Novel antiviral benzofuran-transition metal complexes

Article abstract of DOI:10.1016/j.ejmech.2010.03.034

(5-(1H-benzo[d]imidazol-2-yl)-1H-pyrrol-3-yl)(6-hydroxy-4, 7-dimethoxybenzofuran-5-yl)methanone (4) and 3-(6-hydroxy-4,7- dimethoxybenzofuran-5-carbonyl)-6H-pyrimido[1,6-a]pyrimidine-6,8(7H)-dione (5) were synthesized by the reaction of 4,7-dimethoxy-5-oxo-5H-furo[3,2-g]chromene- 6-carbaldehyde (1) with (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride and 4-amino-2,6-dihydroxypyrimidine, respectively, via ROR in the presence of alcoholic KOH. The metal complexes 6-9 of compound 4; H₂L¹ with (CuCl₂, FeCl₃, ZnCl₂, and LaCl ₃) and the metal complexes 10-13 of compound 5; H₂L ² with (CuCl₂, FeCl₃, CoCl₂ and LaCl₃) were synthesized to form 1:1 or 1:2 (metal: ligand) complexes. The HIV inhibitory activity of all new compounds was tested. The EC50 values showed that, all of tested compounds were more potent than Atevirdine. Moreover, the benzoimidazolylpyrrole derivative 4 (EC₅₀=9×10 ⁶mM) had higher therapeutic index than the standard. The HIV-1 RT inhibitory activity showed that all of the tested compounds showed significant potency but none of them showed higher activity than Atevirdine. The HCV NS3-4A protease inhibitor activity of the tested compounds revealed that the complex formation had great positive effect on the bioactivity, where the Fe-complex 7 was the most potent compound with higher therapeutic index than VX-950, the standard. Also, the cytotoxicity of the synthesized compounds on hepatocyte cell line, showed that Cu-complex 10 was the most potent compound with potency nearly to that of the standard.

Full text of DOI:10.1016/j.ejmech.2010.03.034

European Journal of Medicinal Chemistry 45 (2010) 3035e3046
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel antiviral benzofuran-transition metal complexes
Shadia A. Galal a , Amira S. Abd El-All , Khaled H. Hegab , Asmaa A. Magd-El-Din , Nabil S. Youssef ,
,
a
b
a
b
*
a
Hoda I. El-Diwani
a
Division of Pharmaceutical and Drug Industries, Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo 12622, Egypt
Division of Inorganic Chemical Industries and Mineral Resources, Department of Inorganic Chemistry, National Research Center, Dokki, 12622, Cairo, Egypt
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 August 2009
Received in revised form
(5-(1H-benzo[d]imidazol-2-yl)-1H-pyrrol-3-yl)(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)methanone
(
(
(
4) and 3-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-6H-pyrimido[1,6-a]pyrimidine-6,8(7H)-dione
5) were synthesized by the reaction of 4,7-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-6-carbaldehyde
21 February 2010
1) with (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride and 4-amino-2,6-dihydroxypyrimidine,
Accepted 17 March 2010
Available online 27 March 2010
1
respectively, via ROR in the presence of alcoholic KOH. The metal complexes 6-9 of compound 4; H
2
L with
2
(
CuCl
CoCl
of all new compounds was tested. The EC50 values showed that, all of tested compounds were more potent
2
, FeCl
3
, ZnCl
2
, and LaCl
3
) and the metal complexes 10-13 of compound 5; H
2
L
2
with (CuCl , FeCl
3
,
2
3
and LaCl ) were synthesized to form 1:1 or 1:2 (metal: ligand) complexes. The HIV inhibitory activity
Keywords:
Benzofurans
Transition metal complexes
Antiviral activity
HIV and HIV-1 RT inhibitory activity
HCV NS3-4A protease inhibitor activity
ꢁ
6
than Atevirdine. Moreover, the benzoimidazolylpyrrole derivative 4 (EC50 ¼ 9 ꢀ 10
mM) had higher
therapeutic index than the standard. The HIV-1 RT inhibitory activity showed that all of the tested
compounds showed significant potency but none of them showed higher activity than Atevirdine. The
HCV NS3-4A protease inhibitor activity of the tested compounds revealed that the complex formation had
great positive effect on the bioactivity, where the Fe-complex 7 was the most potent compound with
higher therapeutic index than VX-950, the standard. Also, the cytotoxicity of the synthesized compounds
on hepatocyte cell line, showed that Cu-complex 10 was the most potent compound with potency nearly to
that of the standard.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
host cell chromosomes. Reverse transcriptase is multifunctional
enzyme. This enzyme exhibits an RNA and DNA directed poly-
In a previous manuscript, a new class of highly potent antitumor
and antiviral agents, having the benzofuran [1e7] skeleton related
to heterocyclic moieties by a carbonyl group at the 5-position, was
successfully discovered by Galal et al. [8]. This was quite evidence
that hetero-moieties joined to the benzofuranylcarbonyl skeleton
obtained via the ring opening reaction (ROR) of furochromones
merase activity. In addition reverse transcriptase catalyzes the
degradation of RNA in an RNA-DNA hybrid. Functional HIV1-RT is
a heterodimer contains two domains, the N-terminal polymerase
domain and the C-terminal RNase H domain [11]. Because of the
importance of RT to HIV replication, inhibitors of this enzyme are
potential theraputic agents in the battle against HIV.
[
9,10], mostly have positive effect on activity and selectivity. The
The NS3-4A serine protease of hepatitis C virus (HCV) is essential
for viral replication and therefore has been one of the most attrac-
tive targets for developing specific antiviral agents against HCV.
Functional HCV NS3-4A serine protease is a heterodimeric enzyme
comprising the N-terminal domain of the NS3 protein as well as the
NS4A protein. Fig. 2 shown the X-ray crystallographic structures of
the HCV NS3-4A protease. Based on the enzyme features [12], there
are three alternative strategies for developing inhibitors of the HCV
serine protease were initially envisioned: tampering with the NS3/
NS4A interaction, interfering with zinc binding and preventing
substrate binding in the active site. However, the first two strategies
are currently viewed as extremely difficult [13] and the develop-
ment of active site inhibitors of the NS3-4A enzyme is considered
the most promising approach.
HIV inhibitory activity of the previously synthesized compounds as
IeIII (c.f. Fig. 1), indicated that all the compounds were distinctly
potent and the evaluation of hepatitis C virus (HCV) NS3-4A
protease inhibitory activity showed that some of these compounds
were significantly active.
The enzyme reverse transcriptase (RT) is used by retroviruses to
transcribe their single-stranded RNA genome into single-stranded
DNA and to subsequently construct a complementary strand of
DNA, providing a DNA double helix capable of integration into
*
Corresponding author. Tel.: þ20 233371718; fax: þ20 233370931.
E-mail address: sh12galal@yahoo.com (S.A. Galal).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.03.034

3036
S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
OMe
O
N
O
N
OH
OMe
OMe
O
OMe
OMe
O
II
N
O
N
N
OH
N
OMe
O
N
OH
N
H
I
III
Fig. 1. Structural representations of potent antiviral benzofuran derivatives.
Our aim in this work was to design new ligands 4 and 5, bearing
having potential medicinal applications that could be comple-
mentary to organic compounds [14].
good resemblance to the HIV reverse transcriptase inhibitor drug
atevirdine and the protease inhibitor drug VX-950 (Fig. 3), and
the formation of their transition metal complexes, aiming to study
the HIV and HIV reverse transcriptase inhibitory activity besides
the hepatitis C virus (HCV) NS3-4A protease inhibitory activity.
Compounds 4 and 5 may serve as good ligands for transition metal
ion due to the large conjugated pi-system, the azamethine nitrogen,
the hydroxyl and the carbonyl groups which can positively affect
the structures of the complexes. Where the unique redox and
spectroscopic properties result in metal ions and their complexes
2. Results and discussion
2.1. Chemistry
The reaction of 4,7-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-
6-carbaldehyde 1 [8,15,16] with (1H-benzimidazol-2-yl)methan-
amine dihydrochloride (2) [17] was previously studied by using
a catalytic amount of triethyl amine or piperidine to give
Fig. 2. X-ray crystallographic structures of the HCV NS3-4A protease. (A) NS3-4A protease domain. The side chains of the residues forming the catalytic triad (His-57, Asp-81, and
Ser-139) are shown in red. The NS4A cofactor is shown in purple, the bound zinc ion is modeled in light green. (B) A decapeptide substrate (backbone only) is modeled in the NS3-4A
protease active site. The NS4A cofactor is shown in purple. The side chains of the residues forming the catalytic triad are shown in red. The side chains of the residues forming the S1
specificity pocket of the enzyme (Val-132, Leu-135, and Phe-154) are shown in yellow. (C) Product inhibition in the NS3-4A complex. The NS4A peptide (red) has been artificially
linked to the N-terminus of NS3 protease domain (gray) in the single-chain construct used for crystallization. The side chains of the residues forming the protease catalytic triad are
shown in red. The helicase domain (top portion of the molecule) is depicted in light green. The C-terminus of NS3 generated as product of the NS3/NS4A cleavage reaction, forms
a
b-strand that occupies the proteinase active site (shown in light blue) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.).

