Synthesis and biological evaluation of 2-amino-3-(4-chlorobenzoyl)-4-[(4- arylpiperazin-1-yl)methyl]-5-substituted-thiophenes. Effect of the 5-modification on allosteric enhancer activity at the A1 adenosine receptor

Article abstract of DOI:10.1021/jm3007504

We have recently reported a detailed structure-activity relationship study around a wide series of 2-amino-3-(4-chlorobenzoyl)-4-[(4-arylpiperazin-1-yl) methyl]thiophene derivatives as potent allosteric enhancers of the A₁ adenosine receptor. In the current study, we have continued to explore the potential of these molecules by synthesizing of a novel series of analogues that share a common 2-amino-3-(4-chlorobenzoyl)thiophene nucleus. Modifications were focused on varying the nature and the position of electron-withdrawing or electron-releasing groups on the phenyl of an arylpiperazine moiety attached at the 4-position of the thiophene ring by a methylene chain, combined with the presence of small alkyl groups (methyl or ethyl), bromine, or aryl moieties at the thiophene C-5 position. In this series of compounds, substitution at the 5-position had a fundamental effect on activity, with the 5-aryl group contributing additively to the allosteric enhancer activity. The thiophene C-5 aryl derivatives 4ad, 4ak, and 4al were the most active compounds in binding and functional experiments.

Full text of DOI:10.1021/jm3007504

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 2‑Amino-3-(4-chlorobenzoyl)-
4-[(4-arylpiperazin-1-yl)methyl]-5-substituted-thiophenes. Effect of
the 5‑Modification on Allosteric Enhancer Activity at the A1
Adenosine Receptor
Romeo Romagnoli,*,† Pier Giovanni Baraldi,*,† Maria Dora Carrion,† Carlota Lopez Cara,†
Olga Cruz-Lopez,† Maria Kimatrai Salvador,† Delia Preti,† Mojgan Aghazadeh Tabrizi,†
Allan R. Moorman,‡ Fabrizio Vincenzi,§ Pier Andrea Borea,§ and Katia Varani§
†Dipartimento di Scienze Chimiche e Farmaceutiche, Universita
‡Research and Development, King Pharmaceuticals Inc., 4000 CentreGreen Way, Suite 300, Cary, North Carolina, United States
§Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Universita
di Ferrara, Ferrara, Italy
̀
di Ferrara, Via Fossato di Mortara 17-19, 44121, Ferrara, Italy
̀
ABSTRACT: We have recently reported a detailed structure−activity relationship study around
a wide series of 2-amino-3-(4-chlorobenzoyl)-4-[(4-arylpiperazin-1-yl)methyl]thiophene deriva-
tives as potent allosteric enhancers of the A1 adenosine receptor. In the current study, we have
continued to explore the potential of these molecules by synthesizing of a novel series of analo-
gues that share a common 2-amino-3-(4-chlorobenzoyl)thiophene nucleus. Modifications were
focused on varying the nature and the position of electron-withdrawing or electron-releasing
groups on the phenyl of an arylpiperazine moiety attached at the 4-position of the thiophene
ring by a methylene chain, combined with the presence of small alkyl groups (methyl or ethyl),
bromine, or aryl moieties at the thiophene C-5 position. In this series of compounds, substitu-
tion at the 5-position had a fundamental effect on activity, with the 5-aryl group contributing
additively to the allosteric enhancer activity. The thiophene C-5 aryl derivatives 4ad, 4ak, and 4al were the most active com-
pounds in binding and functional experiments.
side effects due to their ubiquitous nature and poor receptor
subtype selectivity. In contrast to ligands that target the ortho-
steric site, allosteric ligands interact with binding domains that
are topographically distinct from the orthosteric site.7 The
binding of an allosteric ligand to its site causes a conformational
change in the receptor protein that is transmitted to the ortho-
steric site (and vice versa), enhancing or inhibiting activation of
the receptor by its natural ligand.7a Allosteric enhancers (AEs)
at the A1AR have potential as therapeutic agents because they
increase the efficacy of potent and highly selective exogenous
agonists, thereby allowing lower doses, or they may amplify the
action of endogenous adenosine by stabilizing the agonist−
receptor−G protein ternary complex that forms when local
adenosine concentrations increase under conditions of meta-
bolic stress, such as hypoxia.8
One of the first compounds recognized as an AE at the A1AR
was the 2-amino-3-aroylthiophene derivative named PD 81,723
(1, Chart 1)9 that selectively enhanced agonist radioligand
binding at the A1AR with no major effect on agonist binding at
the other adenosine receptor subtypes.10
Several studies emphasized the presence of two regions in
the allosteric binding site in the hA1AR: one region able to
interact with the 2-amino-3-aroyl thiophene moiety, while the
INTRODUCTION
■
Adenosine is an endogenous nucleoside modulator released
from almost all cells and is generated in the extracellular space
by breakdown of ATP.1 This nucleoside mediates its effects
through activation of a family of four G protein-coupled
adenosine receptors (ARs), named A1, A2A, A2B, and A3.2 These
receptors differ in their affinity for adenosine, in the type of G
proteins that they recruit, and finally in the downstream signal-
ing pathways that are activated in the target cells. The A1AR is
coupled to members of the Gi/Go family of G proteins,
inducing inhibition of adenylate cyclase activity.3 The A1AR is
widely distributed in the central nervous system (CNS), with
high levels in brain, cortex, cerebellum, hippocampus, and the
dorsal horn of the spinal cord. It modulates the activity of the
nervous system at the cellular level and is responsible for
sedative, anticonvulsant, anxiolytic, and locomotor depressant
effects induced by adenosine. Importantly, the A1AR has been
reported to play a role in adenosine-mediated analgesia.4 Stimula-
tion of A1ARs in the heart exerts a cardioprotective effect by
inhibiting norepinephrine release from sympathetic nerve end-
ings.5 Adenosine also protects tissues through its effect in ischemic
preconditioning (IPC), a brief period of ischemia and reperfu-
sion, which can protect myocardium from a subsequent prolonged
ischemic insult.6
Efforts to selectively target the A1AR with modified adeno-
sine analogues have resulted in therapeutics that are limited by
Received: May 30, 2012
Published: August 13, 2012
© 2012 American Chemical Society
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Chart 1. Chemical Structures of 2-Amino-3-aroylthiophene Derivatives 1, 2, 3a−e, and 4a-ao, Discovered as Allosteric
Modulators for A1 Adenosine Receptor
second wide lipophilic domain is able to interact with thio-
phene C-4 and C-5 substituents.8,11 The presence of a carbonyl
at C-3 and an adjacent primary amino group at C-2 of the
thiophenes is an important feature of the A1AR allosteric enhancer
activity.9 The benzoyl group was reported to be essential for
activity, and hydrophobic substituents at either the meta or para
position on the phenyl of the benzoyl moiety [such as CF3, Cl, or
C(CH3)3] are critical for high AE activity.12 Replacing the aroyl
with a naphthoyl group was well-tolerated.12c,13
In order to identify highly potent and selective AEs at the
A1AR, a wide range of 2-amino-3-aroylthiophene derivatives
modified at the C-4 and C-5 positions of the thiophene ring
have been reported.12−14 Structure−activity relationship studies
indicated that alkyl12−14 and aryl15 groups at the C-4 position
favored AE activity. Only small alkyl groups were thought to be
tolerated at the C-5 position, while bulky substitutents favored
antagonistic properties.16
In two previous articles,18 we have reported the potent AE
activity at the A1AR of a series of 2-amino-3-(4-chlorobenzoyl)-
4-[(4-arylpiperazin-1-yl)methyl]thiophene derivatives with gen-
eral structure 3. The enhancement was influenced by the
number and position of electron-withdrawing or electron-
releasing groups (EWGs or ERGs, respectively) on the phenyl
tethered to the piperazine moiety. Among them, the 4-chloro
(3a), 4-trifluoromethyl (3b), 3,4-difluoro (3c), 3-chloro-4-fluoro
(3d), and 4-trifluoromethoxy (3e) derivatives were reported to
be the most active compounds in binding (saturation and com-
petition) and functional cAMP studies.
We hypothesized that the A1AR contains an allosteric
binding site able to accommodate the phenylpiperazine moiety,
in which appropriate substituents on the phenyl ring influenced
this interaction and contributed importantly to increase AE
activity. On the basis of these findings, we continued the search
for more active AEs for the A1AR, by refining the structure−
activity relationship around this class of compounds through
the synthesis and biological evaluation of a new series of 2-
amino-3-(4-chlorobenzoyl)-4-[(arylpiperazinyl)methyl]-
thiophene derivatives with general structure 4, characterized by
the insertion of different groups at the 5-position. In detail, the
substituents at the 5-position were selected from small alkyl
Among the synthesized compounds, derivative 2 [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 Phar-
maceuticals for the treatment of neuropathic pain associated
with hyperalgesia and allodynia.17
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a
Scheme 1
a
Reagents: (a) 2-Butanone or 2-pentanone, S8, TEA, EtOH, reflux, 2 h; (b) phthalic anhydride, AcOH, reflux; (c) NBS, CH3CN, reflux; (d) N-
arylpiperazine, TEA, CH2Cl2; (e) NH2NH2, EtOH.
groups such as methyl and ethyl for derivatives 4a−o and 4p−r,
respectively, bromine for compounds 4s−t, or an aryl moiety
for the analogues 4u−ao. For this latter class of compounds,
we have also explored the effect on AE activity of electron-
withdrawing fluorine (4ad and 4ae) or chlorine (4af−ao)
groups at the 4-position of the phenyl at the thiophene
C-5 position. For the phenyl group linked to the piperazine
moiety, a panel of substituents that had been reported to confer
high AE activity in the previous series of thiophene 5-unsub-
stituted derivatives18 with general structure 3 was chosen for
this purpose.
By the preparation of this new series of 2-amino-3-(4-
chlorobenzoyl)-4-[(arylpiperazinyl)methyl]-5-substituted-thio-
phene derivatives with general structure 4, we are interested in
investigating if modifications at the 5-position of this class of
compounds are beneficial for interaction with the allosteric site
of the A1AR and if manipulation at this site could improve
enhancer activity, separating this effect from the competitive
antagonism at the same receptor subtype.
4-methyl-5-ethylthiophene counterpart 5b were directly obtained
by the classical Gewald reaction19 applied to butanone and
2-pentanone, respectively, and 3-(4-chlorophenyl)-3-oxopropa-
nenitrile with elemental sulfur and triethylamine (TEA) as base
in ethanol at reflux. Both of these compounds were transformed
in excellent yields into the corresponding N-phthalimido
derivatives 6a and 6b using phthalic anhydride in refluxing
acetic acid. Regioselective monobromination at the C-4 methyl
of 6a and 6b with N-bromosuccinimide proceeded in good
yield to afford the 4-bromomethyl thiophene derivatives 7a and
7b, which were coupled with the appropriate N-arylpiperazines
(2 equiv) in dichloromethane (DCM) to afford the derivatives
8a−o and 8p−r. Removal of the N-protected phthaloyl group
was accomplished by the use of hydrazine in ethanol at reflux,
to afford the final compounds 4a−o and 4p−r.
The 5-bromo derivatives 4s and 4t were prepared starting
from the 4-bromomethyl-5-bromothiophene 7c,18a,b which was
condensed with 1-phenylpiperazine or 1-(4-fluorophenyl)-
piperazine to yield 8s and 8t, respectively, and then subjected
to treatment with ethanolic hydrazine.
The previously reported 5-bromothiophene derivative 918a
served as the starting material in the sequence shown in Scheme 2
for the preparation of derivatives 4u−ao. With our optimized
CHEMISTRY
■
Compounds 4a−r were synthesized by the method depicted
in Scheme 1. 4,5-Dimethyl thiophene derivative 5a12a and its
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a
Scheme 2
a
Reagents: (a) PdCl2(dppf), ArB(OH)2, CsF, 1,4-dioxane, 65 °C; (b) NBS, 1,2-dichloroethane, reflux; (c) N-arylpiperazine, TEA, CH2Cl2; (d)
NH2NH2, EtOH.
conditions in hand, intermediate 9 was subjected to palladium-
catalyzed cross-coupling conditions in the presence of the appro-
priate arylboronic acid under heterogeneous conditions [PdCl2-
(dppf), CsF] in 1,4-dioxane at 65 °C to furnish the 5-arylthiophene
derivatives 10a−c in good yields. These intermediates were bromi-
nated on the 4-methyl side chain with NBS in 1,2-dichloroethane
to afford the 4-bromomethyl analogues 11a−c, which were sub-
sequently transformed into derivatives 12u−ao by treatment with
the appropriate N-arylpiperazines in DCM. The isolated com-
pounds 12u−ao were converted to the final compounds 4u−ao by
treatment with hydrazine in ethanol at reflux.
Allosteric enhancement was measured as the ability of the
new synthesized compounds 4a−ao and the reference deriva-
tive PD 81,723 to reduce the cAMP content of CHO:hA1 cells
in an in vitro assay on CHO:hA1 cells. Two test paradigms
were utilized: the test compound at a concentration of 10 μM
alone and the test compound at 100-fold lower concentration
(100 nM) in the presence of the selective hA1AR agonist CCPA
(1 pM). A decrease of cAMP content is indicated in Table 1
as a percentage inhibition of cAMP production from control
(absence of test compound) or in the presence of the tested
derivative. A similar reduction of cAMP content was observed
with each of the tested compounds under both of the two test
conditions.
Among the 41 synthesized derivatives, only three compounds
(4b, 4k, and 4l) were less active than PD 81,723, and one com-
pound (4j) was comparable to PD 81,723. All of the remain-
ing compounds were considerably more active at 10 μM than
PD 81,723 and decreased the percentage of cAMP production
from 40% to 89% (Table 1).
BIOLOGICAL RESULTS AND DISCUSSION
■
Functional Assays. Functional assays of allosteric enhance-
ment activity were performed using Chinese hamster ovary (CHO)
cells stably transfected to express the recombinant hA1ARs. Activa-
tion of these receptors causes a detectable inhibition of adenylate
cyclase activity and a reduction of cAMP content of CHO cells.
Compounds with the potential to be allosteric enhancers of
activation of hA1ARs decrease the content of cAMP in CHO
cells expressing hA1ARs.
Comparing compounds characterized by the presence of the
same arylpiperazine moiety at the 4-position of the thiophene
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Within the series of 5-methylthiophene derivatives, 4a−o,
the introduction of a halogen at the 4-position of the phenyl
moiety linked to the piperazine (4c, 4d, 4g, and 4h for F,
Cl, Br, and I, respectively) reduced the activity relative to the
unsubstituted phenylpiperazine derivative 4a, although there
was no significant difference in activity between the four halo-
gens. This may suggest that the adenosine A1 receptor contains
a large lipophilic binding domain around the arylpiperazine
moiety able to accommodate these substituents. Moving the
chlorine atom from the 4- to the 3-position, to furnish 4e, resulted
in activity comparable to that of 4a. Interestingly, the 3,4-dichloro
derivative (4f) was equally active with the 3-chloro derivative 4e,
but superior to the 4-chloro analogue 4d. Replacement of the
N-phenyl with a benzyl group (compound 4b) was detrimental to
the allosteric enhancement activity at 10 μM of concentration.
The N-(4-methylphenyl)piperazine derivative 4i showed an
inhibitory activity on cAMP production similar to the unsub-
stituted phenylpiperazine counterpart 4a, suggesting that the
presence of a lipophilic, electron-releasing moiety on the phenyl
ring retained the activity and seemed to have no effect on
the enhancement. In contrast, replacement of the 4-methyl group
with a less lipophilic and more electron-releasing methoxy group
(compound 4j) significantly reduced the activity with respect to
4i, even though it proved more active than PD 81,723. For
derivative 4j, the reduced enhancement activity may be attributed
both to steric and electronic factors.
The introduction of a nitrile function (compound 4k), which
has a stronger electron-withdrawing effect but is less lipophilic
than the halogens, led to a remarkable reduction in activity with
respect to the halogen derivatives. A similar effect was observed
by replacing the nitrile with the strongly electron-withdrawing
but more hydrophilic nitro group (derivative 4l). In contrast,
derivative 4m, which possesses a lipohilic and strongly electron-
withdrawing trifluoromethyl moiety at the 4-position of the
phenyl ring, showed an activity comparable to that of 4-methyl
counterpart 4i. Moving the trifluoromethyl from the 4-position
to the 3-position (compound 4n) led to a slight decrease in
activity. Introduction of a chlorine in the 4-position, to furnish
the 4-Cl, 3-CF3 derivative 4o, maintained activity relative to the
3-CF3 derivative.
In the small series of 5-ethylthiophene derivatives 4p−r, the
unsubstituted phenylpiperazine derivative, 4p, showed activity
at least as high or equivalent to that of the 5-methyl counter-
part, 4a. The 4-fluoro- and 4-chlorophenylpiperazine (4q and
4r, respectively) were more active than the corresponding
5-methyl analogues, 4c and 4d.
In the series of 5-phenylthiophene derivatives 4u−ac, the
phenyl and 4-fluorophenylpiperazine derivatives (4u and 4v,
respectively) were the most active compounds of the series.
Replacement of fluorine with a chlorine (compound 4w) caused
a decrease of enhancement, which was more pronounced for the
3,4-dichloro analogue, 4x. Among the isomeric trifluoromethyl-
phenylpiperazine derivatives 4y, 4z, and 4ab, the 4-CF3 derivative
4y was more active than the 3-CF3 and 2-CF3 counterparts (4z
and 4ab, respectively). Starting from 4z, the insertion of chlorine
at the 4-position, to furnish the 4-Cl, 3-CF3 derivative 4aa,
maintained the same activity of 4z. The replacement of the
4-trifluoromethyl group of 4y with a less lipophilic and electron-
releasing trifluoromethoxy moiety (compound 4ac) slightly
decreased the enhancement.
Table 1. Effect of the Novel Allosteric Enhancers 4a−ao and
of PD 81,723 in cAMP Assay in hA1CHO Cells
% inhibn of cAMP
% inhibn of cAMP production +
a
b
compd
4a
production
CCPA
59
14
50
44
58
56
49
54
61
31
11
9
6
1
5
4
6
5
5
5
6
3
1
1
5
5
5
6
6
6
5
5
8
8
7
5
7
7
6
6
8
9
9
7
6
7
9
7
8
8
8
7
8
2
57
19
53
40
54
51
46
45
58
30
15
13
61
52
48
50
55
53
43
41
74
71
67
56
73
60
65
66
69
81
77
72
63
68
71
67
75
78
70
62
72
22
6
2
5
4
6
5
5
4
6
3
2
1
6
5
5
5
6
5
4
4
7
8
7
6
7
6
6
7
7
8
8
7
6
7
7
7
8
8
7
6
8
2
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
60
51
52
57
63
60
48
46
79
77
68
55
72
63
64
62
67
89
82
75
65
71
85
69
83
81
76
66
78
19
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
PD 81,723
a
Inhibition of the forskolin-stimulated cAMP production (in
b
percentage) by the novel allosteric enhancers (10 μM). Inhibition
of the cAMP production (in percentage) by the novel allosteric
enhancers (100 nM) in the presence of CCPA (1 pM). The values are
expressed as the mean SEM, n = 3 independent experiments.
ring (4s vs 4a, 4p, 4u, 4ad, and 4af; 4t vs 4c, 4q, 4v, 4ae, and
4ah), it is apparent that the 5-bromothiophene derivatives 4s
and 4t were less active than the 5-methyl ones (4a and 4c),
which were less active than the 5-ethyl (4p and 4q) and 5-aryl
(4u, 4ad, 4af, 4v, 4ae, and 4ah) counterparts as allosteric
enhancers, although 4s and 4t were effective and more active
than PD 81,723 at 10 μM. The greatest percentage reduction of
cAMP production was achieved by compounds 4u−ao that
contained an aryl substituent at the C-5 position of the
thiophene ring.
The effect of the substitution on the phenyl ring attached to
the piperazine moiety, as well as on the phenyl at the thiophene
C-5 position, to reduce the cAMP levels was also investigated
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Table 2. A1AR Density Expressed as Bmax Values Obtained by [3H]CCPA Binding Assays in hA1CHO Membranes in the
Presence of 3a, 3b, 3e, 4a−ao, and Reference Compound PD 81,723 (10 μM) (A) and Modulation by the Novel Allosteric
a
Enhancers (10 μM) on the CCPA Affinity (CCPA Ki shift) in [3H]DPCPX Competition Binding Experiments (B)
b
c
b
c
A
B
A
B
Bmax (fmol/mg Bmax shift (fold CCPA Ki
CCPA Ki shift
Bmax (fmol/mg Bmax shift (fold CCPA Ki
CCPA Ki shift
compd
protein)
of increase)
(nM)
(fold of increase)
compd
4s
protein)
of increase)
(nM)
(fold of increase)
PD 81,723
3a
685 63
3626 332
3989 377
3502 341
3584 323
949 85
1.3 0.1
7.0 0.6
7.7 0.7
6.8 0.6
6.8 0.6
1.8 0.1
5.9 0.6
4.7 0.4
6.5 0.6
5.9 0.6
4.9 0.5
5.4 0.5
6.6 0.6
3.2 0.3
1.4 0.1
1.3 0.1
7.6 0.7
6.2 0.6
5.1 0.5
5.5 0.5
6.7 0.7
6.5 0.5
9.6 0.7
2.7 0.3
2.4 0.2
2.9 0.3
2.4 0.1
10.9 0.9
2.9 0.1
3.6 0.3
2.5 0.2
2.8 0.2
3.4 0.3
3.2 0.3
2.6 0.2
5.9 0.5
12.8 1.0
13.9 1.1
2.3 0.1
2.7 0.2
3.3 0.3
3.1 0.2
4.0 0.3
2.6 0.2
1.6 0.1
5.6 0.5
6.3 0.5
5.3 0.5
6.4 0.5
1.4 0.1
5.2 0.4
4.2 0.3
6.1 0.5
5.4 0.4
4.5 0.4
4.8 0.4
5.9 0.5
2.6 0.2
1.2 0.1
1.1 0.1
6.8 0.6
5.7 0.5
4.6 0.3
4.9 0.4
3.8 0.3
6.0 0.5
2477 213
2319 211
6061 575
5850 527
5112 485
3584 321
5639 516
3953 329
4480 405
4321 412
4954 472
7431 653
6166 587
5375 498
4058 384
5007 467
6008 512
4374 418
5902 498
6219 519
5323 503
4269 375
5639 526
4.7 0.5
4.4 0.4
11.5 1.2
11.1 1.2
9.7 0.9
6.8 0.6
10.7 1.1
7.5 0.7
8.5 0.8
8.2 0.8
9.4 0.9
14.1 1.2
11.7 1.1
10.2 1.0
7.7 0.8
9.5 0.9
11.4 1.1
8.3 0.8
11.2 1.1
11.8 1.1
10.1 1.0
8.1 0.8
10.7 1.0
3.4 0.3
3.7 0.3
1.8 0.1
1.6 0.1
1.9 0.1
2.4 0.2
1.7 0.1
2.2 0.2
1.9 0.1
2.0 0.2
1.8 0.1
1.2 0.1
1.6 0.1
1.6 0.1
2.3 0.1
1.8 0.2
1.8 0.1
2.0 0.1
1.5 0.1
1.2 0.1
1.9 0.1
2.1 0.1
1.7 0.1
4.5 0.3
4.1 0.3
8.3 0.7
9.4 0.8
7.9 0.7
3.0 0.2
8.9 0.7
7.1 0.7
7.9 0.7
7.6 0.8
8.4 0.8
13.3 1.1
9.5 0.8
9.5 0.8
6.8 0.7
8.3 0.6
8.7 0.7
7.7 0.8
10.5 0.9
13.0 1.1
8.0 0.7
7.2 0.6
8.9 0.7
4t
4u
3b
3e
4v
4w
4x
4a
4b
4c
4y
3109 302
2477 238
3426 287
3109 273
2582 227
2846 273
3478 326
1686 154
738 68
4z
4d
4e
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
4f
4g
4h
4i
4j
4k
4l
685 66
4m
4n
4o
4p
4q
4r
4005 384
3267 292
2688 234
2899 222
3531 332
3426 311
a
b
The values are expressed as the mean SEM, n = 3 independent experiments. Bmax (fmol/mg protein) and Bmax shift obtained in [3H]CCPA
c
saturation binding experiments performed in the absence (Bmax = 527 48 fmol/mg protein) or in the presence of 10 μM enhancers. Ki values of
CCPA in the presence of 10 μM test compounds and CCPA shift = Ki(CCPA)/Ki(CCPA + 10 μM enhancers), where the Ki of CCPA was 15.3
1.5 nM.
(compounds 4ad−ao). The presence of an electron-with-
drawing fluorine or chlorine group at the 4-position of the
phenyl at the thiophene C-5 position generally had a positive
influence on the activity (see 4u vs 4ad, 4v vs 4ae, 4w vs 4ai, 4x
vs 4aj, 4y vs 4ak, 4z vs 4al, 4aa vs 4am, 4ab vs 4an, 4ac vs
4ao). The 4-fluorophenyl derivative with an N-phenylpiper-
azine at the 4-position, 4ad, was the most active compound of
the entire series at the concentration tested, reducing the cAMP
level by 82% at a concentration of 10 μM. We assume that the
receptor environment around the thiophene 5-position binding
site is lipophilic, and for this reason, substitution with a phenyl
bearing an electron-withdrawing fluorine or chlorine group at
this position is optimal.
Among the thiophene 5-(4-chlorophenyl) series of deriva-
tives 4af−ao, compounds bearing a 4-chlorophenyl (4ai), 4-
trifluoromethylphenyl (4ak), and the isomeric 3-trifluorome-
thylphenyl (4al) moiety on the piperazine were the most active
compounds of this series. As shown with the 5-methyl series,
replacement of the N-phenyl (4af) with an N-benzyl group
(4ag) was detrimental to the AE activity at a concentration of
10 μM. While 4af and the 4-fluoro analogue (4ah) appeared to
have similar activities, replacement of fluorine with chlorine
(compound 4ai) increased the reduction of cAMP production.
By the synthesis of the 3,4-dichlorophenyl analogue 4aj, it was
confirmed that the addition of a second chlorine atom was
detrimental for activity. The strong electron-withdrawing
trifluoromethyl group at the 4-position and 3-position of the
phenyl led to compounds 4ak and 4al, respectively, appearing
to have similar activities, while the 2-CF3 derivative 4an has
similar activity to the benzyl analogue 4ag. As reported pre-
viously for derivative 4aa, the 4-Cl, 3-CF3 analogue 4am dis-
played a similar pattern, being slightly less active than the 3-CF3
counterpart. The replacement of the 4-CF3 group with a 4-OCF3
(compound 4ao) maintained the level of cAMP reduction, which
is comparable to that of the 4-Cl, 3-CF3 derivative 4am.
Antagonistic Activity. A characteristic feature of allosteric
enhancers at the A1AR is the propensity to also cause antagonism
at higher concentrations. The ability of compounds 4a−ao
to displace the binding of [3H]DPCPX, [3H]ZM241385, and
[3H]MRE-3008-F20 at human A1-, A2A-, and A3ARs was
evaluated in CHO cells at a concentration of 10 μM. The
prototype enhancer PD 81,723 did not inhibit the binding of the
radiolabeled antagonists to A1- and A2AARs, but at 10 μM, it
reduced by 21% the binding of [3H]MRE-3008-F20 to A3ARs.20
None of the examined derivatives significantly inhibited the
specific binding of the radioligands to A1-, A2A-, and A3ARs,
reaching a very low percentage inhibition of 12% or less at 10 μM
concentration, suggesting that these novel enhancers are not able
to bind to the orthosteric site. Substitution of the 5-position of
the thiophene with small alkyl groups (methyl and ethyl), bro-
mine or aryl moieties seemed to maintain or increase allosteric
enhancement with a concomitant absence of antagonistic activity
against A1AR, as suggested from competition binding experi-
ments.22
Effect of Enhancers on A1AR Binding Parameters.
Saturation and competition experiments of the selective
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adenosine A1 agonist [3H]CCPA to A1 receptors were per-
formed to verify if the novel compounds 4a−ao modified the
agonist binding parameters. From these experiments, A1 recep-
tor affinity (KD) and density (Bmax) were evaluated in the
presence and in the absence of the examined compounds (PD
81,723, 3a, 3b, 3e, and 4a−4ao at a concentration of 10 μM)
and were used to calculate the fold increase of the affinity (KD
shift, column B) and density (Bmax shift, column A) reported in
Table 2. In [3H]CCPA saturation binding experiments, the
reference compound PD 81,723 induced a Bmax shift to hA1ARs
of 1.3 fold. Under the same experimental conditions, with the
exception of 4b, 4k, and 4l, all of the new tested compounds
proved to be superior to PD 81,723. From the receptor density
calculated in the presence and in the absence of the novel
enhancers, the derivatives 4u, 4v, 4y, 4ad, 4ae, 4af, 4ai, 4ak,
4am, and 4ao were the most active compounds, each causing a
Bmax shift of more than 10-fold. Interestingly, no differences
were found in the affinity of [3H]CCPA in the presence or
in the absence of the tested compounds, suggesting that the
enhancers were not able to modify the KD values that ranged
from 1.0 0.1 to 1.2 0.1 nM.
Table 2 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. The enhancers were able
to mediate a shift of the A1 receptors toward the active state, as
suggested by the increase of CCPA affinity, expressed as Ki values
(Table 2). This table also shows the CCPA shift representing the
ratio of apparent Ki values in the absence and in the presence of
the tested compounds at 10 μM concentration. In the hA1CHO
membranes, by using [3H]DPCPX as radioligand, the Ki value
of CCPA was 15.3 1.5 nM as evaluated by a one-state model
of analysis. Interestingly, a significant decrease in the apparent
Ki value was observed in 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 1.6-fold. With the exception of derivatives 4b, 4k, and
4l, the CCPA affinity data in the presence of the new tested
derivatives 4a−ao reveal that the displacement curves are
shifted left, suggesting lower Ki values for CCPA. The largest
affinity shift has been observed for compounds 4ad, 4ak, and
4al, which decreased the apparent Ki values of CCPA approxi-
mately 13.3-, 10.5-, and 13.0-fold, respectively, resulting in
these compounds being 2-fold more active than the 5-unsub-
stituted thiophene derivatives 3a, 3b, and 3e (Table 2). Com-
pounds 4u, 4v, 4y, 4ac, 4ae, 4af, 4ah, 4ai, 4am, and 4ao caused
a similar shift of the apparent CCPA affinity, ranging from 8- to
9-fold, while derivatives 4a, 4e, 4m, 4w−x, 4aa−ab, 4ag, and
4an afforded an increase ranging from 6- to 7-fold.
In Figure 1, we have shown the effect of the allosteric modulators
PD 81,723, 4y, 4ad, and 4ai at 10 μM concentration in [3H]CCPA
saturation binding experiments on A1AR binding parameters such
as affinity and density. The novel enhancers were able to signifi-
cantly increase the A1AR receptor density. In Figure 2A, represen-
tative binding curves for the displacement of [3H]DPCPX by
different concentrations of CCPA alone and in the presence of PD
81,723, 4y, 4ad, and 4ai at 10 μM concentration were reported,
showing the increase of the CCPA affinity in the presence of novel
enhancers. Figure 2B,C reports the functional effect of the novel
enhancers on cAMP experiments performed in hA1CHO cells.
In particular, Figure 2B shows the inhibitory effect of PD
81,723, 4y, 4ad, and 4ai at 10 μM concentration on cAMP
stimulated by 1 μM forskolin. The effect of the same com-
pounds at 100 nM concentration in the presence of a low
Figure 1. [3H]CCPA saturation binding curves to human A1
adenosine receptors (A). Under control conditions, the KD value
was 1.1 0.1 nM and the Bmax was 527 48 fmol/mg protein. In the
presence of novel enhancers (10 μM), KD values were similar to those
obtained in controls, and Bmax values are reported in Table 2.
Scatchard plots of the same experimental data (B). Values are the
means and vertical lines are the SEM of three separate experiments as
described in Experimental Section.
concentration of CCPA (1 pM) is depicted in Figure 2C.
Interestingly, the novel enhancers were able to mediate a sig-
nificant inhibition of the cAMP accumulation at lower con-
centration in the presence of the A1AR agonist CCPA relative
to the experiment in the absence of CCPA, confirming their
specific role as A1 allosteric enhancers.
CONCLUSIONS
■
In this new series of compounds, 4a−ao, we have maintained
the 2-amino-3-(4-chlorobenzoyl)thiophene nucleus unchanged,
which is thought to be essential for allosteric modulation at
the A1AR, while evaluating the effect on the allosteric activity
of electron-releasing or electron-withdrawing groups on the phenyl
of the arylpiperazine moiety at the C-4 position of thiophene ring.
This modification was combined with the presence of methyl,
ethyl, bromine, and aryl moieties at its C-5 position. The potential
of these compounds to interact allosterically with the A1AR was
initially screened in an in vitro functional assay by evaluating their
ability to reduce the cAMP production in CHO cells.
Comparing compounds characterized by the presence of
same arylpiperazine moiety at the 4-position of the thiophene
ring, it appeared that the 5-aryl substituted derivatives were
more active than the methyl, ethyl, and bromine counterparts
(i.e., 4u, 4ad, and 4af vs 4a, 4p, and 4s). While it was previously
reported that bulky 5-alkyl substitution favored increased
antagonistic properties, our series of 5-aryl derivatives 4u−ao
showed an absence of antagonistic activity against A1AR. This
presumably results from a favorable interaction of this portion
of the molecule with the allosteric binding site of the A1AR.
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Figure 2. Inhibition curves of specific [3H]DPCPX binding to human A1ARs of CCPA in the absence and in the presence of novel enhancers
(10 μM). Affinity values were calculated by using a one-state model of analysis. Values are the means and vertical lines are the SEM of three separate
experiments, as described in the Experimental Section (A). Histograms relative to the cAMP inhibition, expressed in pmol/106 cells, mediated by
novel allosteric enhancers (10 μM). The effect of the examined compounds (100 nM) was also studied in the presence of CCPA (1 pM) (B).
1H NMR spectra were determined in CDCl3 solutions and recorded
Although the series of 5-ethyl- and 5-bromothiophene deriva-
with a Bruker AC-200 spectrometer or a Varian Mercury Plus 400
spectrometer. Chemical shifts (δ) are given in parts per million (ppm)
tives 4p−r and 4s,t, respectively, were too small to draw many
firm conclusions about the SAR, the biological data demon-
downfield and J values are given in hertz. All products reported
strated that the insertion of a bromo or ethyl group at the C-5
1
showed H NMR spectra in agreement with the assigned structures.
position of the 2-amino-3-(4-chlorobenzoyl)-4-[(4-arylpiperazin-
Positive-ion electrospray ionization (ESI) mass spectra were recorded
1-yl)methyl]thiophene nucleus maintained significant activity.
The therapeutic potential of selective AEs for the A1AR is
hampered by the fact that many of these molecules also possess
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 Micro-
antagonistic properties against this receptor subtype. None of the
analytical Laboratory of the Chemistry Department of the University
synthesized compounds (4a−ao) significantly inhibited antagonist
binding at the hA1AR, hA2AR, or hA3AR. Of the 41 newly syn-
thesized molecules, only three compounds (4b, 4k, and 4l) were
less active than PD 81,723. Those thiophene derivatives bearing
a 5-aryl group, 4u−ao, have substantially higher activity than
PD 81,723. Among these, derivatives 4v, 4ad−ae, 4ak, and 4al,
were the most active compounds in binding (saturation and dis-
placement experiments) and functional cAMP assays. Saturation
and competition experiments have also shown that the series of
5-aryl-substituted thiophene derivatives 4u−ao were more active
than the corresponding 5-unsubstituted analogues 3a, 3b, and 3e.
In competition binding experiments, the Ki values of CCPA in
the presence of compounds 4ad, 4ak, and 4al were decreased
approximately 13.3-, 10.5-, and 13.0-fold, respectively, almost
2-fold more active at 10 μM than 3a, 3b, and 3e.
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 F254 Merck plates) and
visualized with aqueous KMnO4. Flash chromatography was per-
formed using 230−400 mesh silica gel and the solvent system indi-
cated in the procedure. All commercially available compounds were
used without further purification. Organic solutions were dried over
anhydrous Na2SO4. Dichloromethane (DCM) was distilled from cal-
cium chloride and stored over molecular sieves (3 Å). Petroleum ether
refers to the fractions boiling at 40−60 °C.
Synthesis of (2-Amino-5-ethyl-4-methylthiophen-3-yl)(4-
chlorophenyl)methanone (5b). A stirred suspension of 3-(4-chloro-
phenyl)-3-oxopropionitrile (1.8 g, 10 mmol), sulfur (384 mg, 12
mmol), TEA (10 mmol, 1.4 mL), and pentan-2-one (861 mg, 1.1 mL,
10 mmol) in absolute EtOH (20 mL) was refluxed for 2 h. The solu-
tion was evaporated and the residue was dissolved in EtOAc (20 mL).
The organic phase was subsequently washed with 1% (w/v) HCl
EXPERIMENTAL SECTION
■
Chemistry. Materials and Methods. 3-(4-Chlorophenyl)-3-
oxopropionitrile and all arylpiperazines were commercially available.
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(5 mL), a saturated solution of NaHCO3 (5 mL), water (5 mL), and
brine (5 mL); dried (Na2SO4); filtered; and concentrated. The residue was
purified by column chromatography using a mixture of EtOAc:petroleum
2-[4-Bromomethyl-3-(4-chlorobenzoyl)-5-methylthiophen-2-yl]-
isoindoline-1,3-dione (7a). Following general procedure C, com-
pound 7a was purified by chromatography eluting with EtOAc:petro-
leum ether 2:8 to give a yellow solid. Yield: 53%. Mp: 173−175 °C. 1H
NMR (CDCl3): δ 2.53 (s, 3H), 4.65 (s, 2H), 7.17 (d, J = 8.4 Hz, 2H),
7.63 (d, J = 8.4 Hz, 2H), 7.63 (m, 2H), 7.73 (m, 2H). MS (ESI):
[M]+ = 472.9, [M + 2]+ = 474.8.
1
ether 1:9 as eluent, to give 5 as a yellow oil. Yield: 57%. H NMR
(CDCl3): δ 1.18 (t, J = 7.6 Hz, 3H), 2.18 (s, 3H), 2.55 (q, J = 7.6 Hz, 2H),
5.77 (bs, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H). MS
(ESI): [M]+ = 279.8.
2-[4-Bromomethyl-3-(4-chlorobenzoyl)-5-ethylthiophen-2-yl]-
isoindoline-1,3-dione (7b). Following general procedure C, com-
pound 7b was purified by chromatography eluting with EtOAc:petro-
leum ether 2:8 to give an orange oil. Yield: 37%. 1H NMR (CDCl3): δ
1.39 (t, J = 7.6 Hz, 3H), 2.96 (q, J = 7.6 Hz, 2H), 4.66 (s, 2H), 7.17 (d,
J = 7.6 Hz, 2H), 7.64 (d, J = 7.6 Hz, 2H), 7.73 (m, 2H), 7.76 (m, 2H).
MS (ESI): [M]+ = 486.8, [M + 2]+ = 488.8.
General Procedure D for the Synthesis of Compounds 11a−c. To
a solution of 10a, 10b, or 10c (2 mmol) in 1,2-dichloroethane (10 mL)
was added benzoyl peroxide (48 mg, 0.2 mmol) and N-bromosuccini-
mide (356 mg, 2 mmol), and the mixture was heated under reflux. After
2 h, additional N-bromosuccinimide (178 mg, 1 mmol) and benzoyl
peroxide (24 mg, 0.1 mmol) were added, and heating at reflux was
continued for 1 h further. The mixture was cooled to room temperature,
diluted with DCM (10 mL), and washed with 5% NaHCO3 solution
(5 mL), water (5 mL), and brine (5 mL). The organic layer was dried
(Na2SO4), filtered, and evaporated, to give a residue that was purified by
column chromatography on silica gel.
2-[4-Bromomethyl-3-(4-chlorobenzoyl)-5-phenylthiophen-2-yl]-
isoindoline-1,3-dione (11a). Following general procedure D,
compound 11a was purified by column chromatography eluting with
EtOAc:petroleum ether 2:8 to give a light brown solid. Yield: 95%.
Mp: 166−168 °C. 1H NMR (CDCl3): δ 4.73 (s, 2H), 7.21 (d, J = 8.6
Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.52 (m, 1H), 7.68 (m, 8H). MS
(ESI): [M]+ = 534.8, [M + 2]+ = 536.8.
2-[4-Bromomethyl-3-(4-chlorobenzoyl)-5-(4-fluorophenyl)-
thiophen-2-yl]isoindoline-1,3-dione (11b). Following general proce-
dure D, compound 11b was purified by column chromatography
eluting with EtOAc:petroleum ether 1.5:8.5 to give a yellow solid.
Yield: 66%. Mp: 186−188 °C. 1H NMR (CDCl3): δ 4.67 (s, 2H), 7.16
(d, J = 6.6 Hz, 2H), 7.56 (d, J = 6.6 Hz, 2H), 7.72 (d, J = 6.4 Hz, 2H),
7.76 (m, 6H). MS (ESI): [M]+ = 552.8, [M + 2]+ = 554.8.
2-[4-Bromomethyl-3-(4-chlorobenzoyl)-5-(4-chlorophenyl)-
thiophen-2-yl]isoindoline-1,3-dione (11c). Following general proce-
dure D, compound 11c was purified by column chromatography
eluting with EtOAc:petroleum ether 1.5:8.5 to give a yellow solid.
Yield: 88%. Mp: 178−180 °C. 1H NMR (CDCl3): δ 4.68 (s, 2H), 7.17
(d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H),
7.67 (d, J = 8.4 Hz, 2H), 7.76 (m, 4H). MS (ESI): [M]+ = 569.1,
[M + 2]+ = 571.1.
General Procedure A for the Synthesis of Compounds 6a,b. To a
solution of thiophene derivative 5a or 5b (4 mmol) in acetic acid
(25 mL) was added phthalic anhydride (740 mg, 5 mmol) and the
mixture was heated to reflux for 18 h. The solvent was evaporated in
vacuo and the residue was dissolved in DCM (30 mL). The organic
solution was washed with water (10 mL) and brine (10 mL), dried
(Na2SO4), filtered, and concentrated. The crude product was stirred
for 1 h in petroleum ether (20 mL) and then filtered to afford 6a,b.
2-[3-(4-Chlorobenzoyl)-4,5-dimethylthiophen-2-yl]isoindoline-
1,3-dione (6a). Following general procedure A, compound 6a was
1
isolated as a yellow powder. Yield: 92%. Mp: 154−156 °C. H NMR
(CDCl3): δ 2.10 (s, 3H), 2.43 (s, 3H), 7.24 (d, J = 8.4 Hz, 2H), 7.64
(d, J = 8.4 Hz, 2H), 7.78 (m, 4H). MS (ESI): [M]+ = 395.9.
2-[3-(4-Chlorobenzoyl)-5-ethyl-4-methylthiophen-2-yl]-
isoindoline-1,3-dione (6b). Following general procedure A, com-
pound 6b was obtained as a white solid. Yield: 88%. Mp: 115−117 °C.
1H NMR (CDCl3): δ 1.34 (t, J = 7.6 Hz, 3H), 2.17 (s, 3H), 2.82 (q,
J = 7.6 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H),
7.77 (m, 2H), 7.79 (m, 2H). MS (ESI): [M]+ = 409.9.
General Procedure B for the Synthesis of Compounds 10a−c. A
stirred suspension of 9 (461 mg, 1 mmol) and the appropriate
arylboronic acid (1.5 mmol) in 1,4-dioxane (10 mL containing 2−3
drops of water) was degassed under a stream of nitrogen over 10 min
and then treated with PdCl2(dppf) (82 mg, 0.1 mmol) and CsF
(380 mg, 2.5 mmol). The reaction mixture was heated under nitrogen
at 45 °C for 30 min and then at 75 °C for 5 h. The reaction mixture
was cooled to ambient temperature, diluted with DCM (15 mL),
filtered on a pad of Celite, and evaporated in vacuo. The residue was
dissolved with DCM (20 mL), and the resultant solution was washed
sequentially with water (5 mL) and brine (5 mL). The organic layer
was dried, filtered, and evaporated, and the residue was purified by
flash chromatography on silica gel.
2-[3-(4-Chlorobenzoyl)-4-methyl-5-phenylthiophen-2-yl]-
isoindoline-1,3-dione (10a). Following general procedure B with
phenylboronic acid, the residue was purified by chromatography on
silica gel eluting with EtOAc:petroleum ether 2:8, to furnish 10a as a
1
white solid. Yield: 74%. Mp: 124−126 °C. H NMR (CDCl3): δ 2.21
(s, 3H), 7.32 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.78 (m,
4H), 7.78 (m, 5H). MS (ESI): [M]+ = 457.9.
2-[3-(4-Chlorobenzoyl)-5-(4-fluorophenyl)-4-methylthiophen-2-
yl]isoindoline-1,3-dione (10b). Following general procedure B with 4-
fluorophenylboronic acid, the residue was purified by chromatography
on silica gel eluting with EtOAc:petroleum ether 2:8, to furnish 10b as
General Procedure E for the Synthesis of Compounds 8a−t. To a
stirred solution of compound 7a−c or 11a−c (1 mmol) in dry DCM
(5 mL) was added TEA (168 μL, 1.2 mmol). The mixture was cooled
with a bath of ice/water, and then the appropriate arylpiperazine
(2 equiv, 2 mmol) dissolved in DCM (1 mL) was slowly added. The
mixture was then stirred at room temperature for 3 h, diluted with
DCM (5 mL), and washed sequentially with water (5 mL) and brine
(5 mL). The organic phase was dried over Na2SO4, filtered, and con-
centrated in vacuo to give a residue that was purified by column
chromatography on silica gel.
1
a yellow solid. Yield: 78%. Mp: 178−180 °C. H NMR (CDCl3): δ
2.18 (s, 3H), 7.14 (d, J = 6.4 Hz, 2H), 7.22 (d, J = 6.4 Hz, 2H), 7.46
(m, 2H), 7.72 (m, 6H). MS (ESI): [M]+ = 475.8.
2-[3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-methylthiophen-2-
yl]isoindoline-1,3-dione (10c). Following general procedure B with 4-
chlorophenylboronic acid, the residue was purified by chromatography
on silica gel eluting with EtOAc:petroleum ether 3:7, to furnish 10b as
1
a yellow solid. Yield: 72%. Mp: 194−196 °C. H NMR (CDCl3): δ
2-[3-(4-Chlorobenzoyl)-5-methyl-4-[(4-phenylpiperazin-1-yl)-
methyl]thiophen-2-yl]isoindoline-1,3-dione (8a). Following general
procedure E using N-phenylpiperazine, compound 8a was purified by
chromatography eluting with EtOAc:DCM 1:9 to give a yellow solid.
2.21 (s, 3H), 7.27 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.72
(d, J = 8.4 Hz, 2H), 7.78 (m, 6H). MS (ESI): [M]+ = 492.4.
General Procedure C for the Synthesis of Compounds 7a,b. To
a solution of thiophene derivative 6a or 6b (2 mmol) in acetonitrile
(10 mL) was added N-bromosuccinimide (356 mg, 2 mmol), and the
mixture was heated at reflux for 2 h. After this period, another portion
of N-bromosuccinimide (356 mg, 2 mmol) was added and the reflux
was continued for another 2 h and then the mixture was evaporated in
vacuo. The residue was dissolved in DCM (15 mL) and the resultant
solution was washed sequentially with water (5 mL) and brine (5 mL).
The organic layer was dried (Na2SO4), filtered, and evaporated, and
the residue was purified by flash chromatography on silica gel.
1
Yield: 92%. Mp: 103−105 °C. H NMR (CDCl3): δ 2.03 (t, J = 4.8
Hz, 4H), 2.24 (s, 3H), 2.88 (t, J = 4.8 Hz, 4H), 3.06 (s, 2H), 6.83 (d,
J = 8.8 Hz, 2H), 6.90 (m, 3H), 7.20 (d, J = 8.4 Hz, 2H), 7.29 (d, J =
8.4 Hz, 2H), 7.39 (d, J = 6.8 Hz, 1H), 7.53 (t, J = 6.8 Hz, 2H), 7.82 (d,
J = 6.8 Hz, 1H). MS (ESI): [M]+ = 556.1.
2-[4-[(4-Benzylpiperazin-1-yl)methyl]-3-(4-chlorobenzoyl)-5-
methylthiophen-2-yl]isoindoline-1,3-dione (8b). Following general
procedure E using N-benzylpiperazine, compound 8b was purified by
chromatography eluting with EtOAc to give a yellow solid. Yield: 87%.
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Mp: 92−93 °C. 1H NMR (CDCl3): δ 1.84 (t, J = 5.2 Hz, 4H), 2.33 (s,
3H), 3.23 (t, J = 5.2 Hz, 4H), 3.39 (s, 2H), 3.49 (s, 2H), 7.28 (m, 5H),
7.37 (d, J = 8.8 Hz, 2H), 7.43 (t, J = 7.2 Hz, 2H), 7.53 (d, J = 7.2 Hz,
1H), 7.58 (d, J = 8.8 Hz, 2H), 7.82 (t, J = 7.2 Hz, 1H). MS (ESI):
[M]+ = 570.1.
(t, J = 4.8 Hz, 4H), 3.06 (s, 2H), 6.74 (d, J = 8.4 Hz, 2H), 6.82 (d, J =
8.4 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.2 Hz, 2H), 7.39
(d, J = 7.2 Hz, 1H), 7.49 (t, J = 7.2 Hz, 2H), 7.80 (d, J = 7.2 Hz, 1H).
MS (ESI): [M]+ = 570.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-methoxyphenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8j). Following
general procedure E using N-(4-methoxyphenyl)piperazine, com-
pound 8j was purified by chromatography eluting with EtOAc:DCM
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-fluorophenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8c). Follow-
ing general procedure E using N-(4-fluorophenyl)piperazine, com-
pound 8c was purified by chromatography eluting with EtOAc:DCM
1
1:9 to give a yellow solid. Yield: 82%. Mp: 108−110 °C. H NMR
1
1:9 to give a yellow solid. Yield: 92%. Mp: 103−105 °C. H NMR
(CDCl3): δ 1.98 (t, J = 4.8 Hz, 4H), 2.33 (s, 3H), 2.77 (t, J = 4.8 Hz,
4H), 3.06 (s, 2H), 3.77 (s, 3H), 6.85 (d, J = 8.8 Hz, 2H), 6.88 (d, J =
8.8 Hz, 2H), 7.38 (d, J = 7.2 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.52 (t,
J = 7.2 Hz, 2H), 7.58 (m, 3H), 7.78 (d, J = 7.2 Hz, 1H). MS (ESI):
[M]+ = 586.1.
(CDCl3): δ 2.03 (t, J = 4.8 Hz, 4H), 2.24 (s, 3H), 2.88 (t, J = 4.8 Hz,
4H), 3.06 (s, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.90 (m, 3H), 7.20 (d, J =
8.4 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 6.8 Hz, 1H), 7.53 (t,
J = 6.8 Hz, 2H), 7.82 (d, J = 6.8 Hz, 1H). MS (ESI): [M]+ = 574.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-chlorophenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8d). Follow-
ing general procedure E using N-(4-chlorophenyl)piperazine, com-
pound 8d was purified by chromatography eluting with EtOAc:DCM
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-cyanophenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8k). Follow-
ing general procedure E using N-(4-cyanophenyl)piperazine, com-
pound 8k was purified by chromatography eluting with EtOAc:DCM
1
1
1:9 to give a yellow solid. Yield: 89%. Mp: 135−136 °C. H NMR
1:9 to give a yellow solid. Yield: 82%. Mp: 133−135 °C. H NMR
(CDCl3): δ 1.97 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.84 (t, J = 5.2 Hz,
4H), 3.06 (m, 2H), 6.73 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 9.2 Hz, 2H),
7.16 (d, J = 9.2 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 7.6 Hz,
1H, 1H), 7.60 (t, J = 7.6 Hz, 2H), 7.88 (d, J = 7.6 Hz, 1H). MS (ESI):
[M]+ = 590.5.
(CDCl3): δ 2.03 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.96 (t, J = 4.8 Hz,
4H), 3.08 (m, 2H), 6.75 (d, J = 9.2 Hz, 2H), 6.84 (d, J = 9.2 Hz, 2H),
7.42 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.63 (m, 4H), 7.82
(d, J = 7.6 Hz, 1H). MS (ESI): [M]+ = 581.1.
2-{3-(4-Chlorobenzoyl)-5-methyl-4-[(4-(4-nitrophenyl)piperazin-
1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione (8l). Following gen-
eral procedure E using N-(4-nitrophenyl)piperazine, compound 8l was
purified by chromatography eluting with EtOAc:DCM 1:9 to give a
yellow solid. Yield: 88%. Mp: 110−112 °C. 1H NMR (CDCl3): δ 1.99
(t, J = 5.4 Hz, 4H), 2.32 (s, 3H), 3.09 (s, 2H), 3.12 (t, J = 5.4 Hz, 4H),
6.71 (d, J = 9.6 Hz, 2H), 6.82 (d, J = 9.2 Hz, 2H), 7.33 (d, J = 7.6 Hz,
1H), 7.42 (d, J = 8.4 Hz, 2H), 7.53 (t, J = 7.6 Hz, 2H), 7.60 (d, J =
8.4 Hz, 2H), 8.82 (d, J = 7.6 Hz, 1H). MS (ESI): [M]+ = 601.1.
2-{3-(4-Chlorobenzoyl)-5-methyl-4-[(4-(4-(trifluoromethyl)-
phenyl)piperazin-1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione
(8m). Following general procedure E using N-(4-(trifluoromethyl)-
phenyl)piperazine, compound 8m was purified by chromatography
2-{3-(4-Chlorobenzoyl)-4-[(4-(3-chlorophenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8e). Follow-
ing general procedure E using N-(3-chlorophenyl)piperazine, com-
pound 8e was purified by chromatography eluting with EtOAc:DCM
1
1:9 to give a yellow solid. Yield: 92%. Mp: 110−112 °C. H NMR
(CDCl3): δ 1.96 (t, J = 5.4 Hz, 4H), 2.32 (s, 3H), 2.88 (t, J = 5.4 Hz,
4H), 3.06 (s, 2H), 6.66 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H),
6.84 (s, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.40 (d,
J = 8.4 Hz, 2H), 7.52 (t, J = 7.2 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.82
(d, J = 7.2 Hz, 1H). MS (ESI): [M]+ = 590.5.
2-{3-(4-Chlorobenzoyl)-4-[(4-(3,4-dichlorophenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8f). Follow-
ing general procedure E using N-(3,4-dichlorophenyl)piperazine,
compound 8f was purified by chromatography eluting with EtOAc:
DCM 0.5:9.5 to give a yellow solid. Yield: 88%. Mp: 130−132 °C. 1H
NMR (CDCl3): δ 1.96 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.86 (t, J =
4.8 Hz, 4H), 3.07 (s, 2H), 6.62 (dd, J = 9.2 and 3.2 Hz, 1H), 6.94 (d,
J = 2.8 Hz, 1H), 7.22 (d, J = 9.2 Hz, 1H), 7.37 (d, J = 7.2 Hz, 1H),
7.42 (d, J = 8.6 Hz, 2H), 7.53 (t, J = J = 7.2 Hz, 2H), 7.59 (d, J =
8.6 Hz, 2H), 7.82 (d, J = 7.2 Hz, 1H). MS (ESI): [M]+ = 624.1.
2-{4-[(4-(4-Bromophenyl)piperazin-1-yl)methyl]-3-(4-chloroben-
zoyl)-5-methylthiophen-2-yl}isoindoline-1,3-dione (8g). Following
general procedure E using N-(4-bromophenyl)piperazine, compound
8g was purified by chromatography eluting with EtOAc:DCM 1:9 to
give a yellow solid. Yield: 94%. Mp: 135−137 °C. 1H NMR (CDCl3):
δ 1.97 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.85 (t, J = 4.8 Hz, 4H), 3.06
(m, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 9.2 Hz, 2H), 7.30 (d,
J = 9.2 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 7.6 Hz, 1H, 1H),
7.58 (t, J = 7.6 Hz, 2H), 7.82 (d, J = 7.6 Hz, 1H). MS (ESI): [M]+ =
633.0, [M + 2]+ = 635.0.
1
eluting with EtOAc:DCM 1:9 to give a yellow oil. Yield: 89%. H
NMR (CDCl3): δ 1.98 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.97 (t, J =
4.8 Hz, 4H), 3.07 (s, 2H), 6.81 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz,
2H), 7.42 (m, 3H), 7.48 (t, J = 6.8 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H),
7.86 (d, J = 6.8 Hz, 1H). MS (ESI): [M]+ = 624.1.
2-{3-(4-Chlorobenzoyl)-5-methyl-4-[(4-(3-(trifluoromethyl)-
phenyl)piperazin-1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione
(8n). Following general procedure E using N-(3-(trifluoromethyl)-
phenyl)piperazine, compound 8n was purified by chromatography
eluting with EtOAc:DCM 1:9 to give a yellow solid. Yield: 94%. Mp:
1
107−109 °C. H NMR (CDCl3): δ 1.99 (t, J = 4.8 Hz, 4H), 2.32 (s,
3H), 2.93 (t, J = 5.4 Hz, 4H), 3.08 (s, 2H), 6.92 (d, J = 7.8 Hz, 1H),
7.00 (s, 1H), 7.06 (m, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.4
Hz, 2H), 7.43 (d, J = 7.2 Hz, 1H), 7.52 (t, J = 7.2 Hz, 2H), 7.61 (d, J =
8.4 Hz, 2H), 7.82 (d, J = 7.2 Hz, 1H). MS (ESI): [M]+ = 624.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-chloro-3-(trifluoromethyl)-
phenyl)piperazin-1-yl)methyl]-5-methylthiophen-2-yl}isoindoline-
1,3-dione (8o). Following general procedure E using N-(4-chloro-3-
(trifluoromethyl)phenyl)piperazine, compound 8o was purified by
chromatography eluting with EtOAc:DCM 0.5:9.5. Yield: 92%. Mp:
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-iodophenyl)piperazin-1-yl)-
methyl]-5-methylthiophen-2-yl}isoindoline-1,3-dione (8h). Follow-
ing general procedure E using N-(4-iodophenyl)piperazine, compound
8h was purified by chromatography eluting with EtOAc:DCM
1
118−120 °C. H NMR (CDCl3): δ 1.99 (t, J = 5.2 Hz, 4H), 2.32 (s,
3H), 2.90 (t, J = 5.2 Hz, 4H), 3.08 (s, 2H), 6.81 (dd, J = 8.8 and
3.0 Hz, 1H), 6.78 (d, J = 3.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.35
(d, J = 8.6 Hz, 2H), 7.39 (d, J = 7.4 Hz, 1H), 7.44 (d, J = 7.4 Hz, 1H),
7.53 (t, J = 7.4 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 7.4 Hz,
1H). MS (ESI): [M]+ = 658.5.
1
0.25:9.75 to give a yellow solid. Yield: 94%. Mp: 133−135 °C. H
NMR (CDCl3): δ 1.98 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H), 2.87 (t, J = 4.8
Hz, 4H), 3.06 (m, 2H), 6.58 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 9.2 Hz,
2H), 7.33 (d, J = 9.2 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 7.6
Hz, 1H, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.84 (d, J = 7.6 Hz, 1H). MS
(ESI): [M]+ = 681.1.
2-[3-(4-Chlorobenzoyl)-5-ethyl-4-[(4-phenyl-piperazin-1-yl)-
methyl]thiophen-2-yl]-isoindoline-1,3-dione (8p). Following general
procedure E using N-phenylpiperazine, compound 8p was purified by
chromatography eluting with EtOAc:petroleum ether 1.5:8.5 to give an
orange solid. Yield: 34%. Mp: 124−126 °C. 1H NMR (CDCl3): δ 1.34
(t, J = 7.6 Hz, 3H), 2.27 (m, 4H), 2.85 (m, 6H), 3.32 (s, 2H), 6.79 (d,
J = 8.4 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.71
(m, 5H), 7.81 (m, 2H). MS (ESI): [M]+ = 570.1.
2-[3-(4-Chlorobenzoyl)-5-methyl-4-[(4-(4-methylphenyl)-
piperazin-1-yl)methyl]thiophen-2-yl]isoindoline-1,3-dione (8i). Fol-
lowing general procedure E using N-(4-methylphenyl)piperazine,
compound 8i was purified by chromatography eluting with EtOAc:
1
DCM 1:9 to give a yellow solid. Yield: 83%. Mp: 110 °C. H NMR
(CDCl3): δ 1.98 (t, J = 4.8 Hz, 4H), 2.25 (s, 3H), 2.32 (s, 3H), 2.83
7728
dx.doi.org/10.1021/jm3007504 | J. Med. Chem. 2012, 55, 7719−7735

Journal of Medicinal Chemistry
Article
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-fluorophenyl)piperazin-1-yl)-
methyl]-5-ethylthiophen-2-yl}isoindoline-1,3-dione (8q). Following
general procedure E using N-(4-chlorophenyl)piperazine, compound
8q was purified by chromatography eluting with EtOAc:petroleum
(d, J = 2.8 Hz, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H),
7.72 (m, 7H), 7.82 (m, 4H). MS (ESI): [M]+ = 687.1.
2-{3-(4-Chlorobenzoyl)-5-phenyl-4-[(4-(4-(trifluoromethyl)-
phenyl)piperazin-1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione
(12y). Following general procedure E, compound 12y was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 67%. Mp: 163−164 °C. 1H NMR (CDCl3): δ 2.20
(t, J = 4.8 Hz, 4H), 2.88 (t, J = 4.8 Hz, 4H), 3.42 (s, 2H), 6.75 (d, J =
8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.45
(m, 5H), 7.83 (m, 6H). MS (ESI): [M]+ = 686.1.
2-{3-(4-Chlorobenzoyl)-5-phenyl-4-[(4-(3-(trifluoromethyl)-
phenyl)piperazin-1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione
(12z). Following general procedure E using N-(3-(trifluoromethyl)-
phenyl)piperazine, compound 12y was purified by chromatography
eluting with EtOAc:petroleum ether 3:7 to give a yellow solid. Yield:
81%. Mp: 118−120 °C. 1H NMR (CDCl3): δ 2.18 (t, J = 4.8 Hz, 4H),
2.84 (t, J = 4.8 Hz, 4H), 3.42 (s, 2H), 6.94 (m, 3H), 7.34 (d, J =
8.2 Hz, 2H), 7.46 (m, 4H), 7.83 (m, 8H). MS (ESI): [M]+ = 686.1.
2-{3-(4-Chlorobenzoyl)-5-phenyl-4-[(4-(4-chloro-3-
(trifluoromethyl)phenyl)piperazin-1-yl)methyl]thiophen-2-yl}-
isoindoline-1,3-dione (12aa). Following general procedure E using N-
(4-chloro-3-(trifluoromethyl)phenyl)piperazine, compound 12aa was
purified by chromatography eluting with EtOAc:petroleum ether 3:7
to give a white solid. Yield: 78%. Mp: 228−230 °C. 1H NMR
(CDCl3): δ 2.18 (t, J = 4.6 Hz, 4H), 2.80 (t, J = 4.6 Hz, 4H), 3.42 (s,
2H), 6.80 (dd, J = 8.8 and 2.8 Hz, 1H), 7.00 (d, J = 2.8 Hz, 1H), 7.24
(d, J = 8.8 Hz, 1H), 7.35 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H),
7.46 (m, 5H), 7.87 (m, 4H). MS (ESI): [M]+ = 720.6.
1
ether 1.5:8.5 to give a white solid. Yield: 43%. Mp: 122−124 °C. H
NMR (CDCl3): δ 1.20 (t, J = 7.6 Hz, 3H), 1.99 (m. 4H), 2.68 (q, J =
7.6 Hz, 2H), 3.02 (m, 4H), 3.22 (s, 2H), 6.84 (d, J = 8.4 Hz, 2H), 7.21
(d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H),
7.72 (m, 2H), 7.84 (m, 2H). MS (ESI): [M]+ = 588.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-chlorophenyl)piperazin-1-yl)-
methyl]-5-ethylthiophen-2-yl}isoindoline-1,3-dione (8r). Following
general procedure E using N-(4-chlorophenyl)piperazine, compound
8r was purified by chromatography eluting with EtOAc:petroleum
1
ether 1.5:8.5 to give a white solid. Yield: 37%. Mp: 142−144 °C. H
NMR (CDCl3): δ 1.19 (t, J = 7.6 Hz, 3H), 2.32 (m. 4H), 2.77 (q, J =
7.6 Hz, 2H), 3.02 (m, 4H), 3.32 (s, 2H), 6.82 (d, J = 8.4 Hz, 2H), 7.17
(d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H),
7.71 (m, 2H), 7.81 (m, 2H). MS (ESI): [M]+ = 604.5.
2-[5-Bromo-3-(4-chlorobenzoyl)-4-[(4-phenylpiperazin-1-yl)-
methyl]thiophen-2-yl]isoindoline-1,3-dione (8s). Following general
procedure E using N-phenylpiperazine, compound 8s was purified by
chromatography eluting with EtOAc:petroleum ether 4:6 to give a
yellow solid. Yield: 86%. Mp: 191−193 °C. 1H NMR (CDCl3): δ 2.85
(t, J = 5.0 Hz, 2H), 3.14 (s, 2H), 3.34 (t, J = 5.0 Hz, 2H), 3.42 (t, J =
5.2 Hz, 2H), 3.94 (t, J = 5.2 Hz, 2H), 6.80−6.93 (m, 4H), 6.93−7.28
(m, 4H), 7.39 (d, J = 8.4 Hz, 2H), 7.58−7.62 (m, 2H), 7.82 (d, J =
6.8 Hz, 1H). MS (ESI): [M]+ = 619.1, [M + 2]+ = 621.1.
2-{5-Bromo-3-(4-chlorobenzoyl)-4-[(4-(4-fluorophenyl)piperazin-
1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione (8t). Following gen-
eral procedure E using N-(4-fluorophenyl)piperazine, compound 8t
was purified by chromatography eluting with EtOAc:petroleum ether
2-{3-(4-)Chlorobenzoyl)-5-phenyl-4-[(4-(2-(trifluoromethyl)-
phenyl)piperazin-1-ylmethyl]thiophen-2-yl}isoindoline-1,3-dione
(12ab). Following general procedure E using N-(2-(trifluoromethyl)-
phenyl)piperazine, compound 12ab was purified by chromatography
eluting with EtOAc:petroleum ether 3:7 to give a white solid. Yield:
89%. Mp: 201−202 °C. 1H NMR (CDCl3): δ 2.18 (t, J = 4.4 Hz, 4H),
2.46 (t, J = 4.4 Hz, 4H), 3.41 (s, 2H), 6.91 (d, J = 8.8 Hz, 1H), 7.12 (d,
J = 8.8 Hz, 1H), 7.44 (m, 7H), 7.56 (d, J = 8.4 Hz, 2H), 7.76 (m, 6H).
MS (ESI): [M]+ = 686.1.
1
3:7 to give a white solid. Yield: 89%. Mp: 203−204 °C. H NMR
(CDCl3): δ 2.29 (t, J = 4.8 Hz, 4H), 2.78 (t, J = 4.8 Hz, 4H), 3.38 (s,
2H), 6.72 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 8.6
Hz, 1H), 6.91 (d, J = 8.6 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.34 (d,
J = 8.4 Hz, 2H), 7.672 (m, 2H),. 7.82 (m, 2H). MS (ESI): [M]+ =
637.1, [M + 2]+ = 639.1.
2-[5-Phenyl-3-(4-chlorobenzoyl)-4-[(4-phenylpiperazin-1-yl)-
methyl]thiophen-2-yl]isoindoline-1,3-dione (12u). Following general
procedure E using N-(4-phenyl)piperazine, compound 12u was
purified by chromatography eluting with EtOAc:petroleum ether
1.5:8.5 to give a yellow solid. Yield: 78%. Mp: 200−202 °C. 1H NMR
(CDCl3): δ 2.19 (t, J = 5.0 Hz, 4H), 2.79 (t, J = 5.0 Hz, 4H), 3.41 (s,
2H), 6.78 (d, J = 8.8 Hz, 2H), 7.18 (7, J = 8.8 Hz, 2H), 7.37 (d, J = 6.8
Hz, 2H), 7.45 (m, 5H), 7.83 (m, 7H). MS (ESI): [M]+ = 618.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-fluorophenyl)piperazin-1-yl)-
methyl]-5-phenylthiophen-2-yl}isoindoline-1,3-dione (12v). Follow-
ing general procedure E using N-(4-fluorophenyl)piperazine, com-
pound 12v was purified by chromatography eluting with EtOAc:pe-
troleum ether 3:7 to give a yellow solid. Yield: 59%. Mp: 160−161 °C.
1H NMR (CDCl3): δ 2.18 (t, J = 4.8 Hz, 4H), 2.70 (t, J = 4.8 Hz, 4H),
2-{3-(4-Chlorobenzoyl)-5-phenyl-4-[(4-(4-(trifluoromethoxy)-
phenyl)piperazin-1-yl)methyl]thiophen-2-yl}isoindoline-1,3-dione
(12ac). Following general procedure E using N-(4-(trifluoromethoxy)-
phenyl)piperazine, compound 12ac was purified by chromatography
eluting with EtOAc:petroleum ether 3:7 to give a yellow oil. Yield:
68%. 1H NMR (CDCl3): δ 1.88 (t, J = 4.8 Hz, 4H), 2.85 (t, J = 4.8 Hz,
4H), 3.12 (s, 2H), 6.82 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H),
7.34 (m, 3H), 7.38 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.68
(d, J = 8.2 Hz, 2H), 7.84 (m, 4H). MS (ESI): [M]+ = 702.1.
2-[5-(4-Fluorophenyl)-3-(4-chlorobenzoyl)-4-[(4-phenylpiperazin-
1-yl)methyl]thiophen-2-yl]isoindoline-1,3-dione (12ad). Following
general procedure E using N-phenylpiperazine, compound 12ad was
purified by chromatography eluting with EtOAc:petroleum ether 2:8
to give a yellow solid. Yield: 98%. Mp: 188−190 °C. 1H NMR
(CDCl3): δ 2.16 (t, J = 4.8 Hz, 4H), 2.78 (t, J = 4.8 Hz, 4H), 3.36 (s,
2H), 6.77 (t, J = 8.8 Hz, 2H), 7.13 (m, 4H), 7.35 (d, J = 8.6 Hz, 2H),
7.42 (m, 2H), 7.94 (m, 7H). MS (ESI): [M]+ = 636.1.
3.41 (s, 2H), 6.71 (d, J = 8.6 and 4.4 Hz, 2H), 6.88 (t, J = 8.4 Hz, 2H),
7.34 (d, J = 8.4 Hz, 2H), 7.43 (m, 5H), 7.73 (m, 2H), 7.81 (d, J =
8.4 Hz, 2H), 7.86 (m, 2H). MS (ESI): [M]+ = 636.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-chlorophenyl)piperazin-1-yl)-
methyl]-5-phenylthiophen-2-yl}isoindoline-1,3-dione (12w). Follow-
ing general procedure E using N-(4-chlorophenyl)piperazine, com-
pound 12w was purified by chromatography eluting with EtOAc:pe-
troleum ether 2:8 to give a yellow solid. Yield: 81%. Mp: 201−202 °C.
1H NMR (CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H), 2.75 (t, J = 5.2 Hz, 4H),
3.42 (s, 2H), 6.64 (d, J = 9.0 Hz, 2H), 7.12 (d, J = 9.0 Hz, 2H),
7.33 (d, J = 9.0 Hz, 2H), 7.47 (m, 5H), 7.86 (m, 6H). MS (ESI):
[M]+ = 652.5.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-fluorophenyl)piperazin-1-yl)-
methyl]-5-(4-fluorophenyl)thiophen-2-yl}isoindoline-1,3-dione
(12ae). Following general procedure E using N-(4-fluorophenyl)-
piperazine, compound 12ae was purified by chromatography eluting
with EtOAc:petroleum ether 1.5:8.5 to give a yellow solid. Yield: 67%.
Mp: 164−166 °C. 1H NMR (CDCl3): δ 2.18 (t, J = 4.8 Hz, 4H), 2.70
(t, J = 4.8 Hz, 4H), 3.37 (s, 2H), 6.68 (t, J = 8.6 Hz, 2H), 6.73 (t, J =
8.6 Hz, 2H), 7.16 (t, J = 8.6 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.37
(m, 4H), 7.78 (m, 4H). MS (ESI): [M]+ = 654.1.
2-[5-(4-Chlorophenyl)-3-(4-chlorobenzoyl)-4-[(4-phenylpipera-
zin-1-yl)methyl]thiophen-2-yl]isoindoline-1,3-dione (12af). Follow-
ing general procedure E using N-phenylpiperazine, compound 12af
was purified by chromatography eluting with EtOAc:petroleum ether 3:7
to give a white solid. Yield: 76%. Mp: 171−172 °C. 1H NMR (CDCl3):
δ 2.22 (t, J = 5.0 Hz, 4H), 2.83 (t, J = 5.0 Hz, 4H), 3.38 (s, 2H), 6.74
(d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H),
2-{3-(4-Chlorobenzoyl)-4-[(4-(3,4-dichlorophenyl)piperazin-1-yl)-
methyl]-5-phenylthiophen-2-yl}isoindoline-1,3-dione (12x). Follow-
ing general procedure E using N-(3,4-dichlorophenyl)piperazine,
compound 12x was purified by chromatography eluting with
EtOAc:petroleum ether 3:7 to give a white solid. Yield: 81%. Mp:
153−155 °C. H NMR (CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H), 2.76 (t,
J = 4.8 Hz, 4H), 3.41 (s, 2H), 6.62 (dd, J = 8.8 and 2.8 Hz, 1H), 6.82
1
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7.37 (d, J = 8.4 Hz, 2H), 7.43 (m, 5H), 7.75 (m, 4H). MS (ESI):
[M]+ = 652.6.
give a yellow solid. Yield: 78%. Mp: 208−210 °C. 1H NMR (CDCl3):
δ 2.12 (t, J = 4.8 Hz, 4H), 2.98 (t, J = 4.8 Hz, 4H), 3.22 (s, 2H), 6.82
(d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 7.42 (m, 4H), 7.56 (d,
J = 6.8 Hz, 2H), 7.70 (m, 6H). MS (ESI): [M]+ = 720.6.
2-[(4-(4-Benzylpiperazin-1-yl)methyl]-3-(4-chlorobenzoyl)-5-(4-
chlorophenyl)thiophen-2-yl]isoindoline-1,3-dione (12ag). Following
general procedure E using N-benzylpiperazine, compound 12ag was
purified by chromatography eluting with EtOAc:petroleum ether 4:6
to give a yellow solid. Yield: 77%. Mp: 198−200 °C. 1H NMR
(CDCl3): δ 2.12 (t, J = 5.2 Hz, 4H), 3.08 (t, J = 5.2 Hz, 4H), 3.44 (s,
2H), 3.89 (s, 2H), 7.28 (m, 5H), 7.34 (d, J = 8.8 Hz, 2H), 7.42 (d, J =
7.2 Hz, 2H), 7.46 (d, J = 7.2 Hz, 2H), 7.52 (d, J = 8.8 Hz, 2H), 7.84
(m, 4H). MS (ESI): [M]+ = 666.6.
2-{3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-[(4-(4-
(trifluoromethoxy)phenyl)piperazin-1-yl)methyl]thiophen-2-yl}-
isoindoline-1,3-dione (12ao). Following general procedure E using
N-(4-(trifluoromethoxy)phenyl)piperazine, compound 12ao was
purified by chromatography eluting with EtOAc:petroleum ether 3:7
to give a brown solid. Yield: 76%. Mp: 146−147 °C. 1H NMR
(CDCl3): δ 2.08 (t, J = 4.8 Hz, 4H), 2.78 (t, J = 4.8 Hz, 4H), 3.42 (s,
2H), 6.76 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.8
Hz, 2H), 7.43 (m, 4H), 7.76 (m, 6H). MS (ESI): [M]+ = 736.6.
General Procedure F for the Synthesis of Compounds 4a−ao. A
stirred suspension of 8a−t or 12u−ao (0.5 mmol) and hydrazine
monohydrate (58 μL, 1.2 mmol, 1.2 equiv) in absolute EtOH (10 mL)
was refluxed for 1 h. The solvent was then evaporated, the residue
suspended in DCM (10 mL), and the suspension filtered through
Celite. The filtrate was concentrated in vacuo to obtain a residue that
was purified by column chromatography.
[2-Amino-5-methyl-4-[(4-phenylpiperazin-1-yl)methyl]thiophen-
3-yl](4-chlorophenyl)methanone (4a). Following general procedure
F, compound 4a was purified by chromatography eluting with
EtOAc:DCM 1:9 to give a yellow solid. Yield: 96%. Mp: 78−80 °C.
1H NMR (CDCl3): δ 1.98 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H), 2.91 (t, J =
4.8 Hz, 4H), 2.95 (s, 2H), 5.80 (bs, 2H), 6.83 (d, J = 8.4 Hz, 2H), 7.23
(t, J = 8.4 Hz, 3H), 7.35 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H).
MS (ESI): [M]+ = 426.0. Anal. (C23H24ClN3OS): C, H, N.
[2-Amino-4-[(4-benzylpiperazin-1-yl)methyl]-5-methylthiophen-
3-yl](4-chlorophenyl)methanone (4b). Following general procedure
F, compound 4b was purified by chromatography eluting with EtOAc
to give a yellow solid. Yield: 73%. Mp: 48−50 °C.1H NMR (CDCl3):
δ 1.84 (t, J = 5.2 Hz, 4H), 2.18 (s, 3H), 2.86 (t, J = 5.2 Hz, 4H), 3.40
(s, 2H), 3.49 (s, 2H), 5.79 (bs, 2H), 7.26 (m, 5H), 7.33 (d, J = 8.4 Hz,
2H), 7.51 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 440.0. Anal.
(C24H26ClN3OS): C, H, N.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-fluorophenyl)piperazin-1-yl)-
methyl]-5-(4-chlorophenyl)thiophen-2-yl}isoindoline-1,3-dione
(12ah). Following general procedure E using N-(4-fluorophenyl)-
piperazine, compound 12ah was purified by chromatography eluting
with EtOAc:petroleum ether 3:7 to give a white solid. Yield: 68%. Mp:
1
168−170 °C. H NMR (CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H), 2.70 (t,
J = 4.8 Hz, 4H), 3.38 (s, 2H), 6.62 (d, J = 8.6 Hz, 1H), 6.64 (d, J =
8.6 Hz, 1H), 6.88 (t, J = 8.6 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.37 (d,
J = 8.4 Hz, 2H), 7.44 (m, 2H), 7.83 (m, 6H). MS (ESI): [M]+ = 670.6.
2-{3-(4-Chlorobenzoyl)-4-[(4-(4-chlorophenyl)piperazin-1-yl)-
methyl]-5-(4-chlorophenyl)thiophen-2-yl}isoindoline-1,3-dione
(12ai). Following general procedure E using N-(4-chlorophenyl)-
piperazine, compound 12ai was purified by chromatography eluting
with EtOAc:petroleum ether 2:8 to give a white solid. Yield: 78%. Mp:
1
144−145 °C. H NMR (CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H), 2.74 (t,
J = 5.2 Hz, 4H), 3.38 (s, 2H), 6.66 (d, J = 9.0 Hz, 2H), 7.12 (d, J =
9.0 Hz, 2H), 7.33 (m, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.42 (d, J =
7.6 Hz, 2H), 7.76 (m, 6H). MS (ESI): [M]+ = 687.1.
2-{3-(4-Chlorobenzoyl)-4-[(4-(3,4-dichlorophenyl)piperazin-1-yl)-
methyl]-5-(4-chlorophenyl)thiophen-2-yl}isoindoline-1,3-dione
(12aj). Following general procedure E using N-(3,4-dichlorophenyl)-
piperazine, compound 12aj was purified by chromatography eluting
with EtOAc:petroleum ether 3:7 to give a white solid. Yield: 81%. Mp:
1
146−147 °C. H NMR (CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H), 2.82 (t,
J = 4.8 Hz, 4H), 3.42 (s, 2H), 6.64 (dd, J = 9.2 and 3.2 Hz, 1H), 6.68
(d, J = 2.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 2H), 7.43 (m, 5H), 7.83 (m,
6H). MS (ESI): [M]+ = 721.5.
{2-Amino-4-[(4-(4-fluorophenyl)piperazin-1-yl)methyl]-5-methyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4c). Following general
procedure F, compound 4c was purified by chromatography eluting
with EtOAc:DCM 0.5:9.5 to give a yellow solid. Yield: 55%. Mp: 70−
2-{3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-[(4-(4-
(trifluoromethyl)phenyl)piperazin-1-yl)methyl]thiophen-2-yl}-
isoindoline-1,3-dione (12ak). Following general procedure E using N-
(4-(trifluoromethyl)phenyl)piperazine, compound 12ak was purified
by chromatography eluting with EtOAc:petroleum ether 1.5:8.5 to
give a yellow solid. Yield: 78%. Mp: 240−242 °C. 1H NMR (CDCl3):
δ 2.17 (t, J = 4.8 Hz, 4H), 2.88 (t, J = 4.8 Hz, 4H), 3.36 (s, 2H), 6.77
(d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.42 (m, 4H), 7.43 (t, J =
6.8 Hz, 2H), 7.77 (m, 6H). MS (ESI): [M]+ = 720.6.
1
72 °C. H NMR (CDCl3): δ 1.99 (t, J = 4.8 Hz, 4H), 2.23 (s, 3H),
2.83 (t, J = 4.8 Hz, 4H), 2.96 (s, 2H), 5.82 (bs, 2H), 6.76 (d, J = 8.6
Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.94 (t, J = 8.6 Hz, 2H), 7.36 (d, J =
8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 444.1. Anal.
(C23H23ClFN3OS): C, H, N.
{2-Amino-4-[(4-(4-chlorophenyl)piperazin-1-yl)methyl]-5-methyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4d). Following general
procedure F, compound 4d was purified by chromatography eluting
with EtOAc:DCM 1:9 to give a yellow solid. Yield: 62%. Mp: 83−85
2-{3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-[(4-(3-
(trifluoromethyl)phenyl)piperazin-1-yl)methyl]-thiophen-2-yl}-
isoindoline-1,3-dione (12al). Following general procedure E using N-
(3-(trifluoromethyl)phenyl)piperazine, compound 12al was purified
by chromatography eluting with EtOAc:petroleum ether 3:7 to give a
1
°C. H NMR (CDCl3): δ 1.98 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H), 2.87
(t, J = 4.8 Hz, 4H), 2.95 (s, 2H), 5.80 (bs, 2H), 6.73 (d, J = 9.2 Hz,
2H), 7.16 (d, J = 9.2 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4
Hz, 2H). MS (ESI): [M]+ = 460.5. Anal. (C23H23Cl2N3OS): C, H, N.
{2-Amino-4-[(4-(3-chlorophenyl)piperazin-1-yl)methyl]-5-methyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4e). Following general
procedure F, compound 4e was purified by chromatography eluting
with EtOAc:DCM 0.75:9.25 to give a yellow solid. Yield: 56%. Mp:
58−60 °C. 1H NMR (CDCl3): δ 1.96 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H),
2.92 (t, J = 4.8 Hz, 4H), 3.00 (s, 2H), 5.82 (bs, 2H), 6.68 (d, J = 7.6
Hz, 1H), 6.76 (d, J = 7.6 Hz, 1H), 6.78 (s, 1H), 7.12 (t, J = 8.4 Hz,
1H), 7.36 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H). MS (ESI):
[M]+ = 460.4. Anal. (C23H23Cl2N3OS): C, H, N.
1
yellow oil. Yield: 78%. H NMR (CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H),
2.95 (t, J = 4.8 Hz, 4H), 3.39 (s, 2H), 6.82 (d, J = 8.8 Hz, 2H), 6.94 (d,
J = 8.8 Hz, 2H), 7.39 (m, 3H), 7.44 (d, J = 6.8 Hz, 2H), 7.74 (m, 6H).
MS (ESI): [M]+ = 720.6.
2-{3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-[(4-(4-chloro-3-
(trifluoromethyl)phenyl)piperazin-1-yl)methyl]thiophen-2-yl}-
isoindoline-1,3-dione (12am). Following general procedure E using
N-(4-chloro-3-(trifluoromethyl)phenyl)piperazine, compound 12am
was purified by chromatography eluting with EtOAc:petroleum ether
1
2:8 to give a yellow solid. Yield: 76%. Mp: 235−237 °C. H NMR
(CDCl3): δ 2.17 (t, J = 4.8 Hz, 4H), 2.82 (t, J = 4.8 Hz, 4H), 3.039 (s,
2H), 6.82 (dd, J = 8.8 and 1.2 Hz, 1H), 6.92 (d, J = 1.2 Hz, 2H), 7.35
(m, 3H), 7.42 (t, J = 6.8 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.82 (m,
4H). MS (ESI): [M]+ = 755.1.
2-{3-(4-Chlorobenzoyl)-5-(4-chlorophenyl)-4-[(4-(2-
(trifluoromethyl)phenyl)piperazin-1-yl)methyl]thiophen-2-yl}-
isoindoline-1,3-dione (12an). Following general procedure E using
N-(2-(trifluoromethyl)phenyl)piperazine, compound 12an was puri-
fied by chromatography eluting with EtOAc:petroleum ether 3:7 to
{2-Amino-4-[(4-(3,4-dichlorophenyl)piperazin-1-yl)methyl]-5-
methylthiophen-3-yl}(4-chlorophenyl)methanone (4f). Following
general procedure F, compound 4f was purified by chromatography
eluting with EtOAc:DCM 1:9 to give a yellow solid. Yield: 51%. Mp:
63−65 °C. 1H NMR (CDCl3): δ 1.96 (t, J = 4.8 Hz, 4H), 2.22 (s, 3H),
2.88 (t, J = 4.8 Hz, 4H), 2.94 (s, 2H), 5.82 (bs, 2H), 6.63 (dd, J = 9.2
and 2.8 Hz, 1H), 6.86 (d, J = 2.8 Hz, 1H), 7.22 (d, J = 9.2 Hz, 1H),
7.36 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ =
494.9. Anal. (C23H22Cl3N3OS): C, H, N.
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{2-Amino-4-[(4-(4-bromophenyl)piperazin-1-yl)methyl]-5-meth-
ylthiophen-3-yl}(4-chlorophenyl)methanone (4g). Following general
procedure F, compound 4g was purified by chromatography eluting
with EtOAc:DCM 1:9 to give a yellow solid. Yield: 72%. Mp: 73−75
chromatography eluting with EtOAc:DCM 0.5:9.5 to give a yellow
solid. Yield: 54%. Mp: 133−135 °C. 1H NMR (CDCl3): δ 1.98 (t, J =
4.4 Hz, 4H), 2.22 (s, 3H), 2.93 (t, J = 4.4 Hz, 4H), 2.96 (s, 2H), 5.82
(bs, 2H), 6.84 (dd, J = 8.8 and 2.8 Hz, 1H), 7.07 (d, J = 2.8 Hz, 1H),
7.29 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz,
2H). MS (ESI): [M]+ = 528.5. Anal. (C24H22Cl2F3N3OS): C, H, N.
{2-Amino-5-ethyl-4-[(4-phenylpiperazin-1-yl)methyl]thiophen-3-
yl}(4-chlorophenyl)methanone (4p). Following general procedure F,
compound 4p was purified by chromatography eluting with
EtOAc:DCM 1.5:8.5 to give a yellow solid. Yield: 86%. Mp: 104−
106 °C. 1H NMR (CDCl3): δ 1.22 (t, J = 7.6 Hz, 3H), 1.98 (m. 4H),
2.64 (q, J = 7.6 Hz, 2H), 2.92 (m, 4H), 2.96 (s, 2H), 5.78 (bs, 2H),
6.85 (m 3H), 7.23 (m, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.4
Hz, 2H). MS (ESI): [M]+ = 440.1. Anal. (C24H26ClN3OS): C, H, N.
{2-Amino-4-[(4-(4-chlorophenyl)piperazin-1-yl)methyl]-5-ethyl-
thiophen-3-yl}(4-fluorophenyl)methanone (4q). Following general
procedure F, compound 4q was purified by chromatography eluting
with EtOAc:petroleum ether 2:8 to give a yellow solid. Yield: 82%.
Mp: 123−125 °C. 1H NMR (CDCl3): δ 1.21 (t, J = 7.6 Hz, 3H), 1.98
(m, 4H), 2.61 (q, J = 7.6 Hz, 2H), 2.84 (m, 4H), 2.96 (s, 2H), 5.77
(bs, 2H), 6.82 (m, 2H), 6.93 (d J = 9.0 Hz, 2H), 7.34 (d J = 8.6 Hz,
2H), 7.57 (d, J = 8.6 Hz, 2H). MS (ESI): [M]+ = 458.0. Anal.
(C24H25ClFN3OS): C, H, N.
{2-Amino-4-[(4-(4-chlorophenyl)piperazin-1-yl)methyl]-5-ethyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4r). Following general
procedure F, compound 4r was purified by chromatography eluting
with EtOAc:petroleum ether 1.5:8.5 to give a yellow solid. Yield: 69%.
Mp: 115−117 °C. 1H NMR (CDCl3): δ 1.23 (t, J = 7.6 Hz, 3H), 1.97
(m. 4H), 2.61 (q, J = 7.6 Hz, 2H), 2.88 (m, 4H), 2.96 (s, 2H), 5.77
(bs, 2H), 6.72 (d, J = 9.0 Hz, 2H), 7.19 (d, J = 9.0 Hz, 2H), 7.34 (d J =
8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H). MS (ESI): [M]+ = 474.4. Anal.
(C24H25Cl2N3OS): C, H, N.
{2-Amino-5-bromo-4-[(4-phenylpiperazin-1-yl)methyl]thiophen-
3-yl}(4-chlorophenyl)methanone (4s). Following general procedure
F, compound 4s was purified by chromatography eluting with
EtOAc:petroleum ether 3:7 to give a yellow solid. Yield: 61%. Mp:
63−64 °C. 1H NMR (CDCl3): δ 2.04 (d, J = 4.8 Hz, 4H), 2.93 (d, J =
4.8 Hz, 4H), 3.00 (s, 2H), 6.07 (bs, 2H), 6.14 (s, 1H), 6.83 (m, 2H),
7.23 (m, 2H), 7.39 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 7.8 Hz, 2H). MS
(ESI): [M]+ = 490.8, [M + 2]+ = 492.9. Anal. (C22H21BrClN3OS): C,
H, N.
1
°C. H NMR (CDCl3): δ 1.96 (t, J = 5.2 Hz, 4H), 2.22 (s, 3H), 2.87
(t, J = 5.2 Hz, 4H), 2.94 (s, 2H), 5.81 (bs, 2H), 6.68 (d, J = 9.2 Hz,
2H), 7.29 (d, J = 9.2 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.53 (d, J =
8.4 Hz, 2H). MS (ESI): [M]+ = 504.9, [M + 2]+ = 506.9. Anal.
(C23H23BrClN3OS): C, H, N.
{2-Amino-4-[(4-(4-iodophenyl)piperazin-1-yl)methyl]-5-methyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4h). Following general
procedure F, compound 4g was purified by chromatography eluting
with EtOAc:DCM 1:9 to give a yellow solid. Yield: 75%. Mp: 72−73
1
°C. H NMR (CDCl3): δ 1.99 (t, J = 5.2 Hz, 4H), 2.04 (s, 3H), 2.89
(t, J = 5.2 Hz, 4H), 2.98 (s, 2H), 5.80 (bs, 2H), 6.58 (d, J = 8.8 Hz,
2H), 7.36 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4
Hz, 2H). MS (ESI): [M]+ = 551.9. Anal. (C23H23ClIN3OS): C, H, N.
[2-Amino-5-methyl-4-[(4-(4-methylphenyl)piperazin-1-yl)-
methyl]thiophen-3-yl](4-chlorophenyl)methanone (4i). Following
general procedure F, compound 4i was purified by chromatography
eluting with EtOAc:DCM 1:9 to give a yellow solid. Yield: 55%. Mp:
77−79 °C. 1H NMR (CDCl3): δ 1.98 (t, J = 5.2 Hz, 4H), 2.22 (s, 3H),
2.25 (s, 3H), 2.85 (t, J = 5.2 Hz, 4H), 2.94 (s, 2H), 5.81 (bs, 2H), 6.75
(d, J = 8.8 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H),
7.52 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 440.0. Anal.
(C24H26ClN3OS): C, H, N.
{2-Amino-4-[(4-(4-methoxyphenyl)piperazin-1-yl)methyl]-5-
methylthiophen-3-yl}(4-chlorophenyl)methanone (4j). Following
general procedure F, compound 4j was purified by chromatography
eluting with EtOAc:DCM 1.5:8.5 to give a yellow solid. Yield: 67%.
Mp: 63−65 °C. 1H NMR (CDCl3): δ 1.98 (t, J = 5.4 Hz, 4H), 2.21 (s,
3H), 2.80 (t, J = 5.4 Hz, 4H), 2.94 (s, 2H), 3.75 (s, 3H), 5.81 (bs, 2H),
6.81 (s, 4H), 7.34 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H). MS
(ESI): [M]+ = 456.1. Anal. (C24H26ClN3O2S): C, H, N.
{2-Amino-4-[(4-(4-cyanophenyl)piperazin-1-yl)methyl]-5-methyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4k). Following general
procedure F, compound 4k was purified by chromatography eluting
with EtOAc:DCM 1:9 to give a yellow solid. Yield: 54%. Mp: 76−77
1
°C. H NMR (CDCl3): δ 1.96 (t, J = 4.8 Hz, 4H), 2.21 (s, 3H), 2.95
(s, 2H), 3.04 (t, J = 4.8 Hz, 4H), 5.84 (bs, 2H), 6.74 (d, J = 8.8 Hz,
2H), 7.36 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4
Hz, 2H). MS (ESI): [M]+ = 451.0. Anal. (C24H23ClN4OS): C, H, N.
{2-Amino-5-methyl-4-[(4-(4-nitrophenyl)piperazin-1-yl)methyl]-
thiophen-3-yl}(4-chlorophenyl)methanone (4l). Following general
procedure F, compound 4l was purified by chromatography eluting
with EtOAc:DCM 1:9 to give a yellow solid. Yield: 53%. Mp: 85−87
°C. 1H NMR (CDCl3): δ 1.98 (t, J = 5.4 Hz, 4H), 2.331 (s, 3H), 3.08
(s, 2H), 3.11 (t, J = 5.4 Hz, 4H), 6.26 (bs, 2H), 6.70 (d, J = 8.8 Hz,
2H), 6.80 (d, J = 9.2 Hz, 2H), 8.05 (d, J = 9.2 Hz, 2H), 8.13 (d, J = 8.8
Hz, 2H). MS (ESI): [M]+ = 471.0. Anal. (C23H23ClN4O3S): C, H, N.
{2-Amino-5-methyl-4-[(4-(4-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4m). Follow-
ing general procedure F, compound 4m was purified by chromatog-
raphy eluting with EtOAc:DCM 1:9 to give a yellow solid. Yield: 54%.
{2-Amino-5-bromo-4-[(4-(4-fluorophenyl)piperazin-1-yl)methyl]-
thiophen-3-yl}(4-chlorophenyl)methanone (4t). Following general
procedure F, compound 4t was purified by chromatography eluting
1
with EtOAc:petroleum ether 3:7 to give a yellow oil. Yield: 52%. H
NMR (CDCl3): δ 2.03 (t, J = 4.8 Hz, 4H), 2.87 (t, J = 4.8 Hz, 4H),
3.00 (s, 2H), 6.07 (bs, 2H), 6.14 (d, J = 8.6 Hz, 1H), 6.79 (t, J = 8.6
Hz, 1H), 6.88 (t, J = 8.6 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.54 (d, J =
8.4 Hz, 2H). MS (ESI): [M]+ = 508.8, [M + 2]+ = 510.9. Anal.
(C22H20BrClFN3OS): C, H, N.
{2-Amino-4-[(4-phenylpiperazin-1-yl)methyl]-5-phenylthiophen-
3-yl}(4-chlorophenyl)methanone (4u). Following general procedure
F, compound 4u was purified by chromatography eluting with
EtOAc:petroleum ether 3:7 to give a yellow solid. Yield: 46%. Mp:
74−76 °C. 1H NMR (CDCl3): δ 1.91 (t, J = 4.8 Hz, 4H), 2.87 (t, J =
4.8 Hz, 4H), 3.12 (s, 2H), 5.79 (bs, 2H), 6.78 (d, J = 8.4 Hz, 2H), 7.21
(7, J = 8.4 Hz, 2H), 7.32 (d, J = 7.2 Hz, 2H), 7.41 (m, 6H), 7.67 (d,
J = 8.4 Hz, 2H). MS (ESI): [M]+ = 488.1. Anal. (C28H26ClN3OS): C,
H, N.
1
Mp: 76−78 °C. H NMR (CDCl3): δ 2.04 (t, J = 4.8 Hz, 4H), 2.22
(s, 3H), 2.95 (s, 2H), 3.00 (t, J = 4.8 Hz, 4H), 5.82 (bs, 2H), 6.82
(d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H),
7.53 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 494.0. Anal.
(C24H23ClF3N3OS): C, H, N.
{2-Amino-4-[4-[(4-fluorophenyl)piperazin-1-yl]methyl]-5-phenyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4v). Following general
procedure F, compound 4v was purified by chromatography eluting
with EtOAc:petroleum ether 3:7 to give a yellow solid. Yield: 88%.
Mp: 70−71 °C. 1H NMR (CDCl3): δ 1.91 (t, J = 4.8 Hz, 4H), 2.79 (t,
J = 4.8 Hz, 4H), 3.12 (s, 2H), 5.82 (bs, 2H), 6.76 (dd, J = 8.8 and
4.8 Hz, 2H), 6.90 (t, J = 8.4 Hz, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7.39
(m, 5H), 7.65 (d, J = 8.0 Hz, 2H). MS (ESI): [M]+ = 506.1. Anal.
{2-Amino-5-methyl-4-[(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4n). Follow-
ing general procedure F, compound 4n was purified by chromatog-
raphy eluting with EtOAc:DCM 0.5:9.5 to give a yellow solid. Yield:
1
68%. Mp: 60−61 °C. H NMR (CDCl3): δ 1.99 (t, J = 5.2 Hz, 4H),
2.22 (s, 3H), 2.96 (m, 6H), 5.81 (bs, 2H), 6.96 (d, J = 7.6 Hz, 1H),
7.00 (s, 1H), 7.03 (d, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.37 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 494.1.
Anal. (C24H23ClF3N3OS): C, H, N.
(C28H25FClN OS): C, H, N.
3
{2-Amino-4-[(4-(4-chlorophenyl)piperazin-1-yl)methyl]-5-phenyl-
thiophen-3-yl}(4-chlorophenyl)methanone (4w). Following general
procedure F, compound 4w was purified by chromatography eluting
{2-Amino-4-[(4-(4-chloro-3-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]-5-methylthiophen-3-yl}(4-chlorophenyl)methanone
(4o). Following general procedure F, compound 4o was purified by
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Journal of Medicinal Chemistry
Article
with EtOAc:petroleum ether 3:7 to give a yellow solid. Yield: 67%.
Mp: 79−80 °C. 1H NMR (CDCl3): δ 1.89 (t, J = 4.8 Hz, 4H), 2.02 (t,
J = 4.8 Hz, 4H), 2.11 (s, 2H), 5.83 (bs, 2H), 6.71 (d, J = 9.2 Hz, 2H),
7.16 (d, J = 9.2 Hz, 2H), 7.34 (m, 7H), 7.64 (d, J = 8.4 Hz, 2H). MS
(ESI): [M]+ = 522.5. Anal. (C28H25Cl2N3OS): C, H, N.
(dd, J = 8.8 and 4.8 Hz, 2H), 6.93 (t, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8
Hz, 2H), 7.24 (m, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz,
2H). MS (ESI): [M]+ = 524.1. Anal. (C28H24ClF2N3OS): C, H, N.
[2-Amino-5-(4-chlorophenyl)-4-[(4-phenylpiperazin-1-yl)methyl]-
thiophen-3-yl](4-chlorophenyl)methanone (4af). Following general
procedure F, compound 4af was purified by chromatography eluting
with EtOAc:petroleum ether 3:7 to give a yellow solid. Yield: 74%.
Mp: 90−91 °C. 1H NMR (CDCl3): δ 1.88 (t, J = 5.0 Hz, 4H), 2.87 (t,
J = 5.0 Hz, 4H), 3.08 (s, 2H), 5.86 (bs, 2H), 6.79 (d, J = 8.4 Hz, 2H),
6.82 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.27 (m, 3H), 7.37
(d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 522.5.
Anal. (C28H25Cl2N3OS): C, H, N.
[2-Amino-4-[(4-benzylpiperazin-1-yl)methyl]-5-(4-chlorophenyl)-
thiophen-3-yl]-(4-chlorophenyl)methanone (4ag). Following gen-
eral procedure F, compound 4ag was purified by chromatography
eluting with EtOAc:petroleum ether 3:7 to give a yellow oil. Yield:
68%. 1H NMR (CDCl3): δ 1.82 (t, J = 5.2 Hz, 4H), 2.42 (t, J = 5.2 Hz,
4H), 3.06 (s, 2H), 3.54 (s, 2H), 5.78 (bs, 2H), 7.17 (d, J = 8.6 Hz,
2H), 7.26 (m, 5H), 7.32 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H),
7.63 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 536.5. Anal.
(C29H27Cl2N3OS): C, H, N.
{2-Amino-5-(4-chlorophenyl)-4-[(4-(4-fluorophenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ah). Follow-
ing general procedure F, compound 4ah was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 63%. Mp: 90−91 °C. 1H NMR (CDCl3): δ 1.85 (t,
J = 4.8 Hz, 4H), 2.78 (t, J = 4.8 Hz, 4H), 3.08 (s, 2H), 5.82 (bs, 2H),
6.74 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.90 (t, J = 8.6 Hz,
2H), 7.26 (m, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H),
7.67 (d, J = 8.6 Hz, 2H). MS (ESI): [M]+ = 540.5. Anal.
(C28H24Cl2FN3OS): C, H, N.
{2-Amino-4-[(4-(3,4-dichlorophenyl)piperazin-1-yl)methyl]-5-
phenylthiophen-3-yl}(4-chlorophenyl)methanone (4x). Following
general procedure F, compound 4x was purified by chromatography
eluting with EtOAc:petroleum ether 3:7 to give a yellow solid. Yield:
1
62%. Mp: 70−71 °C. H NMR (CDCl3): δ 1.85 (t, J = 5.0 Hz, 4H),
2.88 (t, J = 5.0 Hz, 4H), 3.12 (s, 2H), 5.82 (bs, 2H), 6.58 (dd, J = 9.0
and 3.0 Hz, 1H), 6.81 (d, J = 3.0 Hz, 1H), 7.18 (d, J = 9.0 Hz, 1H),
7.32 (d, J = 8.4 Hz, 2H), 7.39 (m, 5H), 7.64 (d, J = 8.2 Hz, 2H). MS
(ESI): [M]+ = 556.9. Anal. (C28H24Cl3N3OS): C, H, N.
{2-Amino-5-phenyl-4-[(4-(4-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4y). Follow-
ing general procedure F, compound 4y was purified by chromatog-
raphy eluting with EtOAc:petroleum ether 3:7 to give a yellow solid.
Yield: 65%. Mp: 90−91 °C. 1H NMR (CDCl3): δ 1.89 (t, J = 4.8 Hz,
4H), 2.95 (t, J = 4.8 Hz, 4H), 3.12 (s, 2H), 5.83 (bs, 2H), 6.76 (d, J =
8.4 Hz, 2H), 7.39 (m, 9H), 7.68 (d, J = 8.4 Hz, 2H). MS (ESI):
[M]+ = 556.1. Anal. (C29H25ClF3N3OS): C, H, N.
{2-Amino-5-phenyl-4-[(4-(3-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4z). Follow-
ing general procedure F, compound 4z was purified by chromatog-
raphy eluting with EtOAc:petroleum ether 3:7 to give a yellow solid.
Yield: 67%. Mp: 77−78 °C. 1H NMR (CDCl3): δ 1.92 (t, J = 4.8 Hz,
4H), 2.92 (t, J = 4.8 Hz, 4H), 3.12 (s, 2H), 5.82 (bs, 2H), 6.82 (d, J =
8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 7.36 (m, 7H), 7.63 (d, J =
8.4 Hz, 2H). MS (ESI): [M]+ = 556.1. Anal. (C29H25ClF3N3OS): C, H, N.
{2-Amino-4-[(4-(4-chloro-3-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]-5-phenylthiophen-3-yl}(4-chlorophenyl)methanone
(4aa). Following general procedure F, compound 4aa was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 63%. Mp: 80−82 °C. 1H NMR (CDCl3): δ 1.89 (t,
J = 4.8 Hz, 4H), 2.87 (t, J = 4.8 Hz, 4H), 3.12 (s, 2H), 5.82 (bs, 2H),
6.84 (d, J = 8.4 and 2.4 Hz, 1H), 7.02 (d, J = 2.4 Hz, 1H), 7.36 (m,
6H), 7.43 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H). MS (ESI):
[M]+ = 590.5. Anal. (C29H24Cl2F3N3OS): C, H, N.
{2-Amino-4-[(4-(4-chlorophenyl)piperazin-1-yl)methyl]-5-(4-
chlorophenyl)thiophen-3-yl}(4-chlorophenyl)methanone (4ai). Fol-
lowing general procedure F, compound 4ai was purified by chro-
matography eluting with EtOAc:petroleum ether 3:7 to give a yellow
1
solid. Yield: 74%. Mp: 77−78 °C. H NMR (CDCl3): δ 1.89 (t, J =
4.8 Hz, 4H), 2.81 (t, J = 4.8 Hz, 4H), 3.09 (s, 2H), 5.80 (bs, 2H), 6.70
(d, J = 9.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H),
7.34 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.6 Hz,
2H). MS (ESI): [M]+ = 556.8. Anal. (C28H24Cl3N3OS): C, H, N.
{2-Amino-5-(4-chlorophenyl)- 4-[(4-(3,4-dichlorophenyl)-
piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone
(4aj). Following general procedure F, compound 4aj was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 64%. Mp: 108−110 °C. 1H NMR (CDCl3): δ 2.17
(t, J = 4.8 Hz, 4H), 2.82 (t, J = 4.8 Hz, 4H), 3.22 (s, 2H), 5.83 (bs,
2H), 6.62 (dd, J = 9.2 and 2.8 Hz, 2H), 6.82 (d, J = 2.8 Hz, 1H), 7.11
(d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 7.4 Hz, 2H),
7.68 (d, J = 7.4 Hz, 2H). MS (ESI): [M]+ = 591.4. Anal.
(C28H23Cl4N3OS): C, H, N.
{2-Amino-5-phenyl-4-[(4-(2-(trifluoromethyl)phenyl)piperazin-1-
yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ab). Follow-
ing general procedure F, compound 4ab was purified by chromatog-
raphy eluting with EtOAc:petroleum ether 3:7 to give a yellow solid.
Yield: 67%. Mp: 80−81 °C. 1H NMR (CDCl3): δ 1.89 (t, J = 4.8 Hz,
4H), 2.61 (t, J = 4.8 Hz, 4H), 3.12 (s, 2H), 5.75 (bs, 2H), 6.84 (d, J =
8.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.46 (m, 8H), 7.70 (d, J = 8.4
Hz, 2H). MS (ESI): [M]+ = 556.2. Anal. (C29H25ClF3N3OS): C, H, N.
{2-Amino-5-phenyl-4-[(4-(4-(trifluoromethoxy)phenyl)piperazin-
1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone (4ac). Fol-
lowing general procedure F, compound 4ac was purified by chro-
matography eluting with EtOAc:petroleum ether 3:7 to give a yellow
{2-Amino-5-(4-chlorophenyl)-4-[(4-(4-(trifluoromethyl)phenyl)-
piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone
(4ak). Following general procedure F, compound 4ak was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 72%. Mp: 75−77 °C. 1H NMR (CDCl3): δ 1.89 (t,
J = 4.8 Hz, 4H), 2.94 (t, J = 4.8 Hz, 4H), 3.09 (s, 2H), 5.82 (bs, 2H),
6.78 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.28 (m, 2H), 7.34
(d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H).
MS (ESI): [M]+ = 556.1. Anal. (C29H25ClF3N3OS): C, H, N.
{2-Amino-5-(4-chlorophenyl)-4-[(4-(3-(trifluoromethyl)phenyl)-
piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone
(4al). Following general procedure F, compound 4al was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 62%. Mp: 94−96 °C. 1H NMR (CDCl3): δ 2.02 (t,
J = 4.8 Hz, 4H), 2.92 (t, J = 4.8 Hz, 4H), 3.04 (s, 2H), 5.82 (bs, 2H),
6.84 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 7.44 (m, 6H),
7.63 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 556.1. Anal.
(C29H25ClF3N3OS): C, H, N.
1
solid. Yield: 64%. Mp: 68−69 °C. H NMR (CDCl3): δ 1.87 (t, J =
4.8 Hz, 4H), 2.84 (t, J = 4.8 Hz, 4H), 3.11 (s, 2H), 5.82 (bs, 2H), 6.75
(d, J = 9.0 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 7.33 (m, 3H), 7.38 (d,
J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H). MS
(ESI): [M]+ = 572.1. Anal. (C29H25ClF3N3O2S): C, H, N.
{2-Amino-4-[(4-phenylpiperazin-1-yl)methyl]-5-(4-fluorophenyl)-
thiophen-3-yl}(4-chlorophenyl)methanone (4ad). Following general
procedure F, compound 4ad was purified by chromatography eluting
with EtOAc:petroleum ether 2:8 to give a yellow solid. Yield: 53%.
Mp: 113−114 °C. 1H NMR (CDCl3): δ 1.89 (t, J = 4.8 Hz, 4H), 2.87
(t, J = 4.8 Hz, 4H), 3.05 (s, 2H), 5.79 (bs, 2H), 6.80 (d, J = 8.4 Hz,
2H), 7.07 (t, J = 8.4 Hz, 2H), 7.21 (t, J = 8.4 Hz, 2H), 7.22 (m, 3H),
7.28 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H). MS (ESI): [M]+ =
506.1. Anal. (C28H25ClFN3OS): C, H, N.
{2-Amino-4-[(4-(4-fluorophenyl)piperazin-1-yl)methyl]-5-(4-
fluorophenyl)thiophen-3-yl}(4-chlorophenyl)methanone (4ae). Fol-
lowing general procedure F, compound 4ae was purified by chro-
matography eluting with EtOAc:petroleum ether 2:8 to give a yellow
solid. Yield: 86%. Mp: 105−107 °C. 1H NMR (CDCl3): δ 1.89 (t, J =
4.8 Hz, 4H), 2.78 (t, J = 4.8 Hz, 4H), 3.06 (s, 2H), 5.82 (bs, 2H), 6.74
{2-Amino-5-(4-chlorophenyl)-4-[(4-(4-chloro-3-(trifluoromethyl)-
phenyl)piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)-
methanone (4am). Following general procedure F, compound 4am
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Journal of Medicinal Chemistry
Article
was purified by chromatography eluting with EtOAc:petroleum ether
3:7 to give a yellow solid, yield: 63%. Mp: 170−172 °C. H NMR
(CDCl3): δ 1.89 (t, J = 4.8 Hz, 4H), 2.89 (t, J = 4.8 Hz, 4H), 3.09 (s,
2H), 5.84 (bs, 2H), 6.82 (dd, J = 8.4 and 1.2 Hz, 2H), 7.00 (d, J = 1.2
Hz, 2H), 7.27 (m, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz,
2H), 7.63 (d, J = 8.4 Hz, 2H). MS (ESI): [M]+ = 624.9. Anal.
(C29H23Cl3F3N3OS): C, H, N.
different tested compounds at a concentration of 10 μM on the CCPA
curve (0.01 nM −1 μM) was investigated.22 Nonspecific binding was
defined as binding in the presence of 1 μM DPCPX.
1
Assay of the Antagonist Activity versus A1-, A2A- and A3ARs. A1-,
A2A-, and A3AR competition binding experiments were performed using
1 nM [3H]DPCPX, 1 nM [3H]ZM 241385, and 2 nM [3H]MRE-3008-
F20 as radioligands, respectively.22−24 Membrane suspensions were
incubated in 50 mM Tris HCl (pH 7.4) at 25 °C for 120 min, in 50 mM
Tris HCl, 10 mM MgCl2 (pH 7.4) at 4 °C for 60 min, and in 50 mM
Tris HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4) at 4 °C for 120 min
to study A1-, A2A-, and A3ARs, respectively. Nonspecific binding was
defined as the binding in the presence of 1 μM DPCPX or ZM 241385
or MRE-3008-F20 for A1-, A2A-, and A3ARs, respectively. Inhibition was
expressed as percentage of control specific binding (100%). Test agents
were dissolved in DMSO and added to the assay from a 100-fold concen-
trated solution in DMSO. Control incubations also contained 1% DMSO.
Bound and free radioactivity were separated by filtering the assay
mixture through Whatman GF/B glass fiber filters using a Brandel cell
{2-Amino-5-(4-chlorophenyl)-4-[(4-(2-(trifluoromethyl)phenyl)-
piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone
(4an). Following general procedure F, compound 4an was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
1
yellow oil. Yield: 67%. H NMR (CDCl3): δ 1.88 (t, J = 4.8 Hz, 4H),
2.62 (t, J = 4.8 Hz, 4H), 3.08 (s, 2H), 5.77 (bs, 2H), 7.22 (m, 4H),
7.34 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz,
2H), 7.73 (d, J = 8.2 Hz, 2H). MS (ESI): [M]+ = 556.0. Anal.
(C29H25ClF3N3OS): C, H, N.
{2-Amino-5-(4-chlorophenyl)-4-[(4-(4-(trifluoromethoxy)phenyl)-
piperazin-1-yl)methyl]thiophen-3-yl}(4-chlorophenyl)methanone
(4ao). Following general procedure F, compound 4ao was purified by
chromatography eluting with EtOAc:petroleum ether 3:7 to give a
yellow solid. Yield: 64%. Mp: 90−91 °C. 1H NMR (CDCl3): δ 1.89 (t,
J = 4.8 Hz, 4H), 2.85 (t, J = 4.8 Hz, 4H), 3.09 (s, 2H), 5.80 (bs, 2H),
6.75 (d, J = 9.4 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 7.22 (m, 2H), 7.34
(d, J = 8.6 Hz, 2H), 7.42 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H).
MS (ESI): [M]+ = 606.5. Anal. (C29H24Cl2F3N3O2S): C, H, N.
Biology Experiments. Materials. [3H]DPCPX ([3H]-1,3-diprop-
yl-8-cyclopentylxanthine; specific activity, 120 Ci/mmol) and
[3H]CCPA ([3H]-2-chloro-N6-cyclopentyladenosine; specific activity,
55 Ci/mmol) were obtained from Perkin-Elmer Research Products
(Boston, MA); [3H]ZM 241385 ([3H](4-(2-[7-amino-2-(2-furyl)-
[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol); specific
activity, 17 Ci/mmol) was obtained from Biotrend (Cologne, Germany);
[3H]MRE-3008-F20 ([3H]-5-N-(4-methoxyphenylcarbamoyl)amino-8-
propyl-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; specific
activity, 67 Ci/mmol) was obtained from Amersham International
(Buckinghamshire, UK). DPCPX (1,3-dipropyl-8-cyclopentylxanthine),
R-PIA ((R)-N6-(L-2-phenylisopropyl)adenosine), and CPA (N6-cyclo-
pentyladenosine) were obtained from Sigma (St. Louis, MO). All other
reagents were of analytical grade and obtained from commercial sources.
Cell Membrane Preparation. The hA1CHO, hA2ACHO, and
hA3CHO cells were grown adherently and maintained in Dulbecco’s
modified Eagle’s medium with nutrient mixture F12, containing 10%
fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL),
L-glutamine (2 mM), geneticine (G418; 0.2 mg/mL) at 37 °C in 5%
CO2/95% air. For membrane preparation the culture medium was
removed, and the cells were washed with phosphate-buffered saline
and scraped off T75 flasks in ice-cold hypotonic buffer (5 mM Tris
HCl, 1 mM EDTA, pH 7.4). The cell suspension was homogenized
with a Polytron, the homogenate was spun for 10 min at 1000g, and
the supernatant was then centrifuged for 30 min at 100 000g. The
membrane pellet was suspended in 50 mM Tris HCl buffer (pH 7.4)
for A1ARs; in 50 mM Tris HCl, 10 mM MgCl2 (pH 7.4) for A2AARs;
and in 50 mM Tris HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4)
for A3ARs. The membranes were incubated with 2−3 IU/mL of
adenosine deaminase to reduce the endogenous adenosine. The pro-
tein concentration was determined according to a Bio-Rad method
with bovine albumin as a standard reference.21
harvester (Brandel Instruments, Unterfohring, Germany). The filter
bound radioactivity was counted by a Packard Tri Carb 2810 TR
scintillation counter (Perkin-Elmer).
̈
Effect of the Novel Compounds in Cyclic AMP Assays. Human A1
CHO cells (106 cells/mL) were prepared as described above and were
suspended in 0.5 mL incubation mixture phosphate buffer, containing
1.0 IU adenosine deaminase/mL and 0.5 mM 4-(3-butoxy-4-
methoxybenzyl)-2-imidazolidinone (Ro 20−1724) as a phosphodies-
terase inhibitor and preincubated for 10 min in a shaking bath at
37 °C. The effect of allosteric enhancers were studied at 10 μM con-
centration that was added to the mixture for a further 10 min. The
effect of allosteric enhancers (100 nM) was also studied in the pre-
sence of a low concentration of CCPA (1 pM). Forskolin (1 μM) was
added for 5 min and was used to stimulate the activity of adenylate
cyclase. The reaction was terminated by the addition of cold 6%
trichloroacetic acid (TCA). The TCA suspension was centrifuged at
2000g for 10 min at 4 °C and the supernatant was extracted four times
with water-saturated diethyl ether. The final aqueous solution was
tested for cAMP levels by a competition protein binding assay.24
Samples of cAMP standards (0−10 pmol) were added to each test
tube containing Trizma base (0.1 M), aminophylline (8.0 mM), mer-
captoethanol (6.0 mM) (pH 7.4), and [3H]cAMP (at the final con-
centration of 1 nM). The binding protein, previously prepared from
beef adrenals, was added to the samples and incubated at 4 °C for
150 min. At the end of the incubation time and after the addition of
charcoal, the samples were centrifuged at 2000g for 10 min. The clear
supernatant was mixed with 4 mL of Ultima Gold (Perkin-Elmer) and
counted in a Packard Tri Carb 2810 TR scintillation counter (Perkin-
Elmer).
Data Analysis. Saturation and competition binding experiments
were analyzed with the program LIGAND, which performed a weighted,
nonlinear, least-squares curve fitting.25 Inhibitory binding constants, Ki,
were also calculated from the IC50 values according to the Cheng and
Prusoff equation Ki = IC50/(1 + [C*]/KD*), where [C*] is the
concentration of the radioligand and KD* its dissociation constant.26
All experimental data are expressed as mean standard error of the
mean (SEM.) of three or four independent experiments performed in
duplicate.
Binding Experiments in hA1CHO Membranes. [3H]CCPA
Binding Experiments. Saturation binding experiments of [3H]CCPA
(0.05−20 nM) to hA1CHO membranes were performed in triplicate
at 25 °C for 90 min in 50 mM Tris-HCl, pH 7.4, in the absence
and presence of the tested compounds at the final concentration of
10 μM.18a Binding experiments were carried out in triplicate in a final
volume of 250 μL containing diluted membranes, 1 nM [3H]CCPA,
50 mM Tris-HCl (pH 7.4), and the tested compounds at 10 μM
concentration for 90 min at 25 °C.18a Nonspecific binding was defined
as binding in the presence of 1 μM R-PIA.
AUTHOR INFORMATION
Corresponding Author
■
*R.R.: phone, 39-(0)532455303; fax, 39-(0)532455953; e-mail,rmr@
unife.it. P.G.B.: phone, 39-(0)532455293; fax, 39-(0)532-455953;
e-mail, baraldi@unife.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We wish to thank Dr. Alberto Casolari and Dr.ssa Elisa Durini
for technical assistance.
[3H]DPCPX Competition Binding Experiments. Competition bind-
ing experiments of 1 nM [3H]DPCPX were performed in triplicate in
50 mM Tris-HCl (pH 7.4) for 90 min at 25 °C. The effect of the
■
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Journal of Medicinal Chemistry
Article
446−454. (b) Kourounakis, A. P.; Visser, C.; De Groote, M.; IJzerman,
A. P. Differential effects of the allosteric enhancer (2-amino-4,5-
dimethyl-thienyl)][3-(trifluoromethyl)phenyl]-methanone (PD
81,723) on agonist and antagonist binding and function at the
human wild-type and a mutant (T277A) adenosine A1 receptor.
Biochem. Pharmacol. 2001, 61, 137−144.
ABBREVIATIONS USED
■
GPCRs, G protein-coupled receptors; [3H]DPCPX, [3H]1,3-
dipropyl-8-cyclopentylxanthine; [3H]MRE-3008F20, [3H]-5-N-
(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl)-
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; [3H]CCPA,
[3H]-2-chloro-N6-cyclopentyladenosine; [3H]ZM 241385,
[3H](4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]-
triazin-5-ylamino]ethyl)phenol); CPA, N6-cyclopentyladeno-
sine; CHO, Chinese hamster ovary; cAMP, adenosine 3′,5′-
cyclic monophosphate; AEs, allosteric enhancers; NBS, N-
bromosuccinimide; PdCl2(DPPF), [1,1′-bis(diphenylphosphino)-
ferrocene]dichloropalladium(II) complex with dichloromethane;
CsF, cesium fluoride; EWG, electron-withdrawing group; ERG,
electron-releasing group; CNS, central nervous system.
(11) Goblyos, A.; IJzerman, A. P. Allosteric modulation of adenosine
̈
̈
receptors. Purinergic Signalling 2009, 5, 51−61.
(12) (a) Van der Klein, P. A. M.; Kourounakis, A. P.; IJzerman, A. P.
Allosteric modulation of the adenosine A1 receptor. Synthesis and
biological evaluation of novel 2-amino-3-benzoylthiophenes as
allosteric enhancers of agonist binding. J. Med. Chem. 1999, 42,
3629−3635. (b) Baraldi, P. G.; Zaid, A. N.; Lampronti, I.; Fruttarolo,
F.; Pavani, M. G.; Tabrizi, M. A.; Shryock, J. C.; Leung, E.; Romagnoli,
R. Synthesis and biological effects of a new series of 2-amino-3-
benzoylthiophenes as allosteric enhancers of A1 adenosine receptor.
Bioorg. Med. Chem. Lett. 2000, 10, 1953−1957. (c) Tranberg, C. E.;
Zickgraf, A.; Giunta, B. N.; Luetjens, H.; Figler, H.; Murphree, L. J.;
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