine (9)
Thus, the present study demonstrated that compounds 14 and 15
can be regarded as promising compounds for development as
novel HIV inhibitors.
A mixture of 1-Cl-3-Br-azulene (242 mg, 1 mmol), Pd2(dba)3,
(12 mg, 2 mol%), 2,20-bis(diphenylphosphino)-1,10-binaphthyl
(19 mg, 3 mol%), sodium tert-butoxide (240 mg, 2.5 mmol), and
morpholine (174 mg, 2 mmol) in toluene (2 mL) was heated to
100 °C under argon atmosphere. After stirring for 2 h, the reaction
mixture was poured into water and extracted with toluene. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified using column chro-
matography (eluent: EtOAc/PE, 1:4) to give 9 as a green solid
(23 mg, 10%). Mp 107–108 °C. IR (ATR, neat): 3047, 3024, 2974,
2940, 2835, 1574, 1501, 1447, 1350, 1261, 1150, 1111, 907, 741.
1H NMR (400 MHz, CDCl3): d = 8.26 (d, J = 9.5 Hz, 1H), 8.20 (d,
J = 9.5 Hz, 1H), 7.50–7.45 (m, 2H), 6.94 (q, J = 9.2 Hz, 2H), 3.96–
3.94 (m, 4H), 3.11–3.08 (m, 4H). 13C NMR (100 MHz, CDCl3):
d = 139.8, 135.1, 134.0, 130.1, 129.8, 124.6, 121.7, 121.4, 114.3,
67.2, 54.8. HRMS (ESI): m/z [M+H]+ calcd for C14H14ClO:
248.0837; found: 248.0834.
4. Experimental section
4.1. Chemistry
All reagents were purchased from Sigma–Aldrich and used
without further purification. All reactions with organometallic
reagents were carried out under an argon atmosphere using dried
glassware. The syringes used to transfer anhydrous solvents or
reagents were purged with argon prior to use. The tetrahydrofuran
was continuously refluxed and freshly distilled from sodium ben-
zophenone ketyl under nitrogen. The ZnCl2 solution was freshly
prepared and stored under argon for not more than one week. Pet-
roleum ether (PE) refers to the fraction boiling at a temperature
range of 40–65 °C. NMR spectroscopy was performed on a Bruker
AVANCE II 400 MHz spectrometer using the residual solvent peak
(CDCl3, 7.26 ppm for 1H and 77.16 for 13C spectra) as the internal
standard. The infrared (IR) spectra were measured on Shimadzu
IRAffinity-1 FTIR spectrometer using ATR module with a ZnSe crys-
tal. The reactions were monitored using thin-layer chromatogra-
phy (TLC) and visualized with ultraviolet (UV) light. The products
were purified using flash chromatography and silica gel 60
(0.040–0.063 mm, 230–400 mesh ASTM).
4.2. Biological assays
4.2.1. Materials
DMSO, polybrene, phorbol 12-myristate 13-acetate (PMA),
nevirapine and trichloracetic acid (TCA) were purchased from
Sigma–Aldrich (St. Louis, MO, USA). Na4P2O7 was purchased from
Applichem (Germany). HIV-1 reverse transcriptase was purchased
from Calbiochem (USA). All reagents and media used for cell culti-
vation were purchased from Naxo OÜ (Estonia).
4.1.1. Sodium gualenate (3)
4.2.2. Cells and growth media
Sulfuric acid (370 lL, 7 mmol) was added dropwise to a solution
The ACH-2 and TZM-bl cell lines were obtained through the
National Institutes for Health (NIH) AIDS Reagent Program,
Division of AIDS, National Institute of Allergy and Infectious
Diseases (NIAID), NIH: ACH-227,28 was from Dr. Thomas Folks and
TZM-bl29–32 was from Dr. John C. Kappes, Dr. Xiaoyun Wu, and
Tranzyme Inc.
The U2OS (human osteosarcoma) cell line was purchased from
the American Type Culture Collection (ATCC, Manassas, VA, USA)
and maintained in Iscove’s Modified Dulbecco’s Medium (IMDM)
supplemented with 10% fetal bovine serum (FBS), 100 U penicillin,
of guaiazulene (198 mg, 1 mmol) in acetic anhydride (2 mL,
20 mmol) at 0 °C, then cooling was removed and the resulting mix-
ture was stirred at room temperature for 4 h. Then, a 40% aqueous
solution of sodium hydroxide (10 g) was added dropwise, and the
resulting mixture was extracted with dichloromethane and the
precipitated product was quickly separated using a glass filter to
give 3 as dark purple colored crystals (176 mg, 57%). Mp 159–
161 °C (dec.). IR (ATR, neat): 3410, 3086, 2955, 2924, 1578, 1447,
1373, 1192, 1142, 1119, 1049, 1011, 637. 1H NMR (400 MHz,
CDCl3): d = 8.28 (s, 1H), 8.03 (s, 1H), 7.56 (d, J = 10.8 Hz, 1H), 7.30
(d, J = 10.8 Hz, 1H), 3.26 (s, 3H), 3.03 (dquin, J = 13.7 Hz, 1H), 2.51
(s, 3H), 1.24 (d, J = 7.0 Hz, 6H). 13C NMR (100 MHz, CDCl3):
d = 148.3, 144.1, 139.6, 138.8, 137.3, 135.5, 132.5, 131.0, 124.7,
123.1, 37.1, 26.9, 23.6, 23.3, 11.9.
and 100 lg/mL streptomycin (Pen/Strep).
