High affinity ligands and potent antagonists for the α1D- adrenergic receptor. Novel 3,8-disubstituted [1]benzothieno[3,2-d]pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.06.057

A new series of high affinity ligands and antagonists for the α_1D-adrenergic receptor (AR) has been discovered. New molecules present a [1]benzothieno[3,2-d]pyrimidin-2,4(1H,3H)-dione or a [1]benzothieno[3,2-d]pyrimidin-4(3H)-one scaffold and bear a 2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl moiety in the 3-position and various amide substituents in the 8-position. In binding assays at the three human cloned α_1A-, α_1B-, and α_1D-AR subtypes, they showed high affinity values, particularly for the α_1D-AR subtype. Compound 22 (RX18), N¹-methyl-N ⁵-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3, 4-tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-N¹-(phenylmethyl) pentanediamide, was the most interesting in the series displaying very high affinity (pK_i = 10.25) and potent antagonism (pK_b = 9.15) when tested in a functional assay at the α_1D-AR.

Full text of DOI:10.1016/j.ejmech.2014.06.057

Accepted Manuscript
High Affinity Ligands and Potent Antagonists for the α -Adrenergic Receptor. Novel
1D
3,8-Disubstituted [1]Benzothieno[3,2-d]pyrimidine Derivatives
Giuseppe Romeo, Loredana Salerno, Valeria Pittalà, Maria N. Modica, Maria A.
Siracusa, Luisa Materia, Michela Buccioni, Gabriella Marucci, Kenneth P. Minneman
PII:
S0223-5234(14)00586-8
10.1016/j.ejmech.2014.06.057
EJMECH 7100
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 March 2014
Revised Date: 13 June 2014
Accepted Date: 25 June 2014
Please cite this article as: G. Romeo, L. Salerno, V. Pittalà, M.N. Modica, M.A. Siracusa, L. Materia,
M. Buccioni, G. Marucci, K.P. Minneman, High Affinity Ligands and Potent Antagonists for the α
-
D
1
Adrenergic Receptor. Novel 3,8-Disubstituted [1]Benzothieno[3,2-d]pyrimidine Derivatives, European
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.057.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
High Affinity Ligands and Potent Antagonists for the α -Adrenergic Receptor.
1
D
Novel 3,8-Disubstituted [1]Benzothieno[3,2-d]pyrimidine Derivatives.
a,
a
a
a
a
Giuseppe Romeo *, Loredana Salerno , Valeria Pittalà , Maria N. Modica , Maria A. Siracusa ,
a
b
b
c
Luisa Materia , Michela Buccioni , Gabriella Marucci , Kenneth P. Minneman
a
Dipartimento di Scienze del Farmaco, Università di Catania, viale A. Doria 6, 95125 Catania, Italy,
b
Dipartimento di Scienze Chimiche, Università di Camerino, via S. Agostino 1, 62032 Camerino,
c
Italy, Department of Pharmacology, Emory University, 1510 Clifton Road, Atlanta, GA 30322
USA.
*
6
Corresponding author. Dipartimento di Scienze del Farmaco, Università di Catania, viale A. Doria
, 95125 Catania, Italy. Tel.: +39 095 738 4024.
E-mail address: gromeo@unict.it (G. Romeo)
1

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Abstract
A new series of high affinity ligands and antagonists for the α -adrenergic receptor (AR) has been
1
D
discovered. New molecules present a [1]benzothieno[3,2-d]pyrimidin-2,4(1H,3H)-dione or a
1]benzothieno[3,2-d]pyrimidin-4(3H)-one scaffold and bear a 2-[4-(2-methoxyphenyl)piperazin-1-
[
yl]ethyl moiety in the 3-position and various amide substituents in the 8-position. In binding assays
at the three human cloned α -, α -, and α -AR subtypes, they showed high affinity values,
1
A
1B
1D
1
5
particularly for the α -AR subtype. Compound 22 (RX18), N -methyl-N -[3-[2-[4-(2-
1
D
methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-tetrahydro[1]benzothieno[3,2-d]pyrimidin-
1
8
-yl]-N -(phenylmethyl)pentanediamide, was the most interesting in the series displaying very high
affinity (pK = 10.25) and potent antagonism (pK = 9.15) when tested in a functional assay at the
i
b
α -AR.
1
D
Keywords
α -Adrenergic Receptor; Antagonist; Ligand; [1]Benzothieno[3,2-d]pyrimidine.
1
D
Abbreviations
AR, adrenergic receptor; BPH, benign prostatic hypertrophy; CNS, central nervous system; TM,
transmembrane domain; IL, intracellular loop; XL, extracellular loop, PP, phenylpiperazine;
HEPES, 4-(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic acid; NA, noradrenaline.
1. Introduction
2

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α -Adrenergic receptors (α -ARs) are class A (rhodopsin-like) members of the G protein-coupled
1
1
receptor (GPCR) super-family. They are widely distributed in the human body and, along with α2-
ARs and β-ARs, mediate the functional effects of the endogenous catecholamines, noradrenaline
and adrenaline. α -ARs are cell-surface receptors that are involved in several physiological
1
functions (vascular smooth muscle contraction, cardiac inotropy and chronotropy, hepatic glucose
metabolism) and in a number of pathological conditions such as myocardial hypertrophy and benign
prostatic hypertrophy (BPH). They also represent the biological target of currently used drugs
useful in the clinical treatment of arterial hypertension and male lower urinary tract symptoms
associated to benign prostatic hypertrophy (BPH) [1].
α -ARs do not constitute a homogeneous population: three different subtypes are known, viz. α -,
1
1A
α -, and α -ARs, encoded by distinct genes located on human chromosomes 8, 5, and 20,
1
B
1D
respectively. All three subtypes have been cloned and pharmacologically characterized [2]. Each
subtype possesses a discrete tissue distribution, a fact suggesting unique physiological roles for each
of them. In the CNS, all three α -AR subtypes are widely represented but with distinct patterns of
1
distribution in the different brain and spinal cord areas [3].
From a topological point of view, α -ARs share with other GPCRs a common architecture based on
1
a bundle of seven hydrophobic transmembrane α-helices (TM1–TM7) connected by alternating
intracellular (IL1–IL3) and extracellular (XL1–XL3) loops; the N-terminal and C-terminal regions
of the protein lie in the extracellular and in the intracellular environment, respectively. The
sequence identity among the three α -AR subtypes is higher in TM regions (α -α : 75 %; α -
1
1A 1B
1A
α : 66 %; α -α : 73 %) and much lower in connecting loops and N- and C-termini [4].
1
D
1B 1D
3

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As for other class A receptors, the so-called orthosteric binding site in α -ARs is believed to be
1
located within the bundle of α-helices. This is strongly supported by site-directed mutagenesis
studies, which identified within TMs specific amino acid residues involved in ligand recognition,
and by the recent X-rays high-resolution structure of the complexes between the class A β2-
adrenergic receptor and agonists, inverse agonists or antagonists [5-7].
A conserved aspartate in TM3 (Asp3.32 in the Ballesteros-Weinstein notation [8]) of α -ARs is
1
essential for ligand binding. Site-directed mutagenesis studies on α -AR and α -AR in which
1
B
1D
Asp3.32 has been mutated into alanine have shown that Asp3.32Ala receptor mutants are not able
to bind either agonists or antagonists [9, 10]. These data strongly suggests the formation of an
essential ionic interaction between the negatively charged side chain of Asp3.32 and the protonated
amino group of the ligand.
In addition to the classic orthosteric binding site, topographically distinct allosteric binding sites
have been identified in a number of class A receptors [11]. At the α -AR subtype in particular, ρ-
1
B
TIA, a small peptide from marine cone snail Conus tulipa, behaves as a subtype-selective allosteric
antagonist and interacts with the extracellular receptor surface at the base of XL3 [12], defining an
allosteric binding site different from that located within the bundle of α-helices. This suggests that
subtype-selectivity, which can hardly be reached by ligands acting only within the orthosteric site,
could be achieved exploiting the higher sequence dissimilarity of extracellular domains in α -AR
1
subtypes and taking advantage of possible subtype-specific interactions between the ligand and
amino acid residues belonging to such domains [13].
In the last decade, our research group has extensively worked on the development of new α -AR
1
ligands containing a phenylpiperazine (PP) substructure as pharmacophoric moiety [14-19]. In most
of these ligands, the PP moiety was linked, through an alkyl chain, to a heterobicyclic or
4