S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
3037
Fig. 3. The structure of Atevirdine, VX-950, compounds 4 and 5.
imidazodiazepine derivative 3 [8]. On the other hand, when
potassium hydroxide is used as basic medium, benzimidazo-
lylpyrrolyl derivative 4 was produced by the reaction of 1 and 2 (c.f.
Scheme 1). The mass spectra and elemental analyses of both 3 and 4
Reaction of 4-amino-2,6-dihydroxypyrimidine with compound
1 led to pyrimidopyrimidine derivative 5 (c.f. Scheme 3).
The metal complexes 6e9 of compound 4 with (CuCl
ZnCl , and LaCl ) and the metal complexes 10e13 of compound 5
with (CuCl , FeCl , CoCl and LaCl ), respectively, were synthesized
2 3
, FeCl ,
2
3
showed that they have the same molecular weight but different
2
3
2
3
1
melting points and R
f
values. The H-NMR spectrum of 4 showed
according to the reported methods by Merchán et al [18] to form
1:1 or 1:2 (metal: ligand) complexes. These complexes were char-
the presence of some different in the chemical shifts of both
compounds as well as the presence of NH broad signal at 5.9 ppm
exchangeable with D O. Besides, the IR spectrum of 4 revealed the
1
13
d
acterized by elemental analyses, IR, H-NMR and C-NMR (for
diamagnetic complexes), electronic and mass spectra, magnetic
moments and conductance measurements as well as thermo-
2
ꢁ
1
NH band at 3230 cm
.
The suggested mechanism of the reaction of 1 and 2 in the
presence of potassium hydroxide to afford 4 is explained in
Scheme 2.
gravimetric analyses (TG). Based upon this characterization, the
1
prepared complexes have the structural formulae: [Cu(HL )(H
2
O)
4
]
1
1
Cl.2.H
2
O (6), [Fe(HL )Cl
2
(H
2
O)
2
]2.H
2
O (7), [Zn(HL )Cl(H
2
O)] (8), [La
OMe
O
O
N
CHO
NH2 .2 HCl
+
N
H
O
OMe
2
1
T.E.A / methanol
KOH / methanol
O
OMe
O
OMe
OMe
N
N
H
H
O
O
N
OH
N
OH
N
H
N
R
4
3
Scheme 1. The reaction of 4,7-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-6-carbaldehyde (1) and (1H-benzimidazol-2-yl)methanamine dihydrochloride (2) in basic medium.

3038
S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
H
O
OMe
OMe
OMe
O
O
H
OMe
OMe
O
O
O
OMe
OMe
O
O
H
H
O
+
H
O
+
OH
H
O
O
O
H
H
H
O
O
OH
O
O
OH
H
OMe
A
1
N
NH2
N
NH2
OH-
N
NH2
.
2 HCl
-
N
H
N
H
N -
B
2
C
A + B
A + C
-
H O
2
-
H O
2
OMe
O
H
O
H
N
OMe
OMe
N
O
N
OH
O
N
HO
N
OH
HO
R
N
H
-
H O
2
-
H O
2
O
OMe
OMe
OMe
O
N
H
N
O
N
H
OH
O
OH
N
H
N
N
R
H
OMe
O
O
OMe
OMe
N
H
N
N
O
O
OH
N
OH
N
H
R
N
H
4
3
Scheme 2. The suggested mechanism of the reaction of 1 and 2 in the presence of potassium hydroxide to afford 4.
O
OMe
OMe
O
O
OMe
OMe
NH2
N
N
OHC
KOH / methanol
O
N
HO
+
O
HO
N
OH
O
N
H
O
1
5
Scheme 3. The synthesis of pyrimidopyrimidine derivative 5.

S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
3039
1
(
HL1)
other hand, [Cu(HL )Cl(H
2
(H
2
O)
2
]Cl.3.H
2
O (9) for ligand 4; H
2
L (c.f. Scheme 4). On the
[20]. The bands due the stretching frequency of
n
(CeO) and that of
2
2
2
O)
]3Cl.2.H O (12) and [La(H
L (c.f. Scheme 5).
2
].H
2
O (10), [Fe(H
2
2
L )Cl(H
2
O)
2
]$2Cl.3.
n
(C]O) groups were not greatly affected by complex formation in
all complexes 6e13, suggesting the non-bonding of these groups.
For the complexes 6e9, considerable frequency shifts of (C]N) in
2
H
2
O (11), [Co(H
L )
2 2
2
2
L )
2
].3Cl.H
2
O (13) for
2
ligand 5; H
2
n
The IR broad bands in the range (3500e3200 cmꢁ1) indicated
the presence of lattice water in the complexes 6e13 [19], which was
further supported by the percents of weight loss observed from
the wave numbers of the spectra of these complexes with respect to
compound 4 were observed, which indicated the participation of
azomethine nitrogen in the chelation of these complexes. The IR
bands of complexes 11e13 showed that the two bands of the amide
ꢂ
thermo-gravimetric analyses in the temperature range 25e204 C
O
OMe
OMe
H
N
O
N
N
OH
H
4
FeCl / methanol
3
CuCl / methanol
2
OH2
OH2
Cl
OH2
Cl
OH2
OH2
OMe
OMe
O
O
Fe
OMe
OMe
Cu
N
2. H O
2
N
Cl. 2. H O
OH2
2
O
N
O
OH
N
OH
N
N
H
H
6
7
LaCl / methanol
ZnCl / methanol
3
2
OH2
Zn
O
Cl
OMe
N
O
O
N
OH
OMe
OH
OMe
N
MeO
H
O
H
8
N
N
OH2
Cl. 3. H O
2
N
O
OMe
OMe
La
N
OH2
O
OH
N
N
H
9
Scheme 4. The synthesis of metal complexes 6e9 by coordination of compound 4 to CuCl
2 3
, FeCl , ZnCl
2 3
, and LaCl , respectively.

3040
S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
O
OMe
OMe
N
O
N
HO
O
N
H
O
5
CuCl2 / methanol
FeCl
3
/ methanol
O
OMe
OMe
O
OMe
N
N
N
O
HO
N
HO
O
OMe
2 Cl. 3. H O
2
N
.H O
O
N
H
O
O
O
2
Cu
Fe
Cl
OH2 Cl OH
OH
2
2
OH2
1
1
1
0
LaCl3 / methanol
CoCl /methanol
3
O
OMe
OMe
O
OMe
N
N
O
O
N HO
O
N
HO
OMe
O
N
H
O
N
H
O
La
3
Cl. 2 H O
3 Cl. H O
2
2
Co
H
N
H
N
O
O
OMe
OH N
O
O
OMe
O
O
OH
N
N
N
OMeO
O
OMe
1
3
1
2
2 3 2 3
Scheme 5. The synthesis of metal complexes 10e13 by coordination of 5 to CuCl , FeCl , CoCl and LaCl , respectively.
groups in the free ligand 5 at 1725 and 1662 cmꢁ1, were shifted to
higher or lower wave numbers suggesting, the involvement of
these groups in chelation. On the other hand, in the case of
were centered at 7.17 ppm. These signals were shifted towards
lower frequencies, comparable to H-5 and H-6 at 7.50 ppm of
benzimidazole moiety of compound 4, giving coordination shifts
ꢁ
1
compound 10, only one band appeared at 1696 cm due to amide
D
coord: þ0.33 ppm. Also, calculated coordination shifts
of pyrrole moiety: 7.63 ppm (for free ligand 4) ꢁ7.17 ppm (for
complexes
Dcoord of H-4
ꢁ1
group and a new band at 1274 cm , due to n(CeO), suggesting the
chelation via carbonyl group and enolic (OH) groups of pyrimidine
moiety. The FT-IR spectra of all chelates exhibited a band in the
8
and 9) ¼ þ0.46. Furthermore, the multiplet at
7.75 ppm due to H-4 and H-7 in benzimidazole and H-2 in pyrrole
moieties of compound 4 exhibited considerable chemical shifts
upon coordination. H-4 in benzimidazole moiety shifted to
7.44 ppm in complex 8 and to 7.39 ppm in compound 9, whereas H-
7 in benzimidazole moiety showed lower field shift to 7.20 ppm in
complex 8 and to 7.17 ppm in compound 9. H-2 in pyrrole moiety of
ꢁ
1
range of 590e670 cm , assigned to
n(MeO). Also, new bands
ꢁ1
appeared at 485e491, 385e395 and 310e325 cm which may be
1
2
due to
n(CueO), n(CueN) and n(CueCl) for CueHL and CueHL
complexes, respectively. In addition, two new bands displayed at
ꢁ
1
2
4
75 and 370 cm assignable for
2
n(CoeO) and n(CoeN) in CoeH L
complex [21].
compound
appeared as singlet at 8.17 ppm for complex 8 and at 8.15 ppm for
complex 9, giving coordination shifts
ꢁ0.40 ppm for compounds 8 and 9, respectively. Moreover,
exchangeable signal due to NH benzimidazole moiety in compound
4 appeared at 9.5 ppm was shifted to higher frequency, at
12.59 ppm and 11.90 ppm, respectively, due to coordination in both
4 signal shifted towards higher frequencies and
1
Upon coordination, the H-NMR spectra of the complexes 8 and
with respect to the free ligand 4 showed that the exchangeable
9
D
coord:ꢁ0.42 ppm and
signal from NH of pyrrole moiety at 5.90 ppm, disappeared indi-
cating the deprotonation of imino group due to ZneN and LaeN
bond formation, respectively. Multiplets from H-5 and H-6 of
benzimidazole and H-4 of pyrrole moieties in complexes 8 and 9

