Substituted thieno[2,3-b]thiophenes and related congeners: Synthesis, β-glucuronidase inhibition activity, crystal structure, and POM analyses

Article abstract of DOI:10.1016/j.bmc.2014.08.014

A series of 15 novel compounds incorporating the thieno[2,3-b]thiophene moiety were synthesized. The chemical structures of these compounds were deduced from elemental analyses, ¹H NMR, ¹³C NMR, and ESI-mass spectral data. The enzyme

Full text of DOI:10.1016/j.bmc.2014.08.014

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Substituted thieno[2,3-b]thiophenes and related congeners:
Synthesis, b-glucuronidase inhibition activity, crystal structure,
and POM analyses
Yahia Nasser Mabkhot a, , , Assem Barakat a,b, , , Sammer Yousuf c, , M. Iqbal Choudhary a,c,
,
⇑
⇑
Wolfgang Frey d, Taibi Ben Hadda e, , Mohammad S. Mubarak f
a Department of Chemistry, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
b Department of Chemistry, Faculty of Science, Alexandria University, PO Box 426, Ibrahimia, Alexandria 21321, Egypt
c H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d Institut für Organische Chemie, Universitat Stuttgart, Pfaffenwaldring 55, Stuttgart 70569, Germany
e Laboratory of Chemical Material, FSO, University Mohammed Premier, Oujda 60000, Morocco
f Department of Chemistry, The University of Jordan, Amman 11942, Jordan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 15 novel compounds incorporating the thieno[2,3-b]thiophene moiety were synthesized. The
chemical structures of these compounds were deduced from elemental analyses, 1H NMR, 13C NMR, and
ESI-mass spectral data. The enzyme inhibition potential of these compounds was evaluated, in vitro,
against b-glucuronidase, xanthine oxidase, and a-chymotrypsin enzymes. The cytotoxicity was evaluated
by a cell viability assay utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Received 2 June 2014
Revised 4 August 2014
Accepted 12 August 2014
Available online xxxx
(MTT) dye. Among the compounds tested, compound 3 was the most potent b-glucuronidase inhibitor
Keywords:
Thienothiophene
b-Glucuronidase inhibition
with an IC50 value of 0.9 0.0138
1,4-lactone (IC50 = 45.75 2.16 M). Compound 12, on the other hand, was the most potent as a xanthine
oxidase inhibitor with an IC50 of 14.4 1.2 M. With the characterization of their mechanism of action
lM; it was much more active than the standard, D-saccharic acid
l
l
a-Chymotrypsin inhibition
and with further testing, these compounds could be useful candidates as anticancer drugs. In addition,
the newly synthesized compounds were subjected to POM analyses to get insights about their degree
of their toxicity.
Cytotoxicity
Cancer cell lines
Petra/Osiris/Molinspiration (POM) analyses
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
their reactivity and biological properties, thienothiophenes have
drawn considerable attention from organic and medicinal chem-
One cannot overlook the significance of synthesizing and test-
ing new compounds for potential therapeutic purposes, especially
given the fact that the majority of drugs in use today came as a
direct outcome of this process.1 Undoubtedly, cancer ranks high
among human diseases which is still in dire need of effective ther-
apy.2 Lack of a wide array of anticancer drugs able to capitalize on
new discoveries regarding tumor genesis, coupled with the unique
growth patterns of the diverse repertoires of cancer, makes all
approaches including the shotgun ones the more acceptable.3
Due to their structural and therapeutic diversity, thienothioph-
ene derivatives have attracted much synthetic interest. Because of
ists. They have been studied as potential antitumor, antiviral,4–10
antibacterial, and anti-glaucoma drugs, and as inhibitors of platelet
aggregation.11–15 Examples include compounds shown in Figure 1.
Mashraqui et al.5 described the first application of thieno
[2,3-b]thiophene in the design of a novel NLO (non liner optical
properties) system by incorporating this nucleus within an
unsymmetrically functionalized cyclophane. On the other hand,
thienothiophene building blocks were used in the synthesis of
two series of HIV protease inhibitors, possessing
a-hydroxyamin-
opentanamide transition state isoester. These two series were
found to be effective at low concentrations in inhibiting the spread
of AIDS virus.16 In addition, Egbertson and his colleagues have
reported the synthesis and pharmacology of
a
potent
⇑
thienothiophene non-peptide fibrinogen receptor antagonist.17
Thienothiophenes also have potential applications in a wide vari-
ety of optical and electronic systems.18–21 In 2010, Paek et al.22
have designed and synthesized six organic sensitizers containing
Corresponding authors. Tel.: +966 1467 5898; fax: +966 1467 5992 (Y.N.M.);
tel.: +966 1467 5884; fax: +966 1467 5992 (A.B.).
E-mail addresses: Yahia@ksu.edu.sa (Y.N. Mabkhot), ambarakat@ksu.edu.sa
(A. Barakat).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.08.014
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mabkhot, Y. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.014

2
Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
O
O
OH
NH
S
O
S
S
RN
HN
CO2H
NH
O
N
O
O
H
S
O
N
O
R
O
OH
Ph
OH
Inhibitors of Platelet Aggregation
N
O
R
NH
N
S
NH
N
S
Ph
Me
NH2
H2N
N
N
R
N
X
R
X
S
S
O
S
S
S
S
Antitumor
HIV -1 Protease
HIV -1 Protease
inhibitors
α-Glucusidase
Inhibitors
inhibitors
H3C
O
O
O
OH
HO
O
CH3
OH
O
H
OH
O
O
N
O
O
O
O
S
NH
NH
NH
NH
O
H
O
OH
O
CH3
OH
H3C
NH2
HN
N
NH2
H2N
Doxorubicin
Zonisamide
Chymostatin
R1
R2
OH
O
O
NH
NH
O
O
S
S
N
HO
HO
Z
Z
R1, R2 = Alkyl, Aryl
N
OH
Z= Functionnalised group
Saccharic acid 1-4
lactone
Allopurinol
2-16
Figure 1. Structures of some bioactive compounds, containing thieno[2,3-b]thiophenyl moiety.
3,4-ethylenedioxythiophene and thienothiophene.22 These com-
pounds have shown high efficiency and excellent stability.22–24
Recently, Mabkhot et al. have reported for the first time the anti-
2. Results and discussion
2.1. Chemistry
cancer, anti-oxidant, b-glucuronidase and a-glucosidase inhibitory
activities of thieno[2,3-b]thiophene derivatives.25,26
Syntheses of compounds 2–16 were carried out via the routes
shown in Schemes 1–3. The key intermediate 1,10-(3-methyl-4-
phenylthieno[2,3-b]thiophene-2,5-diyl)diethanone (1), required
in this study, was prepared from commercially available starting
materials according to the procedure, described by Mabkhot
et al.,27. Condensation of 1 with dimethylformamide dimethyl ace-
tal (DMF-DMA) in refluxing xylene for 10 h afforded intermediate 2
in good yield (73%). Treatment of compound 2 with triethoxyme-
thane yielded compound 3 in a 57% yield. On the other hand, reac-
tion of compound 2 with hydroxylamine hydrochloride, in the
presence of TEA, yielded compound 4 with a 59% yield. Similarly,
compound 5 was obtained in a 77% yield when compound 2 was
In view of the wide interest in the activity of thienothiophenes,
we describe herein the synthesis and characterization of a number
of new substituted thieno[2,3-b]thiophenes (2–16) (Figure 1),
which, to the best of our knowledge have not previously been
described in the literature. In addition, the in vitro xanthine oxidase,
a-chymotrypsin, and b-glucuronidase inhibitory activities of these
newly synthesized compounds were investigated, along with their
cytotoxicity against PC-3 cancer cell lines. Moreover, these new
compounds 2–16 and five standard drugs were subjected to Petra/
Osiris/Molinspiration (POM) analyses to obtain insights on the
degree of their toxicity, where they were found to be non-cytotoxic.
Please cite this article in press as: Mabkhot, Y. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.014

Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Ph Me
3
O
O
Ph
S
Me
S
S
S
CN
NC
EtO
EtO OEt
OEt
OEt
NC
CN
3
EtO
CH(OEt)3
Fusion
7
Me2N
NMe2
57%
-2NHMe2
DMF-DMA
Reflux 4 h
64%
HOHN
Me2N
CN
Me
Me
O
O
O
O
NC
NH2OH.HCl
Dioxan/TEA
-2NHMe2
DMF-DMA,
Xylene,
Ph
Me
Ph
Ph
CH2(CN)2
EtOH, Reflux 4 h
Me
S
S
S
S
S
S
reflux 10 h
Reflux 7 h
59%
Ph
S
S
65%
73%
Me
Me
O
NC
Me
Me
O
CN
6
HOHN
Me2N
77%
1
2
4
AcOH/AcONH4
Reflux 3 h
Ph
Me
Ph
O
Me
S
O
O
S
S
S
N
Me2N
NMe2
5
Scheme 1. Synthesis of compounds 2–7.
Ph
Me
Me2N
NMe2
Ph
Me
S
S
NC
EtO
CN
OEt
NC
CN
S
S
N
N
CONH2
H2NOC
13
Et
12
O
Reflux 1 h, 62%
C
H
)
2
H
/
N
P
ipe
2
C
(
(
CN
2
r
4 h
Na/EtOH,
flu
i
d
)
2
x
CH
i
n
e
EtO
COCH3
EtO2C
O
Re
N
H3C
N
52%
Ph
O
N
N
ph
O
O
EtO
N
H
Me
Ph
S
S
Ph
Cl
H3C
CH3
S
S
2
Ph-H/TEA,
Reflux 8 h
62%
AcOH/AcONH4
Reflux 3 h
72%
Me
O
Ph
N
N
N
8
EtO2C
H3C
COCH3
11
N+ Cl-
N
CN
NH
Ph
O
O
N
Ph
Me
N
N
O
O
CH2(CN)2
Me
S
S
EtOH, Reflux 6 h
63%
CHO
S
S
OHC
Ph
N
N
Ph
PhHN
NHPh
N
9
NH
CN
10
Scheme 2. Synthesis of compounds 8–13.
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4
Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Ph
Me
NMR spectra of all synthesized compounds were in total agree-
ment with the proposed structures. In addition, mass spectral data
of the synthesized compounds were also in total agreement with
the assigned structures and show the expected molecular ions,
M+, as suggested by their molecular formulas.
NC
CN
S
S
NH
HN
O
O
15
2.2. Description of crystal structure of 2
Yellow colored needle-like single crystals of 2, suitable for X-ray
diffraction, were grown from DMF/EtOH using the slow evapo-
ration technique. Diffraction intensity data were collected with
COOH
CN
O
H2NOC
O
O
NH
N
NMe2
Ph
a
Bruker Kappa APEXII Duo single crystal diffractometer,
Method A: NaNO2/HCl
Me
Ph
using Mo K monochromatic radiation (k = 0.71073 Å) at low tem-
perature (100°K). Crystal data collection and structure refinement
data of 1,10-(3-phenyl-4-methylthieno[2,3-b]thiene-2,5-diyl)bis-
[3-(dimethylamino)prop-2-en-1-one (2) are listed in Table 1.
A multi-scan type of absorption correction was applied to data sets.
The structure was resolved by direct methods using the solution
program SHELXS9729 in the SHELXTL-plus package, and refined
by a full-matrix least-squares procedure on F2 using SHELXS97.
Additional data pertaining to compound 2 are available at the
Cambridge Crystallographic Data Centre (CCDC 994879). All non-
hydrogen atoms were refined, first with isotropic and then with
anisotropic parameters. Hydrogen atoms bonded to carbon were
included using a riding model, starting from calculated positions.
In the geometry of aromatic substituted 2,5-bis-armed-thie-
no[2,3-b]thiophene ring, the C2 and C5 are the nodal atoms. The
molecular structure shows a planar thieno[2,3-b]thiophene geom-
etry. The phenyl ring corresponding to C4 being slightly twisted
with respect to that moiety and the C3–C4–C8–C13 dihedral angle
is ꢀ74.62 degree. In the crystal, there was no sign of intermolecular
and intramolecular hydrogen bonding. The final atomic coordi-
nates for all atoms and a complete listing of bond distance and
angles are given in Tables 2 and 3. An ORTEP-drawing of 2 with
the atomic numbering scheme is presented in Figure 2, while
NaOAc/Dioxane
S
S
S
Reflux 4 h, 72%
Cl-
S
Me
N
+
N
Method B:
N
NH
NMe2
Dioxane, 0->5°C
68%
O
COOH
H2NOC
CN
13
14
Ph
Me
HOOC
COOH
S
S
NH
HN
O
O
16
Scheme 3. Synthesis of compounds 14–16.
treated with AcOH, under reflux for 3 h, in the presence of AcONH4.
Furthermore, when 1 was subjected to condensation reaction with
malononitrile in ethanol, the corresponding dimalononitrile
derivative 6 was obtained which upon treatment with DMF-DMA
in ethanol under reflux condition for 4 h, afforded bis-enamine
derivative 7 in a 64% yield (Scheme 1).
Table 1
Crystal and experimental data for compound 2
Treatment of compound 2 with ethyl-2-(2-arylhydrazono)-2-
chloroacetate in benzene, under refluxed for 8 h, in the presence
of TEA afforded diethyl-4,40-(3-methyl-4-phenylthieno[2,3-b]
thiophene-2,5-dicarbonyl)bis(1-phenyl-1H-pyrazole-3-carboxylate)
(8) in a 62% yield (Scheme 2). Reaction of 2 with benzenediazonium
chloride afforded 9 which upon treatment with malononitrile in
refluxing ethanol gave the bis(pyridazines) derivative 10, in a 63%
yield (Scheme 2). Similarly, reaction of 2 with acetoacetone in
refluxing AcOH and in the presenceof ammonium acetate (NH4OAc),
and with malononitrile in refluxing ethanol and in the presence of
sodium ethoxide yielded the new bis-(pyridine) derivative 11 and
the bis-(heterocycle) 12, respectively, in good to moderate yields.
In addition, the key intermediate 8, used for the synthesis of a wide
variety of bis-(pyridazinones), and bis-(pyridinones), was obtained
when enaminone 1 was treated with malononitrile in ethanolic
piperidine under reflux for 1 h in 62% yield (Scheme 2).
The bis(pyridazinone) derivatives 14 was obtained from the bis-
dienamide 13,28 by either reacting 13 with NaNO2/HCl in refluxing
dioxane in the presence of NaOAc for 4 h (in a 72% yield) or by
coupling of 13 with phenyldiazonium chloride (in a 68% yield), as
shown in Scheme 3; compound 13 in turn was prepared from
the key intermediate 2, as shown in Scheme 2. Similarly, the nico-
tinic nitrile derivative 15 and the nicotinic acid derivative 16 were
obtained, respectively, by heating 13, under reflux, in AcOH and by
boiling 13 in EtOH/HCl as depicted in Scheme 3.
Parameter
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system, space group
Unit cell dimensions
C23 H24 N2 O2 S2
424.56
100(2) K
0.71073 Å
Monoclinic, C2/c
a = 25.370(2) Å
b = 17.627(2) Å
c = 10.9674(11) Å
a
= 90°
b = 108.028(4)°
c
= 90°
Volume
4663.8(8) Å3
Z, calculated density
Absorption coefficient
F(000)
Crystal size
h Range for data collection
Limiting indices
8, 1.209 Mg/m3
0.248 mmꢀ1
1792
0.94 ꢁ 0.55 ꢁ 0.39 mm
2.20–30.55°
ꢀ36 6 h 6 35, ꢀ25 6 k 6 22, ꢀ12 6 l 6 15
6457/7110 [R(int) = 0.0356]
=30.55 99.4%
Reflections collected/unique
Completeness to h
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on
Final R indices [I > 2sigma(I)]
R indices (all data)
Extinction coefficient
Largest diff. peak and hole
Semi-empirical from equivalents
0.7461 and 0.6823
Full-matrix least-squares on F2
7110/0/268
F2 1.030
R1 = 0.0392, wR2 = 0.1104
R1 = 0.0467, wR2 = 0.1154
0.0030(3)
0.444 and ꢀ0.329 e Åꢀ3 (⁄)
The newly synthesized compounds were characterized by
elemental analyses, MS and NMR spectral data. 1H NMR and 13C
(⁄) Remark: disordered solvent electron density squeezed by PLATON.
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Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 2
(IC50 = 121 1.32
l
M) showed a promising xanthine oxidase inhib-
Selected bond lengths (in angstrom) of 1,10-(3-phenyl-4-methylthieno [2,3-b]thiene-
2,5-diyl)bis[3-(dimethylamino)prop-2-en-1-one (2)
itory activity, when compared with the standard, allopurinol
(IC50 = 14.4 1.2 M). The potent activity of compound 12 may
l
A–B
Distance
A–B
Distance
A–B
Distance
be due to the presence of bis-2-ethoxynicotinonitrile rings,
attached to the central thieno[2,3-b]thiophene backbone. Com-
pounds 9 and 10, on the other hand, were identified as moderate
cytotoxic agents (IC50 = 13.85 0.53 and 2.60 0.19
tively) against PC-3 cell lines in comparison with the standard drug
doxorubicin (IC50 = 0.912 0.12 M). Compounds 2–9 and 11–16
S1–C1
S1–C6
O1–C14
N1–C16
S2–C6
S2–C5
1.758(1)
1.706(1)
1.243(1)
1.334(2)
1.714(1)
1.751(1)
O2–C19
N2–C21
C1–C2
C2–C3
C3–C4
C3–C6
1.252(1)
1.326(2)
1.378(2)
1.439(2)
1.441(2)
1.395(2)
C4–C5
1.378(2)
1.489(2)
1.489(2)
1.367(2)
1.424(2)
1.440(2)
C1–C14
C5–C19
C15–C16
C19–C20
C14–C15
lM, respec-
l
were found to be non-cytotoxic with >30% growth inhibition of
PC-3 cancer cell lines.
Table 3
2.4. POM virtual screenings and molecular properties
calculations
Selected bond angles (in degrees) of 1,10-(3-phenyl-4-methylthieno[2,3-b]thiene-2,5-
diyl)bis[3-(dimethylamino)prop-2-en-1-one (2)
A–B–C
Angle
A–B–C
Angle
A–B–C
Angle
We have employed Petra/Osiris/Molinspiration (POM) analyses
to investigate the potential pharmacophores of various heterocy-
clic compounds for their antimicrobial bioactivities.30–38 Based
on the structures, in the series 2–16, bearing carbonyl or oxo
groups and two-fused bicyclic system, the compounds contain
two potential O–S pharmacophoric sites and this is shown in
Figure 1.
S1–C1–C14
S2–C5–C19
S1–C6–S2
C1–C2–C3
C3–C4–C5
117.98(9)
113.78(9)
133.04(8)
111.1(1)
S1–C1–C2
S2–C5–C4
S2–C6–C3
S1–C6–C3
C1–S1–C6
112.75(9)
113.18(9)
113.97(9)
112.93(9)
90.80(6)
C5–S2–C6
C4–C3–C6
C2–C3–C6
C5–C19–O2
C1–C14–O1
90.06(6)
111.2(1)
112.4(1)
116.6(1)
119.5(1)
111.6(13
packing presentation of compound 2 is shown in Figure 3. The sin-
gle-crystal X-ray diffraction analysis reveals that compound 2 pre-
sents three coordinative pockets which together can furnish
trimetallic organometallic system, as depicted in Figure 4.
2.4.1. Molinspiration calculations
The method is very robust and able to process practically all
organic and most organometallic molecules. Molecular Polar Sur-
face Area (PSA) is calculated as a sum of fragment contributions;33
O- and N-centered polar fragments are considered. PSA has been
shown to be a very good descriptor, characterizing drug absorp-
tion, including intestinal absorption, bioavailability, Caco-2 perme-
ability and blood-brain barrier penetration. Prediction results of
compounds 2–16 molecular properties are shown in Table 5. Lipo-
philicity (clogP value) and polar surface area (PSA) values are two
important properties for the prediction of per oral bioavailability of
drug molecules.32–40 Therefore, clogP and PSA values for com-
pounds 2–16 were calculated using molinspiration and Osiris soft-
wares. For all investigated compounds, with the exception of six
compounds 3, 5, 8, 9, 11 and 12, the calculated clogP values were
around ꢀ0.22 to 4.7 (<5), which is the upper limit for the drugs to
be able to penetrate through bio-membranes according to Lipin-
ski’s rule. Therefore, these compounds (2, 4, 6, 7, 10, 13, 14, 15
and 16) are expected to present good bioavailability. The lowest
degree of lipophilicity was exhibited for compounds 2, 4, 6, 7, 10,
13, 14, 15 and 16 which is an indication of good water solubility.
The polar surface area (PSA) is calculated from the surface areas
that are occupied by oxygen and nitrogen atoms and by hydrogen
atoms attached to them. Thus, the PSA is closely related to the
2.3. Biological activity evaluation
Compounds 2–16 were evaluated for biological activities
through different in vitro biochemical assays, including cytotoxic
activity against PC-3 cell lines, b-glucuronidase, xanthine oxidase,
and
sented in Table 4. Among a series of 15 compounds 2–16, com-
pound (IC50 = 0.9 0.0138 M) displayed remarkable b-
glucuronidase inhibitory activity, several fold more active than
the standard -saccharic acid 1,4-lactone (IC50 = 45.75 2.16 M).
a-chymotrypsin enzyme inhibition assays; results are pre-
3
l
a
D
l
All other compounds were found to be inactive. The activity of
compound 3 may be due to the presence of the triethoxymethyl
substituents, attached to the prop-2-en-1-one side chain of the
central thieno[2,3-b]thiophene moiety. In addition, bis(3-(dimeth-
ylamino)prop-2-ene-1-yl-1-ylidene)dimalononitrile moiety-con-
taining compound 7 (IC50 = 202.4 3.4
lM) was found to be a
moderate inhibitor of -chymotrypsin enzyme. This may be attrib-
a
uted to the presence of bis(3-(dimethylamino)prop-2-ene-1-yl-
1-ylidene)dimalononitrile substituent. Similarly, compounds 12
(IC50 = 14.4 1.2
lM),
13
(IC50 = 43.1 0.9
l
M),
and
14
Figure 2. An ORTEP diagram of final X-ray structure of 1,10-(3-phenyl-4-methylthieno[2,3-b]thiene-2,5-diyl)bis[3-(dimethylamino)prop-2-en-1-one (2).
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6
Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 3. Crystal packing presentation of compound 2 showing Z = 8.
OH2
O
H2O
O
M1
M2
M3
O
O
O
O
S
S
S
S
H3C
N
CH3
N
CH3
H3C
N
CH3
N
CH3
CH3
CH3
H3C
H3C
Potential Bioactive
Trimettalic Compound
of ligend 2
Figure 4. Potential of trimetallic building of ligand 2.
Table 4
Bioactivity (IC50 in lM) of compounds 3–16 against various disease targets
Compd Cytotoxicity assay (PC-3 cancer cell
line)
Carbonic anhydrase
Inhibition
a
-Chymotrypsin
Xanthine oxidase
inhibition
b-Glucuronidase
inhibition
inhibition
2
3
>30
>30
NA
NA
NA
NA
NA
—
NA
0.9 0.0138
4
5
6
7
8
9
10
11
12
13
14
15
16
SD
>30
>30
>30
>30
—
NA
—
NA
NA
202.4 3.4
NA
NA
—
ppt
NA
ppt
NA
ppt
—
—
NA
—
—
NA
—
—
—
—
—
—
NA
—
—
—
—
—
—
—
—
441.72 8.10
—
—
NA
—
NA
NA
NA
>30
13.85 0.53
2.607 0.193
>30
>30
>30
>30
>30
>30
Doxorubicin
0.912 0.12
14.4 1.2
43.1 0.9
121.00 1.32
NA
NA
Allopurinol
2.0 0.01
NA
270.13 2.96
NA
Zonisamide
1.86 0.03
NA
Chymostatin
5.7 0.1
D-Saccharic acid
1-4 lactone 45.75 2.16
SEM = standard error of mean; NA = not active; failed to show 50% or more inhibition at 500 lM or more. SD: standard drugs.
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Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Table 5
POM calculations of compounds 2–16
Compd
Molinspiration calculationsa
Osiris calculationsb
TUM
TPSA
ONH
VIOL
VOL
CLP
2.58
MUT
IRRIT
RE
2
41
90
99
0
0
4
0
0
0
2
4
2
0
0
4
4
2
4
7
2
8
2
4
0
2
0
2
1
2
2
2
1
1
2
1
2
1
1
3
0
3
0
0
382
577
331
657
353
466
537
504
562
436
450
493
441
382
402
459
165
562
109
148
3
6.65
1.11
7.69
4.65
3.65
5.17
6.59
3.96
6.18
6.69
2.71
ꢀ0.22
3.08
4
5
58
95
6
7
102
144
158
165
60
8
9
10
11
12
13
14
15
16
SD-1
SD-2
SD-3
SD-4
SD-5
92
140
183
113
140
206
68
204
74
124
2.04
0.17
0.44
ꢀ0.01
ꢀ3.05
ꢀ3.24
SD-1: Doxorubicin; SD-2: Zonisamide; SD-3: Chymostatin; SD-4: Allopurinol; SD-5:
D
-Saccharic acid 1-4 lactone.
a
TPSA: Total of Polar surface area; ONH : OH—N or O—HN interaction; VOL: volume.
b
: not toxic;
: slightly toxic;
: highly toxic.
hydrogen bonding potential of a compound.30–38 Molecules with
PSA values around 160 Å or more are expected to exhibit poor intes-
tinal absorption. Results shown in Table 5 reveal that all tested
compounds, except 10 and 14, are within this limit. cLogP and
PSA values are important, although not sufficient criteria, for pre-
dicting oral absorption of a drug. To support this inference, all com-
pounds, except 3, 5, 7, 8, 9, 12, and 14 have zero or one violation of
the Rule of 5.39 Two or more violations of the Rule of 5 suggest the
probability of problems in bioavailability of standard drugs.30–39
These properties, mainly hydrophobicity, electronic distribution,
hydrogen bonding characteristics, molecule size and flexibility
and presence of various pharmacophoric features, influence the
behavior of molecule in a living organism, including bioavailability,
transport properties, affinity to proteins, reactivity, toxicity, meta-
bolic stability and many others. Results for all compounds are dis-
played in Table 5 by means of numerical assignment. Likewise, all
compounds have consistent negative values in all categories and
numerical values conforming and comparable to that of standard
drugs SD-1–6 (SD-1: Doxorubicin; SD-2: Zonisamide; SD-3: Chy-
with no reproductive effects when run through the mutagenicity
assessment system in comparison with the standard drugs. Low
hydrophilicities and therefore, high clogP values of most in vitro
active compound 3 may cause poor in vivo absorption or perme-
ation.39 It has been shown that for compounds to have a reasonable
probability of good absorption, their clogP values must not be
greater than 5.0. On this basis, the majority of compounds 2–16
were found to possess clogP values in the acceptable range.
3. Conclusions
In summary, the present investigation describes an efficient
method for the synthesis of substituted thieno[2,3-b]thiophenes,
many of which may display interesting biological activities. The
prepared compounds were tested in vitro for their inhibition
potential against b-glucuronidase, xanthine oxidase, and a-chymo-
trypsin enzymes by using mechanism-based biochemical assay.
Among a series of 15 tested derivatives, compound 3 displayed
remarkable b-glucuronidase inhibitory activity with an IC50 value
of 0.9 0.0138
-saccharic acid 1-4-lactone (IC50 = 45.75 2.16
12, on the other hand, was the most potent as a xanthine oxidase
inhibitor with an IC50 of 14.4 1.2 M. POM analyses were per-
lM; it was more active than the standard
mostatin; SD-4: Allopurinol; SD-5: D-Saccharic acid 1-4 lactone).
Therefore, it is readily seen that all of the tested compounds are
expected to have similar activity to the standard drugs.
D
l
M). Compound
l
formed on the newly synthesized compounds. Based on these
observations, we believe that this series deserves to be explored
as new tris-bidentate ligands that may lead to more efficient and
selective drugs.40
2.4.2. Osiris calculations
The remarkably well-behaved mutagenicity of divers synthetic
molecules, classified in the database of Celeron Company of Swiss,
can be used to quantify the role played by various organic groups
in promoting or interfering with the way a drug can associate with
DNA. The Osiris calculations pertaining to the newly synthesized
compounds are presented in Table 5. Toxicity risks (mutagenicity,
tumorogenicity, irritation, reproduction) of compounds 2–16 were
calculated by the methodology developed by Osiris. The toxicity
risk predictor locates fragments within a molecule, which indicate
a potential toxicity risk. Toxicity risk alerts are an indication that
the drawn structure may be harmful concerning the risk category
specified. From the data displayed in Table 5, it is obvious that a
majority of structures (10/15) are non-mutagenic, non-irritating
4. Experimental section
4.1. Materials and equipments
All chemicals used throughout this work were obtained from
commercial sources and were used as received without further
purification. Melting points (uncorrected) were measured with a
Gallenkamp melting point apparatus. Infrared (IR) spectra were
recorded as KBr discs on a Perkin Elmer FT-IR 1000 spectrometer.
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8
Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
1H and 13C NMR spectra were obtained with the aid of a Varian
Mercury Jeol-400 NMR spectrometer and are reported in ppm (d),
relative to TMS as an internal standard and with DMSO-d6 as sol-
vent. Chemical shifts are expressed in d units; coupling constants
(J-values) for 1H–1H coupling are given in Hertz. Abbreviations for
multiplicity are as follows: s (singlet), d (doublet), t (triplet), q
(quadruplet), and m (multiplet). Mass spectra were recorded on a
Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 eV. Elemen-
tal analyses were carried out on an Elementar Vario EL analyzer.
1640 (C@O) cmꢀ1 1H NMR (400 MHz, DMSO-d6) (ppm): 1.96 (s,
;
3H, CH3), 2.0 (s, 3H, CH3), 3.00 (s, 12H, CH3), 4.55 (d, 1H,
J = 12.0 Hz, CH), 5.43 (d, 1H, J = 12.0 Hz, CH), 7.5–7.7 (m, 5H,
C6H5), 7.98–8.10 (m, 3H, C5H3N),; 13C NMR (100 MHz, DMSO-d6)
(ppm):d 14.2, 22.3, 44.0, 129.3, 129.8, 130.4, 131.5, 133.3, 142.0,
182.41, 183.5; MS m/z (%): 758 [M+, 1.2%]; Anal. Calcd for C42H35-
N3O3S4: C, 66.55; H, 4.65; N, 5.54; O, 6.33; S, 16.92. Found: C,
66.58; H, 4.60; N, 5.58; S, 16.89.
4.2.5. 2,20-(1,10-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(ethan-1-yl-1-ylidene)) dimalononitrile (6)
4.2. Synthesis of compounds 2–16
A mixture of compound 1 (314 mg, 1 mmol) with malononitrile
(132 mg, 2 mmol) in absolute ethanol (15 mL) was heated under
reflux for 4 h. The solid product was collected by filtration to afford
4.2.1. 1,10-(3-Phenyl-4-methylthieno[2,3-b]thiene-2,5-
diyl)bis[3-(dimethylamino)prop-2-en-1-one (2)
A mixture of compound 1 (1.75 g, 5 mmol), DMF-DMA (1.19 mL,
0.01 mol) was refluxed in m-xylene (15 mL) for 10 h. After cooling,
the solid product was collected by filtration as dark yellow crystals.
6 as deep red crystals. Yield (65%); mp >320 °C; IR mmax (KBr): 2191
(CN) cmꢀ1; 1H NMR (400 MHz, DMSO-d6) (ppm): 1.63 (s, 6H, CH3),
1.84 (s, 3H, CH3), 7.29–7.55 (m, 5H, Ar-H); 13C NMR (100 MHz,
DMSO-d6) (ppm): d 178.6, 137.0, 136.5, 135.2, 132.1, 129.0,
128.1, 125.4, 112.4, 45.5, 22.2; MS m/z (%): 410 [M+, 2%]; Anal.
Calcd for C23H14N4S2: C, 67.29; H, 3.44; N, 13.65; S, 15.62. Found:
C, 67.12; H, 3.49; N, 13.68; S, 15.59.
Yield: 1.55 g, 3.75 mmol, 73%; mp 250 °C; IR (
mmax): 1622 (C@O)
cmꢀ1 1H NMR ä (ppm): 1.96 (s, 3H, CH3), 2.99 (s, 12H, CH3), 4.53
;
(d, 1H, J = 12.0 Hz, CH), 5.38 (d, 1H, J = 12.0 Hz, CH), 7.41–7.65
(m, 5H, C6H5); 13C NMR d (ppm): 14.9 (CH3), 44.79 (AN@(CH3)2),
109.8 (ACOACH@), 153.9 (@CHAN), 180 (C@O); MS m/z (%): 424
[M]+ (57%), 380 (51), 336 (18), 309 (18), 98 (100); Anal. Calcd for
4.2.6. 2,20-1,10-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(3-(dimethylamino)prop-2-ene-1-yl-1-
ylidene)dimalononitrile (7)
C23H24N2O2S2: C, 65.06; H, 5.70; N, 6.60; S, 15.10. Found: C,
65.10; H, 5.68; N, 6.63; S, 15.07.
A mixture of compound 6 (205 mg, 0.5 mmol) with DMF-DMA
(1 mmol, 77 lL) in absolute ethanol (15 mL) was heated under
reflux for 4 h. The formed solid product was collected by filtration
4.2.2. 1,10-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(4,4,4-triethoxybut-2-en-1-one), (3)
Compound 3 was prepared by fusion of the enaminone deriva-
to afford 7 as deep brown powder. Yield (64%); mp >320 °C; IR mmax
tive
2
(212 mg, 0.5 mmol) with triethylorthoformate (TEOF)
(KBr): 2193 (CN) cmꢀ1 1H NMR (400 MHz, DMSO-d6) (ppm): 1.83
;
(148 mg, 1 mmol). Ethanol was added and the solid product was
(s, 3H, CH3), 3.24–3.13 (s, 12H, CH3), 7.9–8.10 (d, 1H, J = 12.0 Hz,
a
filtered off to afford 3 as pale red crystals. Yield: 57%; mp 315–
CH), 7.4–7.6 (m, 5H, Ar-H), 8.15 (d, 1H, J = 12.0 Hz, bCH); 13C
317 °C; IR mmax (KBr): 1641 (C@O) cmꢀ1
;
1H NMR (400 MHz,
NMR (100 MHz, DMSO-d6) (ppm): d 179.7, 140.6, 137.0, 135.0,
134.2, 129.1, 128.4, 115.8, 100.8, 72.5, 44.7, 13.9; MS m/z (%):
520 [M+, 5%]; Anal. Calcd for C29H24N6S2: C, 66.90; H, 4.65; N,
16.14; S, 12.32. Found: C, 66.85; H, 4.72; N, 16.17; S, 12,39.
DMSO-d6) (ppm): d 1.05 (t, 3H, J = 6.6 Hz, CH3), 1.96 (s, 3H, CH3),
a
(q, 2H, J = 8.0 Hz, CH2), 5.69 (d, 1H, J = 12.0 Hz, CH), 6.53 (d, 1H,
J = 12.0 Hz, bCH), (m, 5H, Ar-H); 13C NMR (100 MHz, DMSO-d6)
(ppm): d 14.2, 22.3, 44.0, 129.3, 129.8, 130.4, 131.5, 133.3, 142.0,
182.41; MS m/z (%): 630 [M+, 35%]; Anal. Calcd for C33H42O8S2: C,
62.83; H, 6.71; O, 20.21; S, 10.17. Found: C, 62.80; H, 6.75; S, 20.18.
4.2.7. Diethyl-4,40-(3-Methyl-4-phenylthieno[2,3-b]thiophene-
2,5-dicarbonyl)bis(1-phenyl-1H-pyrazole-3-carboxylate) (8)
To a solution of 1 (0.5 mmol, 0.21 g) in dry benzene (20 mL),
ethyl 2-(2-arylhydrazono)-2-chloro acetate (2 equiv, 1 mmol) was
added in the presence of a few drops of triethylamine. The reaction
mixture was then heated under reflux for 8 h. After cooling to room
temperature, the solid product was collected by filtration and
recrystallized from EtOH to give pale brown crystals. Yield (62%);
4.2.3. 1,10-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(3-(hydroxyamino)prop-2-en-1-one) (4)
A mixture of compound 2 (212 mg, 0.5 mmol) and hydroxyl-
amine hydrochloride (NH2OHꢂHCl) (70 mg, 1 mmol) in dioxane
(15 mL) was refluxed for 7 h in the presence of triethylamine
(TEA). The reaction mixture was left to cool to room temperature.
The formed solid product was filtered off, washed with ethanol,
dried, and recrystallized from EtOH to afford the corresponding
hydroxylamine derivative 4 as white crystals. Yield (59%); mp
mp 206–208 °C; IR (m
max): 1716–1732 (C@O), 1627 (C@N) cmꢀ1;1H
NMR (400 MHz, DMSO-d6): 1.30 (t, 3H, J = 6.9 Hz, CH3, ester), 1.96
(s, 3H, CH3), 4.25 (q, 2H, J = 16.6 Hz, CH2, ester), 7.36–7.76 (m, 15H,
Ar-H), 10.6 (s, 1H, pyrazolo-H); 13C NMR (100 MHz, DMSO-d6) d
(ppm): d 14.5, 21.7, 66.2, 168.3, 192.1; MS m/z (%): 714 [M+, 1%],
240 (74), 166 (100), 98 (72); Anal. Calcd for C39H30N4O6S2: C,
65.53; H, 4.23; N, 7.84; S, 8.97. Found: C, 65.57; H, 4.21; N, 7.82;
S, 9.
165–166 °C; IR mmax (KBr): 1653 (C@O), 3420 (OH) cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6) (ppm): d 2.2 (br s, 2H, OH & NH),1.96
(s, 3H, CH3), 4.53 (d, 1H, J = 12.0 Hz, CH), 5.38 (d, 1H, J = 12.0 Hz,
CH), (m, 5H, C6H5); 13C NMR (100 MHz, DMSO-d6) (ppm): d 180,
153.9, 109.8, 44.79, 14.9; MS m/z (%): 400 [M+, 15%]; Anal. Calcd
for C19H16N2O4S2: C, 56.98; H, 4.03; N, 7.00; O, 15.98; S, 16.01.
Found: C, 56.91;H, 4.09; N, 7.02; S, 16.04.
4.2.8. 3,30-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(3-oxo-2-(2-phenylhydrazono)propanal) (9)
To a stirred cold solution (0–5 °C) of 1 (0.5 mmol, 0.21 g) in
dioxane (15 mL) was added, drop-wise over a period of 20 min, a
solution of the freshly prepared benzenediazonium chloride
[Freshly prepared by diazotization of aniline (1 mmol) in HCl
(0.28 mL) with NaNO2 (2 mmol) in H2O (4 mL)] and the reaction
mixture was kept in a refrigerator overnight. The solid product
was collected by filtration and recrystallized from EtOH to give
the title compound 9 as yellow crystals. Yield (87%); mp 176–
4.2.4. 3-(Dimethylamino)-1-(5-(6-(5-((E)-3-
(dimethylamino)acryloyl)-3-methyl-4-phenylthieno[2,3-
b]thiophen-2-yl)nicotinoyl)-3-methyl-4-phenylthieno[2,3-
b]thiophen-2-yl)prop-2-en-1-one (5)
A mixture of compound 2 (212 mg, 0.5 mmol) with glacial
acetic acid (15 mL) was refluxed for 3 h in the presence of ammo-
nium acetate. The solid product formed was filtered, washed with
ethanol, dried, and recrystallized from DMF/EtOH to afford 5 as
deep yellow crystals. Yield (77%); mp >330 °C; IR mmax (KBr):
178 °C; IR (
mmax): 1591 (C@N), 1627–1639 (C@O), 3435 (NH)
cmꢀ1 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.08 (s, 3H, CH3),
;
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Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
7.19–7.61 (m, 15H, Ar-H), 7.69 (s, 1H, N–H), 8.66 (s, 1H, CHO); 13
C
(400 MHz, DMSO-d6) d (ppm): 1.95 (s, 3H, CH3), 2.99–3.00 (s,
12H, N-CH3), 5.9 (d, 1H, J = 12.8 Hz, CH), 7.2 (d, 1H, J = 12.8 Hz,
CH), 7.32–7.90 (m, 5H, Ar-H), 8.2 (s, 2H, NH2); 13C NMR
(100 MHz, DMSO-d6) d (ppm): d 180.04, 176.68, 160.58, 149.18,
148.6, 133.6, 129.98, 128.80, 128.68, 119.2, 103, 79.1, 43.2, 14.8;
MS m/z (%): 556 [M+, 1.2], 191 (6.1), 149 (100), 73 (34); Anal. Calcd
for C29H28N6O2S2: C, 62.57; H, 5.07; N, 15.10; S, 11.52. Found: C,
62.60; H, 5.04; N, 15.08; S, 11.48.
NMR (100 MHz, DMSO-d6) d (ppm):14.1, 122.5, 128.6, 129.3, 130.0,
130.34, 130.54, 136.37, 145.8, 147.2, 187.1, 188.9. MS m/z (%):578
[M+, 45%]; Anal. Calcd for C31H22N4O4S2: C, 64.34; H, 3.83; N, 9.68;
O, 11.06; S, 11.08. Found: C, 64.37; H, 3.80; N, 9.63; S, 11.14.
4.2.9. 6-(5-(5-Cyano-6-imino-1,6-dihydropyridazine-3-
carbonyl)-3-methyl-4-phenylthieno [2,3-b]thiophene-2-
carbonyl)-3-imino-2,3-dihydropyridazine-4-carbonitrile (10)
To a stirred solution of 1 (0.5 mmol, 0.21 g) in EtOH (10 mL),
malononitrile (1 mmol, 0.07 g) was added and the resulting reac-
tion mixture was heated under reflux for 6 h. After cooling, the
solid product was collected, by filtration, and recrystallized from
EtOH; compound 10 was obtained as yellow crystals. Yield (63%);
4.2.13. 6,60-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
dicarbonyl)bis(3-oxo-2,3-dihydropyridazine-4-carboxylic acid)
(14)
Procedure A: To a solution of compound 13 (0.5 mmol, 0.278 g)
in a mixture of dioxane (15 mL) and HCl (2 mL), a solution of
NaNO2 (0.069 g, 1 mmol) and sodium acetate (1.5 mmol) in water
(6 mL) was added drop-wise. The reaction mixture was heated
under reflux for 4 h. After cooling to room temperature, the solid
product was collected by filtration and recrystallized from dioxane
to afford the desired product as brown crystals in a 72% yield.
Procedure B: To a cold (0–5 °C, in an ice bath) stirred solution of
compound 13 (0.5 mmol, 0.28 g) in dioxane (15 mL),a solution of
the benzenediazonium chloride [freshly prepared by diazotizing
the respective aniline (1 mmol) in hydrochloric acid (0.28 mL) with
sodium nitrite solution (2 mmol) in (4 mL) water] was added drop
wise over a 20 min period and the mixture was left in a refrigerator
overnight. The solid product was collected by filtration and recrys-
tallized from EtOH to afford 14 as brown crystals. Yield (72%a,
mp >320–322 °C; IR (
mmax): 1593 (C@N), 1627 (C@O), 2187 (CN),
3305 (NH) cmꢀ1 1H NMR (400 MHz, DMSO-d6) d (ppm): 2.01 (s,
;
3H, CH3), 7.23–7.63 (m, 15H, Ar-H), 7.89 (s, 1H, CH, pyridyl), 8.16
(s, 1H, N–H); 13C NMR (100 MHz, DMSO-d6) d (ppm): 14.1, 108.0,
116.0, 122.4, 128.4, 129.4, 130.0, 130.4, 130.5, 136.4, 145.8,
147.2, 154.2, 187.3; MS m/z (%): 674 [M+, 45%]; Anal. Calcd for C37-
H22N8O2S2: C, 65.86; H, 3.29; N, 16.61; O, 4.74; S, 9.50. Found: C,
65.84; H, 3.29; N, 16.63; S, 9.50.
4.2.10. 1,10-(6,60-(3-Methyl-4-phenylthieno[2,3-b]thiophene-
2,5-diyl)bis(2-methylpyridine-6,3-diyl))diethanone (11)
To a solution of 1 (0.5 mmol, 0.21 g) in glacial acetic acid
(10 mL), acetyl acetone (2 equiv, 1 mmol) was added in the pres-
ence of catalytic amount of ammonium acetate. The reaction mix-
ture was heated under reflux for 3 h. The solid product was
collected by filtration, and recrystallized from EtOH to afford the
title compound as dark yellow crystals. Yield (72%); mp 241–
68%b); mp >320–322 °C; IR
(mmax): 1579 (C@N), 1666–1643
(C@O), 3444 (OH), 3444 (NH) cmꢀ1;1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.08 (s, 3H, CH3), 6.68 (s, 1H, NH) 7.42–7.80 (m, 5H, Ar-
H), 8.09 (s, 1H, CH), 8.35 (s, 1H, OH); 13C NMR (100 MHz, DMSO-
d6) d (ppm): 14.9, 125.7, 128.9, 130.0, 131.6, 149.8, 152.2, 163.8,
178.2, 191.6; MS m/z (%): 562 [M+, 45%]; Anal. Calcd for
243 °C; IR (m ;
max): 1682 (C@O), 1624 (C@N) cmꢀ1 1H NMR
(400 MHz, DMSO-d6) d (ppm): 1.96 (s, 3H, CH3), 2.51 (s, 3H,
COCH3), 2.62 (s, 3H, CH3, pyridyl), 7.29–7.59 (m, 5H, C6H5), 7.95
(d, 1H, J = 6.6 Hz, CH, pyridyl), 8.25 (d, 1H, J = 8.0 Hz, CH, pyridyl);
13C NMR (100 MHz, DMSO-d6) d (ppm): 14.5, 29.4, 30.5, 129.2,
129.5, 129.9, 134.8, 138.8, 141.8, 147.7, 151.2, 166.1, 194.1; MS
m/z (%): 496 [M+, 3%]; Anal. Calcd for C29H24N2O2S2: C, 70.13; H,
4.87; N, 5.64; S, 12.91. Found: C, 70.18; H, 4.84; N, 5.6; 1; S, 12.96.
C25H14N4O8S2: C, 53.38; H, 2.51; N, 9.96; S, 11.40. Found: C,
53.40; H, 2.50; N, 9.94; S, 11.42.
4.2.14. 6-(5-(5-Cyano-6-oxo-1,6-dihydropyridin-2-yl)-3-methyl-
4-phenylthieno[2,3-b]thiophen-2-yl)-2-oxo-1,2-
dihydropyridine-3-carboxylic acid (15)
A solution of 13 (0.5 mmol, 0.28 g) in a mixture of EtOH/HCl
(3:1, 10 mL) was refluxed for 1 h. The solid product was collected
by filtration and recrystallized from EtOH to give the title com-
4.2.11. 6,60-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(2-ethoxynicotinonitrile) (12)
To a solution of 1 (0.5 mmol, 0.21 g) in ethanol/ sodium ethox-
ide (EtONa0 (Na 0.02 g/EtOH 10 mL), malononitrile (1 mmol,
0.07 g) was added and the resulting reaction mixture was heated
under reflux for 4 h. The solid product was collected by filtration
and compound 12 was obtained as red crystals. Yield (52%); mp
pound as deep red crystals. Yield (67%); mp 287–289 °C; IR (mmax):
1722–1631 (C@O), 3174 (OH), 3450 (NH) cmꢀ1; 1H NMR (400 MHz,
DMSO-d6) d (ppm): 1.95 (s, 3H, CH3), 5.86 (d, 1H, J = 7.5 Hz, CH),
7.02 (s, 1H, NH), 7.14–7.90 (m, 5H, Ar-H), 8.34 (d, 1H, J = 7.5 Hz,
CH), 11.35 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) d (ppm):
14.4, 105.9, 123.7, 128.6, 129.9, 151.4, 162.9, 175.1; MS m/z (%):
485 [M+, 45%]; Anal. Calcd for C25H15N3O4S2: C, 61.84; H, 3.11; N,
8.65; S, 13.21. Found: C, 61.85; H, 3.10; N, 8.67; S, 13.24.
>330–332 °C; IR (m ;
max) : 1573 (C@N), 2200 (CN) cmꢀ1 1H NMR
(400 MHz, DMSO-d6) d (ppm): 1.32 (t, 3H, J = 8.0 Hz, CH3), 1.89
(s, 3H, CH3), 5.15–5.90 (q, 2H, J = 8.0 Hz, CH2), 7.22 (d, 1H,
J = 7.5 Hz, CH), 7.36–7.43 (m, 5H, Ar-H), 8.44 (d, 1H, J = 7.5 Hz,
CH). 13C NMR (100 MHz, DMSO-d6) d (ppm): 14.8, 29.58, 69.92,
103, 115, 119, 128.68, 128.81, 129.98, 133.61, 143.8, 149.1, 176,
180; MS m/z (%): 522 [M+, 12%], 492 (6), 403 (14), 149 (100), 91
(64); Anal. Calcd for C29H22N4O2S2: C, 66.64; H, 4.24; N, 10.72; S,
12.27. Found: C, 66.67; H, 4.23; N, 10.71; S, 12.25.
4.2.15. 6-(5-(5-Cyano-6-oxo-1,6-dihydropyridin-2-yl)-3-methyl-
4-phenylthieno[2,3-b]thiophen-2-yl)-2-oxo-1,2-
dihydropyridine-3-carbonitrile (16)
A solution of 13 (0.5 mmol, 0.28 g) in acetic acid glacial (10 mL)
was refluxed for 30 min. The solid product was collected by filtra-
tion and recrystallized from EtOH to afford the desired product,
4.2.12. 5,50-(3-Methyl-4-phenylthieno[2,3-b]thiophene-2,5-
diyl)bis(2-cyano-5-(dimethylamino)penta-2,4-dienamide) (13)
To a solution of 1 (0.5 mmol, 0.21 g) in EtOH (10 mL), malono-
nitrile (1 mmol, 0.07 g) was added in the presence of catalytic
amount of piperidine and the reaction mixture was heated under
reflux for 1 h. The solid product was collected by filtration to afford
compound 13 as red crystals. Yield (62%); mp 201–203 °C; IR
compound 16, as red crystals. Yield (67%); mp >320 °C; IR (mmax):
1737 (C@O), 2206 (CN), 3390 (C@N), cmꢀ1;1H NMR (400 MHz,
DMSO-d6) d (ppm):1.91 (s, 3H, CH3), 6.70 (d, 1H, J = 8.6 Hz, CH),
7.42–7.90 (m, 5H, Ar-H), 8.13 (d, 1H, J = 8.6 Hz, CH), 11.97 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) d (ppm): 14.46, 106.18,
98.86, 115.20, 129.86, 151.36, 162.89. MS m/z (%): 466 [M+, 45%];
Anal. Calcd for C25H14N4O2S2: C, 64.36; H, 3.02; N, 12.01; S,
13.75. Found: C, 64.38; H, 3.02; N, 12.03; S, 13.75.
(m ;
max): 1616 (C@O), 2193 (CN), 3388 (NH2) cmꢀ1 1H NMR
Please cite this article in press as: Mabkhot, Y. N.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.08.014

10
Y.N. Mabkhot et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.3. Biological activities
4.3.3. In vitro b-glucuronidase inhibition assay
b-Glucuronidase inhibitory activity was determined spectro-
photometrically by measuring the absorbance at 405 nm of p-
Results are presented here as means standard error mean
from triplicate (n = 3) observation. IC50 Values were determined
by using EZ-FIT, enzyme kinetics software obtained from Perrella
Scientific, Inc., USA.
nitrophenol formed from the substrate p-nitrophenyl-b-
nide N1627-250 mg (Sigma Aldrich CO.3050 Spruce Street, St.
Louis, MO 63103, USA). The total reaction volume was 250 L.
The compound was dissolved in DMSO (100%), which became 2%
in the ultimate assay (250 L) and similar conditions were used
for standard ( -Saccharic acid 1,4-lactone, Sigma Aldrich CO.3050
spruce street, St. Louis, MO 63103, USA). The reaction mixture con-
tained 185 L of 0.1 M acetate buffer, 5 L of test compound solu-
tion, 10 L of (1U) enzyme solution (G7396-25KU, Sigma Aldrich)
D-glucuro-
l
4.3.1. Cytotoxicity
l
Cytotoxic activity of the prepared compounds was evaluated by
using standard MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-
tetrazolium bromide) colorimetric assay. PC3 cells (Prostrate
Cancer cells) were cultured in a 75 cm2 flask having Dulbecco’s
Modified Eagle Medium, followed by the addition of 5% FBS (fetal
D
l
l
l
was incubated at 37° C for 30 min. The plates were read on a mul-
bovine serum), 100 IU/mL penicillin and streptomycin (100
lg/
tiplate reader (SpectraMax plus 384) at 405 nm after the addition
mL), and were then incubated at 37 °C in 5% CO2 incubator. The cell
growth was monitored exponentially and counted using a haemo-
cytometer. The diluted cell culture (concn 5 ꢁ 104 cells/mL) was
of 50 lL of 0.4 mM of p-nitrophenyl-b-D-glucuronide. All assays
were performed in triplicate. IC50 Values were calculated by using
EZ-Fit software (Perrella Scientific Inc., Amherst, MA, USA). These
values are the mean of three independent readings.25
then added in a 96-well plate (100 lL/well) and left overnight.
The fresh media was replaced on next day, followed by the
addition of test compounds in concentrations of 5–50 M. After
48 h, 200 L of MTT (0.5 mg/mL) was added to each well, the cells
were allowed to stand for 4 h, and then 100 L of DMSO was
added. The amount of formation of formazan from MTT was calcu-
lated by measuring the absorbance at 540 nm on a micro plate
reader (Spectra Max plus, Molecular Devices, CA, USA). The
cytotoxicity was measured as IC50 for PC3 cells [Mosmann, T.
1983]. The percent inhibition was calculated by using the following
equation:
l
4.3.4. In vitro xanthine oxidase inhibition assay
l
Xanthine oxidase inhibition assay was performed spectrophoto-
metrically. Xanthine oxidase (0.003 units/well) from buttermilk
was dissolved in phosphate buffer (pH 7.4, 50 mM) while test com-
l
pounds were dissolved in DMSO. In a 96-well plate, 10
sample, 20 L of enzyme and 150 L of buffer were added and
incubated for 10 min at 30 °C. After incubation, 20 L of xanthine
lL of test
l
l
l
(0.15 mM) was added and the change in absorbance was
monitored for 30 min at 295 nm; allopurinol was used as positive
% Inhibition ¼ 100 ꢀ ððmean of OD of test compound ꢀ mean of OD of negative controlÞ=ðmean of OD of positive control
ꢀ mean of OD of negative controlÞ ꢁ 100Þ:
The results (% inhibition) were processed by using SoftMax Pro
software (Molecular Devices, USA).
control.
equation:
% inhibition was calculated by using the following
% Inhibition ¼ 100 ꢀ ðOD of test=OD of controlÞ ꢁ 100
4.3.2. In vitro
a-chymotrypsin activity
The -chymotrypsin inhibitory activity was evaluated in 50 mM
tris–HCl buffer pH 7.6 with 10 mM CaCl2 as described by Carnell
a
4.3.5. Statistical analysis
All reactions were performed in triplicate in a final volume of
et al., (1988) with slight modification. A mixture of the enzyme
200 lL. The results were processed using SoftMax Pro 4.8 software
(Molecular Devices, CA, USA) and then using MS Excel. The per-
a
-chymotrypsin (12 Units/mL prepared using the aforementioned
buffer) and the test compound (0.5 mM) dissolved in DMSO (final
centage of inhibition was calculated by the following equation:
concentration 7%) was incubated at 30 °C for 25 min. The reaction
was initiated by adding the substrate, N-succinyl-L-phenylalanine-
% Inhibition ¼ 100ꢀðOD of test sample=OD of the controlÞꢁ100
p-nitroanilide (SPpNA; 0.4 mM prepared using the buffer above).
The change in absorbance due to the release of p-nitroanilide
was continuously monitored at 410 nm. The positive control, pre-
pared without the test compound, and the negative control pre-
pared without the enzyme or with standard inhibitor were run in
parallel. The % of inhibition was calculated using the following
equation:
Acknowledgments
This project was supported by the NSTIP strategic technologies
program, Grant number 13-BIO1382-02, Kingdom of Saudi Arabia.
References and notes
% Inhibition ¼ 100ꢀ½ðOD of Test Compound=OD of Positive ControlÞꢁ100ꢃ
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