Design and synthesis of novel annulated thienopyrimidines as phosphodiesterase 5 (PDE5) inhibitors

Article abstract of DOI:10.1002/ardp.201800018

Novel cycloalkene-fused thienopyrimidine analogues with enhanced phosphodiesterase 5 (PDE5) inhibitory properties are presented. The structure of the reported scaffold was modulated through variation of the terminal cycloalkene ring size, as well as by va

Full text of DOI:10.1002/ardp.201800018

Received: 23 January 2018
Revised: 14 March 2018
Accepted: 19 March 2018
|
|
DOI: 10.1002/ardp.201800018
FULL PAPER
Design and synthesis of novel annulated thienopyrimidines as
phosphodiesterase 5 (PDE5) inhibitors
Lina Y. El-Sharkawy1
Rowaida A. El-Sakhawy1
Mohammad Abdel-Halim1
|
|
|
Kevin Lee2
Gary A. Piazza2
Christian Ducho3
Rolf W. Hartmann4
|
|
|
|
Ashraf H. Abadi1
1 Faculty of Pharmacy and Biotechnology,
Department of Pharmaceutical Chemistry,
German University in Cairo, Cairo, Egypt
Abstract
Novel cycloalkene-fused thienopyrimidine analogues with enhanced phosphodiester-
ase 5 (PDE5) inhibitory properties are presented. The structure of the reported scaffold
was modulated through variation of the terminal cycloalkene ring size, as well as by
varying the substituents at position 4 through the attachment of different groups
including aniline, benzylamine, cyclohexylethylamine, methyl/acetyl/aryl piperazines,
and aryl hydrazones. Compound 15Y with a benzylaminesubstituent and cycloheptene
as terminal ring showed the highestPDE5 inhibitory activity with anIC50 value as low as
190 nM and with good selectivity versus PDE7 and PDE9.
2 Drug Discovery Research Center, Mitchell
Cancer Institute, University of South Alabama,
Mobile, Alabama, USA
3 Department of Pharmacy, Pharmaceutical
and Medicinal Chemistry, Saarland University,
Saarbrücken, Germany
4 Department of Drug Design and
Optimization, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS),
Saarbrücken, Germany
Correspondence
K E Y W O R D S
Prof. Ashraf H. Abadi, Faculty of Pharmacy
and Biotechnology, Department of
Pharmaceutical Chemistry, German University
in Cairo, Cairo 11835, Egypt.
annulated thienopyrimidines, cycloalkene-fused thienopyrimidines, phosphodiesterase
inhibitor, planarity
Email: ashraf.abadi@guc.edu.eg
Funding information
National Cancer Institute (USA),
Grant number: NCI 5 R01 CA155638
|
1
INTRODUCTION
100 distinct isoforms. PDE isozyme families differ in structure, catalytic
property, substrate specificity, regulatory properties, tissue localization,
Phosphodiesterases (PDEs) are a group of enzymes that act by breaking
down the secondary messengers cAMP (cyclic adenosine mono-
phosphate) and cGMP (cyclic guanosine monophosphate) in cells to
regulatesignaltransduction.[1] Thesesecondarymessengersareinvolved
in diverse biological functions, including inflammation, skeletal muscle
contraction, steroid hormone function, and memory. There are currently
11 PDE enzymes with multiple subtypes and splice variants yielding over
and inhibitor sensitivity.[2,3] The different gene products for PDEs are
indicatedby lettersafterthenumbersandfinally theArabicnumeralafter
the letter is indicative of the splice variant present.[4]
Four PDE5 inhibitors have been approved by the FDA as shown in
Figure 1. PDE5 inhibitors are mainly indicated for the treatment of
erectile dysfunction and pulmonary arterial hypertension. Although
PDE5 inhibitors are widely used and generally well tolerated, these
drugs can have serious side effects such as disturbance of vision. On
the other hand, there are many emerging uses for PDE5 inhibitors
including potential treatment of female sexual dysfunction, essential
hypertension, benign prostatic hyperplasia, endothelial dysfunction,
congestive heart failure, and Raynaud's phenomenon.[5,6] Additionally,
Abbreviations: cAMP, cyclic adenosine monophosphate; CC, column chromatography;
cGMP, cyclic guanosine monophosphate; CREB, cAMP responsive element binding;
N-terminal, amino terminal; PDE, phosphodiesterase; UV, ultraviolet.
Lina Y. El-Sharkawy and Rowaida A. El-Sakhawy contributed equally to the work.
|
Arch Pharm Chem Life Sci. 2018;e1800018.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
1 of 17
https://doi.org/10.1002/ardp.201800018

2 of 17
EL-SHARKAWY ET AL.
|
FIGURE 1 Structures of the approved PDE5 inhibitors
|
PDE5 inhibition was shown to play an important role in treatment of
schizophrenia, improvement of cognitive function as well as improve-
ment of hearing.[7,8] In the field of cancer therapy, PDE5 inhibitors
showed the ability to reverse multiple drug resistance to several
anticancer agents. Recent studies in a mouse model of colitis-induced
colorectal cancer suggest that PDE5 inhibitors may provide chemo-
preventive benefits for individuals with inflammatory bowel disease
who are at high risk of developing colorectal cancer.[9]
2
RESULTS AND DISCUSSION
|
2.1 Chemistry
Thieno[2,3-d]pyrimidine derivatives have been reported as inhibitors
of various PDE isozymes including PDE4, PDE5, PDE7, PDE9, and
PDE10 (Figure 2).[19–23]
Thus, it is of considerable interest to design and synthesize novel
thieno[2,3-d]pyrimidine derivatives for discovery of the scope and
limitations of this class of compounds to inhibit PDE5. Our design
strategy was aimed at synthesizing thieno[2,3-d]pyrimidines annulated
to different rings of different sizes and lipophilicities, including
cyclopentene and cycloheptene. Derivatization of this scaffold was
examined through the attachment of different substituents to position
4 of the annulated thienopyrimidine. Substituents included unsub-
stituted or substituted aryl, alkyl, cycloalkyl that are separated from the
main scaffold by varying spacer lengths from one atom, including an
amino group, two atoms like the methylamino spacer, three atoms
including an ethylamino, as well as a hydrazone spacer, and four atoms
including a cyclized ethylenediamine moiety (i.e., piperazine).
One of the essential features required for PDE5 inhibition is the
presence of a planar ring structure to interact with Phe820 by π–π
stacking and a hydrogen bonding interaction with Gln817. Binding
pockets near the invariant glutamine residue differ in both shape and
hydrophobicity which is why inhibitors show variable selectivity for
PDE5.[12,13,24]
The chemical structure for PDE isozymes includes three domains:
a variable regulatory domain, a highly conserved catalytic core, and the
amino acid terminal showing isoform specificity. The N-terminus of the
mRNA transcript is responsible for intracellular targeting domains,
signalosomes, responsible for localization of PDEs to different
organelles and membranes.[3,10]
Mammalian PDEs exist as homodimers, with the exception of
PDE1 and PDE6, which occur as heterotetramers under physiological
conditions. Biochemical observations showed dimer formation for
both catalytic and regulatory domains. The catalytic domain, contain-
ing a deep hydrophobic pocket is folded into 16 helices. In the active
site, the presence of glutamine residue is involved in recognition and
selectivity to cyclic nucleotides. A histidine moiety is also important for
catalysis as well as divalent zinc metal ions, showing co-ordination
between conserved amino acids histidine and aspartate and magne-
sium occupying the M pocket.[11–13]
Glutamine, phenylalanine and either cyclic nucleotide substrate,
product or inhibitor and a hydrophobic residue form the hydrophobic P
clamp (part of the Q pocket). Glutamine and phenylalanine residues are
responsible for anchorage of inhibitors in the pocket. Along with the M
pocket, the active site also contains the S pocket (solvent-filled pocket).
Sildenafil and tadalafil were co-crystallized with PDE5 (PDB 1UDU and
2H42) and showed common interactions with the enzyme, including:
hydrogen bonding with Gln817 and π–π stacking with Phe820.[11–15]
Many thienopyrimidines have been developed and evaluated for
different pharmacological effects including anti-viral, anti-inflamma-
tory, anti-microbial, anti-cancer activities and most importantly
potential cGMP PDE inhibitors as well as cAMP PDE inhibitors.[16–
23] In this work, we present a novel series of annulated thieno[2,3-d]-
pyrimidine derivatives with a detailed study of the structure–activity
relationship of this class of compounds as PDE5 inhibitors. A docking
experiment is done to check the interactions of potent molecules with
the protein.
|
2.1.1 Synthesis
Two parallel sets of compounds were synthesized; one processes the
thieno[2,3-d]pyrimidine fused to cyclopentene, while the second one
has a cycloheptene ring. The synthesis of the starting 2-substituted
aminothiophene was performed using the multicomponent single-pot
Gewald reaction. Generally, the reaction involves an aldehyde or
ketone, sulfur, α-activated nitrile, and a base usually diethylamine or
morpholine in the presence of ethanol or methanol as a solvent at
temperature of 50°C.[25] Compounds 1A and 1B (Scheme 1) were
synthesized using a mixture of cyclopentanone or cycloheptanone,
respectively, ethyl cyanoacetate and sulfur in the presence of
triethylamine in ethanol and left overnight. Reaction of compounds
1A and 1B with formamidine acetate was carried out according to the

EL-SHARKAWY ET AL.
3 of 17
|
FIGURE 2 Structures of thienopyrimidines as PDE inhibitors
|
reported procedures, under reflux to give the cyclized intermediates,
compounds 2A and 2B, respectively (Scheme 1).
2.2 Biological activity
|
Conversion of the hydroxyl group in compounds 2A and 2B to
the chloro substituent in compounds 3A and 3B, respectively, was
a crucial step to have an effective leaving group for further
aromatic nucleophilic substitution to occur in the following step. In
order to optimize the reaction conditions for chlorination several
trials were carried out including the use of POCl3 only, POCl3/TEA
in a ratio 1:3. It is worth mentioning that chlorination did not
succeed when only phosphoryl chloride was used. The mixture
POCl3/TEA in a ratio 1:3 was shown to be optimum. The reaction
was left for 24 h at 60°C. Aromatic nucleophilic substitution
reaction between the different amine derivatives with compounds
3A and 3B produced compounds 1X–15X, 17X, and 1Y–16Y,
respectively (Scheme 1).
2.2.1 Structure–activity relationship of PDE5
inhibitory activity
The synthesized compounds were tested for PDE5 inhibition at
25 µM, IC50 was determined for compounds having a percentage
inhibition of greater than 50%. Results are shown in Tables 1 and 2.
According to the substitution type at position 4, the compounds
synthesized can be divided into three main clusters; the piperazine
derivatives, aryl, aralkyl as well as cycloalkyl amines and aryl
hydrazones.
The 4-phenylpiperazine derivatives
Results showed that the presence of plain phenyl in compounds 1X
and 1Y gave weak inhibition of less than 50% regardless of the terminal
ring size being five-membered or seven-membered. Adding electron-
withdrawing lipophilic halogens such as the m-chloro, p-chloro
(compounds 2X–3X and 2Y–3Y), and p-fluoro (compounds 4X and
4Y) did not boost the activity where all of these derivatives showed
percentage inhibition of less than 30%. On the other hand, the
presence of the electron-donating methoxy in both meta and para
positions (compounds 7X, 7Y and 8X, 8Y) generally gave more active
compounds, where the % inhibition observed was between 52 and
68% with the best activity shown by the m-methoxyphenylpiperazine
compound 7Y (IC50 = 8.5 μM). Conversely, shifting the methoxy group
to the ortho position remarkably reduced the activity as shown in
Reaction of the chloro derivatives, compounds 3A and 3B with
hydrazine monohydrate yielded compounds 4X and 4Y, respectively
(Scheme 2). Compounds 18X–26X and 18Y–26Y (Scheme 2) were
obtained by the reaction of different aryl aldehydes with compounds
4A and 4B to give the respective aryl hydrazone derivatives through an
addition–elimination mechanism. The possibility of having one or two
geometric isomers, E and Z, was possible. Some of the hydrazone
derivatives were found to be E/Z mixtures. The 13C NMR in some of
the compounds was confirmatory evidence since there is a doubling in
the aliphatic and the aromatic carbons. However, other compounds
were less likely to be mixture of isomers as indicated by their NMR
spectra.

