Synthesis and Screening of Phosphodiesterase 5 Inhibitory Activity of Fused and Isolated Triazoles Based on Thieno[2,3-d]pyrimidines

Article abstract of DOI:10.1002/jhet.3560

Novel fused and isolated triazoles based on thieno[2,3-d]pyrimidines were synthesized starting from 2-aminothiophene ester and structurally confirmed by spectral and analytical data. The synthesized triazolo compounds were screened for their phosphodieste

Full text of DOI:10.1002/jhet.3560

Month 2019
Synthesis and Screening of Phosphodiesterase 5 Inhibitory Activity of
Fused and Isolated Triazoles Based on Thieno[2,3-d]pyrimidines
a
Mohamed A. Ameen,
Essam Kh. Ahmed,a Hemdan I. Mahmoud,b and Mohamed Ramadanc
*
aChemistry Department, Faculty of Science, Minia University, El Minia, 61519, Egypt
bAgricultural Chemistry Department, Faculty of Agriculture, Minia University, El Minia, 61519, Egypt
cOrganic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt
*E-mail: m_ameen10@yahoo.com
Received December 1, 2018
DOI 10.1002/jhet.3560
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
Novel fused and isolated triazoles based on thieno[2,3-d]pyrimidines were synthesized starting from 2-
aminothiophene ester and structurally confirmed by spectral and analytical data. The synthesized triazolo
compounds were screened for their phosphodiesterase 5 inhibitory activity and showed encouraging activity
than did sildenafil as a reference drug.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
coordination compounds can be considered as model
systems for metal–ligand interactions observed in
biological systems [12,13]. The simple molecule of
trapidil, the most widely known TP derivative, acts as a
The condensation of 1,2,4-triazole ring and pyrimidine
ring gives rise to the formation of bicyclic heterocycles
known as 1,2,4-triazolopyrimidines. Four different
possibilities exist for the relative orientation of both
rings, so four different isomeric families of compounds
are defined. Among these, 1,2,4-triazolo[1,5-a]pyrimidine
derivatives are thermodynamically more stable and, thus,
the most studied ones [1], with a few of them being
commercially available. Revisions surveying the
synthesis, reactivity, spectroscopic characterization, and
platelet-derived growth factor antagonist and as
phosphodiesterase (PDE) inhibitor [6].
a
1,2,3-Triazoles are highly stable under basic and acid
hydrolysis and reductive and oxidative conditions,
indicative of a high aromatic stabilization [14,15].
Moreover, this heterocycle has a high dipole moment and
is capable of hydrogen bonding, which could be
favorable in the binding of biomolecular targets [16].
1,2,3-Triazole is one of the key structural units found in a
large variety of bioactive molecules such as antifungal
[17], antibacterial [18,19], anti-allergic [20], anti-HIV
[21,22], anti-tubercular [23,24], and anti-inflammatory
agents [25]. Several 1,2,3-triazole-containing drug
molecules including tazobactam [26], cefatrizine [14],
and carboxyamidotriazole [27] are now available in the
market. In recent years, people are increasingly focused
on their anticancer activity [28–32].
crystallographic
studies
of
1,2,4-triazolo[1,5-c]
pyrimidines [2], 1,2,4-triazolo[4,3-a]pyrimidines [3], and
1,2,4-triazolo[4,3-c]pyrimidines [4] have also been
reported.
Triazolopyrimidines (TPs), a subtype of purine analogs,
have been the subject of chemical and biological studies
owing to their interesting pharmacology including
antihypertensive,
cardiac
stimulant,
antimalarial,
antifungal, anti-hepatitis
B
virus, antimicrobial,
anticancer, antipyretic, analgesic, anti-inflammatory,
potential herbicidal, and leishmanicidal activities [5–12].
In addition, TPs are versatile ligands, and their derived
Male erectile dysfunction could be a common public
pathological state touching innumerable men worldwide.
PDE 5 inhibitors are very attractive therapeutic agents for
© 2019 Wiley Periodicals, Inc.

M. A. Ameen, E. K. Ahmed, H. I. Mahmoud, and M. Ramadan
the treatment of male erectile dysfunction [33]. Sildenafil RESULTS AND DISCUSSION
Vol 000
(Viagra, IC50 = 3 nM) is the first and most famous PDE 5
inhibitor; however, there are still some side effects due to
the low selectivity against other PDEs, mainly toward
PDE 6, which is distributed in the retina, and inhibition
of it may cause visual disturbances, perception of bluish
haze, and increased light sensitivity [34]. In recent years,
numerous efforts have been made toward the discovery
of selective PDE 5 inhibitors. PDE 5 belongs to a
superfamily of enzymes that catalyzes the hydrolysis of
cyclic nucleotides cAMP and cGMP to the corresponding
5-nucleoside monophosphate. Currently, 11 different
isoforms of PDEs (PDE 1–11) and their subtypes are
known, distinguished by substrate specificities and tissue
concentration. The PDE plays a pivotal role in the
cellular signaling process [35].
