Amino acid derivatives. Part 6. Synthesis, in vitro antiviral activity and molecular docking study of new N-α-amino acid derivatives conjugated spacer phthalimide backbone

Article abstract of DOI:10.1007/s00044-016-1693-9

A series of phthalimido-(substituted-alkyl-2-thioureido)alkyl carboxylic acid derivatives 5–13 and methyl 2-(4-(phthalimido-2-yl)butanamido)alkyl carboxylates 14–23 were synthesized with the aim to develop newly non-nucleoside reverse transcriptase inhibi

Full text of DOI:10.1007/s00044-016-1693-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1693-9
ORIGINAL RESEARCH
Amino acid derivatives. Part 6. Synthesis, in vitro antiviral activity and
molecular docking study of new N-α-amino acid derivatives conjugated
spacer phthalimide backbone
1 ●
2 ●
2 ●
3 ●
Najim A. Al-Masoudi Einas Abood Ziyad T. Al-Maliki Wasfi A. Al-Masoudi
4
Christophe Pannecouque
Received: 24 August 2015 / Accepted: 15 July 2016
©
Springer Science+Business Media New York 2016
Abstract A series of phthalimido-(substituted-alkyl-2-
thioureido)alkyl carboxylic acid derivatives 5–13 and
methyl 2-(4-(phthalimido-2-yl)butanamido)alkyl carbox-
ylates 14–23 were synthesized with the aim to develop
newly non-nucleoside reverse transcriptase inhibitors
some amino acids of human immunodeficiency virus
reverse transcriptase has been studied.
●
●
●
Keywords Antiviral activity Amino acids Phthalimide
Molecular docking study
(
NNRTIs). The new synthesized compounds were assayed
against human immunodeficiency virus-1 and human
immunodeficiency virus-2 in MT-4 cells. The results
showed that 22 and 23 were the only compounds in the
series inhibiting human immunodeficiency virus-1 (IIIB
strain) replication in cell culture with an EC50 value of
Introduction
>
2.07 and >4.23 μM (SI = 7 and 5), respectively. In addi-
Phthalimide derivatives have received considerable atten-
tions in recent years because of their diverse biological
activities (Sharma et al., 2010). Various phthalimide deri-
vatives have shown anti-inflammatory (Lima et al., 2002),
anticancer (Dredge et al., 2003; Little et al., 2002; Teo et al.,
2005; Al-Soud and Al-Masoudi, 2001), anti-human immu-
nodeficiency virus (Moreira et al., 1997; Derpoorten et al.,
tion, the new analogs were screened against hepatitis virus
C genotype 1b in the Huh-5-2 replicon system. Compounds
9
exhibited activity with EC50 = 26.7 and 22.0 μM (SI > 1.87
and >2.28, respectively). The molecular docking of 22 with
and 12 were the most active analogs of the series and
1997; Al-Masoudi et al., 2010), tumor necrosis factor-α
(TNF-α) regulating activity (Miyachi et al., 1997), antic-
onvulsant (Usifoh et al., 2011; Ragavendran et al., 2007),
hypoglycemic (Sou et al., 2001), antiandrogenic (Miyachi et
al., 1997), antiangiogenic (Shimazawa et al., 1999), hypo-
lipidimic (Chapman et al., 1984), and antimicrobial (Sosa
and Cvetnic, 2005) activities. Thalidomide (1) (Fig. 1) is the
best-known phthalimide derivative, a hypnotic/sedative
drug with teratogenic effect (Hales, 1999), as well as a
powerful inhibitor for cytokine TNF-α (Hashimoto, 2002;
Noguchi et al., 2002; Klausner et al., 1996), in addition to
its effectiveness for the treatment of HIV (Makonkawke-
toon et al., 1993). Hashimoto (2002) have reported the
activity of thalidomide and its analogs as cyclooxygenase
inhibitors. Furthermore, naphthalimides such as amonafide
and mitonafide, exhibited substantial anticancer activities
against various animal tumors (Samanta et al., 2001).
N.A. Al-Masoudi’s present address: Am Tannenhof 8, 78464
Konstanz, Germany
E. Abood’s Present address: School of Chemistry, University of
Newcastle upon Tyne, NE1 7RU, UK
*
Najim A. Al-Masoudi
najim.al-masoudi@gmx.de
1
2
3
4
College of Science, Department of Chemistry, University of
Basrah, Basrah, Iraq
Department of Chemistry and Polymer Technology, Polymer
Research Center, University of Basrah, Basrah, Iraq
Department of Physiology and Chemistry, College of Veterinary,
University of Basrah, Basrah, Iraq
Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven B-3000, Belgium

Med Chem Res
On the other hand, amino acids coupled derivatives are a
major of interest, since the amino acid itself had emerged as
the key determinant of intracellular phosphate delivery.
McGuigan et al. (1998) have described the synthesis of
phosphoamidate derivatives of stavudine bearing unusual
amino acids as inhibitors for HIV.
interest (Hamad et al., 2010), we described here the
development of a new series of amino acids or their ester
analogs bearing side-chain phthalimide backbone with
evaluation of their HIV and HCV inhibitory activity.
Hepatitis C virus (HCV) belongs to a member of Fla-
viviridae and is a worldwide infectious pathogen causing
chronic hepatitis that can progress further to hepatocellular
carcinoma (Onji and Ohta, 1990). The current therapeutic
protocol for HCV infection consists mainly of interferon in
combination with ribavirin that usually accompanies with
strong side effects and moderate successful rate (Mangia
et al., 2005; Lai, 2006). Hence, there is an urgent need to find
new regimens to increase the efficacy of anti-HCV therapy.
In view of the various activities of phthalimides, and in
continuation of our ongoing research on amino acid deri-
vatives in the synthesis of new analogs of pharmacological
Results and discussion
Chemistry
Recently, Herrewege et al. (2008) reported that the antiviral
activity was positively correlated with the carbon spacer
length during the synthesis of new potent HIV agents. In
our present work, 4-(phthalimido-2-yl)butyric acid (2),
having three carbon atoms spacer, has been selected as a
key intermediate for the synthesis of new derivatives
of phthalimido-(substituted-alkyl-2-thioureido)alkyl car-
boxylic acid, aiming for evaluation of their in vitro anti-HIV
and HCV activity. Thus, treatment of 2 with NH SCN in
4
acetone, following Kabbani et al. (2005) approach afforded
5, which directly treated with the desired amino acid deri-
vatives to give, after purification, the phthalimido thiour-
eido-amino acid derivatives in 80–54 % yield. The synthetic
reactions are summarized in Scheme 1.
