Molecular design and synthesis of 1,4-disubstituted piperazines as α1-adrenergic receptor blockers

Article abstract of DOI:10.1016/j.bioorg.2014.03.005

A new series of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide and 3,5,6,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin- 4-one derivatives were designed, synthesized, their binding and functional properties as α₁-adrenorec

Full text of DOI:10.1016/j.bioorg.2014.03.005

Accepted Manuscript
Molecular design and synthesis of 1,4-disubstituted piperazines as α1-adrenergic
receptor blockers
Dalal A. Abou El-Ella, Mohammed M. Hussein, Rabah A.T. Serya, Rana M.
Abdel Naby, Ahmed M. Al-Abd, Dalia O. Saleh, Wafaa I. El-Eraky, Khaled M.
Abouzid
PII:
S0045-2068(14)00017-0
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.03.005
YBIOO 1706
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
25 October 2013
Please cite this article as: D.A. Abou El-Ella, M.M. Hussein, R.A.T. Serya, R.M. Abdel Naby, A.M. Al-Abd, D.O.
Saleh, W.I. El-Eraky, K.M. Abouzid, Molecular design and synthesis of 1,4-disubstituted piperazines as α1-
adrenergic receptor blockers, Bioorganic Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.03.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular design and synthesis of 1,4-disubstituted piperazines as α -adrenergic
1
receptor blockers
Dalal A. Abou El-Ella1**, Mohammed M. Hussein2, Rabah A.T. Serya1, Rana M. Abdel
Naby3, Ahmed M. Al-Abd4 , Dalia O. Saleh4, Wafaa I. El-Eraky4 and Khaled M. Abouzid1*
1Department of Pharmaceutical Chemistry, faculty of pharmacy, Ain Shams University, Abbasia 11566,
Cairo, Egypt.2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El
Aini 11562, Cairo, Egypt.3Department of Pharmaceutical Chemistry, Misr University for Science and
Technology, Al-Motamayez, 6Th October, Egypt.4Department of Pharmacology, Medical Division, National
Research Center, Dokki, Cairo, Egypt.
Abstract:
A new series of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide and 3,5,6,8-
tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4-one derivatives were designed, synthesized,
their binding and functional properties as α -adrenoreceptors blockers were evaluated. A new
1
validated α -adrenoreceptor blocker pharmacophore model (hypothesis) was generated using
1
Discovery Studio 2.5. The compare-fit study for the designed molecules with the generated
hypothesis was fulfilled and several compounds showed significant high fit values. Compounds
IVa-c, VIIa-d, VIIIa-c, Xa-c, XIa-d have shown blocking activity ranging from 46.73% up to
94.74% compared to 99.17% for prazosin.
Key words:
Phenylpiperazines, Pyridothienopyrimidine, α -Adrenoreceptors blockers.
1
Corresponding authors*:
1) Emails: abouzid@yahoo.com, khaled.abouzid@pharm.asu.edu.eg, Tel.: +201222165624
2) Emails: dalal999@hotmail.com, dalal@pharm.asu.edu.eg, Tel.: +201222745745

1-Introduction:
Elevated blood pressure is a common disorder affecting high proportion of the community which
may amounts up to 15 % or more. Many of these individuals have no apparent signs which may
lead to congestive heart failure, myocardial infarction, renal and even cerebrovascular damages.
When dealt with early, the incidence of these morbidity and mortality may be decreased. Thus, the
need for selective and safe antihypertensive drug became an important research point. By studying
different classes of antihypertensive drugs, the α - adrenergic receptors (ARs) antagonists emerged
1
as promising effective class with fewer side effects. The α1- adrenergic subtypes are members of
the G-protein-coupled receptor (GPCR) superfamily. At least three distinct α - receptor subtypes
1
have been reported on the basis of both pharmacology and molecular biology [1-7]. The GPCRs are
integral membrane proteins and contain seven hydrophobic domains which are believed to be
arranged in seven membrane spanning helices [7-9]. These helices are responsible for passing
signals across the cell membrane after interaction with their ligand on the extracellular side. The
binding of the ligand elicits a response felt by the G-protein on the intracellular side. The activated
G-protein then sets a second messenger system in train. Different GPCRs recognize different
ligands. For adrenergic receptors, the activating ligands are norepinephrine and epinephrine..
Adrenoreceptors (AR) are classified into α-AR and β-AR [7]. α-ARs play a pivotal role in the
regulation of a variety of physiological processes, particularly within the cardiovascular system and
are divided into two main subtypes α1- and α2-ARs [2,8]. The α1 and α2-ARs are located in the
vascular smooth muscle cell membrane, and upon stimulation by an appropriate agonist, mediate
vasoconstriction. The simultaneous occurrence of both receptor subtypes on vascular smooth
muscles indicate that α1and α2-ARs could contribute to the maintenance of peripheral arterial tone
and may play an important role in resistance seen in hypertension. α1-ARs modulate intercellular
biochemical processes in response to changes in extracellular concentrations of the neurotransmitter
norepinephrine and the circulating hormone epinephrine [9-11] . The α -adrenergic receptor
1
subtypes (α A-, α B-, α D) are the prime mediators of smooth muscle contraction and hypertrophic
1
1
1
growth, but their characterization in both binding and function have lagged the other adrenergic
family members. The α A-ARs are the predominant receptor subtypes, causing vasoconstriction in
1
many vascular beds, including the following arteries: mammary, mesenteric, splenic, hepatic,
omental, renal, pulmonary, and epicardial coronary. It is also the predominant subtype in the vena
cava and the saphenous and pulmonary veins. Together with the α B receptor subtype, it promotes
1
cardiac growth and structure. The α B receptor subtype is the most abundant type in the heart,
1
whereas the α D receptor subtype is the predominant receptor, causing vasoconstriction in the
1
aorta.[3]. Studies using young prehypertensive and adult spontaneously hypertensive rats as well as

knockout mice suggest that vascular α D-adrenoceptors are involved in the genesis/maintenance of
1
hypertension [26]. Compounds acting as antagonists at various post-junctional α1A-ARs are
frequently used in the therapy of high blood pressure, most common drug are prazosin (1) [7,12] ,
doxazosin (2) [6] and terazosin (3) [9]. They cause arterial and venous vasodilatation and
consequently lower total peripheral resistance, lower arterial blood pressure, and decrease platelet
aggregation. Also, they may be useful in the treatment of benign prostatic hyperplasia (BPH), lower
urinary tract symptoms (LUTS), and cardiac arrhythmia [1-5]. But the available subtype-selective
drugs such as BMY-7378 [6, 12] and WAY-100635 (5) [10] are only moderately selective as native
tissues can express all the three subtypes. Thus, the aim in this research is to develop more subtype
selective antagonists with higher efficacy, minimum side effects, and better patient compliance.
In order to design such ligands, structural information on the receptor subtypes, especially their
binding sites, would be invaluable. There is, however, no X-ray structure available for any GPCRs.
Many GPCR transmembrane domains have been modeled on the basis of the known structure of
bacteriorhodopsin, but these models are only qualitative in nature and are still under development
[7-9].
NH2
N
NH2
H3CO
H3CO
H3CO
H3CO
N
N
O
O
N
N
O
N
N
N
Prazosin (1)
Doxazosin (2)
O
O
NH2
H3CO
H3CO
N
N
N
O
N
Terazosin (3)
O
O
N
O
H3CO
N
H3CO
O
N
N
N
N
N
WAY-100635 (5)
BMY-7378 (4)
The most recent models elaborated using radioligand binding data of selective α -ARs
1
antagonists and computer aided methods with Catalyst have been proposed by Bremner et al. [6]
and Barbaro et al. [7]. Barbaro’s model, especially useful for phenylpiperazine derivatives had
postulated five pharmacophore features [2,7]: a positive ionizable atom (PI), three hydrophobic
regions (HY1–HY3), and a hydrogen bond acceptor (HBA). As 2-substituted phenylpiperazines
derivatives, possess a positive ionizable nitrogen atom and an ortho-substituted phenyl ring
corresponding to PI (A) and both HY1 and HY2 (B), respectively. These compounds also contain

an additional hydrophobic moiety (ending the heterocyclic fragment) and a carbonyl group, which
are corresponding to the HY3 and HBA features, respectively (C). Additionally, they possess an
alkyl spacer between piperazine nitrogen and nitrogen placed close to carbonyl oxygen (HBA) at a
chain or at heterocyclic rings. The alkyl spacer may be 2–7 carbons in length and may additionally
be substituted with alkyl or hydroxyl moieties (Fig. 1) [8,9].
Based on the previously reported SAR, the target compounds (IVa-c, VIa-d, VIIIa-c, Xa-c, and
XIa-d) were designed according to the following bioisosteric modification carried on reference
compounds REC15/2739 (6) [6] and RN17 (7) [12] (Fig. 2 and 3). The influence of these
bioisosteric modifications on their α -ARs affinity will be studied.
1
2. Results and Discussion:
2.1. Molecular modeling study:
Molecular modeling studies were used to design a selective α A antagonists using pharmacophore
1
modeling.
Pharmacophore modeling method has been widely used as a key tool of computer aided drug design
in the lead discovery and optimization and also, to rationalize the relationship between the structural
features and pharmacological activity. Molecular modeling simulation studies were performed by
running the “common feature pharmacophore model” protocol in Discovery Studio 2.5 software
and the generated pharmacophore model (hypothesis) for α1A-ARs antagonists was validated and
used for virtual screening of the designed compounds that were proposed through SAR
manipulation [2,6,7]. Lead compounds used to generate common feature pharmacophore are listed
in (Table 1). The generated α A-AR antagonist hypotheses were subjected to simulation compare/fit
1
studies (using the best-fit algorithmic method) with the conformational model of the proposed
training set compounds to predict their α A-AR antagonist activities. The simulated fitting values of
1
the best-fitted conformer may be a guide for prioritizing relative affinities of these compounds with
the receptor. Consequently, the fit values between the reference drug prazosin (1) which is reported
to posses high α A antagonistic activity [7,12], IVa-c, VIa-d, VIIIa-c, Xa-c, XIa-d, and the α A-AR
1
1
antagonist hypothesis were calculated separately.
(Table 1) shows of the results of the best fitting value of the best-fitted conformer of leads used to
generate this hypothesis
The training set used above are reported to have more potent antagonistic activity at α1A-AR over
other subtypes [6,7] which might support the idea that the generated hypothesis will be more
selective for α1A-AR antagonists. The common feature hypotheses generation gave 10 hypotheses.

The assessment of the generated α -ARs antagonists hypotheses revealed that hypothesis ranking
1
number 4 (Fig. 4) was proved to be the ideal one rather than the other generated hypothesis
numbers. This was because hypothesis number 4 has more matched simulation data with
experimental biological activity.
2.1.1. Validation of the generated pharmacophore model:
The generated hypothesis encompassed five features namely; positive ionizable (PI), hydrogen
bonding acceptor (HBA), and three hydrophobic features (HY1, HY2, and HY3) (Fig. 4), which
was consistent with the reported literature [6, 7].
1) Mapping of prazosin (1) to the generated pharmacophore model:
This hypothesis showed full mapping of all of its five features with the lead structure of the
Prazosin. (Fig.5).
2) Validation through mapping of certain reported active α A-AR antagonists (test set):
1
Further validation included testing a reported highly selective α A-ARs antagonists [6,7] with the
1
generated hypothesis and the simulation fit values showed good mapping with high fit values which
validate the efficiency of our hypothesis as shown in (Table 2)
2.1.2. Mapping of the target compounds IVa-c, VIa-d, VIIIa-c, Xa-c, and XIa-d:
The newly designed molecules were subjected to molecular modelling study through ligand-
pharmacophore mapping, calculating the fitting value and comparing them to that of the reference
compound. The results of the simulation study are summarized in (Table 3). The results of such
simulation studies have revealed that most of the designed compounds would be promising active
hit molecules.
The highest score compounds IVb, VIIIb and XIb mapping to the newly generated pharmacophore
is illustrated in (Fig. 6).
2.1.3 Structure activity relationship of the investigated compounds:
1- The substitution on the phenyl ring of the arylpiperazine is crucial element for the α1-ARs
blocking activity. As in the IXa-d series, there was a decrease in the activity by changing the
substitution on the phenyl ring in the following order o-methoxy (94.74 % )
m-chloro
(86.46%) unsubstituted (77.93%) o-flouro (65.68%). The same was observed in the
VIIIa-c series; where compound VIIIb showed the highest activity among this series
(78.27%) compared to 46.37% for the unsubstituted VIIIa. Also, in VIa-c series, o-methoxy

has great effect on the activity where compound VIb showed 76.76% blocking activity
compared to 69.93% for the m-chloro substituted VId. Unexpectedly in Va-c series, Vc with
o-flouro substitution showed 88.88% blocking activity compared to o-methoxy Vb
(81.15%).Thus the presence of a methoxy group at the ortho position seems optimum for the
activity.
2- The length of the alkyl spacer between N1 of the phenyl piperazine and the
thienopyrimidine or thienopyridines rings is considered important for the α1- ARs blocking
activity. A gradual decrease in the activity was observed by lengthening the spacer in the 6-
methyl-4, 5, 6, 7-tetrahydrothieno [2, 3-c]pyridine-3-carboxylic acid amide analogs Va-c,
VIa-d, and VIIa-c. Where the highest activity was found in Va-c series compound Vc
(88.88%), Vb (81.15%) characterized by two-carbon alkyl chain (2-oxo-ethylamino). For
5,6,7,8-tetrahydropyrido[4’,3’:4,5]-thieno[2,3-d]-pyrimidin-4(3H)-one analogs the opposite
occur; changing the linker from 2carbon-chain (2-oxo-ethylamino-thio) in VIIIa-c series to
propyl saturated chain in IXa-d series showed increase in the α1- ARs blocking activity,
where compound IXb possesses 94.74% blocking activity compared to 78.27% for
compound VIIIb. This may be contributed to placing the important features at good spacial
distances that allowed good fitting with receptor.
3- The thienopyrimidine and thienopyridine rings both have good effect on the activity as they
fit in the third hydrophobic (HY-3) pocket in the receptor. By comparing the 6-methyl-4, 5,
6, 7-tetrahydrothieno [2,3-c]pyridine-3-carboxylic acid amide
and 5, 6, 7, 8-
tetrahydropyrido [4’,3’:4,5]-thieno [2, 3-d]-pyrimidin-4(3H)-one analogs, it was obvious
that they have comparable activities.
.2.2. Chemistry:
In scheme 1, the o-amino amide (II) was prepared by the method reported by Gewald et al [13]
using 1-methyl-piperidin-4-one (I) as the starting reagent and 2-cyanoacetamide [14]; this o-amino
amide (II) was N-alkylated in dry acetone and anhydrous K2CO3 with1-(2-chloroacetyl)-4-(2-
substitutedphenyl)piperazines (IIIa-c) prepared as reported by Bourdais et al [15] to give the target
compounds IVa-c [16-18]. Compounds VIa-d were prepared in the same manner as compounds
IVa-c by alkylating o-amino amide (II) with 1-(2-substitutedphenyl)-4-(3-chloropropyl)-
piperazines (Va-c) prepared in situ as hydrochloride salt for compounds VIa-c [15] or with the
commercially available 1-(3-chlorophenyl)-4-(3-chloropropyl)-piperazine.2HCl for compound VId
c [16-18]. Compounds VIIIa-c was prepared by refluxing compound (VII) with commercially
available 2-substituted-phenylpiperazines in DMF and anhydrous K2CO3 [16, 19].

In scheme 2, the o-amino amide (II) was cyclized into compound (IX) [20, 21] using CS2 in
NaOH; this key intermediate (IX) was furthe alkylated with phenylpiperazine derivatives (IIIa-c),
(Va-c) or the commercially available 1-(3-chlorophenyl)-4-(3-chloropropyl)-piperazine.2HCl in
ethanolic KOH [22, 23] to give compounds Xa-c, XIa-d, respectively. All the proposed structures
were confirmed by Elemental Analysis, IR, and 1H-NMR data.
2.3. Biological Evaluation:
2.3.1. In vivo vasodilatation activity (functional bioassay):
Based on the fitting values obtained from Discovery studio, the active hits with best fitting
values such as IVb, IVc, VIb, VId, VIIIb, Xa-c and XIa-d were subjected to functional bioassay to
evaluate their α -adrenoreceptors antagonistic activity, according to the standard procedure [24].
1
The antagonistic activity was assessed by the inhibition of the norepinephrine (NE)-induced
contraction on isolated rat aorta tissues which predominantly express the α D-AR subtype which
1
mediate vasoconstriction in rat aorta [2, 25] and using prazosin (1) as the reference drug.
Stimulation of α D -AR subtype is known to cause blood vessels contraction and control blood
1
pressure.
Prazosin (1) completely abolished norepinephrine induced aortic contraction (B, 0.83%).
Compounds IVc, XIb, and XId showed the most potent α -blocking activity by inhibiting aortic
1
tension to 11.1, 5.3 and 13.5% its norepinephrine contracted tone (Table 4). Compounds IVb, VIb,
VId, Xb, Xc, XIa, XIc, and VIIIb showed potentially strong α -blocking activity by decreasing the
1
aortic tension to 18.9 - 34.3% of norepinephrine contracted aortic tension (Table 4). Only
compounds Xa showed mild α -blocking activity by decreasing the aortic tension to 53.6% (Table
1
4).
2.3.2. Aortic ring tension recording:
The change in pre-treated aortic muscle tension after nor epinephrine exposure was recorded (Table
4). This change in tension is expressed as average muscle tone over duration of 1-2 minutes of
recording
3. Conclusion:
Novel series of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide and 3,5,6,8-
tetrahydropyrido[4’,3’:4,5]-thieno [2,3-d]pyrimidin-4-one derivatives were designed and

synthesized. These 2 series were characterized by variable linker chain and substituents on the
phenylpiperazine ring. The molecular modeling study helped to clarify molecular requirements for
the design of high selective α -adrenergic ligands. The compounds that gave good fitting values
1
through the pharmacophoric model were tested for their a1-ARs antagonistic activity on isolated
thoracic rat aorta. The tested compounds IVb, IVc, VIb, Xb, XIa, XIb, and XId showed comparable
activity to prazosin (1) as reference standard and the results were matched with their high fitting
values ( table 3). Some structural features have been proven to markedly affect the affinity to α -
1
ARs blocking activity. In particular, the addition of o-methoxyphenyl on piperazine ring and
presence of propyl spacer led to the best α -ARs affinity profile (high fitting value= 4.88; Figure 6)
1
and the highest α -ARs blocking activity (94.74 % aortic ring inhibition) as in compound XIb.
1
4. Experimental
4.1. Molecular Modeling study:
In this work, the pharmacophore hypothesis was produced using Accelrys Discovery Studio 2.5
in the computer drug design lab in Pharmaceutical Chemistry Department, Faculty of pharmacy,
Ain Shams University. The set of conformational models of each structure of the lead compounds
(table 1) was performed and was used to generate the common feature hypotheses. The ideal
hypothesis encompassed five features; positive ionizable nitrogen, three hydrophobic pockets and a
hydrogen bond acceptor group.
4.1.1. Generation of the pharmacophore model for α -ARs antagonists:
1
1) The training set for the development of the pharmacophore has been chosen according to the
catalyst guidelines and loaded into discovery studio.
2) Generate hypothesis from this set using Protocol/ Pharmacophore/ Common Feature
Pharmacophore Generation.
3) Catalyst produces 10 hypotheses and Hypo5 is the best simulated pharmacophore hypothesis
in this study, characterized by the highest cost difference, lowest error cost and lowest root mean-
square divergence and has the best correlation coefficient. All 10 hypotheses have the same
features: one positive ionizable, one hydrogen-bond donor, one aromatic ring, and one hydrophobic
group.

4.1.2. Mapping of the test compounds (IVa-c, VIa-d, VIIIa-c, Xa-c, and XIa-d):
1. The structures of the test set of the target and tetrahydrothienopyridines
pyridothienopyrimidines were built using the Discovery Studio software.
2. Their conformational models were generated in the energy range of 20 kcal/mol above the
estimated global energy minimum to ensure conformational diversity.
3. The fitting of the tested compounds was performed using ligand pharmacophore mapping
protocol. The best fit option has been selected which manipulate conformers of each compound to
find, when possible, different mapping modes of the ligand within the model. Mappings for all the
test compounds to the hypothesis were visualized.
4.2. Chemistry:
Melting points were determined by open capillary method using Electro thermal capillary
melting point apparatus 9100 and were uncorrected. FT-IR spectra were recorded on a Shimadzu IR
1
Affinity-1 Spectrophotometer, using KBr discs and measured by , cm-1. H-NMR spectra were
recorded in δ scale given in ppm, performed on a JEOL ECA 300, 500 MHz spectrometer using
CDCl3 or DMSO as stated, using TMS as internal standard at National center for research and Cairo
University. Mass spectra were performed on Shimadzu Qp-2010 plus (70 eV) spectrometer at
Cairo University. Elemental analysis was performed at Azhar University. Progress of the reactions
was monitored by TLC using pre-coated aluminium sheets silica gel ALUGRAM® SIL GLUV254
and was visualized under Vilber Lourmat UV lamp at 254nm.
Compound II was prepared adopting the reported method [13, 14] as follows:
4.1.1. 2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide (II):
To a mixture of N-methylpiperdine-4-one (1.92 g, 0.0177 mol) and Sulphur (0.62 g, 0.0195 mol) in
(25 mL) absolute ethanol, cyanoacetamide (1.83 g, 0.0195 mol) was added, followed by slow
addition of morpholine (2.5 mL). The reaction was heated at 40-50°C for18 hrs, after which an
equivalent amount of water was added and the mixture was refrigerated. The separated product was
filtered, dried, and crystallized from ethanol/water mixture. Yield: 69%, found m.p: 188-190 ºC;
reported m.p.:190 °C [14], IR (KBr, cm-1): 3255-3410 (2 NH2 stretching), 1674 (C=O).
4.1.2. 2-(2-Chloroacetylamino)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylic
acid amide (VII): A solution of II (2.11g, 0.0100 mol) and sodium acetate (0.82g) in DMF (17

mL) was kept in ice bath and chloroacetyl chloride (1.12 mL, 0.0100 mol) was added dropwise.
The reaction was stirred at 20˚C for 3 hrs, and then poured into crushed ice. The separated solid
was filtered, dried and crystallized from absolute ethanol. Yield: 59%; m.p: 256-263°C; FT-IR
(
max, cm-1): 1724 (C=O); MS (m/z: M+:286, M+2: 288); 1H-NMR (300 MHz, CDCl3): δ=4.58
(O=C-CH2-Cl).
4.1.3. 2-Mercapto-7-methyl-3,5,6,8-tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4-one
(IX): A solution of I (2.11g, 0.0100 mol) and NaOH (0.43g, 0.0100 mol) in 10 mL ethanol/ water
mixture (5:1) was stirred for 30 minutes. CS2 (0.90 mL, 0.0110 mol) was then added and stirring
was continued for 20 hrs on cold. The reaction was diluted with an equivalent amount of water and
acetic acid was added dropwise to render the medium slightly acidic. The solid formed was filtered,
dried and crystalized from absolute ethanol. Yield: 95%; m.p: 290-292°C; FT-IR ( max, cm-1):
3506 cm-1 (NH, 2 amine).
4.1.4. 2-Chloro-1-[4-(2-substitutedphenyl)-piperazin-1-yl]-ethanone derivatives (IIIa-c):
Prepared according to the general procedure reported [15]
General Procedure: To a solution of the corresponding arylpiperazines (0.0100 mol) in benzene
(20 mL) and 2.5 mL 20% NaOH, chloroacetyl chloride (1.13 g, 0.0100 mole) was added dropwise
with stirring at 0˚C. After complete addition, stirring was continued for 3 hrs at 0-10˚C. The
benzene layer was washed with water and dried over anhydrous MgSO4. The solvent was
evaporated under vacuum leaving a solid material, which was collected, washed with ether and
crystallized from n-hexane-benzene mixture, as reported [15].
5.1.4.1. 2-Chloro-1-[4-(2-substituted-phenyl)-piperazin-1-yl]-ethanone (IIIa): Yield: 59 %; found
m.p: 76 ºC, reported m.p.: 75°C[15].
5.1.4.2. 2-Chloro-1-[4-(2-substitutedphenyl)-piperazin-1-yl]-ethanone (IIIb): Yield: 70%; found
m.p: 100 ºC, reported m.p.: 100°C.[15].
5.1.4.3. 2-Chloro-1-[4-(2-substitutedphenyl)-piperazin-1-yl]-ethanone (IIIc): Yield: 49 %; found
m.p: 112-114 ºC, reported m.p.: 115°C.[15].
4.1.5. 1-(2-Substitutedphenyl)-4-(3-chloropropyl)-piperazine. Dihydrochloride derivatives (Va-c):
General Procedures: A solution of the appropriate arylpiperazine (0.1000 mole), 1-bromo-3-
chloropropane (18.9 mL, 0.1200 mole) and anhydrous K2CO3 (16.65 g, 0.1200 mole) in dry DMF

(100 mL) was stirred at 20 ºC for 24 hrs, after which it was filtered. To the filtrate, ethanolic HCl
and diethyl ether was added portionwise till complete precipitation of the dihydrochloride
derivatives. The solid was filtered and washed with ether to give compounds Va-c as reported [15].
4.1.5.1. 1-(3-Chloropropyl)-4-phenylpiperazine.dihydrochloride (Va): Yield: 75%; found m.p:160
ºC, reported m.p. 160°C.[15].
4.1.5.2. 1-(3-Chloropropyl)-4-(2-methoxyphenyl)piperazine. dihydrochloride (Vb): Yield: 70%;
found m.p: 118-120 ºC, reported m.p.: 120°C.[15].
4.1.5.3. 1-(3-Chloropropyl)-4-(2-fluorophenyl)piperazine. dihydrochloride (Vc): Yield: 49 %;
found m.p: 165 ºC, reported m.p.:165°C.[15].
4.1.6.
2-{2-[4-(2-Substitutedphenyl)piperazin-1-yl]-2-oxo-ethylamino}-6-methyl-4,5,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide derivatives (IVa-c): General
Procedures: The titled compounds were prepared by refluxing a mixture of II (2.11g, 0.0100 mol),
anhydrous K2CO3 (1.38 g, 0.0100 mol) and (0.0120 mol) of 2-chloro-1-[4-(2-substituted phenyl)-
piperazin-1-yl]-ethanone (IIIa-c) in (30 mL) dry acetone for 12 hrs. The reaction was then filtered,
washed with ethanol. The filtrate was concentrated and the formed precipitate was collected, dried
and crystallized from methanol/ethyl acetate mixture to give pure solids of IVa-c.
4.1.6.1.
6-Methyl-2-[2-oxo-2-(4-phenylpiperazin-1-yl)-ethylamino]-4,5,7-tetrahydrothieno[2,3-
1
c]pyridine-3-carboxylic acid amide (IVa): Yield: 63%; m.p: 77-80ºC; H-NMR (500 MHz,
DMSO): δ 2.29 (s,3H, CH3-N), δ 2.7 (t, J= 5.2 Hz, 2H, CH2-N of pyridine), δ 2.59 (t, J= 5.2 Hz,
2H, CH2CH2-N of pyridine), δ 3.6 (s, 2H, CH2-N of pyridine), δ 3.5 (t, J= 5.2 Hz, 4H, 2CH2-N of
piperazine), δ 3.8 (t, J= 5.1 Hz, 4H, 2CH2-N of piperazine), δ 4.09 (s, 2H, NH-CH2-CO), δ 6.6-7.1
(m, 5H, Ar); FT-IR ( max, cm-1): 1597&1635 (2 C=O), 2819-2937 (CH aliphatic), 3026-3055
(CH Ar), 3388 (NH amide); MS (M. Wt.: 413): m/z 413 (M+, 100%). Anal. Calcd for
C21H27N5O2S: C, 60.99; H, 6.58; N, 16.94 Found: C, 61.25; H, 6.53; N, 17.32.
4.1.6.2.
2-{2-[4-(2-Methoxyphenyl)-piperazin-1-yl]-2-oxo-ethylamino}-6-methyl-4,5,7-
1
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide (IVb): Yield: 91%; mp: 94-95ºC; H-
NMR (300 MHz, DMSO): δ 2.28 (s,3H, CH3-N), δ 3.0 (t, J= 5.8 Hz, 2H, CH2-N of pyridine), δ
2.79 (t, J= 5.8 Hz, 2H, CH2CH2-N of pyridine), δ 3.69 (s, 2H, CH2-N of pyridine), δ 3.47 (t, 4H,
2CH2-N of piperazine), δ 3.6 (t, 4H, 2CH2-N of piperazine), δ 4.11 (s, 2H, NH-CH2-CO), δ 3.78 (s,
3H, Ph-OCH3), δ 6.7-7.1 (m, 4H, Ar); FT-IR( max, cm-1): 1641&1656 (2C=O), 2831-2997 (CH,

aliphatic), 3061 (CH Ar), 3388 (NH); MS (M. Wt.: 443): m/z 441 (M+, 100%). Anal. Calcd for
C22H29N5O3S: C, 59.57; H, 6.59; N, 15.79 Found: C, 59.91; H, 6.45; N, 16.11.
4.6.1.3.
2-{2-[4-(2-Flourophenyl)-piperazin-1-yl]-2-oxo-ethylamino}-6-methyl-4,5,7-
1
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide (IVc): Yield: 67%; mp: 85-87ºC; H-
NMR (300 MHz, DMSO): δ 2.26 (s,3H, CH3-N), δ 2.93 (t, J= 5.9 Hz, 2H, CH2-N of pyridine), δ
2.61 (t, J= 5.9 Hz, 2H, CH2CH2-N of pyridine), 3.68 (s, 2H, CH2-N of pyridine), δ 3.31 (t, 4H,
2CH2-N of piperazine), δ 3.69 (t, 4H, 2CH2-N of piperazine),δ 4.10 (s, 2H, NH-CH2-CO), δ 6.6-
6.88 (m, 4H, Ar); FT-IR ( max, cm-1): 1629&1641 (2C=O), 2829-2945 (CH, aliphatic), 3039-
3066 (CH, Ar), 3396 (NH); MS (M.Wt: 431): m/z 431(M+, 100%). Anal. Calcd for C21H26FN5O2S:
C, 58.45; H, 6.07; N, 16.23 Found: C, 58.63; H, 6.15; N, 16.70.
4.1.7.
2-{3-[4-(2-or3-Substitutedphenyl)-piperazin-1-yl]-propylamino}-6-methyl-4,5,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide derivatives (VIa-d). General
Procedures: To a stirred solution of II (2.11g, 0.0100 mol) and anhydrous K2CO3 (4.14 g, 0.0300
mol) in (30 mL) dry acetone, (0.0120 mol) of 1-(3-chloro-propyl)-4-(2-substituted-phenyl)-
piperazines (Va-c) were added for compounds VIa-c or (3.7 g, 0.0120 mol) of 1-(3-chloropropyl)-4-
(3-chlorophenyl)-piperazine was added for compound VId. Stirring and reflux were continued for
12 hrs. The reaction mixture was then filtered while hot, washed with ethanol and the filtrate was
concentrated under vacuum. The solid formed was filtered and crystallized from methanol/ethyl
acetate mixture to yield the titled compounds.
4.1.7.1.
6-Methyl-2-[3-(4-pheny-piperazin-1-yl)-propylamino]-4,5,7-tetrahydrothieno[2,3-
1
c]pyridine-3-carboxylic acid amide (VIa): Yield: 85%; m.p: 73-74°C; H-NMR (500 MHz,
DMSO): δ 3.04 (t, J= 5.2 Hz, 2H, N-CH2-CH2-CH2), δ1.9 (m, J= 5.2 Hz, 2H, N-CH2-CH2-CH2), δ
2,34 (t, J= 5.2 Hz, 2H, N-CH2-CH2-CH2), δ 2.36 (s, 3H, CH3-N), δ 2.84 (t, J= 5.7 Hz, 2H, CH2-N
of pyridine), δ 2.73 (t, J= 5.7 Hz, 2H, CH2-CH2-N of pyridine), δ 3.68 (s, 2H, CH2-N of pyridine),δ
2.71 (t, 4H, 2CH2-N of piperazine), δ 3.53 (t, 4H, 2CH2-N of piperazine), δ 6.63-7.01 (m, 5H, Ar);
FT-IR ( , max, cm-1): 1635 &1650 (C=O amide), 2823-2924 (CH aliphatic), 3032-3062 (CH
aromatic), 3390-3450 (NH2 amide); MS: 413(M+, 100%); Anal. Calcld for C22H31N5OS: C, 63.89;
H, 7.56; N, 16.93 Found C, 64.08; H, 7.63; N, 17.14.
4.1.7.2.
2-{3-[4-(2-Methoxyphenyl)-piperazin-1-yl]-propylamino}-6-methyl-4,5,7-
1
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide VIb: Yield: 83%; m.p: 98-100ºC; H-
NMR (500 MHz, CDCl3): δ 3.08 (t, J= 5.4 Hz, 2H, N-CH2-CH2-CH2), δ1.26 (m, J= 5.4 Hz, 2H, N-
CH2-CH2-CH2), δ2,47 (t, J= 5.4 Hz, 2H, N-CH2-CH2-CH2), δ 2.17 (s, 3H, CH3-N), δ 2.78 (t, J= 5.9

Hz, 2H, CH2-N of pyridine), δ 2.74 (t, J= 5.9 Hz, 2H, CH2-CH2-N of pyridine), δ 3.68 (s, 2H, CH2-
N of pyridine),δ 2.72 (t, 4H, 2CH2-N of piperazine), δ 3.38 (t, 4H, 2CH2-N of piperazine), δ 3.85 (s,
3H, Ph-OCH3), δ 6.74-6.90 (m, 4H, Ar); FT-IR ( max, cm-1): 1650 (C=O amide), 2831-2927 (CH
aliphatic), 3032-3066 (CH Ar), 3394-3417 (NH, NH2); MS: (MWt.: 431): m/z 443(M+, 100%).
Anal. Calcd for C23H33N5O2S: C, 62.27; H, 7.50; N, 15.79 Found C, 62.41; H, 7.13; N, 16.02.
4.1.7.3. 2-{3-[4-(2-Flourophenyl)-piperazin-1-yl]-propylamino}-6-methyl-4,5,7-tetrahydrothieno
1
[2,3-c]pyridine-3-carboxylic acid amide (VIc): Yield: 59%; m.p: 67-68ºC; H-NMR (500 MHz,
CDCl3): δ 3.35 (t, J= 5.3 Hz, 2H, N-CH2-CH2-CH2), δ 1.96 (m, J= 5.3 Hz, 2H, N-CH2-CH2-CH2), δ
2,48 (t, J= 5.3 Hz, 2H, N-CH2-CH2-CH2), δ 2.17 (s, 3H, CH3-N), δ 2.78 (t, J= 6.6 Hz, 2H, CH2-N
of pyridine), δ 2.72 (t, J= 6.6 Hz, 2H, CH2-CH2-N of pyridine), δ 3.74 (s, 2H, CH2-N of pyridine),δ
2.64 (t, 4H, 2CH2-N of piperazine), δ 3.57 (t, 4H, 2CH2-N of piperazine), δ 6.61-7.08 (m, 4H, Ar);
FT-IR ( max, cm-1): 1650 (C=O amide), 2831-2927 (CH aliphatic), 3032-3066 (CH Ar), 3394-
3417 (NH, NH2); MS (M.Wt.: 431): m/z 443 (M+, 100%). Anal. Calcd for C22H30FN5OS: C, 61.23;
H, 7.01; N, 16.23 Found C, 61.35; H, 7.12; N, 16.58.
4.1.7.4. 2-{3-[4-(3-Chlorophenyl)-piperazin-1-yl]-propylamino}-6-methyl-4,5,7-tetrahydrothieno
1
[2,3-c]pyridine-3-carboxylic acid amide (VId): Yield: 40%); m.p: 110-113ºC; H-NMR (500
MHz, CDCl3): δ 3.21 (t, J= 5.2 Hz, 2H, N-CH2-CH2-CH2), δ 1.25 (m, J= 5.2 Hz, 2H, N-CH2-CH2-
CH2), δ 2,47 (t, J= 5 Hz, 2H, N-CH2-CH2-CH2), δ 2.15 (s, 3H, CH3-N), δ 2.93 (t, J= 5.6 Hz, 2H,
CH2-N of pyridine), δ 2.81 (t, J= 5.4 Hz, 2H, CH2-CH2-N of pyridine), δ 3.65 (s, 2H, CH2-N of
pyridine), δ 2.62 (t, 4H, 2CH2-N of piperazine), δ 3.69 (t, 4H, 2CH2-N of piperazine), δ 6.50-7.02
(m, 4H, Ar); MS (M.Wt.: 447): m/z 447 (M+, 100%), 449 (M+2, 24.5%); Anal. Calcd for
C22H30ClN5OS: C, 58.98; H, 6.75; N, 15.63 Found C, 58.68; H, 6.52; N, 15.97.
4.1.8. 2-{2-[4-(2-substitutedphenyl)-piperazin-1-yl]-acetylamino}-6-methyl-4,5,7-tetrahydrothieno
[2,3-c]pyridine-3-carboxylic acid amide derivatives (VIIIa-c): General Procedures: The titled
compounds VIIIa-c were prepared by refluxing compound VII (2.87g, 0.0100 mol) with different
(1or 2-substitutedphenyl)-piperazines (0.0120 mol) and anhydrous K2CO3 (1.38g, 0.0100 mol) in
DMF (20 mL) for 12 hrs. The reaction was diluted with iced cold water. The yellow solid formed
was filtered, dried and crystallized from absolute ethanol.
4.1.8.1.
6-Methyl-2-[2-(4-phenylpiperazin-1-yl)-acetylamino]-4,5,7-tetrahydrothieno[2,3-
1
c]pyridine-3-carboxylic acid amide (VIIIa): Yield: 68%; m.p: 200ºC; H-NMR (300 MHz,
CDCl3): δ 2.55 (s,3H, CH3-N), δ 2.85 (t, J= 5.9 Hz, 2H, CH2-N of pyridine), δ 2.87 (t, J= 5.9 Hz,
2H, CH2-CH2-N of pyridine), δ 3.65 (s, 2H, CH2-N of pyridine),δ 2.87 (t, 4H, 2CH2-N of

piperazine), δ 3.42 (t, 4H, 2CH2-N of piperazine), δ 6.60-7.01 (m, 5H, Ar) δ 3.30 (s, 2H, CO-CH2-
N); FT-IR ( max, cm-1): 1674&1645 (C=O), 2945-2819 (CH aliphatic), 3157-3024 (CH Ar),
3502-3307 (NH amide); MS (M.Wt.: 413): m/z 414 (M+, 100%); Anal. Calcd for C21H27N5O2S: C,
60.99; H, 6.58; N, 16.94 Found C, 61.14; H, 6.83; N, 17.32.
4.1.8.2.
2-{2-[4-(2-Methoxyphenyl)-piperazin-1-yl]-acetylamino}-6-methyl-4,5,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide (VIIIb): Yield: 64%; m.p: 111-112ºC;
1H-NMR (300 MHz, CDCl3): δ 2.38 (s,3H, CH3-N), δ 2.78 (t, J= 5.9 Hz, 2H, CH2-N of pyridine), δ
2.74 (t, J= 5.9 Hz, 2H, CH2-CH2-N of pyridine), δ 3.54 (s, 2H, CH2-N of pyridine),δ 2.48 (t, 4H,
2CH2-N of piperazine), δ 3.18 (t, 4H, 2CH2-N of piperazine), δ 6.70-6.88 (m, 4H, Ar) δ 2.95 (s, 2H,
CO-CH2-N); δ 3.86 (s, 3H, Ph-OCH3); FT-IR ( max, cm-1): 1654 (C=O), 2819-2993 (CH
aliphatic), 3061-3157 (CH Ar), 3427-3498 (NH amide); MS: (M.Wt.: 443): m/z 443(M+, 100%);
Anal. Calcd for C22H29N5O3S: C, 59.57; H, 6.59; N, 15.79 Found C, 59.78; H, 6.86; N, 16.13.
4.1.8.3.
2-{2-[4-(2-Fluorophenyl)-piperazin-1-yl]-acetylamino}-6-methyl-4,5,7-
1
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid amide (VIIIc): Yield: 67%; m.p: 185ºC; H-
NMR (300 MHz, CDCl3): δ 2.60 (s, 3H, CH3-N), δ 2.69 (t, 2H, CH2-N of pyridine), δ 2.73 (t, J=
5.9 Hz, 2H, CH2-CH2-N of pyridine), δ 3.78 (s, 2H, CH2-N of pyridine),δ 2.87 (t, 4H, 2CH2-N of
piperazine), δ 3.31 (t, 4H, 2CH2-N of piperazine), δ 6.62-7.08 (m, 4H, Ar) δ 3.31 (s, 2H, CO-CH2-
N); FT-IR ( max, cm-1): 1674&1647 (C=O), 2991-2825 (CH aliphatic), 3159-3034 (CH Ar),
3506-3307 (NH amide); MS (M.Wt.: 431): m/z 431 (M+, 100%); Anal. Calcd for C21H26FN5O2S: C,
58.45; H, 6.07; N, 16.23 Found C, 58.57; H, 6.14; N, 16.47.
4.1.9.
7-Methyl-2-[(2-oxo-2-(4-(2-substitutedphenyl)piperazin-1-yl)ethyl)thio]-5,6,7,8-
derivatives (Xa-c): General
tetrahydropyrido[4’,3’:4,5]-thieno[2,3-d]-pyrimidin-4-one
procedures: Compounds Xa-c were prepared by refluxing IX (2.53g, 0.0100 mol) with KOH
(0.56g, 0.0100 mol) in absolute ethanol (32 mL) for 30 minutes. After which the phenylpiperazines
IIIa-c (0.0120 mol) were added. Stirring and reflux were continued for 20 hrs. The reaction mixture
was filtered while hot, washed with ethanol and concentrated under vacuum. The formed solid was
filtered and crystallized from absolute ethanol.
4.1.9.1.
7-Methyl-2-[(2-oxo-2-(4-(phenyl)piperazin-1-yl)ethyl)thio]-5,6,7,8-
tetrahydropyrido[4’,3’:4,5]-thieno[2,3-d]-pyrimidin-4-one (Xa): Yield: 70%; m.p: 128-129 ºC;
1H-NMR (300 MHz, DMSO): δ 2.30 (s,3H, CH3-N), δ 2.77 (t, J= 5.8 Hz, 2H, CH2-N of pyridine),

δ 2.61 (t, J= 5.8 Hz, 2H, CH2-CH2-N of pyridine), δ 3.78 (s, 2H, CH2-N of pyridine),δ 3.31 (t, 4H,
2CH2-N of piperazine), δ 3.78 (t, 4H, 2CH2-N of piperazine), δ 6.50-7.02 (m, 5H, Ar), δ 3.77 (s,
2H, S-CH2-CO); FT-IR ( max, cm-1): 1631&1597 (C=O), 2821-2916 (CH aliphatic), 3001-3107
(CH aromatic), 3439 (NH amide); MS (M.Wt.: 455): m/z 455 (M+, 100%); Anal. Calcd for
C22H25N5O2S2: C, 58.00; H, 5.53; N, 15.37 Found C, 58.4; H, 5.61; N, 15.56.
4.1.9.2.
7-Methyl-2-[(2-oxo-2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl)thio]-5,6,7,8-
tetrahydropyrido[4’,3’:4,5]-thieno[2,3-d]-pyrimidin-4-one (Xb): Yield: 57%; m.p: 140-142ºC; 1H-
NMR (300 MHz, DMSO): ): δ 2.28 (s,3H, CH3-N), δ 2.92 (t, J= 5.7 Hz, 2H, CH2-N of pyridine), δ
2.74 (t, J= 5.7 Hz, 2H, CH2-CH2-N of pyridine), δ 3.72 (s, 2H, CH2-N of pyridine),δ 3.54 (t, 4H,
2CH2-N of piperazine), δ 3.64 (t, 4H, 2CH2-N of piperazine), δ 6.62-7.01 (m, 4H, Ar), δ 3.97 (s,
2H, S-CH2-CO), δ 3.84 (s, 3H, Ph-OCH3); FT-IR ( max, cm-1): 1639 (C=O), 2827-2908 (CH
aliphatic), 3421 (NH amide); MS (M.Wt.: 485): m/z 485 (M+, 100%); Anal. Calcd for
C23H27N5O3S2: C, 56.88; H, 5.60; N, 14.42 Found C, 57.02; H, 5.62; N, 14.83.
5.1.9.3.7-Methyl-2-[(2-oxo-2-(4-(2-flourophenyl)piperazin-1-yl)ethyl)thio]-5,6,7,8-
1
tetrahydropyrido[4’,3’:4,5]-thieno[2,3-d]-pyrimidin-4-ones (Xc): Yield: 53%; m.p: 110ºC; H-
NMR (300 MHz, DMSO): δ 2.3 (s, 3H, CH3-N), δ 3.03 (t, 2H, CH2-N of pyridine), δ 2.73 (t, J= 5.7
Hz, 2H, CH2-CH2-N of pyridine), δ 3.62 (s, 2H, CH2-N of pyridine),δ 3.66 (t, 4H, 2CH2-N of
piperazine), δ 3.71 (t, 4H, 2CH2-N of piperazine), δ 6.70-7.02 (m, 4H, Ar), δ 4.14 (s, 2H, S-CH2-
CO); FT-IR ( max, cm-1): 1631&1612 (2 C=O), 2825-2995 (CH aliphatic), 3385 (NH amide);
MS (M.Wt.: 473): m/z 473 (M+, 100%); Anal. Calcd for C22H24FN5O2S2: C, 55.79; H, 5.11; N,
14.79; Found C, 55.86; H, 5.19; N, 15.18.
4.1.10.
7-Methyl-2-[[3-[4-(2or3-substitutedphenyl)-1-piperazinyl]propyl]thio]-3,5,6,8-
tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4-onederivatives (XIa-d): General procedures:
Compounds XIa-d were prepared by refluxing IX (2.53g, 0.0100 mol) with KOH (0.56g, 0.0100
mol) in absolute ethanol (32 mL) for 30 minutes, after which (0.0120 mol) of 1-(3-chloropropyl)-4-
(2-substitutedphenyl)-piperazines (Va-c) were added for compounds XIa-c or (3.7g, 0.0120 mol) of
1-(3-chloropropyl)-4-(3-chlorophenyl)-piperazine for compound XId. The solution was refluxed
for 20 hrs. The reaction mixture was filtered while hot, washed with ethanol and concentrated under
vacuum. The formed solid was filtered and crystallized from absolute ethanol.

4.1.10.1. 7-Methyl-2-[[3-[4-(phenyl)-1-piperazinyl] propyl] thio]-5,6,7,8-pyrido[4’,3’:4,5]thieno
[2,3-d]pyrimidin-4-one (XIa): Yield: 64%; m.p: 95-97ºC; 1H-NMR (300 MHz, DMSO): δ 3.07 (t,
J= 4.9 Hz, 2H, S-CH2-CH2-CH2), δ 1.87 (m, J= 4.9 Hz, 2H, S-CH2-CH2-CH2), δ 2,51 (t, J= 5.2 Hz,
2H, S-CH2-CH2-CH2), δ 2.34 (s, 3H, CH3-N), δ 2.81 (t, 2H, CH2-N of pyridine), δ 2.74 (t, 2H,
CH2-CH2-N of pyridine), δ 3.48 (s, 2H, CH2-N of pyridine),δ 2.82 (t, 4H, 2CH2-N of piperazine), δ
3.32 (t, 4H, 2CH2-N of piperazine), δ 6.69-7.06 (m, 5H, Ar); FT-IR ( max, cm-1): 1647 (C=O),
2796-2964 (CH aliphatic), 3037-3165 (CH Ar), 3508 (NH amide); MS (M.Wt.: 455): m/z 455 (M+,
100%); Anal. Calcd for C23H29N5OS2: C, 60.63; H, 6.42; N, 15.37 Found C, 61.11; H, 6.38; N,
15.61.
4.1.10.2
.7-Methyl-2-[[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]thio]-5,6,7,8-
pyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (XIb): Yield: 55%; m.p: 114ºC; 1H-NMR (500
MHz, DMSO): δ 3.15 (t, J= 5.6 Hz, 2H, S-CH2-CH2-CH2), δ 2.06 (m, J= 5.6 Hz, 2H, S-CH2-CH2-
CH2), δ 2,38 (t, J= 5.6 Hz, 2H, S-CH2-CH2-CH2), δ 2.27 (s, 3H, CH3-N), δ 2.90 (t, J= 5.4 Hz, 2H,
CH2-N of pyridine), δ 2.75 (t, J= 5.4 Hz, 2H, CH2-CH2-N of pyridine), δ 3.73 (s, 2H, CH2-N of
pyridine),δ 2.83 (t, 4H, 2CH2-N of piperazine), δ 3.32 (t, 4H, 2CH2-N of piperazine), δ 6.73-6.89
(m, 4H, Ar), δ 3.70 (s, 3H, Ph-OCH3); FT-IR ( max, cm-1): 1624 (C=O), 2812-2939 (CH-
aliphatic), 3090-3167 (CH-Ar), 3425 (NH-); MS (M.Wt.: 485): m/z 485(M+, 100%); Anal. Calcd
for C24H31N5O2S2: C, 59.35; H, 6.43; N, 14.42 Found C, 59.32; H, 6.52; N, 14.81.
4.1.10.3.
7-Methyl-2-[[3-[4-(2-flouro-phenyl)-1-piperazinyl]propyl]thio]-5,6,7,8-
1
pyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (XIc): Yield: 77%; m.p: 116-117ºC; H-NMR
(500 MHz, DMSO): δ 2.98 (t, J= 6.9 Hz, 2H, S-CH2-CH2-CH2), δ 1.81 (m, J= 6.9 Hz, 2H, S-CH2-
CH2-CH2), δ 2,46 (t, J= 6.9 Hz, 2H, S-CH2-CH2-CH2), δ 2.29 (s, 3H, CH3-N), δ 2.94 (t, J= 5.9 Hz,
2H, CH2-N of pyridine), δ 2.74 (t, J= 5.9 Hz, 2H, CH2-CH2-N of pyridine), δ 3.76 (s, 2H, CH2-N of
pyridine),δ 2.78 (t, 4H, 2CH2-N of piperazine), δ 3.56 (t, 4H, 2CH2-N of piperazine), δ 6.63-6.99
(m, 4H, Ar); FT-IR ( max, cm-1): 1685 (C=O), 2812-2943 (CH aliphatic), 3039-3062 (CH Ar),
3410 (NH); MS (M.Wt.: 473), m/z: (473, M+, 100%); Anal. Cacld for C23H28FN5OS2: C, 58.33; H,
5.96; N, 14.79 Found C, 58.68; H, 5.94; N, 15.13.
5.1.10.4.
7-Methyl-2-[[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]thio]-3,5,6,8-
1
tetrahydropyrido[4’,3’:4,5]thieno[2,3-d]pyrimidin-4-one (XId): Yield: 63%; m.p: 122ºC; H-
NMR (300 MHz, CDCl3): δ 3.06 (t, J= 5.4 Hz, 2H, S-CH2-CH2-CH2), δ 1.96 (m, J= 5.4 Hz, 2H, S-
CH2-CH2-CH2), δ 2,52 (t, J= 5.4 Hz, 2H, S-CH2-CH2-CH2), δ 2.46 (s, 3H, CH3-N), δ 2.99 (t, Hz,
2H, CH2-N of pyridine), δ 2.67 (t, 2H, CH2-CH2-N of pyridine), δ 3.74 (s, 2H, CH2-N of pyridine),δ
2.54 (t, 4H, 2CH2-N of piperazine), δ 3.13 (t, 4H, 2CH2-N of piperazine), δ 6.52-7.02 (m, 4H, Ar);

FT-IR ( max, cm-1): 1697 (C=O), 2787-2940 (CH aliphatic), 3388 (NH amide); MS: (M.Wt:
490), m/z: 489 (M+, 100%), 491 (M+2, 41%); Anal. Calcld for C23H28ClN5OS2: C, 56.37; H, 5.76;
N, 14.29 Found C, 56.39; H, 5.84; N, 14.53.
4.3. Pharmacology:
Rats were euthanized by cervical dislocation and the thoracic aorta was carefully exposed and
isolated. The isolated aorta was cut into rings (3-5 mm width) and each ring was then vertically
mounted between two stainless steel hooks passed through its lumen. The lower hook was fixed
between two plates, while the upper one was attached to a force displacement transducer (Model no.
MLT0201, Panlab, Spain) connected to an amplifier (PowerLab, AdInstruments Pty. Ltd.) which is
connected to a computer. The Chart for windows (v 3.4) software was used to record and elaborate
data.
The isolated aortic ring was mounted in 10 ml water jacketed automatic multi-chamber organ
bath system (Model no. ML870B6/C, Panlab, Spain). The organ bath contained Krebs’ solution of
the following composition (g/l): NaCl 6.9, KCl 0.35, KH2PO4 0.16, MgSO4.7H2O 0.3, CaCl2.2H2O
0.37, NaHCO3 2.1 and glucose 1.05. The organ bath solution was continuously aerated with
carbogen (a mixture of 95% O2 and 5% CO2) and its temperature was kept at 37˚C. The preparation
was allowed to equilibrate for about 120 min under a resting tension of 2 g during that time any
change in the resting tension was readjusted. The aortic ring was contracted by norepinephrine
(10M-6).
Pre-exposure of the aortic ring to α-blocker is supposed to suppress norepinephrine induced
aortic contraction. Percent change in aortic tension was calculated for compounds under
investigation (10M-6) and compared to standard α-blocker, prazosin (1) (10M-6). Change in aortic
tension was recorded and calculated over a period of one minute and average tension was used for
calculation [24].
5. References:
[1] Hirasawa A., Shibata K., Horie K., Takei Y., Obika K., Tanaka T., Muramoto N., Takagaki
K., Yana J., and Tsujimoto G. (1995). “Cloning, functional expression and tissue
distribution of human α -adrenoceptor splice variants”. FEBS Lett., 363: 256–260
1c
[2] Handzlik J., Macia D., Kubacka M., Mogilski S., Filipek B., Stadnickad K., and Kiec´-
Kononowicz K. (2008). “Synthesis, α -adrenoceptor antagonist activity, and SAR study of
1
novel arylpiperazine derivatives of phenytoin”. Bioorg. Med. Chem. 16: 5982–5998.

[3] Rosini, M.; Bolognesi, M. L.; Giardinà, D.; Minarini, A.; Tumiatti, V.; Melchiorre, C.
(2007). “Recent advances in alpha1-adrenoreceptor antagonists as pharmacological tools
and therapeutic agents”. Curr. Top. Med. Chem., 7: 147–162.
[4] Romeo, G.; Materia, L.; Salerno, L.; Russo, F.; Minneman, K. P. (2004). “Novel antagonists
for α adrenoreceptor subtypes”. .Expert Opin. Ther. Patents, 14: 619–637.
1-
[5] Cotecchia, S. J. Recept. (2010). “The α1-adrenergic receptors: diversity of signaling
networks and regulation”. Signal Transduct., 30: 410–419.
[6] Bremner J. B., Coban B., Grith R., Groenewoud K. M., and Yates B. F. (2000). “Ligand
Design for α Adrenoceptor Subtype Selective Antagonists”. Bioorg. Med. Chem. 8: 201-
1
214.
[7] Barbaro R., Betti L., Botta M., Corelli F., Giannaccini G., Maccari L., Manetti F.,
Strappaghetti G., and Corsano S. (2001). “Synthesis, Biological Evaluation, and
Pharmacophore Generation of New Pyridazinone Derivatives with Affinity toward α - and
1
α -Adrenoceptors”. J. Med. Chem. 44: 2118-2132.
2
[8] Hassan G. S. (2011). “Design and Synthesis of some piperazinomethyl benzofuran
derivatives and preliminary investigation of their hypotensive activity as α1 adrenoreceptor
antagonists”. Inter. J. Pharm. Pharmaceut. Sci. 3: 441-449.
[9] MacDougall I. J. A., Griffith R. (2006). “Selective pharmacophore design for α1-
adrenoceptor subtypes”. J. Mole. Graphics. Modell. 25: 146–157.
[10]
Handzlik J., Pertz H. H., Görnemann T., Jähnichen S., Kiec´-Kononowicz K. (2010).
“Search for influence of spatial properties on affinity at α -adrenoceptor subtypes for
1
phenylpiperazine derivatives of phenytoin”. Bioorg. Med. Chem. Lett., 20: 6152–6156.
[11]
Li M., Tsaib K., and Xia L. (2005). “Pharmacophore identification of α -
1A
adrenoceptor antagonists”. Bioorg. Med. Chem. Lett. 15 :657–664.
[12]
Handzlik J., Macia D., Kubacka M., Mogilski S., Filipek B., Stadnickad K., and
Kiec´-Kononowicz K. (2008). “Synthesis, α -adrenoceptor antagonist activity, and SAR
1
study of novel arylpiperazine derivatives of phenytoin”. Bioorg. Med. Chem. 16: 5982–
5998.
[13]
Gewald K., Schinke E. And Bottcher H. (1966). “2-Amino-thiophene aus
methylenaktiven Nitrilen, Carbonylverbindungen und Schwefel”. Chem. Ber., 99: 94-100.
[14]
Aurelio L., Valant C., Figler H., Flynn B. L., Linden J., Sexton P. M., Christopoulos
A., Scammells P. J. (2009). “3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-
c]pyridines as A1 adenosine receptor allosteric modulators and antagonists”. Bioorg. Med.
Chem. 17: 7353-7361.

[15]
Bourdais. J. (1968). “Alkylation of piperazines in N,N-dimethylformamide ”. Bull.
Soc. Chim. Fr. 8 : 3246.
[16]
Modica M., Santagati M., Russo F., Selvaggini C., Cagnotto A., and Mennini T.
(2000). “High affinity and selectivity of [[(arylpiperazinyl) alkyl] thio] thieno[2,3-d]
pyrimidinone derivatives for the 5-HT1A receptor. Synthesis and structure–affinity
relationships” Eur. J. Med. Chem. 35: 677-689.
[17]
Carrieri A., Piergentili A., Del Bello F., Giannella M., Pigini M., Leonardi A.,
Fanelli F., and Quaglia W. (2010). “Structure–activity relationships in 1,4-benzodioxan-
related compounds. Novel α₁-adrenoreceptor antagonists related to openphendioxan:
Synthesis, biological evaluation, and α₁d computational study”. Bioorg. Med. Chem. 18:
7065–7077.
[18]
Romeo G., Materia L., Modica M., Pittalà V., Salerno L., Siracusa M. A., Manetti F.,
Botta M., and Minneman K. P. (2011). “Novel 4-phenylpiperidine-2,6-dione derivatives.
Ligands for α₁-adrenoceptor subtypes”. Eur. J. Med. Chem. 46: 2676-2690.
[19]
Ameen M. A., Karsten S., and Liebscher J. (2010). “A convenient method for
constructing novel tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidinones-carbohydrate and
amino acid conjugates via copper(I)-catalyzed alkyne-azide ‘Click Chemistry”. Tetrahedron
66: 2141–2147.
[20]
Bakhite E. A., Abdel-Rahman A. E., Mohamed O. S., and Thabet E. A. (2002).
“Synthesis and Reactions of New Thienopyridines, Pyridothienopyrimidines and
Pyridothienotriazines”. Bull. Korean Chem. Soc., 23: 1709-1714.
[21]
Hassan A. Y. and Mohamed H. A. (2009). “Synthesis and biological evaluation of
thieno[2,3-c]pyridines and related heterocyclic systems”. Asian J. Chem. 21: 3947-3961.
[22]
Guccione S., Modica M., Longmore J., Shaw D., Barretta G. U., Santagati A.,
Santagati M., and Russo F. (1996). “Synthesis and NK-2 antagonist effect of 1,6-diphenyl-
pyrazolo [3,4-d]-thiazolo[3,2-a]-4H-pyrimidin-4-one”. Bioorg. Med. Chem. Lett. 6: 59–64.
[23]
Santagati A., Longmore J., Guccione S., Langer T., Tonnel E., Modica M., Santagati
M., Scolaro L. M., and Russo F. (1998). “Building a model of interaction at the NK-2
receptors: Polycondensed heterocycles containing the pyrimidoindole skeleton”. Eur. J.
Med. Chem. 32: 973–985.
[24]
Girgis AS,Ismail NS, Farag H,el-Eraky WI, Saleh DO, Tala SR, Katritzky
AR.(2010). “Regioselective synthesis and molecular modeling study of vasorelaxant active
7,9-dioxa-1,2-diaza-spiro[4.5]dec-2-ene-6,10-diones”. Eur. J. Med. Chem. 45: 4229-4238.
[25]
Kuo G.H. , Prouty C. , Murray W.V. , Pulito V. , Jolliffe L., Cheung P. , Varga S. ,
Evangelisto M. , Shaw C. (2000). “Design, synthesis and biological evaluation of pyridine-

phenylpiperazines: a novel series of potent and selective alpha1a-adrenergic receptor
antagonist”. Bioorg. Med. Chem. 8: 2263-2275.
[26]
Garcia-Sainz J A, Villalobos-Molina R. (2004). “The elusive alpha (1D)-
adrenoceptor: molecular and cellular characteristics and integrative roles”. Eur.J.Pharmacol,
1-3: 113-120

Tables, Figures and Schemes captions
Table 1: Lead compounds used as training set in creating α A- ARs hypothesis
1
Table 2: Mapping of the test set (selective α A-AR antagonists ) to the generated pharmacophore
1
model.
Table 3: Fitting and relative energy values of the best-fitted conformers of compounds IVa-c,
VIa-d, VIIIa-c, Xa-c, XIa-d through mapping onto the pharmacophore model of the a1-AR
antagonist.
Table 4: Change in pre-treated aortic muscle tension after NE exposure. Change in tension is
expressed as average muscle tone over duration of 1-2 minutes of recording.
Fig. 1: SAR of α1-ARs antagonists
Fig. 2: The design of the target compounds IVa-c, VIa-d, and VIIIa-c by bioisosteric
modification of the reference compound REC 15/2739 (6). The green dots represent the
heteroaromatic moiety with carbonyl group (HY-3, HBA, respectively), the red dots represent the
linker, and the blue dots represent the aryl group (HY-1, HY-2).
Fig. 3: The design of the target compounds Xa-d and XIa-d by bioisosteric modification of the
reference compound RN17 (7). The dots colors are described in the legend of Figure 2.
Fig. 4: Pharmacophore model of selective α1A- ARs antagonists generated by discovery studio
2.5. Pharmacophoric features are color coded: cyan for hydrophobic regions (HY1-3), green for
hydrogen bond acceptor (HBA), and red for positive ionizable feature (PI).
Fig. 5: Mapping of a1A-AR antagonist hypothesis and prazosin. The two o-methoxy groups of
quinazoline moiety occupied both HY1 and HY2 (blue sphere) and the quinazoline N1 atom is
located inside the positive ionizable feature PI (red sphere). The oxygen of the carbonyl group
attached to piperazine overlapped HBA (green sphere), while the furan ring matched YH3 (blue
sphere).The sphere colors are described in the legend of figure 4.
Fig. 6: Mapping of a1-AR antagonist hypothesis and compounds IVb, VIIIb, and XIb (fit value =
4.93, 4.89, and 4.88 respectively). o-Methoxyphenyl attached to piperazine moiety occupied both
HY1 and HY2 (blue spheres) and the piperazinyl N-1 atom is located inside the positive ionizable

feature PI (red sphere). The carbonyl oxygen of tetrahydropyridothienopyrimidine system
overlapped HBA (green sphere), while its thiophene ring matched HY3 (blue sphere).
Scheme 1: Synthesis of target compounds IVa-c, VIa-d, VIIIa-c
Scheme 2: Synthesis of target compounds Xa-c and XIa-d
Table 1: Lead compounds used as training set in creating α A- ARs hypothesis
1
Fitting
Values
3.83
Fitting
Values
1.02
Lead
Lead
Prazosin[7, 12] (1)
BMY-7378[6, 12]
WAY-100635[10]
Abanoquil[6]
REC-15/2739[6]
Cyclazosin[6, 10]
5-Me-urapidil[10]
Benzoxathion[6]
A131701[6]
SNAP-8719 [6, 8]
Terazosin [ 9]
NAN-190 [7, 11]
KMD-3213[6,7]
RN17[12]
REC-15/2615[6]
WB4101[6]
Amiodarone[8]
SGB-1534[7]
Cdm-12[7, 8]
3.60
3.22
4.26
4.99
2.39
3.54
4.20
3.56
4.01
3.21
3.90
3.34
4.26
2.06
2.63
4.06
Tamsulosin[11]
1.33
3.10

Table 2: Mapping of the test set (selective α A-AR antagonists ) to the generated pharmacophore
1
model.
Lead
Fitting value
H
H3C
H3CO
N
CH3
O
N
4.71
O
O
Niguldidpine [6]
NO2
NH2
H3CO
H3CO
N
4.84
N
N
N
Bunazosin [6]
O
O
N
N
H
HO
O
O
HN
4.15
4.65
N
O
H
N
N
H
ergotamine [7]
Cl
N
Et
Et
N
N
N
O
etopridone [7]

Table 3: Fitting and relative energy values of the best-fitted conformers of compounds IVa-c,
VIa-d, VIIIa-c, Xa-c, XIa-d through mapping onto the pharmacophore model of the a1-AR
antagonist.
Test Compounds
Fit Values
Relative energy Kcal/mol
IVa
IVb
IVc
VIa
VIb
VIc
VId
VIIIa
VIIIb
VIIIc
Xa
Xb
Xc
XIa
XIb
XIc
XId
4.69
4.93
4.72
1.68
4.81
2.02
4.81
1.80
4.89
1.90
4.49
56.8
51.27
44.98
37.38
7725
38.60
38.69
36.60
196.53
16.40
77.50
23.18
46.56
19.90
13.25
32.38
14.13
4.56
4.54
4.57
4.88
4.58
4.73

Table 4: Change in pre-treated aortic muscle tension after NE exposure. Change in tension is
expressed as average muscle tone over duration of 1-2 minute of recording.
Induced change in aortic Calculated α -ARs blocking
1
muscle tension (%)
activity (%)
Control
100
(Norepinephrine)
Prazosin (1)
IVb
99.17
0.83
18.85
11.12
23.24
30.07
29.45
21.73
28.53
53.63
5.26
81.15
88.88
76.76
69.93
70.55
78.27
71.47
46.37
94.74
86.46
77.93
65.68
IVc
VIb
VId
VIIIb
Xb
Xc
Xa
XIb
XId
13.54
22.07
34.32
XIa
XIc

Alkyl spacer
C2-C7 in length
C
heterocyclic
carbonyl
N
N
n
B
R
Hydrophobic feature-3
(HY-3)
Positive ionizable
(PI)
o-substituted phenyl
(HY1-HY2)
Hydrogen bond acceptor
HBA
A
Fig. 1: SAR of α1-ARs antagonists

Reference compound
REC-15/2739 (6)
3
3
Target compounds
1) IVa-c series
2
3
2
2) VIa-d series
3
2
3) VIIIa-c series
3
Fig. 2: The design of the target compounds IVa-c, VIa-d, and VIIIa-c by bioisosteric
modification of the reference compound REC 15/2739 (6). The green dots represent the
heteroaromatic moiety with carbonyl group (HY-3, HBA, respectively), the red dots represent
the linker, and the blue dots represent the aryl group (HY-1, HY-2).

Refrence compound
RN17 (7)
H
N
O
N
S
N
N
O
H3CO
Target compounds
4- Xa-c series
O
N
H3C
N
NH
N
N
S
S
X
O
5- XIa-d series
O
N
H3C
N
NH
N
N
S
S
X
Fig. 3: The design of the target compounds Xa-d and XIa-d by bioisosteric modification of the
reference compound RN17 (7). The dots colors are described in the legend of Figure 2.

Fig. 4: Pharmacophore model of selective α1A- ARs antagonists generated by discovery
studio 2.5. Pharmacophoric features are color coded: cyan for hydrophobic regions (HY1-3),
green for hydrogen bond acceptor (HBA), and red for positive ionizable feature (PI).
,
Fig. 5: Mapping of a1A-AR antagonist hypothesis and prazosin. The two o-methoxy groups of
quinazoline moiety occupied both HY1 and HY2 (blue sphere) and the quinazoline N1 atom is
located inside the positive ionizable feature PI (red sphere). The oxygen of the carbonyl group
attached to piperazine overlapped HBA (green sphere), while the furan ring matched YH3
(blue sphere).The sphere colors are described in the legend of figure 4.