Design, Synthesis, and Biological Evaluation of Some Cyclohepta[b]Thiophene and Substituted Pentahydrocycloheptathieno[2,3-d]Pyrimidine Derivatives

Article abstract of DOI:10.1002/jhet.2678

This investigation describes the design of a series of cycloheptathieno[2,3-d]pyrimidines along with their synthetic strategy. The target compounds were screened for their PARP-1 inhibitory activity. The modeling study declared that most of the docked compounds showed the same interactions as 3-aminobenzamide, where Gly 894, His 862, Tyr 896, Arg 878, and Ser 864 were the main residues involved in hydrogen bond formation. Compounds eliciting the top ranked docking results were screened for their PARP-1 inhibitory activity giving promising results, and three representative compounds were tested for their cytotoxic activity using Doxorubicin as a reference standard. The target compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 including cyclohepta[b]thiophene and pentahydrocycloheptathieno[2,3-d]pyrimidine cores were designed, prepared, and tested for their PARP-1 inhibitory activity. Compounds 16 (R: ―NHC(S)NH2) and 11 (R: ―C S) were the most potent ones.

Full text of DOI:10.1002/jhet.2678

Month 2016
Design, Synthesis, and Biological Evaluation of
Some Cyclohepta[b]Thiophene and Substituted
Pentahydrocycloheptathieno[2,3-d]Pyrimidine Derivatives
a
a
a
b
c
*
E. I. El-Mongy, Mohammed A. Khedr, Nageh A. Taleb, Hanem M. Awad, and Safinaz E.-S. Abbas
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Ain Helwan, P.O. Box 11795,
Cairo, Egypt
bDepartment of Tanning Materials and Leather Technology, National Research Centre, P.O. Box 12622, Dokki, Cairo, Egypt
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, P.O. Box 11562,
Cairo, Egypt
*
E-mail: drshimo.mongy@gmail.com
Received April 20, 2015
DOI 10.1002/jhet.2678
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
This investigation describes the design of a series of cycloheptathieno[2,3-d]pyrimidines along with their
synthetic strategy. The target compounds were screened for their PARP-1 inhibitory activity. The modeling
study declared that most of the docked compounds showed the same interactions as 3-aminobenzamide,
where Gly 894, His 862, Tyr 896, Arg 878, and Ser 864 were the main residues involved in hydrogen bond
formation. Compounds eliciting the top ranked docking results were screened for their PARP-1 inhibitory
activity giving promising results, and three representative compounds were tested for their cytotoxic activity
using Doxorubicin as a reference standard. The target compounds 1–17 including cyclohepta[b]thiophene and
pentahydrocycloheptathieno[2,3-d]pyrimidine cores were designed, prepared, and tested for their PARP-1
inhibitory activity. Compounds 16 (R: ―NHC(S)NH2) and 11 (R: ―C¼S) were the most potent ones.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
causing oncogenic chromosomal translocation and eventually
to cancer [7]. An inspection of the available PARP–ligand
complex structures [8] showed that the NAD+ binding pocket
is made up of three parts: a base formed by β-sheet 3 and a
loop connecting to α-helix 3, walls formed by β-sheet 4 and
α-helix 4, and a lid formed by a loop between α-helix 3 and
β-sheet 4 (the D-loop).
Because PARP activation plays a role in DNA repair and
cell survival as well; therefore, PARP inhibition has a cyto-
toxic effect [9]. Accordingly, PARP inhibitors may be used ei-
ther in combination with anticancer drugs or in potentiating
their effects [10] as exemplified by the use of Rucaparib
(AG014699) (Fig. 2a) which is a known PARP inhibitor in
combination with Temozolomide [11] (Fig. 2b). Several
chemical classes of PARP-1 inhibitors have been reported
including the NAD structurally similar compound
3-aminobenzamide [12,13] which is mostly used to investi-
gate both in-vivo and in-vitro effects of PARP inhibition be-
cause of its availability commercially and apparent less
toxicity (Fig. 3a). Different isoquinolinones and
dihydroisoquinolinones were also described as effective
Poly(ADP-ribose)polymerases are a family of enzymes that
uses NAD+ as a substrate. They tend to cause chromatin
decondensation in response to DNA damage so as to enhance
the repair of the damaged DNA binding protein [1]. DNA
damage may be induced by oxidative stress, eroded telomers,
genotoxic medication, irradiation, depurination, reactive nitro-
gen species, alkylating agents, and lipid peroxidation products
[2]. The activated PARP enzymes in response to DNA dam-
age cause cleavage of nicotinamide from ADP-ribose, the lat-
ter is then polymerized on glutamic residues onto the nuclear
acceptor protein, the resulted polymer is used to repair DNA at
the damaged site [3] (Fig. 1). Eventually ADP-ribose poly-
mers are rapidly hydrolyzed by the action of PARG (poly
ADP-ribose glucohyrolase) [4]. However, severe DNA dam-
age may lead to PARP over activation causing ATP depletion
eventually causing several inflammatory pathogenesis [5,6].
In parallel to the repair of DNA damaged protein, activation
of cell cycle check points at the lesion site must be coordinated
otherwise accumulation of DNA damage would display
© 2016 Wiley Periodicals, Inc.

E. I. El-Mongy, M. A. Khedr, N.A. Taleb, H. M. Awad, and S. E.-S. Abbas
Vol 000
Figure 1. PARP enzyme in response to DNA damage.
Figure 2. a: Rucaparib (structure). b: Temozolamide (structure).
Figure 5. MK4827, Olaparib, and Velaparib.
over PARP-2 at R¼H with EC50s of 5.5 and 41μM respec-
tively [17] (Fig. 4a), the thieno[3,4-d]pyrimidine derivative
(Fig. 4b) expressed 93% inhibition of PARP at a dose of
10 μM [18]. Additionally, many PARP-1 inhibitors were de-
clared to be used in clinical trials as: MK4827 [19] (Fig. 5),
Olaparib (Fig. 5)+Dacarbazine [20], and also Velaparib
(Fig. 5)+Cyclophosphamide or Topotecan [21,22].