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heterotricyclic planar scaffold. Notable examples of such a kind of molecules are RN17 and RC23,
two potent α -AR ligands with antagonistic properties [14, 16, 20]. In particular, RN17 and RC23
1
possess a 4-(2-methoxyphenyl)piperazin-1-yl moiety connected through an ethylene chain to the 3-
position of a tricyclic [1]benzothieno[3,2-d]pyrimidin-2,4(1H,3H)-dione or a [1]benzothieno[3,2-
d]pyrimidin-4(3H)-one system, respectively (Figure 1). The two molecules act as high affinity
ligands at all three human cloned α -, α , and α -ARs with a slight preference for the last
1
A
1B
1D
subtype (Table 1). In both structures, the protonatable amino group of the PP moiety can assure the
ionic interaction with Asp3.32 side chain of the binding site.
In an effort to expand our previous work and with the aim to obtain α -AR ligands with higher
1
affinity and greater subtype selectivity, here we now report the preparation of a series of new α -AR
1
ligands (7-10, 20-23, 26, 27, 33-36, and 38) based on structures of RN17 and RC23 (Figure 1). In
particular, the 8-position of the two tricyclic systems in RN17 and RC23 was chosen for chemical
manipulation. Through an amide linker, a number of side chains of increasing length and different
natures were inserted at this position. At one end, these side chains bear a phenyl group or a
protonatable amine function in order to take advantage of additional possible aromatic/aromatic,
cation/aromatic, or ionic interactions that could take place between these groups and the
extracellular surface of the receptor proteins, particularly the second loop (XL2). In fact, XL2
presents the highest divergence in sequence among the three human α -AR subtypes and it is rich in
1
polar residues, especially negatively charged aspartate and glutamate residues.
The choice of the 8-position for the insertion of side chains was made taking into account the
structure-activity relationships previously seen in a series of α -AR ligands designed using RN17 as
1
template and developed at Abbott Laboratories [21]. These compounds, based on a
pyrazino[2’,3’:4,5]thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione tricyclic scaffold, bear different
substituents (hydrogen, methyl, methoxy, chloro, phenyl) at the 7- and/or 8-positions. Compound
5

ACCEPTED MANUSCRIPT
39 (Figure 1) is one of them: the presence in it of a bulky substituent such as a phenyl ring at the 8-
position is well tolerated whereas the shifting of the same substituent to the 7-position causes a
marked drop in affinity for all three α -AR subtypes [21]. This suggests that, mutatis mutandis, the
1
8-position could represent a site in the planar tricyclic systems of RN17 and RC23 useful for the
introduction of additional structural moieties.
2. Chemistry
Synthetic pathways to the final [1]benzothieno[3,2-d]pyrimidin-2,4(1H,3H)-diones 7-10, 20-23, 26,
and 27 are shown in Schemes 1, 2, and 3, respectively.
Ethyl 3-amino-5-nitro[1]benzothiophene-2-carboxylate (1) [21] was treated with ethyl
chlorocarbonate to give in good yield urethane 2, which in turn was transformed into the tricyclic
alcohol 3 by reaction with an excess of 2-aminoethanol (Scheme 1). Compound 4, obtained by the
action of thionyl chloride on alcohol 3, was allowed to react with 1-(2-methoxyphenyl)piperazine to
give tricyclic nitro derivative 5. Successive reduction of the nitro group with hydrazine
monohydrate in DMF in the presence of Raney Nickel afforded the 8-amino derivative 6 which
represents the key intermediate in the preparation of all desired [1]benzothieno[3,2-d]pyrimidin-
2,4(1H,3H)-diones. Reaction of 6 with benzoyl chloride or phenylalkanoyl chlorides gave final
compounds 7-10 in good yields (Scheme 1).
In Scheme 2 is outlined the preparation of the final derivatives 20-23 bearing diamide side chains at
the 8-position. The suitable acidic intermediates 16-19 were synthesized by reaction of amines 11-
13 with succinic anhydride (14) or glutaric anhydride (15) in anhydrous toluene. Acids 16-19,
activated by ethyl chlorocarbonate, were treated with the 8-amino derivative 6 to afford the desired
products 20-23.
Worthy of note, due to the hindered rotation around the C(O)-N bond of the tertiary N-
1
13
phenylmethyl-N-methylamide group, in H NMR and C NMR spectra of acid intermediate 18 and
6

ACCEPTED MANUSCRIPT
of the corresponding final derivative 22, it is possible to observe signals for the two rotational
conformers E and Z, according to literature data for similar compounds [22].
Preparation of final compounds 26 and 27, having basic side chains in the 8-position, was
accomplished by reaction of amino acid hydrochloride 24, obtained following the procedure of
Gittos and Wilson [23], or the commercially available 25, activated by 1,1’-carbonyldiimidazole,
and the amino derivative 6 (Scheme 3).
The synthetic pathway for the preparation of the 8-amino derivative 32, the key intermediate in the
synthesis of the final [1]benzothieno[3,2-d]pyrimidin-4(3H)-ones 33-36 and 38 is presented in
Scheme 4. Ethyl 3-amino-5-nitro[1]benzothiophene-2-carboxylate (1) was treated with 1,1,1-
triethoxyethane to give the iminoether 28 which was then cyclized to the alcohol 29 by reaction
with 2-aminoethanol. Conversion to alkyl chloride 30, successive reaction with 1-(2-
methoxyphenyl)piperazine to give the tricyclic nitro derivative 31, and final reduction of the nitro
group provided the 8-amino intermediate 32.
As shown in Scheme 5, 32 was allowed to react with benzoyl chloride and phenylacetyl chloride to
give 33 and 34 or with acids 17 and 19, activated by ethyl chlorocarbonate, to provide diamides 35
and 36, respectively.
Final compound 38 is an isomer of 35 and presents an inverse amide function on the side chain in
the 8-position. Activation with ethyl chloroformate was also used in its preparation, which was
accomplished starting from acid 37 and amino derivative 32 (Scheme 5). Acid 37 was synthesized
by reaction of phenylacetyl chloride with 4-aminobutanoic acid in basic conditions [24].
3. Results and Discussion
3.1. Binding at the α1-AR subtypes
7

ACCEPTED MANUSCRIPT
Compounds 7-10, 20-23, 26, 27, 33-36, and 38 were evaluated in binding assays on human α -AR,
1
A
1
25
α -AR, and α -AR subtypes stably expressed in HEK293 cells using [ I]BE2254 as radioligand
1
B
1D
[
18]. Their affinity values, expressed as pK , are summarized in Table 1. For sake of clarity, affinity
i
values of parent compounds RN17 and RC23 are also reported.
All final compounds displayed good to excellent affinities for α -ARs, indicating that the
1
introduction of long and bulky side chains in the 8-position of the tricyclic system is tolerated by
receptor binding sites. Most of them showed affinities in the order α - ≥ α - > α -AR and in
1
D
1A
1B
some cases an α -/α -AR selectivity over the α -AR subtype appeared.
1
A
1D
1B
In the series of [1]benzothieno[3,2-d]pyrimidine-2,4(1H,3H)-diones, derivatives 26 and 27, bearing
a basic tertiary amine as terminal group of the side chain, were the least interesting. They displayed
no improvement in affinity with respect to parent compound RN17 and even selectivity among
subtypes was completely absent; compound 27 showed the same pK value (8.78) for the three α -
i
1
ARs (Table 1).
On the other hand, derivatives 7-10 and 20-23, having neutral side chains with a terminal aromatic
ring, gave better results, at least in terms of discrimination among receptor subtypes.
Compounds 7-10 bear simple side chains, constituted by a phenyl ring bound directly or through an
alkyl chain to the amide linker in the 8-position. These compounds displayed receptor preferences
in the order α - > α - > α -AR and their affinity values were dependent on the distance between
1
D
1A
1B
the phenyl ring and the tricyclic system. The best result was obtained with the benzoylamide
derivative 7, having the shortest side chain; lower affinities were displayed by homologues 8-10, as
the phenyl ring was moved farther away (Table 1).
Compound 7 showed higher pK values for both α - and α -AR than the parent ligand RN17
i
1D
1A
(
9.24 and 8.96 vs 8.90 and 8.48, respectively) while it maintained the same pK value as RN17 for
i
the α -AR subtype (8.00 vs 8.05). These binding features make 7 a better ligand than RN17 with a
1
B
selectivity α -/α -AR over α -AR.
1
D
1A
1B
8

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More promising results were obtained with the diamide derivatives 20-23. They all proved to be
better ligands than RN17 for the α -AR with affinity values in the subnanomolar range. The length
1
D
of the alkyl spacer between the two amide groups influences affinity, with pentanediamide
derivative 21 being a better ligand for all three receptor subtypes than its succinamide homologue
20. Unlike monoamides 7-10 (where the shorter the side chain is, the better is the affinity), in
diamides 20-23, longer alkyl spacer resulted in ligands with higher affinity. It is possible to
speculate that the binding poses of the side chains in the two families of compounds could be
different and this could explain the different SARs. In compounds 20-23 the second distal amide
group in the side chain could exert possible additional interactions that are not available to
monoamides 7-10.
In pentanediamide derivatives 21-23, the structure of the distal amide greatly affects the strength of
the receptor/ligand interaction, in particular for the α -AR. When the N-phenylmethylamide
1
D
moiety in 21 was replaced by a N-phenylethylamide in 23, a drop in affinity was observed (pK =
i
9.85 and 9.00, respectively). However, when the secondary amide moiety in 21 was transformed
into the tertiary N-methyl-N-phenylmethylamide (22), affinity for the α -AR increased to really
1
D
high values (pK = 10.25) whereas those for the α - and α -AR subtypes underwent a decrease
i
1A
1B
(Table 1). Thus, compound 22 (RX18) emerged as the most interesting molecule in the series; it
displays very high affinity for the α -AR coupled to selectivity over the other two subtypes.
1
D
Although 2-methyl[1]benzothieno[3,2-d]pyrimidin-4(3H)-ones 33-36 and 38 showed good affinities
for the α -ARs, generally they are poorer ligand for the α -AR than the corresponding
1
1D
[
1]benzothieno[3,2-d]pyrimidine-2,4(1H,3H)-dione analogues (compare 7 vs 33, 8 vs 34, 21 vs 35).
Only the N-phenylethylamide derivative 36 maintained comparable affinities to its analogue 23.
This general trend was somewhat surprising because in RC23, the parent ligand of compounds 33-
9

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36 and 38, the presence of a methyl group in 2-position of the tricyclic system had enhanced affinity
for α -AR (pK = 9.40) with respect to RN17 (pK = 8.90).
1
D
i
i
Worth of note and inversely to what has been seen in the [1]benzothieno[3,2-d]pyrimidine-
,4(1H,3H)-dione series, here the N-phenylethylamide derivative 36 possesses the highest affinity
2
(
pK = 9.12) at α -AR whereas the N-phenylmethylamide derivative 35 displays the lowest (pK =
i
1D
i
8.26).
In order to explore if the arrangement of the distal amide moiety in 35 could affect affinity, its
isomer 38, having an inverted amide function, was synthesized. With respect to 35, it shows
increased affinity for all three α -ARs with and a slight preference for the α -AR subtype (Table
1
1A
1).
The observation that the 2,4(1H,3H)-dione derivatives 7, 8, and 27 are better ligands than the
corresponding 2-methyl-4(1H)-one analogues 33, 34, and 35 indicates that the carbonyl group in the
2
-position of the tricyclic scaffold is important for an optimal binding at α -AR site and suggests
1D
its probable influence in inducing the correct disposition of the flexible side chains in the ligands of
the [1]benzothieno[3,2-d]pyrimidine-2,4(1H,3H)-dione series.
3
.2. Functional activity at the α1-AR subtypes
1]Benzothieno[3,2-d]pyrimidine-2,4(1H,3H)-diones 21 and 22, which, within the series, had
showed the best affinities for the α -AR, were also tested in functional assays for their activity at
[
1
D
α -AR subtypes in isolated rat prostatic vas deferens (α -AR), spleen (α -AR) and thoracic aorta
1
1A
1B
(
α -AR) [25]. Their pK values, along with those of the parent compound RN17, are summarized
1D b
in Table 2.
Mimicking the functional activity of RN17, both compounds behaved as antagonists; in particular
they displayed a competitive antagonism. In fact, the concentration-response curves of reference
1
0

ACCEPTED MANUSCRIPT
agonists after and before incubation with compounds 21 and 22 were parallel without reduction of
the maximal effect and the shift produced was proportional to the concentrations used.
Both compounds show a functional activity trend similar to that seen in binding studies; in fact,
they are more active at α -AR (pK = 9.15 and pK = 8.64) and display a lower and similar
1
D
b
b
potency at α1A (pK = 7.70 and pK = 7.82) and α -AR (pK = 7.94 and pK = 7.77), respectively.
b
b
1B
b
b
Moreover, compound 22 is a more potent antagonist (pK = 9. 15) than 21 (pK = 8.64) at α -AR.
b
b
1D
Thus, diamide 22 can be regarded as the most interesting compound in the series, being a high
affinity ligand and a potent competitive antagonist for the α -AR subtype.
1
D
4. Conclusions
Structure modifications of α -AR ligands RN17 and RC23 gave a series of novel
1
D
[
1]benzothieno[3,2-d]pyrimidine derivatives. These compounds, bearing side chains in the 8-
position, were tested for their affinity at the three α -AR subtypes. In binding assays on human
1
cloned receptors, some derivatives showed very high affinity and a preference for the α -AR
1
D
subtype. Among them, compound 22 (RX18) emerged and revealed very high affinity (pK = 10.25)
i
for the α -AR; when tested in a functional assay, it displayed potent and competitive antagonism
1
D
coupled to selectivity for the above-cited subtype.
In general, it can be stated that introduction of diamide side chains in the 8-position of the tricyclic
scaffolds of RN17 and RC23 generates very high affinity α -AR ligands with antagonistic
1
D
properties. The nature of the distal amide group in the side chain influences affinity and additional
investigations of the chemical space around this moiety can be imagined to further improve
selectivity among α -AR subtypes.
1
Intriguingly, orthosteric ligands such as 22 (RX18) could represent a base for the future design,
through an appropriate chemical manipulation of the distal amide group, of new
1
1

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orthosteric/allosteric bitopic ligands which, as seen in other class A GPCRs [26, 27], can present
enhanced binding and functional properties.
5. Experimental Section
5.1. Chemistry
Melting points were determined in a IA9200 Electrothermal apparatus equipped with a digital
thermometer in glass capillary tubes and are uncorrected. Infrared spectra were recorded on a
Perkin Elmer 281 FTIR spectrometer in KBr disks or NaCl crystal windows. Elemental analyses for
C, H, N, and S were within ± 0.4% of theoretical values and were performed on a Carlo Erba
1
13
Elemental Analyzer Mod. 1108 apparatus. H NMR and C NMR spectra were recorded on a
Varian Inova Unity 200 spectrometer in DMSO-d solution. Chemical shifts are given in δ values
6
(
ppm), using tetramethylsilane as the internal standard; coupling constants (J) are given in Hz.
Signal multiplicities are characterized as s (singlet), d (doublet), t (triplet), q (quartet), m
multiplet), br (broad signal). Microwave reaction were performed with a CEM Discovery
instrument in sealed vessels. All the synthesized compounds were tested for purity on TLC
aluminium sheet coated with silica gel 60 F , Merck) and visualized by UV (λ = 254 and 366
(
(
2
54
nm). Purification of synthesized compounds by column chromatography was performed using silica
gel 60 (Merck). All chemicals and solvents were reagent grade and were purchased from
commercial vendors.
5
.1.1. Ethyl 3-amino-5-nitro-1-benzothiophene-2-carboxylate (1)
This compound [21] was prepared following a published procedure [28] for the homologous methyl
-amino-5-nitro-1-benzothiophene-2-carboxylate, starting from 2-chloro-5-nitrobenzonitrile and
ethyl thioglycolate in the presence of K CO in EtOH. Recristallization from EtOH gave 1 as a pure
3
2
3
1
orange solid (93%), mp 200–201 °C; H NMR (DMSO-d ): δ 9.23 (d, J = 2.2 Hz, 1H, aromatic),
6
1
2

ACCEPTED MANUSCRIPT
8.29 (dd, J = 9.0 Hz, J = 2.2 Hz, 1H, aromatic), 8.11 (d, J = 9.0 Hz, 1H, aromatic), 7.45 (br s, 2H,
NH which exchanges with D O), 4.29 (q, J = 7.0 Hz, 2H, COOCH CH ), 1.31 (t, J = 7.0 Hz, 3H,
2
2
2
3
COOCH CH ). Anal. (C H N O S) C, H, N, S.
2
3
11 10
2
4
5.1.2. Ethyl 3-[(ethoxycarbonyl)amino]-5-nitro-1-benzothiophene-2-carboxylate (2)
Amino ester 1 (7.00 g, 26.29 mmol) was dissolved in hot toluene (150 mL). Solution was cooled to
room temperature, ethyl chlorocarbonate (4.28 g, 39.44 mmol) was added and the mixture refluxed
under stirring. After 12 h, ethyl chlorocarbonate (4.28 g, 39.44 mmol) was added again and the
mixture refluxed for further 14 h. After being cooled, reaction mixture was poured into petroleum
ether 40-60 °C (200 mL) and the precipitate was filtered off and dried to give crude 2 (7.66 g, 86
%). A small portion was recrystallized from ethanol affording 2 as a pure product: mp 144–145 °C;
1
H NMR (DMSO-d ): δ 10.00 (s, 1H, NH which exchanges with D O), 8.81–8.79 (m, 1H,
6
2
aromatic), 8.33–8.29 (m, 2H, aromatic), 4.32 (q, J = 7.2 Hz, 2H, COOCH CH ), 4.16 (q, J = 7.2 Hz,
2
3
2
H, NHCOOCH CH ), 1.31 (t, J = 7.2 Hz, 3H, COOCH CH ), 1.26 (t, J = 7.2 Hz, 3H,
2 3 2 3
NHCOOCH CH ). Anal. (C H N O S) C, H, N, S.
2
3
14 14
2
6
5.1.3. 3-(2-Hydroxyethyl)-8-nitro[1]benzothieno[3,2-d]pyrimidine-2,4(1H,3H)-dione (3)
Compound 2 (5.00 g, 14.78 mmol) was dissolved in 2-aminoethanol (100 mL) and the obtained
solution was heated at 50 °C for 40 min. The reaction mixture was then poured into ice-water and
acidified to pH 5 with HCl 0.5 M. The obtained precipitate was filtered off, thoroughly washed with
water and dried. Recrystallization from ethanol gave 3 (3.95 g, 87 %) as a pure product: mp 290–
1
2
91 °C; H NMR (DMSO-d ): δ 12.71 (s, 1H, NH which exchanges with D O), 9.46–9.38 (m, 1H,
6
2
aromatic), 8.40–8.35 (m, 2H, aromatic), 4.80 (t, J = 5.8 Hz, 1H, OH), 4.05 (t, J = 6.4 Hz, 2H,
CH CH OH), 2.56–2.46 (m, 2H, CH CH OH). Anal. (C H N O S) C, H, N, S.
2
2
2
2
12
9
3
5
1
3