The synthesis of novel 1,2,3-triazole-containing N-aryl
conjugate is depicted in Scheme 1. The 2-
isothiocyanothiophene ester 2 used as a starting material
was synthesized by treatment of 2-aminothiophene
carboxylate 1 with thiophosgen [36]. Hydrazinolysis of
8
isothiocyano
2 with ethyl carbazate produced the
thioureido product 4, which was subsequently cyclized to
produce compound 6 followed by hydrolysis in basic
medium to yield the target product 5. The application of
an alternative method for the synthesis of the target 3-
amino-2-thioxopyrimidinone 5 was desirable here with
shorter pathway. Therefore, N-alkylation of the readily
prepared o-aminothiophene carboxylate 1 with dimethyl
sulfate and carbon disulfide gave (((methylthio)carbonot-
hioyl)amino)-4,5-dihydrothieno[2,3-c]pyridine 3, which
underwent cyclization by treatment with hydrazine
hydrate in benzene to afford compound 5. S-Alkylation of
compound 5 with propargyl bromide proceeded smoothly
and in significantly high yield to give ethyl-3-amino-4-
oxo-2-(prop-2-yn-1-ylthio)-3,4,5,6-tetrahydropyrido[40,30:
4,5]thieno[2,3-d]pyrimidin-7(8H)-carboxylate (7). Enc-
ouraged by the advantages of the application of the one-
pot processes, and convergent construction of complex
molecules without the isolation of intermediates, we
employed a convenient one-pot method for the synthesis
of 1,4-disubstituted 1,2,3-triazoles via the copper(I)-
As a part of our continuous research in the area of click
approach, we have designed and synthesized new S-
methylene bridge 1,2,3-triazolyl pyrimidines by
incorporating triazole moiety with the pyrimidine ring,
which are expected to have biological activity.
Scheme 1. Synthesis of isolated triazolo pyrimidine.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and Screening of PDE 5 inhibitory activity of Fused and Isolated
Triazoles Based on Thieno[2,3-d]Pyrimidines
catalyzed Huisgen 1,3-dipolar cycloaddition. Therefore,
the target regioselective 1,4-disubstituted 1,2,3-triazole 8
was prepared via the coupling of a terminal alkyne in
compound 7 and p-nitrobenzene azide under the standard
reported alkyne-azide click conditions.
The structures of compound 8 and other compounds
were confirmed by NMR measurements. The 1H-NMR
and 13C-NMR chemical shifts for all the products are
reported in the Experimental section.
direct interaction of the very active NSC group in
compound 2 and ammonia or methyl amine in methylene
chloride led to the formation of thioureido derivatives 9a,b.
The pyrimidine ring formation of 2,3-diaminothieno[2,3-
d]pyrimidin-4(3H)-ones derivatives 10–12 were performed
by heterocyclic intramolecular cyclo-condensation
of thioureido compounds 9a,b with hydrazine hydrate or
methyl hydrazine as precursor for the synthesis of the
target compounds. To accomplish our aims to
synthesize new triazolopyrimidinone derivatives we
carried out in a study of the reaction between the
The synthesis of thieno[2,3-d]pyrimidine fused to 1,2,4-
triazolo derivatives is illustrated in Schemes 2 and 3. The
Scheme 2. Building the pyrimidine moieties in 10–12. [Color figure can be viewed at wileyonlinelibrary.com]
Scheme 3. Synthesis of fused triazolopyrimidines 13–15. [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

M. A. Ameen, E. K. Ahmed, H. I. Mahmoud, and M. Ramadan
Vol 000
Table 1
EXPERIMENTAL
Percent of phosphodiesterase 5 inhibition of compounds 5–8, 10, 11, and
13–15 at conc. 3 nM.
General remarks. Melting points were determined on a
Boetius melting-point apparatus and are uncorrected. Vario
EL III German CHN elemental analyzer model was used
for elemental analysis, (Euro EA analyser, HU, Berlin,
Germany). The NMR spectra were obtained using a Bruker
Avance (400 MHz for 1H, and 100 MHz for 13C; Institute
of Organic Chemistry, Karlsruhe University, Karlsruhe,
Germany) with deuterated DMSO-d6 as solvent chemical
Compound
% of PDE 5 inhibition
5
6
7
8
100
100
100
4.6
10
11
13
14
15
100
25.3
100
100
100
shifts are quoted in
δ
and are referenced to
tetramethylsilane. Mass spectrometry was performed by
electron impact at 70 eV (fast atom bombardment–mass
spectrometry; Finnigan MAT 95, Institute of Organic
Chemistry, Karlsruhe Institute of Technology, Karlsruhe,
Germany). The reactions and purity were monitored by
thin-layer chromatography (TLC) on aluminum plates
coated with silica gel with fluorescent indicator (Merck,
Kenilworth, NJ; 60 F254).
corresponding thieno[2,3-d]pyrimidin-4(3H)-ones derivati-
ves 10–12 and triethyl orthoformate building the linear
1,2,4-triazole moiety fused to thienopyrimidinone in
compounds 13–15.
The chemical structures of the new compounds were
established by elemental analyses and NMR spectros-
copy, and the results are presented in the Experimental
Diethyl 2-(((methylthio)carbonothioyl)amino)-4,5-dihydrothi
eno[2,3-c]pyridin-3,6(7H)dicarboxylate (3).
To a solution
1
part. In the H-NMR and 13C-NMR spectra, particularly
of o-aminoester 1 (0.9 g, 3 mmol) in DMSO (5 mL),
carbon disulfide (0.3 mL), saturated sodium hydroxide
(0.2 mL), and dimethyl sulfate (0.5 mL) were added. The
mixture was stirred overnight (TLC showed complete
conversion). The precipitate that formed was filtered off,
washed with ethanol, and crystallized from methanol to
give compound 3 as yellow crystals (0.72 g, 62%). mp:
CH-triazole signals are characteristic of triazolo
compounds 13–15. The chemical shift values varied in
the range from 6.97 to 7.08 and 136.8 to 136.9 ppm,
respectively.
Screening of PDE 5 inhibition activity.