4 of 17
EL-SHARKAWY ET AL.
|
SCHEME 1 Reagents and conditions: (a) CH3CH2OH, Et3N, reflux 24 h, 100°C, (b) formamidine acetate, DMF, reflux 24 h, 80°C, (c) POCl3,
Et3N, 70°C, 24 h, (d) amine derivative, EtOH, reflux 24 h
compounds 6X and 6Y. Demethylation of the p-methoxy in
compounds 8X and 8Y to give a p-hydroxyl substituent in compounds
5X and 5Y maintained comparable potency. However, using the
lipophilic electron donating methyl in the meta and para positions
(compounds 10X, 11X and 10Y, 11Y) instead of the more polar
methoxy group at similar positions failed to maintain the same level of
potency indicating that the methoxy oxygen might play an important
role via its +m effect and enriching the electron density of the aromatic
system.
compounds 12X and 12Y, this might be due to additional polar
interactions by the carbonyl oxygen.
Phenylamino/cycloalkylamino
Using the aniline moiety at position 4 (compounds 14X and 14Y) gave
inhibition of 53 and 46%, respectively, at 25 µM with superior activity
to the non-substituted phenyl piperazine derivatives (compounds 1X
and 1Y), suggesting that the phenyl group in close proximity to position
4 is preferable for PDE5 inhibitory activity. Inserting a carbon spacer
between the 4-amino group and the phenyl (i.e., using benzylamine) led
to a significant increase in potency by more than 60-fold in the
cyclopentene series and 130-fold in the cycloheptene series (com-
pounds 15X and 15Y) to give the most two potent compounds in
present series with IC50 values of 0.42 and 0.19 μM, respectively.
4-Methyl/acetyl piperazines
Both acetyl and methyl piperazines were tried at position 4 of the
thienopyrimidine scaffold. Only the acetyl could maintain the marginal
activity with % inhibition slightly greater than 50% at 25 μM as seen in

EL-SHARKAWY ET AL.
5 of 17
|
SCHEME 2 Reagents and conditions: (a) N2H4.H2O reflux 24 h, MeOH, reflux 2 h, (b) RCHO, EtOH, reflux 24 h
Employing the cyclohexylmethyl amine gave the saturated analog of
compound 15X which has similar activity (compound 17X,
IC50 = 0.58 μM), suggesting that it is the effect of the hydrophobic
interactions, rather than the π-mediated interactions, that predomi-
nantly improved the potency. However, increasing the spacer length
between N4 and the cyclohexyl into two carbons reduced the potency
five times. This was revealed by comparing compound 16Y
(IC50 = 0.96 μM) with compound 15Y (IC50 = 0.19μM), which might
be due to the shift of the cyclohexyl from the optimum position for
interaction with PDE5 pocket as well as the change in conformation of
16Y. This can be shown in the energy minimized forms where the
cyclohexylethyl is in the coplanar conformation to the cycloheptene-
thienopyrimidine scaffold relative to the non-coplanar conformation
of the benzyl in compound 15Y with the cycloheptenethienopyr-
imidine scaffold, revealing the effect of the size of carbon spacer on the
planarity of the molecule and its activity (Figure 3).
% inhibition of greater than or equal to 50% at the screening
concentration, namely compound 20X (with 4-bromo substitution),
21X (with 2-fluoro substitution), and 23X (with 2-fluoro-4-methoxy
substitution). Compounds 20X, 21X, and 23X showed IC50 values of 25,
19.3, and 21.8 µM, respectively. This modest activity indicated that the
rigid and long hydrazone spacer adopted in the 4-arylhydrazone
derivatives was deleterious to the activity. Additionally, it was also
observed in this group of compounds that only the cylopentene analogs
but not the respective cycloheptene compounds exhibited significant
activity. Furthermore, all of the bulkier naphthylhydrazone analogues
were totally inactive whether they belong to the cyclopentene or the
cycloheptene derivatives (compounds 24X–26X and 24Y–26Y).
Cylcopentene versus cycloheptene derivatives
By comparing the % PDE5 inhibition of the piperazine derivatives
(Table 1), it can be seen that many cyclopentene derivatives showed
similar inhibition to the respective cycloheptene analogues, giving
indication that the terminal ring size and its lipophilicity are not major
determinants for activity among these compounds. However, by
comparing the most two potent compounds, the benzylamine derivatives
4-Arylhydrazones
Several arylhydrazone moieties were connected to position 4 of the
thienopyrimidine scaffold, three of the halo phenylhydrazones showed

6 of 17
EL-SHARKAWY ET AL.
|
TABLE 1 Inhibition of PDE5 by compounds 1X–17X and 1Y–17Y
X
Cpd#
1X
PDE5 % inhibition at 25 μM
PDE5 IC50 (μM)
Cpd#
1Y
PDE5 % inhibition at 25 μM
PDE5 IC50 (μM)
23
ND
39
ND
2X
28
ND
2Y
19
ND
3X
4X
28
30
ND
ND
3Y
4Y
0
ND
ND
27
5X
6X
47
27
ND
ND
5Y
6Y
77
0
19.2
ND
7X
64
12.6
7Y
52
8.5
8X
9X
68
22
21
8Y
9Y
55
40
24.5
ND
ND
10X
25
ND
10Y
19
ND
11X
12X
13X
14X
15X
10
57
40
53
98
ND
22
11Y
12Y
13Y
14Y
15Y
39
55
31
46
88
ND
21.1
ND
ND
25
ND
0.42
0.19
16X
17X
–
–
16Y
17Y
87
0.96
ND
100
0.58
–
|
(15X and 15Y), the cycloheptene derivative (15Y) showed more than
twofold potency of the corresponding cyclopentene congener, which
could be due to a better positioning of the benzyl group in its critical
interaction with the pocket as an effect of the expanded terminal ring
(Figure 3). On thecontrary, the expanded cycloheptene ring decreased the
activity of the hydrazone analogues (Table 2) as described above.
2.2.2 Docking and molecular modeling
Docking was performed on sildenafil and one of the most active
compounds, compound 15Y, using MOE software (version 2016.08)
on the PDE5 crystal structure 2H42.[15,26] Comparing the 2D
interaction maps of sildenafil in the pocket (Figure 4A) and compound

EL-SHARKAWY ET AL.
7 of 17
|
TABLE 2 Inhibition of PDE5 by the arylhydrazones
X
Cpd#
18X
PDE5 % inhibition at 25 μM
PDE5 IC50 (μM)
Cpd#
18Y
PDE5 % inhibition at 25 μM
PDE5 IC50 (μM)
25
ND
0
ND
19X
20X
21X
22X
40
50
60
29
ND
25
19Y
20Y
21Y
22Y
4
ND
ND
ND
ND
15
28
19
19.3
ND
23X
55
21.8
23Y
23
ND
24X
25X
0
0
ND
ND
24Y
25Y
2
0
ND
ND
26X
12
ND
26Y
1
ND
15Y (Figure 4B), compound 15Y maintained some of the critical
interactions made by sildenafil, importantly hydrogen bonding with the
invariant glutamine residue Gln817 through the NH at position 4 of the
cycloheptenethienopyrimidine scaffold and secondly π–π stacking
interaction was observed for compound 15Y with Phe820 similar to
that made with sildenafil. The energy minimized form for compound
15Y inside the PDE5 pocket indicates that the phenyl ring is present in
a non-coplanar conformation to the cycloheptenethienopyrimidine
scaffold as can be seen in Figure 5. The non-coplanarity appears to be
required for interaction of the phenyl ring inside the pocket. Some of
the hydrophobic residues in the pocket include Phe786, Leu804, and
Ile813 (Figure 4). Figure 6 shows similar interactions made by
compound 15Y and sildenafil with essential PDE5 residues upon
overlay of the two structures.
|
2.2.3 Selectivity for PDE5 against PDE7 and PDE9
inhibition
The most active compounds for PDE5 inhibition including compounds 15X,
15Y, 16Y, and 17X with percentages of inhibition at 98, 88, 87, and 100%,
respectively, showed weak activity of less than 33% for both PDE7 and
PDE9 inhibition at 25 μM. For % PDE7 inhibition, percentages were 28 and
22% for compounds 15X and 15Y, respectively, and for 16Y and 17X it was
6 and 28%, respectively. For PDE9, the % inhibition values were 23 and 11%

8 of 17
EL-SHARKAWY ET AL.
|
FIGURE 3 Energy minimized forms of the most active compounds on PDE5 showing the effect of ring size on the planarity for compounds
15X and 15Y and of variation of carbon spacer on the planarity of compounds 16X and 17Y
for compounds 15X and 15Y,respectively,and3and32%inhibitionfor16Y
and 17X, respectively. Thus, the most active compounds for PDE5 inhibition
showed greater selectivity toward PDE5 compared to PDEs 7 and 9.
gel 60 (0.063–0.200 mm). Reaction progress was monitored by TLC
using fluorescent pre-coated silica gel plates and detection of the
components was made by short UV light (λ = 254 nm). 1H NMR and
13C NMR spectra were recorded using a Bruker Fourier 300, Varian
Mercury 400 or Bruker DRX 500. 1H shifts are referenced to the
residual protonated solvent signals δ 2.50 for dimethylsulfoxide
(DMSO-d6) and δ 7.2 for chloroform-d (CDCl3). 13C shifts are
referenced to the deuterated solvent signal δ 39.5 for DMSO-d6
and δ 77.0 for CDCl3. Coupling constants (J) are given in hertz (Hz).
Multiplicities are abbreviated as s: singlet; d: doublet; t: triplet; q:
quartet; m: multiplet; dd: doublet of doublet; dt: doublet of triplet; brs:
broad singlet. Mass spectrometric analysis (HPLC-ESI-MS) was
|
3
CONCLUSION
Compound 15Y (IC50 = 190 nM) can serve as a promising lead compound
for further optimization as PDE5 inhibitor through using different
substituted benzyl derivatives to reach the single digit nM range, this is
encouraged by the advantageous selectivity over PDE7 and PDE9.
performed on
a TSQ quantum (Thermo Electron Corporation)
|
4
EXPERIMENTAL
instrument equipped with an ESI source and a triple quadrupole
mass detector (Thermo Finnigan, San Jose, CA). The MS detection was
carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas pressure
of 4.0 × 105 Pa, an auxiliary gas pressure of 1.0 × 105 Pa, a capillary
temperature of 400°C, capillary voltage of 35 V, and source CID of
10 V. All samples were injected by autosampler (Surveyor, Thermo
Finnigan) with an injection volume of 10 µL. A RP C18 NUCLEODUR
100-3 (125 mm × 3 mm) column (Macherey-Nagel) was used as
|
4.1 Chemistry
|
4.1.1 General
Solvents and reagents were obtained from commercial suppliers and
were used without further purification. All solvents used were of pure
analytical grade. Column chromatography was carried out using silica