Figure 3. a: 3-Aminobenzamide. b: Phthalazin-1(2H)-one.
Based on the aforementioned facts, the search for simple
readily accessible compounds that act as PARP inhibitors
prompted our interest to synthesize
a series of
cycloheptathieno[2,3-d]pyrimidines, and screen their in-
vitro PARP-1 inhibitory activity.
RESULTS AND DISCUSSION
Figure 4. Some thienopyrimidines as PARP inhibitors.
Molecular modeling and docking studies.
Molecular
PARP inhibitors [14,15]. Phthalazin-1(2H)-one was cited to
have IC50 of 12μM [16], (Fig. 3b). Moreover, Pfizer
docking study of the thienopyrimidine scaffold with
PARP-1 crystal structure revealed that both Gly 894 and
Tyr 896 residues were involved in both hydrogen bond
and hydrophobic interactions, respectively. The molecular
surface of the active site of PARP-1 (pdb code= 1 UK1)
was determined by using MOE 2012.10 [23] in order to
(Warner-Lambert)
disclosed
5-methoxy-4-methyl-1(2)
phthalizinone with an IC50 of 80nM [14,15].
Furthermore, some thienopyrimidines were reported as se-
lective PARP-1 inhibitors showing selectivity for PARP-1
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Substituted Cycloheptathienopyrimidine Derivatives
Figure 6. Binding site identification of PARP-1 enzyme. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 7. a: Docking of thienopyrimidine scaffold inside the PARP-1 active site. b: Docking of cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-one scaffold
inside PARP active site. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 8. a: Docking of compound 10 inside PARP-1 active site. b: Docking of compound 13 inside PARP-1 active site. c: Docking of compound 16
inside PARP-1 active site. d: Docking of 3-Aminobenzamide inside PARP-1 active site. [Color figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E. I. El-Mongy, M. A. Khedr, N.A. Taleb, H. M. Awad, and S. E.-S. Abbas
Vol 000
Table 1
Docking results of the synthesized compounds.
Compound no.
Affinity by MOE
Residues involved in interactions
Affinity by Leadit
Residues involved in interactions
kcal/mol
using MOE
kcal/mol
using Leadit
1
2
3
4
5
6
7
8
10
11
12
13
15
16
17
ꢀ3.64
ꢀ3.58
ꢀ3.70
ꢀ3.98
ꢀ3.60
ꢀ3.50
ꢀ4.00
ꢀ3.55
ꢀ4.50
ꢀ4.56
ꢀ3.85
ꢀ3.90
ꢀ3.51
ꢀ4.57
ꢀ3.80
ꢀ3.75
Ser 864
Arg 865, Ser 864
Gly 863
ꢀ13.66
ꢀ13.60
ꢀ13.55
ꢀ12.74
ꢀ14.11
ꢀ13.33
ꢀ16.74
ꢀ13.14
ꢀ19.86
ꢀ18.57
ꢀ18.20
ꢀ15.22
ꢀ13.12
ꢀ21.91
ꢀ18.36
ꢀ15.86
Arg 878, Tyr 896, His 862
Gly 863
Arg 878, Tyr 896, Gly 894
Arg 878
Tyr 896
Gly 863
Gly 863, Ala 898
Gly 863
Ala 880, Arg 878, Gly894
Gly 863
Ser 864
Ser 864
Gly 863
Tyr 907, Glu 988
His 862, Ser 864
Ser 864
His 864
Gly 863
Gly 863
His 862
Ser 864
Gly 862
Gly 863
Arg 878, Tyr 896, His 862
Arg 878, Asp 770, Asp 766
Arg 878, Tyr 896, Gly 894
Gly 863, Glu 988
Gly 894, Arg 878, Tyr 896, Ser
864, His 862
3-Amino-
benzamide
Arg 878, Ser 864
Scheme 1. Synthesis of compounds 1 to 7.
Scheme 3. Synthesis of compounds 12 to 17.
get the hydrophobic regions of the site (Fig. 6). By
computing the molecular surface, we were able to
determine the large hydrophobic space in the binding site
that required to be filled. As a result, a cycloheptane ring
was introduced to the thienopyrimidine scaffold, and the
designed structure was then re-docked in the PARP-1
Scheme 2. Synthesis of compounds 8 to 11.
active site. It was
a successful trial where the
hydrophobic space was filled and the main interactions
with the conserved residues were not affected (Fig. 7a,
7b). Derivatives of the cyclohepta[4,5]thieno[2,3-d]
pyrimidine scaffold were subjected to molecular docking
study to predict their affinity against PARP-1 enzyme.
The interactions of the docked compounds were
compared to 3-aminobenzamide. Most of the docked
compounds showed the same interactions of 3-
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Substituted Cycloheptathienopyrimidine Derivatives
Table 2
IC50 values, docking results, and % inhibition of PARP-1 activity of the tested compounds.
Compound no.
Affinity by MOE
Affinity by Leadit
IC50 μM Means SD
% inhibition at min
% inhibition at max
kcal/mol
kcal/mol
(n = 3)
conc. 50 μM
conc. 200 μM
1
2
3
4
5
6
7
8
10
11
12
13
15
16
17
ꢀ3.64
ꢀ3.58
ꢀ3.70
ꢀ3.98
ꢀ3.60
ꢀ3.50
ꢀ4.00
ꢀ3.55
ꢀ4.50
ꢀ4.56
ꢀ3.85
ꢀ3.90
ꢀ3.51
ꢀ4.57
ꢀ3.80
ꢀ13.66
ꢀ13.60
ꢀ13.55
ꢀ12.74
ꢀ14.11
ꢀ13.33
ꢀ16.74
ꢀ13.14
ꢀ19.86
ꢀ18.57
ꢀ18.20
ꢀ15.22
ꢀ13.12
ꢀ21.91
ꢀ18.36
80.007
102.516
86.744
105.126
79.275
92.235
74.379
108.438
64.782
58.206
72.952
75.242
94.934
50.392
73.563
73.399
55.515
73.473
58.228
72.647
56.041
74.214
64.790
69.200
75.304
70.516
67.189
67.648
77.491
79.132
63.644
68.756
58.042
63.803
66.326
74.175
68.231
51.850
79.457
78.903
74.443
75.762
78.454
82.458
60.137
aminobenzamide where Gly 894, His 862, Tyr 896, Arg
878, and Ser 864 were found to be the main residues
involved in hydrogen bond formation (Fig. 8a–d).