ACCEPTED MANUSCRIPT
5
2
.1.4. 3-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-8-nitro[1]benzothieno[3,2-d]pyrimidine-
,4(1H,3H)-dione (5)
Alcohol 3 (0.50 g, 1.63 mmol) was dissolved in hot toluene (13 mL). The solution was allowed to
cool to room temperature and thionyl chloride (0.58 g, 4.88 mmol) was added. The reaction mixture
was refluxed under stirring for 12 h; then, a further amount of thionyl chloride (0.10 g, 0.84 mmol)
was added and the mixture refluxed for further 10 h. After being cooled, liquids were eliminated in
vacuo and solids were suspended in cyclohexane, then filtered off and dried. Obtained crude 4 (0.53
g, quantitative yield) was successively used without further purification.
A mixture of 4 (0.50 g, 1.53 mmol) and 1-(2-methoxyphenyl)piperazine (1.47 g, 7.65 mmol) was
heated in an oil bath at 160 °C for 30 min. After being cooled, ethanol (10 mL) was added to the
reaction mixture. Solids were filtered off, washed with cold ethanol and dried. The crude product
was triturated in ethanol and then filtered off an dried to give 5 (0.62 g, 67 %) as a pure product: mp
-1
1
2
49-251 °C; IR (KBr) cm 3446, 3086, 2830, 1710, 1651, 1501, 1339, 1018; H NMR (DMSO-d ):
6
δ 9.44–9.36 (m, 1H, aromatic), 8.45–8.30 (m, 2H, aromatic), 6.98–6.78 (m, 4H, aromatic), 4.70 (t, J
=
+
6.6 Hz, 2H, CONCH CH ), 3.77 (s, 3H, OCH ), 3.05-2.88 (m, 4H, piperazine), 2.72–2.54 (m, 4H
2 2 3
2H, piperazine + CONCH CH ). Anal. (C H N O S) C, H, N, S.
2
2
23 23
5
5
5.1.5. 8-Amino-3-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl][1]benzothieno[3,2-d]pyrimidine-
2,4(1H,3H)-dione (6)
A mixture of 5 (0.50 g, 1.04 mmol), hydrazine hydrate (0.26 g, 5.19 mmol), and Raney Ni (0.26 g)
in DMF (12 mL) was stirred at room temperature for 24 h. Solids were eliminated by filtration and
water (40 mL) was added to the filtrate. Obtained precipitate was filtered off, washed with water
and dried. Recrystallization from ethanol gave 6 (0.35 g, 75 %) as a pure product: mp 249–251 °C;
-1
1
IR (KBr) cm 3328, 2930, 1711, 1650, 1440, 1241, 745, 687; H NMR (DMSO-d ): δ 12.33 (s, 1H,
6
NH which exchange with D O), 7.76–7.60 (m, 1H, aromatic), 7.50–7.38 (m, 1H, aromatic), 7.04–
2
6
.80 (m, 4H + 1H, aromatic), 5.32 (s, 2H, NH which exchange with D O), 4.06 (t, J = 6.0 Hz, 2H,
2 2
1
4

ACCEPTED MANUSCRIPT
CONCH ), 3.76 (s, 3H, OCH ), 2.98–2.88 (m, 4H, piperazine), 2.66-2.52 (m, 4H + 2H, piperazine +
2
3
CONCH CH ). Anal. (C H N O S) C, H, N, S.
2
2
23 25
5
3
5.1.6. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
tetrahydro[1]benzothieno [3,2-d]pyrimidin-8-yl]benzamide (7)
A mixture of compound 6 (0.20 g, 0.44 mmol), anhydrous potassium carbonate (0.31 g, 2.24 mmol)
and benzoyl chloride (0.074 g, 0.53 mmol) in THF (10 mL) was stirred for 5 h at room temperature.
An additional amount of benzoyl chloride (0.03 g, 0.21 mmol) was added and the mixture was
stirred for further 24 h. Volatiles were eliminated under reduced pressure and the solid residue was
suspended in water, then filtered off and dried. Recrystallization from DMF/water gave 7 (0.15 g,
-1
6
1 %) as a pure compound: mp 285–287 °C; IR (KBr) cm 3287, 3003, 1710, 1636, 1554, 1241,
1
7
49, 681; H NMR (DMSO-d ): δ 12.71 (br s, 1H, NH which exchanges with D O), 10.55 (s, 1H
6
2
C H CONH which exchanges with D O), 9.02–8.98 (m, 1H, aromatic), 8.16–7.98 (m, 1H + 2H,
6
5
2
aromatic), 7.78–7.52 (m, 1H, aromatic), 7.44–7.28 (m, 3H, aromatic), 6.98–6.80 (m, 4H, aromatic),
.08 (t, J = 6.0 Hz, 2H, CONCH ), 4.08 (s, 3H, OCH ), 2.98–2.80 (m, 4H, piperazine), 2.68–2.45
4
2
3
1
3
(
m, 4H + 2H, piperazine + CONCH CH ). C NMR (DMSO-d ): δ 165.57, 159.00, 151.94, 151.50,
2 2 6
141.21, 140.07, 136.57, 135.45, 134.65, 131.76, 128.80, 128.49, 127.72, 124.05, 123.64, 122.32,
1
20.82, 117.87, 115.27, 111.84, 110.29, 55.28, 55.09, 53.15, 50.06, 37.68. Anal. (C H N O S) C,
3
0
29
5
4
H, N, S.
5.1.7. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-2-phenylacetamide (8)
A mixture of compound 6 (0.10 g, 0.22 mmol), anhydrous potassium carbonate (0.15 g, 1.08 mmol)
and phenylacetyl chloride (0.04 g, 0.26 mmol) in THF (5 mL) was stirred for 4 h at room
temperature. Volatiles were eliminated under reduced pressure, the solid residue was suspended in
water, then filtered off and dried to give 8 (0.10 g, 79 %) as a pure compound: mp 227–229 °C. IR
1
5

ACCEPTED MANUSCRIPT
-1
1
(
KBr) cm 2940, 1703, 1646, 1446, 1241, 1025, 756, 690. H NMR (DMSO-d ): δ 12.65 (br s, 1H,
6
NH which exchanges with D O), 10.44 (s, 1H, CH CONH which exchanges with D O), 8.84–8.75
2
2
2
(m, 1H, aromatic), 8.08–7.98 (m, 1H, aromatic), 7.65–7.58 (m, 1H, aromatic), 7.42-7.22 (m, 5H,
aromatic), 6.98–6.78 (m, 4H, aromatic), 4.06 (t, J = 7.8 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ),
2
3
3
.70 (s, 2H, C H CH ), 3.01–2.88 (m, 4H, piperazine), 2.68–2.55 (m, 4H + 2H, piperazine +
6
5
2
1
3
CONCH CH ). C NMR (DMSO-d ): δ 169.54, 159.27, 152.13, 151.68, 141.32, 140.13, 136.75,
2
2
6
1
36.04, 135.24, 129.37, 128.99, 128.58, 126.87, 124.44, 122.65, 121.05, 118.11, 114.14, 112.02,
10.57, 55.48, 55.21, 53.27, 50.16, 43.35, 38.24. Anal. (C H N O S) C, H, N, S.
1
3
1
31
5
4
5.1.8. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-4-phenylbutanamide (9)
A mixture of compound 6 (0.23 g, 0.51 mmol), anhydrous potassium carbonate (0.35 g, 2.53 mmol)
and 4-phenylbutyryl chloride (0.10 g, 0.55 mmol) in THF (15 mL) was stirred for 6 h at room
temperature. Volatiles were eliminated in vacuo and the solid residue was suspended in water, then
filtered off and dried. Recrystallization from DMF/water gave 9 (0.18 g, 59 %) as a pure compound:
-1
1
mp 226–228 °C. IR (KBr) cm 3529, 3025, 1774, 1710, 1240, 1179, 743, 684; H NMR (DMSO-
d ): δ 12.68 (br s, 1H, NH which exchanges with D O), 10.16 (s, 1H, CONH which exchanges with
6
2
D O), 8.82–8.74 (m, 1H, aromatic), 8.01–7.97 (m, 1H, aromatic), 7.62–7.57 (m, 1H, aromatic),
2
7
.38–7.18 (m, 5H, aromatic), 6.98–6.78 (m, 4H, aromatic), 4.08 (t, J = 6.0 Hz, 2H, CONCH ), 3.76
2
(
s, 3H, OCH ), 3.15–2.80 (m, 4H, piperazine), 2.63–2.53 (m, 4H + 2H + 2H, piperazine + C H CH
3
6
5
2
+
CONCH CH ), 2.40 (t, J = 7.2 Hz, 2H, CH CONH), 2.02–1.89 (m, 2H, C H CH CH ). Anal.
2 2 2 6 5 2 2
(
C H N O S) C, H, N, S.
33 35 5 4
5.1.9. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-5-phenylpentanamide (10).
1
6

ACCEPTED MANUSCRIPT
A mixture of compound 6 (0.23 g, 0.51 mmol), anhydrous potassium carbonate (0.35 g, 2.53 mmol)
and 5-phenylpentanoyl chloride (0.15 g, 0.76 mmol) in THF (15 mL) was stirred for 20 h at room
temperature. Volatiles were eliminated in vacuo and the solid residue was suspended in a solution
(25 mL) of potassium carbonate (5% w/v), then filtered off, washed with water and dried to give 10
-1
(
0.28 g, 90 %) as a pure compound: mp 214–216 °C. IR (KBr) cm 3487, 2930, 1711, 1637, 1240,
1
1
1
7
6
+
177, 741, 684; H NMR (DMSO-d ): δ 12.68 (br s, 1H, NH which exchanges with D O), 10.16 (s,
6
2
H, CONH which exchanges with D O), 8.84–8.76 (m, 1H, aromatic), 8.02–7.78 (m, 1H, aromatic),
2
.63–7.57 (m, 1H, aromatic), 7.38–7.15 (m, 5H, aromatic), 6.98–6.80 (m, 4H, aromatic), 4.08 (t, J =
.0 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ), 3.02-2.80 (m, 4H, piperazine), 2.70-2.56 (m, 4H + 2H
2
3
2H, piperazine + C H CH + CONCH CH ), 2.40 (t, J = 7.2 Hz, 2H, CH CONH), 1.70–1.57 (m,
6
5
2
2
2
2
1
3
2
1
1
H + 2H, CH CH CH CONH). C NMR (DMSO-d ): δ 171.33, 159.11, 151.97, 151.56, 142.11,
2 2 2 6
40.99, 140.04, 136.80, 134.77, 128.85, 128.35, 128.33, 125.74, 124.15, 122.54, 122.41, 120,88,
17.98, 113.74, 111.90, 110.33, 55.33, 54.95, 49.72, 37.42, 36.11, 35.00, 30.70, 24.81. Anal.
(
C H N O S) C, H, N, S.
34 37 5 4
5.1.10. 4-Oxo-4-[(phenylmethyl)amino]butanoic acid (16) [29].
A solution of benzylamine (0.20 g, 1.87 mmol) in anhydrous toluene (6 mL) was added to a
solution of succinic anhydride (0.19 g, 1.87 mmol) in anhydrous toluene (14 mL) at 0 °C and the
mixture was stirred at the same temperature for 1 h. Then, solids were filtered off washed with cold
toluene and dried. Obtained crude 16 (0.37 g, 94 %) was used successively without further
-1
1
purification: mp 127–130 °C. IR (KBr) cm 3299, 2918, 1693, 1640, 1546, 1231, 734, 660; H
NMR (DMSO-d ): δ 8.37 (t, J = 6.0 Hz, 1H, NH which exchanges with D O), 7.42–7.18 (m, 5H,
6
2
aromatic), 4.26 (d, J = 6.0 Hz, 2H, C H CH ), 2.44–2.38 (m, 2H + 2H, CH CH COOH).
6
5
2
2
2
5.1.11. 5-Oxo-5-[(phenylmethyl)amino]pentanoic acid (17) [29].
1
7

ACCEPTED MANUSCRIPT
A solution of benzylamine (0.50 g, 4.67 mmol) in anhydrous toluene (15 mL) was added to a
solution of glutaric anhydride (0.53 g, 4.67 mmol) in anhydrous toluene (40 mL) at 0 °C and the
mixture was stirred at the same temperature for 30 min. Then, solids were filtered off, washed with
cold toluene and dried. Obtained crude 17 (0.80 g, 78 %) was used successively without further
-1
1
purification: mp 84–85 °C. IR (KBr) cm 3295, 3030, 1699, 1635, 1548, 1241, 746, 694; H NMR
(
DMSO-d ): δ 8.34 (t, J = 6.0 Hz, 1H, NH which exchanges with D O), 7.38–7.18 (m, 5H,
6
2
aromatic), 4.25 (d, J = 6.0 Hz, 2H, C H CH ), 2.22–2.08 (m, 2H + 2H, CH CH CH COOH), 1.80–
6
5
2
2
2
2
1
.62 (m, 2H, CH CH COOH).
2 2
5.1.12. 5-[Methyl(phenylmethyl)amino]-5-oxopentanoic acid (18)
A solution of N-benzylmethylamine (0.50 g, 4.12 mmol) in anhydrous toluene (5 mL) was added to
a solution of glutaric anhydride (0.47 g, 4.12 mmol) in anhydrous toluene (40 mL) and the mixture
was stirred at 40 °C for 42 h. Then, volatiles were eliminated under reduced pressure and the oily
residue was dissolved in NaOH 1 M (9 mL). The alkaline phase was extracted with ethyl ether (2 ×
20 mL) and the organic phase discharged. Aqueous phase was then acidified with HCl 1 M (14 mL)
and extracted with ethyl ether (3 × 20 mL). Combined organic phases were dried on anhydrous
sodium sulfate and evaporated at reduced pressure to give an oily residue. Obtained crude 18 (0.54
-1
g, 53 %) was used successively without further purification. IR (KBr) cm 2939, 1726, 1608, 1450,
1
1
229, 1409, 734, 699; H NMR (DMSO-d ): δ 12.03 (s, 1H, COOH which exchanges with D O),
6
2
7
.42–7.13 (m, 5H, aromatic), 4.55 (s, 2H, C H CH , conformer E (or Z)), 4.49 (s, 2H, C H CH ,
6
5
2
6
5
2
conformer Z (or E)), 2.88 (s, 3H, CH , conformer Z (or E)), 2.79 (s, 3H, CH , conformer E (or Z)),
3
3
2
.45–2.18 (m, 2H + 2H, COCH CH CH COOH), 1.82–1.64 (m, 2H, CH CH COOH).
2 2 2 2 2
5.1.13. 5-Oxo-5-[(2-phenylethyl)amino]pentanoic acid (19) [30]
A solution of 2-phenylethylamine (0.50 g, 4.12 mmol) in anhydrous toluene (4 mL) was added to a
solution of glutaric anhydride (0.47 g, 4.12 mmol) in anhydrous toluene (20 mL) and the mixture
1
8

ACCEPTED MANUSCRIPT
was stirred under reflux for 3 h. Then, solids were filtered off, washed with cold toluene and dried
-1
to obtain 19 (0.32 g, 33 %): mp 87–90 °C. IR (KBr) cm 3297, 2935, 1697, 1638, 1546, 863, 748,
1
6
5
2
98; H NMR (DMSO-d ): δ 7.91 (br t, J = 5.4 Hz, 1H, NH which exchanges D O), 7.35–7.16 (m,
6
2
H, aromatic), 3.25–3.17 (m, 2H, CH NH), 2.69 (t, J = 7.4 Hz, 2H, C H CH ), 2.20–2.00 (m, 2H +
2
6
5
2
H, CH CH CH ), 1.78–1.58 (m, 2H, CH CH CH ). Anal. (C H NO ) C, H, N.
2
2
2
2
2
2
13 17
3
1
5
.1.14. N -[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
4
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-N -(phenylmethyl)succinamide (20)
Triethylamine (0.073 g, 0.72 mmol) was added to a solution of the acid 16 (0.15 g, 0.72 mmol) in
THF (4 mL) and the mixture was placed under stirring in an ice bath. After five minutes ethyl
chlorocarbonate (0.078 g, 0.72 mmol) was added. After further fifteen minutes, a solution of 6
(0.325 g, 0.72 mmol) in THF (21 mL) was added and then the mixture was left under stirring at
room temperature for 24 h. Volatiles were eliminated under reduced pressure and the obtained
residue was purified by flash chromatography on silica gel 60 using a mixture of
chloroform/methanol (9/1, v/v) as eluent. Homogeneous fractions were combined and evaporated
under reduced pressure to give 20 as a pure compound (0.033 g, 7 %): mp 251–253 °C. IR (KBr)
-1
1
cm 3321, 2938, 1698, 1640, 1241, 1024, 747, 688; H NMR (DMSO-d ): δ 12.70 (br s, 1H, NH
6
which exchanges with D O), 10.24 (s, 1H, NH which exchanges with D O), 8.76 (s, 1H, aromatic),
2
2
8
.44 (t, J = 5.8 Hz, 1H, C H CH NHCO which exchanges with D O), 8.08–7.98 (d, J = 9.2 Hz, 1H,
6 5 2 2
aromatic), 7.66–7.58 (m, 1H, aromatic), 7.38–7.18 (m, 5H, aromatic), 7.00–6.82 (m, 4H, aromatic),
4.29 (d, J = 5.6 Hz, 2H, C H CH NHCO), 4.07 (t, J = 6.6 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ),
6 5 2 2 3
1
3
2
.98–2.88 (m, 4H, piperazine), 2.72–2.58 (m, 4H + 2H, piperazine + CONCH CH ). C NMR
2
2
(
DMSO-d ): δ 171.28, 170.61, 159.03, 151.97, 151.51, 141.24, 140.02, 139.60, 136.87, 134.64,
6
1
28.82, 128.27, 127.19, 126.73, 124.12, 122.35, 122.19, 120.86, 117.89, 113.42, 111.89, 110.27,
5.29, 55.11, 53.15, 50.07, 42.08, 37.66, 31.54, 30.20. Anal. (C H N O S) C, H, N, S.
5
3
4
36
6
5
1
9

ACCEPTED MANUSCRIPT
1
5
.1.15. N -[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
5
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-N -(phenylmethyl)pentanediamide (21)
A solution of the acid 17 (0.20 g, 0.90 mmol) in THF (15 mL) was prepared by gentle heating.
Then, it was placed under stirring in an ice bath and triethylamine (0.091 g, 0.90 mmol) was added.
After five minutes ethyl chlorocarbonate (0.098 g, 0.90 mmol) was added and the mixture was
stirred for 3 h at 0 – 5 °C. The mixture was allowed to reach room temperature and a solution of 6
(0.33 g, 0.73 mmol) in THF (22 mL) was added; the mixture was left under stirring at room
temperature for 40 h. Volatiles were eliminated under reduced pressure and the obtained residue
was purified by flash chromatography on silica gel 60 using a mixture of chloroform/methanol (9/1,
v/v) as eluent. Homogeneous fractions were combined and evaporated under reduced pressure to
-
1
give 21 as a pure compound (0.14 g, 29 %): mp 232–235 °C. IR (KBr) cm 3261, 2934, 1709,
1
1
650, 1238, 1024, 753, 685; H NMR (DMSO-d ): δ 12.69 (br s, 1H, NH which exchanges with
6
D O), 10.21 (s, 1H, CONH which exchanges with D O), 8.82–8.76 (m, 1H, aromatic), 8.41 (t, J =
2
2
6
.0 Hz, 1H, C H CH NH), 8.08–7.98 (m, 1H, aromatic), 7.65–7.56 (m, 1H, aromatic), 7.38–7.18
6 5 2
(
m, 5H, aromatic), 6.98–6.80 (m, 4H, aromatic), 4.30 (d, J = 5.6 Hz, 2H, C H CH ), 4.08 (t, J = 6.0
6 5 2
Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ), 3.05–2.90 (m, 4H, piperazine), 2.16–2.06 (m, 4H + 2H,
2
3
piperazine + CONCH CH ), 2.45 (t, J = 7.4 Hz, 2H, CH CONH), 2.25 (t, J = 7.4 Hz, 2H,
2
2
2
1
3
NHCOCH ), 1.98–1.72 (m, 2H, CH CH CH ). C NMR (DMSO-d ): δ 171.79, 171.02, 159.05,
2
2
2
2
6
1
1
3
51.97, 151.52, 141.23, 140.03, 139.68, 136.81, 134.75, 128.87, 128.33, 127.22, 126.76, 124.12,
22.38 (2C), 120.87, 117.91, 113.69, 111.88, 110.30, 55.31, 55.11, 53.18, 50.09, 42.05, 37.71,
5.63, 34.64, 21.28. Anal. (C H N O S) C, H, N, S.
3
5
38
6
5
1
5
5
.1.16. N -Methyl-N -[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
1
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-N -(phenylmethyl)pentanediamide (22)
2
0

ACCEPTED MANUSCRIPT
Triethylamine (0.11 g, 1.10 mmol) was added to a solution of the acid 18 (0.26 g, 1.10 mmol) in
THF (10 mL) and the mixture was placed under stirring in an ice bath. After five minutes ethyl
chlorocarbonate (0.12 g, 1.10 mmol) was added. After 1 h, a solution of 6 (0.496 g, 1.10 mmol) in
THF (33 mL) was added and then the mixture was left under stirring at room temperature for 30 h.
Volatiles were eliminated under reduced pressure and the obtained residue was purified by flash
chromatography on silica gel 60 using a mixture of chloroform/methanol (9/1, v/v) as eluent.
Homogeneous fractions were combined and evaporated under reduced pressure to give 22 as a pure
-1
compound (0.16 g, 22 %): mp 207–208 °C. IR (KBr) cm 3487, 3301, 2933, 1708, 1649, 1238,
1
7
43, 681; H NMR (DMSO-d ): δ 12.68 (br s, 1H, NH which exchanges with D O), 10.19 (s, 1H,
6
2
CONH which exchanges with D O, conformer Z (or E)), 10.15 (s, 1H, CONH which exchanges
2
with D O, conformer E (or Z)), 8.79 (s, 1H, aromatic, conformer Z (or E)), 8.76 (s, 1H, aromatic,
2
conformer E (or Z)), 8.08-7.92 (m, 1H, aromatic), 7.68-7.56 (m, 1H, aromatic), 7.42-7.12 (m, 5H,
aromatic), 7.01-6.80 (m, 4H, aromatic), 4.58 (s, 2H, C H CH , conformer E (or Z)), 4.52 (s, 2H,
6
5
2
C H CH , conformer Z (or E)), 4.08 (t, J = 6.0 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ), 3.04–2.88
6
5
2
2
3
(
m, 4H + 3H, piperazine + CH , conformer Z (or E)), 2.81 (s, 3H, CH , conformer E (or Z)), 2.70–
3
3
2
.55 (m, 4H + 2H + 2H, piperazine + CONCH CH + CH CONH), 2.46–2.40 (m, 2H,
2 2 2
13
NHCOCH CH CH ), 1.90 (t, J = 8.8 Hz, CH CH CH ). C NMR (DMSO-d ): (conformer E (or
2
2
2
2
2
2
6
Z)) δ 172.04, 171.24, 159.09, 152.00, 151.57, 141.26, 140.05, 137.98, 136.82, 134.81, 128.90,
1
5
1
1
3
28.56, 127.50, 127.07, 124.16, 122.43 (2C), 120.91, 117.94, 113.78, 111.91, 110.36, 55.34, 55.15,
3.21, 50.12, 49.95, 37.74, 35.55, 34.76, 31.90, 20.69; (conformer Z (or E)) δ 172.04, 171.19,
59.09, 152.00, 151.57, 141.26, 140.05, 137.53, 136.82, 134.81, 128.90, 128.81, 127.28, 126.52,
24.16, 122.43 (2C), 120.91, 117.94, 113.78, 111.91, 110.36, 55.34, 55.15, 53.21, 52.40, 50.12,
7.74, 35.55, 33.50, 31.59, 20.89. Anal. (C H N O S) C, H, N, S.
3
6
40
6
5
2
1

ACCEPTED MANUSCRIPT
1
5
.1.17. N -[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
5
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-N -(2-phenylethyl)pentanediamide (23)
Triethylamine (0.086 g, 0.85 mmol) was added to a solution of the acid 19 (0.20 g, 0.85 mmol) in
THF (15 mL) and the mixture was placed under stirring in an ice bath. After five minutes ethyl
chlorocarbonate (0.092 g, 0.85 mmol) was added. After 2 h, a solution of 6 (0.38 g, 0.85 mmol) in
THF (25 mL) was added and then the mixture was left under stirring at room temperature for 28 h.
Volatiles were eliminated under reduced pressure and the obtained residue was purified by flash
chromatography on silica gel 60 using a mixture of chloroform/methanol (9/1, v/v) as eluent.
Homogeneous fractions were combined and evaporated under reduced pressure to give 23 as a pure
-1
compound (0.11 g, 20 %): mp 227–230 °C. IR (KBr) cm 3298, 2934, 1708, 1649, 1239, 1023,
1
7
51, 699; H NMR (DMSO-d ): δ 12.69 (br s, 1H, NH exchanges with D O), 10.16 (s, 1H, NH
6
2
which exchanges with D O), 8.78 (s, 1H, aromatic), 8.08–7.90 (m, 1H + 1H, aromatic + CH NHCO
2
2
which exchanges with D O), 7.61 (d, J = 8.4 Hz, 1H, aromatic), 7.38–7.16 (m, 5H, aromatic), 6.98-
2
6
.80 (m, 4H, aromatic), 4.07 (t, J = 6.0 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ), 3.32-3.21 (m, 2H,
2 3
CH NHCO), 3.00–2.88 (m, 4H, piperazine), 2.78–2.48 (m, 4H + 2H + 2H, piperazine +
2
CONCH CH + C H CH ), 2.36 (t, J = 7.6 Hz, 2H, CH CH CH ), 2.14 (t, J = 7.6 Hz, 2H,
2
2
6
5
2
2
2
2
13
CH CH CH ), 1.92–1.74 (m, 2H, CH CH CH ). C NMR (DMSO-d ): δ 171.58, 170.95, 158.99,
2
2
2
2
2
2
6
1
1
3
51.93, 151.47, 141.20, 139.99, 139.51, 136.47, 134.69, 128.84, 128.61, 128.29, 126.05, 124.07,
22.31, 120.81, 117.85, 113.62, 111.84, 110.25, 55.27, 55.09, 53.14, 50.05, 40.14, 37.66, 35.56,
5.24, 34.64, 21.19. Anal. (C H N O S) C, H, N, S.
3
6
40
6
5
5.1.18. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-4-[methyl(phenylmethyl)amino]butanamide (26)
,1’-Carbonyldiimidazole (0.09 g, 0.55 mmol) was added to a solution of 4-
methyl(phenylmethyl)amino]butanoic acid hydrochloride 24 (0.07 g, 0.29 mmol) in anhydrous
1
[
2
2

ACCEPTED MANUSCRIPT
THF (8 mL) and the mixture was stirred under reflux. After 1 h, compound 6 (0.10 g, 0.22 mmol)
was added and the reaction mixture was stirred under reflux for 28 h. Then, the solvent was
evaporated under reduced pressure and sodium carbonate 5 % (15 mL) was added to the residue.
The mixture was extracted with CHCl (3 × 15 mL), collected organic phase was dried on
3
anhydrous sodium sulfate and solvent eliminated under reduced pressure. Obtained residue was
purified by flash chromatography on silica gel 60 using a mixture of chloroform/methanol (8/2, v/v)
as eluent. Homogeneous fractions were combined and evaporated under reduced pressure to give 26
1
as a pure compound (0.02 g, 14 %): mp 148–151 °C dec. H NMR (CDCl ): δ 10.20 (br s, 1H, NH
3
exchanges with D O), 8.70 (s, 1H, aromatic), 7.61 (d, J = 8.6 Hz, 1H, aromatic), 7.40–7.22 (m, 5H,
2
aromatic), 7.14 (d, J = 8.6 Hz, 1H, aromatic), 7.04–6.78 (m, 4H, aromatic), 4.35 (t, J = 7.4 Hz, 2H,
CONCH ), 3.81 (s, 3H, OCH ), 3.58 (s, 2H, C H CH ), 3.10–2.94 (m, 4H, piperazine), 2.86–2.68
2
3
6
5
2
(
m, 4H + 2H, piperazine + CONCH CH ), 2.66–2.50 (m, 2H + 2H, CH CH CH ), 2.27 (s, 3H,
2 2 2 2 2
13
NCH ), 2.05–1.88 (m, 2H, CH CH CH ). C NMR (DMSO-d ): δ 172.47, 159.34, 152.62,152.14,
3
2
2
2
6
1
1
2
41.24, 139.17, 137.41, 136.34, 136.18, 129.44, 128.83, 128.52, 127.57, 123.71, 122.76, 121.88,
20.89, 118.08, 113.23, 111.06, 62.56, 57.34, 55.59, 55.26, 53.46, 50.52, 41.62, 38.24, 36.86,
2.53. Anal. (C H N O S) C, H, N, S.
3
5
40
6
4
5.1.19. 4-(Dimethylamino)-N-[3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-2,4-dioxo-1,2,3,4-
tetrahydro[1]benzothieno[3,2-d]pyrimidin-8-yl]butanamide (27)
,1’-Carbonyldiimidazole (0.26 g, 1.60 mmol) was added to a solution of 4-(dimethylamino)
1
butanoic acid hydrochloride 25 (0.14 g, 0.83 mmol) in anhydrous 1,4-dioxane (23 mL) and the
mixture was stirred under reflux. After 1 h, compound 6 (0.30 g, 0.66 mmol) was added and the
reaction mixture was stirred under reflux for 4 h. Then, the solvent was evaporated under reduced
pressure and sodium carbonate 5 % solution (45 mL) was added to the residue. The mixture was
extracted with CHCl (3 × 20 mL), collected organic phase was dried on anhydrous sodium sulfate
3
and solvent eliminated under reduced pressure. Obtained residue was purified by flash
2
3

ACCEPTED MANUSCRIPT
chromatography on silica gel 60 using a mixture of methanol/NH OH 30 % (9.9/0.01, v/v) as
4
eluent. Homogeneous fractions were combined and evaporated under reduced pressure to give 27 as
-1
a pure compound (0.16 g, 43 %): mp 187–189 °C dec. IR (KBr) cm 3241, 2942, 1707, 1647, 1501,
1
1
242, 754, 684; H NMR (CDCl ): δ 10.84 (br s, 1H, NH which exchanges with D O), 8.82 (d, J =
3
2
1.6 Hz, 1H, aromatic), 7.69 (m, J = 8.8 Hz, 1H, aromatic), 7.22 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H,
aromatic), 7.00–6.78 (m, 4H, aromatic), 4.32 (t, J = 6.6 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ),
2
3
3
.06-2.94 (m, 4H, piperazine), 2.82–2.68 (m, 4H + 2H, piperazine + CONCH CH ), 2.57 (t, J = 6.6
2 2
Hz, 2H, CH CONH), 2.45 (t, J = 5.8 Hz, 2H, CH CH CH CONH), 2.30 (s, 3H + 3H, CH + CH ),
2
2
2
2
3
3
1
.96–1.78 (m, 2H, CH CH CO). Anal. (C H N O S) C, H, N, S.
2 2 29 36 6 4
5.1.20. Ethyl 3-[(1-ethoxyethylidene)amino]-5-nitro-1-benzothiophene-2-carboxylate (28)
Amino ester 1 (1.00 g, 3.75 mmol) and 1,1,1-triethoxyethane (6.98 g, 43.02 mmol) were inserted in.
a 10 mL vessel. The vessel was sealed with the appropriate stopper septum and irradiated under
stirring in a CEM Discovery microwave instrument set at power = 60 W and temperature = 200 °C,
for 60 min. After being cooled, the reaction mixture was transferred in a flask, cyclohexane (20 mL)
was added and the obtained suspension was gentle heated under stirring for 30 min. The warm
mixture was filtered and the filtrate collected. Volatiles were eliminated under reduced pressure and
the obtained residue was purified by flash chromatography on silica gel 60 using a mixture of
cyclohexane/ethyl acetate (9/1, v/v) as eluent. Homogeneous fractions were combined and
evaporated under reduced pressure to give 28 as a pure compound (1.09 g, 86 %): mp 113 °C. IR
-1
1
(
KBr) cm 2983, 2931, 1711, 1513, 1441, 1257, 1051, 836; H NMR (DMSO-d ): δ 8.41–8.22 (m,
6
3
H, aromatic), 4.38 (q, J = 7.2 Hz, 2H, CH CH ), 4.28 (q, J = 7.2 Hz, 2H, CH CH ), 1.82 (s, 3H,
2 3 2 3
CH ), 1.38 (t, J = 7.2 Hz, 3H, CH CH ), 1.28 (t, J = 7.2, 3H, CH CH ). Anal. (C H N O S) C, H,
3
2
3
2
3
15 16
2
5
N, S.
2
4

ACCEPTED MANUSCRIPT
5.1.21. 3-(2-Hydroxyethyl)-2-methyl-8-nitro[1]benzothieno[3,2-d]pyrimidin-4(3H)-one (29)
A mixture of 28 (0.75 g, 2.23 mmol) and 2-aminoethanol (3.00 g, 49.1 mmol) was heated at 50 °C
for 2 h. Then, water was added and the precipitate was filtered off, washed with water and dried.
Obtained crude 29 (0.52 g, 76 %) was used in the successive synthetic step without any further
-1
purification. An analytical sample was recrystallized from ethanol: mp 235 °C. IR (KBr) cm 3382,
1
1
650, 1550, 1461, 1334, 1181, 1049, 830; H NMR (DMSO-d ): δ 8.86–8.84 (m, 1H, aromatic),
6
8
.45–8.40 (m, 2H, aromatic), 5.09 (t, J = 5.8 Hz, 1H, OH which exchanges with D O), 4.23 (t, J =
2
5
.2 Hz, 2H, CH CH OH), 3.80–3.68 (m, 2H, CH CH OH), 2.81 (s, 3H, CH ). Anal. (C H N O S)
2
2
2
2
3
13 11
3
4
C, H, N, S.
5
.1.22. 3-(2-Chloroethyl)-2-methyl-8-nitro[1]benzothieno[3,2-d]pyrimidin-4(3H)-one
hydrochloride (30)
Thionyl chloride (0.53 g, 4.45 mmol) was added to a solution of 29 (0.45 g, 1.47 mmol) in toluene
12 mL) and the mixture was refluxed under stirring for 3 h. After being cooled, volatiles were
(
eliminated under reduced pressure and cyclohexane (20 mL) was added to the residue. The obtained
suspension was filtered under vacuum and the solid residue was washed with cyclohexane and
dried. Obtained compound 30 (0.49 g, 92 %) was used successively without further purification: mp
-1
1
2
10–212 °C. IR (KBr) cm 3068, 1785, 1629, 1564, 1381, 1133, 955; H NMR (DMSO-d ): δ
6
8.88–8.85 (m, 1H, aromatic), 8.49–8.44 (m, 2H, aromatic), 5.05 (br s, 1H, NH which exchanges
with D O), 4.50 (t, J = 6.8 Hz, 2H, CH CH Cl,), 4.01 (t, J = 6.8 Hz, 2H, CH CH Cl,), 2.82 (s, 3H,
2
2
2
2
2
CH3).
5.1.23. 3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2-methyl-8-nitro[1]benzothieno[3,2-
d]pyrimidin-4(3H)-one (31)
A mixture of 30 (0.49 g, 1.36 mmol) and 1-(2-methoxyphenyl)piperazine (1.31 g, 6.81 mmol) was
heated in an oil bath at 160 °C for 3 h. After being cooled, ethanol (10 mL) was added to the
2
5

ACCEPTED MANUSCRIPT
reaction mixture. Solids were filtered off, washed with cold ethanol and dried. The crude product
was triturated in ethanol and then filtered off and dried to give 31 (0.53 g, 81 %) as a pure product:
-
1
1
mp 210 °C; IR (KBr) cm 2951, 1676, 1555, 1341, 1245, 1035, 929; H NMR (DMSO-d ): δ 9.00–
6
8
7
2
.82 (m, 1H, aromatic), 8.52–8.40 (m, 1H, aromatic), 7.00–6.81 (m, 4H + 1H, aromatic), 4.30 (t, J =
.2 Hz, 2H, CONCH ), 3.77 (s, 3H, OCH ), 3.00–2.93 (m, 4H, piperazine), 2.85 (s, 3H, CH ), 2.78–
2
3
3
.60 (m, 4H + 2H, piperazine + CONCH CH ). Anal. (C H N O S) C, H, N, S.
2
2
24 25
5
4
5.1.24. 8-Amino-3-]2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-2-methyl[1]benzothieno[3,2-
d]pyrimidin-4(3H)-one (32)
A mixture of 31 (0.53 g, 1.10 mmol), hydrazine hydrate (0.28 g, 5.59 mmol), and Raney Ni (0.26 g)
in DMF (40 mL) was stirred at room temperature. Two further amounts of hydrazine hydrate were
added after 24 h (0.14 g, 2.80 mmol) and 48 h (0.055 g, 1.10 mmol) from the starting of the
reaction. After further 48 h, solids were eliminated by filtration and water (60 mL) was added to the
filtrate. The mixture was extracted with ethyl acetate (3 × 25 mL). Combined organic phases were
dried on anhydrous sodium sulfate and evaporated at reduced pressure to give a residue which was
suspended in diethyl ether. Solids were filtered off, washed with diethyl ether and dried to give 32
-1
(
0.43 g, 87 %) as a pure product: mp 230 °C; IR (KBr) cm 3448, 2816, 1671, 1553, 1449, 1335,
1
1
144, 1011, 926; H NMR (DMSO-d ): δ 7.70 (d, J = 8.4 Hz, 1H, aromatic), 7.40–7.34 (m, 1H,
6
aromatic), 7.05–6.84 (m, 4H + 1H, aromatic), 5.44 (br s, 2H, NH which exchange D O), 4.25 (t, J
2
2
=
7.2 Hz, 2H, CONCH ), 3.77 (s, 3H, OCH ), 3.10–2.90 (m, 4H, piperazine), 2.76 (s, 3H, CH ),
2 3 3
2
.70–2.58 (m, 4H + 2H, piperazine + CONCH CH ). Anal. (C H N O S) C, H, N, S.
2 2 24 27 5 2
5.1.25. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2-methyl-4-oxo-3,4-
dihydro[1]benzothieno[3,2-d]pyrimidin-8-yl]benzamide (33)
A mixture of compound 32 (0.20 g, 0.44 mmol), anhydrous potassium carbonate (0.31 g, 2.24
mmol) and benzoyl chloride (0.074 g, 0.53 mmol) in THF (10 mL) was stirred for 24 h at room
2
6

ACCEPTED MANUSCRIPT
temperature. Then, water was added and the mixture extracted with ethyl acetate (3 × 20 mL).
Combined organic phases were dried on anhydrous sodium sulfate and evaporated at reduced
pressure to give a residue which was suspended in water, then filtered off and dried.
Recrystallization from ethanol gave 33 (0.11 g, 45 %) as a pure compound: mp 202 °C; IR (KBr)
-1
1
cm 3254, 2946, 1674, 1454, 1236, 1027, 927, 805, 745; H NMR (DMSO-d ): δ 10.55 (br s, 1H,
6
NH which exchanges with D O), 8.90–8.82 (m, 1H, aromatic), 8.14–7.94 (m, 2H + 2H, aromatic),
2
7
.68–7.54 (m, 3H, aromatic), 6.95–6.82 (m, 4H, aromatic), 4.28 (t, J = 6.0 Hz, 2H, CONCH ), 3,77
2
(
s, 3H, OCH ), 2.99–2.90 (m, 4H, piperazine), 2.81 (s, 3H, CH ), 2.74–2.60 (m, 4H + 2H,
3
3
1
3
piperazine + CONCH CH ). C NMR (DMSO-d ): δ.169.22, 165.87, 158.18, 157.95, 152.04,
2
2
6
1
51.35, 141.18, 137.22, 135.44, 134.76, 134.47, 131.90, 128.59, 127.80, 124.04, 122.58, 120.95,
18.04, 113.91, 111.95, 55.59, 55.38, 53.34, 50.13, 42.25, 23.00. Anal. (C H N O S) C, H, N, S.
1
3
1
31
5
3
5.1.26. N-[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2-methyl-4-oxo-3,4-
dihydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-2-phenylacetamide (34)
A mixture of compound 32 (0.10 g, 0.22 mmol), anhydrous potassium carbonate (0.15 g, 1.08
mmol) and phenylacetyl chloride (0.040 g, 0.26 mmol) in THF (5 mL) was stirred for 4 h at room
temperature. Then, volatiles were eliminated under reduced pressure and sodium carbonate 5 %
solution (10 mL) was added. After stirring for 30 min, suspension was filtered under vacuum, solids
washed with water and dried. Obtained solid product was suspended again in petroleum ether (40-
60 °C, 15 mL). After stirring for 30 min, suspension was filtered under vacuum and the obtained
solid product was dried to give 34 (0.075 g, 59 %) as a pure compound: mp 187–189 °C; IR (KBr)
-1
1
cm 3240, 2938, 2817, 1667, 1512, 1453, 1239, 1019, 747; H NMR (DMSO-d ): δ 10,50 (br s,
6
1
H, NH which exchanges with D O), 8.72-8.69 (m,1H, aromatic), 8.04 (d, J = 8.8 Hz, 1H,
2
aromatic), 7.75–7.67 (m, 1H, aromatic), 7.40–7.20 (m, 5H, aromatic), 6.95-6.80 (m, 4H, aromatic),
.25 (t, J = 6.0 Hz, 2H, CONCH ), 3.76 (s, 3H, OCH ), 3.70 (s, 2H, CH CO), 3.00–2.90 (m, 4H,
4
2
3
2
2
7

ACCEPTED MANUSCRIPT
1
3
piperazine), 2.78 (s, 3H, CH ), 2.74–2.58 (m, 4H + 2H, piperazine + CONCH CH ). C NMR
3
2
2
(
DMSO-d ): δ 169.50, 158.10, 157.86, 151.98, 151.21, 147.38, 141.13, 137.15, 135.87, 134.95,
6
1
34.48, 129.19, 128.40, 126.67, 124.13, 122.49, 121.48, 120.87, 117.98, 112.66, 111.92, 55.54,
5.34, 53.28, 50.07, 43.42, 42.16, 22.92. Anal. (C H N O S) C, H, N, S.
5
3
2
33
5
3
1
5
.1.27. N -[3-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-2-methyl-4-oxo-3,4-
5
dihydro[1]benzothieno[3,2-d]pyrimidin-8-yl]-N -(phenylmethyl)pentanediamide (35)
A solution of the acid 17 (0.21 g, 0.95 mmol) in THF (15 mL) was prepared by gentle heating.
Then, it was placed under stirring in an ice bath and triethylamine (0.096 g, 0.95 mmol) was added.
After five minutes ethyl chlorocarbonate (0.103 g, 0.95 mmol) was added and the mixture was
stirred for 3 h at 0–5 °C. The mixture was allowed to reach room temperature and a solution of 32
(0.35 g, 0.78 mmol) in THF (30 mL) was added dropwise; the mixture was left under stirring at
room temperature for 5 days. Then, solids were filtered off; on the filtrate, volatiles were eliminated
under reduced pressure and the obtained residue was dissolved in ethyl acetate. The organic phase
was washed with sodium carbonate 5 % solution, then with brine and dried on anhydrous sodium
sulfate. Volatiles were eliminated under reduced pressure and the obtained residue was purified by
flash chromatography on silica gel 60 using a mixture of ethyl acetate/methanol (9/1, v/v) as eluent.
Homogeneous fractions were combined and evaporated under reduced pressure to give 35 (0.20 g,
-1
3
9 %) as a pure compound: mp 180 °C. IR (KBr) cm 3305, 2955, 1581, 1453, 1234, 1146, 1023,
1
8
29; H NMR (DMSO-d ): δ 10.23 (s, 1H, CONH which exchanges with D O), 8.76–8.73 (m, 1H,
6
2
aromatic), 8.39 (t, J = 6.0 Hz, 1H, ArCH NHCO which exchanges with D O), 8.02 (d, J = 8.8 Hz,
2
2
1H, aromatic), 7.75–7.65 (m, 1H, aromatic), 7.36-7.17 (m, 5H, aromatic), 6.93–6.87 (m, 4H,
aromatic), 4.30–4.25 (m, 2H + 2H, ArCH NH + CONCH ), 3.77 (s, 3H, OCH ), 2.98–2.90 (m, 4H,
2
2
3
piperazine), 2.80 (s, 3H, CH ), 2.72–2.60 (m, 4H + 2H, piperazine + CONCH CH ), 2.40 (t, J = 7.2
3
2
2
13
Hz, 2H, CH CH CH ), 2.24 (t, J = 6.8 Hz, 2H, CH CH CH ), 1.93-1.84 (m, 2H, CH CH CH ). C
2
2
2
2
2
2
2
2
2
2
8