1
13
The structures of 5–13 were determined by their H,
C
nuclear magnetic resonance (NMR) and by mass spectra.
The phthalimide and its propyl side chain protons showed a
similar pattern. Compound 8 was selected for further NMR
experiment. From a gradient heteronuclear multiple bond
correlation (HMBC) (Willker et al., 1993) NMR spectrum,
Fig. 1 Chemical structure of thalidomide (1)
Scheme 1 Reagents and
conditions: (i) SOCl
0 °C; (ii) NH NCS, acetone,
reflux, 6 h; (iii) R–CH(NH
CO H, acetone, reflux, 6 h
2
, 1 h,
7
4
2
)
2

Med Chem Res
Fig. 2 JC,H correlations in the
HMBC NMR spectrum of 8
2 2
Scheme 2 Reagents and conditions: (i) R–CH(NH )CO Me, DCC, HOBt,5 °C, MeCN; (ii) then stirring at 0 °C, 1 h, 5 °C, 1 h, 23 °C, 16 h
3
two J
couplings between 1′-CH and 2′-CH protons at
suppresses racemization, especially in the presence of DCC
(Katritzky et al., 2004).
C,H
2
2
δ = 4.21 and 1.83 ppm and carbonyl carbon atoms of the
phthalimide group and NHCO at δ = 168.4 and 175.7 ppm,
respectively, were observed. Further, 8′-H at δ = 3.41 ppm
Amides 14–23 were prepared by coupling 2 with the
appropriate methyl ester derivatives of the amino acids
(L-alanine, L-valine, L-serine, L-cysteine, L-methionine,
L-leucine, L-threonine, L-phenylalanine, L-tryptophane,
and L-proline) in the presence of HOBt and DCC as cou-
pling reagents to give 14–23 in 80-59 % yield (Scheme 2).
3
showed a JC,H coupling with C=S at δ = 187.8 ppm,
whereas 10′-CH protons revealed the same coupling with
2
C-8′ at δ = 61.1 ppm. Similarly, 1′-CH and 2′-CH protons
2
2
2
at δ = 4.21 and 1.83 ppm showed two J
couplings with
C,H
1
carbon atoms 2′ and 3′ at δ = 24.1 and 30.7 ppm, respec-
tively. In addition, 3′-CH at δ = 2.29 ppm revealed a J
The structures of 14–23 were determined by their H,
C NMR and mass spectra. Phthalimide and propyl side
2
13
2
C,H
coupling with carbonyl carbon atom (NHCO) at δ = 175.7
ppm, while the 9′-CH and 10′-CH protons showed two
chain carbon atoms showed a similar pattern and almost
identical chemical shifts for those of 5–13. In addition, all
the synthesized compounds were confirmed from their
2
2
2
JC,H couplings with C-8′ and C-9′ at δ = 61.1 and 29.6
1
13
ppm, respectively (Fig. 2).
H, C HSQC NMR spectra (Davis et al., 1992).
A suitable coupling method (Davis and Mohammed,
1
981) was employed for the formation of peptides by
Biological activities
reaction of the carboxylic acid group with acylated amino
acid, using 1-hydroxybenzotriazole (HOBt) (Konig and
Geiger, 1970) and N,N′-dicyclohexylcarbodiimide (DCC)
In vitro anti-HIV activity Compounds 3 and 5–23 were
(
Sheeban and Hlavka, 1956) as coupling reagents. HOBt is
tested for their anti-HIV-1 activity in vitro, using III strain
B
currently the most frequently used activating agent for the
carboxyl group of amino acids. The procedure is fast and
in human MT-4 cells, based on a Microculture Tetrazolium
(MTT) assay (Pannecouque et al., 2008). Cytotoxicity was

Med Chem Res
a
b
Table 1 In vitro anti-HIV-1 and HIV-2 of new phthalimide
derivatives 3, 5–23
also measured on MT-4 cells, where the results are sum-
marized at Table 1. None of the in vitro tested compounds
were found to inhibit HIV-replication at EC50 lower than
the CC50 compared to the antiviral agents navirapine
c
d
e
Compd.
Virus strain
EC50 (μM)
CC50 (μM)
SI
3
III
ROD
III
ROD
III
B
>52.80
>52.80
>125.0
>125.0
>89.50
>89.50
>90.90
>90.90
>78.22
>78.22
>26.73
>26.73
>28.54
>28.54
>47.26
>47.26
>12.60
>12.60
>37.82
>37.82
>100.76
>100.76
>125.0
>125.0
>96.11
>96.11
>60.33
>60.33
>18.85
>18.85
>10.89
>10.89
>37.82
>37.82
>37.82
>37.82
>4.23
52.80
52.80
125.0
125.0
89.50
89.50
90.90
90.90
78.22
78.22
26.73
26.73
28.54
28.54
47.26
47.26
12.60
12.60
37.82
37.82
100.76
100.76
125.0
125.0
96.11
96.11
60.33
60.33
18.85
18.85
10.89
10.89
37.82
37.82
37.82
37.82
29.61
29.61
101.90
101.90
>25
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
7
(
Hargrave et al., 1991) and azidothymidine (AZT) (Mitsuya
et al., 1985). It is interesting to report that compounds 22
and 23 were effected a 50 % reduction of the cytopatho-
5
6
7
8
9
B
genicity induced by HIV-1 (III strain) at a concentration of
B
B
EC50 > 4.23 and 20.38 μM, and CC50 of 29.61 and 101.90
μM, resulting in SI of 7 and 5, respectively, at non-toxic
concentration. However, the anti-HIV activity and selec-
tivity of 22 and 23 are too limited to perform extensive
mode of action studies. The synthesis of new analogs of
these phthalimide derivatives could lead to the discovery of
more potent and selective analogs that will subject for fur-
ther structural development.
ROD
IIIB
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
B
B
1
1
1
0
1
2
B
In vitro anti-HCV activity The establishment of stable
HCV subgenomic replicon systems (Lohmann et al., 1999;
Blight et al., 2000) in the human hepatoblastoma cell line
Huh-7 has provided a useful system for the development of
new antiviral approaches against HCV (Gao et al., 2001;
Llinàs-Brunet et al., 2004; Po et al., 2012). The overriding
aims of the new therapeutic strategies are higher efficacy
associated with shortened duration of treatment, favorable
mode of administration, and thus improved tolerability and
adherence. Compounds 3, 7–9 and 11–13 have been
selected for evaluation in vitro for antiviral activity against
hepatitis C virus in the Huh-5-2 replicon system (type 1b,
Con1 strain). The results are shown in Table 2. Even though
for many compounds an EC50 was obtained with selectivity
index (SI) up to 2.28 and inhibition of 87.3 % (e.g.,: com-
pound 12), none of the compounds matched the selection
criteria of a selective inhibitor of virus replication in this
assay (i.e., >70 % inhibition at concentrations that do not
elicit an antimetabolic effect on the host cells).