The docking score was calculated by MOE 2012.10, and
all affinities were calculated in kcal/mol (Table 1). Accord-
ing to the docking outputs, 15 compounds were selected to
be tested for their PARP-1 inhibitory activity. Another mo-
lecular docking process was accomplished by Leadit 2.1.2
software [24]. The ranking of the docking outputs in both
cases was nearly consistent. Docking with Leadit 2.1.2
software added some binding modes and suggested more
residues that could be a good explanation for the biological
activities.
moiety, and a singlet signal at δ 11.32 ppm equivalent to
NH proton that disappeared upon deuteration. In addition,
13C-NMR spectrum confirmed a signal at δ 60.67 ppm
corresponding to the CH2 carbons of the chloroacetamido
group. Furthermore, mass spectrum disclosed [M+Cl35] at
m/z 325 and [M+Cl37] at m/z 327.
Heating the 3-amino derivative 3 with phenyl isothiocy-
anate in dichloromethane at 70°C on a water bath gave the
thiosemicarbazide derivative 6.1H-NMR spectrum indi-
cated the presence of a multiplet representing the aromatic
protons at δ 7.12–7.45 ppm and two singlet signals at
δ10.83 and δ 11.50 ppm assigned for the two NH protons,
which disappeared upon deuteration. Moreover, 13C-NMR
spectrum disclosed a signal at δ 175.64 ppm attributed to
the C¼S carbon and signals equivalent to the aromatic ring
carbons at the range δ 124.23–139.40 ppm. The condensed
triazinothienopyrimidine derivative 7 was prepared by
refluxing the key compound 3 with chloroacetamide in
DMF.1H-NMR spectrum illustrated a singlet signal at δ
3.23 ppm attributed to the exchangeable NH proton and a
singlet signal at δ 5.79 ppm equivalent to CH2 protons of
the triazine ring (Scheme 1).
Refluxing the aminothiophene ester 1 with excess
formamide as reported [20] gave the cycloheptathieno
[2,3-d]pyrimidin-4(3H)-one derivative 8 which afforded
the corresponding 4-chloro derivative 9 upon treatment
with thionyl chloride as described in literature [11].
Refluxing 9 with hydrazine hydrate in dioxane or etha-
nol produced the 4-hydrazino 10.1H-NMR spectrum of
10 disclosed two exchangeable singlet signals at δ
3.81 and δ12.28 ppm attributed to NH2 and NH protons,
respectively, whereas the 13C-NMR spectrum indicated
two signals at δ 158.99 ppm and 169.21 ppm corre-
sponding to C-2 and C-4 of the pyrimidine ring respec-
tively. The 4-thioxo derivative 11 was obtained via
Chemistry.
The synthetic strategy to synthesize the
target thienopyrimidines 1–17 is depicted in Schemes 1–3.
The aminothiophene ester [25] 1 was readily available
through Gewald reaction [26] using cycloheptanone,
ethyl cyanoacetate, sulfur, and a secondary amine.
Reaction of the aminothiophene ester 1 with acetic
anhydride afforded the corresponding acetylated
derivative 2 [25]. Refluxing 2 with hydrazine hydrate in
ethanol gave the 3-amino-2-methyl thienopyrimidine
derivative 3 [25] which acted as a key intermediate in
(Scheme 1). Reaction of the amino derivative 3 with
excess acetic anhydride afforded the acetamido 4.1H-
NMR spectrum revealed a singlet signal at δ 2.30 ppm
corresponding to the CH3 protons of the acetamido
group, and also a singlet signal at
δ 11.00 ppm
representing the NH proton that disappeared upon
deuteration. Furthermore, mass spectrum showed M+. at
m/z 291. Yet intermediate 3 reacted with chloroacetyl
chloride upon stirring in dry benzene at room temperature
overnight to give the chloroacetamido derivative 5.1H-
NMR spectrum indicated a singlet signal at δ 4.30ppm
corresponding to CH2 protons of the chloroacetamido
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E. I. El-Mongy, M. A. Khedr, N.A. Taleb, H. M. Awad, and S. E.-S. Abbas
Vol 000
refluxing the 4-chloro derivative 9 with thiourea in eth-
anol and in the presence of TEA. The structure of 11
was established by IR spectrum that revealed the pres-
group.1H-NMR spectrum of 17 revealed two singlet sig-
nals at δ12.23 and δ13.21 ppm assigned to the two NH pro-
tons that were exchanged upon deuteration.13C-NMR
spectrum of 17 disclosed two signals at δ 157.37 ppm and
δ 172.46 ppm assigned for C¼O and C¼S respectively
(Scheme 3).
ence of a band equivalent to NH group at 3396 cmꢀ1
,
and a band for C¼S at 2020 cmꢀ1. Furthermore, 1H-
NMR spectrum disclosed
a singlet signal at δ
12.15 ppm assigned for the NH proton which was ex-
changed upon deuteration, in addition to the lack of ex-
changeable signal of NH2 protons; moreover, 13C-NMR
spectrum showed a signal at δ 158.10 ppm assigned for
the C¼N and a signal attributed to C¼S at δ
173.78 ppm. Also, mass spectrum of 11 indicated the
molecular ion peak at m/z 236 corresponding to the mo-
lecular weight of the 4-thioxo derivative (Scheme 2).