S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
3041
Table 1
The observed chemical shifts of some 1H-NMR signals of free ligand 4 and its complexes 8 and 9.
0
0
00
00
Compounds #
NH pyrrol
H5 and H6 Benzimi-dazole
H4 pyrrol
H4 benzimi-dazole
H7 Benzimi-dazole
H2 pyrrol,
NH benzimi-dazole
4
8
9
5.90
e
7.50
7.17
7.17
7.63
7.17
7.17
7.75
7.44
7.39
7.75
7.20
7.17
7.75
8.17
8.15
9.29
12.59
11.90
e
complexes 8 and 9. The presence of the exchangeable protons (NH
of benzimidazole and OH of hydroxy benzofuran) signals and the
disappearance of (NH of pyrrole) signal was in agreement with the
IR data of these complexes. Besides, the 13C-NMR spectra of these
complexes 8 and 9 showed strong chemical shifts of (C]N) signals
upon coordination, from 148.76 ppm for the free ligand 4 to
observed for octahedral Cu (II) complexes and corresponding to one
unpaired electron, consequently confirming the monomeric nature
of these complexes [23]. The observed magnetic moment of the
complex 12 was 4.7 BM. The molar conductance of this complex in
ꢁ ꢁ1
1
2
DMF is 79
U
cm mol , indicating the electrolytic nature of this
complex. The electronic spectrum of this complex displayed two
electronic spectral bands at 708 and 530 nm assignable to
1
55.76 ppm and 152.81 ppm for complexes 8 and 9, respectively.
The 1H-NMR spectra of the complexes 12 and 13 exhibited
remarkable downfield shift of pyrimidopyrimidinone signals as
4
4
4
4
T
1g(F) / A2g(F) and
T1g(F) / T2g(P) transitions, respectively,
d
characteristic of octahedral geometry [12]. In case of the Fe
well as high downfield shift of the exchangeable proton NH of pyr-
imidopyrimidinone at 11.67 ppm (for complex 12) and 10.35 ppm
complexes 7 and 11, the broad band’s observed at 527e720 and
6
4
705e765, respectively, can be assigned to the
A1g / T2g(G) multiplicity forbidden transitions suggesting an
octahedral field around iron (III) ion similar to other high spin d -
A
1g / T1g(G) and
6
4
(
for complex 13) compared to the free ligand 5 at 6.71 ppm. Also, the
0
5
H-9 pyrimidopyrimidinone signal appeared at 8.23 ppm in free
ligand 5 exhibited higher frequency shift at 8.93 ppm for complex 12
and lower frequency shift at 7.95 ppm for complex 13. Furthermore,
chelates [24]. The magnetic moment of this complex was 5.18 B.M.
0
the H-2 pyrimidopyrimidinone signal appeared at 8.81 ppm in free
2.2. Antiviral activity
ligand 5 exhibited higher frequency shift at 9.56 ppm for complex 12
and lower frequency shift at 8.42 ppm for complex 13. On the other
hand, the presence of the exchangeable proton (OH of hydroxy
benzofuran) without notable shift with respect to compound 5 was in
accordance with the IR data. Moreover, the C-NMR of complexes 12
and 13 showed significant coordination shifts in C]O groups of
2.2.1. HIV inhibitory activity and reverse transcriptase inhibition
with therapeutic windows
Antiviral and toxicity data were reported as compound
concentration required to inhibit the virus induced cell killing by
50%. (EC50). The compounds 1, 4e13 were tested for RT inhibitory
activity against purified recombinant HIV-1 RT using the cell-free
Quan-T-RT assay system (Amersham Corp., Arlington Heights, IL),
which utilizes the scintillation proximity assay (SPA) principle
[25,26]. IC50[RT] values (concentration at which the compound
inhibits recombinant RT by 50%) were calculated by comparing the
measurements to untreated sample [27]. Results of HIV and HIV-RT
inhibitory activity of the tested compounds and Atevirdine, the
standard drug used, were evaluated (c.f. Table 4).
13
pyrimidopyrimidinone. Calculated coordination shift Dcoord of C]O:
1
3
48.86 ppm (for free ligand 5) e 144.96 ppm (for comple 12) ¼ þ
.90 ppm, whereas the (C]O) signal appeared at 146.46 ppm for
complex 9 upon coordination, with
D
coord: þ2.20 ppm. Tables 1 and 2
1
exhibited the remarkable chemical shifts of the H-NMR signals of
free ligands 4 and 5 and their diamagantic complexes that affected by
coordination.
The molar conductance of complexes 6, 9 and 11e13 are around
ꢁ1
2
ꢁ1
6
7e85
U
cm mol , indicating the electrolytic nature of these
The EC50 values listed inTable 4 showed that, the parent compound
ꢁ
6
ꢁ6
m
complexes, whereas, 7, 8 and 10 have molar conductance values of
1 (EC50 ¼ 2.0 ꢀ 10 ), the free ligands 4 (EC50 ¼ 9 ꢀ 10
M) and 5
ꢁ
1
2
ꢁ1
ꢁ6
16e28
U
cm mol , respectively, confirming their non-electro-
(EC50 ¼ 10 ꢀ 10
(EC50 ¼ 10 ꢀ 10
mM) are more potent than Atevirdine
M). Moreover the benzimidazolylpyrrol derivative
ꢁ
4
lytic nature [22].
m
The thermogravimetric data obtained were in a good agreement
with the theoretical formulae as suggested from the elemental
analyses.
The electronic transitions for 4 and 5 and their metal complexes,
with their magnetic moments (B.M.) and the electronic of these
complexes are studied (c.f. Table 3). The absorption spectra of the
two ligands showed bands in the region 245e400 nm, which can be
4 has much better therapeutic index (TI ¼ 110,000) than the standard
drug (TI ¼ 100,000). On the other hand, the complexes 6e13 and
Atevirdine have comparable potency. Also, the complexes 6 (TI ¼ 52,
665) and 13 (TI ¼ 56, 234) presented relative interesting therapeutic
index. Studying the bioactivity of new synthesized compounds indi-
cated that the coordination of 4 and 5 to a transition metal ion to form
the complexes 6e13 decrease the potency relatively.
assigned to
The spectra of compound 8 exhibited two bands at 293 and 350 nm
which can be assigned to * transition of the conjugated system
and only one peak about 410 nm which can be attributed to MLCT
), which confirming the tetrahedral structure of this
pep* and n- p* transitions of the conjugated system.
pep
Table 3
Electronic Absorption Spectral Bands and Magnetic Moment of the Ligands 4 and 5
and their Complexes 6-13.
M L
(d - p*
complex. On the other hand, the Cu complexes 6 and 10 have
magnetic moments 1.78e1.71 B.M, lying in the normal range
Compounds # Intraligand & Charge
Transfer bands (nm)
d-d Bands
(nm)
Magnetic
Moment B.M.
4
5
6
7
8
9
1
295, 352, 360, 395
245, 260, 350, 400, 440
262, 289, 336, 363, 399, 443 530, 730
260, 277, 324, 398, 414
293, 350, 400
e
e
e
e
Table 2
1.78
1.68
Dia
Dia
The observed chemical shifts of some 1H-NMR signals of free ligand 5 and its
complexes 12 and 13.
527, 720
e
290, 350, 400, 423
263, 291, 335, 354, 362, 385 530, 706, 735 1.71
e
0
0
Compounds # NH
H9 pyrimido-pyrimidinone H2 pyrimido-pyrimidinone
0
5
1
1
6.71 8.23
11.67 8.93
10.35 7.95
8.81
9.56
8.42
11
12
13
260, 285, 330, 350, 362
263, 277, 289, 355, 364
268, 295, 350
705, 730, 765 1.70
530, 708, 735 4.7
2
3
500, 550
Dia

3042
S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
Table 4
The activity of the potent complexes may be due to intercalation of
the metals with the DNA of the virus and hence inhibit its repli-
cation. Also, the activity indicates that the complexes have a suit-
able molecular size and stereochemistry so that metal ions can bind
to the enzyme at its active site.
The HIV inhibitory activity and HIV- reverse transcriptase inhibition with thera-
peutic windows of the tested compounds 1 and 4e13 and standard.
Compounds #
EC50d/
m
Ma
6
IC50[RT]
/m
Mb
Therapeutic indexc
1
4
5
6
7
8
9
2.0 ꢀ 10ꢁ
17.53
34.12
40.31
87.98
90.17
92.27
82.27
80.16
90.31
96.18
78.67
10
32 567
110 000
45 786
52 665
12 768
23 498
43 498
22 567
44 324
23 458
56 234
100 000
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
6
6
4
4
4
4
4
4
4
4
4
9 ꢀ 10
10 ꢀ 10
2.2.2. Hepatitis C virus (HCV) NS3-4A protease inhibitor activity
6.8 ꢀ 10
The development of potent competitive inhibitors of the HCV
7.1 ꢀ 10
8.5 ꢀ 10
serine protease and identification of non-peptidic small molecule
inhibitors of NS3-4A enzyme is significantly important. Studying of
the hepatitis C virus (HCV) NS3-4A protease inhibitor activity of the
compounds 1, 4e13 was performed. For comparison, the non-
covalent peptidomimetic NS3-4A protease inhibitor, VX-950 was
used as standard drug. The cytotoxicity of the tested compounds
was measured under the same experimental settings using
a tetrazolium (MTS)-based cell viability assay (Promega, Madison,
WI). Cells were identical in sequence as described by Lohmann et al.
5.5 ꢀ 10
1
1
1
1
0
1
2
3
3.1 ꢀ 10
5.4 ꢀ 10
ꢁ
ꢁ
6.5 ꢀ 10
2.1 ꢀ 10
Atevirdine
10 ꢀ 10ꢁ
a
Compound concentration required to inhibit the virus induced cell killing by
5
0%.
b
Compound concentration required to achieve 50% inhibition of recombinant
HIV-RT activity.
[
28]. Determination of 50% inhibitory concentration (IC50), 90%
inhibitory concentration (IC90), and 50% cytotoxic concentration
CC50) of all the tested compounds in HCV replicon cells was per-
c
A therapeutic index is the lethal dose of a drug for 50% of the population (LD50
)
divided by the minimum effective dose for 50% of the population (ED50). (LD50) is the
dose required to kill half the members of the tested population. (ED50) is the dosage
that produces a desired effect in half the test.
(
formed using a quantitative RT-PCR (QRT-PCR) assay [29,30]. The
results of HCV NS3-4A protease inhibitor activity and the cytotoxic
activity of the tested compounds and standard were listed (c.f.
Table 5)
d
EC50 & IC50 values were estimated by logistic regression analysis. One way
ANOVA (P < 0.01) was used to test treatment difference in EC50 & IC50. After
significant factor by ANOVA, individual group differences were analyzed using
Holm-Sidak’s procedure for multiple comparisons versus control.
Screening of the 90% inhibitory concentration (IC90) values
revealed that all of the synthesized compounds have potent
Screening of HIV-1 reverse transcriptase inhibition of the
synthesized compounds showed that the starting material, furo-
chromone derivative 1, was the most potent compound with lowest
activity. The Fe-complex 7 (IC90 ¼ 0.345 ꢃ 0.01
potent than the standard drug, VX-95, (IC90 ¼ 0.45 ꢃ 0.0003
and its free ligand 4 (IC90 ¼ 1.654 ꢃ 0.14 M) besides the parent
M). Interestingly, compound 7
m
M) was more
mM)
m
IC50 (17
compound 4 showed high potency against HIV-1 reverse tran-
M, that was paral-
mM). This potency was correlated by high HIV activity. Also
compound 1 (IC90 ¼ 0.796 ꢃ 0.08
m
exhibited also wider therapeutic index than VX-950 (TI ¼ 1200)
scriptase inhibition activity with IC50 ¼ 34.12
m
and compound 4 (TI ¼ 462.52) besides compound 1 (TI ¼ 555.82).
leled by its potent HIV activity, as the therapeutic index. This
feature made compound 4 very interesting candidate for in vivo
study. Furthermore, compound 5 showed high activity toward HIV-
Also, the Zn complex 8 (IC ¼ 1.23 ꢃ 0.12
mM) and La complex 9
M) have higher potency than the ligand 4.
9
0
(IC90 ¼ 1.32 ꢃ 0.13
m
Whereas, the Cu complex 6 (IC ¼ 1.8 ꢃ 0.09
9
0
mM) has less potency
1
reverse transcriptase inhibition with IC50 ¼ 40.31
m
M. Upon
than 4. By comparing the efficacy of complex formation 6e9 in the
inhibition of hepatitis C virus (HCV) NS3-4A protease with respect
to their free ligand 4. The octahedral geometrical structure with of
Fe-complex 7 is the most potent one, followed by the square planar
Zn complex 8 then octahedral La complex 9 and finely the octa-
hedral Cu complex 6.
coordination of compound 4 and 5 to transition metal ions as Cu(II),
Fe(III), Zn(II), Co (II) or La(II) to form the complexes 6e13, The HIV-1
reverse transcriptase inhibition activity decreased which was also
paralleled by HIV inhibitory activity reduction.
From the above screening of HIV-1 reverse transcriptase inhib-
itory activity with the HIV activity, the synthesized compounds
showed high efficacy in inhibition of HIV reverse transcriptase
enzyme which is the enzyme responsible about for viral replication.
Furthermore, the pyrimidopyrimidine derivative 5 (IC90
0.844 ꢃ 0.07 M) and the Cu complex 10 (IC90 ¼ 0.876 ꢃ 0.09 M)
have convergent potent activity, but the Co complex 12
¼
m
m
Table 5
HCV NS3-4A protease inhibitory activity and the cytotoxic activity of the tested compounds (1 and 4e13) and standard.
a
f
b
f
c
f
Therapeutic indexd
Compounds #
IC50 (ꢃSD )
m
M After 48 Hours
IC90 (ꢃSD )
mM After 48 Hours
CC50 (ꢃSD )
mM
1
4
5
6
7
8
9
0.421 ꢃ 0.03
0.657 ꢃ 0.05
0.388 ꢃ 0.04
0.432 ꢃ 0.04
0.112 ꢃ 0.01
0.643 ꢃ 0.04
0.564 ꢃ 0.04
0.445 ꢃ 0.034
0.987 ꢃ 0.09
0.456 ꢃ 0.05
0.765 ꢃ 0.07
0.796 ꢃ 0.08
1.654 ꢃ 0.14
0.844 ꢃ 0.07
1.8 ꢃ 0.09
234 ꢃ 5
555.82
462.52
878.87
592.59
3080.36
346.81
414.89
208.99
348.53
337.72
278.43
1200
765 ꢃ 22
341 ꢃ 8
256 ꢃ 10
345 ꢃ 17
223 ꢃ 11
234 ꢃ 6
0.345 ꢃ 0.01
1.23 ꢃ 0.12
1.32 ꢃ 0.13
0.876 ꢃ 0.09
1.98 ꢃ 0.09
0.987 ꢃ 0.08
1.78 ꢃ 0.09
1
1
1
1
0
1
2
3
93 ꢃ 10
330 ꢃ 13
154 ꢃ 12
213 ꢃ 12
90 ꢃ 1.20
e
VX-950
0.20 ꢃ 0.0001
0.45 ꢃ 0.0003
a
Compound concentration required to achieve 50% inhibition of HCV NS3-4A protease activity.
Compound concentration required to achieve 90% inhibition of HCV NS3-4A protease activity.
Compound concentration required to reduce the cell viability by 50% as determined by MTT method.
b
c
d
A therapeutic index is the lethal dose of a drug for 50% of the population (LD50) divided by the minimum effective dose for 50% of the population (ED50). (LD50) is the dose
required to kill half the members of the tested population. (ED50) is the dosage that produces a desired effect in half the test.
e
The standard used in comparison.
Data expressed as means ꢃ SD for three independent experiments.
f

S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
3043
Table 6
a transition metal ion as Cu(II), Fe(III), Zn(II), Co (II) or La(II). The
HIV inhibitory activity of the tested compounds were more potent
than Atevirdine. Moreover, compound 4 had much better thera-
peutic index than the standard and its derived complexes 6e9. On
the other hand, the HIV-1 reverse transcriptase inhibition of the
synthesized compounds showed significant potency but none of
them showed higher activity than the standard. The HCV-NS3-4A
protease inhibitor activity of the tested compounds showed high
potency. The complex formation positively affected the bioactivity;
furthermore, the Fe-complex 7 was the most potent compound
with higher therapeutic index than the standard drug. Also, the
cytotoxicity of the synthesized compounds on hepatocyte cell line,
showed that Cu complex 10 was the most potent compound
having almost the same potency as VX-950. Studying the median
lethal dose LD50 showed that the complex formation have positive
effect on the reduction of toxicity of free ligands 4 and 5. These
features transform them in interesting candidate to further in vivo
testing.
The median lethal dose of the tested compounds (1 and 4e13).
a
b c
Compound no.
(LD50 ꢃ SD ) mg/kg
1
4
5
6
7
8
9
113.29 ꢃ 1
166.15 ꢃ 1
152.23 ꢃ 1
169.4 ꢃ 2
256 ꢃ 2
263 ꢃ 2
225.98 ꢃ 1
416.18 ꢃ 8
248 ꢃ 2
1
1
1
1
0
1
2
3
243 ꢃ 2
516 ꢃ 5
a
b
c
Dose required to kill half the members of the tested population.
Data expressed as means ꢃ SD for three independent experiments.
The median lethal dose of the tested compounds was determined according to
the procedure described by Lorke.
(
IC90 ¼ 0.987 ꢃ 0.08
Fe-complex 11(IC90 ¼ 1.98 ꢃ 0.09
. Regarding to the cytotoxicity of the synthesized compounds on
hepatocyte cell lines, the Cu complex 10 (CC50 ¼ 93 ꢃ 10 M) is the
most potent compound. The Co complex 12 (CC50 ¼ 154 ꢃ12 M),
La complex 13 (CC50 ¼ 213 ꢃ12 M) and Fe-complex 11
CC50 ¼ 330 ꢃ 13 M) have higher potency than their correspond-
m
M), La complex 13 (IC90 ¼ 1.23 ꢃ 0.12
mM) and
m
M) exhibited lower potency than
4. Experimental
5
m
4.1. Physical measurements
m
m
Microanalyses of the ligands and complexes were performed in
(
m
the Microanalytical Laboratory Center, Faculty of Science, Cairo
University, Egypt. Molar conductance was measured at room
temperature on electronic conductivity model 19000-05 (USA). The
ing ligand 5. This increased potency was not paralleled by higher
inhibitory concentration (IC50), as the therapuetic index. On the
other hand, the complexes 6e9 have higher bioactivity than the
ligand 4. This increased potency was paralleled by higher inhibitory
concentration (IC50) (c.f. Table 5). The activity of the prepared
complexes may be attributed to the binding of the metal ion with
the active site of the NS3-4A enzyme.
ꢁ1
IR spectra (4000e400 cm ) were recorded using KBr pellets in
a Jasco FT/IR 300E Fourier transform infrared spectrophotometer
ꢁ1
and in the 500e100 cm region using Polyethylene-Sandwiched
Nujol mulls on a Perkin Elmer FT-IR 1650 (spectrophotometer). The
1
13
H and C NMR spectra were recorded using Joel EX-270 MHz and
00 MHz NMR spectrophotometers. The electronic absorption
5
2.3. The median lethal dose (LD50
)
spectra were recorded using a Shimadzu UV-240 UVeVisible
recording spectrometer. The mass spectra were carried out using
Finnigan mat SSQ 7000 (Thermo. Inst. Sys. Inc., USA) spectroscopy
at 70 eV. Thermo-gravimetric analyses were carried out using
a DTA-7 and TGA-7 perkin Elmer 7 series thermal analyses system.
The magnetic moments were measured using Gouy method with
The median lethal dose (LD50) which is the dose of the tested
compounds required to kill half the members of the tested pop-
ulation was determined according to the procedure described by
Lorke [31e33]. The results of the LD50 in mg/kg of the synthesized
compounds were listed (c.f. Table 6). Screening the median lethal
dose (LD50) of the tested compounds 1, 4e13 showed that, all
the compounds were found to have higher LD50 than compound 1.
These results indicated that the heteromoieties included into
benzofuranyl skeleton decreased the toxicity of starting material
4
Hg[Co(SCN) ] as calibrant.
4.1.1. Preparation of ((1H-benzo[d]imidazol-2-yl)methanamine)
dihydrochloride (2) [17]
A solution of glycine (150 mmol) and o-phenylenediamine
1
(LD50 ¼ 113.29 ꢃ 1 mg/kg). Screening of LD50, of the synthe-
(100 mmol) were refluxed in 5.5 N hydrochloric acid (100 mL) for
sized complexes with respect to their free ligand indicated
30 h. After the reaction mixture was left overnight, the hydro-
that, complexes 8 (LD50 ¼ 263 ꢃ 2 m/kg), 7 (LD50 ¼ 256 ꢃ 2 mg/kg)
chloride salt was removed as white crystals which recryslallized
9
(LD50 ¼ 225.98 ꢃ 1 mg/kg), and 6(LD50 ¼ 166.15 ꢃ1 mg/kg),
ꢂ
from ethanol. Yield 89%, m.p. 263e265 C (dec.) (lit. m.p.
respectively,
have
higher
LD50
than
compound
4
ꢂ
1
2
63e265 C dec.). H-NMR (270 MHz, DMSO-d
6 2
): 4.55 (s, 2H, CH ),
(
LD50 ¼ 166.15 ꢃ1 mg/kg). Moreover, complex 13 showed highest
4
.96 (br., NH , D O exchangeable), 7.49(m, 2H, H4 and H7 benzi-
2
2
median lethal dose (LD50 ¼ 516 ꢃ 5 mg/kg), followed by 10
2
midazle), 7.79(m, 2H, H5 and H6 benzimidazole), 9.11(br., NH, D O
(
(
LD50 ¼ 416.18 ꢃ 8 mg/kg), 11 (LD50 ¼ 248 ꢃ 2 mg/kg), and 12
LD50 ¼ 243 ꢃ 2 mg/kg) and compound 5 (LD50 ¼ 152.23 ꢃ1 mg/kg).
13
exchangeable). C-NMR (270 MHz, DMSO-d
1
6
): 34.54,114.47,126.11,
ꢁ1
31.34, 146.90. IR (cm ): 3492.45(NH benzimidazole), 3288, 3208,
So the coordination of compound 4 and 5 to transition metal ion to
form the complexes 6e13 have positive effect on the reduction of
toxicity with respect to the free ligands 4 and 5.
3157 (NH
2
), 3052-2596 (br., CH (aromatic, aliphatic) and intermo-
lecular H bonded NH), 1624.73(C]N), 1588, 1573 (C]C). MS: m/z
þ þ
20 (M , 0.3%), 147 (M - 2HCl, 100%).
2
3
. Conclusion
4.1.2. Preparation of (11H-benzo[4,5]imidazo[1,2-a][1,4]diazepin-4-
yl)(6-hydroxy-4,7-dimethoxy-benzofuran-5-yl)methanone (3) [8]
A mixture of 4,7-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-
The reaction of 5-oxo-5H-furo[3,2-g]chromene-6-carbalde-
13,14
hydes
1
with (1H-benzimidazol-2-yl)methanamine dihydro-
6-carbaldehyde (1)
(100 mmole), compound 2 (100 mmole)
chloride (2) was reliant on the reaction conditions and the
mechanism of reaction had been suggested.
New potent antiviral benzofuran derivatives 4 and 5 besides,
new potent complexes formed by the coordination of 4 and 5 to
and triethylamine or piperidine (few drops) in methanol (60 mL)
was refluxed for 7 h. The reaction mixture was filtered off and
crystallized from DMF/methanol to give 3. Yield: 83%, m.p.
ꢂ
1
6 3
315e317 C. H-NMR (500 MHz, DMSO-d ): 3.94(s, 3H, CH ), 4.0

3044
S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
(
s, 3H, CH
3
), 7.14(d, 1H, J ¼ 1.5 Hz, H3 benzofuran), 7.47 (m, 2H,
4.1.7. (2-(1H-benzo[d]imidazol-2-yl)-4-(6-hydroxy-4,7-
dimethoxybenzofuran-5-carbonyl)-1H-pyrrol-1-yl)copper(II)
0
0
H9 and H10 benzimidazodiazepine), 7.56(d, 1H, J ¼ 1.5 Hz, H2
0
0
0
0
1
benzofuran), 7.72(m, 4H, H2 , H6 , H8 and H11 benzimidazo-
chloride hexahydrate;[Cu(HL )(H
2
O)
4
]Cl$2$H
2
O complex (6)
0
ꢁ1
diazepine), 7.91 (s, 1H, H4 benzimidazodiazepine), 9.23(br., NH,
D
n
(
Pale green solid. Yield: 71%. IR (KBr,
O) 3432br&s, (C]O)1639s,
(CeO) 1261 m, FT-IR(KBr,
(CueCl) 327 m. Molar conductance Lm (
n
/cm ):
n
(OH, NH and
(C]C) 1535 m,
(CueO) 490 m, (CueN) 385 m,
2
O exchangeable), 12.75(s, 1H, OH, D
2
n
O exchangeable), IR (KBr,
(CH, aromatic) 3064,
(intermolecular H bonded OH and NH)
H
2
n
n(C]N)1607s,
n
n
ꢁ
1
ꢁ1
/cm ):
n
(OH) 3503,
n
(NH) 3415,
n
n
/cm ):
n
n
ꢁ
1
2
ꢁ1
CH, aliphatic) 2970,
164-2655s,
n
n
U
cm mol ) ¼ 75. TG:
ꢂ
3
n
(C]O) 1641s,
n
(C]N) 1608 m,
n
(C]C) 1540 m.
showed weight loss of 5.87% in temperature range 25e100 C, calc.
þ
1
MS: m/z 403 (M , 45%). 220 (m
for C22
H, 4.19; N, 10.30.
1
, 100%), 183 (m , 93%), Anal. Calcd
2
for 2(H
2
O), 5.92% corresponding to dehydration process [Cu(HL )
1
H
17
N
3
O
5
: C, 65.50; H, 4.25; N, 10.42; Found: C, 65.40;
(H O) ]Cl.2.H
2
4
2
O / [Cu(HL ) (H
2
O)
4
]Cl. Also, TG: found weight loss
ꢂ
of 11.54% in temperature range 100.5e204 C, calcd. for 4(H
2
O),
1
1.84% corresponding to dehydration process of coordinated water
1
1
4.1.3. Preparation of compounds 4 and 5
2 4 3 11
[Cu(HL )(H O) ]Cl / [Cu(HL )]Cl. Anal. Calcd for C22H28ClCuN O ;
General Procedure: A mixture of 4,7-dimethoxy-5-oxo-5H-furo
C, 43.35; H, 4.63; Cl, 5.82; Cu, 10.43; N, 6.89. Found; C, 43.47;
H, 4.86; N, 6.75.
[
3,2-g]chromene-6-carbaldehyde (1) (100 mmole), potassium
hydroxide (100 mmole) in methanol (80 mL) and compound 2 or
-amino-2,6-dihydropyrimidine (100 mmole) was refluxed. After
4
4.1.8. (2-(1H-benzo[d]imidazol-2-yl)-4-(6-hydroxy-4,7-
the reaction completion, the reaction mixture was filtered off and
crystallized from DMF/methanol to give 4 and 5, respectively.
dimethoxybenzofuran-5-carbonyl)-1H-pyrrol-1-yl)iron(III) chloride
1
tetrahydrate; [Fe(HL )Cl
2
(H
2
O)
2
]2$H
2
O complex (7)
ꢁ
Dark brown solid. Yield: 78%. IR (KBr,
O) 3571e3159s., (C]O)1642s, (C]N)1581s,
(FeeO) 670 m, (FeeCl) 590 m. Molar conductance Lm
n
/cm 1):
n(OH, NH and
4
4
.1.4. (5-(1H-benzo[d]imidazol-2-yl)-1H-pyrrol-3-yl)(6-hydroxy-
,7-dimethoxybenzofuran-5-yl)methanone (4)
H
2
n
n
n(CeO) 1266s, n
n
Yield: 73%, m.p. 285-287 C. 1H-NMR (500 MHz, DMSO- d
ꢂ
):
O
(U
ꢁ1
cm mol ) ¼ 23. TG: found weight loss of 5.81% in tempera-
2
ꢁ1
6
ꢂ
3
.91(s, 3H, CH
3
), 3.97(s, 3H, CH
3
), 5.90(br, NH pyrrol, D
2
ture range 25e100 C, calc. for 2(H
2
O), 6.0% corresponding to
1
1
exchangeable), 7.17(d, 1H, J ¼ 1.5 Hz, H3 benzofuran), 7.50(m, 2H,
dehydration
Cl $(H O)
range 100.5e204 C, calcd. for 2(H
process
2 2 2 2
[Fe(HL )Cl .(H O) ]$2H O / [Fe(HL )
0
0
00
00
H5 and H6 benzimidazole), 7.63(s,1H, H4 pyrrol), 7.75(m, 3H, H2
pyrrol, H4 and H7 benzimidazole), 7.94 (d, 1H, J ¼ 1.5 Hz, H2
benzofuran), 9.29(br, NH benzimidazole D O exchangeable), 13.02
O exchangeable). C-NMR(500 MHz, DMSO- d ):
), 61.47 (OCH ), 106.04, 112.18, 114.08, 115.91, 118.03,
18.24, 125.91, 129.142, 129.20, 131.28, 132.17, 142.99, 144.36, 144.81,
2
2
2
]. Also, TG: showed weight loss of 6.21% in temperature
ꢂ
2
O), 6.0% corresponding process
1
2
to dehydration of coordinated water [Fe(HL )Cl
2
$(H
2 2
O) ] / [Fe
13
1
(
6
1
s, 1H, OH, D
1.03 (OCH
2
6
(HL )Cl ]. Anal. Calcd for C22H24Cl FeN O : C, 43.95; H, 4.02; Cl,
2 2 3 9
3
3
11.79; Fe, 9.29; N, 6.99. Found: C, 43.81; H, 4.23; Cl, 11.99; Fe, 9.03;
N, 6.74.
ꢁ
1
1
45.36, 148.76(C]N), 188.37(C]O). IR (
NH) 3415br., (NH) 3230, (CH, aromatic) 3064 m,
(intermolecular H-bonded OH and NH) 3200e2655,
(C]N) 1628 m, (C]C) 1539 m, (CeO) 1263s . MS: m/z
, 5.64%), 220(m , 100%), 183 (m , 98%), Anal.
: C, 65.50; H, 4.25; N, 10.42; Found: C, 65.37
n
/cm ):
n
(OH)3520br.,
(CH, aliphatic)
(C]O)
n
(
n
n
n
4.1.9. (2-(1H-benzo[d]imidazol-2-yl)-4-(6-hydroxy-4,7-
2
970,
641s,
n
n
dimethoxybenzofuran-5-carbonyl)-1H-pyrrol-1-yl)zinc(II) chloride
1
1
4
n
n
n
hydrate; [Zn(HL )Cl H
2
O] complex (8)
þ
Greenish yellow solid. Yield: 76%. 1H-NMR (500 MHz, DMSO-
03(M , 6.5%), 372(m
Calcd for C22
H, 4.17; N, 10.48.
1
2
3
17
H N
3
O
5
d
6
): 3.89(s, 3H, CH
3
), 3.97(s, 3H, CH
3
), 7.17(m, 4H, H3 benzofuran,
0
0
00
H5 and H6 benzimidazole, H4 pyrrol), 7.20(m, 1H, H7 benz-
0
imidazole), 7.44(m, 1H, H4 benzimidazole), 7.91 (d, 1H, J ¼ 1.5 Hz,
00
4.1.5. 3-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-6H-
H2 benzofuran), 8.17(s, 1H, H2 pyrrol), 12.59(br, NH benzimidazole
13
pyrimido[1,6-a]pyrimidine-6,8(7H)-dione (5)
D
2
O exchangeable), 12.69(s, 1H, OH, D
2
O exchangeable). C-NMR
), 106.12, 112.28,
ꢂ
1
Yield: 88%. 290e292 C. H-NMR (270 MHz, DMSO- d
6
): 3.85
(500 MHz, DMSO-d ): 61.1 (OCH ), 61.45 (OCH
6
3
3
(
s, 3H, OCH
3
), 3.93(s, 3H, OCH
3
), 6.71(s, 1H, NH, D
2
O exchangeable),
114.15, 115.81, 118.03, 118.24, 125.79, 129.14, 129.45, 131.01, 132.43,
7
.13(d, 1H, J ¼ 2 Hz, H3 benzofuran), 7.96(d, 1H, J ¼ 2 Hz, H2
142.01, 144.35, 144.89, 145.37(Ar-C), 155.76(C]N), 188.37(C]O). IR
0
0
ꢁ1
benzofuran), 8.15(s, 1H, H4 pyrimidopyrimidinone), 8.23(s, 1H, H9
pyrimidopyrimidinone), 8.81(s, 1H, H2 pyrimidopyrimidinone),
(KBr,
n
/cm ):
n
(OH, NH and H
2
O) 3201br
n
(CH, aromatic) 3128 m,
(C]N) 1615s, (C]C)
(ZneN) 465 m,
0
n(CH, aliphatic) 2978 m,
n(C]O) 1641 s,
n
n
ꢁ
1
9
.89 (s, 1H, OH,
D
2
O
exchangeable), 12.16(s, 1H, OH,
exchangeable). C-NMR(270 MHz, DMSO- d ): 61.31, 60.83, 105.56,
08.20, 109.69, 110.87, 111.45, 112.12, 114.47, 127.12, 128.39, 136.50,
37.76, 143.23, 144.01, 144.63, 144.92, 145.68, 148.86(C]O),153.56,
57.15, 158.23(C]N), 165.44 (C]O), 191.23 (C]O, ketone). (KBr,
D
2
O
1529 m, (CeO) 1265s. FT-IR (KBr,
(ZneCl) 390 m. Molar conductance Lm (
showed no weight loss in temperature range 25e100 C, whereas,
n
n
/cm ):
n
n
13
ꢁ1
2
ꢁ1
6
U
cm mol ) ¼ 16. TG:
ꢂ
1
1
1
TG: found weight loss of 3.69% weight in temperature range
ꢂ
100.5e204 C calcd. for (H
2
O), 3.46% corresponding to dehydration
ꢁ1
1
1
n
/cm ):
aliphatic) 2992 m,
(C]N) 1605s, (C]C) 1542 m,
00%). Anal. Calcd for C18
Found: C, 56.21; H, 3.88; N, 10.89.
n(OH) 3577,
n(NH) 3414br.,
n(CH, aromatic) 3164 m,
n(CH
process
18ClN
Found: C, 50.42; H, 3.51; Cl, 6.69; N, 8.35; Zn, 12.32.
[Zn(HL )Cl.H
2
O] / [Zn(HL )Cl].
Anal.
Calcd
for
n
(C]O) 1725s,
n
(C]O) 1662s,
n
(C]O) 1640s,
C
22
H
3 6
O Zn: C, 50.69; H, 3.48; Cl, 6.80; N, 8.06; Zn, 12.55.
þ
n
1
n
n
(CeO) 1213s. MS: m/z 383 (M ,
13 3 7
H N O : C, 56.40; H, 3.42; N, 10.96.
4.1.10. Bis(2-(1H-benzo[d]imidazol-2-yl)-4-(6-hydroxy-4,7-
dimethoxybenzofuran-5-carbonyl)-1H-pyrrol-1-yl)lanthanum(III)
1
4
.1.6. Preparation of the metal complexes (6e13)
General procedure: A solution of the metal ion in ethanol was
chloride pentahydrate [La(HL )
2
(H
2
O)
2
]Cl.3.H
2
O complex (9)
1
Pale green solid. Yield: 71%. H-NMR (500 MHz, DMSO- d
6
): 3.90
0
added to a solution of the corresponding ligand in ethanol to form
:1 or 1:2 M: L complexes. The reaction mixture was heated under
(s, 3H, CH
3
), 3.95(s, 3H, CH
3
), 7.17(m, 4H, H3 benzofuran, H5 and
0
00
1
H6 benzimidazole, H4 pyrrol), 7.22(m, 1H, H7 benzimidazole),
reflux temperature, after reaction completion. The resulting
precipitates were filtered off, washed with diethyl ether followed
by hot ethanol. Then, recrystallized from methanol/DMF. All of the
7.39(m, 1H, H4 benzimidazole), 7.91 (d, 1H, J ¼ 1.5 Hz, H2 benzo-
0
0
2
furan), 8.15(s, 1H, H2 pyrrol), 11.90(br, NH benzimidazole D O
13
exchangeable), 13.07(s, 1H, OH, D
2
O exchangeable). C-NMR
), 106.5, 112.21,
ꢂ
complexes decomposed without melting with m.p. >300 C.
(200 MHz, DMSO-d ): 61.1 (OCH ), 61.47 (OCH
6
3
3

S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
3045
1
14.13, 115.91, 118.03, 118.31, 126.12, 128.87, 129.20, 131.31, 132.21,
found no weight loss of coordinated water in temperature range
ꢂ
143.11, 144.44, 145.02, 145.48, 152.81(C]N), 188.41 (C]O). IR (KBr,
3 6
100.5e204 C. Anal. Calcd. for C36H30Cl CoN O16: C, 44.67; H, 3.12;
ꢁ
1
n
1
3
/cm ):
579s, (CeO)1261s, FT-IR (KBr,
90 m. Molar conductance Lm (
n
(OH, NH and H
2
O) 3503-3165s,
n
(C]O) 1641s,
n
n
(C]N)
(LaeCl)
Cl,10.99; Co, 6.09; N, 8.68. Found: C, 44.32; H, 3.08; Cl,10.71; Co, 6.32;
N, 8.39.
ꢁ1
n
n
/cm ):
n
(LaeN) 470 m,
ꢁ
1
2
ꢁ1
U
cm mol ) ¼ 68. TG: found
ꢂ
weight loss of 5.23% in temperature range 25e100 C, calcd. for 3
4.1.14. (3-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-8-
(
(
H
HL )
2
O), 5.05% corresponding to dehydration process [La
oxo-8H-pyrimido[1,6-a]pyrimidin-6-yloxy)lanthanum(III) chloride
1
1
2
2
(H
2
O)
2
]Cl.3.H
2
O / [La(HL )
2
(H
2
O)
2
]Cl. Also, TG: exhibited
hydrate; [La(H
Yield: 66%. H-NMR (270 MHz, DMSO-d
3.96(s, 3H, OCH
H2 benzofuran, H4 pyrimidopyrimidinone, H9 pyrimidopyr-
imidinone), 8.42(s, 1H, H2 pyrimidopyrimidinone), 10.35 (s, 1H,
2
L )
2
]3Cl$H
2
O complex (13)
ꢂ
1
weight loss of 3.57% in temperature range 100.5e204 C, calc. for 2
(
6 3
): 3.85(s, 3H, OCH ),
H
2
O), 3.37% corresponding to dehydration of coordinated water
3
), 7.17(d, 1H, J ¼ 2 Hz, H3 benzofuran), 7.95(m, 3H,
1
1
0
0
process [La(HL )
42ClLaN 15: C, 49.43; H, 3.96; Cl, 3.32; La, 12.99; N, 7.86;.
Found: C, 49.71; H, 3.81; Cl, 3.22; La, 12.78; N, 7.98.
2
(H
2
O)
2
]Cl / [La(HL )
2
]Cl. Anal. Calcd. for
0
C
44
H
6
O
NH, D
2
O exchangeable), 12.16(s, 1H, OH, D
2
O exchangeable).
13
C-NMR(270 MHz, DMSO-d ): 61.26, 60.83, 105.55, 108.11, 109.70,
6
4.1.11. (3-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-8-
110.98, 111.41, 112.09, 114.42, 127.08, 128.41, 136.52, 137.79, 143.21,
oxo-8H-pyrimido[1,6-a]pyrimidin-6-yloxy)copper(II) chloride
143.98, 144.65, 144.99, 145.63, 146.46(C]O), 153.44, 157.29, 158.57
2
ꢁ1
trihydrate [Cu(HL )Cl(H
Yield: 75%. IR (KBr,
691vs, (C]O)1641s,
214s. FT-IR(KBr,
2
O)
/cm ):
(C]N) 1609 m,
2
]$H
2
O complex(10)
(C]N), 161.98(C]O), 191.37(C]O). IR (KBr,
O) 3362br., (NH) 3160b,
aliphatic), (C]O, amide) 1738s,
1639s, (C]N) 1607s, (CeO)1213s,
Molar conductance Lm (
n
/cm ):
n
(OH, NH and
ꢁ
1
n
n
(OH, H
2
O) 3396br&s,
n
(C]O)
(CeO)
(CueN) 395 m,
H
2
n
n
(CH, aromatic)3026 m, 2936 (CH,
(C]O, amide) 1672s, (C]O)
(LaeO) 650 m, (La-N) 475 m.
1
1
n
n
n
(CeO) 1274 m,
n
n
n
n
n
ꢁ
1
n
/cm ):
n
(CueO) 485 m,
n
n
n
n
n
ꢁ
1
2
ꢁ1
ꢁ1
2
ꢁ1
(
CueCl) 325 m. Molar conductance Lm (
U
cm mol ) ¼ 28. TG:
U
cm mol ) ¼ 85. TG: found weight loss
ꢂ
ꢂ
found weight loss of 3.43% in temperature range 25e100 C, calcd.
of 1.91% in temperature range 25e100 C, calcd. for 2(H
2
O), 1.75%
2
2
for 1(H
2
O), 3.37% corresponding to dehydration process [Cu(HL )Cl
2 2 2
corresponding to dehydration process [La(H L ) ]3Cl$H O / [La
2
2
(
H
2
2
O) ]$H
2
O / [Cu(HL )Cl(H
2
O)
2
]. Also, TG: found weight loss of
(H
2
L )
2
]3Cl. Also, TG: found no loss of coordinated water in
ꢂ
ꢂ
6
.77% in temperature range 100.5e204 C, calc. for 2(H
2
O), 6.74%
temperature range 100.5e204 C. Anal. Calc. for C36
H28Cl
3
LaN
6
O
15
:
corresponding to dehydration of coordinated water process
C, 41.98; H, 2.74; Cl, 10.33; La, 13.49; N, 8.16. Found: C, 42.08;
H, 2.67; Cl, 10.64; La, 13.56; N, 8.25.
2
2
2 2 3 10
[Cu(HL )Cl(H O) ] / [Cu(HL )Cl]. Anal. Calc. for C18H18ClCuN O :
C, 40.38; H, 3.39; Cl, 6.62; Cu, 11.87; N, 7.85. Found: C, 40.03;
H, 3.28; Cl, 6.52; Cu, 11.69; N, 7.51.
4.2. Biological assays
4
.1.12. (3-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-8-
4.2.1. HIV inhibitory activity and reverse transcriptase inhibition
with therapeutic windows
4.2.1.1. Materials and methods
4.2.1.1.1. Cells and viruses. CEM-SS cells, laboratory-derived
virus isolates (including drug-resistant virus isolates), and low-
passage clinical virus isolates used in these evaluations were
previously described in detail [25]. These cells were maintained in
RPMI 1640 medium supplemented with 10% fetal bovine serum,
oxo-8H-pyrimido[1,6-a]pyrimidin-6-yloxy)iron(III) chloride
2
pentahydrate; [Fe(H
Yield: 76%. IR (KBr,
409e3208br, (C]O) 1709s,
N) 1606s, (CeO) 1213s. FT-IR(KBr,
80, (FeeCl) 397. Molar conductance Lm (
2
L )Cl$(H
/cm ):
(C]O) 1655s,
2
O)
2
]$2Cl$3H
2
O (11)
(OH) 3509br,
(C]O) 1641s,
(FeeO) 660, (FeeN)
ꢁ
1
n
n
n
(NH,
H
(C]
2
O)
3
n
n
n
n
ꢁ
1
n
n
/cm ):
n
n
ꢁ
1
2
ꢁ1
4
n
U
cm mol ) ¼ 67. TG:
ꢂ
found weight loss of 8.21% in temperature range 25e100 C, calc.
2
for 3(H
Cl$(H O)
weight loss of 5.41% in temperature range 100.5e204 C, calc. for 2
O), 5.68% corresponding to dehydration process of coordinated
2
O), 8.52% corresponding to dehydration process [Fe(H
2
L )
2 mM glutamine, penicillin (100 U/mL), and streptomycin (100 mg/
mL). Fresh human cells were obtained from the American Red Cross
(Baltimore, Md.).
2
2
2
]$2Cl$3H
2
O / [Fe(H
2
L )Cl$(H
2
O)
2
]$2Cl. Also, TG: found
ꢂ
(
H
2
4.2.1.1.2. Antiviral and cross-resistance assays. The inhibitory
activities of the tested compounds against HIV were evaluated by
microtiter anti-HIV assays with CEM-SS cells or fresh human
peripheral blood mononuclear cells (PBMCs); these assays quantify
the ability of a compound to inhibit HIV-induced cell killing or HIV
replication. Quantification was performed by the tetrazolium dye
XTT assay, which is metabolized to a colored formazan product by
viable cells. Antiviral and toxicity data were reported as the quan-
tity of drug required to inhibit virus-induced cell killing or virus
production by 50% (EC50).
4.2.1.1.3. Reverse transcriptase (RT) enzyme inhibition assays.
Each of the newly synthesized compounds was tested for RT
inhibitory activity against purified recombinant HIV-1 RT using the
cell-free Quan-T-RT assay system (Amersham Corp., Arlington
Heights, IL), which utilizes the scintillation proximity assay (SPA)
principle [26,27]. In the assay, a DNA/RNA template is bound to SPA
beads via a biotin/streptavidin linkage. The primer DNA is a 16-
meroligo (T), which has been annealed to a poly(rA) template. The
2
2
water [Fe(H
l3FeN
Found: C, 33.93; H, 3.33; Cl, 16.65; Fe, 8.91; N, 6.36.
2
L )Cl$(H
2 2 2
O) ]$2Cl / [Fe(H L )Cl]$2Cl. Anal. Calcd. for
C
H
18 23
C
3
O
12: C, 34.01; H, 3.65; Cl, 16.73; Fe, 8.79; N, 6.61.
4.1.13. Bis-(6-hydroxy-4,7-dimethoxybenzofuran-5-carbonyl)-8-
oxo-8H-pyrimido[1,6-a]pyrimidin-6-yloxy)coblt(III) chloride
2
dihydrate [Co(H
2
L )
2
]3Cl$ 2$H
Yield: 85%. H-NMR (270 MHz, DMSO-d
s, 3H, OCH
2
O (12)
1
6 3
): 3.85(s, 3H, OCH ), 3.93
(
3
), 7.18(d,1H, J ¼ 2 Hz, H3 benzofuran), 7.96(d,1H, J ¼ 2 Hz,
0
H2 benzofuran), 8.30(s, 1H, H4 pyrimidopyrimidinone), 8.93(s, 1H,
H9 pyrimidopyrimidinone), 9.56(s, 1H, H2 pyrimidopyrimidinone),
0
1
1.67 (s, 1H, NH, D
2
O exchangeable), 12.16(s, 1H, OH, D
able). C-NMR(270 MHz, DMSO-d ): 61.24 (OCH ), 60.83(OCH
05.53, 108.11, 109.65, 110.78, 111.38, 112.12, 114.34, 127.17, 128.27,
35.92, 136.69, 143.15, 143.82, 144.31, 144.53, 145.08, 144.96 (C]O),
53.56, 157.15, 158.23(C]N), 161.26 (C]O), 191.37(C]O). IR (KBr,
2
O exchange-
13
6
3
3
),
1
1
1
n/
ꢁ
1
cm ):
3
amide) 1682s,
n
(OH, and H
2
O) 3397br&s,
n
n
(NH)3164br&s,
n
(CH, aromatic)
(C]O, amide) 1734s, (C]O,
(CeO) 1210s. FT-IR(KBr,
3
061 m, 2931.36 (CH, aliphatic),
n
primer-template is bound to a streptavidin-coated SPA bead. [ H]
0
n
(C]O)1641s, 1610
n
(C]N)s,
n
TTP (thymidine 5 triphosphate) is incorporated into the primer by
ꢁ
1
3
n/cm ):
n
(CoeO) 475,
n(CoeN) 370. Molar conductance Lm
reverse transcription. In brief, [ H]TTP, at a final concentration of
ꢁ1
2
ꢁ1
(U
cm mol ) ¼ 79. TG: found weight loss of 3.58% in temperature
0.5
pH 8.0, 80
0.05% Nonidet P-40) and added to annealed DNA/RNA bound to SPA
mCi/sample, was diluted in RT assay buffer (49.5
mM TriseHCl,
ꢂ
range 25e100 C, calc. for 2(H
tion process [Co(H L ) ]$3Cl$2$H O / [Co(H L ) ]3Cl. Also, TG:
2 2 2 2 2
2
O), 3.73% corresponding to dehydra-
m
M KCl, 10 M MgCl , 10 M dithiothreitol, 2.5
m
2
m
mM EGTA,
2
2

3046
S.A. Galal et al. / European Journal of Medicinal Chemistry 45 (2010) 3035e3046
beads. The compound being tested was added to the reaction
doses, based on the findings of phase 1, the mice were again
monitored for 24 h. The geographic mean of the least dose that
killed mice and the highest dose that did not kill mice was taken as
the median lethal dose.
mixture at 0.001e100
recombinant HIV RT and incubation at 37 C for 1 h resulted in the
m
M concentrations. Addition of 10
m
U of
ꢂ
3
extension of the primer by incorporation of [ H]TTP. The reaction
was stopped by addition of 0.2 mL of 120 mM EDTA. The samples
were counted in an open window using a Beckman LS 7600
instrument and IC50[RT] values (concentration at which the
compound inhibits recombinant RT by 50%) were calculated by
comparing the measurements to untreated sample.
Acknowledgements
The authors express their deep thanks to “Sanofi-Aventis (Paris,
FR)” for the evaluation of: the HIV & HIV RT inhibitory activity, the
HCV NS3-4A protease inhibitor activity and the median lethal dose
of the synthesized compounds and we also thank Dr. Mohamed M.
Abdalah.
4.3. Hepatitis C virus (HCV) NS3-4A protease inhibitory activity
4
4
.3.1. Materials and methods
.3.1.1. Cells. Parental Huh-7 and HepG2 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-
inactivated fetal bovine serum (FBS), 2 -glutamine, and
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ꢂ
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[
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[
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3
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_
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