EL-SHARKAWY ET AL.
9 of 17
|
FIGURE 4 (A) 2D image of the detailed mode view showing the docked pose of sildenafil with human PDE5. (B) 2D image of the detailed
mode view showing the docked pose of compound 15 with human PDE5 enzyme
|
stationary phase. The solvent system consisted of water containing
0.1% TFA (A) and 0.1% TFA in acetonitrile (B). HPLC Experimental 63
method: flow rate 400 µL/min. The percentage of B started at an initial
of 5%, was increased up to 100% during 16 min, kept at 100% for
2 min, and flushed back to 5% in 2 min. All masses were reported as
those of the protonated parent ions. The purities of the tested
compounds were determined by HPLC coupled with mass spectrome-
try and were higher than 95% purity. Measurements for melting point
were not corrected and were recorded using capillary Buchi B-540
melting point apparatus.
4.1.2 General procedure for Gewald reaction for the
synthesis of compounds 1A and 1B
A 100 mL round bottom flask was charged with cyclopentanone or
cycloheptanone (10 mmol), sulfur (0.32 g, 10 mmol), ethylcyano acetate
(1.31 g, 4 mmol), morpholine (5 mL), and ethanol (10 mL). The reaction
solution was stirred overnight at temperature not exceeding 65°C. The
solventwas removedunderreducedpressure, thenbrinewasaddedand
extractionwas doneusing methylene chloride (3 × 50 mL). The collected
organic layers were dried over anhydrous MgSO4, evaporated under
reduced pressure, and the residue was purified using column
chromatography to yield the desired product compound 1A as a brown
powder, 92% yield, mp of 91–92°C and compound 1B as a yellow solid,
63% yield, mp of 88–89°C.[27,28]
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting
Information.
FIGURE 5 Detailed mode view of 15Y in PDE5 pocket showing
the non-coplanarity between the phenyl ring and the
cycloheptenethienopyrimidine scaffold (PDB: 2H42). PDE5 binding
site–red color: hydrophilic region, blue color: hydrophobic region,
white color: solvent exposed region
FIGURE 6 Graphical representation of the binding modes of
sildenafil (brown) and compound 15Y (gold) in the pocket of human
PDE5 enzyme (PDB: 2H42). PDE5 binding site–red color: hydrophilic
region, blue color: hydrophobic region, white color: solvent exposed
region

10 of 17
EL-SHARKAWY ET AL.
|
|
4.1.3 General procedure for thieno[2,3-d]pyrimidin-
was purified by CC (hexane/EtOAc, 85:15) to give orange crystals:
yield 80%; mp 157–159°C; 1H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H),
7.33–7.27 (m, 2H), 7.00 (t, J = 1.5 Hz, 1H), 6.99–6.97 (m, 1H), 6.91 (tt,
J = 7.4, 1.0 Hz, 1H), 3.80–3.64 (m, 4H), 3.34–3.44 (m, 4H), 3.12–3.06
(m, 2H), 3.05–2.99 (m, 2H), 2.51–2.43 (m, 2H); 13C NMR (126 MHz,
CDCl3) δ 173.43, 160.73, 151.69, 151.28, 140.23, 135.75, 129.35,
120.38, 117.55, 116.49, 49.44, 49.41, 31.85, 29.99, 28.36; MS (ESI)
m/z = 337.18 (M+H)+.
4-ones synthesis of compounds 2A and 2B
Compound 1A or 1B (56.7 mmol) and formamidine acetate (8.85 g,
85.0 mmol) in DMF (100 mL) were heated at 80°C for 3 days. The
reaction mixture was cooled, DMF removed in vacuum, and the solid
obtained was washed thoroughly with water, extracted with ethyl
acetate, and the crude product was purified by silica gel flash CC to
give the desired products; 2A, white solid: yield 40%, mp
242–244°C and 2B, light brown solid: yield 59%, mp 118–220°C.[28,29]
4-(4-Phenylpiperazine-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (1Y)
|
4.1.4 General procedure for chlorination to
The title compound was prepared by reaction of compound 3B and
4-phenylpiperazine according to the general procedure. The product
was purified by CC (hexane/EtOAc, 8:1) to give light brown crystals:
yield 63%; mp 174.3–176.2°C; 1H NMR (300 MHz, CDCl3) δ 8.47 (s,
1H), 7.27–7.17 (m, 2H), 6.92 (dd, J = 8.7, 0.9 Hz, 2H), 6.87–6.80 (m,
1H), 3.48 (s, 4H), 3.29 (s, 4H), 3.07–3.00 (m, 2H), 2.86 (m, 2H), 1.92–
1.82 (m, 2H), 1.74–1.56 (m, 4H); 13C NMR (75 MHz, CDCl3) δ
161.84, 151.24, 151.20, 144.8 139.43, 132.08, 129.24, 121.15,
120.26, 116.36, 50.25, 48.99, 32.68, 30.46, 28.74, 27.54, 27.33; MS
(ESI) m/z = 364.17 (M+H)+.
synthesize compounds 3A and 3B
To a cooled mixture of POCl3 (3 mL) and triethylamine (9 mL) was added
compound 2A or 2B (4.54 mmol). The mixture was then heated at
70°C for 24 h. Ice water was poured into the mixture. The resulting
mixture was extracted with ethyl acetate, the organiclayer was separated,
dried over MgSO4, and concentrated in vacuum to obtain the desired
product that was used in the next step without further purification.
Compound 3A gave a yellow powder: yield 45%; mp 130–132°C, while
compound 3B gave a dark brown solid: yield 95%; mp 68–71°C.[28]
4-(4-(m-Chlorophenyl)piperazin-1-yl)-6,7-dihydro-5H-
|
4.1.5 General procedure for hydrazination to
cyclopenta[4,5]thieno[2,3-d]pyrimidine (2X)
synthesize compounds 4A and 4B
The title compound was prepared by reaction of compound 3A and
4-(m-chlorophenyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 80:20) to give beige oil: yield
3.4%; mp 157–159°C; 1H NMR (500 MHz, MeOD) δ 8.41 (s, 1H), 7.22
(t, J = 8.1 Hz, 1H), 7.01 (t, J = 2.2 Hz, 1H), 6.96–6.93 (m, 1H), 6.83 (ddd,
J = 7.9, 1.9,0.8 Hz, 1H), 3.80–3.73 (m, 4H), 3.36 (dd, J = 9.6, 4.6 Hz, 4H),
3.17–3.13 (m, 2H), 3.05 (dd, J = 10.4, 4.2 Hz, 2H), 2.50(dt, J = 14.3,7.3 Hz,
2H); 13C NMR (126 MHz, MeOD) δ 173.71, 161.84, 153.94, 152.21,
141.46, 137.47, 136.01, 131.30, 120.54, 118.44, 116.98, 115.49, 50.18,
49.87, 32.79, 30.53, 29.33; MS (ESI) m/z = 371.14 (M+H)+.
A mixture of the chloro derivative 3A or 3B (2 mmol) and hydrazine
monohydrate (5 equiv.) was dissolved in (10 mL) methanol. The
reaction mixture was kept under reflux for 3 h. After completion of the
reaction (monitored by TLC), the precipitate formed was purely
obtained by vacuum filtration and then washed with diethyl ether
without any further purification. Compound 4A gave a yellow powder:
yield 86.8%, mp 223–225°C whereas compound 4B gave a reddish
brown solid: yield 78%; mp 145–147°C.[30]
4-(4-(m-Chlorophenyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (2Y)
|
4.1.6 General procedures for synthesis of N-
(substituted)-cyclopentenethienopyrimidine-4-amine
and N-(substituted)-cycloheptenethienopyrimidine-4-
amine
The title compound was prepared by reaction of compound 3B and
4-(m-chlorophenyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 5:1) to give a brown solid:
yield 5.9%; mp 134.8–136.1°C; 1H NMR (300 MHz, CDCl3) δ 8.46 (s,
1H), 7.13 (t, J = 8.1 Hz, 1H), 6.86 (s, 1H), 6.78 (d, J = 8.1 Hz, 2H), 3.46 (s,
4H), 3.29 (s, 4H), 3.03 (m, 2H), 2.89–2.83 (m, 2H), 1.91–1.82 (m, 2H), δ
1.71–1.58 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.60, 161.73,
152.25, 151.21, 139.63, 135.07, 131.98, 130.14, 121.19, 119.89,
116.12, 114.27, δ 50.06, 48.52, 32.65, 30.46, 29.71, 28.72, 27.30; MS
(ESI) m/z = 379.21 (M+H)+.
A mixture of compound 3A or 3B (45 mg, 0.2 mmol) and the different
amines (2.0 equiv, 0.4 mmol) in ethanol (3 mL) was refluxed for 24 h.
The reaction mixture was concentrated in vacuum and the residue was
partitioned between water and dichloromethane or ethyl acetate. The
organic layers were dried over MgSO4 and concentrated in vacuum.
The crude product was purified by silica gel flash column chromatog-
raphy to obtain compounds 1X–15X, 17X and 1Y–16Y from 3A and
3B, respectively.[28]
4-(4-(p-Chlorophenyl)piperazin-1-yl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (3X)
4-(4-Phenylpiperazine-1-yl)-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidine (1X)
The title compound was prepared by reaction of compound 3A and
4-(p-chlorophenyl)piperazine according to a general procedure. The
product was purified by CC (hexane/EtOAc, 80:20) to give yellow
The title compound was prepared by reaction of compound 3A and
4-phenylpiperazine according to the general procedure. The product