Enzyme specific assay against PARP-1. The designed
compounds were screened as PARP-1 inhibitors at
different concentrations, and the PARP-1 inhibitory activity
reflected by their IC50 was expressed as %inhibition values
and provided in Table 2.
Biological discussion. The results presented in Table 2
indicate that the most active compounds reflected by their
IC50 values are the bicyclic 2-thioureidocyclohepta[b]
thiophene 16 (IC50 50.392 μM) followed by the 4-
thioxothienopyrimidine derivative 11 (IC50 58.206μM),
then the 4-hydrazinocycloheptathienopyrimidine 10 (IC50
64.782μM).
Refluxing the aminothiophene ester 1 with phenyl isothio-
cyanate in ethanol or acetonitrile on a water bath at 90°C for
15h gave 3-phenyl-2-thioxothienopyrimidine derivative 12;
1H-NMR spectrum disclosed the presence of multiplet at δ
7.19–7.47ppm representing the aromatic protons, and a sin-
glet signal at δ 13.59ppm assigned to the exchangeable NH
proton. Mass spectrum revealed M+. at m/z 328. Decreasing
the reflux time to 2h in ethanol afforded the ethyl (3-phenyl-
thioureido)cycloheptathiophene carboxylate derivative
14.1H-NMR spectrum indicated multiplet corresponding to
the aromatic protons that appeared at δ 7.12–7.49ppm and
two exchangeable singlet signals at δ 10.83 and δ
11.50ppm representing the two NH protons. The two previ-
ously mentioned derivatives 12 and 14 were allowed to react
with hydrazine hydrate, the former in pyridine and the latter
in ethanol under reflux to afford the thienopyrimidine deriv-
atives 13 and 15 respectively.1H-NMR spectrum of 13 indi-
cated the two singlet signals at δ 4.28 ppm and δ 6.80ppm
assigned for NH2 protons and NH proton, respectively, that
disappeared upon deuteration; moreover, 13C-NMR spec-
trum revealed signals corresponding to the aromatic carbons
at δ 124.23–130.03ppm and signals at δ 161.13 and
164.84ppm equivalent to C-4 and C-2 of the pyrimidine
ring, respectively. Also, mass spectrum of 13 showed M+.
at m/z 326. The 1H-NMR spectrum of 15 indicated two ex-
changeable singlet signals at δ 2.60ppm and at δ
13.85ppm equivalent to NH2 and NH protons, respectively
(Scheme 3).
Regarding Scheme 1, compounds bearing 2-methyl
cycloheptathienopyrimidine skeleton, the 3-chloroacetylated
derivative 5 and the rigid triazino derivative 7, exhibited
IC50s 79.275 and 74.379 μM respectively. On the other hand,
out of the 4-substituted thienopyrimidine series (Scheme 2),
4-hydrazino 10 and 4-thioxocycloheptathienopyrimidine 11
derivatives afforded distinguished elevation in activity re-
cording IC50s 64.782 and 58.206 μM with respect to their
former congener 8 (IC50 108.438μM).
Concerning Scheme 3, the most potent compound was
the 2-thioureidothiophene derivative 16 (IC50 50.392μM)
whose activity decreased upon cyclization into the
thioxo derivative 17 (IC50 73.563 μM), while, the 2-
thioxothienopyrimidine 12 (IC50 72.952μM) was more ac-
tive than its 2-hydrazino congener 13 (IC50 75.242 μM).
Cytotoxic activity against human Caucasian breast
adenocarcinoma (MCF-7). The Cytotoxic activity against
Human Caucasian breast adenocarcinoma (MCF-7) of three
compounds were tested in order to see if they possess
anticancer effect against breast cancer cell line or not.
The examined compounds were compound 11 as a
representative compound for thioxothienopyrimidines,
compound 15 representing the thienopyrimidine derivatives,
and compound 16 on behalf of the thiophene derivatives.
According to the results, compound 11 showed 22.9% of
inhibition at 50 μM/mL and compound 15 showed 20.8%
of inhibition at 50μM/mL the thiophene derivative 16
showed 30.65% of inhibition at 50μM/mL, and were
compared to Doxorubicin that showed 45.02% of
inhibition at 50 μM/mL.
Yet, treatment of the aminothiophene ester 1 with potas-
sium thiocyanate in the presence of HCl 10% afforded the
ethyl-2-thioureido-cycloheptathiophene-3-carboxylate 16,
which was cyclized into 2-thioxocycloheptathieno[2,3-d]
1
pyrimidine 17 upon refluxing with sodium ethoxide. H-
NMR spectrum of the thioureido 16 showed the presence
of two exchangeable singlet signals at δ 8.30 and δ
11.00 ppm assigned to NH2 and NH protons, respectively;
moreover, mass spectrum of 16 showed M+. at m/z 298
(11.19%), whereas, IR spectrum of 2-thioxo 17 confirmed
the absence of the forked band of NH2 and elicited the
presence of a band at 3416 cmꢀ1 representing the NH
CONCLUSION
The previous enzyme specific assay demonstrated that,
all the designed compounds were found to be moderately
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Substituted Cycloheptathienopyrimidine Derivatives
potent and efficacious as PARP-1 inhibitors with IC50
values (50.392–108.438 μM), when compared to 3-
aminobenzamide as a positive control. The highest activity
was associated with the 2-thioureidothiophene derivative
16 (IC50 50.392 μM). The least potent one was the
cycloheptathienopyrimidin-4-one 8 (IC50 108.438 μM).
In general, it should be noted that almost all structural
modifications on thiophene derivative 1 concerning the syn-
thesis of mono- or di-substituted cycloheptathienopyrimidine
at positions 2-, 2, 3-, or 4, were found to produce com-
pounds with promising PARP-1 inhibitory activity. In-
terestingly, the biological activity profile of these
compounds for both the enzyme inhibition and the cyto-
toxic activity assays was nearly consistent with the
docking results.