The ACH-2 cell line was maintained in Roswell Park Memorial
Institute medium 1640 (RPMI 1640), supplemented with 25 mM
HEPES, 0.3 g/L L-glutamine, 10% heat-inactivated FBS, and Pen/
Strep. The TZM-bl cell line was maintained in Dulbecco’s Modified
Eagle’s medium (DMEM) supplemented with 10% heat-inactivated
FBS and Pen/Strep. All cells were maintained at 37 °C in the
presence of 5% CO2.
4.1.2. 3-(3-Chloroazulen-1-yl) benzoic acid (8)
To
a
solution of ethyl 4-(3-chloroazulen-1-yl) benzoate20
(700 mg, 2.25 mmol) in a mixture of methanol (55 mL) and 1,2-
dimethoxyethane (20 mL) 10% aqueous solution of NaOH (28 mL)
was added and the reaction mixture was heated under reflux for
2 h. Then, the organic solvents were evaporated, and the aqueous
residue was treated with 10% hydrochloric acid to obtain a solution
pH 2–3, and finally extracted with ethyl acetate. The organic
extracts were combined, washed with brine, dried over MgSO4,
and evaporated in vacuo to give 8 as a green solid (564 mg, 87%).
Mp 186–187 °C (dec.). IR (ATR, neat): 2959, 2874, 2661, 2546,
1697, 1686, 1574, 1454, 1412, 1393, 1346, 1300, 1265, 910, 837,
732, 682. 1H NMR (400 MHz, CDCl3): d = 8.48 (d, J = 9.7 Hz, 1H),
8.39 (d, J = 9.7 Hz, 1H), 8.15 (s broad, 1H), 8.09 (s, 1H), 7.97
4.2.3. Cytotoxicity assay
The cytotoxicity of all the test compounds was measured using
the xCELLigence RTCA DP Instrument (ACEA Biosciences, Inc.) as
described previously.26
4.2.4. Analysis of the antiretroviral activity of compounds using
HIV-1-based VLPs
The antiretroviral activity of the test compounds was tested
using a ViraPower Lentiviral Expression System (Invitrogen) as
described previously.26
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J. Peet et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
4.2.5. Analysis of the antiretroviral activity of compounds
infectious HIV-1
3.2.0501.10-0004) and the European Union through the European
Regional Development Fund via the Center of Excellence in
Chemical Biology for financial support.
4.2.5.1. HIV-1 production.
The HIV-1 was produced using the
ACH-2 cell line with a T cell clone caring an HIV-1 provirus in its
genome. To induce HIV-1 production, 6 ꢂ 106 ACH-2 cells were
seeded in 10 mL of full media with 100 nM PMA and incubated
for 48 h. Then, the virus-containing media was collected, filtered
through a 0.45-lM filter (Sarstedt) and the amount of viral p24
protein was measured using an enzyme-linked immunosorbent
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.02.038.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
assay (ELISA) developed in house.
4.2.5.2. Tests for antiretroviral activity.
TZM-bl cell line was
References and notes
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reporter gene under the control of the HIV-1 LTR. The HIV-1 Tat
protein, which is produced after HIV-1 integration into the host
genome, is required for effective reporter gene expression. There-
fore, the firefly luciferase activity in cell lysate correlates with
the efficiency of infection. First, 5 ꢂ 104 TZM-bl cells per well were
seeded in a 24-well plate and incubated for 24 h. The cells were
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4.2.6. Cell-free assay for HIV-1 reverse transcriptase activity
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(Calbiochem). First, 1.25
required concentrations (or DMSO as a negative control) were
mixed with 21.75 L of the reaction mixture containing 50 mM
Tris–HCl, pH 7.8, 1 mM DTT, 6 mM MgCl2, 80 mM KCl, 0.3% PEG
4000, 50 M of dATP, dGTP and dTTP, 5 M dCTP, 1 unit of HIV-1
reverse transcriptase and 1 Ci (0.33 pmol)
[P33]dCTP (Perkin
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l
l
l
a
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7.5, 1 mM EDTA). Samples were incubated for 40 min at 37 °C. The
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Acknowledgments
The authors would like to thank the Estonian Science Founda-
tion (grant ETF9198), the Archimedes Foundation (Project No.
Please cite this article in press as: Peet, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.038