EL-SHARKAWY ET AL.
11 of 17
|
crystals: yield 94%; mp 172–174°C; 1H NMR (500 MHz, CDCl3) δ 8.50
(s, 1H), 7.25–7.21 (m, 2H), 6.91–6.87 (m, 2H), 3.74–3.70 (m, 4H), 3.33–
3.29 (m, 4H), 3.10–3.06 (m, 2H), 3.05–3.00 (m, 2H), 2.50–2.43 (m, 2H);
13C NMR (126 MHz, CDCl3) δ 173.47, 160.67, 151.67, 149.90, 140.40,
135.66, 129.20, 125.24, 117.68, 117.58, 49.42, 49.30, 31.82, 30.00,
28.36; MS (ESI) m/z = 371.05 (M+H)+.
3.62 (m, 4H), 3.14–3.09 (m, 4H), 3.06 (t, J = 7.0 Hz, 2H), 2.98 (t,
J = 7.3 Hz, 2H), 2.41–2.35 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ
172.34, 159.83, 151.30, 151.25, 143.98, 138.89, 135.94, 118.18,
116.27, 115.50, 50.12, 48.79, 31.32, 29.35, 27.76; MS (ESI)
m/z = 353.20 (M+H)+.
4-(4-(6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)piperazin-1-yl)phenol (5Y)
4-(4-(p-Chlorophenyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (3Y)
The title compound was prepared by reaction of compound 3B and 4-
(p-hydroxyphenyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 2:1) to give a dark brown
solid: yield 76.2%; mp 230–233.6°C; 1H NMR (300 MHz, DMSO-d6) δ
8.90 (s, 1H), 8.48 (s, 1H), 6.84 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H),
3.10 (m, 2H), 2.90 (d, J = 4.7 Hz, 2H), 1.88 (m, 2H), 1.64 (m, 4H);
13C NMR (75 MHz, DMSO-d6) δ 165.37, 161.18, 151.15, 150.92,
143.84, 138.37, 132.12, 120.02, 118.06, 115.41, 49.87, 49.62, 31.85,
29.49, 28.06, 26.80, 26.79; MS (ESI) m/z = 381.17 (M+H)+.
The title compound was prepared by reaction of compound 3B and
4-(p-chlorophenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 8:1) to give an off-
white solid: yield 48%; mp 168.8–171°C; 1H NMR (300 MHz, CDCl3)
δ 8.47 (s, 1H), 7.18–7.14 (m, 2H), 6.84–6.80 (m, 2H), 3.46 (s, 4H),
3.24 (s, 4H), 3.06–3.00 (m, 2H), 2.86 (m, 2H), 1.92–1.83 (m, 2H),
1.69–1.59 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.55, 161.73,
151.22, 149.80, 139.57, 131.99, 129.07, 125.12, 121.16, 117.56,
50.10, 48.97, 32.66, 30.46, 28.72, 27.54, 27.30; MS (ESI)
m/z = 379.21 (M+H)+.
4-(4-(o-Methoxyphenyl)piperazin-1-yl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (6X)
4-(4-(p-Fluorophenyl)piperazin-1-yl)-6,7-dihydro-5H-
The titlecompound was prepared by reactionofcompound 3A and4-(o-
methoxyphenyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 70:30) to give beige
crystals: yield 69.2%; mp 140–142°C; 1H NMR (500 MHz, DMSO-d6) δ
8.43 (s, 1H), 7.00–6.93 (m, 3H), 6.89 (ddd, J = 7.9, 6.5, 2.2 Hz, 1H), 3.80
(s, 3H), 3.68 (dd, J = 11.8, 7.0 Hz, 4H), 3.11 (dd, J = 9.9, 5.2 Hz, 4H), 3.07
(t, J = 7.0 Hz, 2H), 2.98 (t, J = 7.3 Hz, 2H), 2.38 (q, J = 7.4 Hz, 2H);
13C NMR (126 MHz, DMSO-d6) δ 172.35, 159.76, 152.02, 151.30,
140.86, 138.73, 136.02, 122.82, 120.85, 118.24, 116.16, 111.92,
55.39, 50.06, 48.86, 31.38, 29.36, 27.78; MS(ESI) m/z = 367.21 (M+H)+.
cyclopenta[4,5]thieno[2,3-d]pyrimidine (4X)
The title compound was prepared by reaction of compound 3A and
4-(p-fluorophenyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 85:15) to give yellow
crystals: yield 40%; mp 142–144°C; 1H NMR (500 MHz, DMSO-d6) δ
8.44 (s, 1H), 7.11–7.04 (m, 2H), 7.04–6.98 (m, 2H), 3.69–3.64 (m, 4H),
3.26–3.22 (m, 4H), 3.07 (t, J = 7.1 Hz, 2H), 2.99 (t, J = 7.3 Hz, 2H), 2.43–
2.35 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 172.37, 159.78, 156.22
(d,
JCF = 236.0 Hz), 151.34, 147.76, 139.03, 135.82, 117.47 (d,
J
CF = 7.6 Hz), 116.29, 115.34 (d, JCF = 21.9 Hz), 49.01, 48.57, 31.29,
29.35, 27.75; MS (ESI) m/z = 355.18 (M+H)+.
4-(4-(o-Methoxyphenyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (6Y)
4-(4-(p-Fluorophenyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (4Y)
The title compound was prepared by reaction of compound 3B and
4-(o-methoxyphenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 2:1) to give a white
solid: yield 48.4%; mp 180.9–182.1°C; 1H NMR (300 MHz, CDCl3) δ
8.46 (d, J = 1.5 Hz, 1H), 7.00–6.79 (m, 4H), 3.53 (s, 4H), 3.17 (s, 4H),
3.07 (m, 2H), 2.88–2.80 (m, 2H), 1.86 (m, 2H), 1.64 (m, 4H); 13C NMR
(75 MHz, CDCl3) δ 166.50, 161.81, 160.68, 152.58, 151.23, 139.45,
132.06, 129.92, 121.15, 109.10, 104.94, 102.88, 55.26, 50.18, 48.89,
32.67, 30.46, 28.73, 27.53, 27.32; MS (ESI) m/z = 331.17 (M+H)+.
The title compound was prepared by reaction of compound 3B and
4-(p-fluorophenyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 6:1) to give a white solid:
yield 59.2%; mp 168.4–170.2°C; 1H NMR (300 MHz, CDCl3) δ 8.47 (s,
1H), 6.96–6.91 (m, 2H), 6.89–6.84 (m, 2H), 3.48 (s, 4H), 3.20 (s, 4H),
3.06–3.01 (m, 2H), 2.89–2.80 (m, 2H), 1.94–1.82 (m, 2H), 1.69–1.57
(m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.53, 161.77, 157.46 (d,
J
CF = 239.5 Hz), 151.23, 147.82, 139.48, 132.03, 121.13, 118.18 (d,
J
CF = 7.7 Hz), 115.65 (d, JCF = 22.1 Hz), 50.25, 49.96, 32.67, 30.46,
4-(4-(m-Methoxyphenyl)piperazin-1-yl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (7X)
28.74, 27.54, 27.31; MS (ESI) m/z = 383.18 (M+H)+.
The title compound was prepared by reaction of compound 3A and
4-(m-methoxyphenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 80:20) to give beige
powder: yield 63%; mp 112–114°C; 1H NMR (500 MHz, CDCl3) δ 8.50
(s, 1H), 7.23–7.19 (m, 1H), 6.60 (ddd, J = 8.3, 2.4, 0.7 Hz, 1H), 6.52 (t,
J = 2.3 Hz, 1H), 6.46 (ddd, J = 8.2, 2.4, 0.7 Hz, 1H), 3.81 (s, 3H), 3.75–
3.69 (m, 4H), 3.43–3.30 (m, 4H), 3.11–3.06 (m, 2H), 3.05–3.00 (m, 2H),
2.50–2.43 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 173.18, 160.53,
4-(4-(6,7-Dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4-
yl)piperazin-1-yl)phenol (5X)
The title compound was prepared by reaction of compound 3A and
4-(p-hydroxyphenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 60:40) to give brown
powder; yield 35.82%; mp 242–244°C; 1H NMR (500 MHz, DMSO-d6)
δ 8.89 (s, 1H), 8.43 (s, 1H), 6.88–6.81 (m, 2H), 6.70–6.64 (m, 2H), 3.68–

12 of 17
EL-SHARKAWY ET AL.
|
160.44, 152.40, 151.42, 140.00, 135.48, 129.79, 117.28, 108.95,
104.81, 102.70, 55.12, 49.08, 31.60, 29.74, 28.11; MS (ESI)
m/z = 366.95 (M+H)+.
132.88, 131.33, 126.78, 123.70, 119.29, 117.53, 51.92, 49.97, 31.94,
30.00, 28.38, 18.01; MS (ESI) m/z = 350.95 (M+H)+.
4-(4-(o-Tolyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-cyclohepta
[4,5]thieno[2,3-d]pyrimidine (9Y)
4-(4-(m-Methoxyphenyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (7Y)
The title compound was prepared by reaction of compound 3B and
4-(o-tolyl)piperazine according to the general procedure. The product
was purified by CC (hexane/EtOAc, 6:1) to give a white solid: yield
59.7%; mp 127.5–131.4°C; 1H NMR (300 MHz, CDCl3) δ 8.46 (s, 1H),
7.14 (s, 1H), 7.11 (d, J = 7.4 Hz, 1H), 7.02–6.97 (m, 1H), 6.97–6.90 (m,
1H), 3.48 (s, 4H), 3.02 (s, 4H), 2.89–2.80 (m, 2H), 2.26 (s, 3H), 1.86 (m,
4H), 1.74–1.56 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 165.36, 160.96,
150.16, 150.12, 138.16, 131.71, 131.14, 130.17, 125.60, 122.50,
120.06, 118.06, 50.39, 49.75, 31.65, 29.41, 27.76, 26.50, 26.29,
16.83; MS (ESI) m/z = 379.21 (M+H)+.
The title compound was prepared by reaction of compound 3B and
4-(m-methoxyphenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 4:1) to give an off-
white solid: yield 54.4%; mp 118–119°C; 1H NMR (300 MHz, CDCl3) δ
8.47 (s, 1H), 7.20–7.09 (m, 1H), 6.52 (dd, J = 8.2, 2.2 Hz, 1H), 6.45 (t,
J = 2.1 Hz, 1H), 6.39 (dd, J = 8.1, 2.2 Hz, 1H), 3.73 (s, 3H), 3.46 (s, 4H),
3.28 (s, 4H), 3.06–3.00 (m, 2H), 2.84 (m, 2H), 1.92–1.81 (m, 2H), 1.65
(m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.50, 161.81, 160.68, 152.58,
151.23, 139.45, 132.06, 129.92, 121.15, 109.10, 104.94, 102.88,
55.26, 50.18, 48.89, 32.67, 30.46, 28.73, 27.53, 27.32; MS (ESI)
m/z = 395.17 (M+H)+.
4-(4-(m-Tolyl)piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidine (10X)
4-(4-(p-Methoxyphenyl)piperazin-1-yl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (8X)
The title compound was prepared by reaction of compound 3A and
4-(m-tolyl)piperazine according to the general procedure. The product
was purified by CC (hexane/EtOAc, 90:10) to give yellow crystals: yield
73%; mp 104–106°C; 1H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H), 7.19
(dd, J = 10.0, 5.6 Hz, 1H), 6.83–6.78 (m, 2H), 6.75–6.72 (m, 1H), 3.76–
3.70 (m, 4H), 3.37–3.31 (m, 4H), 3.11–3.06 (m, 2H), 3.06–3.00 (m, 2H),
2.50–2.43 (m, 2H), 2.34 (s, 3H); 13C NMR (126 MHz, CDCl3) δ 173.43,
160.75, 151.69, 151.34, 140.22, 139.09, 135.77, 129.19, 121.32,
117.55, 117.35, 113.65, 49.55, 49.44, 31.87, 29.99, 28.37, 21.91; MS
(ESI) m/z = 351.08 (M+H)+.
The title compound was prepared by reaction of compound 3A and
4-(p-methoxyphenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 70:30) to give beige
powder:yield74.9%;mp151–155°C; 1H NMR(500 MHz, CDCl3) δ 8.49
(s, 1H), 7.00–7.1 (m, 2H), 6.88–6.84 (m, 2H), 3.78 (d, J = 3.6 Hz, 4H), 3.76
(s, 3H), 3.32–3.17 (m, 4H), 3.16–3.04 (m, 2H), 3.02 (t, J = 7.3 Hz, 2H),
2.55–2.38 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 173.46, 160.68,
151.68, 140.21, 135.78, 118.85, 117.51, 114.72, 55.73, 51.08, 49.40,
31.88, 29.99, 28.37; MS (ESI) m/z = 367.22 (M+H)+.
4-(4-(m-Tolyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-cyclohepta
[4,5]thieno[2,3-d]pyrimidine (10Y)
4-(4-(p-Methoxyphenyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-
cyclohepta[4,5]thieno[2,3-d]pyrimidine (8Y)
The title compound was prepared by reaction of compound 3B and
4-(m-tolyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 5:1) to give a white
solid: yield 28.3%; mp 130.6–131.8°C; 1H NMR (300 MHz, CDCl3) δ
8.47 (s, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.74 (s, 1H), 6.71 (s, 1H), 6.66 (d,
J = 7.2 Hz, 1H), 3.47 (s, 4H), 3.28 (s, 4H), 3.08–3.00 (m, 2H), 2.88–
2.83 (m, 2H), 2.27 (s, 3H), 1.93–1.83 (m, 2H), 1.69–1.56 (m, 4H);
13C NMR (75 MHz, CDCl3) δ 165.44, 160.81, 150.21, 150.19,
138.37, 137.92, 131.05, 128.03, 120.15, 120.11, 116.17, 112.48,
49.23, 48.05, 31.64, 29.42, 27.70, 26.49, 26.29, 20.75; MS (ESI)
m/z = 379.14 (M+H)+.
The title compound was prepared by reaction of compound 3B and
4-(p-methoxyphenyl)piperazine according to the general procedure.
The product was purified by CC (hexane/EtOAc, 4:1) to give yellow
powder: yield 54.4%; mp 131.2–132°C; 1H NMR (300 MHz, CDCl3) δ
8.47 (s, 1H), 6.88 (d, J = 8.9 Hz, 2H), 6.79 (d, J = 8.7 Hz, 2H), 3.71 (s, 3H),
3.48 (s, 4H), 3.17 (s, 4H), 3.06–2.99 (m, 2H), 2.88–2.81 (m, 2H), 1.86 (m,
2H), 1.72–1.56 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.47, 161.83,
154.17, 151.24, 145.52, 139.33, 132.10, 121.10, 118.49, 114.55,
55.62, 50.42, 50.35, 32.68, 30.46, 28.76, 27.54, 27.33; MS (ESI)
m/z = 395.17 (M+H)+.
4-(4-(o-Tolyl)piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidine (9X)
4-(4-(p-Tolyl)piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidine (11X)
The title compound was prepared by reaction of compound 3A and
4-(o-tolyl)piperazine according to the general procedure. The product
was purified by CC (hexane/EtOAc, 80:20) to give white crystals: yield
72.89%; mp 124–126°C; 1H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H),
7.23–7.17 (m, 2H), 7.07 (dd, J = 7.9, 1.2 Hz, 1H), 7.02 (td, J = 7.4,
1.2 Hz, 1H), 3.77–3.67 (m, 4H), 3.11 (dd, J = 5.1, 3.5 Hz, 2H), 3.09–3.06
(m, 4H), 3.05–3.00 (m, 2H), 2.50–2.43 (m, 2H), 2.36 (s, 3H); 13C NMR
(126 MHz, CDCl3) δ 173.40, 160.90, 151.70, 151.26, 140.00, 135.89,
The title compound was prepared by reaction of compound 3A and
4-(p-tolyl)piperazine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 80:20) to give white
crystals: yield 68.98%; mp 164–166°C; 1H NMR (500 MHz, CDCl3) δ
8.49 (s, 1H), 7.14–7.08 (m, 2H), 6.93 (d, J = 8.5 Hz, 2H), 3.77–3.73
(m, 4H), 3.33–3.28 (m, 4H), 3.10–3.05 (m, 2H), 3.04–2.99 (m, 2H),
2.49–2.43 (m, 2H), 2.29 (s, 3H); 13C NMR (126 MHz, CDCl3) δ
173.33, 160.63, 151.60, 148.81, 140.22, 135.74, 130.34, 129.90,

EL-SHARKAWY ET AL.
13 of 17
|
117.48, 116.98, 50.15, 49.28, 31.84, 29.96, 28.35, 20.59; MS (ESI)
m/z = 351.20 (M+H)+.
4-(4-Methylpiperazin-1-yl)-6,7,8,9-tetrahydro-5H-cyclohepta
[4,5]thieno[2,3-d]pyrimidine (13Y)
The title compound was prepared by reaction of compound 3B and 4-
methylpiperazine according to the general procedure. The product was
purified by CC (1% TEA in EtOAc) to give a white solid: yield 67%; mp
115.2–116°C; 1H NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 3.36 (s, 4H),
3.04–2.90 (m, 2H), 2.84 (m, 2H), 2.51 (s, 4H), 2.29 (s, 3H), 1.93–1.77 (m,
2H), 1.73–1.53 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 166.37, 161.77,
151.19, 139.02, 132.15, 120.92, 54.66, 50.11, 46.18, 32.69, 30.44,
28.75, 27.51, 27.33; MS (ESI) m/z = 395.17 (M+H)+.
4-(4-(p-Tolyl)piperazin-1-yl)-6,7,8,9-tetrahydro-5H-cyclohepta
[4,5]thieno[2,3-d]pyrimidine (11Y)
The title compound was prepared by reaction of compound 3B and 4-
(p-tolyl)piperazine according to the general procedure. The product
was purified by CC (hexane/EtOAc, 8:1) to give a white solid: yield
47.3%; mp 163.2–164.8°C; 1H NMR (300 MHz, CDCl3) δ 8.47 (s,
J = 4.7 Hz, 1H), 7.03 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H), 3.47 (s,
4H), 3.23 (s, 4H), 3.08–2.99 (m, 2H), 2.90–2.72 (m, 2H), 2.21 (s, 3H),
1.88–1.81 (m, 2H), 1.72–1.56 (m, 4H); 13C NMR (75 MHz, CDCl3) δ
166.48, 161.85, 151.24, 149.09, 139.36, 132.10, 129.82, 129.75,
121.12, 116.69, 50.27, 49.53, 32.68, 30.46, 28.75, 27.53, 27.34,
20.46; MS (ESI) m/z = 379.21 (M+H)+.
N-Phenyl-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-amine (14X)
The title compound was prepared by reaction of compound 3A and
aniline according to the general procedure. The product was purified
by CC (hexane/EtOAc, 80:20) to give brown crystals: yield 79.3%; mp
194–196°C; 1H NMR (500 MHz, CDCl3) δ 8.51 (s, 1H), 7.71–7.62 (m,
2H), 7.40 (dd, J = 10.8, 5.2 Hz, 2H), 7.14 (t, J = 7.4 Hz, 1H), 7.00 (s, 1H),
3.13 (t, J = 7.2 Hz, 2H), 3.05 (dd, J = 10.6, 4.2 Hz, 2H), 2.60 (dt, J = 14.5,
7.3 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 171.45, 154.50, 152.41,
140.44, 138.51, 133.91, 129.31, 124.26, 121.17, 114.21, 29.76,
29.48, 28.19; MS (ESI) m/z = 268.04 (M+H)+.
1-(4-(6,7-Dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-pyrimidin-4-
yl)piperazin-1-yl)ethan-1-one (12X)
The title compound was prepared by reaction of compound 3A and
N-acetylpiperazine according to the general procedure. The product
was purified by CC (EtOAc/TEA, 100:1) to give yellow oil: yield 80%;
1H NMR (500 MHz, DMSO-d6) δ 8.42 (s, 1H), 3.60 (dd, J = 6.8,
3.2 Hz, 4H), 3.55 (dd, J = 6.6, 2.7 Hz, 2H), 3.49 (dd, J = 6.4, 3.9 Hz,
2H), 3.03 (dd, J = 13.0, 5.8 Hz, 2H), 2.97 (dd, J = 7.9, 6.7 Hz, 2H),
2.43–2.32 (m, 2H), 2.04 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ
172.40, 168.49, 159.65, 151.23, 138.96, 135.85, 116.19, 48.63,
48.51, 45.33, 40.63, 31.22, 29.35, 27.75, 21.27; MS (ESI)
m/z = 303.12 (M+H)+.
N-Phenyl-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]-
pyrimidin-4-amine (14Y)
The title compound was prepared by reaction of compound 3B and
aniline according to the general procedure. The product was purified
by CC (hexane/EtOAc, 8:1) to give a light brown powder: yield 47.2%;
mp 153.9–155.5°C; 1H NMR (300 MHz, CDCl3) δ 8.40 (s, 1H), 7.52 (d,
J = 8.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.09–7.00 (m, 1H), 3.12–3.02
(m, 2H), 2.92–2.80 (m, 2H), 1.81 (m, 6H); 13C NMR (75 MHz, CDCl3) δ
165.37, 154.95, 152.21, 138.55, 138.53, 129.78, 129.10, 124.15,
121.64, 117.87, 30.41, 30.36, 29.07, 26.97, 26.36; MS (ESI)
m/z = 379.21 (M+H)+.
1-(4-(6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)piperazin-1-yl)ethanone (12Y)
The title compound was prepared by reaction of compound 3B and
N-acetylpiperazine according to the general procedure. The product
was purified by CC (EtOAc) to give a white solid: yield 35.7%; mp
144.4–146.3°C; 1H NMR (300 MHz, CDCl3) δ 8.45 (s, 1H), 3.77–3.52
(m, 4H), 3.32 (m, 4H), 3.02–2.97 (m, 2H), 2.89–2.82 (m, 2H), 2.08 (s,
3H), 1.92–1.83 (m, 2H), 1.72–1.55 (m, 4H); 13C NMR (75 MHz,
CDCl3) δ 169.26, 166.71, 161.48, 151.13, 139.91, 131.77, 121.15,
50.89, 49.53, 45.78, 40.97, 32.62, 30.45, 28.63, 21.39; MS (ESI)
m/z = 331.17 (M+H)+.
N-Benzyl-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-amine (15X)
The title compound was prepared by reaction of compound 3A and
benzylamine according to the general procedure. The product was
purified by CC (hexane/EtOAc, 90:10) to give beige powder; yield
87%; mp 155–157°C; 1H NMR (500 MHz, CDCl3) δ 8.42 (s, 1H), 7.39–
7.33 (m, 4H), 7.32–7.28 (m, 1H), 5.37 (t, J = 4.8 Hz, 1H), 4.82 (d,
J = 5.6 Hz, 2H), 3.02–2.93 (m, 4H), 2.62–2.43 (m, 2H); 13C NMR
(126 MHz, CDCl3) δ 170.78, 156.72, 153.11, 139.06, 138.64, 134.39,
128.96, 127.73, 127.67, 113.40, 44.82, 29.63, 29.34, 28.10; MS (ESI)
m/z = 282.094 (M+H)+.
4-(4-Methylpiperazin-1-yl)-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidine (13X)
The title compound was prepared by reaction of compound 3A and
4-methylpiperazine according to the general procedure. The product
was purified by CC (EtOAc/TEA, 100:1) to give beige crystals: yield
77%; mp 100–102°C; 1H NMR (500 MHz, CDCl3) δ 8.46 (s, 1H),
3.67–3.59 (m, 4H), 3.06–3.02 (m, 2H), 3.00 (ddd, J = 8.3, 3.3, 1.6 Hz,
2H), 2.63–2.59 (m, 4H), 2.47–2.41 (m, 2H), 2.39 (s, 3H); 13C NMR
N-Benzyl-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]-
pyrimidin-4-amine (15Y)
The title compound was prepared by reaction of compound 3B and
benzylamine according to the general procedure. The product was
purified by CC (hexane/EtOAc, 4.5:1) to give white crystals: yield
53.9%; mp 98.5–100.7°C; 1H NMR (300 MHz, CDCl3) δ 8.41 (s, 1H),
(126 MHz, CDCl3)
δ 173.40, 160.56, 151.65, 140.00, 135.81,
117.38, 54.91, 49.04, 46.15, 31.85, 29.97, 28.37; MS (ESI)
m/z = 275.08 (M+H)+.

14 of 17
EL-SHARKAWY ET AL.
|
7.38–7.27 (m, 5H), 5.58 (s, 1H), 4.82 (d, J = 5.4 Hz, 2H), 2.97 (m, 2H),
2.89 (m, 2H), 1.82 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 164.35,
157.14, 152.65, 138.69, 137.17, 130.14, 128.83, 127.66, 127.55,
117.19, 45.07, 30.34, 30.30, 28.88, 27.03, 26.25; MS (ESI)
m/z = 310.15 (M+H)+.
(E)-4-(2-(o-Bromobenzylidene)hydrazinyl)-6,7,8,9-tetrahydro-
5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (18Y)
The title compound was prepared by reaction of compound 4B and
o-bromobenzaldehyde according to the general procedure for
hydrazone synthesis. The product was purified by CC (hexane/
EtOAc, 3:1) to give orange solid: yield 33.8%; mp 180.5–183.1°C;
1H NMR (300 MHz, CDCl3) δ 10.45 (s, 1H), 8.76 (s, 1H), 7.97 (d,
J = 7.7 Hz, 1H), 7.63 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.25 (t, J = 7.5 Hz,
1H), 7.18–7.11 (m, 1H), 3.43 (m, 2H), 2.79 (m, 2H), 1.83 (m, 2H),
1.63–1.53 (m, 4H); 13C NMR (75 MHz, CDCl3) δ 153.01, 140.71,
138.63, 135.87, 134.20, 133.41, 132.06, 130.94, 129.95, 128.06,
127.34, 124.93, 124.58, 32.63, 30.06, 28.47, 27.73, 27.28; MS (ESI)
m/z = 402.76 (M+H)+.
N-(2-Cyclohexylethyl)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]-
thieno[2,3-d]pyrimidin-4-amine (16Y)
The title compound was prepared by reaction of compound 3B and 2-
cyclohexylethylamine according to the general procedure. The product
was purified by CC (hexane/EtOAc, 5:1) to give a yellow solid: yield
50.67%; mp 113.4–115.6°C; 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H),
5.17 (s, 1H), 3.56–3.47 (m, 2H), 2.97–2.88 (m, 2H), 2.83–2.76 (m, 2H),
1.88–1.43 (m, 14H), 1.37–1.02 (m, 5H); 13C NMR (75 MHz, CDCl3) δ
163.90, 157.37, 152.67, 136.79, 130.20, 117.10, 39.11, 36.93, 35.78,
33.30, 30.42, 30.29, 28.87, 27.07, 26.50, 26.29, 26.27; MS (ESI)
m/z = 330.20 (M+H)+.
(E,Z)-4-(2-(m-Bromobenzylidene)hydrazinyl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (19X)
The title compound was prepared by reaction of compound 4A and m-
bromobenzaldehyde according to the general procedure for hydra-
zone synthesis. The product was purified by CC (hexane/EtOAc,
50:50) to give yellow powder: yield 87.73%; mp 193–195°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.20–11.09 (m, 1H), 8.47–8.24 (m, 2H), 7.90–
7.80 (m, 1H), 7.68–7.52 (m, 2H), 7.40 (dt, J = 13.8, 7.8 Hz, 1H), 3.12 (t,
J = 7.2 Hz, 1H), 2.95–3 (m, 3H), 2.42–2.30 (m, 2H); 13C NMR
(126 MHz, DMSO-d6) δ 173.21, 162.83, 154.72, 151.57, 151.06,
149.05, 143.63, 143.26, 139.59, 138.46, 137.93, 136.83, 136.71,
132.05, 131.86, 131.00, 130.61, 129.29, 129.11, 127.22, 126.00,
122.20, 122.15, 116.61, 112.26, 32.38, 29.67, 29.48, 29.09, 27.46,
27.05; MS (ESI) m/z = 373.06 (M+H)+.
N-(Cyclohexylmethyl)-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidin-4-amine (17X)
The title compound was prepared by reaction of compound 3A and
cyclohexylmethylamine according to the general procedure. The
product was purified by CC (hexane/EtOAc, 70:30) to give white
powder: yield 100%; mp 122–124°C; 1H NMR (500 MHz, CDCl3) δ
8.37 (s, 1H), 5.14 (s, 1H), 3.43 (dd, J = 6.8, 5.8 Hz, 2H), 3.05–2.92 (m,
4H), 2.58–2.49 (m, 2H), 1.83–1.72 (m, 4H), 1.71–1.57 (m, 2H), 1.31–
1.11 (m, 3H), 1.01 (qd, J = 12.0, 2.7 Hz, 2H); 13C NMR (126 MHz,
CDCl3) δ 170.64, 157.17, 153.30, 138.57, 134.39, 113.41, 47.01,
37.94, 31.07, 29.62, 29.40, 28.12, 26.57, 25.98; MS (ESI) m/z = 288.74
(M+H)+.
(E)-4-(2-(m-Bromobenzylidene)hydrazinyl)-6,7,8,9-tetrahydro-
5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (19Y)
|
The title compound was prepared by reaction of compound 4B and m-
bromobenzaldehyde according to the general procedure for hydra-
zone synthesis. The product was purified by CC (hexane/EtOAc, 3:1) to
give a brown solid: yield 28.3%; mp 214.8–217.8°C; 1H NMR
(300 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.41 (s, 1H), 8.31–8.29 (m,
1H), 7.87 (d, J = 7.7 Hz, 1H), 7.83 (d, J = 3.7 Hz, 1H), 7.59 (ddd, J = 8.0,
1.9, 0.9 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 3.52–3.42 (m, 2H), 2.92–2.77
(m, 2H), 1.86 (m, 2H), 1.70–1.54 (m, 4H); 13C NMR (75 MHz, DMSO-d6)
δ 156.78, 151.86, 150.47, 143.92, 138.38, 137.44, 137.15, 132.61,
131.11, 129.84, 127.81, 122.69, 119.73, 32.57, 29.68, 28.33, 27.80,
27.36; MS (ESI) m/z = 400.97 (M+H)+.
4.1.7 General procedure for the synthesis of
hydrazones
Compound 4A or 4B (0.2 g, 0.85 mmol) and the respective aldehyde
(1.18 equiv, 1 mmol) were dissolved in EtOH (10–15 mL). The
mixture was refluxed for 24 h. After cooling to room temperature,
ethanol was removed in vacuum and silica gel flash CC was done on
most of the compounds followed by washing of the compounds with
81% EtOH to obtain compounds 18X–26X and 18Y–26Y, respec-
[30]
tively.
(E)-4-(2-(o-Bromobenzylidene)hydrazinyl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (18X)
(E,Z)-4-(2-(p-Bromobenzylidene)hydrazinyl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (20X)
The title compound was prepared by reaction of compound 4A and o-
bromobenzaldehyde according to the general procedure for hydra-
zone synthesis. The product was purified by CC (hexane/EtOAc,
70:30) to give orange crystals: yield 75.5%; mp 199–201°C; 1H NMR
(500 MHz, CDCl3) δ 8.42 (d, J = 7.5 Hz, 2H), 8.06 (d, J = 7.7 Hz, 1H),
7.59–7.50 (m, 1H), 7.35–7.26 (m, 1H), 7.20 (td, J = 8.0, 1.7 Hz, 1H),
3.15 (dd, J = 17.1, 10.2 Hz, 2H), 3.04–2.96 (m, 2H), 2.55–2.42 (m, 2H);
MS (ESI) m/z = 372.90 (M+H)+.
The title compound was prepared by reaction of compound 4A and p-
bromobenzaldehyde according to the general procedure for hydra-
zone synthesis. The product was purified by CC (hexane/EtOAc,
50:50) to give yellow powder: yield 80%; mp 205–209°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.05–11.08 (m, 1H), 8.44–8.22 (m, 2H), 7.92–
7.79 (m, 1H), 7.66–7.59 (m, 3H), 3.10 (t, J =7.2 Hz, 1H), 2.96 (dd,
J = 15.6, 8.2 Hz, 2H), 2.90 (t, J = 7.2 Hz, 1H), 2.35 (tt, J = 14.7, 7.4 Hz,

EL-SHARKAWY ET AL.
15 of 17
|
2H); 13C NMR (126 MHz, DMSO-d6) δ 173.17, 162.71, 154.78,
151.57, 151.43, 148.87, 143.87, 143.68, 139.60, 138.34, 137.86,
136.82, 134.75, 133.65, 131.84, 131.47, 129.52, 128.82, 122.86,
122.59, 116.64, 112.27, 32.32, 29.67, 29.47, 29.08, 27.46, 27.09; MS
(ESI) m/z = 373.08 (M+H)+.
(E)-4-(2-(5-Bromo-2-methoxybenzylidene)hydrazinyl)-6,7-
dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (22X)
The title compound was prepared by reaction of compound 4A and 5-
bromo-2-methoxybenzaldehyde according to the general procedure
for hydrazone synthesis. The product was purified by CC (DCM/
MeOH, 100:0.5) to give an orange powder: yield 77%; mp 258–260°C;
1H NMR (500 MHz, DMSO-d6) δ 11.63–11.52 (m, 1H), 8.55–8.48 (m
2H), 7.92–7.84 (m, 1H), 7.58–7.50 (m, 1H), 7.13–7.02 (m, 1H), 3.87 (d,
J = 2.8 Hz, 3H), 3.15–3.04 (m, 1H), 3.02–2.89 (m, 3H), 2.36 (dt, J = 11.7,
4.9 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ 156.58, 154.64, 151.67,
143.67, 140.82, 138.86, 132.93, 127.99, 127.80, 127.61, 114.27,
114.14, 113.57, 56.07, 29.49, 27.34, 27.05; MS (ESI) m/z = 402.98
(M+H)+.
(E)-4-(2-(p-Bromobenzylidene)hydrazinyl)-6,7,8,9-tetrahydro-
5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (20Y)
The title compound was prepared by reaction of compound 4B and
p-bromobenzaldehyde according to the general procedure for
hydrazone synthesis. The product was purified by CC (hexane/
EtOAc, 4:1) to give an off-white solid: yield 76.09%; mp 230.3–
232.5°C; 1H NMR (300 MHz, DMSO-d6) δ 11.93 (d, J = 2.9 Hz, 1H),
8.42 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 3.6 Hz, 1H), 7.65 (d,
J = 8.4 Hz, 2H), 3.47 (m, 2H), 2.87–2.77 (m, 2H), 1.86 (m, 2H), 1.74–
1.39 (m, 4H); 13C NMR (75 MHz, DMSO-d6) δ 156.65, 152.22,
150.28, 143.96, 137.37, 137.15, 135.20, 131.96, 130.08, 123.43,
119.75, 32.58, 29.67, 28.32, 27.80, 27.36; MS (ESI) m/z = 400.99
(E)-4-(2-(5-Bromo-2-methoxybenzylidene)hydrazinyl)-6,7,8,9-
tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (22Y)
The title compound was prepared by reaction of compound 4B and 5-
bromo-2-methoxybenzaldehyde according to the general procedure
for hydrazone synthesis. The product was purified by CC (hexane/
EtOAc, 5:1) to give an yellow solid: yield 16%; mp 237.2–238.5°C;
1H NMR (300 MHz, CDCl3) δ 10.50 (s, 1H), 8.70 (s, 1H), 8.05 (s, 1H),
7.65 (s, 1H), 7.35 (dd, J = 8.8, 2.5 Hz, 1H), 6.75 (t, J = 14.2 Hz, 1H), 3.80
(s, 3H), 3.41 (m, 2H), 2.78 (m, 2H), 1.83 (m, 2H), 1.67 (m, 4H); 13C NMR
(101 MHz, CDCl3) δ 157.27, 156.04, 148.50, 143.22, 140.90, 138.39,
137.19, 135.52, 133.35, 128.94, 125.79, 120.25, 112.99, 55.82,
32.36, 29.97, 29.68, 27.64, 27.20; MS (ESI) m/z = 430.98 (M+H)+.
(M+H)+.
(E,Z)-4-(2-(2-Fluorobenzylidene)hydrazinyl)-6,7-dihydro-5H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine (21X)
The title compound was prepared by reaction of compound 4A and o-
fluorobenzaldehyde according to the general procedure for hydrazone
synthesis. The product was purified by CC (hexane/EtOAc, 60:40) to
give yellow powder: yield 80%; mp 190–192°C; 1H NMR (500 MHz,
DMSO-d6) δ 11.64–11.55 (m, 1H), 8.50 (d, J = 15.2 Hz, 1H), 8.43–8.32
(m, 1H), 7.92–7.79 (m, 1H), 7.49–7.39 (m, 1H), 7.33–7.22 (m, 2H), 3.11
(t, J = 7.2 Hz, 1H), 2.93 (dt, J = 13.6, 7.3 Hz, 3H), 2.40–2.28 (m, 2H);
13C NMR (126 MHz, DMSO-d6) δ 173.25, 160.59 (d, JCF = 249.3 Hz),
154.68, 151.63, 149.18, 144.69 (d, JCF = 3.1 Hz), 143.72, 139.72,
(E)-4-(2-(o-Fluoro-p-methoxybenzylidene)hydrazinyl)-6,7-
dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (23X)
The title compound was prepared by reaction of compound 4A and o-
fluoro-4-methoxybenzaldehyde according to the general procedure
for hydrazone synthesis. The product was purified by CC (hexane/
EtOAc, 70:30) to give a brown powder: yield 87%; mp 207–209°C;
1H NMR (500 MHz, DMSO-d6) δ 11.60–11.51 (m, 1H), 8.40 (d,
J = 5.5 Hz, 2H), 7.79 (s, 1H), 6.90 (t, J = 11.1 Hz, 2H), 3.82 (s, 3H), 3.36
(s, 1H), 3.11 (s, 1H), 2.95 (s, 2H), 2.38–2.28 (m, 2H); MS (ESI)
m/z = 343.15 (M+H)+.
138.48, 138.02, 137.58 (d,
JCF = 4.7 Hz), 136.90, 131.56 (d,
J
CF = 9.2 Hz), 131.37 (d, JCF = 8.6 Hz), 127.45 (d, JCF = 1.9 Hz),
126.62 (d, JCF = 2.0 Hz), 124.97 (d, JCF = 3.2 Hz), 124.54, 122.95,
122.87, 122.14, 122.06, 116.65, 116.04 (d, JCF = 20.6 Hz), 115.80,
112.33, 32.41, 29.71, 29.54, 29.16, 27.52, 27.14; MS (ESI)
m/z = 313.13 (M+H)+.
(E)-4-(2-(o-Fluoro-p-methoxybenzylidene)hydrazinyl)-6,7,8,9-
tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (23Y)
The title compound was prepared by reaction of compound 4B and
o-fluoro-p-methoxybenzaldehyde according to the general proce-
dure for hydrazone synthesis. The product was purified by CC
(hexane/EtOAc, 4:1) to give an off-white solid: yield 73%; mp 230.3–
235.8°C; 1H NMR (300 MHz, DMSO-d6) δ 11.86 (d, J = 2.5 Hz, 1H),
8.46 (s, 1H), 8.30 (dd, J = 11.3, 6.6 Hz, 1H), 7.80 (d, J = 3.7 Hz, 1H),
6.96–6.90 (m, 1H), 6.90–6.87 (m, 1H), 3.84 (s, 3H), 3.50–3.43 (m,
2H), 2.87–2.76 (m, 2H), 1.85 (m, 2H), 1.61 (m, 4H); 13C NMR
(75 MHz, DMSO-d6) 162.65, 162.50, 162.30 (d, JCF = 253.1 Hz),
156.36, 149.88, 145.50 (d, JCF = 3.2 Hz), 143.97, 137.21, 128.79 (d,
(E)-4-(2-(o-Fluorobenzylidene)hydrazinyl)-6,7,8,9-tetrahydro-
5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (21Y)
The title compound was prepared by reaction of compound 4B and o-
fluorobenzaldehyde according to the general procedure for hydrazone
synthesis. The product was purified by CC (hexane/EtOAc, 4:1) to give
a light brown solid: yield 32.37%; mp 173.7–178.3°C; 1H NMR
(300 MHz, CDCl3) δ 8.63 (s, 1H), 7.97 (td, J = 7.6, 1.5 Hz, 2H), 7.27 (m,
1H), 7.05 (m, 2H), 3.39 (m, 2H), 2.84–2.72 (m, 2H), 1.88–1.80 (m, 2H),
1.69–1.61 (m, 4H); 13C NMR (101 MHz, CDCl3) δ 162.78, 160.91, δ
159.70 (d, JCF = 244.2 Hz), 156.79, 143.67, 135.50, 133.08 (d, JCF
=8.3 Hz), 132.05 (d, JCF = 9.5 Hz), 131.30 (d, JCF = 8.5 Hz), 127.30,
124.18 (d,
JCF = 3.5 Hz), 116.44 (d, JCF = 20.7 Hz), 115.94 (d,
J
CF = 4 Hz), 119.70, 115.68 (d, JCF =10.4 Hz), 112.01, 101.60 (d,
JCF = 21.1 Hz), 30.52, 29.68, 28.90, 27.81, 27.29; MS (ESI)
m/z = 341.1 (M+H)+.
J
CF = 25.1 Hz), 56.37, 32.57, 29.65, 28.31, 27.82, 27.33; MS (ESI)
m/z = 371.1 (M+H)+.

16 of 17
EL-SHARKAWY ET AL.
|
(E,Z)-4-(2-(Naphthalen-1-ylmethylene)hydrazinyl)-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (24X)
J = 8.5 Hz, 1H), 3.52 (m, 2H), 2.85 (m, 2H), 1.87 (m, 2H), 1.65 (m, 4H);
13C NMR (101 MHz, DMSO-d6) δ 156.86, 156.17, 154.44, 147.82,
144.05, 137.41, 137.15, 135.00, 128.15, 128.03, 127.60, 126.09,
124.76, 122.95, 119.65, 119.42, 113.51, 32.53, 29.63, 28.40, 27.75,
27.33; MS (ESI) m/z = 389.10 (M+H)+.
The title compound was prepared by reaction of compound 4A and
naphthaldehyde according to the general procedure for hydrazone
synthesis. The product was purified by CC (DCM/MeOH, 100:1) to give
an orange powder: yield 80%; mp 210–212°C; 1H NMR (500 MHz,
DMSO-d6) δ 12.01–11.20 (m, 1H), 8.54–8.31 (m, 2H), 8.16–8.08 (m,
1H), 8.03–7.78 (m, 4H), 7.58–7.53 (m, 2H), 3.21 (t, J = 7.2 Hz, 1H), 2.99
(t, J = 7.3 Hz, 2H), 2.92 (t, J = 7.2 Hz, 1H), 2.42–2.33 (m, 2H); 13C NMR
(126 MHz, DMSO-d6) δ 173.55, 173.17, 162.60, 154.84, 152.75,
151.64, 148.70, 145.27, 143.78, 139.67, 138.24, 137.84, 136.94,
133.70, 133.36, 133.30, 132.96, 132.11, 128.88, 128.51, 128.27,
128.19, 128.14, 127.97, 127.79, 127.74, 126.95, 126.83, 126.77,
126.63, 123.94, 123.00, 116.72, 112.27, 32.43, 29.72, 29.51, 29.11,
27.50, 27.12; MS (ESI) m/z = 345.17 (M+H)+.
(E)-6-((2-(6,7-Dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)hydrazono)methyl)naphthalen-2-ol (26X)
The title compound was prepared by reaction of compound 4A and 6-
hydroxy-2-naphthaldehyde according to the general procedure for
hydrazone synthesis. The product was purified by CC (hexane/EtOAc,
70:30) to give a green powder: yield 38%; mp 299–301°C; 1H NMR
(500 MHz, DMSO-d6)δ 10.03 (s, 1H), 8.65 (s, 1H), 8.39 (s, 1H), 8.07 (s, 2H),
7.84 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.20–7.10 (m, 2H), 3.19 (t,
J = 6.8 Hz, 2H), 2.99 (t, J = 7.1 Hz, 2H), 2.47–2.37 (m, 2H); 13C NMR
(126 MHz, DMSO-d6) δ 157.00, 151.26, 148.86, 145.65, 137.35, 135.95,
130.30, 130.12, 130.10, 128.23, 127.24, 126.75, 123.50, 119.34, 114.35,
110.31, 109.16, 30.28, 29.47, 27.45; MS (ESI) m/z = 361.15 (M+H)+.
(E)-4-(2-(Naphthalen-1-ylmethylene)hydrazinyl)-6,7,8,9-
tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidine (24Y)
The title compound was prepared by reaction of compound 4B and
naphthaldehyde according to the general procedure for hydrazone
synthesis. The product was purified by CC (hexane/EtOAc, 3:1) to give
white solid: yield 67%; mp 185.7–187.1°C; 1H NMR (300 MHz, CDCl3) δ
10.47 (s, 1H), 9.17 (s, 1H), 8.53 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 7.2 Hz, 1H),
7.83 (s, 1H), 7.81 (s, 1H), 7.62 (s, 1H), 7.52 (d, J = 6.8 Hz, 1H), 7.47 (dd,
J = 4.8, 2.8 Hz, 1H), 7.43 (dd, J = 7.2, 3.2 Hz, 1H), 3.61–3.49 (m, 2H),
2.93–2.78 (m, 2H), 1.93 (m, 2H), 1.75 (m, 4H); 13CNMR(75 MHz, CDCl3)δ
155.91, 153.09, 149.90, 140.93, 138.47, 137.21, 133.92, 131.30, 130.98,
130.42, 128.83, 126.87, 126.64, 126.06, 125.39, 123.88, 120.27, 32.68,
30.09, 28.54, 27.75, 27.34; MS (ESI) m/z = 402.76 (M+H)+.
(E)-6-((2-(6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)hydrazono)methyl)naphthalen-2-ol (26Y)
The title compound was prepared by reaction of compound 4B and 6-
hydroxy-2-naphthaldehyde according to the general procedure for
hydrazone synthesis. The product was purified by CC (5% acetone in
DCM) to give brown solid: yield 33%; mp 232.3–237°C; 1H NMR
(300 MHz, DMSO-d6) δ 11.92 (s, 1H), δ 10.01 (s, 1H), 8.54 (d,
J = 13.7 Hz, 1H), 8.28 (d, J = 8.6 Hz, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 7.81
(d, J = 10.8 Hz, 1H), 7.77 (s, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.17 (dd,
J = 8.7, 2.4 Hz, 1H), 3.55 (m, 2H), 2.87 (m, 2H), 1.92 (m, 2H), 1.68 (m,
4H); 13C NMR (101 MHz, DMSO-d6) δ 156.80, 156.13, 153.88,
149.56, 144.02, 137.15, 137.11, 135.98, 130.57, 130.40, 129.53,
127.84, 126.72, 124.68, 119.78, 119.46, 109.58, δ 32.56, 29.64,
28.30, 27.80, 27.37; MS (ESI) m/z = 389.14 (M+H)+.
(E)-2-((2-(6,7-Dihydro-5H-cyclopenta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)hydrazono)methyl)naphthalen-1-ol (25X)
The title compound was prepared by reaction of compound 4A and 1-
hydroxy-2-naphthaldehyde according to the general procedure for
hydrazone synthesis. The product was purified by CC (hexane/EtOAc,
70:30) to give a yellow powder: yield 72%; mp 266–268°C; 1H NMR
(500 MHz, DMSO-d6) δ 12.88 (s, 1H), 10.54 (s, 1H), 8.75 (s, 1H), 8.55 (s,
1H), 8.32(d, J = 7.2 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.57 (dd, J = 5.5, 4.2 Hz,
1H), 7.55 (d, J = 7.5 Hz, 1H), 7.53–7.49(m,1H),7.43(d,J = 8.5 Hz, 1H), 3.19
(s, 2H), 2.97 (s, 2H), 2.44 (d, J = 5.9 Hz,2H);13C NMR (126 MHz, DMSO-d6)
δ 172.57, 157.34, 155.13, 153.55, 153.36, 148.60, 139.69, 136.19,
135.09, 128.45, 128.36, 127.10, 126.45, 125.32, 123.36, 119.62, 112.85,
30.21, 30.14, 28.15; MS (ESI) m/z = 361.16 (M+H)+.
|
4.2 PDE assay
Purified PDE5, 7 and 9 (BPS Biosciences) was added to the wells of
black 96-well non-binding plates at concentrations required to exhibit
80% activity. Immediately, the protein was treated with compound or
vehicle control and allowed to incubate for 30 min at 37°C. Following
this incubation, 25 nM FL-cGMP (Molecular Devices) was added to
each assay well and allowed to incubate for 1.5 h at 30°C. After
incubation, IMAP FP Phosphodiesterase Evaluation Assay (Molecular
Devices) binding reagent was added to each well and the plates were
incubated for an additional 10 min at 30°C. FP was measured
according to manufacturer's specifications using a Biotek Synergy 4
plate reader. All synthesized compounds were tested for their ability to
inhibit recombinant PDE5 and PDE9 using cGMP as a substrate and
PDE7 using cAMP as a substrate, respectively. For enzyme assays, the
IC50 value was determined by testing a range of four concentrations
with at least two replicates per concentration, which was repeated at
least twice to confirm reproducibility of IC50 values and extrapolation
of the curve. Concentration-dependent curves were analyzed using
(E)-2-((2-(6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]-
pyrimidin-4-yl)hydrazono)methyl)naphthalen-1-ol (25Y)
The title compound was prepared by reaction of compound 4B and
1-hydroxy-2-naphthaldehyde according to the general procedure for
hydrazone synthesis. The product was purified by CC (hexane/EtOAc,
2.5:1) to give brown solid: yield 33%; mp 233.2–234.9°C; 1H NMR
(300 MHz, DMSO-d6) δ 11.81 (s, 1H), 11.45 (s, 1H), 8.80 (s, 1H), 8.31
(dd, J = 6.6, 2.9 Hz, 1H), 7.88 (dd, J = 6.4, 2.9 Hz, 1H), 7.85 (d, J = 4.4 Hz,
1H), 7.75–7.68 (m, 1H), 7.62–7.57 (m, 1H), 7.57–7.53 (m, 1H), 7.45 (d,

EL-SHARKAWY ET AL.
17 of 17
|
Prism™ 4 software (GraphPad) to calculate IC50 values using a four
[12] D. H. Maurice, H. Ke, F. Ahmad, Y. Wang, J. Chung, V. C. Manganiello,
Nat. Rev. Drug Discov. 2014, 13(4), 290.
parameter logistic equation.
[13] S. H. Francis, M. A. Blount, J. D. Corbin, Physiol. Rev. 2011, 91(2),
651.
[14] B. J. Sung, K. Y. Hwang, Y. H. Jeon, J. I. Lee, Y. S. Heo, J. H. Kim, J.
Moon, J. M. Yoon, Y. L. Hyun, E. Kim, Nature 2003, 425(6953), 98.
[15] H. Wang, Y. Liu, Q. Huai, J. Cai, R. Zoraghi, S. H. Francis, J. D. Corbin,
H. Robinson, Z. Xin, G. Lin, J. Biol. Chem. 2006, 281(30), 21469.
[16] A. E. Rashad, A. H. Shamroukh, R. E. Abdel-Megeid, A. Mostafa, R. El-
Shesheny, A. Kandeil, M. A. Ali, K. Banert, Eur. J. Med. Chem. 2010,
45(11), 5251.
|
4.3 Molecular modeling
Molecular docking of the most active compound on PDE5, compound 15Y,
was implemented on PDE5 active site using MOE 2010. First, construction
of the compound was carried out on MOE window followed by
minimization of the compound. Then 2H42 code, PDE5 co-crystallized
with sildenafil, was imported into MOE window and the structure was
protonated. Following this, isolation of the pocket was carried out and
docking of the compound was carried out after removal of the co-
crystallized ligand from the pocket. The poses from the ligand conformation
were generated using triangle matcher, the scoring function used was ASE
withalphatrianglerefinement. Theposewithahighscoreshowingessential
features required for PDE5 activity was chosen. Self-docking was carried
out for the co-crystallized ligand, sildenafil, using the same docking
procedure settings to ensure a more accurate docking process.
[17] M. B. Dewal, A. S. Wani, C. Vidaillac, D. Oupický, M. J. Rybak, S. M.
Firestine, Eur. J. Med. Chem. 2012, 51, 145.
[18] E. Z. Elrazaz, R. A. T. Serya, N. S. M. Ismail, D. A. Abou El Ella, K. A. M.
Abouzid, Future J. Pharm. Sci. 2015, 1(2), 33.
[19] R. Mannhold, H. Kubinyi, G. Folkers, Methods and Principles in
Medicinal Chemistry, Vol. 22, WILEY-VCH, New Jersey, USA 2006, pp.
277–278.
[20] K. Kawai, Y. Endo, T. Asano, S. Amano, K. Sawada, N. Ueo, N.
Takahashi, S. Y. onoda, M. Nagai, N. Kamei, N. Nagata, J. Med. Chem.
2014, 57(23), 9844.
[21] M. I. Crespo, L. Pagès, A. Vega, V. Segarra, M. López, T. Doménech, M.
Miralpeix, J. Beleta, H. Ryder, J. M. Palacios, J. Med. Chem. 1998,
41(21), 4021.
[22] N. J. Brandon, D. P. Rotella, Annual Reports in Medicinal Chemistry, Vol.
42 (Ed: J. E. Macor), Academic Press, USA 2007, pp. 3–12.
[23] N. S. Vyawahare, A. A. Hadambar, A. S. Chothe, R. R. Jalnapurkar,
A. M. Bhandare, M. K. Kathiravan, J. Chem. Biol. 2011, 5, 35.
[24] G. L. Card, B. P. England, Y. Suzuki, D. Fong, B. Powell, B. Lee, C. Luu,
M. Tabrizizad, S. Gillette, P. N. Ibrahim, D. R. Artis, G. Bollag, M. V.
Milburn, S. H. Kim, J. Schlessinger, K. Y. Zhang, Structure 2004, 12(12),
2233.
ACKNOWLEDGMENTS
We thank Dr. Mostafa Hamed's assistance in the analytical measure-
ments for the compounds. The biological testing is partially funded by
NCI 5 R01 CA155638 to Gary A. Piazza.
CONFLICT OF INTEREST
[25] Z. Puterová, A. Krutošíková, D. Végh, ARKIVOC 2010 2010(i), 209.
[26] M.O.E., Chemical computing group Inc., 1010 Sherbrooke St. West,
Suite#910, Montreal, QC, Canada, H3A2R7, 2017.
[27] G. Durgareddy, R. Ravikumar, S. Ravi, S. R. Adapa, J. Pharma. Chem.
2013, 5(6), 294.
The authors have declared no conflict of interest.
ORCID
[28] C. H. Wu, M. S. Coumar, C. Y. Chu, W. H. Lin, Y. R. Chen, C. T. Chen,
H. Y. Shiao, S. Rafi, S. Y. Wang, H. Hsu, J. Med. Chem. 2010, 53(20),
7316.
Ashraf H. Abadi
http://orcid.org/0000-0002-7433-261X
[29] F. E. El-Baih, H. A. Al-Blowy, H. M. Al-Hazimi, Molecules 2006, 11(7),
498.
REFERENCES
[30] Z. Li, X. Lu, L. J. Feng, Y. Gu, X. Li, Y. Wu, H. B. Luo, Mol. BioSyst. 2015,
11(1), 115.
[1] P. H. Hutson, E. N. Finger, B. C. Magliaro, S. M. Smith, A. Converso, P. E.
Sanderson, D. Mullins, L. A. Hyde, B. K. Eschle, Z. Turnbull, H. Sloan, M.
Guzzi, X. Zhang, A. Wang, D. Rindgen, R. Mazzola, J. A. Vivian, D.
Eddins, S. Parmentier-Batteur, J. Neuropharm. 2011, 61(4), 665.
[2] M. F. Azevedo, F. R. Faucz, E. Bimpaki, A. Horvath, I. Levy, R. B. de
Alexandre, F. Ahmad, V. Manganiello, C. A. Stratakis, Endocr. Rev.
2014, 35(2), 195.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article.
[3] A. Garcia-Osta, M. Cuadrado-Tejedor, C. Garcia-Barroso, J. Oyarza-
bal, R. Franco, ACS Chem. Neurosci. 2012, 3(11), 832.
[4] C. Lugnier, Pharmacol. Ther. 2006, 109(3), 366.
[5] N. M. Kerr, H. V. Danesh-Meyer, Clin. Exp. Ophthalmol. 2009, 37(5), 514.
[6] T. Mostafa, J. Sex. Med. 2008, 5(11), 2502.
[7] M. Van Duinen, R. Oah, L. Lambrecht, A. Sambeth, B. Rutten, J. van
Os, A. Blokland, J. Prickaerts, Curr. Pharm. Des. 2015, 21(26), 3813.
[8] W. Z. Layman, J. Nat. Med. 2018, 18(2), 206.
How to cite this article: El-Sharkawy LY, El-Sakhawy RA,
Abdel-Halim M, et al. Design and synthesis of novel
annulated thienopyrimidines as phosphodiesterase 5 (PDE5)
inhibitors. Arch Pharm Chem Life Sci. 2018;1–17.
https://doi.org/10.1002/ardp.201800018
[9] J. J. Chen, Y. L. Sun, A. K. Tiwari, Z. J. Xiao, K. Sodani, D. H. Yang, S. G.
Vispute, W. Q. Jiang, S. D. Chen, Z. S. Chen, Cancer Sci. 2012, 103(8), 1531.
[10] Y. H. Jeon, Y. S. Heo, C. M. Kim, Y. L. Hyun, T. G. Lee, S. Ro, J. M. Cho,
Cell Mol. Life Sci. 2005, 62(11), 1198.
[11] P. Wang, P. Wu, R. W. Egan, M. M. Billah, Gene 2003, 314, 15.