Cairo University, Cairo, Egypt. Elemental analyses were
carried out at the Regional Center for Mycology and
Biotechnology, Al-Azhar University, and the results were
within the accepted range ( 0.40) of the calculated values.
The starting materials, namely, ethyl-2-amino-4,5,6,7,8-
pentahydrocyclohepta[b]thiophene-3-carboxylate 1, ethyl
2-acetamido-4,5,6,7,8-pentahydrocyclohepta[b]thiophene-
3-carboxylate 2, and 3-amino-2-methyl-5,6,7,8,9-pentahydro
cyclohepta[4,5]thieno-[2,3-d] pyrimidin-4(3H)-one 3 were
prepared following the corresponding literature procedures
[25] in (Scheme 1). 5,6,7,8,9-Pentahydro-cyclohepta[4,5]
thieno[2,3-d]pyrimidin-4-one
8 and 4-chloro-5,6,7,8,9-
pentahydrocyclo-hepta[4,5]thieno[2,3-d]pyrimidine 9 were
synthesized according to the reported methods [20]
(Scheme 2).
N-(2-methyl-4-oxo-5,6,7,8,9-pentahydrocyclohepta[4,5]
thieno[2,3-d]pyrimidin-3(4H)-yl) acetamide (4).
A mixture
of 3-aminothienopyrimidine 3 (2.49g, 10mmol) and acetic
anhydride (15mL) was refluxed for 3h. The mixture was
then left at room temperature for 24h, where a solid
product was separated, filtered, and recrystallized from
absolute ethanol to give yellow needles. Yield: 71%, m.p.
(110–111°C). IR (KBr disc) (cmꢀ1): 3413 (NH), 2920
EXPERIMENTAL
Molecular modeling study. Preparation of macromolecule.
Protein data bank (www.pdb.org) was used for downloading
the crystal structure of PARP-1 enzyme with pdb
code=1UK1 along with a potent quinazolinone derivative.
The active site of the enzyme was determined, and important
pharmacophores were identified.
Preparation of ligands. All compounds were built using
MOE 2012.10 builder, minimized, and saved as mol2
format. The gradient for energy minimization was set to
be =0.05, and the force field was selected as
“MMFF94X” to be suitable for organic molecules. All
bonded, Van der Waals, electrostatics, and restraints were
enabled. Partial charges were calculated.
1
(CH2), 2851 (CH3), 1738 (C¼O), 1669 (C¼O). H-NMR
(DMSO) δ (ppm): 1.21 (s, CH3 at C-2 of pyrimidine), 2.30
(s, CH3 acetamido group), 1.26–3.34 (m, 10H
cycloheptane), 11.00 (s, NH, D2O exchangeable).13C NMR
(DMSO-d6) δ ppm: 20.39, 20.76, 26.67, 26.93, 27.20,
28.91, 31.76, 120.93, 136.39, 136.85, 155.76, 155.87,
158.99, 169.21. m/z (%): M+.: 291 (17.2), 43 (100). Anal.
for C14H17N3O2S (291.369), Calcd %: C, 57.71; H, 5.88;
N,14.42 Found %: C, 57.42; H, 5.63; N, 14.21.
2-Chloro-N-(2-methyl-4-oxo-5,6,7,8,9-pentahydrocyclohepta
[4,5]thieno[2,3-d] pyrimidin-3(4H)-yl) acetamide (5).
Chloroacetylchloride (1.12mL, 10mmol) was added drop
wise to 3-aminothienopyrimidine 3 (2.49g, 10mmol) in
DMF (10 mL), and stirring was continued overnight at
room temperature. The reaction mixture was then poured
onto ice/water. The separated solid was filtered, left to dry
then recrystallized from absolute ethanol. Yellowish white
crystals were resulted in a yield of 67%, m.p. (140–141°C).
IR (KBr disc) (cmꢀ1): 3337 (NH), 1705, 1655 (C¼O),
2922 (CH-aliphatic).1H-NMR (DMSO) δ (ppm), 1.26–
3.34 (m, 3H, C2―H, 10H of cycloheptane), 4.30 (s, 2H,
COCH2), 11.32 (s, 1H, NH D2O exchangeable). 13C NMR
(DMSO-d6) δ ppm: 13.97, 20.60, 26.50, 27.81, 28.92,
31.74, 60.67, 120.89, 136.34, 138.08, 155.53, 158.90,
164.69. m/z (%): 325 (M 35Cl, 10.85), 327 (M 37Cl,
4.01),77 (100). Anal. for C14H16N3O2S (325.81) Calcd %:
C, 51.61; H, 4.95; N, 12.90. Found %: C, 51.46; H, 4.74;
N, 12.74
Molecular docking using Leadit 2.1.2. Preparation of
receptor.
The crystal structure of PARP-1 was
prepared using Leadit 2.1.2 software. Quinazolinone
derivative inhibitor was selected as a reference ligand.
Leadit docking.
Docking strategy was selected to be
Enthalpy and Entropy (Hybrid approach). Scoring was set
as default. Clash factor was set to be medium; maximum
number of solution per iteration was =200.
Chemistry. General methods. All melting points were
measured using electro thermal IA9100 apparatus
(Shimadzu, Japan). Infrared spectra were recorded on
Bruker FT-IR spectrophotometer and were expressed as
wave number (cmꢀ1) using potassium bromide discs, at
the Micro-analytical center, Faculty of Science, Cairo
University. The 1H-NMR spectra were determined on
Varian Mercury VX-300 NMR spectrophotometer at
300MHZ at the Faculty of Science, Cairo University.
The 13C-NMR spectra were recorded on Varian Mercury
VX-300 NMR spectrophotometer and were run at
75.446 MHZ in (DMSO-d6), at main defense chemical
laboratories. Mass spectra were recorded on 70eV EL
Ms-QP 1000 EX (Shimadzu-Japan), Faculty of Science
1-(2-Methyl-4-oxo-5,6,7,8,9-pentahydrocyclohepta[4,5]thieno
[2,3-d]pyrimidin-3(4H)-yl)-3-phenylthiourea (6).
Phenyl
isothiocyanate (5.6mL, 40mmol) was added to 3-
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E. I. El-Mongy, M. A. Khedr, N.A. Taleb, H. M. Awad, and S. E.-S. Abbas
Vol 000
aminothienopyrimidine
3
(2.49g,
10mmol)
in
under reduced pressure, and the obtained solid was
recrystallized from methanol. Dark brown powder was
obtained in a 57% yield, m.p. (150–151°C). IR (KBr)
(cmꢀ1): 3396 (NH), 3100 (CH-aromatic), 1500 (C¼C).
dichloromethane (20 mL). The mixture was heated at (70°C)
on water bath for 12h, and excess solvent was removed
under reduced pressure. The remained solid was collected,
dried, and recrystallized from absolute ethanol. Light
brown powder was obtained in a yield of 73%, m.p. (138–
140°C). IR (KBr disc) (cmꢀ1): 3340 (NH broad), 3090
(CH aromatic), 1656 (C¼O), 2922 (CH-aliphatic). 1H-
NMR (DMSO), δ (ppm), 1.22 (s, 1H, CH3, 1.26–2.98 (m,
10H of cycloheptane ring), 7.12–7.45 (m, 5H of aromatic
ring), 10.83 (s, 1H, NH, D2O exchangeable), 11.50 (s, 1H,
NH, D2O exchangeable). 13C NMR (DMSO-d6) δ ppm:
14.20, 26.52, 27.49, 27.90, 29.10, 31.76, 120.81, 123.51,
124.23, 127.90, 130.03, 136.34, 137.68, 139.40, 155.36,
161.13, 165.37, 175.64, m/z (%): 384M+. (62.76). Anal.
for C19H20N4OS2 (384.52) Calcd %: C, 59.35; H, 5.24; N,
14.57. Found %: C, 59.48; H, 5.46; N, 14.88.
3,4-Dihydro-6-methyl[1,2,4]triazino[2,3c]9,10,11,12,13
pentahydrocyclohepta [4,5]thieno[3,2-e]pyrimidin-2-one (7).
An equimolar amount of 3-aminothienopyrimidine
derivative 3 (2.49g, 10mmol) and chloroacetamide (~1g,
10mmol) in DMF (10mL) was heated under reflux for
12h, left to cool then poured onto ice/water. The formed
solid was filtered, dried, and recrystallized from
ethanol/DMF. Brownish powder was obtained in a yield of
65%, m.p. (188–190°C). IR (KBr disc) (cmꢀ1): 3420
1H-NMR (DMSO)
δ (ppm), 1.21–2.90 (m, 10H
cycloheptane), 8.00 (s, 1H, CH pyrimidine), 12.15 (s, 1H,
NH, D2O exchangeable). 13C NMR (DMSO-d6) δ ppm:
26.78, 27.23, 27.79, 29.07, 31.88, 123.17, 136.38,
143.20, 144.39, 158.10, 173.78. m/z (%): 236 (M+.,
5.21), 220 (100). Anal. for C11H12N2S2 (236.356), Calcd
%: C, 55.90; H, 5.12; N, 11.85; S, 27.13. Found %: C,
55.96; H, 5.10; N, 12.02; S, 27.22.
3-Phenyl-2-thioxo-5,6,7,8,9-pentahydrocyclohepta[4,5]thieno
[2,3-d]pyrimidin-4(1H)one (12).
Equimolar amounts of
aminothiophene
1
(2.40g, 10mmol), phenyl
isothiocyanate (1.36mL, 10mmol), and anhydrous
potassium carbonate (1.4g, 10mmol) in acetonitrile
(20mL) were refluxed on a water bath for 15h. The
reaction mixture was then cooled, filtered, and neutralized
with 2M hydrochloric acid. The separated product was
filtered, washed with water, dried, and recrystallized from
acetic acid. Yellowish white crystals were obtained in a
yield of 81%, m.p. >250°C. IR (KBr disc) (cmꢀ1): 3416
(NH), 3109 (CH-aromatic), 1656 (C¼O). 1H-NMR
(DMSO), δ (ppm) 1.54–3.07 (m, 10H cycloheptane ring),
7.19–7.47 (m, 5H of aromatic ring), 13.59 (s, NH, D2O
exchangeable), m/z (%): 328 (M+., 64.04), 252 (100).
Anal. for C17H16N2OS2 (328.452), Calcd %: C, 62.16; H,
4.91; N, 8.53. Found %: C, 62.31; H, 4.88; N: 8.35.
3-Phenyl-2-hydrazino-5,6,7,8,9-pentahydrocyclohepta[4,5]
thieno[2,3-d]pyrimidin-4-one (13). A mixture of 2-thioxo
12 (3.2 g, 10mmol) and hydrazine hydrate (~2 mL,
20mmol) was refluxed in pyridine (10 mL) for 15h, and
the reaction mixture was then poured onto ice/water. The
solid product was filtered, washed with ethanol, and
recrystallized from ethanol. Yellow crystals were
obtained in a yield 74%, m.p. (205–208°C). IR (KBr)
(cmꢀ1): 3425 (NH2), 3336 (NH), 1679 (C¼O), 3103
(CH-aromatic).1H-NMR (DMSO) δ (ppm): 1.29–3.09 (m,
10H cycloheptane), 4.28 (s, 2H, NH2 D2O
exchangeable), 6.80 (s, 1H, NH, D2O exchangeable),
7.00–8.00 (m, 5H Ar―H). 13C NMR (DMSO-d6) δ ppm:
26.78, 27.19, 27.23, 28.94, 31.87, 117.12, 119.24,
129.63, 136.55, 141.20, 144.31, 150.53, 155.49, 157.37,
161.13, 164.84. m/z (%): 326 (M+., 60.04), 77 (100).
Anal. for C17H18N4OS (326.416), Calcd %: C, 62.55; H,
5.56; N, 17.16. Found %: C, 62.63; H, 5.95; N, 17.31.
Ethyl-2-(3-phenylthioureido)-4,5,6,7,8-pentahydrocyclohepta[b]
thiophene-3-carboxylate (14). 2-Aminothiophene derivative
1 (2.40g,10 mmol) was dissolved in hot ethanol (10mL),
then phenyl isothiocyanate (~2 mL, 15mmol) was added
drop wise while stirring. The reaction mixture was heated
under reflux on a water bath for 2h and left to stand
overnight at room temperature. The separated solid was
1
(NH), 2922 (C-aliphatic), 1667 (C¼O), 1641 (C¼N). H-
NMR (δppm) (DMSO), 1.26–3.34 (m, 3H of C-2
pyrimidine ring, 10H of cycloheptane), 3.23 (s, 1H, NH
triazine ring, D2O exchangeable), 5.79 (s, 2H, CH2 of
triazine ring), m/z (%); 288 (M+, 0.56), 249(100). Anal. for
C14H16N4OS (288.37) Calcd %: C, 58.31; H:, 5.59; N,
19.43. Found %: C, 58.42; H, 5.57; N, 19.75.
4-Hydrazino-5,6,7,8,9-pentahydrocyclohepta[4,5]thieno[2,3-
d]pyrimidine (10). A mixture of 4-chlorothienopyrimidine
derivative 9 (2.4 g, 10 mmol) and hydrazine hydrate
(~2 mL, 20mmol) was refluxed in dioxane (10 mL) for
4 h. The separated solid was filtered off, dried, and
recrystallized from dioxane. Reddish brown powder was
resulted in a yield of 58%, m.p. (145–147°C). IR (KBr)
(cmꢀ1): 3410 (NH2), 3387 (NH), 3109 (CH-aromatic),
1554 (C¼C).1H-NMR (DMSO) δ (ppm) 1.21–2.90 (m,
10H cycloheptane) 3.81 (s, 2H, NH2, D2O exchangeable),
7.98 (s, 1H, CH pyrimidine), 12.28 (s, NH, D2O
exchangeable). 13C NMR (DMSO-d6) δ ppm: 26.42,
27.36, 28.22, 27.17, 31.76, 123.17, 136.37, 144.47,
158.99, 169.21. m/z (%): 234 (M+., 1.05), 64 (100). Anal.
for C11H14N4S (234.321), Calcd %: C, 56.38; H, 6.02; N,
23.91. Found %: C, 56.44; H, 6.09; N, 24.04
4-Thioxo-5,6,7,8,9-pentahydrocyclohepta[4,5]-thieno[2,3-d]
pyrimidine (11). A mixture of 4-chloro thienopyrimidine
derivative 9 (2.4 g, 10 mmol), thiourea (0.76 g, 10mmol)
in ethanol (10 mL), and in the presence of 3 drops of
TEA was refluxed for 4h. The solvent was evaporated
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Substituted Cycloheptathienopyrimidine Derivatives
filtered, washed with ethanol, and recrystallized from
ethanol. Yellow crystals were obtained in a yield of 96%,
m.p. (150–153°C). IR (KBr disc) (cmꢀ1): 3330 (NH),
2223 (―S¼C¼N―), 1702 (C¼O). H-NMR (DMSO) δ
yield of 78%, m.p. (230–232°C). IR (KBr) (cmꢀ1):
3416 (NH), 1662 (C¼O), 1560 (C¼C). 1H-NMR
(DMSO) δ (ppm) 1.29–3.40 (m, 10H (cycloheptane)),
12.23 (s, 1H, NH, D2O exchangeable), 13.21 (s, 1H,
NH, D2O exchangeable). 13C NMR (DMSO-d6) δ
ppm: 26.59, 27.19, 28.28, 31.67, 116.90, 131.94,
136.27, 148.27, 157.37, 172.43. m/z (%): 252 (M+.,
4.05), 80 (100). Anal. for C11H12N2OS2 (252.356),
Calcd %: C, 52.35; H, 4.79; N, 11.10. Found %: C,
52.42; H, 4.81; N, 10.23.
1
(ppm) 1.22 (t, 3H, CH3), 1.34–2.97 (m, 10H
cycloheptane) 4.22 (q, 2H, CH2― CH3), 7.12–7.49 (m,
5H, aromatic H), 10.83 (s, 1H, NH, D2O exchangeable),
11.50 (s, 1H, NH, D2O exchangeable). 13C NMR
(DMSO-d6) δ ppm: 14.29, 26.53, 27.41, 27.79, 27.90,
31.50, 60.45, 113.87, 118.87, 123.53, 124.23, 128.33,
136.81, 146.61, 155.21, 161.13, 175.64. m/z (%): 374
(M+., 62.76), 193 (100). Anal. for C19H22N2O2S2
(374.52), Calcd %: C, 60.93; H, 5.92; N, 7.48. Found %:
C, 60.68; H; 5.89; N, 7.71.
Inhibition of PARP-1 activity.
The enzyme specific
assay was carried out at the National Research Centre,
Dokki, Giza, Egypt.
Materials and methods. Na2HPO4, NaH2PO4, and NaCl
were purchased from Sigma Chem. Co. (St. Louis, MO,
USA). The investigated compounds were dissolved in
DMSO and diluted at specified concentrations (from 50
to 200 μM). The PARP inhibitory activities of the
investigated compounds were determined using
Trevigen’s HT Universal 96-well PARP Assay Kits
(Trevigen, Inc., Gaithersburg, Maryland, USA) to
measure the incorporation of biotinylated poly (ADP-
ribose) onto histone proteins in a 96-well strip well
format. This assay is ideal for the screening of PARP
inhibitors and the determination of IC50 values. The
measurements were performed according to the
manufacturer’s protocol. The decrease in absorbance of
each solution was measured at 450nm using an ELISA
micro plate reader (Model 550, Bio-Rad Laboratories
Inc., California, USA). Absorbance of blank sample
containing the same amount of DMSO was also prepared
and measured. A negative control without PARP was
prepared to determine background absorbance. 3-
Aminobenzamide was used as a positive control for
PARP inhibitory activity with a reported IC50 of 30 μM
[27] (Table 2).
3-Amino-2-phenylamino-5,6,7,8,9-pentahydrocyclohepta
[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (15).
A mixture of
the thioureido derivative 14 (3.70 g, 10 mmol) and
hydrazine hydrate (~1mL, 20 mmol) in ethanol (15 mL)
was refluxed for 8h and left to cool. The separated solid
was filtered, washed with water, and recrystallized from
ethanol/DMF. Yellow powder was obtained in a yield of
64%, m.p. (200–203°C). IR (KBr) (cmꢀ1): 3324 (NH2),
1
3310 (NH), 3105 (CH-aromatic), 1690 (C¼O). H-NMR
(DMSO) δ (ppm): 1.29–2.99 (m, 10H cycloheptane),
2.60 (s, 2H, NH2, D2O exchangeable), 7.16–7.49 (m, 5H,
Ar―H), 13.85 (s, 1H, NH, D2O exchangeable). m/z (%):
326 (M +., 100). Anal. for C17H18N4OS (326.416), Calcd
%: C, 62.55; H, 5.56; N, 17.16. Found %: C, 62.61; H,
5.58; N, 17.29.
Ethyl-2-thioureido-4,5,6,7,8-pentahydrocyclohepta[b]thiophene-
3-carboxylate (16).
A mixture of 2-aminothiophene 1
(2.40 g, 10 mmol) and potassium thiocyanate (~0.15 g,
15mmol) was refluxed in 10% HCl (10 mL) for 12 h. The
reaction mixture was allowed to stand overnight at room
temperature. The formed solid was filtered, washed with
ethanol, dried, and recrystallized from ethanol. Brown
powder was obtained in a yield of 59% m.p. (180–182°C).
IR spectrum: 3430 (NH2), 2982 (CH-aliphatic), 2259
Statistical analysis. All experiments were conducted in
triplicate (n =3), and all the values were represented as
mean SD. Significant differences between the means of
parameters as well as IC50 values were determined by
probit analysis using SPSS software program (SPSS Inc.,
Chicago, IL) (Table 2).
Cytotoxic effect on human Caucasian breast
adenocarcinoma (MCF7). Cell viability was assessed by
the mitochondrial dependent reduction of yellow MTT
1
(―N¼C¼S), 1661 (C¼O). H-NMR spectrum (DMSO) δ
(ppm): 1.22–3.40 (m, 3H, CH3, and 10H cycloheptane),
4.26 (q, 2H, CH2), 8.30 (s, 2H, NH2, D2O exchangeable),
11.00 (s, 1H, NH, D2O exchangeable). m/z (%): M+.: 298
(M+., 11.19), 60 (100). Anal. for C13H18N2O2S2 (298.42),
Calcd %: C, 52.32; H, 6.08; N, 9.39. Found %: C, 52.46;
H, 6.13; N, 9.52.
2-Thioxo-5,6,7,8,9-pentahydrocyclohepta[4,5]thieno[2,3-d]
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
bromide) to purple formazan [28].
tetrazolium
pyrimidin-4[1H]-one (17).
A
solution of thiourea
derivative 16 (2.60 g, 10 mmol) and sodium ethoxide
(0.23 g of sodium metal in ethanol 5 mL) was refluxed
in ethanol (15 mL) for 6 h and left to cool. The
reaction mixture was neutralized with 10% HCl where
a solid was formed, collected by filtration, washed
The experiment was done in a sterile area using a Lam-
inar flow cabinet biosafety class II level (Baker,
SG403INT, Sanford, ME, USA). Cells were suspended in
RPMI 1640 medium for MCF7. The media are supple-
mented with 1% antibiotic–antimycotic mixture
(10 000 U/mL potassium penicillin, 10000 μg/mL strepto-
mycin sulfate, and 25 μg/mL amphotericin B), 1% L-
with
water,
dried,
and
recrystallized
from
ethanol/DMF. Light brown powder was obtained in a
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E. I. El-Mongy, M. A. Khedr, N.A. Taleb, H. M. Awad, and S. E.-S. Abbas
Vol 000
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glutamine, and 10% fetal bovine serum and kept at 37°C
under 5% CO2. Cells were batch cultured for 10 days, then
seeded at concentration of 10 × 103 cells/well in fresh com-
plete growth medium in 96-well microtiter plastic plates at
37°C for 24h under 5% CO2 using a water jacketed carbon
dioxide incubator (Sheldon, TC2323, Cornelius, OR,
USA). Media was aspirated, fresh medium (without serum)
was added, and cells were incubated either alone (negative
control) or with different concentrations of sample to give a
final concentration of (100–50–25–12.5–6.25–3.125–0.78
and 1.56 μM/mL). After 48 h of incubation, medium was
aspirated, 40-μL MTT salt (2.5 μg/mL) was added to each
well and incubated for further 4 h at 37°C under 5% CO2.
To stop the reaction and dissolving the formed crystals,
200μL of 10% sodium dodecyl sulfate (SDS) in deionized
water was added to each well and incubated overnight at
37°C. A positive control which composed of 100 μM/mL
was used as a known cytotoxic natural agent who gives
100% lethality under the same conditions [29].
The absorbance was then measured using a microplate
multi-well reader (Bio-Rad Laboratories Inc., model
3350, Hercules, California, USA) at 595 nm and a refer-
ence wavelength of 620 nm. A statistical significance was
tested between samples and negative control (cells with ve-
hicle) using independent t-test by SPSS 11 program.
DMSO is the vehicle used for dissolution of plant extracts,
and its final concentration on the cells was less than 0.2%.
The percentage of change in viability was calculated ac-
cording to the formula:
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet