Synthesis and biological effects of novel 2-amino-3-(4-chlorobenzoyl)-4- substituted thiophenes as allosteric enhancers of the A1 adenosine receptor

Article abstract of DOI:10.1016/j.ejmech.2013.07.002

Allosteric enhancers for the A₁ adenosine receptor represent a novel and unique drug design strategy to augment the response to endogenous adenosine in a site- and event-specific manner.We have previously investigated a detailed structure-activity relationship study around a wide series of 2-amino-3-aroyl-4-[(4-arylpiperazin-1-yl)methyl]thiophene derivatives as potent allosteric enhancers of the A₁ adenosine receptor. In this manuscript we report our investigation on the influence on allosteric enhancer activity of further substitution at the 4-position of the 2-amino-3-(4-chlorobenzoyl)- thiophene system to explore bulk tolerance by replacement of the arylpiperazine moiety with a series of fused indole nuclei corresponding to 1,2,3,4-tetrahydropyrazino[1,2-a ]indole, 1,2,3,4,10,10a-hexahydropyrazino[1,2- a]indole, tet-rahydro- γ-carboline, tetrahydroisoquinoline, spiro-1,3-benzodioxolepiperidine, aliphatic tertiary amine, N-alkylaniline, aryl ether and aryl thioether templates. The 1,2,3,4-tetrahydropyrazino[1,2- a]indole derivatives 3-ec and 3- were the most active compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [³H]CCPA binding to the A₁ receptor. This study also shows that it is possible to obtain a good separation between allosteric enhancement and antagonistic activity at the A₁ adenosine receptor.

Full text of DOI:10.1016/j.ejmech.2013.07.002

Accepted Manuscript
Synthesis and Biological Effects of Novel 2-Amino-3-(4-Chlorobenzoyl)-4-Substituted
Thiophenes as Allosteric Enhancers of the A Adenosine Receptor
1
Romeo Romagnoli, Pier Giovanni Baraldi, Maria Dora Carrion, Carlota Lopez Cara,
Maria Kimatrai Salvador, Delia Preti, Mojgan Aghazadeh Tabrizi, Allan R. Moorman,
Fabrizio Vincenzi, Pier Andrea Borea, Katia Varani
PII:
S0223-5234(13)00440-6
10.1016/j.ejmech.2013.07.002
EJMECH 6289
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2013
Revised Date: 1 July 2013
Accepted Date: 6 July 2013
Please cite this article as: R. Romagnoli, P.G. Baraldi, M.D. Carrion, C.L. Cara, M.K. Salvador, D.
Preti, M.A. Tabrizi, A.R. Moorman, F. Vincenzi, P.A. Borea, K. Varani, Synthesis and Biological
Effects of Novel 2-Amino-3-(4-Chlorobenzoyl)-4-Substituted Thiophenes as Allosteric Enhancers
of the A Adenosine Receptor, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
1
j.ejmech.2013.07.002.
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ACCEPTED MANUSCRIPT
Synthesis and Biological Effects of Novel 2-Amino-3-(4-Chlorobenzoyl)-
4
-Substituted Thiophenes as Allosteric Enhancer of the A Adenosine
1
Cl
a
Receptor a
a
Romeo Romagnoli *, Pier Giovanni Baraldi *, Maria Dora Carrion , Carlota
a
a
a
Lopez Cara , Maria Kimatrai Salvador , Delia Preti , Mojgan Aghazadeh
Tabrizi , Allan R. Moorman , Fabrizio Vincenzi , Pier Andrea Borea ,
a
b
c
c
R1
O
c
Katia Varani
a
Dipartimento di Scienze Farmaceutiche, Via Fossato di Mortara 17-19,
Università di Ferrara, 44121, Ferrara, Italy,
King Pharmaceuticals Inc., Research and Development, 4000 CentreGreen
Way, Suite 300, Cary, North Carolina, USA
R2
S
NH
2
b
R =spiro-and fused-piperidines,
anilines, acyclic alkyl amines, substituted phenol
and thiophenol.
1
c
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, R =H, Br or C H
2
6 5
Università di Ferrara, Ferrara, Italy

ACCEPTED MANUSCRIPT
Synthesis and Biological Effects of Novel 2-Amino-3-(4-Chlorobenzoyl)-4-
Substituted Thiophenes as Allosteric Enhancers of the A Adenosine Receptor
1
a
a
a
a
Romeo Romagnoli *, Pier Giovanni Baraldi *, Maria Dora Carrion , Carlota Lopez Cara , Maria
a
a
a
b
Kimatrai Salvador , Delia Preti , Mojgan Aghazadeh Tabrizi , Allan R. Moorman , Fabrizio
c
c
c
Vincenzi , Pier Andrea Borea , Katia Varani
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Via Fossato di Mortara 17-19, Università di
Ferrara, 44121, Ferrara, Italy
b
King Pharmaceuticals Inc., Research and Development, 4000 CentreGreen Way, Suite 300, Cary,
North Carolina, USA
c
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Ferrara,
Ferrara, Italy
*
To whom correspondence should be addressed. Phone: 39-(0)532455303. Fax: 39-(0)532455953.
E-mail:rmr@unife.it (R.R.); Phone: 39-(0)532455293. Fax: 39-(0)532-455953. E-
mail:baraldi@unife.it (P.G.B.).
1

ACCEPTED MANUSCRIPT
Abstract: Allosteric enhancers for the A adenosine receptor represent a novel and unique drug
1
design strategy to augment the response to endogenous adenosine in a site- and event- specific
manner. We have previously investigated a detailed structure-activity relationship study around a
wide series of 2-amino-3-aroyl-4-[(4-arylpiperazin-1-yl)methyl]thiophene derivatives as potent
allosteric enhancers of the A
the influence on allosteric enhancer activity of further substitution at the 4-position of the 2-amino-
-(4-chlorobenzoyl)-thiophene system to explore bulk tolerance by replacement of the
1
adenosine receptor. In this manuscript we report our investigation on
3
arylpiperazine moiety with a series of fused indole nuclei corresponding to 1,2,3,4-
tetrahydropyrazino[1,2-a]indole, 1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indole, tetrahydro-γ-
carboline, tetrahydroisoquinoline, spiro-1,3-benzodioxolepiperidine, aliphatic tertiary amine, N-
alkylaniline, aryl ether and aryl thioether templates. The 1,2,3,4-tetrahydropyrazino[1,2-a]indole
derivatives 3a-c and 3e were the most active compounds in binding (saturation and competition)
3
and functional cAMP studies, being able to potentiate agonist [ H]CCPA binding to the A
1
receptor.
This study also shows that it is possible to obtain a good separation between allosteric enhancement
and antagonistic activity at the A adenosine receptor.
1
Keywords: A adenosine receptor; allosteric enhancer; G protein-coupled receptors; 2-amino-3-
1
benzoylthiophene.
1
. Introduction
Adenosine is a physiological extracellular modulator acting via four distinct G protein-coupled
receptors, named A , that are widely distributed throughout the body [1].
Activation of A receptors induces inhibition of the enzyme adenylate cyclase mediated by an
1
, A2A, A2B and A
3
1
inhibitory G-protein (Gi) with a consequent reduction in cAMP levels [2]. The A
receptors (A AR) are expressed in high density in the brain (cortex, hippocampus, cerebellum and
thalamus) and fat cells, and in moderate to low levels in many other tissues, such as bladder, lung,
1
adenosine
1
1
kidney and heart [3]. Effects mediated by selective activation of the A AR include neuro- and
2

ACCEPTED MANUSCRIPT
cardioprotection, an antiarrhythmic effect, hypotension, reduction of neuropathic pain and
inhibition of lipolysis in adipose tissue [4].
Due to the ubiquitous presence of adenosine receptors, agents which can selectively activate A
within specific tissues have attracted much attention as potential therapeutic agents [5]. Although
numerous full agonists with high affinity for A AR have been developed, their clinical use is
1
ARs
1
hampered by severe cardiovascular side effects caused by their strong hypotensive action,
depression of the central nervous system (CNS) and numerous peripheral side effects on other
organs. Unfortunately, agonists that selectively activate A
been found. An alternative approach toward selective A AR activation is provided by the concept of
allosteric modulation [6]. The binding of an allosteric modulator to the allosteric site of the A AR,
1
AR in a specific target tissue have not
1
1
structurally distinct from the orthosteric binding site, induces a reversible change of the A AR
1
conformation, that increases the action of endogenous adenosine or selective full agonists [7].
1
Allosteric enhancers (AEs) of A AR function might provide a more selective therapeutic effect than
full agonists [8]. Such agents might synergize with endogenous adenosine, with minimal effects in
the absence of adenosine. Their action would therefore be limited to times and locations at which
significant release of adenosine occurred [9].
1
PD 81,723 (1a, Chart 1) was one of the first allosteric enhancers acting at the A AR, enhancing the
functional effects of adenosine or its analogues, and having no effect on agonist binding at the other
adenosine receptor subtypes [10]. Among the synthesized compounds [11], derivative 1b {T-62, (2-
amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)(4-chlorophenyl)methanone}, was taken into
Phase II clinical trials by King Pharmaceuticals for the treatment of neuropathic pain associated
with hyperalgesia and allodynia [12].
A wide series of 2-amino-3-aroyl thiophene derivatives with general structure 2, characterized by
the presence of different phenyl-substituted piperazine moieties attached to the 4-position of the
1
thiophene ring by a methylene unit, have been identified as potent AEs at the A AR [13]. The
nature and the position of substituents on the phenyl ring tethered to the piperazine seemed to exert
3

ACCEPTED MANUSCRIPT
a fundamental influence on the AE activity. Based on this information, chemical modifications of
compounds with general formula 2 were started. In the present article, we have focused our
attention on the systematic modification of the 4-position of the 2-amino-3-(4-chlorobenzoyl)
thiophene ring, examining the effects on AE activity due to the replacement of the arylpiperazine
moiety with different chemical functionalities. In this new series of compounds, the 2-amino-3-(4-
chlorobenzoyl) thiophene nucleus was maintained, as this was shown to be an essential feature for
AE activity.
In the first series of compounds, the arylpiperazine moiety was replaced by 1,2,3,4-
tetrahydropyrazino[1,2-a]indole or 1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indole moieties to
furnish derivatives 3a-d and 3e-g, respectively. The pyrazino-indole system might be viewed as a
conformationally restricted aryl piperazine, in which the ortho position of the phenyl ring is
tethered to the carbon at the 2’-position of the piperazine moiety by a methanylylidene (-CH=) or
2
methanediyl (-CH -) linker. In this way, a five-membered ring was introduced to freeze the free
rotation of the C-N bond between the phenyl and the anilinic nitrogen of the piperazine moiety.
Thus, derivatives 3a-g have been synthesized in order to restrict the conformational freedom of the
phenylpiperazine system, thereby constraining the corresponding “open” counterparts with general
structure 2 into a near planar conformation. It is noteworthy that the 8-position of the pyrazino[1,2-
a]indole nucleus corresponds to the 4’-position of the phenyl ring linked to the piperazine moiety in
compounds with general structure 2. A further simplification of the phenylpiperazine moiety was
represented by the 1,2,3,4-tetrahydroisoquinoline system (TIQ), exemplified by compound 3h.
Derivatives 3i-l are characterized by the presence of a 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole
(or tetrahydro-γ-carboline) moiety, as regioisomeric analogues of the 1,2,3,4-
tetrahydropyrazino[1,2-a]indole system that characterized analogues 3a-d.
While compounds 3a-g and 3i-l were characterized by the presence of an indole nucleus fused with
a piperidine ring, joining the 4’-position of the piperidine nucleus to the 2-position of a
benzodioxole ring, furnished the spiropiperidine derivatives 3m-q. As a further molecular
4

ACCEPTED MANUSCRIPT
simplification of derivatives with general structure 2, the phenylpiperazine moiety was replaced
with structurally simpler and less bulky substituents, such as N-alkylanilines (3r-v), N-methyl
benzylamine (3w) or aliphatic tertiary amines (3x-z) (Chart 2). The influence of substituents at the
5
-position of the thiophene ring (phenyl or bromine) on AE activity, combined with a tertiary amine
as the 4-substituent, was evaluated by the synthesis of compounds 3aa-aj and 3ak-al, respectively.
Finally, the nitrogen of aniline derivatives 3r-v was replaced with an oxygen or a sulphur, to yield
aryl ethers (3am-ap) and aryl thioether (3aq), respectively.
2
. Chemistry
The 4-bromomethyl-5-bromothiophene analogue 4 [13a] was employed as a common intermediate
for the synthesis of compounds 3a-z by a three-step procedure (Scheme 1). Thiophene derivative 4
was
hexahydropyrazino[1,2-a]indoles 5e-g [15], tetrahydroisoquinoline 5h, tetrahydro-γ-carbolines 5i-l
16], spiropiperidines 5m-q [17] and secondary amines 5w-z in dichloromethane, to afford the
coupled
with
the
appropriate
tetrahydropyrazino[1,2-a]indoles
5a-d
[14],
[
derivatives 6a-q and 6w-z, respectively, in acceptable yields. Compounds 6r-v were prepared by
condensation of 4 with anilines 5r-v in acetonitrile in the presence of potassium carbonate at 60 °C.
Starting from the 5-bromothiophene derivatives 6a-z, dehalogenation by catalytic hydrogenation
using 10% Pd/C furnished the thiophene derivatives 7a-z. The subsequent treatment with ethanolic
hydrazine furnished the final compounds 3a-z.
The 4-bromomethyl-5-phenyl analogue 8 [18] was transformed into derivatives 6aa-aj by treatment
with the appropriate amine in dichloromethane in the presence of TEA. The isolated compounds
6
aa-aj were converted to the final compounds 3aa-aj by treatment with hydrazine in ethanol
(Scheme 2).
Compound 3ak was prepared by the condensation of 4 with diallyl amine, to furnish the 5–bromo
derivative 6ak, followed by removal of the N-protected phthaloyl group by the use of hydrazine
(Scheme 3). Compound 3al was prepared by treatment of 6z with hydrazine in ethanol at reflux.
5

ACCEPTED MANUSCRIPT
Finally, reaction of 4 with the appropriate phenol or thiophenol in acetonitrile in the presence of
potassium carbonate at 60 °C afforded the intermediates 6am-aq (Scheme 3). The subsequent
hydrogenation in the presence of 10% Pd/C furnished the 5-unsubstituted thiophene derivatives
7
am-aq, which were converted to the final compounds 3am-aq by treatment with hydrazine in
ethanol.
3
. Results and Discussion
3
.1. Functional assays
To assess the biological activity of the novel series of synthesized compounds 3a-aq, we initially
screened all molecules using a functional assay, evaluating their ability to inhibit forskolin-
1
stimulated cAMP production via the hA -AR in intact Chinese hamster ovary (CHO) cells. When
this receptor is in an active conformation in CHO , it cause a measurable inhibition of adenylyl
cyclase activity. Allosteric enhancers are thought to stabilize the active conformation of the A
1
-
AR, leading to a reduction in the cAMP content of the cells.
The reference compound PD 81,723 and the new derivatives 3a-aq were evaluated at a
concentration of 10 µM alone (Figure 1A) or at a concentration of 100 nM in the presence of the
orthosteric agonist CCPA (1 pM) to assess enhancement of the A -AR agonist activity (Figure 1B).
1
A reduction in cAMP content is indicated in Table 1 as a percentage inhibition of cAMP
production relative to control (absence of the test compound), in the absence or presence of the
orthosteric agonist. The degree of inhibition of cAMP production was similar under the two
conditions tested.
The tetrahydropyrazino[1,2-a]indoles 3a-e, tetrahydroisoquinoline 3h, spiro-1,3-benzodioxole-
pipridines 3m-q and aliphatic amines 3w and 3ad represent the most active compounds to emerge
from this investigation, inhibiting the percentage of cAMP production from 36% to 72% (Table 1).
In the series of 5-phenyl derivatives 3aa-aj, greater allosteric enhancement was seen with longer
aliphatic chains among the symmetrically di-substituted aliphatic amines attached to a methylene
6

ACCEPTED MANUSCRIPT
at the 4-position of the thiophene ring. Thus the N,N-di-n-propylamine (3ac) and N,N-diallylamine
(3ad) are more active than the N,N-diethylamine (3ab) and significantly more active than the N,N-
dimethylamine (3aa). The piperidine derivative (3ah) had activity similar to that of the N,N-diethyl
analogue (3ab). Replacing the N,N-di-n-propylamine (3ac) with the branched N,N-di-
isopropylamine (3ae) led to a significant reduction in activity, while the sterically more demanding
N,N-dicyclohexylamine (3ag) was more active. Interestingly, disubstituted amines attached to a
with two different substituents on nitrogen (3af, 3ai, 3aj), one being a methyl group, were
essentially equipotent to the N,N-dimethyl analogue (3aa).
As shown in Table 1, compound 3a, characterized by the presence of a tetrahydropyrazino[1,2-
a]indole unit linked to the 4-position of the thiophene ring by a methylene unit, had a significantly
greater effect than PD 81,723. Introduction of an electron-withdrawing fluorine or chlorine at the
8
’-position of the tetrahydropyrazino[1,2-a]indole nucleus (compounds 3b and 3c, respectively)
maintained the activity, which was reduced by the insertion of an electron-releasing methoxy group
derivative 3d). Replacing the tetrahydropyrazino[1,2-a]indole nucleus with
tetrahydroisoquinoline afforded derivative 3h, with activity similar to that of compound 3d.
Reduction of the C -C double bond of the tetrahydropyrazino[1,2-a]indole nucleus had a
(
a
10
10a
contrasting effect on the activity (compounds 3e-g). While there was no difference in activity
between the unsubstituted derivatives 3a and 3e, the 8-chloro-substituted indoline derivative 3f was
considerably less active than the corresponding indole analogue 3c. The data reported in Table 1
show that incorporation of a chlorine or methyl substituent at the indoline C-8 position (compounds
3
f and 3g, respectively) led to a dramatic drop in activity relative to the unsubstituted analogue 3e.
It would be interesting to evaluate the individual optical isomers of 3e to determine the effect of
stereochemistry on activity.
In the series of tetrahydro-γ-carboline derivatives 3i-l, the 5’-unsubstituted derivatives 3i-j showed
the least activity, while the corresponding 5’-methyl analogues 3k and 3l were 9- and 3-fold more
7

ACCEPTED MANUSCRIPT
active, respectively, at a concentration of 10 µM. These latter compounds were also 1.5-fold more
active than PD 81,723 when tested alone or in presence of CCPA.
Several chemically and sterically different substituents, such as fluorine (3n), methyl (3o-p) and
tert-butyl (3q) in either the 4’- or 5’-position of the benzene portion of the spiro[1,3-benzodioxole-
2
,4'-piperidine] system were investigated. Although these modifications alter the electronic, steric
and lipophilic features of this residue, there was no apparent difference in activity between
compounds 3n-q. The 4’- and 5’-methyl analogues (3o and 3p, respectively) showed essentially
identical activity, suggesting a lack of regiochemical preference. In addition, compound 3p was
equiactive to the more bulky and lipophilic tert-butyl derivative 3q at the concentration of 10 µM.
Replacement of the electron-releasing methyl in 3o with an electron-withdrawing fluorine (3n),
may have caused a slight increase in activity.
In the series of 5-unsubstituted thiophene derivatives 3r-z, the aniline derivatives 3r-t were found
to be amenable to modification. The N-methyl-N-phenyl (3r) and N-ethyl-N-phenyl (3s)
homologues were equiactive. Introduction of electron withdrawing substituents at the 4-position of
the anilino-phenyl led to an increase in activity in the order 3v (CF ) = 3t (F) > 3u (Cl).
3
Homologation of the N-methyl aniline derivative 3r furnished the N-methyl-N-benzylamine
analogue 3w, with 2-fold improved activity at the two concentrations tested. In contrast, the
presence of short-chain dialkylamines, such as N,N-diethylamine (3x), N,N-di-n-propylamine (3y),
as well as branched-chain dialkylamines, such as N,N-di-iso-propylamine (3z) was not desirable,
conferring low activity. However, when the N,N-diethylamine and N,N-di-n-propylamine moieties
were combined with a phenyl moiety at the 5-position of the thiophene ring (compounds 3ab and
3
ac, respectively), an increase of activity was observed that was superior to that of PD 81,723. This
was not true for the N-methyl-N-benzylamine derivative 3w, in which the corresponding 5-
phenylthiophene analogue, 3aj, showed a 10-fold reduced activity compared to 3w when tested
alone at 10 µM.
8

ACCEPTED MANUSCRIPT
Replacing the N-methyl-N-phenyl moiety of 3r with an arylether or arylthioether afforded
derivatives 3am-aq. The unsubstituted phenyl thioether (3aq) maintained activity equivalent to 3r,
while the phenyl ether (3am) was two fold less active than 3r. The substituted aryl ether
3
derivatives 3an (p-CH ) and 3ap (p-F) appeared almost 2-fold less active than the corresponding
N-methyl aniline analogues 3u and 3t, respectively.
As noted above, introducing a 5-phenyl moiety on 4-(N,N-dialkylamino)methyl thiophenes led to
increased allosteric enhancer activity. Interestingly , the N,N-dicyclohexylamine and piperidine
analogues 3ag and 3ah, respectively, displayed activity similar to 3ac. Replacement of the N,N-di-
n-propylamine moiety of 3ac with the unsaturated N,N-diallylamine (3ad) retained the same level
of activity. Replacing the 5-phenyl moiety of 3ad with a bromine (3ak) resulted in a significant
loss of activity. Compared to the N,N-di-iso-propylamine derivative 3z, the corresponding 5-phenyl
and 5-bromo analogues 3ae and 3al, respectively, showed two- to three-fold increased activity
relative to 3z. This suggests that activity is not only dependent on the presence of phenyl at the 5-
position of thiophene ring, but also the nature of the substituent at the 4-position. This was
confirmed by the reduced activity of N,N-dimethylamino, N-tert-butyl-N-methylamino and N-n-
butyl-N-methylamino derivatives 3aa, 3af and 3ai, respectively.
3
.2. Antagonistic activity
Many of the currently available allosteric enhancers of agonist binding to the hA
non-specific actions. These non-specific actions include antagonism at the A adenosine receptor,
especially at higher concentrations. The ability of compounds 3a-aq to displace the binding of
1
AR have several
1
3
3
3
[
H]DPCPX, [ H]ZM241385 and [ H]MRE-3008-F20 at human A , A and A ARs were
1 2A 3
evaluated in CHO cells at a concentration of 10 µM (Table 2). The prototype enhancer PD 81,723
did not inhibit the binding of the radiolabeled antagonists to A and A2A ARs, but at 10 µM, it
1
3
reduced by 21% the binding of [ H]MRE-3008-F20 to A
3
ARs [19]. None of the examined
derivatives significantly inhibited the specific binding of the radioligands to A , A and A ARs,
1
2A
3
9

ACCEPTED MANUSCRIPT
causing inhibition of radioligand binding of 12% or less. For the most active compounds in a
functional assay, such as 3a-c, 3e, 3n and 3q, it was possible to achieve a good separation between
high efficacy in the inhibition of cAMP production and binding to the orthosteric site.
3
1
.3. Effect of enhancers on A AR binding parameters
3
Saturation and competition experiments of the selective adenosine A
receptors were performed to determine if the novel compounds modified the agonist binding
parameters. From these experiments, A receptor affinity (K ) and density (Bmax) were evaluated
1 1
agonist [ H]CCPA to A
1
D
in the presence and in the absence of the examined compounds (PD 81,723 and 3a-aq at a
concentration of 10 µM) and were used to calculate the increase of A density (Bmax shift) (Table
1
3
). The reference compound PD 81,723 induced a Bound and BMAX shift to human A
receptors of 1.3- and 1.2-fold, respectively. Under the same experimental conditions, compounds
a-e, 3h, 3k-w, 3ab-ad and 3ag-ah were significantly more potent than PD 81,723. From the
1
adenosine
3
receptor density calculated in the presence and in the absence of the novel enhancers, the
derivatives 3a-c, 3e, 3n, and 3q were the most active compounds, each causing a BMAX shift of
more than 5-fold. Figure 2 shows the effect of the allosteric modulators PD 81,723, 3a, 3b and 3n
3
at 10 µM concentration in [ H]CCPA saturation binding experiments on A
1
AR binding parameters
such as affinity and density.
Interestingly, no differences were found in K
D
shift of the tested compounds, suggesting that the
enhancers were not able to modify the K
D
values of the high affinity binding sites labeled by
3
[
D
H]CCPA (K shift ranged from 1.0 ± 0.1 to 1.2± 0.1).
Table 3 also reports the derived apparent affinity (Ki) values for CCPA, based on a one-state model
of analysis, in the absence and in the presence of tested enhancers. This table also shows the CCPA
shift representing the ratio of apparent Ki values in the absence and in the presence of the tested
3
compounds at 10 µM concentration. In the hA
1
CHO membranes, by using [ H]DPCPX as
radioligand, the Ki value of CCPA was 15.2 ± 1.3 nM. Interestingly, a significant decrease in the
1
0

ACCEPTED MANUSCRIPT
apparent Ki value was due to the presence of the putative allosteric enhancers, suggesting an
increase in the high-affinity binding sites. In the presence of PD 81,723, the affinity of CCPA
increased by 1.5-fold. The CCPA affinity data in the presence of the derivatives 3a-e, 3h, 3k-w,
3
ab-ad and 3ag-ah reveal that the displacement curves are shifted left, suggesting lower Ki values
for CCPA. In particular, the largest affinity shift has been observed for compounds 3a, 3b, 3c, 3e
and 3n. These molecules enhanced the apparent affinity of CCPA approximately 6.6-, 7.2-, 6.9-,
7
.6- and 6.3-fold, respectively (Table 3). Thus, the enhancers were able to mediate a shift of the A
1
receptors towards the high affinity state as suggested from the increase of the CCPA affinity
expressed as Ki values (Table 3).
The results obtained from competition and saturation experiments confirmed the potency of most
of the synthesized compounds, in agreement with the cAMP functional assay.
4
. Conclusions
Encouraged by the results obtained for the arylpiperazine derivatives with general structure 2, we
continued to follow our strategy of incorporating other substituents attached to the methylene at the
thiophene 4-position. Compounds 3a-d, characterized by the presence of a tetrahydropyrazino[1,2-
a]indole nucleus as conformationally constrained arylpiperazine analogues, appeared to be
significantly more active than PD 81,723 in the functional assay. Biological data confirmed that the
replacement of the arylpiperazine moiety by a tetrahydropyrazino[1,2-a]indole nucleus in the 4-
position of thiophene ring is well tolerated. In terms of activity, reduction of the C10-C10a double
bond of the tetrahydropyrazino[1,2-a]indole nucleus was tolerated only for the 8-unsubstituted
hexahydropyrazino[1,2-a]indole derivative 3e, which showed the same activity as the
corresponding analogue 3a.
A characteristic feature of AE at the A
concentrations. None of the synthesized compounds (3a-aq) significantly inhibited antagonist
binding at the hA AR, hA AR, or hA AR. Among these, derivatives 3a, 3b, 3c, 3e and 3n were the
1
AR is the propensity to also cause antagonism at higher
1
2
3
1
1

ACCEPTED MANUSCRIPT
most active compounds in binding (saturation and displacement) experiments and functional cAMP
assays.
5
. Experimental Section
5
.1. Chemistry.
5
.1.1. Materials and Methods.
1
13
H NMR and C NMR spectra were determined in CDCl or d -DMSO solutions and recorded with
3
6
a Varian VXR-200 spectrometer or a Varian Mercury Plus 400 spectrometer. Chemical shifts (δ)
are given in parts per million (ppm) downfield and J values are given in hertz. All products reported
1
13
showed H NMR and C NMR spectra in agreement with the assigned structures. Positive-ion
electrospray ionization (ESI) mass spectra were recorded on a double-focusing ESI Micromass
ZMD 2000 mass spectrometer. Melting points (mp) were determined on a Buchi-Tottoli apparatus
and are uncorrected. Elemental analyses were conducted by the Microanalytical Laboratory of the
Chemistry Department of the University of Ferrara and were performed on a Yanagimoto MT-5
CHN recorder analyzer. All tested compounds yielded data consistent with a purity of at least 95%
as compared with the theoretical values. All reactions were performed under an inert atmosphere of
dry nitrogen, unless otherwise described. Standard syringe techniques were applied for transferring
dry solvents. Reaction courses and product mixtures were routinely monitored by TLC on silica gel
(
precoated F
Merck plates) and visualized with aqueous KMnO . Flash chromatography was
54 4
2
performed using 230-400 mesh silica gel and the solvent system indicated in the procedure. All
commercially available compounds were used without further purification. Organic solutions were
dried over anhydrous Na SO . Dichloromethane (DCM) was distilled from calcium chloride and
2
4
stored over molecular sieves (3 Å). Petroleum ether refers to the fraction boiling at 40-60 °C.
5
.1.2. General procedure (A) for the synthesis of compounds 6a-q and 6w-ak.
1
2

ACCEPTED MANUSCRIPT
To a stirred solution of compound 4 or 8 (2 mmol) in dry dichloromethane (10 mL) was added
2 3
K CO (1.1 equiv., 2.2 mmol, 304 mg). The mixture was cooled with a bath of ice/water, and then
the appropriate amine (1.2 equiv., 2.4 mmol), dissolved in dichloromethane (3 mL), was added
slowly over 5 min. The mixture was then stirred at room temperature for two hours, diluted with
dichloromethane (10 mL), washed with water (5 mL) and then with brine (5 mL). The organic layer
was dried (Na SO ), filtered and concentrated in vacuo to give a residue that was purified by
2
4
column chromatography to furnish the derivatives 6a-q and 6w-ak.
5
.1.2.1.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6a). Following general procedure (A), derivative 6a
was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 69%. Yellow solid,
1
mp 148-150 °C. H NMR (CDCl
3
) δ: 3.00 (s, 2H), 3.39 (t, J=5.8 Hz, 2H), 4.10 (t, J=5.8 Hz, 2H),
+
4
.24 (s, 2H), 6.05 (s, 1H), 7.19 (m, 4H), 7.42 (m, 4H), 7.54 (m, 4H). MS (ESI): [M] =629.1,
+
[
M+2] =631.1.
5
2
6
.1.2.2. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-fluoro-1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-
-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6b). Following general procedure (A), derivative
b was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 78%. Yellow
1
solid, mp 163-165 °C. H NMR (CDCl
3
) δ: 2.52 (t, J=5.4 Hz, 2H), 3.12 (s, 2H), 3.22 (s, 2H), 3.78
+
(
t, J=5.6 Hz, 2H), 6.00 (s, 1H), 7.13 (m, 3H), 7.39 (m, 4H), 7.56 (m, 4H). MS (ESI): [M] =647.0,
+
[
M+2] =649.1.
5
2
.1.2.3. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-chloro-1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-
-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6c). Following general procedure (A), derivative 6c
was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 82%. Yellow solid,
1
mp 220-222 °C. H NMR (CDCl ) δ: 2.46 (t, J=5.4 Hz, 2H), 3.09 (s, 2H), 3.34 (s, 2H), 3.78 (t,
3
13
J=5.4 Hz, 2H), 6.00 (s, 1H), 7.11 (m, 4H), 7.33 (d, J=8.6 Hz, 2H),), 7.50 (s, 1H), 7.56 (m, 4H). C
NMR (100 MHz, CDCl ) δ: 41.0, 48.8, 50.4, 53.6, 96.6, 109.6, 119.5, 120.9, 124.3 (2C), 125.3,
3
1
3

ACCEPTED MANUSCRIPT
1
1
25.6, 128.5 (2C), 128.7, 129.2, 130.1 (2C), 131.0, 134.3, 135.0 (2C), 135.3, 135.8 (2C), 136.1,
+
+
38.9, 139.2, 165.5 (2C), 189.2. MS (ESI): [M] =663.1, [M+2] =665.3.
5
.1.2.4.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-methoxy-1,2,3,4-tetrahydro-1H-pyrazino[1,2-
a]indol-2-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6d). Following general procedure (A),
derivative 6d was purified by column chromatography (EtOAc:dichloromethane 0.5:9.5). Yield:
1
8
2%. Yellow solid, mp 133-135 °C. H NMR (CDCl
3
) δ: 2.47 (t, J=5.6 Hz, 2H), 3.12 (s, 2H), 3.36
(s, 2H), 3.83 (t, J=5.6 Hz, 2H), 3.87 (s, 3H), 6.02 (s, 1H), 7.13 (m, 4H), 7.35 (d, J=8.6 Hz, 2H),),
+
+
7
.53 (s, 1H), 7.54 (m, 4H). MS (ESI): [M] =659.1, [M+2] =661.1.
5
.1.2.5.
(R,S)-2-[5-Bromo-3-(4-chlorobenzoyl)-4-(1,2.3,4,10,10a-hexahydro-1H-pyrazino[1,2-
a]indol-2-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6e). Following general procedure (A),
derivative 6e was purified by column chromatography (EtOAc:dichloromethane 1:9). Yield: 68%.
1
Yellow solid, mp 138-140 °C. H NMR (CDCl
3
) δ: 2.56 (dd, J=15.6 and 9.6 Hz, 2H), 2.88 (m, 2H),
2
1
.97 (m, 4H), 3.09 (d, J=11.6 Hz, 1H), 3.53 (m, 2H), 6.43 (d, J=7.6 Hz, 1H), 6.66 (d, J=8.0 Hz,
H), 7.09 (m, 2H), 7.36 (d, J=8.8 Hz, 2H), 7.48 (d, J=8.8 Hz, 2H), 7.65 (m, 4H). MS (ESI):
+
+
[
M] =631.1, [M+2] =633.1.
5
.1.2.6.
(R,S)-2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-chloro-1,2.3,4,10,10a-hexahydro
-1H-
pyrazino[1,2-a]indol-2-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6f). Following general
procedure (A), derivative 6f was purified by column chromatography (EtOAc:dichloromethane
1
0
.5:9.5). Yield: 74%. Yellow solid, mp 153-155 °C. H NMR (CDCl
3
) δ: 2.56 (dd, J=15.6 and 9.6
Hz, 2H), 2.84 (m, 2H), 2.95 (m, 4H), 3.04 (d, J=11.6 Hz, 1H), 3.48 (m, 2H), 6.33 (d, J=8.0 Hz, 1H),
.00 (d, J=8.0 Hz, 1H), 7.02 (s, 1H), 7.44 (d, J=8.8 Hz, 2H), 7.58 (d, J=8.8 Hz, 2H), 7.62 (m, 4H).
7
+
+
MS (ESI): [M] =665.0, [M+2] =667.1.
5
.1.2.7.
(R,S)-2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-methyl-1,2.3,4,10,10a-hexahydro-1H-
pyrazino[1,2-a]indol-2-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6g). Following general
1
4

ACCEPTED MANUSCRIPT
procedure (A), derivative 6g was purified by column chromatography (EtOAc:dichloromethane
1
0
.5:9.5). Yield: 77%. Yellow solid, mp 141-143 °C. H NMR (CDCl
3
) δ: 2.44 (s, 3H), 2.52 (dd,
J=15.6 and 9.6 Hz, 2H), 2.84 (m, 2H), 2.98 (m, 4H), 3.06 (d, J=11.6 Hz, 1H), 3.52 (m, 2H), 6.10 (s,
1
4
H), 6.96 (d, J=8.0 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 7.34 (m, 2H), 7.48 (d, J=8.4 Hz, 2H), 7.62 (m,
+
+
H). MS (ESI): [M] =645.0, [M+2] =647.1.
5
.1.2.8.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(1,2,3,4-tetrahydro-1H-isoquinolin-2-ylmethyl)-
thiophen-2-yl]-isoindole-1,3-dione (6h). Following general procedure (A), derivative 6h was
purified by column chromatography (EtOAc:petroleum ether 3:7). Yield: 76%. Yellow solid, mp
1
1
01-103 °C. H NMR (CDCl
3
) δ: 2.23 (t, J=5.4 Hz, 2H), 2.61 (t, J=5.6 Hz, 2H), 2.89 (s, 2H), 3.28
(s, 2H), 6.87 (d, J=7.8 Hz, 1H), 7.19 (m, 3H), 7.33 (m, 6H), 7.50 (t, J=8.8 Hz, 1H), 7.73 (t, J=8.8
1
3
Hz, 1H). C NMR (100 MHz, CDCl
3
) δ: 28.2, 49.3, 54.3, 54.9, 124.3 (2C), 125.3, 125.5, 125.8,
1
1
26.1, 126.5, 127.9, 128.4 (2C), 128.6 (2C), 128.9, 130.1 (2C), 131.1, 133.8, 134.1, 134.9, 136.1,
+
+
38.4, 138.7, 165.7 (2C), 189.0. MS (ESI): [M] =590.0, [M+2] =592.1.
5
.1.2.9.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-ylmethyl)-
thiophen-2-yl]-isoindole-1,3-dione (6i). Following general procedure (A), derivative 6i was purified
by column chromatography (EtOAc:petroleum ether 2:8). Yield: 72%. Yellow solid, mp 152-154
1
°
C. H NMR (CDCl ) δ: 2.76 (t, J=5.8 Hz, 2H), 3.22 (t, J=5.8 Hz, 2H), 3.44 (s, 2H), 4.06 (s, 2H),
3
7
7
.12 (m, 4H), 7.24 (d, J=8.6 Hz, 1H); 7.36 (d, J=8.8 Hz, 2H), 7.42 (d, J=7.6 Hz, 1H), 7.65 (m, 4H),
+
+
.82 (bs, 1H). MS (ESI): [M] =629.0, [M+2] =631.2.
5
.1.2.10.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-chloro-2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6j). Following general procedure (A), derivative 6j
was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 55%. Yellow solid,
1
mp 150-152 °C. H NMR (CDCl
3
) δ: 2.76 (t, J=5.8 Hz, 2H), 3.22 (t, J=5.6 Hz, 2H), 3.72 (s, 2H),
4
7
.01 (s, 2H), 7.03 (d, J=8.6 Hz, 1H), 7.17 (d, J=8.6 Hz, 2H), 7.25 (d, J=8.6 Hz, 1H), 7.37 (s, 1H);
+
+
.46 (d, J=8.6 Hz, 2H), 7.62 (m, 4H), 7.82 (bs, 1H). MS (ESI): [M] =663.0, [M+2] =665.1.
1
5

ACCEPTED MANUSCRIPT
5
.1.2.11.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(5-methyl-2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6k). Following general procedure (A), derivative 6k
was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 73%. Yellow solid,
1
mp 134-136 °C. H NMR (CDCl ) δ: 2.76 (t, J=5.6 Hz, 2H), 3.27 (t, J=5.6 Hz, 2H), 3.62 (s, 3H),
3
4
.22 (m, 4H), 7.10 (m, 4H), 7.26 (d, J=8.6 Hz, 2H); 7.44 (d, J=8.6 Hz, 2H), 7.65 (m, 4H). MS
+
+
(
ESI): [M] =643.1, [M+2] =645.3.
5
.1.2.12.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(8-chloro-5-methyl-2,3,4,5-tetrahydro-pyrido[4,3-
b]indol-2-ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (6l). Following general procedure (A),
derivative 6l was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 81%.
1
Yellow solid, mp 155-157 °C. H NMR (CDCl
3
) δ: 2.74 (t, J=5.6 Hz, 2H), 3.25 (t, J=5.6 Hz, 2H),
3
4
.60 (s, 3H), 3.62 (s, 2H), 4.01 (s, 2H), 7.16 (m, 4H), 7.37 (s, 1H); 7.46 (d, J=8.6 Hz, 2H), 7.64 (m,
+
+
H). MS (ESI): [M] =677.1, [M+2] =679.2.
5
.1.2.13. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(spiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-ylmethyl-
thiophen-2-yl]-isoindole-1,3-dione (6m). Following general procedure (A), derivative 6m was
purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 58%. White solid, mp
1
2
29-231 °C. H NMR (CDCl
3
) δ: 1.98 (t, J=6.0 Hz, 4H), 3.06 (t, J=6.0 Hz, 4H), 3.49 (s, 2H), 6.78
1
3
(
m, 4H), 7.36 (d, J=8.6 Hz, 2H), 7.43 (d, J=8.6 Hz, 2H), 7.63 (m, 4H). C NMR (100 MHz,
CDCl ) δ: 34.3 (2C), 49.5 (2C), 54.3, 108.6 (2C), 110.3, 115.9, 121.2 (2C), 124.3 (2C), 128.7 (2C),
30.1 (2C), 131.2 (2C), 135.0 (2C), 135.9 (2C), 136.3, 138.0, 138.9, 147.0 (2C), 165.6 (2C), 188.9.
3
1
+
+
MS (ESI): [M] =648.1, [M+2] =650.1.
5
.1.2.14. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(4-fluorospiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-
ylmethyl-thiophen-2-yl]-isoindole-1,3-dione (6n). Following general procedure (A), derivative 6n
1
was purified by column chromatography (EtOAc:petroleum ether 2:8). Yield: 56%. Yellow oil. H
3
NMR (CDCl ) δ: 2.19 (t, J=6.0 Hz, 4H), 3.07 (t, J=6.0 Hz, 4H), 3.44 (s, 2H), 6.56 (d, J=8.4 Hz,
1
6

ACCEPTED MANUSCRIPT
1
H), 6.62 (m, 1H), 6.76 (m, 1H), 7.37 (d, J=8.6 Hz, 2H), 7.52 (d, J=8.6 Hz, 2H), 7.84 (m, 4H). MS
+
+
(
ESI): [M] =666.0, [M+2] =668.1.
5
.1.2.15. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(4-methylspiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-
ylmethyl-thiophen-2-yl]-isoindole-1,3-dione (6o). Following general procedure (A), derivative 6o
was purified by column chromatography (EtOAc:petroleum ether 4:6). Yield: 74%. Yellow solid,
1
mp 194-196 °C. H NMR (CDCl
3
) δ: 1.97 (t, J=5.6 Hz, 4H), 2.20 (s, 3H), 3.02 (m, 4H), 3.49 (s,
2
4
H), 6.62 (m, 2H), 6.68 (t, J=8.0 Hz, 1H), 7.44 (d, J=8.6 Hz, 2H), 7.57 (d, J=8.6 Hz, 2H), 7.68 (m,
+
+
H). MS (ESI): [M] =662.0, [M+2] =664.1.
5
.1.2.16. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(5-methylspiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-
ylmethyl-thiophen-2-yl]-isoindole-1,3-dione (6p). Following general procedure (A), derivative 6p
was purified by column chromatography (EtOAc:petroleum ether 2:8). Yield: 68%. White solid, mp
1
1
42-144 °C. H NMR (CDCl
3
) δ: 1.96 (t, J=6.0 Hz, 4H), 2.26 (s, 3H), 3.02 (t, J=6.0 Hz, 4H), 3.24
(s, 2H), 6.60 (m, 2H), 6.64 (d, J=8.0 Hz, 1H), 7.44 (d, J=8.6 Hz, 2H), 7.56 (d, J=8.6 Hz, 2H), 7.65
+
+
(
m, 4H). MS (ESI): [M] =662.1, [M+2] =664.1.
5
.1.2.17. 2-[5-Bromo-4-(5-t-butylspiro-benzo[1,3-d]-dioxol-2-yl)-3-(4-chlorobenzoyl)-piperidin-1-
ylmethyl-thiophen-2-yl]-isoindole-1,3-dione (6q). Following general procedure (A), derivative 6q
was purified by column chromatography (EtOAc:petroleum ether 3:7). Yield: 77%. White solid, mp
1
1
05-107 °C. H NMR (CDCl ) δ: 1.28 (s, 9H), 2.08 (t, J=6.0 Hz, 4H), 3.24 (t, J=6.0 Hz, 4H), 3.48
3
(s, 2H), 6.66 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.4 Hz, 1H), 6.84 (s, 1H), 7.38 (d, J=8.6 Hz, 2H), 7.45
+
+
(
d, J=8.6 Hz, 2H), 7.66 (m, 4H). MS (ESI): [M] =704.1, [M+2] =706.2.
5
.1.2.18.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N-benzyl-N-methylaminomethyl)-thiophen-2-yl]-
isoindole-1,3-dione (6w). Following general procedure (A), the product 6w was purified by column
chromatography (EtOAc-petroleum ether 3-7 as eluent). Yield 78%. Yellow solid, mp 184-186 °C.
1
3
H NMR (CDCl ) δ: 2.48 (s, 3H), 3.44 (s, 2H), 4.13 (s, 2H), 6.28 (d, J=8.4 Hz, 2H), 6.54 (t, J=7.2
1
7

ACCEPTED MANUSCRIPT
13
Hz, 1H), 7.02 (m, 4H), 7.40 (d, J=8.4 Hz, 2H), 7.72 (m, 4H). C NMR (100 MHz, CDCl ) δ: 40.8,
3
5
1
4.7, 61.0, 110.8, 124.3 (2C), 126.9, 128.1 (2C), 128.7 (2C), 128.8 (2C), 130.4 (2C), 131.2 (2C),
+
34.9 (2C), 135.2, 135.8, 136.0, 138.1, 138.6, 139.0, 165.6 (2C), 189.0. MS (ESI): [M] =578.1,
+
[
M+2] =580.2.
5
1
.1.2.19. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N,N-diethylaminomethyl)-thiophen-2-yl]-isoindole-
,3-dione (6x). Following general procedure (A), the product 6x was purified by column
1
chromatography (EtOAc-petroleum ether 1.5:8.5 as eluent). Yield 54%. Brown oil. H NMR
(
CDCl ) δ: 1.25 (t, J=7.4 Hz, 6H), 2.17 (q, J=7.4 Hz, 4H), 3.43 (s, 2H), 7.32 (d, J=8.6 Hz, 2H), 7.66
3
13
(
d, J=8.6 Hz, 2H), 7.75 (m, 4H). C NMR (100 MHz, CDCl
3
) δ: 9.88 (2C), 44. 6(2C), 50.8, 124.1
(2C), 128.4 (2C), 130.1 (2C), 130.5, 131.1 (2C), 134.8 (2C), 135.1, 135.9, 137.1, 138.6, 139.7,
+
+
1
65.6 (2C), 188.4. MS (ESI): [M] =530.2, [M+2] =532.2.
5
1
.1.2.20. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N,N-dipropylaminomethyl)-thiophen-2-yl]-isoindole-
,3-dione (6y). Following general procedure (A), the product 6y was purified by column
chromatography (EtOAc-petroleum ether 3:7 as eluent). Yield 88%. Yellow solid, mp 143-145 °C.
1
3
H NMR (CDCl ) δ: 0.63 (t, J=7.6 Hz, 6H), 1.19 (m, 4H), 1.83 (t, J=7.4 Hz, 4H), 3.27 (s, 2H), 7.23
+
+
(
d, J=8.6 Hz, 2H), 7.38 (d, J=8.6 Hz, 2H), 7.52 (m, 4H). MS (ESI): [M] =558.1, [M+2] =560.2.
5
.1.2.21.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N,N-diisopropylaminomethyl)-thiophen-2-yl]-
isoindole-1,3-dione (6z). Following general procedure (A), the product 6z was purified by column
chromatography (EtOAc-petroleum ether 1.5:8.5 as eluent). Yield 53%. Yellow solid, mp 144-146
1
°
C. H NMR (CDCl
3
) δ: 0.74 (d, J=6.8 Hz, 12H), 2.66 (m, 2H), 3.64 (s, 2H), 7.26 (d, J=8.6 Hz,
+
+
2
H), 7.61 (d, J=8.6 Hz, 2H), 7.68 (m, 4H). MS (ESI): [M] =558.2, [M+2] =560.3.
5
1
.1.2.22. 2-[3-(4-Chlorobenzoyl)-4-(N,N-dimethylaminomethyl)-5-phenyl-thiophen-2-yl]-isoindole-
,3-dione (6aa). Following general procedure (A), the product 6aa was purified by column
1
chromatography (EtOAc as eluent). Yield 61%. Yellow solid, mp 106-108 °C. H NMR (CDCl
3
) δ:
1
8

ACCEPTED MANUSCRIPT
1
.61 (s, 6H), 3.09 (s, 2H), 7.40 (m, 7H), 7.45 (m, 4H), 7.66 (d, J=8.4 Hz, 2H). MS (ESI):
+
+
[
M] =500.0, [M+2] =502.0.
5
1
.1.2.23. 2-[3-(4-Chlorobenzoyl)-4-(N,N-diethylaminomethyl)-5-phenyl-thiophen-2-yl]-isoindole-
,3-dione (6ab). Following general procedure (A), the product 6ab was purified by column
chromatography (EtOAc:petroleum ether 3:7 as eluent). Yield 62%. Yellow solid, mp 176-178 °C.
1
H NMR (CDCl
H), 7.43 (m, 5H), 7.74 (m, 6H). C NMR (100 MHz, CDCl
2C), 128.4 (2C), 128.7 (2C), 129.6, 130.0 (2C), 130.6, 131.4 (2C), 132.8, 134.7 (2C), 134.9, 136.4,
3
) δ: 0.50 (t, J=7.2 Hz, 6H), 2.11 (q, J=7.2 Hz, 4H), 3.46 (s, 2H), 7.31 (d, J=8.8 Hz,
13
2
3
) δ: 9.68 (2C), 44.4 (2C), 49.9, 124.1
(
+
+
1
36.7, 138.3 (2C), 139.4, 139.6, 166.1 (2C), 189.1. MS (ESI): [M] =528.1, [M+2] =530.2.
5
1
.1.2.24. 2-[3-(4-Chlorobenzoyl)-4-(N,N-dipropylaminomethyl)-5-phenyl-thiophen-2-yl]-isoindole-
,3-dione (6ac). Following general procedure (A), the product 6ac was purified by column
chromatography (EtOAc:petroleum ether 3:7 as eluent). Yield 61%. Yellow solid, mp 175-177 °C.
1
H NMR (CDCl ) δ: 0.55 (t, J=7.2 Hz, 6H), 1.42 (m, 4H), 2.02 (d, J=7.2 Hz, 4H), 3.24 (s, 2H), 7.36
3
+
+
(
d, J=8.8 Hz, 2H), 7.41 (m, 7H), 7.55 (m, 4H). MS (ESI): [M] =556.1, [M+2] =558.2.
5
1
.1.2.25. 2-[3-(4-Chlorobenzoyl)-4-(N,N-diallylaminomethyl)-5-phenyl-thiophen-2-yl]-isoindole-
,3-dione (6ad). Following general procedure (A), the product 6ad was purified by column
chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 66%. Yellow solid, mp 183-185
1
°
C. H NMR (CDCl ) δ: 2.67 (d, J=6.4 Hz, 4H), 3.49 (s, 2H), 4.84 (m, 4H), 5.33 (m, 2H), 7.31 (d,
3
+
+
J=8.8 Hz, 2H), 7.49 (m, 3H), 7.74 (m, 8H). MS (ESI): [M] =552.1, [M+2] =554.2.
5
.1.2.26.
2-[3-(4-Chlorobenzoyl)-4-(N,N-diisopropylaminomethyl)-5-phenyl-thiophen-2-yl]-
isoindole-1,3-dione (6ae). Following general procedure (A), the product 6ae was purified by
column chromatography (EtOAc:petroleum ether 3:7 as eluent). Yield 53%. White solid, mp 168-
1
1
2
70 °C. H NMR (CDCl
3
) δ: 0.57 (d, J=6.6 Hz, 12H), 2.62 (m, 2H), 3.71 (s, 2H), 7.22 (d, J=8.6 Hz,
+
+
H), 7.47 (m, 2H), 7.74 (m, 9H). MS (ESI): [M] =556.1, [M+2] =558.2.
1
9

ACCEPTED MANUSCRIPT
5
.1.2.27.
2-[3-(4-Chlorobenzoyl)-4-(N-t-butyl-N-methylaminomethyl)-5-phenyl-thiophen-2-yl]-
isoindole-1,3-dione (6af). Following general procedure (A), the product 6af was purified by column
chromatography (EtOAc:petroleum ether 3:7 as eluent). Yield 64%. Brown solid, mp 221-223 °C.
1
H NMR (CDCl ) δ: 0.66 (s, 9H), 1.72 (s, 3H), 3.89 (s, 2H), 7.29 (d, J=8.6 Hz, 2H), 7.46 (m, 5H),
3
+
+
7
.72 (m, 2H), 7.82 (m, 4H). MS (ESI): [M] =542.1, [M+2] =544.2.
5
.1.2.28.
2-[3-(4-Chlorobenzoyl)-4-(N,N-dicyclohexylaminomethyl)-5-phenyl-thiophen-2-yl]-
isoindole-1,3-dione (6ag). Following general procedure (A), the product 6ag was purified by
column chromatography (EtOAc:petroleum ether 2:8 as eluent). Yield 52%. Yellow solid, mp 98-
1
1
00 °C. H NMR (CDCl
3
) δ: 0.92 (m, 12H), 1.51 (m, 8H), 2.17 (m, 2H), 3.81 (s, 2H), 7.22 (d, J=8.4
13
Hz, 2H), 7.47 (m, 5H), 7.74 (m, 6H). C NMR (100 MHz, CDCl ) δ: 24.8 (4C), 25.1 (2C), 28.9
3
(4C), 43.2 (2C), 61.2, 123.9 (2C), 128.4 (2C), 129.1, 129.5 (2C), 129.9 (2C), 130.1, 130.5 (2C),
1
30.9, 131.2, 131.5 (2C), 132.3 (2C), 135.1, 135.4, 135.7, 139.9, 165.3 (2C), 185.6. MS (ESI):
+
+
[
M] =636.2, [M+2] =638.2.
5
.1.2.29. 2-[3-(4-Chlorobenzoyl)-5-phenyl-4-((piperidin-1-yl)methyl)-thiophen-2-yl]-isoindole-1,3-
dione (6ah). Following general procedure (A), the product 6ah was purified by column
chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 47%. Yellow solid, mp 187-189
1
°
C. H NMR (CDCl ) δ: 1.25 (m, 6H), 1.65 (m, 4H), 2.98 (s, 2H), 7.22 (d, J=8.8 Hz, 2H), 7.53 (m,
3
+
+
3
H), 7.73 (m, 8H). MS (ESI): [M] =540.1, [M+2] =540.2.
5
.1.2.30.
2-[3-(4-Chlorobenzoyl)-4-(N-n-butyl-N-methylaminomethyl)-5-phenyl-thiophen-2-yl]-
isoindole-1,3-dione (6ai). Following general procedure (A), the product 6ai was purified by column
1
chromatography (EtOAc:petroleum ether 1:1 as eluent). Yield 63%. Yellow solid, mp 75-77 °C. H
NMR (CDCl ) δ: 0.76 (t, J=7.2 Hz, 3H), 0.86 (m, 4H), 1.58 (s, 3H), 1.69 (t, J=7.2 Hz, 2H), 3.16 (s,
3
+
+
2
H), 7.40 (m, 4H), 7.48 (m, 4H), 7.67 (m, 5H). MS (ESI): [M] =542.1, [M+2] =544.2.
2
0

ACCEPTED MANUSCRIPT
5
.1.2.31.
2-[3-(4-Chlorobenzoyl)-4-(N-benzyl-N-methylaminomethyl)-5-phenyl-thiophen-2-yl]-
isoindole-1,3-dione (6aj). Following general procedure (A), derivative 6aj was purified by column
1
chromatography (EtOAc:petroleum ether 4:6). Yield: 61%. Yellow solid, mp 106-107 °C. H NMR
(
CDCl ) δ: 1.56 (s, 3H), 3.27 (s, 2H), 4.75 (s, 2H), 7.18 (d, J=8.4 Hz, 2H), 7.36 (m, 8H), 7.45 (m,
3
13
8
3
H). C NMR (100 MHz, CDCl ) δ: 40.6, 53.6, 60.7, 124.1 (2C), 126.8 (2C), 127.9 (2C), 128.6
(2C), 128.8, 128.9 (2C), 129.7, 130.1 (2C), 130.4, 130.9, 131.3 (2C), 132.6, 134.7 (4C), 135.0,
+
+
1
35.5, 136.2, 137.9, 138.6, 166.1 (2C), 189.8. MS (ESI): [M] =576.1, [M+2] =578.2.
5
1
.1.2.32.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N,N-diallylaminomethyl)-thiophen-2-yl]-isoindole-
,3-dione (6ak). Following general procedure (A), derivative 6ak was purified by column
1
chromatography (EtOAc:petroleum ether 1.5:8.5). Yield: 92%. Yellow oil. H NMR (CDCl
3
) δ:
2
.69 (d, J=6.6 Hz, 4H), 3.92 (s, 2H), 4.89 (m, 4H), 5.29 (m, 2H), 7.26 (d, J=8.6 Hz, 2H), 7.64 (d,
13
J=8.6 Hz, 2H), 7.77 (m, 4H). C NMR (100 MHz, CDCl
3
) δ: 50.9, 55.6 (2C), 110.5, 117.9 (2C),
1
1
24.2 (2C), 128.6 (2C), 130.3 (2C), 131.2, 134.2 (2C), 134.9 (4C), 135.8, 136.8, 138.8, 138.9,
+
+
65.6 (2C), 188.6. MS (ESI): [M] =554.2, [M+2] =556.2.
5
.1.3. General procedure (B) for the synthesis of compounds (6r-v).
To a stirred solution of 4 (540 mg, 1 mmol) in dry acetonitrile (10 mL) was added the appropriate
aniline 5r-v (2 equiv., 2 mmol,) and K CO (207 mg, 3 equiv., 1.5 mmol). The mixture was then
2
3
heated at 60°C for 1 h. The solvent was removed under reduced pressure and the residue partitioned
between a mixture of dichloromethane (15 mL) and water (5 mL). The organic phase was washed
with brine (5 mL), dried (Na SO ), then concentrated in vacuo to give a residue that was purified by
2
4
column chromatography.
5
.1.3.1.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N-methyl-N-phenyl-amino-methyl)-thiophen-2-yl]-
isoindole-1,3-dione (6r). Following general procedure (B), the product 6r was purified by column
chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 95%. Yellow solid, mp 186-188
2
1

ACCEPTED MANUSCRIPT
1
°
C. H NMR (CDCl
3
) δ: 2.56 (s, 3H), 4.29 (s, 2H), 6.33 (d, J=8.6 Hz, 2H), 6.69 (t, J=7.2 Hz, 1H),
1
3
7
1
1
6
.02 (m, 4H), 7.42 (d, J=8.6 Hz, 2H), 7.78 (m, 4H). C NMR (100 MHz, d -DMSO) δ: 50.1, 54.8,
10.7, 113.6 (2C), 117.9, 123.7 (2C), 128.2 (2C), 128.4 (2C), 129.7, 130.6, 131.3, 134.4 (2C),
+
35.1 (2C), 136.7, 137.8 (2C), 142.3, 148.9, 185.4 (2C), 187.8. MS (ESI): [M] =564.0,
+
[
M+2] =566.1.
5
.1.3.2.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N’-ethyl-N-phenyl-aminomethyl)-thiophen-2-yl]-
isoindole-1,3-dione (6s). Following general procedure (B), the product 6s was purified by column
chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 86%. Yellow solid, mp 158-160
1
°
C. H NMR (CDCl
3
) δ: 0.89 (t, J=7.2 Hz, 3H), 2.96 (q, J=7.2 Hz, 2H), 4.24 (s, 2H), 6.29 (d, J=8.8
Hz, 2H), 6.92 (t, J=7.2 Hz, 1H), 7.02 (m, 4H), 7.35 (d, J=8.6 Hz, 2H), 7.73 (m, 4H). MS (ESI):
+
+
[
M] =578.2, [M+2] =580.3.
5
.1.3.3. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N-(4-fluorophenyl)-N-methylaminomethyl)-thiophen-2-
yl]-isoindole-1,3-dione (6t). Following general procedure (B), the product 6t was purified by
column chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 52%. Yellow solid, mp
1
1
7
88-190 °C. H NMR (CDCl
3
) δ: 2.50 (s, 3H), 4.22 (s, 2H), 6.35 (m, 2H), 6.76 (t, J=8.4 Hz, 2H),
+
.11 (d, J=8.6 Hz, 2H), 7.45 (d, J=8.6 Hz, 2H), 7.78 (m, 4H). MS (ESI): [M] =582.1,
+
[
M+2] =584.3.
5
.1.3.4. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N-(4-chlorophenyl)-N-methylaminomethyl)-thiophen-2-
yl]-isoindole-1,3-dione (6u). Following the general procedure (B), the product 6u was purified by
column chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 90%. Yellow solid, mp
1
1
22-124 °C. H NMR (CDCl
3
) δ: 2.54 (s, 3H), 4.29 (s, 2H), 6.28 (d, J=9.0 Hz, 2H), 6.96 (d, J=9.0
+
Hz, 2H), 7.11 (d, J=8.6 Hz, 2H), 7.41 (d, J=8.6 Hz, 2H), 7.43 (m, 4H). MS (ESI): [M] =598.1,
+
[
M+2] =600.1.
2
2

ACCEPTED MANUSCRIPT
5
.1.3.5. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(N-methyl-N-(4-trifluoromethylphenyl)-aminomethyl)-
thiophen-2-yl]-isoindole-1,3-dione (6v). Following the general procedure (B), the product 6v was
purified by column chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 74%.Yellow
1
solid, mp 189-191 °C. H NMR (CDCl ) δ: 2.66 (s, 3H), 4.43 (s, 2H), 6.33 (d, J=8.8 Hz, 2H), 7.04
3
(d, J=8.8 Hz, 2H), 7.27 (d, J=6.8 Hz, 2H), 7.35 (d, J=6.8 Hz, 2H), 7.72 (m, 4H). MS (ESI):
+
+
[
M] =632.1, [M+2] =634.1.
5
.1.4. General procedure (C) for the synthesis of compounds (6am-aq).
To a stirred solution of 4 (270 mg, 0.5 mmol) in dry acetonitrile (5 mL) was added the appropriate
phenol or thiophenol (1 equiv., 1 mmol) and K CO (1 equiv., 69 mg, 0.5 mmol). The mixture was
2
3
then heated at 60°C for 1 h. After this time, the appropriate phenol or thiophenol (1 equiv., 1
mmol,) and K CO (1 equiv., 69 mg, 0.5 mmol) were added and the mixture was heated at 60°C for
2
3
1
h further. The solvent was removed under reduced pressure and the residue partitioned between a
mixture of dichloromethane (15 mL) and water (5 mL). The organic phase was washed with brine
5 mL), dried (Na SO ), then concentrated under reduced pressure to give a residue that was
purified by column chromatography.
(
2
4
5
.1.4.1. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(phenyloxymethyl)-thiophen-2-yl]-isoindole-1,3-dione
(6am). Following general procedure (C), the product 6am was purified by column chromatography
1
(
EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 72%. Colorless oil. H NMR (CDCl ) δ: 4.87 (s,
3
2
4
H), 6.36 (d, J=8.4 Hz, 2H), 6.72 (t, J=7.2 Hz, 1H), 7.12 (m, 4H), 7.44 (d, J=8.6 Hz, 2H), 7.82 (m,
+
+
H). MS (ESI): [M] =551.0, [M+2] =553.1.
5
1
.1.4.2. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(4-(methyl)phenyloxymethyl)-thiophen-2-yl]-isoindole-
,3-dione (6an). Following general procedure (C), the product 6an was purified by column
chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 56%. White solid, mp 97-100 °C.
1
3
H NMR (CDCl ) δ: 2.28 (s, 3H), 4.97 (s, 2H), 6.54 (d, J=8.8 Hz, 2H), 6.94 (d, J=8.8 Hz, 2H), 7.18
2
3

ACCEPTED MANUSCRIPT
13
(
d, J=8.6 Hz, 2H), 7.62 (d, J=8.6 Hz, 2H), 7.78 (m, 4H). C NMR (100 MHz, CDCl ) δ: 62.3,
3
1
1
13.2, 125.2 (2C), 127.8, 128.9 (2C), 129.5 (2C), 130.8 (2C), 131.2 (2C), 132.4, 133.4, 134.0,
+
34.6, 135.3 (2C), 136.1 (2C), 137.3, 159.4, 164.5 (2C), 186.4. MS (ESI): [M] =565.1,
+
[
M+2] =567.2.
5
.1.4.3.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-(4-(isopropyl)phenyloxymethyl)-thiophen-2-yl]-
isoindole-1,3-dione (6ao). Following general procedure (C), the product 6ao was purified by
1
column chromatography (EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 47%. Yellow oil. H
NMR (CDCl ) δ: 1.16 (d, J=6.8 Hz, 6H), 2.81 (m, 1H), 4.98 (s, 2H), 6.58 (d, J=8.8 Hz, 2H), 7.00
3
(d, J=8.8 Hz, 2H), 7.21 (d, J=8.6 Hz, 2H), 7.64 (d, J=8.6 Hz, 2H), 7.78 (m, 4H). MS (ESI):
+
+
[
M] =593.1, [M+2] =595.2.
5
1
.1.4.4. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(4-(fluoro)phenyloxymethyl)-thiophen-2-yl]-isoindole-
,3-dione (6ap). Following general procedure (C), the 6ap product was purified by column
chromatography (EtOAc:petroleum ether 2:8 as eluent). Yield 65%. White solid, mp 112-114 °C.
1
3
H NMR (CDCl ) δ: 4.98 (s, 2H), 6.63 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 7.21 (m, 2H),
+
+
7
.63 (d, J=8.6 Hz, 2H), 7.76 (m, 4H). MS (ESI): [M] =569.1, [M+2] =571.2.
5
.1.4.5. 2-[5-Bromo-3-(4-chlorobenzoyl)-4-(phenylthiomethyl)-thiophen-2-yl]-isoindole-1,3-dione
(
(
(
6aq). Following general procedure (C), the product 6aq was purified by column chromatography
1
EtOAc:petroleum ether 1.5:8.5 as eluent). Yield 69%. Yellow solid, mp 127-129 °C. H NMR
CDCl
3
) δ: 4.14 (s, 2H), 6.32 (d, J=8.4 Hz, 2H), 6.68 (m, 1H), 7.06 (m, 4H), 7.34 (d, J=8.6 Hz, 2H),
13
7
.74 (m, 4H). C NMR (100 MHz, CDCl ) δ: 32.4, 112.5, 124.2 (2C), 127.6, 128.7 (2C), 129.0
3
(2C), 130.6 (2C), 130.8 (2C), 132.1, 133.7, 134.2, 134.8, 135.1 (2C), 135.7 (2C), 137.3, 139.5,
+
+
1
65.5 (2C), 189.1. MS (ESI): [M] =567.2, [M+2] =569.3.
5
.1.5. General procedure (D) for the synthesis of compounds 7a-z and 7am-aq.
2
4

ACCEPTED MANUSCRIPT
A solution of derivative 6a-z or 6am-aq (2 mmol) in DMF (20 mL), containing Et N (0.3 mL, 2
3
mmol, 1 equiv.) was hydrogenated over 120 mg of 10% Pd/C at 60 p.s.i for 3 h. The catalyst was
removed by filtration, the filtrate concentrated, and the residue dissolved in dichloromethane (20
mL), washed with water (5 mL), brine (5 mL), dried (Na
2 4
SO ). After filtering, the solvent was
removed in vacuo to obtain a residue that was purified by column chromatography.
5
.1.5.1.
2-[3-(4-Chlorobenzoyl)-4-(1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-2-ylmethyl)-
thiophen-2-yl]-isoindole-1,3-dione (7a). Following general procedure (D), derivative 7a was
purified by column chromatography (EtOAc:dichloromethane 1:9). Yield: 68%. White solid, mp
1
1
48-150 °C. H NMR (CDCl ) δ: 3.02 (s, 2H), 3.42 (t, J=5.6 Hz, 2H), 4.12 (t, J=5.6 Hz, 2H), 4.36
3
+
(
s, 2H), 6.04 (s, 1H), 6.26 (s, 1H), 7.17 (m, 4H), 7.42 (m, 4H), 7.584 (m, 4H). MS (ESI): [M+1] =
5
52.2.
5
.1.5.2.
2-[3-(4-Chlorobenzoyl)-4-(8-fluoro-1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (7b). Following general procedure (D), derivative 7b
was purified by column chromatography (EtOAc:dichloromethane 1:9). Yield: 68%. Yellow solid,
1
mp 145-147 °C. H NMR (CDCl
3
) δ: 2.48 (t, J=5.4 Hz, 2H), 3.16 (s, 2H), 3.26 (s, 2H), 3.82 (t,
J=5.6 Hz, 2H), 6.04 (s, 1H), 6.26 (s, 1H), 7.17 (m, 4H), 7.42 (m, 4H), 7.58 (m, 4H). MS (ESI):
+
[
M+1] = 570.3.
5
.1.5.3.
2-[3-(4-Chlorobenzoyl)-4-(8-chloro-1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (7c). Following general procedure (D), derivative 7c
was purified by column chromatography (EtOAc:dichloromethane 1:9). Yield: 68%. Yellow solid,
1
mp 145-147 °C. H NMR (CDCl
3
) δ: 2.43(t, J=5.4 Hz, 2H), 3.14 (s, 2H), 3.25 (s, 2H), 3.79 (t, J=5.4
Hz, 2H), 6.01 (s, 1H), 6.63 (s, 1H), 7.12 (m, 4H), 7.39 (m, 2H),), 7.52 (s, 1H), 7.68 (m, 4H). MS
+
(
ESI): [M+1] = 586.2.
2
5

ACCEPTED MANUSCRIPT
5
.1.5.4.
2-[3-(4-Chlorobenzoyl)-4-(8-methoxy-1,2,3,4-tetrahydro-1H-pyrazino[1,2-a]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (7d). Following general procedure (D), derivative 7d
was purified by column chromatography (EtOAc:dichloromethane 2:8). Yield: 74%. Yellow solid,
1
mp 135-137 °C. H NMR (CDCl ) δ: 2.42 (t, J=5.4 Hz, 2H), 3.13 (s, 2H), 3.26 (s, 2H), 3.79 (t,
3
J=5.4 Hz, 2H), 3.84 (s, 3H), 6.02 (s, 1H), 6.64 (s, 1H), 7.15 (m, 4H), 7.35 (m, 2H), 7.56 (s, 1H),
+
7
.71 (m, 4H). MS (ESI): [M+1] = 582.2.
5
.1.5.5. 2-[3-(4-Chlorobenzoyl)-4-(1,2.3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indol-2-ylmethyl)-
thiophen-2-yl]-isoindole-1,3-dione (7e). Following general procedure (D), derivative 7e was
purified by column chromatography (EtOAc:dichloromethane 1:9). Yield: 71%. Yellow solid, mp
1
1
1
30-131 °C. H NMR (CDCl
3
) δ: 2.54 (m, 2H), 2.90 (m, 2H), 2.96 (m, 4H), 3.11 (d, J=11.6 Hz,
H), 3.44 (m, 2H), 6.40 (d, J=7.6 Hz, 1H), 6.66 (m, 2H), 7.12 (m, 2H), 7.24 (d, J=8.8 Hz, 2H), 7.48
+
(
d, J=8.8 Hz, 2H), 7.65 (m, 4H). MS (ESI): [M+1] = 554.3.
5
.1.5.6. 2-[3-(4-Chlorobenzoyl)-4-(8-chloro-1,2.3,4,10,10a-hexahydro-1H-pyrazino[1,2-a]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (7f). Following general procedure (D), derivative 7f
was purified by column chromatography (EtOAc:dichloromethane 2:8). Yield: 70%. Yellow solid,
1
mp 138-140 °C. H NMR (CDCl
3
) δ: 2.44 (m, 2H), 2.82 (m, 2H), 2.92 (m, 4H), 3.10 (d, J=11.4 Hz,
1
H), 3.28 (m, 2H), 6.30 (d, J=8.0 Hz, 1H), 6.62 (s, 1H), 7.00 (d, J=8.0 Hz, 1H), 7.04 (s, 1H), 7.46
+
(
d, J=8.8 Hz, 2H), 7.60 (d, J=8.8 Hz, 2H), 7.68 (m, 4H). MS (ESI): [M+1] = 588.3.
5
.1.5.7. 2-[3-(4-Chlorobenzoyl)-4-(1,2.3,4,10,10a-hexahydro-8-methyl-1H-pyrazino[1,2-a]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (7g). Following general procedure (D), derivative 7g
was purified by column chromatography (EtOAc:dichloromethane 0.5:9.5). Yield: 64%. Yellow
1
solid, mp 134-136 °C. H NMR (CDCl ) δ: 2.21 (s, 3H), 2.42 (m, 2H), 2.74 (m, 2H), 2.90 (m, 4H),
3
3
2
.08 (d, J=11.4 Hz, 1H), 3.22 (m, 2H), 6.12 (s, 1H), 7.00 (d, J=8.0 Hz, 1H), 7.11 (m, 2H), 7.36 (m,
+
H), 7.44 (d, J=8.4 Hz, 2H), 7.64 (m, 4H). MS (ESI): [M+1] = 568.2.
2
6

ACCEPTED MANUSCRIPT
5
.1.5.8. 2-[3-(4-Chlorobenzoyl)-4-(1,2,3,4-tetrahydro-1H-isoquinolin-2-ylmethyl)-thiophen-2-yl]-
isoindole-1,3-dione (7h). Following general procedure (D), derivative 7h was purified by column
1
chromatography (EtOAc:dichloromethane 1:9). Yield: 89%. White solid, mp 98-100 °C. H NMR
(
CDCl ) δ: 2.25 (t, J=5.4 Hz, 2H), 2.64 (t, J=5.6 Hz, 2H), 2.94 (s, 2H), 3.22 (s, 2H), 6.71 (s, 1H),
3
6
.89 (d, J=7.8 Hz, 1H), 7.13 (m, 3H), 7.35 (m, 6H), 7.54 (t, J=8.8 Hz, 1H), 7.78 (t, J=8.8 Hz, 1H).
+
MS (ESI): [M+1] = 513.3.
5
.1.5.9. 2-[3-(4-Chlorobenzoyl)-4-(2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-ylmethyl)-thiophen-2-
yl]-isoindole-1,3-dione (7i). Following general procedure (D), derivative 7i was purified by column
1
chromatography (EtOAc:petroleum ether 1:1). Yield: 52%. Yellow solid, mp 112-114 °C. H NMR
(CDCl
3
) δ: 2.72 (t, J=5.8 Hz, 2H), 3.20 (t, J=5.8 Hz, 2H), 3.46 (s, 2H), 3.84 (s, 2H), 6.84 (s, 1H),
7
7
.06 (m, 4H), 7.22 (d, J=8.6 Hz, 1H); 7.34 (d, J=8.6 Hz, 2H), 7.44 (d, J=7.6 Hz, 1H), 7.62 (m, 4H),
+
.88 (bs, 1H). MS (ESI): [M+1] = 552.2.
5
.1.5.10.
2-[3-(4-Chlorobenzoyl)-4-(8-chloro-2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-ylmethyl)-
thiophen-2-yl]-isoindole-1,3-dione (7j). Following general procedure (D), derivative 7j was purified
by column chromatography (EtOAc:petroleum ether 9:1). Yield: 55%. Yellow solid, mp 165-167
1
°
C. H NMR (CDCl
3
) δ: 2.74 (t, J=5.8 Hz, 2H), 3.24 (t, J=5.6 Hz, 2H), 3.70 (s, 2H), 4.00 (s, 2H),
7
7
.00 (d, J=8.6 Hz, 1H), 7.04 (s, 1H), 7.15 (d, J=8.6 Hz, 2H), 7.24 (d, J=8.6 Hz, 1H), 7.38 (s, 1H);
+
.46 (d, J=8.6 Hz, 2H), 7.60 (m, 4H), 7.82 (bs, 1H). MS (ESI): [M+1] = 586.1.
5
.1.5.11. 2-[3-(4-Chlorobenzoyl)-4-(5-methyl-2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-ylmethyl)-
thiophen-2-yl]-isoindole-1,3-dione (7k). Following general procedure (D), derivative 7k was
purified by column chromatography (EtOAc:petroleum ether 7:3). Yield: 55%. Yellow solid, mp
1
1
24-126 °C. H NMR (CDCl ) δ: 2.76 (t, J=5.6 Hz, 2H), 3.27 (t, J=5.6 Hz, 2H), 3.62 (s, 3H), 4.22
3
(m, 4H), 7.10 (m, 5H), 7.32 (d, J=8.6 Hz, 2H); 7.46 (d, J=8.6 Hz, 2H), 7.66 (m, 4H). MS (ESI):
+
[
M+1] = 566.2.
2
7

ACCEPTED MANUSCRIPT
5
.1.5.12.
2-[3-(4-Chlorobenzoyl)-4-(8-chloro-5-methyl-2,3,4,5-tetrahydro-pyrido[4,3-b]indol-2-
ylmethyl)-thiophen-2-yl]-isoindole-1,3-dione (7l). Following general procedure (D), derivative 7l
was purified by column chromatography (EtOAc:petroleum ether 9:1). Yield: 65%. Yellow solid,
1
mp 145-147 °C. H NMR (CDCl ) δ: 2.72 (t, J=5.6 Hz, 2H), 3.24 (t, J=5.6 Hz, 2H), 3.54 (s, 3H),
3
3
4
.63 (s, 2H), 4.12 (s, 2H), 6.84 (s, 1H), 7.11 (d, J=8.0 Hz, 2H), 7.16 (m, 2H), 7.32 (m, 3H), 7.64 (m,
+
H). MS (ESI): [M+1] = 600.5.
5
2
.1.5.13. 2-[3-(4-Chlorobenzoyl)-4-(spiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-ylmethyl-thiophen-
-yl]-isoindole-1,3-dione (7m). Following general procedure (D), derivative 7m was purified by
column chromatography (EtOAc:dichloromethane 1:9). Yield: 95%. Yellow solid, mp 142-144 °C.
1
H NMR (CDCl
3
) δ: 2.07 (t, J=6.0 Hz, 4H), 3.19 (t, J=6.0 Hz, 4H), 3.49 (s, 2H), 6.76 (m, 5H), 7.38
+
(
d, J=8.6 Hz, 2H), 7.44 (d, J=8.6 Hz, 2H), 7.64 (m, 4H). MS (ESI): [M+1] = 571.2.
5
.1.5.14. 2-[3-(4-Chlorobenzoyl)-4-(4-fluoro-spiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-ylmethyl-
thiophen-2-yl]-isoindole-1,3-dione (7n). Following general procedure (D), derivative 7n was
purified by column chromatography (EtOAc:dichloromethane 0.25:9.75). Yield: 55%. Yellow
1
solid, mp 210-212 °C. H NMR (CDCl
3
) δ: 2.08 (t, J=6.0 Hz, 4H), 3.04 (t, J=6.0 Hz, 4H), 3.34 (s,
2
2
H), 6.56 (d, J=8.4 Hz, 1H), 6.64 (m, 2H), 6.74 (m, 1H), 7.42 (d, J=8.6 Hz, 2H), 7.56 (d, J=8.6 Hz,
+
H),7.64 (m, 4H). MS (ESI): [M+1] = 589.2.
5
.1.5.15. 2-[3-(4-Chlorobenzoyl)-4-(4-methyl-spiro-benzo[1,3-d]-dioxol-2-yl)-piperidin-1-ylmethyl-
thiophen-2-yl]-isoindole-1,3-dione (7o). Following general procedure (D), derivative 7o was
purified by column chromatography (EtOAc:dichloromethane 1:9). Yield: 79%. White solid, mp
1
1
6
66-168 °C. H NMR (CDCl
3
) δ: 1.89 (t, J=5.6 Hz, 4H), 2.20 (s, 3H), 3.13 (m, 4H), 3.43 (s, 2H),
.63 (m, 2H), 6.70 (m, 2H), 7.44 (d, J=8.6 Hz, 2H), 7.52 (d, J=8.6 Hz, 2H), 7.63 (m, 4H). MS
+
(
ESI): [M+1] = 585.3.
2
8