The target
compounds were evaluated for their ability to inhibit
PDE 5, with sildenafil chosen as a standard for comparison
(Table 1). All the compounds were found to exhibit PDE 5
inhibitory activity at concentration of 3 nM (as IC50 of
sildenafil). Despite the fact that this assay is less sensitive
1
168–169°C. H-NMR (400 MHz, DMSO-d6): δ = 1.21 (t,
3H,
J
=
7.1 Hz, COOCH2CH3), 1.32 (t, 3H,
COOCH2CH3), 2.80 (s, 3H, CH3), 2.82 (t, 2H,
J = 6.5 Hz, H-4), 3.62 (t, 2H, J = 6.5 Hz, H-5), 4.03 (q,
2H, J = 7.2 Hz, COOCH2CH3), 4.08 (bs, 1H, NH), 4.30
(s, 2H, H-7), 4.53 (q, 2H, J = 7.2 Hz, COOCH2CH3).
13C-NMR (100 MHz, DMSO-d6): δ = 14.4, 14.7 (2CH3),
25.4 (C-4), 40.7 (C-5), 42.8 (C-7), 62.3, 63.2 (2CH2,
ester), 116.1 (C-7a), 125.1 (C-3), 128.8 (C-3a), 149.3 (C-
2), 164.6, 164.8 (2C═O, ester), 174.4 (C═S). [electron-
impact mass spectrometry (EI-MS)]: m/z calcd.
C15H20N2O4S3 [M]+: 388.06, found: 388.20. Anal. Calcd
for C15H20N2O4S3 (388.53): C, 46.37; H, 5.19; N, 7.21;
Found: C, 46.22; H, 5.09; N, 7.13%.
than radioisotope PDE
5
assays and likely to
underestimate the relative inhibitory potencies of the test
compounds and that further studies needed, the results of
this evaluation indicate that most of the synthesized
derivatives are potent inhibitors of PDE, which is
comparable with sildenafil.
CONCLUSION
In conclusion, we reported the synthesis of fused and
isolated triazoles based on thieno[2,3-d]-pyrimidines. The
synthesized 1,2,4-triazolo compounds were screened for
their PDE 5 inhibitory activity; encouragingly, most of the
synthesized derivatives possess potent PDE 5 inhibitory
activity than do sildenafil as a reference drug. Despite the
fact that this assay is less sensitive than are radioisotope
PDE 5 assays to underestimate the relative inhibitory
potencies of the test compounds, further studies are
needed. The possibility of combining 1,2,4-triazolo
moiety to thieno[2,3-d]pyrimidines should open new
opportunities for the rational design of medicinal agents.
Ethyl
hydropyrido[40,30:4,5]thieno[2,3-d]pyrimidin-7(8H)-carboxylate
(7). mmol) in
3-amino-4-oxo-2-(prop-2-yn-1-ylthio)-3,4,5,6-tetra-
A
solution of
5
(0.98 g,
3
dimethylformamide (DMF) (10 mL) was placed in a two-
necked flask fitted with an efficient reflux condenser.
Na2CO3 (0.63 g, 6 mmol) was then added. The reaction
mixture was stirred for 30 min at room temperature.
Propargyl bromide 80% (0.72 g, 6 mmol) was added. The
reaction mixture was further stirred while its progress was
being followed by TLC. After completion (about 7 h), the
mixture was poured into iced water (100 mL) with stirring.
The resulting precipitate that formed was filtered off,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and Screening of PDE 5 inhibitory activity of Fused and Isolated
Triazoles Based on Thieno[2,3-d]Pyrimidines
washed with water, and dried well to furnish the title product
as yellow powder (0.92 g, 84%). mp: 232–233°C. 1H-NMR
(400 MHz, DMSO-d6): δ = 1.22 (t, 3H, J = 7.1 Hz,
COOCH2CH3), 2.93 (t, 2H, J = 5.1 Hz, H-5), 3.16 (t, 1H,
J = 2.5 Hz, C–CH), 3.67 (t, 2H, J = 5.1 Hz, H-6), 3.84 (d,
2H, J = 2.2 Hz, CH2-C), 4.13 (q, 2H, J = 7.0 Hz,
COOCH2CH3), 4.62 (s, 2H, H-8), 5.82 (s, 2H, NH2), 13C-
NMR (100 MHz, DMSO-d6): δ = 14.6 (CH3), 19.4 (CH2-
C), 26.2 (C-5), 40.5 (C-6), 42.8 (C-8), 61.1 (CH2,
carbamate), 73.5 (C–CH), 79.8 (C–CH), 101.3 (C-4a),
117.5 (C-4b), 128.8 (C-8a), 154.7 (C-9a), 157.2 (C-2),
159.6 (C═O), 161.6 (C═O, carbamate). (EI-MS): m/z
calcd. C15H16N4O3S2 [M]+: 364.07, found: 364.20. Anal.
Calcd for C15H16N4O3S2 (364.44): C, 49.43; H, 4.43; N,
15.37; Found: C, 49.32; H, 4.39; N, 15.33%.
The solid product was filtered off, washed with water,
and recrystallized from ethanol to give compounds 6 as
colorless powder (0.66 g, 82%). mp: 217–218°C. 1H-
NMR (400 MHz, DMSO-d6):
δ = 1.26 (t, 6H,
J = 7.1 Hz, 2COOCH2CH3), 2.92 (t, 2H, J = 5.6 Hz, H-
5), 3.70 (t, 2H, J = 5.1 Hz, H-6), 4.17 (q, 2H, J = 7.0 Hz,
COOCH2CH3), 4.24 (q, 2H, J = 7.0 Hz, COOCH2CH3),
4.54 (s, 2H, H-8), 7.43 (s, 1H, NH), 11.78 (s, 1H, NH).
13C-NMR (100 MHz, DMSO-d6): δ = 14.4 (CH3), 14.7
(CH3), 25.4 (C-5), 40.7 (C-6), 42.8 (C-8), 62.3 (CH2,
carbamate), 63.2 (CH2, carbamate), 116.1 (C-4a), 125.1
(C-4b), 128.8 (C-8a), 147.6 (C-9a), 149.1 (C═O), 164.6
(C═O, carbamate), 164.8 (C═O, carbamate), 174.4
(C═S). (EI-MS): m/z calcd. C15H18N4O5S2 [M]+: 398.07,
found: 398.24. Anal. Calcd for C15H18N4O5S2 (398.46):
C, 45.21; H, 4.55; N, 14.06; Found: C, 45.07; H, 4.39;
N, 14.01%.