B
B
ROD
IIIB
13
14
15
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
B
B
1
1
1
1
2
2
2
2
6
7
8
9
0
1
2
3
B
B
B
B
B
B
Molecular docking study
B
>29.61
>20.38
>101.90
0.0022
0.00094
0.050
<1
5
Docking study with HIV-1 reverse transcriptase enzy-
me In the docking study, the X-ray crystal structure of
HIV-1 reverse transcriptase (RT) enzyme; PDB ID: 3dlg)
was obtained from Protein Data Bank (http://www.rcsb.org)
B
<1
>11363
>26596
>80
<1
AZT
B
>25
(
Ren et al., 2008). The Graphical User Interface program
Nevirapine
B
>4.00
>4.00
AutoDock Tools and AutoDockTools4 were used to study
the interactions and the binding energy of the docked
structure (Morris et al., 2009). Polar hydrogens were added
followed by computing gasteiger charges and nonpolar
hydrogen’s were merged into the receptor PDB file. Com-
pound 22 has been selected for the docking modeling study
as a best dock pose, used as standard inhibitor with the
following energy data (kcal/mol): binding energy = −8.82,
competitive inhibition (Ki) = 343.93 nM, intermolecular
>4.00
a
Anti-HIV-1 activity measured with strain III
B
b
c
anti-HIV-2 activity measured with strain ROD
compound concentration required to achieve 50 % protection of MT-
cells from the HIV-1 and 2-induced cytopathogenic effect
4
d
compound concentration that reduces the viability of mock-infected
MT-4 cells by 50 %
e
SI selectivity index (CC50/EC50
)

Med Chem Res
energy = −8.45, internal energy = −4.24, torsional energy =
Experimental section
2
.68, and unbound extended energy = −1.18. As suggested
by the model and visualized in Fig. 3, docking study of 22
has shown satisfactory results. Three hydrogen bonding
interactions are shown: Lys395 with O atom of the phtha-
limide moiety, Thr377 with NH of the indol residue and O
atom of methoxy group of ester residue with Lys22 of RT
enzyme residues. There are non-bonded amino acid residues
such as Trp24, Glu399, and Thr400 are shown the receptor
activity.
Chemistry
Melting points are uncorrected and were measured on a
Buchi melting point apparatus B-545 (BUCHI Labortechnik
AG, Switzerland). Microanalytical data were obtained with
a Vario, Elementar apparatus (Shimadzu, Japan). NMR
1
spectra were recorded on 400 MHz and 600 MHz ( H) and
1
3
150.91 MHz ( C) spectrometers (Avance III, Bruker,
Germany) with TMS as internal standard and on δ scale in
ppm. Signal assignments for protons were identified by
selective proton decoupling or by correlation spectroscopy
spectra. Heteronuclear assignments were verified by het-
eronuclear single quantum correlation (HSQC), or HMBC
experiments. Mass spectra were recorded on 70 eV EI and
FAB MAT 8200 spectrometers (FinniganMAT, U.S.A.),
using 3-nitrobenzyl alcohol (NBOH) or glycerol as matrix.
Table 2 In vitro anti-HCV-1b activity, cytotoxicity and inhibition [%]
of new phthalimide derivatives 3, 7–9 and 11–13
c
d
Compd. CC50
EC50
(μM)
SI
TI
Inhibition
(%)
a
b
(μM)
3
7
8
9
1
1
1
a
>50
>50
>50
>50
>50
>50
>50
>50
39.9
>50
26.7
43.8
22.0
>50
nd
nd
38.7
>1.25 >0.0254 55.2
General procedure for the synthesis of phthalimido(buta-
noylthioureido)alkyl carboxylic acid derivatives (5–13) A
solution of 4-(phthalimido-2-yl)butyric acid (2) (350 mg,
nd
nd
47.5
88.7
>1.87 >1.52
1
2
3
>1.14 >0.0056 53.4
1.50 mmol) and SOCl (5 mL) was heated for 1 h at 70 °C.
2
>2.28 >2.44
nd nd
87.3
42.6
After cooling, the excess of thionyl chloride was removed
under reduced pressure and the residue 3 was dissolved in
acetone (10 mL). To a stirred solution of NH SCN (174 mg,
CC50 50 % cytostatic/cytotoxic concentration (concentration at which
4
5
b
0 % adverse effect is observed on the host cell)
2.29 mmol) in acetone (10 mL) was added and the reaction
mixture was heated under reflux for 2 h. After cooling and
filtration, a suspension of the desired amino acid (1.17
mmol) in dry acetone (10 mL) was added dropwise and the
mixture was heated under reflux for 6 h. After cooling, the
EC50 50 % effective concentration (concentration at which 50 %
inhibition of virus replication is observed)
c
SI selectivity index (CC50/EC50
)
d
TI thepapeutical index (10 logSI)
Fig. 3 Docked conformation of 22 showing three hydrogen bonds: Lys395 with O atom of the phthalimide moiety, Thr377 with NH of the indole
residue and O atom of OMe ester group and Lys22 of the reverse transcriptase (RT) enzyme residues

Med Chem Res
solvent was evaporated to dryness and the residue was
purified on a column of silica gel (10 g), using, in gradient,
(CH CH SMe), 24.1 (C2′), 13.9 (SMe). MS (FAB) m/z:
2
2
+
446 [M+Na] . Anal. Calcd. for C H N O S (423.51): C,
18 21 3 5 2
MeOH (0–10 %) and CHCl as eluent to give the desired
51.05; H, 5.00; N, 9.92. Found: C, 50.88; H, 4.89; N, 9.75.
3
thioureido derivative.
2-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-4-methyl-
2
-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)propanoic
pentanoic acid (9) From L-leucine (154 mg). Yield: 480
1
acid (5) From L-alanine (104 mg). Yield: 365 mg (67 %);
M.p. 99–102 °C. H NMR (DMSO-d ): δ = 11.98 (br s.,
mg (79 %); M.p. 72–75 °C. H NMR (DMSO-d ):δ = 11.72
6
1
(br s., 1H, CO H), 8.12 (br s., 1H, NH), 7.83 (m, 4H, H–
6
2
1
H, CO H), 8.12 (br s., 1H, NH), 7.86 (m, 4H, H–Ar), 4.25
Ar), 4.29 (m, 2H, 1′-CH ), 3.42 (m, 1H, 8′-H), 2.30 (m, 2H,
2
2
(
1
m, 2H, 1′-CH ), 3.39 (m, 1H, 8′-H), 2.26 (m, 2H, 3′-CH ),
3′-CH ), 1.90 (m, 2H, 2′-CH ), 1.56 (m, 1H, CHMe ), 0.88,
2
2
2
2
2
1
3
13
.81 (m, 2H, 2′-CH ), 1.24 (t, 3H, J = 7.1 Hz, CH ).
C
0.87 (2xs, 6H, CHMe2). C NMR (DMSO-d ): δ = 187.4
2
3
6
NMR (DMSO-d ): δ = 185.8 (C=S), 173.8 (CO H), 171.9
(C=S), 174.1 (CO H), 170.9 (NHCO), 166.9 (Cphthal.=O),
6
2
2
(
6
NHCO), 167.9 (Cphthal.=O), 134.3, 131.6, 122.9 (Carom.),
132.4, 129.9, 122.8 (Carom.), 67.4 (C8′), 38.6 (C1′), 31.8
(C3′), 29.5 (CHMe ), 23.8 (C2′), 21.4 (CHMe ). MS (FAB)
1.5 (C8′), 38.9 (C1′), 30.9 (C3′), 23.3(C2′), 16.3 (Me).
2
2
+
+
MS (FAB) m/z: 364 [M+H] . Anal. Calcd. for
m/z: 406 [M+H] . Anal. Calcd. for C H N O S (405.47):
19 23 3 5
C H N O S (363.39): C, 52.88; H, 4.72; N, 11.56.
C, 56.28; H, 5.72; N, 10.36. Found: C, 56.02; H, 5.81;
N,10.52.
1
6 17 3 5
Found: C, 52.63; H, 4.59; N, 11.29.
-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-3-hydro-
xypropanoic acid (6) From L-serine (123 mg). Yield: 359
2
2-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-3-hydro-
xybutanoic acid (10) From L-threonine (139 mg). Yield:
1
1
(
(
63 %); M.p. 103–106 °C. H NMR (DMSO-d ): δ = 12.00
401 mg (68 %); M.p. 69–72 °C. H NMR (DMSO-d ): δ =
6
6
br s., 1H, CO H), 8.20 (br s., 1H, NH), 7.85 (m, 4H, H–
12.16 (br s., 1H, CO H), 7.97 (m, 4H, H–Ar),4.30 (m, 2H,
2
2
Ar), 4.31 (t, 2H, J = 6.3 Hz, 1′-CH ), 4.14 (t, 2H, J = 6.5
1′-CH ), 4.12 (m, 1H, CH OH), 3.61 (br s., 1H, OH), 3.43
2
2
2
Hz, CH OH), 3.78 (t, 1H, J = 6.7 Hz, 8′-H), 2.27 (t, 2H, J
(m, 2H, 8′-CH ), 2.29 (m, 2H, 3′-CH ), 1.90 (m, 2H, 2′-
2
2
2
1
3
13
=
6.7 Hz, 3′-CH ), 1.83 (m, 2H, 2′-CH ). C NMR
DMSO-d ): δ = 185.6 (C=S), 173.8 (CO H), 171.3
CH ), 1.25 (d, 3H, J = 9.9 Hz, CHMe). C NMR (DMSO-
2
2
2
(
d ): δ = 186.7 (C=S), 173.8 (CO H), 172.6 (NHCO), 167.9
6
2
6
2
(
6
NHCO), 168.0 (Cphthal.=O), 133.9, 131.4, 123.1 (Carom.),
(Cphthal.=O), 134.3, 131.6, 122.9 (Carom.), 70.5 (C8′), 67.3
2.7 (C8′), 51.7 (CH OH), 39.0 (C1′), 30.5 (C3′), 23.1
(CHOH), 39.0 (C1′), 31.0 (C3′), 23.4 (C2′), 18.1 (Me). MS
2
+
+
(
C2′). MS (FAB) m/z: 380 [M+H] . Anal. Calcd. for
(FAB): m/z 394 [M+H] . Anal. Calcd. for C H N O S
1
7 19 3 5
C H N O S (379.39): C, 50.65; H, 4.52; N, 11.08.
(393.41): C, 51.90; H, 4.87; N, 10.68. Found: C, 51.76; H,
4.72; N, 10. 24.
1
6 17 3 6
Found: C, 50.39; H, 4.40; N, 11.17.
-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-3-mercap-
topropanoic acid (7) From L-cysteine (142 mg). Yield:
2
2-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-3-(imida-
zole-4-yl)propanoic acid (11) From L-histidine (183 mg).
1
1
4
1
1
54 mg (75 %); M.p. 95–98 °C. H NMR (DMSO-d ):δ =
Yield: 348 mg (54 %); M.p. 65–69 °C. H NMR (DMSO-
6
1.06 (br s., 1H, CO H), 7.89 (m, 4H, H–Ar),4.24 (m, 2H,
d ): δ = 12.10 (br s., 1H, CO H), 11.83 (br s., 1H,
2
6
2
′-CH ′), 3.58 (m, 2H, 8′-H), 3.32 (m, 2H, CH SH), 2.27
NHimidazole), 8.08 (br s., 1H, NHimidazole), 7.97 (m, 4H, H–
Ar), 7.82 (m, 4H, H–Ar), 7.69 (d, 1H, J = 5.2 Hz,
2
2
1
3
(
m, 2H, 3′-CH ′), 1.78 (m, 2H, 2′-CH ). C NMR (DMSO-
2
2
d ): δ = 186.6 (C=S), 179.6 (CO H), 172.0 (NHCO), 167.9
5-Himidazole), 4.29 (t, 2H, J = 6.5 Hz, 1′-CH ), 3.65 (m, 2H,
6
2
2
(
8
Cphthal.=O), 134.1, 129.7, 128.6, 122.9 (Carom.), 61.3 (C-
′), 38.8 (C1′), 30.1 (C3′), 23.8 (C2′), 23.1 (CH SH). MS
8′-H), 3.16 (s, 2H, CH ), 2.25 (t, 2H, J = 6.6 Hz, 3′-CH ),
2
2
1
3
1.80 (t, 2H, J = 6.6 Hz, 2′-CH2). C NMR (DMSO-d ): δ
= 188.2 (C=S), 173.0 (CO H), 172.7 (NHCO), 167.9
2
6
+
(
(
FAB) m/z: 418 [M+Na] . Anal. Calcd. for C H N O S
1
6
17
3
6
2
2
395.45): C, 48.60; H, 4.30; N, 10.63. Found: C, 48.41; H,
(Cphthal.=O), 134.3 (C(2)imidazole), 131.6, 122.9, (Carom.),
4
.25; N, 10.44.
63.8 (C8′), 39.5 (C1′), 30.8 (C3′), 25.2 (CH ), 23.2 (C2′).
2
+
MS (FAB) m/z: 430 [M+H] . Anal. Calcd. for
2
-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-4-(methyl-
C H N O S (429.45): C, 53.14; H, 4.46; N, 16.31.
1
9 19 5 5
thio)butanoic acid (8) From L-methionine (174 mg).
Yield: 464 mg (73 %); M.p. 134–137 °C. H NMR (DMSO-
Found: C, 52.88; H, 4.32; N, 16.04.
1
d ): δ = 11.52 (br s., 1H, CO H), 7.87 (m, 4H, H–Ar), 4.21
2-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-5-guanidi-
6
2
(
m, 2H, 1′-CH ), 3.41 (m, 1H, 8′-H), 2.56 (m, 2H,
nopentanoic acid (12) From L-arginine (203 mg). Yield:
2
1
CH CH SMe), 2.29 (m, 2H, 3′-CH ), 2.11 (CH CH SMe),
2
δ = 187.8 (C=S), 176.0 (CO H), 175.7 (NHCO), 168.4
404 mg (60 %); M.p. 129–132 °C. H NMR (DMSO-d ): δ
2
2
2
2
2
6
1
3
.07 (SMe), 1.83 (m, 2H, 2′-CH2). C NMR (DMSO-d6):
= 11.72 (s, 1H, CO H), 8.32 (br s., 1H, NH),7.83 (m, 4H,
2
H–Ar), 4.27 (m, 2H, 1′-CH ), 3.48 (m, 1H, 8′-H), 2.59 (m,
2
2
(
8
Cphthal.=O), 133.3, 130.7, 129.1, 122.3 (Carom.), 61.1 (C-
′), 38.9 (C1′), 30.7 (C3′), 29.6 (CH CH SMe), 29.0
2H, CH CH CH N), 2.29 (m, 2H, 3′-CH ), 1.79 (m, 2H, 2′-
2
2
2
2
CH ), 1.69 (m, 2H, CH CH CH N), 1.48 (m, 2H,
2 2 2 2
2
2

Med Chem Res
1
3
CH CH CH N). C NMR (DMSO-d ): δ = 188.3 (C=S),
Methyl 2-(4-(phthalimido-2-yl)butanamido)-3-methylbu-
2
2
2
6
1
74.6 (CO H), 172.1 (NHCO), 167.3 (Cphthal.=O), 158.4
tanoate (15) From L-valine methyl ester (197 mg). Yield:
2
1
(
(
(
[
NC=NH), 132.3, 125.6, 122.5 (Carom.), 63.8 (C8′), 42.5
384 mg (74 %); M.p. 65–69 °C. H NMR (DMSO-d ): δ =
6
CH CH CH N), 38.8 (C1′), 34.9 (CH CH CH N), 29.8
8.05 (m, 4H, H–Ar), 7.88 (d, 1H, J = 7.8 Hz, NH), 4.29 (m,
2
2
2
2
2
2
C3′), 24.2 (C2′+ CH CH CH N).MS (FAB) m/z: 452
M+Na] . Anal. Calcd. for C H N O S (448.50): C, 50.88;
2H, 2′-CH ), 4.11 (t, 1H, J = 6.0 Hz, NCH), 3.54 (s, 3H,
2
2
2
2
+
OAc), 2.25 (m, 3H, 3′-CH +CHMe ), 1.82 (d, 2H, J = 6.6
1
9
24
6
5
2
2
1
3
H, 5.39; N, 18.74. Found: C, 50.65; H, 5.35; N, 18.51.
Hz, 2′-CH ), 0.90, 0.88 (2xs, 6H, Me ). C NMR (DMSO-
d ): δ = 173.8 (NHCO), 170.2 (CO Me), 167.9
2
2
6
2
2
-(3-(4-(Phthalimido-2-yl)butanoyl)thioureido)-3-(4-hydro-
(Cphthal.=O), 134.2, 131.6, 122.9 (Carom.), 57.3
xyphenyl)propanoic acid (13) From L-tyrosine (212 mg).
Yield: 526 mg (77 %); M.p. 112–115 °C. H NMR (DMSO-
(CHCO Me), 51.6 (CO Me), 39.2 (C1′), 33.3 (C3′), 29.8
2
2
1
(CHMe ), 24.4 (C2′), 18.9, 18.3 (CHMe ). MS (FAB) m/z:
2
2
+
d ): δ = 11.21 (br s., 1H, CO H), 7.95 (br s., 1H, NH), 7.79
347 [M+H] . Anal. Calcd. for C H N O (346.38): C,
18 22 2 5
6
2
(
m, 4H, H–Ar), 7.14–6.92 (m, 4H, H–Ar), 5.12 (s, 1H, OH),
62.42; H, 6.40; N, 8.09. Found: C, 62.18; H, 6.48; N, 7.89.
4
6
.31 (m, 2H, 1′-CH ), 3.69 (m, 1H, 8′-H), 2.93 (d, 2H, J =
2
Methyl 2-(4-(phthalimido-2-yl)butanamido)-3-hydroxypro-
.3 Hz, CH ), 2.29 (m, 2H, 3′-CH ), 1.92 (m, 2H, 2′-CH ).
2
2
2
1
3
panoate (16) From L-serine methyl ester (179 mg). Yield:
C NMR (DMSO-d ): δ = 187.1 (C=S), 175.8 (CO H),
6
2
1
3
66 mg (73 %); M.p. 143–145 °C. H NMR (DMSO-d ): δ
6
1
73.4 (NHCO), 167.9 (Cphthal.=O), 155.3 (Carom.-
=
7.92 (m, 4H, H–Ar), 5.02 (t, 1H, J = 5.1 Hz, OH), 4.31
OH),131.1, 129.4, 129.1, 128.8, 128.5, 115.7 (Carom.), 66.5
(
m, 2H, 1′-CH2), 4.15 (m, 2H, CH OH), 3.80 (m, 1H,
2
(
C-8′), 38.9 (C1′), 34.4 (CH Ar), 30.3 (C3′), 22.4 (C2′).
2
+
CHCH OH), 3.54 (s, 3H, OAc),2.25 (m, 2H, 3′-CH ), 1.85
(
(
2
2
MS (FAB) m/z: 456 [M+H] . Anal. Calcd. for
1
3
m, 2H, 2′-CH2). C NMR (DMSO-d ): δ = 173.4
NHCO), 170.8(CO Me), 167.2 (Cphthal.=O), 133.8, 129.8,
6
C H N O S (455.48): C, 58.01; H, 4.65; N, 9.23. Found:
C, 57.83; H, 4.54; N, 8.08.
2
2 21 3 6
2
1
22.7 (Carom.), 60.2 (CH OH), 53.8 (CHCO Me), 51.8
2 2
(
3
5
CO Me), 39.6 (C1′), 32.6 (C3′), 23.8 (C2′). MS (FAB) m/z:
2
General procedure for the synthesis of methyl 2-(4-(phtha-
limido-2-yl)butanamido)alkyl carboxylates (14–23) To a
cold solution of the amino acid ester (1.50 mmol), at - 5 °C,
in MeCN (15 mL), 4-(phthalimido-2-yl)butyric acid (2)
+
57 [M+Na] . Anal. Calcd. for C H18N O (334.32): C,
16 2 6
7.48; H, 5.43; N, 8.38. Found: C, 57.26; H, 5.38; N, 7.87.
Methyl
2-(4-(phthalimido-2-yl)butanamido)-3-mercapto-
(
350 mg, 1.50 mmol), HOBt (203 mg, 1.50 mmol) and
pronoate (17) From L-cysteine methyl ester (203 mg).
Yield: 363 mg (69 %); M.p. 112–115 °C. H NMR (DMSO-
d6): δ = 7.92 (m, 4H, H–Ar), 4.56 (m, 1H, NCH), 4.24 (m,
2
N,N′-dicyclohexylcarbodiimide (DCC) (309 mg, 1.5 mmol)
were added successively. The reaction mixture was stirred
at 0 °C for 1 h, at 5 °C for 1 h, and at 23 °C for 16 h.
Dicyclohexylurea (DCU) was filtered, and the filtrate was
evaporated to dryness and the residue was dissolved in ethyl
acetate, filtered, washed successively with saturated NaCl
solution, 5 % NaHCO3 solution, 1 M HCl, followed by
washing with saturated NaCl solution and finally with
water. The residue was dried (Na SO ), filtered, evaporated
1
H, 1′-CH ), 3.56 (s, 3H, OAc), 3.30 (m, 2H, CH SH), 2.22
2
2
13
(
m, 2H, 3′-CH ), 1.85 (m, 2H, 2′-CH ). C NMR (DMSO-
2
2
d ): δ = 173.2 (NHCO), 171.1 (CO Me), 168.0
6
2
(
(
(
Cphthal.=O), 132.9, 130.1, 122.5 (Carom.), 56.2
CHCO Me), 51.4 (CO Me), 39.8 (C1′), 33.4 (C3′), 25.4
2
2
+
CH SH), 23.4 (C2′). MS (FAB) m/z: 373 [M+Na] . Anal.
2
2
4
Calcd. for C H N O S (350.39): C, 54.85; H, 5.18; N,
1
6 18 2 5
to dryness and purified on a SiO column (10 g). Elution
2
7
.99. Found: C, 54.67; H, 5.02; N, 8.41.
with MeOH (0–10) and CHCl as an eluent afforded the
3
pure amide derivative.
Methyl 2-(4-(phthalimido-2-yl)butanamido)-4-(methythio)
butanoate (18) From L-methionine methyl ester (245 mg).
1
Methyl 2-(4-(phthalimido-2-yl)butanamido)propanoate (14)
Yield: 444 mg (78 %); M.p. 123–125 °C. H NMR (DMSO-
From L-alanine methyl ester (155 mg). Yield: 382 mg (80
d6): δ = 7.86 (br s., 4H, H–Ar), 4.40 (m, 1H, NCH),4.19 (m,
2H, 1′-CH2), 3.56 (s, 3H, OAc), 2.52 (m, 2H,
CH CH SMe), 2.22 (m, 2H, 3′-CH ), 2.14 (br s., 2H,
1
%
); M.p. 79–82 °C. H NMR (DMSO-d ): δ = 8.21 (br s.,
6
1
H, NH), 8.01 (m, 4H, H–Ar), 4.26 (m, 2H, 1′-CH ), 4.12
2
2
2
2
1
3
(
d, 1H, J = 5.1 Hz, NCH), 3.55 (s, 3H, OAc), 2.23 (m, 2H,
′-CH ), 1.76 (m, 2H, 2′-CH ), 1.39 (d, 3H, J = 5.4 Hz,
CH CH SMe), 2.10 (SMe), 1.88 (m, 2H, 2′-CH ).
C
2
2
2
3
NMR (DMSO-d ): δ = 173.6 (NHCO),171.5 (CO Me),
2
2
6
2
1
3
NCHMe). C NMR (DMSO-d ): δ = 73.7 (NHCO), 171.1
166.9 (Cphthal.=O), 133.8, 129.9, 123.1 (Carom.), 54.2
(CHCO Me), 51.8 (CO Me), 38.9 (C1′), 33.2 (C3′), 29.5
6
(
CO Me), 167.4 (Cphthal.=O), 134.0, 130.3, 122.5 (Carom.),
2
2
2
5
2
1.8 (CHCO Me), 51.5 (CO Me), 39.8 (C1′), 32.9 (C3′),
(CH CH SMe), 28.8 (CH CH SMe), 23.2 (C2′), 14.2
2
2
2
2
2
2
+
+
3.2 (C2′), 18.1 (Me). MS (FAB) m/z: 319 [M+H] . Anal.
(SMe). MS (FAB) m/z: 380 [M+H] . Anal. Calcd. for
Calcd. for C H N O (318.32): C, 60.37; H, 5.70; N,
C H N O S (379.44): C, 57.13; H, 5.86; N, 7.40. Found:
18 22 2 5
1
6
18
2
5
8
.80. Found: C, 60.03; H, 5.62; N, 9.09.
C, 57.24; H, 5.77; N, 7.26.

Med Chem Res
Methyl 2-(4-(phthalimido-2-yl)butanamido)-4-methylpen-
C H N O (433.46): C, 66.50; H, 5.35; N, 9.69. Found:
2
4 23 3 5
tanoate (19) From L-leucine methyl ester (218 mg). Yield:
C, 66.37; H, 5.42; N, 9.53.
1
4
05 mg (75 %); M.p. 71–74 °C. H NMR (DMSO-d ): δ =
6
Methyl 1-(4-(phthalimido-2-yl)butanoyl)pyrrolidine-2-car-
boxylate (23) From L-proline methyl ester (194 mg).
Yield: 305 mg (59 %); Mp 103–106 °C. H NMR (DMSO-
7
.89 (br s., 4H, H–Ar), 4.47 (m, 1H, NCH),4.22 (m, 2H, 1′-
CH ), 3.52 (s, 3H, OAc), 2.13 (m, 2H, 3′-CH ), 1.82 (m,
2
2
2
1
H, 2′-CH +CH CHMe ),1.52 (m, 1H, CHMe ), 1.06, 1.04
2
2
13
2
2
d ): δ = 7.79 (br s, 4H, H-Ar), 4.30 (m, 2H, 1′-CH ), 3.97
6
2
(
2xs, 6H, Me2). C NMR (DMSO-d ): δ = 174.0 (NHCO),
6
(m, 1H, 2-CH2pyrrol.), 3.55 (s, 3H, OAc), 3.40 (m, 2H, 5-
1
70.0(CO Me), 168.0 (Cphthal.=O), 134.3, 131.6, 123.0
2
CH2pyrrol.), 2.32 (m, 2H, 3-CH2pyrrol.), 2.25 (m, 2H, 3′-
(
Carom.), 53.8 (CHCO Me), 52.1 (CO Me), 39.1 (C1′+
2
2
1
3
CH ), 1.90 (m, 4H, 2′-CH +4-CH2pyrrol.).
C NMR
2
2
CH CHMe ), 32.0 (C3′), 24.4 (CHMe ), 23.9 (C2′), 21.8
2
2
2
+
(DMSO-d ): δ = 173.8 (NHCO), 170.7 (CO Me), 167.4
(
CHMe ). MS (FAB) m/z: 361 [M+H] . Anal. Calcd. for
6
2
2
(Cphthal.=O), 131.8, 129.9, 123.6 (Carom.), 63.9
C H N O (360.40): C, 63.32; H, 6.71; N, 7.77. Found:
1
9 24 2 5
(
3
CHCO Me), 51.0 (CO Me), 48.3 (C(5)pyrrol.), 39.6 (C1′),
2
2
C, 63.07; H, 6.60; N, 7.49.
1.4 (C3′), 27.8 (C(3)pyrrol.), 24.2 (C(4)pyrrol.), 23.7 (C2′).
+
Methyl
2-(4-(phthalimido-2-yl)butanamido)-3-hydroxy-
MS (FAB) m/z: 345 [M+H] . Anal. Calcd. for C H N O
18 20 2 5
butanoate (20) From L-threonine methyl ester (200 mg).
Yield: 418 mg (80 %); M.p. 91–93 °C. H NMR (DMSO-
(344.36): C, 62.78; H, 5.85; N, 8.13. Found: C, 62.57, H,
5.74; N, 7.95.
1
d6): δ = 7.89 (br s., 4H, H–Ar), 4.47 (m, 1H, NCH), 4.22
(
m, 2H, 1′-CH ), 3.52 (s, 3H, OAc), 2.13 (m, 2H, 3′-CH ),
2
2
In vitro HIV assay
1
1
1
1
5
.82 (m, 2H, 2′-CH +CH CHMe ), 1.52 (m, 1H, CHMe ),
2
2
2
2
13
.06, 1.04 (2xs, 6H, Me2). C NMR (DMSO-d ): δ =
6
Evaluation of the antiviral activity of compounds 5–14
against the HIV-1 strain (III ) and the HIV-2 strain (ROD)
73.6 (NHCO), 170.3(CO Me), 167.8 (Cphthal.=O), 133.8,
2
B
31.0, 123.1 (Carom.), 66.3 (CHOHMe), 60.8 (CHCO Me),
2
in MT-4 cells was performed using an MTT assay as
described previously (Pannecouque et al., 2008). In brief,
stock solutions (10 times final concentration) of test com-
pounds were added in 25 μL volumes to two series of tri-
plicate wells to allow simultaneous evaluation of their
effects on mock and HIV-infected cells at the beginning of
each experiment. Serial 5-fold dilutions of test compounds
were made directly in flat-bottomed 96-well microtiter trays
using a Biomek 3000 robot (Beckman instruments).
Untreated control, HIV and mock-infected cell samples,
1.6(CO2Me), 39.8 (C1′), 32.2 (C3′), 23.8 (C2′), 18.6
+
(
Me). MS (FAB) m/z: 349 [M+H] . Anal. Calcd. for
C H N O (348.35): C, 58.61; H, 5.79; N, 8.04. Found:
C, 58.43; H, 5.68; N, 8.18.
1
7 20 2 6
Methyl 2-(4-(phthalimido-2-yl)butanamido)-3-phenylpro-
panoate (21) From L-phenylalanine methyl ester (269 mg).
Yield: 444 mg (75 %); M.p. 85–88 °C. H NMR (DMSO-
d6): δ = 7.74-7.61 (m, 4H, H–Ar), 7.46-7.16 (m, 5H, H–Ar),
1
4
.46 (m, 1H, NCH), 4.27 (m, 2H, 1′-CH ), 3.56 (s, 3H,
2
were included for each sample. HIV-1 (III ) (Popovic et al.,
1984) or HIV-2 (ROD) (Barré-Sinoussi et al., 1983) stock
B
OAc), 3.18 (d, 2H, J = 7.6 Hz, CH Ph), 2.29 (m, 2H, 3′-
2
1
3
CH ), 1.87 (m, 2H, 2′-CH ). C NMR (DMSO-d ): δ =
2
2
6
(50 μL) at 100–300 CCID50 (50 % cell culture infectious
1
73.8 (NHCO), 170.6 (CO Me), 167.2 (Cphthal.=O), 135.8,
2
dose) or culture medium was added to either of the infected
or mock-infected wells of the microtiter tray. Mock-infected
cells were used to evaluate the effect of test compound on
uninfected cells in order to assess the cytotoxicity of the test
compounds. Exponentially growing MT-4 cells (Witvrouw
et al., 1999) were centrifuged for 5 min at 1000 rpm, and the
supernatant was discarded. The MT-4 cells were resus-
pended at 6 × 105 cells per mL, and 50-μL volumes were
transferred to the microtiter tray wells. Five days after
infection, the viability of the mock-infected cells and HIV-
infected cells was examined spectrophotometrically.
1
32.2, 131.8, 128.3, 128.1, 124.6 (Carom.), 52.7
(
3
CHCO Me), 52.2 (CO Me), 39.1 (C-1′), 35.3 (CH Ph),
2
2
2
+
1.7 (C3′), 23.8 (C2′). MS (FAB) m/z: 395 [M+H] . Anal.
Calcd. for C H N O (394.42): C, 66.99; H, 5.62; N,
2
2 22 2 5
7
.10. Found: C, 66.78; H, 5.57; N, 6.93.
Methyl 2-(4-(phthalimido-2-yl)butanamido)-3-(indol-3-yl)
propanoate (22) From L-tryptophane methyl ester (327
mg). Yield: 507 mg (78 %); M.p. 132–135 °C. H NMR
1
(
DMSO-d ): δ = 7.88 (br s., 4H, H–Ar), 7.56–7.13 (m, 5H,
6
H–Ar+2-Hindole), 4.32 (m, 2H, 1′-CH ), 4.23 (m, 1H,
NCH), 3.60 (s, 3H, OAc), 3.29 (d, 2H, J = 7.1 Hz, CH
dol), 2.23 (m, 2H, 3′-CH2), 1.88 (m, 2H, 2′-CH2). C NMR
2
2
in-
1
3
In vitro HCV assay
(
(
DMSO-d ): δ = 172.7(NHCO), 171.3 (CO Me), 167.8
Cphthal.=O), 136.0, 132.0, 130.7, 128.5, 126.4, 124.1,
6
2
Huh-5-2 cells, containing the HCV genotype 1b I389luc-
ubi-neo/NS3-3′/5.1 replicon (Vrolijk et al., 2003) were
subcultured in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10 % Fetal calf serum (FCS),
1
20.5, 118.9, 111.0, 108.9 (Carom.), 53.9 (CHCO Me), 51.2
2
(
(
CO Me), 39.3 (C1′), 31.7 (C3′), 27.8 (CH2indole), 23.5
2
+
C2′). MS (FAB) m/z: 456 [M+Na] . Anal. Calcd. for

Med Chem Res
1
% non-essential amino acids, 1 % penicillin/streptomycin,
Conclusions
and 2 % Geneticin at a ratio of 1:3–1:4, and grown for 3–4
days in 75 cm² tissue culture flasks. One day before the
addition of the compound, cells were harvested and seeded
in assay medium (DMEM, 10 % FCS, 1 % non-essential
amino acids, 1 % penicillin/ streptomycin) at a density of
A series of phthalimido-(substituted-alkyl-2-thioureido)
alkyl carboxylic acid derivatives 5–13 and methyl 2-(4-
(phthalimido-2-yl)butanamido)alkyl carboxylates 14–23
were synthesized. The new synthesized compounds were
assayed against HIV-1 and HIV-2 in MT-4 cells. Com-
pounds 22 and 23 showed significant inhibition of HIV-1
with EC50 value of >2.07 and >4.23 μM (SI = 7 and 5),
respectively. The anti-HIV activity results suggest that
compounds 22 and 23 might act as new candidates for non-
nucleoside reverse transcriptase inhibitors. Furthermore, the
new analogs were screened against HCV genotype 1b in the
Huh-5-2 replicon system. Compounds 9 and 12 showed a
moderate activity with EC50 = 26.7 and 22.0 μM (SI > 1.87
and >2.28, respectively). These analogs could be promising
agents in HCV inhibition studies after further structural
modification. The molecular docking study of 22 showed
hydrogen bond interactions with some amino acids of HIV
reverse transcriptase enzyme.
6
500 cells/well (100 μL/well) in 96-well tissue culture
microtiter plates for the evaluation of anti-metabolic effect
and cultur plate (Perkin Elmer) for the evaluation of the
antiviral effect. The microtiter plates were incubated over-
night (37 °C, 5 % CO , 95–99 % relative humidity), yield-
2
ing a non-confluent cell monolayer.
The evaluation of the anti-metabolic as well as antiviral
effect of each compound was performed in parallel. Four-
step, 1-to-5 compound dilution series were prepared for the
first screen, to collect data for a more detailed dose-response
curve, an eight-step, 1-to-2 dilution series was used. Fol-
lowing assay setup, the microtiter plates were incubated for
7
2 h. (37 °C, 5 % CO , 95–99 % relative humidity). For the
2
evaluation of anti-metabolic effects, the assay medium was
aspirated, replaced with 75 μL of a 5 % MTS solution in
phenol red-free medium and incubated 45 for 1.5 h. (37 °C,
Acknowledgments We thank Mr. U. Haunz and Miss A. Frimel of
the Chemistry Department, University of Konstanz, Germany, for the
NMR experiments. We thank Prof. B. A. Saeed of Basrah University
for molecular docking study.
5
% CO , 95–99 % relative humidity). Absorbance was
2
measured at a wavelength of 498 nm (Safire², Tecan), and
optical densities (OD values) were converted to percentage
of untreated controls. For the evaluation of antiviral effects,
the assay medium was aspirated and the cell monolayers
were washed with phosphate buffered saline. The wash
buffer was aspirated, and 25 μL of Glo Lysis Buffer (Pro-
mega) was added allowing for cell lysis to proceed for 5 min
at room temperature. Subsequently, 50 μL of Luciferase
Assay System (Promega) was added, and the luciferase
luminescence signal was quantified immediately (1,000 ms
integration time/well, Safire², Tecan). Relative lumines-
cence units were converted into percentage of untreated
controls. The EC50 (values calculated from the dose-
response curve) represent the concentrations at which 50 %
inhibition, respectively, of viral replication is achieved. The
CC50 (value calculated from the dose-response curve)
represents the concentration at which the metabolic activity
of the cells is reduced by 50 % as compared to untreated
cells. A concentration of a compound is considered to elicit
a genuine antiviral effect in the HCV replicon system when
the anti-replicon effect is well above the 70 % threshold at
concentrations where no significant anti-metabolic activity
is observed (Vrolijk et al., 2003).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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