Ethyl 3-amino-2-(((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methyl)thio)-4-oxo-3,4,5,6-tetrahydropyrido-[40,30:4,5]
thieno[2,3-d]pyrimidin-7(8H)carboxylate (8). A mixture of
alkyne core 7 (0.36 g, 1 mmol), sodium ascorbate
(0.4 mmol), and CuSO4•5H2O (0.2 mmol) was suspended
in 10 mL tetrahydrofuran–water (1:1). Then, p-
nitrobenzene azide (0.16 g, 1.0 mmol) was added. The
mixture was stirred at room temperature until complete
consumption of the starting materials, monitored by TLC.
The reaction mixture was diluted with 25 mL of water,
cooled in ice, and extracted with CH2Cl2 (3 × 15 mL).
The combined organic extracts were washed with brine
and dried (MgSO4). After removal of the solvent under
reduced pressure, the residue was purified on silica gel
with n-hexane–ethyl acetate (8:2) yielding the target
compound 8 as colorless powder (0.42 g, 79%). mp:
Synthesis of heterocycles 4 and 9a,b. To a solution of
isothiocyanate 2 (0.68 g, 2 mmol) in dichloromethane
(10 mL), a propriate amine (2 mmol) was added with
stirring. The mixture was stirred for 1–3 h, and then the
solvent was removed under reduced pressure, giving
compounds 4 and 9a,b.
Diethyl 2-(2-(ethoxycarbonyl)hydrazinecarbothioamido)-4,5-
dihydrothieno[2,3-c]pyridin-3,6(7H)dicarboxylate
(4).
Colorless crystals from methanol (0.61 g, 69%). mp:
1
197–198°C. H-NMR (400 MHz, DMSO-d6): δ = 1.16 (t,
6H, J = 7.1 Hz, 2COOCH2CH3), 1.32 (t, 3H, J = 7.1 Hz,
COOCH2CH3), 2.79 (t, 2H, J = 5.6 Hz, H-4), 3.61 (t,
2H, J = 5.1 Hz, H-5), 4.16 (q, 4H, J = 7.0 Hz,
2COOCH2CH3), 4.30 (q, 2H, J = 7.0 Hz, COOCH2CH3),
4.48 (s, 2H, H-7), 9.76 (s, 1H, NH), 10.35 (s, 1H, NH),
12.34 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6):
δ = 13.8 (CH3), 14.4 (CH3), 14.5 (CH3), 25.8 (C-4), 41.0
(C-5), 42.1 (C-7), 60.5 (CH2, ester), 60.8 (CH2,
carbamate), 61.2 (CH2, carbamate), 111.5 (C-3), 122.5
(C-3a), 128.9 (C-7a), 149.5 (C-2), 154.5 (C═O), 155.6
(C═O, carbamate), 165.1 (C═O, carbamate), 176.7
(C═S). (EI-MS): m/z calcd. C17H24N4O6S2 [M]+: 444.11,
found: 444.20. Anal. Calcd for C17H24N4O6S2 (444.53):
C, 45.93; H, 5.44; N, 12.60; Found: C, 45.87; H, 5.39;
N, 12.51%.
1
182–183°C. H-NMR (400 MHz, DMSO-d6): δ = 1.13 (t,
3H, J = 7.1 Hz, COOCH2CH3), 2.87 (t, 2H, J = 5.1 Hz,
H-5), 3.63 (t, 2H, J = 5.1 Hz, H-6), 4.05 (q, 2H,
J = 7.0 Hz, COOCH2CH3), 4.30 (s, 2H, H-8), 4.56 (s,
2H, S-CH2), 5.72 (s, 2H, NH2), 8.14 (d, 2H, J = 9.1 Hz,
Ar-H), 8.30 (d, 2H, J = 9.1 Hz, Ar-H), 8.86 (s, 1H, CH).
13C-NMR (100 MHz, DMSO-d6): δ = 14.5 (CH3), 25.5
(C-5), 40.5 (C-6), 42.8 (C-8), 45.2 (S-CH2), 61.0
(CH2, carbamate), 117.4 (C-4a), 120.5 (2CH-Ar), 122.2
(CH-triazole), 125.5 (2CH-Ar, C-4b), 128.8 (C-8a), 140.7
(Ar-C), 144.8 (Ar-C), 146.6 (Cq-triazole), 154.7 (C-9a),
157.3 (C-2), 159.0 (C═O), 161.6 (C═O, carbamate). (EI-
MS): m/z calcd. C21H20N8O5S2 [M]+: 528.1, found:
529.1. Anal. Calcd for C21H20N8O5S2 (528.56): C, 47.72;
H, 3.81; N, 21.20; Found: C, 47.58; H, 3.72; N, 21.08%.
Ethyl 3-(2-(ethoxycarbonyl)hydrazinyl)-4-oxo-2-thioxo-1,2,3,
4,5,6-hexahydropyrido[40,30:4,5]thieno[2,3-d]-pyrimidin-7(8H)
carboxylate (6). To a sodium ethoxide solution (0.05 g,
2 mmol in 10 mL of abs. ethanol), compound 4 (0.88 g,
2 mmol) was added. The reaction mixture was stirred at
room temperature overnight. After removal of the
solvent, the formed sodium salt was collected, dissolved
in water, and adjusted with hydrochloric acid to pH = 4.
Diethyl
2-thioureido-4,5-dihydrothieno[2,3-c]pyridin-3,6
(7H)dicarboxylate (9a). Colorless crystals from methanol
(0.54 g, 76%). mp: 200–201°C. 1H-NMR (300 MHz,
DMSO-d6): δ = 1.20 (t, 3H, J = 7.1 Hz, COOCH2CH3),
1.31 (t, 3H, J = 7.1 Hz, COOCH2CH3), 2.77 (t, 2H,
J = 5.6 Hz, H-4), 3.59 (t, 2H, J = 5.1 Hz, H-5), 4.08 (q,
2H, J = 7.0 Hz, COOCH2CH3), 4.29 (q, 2H, J = 7.0 Hz,
COOCH2CH3), 4.45 (s, 2H, H-7), 8.55 (s, 2H, NH2),
11.42 (s, 1H, NH). 13C-NMR (DMSO): δ = 13.8 (CH3),
14.3 (CH3), 25.7 (C-4), 40.6 (C-5), 41.9 (C-7), 60.2
(CH2, ester), 60.7 (CH2, carbamate), 110.5 (C-3), 121.8
(C-3a), 128.4 (C-7a), 151.0 (C-2), 154.5 (C═O), 165.0
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M. A. Ameen, E. K. Ahmed, H. I. Mahmoud, and M. Ramadan
Vol 000
(C═O, carbamate), 178.7 (C═S). Anal. Calcd for
C14H19N3O4S2 (357.45): C, 47.04; H, 5.36; N, 11.76;
Found: C, 46.91; H, 5.19; N, 11.58%.
114.9 (C-4a), 123.5 (C-4b), 129.2 (C-8a), 153.9 (C-9a),
154.6 (C-2), 157.1 (C═O), 167.2 (C═O, carbamate).
Anal. Calcd for C13H17N5O3S (323.37): C, 48.28; H,
5.30; N, 21.66; Found: C, 48.14; H, 5.12; N, 21.71%.
Diethyl 2-(3-methylthioureido)-4,5-dihydrothieno[2,3-c]pyrid
in-3,6(7H)dicarboxylate (9b).
Colorless crystals from
Ethyl 3-amino-4-oxo-2-thioxo-1,2,3,4,5,6-hexahydropyrido
[40,30:4,5]thieno[2,3-d]pyrimidin-7(8H)-carboxylate (5) (method
methanol (0.58 g, 78%). mp: 152–153°C. 1H-NMR
(300 MHz, DMSO-d6): δ = 1.21 (t, 3H, J = 7.1 Hz,
COOCH2CH3), 1.33 (t, 3H, J = 7.1 Hz, COOCH2CH3),
2.77 (t, 2H, J = 5.6 Hz, H-4), 2.92 (s, 3H, NCH3), 3.60
(t, 2H, J = 5.1 Hz, H-5), 4.11 (q, 2H, J = 7.0 Hz,
COOCH2CH3), 4.27 (q, 2H, J = 7.0 Hz, COOCH2CH3),
4.45 (s, 2H, H-7), 9.46 (s, H, NH), 11.50 (s, 1H, NH).
13C-NMR (DMSO-d6): δ = 13.9 (CH3), 14.3 (CH3), 25.7
(C-4), 30.7 (NCH3), 40.6 (C-5), 41.9 (C-7), 60.2 (CH2,
ester), 60.8 (CH2, carbamate), 109.8 (C-3), 121.2 (C-3a),
128.4 (C-7a), 151.4 (C-2), 154.5 (C═O), 165.2 (C═O,
carbamate), 174.4 (C═S). Anal. Calcd for C15H21N3O4S2
(371.47): C, 48.50; H, 5.70; N, 11.31; Found: C, 48.32;
H, 5.49; N, 11.41%.
B).
To alcoholic KOH solution (0.29 g, 4 mmol in
10 mL of abs. ethanol), compound 6 (0.8 g, 2 mmol) was
added. The reaction mixture was refluxed for 3 h. After
removal of the solvent, the formed potassium salt was
dissolved in water and neutralized with hydrochloric acid.
The solid product was filtered off and then washed with
water.
Colorless crystals from methanol (0.51 g, 78%). mp:
230-231°C (232–234°C) [38]. 1H-NMR (400 MHz,
DMSO-d6):
δ
=
1.02 (t, 3H,
J
=
7.0 Hz,
COOCH2CH3), 2.66 (t, 2H, J = 5.1 Hz, H-5), 3.16 (s,
2H, NH2), 3.46 (t, 2H, J = 5.1 Hz, H-6), 3.96 (q, 2H,
J = 7.0 Hz, COOCH2CH3), 4.34 (s, 2H, H-8), 5.94 (s,
1H, NH). 13C-NMR (100 MHz, DMSO-d6): δ = 14.5
(CH3), 25.0 (C-5), 40.5 (C-6), 42.6 (C-8), 61.6 (CH2,
carbamate), 114.2 (C-4a), 125.1 (C-4b), 129.2 (C-8a),
148.6 (C-9a), 152.8 (C═O), 164.7 (C═O, carbamate),
166.7 (C═S). (EI-MS): m/z calcd. C12H14N4O3S2 [M]+:
326.05, found: 326.17. Anal. Calcd for C12H14N4O3S2
(326.39): C, 44.16; H, 4.32; N, 17.17; Found: C,
44.07; H, 4.19; N, 17.05%.
Synthesis of 5 (method A), 10, 11, and 12.
To a
suspension
of
2-((methylthio)carbonothioyl)amino
compound 3 or thioureido compound 9a or 9b (2 mmol)
in benzene (10 mL), hydrazine hydrate or methyl
hydrazine (5 mmol) was added; the mixture was stirred
for 5–7 h at 100°C. Solvent was removed under reduced
pressure with a rotary evaporator. The residue was
subjected to crystallization to afford the product.
Ethyl 3-amino-2-(methylamino)-4-oxo-3,4,5,6-tetrahydropyri
do[40,30:4,5-]thieno[2,3-d]pyrimidin-7(8H)-carboxylate (10).
Colorless crystals from methanol (0.49 g, 77%). mp:
Synthesis of 13–15 heterocycles.
2,3-Disubstituted
pyrimidine 10 or 11 (2 mmol) was refluxed in triethyl
orthoformate or triethyl orthoacetate (10 mL) and few
drops of acetic anhydride for 4 h. Solvent was removed
under reduced pressure. The residue was crystallized to
give the triazolo compounds.
1
294–295°C. H-NMR (300 MHz, DMSO-d6): δ = 1.22 (t,
3H, J = 7.1 Hz, COOCH2CH3), 2.85 (t, 2H, J = 5.6 Hz,
H-5), 3.56 (s, 3H, CH3), 3.62 (t, 2H, J = 5.1 Hz, H-6),
4.09 (q, 2H, J = 7.0 Hz, COOCH2CH3), 4.53 (s, 2H, H-
8), 7.13 (s, 1H, NH2), 13.55 (s, 1H, NH). 13C-NMR
(DMSO-d6): δ = 14.3 (CH3), 25.0 (C-5), 36.2 (CH3),
40.1 (C-6), 42.6 (C-8), 60.9 (CH2, carbamate), 119.2 (C-
4a), 126.3 (C-4b), 129.0 (C-8a), 154.6 (C-9a), 156.3 (C-
2), 157.8 (C═O), 163.3 (C═O, carbamate). Anal. Calcd
for C13H17N5O3S (323.37): C, 48.28; H, 5.30; N, 21.66;
Found: C, 48.10; H, 5.13; N, 21.81%.
Ethyl 3-methyl-10-oxo-6,8,9,10-tetrahydropyrido[40,30:4,5]
thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-carboxylate
(13). Colorless crystals from DMF (0.42 g, 63%). mp:
1
289–290°C. H-NMR (300 MHz, DMSO-d6): δ = 1.33 (t,
3H, J = 7.1 Hz, COOCH2CH3), 2.91 (t, 2H, J = 5.6 Hz,
H-9), 3.46 (s, 3H, CH3), 3.67 (t, 2H, J = 5.1 Hz, H-8),
4.12 (q, 2H, J = 7.0 Hz, COOCH2CH3), 4.58 (s, 2H, H-
6), 7.08 (s, 1H, H-2). 13C-NMR (DMSO): δ = 14.4
(CH3), 25.0 (C-9), 29.5 (CH3), 40.7 (C-8), 42.6 (C-6),
60.9 (CH2, carbamate), 116.8 (C-9b), 126.5 (C-9a), 129.1
(C-5a), 136.8 (C-2), 154.6 (C-3a), 157.1 (C-4a), 157.8
(C═O), 161.8 (C═O, carbamate). Anal. Calcd for
C14H15N5O3S (333.37): C, 50.44; H, 4.54; N, 21.01;
Found: C, 50.27; H, 4.42; N, 21.13%.
Ethyl 2,3-diamino-4-oxo-3,4,5,6-tetrahydropyrido[40,30:4,5]
thieno[2,3-d]pyrimidin-7(8H)-carboxylate (11).
mp: 300–
301°C (reported 297–299°C) [37].
Ethyl 2-amino-3-(methylamino)-4-oxo-3,4,5,6-tetrahydropy
rido[40,30:4,5]thieno[2,3-d]pyrimidin-7(8H)-carboxylate (12).
Colorless crystals from methanol (0.45 g, 70%). mp: 164–
1
Ethyl 10-oxo-6,8,9,10-tetrahydropyrido[40,30:4,5]thieno[2,3-
d][1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-carboxylate (14).
Colorless crystals from DMF (0.5 g, 78%). mp: 237–
165°C. H-NMR (300 MHz, DMSO-d6): δ = 1.21 (t, 3H,
J = 7.1 Hz, COOCH2CH3), 2.61 (s, 1H, NH), 2.81 (t,
2H, J = 5.6 Hz, H-5), 3.56 (s, 3H, CH3), 3.61 (t, 2H,
1
J
=
5.1 Hz, H-6), 4.08 (q, 2H,
J
=
7.0 Hz,
238°C. H-NMR (300 MHz, DMSO-d6): δ = 1.27 (t, 3H,
COOCH2CH3), 4.50 (s, 2H, H-8), 10.54 (bs, 1H, NH2).
13C-NMR (DMSO-d6): δ = 14.4 (CH3), 24.9 (C-5), 36.2
(CH3), 40.0 (C-6), 42.3 (C-8), 60.9 (CH2, carbamate),
J = 7.1 Hz, COOCH2CH3), 2.90 (t, 2H, J = 5.6 Hz, H-9),
3.66 (t, 2H, J = 5.1 Hz, H-8), 4.11 (q, 2H, J = 7.0 Hz,
COOCH2CH3), 4.57 (s, 2H, H-6), 6.97 (s, 1H, H-2),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Synthesis and Screening of PDE 5 inhibitory activity of Fused and Isolated
Triazoles Based on Thieno[2,3-d]Pyrimidines
12.65 (s, 1H, NH). 13C-NMR (DMSO-d6): δ = 14.5 (CH3),
25.2 (C-9), 40.1 (C-8), 42.8 (C-6), 61.1 (CH2, carbamate),
118.4 (C-9b), 126.5 (C-9a), 129.2 (C-5a), 136.9 (C-2),
154.8 (C-3a), 156.5 (C-4a), 158.0 (C═O), 163.6 (C═O,
carbamate). Anal. Calcd for C13H13N5O3S (319.34): C,
48.89; H, 4.10; N, 21.93; Found: C, 48.70; H, 3.97; N,
21.76%.
1.5 mL/min; the diode array detector was at 254 nm. The
injection volume was 30 μL.
Peak identities were confirmed by coelution with
standards. Quantitative measurements were carried out by
comparison using standard solutions of known
concentrations.
Ethyl 1-methyl-10-oxo-6,8,9,10-tetrahydropyrido[40,30:4,5]
thieno[2,3-d][1,2,4]triazolo[1,5-a]pyrimidin-7(1H)-carboxylate
(15). Colorless crystals from DMF (0.48 g, 73%). mp:
Acknowledgments. We would like to thank Analytical
Chemistry Unit, Assiut University, for measuring PDE 5
inhibitory activity; we also acknowledge the Institute of Organic
Chemistry, Karlsruhe University, Karlsruhe, Germany, for mea-
suring NMR data of the synthesized compounds.
1
213–214°C. H-NMR (300 MHz, DMSO-d6): δ = 1.23 (t,
3H, J = 7.1 Hz, COOCH2CH3), 2.78 (t, 2H, J = 5.6 Hz,
H-9), 3.13 (s, 3H, CH3), 3.82 (t, 2H, J = 5.1 Hz, H-8),
4.11 (q, 2H, J = 7.0 Hz, COOCH2CH3), 4.78 (s, 2H, H-
6), 9.23 (s, 1H, H-2). 13C-NMR (DMSO): δ = 13.8
(CH3), 14.3 (CH3), 25.1 (C-9), 28.5 (CH3), 40.6 (C-8),
42.5 (C-6), 60.8 (CH2, carbamate), 116.8 (C-9b), 126.5
(C-9a), 129.1 (C-5a), 136.8 (C-2), 154.6 (C-3a), 157.1
(C-4a), 157.8 (C═O), 161.9 (C═O, carbamate). Anal.
Calcd for C14H15N5O3S (333.37): C, 50.44; H, 4.54; N,
21.01; Found: C, 50.28; H, 4.36; N, 20.90%.
REFERENCES AND NOTES
[1] Fischer, G. Adv Heterocycl Chem 1993, 57, 81.
[2] Morgan, A. E. A. Adv Heterocycl Chem 2000, 77, 345.
[3] Shaban, M. A. E.; Morgan, A. E. A. Adv Heterocycl Chem
1999, 73, 131.
[4] Shaban, M. A. E.; Morgan, A. E. A. Adv Heterocycl Chem
2000, 75, 243.
[5] El Ashry, E. S. H.; Rashed, N. Adv Heterocycl Chem 1998,
71, 57.
PDE assay.
Inhibition of human PDE 5 A1 was
determined by the known method [39]. cGMP and PDE
(PDE 5 A1) from human were purchased from (Sigma-
Aldrich). All other reagents were of analytical grade from
Merck (Kenilworth, NJ).
[6] Fischer, G. Adv Heterocycl Chem 2007, 95, 143.
[7] Ohnishi, H.; Yamaguchi, K.; Shimada, S.; Suzuki, Y.;
Kumagai, A. Life Sci 1981, 28, 1641.
[8] Novinson, T.; Springer, R. H.; OíBrien, D. E.; Scholten, M. B.;
Miller, J. P.; Robins, R. K. J Med Chem 1982, 25, 420.
[9] Gujjar, R.; Marwaha, A.; El Mazouni, F.; White, J.; White, K.
L.; Creason, S.; Shackleford, D. M.; Baldwin, J.; Charman, W. N.; Buck-
ner, F. S.; Charman, S.; Rathod, P. K.; Phillips, M. A. J Med Chem 2009,
52, 1864.
[10] Chen, Q.; Zhu, X. L.; Jiang, L. L.; Liu, Z. M.; Yang, G. F. Eur
J Med Chem 2008, 43, 595.
[11] Yu, W.; Goddard, C.; Clearfield, E.; Mills, C.; Xiao, T.; Guo,
H.; Morrey, J. D.; Motter, N. E.; Zhao, K.; Block, T. M.; Cuconati, A.;
Xu, X. J Med Chem 2011, 54, 5660.
[12] El-Gendy, M. M. A.; Shaaban, M.; Shaaban, K. A.; El-
Bondkly, A. M.; Laatsch, H. J Antibiot 2008, 61, 149.
[13] Salas, J. M.; Romero, M. A.; Snchez, M. P.; Quiros, M. Coord
Chem Rev 1999, 193, 1119.
[14] Vatmurge, N. S.; Hazra, B. G.; Pore, V. S.; Shirazi, F.; Chavan,
P. S.; Deshpande, M. V. Bioorg Med Chem Lett 2008, 18, 2043.
[15] Whiting, M.; Muldoon, J.; Lin, Y. C.; Silverman, S. M.;
Lindstron, W.; Olson, A. J.; Kolb, H. C.; Finn, M. G.; Sharpless,
K. B.; Elder, J. H.; Fokin, V. V. Angew Chem Int Ed 2006, 45, 1435.
[16] Bock, V. D.; Speijer, D.; Hiemstra, H.; Maarseveen, J. H. V.
Org Biomol Chem 2007, 5, 971.
[17] Aher, N. G.; Pore, V. S.; Mishra, N. N.; Kumar, A.; Shukla,
P. K.; Sharma, A.; Bhat, M. K. Bioorg Med Chem Lett 2009, 19, 759.
[18] Demaray, J. A.; Thuener, J. E.; Dawson, M. N.; Sucheck, S. J.
Bioorg Med Chem Lett 2008, 18, 4868.
[19] Wang, X. L.; Wan, K.; Zhou, C. H. Eur J Med Chem 2010, 45,
4631.
The enzymatic reaction was carried out in 0.1M Tris–
HCl buffer, pH 8.3, 10 mM MgCl2, and 0.1M KCl at
37°C. Alfa casein (2 mg) may be used as carrier for
protein precipitation when protein concentration is low.
The final volume ranged between 500 and 100 cm3. The
reaction was initiated by the addition of cGMP in a final
concentration of 0.5 × 10ꢀ4M. Enzyme and time of
incubation may vary, and if these are not indicated in the
specific experimental procedure, time of incubation is
60 min. The reaction was terminated by transferring the
tubes with the reaction mixture in boiling water bath for
3 min. The sample was then centrifuged and filtered
through a nylon-66 filter, 0.2 mm (Rainin Corporation).
The clear filtrate obtained may be used directly for high-
performance liquid chromatography assay or stored at
ꢀ20°C. A blank with protein, denaturated by boiling
water bath for 3 min, with and without substrate, was
performed. Both incubation time and enzyme
concentration were adjusted so that no more than 25% of
the substrate was hydrolyzed under the assay conditions.
All assays were performed in duplicate.
[20] Buckle, D. R.; Outred, D. J.; Rockell, C. J. M.; Smith, H.;
Spicer, B. A. J Med Chem 1983, 26, 251.
[21] Whiting, M.; Tripp, J. C.; Lin, Y. C.; Lindstrom, W.; Olson,
A. J.; Elder, J. H.; Sharpless, K. B.; Fokin, V. V. J Med Chem 2006,
49, 7697.
[22] Giffin, M. J.; Heaslet, H.; Brik, A.; Lin, Y. C.; Cauvi, G.;
Wong, C. H.; McRee, D. E.; Elder, J. H.; Stout, C. D.; Torbett, B. E. J
Med Chem 2008, 51, 6263.
[23] Costa, M. S.; Boechat, N.; Rangel, E. A.; Silva, F. D. C. D.;
Souza, A. M. T. D.; Rodrigues, C. R.; Castro, H. C.; Junior, I. N.;
The high-performance liquid chromatography system
was Agilent Technologies 1200 Series, G1315D DAD.
The column used was Zorbax Eclipse AAA rapid
resolution 4.6 × 150 mm 3.5 μM particle size. The
mobile phase employed for the separation (isocratic
elution) consisted of 200 mM ammonium acetate, pH 6.0,
with 2% acetonitrile (v/v). The flow rate was
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

M. A. Ameen, E. K. Ahmed, H. I. Mahmoud, and M. Ramadan
Vol 000
[32] Li, X. L.; Lin, Y. J.; Wang, Q. Q.; Yuan, Y. K.; Zhang, H.;
Qian, X. H. Eur J Med Chem 2011, 45, 1274.
Lourenc, M. C. S.; Wardell, S. M. S. V.; Ferreirab, V. F. Bioorg Med
Chem 2006, 14, 8644.
[33] Rajfer, J.; Aronson, W. J.; Bush, P. A.; Dorey, F. J.; Ignarro, L.
J. N Engl J Med 1992, 326, 90.
[34] Marmor, M. F.; Kessler, R. Surv Ophthalmol 1999, 44, 153.
[35] Carson, C. C. Psychosom Med 2004, 66, 664.
[36] Ahmed, E. K.; Sensfuss, U.; Habicher, W. D. J Heterocyclic
Chem 1999, 36, 1119.
[37] Ameen, M. A. Z Naturforsch 2006, 61B, 1234.
[38] Ahmed, E. K. Heteroatom Chemistry 2002, 13, 280.
[39] Spoto, G.; Berardi, S.; Ajerba, G.; De Laurentiis, V. Adv Exp
Med Biol 1994, 370, 815.
[24] Patpi, S. R.; Pulipati, L.; Yogeeswari, P.; Sriram, D.; Jain, N.;
Sridhar, B.; Murthy, R.; Anjana, D. T.; Kalivendi, S. V.; Kantevar, S. J
Med Chem 2012, 55, 3911.
[25] Simone, R. D.; Chini, M. G.; Bruno, I.; Riccio, R.; Mueller,
D.; Werz, O.; Bifulco, G. J Med Chem 2011, 54, 1565.
[26] Bennet, I. S.; Brooks, G.; Broom, N. J. P.; Calvert, S. H.;
Coleman, K.; Francois, I. J Antibiot. 1991, 44, 969.
[27] Soltis, M. J.; Yeh, H. J.; Cole, K. A.; Whittaker, N.; Wersto, R.
P.; Kohn, E. C. Drug Metab Dispos 1996, 24, 799.
[28] Fray, M. J.; Bull, D. J.; Carr, C. L.; Gautier, E. C. L.;
Mowbray, C. E.; Stobie, A. J Med Chem 2001, 24, 1951.
[29] Imperio, D.; Pirali, T.; Galli, U.; Pagliai, F.; Cafici, L.;
Canonico, P. L.; Sorba, G.; Genazzani, A. A.; Tron, G. C. Bioorg Med
Chem 2007, 15, 6748.
SUPPORTING INFORMATION
[30] Kallander, L. S.; Lu, Q.; Chen, W.; Tomaszek, T.; Yang, G.;
Tew, D.; Meek, T. D.; Hofmann, G. A.; Pritchard, C. K. S.; Smith, W.
W.; Ho, T. F.; Fisher, P. W.; Mattern, M. R.; Johnson, R. K.; Hansbury,
M. J.; Winkler, J. D.; Ward, K. W.; Veber, D. F.; Thompson, S. K. J
Med Chem 2005, 48, 5644.
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
[31] Pagliai, F.; Pirali, T.; Grosso, E. D.; Brisco, R. D.; Tron, G. C.;
Sorba, G.; Genazzani, A. A. J Med Chem 2006, 49, 467.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet