Synthesis and biological effects of novel 2-amino-3-naphthoylthiophenes as allosteric enhancers of the A1, adenosine receptor

Article abstract of DOI:10.1021/jm0210212

The current study describes the synthesis and biological evaluation of a novel series of 2-amino-3-naphthoylthiophenes, with variable modifications at the 4- and 5-position of the thiophene as well as the naphthoyl ring. Allosteric enhancer activity was m

Full text of DOI:10.1021/jm0210212

794
J . Med. Chem. 2003, 46, 794-809
Syn th esis a n d Biologica l Effects of Novel 2-Am in o-3-n a p h th oylth iop h en es a s
Alloster ic En h a n cer s of th e A1 Ad en osin e Recep tor
Pier Giovanni Baraldi,*,† Romeo Romagnoli,† Maria Giovanna Pavani,† Maria del Carmen Nun˜ez,†
Mojgan Aghazadeh Tabrizi,† J ohn C. Shryock,‡ Edward Leung,§ Allan R. Moorman,§ Canan Uluoglu,|
Valeria Iannotta, Stefania Merighi, and Pier Andrea Borea
Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara, 44100 Ferrara, Italy, Department of Medicine, University of
Florida, Gainesville, Florida, King Pharmaceuticals Research and Development Inc., Cary, North Carolina 27513, Department
of Pharmacology, Gazi University, Medical Faculty, Ankara, Turkey, and Dipartimento di Medicina Clinica e Sperimentale,
Sezione di Farmacologia, Universita` di Ferrara, 44100 Ferrara, Italy
Received August 6, 2002
The current study describes the synthesis and biological evaluation of a novel series of 2-amino-
3-naphthoylthiophenes, with variable modifications at the 4- and 5-position of the thiophene
as well as the naphthoyl ring. Allosteric enhancer activity was measured in several ways: (1)
evaluating the effect on forskolin-stimulated cAMP accumulation in the presence of an
A1-adenosine agonist (CPA) in Chinese hamster ovary (CHO) cells expressing the cloned
human A1-adenosine receptor (hA1AR); (2) ability of these compounds to displace the bind-
ing of [3H]DPCPX, [3H]ZM 241385, and [3H]MRE 3008F20 to the ligand binding site of CHO
cells expressing the hA1, hA2A, and hA3 adenosine receptors, respectively; (3) effect on the
binding of [3H]CCPA to membranes from CHO cells expressing hA1AR, to rat brain and human
cortex membrane preparations containing native adenosine A1 receptors; (4) kinetics of the
dissociation of [3H]CCPA from CHO-hA1 membranes. The pharmacological assays compared
the various activities to that of the reference compound PD 81,723 (compound 1). Several
compounds appeared to be better than PD 81,723 to enhance the effect of CPA (and thus reduce
cAMP content) in the CHO:hA1 assay. The effect of these compounds at a concentration of 10
µM was slightly greater than that of the same concentration of the PD 81,723 and substantially
greater than that of PD 81,723 when responses to 1 µM of each compound were compared.
These include compounds 23, 25-29, 31-34, 38, 39, 43, and 58. Cycloalkylthiophenes tended
to be more potent then their 4,5-dimethyl analogues, and in the series of cycloalkylthiophenes,
tetrahydrobenzo[b]thiophene derivatives appeared to be more potent than the dihydrocyclo-
pentadien[b]thiophene counterparts. Some of the most potent compounds were tested at a
concentration of 10 µM for their affinity as competitors to the antagonist binding site of CHO
cells expressing hA1, hA2A, and hA3 adenosine receptors. None inhibited binding at the hA2A-
AR, but most of them inhibited binding to the hA1AR to varying extents (0-19%) as well as to
the hA3AR to a substantial degree (0-57%). At a concentration of 10µM, the compounds 31,
34, 37, 38, and 39 were more active than PD 81,723 to increase the binding of [3H]CCPA to
CHO:hA1, human brain and rat cortex membranes. Compound 37 was the most active compound
increasing the binding to CHO:hA1, human brain, and rat cortex membranes by 149, 43, and
27%, respectively (51, 15, and 22%, respectively, for PD 81,723). A good correlation was found
between the increments [3H]CCPA binding to A1 receptors expressed in different systems. Unlike
the effect on agonist binding, the tested compounds did not increase the binding of the
antagonist [3H]DPCPX on hCHO-A1 membranes. Ligand dissociation studies revealed that two
compounds (22 and 39) were more potent than 1 to slow the dissociation of [3H]CCPA from
CHO:hA1AR membranes. No clear-cut structure-activity relationship can be observed based
on data from the functional assay, but we have identified several compounds, in particular 37
and 39, which appeared to be more potent than 1 and that may be selected for further
development.
In tr od u ction
taneously or sequentially: the binding of one ligand
affects the binding of another ligand. The flexible nature
of the interaction between the receptors and various
allosteric modulators, together with the potential for
subtype selectivity, make allosteric sites attractive for
therapeutic intervention.1 In the case of the GABAA
receptor, which is a transmitter-gated ion channel, the
benzodiazepines acting on an allosteric site on the
receptor showed substantial therapeutic effects and
acceptable side effects.2
Allosteric effects are observed when there are interac-
tions between two binding processes that occur simul-
* To whom correspondence should be addressed., Tel: +39-(0)532-
291293, FAX: +39-(0)532-291296, E-mail: pgb@ifeuniv.unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ University of Florida.
§ King Pharmaceuticals Research and Development Inc.
| Gazi University.
Dipartimento di Medicina Clinica e Sperimentale, Universita` di
Ferrara.
10.1021/jm0210212 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/28/2003

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 795
Adenosine is an ubiquitous autocoid with multiple
effects, which exerts its actions on the human body
interacting with four different P1-purinoreceptor sub-
types classified as A1, A2A, A2B, and A3.3 These receptor
subtypes belong to the superfamily of G-protein-coupled
receptors and are widely distributed throughout the
body.4 Extracellular adenosine, as a breakdown product
of ATP, protects tissues from ischemic damage by
lowering oxygen demand and increasing oxygen supply.
Agents that increase the activation of A1-adenosine
receptor in response to adenosine would be useful in
conditions characterized by a localized oxygen deficit,
such as angina, myocardial infarction, and stroke.5
PD 81,723 showed to enhance agonist binding and the
functional activation of the A1 receptor in both brain
and cardiovascular tissues.15 While the exact molecular
mechanism(s) through which PD 81,723 exerts its allo-
steric actions remain unknown, the available data indi-
cate that PD 81,723 functions to stabilize a high affinity
or agonist-preferring state of the A1 receptor.1c,14b,16
The adenosine A1 receptor is coupled to Gi/Go protein
signal transduction pathways and may mediate a
number of biochemical processes such as the activation
of several types of K+ channels, inactivation of N-, P-,
and Q-type Ca2+ channels, inhibition of adenylyl cy-
clase,6 and activation of phospholipase C.
To study the role of various substitutions on the
phenyl ring and the importance of the 4,5-dimethyl
group on the thienyl ring, Baraldi,17 IJ zerman,18 and
Tranberg19 have described the synthesis and biological
evaluation of mostly novel PD 81,723 analogues. It was
evident from previous SAR studies that substitution
with electron-withdrawing substituents, such as chlo-
rine and trifluoromethyl, on the benzoyl moiety at the
3-position of the thiophene ring resulted in higher
enhancement activity.17-19
A variety of adenosine-mediated effects occurs via the
A1 adenosine receptors, highly and extensively ex-
pressed in the central nervous system (CNS), and in
other tissues such as kidney, lung, bladder, and heart.4
Thus, it would be of great therapeutic importance to
have compounds that are able to enhance the activation
of A1-adenosine receptors by the endogenous ligand,
adenosine, within specific target tissues. Such an op-
portunity of intervention is provided by the concept of
allosteric modulation of G-protein-coupled receptors
(GPCRs). This concept, investigated also in other classes
of GPCRs, such as the muscarinic,1b,1d adrenergic,7 D2
The purpose of our investigation was to synthesize
and evaluate the allosteric enhancer activity of a new
series of 52 derivatives of PD 81,723 (compounds 22-
73 and 77), where various modifications both on the
naphthalene ring and on the 4- and 5-position of the
thiophene system occurred, and to establish the struc-
tural requirements and the structure-activity relation-
ship for enhancement of the action of an agonist at the
human A1-adenosine receptor. Previous studies by
Bruns indicated that the addition of a fused ring on the
thiophene provided improved allosteric enhancer activ-
ity. We sought to examine if this trend would also be
observed with the more complex naphthoyl derivatives.
Moreover, in the same series of 2-amino-3-benzoylthio-
phenes,14a,b the receptor environment neighboring the
benzoyl binding site is lipophilic, and for this reason the
substituents at the 4-position on the naphthalene were
varied from H to groups with a different degree of
lipophilicity (CH3, CH3O, Cl, Br, and I), and at 6-position
from H to Cl. For substituents at the 4- and 5-position
of the thiophene, we chose two methyl (obtaining 4,5-
dimethylthiophenes) and an alkylene linkage between
the 4- and 5-position, which varied from three to four
methylenes to yield the 4,5,6,7-tetrahydrobenzo[b]-
thiophene and 5,6-dihydro-4H-cyclopentadien[b]thio-
phene derivatives, respectively. Finally, by the introduc-
tion of a nitrogen atom into the 6 position of the
tetrahydrobenzo[b]thiophene ring we had the 4,5,6,7-
tetrahydrothieno[2,3-c]pyridine derivatives. Among the
synthesized compounds, some appeared superior to PD
81,723 in their enhancing activity.
dopamine,8 5-HT,9 tachykinin NK-1 and NK-2,10 angio-
12
tensin AT1,11 P2Y
,
and oxytocin13 receptors, is also
demonstrated for the adenosine A1 receptor.14a,b
Allosteric enhancers of the action of adenosine are
believed to stabilize a conformation of the A1-adenosine
receptor that has a high affinity for agonists. This effect
is manifested as a slowing of the rate of dissociation of
agonist from the receptor.14a In addition, an allosteric
enhancer appears to stabilize an active conformation of
the receptor even in the absence of an agonist. Thus, in
cells with A1-adenosine receptors that are active spon-
taneously in the absence of an agonist, such as the
chinese hamster ovary (CHO) cells used in our study,
an allosteric enhancer may increase the number of
receptors that are active at any given time, and thereby
cause a change in cell function.14c,d
The currently available allosteric enhancers of agonist
binding to the A1 adenosine receptor have several
nonspecific actions. These nonspecific actions include
14a,b
antagonism of the A1 adenosine receptor
and
inhibition of the activity of adenylyl cyclase.14d
Bruns and co-workers reported that 2-amino-3-ben-
zoylthiophene derivatives are capable both of enhancing
the binding and activity of reference A1 receptor ago-
nists, such as N6-cyclopentyladenosine (CPA), to the A1-
adenosine receptor and, usually at higher concentra-
tions, of acting as competitive antagonists at the same
receptor.14 Among the compounds tested by Bruns, it
was demonstrated that PD 81,723 (2-amino-4,5-dim-
ethylthien-3-yl)-[3-(trifluoromethyl)phenyl]methanone
(1) represents a specific and selective allosteric enhancer
of agonist binding to the A1 receptor, with the best ratio
of enhancement to antagonistic action at this receptor.14b
Ch em istr y
The formation of 2-amino-3-benzoylthiophenes from
the base-catalyzed condensation of carbonyl compounds
and nitriles is achieved by the Gewald reaction.20 This
method was used for the synthesis of our new 2-amino-
3-naphthoylthiophene derivatives 22-73 and 77
(Schemes 1-4) wherein the appropriate carbonyl com-

796 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
Sch em e 1a
with nicotinoyl chloride produced the amide 19 in good
yield. Reduction with excess lithium aluminum hydride
yielded the amine 20 that was finally transformed to
the desired piperidone 21 after hydrolysis in aqueous
HCl.
The synthesis of compounds 73 and 77 is shown in
Scheme 4. The compound 73 was obtained by the
Gewald procedure applied to â-ketonitrile 2 and 2-(4-
methoxyphenyl)-[1,3]dithian-5-one 74.25 Compound 77
was synthesized by a three-step synthesis, starting from
compound 34. Acetylation of the amino group using a
mixture of acetic anhydride and pyridine gave 75. The
subsequent dehydrogenation with Pd/C with heating
furnished the benzo[b]thiophene derivative 76, which
was transformed by saponification into the desired
product 77.
a
Reagents and conditions. i: CH3CN, NaH, toluene 90 °C, 18
The structures of the synthesized compounds and the
yields of the syntheses are presented in Tables 1 and 2.
The range of yields was 35-67%.
h; ii: Br2, AcOH, 1 h, rt; iii: KCN, EtOH/water, 2 h, rt.
Sch em e 2a
Resu lts a n d Discu ssion
The purpose of our investigation was to assess a series
of new compounds in which the 3-, 4-, and 5-positions
of the thiophene ring were varied and to determine
which among them were potentially allosteric enhancers
of the action of adenosine to activate the human A1-
adenosine receptor. The reference compound for com-
parison was 1 (PD 81,723), and assays of allosteric
enhancement were performed using Chinese hamster
ovary (CHO) cells stably transfected to express the
recombinant human A1-adenosine receptors. Activation
of these receptors causes an inhibition of the activity of
adenylyl cyclase and a reduction of cAMP content of
CHO cells. Allosteric enhancement was measured as the
ability of the compounds 22-73 and 77 at four different
concentrations (0.01, 0.1, 1, and 10 µM) to reduce the
cAMP content of CHO:hA1 cells. The results are shown
in Table 3. It is important to note that compounds 70-
73 were synthesized and biologically evaluated in their
racemic forms.
The effect of each tested compound on cAMP content
was presented as a percentage of the value of cAMP
content in the absence of drug (control, 100%). A
decrease of cAMP content is indicated in Table 3 as a
negative percentage change of cAMP content from
control (absence of compound) in the presence of the
tested compound. Compounds with the potential to be
allosteric enhancers of activation of human A1-adenos-
ine receptors decrease the content of cAMP in CHO cells
expressing human A1-adenosine receptors. Receptors in
an active conformation in CHO:hA1 cells cause a detect-
able inhibition of adenylyl cyclase activity. Allosteric
enhancers are thought to stabilize the active conforma-
tion of the A1-adenosine receptors, leading to a reduction
in the cAMP content of the cells, whereas an increase
of cAMP content is consistent with reduced activity of
these receptors (i.e., receptor antagonism). Allosteric
enhancers cannot be distinguished from agonists by use
of this functional assay alone; then this assay assessed
the overall effect for each tested compound as enhancer
or antagonist at the A1-adenosine receptor.
a
Reagents and conditions. i: Benzyl chloride derivatives, DCM,
TEA, reflux; ii: nicotinoyl chloride, DCM, TEA, reflux; iii: LiAlH4,
THF; iv: 10% HCl THF/water, reflux.
pounds were reacted with naphthoylacetonitrile deriva-
tives 2-11 and sulfur in ethanol in the presence of
morpholine, according to the route shown in Scheme 3.
Only two compounds (43 and 46) of this series were
reported previously by Tranberg.19 The â-ketonitrile
derivatives 2-10 (Scheme 1) were synthesized by
condensation of the acetonitrile anion21 with the ap-
propriate substituted naphthoates with general formula
A22 and B.23 The derivative 11 was synthesized starting
from the commercially available 2-acetylfluorene. This
was achieved by converting it to the corresponding
acetyl bromide with bromine in acetic acid, followed by
treatment with potassium cyanide.
The appropriate carbonyl compounds were commer-
cially available except for the 4-benzylcyclohexanone,24
2-(4-methoxyphenyl)-[1,3]dithian-5-one,25 N-[(3-pyridyl)-
methyl]-4-piperidone 21, and the alkylated piperidones
12-18.26 The synthesis of these latter two series of
compounds is described in Scheme 2. The preparation
of alkylated piperidones 12-18 was performed following
the procedure of IJ zerman,18a which consists of the
reaction of the commercially available hydrochloric salt
of 4-piperidone with the appropriate substituted benzyl
chloride. For the synthesis of N-[(3-pyridyl)methyl]-4-
piperidone 21, acylation of 4-piperidone ethylene acetal
Among the synthesized compounds, the derivatives
22, 29, 35, 37, 46, and 56 were comparable to PD
81,723 as allosteric enhancers, whereas the compound

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 797
Sch em e 3a
a
Reagents and conditions. i: 2-Butanone (compounds with R4 ) R5 ) CH3), cyclopentanone [compounds with R4, R5 ) -(CH2)3-],
cyclohexanone [compounds with R4, R5 ) -(CH2)4-], S8, morpholine, EtOH, 70 °C for 1 h then 18 h at rt; ii: N-benzylpiperidone or
compounds 12-18 or 21, S8, morpholine, EtOH, 70 °C for 1 h then 18 h at rt; iii: compound 8, 4-benzylcyclohexanone or
4-phenylcycloexanone, S8, morpholine, EtOH, 70 °C for 1 h then 18 h at rt; iv: compound 1, 4-benzylcyclohexanone, S8, morpholine,
EtOH, 70 °C for 1 h then 18 h at rt.
77 showed enhancement activity only at high concen-
tration (10 µM). Several molecules appeared to be more
potent than PD 81,723 as allosteric enhancers in the
CHO:hA1 assay. These include compounds 23, 25-29,
31-34, 38, 39, 43, and 58. These latter compounds meet
the following criteria: (1) they cause a decrease of cAMP
content of CHO:hA1 cells by more than 40% when
present at a concentration of 10 µM; (2) they cause a
decrease of cAMP content of at least 22% at a concen-
tration of 1 µM; (3) they do not increase the content of
cAMP of CHO:hA1 cells at any concentration; (4) the
response to the compound is concentration dependent.
Several compounds in the synthesized series appear
to have activity as allosteric enhancer at a concentration
of 10 µM, and most of these compounds have significant
(i.e., causing g10% decrease of cAMP) activity at a
concentration of 1 µM. Very few of the tested compounds
increased the cAMP content of CHO:hA1 cells (indicated
as a positive change of cAMP content in Table 3). In
contrast, DPCPX, the prototype antagonist (which is
actually an inverse agonist of CHO:hA1 receptors)
increased the cAMP content of cells by 69% at a
concentration of 1 µM (data not shown). It thus appears
that none of the tested compounds was active (at 1 µM
of concentration) as an inverse agonist of the human
A1-adenosine receptor as well as DPCPX.
at the 3-position of the thiophene ring (compounds 22,
28, 34, 52, 72, 73, and 77) and which differ in the C4-
and C5-substituents, it appeared that the most potent
compounds had a three- and four-carbon methylene
linkage between the 4- and 5-position of the thiophene
(compounds 28 and 34, respectively). The presence of a
methyl group in both of these positions (compound 22)
decreased slightly the potency at 10 µM, while com-
pound 52, which possess an N-benzyltetrahydropyrido-
[b]thiophene system, showed activity as an allosteric
enhancer at low concentrations (0.01-1 µM) and an-
tagonistic activity at the highest concentration tested
(10 µM). The insertion of two sulfur atoms and a
p-methoxyphenyl ring at the 5-, 7-, and 6-position,
respectively, of compound 34, furnished compound 73
that does not demonstrate enhancement activity at any
concentration. We have also introduced a benzyl group
at the 7-position of the tetrahydrobenzo[b]thiophene
ring of compound 34, obtaining the derivative 72. The
effect of the introduction of this lipophilic substituent
is detrimental for the allosteric enhancement activity.
In fact, compound 72 is less active than 34 at any
concentration studied.
Several chemically different substituents at the 4-po-
sition of the 1-naphthoyl moiety were investigated, and
these modifications will alter the electronic, steric, and
lipophilic features of this residue. In our first series of
compounds, several groups having different lipophilic
In the series of seven derivatives characterized by the
presence of the same unsubstituted 1-naphthoyl moiety

798 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
Ta ble 1. Physical and Synthetic Data for 2-Amino-3-aroylthiophenes 22-51
compd
R1
R2
R4
R5
mp, °C
yielda, %
formulab
anal
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
195-197
171-173
138-140
175-177
152-154
142-144
255-257
233-235
214-215
258-260
210-212
253-255
137-140
211-212
227-229
179-181
176-178
180-182
255-257
120-122
145-147
178-180
65-67
38
59
39
48
32
46
89
56
71
71
67
68
73
79
66
68
68
69
43
35
39
50
38
55
78
61
62
44
68
66
C17H15NOS
C18H17NOS
C18H17NO2S
C17H14ClNOS
C17H14BrNOS
C17H14INOS
C18H15NOS
C19H17NOS
C19H17NO2S
C18H14ClNOS
C18H14BrNOS
C18H14INOS
C19H17NOS
C20H19NOS
C20H19NO2S
C19H16ClNOS
C19H16BrNOS
C19H16INOS
C17H15NOS
C18H17NOS
C18H16ClNOS
C18H15NOS
C19H17NOS
C19H16ClNOS
C19H17NOS
C20H19NOS
C20H18ClNOS
C20H17NOS
C21H17NOS
C22H19NOS
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
CH3
OCH3
Cl
Br
I
H
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
CH3
OCH3
Cl
Br
I
H
CH3
OCH3
Cl
Br
I
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
Cl
H
H
Cl
H
H
Cl
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
CH3
CH3
H
CH3
CH3
195-197
95-97
70-71
157-159
134-136
176-178
146-149
CH3
CH3
-(CH2)3-
-(CH2)4-
a
b
Yield of synthesized compounds after purification by column chromatography. All compounds were analyzed for C, H,N: analytical
results were within 0.4% of theoretical value.
Sch em e 4a
character (CH3, OCH3, Cl, Br, I) were introduced.
Starting from the derivatives 22, 28, 34, and 52, by the
introduction of a lipophilic and electron-releasing meth-
yl group at the 4-position of the naphthoyl ring, to obtain
the derivatives 23, 29, 35, and 53, respectively, the
allosteric enhancing activity was increased for com-
pound 23 but decreased for the derivatives 29 and 35
with respect to the reference compounds 28 and 34,
respectively, and was substantially unchanged for the
derivative 53. However, compound 23 was more active
both than PD 81,723 and the 22 counterparts at any
concentration.
In the series of derivatives 23, 29, 35, and 53, the
replacement of the methyl with a less lipophilic and
more electron-releasing moiety (a methoxy group), to
form derivatives 24, 30, 36, and 54, reduced allosteric
enhancer activity. For these latter compounds, the
reduction in activity may be attributed both to steric
and electronic factors, as we have observed previously
for the methoxybenzoyl counterparts.17 In fact the
methoxy group has an angularity component, i.e., part
of the methoxy group can extend significantly above or
below the plane of the naphthalene ring, and this
introduces a unique steric parameter.
Otherwise, the substitution of the methyl with sub-
stituents showing similar electronic effects and different
lipophilic characters (e.g., Cl, Br, and I) resulted in
improved activity. It is noteworthy that although the
introduction of a chlorine at the 4-position of the
a
Reagents and conditions. i: Ketone 74, S8, morpholine, EtOH,
70 °C for 1 h then 18 h at rt; ii: Ac2O, pyridine , reflux; iii:
10%Pd/C moistened with a 50% of water, heating; iv: KOH, EtOH,
reflux, 2 h.

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 799
Ta ble 2. Physical and Synthetic Data for 2-Amino-3-aroylthiophenes 52-67
compd
R1
R2
R3
mp, °C
yielda, %
formulab
anal
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
H
178-180
62-64
65
82
67
64
63
59
64
68
83
68
70
69
68
89
52
32
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
25H22N2OS
26H24N2OS
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
CH3
OCH3
Cl
Br
I
H
H
H
H
H
H
H
H
CH3
CH3
75-77
26H24N2O2S
25H21ClN2OS
25H21BrN2OS
25H21IN2OS
25H22N2OS
25H21ClN2OS
25H21ClN2OS
25H21ClN2OS
25H21FlN2OS
25H21FlN2OS
25H21N3O3S
29H29N2O4S
26H24N2OS
78-80
68-70
110-112
174-177
169-171
68-70
H
H
H
H
H
H
H
H
H
Cl
H
o-chloro
m-chloro
p-chloro
o-fluoro
p-fluoro
p-nitro
3,4,5-trimethoxy
H
105-108
183-185
94-96
76-78
127-129
73-75
H
148-149
26H23ClN2OS
a
b
Yield of synthesized compounds after purification by column chromatography. All compounds were analyzed for C, H, N: analytical
results were within 0.4% of theoretical value.
Ta ble 3. Percentage Change in CHO Cell CAMP Content in Presence of Compounds 22-67, 73, and 77
change in cAMP content from control
(mean ( SEM)a
change in cAMP content from control
(mean ( SEM)a
concentration of compounds
concentration of compounds
compound
0.01 µM
0.1 µM
1 µΜ
10 µM
compound
0.01 µM
0.1 µM
1 µΜ
10 µM
PD 81,723
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
-1 ( 2
-0.6 ( 2
-5 ( 1
9 ( 6
12 ( 4
-8 ( 4
-5 ( 6
-0.6 ( 3
9 ( 4
-6 ( 3
-10 ( 2
3 ( 3
-6 ( 3
-11 ( 4
17 ( 5
5 ( 4
-7 ( 2
-3 ( 3
-8 ( 3
3 ( 10
-18 ( 5
-1 ( 4
-3 ( 10
-6 ( 1
4 ( 3
-3 ( 4
-15 ( 4
19 ( 5
-8 ( 3
-15 ( 4
13 ( 5
-5 ( 4
3 ( 3
-10 ( 2
-4 ( 4
-4 ( 3
-3 ( 5
0 ( 5
-14 ( 4
-7 ( 4
-14 ( 3
-24 ( 3
-9 ( 5
-13 ( 1
6 ( 5
-50 ( 1
-45 ( 2
-51 ( 4
-38 ( 9
-56 ( 2
-60 ( 3
-63 ( 5
-60 ( 1
-48 ( 2
-35 ( 3
-55 ( 2
-67 ( 3
-75 ( 1
-52 ( 3
-45 ( 5
-17 ( 1
-44 ( 2
-72 ( 1
-67 ( 2
-28 ( 3
-22 ( 2
-24 ( 6
-51 ( 3
-34 ( 1
-17 ( 1
-42 ( 3
-20 ( 5
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
77
0 ( 2
-4 ( 5
-3 ( 5
7 ( 6
1 ( 3
-17 ( 3
-15 ( 3
6 ( 5
-7 ( 4
-7 ( 4
11 ( 12
-1 ( 4
-5 ( 2
1 ( 6
-12 ( 3
-20 ( 4
-12 ( 4
-8 ( 3
-3 ( 5
-9 ( 5
12 ( 9
-1 ( 3
2 ( 10
1 ( 4
-11 ( 5
-10 ( 5
-6 ( 5
7 ( 4
-25 ( 4
-13 ( 3
3 ( 8
-7 ( 6
-12 ( 1
-6 ( 4
-18 ( 3
-10 ( 5
7 ( 3
-21 ( 3
-10 ( 6
-2 ( 4
26 ( 13
-10 ( 7
0 ( 7
-13 ( 2
-19 ( 6
-9 ( 6
3 ( 3
-32 ( 2
-5 ( 9
-47 ( 6
-2 ( 3
-19 ( 10
-29 ( 4
-17 ( 4
-8 ( 4
-27 ( 2
-14 ( 5
-22 ( 4
-22 ( 3
-21 ( 1
-3 ( 3
-21 ( 3
0 ( 3
19 ( 2
-34 ( 1
-12 ( 5
-21 ( 5
-42 ( 3
-13 ( 5
-60 ( 4
-17 ( 5
-3 ( 3
-6 ( 4
-31 ( 4
-8 ( 3
26 ( 12
5 ( 5
0 ( 4
3 ( 4
18 ( 2
-6 ( 3
-11 ( 4
-10 ( 4
9 ( 8
-6 ( 6
-3 ( 4
-11 ( 6
8 ( 6
-3 ( 4
0 ( 5
-1 ( 3
-8 ( 2
4 ( 7
-8 ( 7
7 ( 5
-11 ( 3
-6 ( 3
-3 ( 4
-1 ( 3
4 ( 6
-25 ( 4
-29 ( 2
-24 ( 5
-8 ( 3
-16 ( 10
-9 ( 6
56 ( 14
-1 ( 5
33 ( 13
28 ( 10
-21 ( 3
6 ( 5
-5 ( 3
41 ( 12
8 ( 6
-9 ( 6
-12 ( 3
-7 ( 7
5 ( 3
10 ( 4
-7 ( 4
16 ( 5
9 ( 17
0 ( 5
-18 ( 4
-15 ( 3
-13 ( 3
-6 ( 2
-19 ( 3
0 ( 3
31 ( 8
-9 ( 3
-14 ( 2
4 ( 5
4 ( 3
-8 ( 3
18 ( 6
17 ( 5
46
47
-13 ( 4
-7 ( 5
-28 ( 2
-8 ( 4
1 ( 8
-37 ( 6
a
The results are the average of six experiments at each of four concentrations of tested compound.
1-naphthoyl moiety (compounds 25, 31, 37, and 55) had
not a profound effect on their activity with respect to
the 4-unsubstituted derivatives, the increase of the size
of the halogen atom from chlorine to bromine (deriva-
tives 26, 32, 38, and 56) to end with indole (compounds
27, 33, 39, and 57) caused a large increase of the
allosteric enhancer activity, especially when a lipophilic
substituent (methyl or cycloalkyl moiety) was present
in 4- and 5-position of the thiophene ring.
These findings suggest that the introduction of a
lipophilic halogen or methyl group at the 4-position of
the naphthalene ring were preferred for enhancing
activity, whereas a more hydrophilic group such as the
methoxy was not favorable. The results indicated sig-

800 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
nificant qualitative direct correlation between the al-
losteric enhancer activity and the lipophilicity of the
substitution at the 4-position of the 1-naphthoyl moiety,
where the compounds characterized by the presence of
substituents (Cl, Br, I) with higher lipophilic character
were among the most potent as A1 adenosine allosteric
enhancers.
The electronic effect of the substituents is not relevant
for biological activity, since substituents different in this
respect (e.g., methyl and chlorine) confer similar en-
hancing effects (cf. 23 and 25, 29 and 31, 35 and 37, 53
and 55). This was confirmed comparing substituents
showing similar electronic effect, i.e., chlorine with
bromine and iodine (25 vs 26 and 27, 31 vs 32 and 33,
37 vs 38 and 39, 55 vs 56 and 57).
To determine if changes in activity were correlated
with physicochemical properties of the substituents, we
have analyzed the allosteric enhancement obtained at
10 µM of concentration of each tested compound, using
electronic (σp), lipophilic (π), and steric (molar refractiv-
ity, MR) parameters.28 Unfortunately, a quantitative
structure-activity analysis (QSAR) was not feasible for
each of these parameters (r2 for the regressions of
enhancement activity on σp, π and MR parameters were
<0.1 and were not significant, data not shown).
Molecules with the same substituents at different
positions and which are characterized by the same
lipophilicity have studied. This is the case of two groups
of isomer derivatives, where the first group is consti-
tuted by the thiophene derivatives substituted in 3-posi-
tion with the 1-naphthoyl and 2-naphthoyl moieties
(compounds 22, 28, 34, 52, 72 and 40, 43, 46, 58, 71,
77, respectively). The second group included the deriva-
tives modified on the naphthoyl ring and corresponding
to 4-methyl-1-naphthoyl and 4-methyl-2-naphthoyl
thiophene derivatives (compounds 23, 29, 35, 53 and
41, 44, 47, 66, respectively).
In the first group of molecules, comparing the activi-
ties of derivatives bearing the same substituent on 4-
and 5-position of the thiophene ring (22 vs 40, 28 vs
43, 34 vs 46, 52 vs 58, 72 vs 71), with the exception of
the compound 58, at high concentrations (10 µM) the
derivatives with the 1-naphthoyl moiety are generally
more potent than the corresponding analogues with the
2-naphthoyl substituent. This is confirmed also for the
second group of derivatives, where with the exception
of the compound 66, the molecules which possess the
4-methyl-1-naphthoyl moiety (23, 29, and 35) are more
potent then the analogues characterized by the presence
of the 4-methyl-2-naphthoyl moiety (41, 44, and 47).
Therefore, because molecules with equivalent lipophi-
licity may have very different levels of activity, we can
conclude that lipophilicity is a necessary condition but
not the only determining parameter for the allosteric
enhancer activity.
In the series of 2-naphthoyl derivatives, only two
compounds (43 and 58) are more active than PD 81,-
723. For the derivative 58, the introduction of different
electron-withdrawing, electron-donating and hydropho-
bic substituents in the phenyl ring of the N-benzyl
moiety (compounds 59-65) was responsible for a marked
decrease in their activity as A1 allosteric enhancers.
Only compounds 59 and 62, which have the 2-fluoro and
2-chloro substituent, respectively, on the benzyl group
retained activity. The 4-substituted benzyls (61 and 63-
65) were inactive. Starting from 58, removal of the
nitrogen from the 6-position of the tetrahydropyrido[b]-
thiophene system, to yield 71, resulted in a loss of
allosteric enhancer activity, and this finding suggests
that the nitrogen atom at the 6-position is required for
the activity of compound 58. In contrast, for compound
52 which showed a weak allosteric enhancer activity
with respect to the isomer 58, removal of the nitrogen
from the 6-position has the effect to furnish a compound
(72), which showed moderate enhancer potency at high
concentration (10 µM). For the same compound 58, the
phenyl ring of the N-benzyl group has been replaced by
a bioisosteric pyridine ring (compound 68), Our objective
behind introducing this polar bioisosteric moiety was
to decrease the lipophilicity of 58 without compromising
activity. Unfortunately, the compound 68 exhibited
reduced potency at any concentration when compared
to the counterpart 58, and this result indicated that the
exchange of the phenyl ring in the N-benzyl part of the
molecule with a nitrogen-containing heterocycle did not
result in a good bioisosteric replacement, and it was not
tolerated in terms of allosteric enhancer activity at A1
adenosine receptor.
In the series of derivatives which possess the 4-meth-
yl-2-naphthoyl moiety (40, 44, 47, and 66), the introduc-
tion of a lipophilic chloro atom at the 6-position of the
naphthalene moiety did not improve the allosteric
enhancer potency, which indicates an unfavorable steric
interaction originating from the presence of 6-chloro
substitution in compounds 42, 45, 48, and 67.
An increase of the lipophilic bulk at the 3-position of
the thiophene ring by the introduction of a tricyclic
2-fluorenoyl moiety (compounds 49-51 and 69) led to
a substantial decline in potency.
A series of selected compounds which appeared to be
better than (28, 31, 32-34, 38, 39, 43, and 58) or
comparable (22, 30, 37, 40, 42, 44, 46, 62, and 77) to
PD 81,723 as allosteric enhancers in the CHO:hA1 assay
were tested at a concentration of 10 µM for their affinity
for subtypes of adenosine receptors. The ability of these
compounds to displace the binding of [3H]DPCPX, [3H]-
ZM 241385, and [3H]MRE 3008F20 to the ligand binding
site of CHO:hA1, CHO:hA2A, and CHO:hA3 adenosine
receptors, respectively, is shown in Table 4. None
inhibited binding at the hA2AAR, but most of the subset
inhibited binding to the hA1AR to some extent (0-19%)
and to the hA3AR to a substantial degree (as high as
57%).
The prototype enhancer PD 81,723 did not inhibit the
binding of a radiolabeled antagonist to A1 and A2A
receptors, but it reduced by 21% the binding of [3H]MRE
3008F20 to the A3 receptor. The derivatives 22 and 32
showed the same behavior, whereas compounds 28, 33,
42, 44, and 46 bound only to the A1 receptor. For only
two compounds (40 and 31) it was possible to achieve a
good separation between enhancing activity and binding
to the orthosteric site. Compound 31 was more active
than PD 81,723 in its enhancing activity, and at the
same time was devoid of activity on A1, A2A and A3
receptors. All other compounds inhibited [3H]MRE
3008F20 binding to A3 receptor in a manner which is
better (34, 37-39, 43, and 83) or comparable (77 and
58) to PD 81,723.

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 801
Ta ble 4. Inhibition Activity of a Selected Number of Enhancer
Compounds
a
b
c
compound % inhibition A1 % inhibition A2A % inhibition A3
PD 81,723
22
28
30
31
32
33
34
37
38
39
40
42
43
44
46
58
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
3.0 ( 1.9
3.0 ( 1.7
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
0 ( 0
21.0 ( 1.8
18.0 ( 2.3
0 ( 0
8.0 ( 2.8
19.0 ( 1.3
0 ( 0
57 ( 2.9
0 ( 0
0 ( 0
22.0 ( 4.6
0 ( 0
3.3 ( 0.5
2.0( 1.4
4.8 ( 0.8
12.0 ( 1.4
15.3 ( 1.6
0 ( 0
34.5 ( 3.7
30.5 ( 3.1
48.5 ( 6.3
48.0 ( 4.4
0 ( 0
4.0 ( 1.0
14.5 ( 1.8
6.0 ( 1.2
3.3 ( 0.2
2.5 ( 0.3
3.0 ( 0.7
13.5 ( 1.6
0 ( 0
54.0 ( 8.9
0 ( 0
0 ( 0
22.0 ( 5.1
0 ( 0
62
77
19 ( 1.6
a
Inhibition activity was expressed as percent displacement
value ((STD, n ) 3) of 1 nM of [3H]DPCPX by 10 µM of tested
compound. b Inhibition activity was expressed as percent displace-
ment value ((STD, n ) 3) of 2 nM of [3H]ZM 241385 by 10 µM of
tested compound. c Inhibition activity was expressed as percent
displacement value ((STD, n ) 3) of 2 nM [3H]MRE 3008F20 by
10 µM of tested compound.
Finally, CHO cells expressing the human adenosine
A1 receptor (Coho1), and rat brain and human cortex
membrane preparations containing native adenosine A1
receptors were used to evaluate the effect of selected
allotter enhancers on the binding of the radiolabel led
agonist [3H]CCPA to A1 receptors.
At higher concentrations some compounds produced
inhibition of [3H]CCPA specific binding (data not shown).
It should be mentioned, however, that the limited
solubility of the tested compounds precluded the record-
ing of a full concentration-effect curve. Figure 1 A-C
shows the correlation between increases of [3H]CCPA
binding to A1 receptors in CHO:hA1 and rat cortex
membranes, CHO:hA1 and human brain membranes
and rat cortex and human brain membranes, respec-
tively, caused by 10 µM concentration of a selected series
of tested compounds. A good correlation was found
between the increments [3H]CCPA binding to A1 recep-
tors expressed in different systems. Compound 37 at 10
µM was the most active compound increasing the
binding of [3H]CCPA to CHO:hA1, human brain, and rat
cortex membranes by 149, 43, and 27%, respectively.
Unlike the effect on agonist binding, the tested com-
pounds did not increase the binding of the antagonist
[3H]DPCPX on CHO:hA1 membranes. The results of
these experiments demonstrated that our tested com-
pounds significantly enhanced agonist, but not antago-
nist binding to the recombinant human A1 adenosine
receptors.
F igu r e 1. Comparison between % of bound increase values
of [3H]CCPA binding to CHO:hA1 and rat cortex (A), CHO:
hA1 and human brain (B), and rat cortex and human brain
(C) membranes. The circle indicates PD 81,723 (compound 1).
Ta ble 5. Enhancer’s Effects on [3H]CCPA Dissociation from A1
Adenosine Receptors Transfected in CHO Cellsa
compound (10 µM)
% t1/2 increase
% K-1 decrease
PD 81,723
43 ( 3
85 ( 9
0 ( 0
30 ( 3
46 ( 4
0 ( 0
22
28
31
32
33
34
37
38
39
43
46
43 ( 3
4 ( 1
30 ( 3
4 ( 1
41 ( 2
29 ( 3
36 ( 4
43 ( 3
60 ( 5
42 ( 4
10 ( 1
29 ( 2
23 ( 2
27 ( 2
30 ( 3
38 ( 4
29 ( 3
9 ( 1
a
The results are the average of three experiments at 10 µM
concentration of each tested compound.
37, as shown by the 36% increase in t1/2. This particular
behavior is of great importance because it allows us to
analyze the enhancing activity of the tested compounds
more directly through its influence on the dissociation
rate of [3H]CCPA agonist for the A1 adenosine receptor.
Our data are in agreement with previous reports
showing that the dissociation of agonist from the recep-
tor was, in the presence of enhancer compounds, sig-
nificantly decreased.14,15b,18b,19,27
Ligand dissociation studies were conducted in an
attempt to further elucidate putative mechanism(s)
underlying the effects of the tested compounds to
enhance A1 agonist binding to hCHO-A1 membranes.
Comparison of the dissociation rates (t1/2 values) re-
vealed that tested compounds (10 µM) significantly
slowed the dissociation of [3H]CCPA from hCHO-A1
membranes (Table 5).
Figure 2 shows a typical dissociation curve: the
To determine whether our compounds potentiate
agonist binding to A1 receptors, saturation-binding
dissociation of [3H]CCPA was significantly decreased by

802 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
receptor agonist [3H]CCPA increased up to 159% (in
CHO:hA1 membranes) in the presence of 37 (10 µM).
The KD value of [3H]CCPA was not significantly affected
by the allosteric enhancers. In contrast to the effect of
our tested compounds on agonist binding, the BMAX and
KD of the A1 antagonist [3H]DPCPX was not signifi-
cantly different in the absence and presence of test
compounds (data not shown).
Con clu sion s
We have identified novel allosteric enhancers of
agonist binding to adenosine A1 receptors. Some of the
compounds proved superior to the reference enhancer
PD 81, 723. With the exception of compounds 32 and
38, the tetrahydrobenzo[b]thiophene derivatives ap-
peared to be more potent than the dihydrocyclopenta-
dien[b]thiophene counterparts.
F igu r e 2. Typical dissociation curve of [3H]CCPA from A1
adenosine receptors in CHO cells in the presence (2) and
absence (9) of enhancer 37 (10 µM).
In the series of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine
derivatives 52-69, the only compound, which showed
significant allosteric enhancer activity at any concentra-
tion, was derivative 58. In the series of compounds 58-
65, which bear the 2-naphthoyl moiety and different
benzyl derivatives on position 6, the benzyl moiety was
the best substituent whereas the substitution of the
benzene with a pyridine caused a loss of enhancing
activity (compound 68). Indeed, only the unsubstituted
N-benzyl derivative 58 possessed an activity superior
to PD 81,723 at any concentration. The lack of activity
of 71 (which does not contain the ring nitrogen) supports
the importance of the tetrahydropyridyl ring nitrogen
for the allosteric enhancer activity.
Of all compounds synthesized for this study, only two
(43 and 46) were synthesized and studied by Tranberg19
(see Table 3). Our results are consistent with the results
reported by Tranberg, even though different assays and
different sources of adenosine receptors were used in
these studies. However, it should be noted that our
assay of effects of putative enhancers on cAMP content
of CHO cells expressing human A1-adenosine receptors
is not a specific assay of allosteric enhancement of
agonist binding. Our assay does not directly measure
the interaction between receptor activation and G-
protein activation, and our observations may be com-
plicated by drug actions not related to enhancement,
such as cell toxicity. However, the effect of a tested
compound in the intact cell cAMP assay used in this
study may be a more useful predictor of the effect of
the compound in vivo than a binding assay that more
specifically assesses allosteric enhancement. It is in-
tended that these derivatives may be of help in better
understanding adenosine receptor function. We propose
to continue this study focusing our efforts on the
F igu r e 3. Comparison between % of BMAX increase values of
[3H]CCPA binding to CHO:hA1 and rat cortex (A), CHO:hA1
and human brain (B), and rat cortex and human brain (C)
membranes. The circle indicates PD 81,723 (compound 1).
experiments were performed using membranes pre-
pared from CHO:hA1, rat cortex, and human brain. In
the current study PD 81,723 increased the BMAX of the
agonist [3H]CCPA to recombinant human A1 adenosine
receptors in CHO cells membranes and to A1 receptors
in human brain and rat cortex membranes.
Figure 3 A-C shows the correlation between incre-
ments in the maximum specific binding (BMAX) of [3H]-
CCPA binding to A1 receptors in CHO:hA1 and rat
cortex membranes, CHO:hA1 and human brain mem-
branes, and rat cortex and human brain membranes,
respectively. A good correlation was found between the
increments in BMAX of [3H]CCPA binding to A1 receptors
expressed in different systems. The BMAX of the A1
29
evaluation of their enhancing activity at A2A and A3
adenosine receptors expressed in CHO cells by means
of radioligand binding studies performed with the
agonists [3H]CGS 21680 and [125I]AB-MECA for A2A and
A3, respectively.
Exp er im en ta l Section
Abbr evia tion s. ADA, adenosine deaminase; [3H]DPCPX,
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]CHA, N6-
[adenine-2,8-3H]cyclohexyladenosine; [3H]CCPA, 2-chloro-N6-
cyclopentyladenosine; [3H]ZM 241385, [2-3H](4-(2-[7-amino-

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 803
2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)-
phenol); R-PIA, R-N6-(2-phenylisopropyl)adenosine, CHO, Chi-
nese hamster ovary.
under vacuo, and the product, as a colorless liquid, was used
in the next reaction without any further purification. Yield
54% (594 mg) H NMR (CDCl3) δ: 1.74 (t, 4H, J ) 5.8 Hz),
1
2.53 (t, 4H, J ) 6.2 Hz), 3.53 (s, 2H), 3.96 (s, 4H), 7.28 (m,
1H), 7.67 (d, 1H, J ) 7.8 Hz), 8.49 (d, 1H, J ) 4.4 Hz), 8.54 (s,
1H).
Ch em istr y. Ma ter ia ls a n d Meth od s. Cyclopentanone,
cyclohexanone, 2-butanone, 2-acetylfluorene, 4-phenylcyclo-
hexanone, and 4-benzylpiperidone are commercially available,
and they have been purchased from Aldrich. 1H NMR spectra
were recorded on a Bruker AC 200 spectrometer. Chemical
shifts (δ) are given in ppm upfield from tetramethylsilane as
internal standard, and the spectra were recorded in appropri-
ate deuterated solvents indicated in the procedure. Melting
points (mp) were determined on a Buchi-Tottoli apparatus and
are uncorrected. All products reported showed 1H NMR spectra
in agreement with the assigned structures. Elemental analyses
were conducted by the Microanalytical Laboratory of the
Chemistry Department of the University of Ferrara. All
reactions were carried out under an inert atmosphere of dry
nitrogen, unless otherwise described. Standard syringe tech-
niques were applied for transferring dry solvents. Reaction
courses and product mixtures were routinely monitored by
TLC on silica gel (precoated F254 Merk plates) and visualized
with aqueous KMnO4. Flash chromatography was performed
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. Dioxane was distilled from
calcium hydride, and dry DMF was distilled from calcium
chloride and stored over molecular sieves (3 Å). In high-
pressure hydrogenation experiments, a Parr shaker on a high-
pressure autoclave was used.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
15 a n d 18. To a suspension of 4-piperidone hydrochloride (10
mmol) in CH2Cl2 (20 mL) cooled at 0 °C were added the
appropriate substituted benzyl chloride (11 mmol) and TEA
(22 mmol). The mixture was heated at reflux overnight, diluted
with CH2Cl2 (20 mL), and then washed with water and brine.
The organic layer was separated, dried (Na2SO4), and evapo-
rated to give a residue, which was purified by column chro-
matography on silica gel using EtOAc-petroleum ether (1:1,
v/v) as eluent.
1-(2-F lu or oben zyl)-4-p ip er id on e (15). Yield 56%, oil.1H
NMR (CDCl3) δ: 2.46 (t, 4H, J ) 6.2 Hz), 2.79 (t, 4H, J ) 6.0
Hz), 3.71 (s, 2H), 7.05 (t, 1H, J ) 7.2 Hz), 7.11 (t, 1H, J ) 7.2
Hz), 7.26 (m, 1H), 7.39 (t, 1H, J ) 7.4 Hz).
Syn th esis of 1-[(3-P yr id yl)m eth yl)]-4-p ip er id on e (21).
The compound 20 (920 mg, 4.18 mmol) was taken up in a
mixture of THF (20 mL) and 2 N HCl (10 mL) and stirred at
rt for 20 h. The solution was carefully basified with solid
NaHCO3, the THF was evaporated, and the remainder was
partitioned between EtOAc and water. The organic layer was
dried and evaporated and the residue purified by flash column
chromatography (silica gel) using EtOAc-MeOH 8:2 as eluent.
The compound 21 (455 mg, 57% yield) was obtained as a
1
colorless liquid. H NMR (CDCl3) δ: 2.46 (t, 4H, J ) 6.2 Hz),
2.76 (t, 4H, J ) 6.2 Hz), 3.66 (s, 2H), 7.33 (dd, 1H, J ) 7.8 and
4.8 Hz), 7.77 (d, 1H, J ) 8.0 Hz), 8.53 (d, 1H, J ) 3.6 Hz),
8.60 (s, 1H).
Syn th esis of 2-Br om oa cetylflu or en e. To a solution of
2-acetylfluorene (2.08 g, 10 mmol) in 10 mL of glacial acetic
acid, bromine (10 mmol, 0.51 mL) was added dropwise and
the mixture stirred at room temperature for 2 h. After this
time, the acetic acid was evaporated under reduced pressure
at a temperature lower than 40 °C. The crude product so
obtained was used for the next reaction without purification.
Syn th esis of 2-F lu or en oyla ceton itr ile. The crude 2-bro-
moacetylfluorene previously prepared (2.87 g, 10 mmol) was
dissolved in 95% EtOH (15 mL). A solution of potassium
cyanide (3.6 g, 55 mmol), dissolved in water (10 mL), was
added in one portion, and the mixture was stirred at room
temperature (rt) for 24 h. The mixture was then poured onto
a mixture of crushed ice and water and acidified with glacial
acetic acid (pH ) 5-6). The resulting solid was collected by
filtration and washed with water. Yield ) 83%; mp 52-155
°C. 1H NMR (CDCl3) δ: 3.98 (s, 2H), 4.14 (s, 2H), 7.44 (m, 2H),
7.62 (m, 1H), 7.91 (m, 3H), 8.11 (s, 1H).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
2-10 (Step A). CH3CN (8 mL, 152 mmol) was added dropwise
to a suspension of the appropriate methyl ester with general
formula B or C (50 mmol) and 50% NaH (50 mmol) in toluene
(75 mL) under stirring at 90 °C After 24 h, the mixture was
cooled at rt and the precipitate was filtered and washed with
toluene (25 mL). The solid was then dissolved in water, the
solution cooled at 0 °C, and the pH brought to 2 with 2 N HCl.
The suspension was extracted with EtOAc (4 × 50 mL), the
recombined organic layers were washed with brine, dried over
Na2SO4, and concentrated to give the corresponding naph-
thoylacetonitrile 2-10 as a solid, used without any further
purification for the next reaction.
1-(3,4,5-Tr im eth oxyben zyl)-4-piper idon e (18). Yield 63%,
1
mp 102-104 °C. H NMR (CDCl3) δ: 2.46 (t, 4H, J ) 6.2 Hz),
2.75 (t, 4H, J ) 6.0 Hz), 3.55 (s, 2H), 3.84 (s, 3H), 3.88 (s, 6H),
6.60 (s, 2H).
Syn t h esis of 1-Nicot in oyl-4-p ip er id on e E t h ylen e
Aceta l (19). 4-Piperidone ethylene acetal (1.43 g., 10 mmol)
was dissolved into 15 mL of dry methylene chloride solution
with triethylamine (7 mL, 50 mmol). Nicotinoyl chloride (1.42
g, 10 mmol) dissolved in dry methylene chloride (10 mL) was
then added to the solution in a dropwise manner. The solution
was stirred under nitrogen overnight. The organic phase was
washed with water (5 mL), brine, dried over Na2SO4, and
finally concentrated under vacuo to give a crude product which
was then purified by flash chromatography over a silica gel
column (eluent EtOAc-petroleum ether 1:1, v/v), to yield 19
as a white solid. Yield 82% (1.92 g.), mp ) 113-114 °C. 1H
NMR (CDCl3) δ: 1.68 (m, 2H), 1.77 (m, 2H), 3.50 (m, 2H), 3.86
(m, 2H), 3.99 (s, 4H), 7.37 (dd, 1H, J ) 4.8 and 3 Hz), 7.75 (d,
1H, J ) 7.6 Hz), 8.66 (m, 2H).
2-(Na p h th a len e-1-ca r bon yl)a ceton itr ile (2). Yield: 78%;
1
mp 85-86 °C. H NMR (CDCl3) δ: 4.21 (s, 2H), 7.71 (m, 2H),
7.92 (m, 1H), 8.13 (d, 1H, J ) 7.8 Hz), 8.62 (d, 1H, J ) 8.4
Hz), 8.76 (d, 1H, J ) 8.8 Hz), 8.84 (d, 1H, J ) 8.4 Hz).
2-(4-Met h yln a p h t h a len e-1-ca r b on yl)a cet on it r ile (3).
Yield: 78%; mp 133-134 °C. 1H NMR (CDCl3) δ: 2.77 (s, 3H),
4.18 (s, 2H), 7.38 (d, 1H, J ) 7.4 Hz), 7.66 (m, 2H), 7.79 (d,
1H, J ) 7.6 Hz), 8.06 (d, 1H, J ) 7.4 Hz), 8.91 (d, 1H, J ) 7.4
Hz).
2-(4-Meth oxyn a p h th a len e-1-ca r bon yl)a ceton itr ile (4).
Yield ) 74%, mp 178-180 °C. 1H NMR (CDCl3) δ: 4.06 (s,
3H), 4.47 (s, 2H), 6.88 (d, 1H, J ) 8.2 Hz), 7.67 (m, 2H), 8.10
(d, 1H, J ) 8.4 Hz), 8.31 (m, 1H), 9.01 (d, 1H, J ) 8.4 Hz).
Syn t h esis of 1-[(3-P yr id yl)m et h yl)]-4-p ip er id on e
Eth ylen e Aceta l (20). Lithium aluminum hydride (810 mg,
21 mmol) was suspended in 20 mL of dry tetrahydrofuran
(THF), and the solution was cooled in an ice bath. The
compound 19 (1.17 g, 5 mmol) dissolved in 10 mL of THF was
added dropwise to the cold solution. At the end of the addition,
the mixture was placed at rt, and after 4 h the unreacted
LiAlH4 was quenched by careful addition of an excess amount
of 10% NaOH solution. The solution was filtered, and the
residue was repeatedly washed with an excess amount of
EtOAc. The combined extract was dried and concentrated
2-(4-Ch lor on a p h t h a len e-1-ca r b on yl)a cet on it r ile (5).
1
Yield 74%; mp 123-124 °C. H NMR (CDCl3) δ: 4.19 (s, 2H),
7.73 (m, 4H), 8.38 (d, 1H, J ) 9.8 Hz), 8.84 (d, 1H, J ) 9.8
Hz).
2-(4-Br om on a p h t h a len e-1-ca r b on yl)a cet on it r ile (6).
Yield: 72%; mp 109 °C. 1H NMR (CDCl3) δ: 4.21 (s, 2H), 7.63
(m, 2H)0.7.91 (m, 2H), 8.09 (d, J ) 8.0 Hz, 1H), 8.81 (d, J )
8.4 Hz, 1H).
2-(4-Iod on a p h t h a len e-1-ca r b on yl)a cet on it r ile
(7).
Yield: 66%; mp 123 °C. 1H NMR (CDCl3) δ: 4.23 (s, 2H), 7.64

804 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
(m, 2H)0.7.90 (d, J ) 7.8 Hz, 1H), 8.03 (d, J ) 8.2 Hz, 1H),
8.20 (d, J ) 7.8 Hz, 1H), 8.93 (d, J ) 8.2 Hz, 1H).
1444, 1290, 1255, 1032, 780; 1H NMR (CDCl3): δ 1.28 (m, 2H),
1.87 (t, 2H, J ) 7.0 Hz), 2.54 (t, 2H, J ) 7.2 Hz), 7.41 (d, 1H,
J ) 6.4 Hz), 7.59 (m, 3H), 7.73 (d, 1H, J ) 8.8 Hz), 8.02 (d,
2H, J ) 8.6 Hz), 8.73 (bs, 2H).
(2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(4-m eth yln aph th alen -1-yl)m eth an on e (29). Cyclopentanone,
2-(4-methylnaphthalen-1-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 29. IR (KBr) cm-1: 3354, 1583,
1435, 1290, 1256, 1019, 835. 1H NMR (CDCl3) δ: 1.51 (m, 2H),
1.93 (t, 2H, J ) 7.6 Hz), 2.58 (t, 2H, J ) 7.6 Hz), 2.73 (s, 3H),
7.19 (m, 2H), 7.27 (m, 2H), 7.49 (m, 2H), 7.90 (d, 1H, J ) 7.6
Hz), 8.03 (d, 1H, J ) 7.6 Hz).
2-(Na p h th a len e-2-ca r bon yl)a ceton itr ile (8). Yield: 81%;
1
mp 124 °C. H NMR (d6-DMSO) δ: 4.91 (s, 2H), 7.67 (m, 2H),
8.03 (m, 4H), 8.61 (s, 1H).
2-(4-Met h yln a p h t h a len e-2-ca r b on yl)a cet on it r ile (9).
Yield: 67%; mp 136 °C.1H NMR (CDCl3) δ: 2.73 (s, 3H), 4.46
(s, 2H), 7.48 (s, 1H), 7.57 (m, 2H), 7.83 (s, 1H), 7.88 (d, J ) 8.0
Hz, 1H), 8.03 (d, J ) 8.0 Hz, 1H).
2-(4-Meth yl-6-ch lor on a p h th a len e-2-ca r bon yl)a ceton i-
tr ile (10). Yield: 78%; mp 201 °C.1H NMR (CDCl3) δ: 2.61
(s, 3H), 4.55 (s, 2H), 7.49 (m, 1H), 7.87 (m, 2H), 7.97 (s, 1H),
8.35 (s, 1H).
2-Am in o-5,6-d ih yd r o-4H -cyclop en t a [b]t h iop h en -3-yl-
(4-m eth oxyn a p h th a -len -1-yl)m eth a n on e (30). Cyclopen-
tanone, 2-(4-methoxynaphthalene-1-carbonyl)acetonitrile, mor-
pholine, and sulfur were reacted according to the procedure
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
22-67 (Step B). A mixture of aroylacetonitrile (5 mmol,
prepared by Step A), appropriate ketone (5 mmol), morpholine
(0.44 mL, 5 mmol), and sulfur (164 mg, 5 mmol) was heated
at 70° C for 1 h and then stirred at room temperature for 20
h. At the end of this period, the solvent was evaporated under
reduced pressure and the residue diluted with ethyl acetate.
After washing with water, the organic layer was dried, filtered,
and then evaporated. The crude product was purified by flash
column chromatography and then recrystallized from petro-
leum ether.
of Step B to afford the desired compound 30. IR (KBr) cm-1
:
3351, 3243, 2852, 1581, 1433, 1261, 1242, 1162, 1091, 1023,
822, 762, 712; 1H NMR (CDCl3): δ 1.59 (m, 2H), 1.96 (m, 2H),
2.59 (t, 2H, J ) 7.6 Hz), 4.04 (s, 3H), 6.79 (d, 1H, J ) 8 Hz),
7.12 (bs, 2H), 7.34 (d, 1H, J ) 8 Hz), 7.48 (m, 2H), 7.89 (m,
1H), 8.30 (m, 1H).
2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(4-ch lor on aph th alen -1-yl)m eth an on e (31). Cyclopentanone,
2-(4-chloronaphthalene-1-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 31. IR (KBr) cm-1: 3348, 3229,
3119, 1571, 1443, 1359, 1272, 1253, 1195, 962, 835, 787, 759;
1H NMR (CDCl3): δ 1.06 (m, 2H), 1.84 (t, 2H, J ) 7.2 Hz),
2.52 (m, 2H), 7.37 (d, 1H, J ) 6.8 Hz), 7.71 (m, 4H), 8.24 (d,
1H, J ) 8.8 Hz), 8.70 (bs, 2H).
2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(4-br om on aph th alen -1-yl)m eth an on e (32). Cyclopentanone,
2-(4-bromonaphthalene-1-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 32. IR (KBr) cm-1: 3333, 3110,
2851, 1576, 1443, 1289, 1254, 1032, 804, 780; 1H NMR
(CDCl3): δ 1.29 (m, 2H), 1.81 (t, 2H, J ) 6.8 Hz), 3.31 (m,
2H), 7.34 (m, 1H), 7.53 (m, 4H), 7.71 (m, 1H), 7.95 (m, 1H),
8.64 (m, 1H).
2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(4-iod on a p h th a len -1-yl)m eth a n on e (33). Cyclopentanone,
2-(4-iodonaphthalene-1-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 33. IR (KBr) cm-1: 3329, 3221,
3106, 2852, 1581, 1444, 1290, 1254, 780; 1H NMR (CDCl3): δ
1.28 (m, 2H), 1.81 (t, 2H, J ) 7 Hz), 3.34 (m, 2H), 7.36 (d, 1H,
J ) 6.8 Hz), 7.54 (m, 2H), 7.66 (d, 1H, J ) 9.0 Hz), 7.97 (d,
2H, J ) 8.6 Hz), 8.65 (bs, 2H).
2-Am in o-4,5-d im et h ylt h iop h en -3-yl)n a p h t h a len -1-yl-
m eth a n on e (22). 2-Butanone, 2-(naphthalen-1-carbonyl)ac-
etonitrile, morpholine, and sulfur were reacted according to
the procedure of Step B to afford the desired compound 22.
1
IR (KBr) cm-1: 3358, 3242, 1576, 1426, 1282, 1253, 781; H
NMR (CDCl3): δ 1.16 (s, 3H), 2.07 (s, 3H), 7.17 (bs, 2H), 7.49
(m, 4H), 7.89 (m, 3H).
(2-Am in o-4,5-d im et h ylt h iop h en -3-yl)(4-m et h yln a p h -
th a len -1-yl)m eth a n on e (23). 2-Butanone, 2-(4-methylnaph-
thalen-1-carbonyl)acetonitrile, morpholine, and sulfur were
reacted according to the procedure of Step B to afford the
desired compound 23. IR (KBr) cm-1: 3368, 1576,; 1429, 1256,
759. 1H NMR (CDCl3) δ: 1.17 (s, 3H), 2.06 (s, 3H), 2.73 (s,
3H), 7.04 (bs, 2H), 7.31 (m, 2H), 7.53 (m, 2H), 7.93 (d, 1H, J )
7.8 Hz), 8.05 (d, 1H, J ) 7.8 Hz).
(2-Am in o-4,5-d im eth ylth iop h en -3-yl)(4-m eth oxyn a p h -
th a len -1-yl)m eth a n on e (24). 2-Butanone, 2-(4-methoxynaph-
thalen-1-carbonyl)acetonitrile, morpholine, and sulfur were
reacted according to the procedure of Step B to afford the
desired compound 24. IR (KBr) cm-1: 3354, 1582, 1416, 1242,
1
1087, 763. H NMR (CDCl3) δ: 1.27 (s, 3H), 2.07 (s, 3H), 4.05
(s, 3H), 6.83 (m, 3H), 7.37 (d, 1H, J ) 8 Hz), 7.48 (m, 2H),
7.97 (m, 1H), 8.31 (m, 1H).
(2-Am in o-4,5-dim eth ylth ioph en -3-yl)(4-ch lor on aph th a-
len -1-yl)m eth a n on e (25). 2-Butanone, 2-(4-chloronaphtha-
lene-1-carbonyl)acetonitrile, morpholine, and sulfur were re-
acted according to the procedure of Step B to afford the desired
compound 25. IR (KBr) cm-1: 3248, 1575, 1427, 1252, 965, 760.
1H NMR (CDCl3) δ: 2.06 (s, 9H), 7.19 (bs, 2H), 7.58 (m, 4H),
7.90 (d, 1H, J ) 7.6 Hz), 8.34 (d, 1H, J ) 7.6 Hz).
(2-Am in o-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -3-yl)n aph -
th a len -1-ylm eth a n on e (34). Cyclohexanone, 2-(naphthalen-
1-carbonyl)acetonitrile, morpholine, and sulfur were reacted
according to the procedure of Step B to afford the desired
compound. IR (KBr) cm-1: 3335, 3235, 3124, 1559, 1430, 1290,
781; 1H NMR (CDCl3): δ 1.29 (m, 4H), 1.50 (t, 2H, J ) 6.0
Hz), 2.44 (t, 2H, J ) 6.2 Hz), 7.47 (m, 4H), 7.85 (m, 4H), 8.28
(d, 1H, J ) 6.4 Hz).
(2-Am in o-4,5-dim eth ylth ioph en -3-yl)(4-br om on aph th a-
len -1-yl)m eth a n on e (26). 2-Butanone, 2-(4-bromonaphtha-
lene-1-carbonyl)acetonitrile, morpholine, and sulfur were re-
acted according to the procedure of Step B to afford the desired
1
compound 26. IR (KBr) cm-1: 3241, 1575, 1426, 1251, 781. H
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
m eth yln a p h th a len -1-yl)m eth a n on e (35). Cyclohexanone,
2-(4-methylnaphthalene-1-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 35. IR (KBr) cm-1: 3370, 1567,
NMR (CDCl3) δ: 2.06 (s, 3H), 2.18 (s, 3H), 7.44 (m, 1H), 7.52
(m, 4H), 7.86 (m, 1H), 7.98 (m, 1H), 8.44 (m, 1H).
(2-Am in o-4,5-d im et h ylt h iop h en -3-yl)(4-iod on a p h t h a -
len -1-yl)m eth a n on e (27). 2-Butanone, 2-(4-iodonaphthalene-
1-carbonyl)acetonitrile, morpholine, and sulfur were reacted
according to the procedure of Step B to afford the desired
compound 27. IR (KBr) cm-1: 3044, 1673, 1592, 1415, 1300,
1251, 773. 1H NMR (CDCl3) δ: 2.18 (s, 6H), 7.62 (m, 3H), 7.87
(m, 2H), 7.94 (d, 1H, J ) 8.2 Hz), 8.43 (d, 1H, J ) 8.6 Hz),
9.08 (d, 1H, J ) 8.6 Hz).
2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
n a p h th a len -1-ylm eth a n on e (28). Cyclopentanone, 2-(naph-
thalen-1-carbonyl)acetonitrile, morpholine, and sulfur were
reacted according to the procedure of Step B to afford the
desired compound 28. IR (KBr) cm-1: 3328, 3102, 2853, 1582,
1
1433, 1415, 1256, 756. H NMR (CDCl3) δ: 1.29 (m, 2H), 1.43
(m, 2H), 1.61 (t, 2H, J ) 6 Hz), 2.46 (t, 2H, J ) 6 Hz), 2.74 (s,
3H), 7.21 (m, 3H), 7.30 (m, 2 H), 7.51 (m, 2H), 7.93 (d, 1H, J
) 7.8 Hz), 8.06 (d, 1H, J ) 7.8 Hz).
2-Am in o-4,5,6,7-t et r a h yd r ob en zo[b]t h iop h en -3-yl)(4-
m eth oxyn a p h th a len -1-yl)m eth a n on e (36). Cyclohexanone,
2-(4-iodonaphthalene-1-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 36. IR (KBr) cm-1: 3383, 3275,
2928, 1578, 1436, 1323, 1245, 1093, 733; 1H NMR (CDCl3): δ
1.28 (m, 4H), 1.48 (t, 2H, J ) 7.4 Hz), 2.35 (t, 2H, J ) 6 Hz),

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 805
3.98 (s, 3H), 6.78 (d, 1H, J ) 8 Hz), 7.21 (d, 1H, J ) 8 Hz),
7.40 (m, 2 H), 7.79 (m, 1H), 8.19 (m, 3H).
(CDCl3) δ: 1.61 (m, 2H), 2.05 (m, 4H), 2.68 (s, 3H), 6.97 (bs,
2H), 7.48 (m, 2 H), 7.79 (m, 2H), 7.99 (s, 1H).
2-Am in o-4,5,6,7-t et r a h yd r ob en zo[b]t h iop h en -3-yl)(4-
ch lor on a p h th a len -1-yl)m eth a n on e (37). Cyclohexanone,
2-(4-chloronaphthalene-1-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 37. IR (KBr) cm-1: 3356, 3253,
2-Am in o-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -3-yl)n aph -
th a len -2-ylm eth a n on e (46). Cyclohexanone, 2-(naphthalene-
2-carbonyl)acetonitrile, morpholine, and sulfur were reacted
according to the procedure of Step B to afford the desired
compound 46. IR (KBr) cm-1: 3392, 2929, 1560, 1424, 1292,
1128, 783; 1H NMR (CDCl3): δ 1.43 (m, 4H), 1.75 (m, 2H),
2.53 (t, 2H, J ) 6.2 Hz), 7.53 (m, 3H), 7.62 (d, 1H, J ) 8.4
Hz), 7.89 (m, 5H).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
m eth yln a p h th a len -2-yl)m eth a n on e (47). Cyclohexanone,
2-(4-methylnaphthalene-2-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 47. IR (KBr) cm-1: 3421, 2922,
1560, 1430. 1H NMR (CDCl3) δ: 1.45 (m, 2H), 1.73 (m, 2H),
1.84 (m, 2H), 2.54 (t, 2H, J ) 6.0 Hz), 2.73 (s, 3H), 6.63 (bs,
2H), 7.48 (s, 1H), 7.57 (m, 2H), 7.82 (s, 1H), 7.90 (d, 1H, J )
8.0 Hz), 8.03 (d, 1H, J ) 8.0 Hz).
1
2928, 1573, 1428, 1286, 1254, 1132, 941, 787, 760; H NMR
(CDCl3): δ 1.31 (m, 4H), 1.60 (t, 2H, J ) 6.4 Hz), 2.41 (t, 2H,
J ) 6.4 Hz), 7.29 (m, 3H), 7.56 (m, 3 H), 7.91 (d, 1H, J ) 8
Hz), 8.31 (d, 1H, J ) 6.8 Hz).
2-Am in o-4,5,6,7-t et r a h yd r ob en zo[b]t h iop h en -3-yl)(4-
br om on a p h th a len -1-yl)m eth a n on e (38). Cyclohexanone,
2-(4-bromonaphthalene-1-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 38. IR (KBr) cm-1: 3336, 3234,
2932, 1579, 1558, 1427, 1290, 1253, 1129, 780; 1H NMR
(CDCl3): δ 1.29 (m, 4H), 1.59 (t, 2H, J ) 6 Hz), 2.44 (t, 2H, J
) 6 Hz), 7.26 (m, 2H), 7.48 (m, 3 H), 7.89 (m, 3H).
2-Am in o-4,5,6,7-t et r a h yd r ob en zo[b]t h iop h en -3-yl)(4-
iod on a p h th a len -1-yl)m eth a n on e (39). Cyclohexanone, 2-(4-
iodonaphthalene-1-carbonyl)acetonitrile, morpholine, and sul-
fur were reacted according to the procedure of Step B to afford
the desired compound 39. IR (KBr) cm-1: 3336, 3234, 3123,
2933, 1579, 1558, 1429, 1290, 1254, 1130, 780; 1H NMR
(CDCl3): δ 1.28 (m, 4H), 1.59 (t, 2H, J ) 6.4 Hz), 2.42 (t, 2H,
J ) 6.2 Hz), 7.26 (m, 2H), 7.49 (m, 3 H), 8.87 (m, 3H).
(2-Am in o-4,5-d im eth ylth iop h en -3-yl)n a p h th a len -2-yl-
m eth a n on e (40). 2-Butanone, 2-(naphthalene-2-carbonyl)-
acetonitrile, morpholine, and sulfur were reacted according to
the procedure of Step B to afford the desired compound 40.
IR (KBr) cm-1: 3391, 2922, 1560, 1424, 1263, 1154, 782, 761;
1H NMR (CDCl3): δ 1.55 (s, 3H), 2.16 (s, 3H), 6.42 (bs, 2H),
7.54 (m, 2 H), 7.68 (m, 1H), 7.82 (d, 1H, J ) 8.4 Hz), 7.87 (m,
3H).
(2-Am in o-4,5,6,7-tetr a h yd r o-ben zo[b]th iop h en -3-yl)(6-
ch lor o-4-m eth yln a p h th a len -2-yl)m eth a n on e (48). Cyclo-
hexanone, 2-(4-methyl-6-chloronaphthalene-2-carbonyl)aceto-
nitrile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 48. IR
(KBr) cm-1: 3259, 1559, 1430, 1294, 1090, 787. 1H NMR
(CDCl3) δ: 1.44 (m, 2H), 1.75 (m, 4H), 2.53 (d, 2H, J ) 7.2
Hz), 2.69 (s, 3H), 6.73 (bs, 2H), 7.48 (m, 2 H), 7.80 (m, 2H),
7.98 (s, 1H).
(2-Am in o-4,5-d im eth ylth iop h en -3-yl)(9H-flu or en -2-yl)-
m eth a n on e (49). Butanone, 2-(fluorene-2-carbonyl)acetoni-
trile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 49. IR
(KBr) cm-1: 3360, 2910, 2830, 1660, 1530, 1280, 740; 1H NMR
(CDCl3) δ: 1.16 (s, 3H), 2.07 (s, 3H), 3.86 (s, 2H), 6.82 (bs,
2H), 7.32 (m, 3H), 7.52 (d, 1H, J ) 8 Hz), 7.56 (d, 1H, J ) 7.8
Hz), 7.78 (t, 2H, J ) 7.8 Hz).
(2-Am in o-4,5-d im et h ylt h iop h en -3-yl)(4-m et h yln a p h -
th a len -2-yl)m eth a n on e (41). Butanone, 2-(4-methylnaph-
thalene-2-carbonyl)acetonitrile, morpholine, and sulfur were
reacted according to the procedure of Step B to afford the
desired compound 41. IR (KBr) cm-1: 3431, 1555, 1428, 1277,
(2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(9H-flu or en -2-yl)m eth a n on e (50). Cyclopentanone, 2-(fluo-
rene-2-carbonyl)acetonitrile, morpholine, and sulfur were re-
acted according to the procedure of Step B to afford the desired
compound 50. IR (KBr) cm-1: 3400, 2928, 2846, 1685, 1560,
1
1027, 758. H NMR (CDCl3) δ: 1.58 (s, 3H), 2.18 (s, 3H), 2.74
1
(s, 3H), 6.37 (bs, 2H), 7.53 (s, 1H), 7.57 (m, 2H), 7.87 (m, 2H),
8.07 (d, 1H, J ) 8.2 Hz).
1430, 1268, 737; H NMR (CDCl3) δ: 2.15 (m, 4H), 2.69 (m,
2H), 3.94 (s, 2H), 6.86 (bs, 2H), 7.36 (m, 3H), 7.54 (d, 1H, J )
8 Hz), 7.58 (d, 1H, J ) 7.8 Hz), 7.89 (t, 2H, J ) 7.8 Hz).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(9H-
flu or en -2-yl)m eth a n on e (51). Cyclohexanone, 2-(fluorene-
2-carbonyl)acetonitrile, morpholine, and sulfur were reacted
according to the procedure of Step B to afford the desired
compound 51. IR (KBr) cm-1: 3428, 2927, 1560, 1431, 1267,
(2-Am in o-4,5-d im eth ylth iop h en -3-yl)(6-ch lor o-4-m eth -
yln a p h th a len -2-yl)m eth a n on e (42). 2-Butanone, 2-(4-meth-
yl-6-chloronaphthalene-2-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 42. IR (KBr) cm-1: 3362, 1577,
1424, 1165, 863. 1H NMR (CDCl3) δ: 1.54 (s, 3H), 2.15 (s, 3H),
2.69 (s, 3H), 6.56 (bs, 2H), 7.48 (d, 1H, J ) 8.8 Hz), 7.54 (s,
1H), 7.82 (m, 2H), 8.00 (s, 1H).
2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
n a p h th a len -2-ylm eth a n on e (43). Cyclopentanone, 2-(naph-
thalene-2-carbonyl)acetonitrile, morpholine, and sulfur were
reacted according to the procedure of Step B to afford the
desired compound 43. IR (KBr) cm-1: 3341, 3240, 2926, 1561,
1436, 1285, 1039, 760, 742; 1H NMR (CDCl3) δ: 1.60 (m, 2H),
2.09 (m, 2H), 2.68 (m, 2H), 6.96 (bs, 2H), 7.53 (t, 2H, J ) 4.4
Hz), 7.60 (d, 1H, J ) 6.4 Hz), 7.89 (m, 4H).
(2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(4-m eth yln aph th alen -2-yl)m eth an on e (44). Cyclopentanone,
2-(4-methylnaphthalene-2-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 44. IR (KBr) cm-1: 3447, 1560,
1430, 1289, 1157, 1037, 746. 1H NMR (CDCl3) δ: 1.61 (m, 2H),
2.09 (m, 2H), 2.66 (t, 2H, J ) 6 Hz), 2.73 (s, 3H), 6.94 (bs,
2H), 7.48 (s, 1H), 7.56 (m, 2H), 7.81 (s, 1H), 7.88 (d, 1H, J )
8.0 Hz), 8.03 (d, 1H, J ) 8.0 Hz).
(2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(6-ch lor o-4-m eth yln a p h th a len -2-yl)m eth a n on e (45). Cy-
clopentanone, 2-(4-methyl-6-chloronaphthalene-2-carbonyl)-
acetonitrile, morpholine, and sulfur were reacted according to
the procedure of Step B to afford the desired compound 45.
IR (KBr) cm-1: 3405, 1589, 1428, 1292, 1162, 880. 1H NMR
1
1228, 1139, 760; H NMR (CDCl3) δ: 1.48 (m, 2H), 1.72 (m,
2H), 1.86 (t, 2H, J ) 6 Hz), 2.53 (t, 2H, J ) 6 Hz), 3.94 (s, 2H),
6.53 (bs, 2H), 7.37 (m, 2H), 7.55 (t, 1H, J ) 7.2 Hz), 7.70 (s,
2H), 7.81 (t, 2H, J ) 7.8 Hz).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)n a p h th a len -1-ylm eth a n on e (52). 1-Benzyl-4-pip-
eridone, 2-(naphthalen-1-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 52. IR (KBr) cm-1: 3435, 1576,
1426, 1358, 1253, 784; 1H NMR (CDCl3) δ: 1.53 (m, 2H), 2.30
(m, 2H), 3.38 (s, 2H), 3.54 (s, 2H), 7.43 (m, 11 H), 7.88 (m,
3H).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r o-th ien o[2,3-c]p y-
r id in -3-yl)(4-m et h yln a p h t h a len -1-yl)m et h a n on e
(53).
N-Benzyl-4-piperidone, 2-(4-methylnaphthalene-1-carbonyl)-
acetonitrile, morpholine, and sulfur were reacted according to
the procedure of Step B to afford the desired compound 53.
IR (KBr) cm-1: 3250, 1734, 1574, 1430, 1357, 1255, 698. 1H
NMR (CDCl3) δ: 1.56 (m, 2H), 2.26 (bs, 2H), 2.71 (s, 2H), 2.74
(s, 3H), 3.36 (s, 2H), 3.51 (s, 2H), 7.29 (m, 9H), 7.50 (m, 2H),
7.88 (d, 1H, J ) 7.6 Hz), 7.99 (d, 1H, J ) 7.6 Hz).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)(4-m et h oxyn a p h t h a len -2-yl)m et h a n on e
(54).
N-Benzyl-4-piperidone, 2-(4-methoxynaphthalene-1-carbonyl)-
acetonitrile, morpholine, and sulfur were reacted according to

806 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
the procedure of Step B to afford the desired compound 54.
IR (KBr) cm-1: 3369, 1578, 1432, 1240, 1091, 769. 1H NMR
(CDCl3) δ: 1.65 (bs, 2H), 2.32 (bs, 2H), 3.37 (s, 2H), 3.53 (s,
2H), 4.03 (s, 3H), 6.77 (d, 1H, J ) 8 Hz), 7.10 (bs, 2H), 7.33
(m, 5H), 7.35 (d, 1H, J ) 8 Hz), 7.47 (m, 2H), 7.90 (m, 1H),
8.28 (m, 1H).
procedure of Step B to afford the desired compound 62. IR
(KBr) cm-1: 3420, 3316, 2793, 1579, 1458, 1358, 1285, 1228,
1
1131, 762; H NMR (CDCl3) δ: 1.95 (t, 2H, J ) 5.6 Hz), 2.45
(t, 2H, J ) 5.4 Hz), 3.49 (s, 2H), 3.66 (s, 2H), 6.61 (bs, 2H),
7.08 (m, 3H), 7.31 (m, 1H), 7.35 (d, 1H, J ) 7.2 Hz), 7.54 (t,
2H, J ) 4.2 Hz), 7.63 (d, 1H, J ) 8.6 Hz), 7.86 (m, 2H), 7.97
(s, 1H).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r o-th ien o[2,3-c]p y-
r id in -3-yl)(4-ch lor on a p h t h a len -1-yl)m et h a n on e
(55).
[2-Am in o-6-(4-flu or oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl]n a p h th a len -2-ylm eth a n on e (63). 1-(4-
Fluorobenzyl)-4-piperidone, 2-(naphthalene-2-carbonyl)aceto-
nitrile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 63. IR
(KBr) cm-1: 3401, 2928, 1577, 1508, 1424, 1263, 1221, 1130,
824; 1H NMR (CDCl3) δ: 1.96 (t, 2H, J ) 5.4 Hz), 2.38 (t, 2H,
J ) 5.6 Hz), 3.42 (s, 2H), 3.56 (s, 2H), 6.81 (bs, 2H), 6.98 (t,
2H, J ) 8.8 Hz), 7.27 (t, 2H, J ) 6.2 Hz), 7.56 (m, 2H), 7.90
(m, 5H).
N-Benzyl-4-piperidone, 2-(4-chloronaphthalene-1-carbonyl)ac-
etonitrile, morpholine, and sulfur were reacted according to
the procedure of Step B to afford the desired compound 55.
IR (KBr) cm-1: 3369, 1569, 1430, 1358, 1131, 698. 1H NMR
(CDCl3) δ: 2.46 (t, 2H, J ) 5.4 Hz), 2.75 (t, 2H, J ) 5.4 Hz),
3.34 (s, 2H), 3.52 (s, 2H), 7.34 (m, 7 H), 7.56 (m, 4H), 7.87 (d,
1H, J ) 7.8 Hz), 8.33 (d, 1H, J ) 7.8 Hz).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r o-th ien o[2,3-c]p y-
r id in -3-yl)(4-b r om on a p h t h a len -1-yl)m et h a n on e
(56).
N-Benzyl-4-piperidone, 2-(4-bromonaphthalene-1-carbonyl)ac-
etonitrile, morpholine, and sulfur were reacted according to
the procedure of Step B to afford the desired compound 56.
IR (KBr) cm-1: 3255, 1570, 1424, 1248, 1130, 783.1H NMR
(CDCl3) δ: 2.52 (t, 2H, J ) 5.2 Hz), 2.78 (t, 2H, J ) 5.2 Hz),
3.36 (s, 2H), 3.52 (s, 2H), 7.31 (m, 7 H), 7.48 (m, 4H), 7.85 (d,
1H, J ) 7.8 Hz), 8.36 (d, 1H, J ) 7.8 Hz).
[2-Am in o-6-(4-n itr oben zyl)-4,5,6,7-tetr a h yd r oben zo[b]-
th iop h en -3-yl]n a p h th a len -2-ylm eth a n on e (64). 1-(4-Ni-
trobenzyl)-4-piperidone, 2-(naphthalene-2-carbonyl)-acetoni-
trile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 64. IR
1
(KBr) cm-1: 3422, 1577, 1518, 1424, 1344, 858, 740. H NMR
(CDCl3) δ: 1.94 (t, 2H, J ) 5.4 Hz), 2.42 (t, 2H, J ) 5.4 Hz),
3.45 (s, 2H), 3.68 (s, 2H), 6.80 (bs, 2H), 7.58 (m, 5H), 7.91 (m,
4H), 8.15 (d, 2H, J ) 8.6 Hz).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r o-th ien o[2,3-c]p y-
r idin -3-yl)(4-iodon aph th alen -1-yl)m eth an on e (57). N-Ben-
zyl-4-piperidone, 2-(4-iodonaphthalene-1-carbonyl)acetonitrile,
morpholine, and sulfur were reacted according to the procedure
[2-Am in o-6-(3,4,5-t r im et h oxyb en zyl)-4,5,6,7-t et r a h y-
d r ot h ie n o[2,3-c]p yr id in -3-yl]n a p h t h a le n -2-ylm e t h a -
n on e (65). 1-(3,4,5-Trimethoxybenzyl)-4-piperidone, 2-(naph-
thalene-2-carbonyl)acetonitrile, morpholine, and sulfur were
reacted according to the procedure of Step B to afford the
desired compound 65. IR (KBr) cm-1: 3394, 2933, 2831, 1578,
of Step B to afford the desired compound 57. IR (KBr) cm-1
3108, 1581, 1433, 1359, 1048, 783. H NMR (CDCl3) δ: 1.55
(m, 2H), 2.27 (m, 2H), 3.35 (s, 2H), 3.51 (s, 2H), 7.29 (m, 7 H),
7.45 (m, 4H), 7.86 (d, 2H, J ) 6.8 Hz).
:
1
1
1421, 1358, 1233, 1125, 1005, 782; H NMR (CDCl3) δ: 1.93
2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)n a p h th a -len -2-ylm eth a n on e (58). 1-Benzyl-4-pi-
peridone, 2-(naphthalene-2-carbonyl)acetonitrile, morpholine,
and sulfur were reacted according to the procedure of Step B
to afford the desired compound 58. IR (KBr) cm-1: 3415, 3313,
(t, 2H, J ) 5.6 Hz), 2.40 (t, 2H, J ) 5.6 Hz), 3.46 (s, 2H), 3.52
(s, 2H), 3.81 (s, 3H), 3.83 (s, 6H), 6.53 (s, 2H), 6.62 (bs, 2H),
7.53 (t, 2H, J ) 4 Hz), 7.62 (d, 1H, J ) 8.8 Hz), 7.87 (m, 3H),
7.98 (s, 1H).
1
2926, 1578, 1458, 1407, 1358, 1128, 749; H NMR (CDCl3) δ:
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
din -3-yl)(4-m eth yln aph th alen -2-yl)m eth an on e (66). N-Ben-
zyl-4-piperidone, 2-(4-methylnaphthalene-2-carbonyl)acetoni-
trile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 66. IR
(KBr) cm-1: 3441, 1638, 1455, 1324, 1167, 986. 1H NMR
(CDCl3) δ: 2.47 (t, 2H, J ) 6 Hz), 2.71 (t, 2H, J ) 6 Hz), 2.74
(s, 3H), 3.71 (m, 2H), 3.75 (s, 2H), 6.85 (bs, 2H), 7.31 (m, 5H),
7.46 (s, 1H), 7.56 (m, 2H), 7.82 (s, 1H), 7.87 (d, 1H, J ) 7.8
Hz), 8.02 (d, 1H, J ) 7.8 Hz).
1.94 (t, 2H, J ) 5.2 Hz), 2.41 (t, 2H, J ) 5.8 Hz), 3.42 (s, 2H),
3.60 (s, 2H), 6.79 (bs, 2H), 7.31 (m, 4H), 7.53 (m, 2H), 7.62
(dd, 1H, J ) 9.6 and 1.4 Hz), 7.87 (m, 5H).
[2-Am in o-6-(2-ch lor oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl]n a p h th a len -2-ylm eth a n on e (59). 1-(2-
Chlorobenzyl)-4-piperidone, 2-(naphthalene-2-carbonyl)aceto-
nitrile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 59. IR
(KBr) cm-1: 3419, 3317, 2918, 2792, 1600, 1578, 1461, 1410,
1
1359, 1282, 1135, 984, 781, 759; H NMR (CDCl3) δ: 1.96 (t,
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)(6-ch lor o-4-m eth yln a p h th a len -2-yl)m eth a n on e
(67). N-Benzyl-4-piperidone, 2-(4-methyl-6-chloronaphthalene-
2-carbonyl)acetonitrile, morpholine, and sulfur were reacted
according to the procedure of Step B to afford the desired
compound 67. IR (KBr) cm-1: 3377, 1561, 1438, 1364, 1298,
2H, J ) 5.4 Hz), 2.49 (t, 2H, J ) 5.6 Hz), 3.53 (s, 2H), 3.73 (s,
2H), 6.60 (bs, 2H), 7.19 (m, 4H), 7.46 (d, 1H, J ) 6.6 Hz), 7.52
(t, 2H, J ) 6.4 Hz), 7.61 (d, 1H, J ) 8.4 Hz), 7.88 (m, 2H),
7.99 (s, 1H).
[2-Am in o-6-(3-ch lor oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl]n a p h th a len -2-ylm eth a n on e (60). 1-(3-
Chlorobenzyl)-4-piperidone, 2-(naphthalene-2-carbonyl)aceto-
nitrile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 60. IR
(KBr) cm-1: 3270, 1575, 1523, 1357, 1285, 1129, 780. 1H NMR
(CDCl3) δ: 1.94 (t, 2H, J ) 5.6 Hz), 2.41 (t, 2H, J ) 5.6 Hz),
3.46 (s, 2H), 3.53 (s, 2H), 6.81 (bs, 2H), 7.26 (m, 4H), 7.33 (s,
1H), 7.56 (m, 3H), 8.15 (m, 3H).
[2-Am in o-6-(4-ch lor oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl]n a p h th a len -2-ylm eth a n on e (61). 1-(4-
Chlorobenzyl)-4-piperidone, 2-(naphthalene-2-carbonyl)aceto-
nitrile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 61. IR
(KBr) cm-1: 3392, 2963, 1715, 1577, 1423, 1262, 1088, 1016,
800; 1H NMR (CDCl3) δ: 1.93 (t, 2H, J ) 5.6 Hz), 2.43 (t, 2H,
J ) 5.8 Hz), 3.42 (s, 2H), 3.56 (s, 2H), 6.80 (bs, 2H), 7.26 (s,
4H), 7.59 (m, 3H), 7.92 (m, 4H).
1
1246, 733. H NMR (CDCl3) δ: 1.91 (t, 2H, J ) 5.4 Hz), 2.41
(t, 2H, J ) 5.4 Hz), 2.68 (s, 3H), 3.43 (s, 2H), 3.62 (s, 2H), 6.82
(bs, 2H), 7.28 (m, 5 H), 7.47 (m, 2H), 7.80 (m, 2H), 7.99 (s,
1H).
(2-Am in o-6-pyr idin -3-ylm eth yl-4,5,6,7-tetr ah ydr oth ien o-
[2,3-c]p yr id in -3-yl)n a p h th a len -2-ylm eth a n on e (68). N-(3-
Nicotinyl)-4-piperidone, 2-(naphthalene-2-carbonyl)acetoni-
trile, morpholine, and sulfur were reacted according to the
procedure of Step B to afford the desired compound 68. Yield
57%, mp 189-190 °C. IR (KBr) cm-1: 3370, 1599, 1580, 1458,
1
1230, 1022, 783. H NMR (CDCl3) δ: 1.98 (t, 2H, J ) 5.2 Hz),
2.44 (t, 2H, J ) 5.8 Hz), 3.49 (s, 2H), 3.66 (s, 2H), 6.84 (bs,
2H), 7.26 (d, 2H, J ) 7.8 Hz), 7.56 (m, 3H), 7.72 (d, 1H, J )
7.6 Hz), 7.91 (m, 4H), 8.53 (s, 1H).
(2-Am in o-5,6-d ih yd r o-4H-cyclop en ta [b]th iop h en -3-yl)-
(9H-flu or en -2-yl)m eth a n on e (69). Cyclopentanone, 2-(fluo-
rene-2-carbonyl)acetonitrile, morpholine, and sulfur were re-
acted according to the procedure of Step B to afford the desired
compound 69 as yellow solid. Yield: 68%; mp 197-200 °C
(petroleum ether). IR (KBr) cm-1: 3400, 2928, 2846, 1685,
1560, 1430, 1268, 737; 1H NMR (CDCl3) δ: 2.15 (m, 4H), 2.69
[2-Am in o-6-(2-flu or oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl]n a p h th a len -2-ylm eth a n on e (62). 1-(2-
Fluorobenzyl)-4-piperidone, 2-(naphthalene-2-carbonyl)aceto-
nitrile, morpholine, and sulfur were reacted according to the

2-Amino-3-naphthoylthiophenes as Allosteric Enhancers
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5 807
(m, 2H), 3.94 (s, 2H), 6.86 (bs, 2H), 7.36 (m, 3H), 7.54 (d, 1H,
J ) 8 Hz), 7.58 (d, 1H, J ) 7.8 Hz), 7.89 (t, 2H, J ) 7.8 Hz).
(2-Am in o-6-ph en yl-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -
3-yl)n a p h t h a len -2-ylm et h a n on e (70). 4-Phenylcyclohex-
anone, 2-(naphthalene-2-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 70. Yield 78%; mp 119-120 °C.
1287, 1240, 1201, 781. 1H NMR (CDCl3) δ: 6.03 (d, 1H, J )
7.8 Hz), 6.74 (t, 1H, J ) 7.4 Hz), 6.95 (t, 1H, J ) 7.4 Hz), 7.50
(m, 5H), 7.78 (m, 2H), 7.96 (m, 3H).
Biologica l Ma ter ia ls. [3H]DPCPX (specific activity, 112 Ci/
mmol) and [3H]CCPA (specific activity, 55 Ci/mmol) were
obtained from NEN Research Products (Boston, MA); [3H]ZM
241385 (specific activity, 17 Ci/mmol) was obtained from Tocris
Cookson (Bristol, UK); [3H]MRE 3008F20 (specific activity 67
Ci/mmol) was obtained from Amersham International (Buck-
inghamshire, UK). CHO cells transfected with the human
recombinant A1 adenosine receptor (hCHO-A1) were obtained
by Prof. K. N. Klotz, University of Wu¨rzburg. Human cerebral
cortex was obtained from the University of Ferrara, Medicina
Legale Section, with approval of human tissue use protocol.
Rat cerebral cortex was harvested from adult male Wistar rats.
All tissue culture reagents were obtained from Sigma.
1
IR (KBr) cm-1: 3402, 1560, 1439, 1245, 760. H NMR (CDCl3)
δ: 1.63 (m, 1H), 1.72 (d, 2H, J ) 10.4 Hz), 2.75 (m, 2H), 2.96
(m, 2H), 7.21 (bs, 2H), 7.23 (m, 6H), 7.55 (dd, 1H, J ) 8.4 and
4.6 Hz), 7.63 (d, 1H, J ) 7.6 Hz), 7.89 (m, 3H), 7.99 (s, 1H).
(2-Am in o-6-ben zyl-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -
3-yl]n a p h t h a len -2-ylm et h a n on e (71). 4-Benzylcyclohex-
anone, 2-(naphthalene-2-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 71. Yield: 60%; mp 153 °C
(petroleum ether). IR (KBr) cm-1: 3419, 3315, 2912, 1596, 1568,
Biologica l Meth od s. Cyclic AMP Accu m u la tion in
CHO Cells. Chinese hamster ovary cells expressing human
recombinant A1-adenosine receptors (CHO:hA1 cells) at a
density of approximately 8000 fmol/mg protein were prepared
as previously described,14c and aliquots of these cells at low
passage numbers were frozen and stored in liquid nitrogen.
Upon the arrival of a group of compounds for testing in the
laboratory, an aliquot of cells was removed from liquid nitrogen
storage and grown in Ham’s F-12 culture medium with 10%
fetal bovine serum and 0.5 mg/mL of antibiotic G-418.30 Cells
were passaged thrice weekly. For experiments, aliquots of cells
were placed into 12-well culture plates with culture medium,
serum, and antibiotic for 48 h, by which time the cells had
grown to a confluent monolayer.
1
1456, 1282, 1130, 781, 749, 699. H NMR (CDCl3) δ: 1.04 (m,
2H), 1.62 (m, 2H), 1.81 (m, 1H), 2.19 (m, 2H), 2.59 (d, 2H, J )
7.4 Hz), 6.67 (bs, 2H),7.19 (m, 4H), 7.56 (m, 4H), 7.88 (m, 4H).
(2-Am in o-6-ben zyl-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -
3-yl)n a p h t h a len -2-ylm et h a n on e (72). 4-Benzylcyclohex-
anone, 2-(naphthalene-2-carbonyl)acetonitrile, morpholine, and
sulfur were reacted according to the procedure of Step B to
afford the desired compound 72 as white solid. Yield: 61%;
mp 69-70 C (petroleum ether). IR (KBr) cm-1: 3369, 2914,
1569, 1424, 1285, 1252, 782; 1H NMR (CDCl3): δ 1.17 (m, 2H),
1.27 (m, 1H), 1.56 (m, 1H), 1.81 (m, 1H), 2.19 (m, 2H), 2.49 (d,
2H, J ) 7 Hz), 7.03 (bs, 2H), 7.29 (m, 6H), 7.48 (m, 5H), 7.88
(d, 1H, J ) 8.8 Hz).
[2-Am in o-6-(4-m eth oxyp h en yl)-4H-1,5,7-tr ith ia -in d en -
3-yl]n a p h th a len -1-ylm eth a n on e (73). 2-(4-Methoxyphenyl)-
1,3-dithian-5-one, 2-(naphthalene-1-carbonyl)acetonitrile, mor-
pholine, and sulfur were reacted according to the procedure
of Step B to afford the desired compound 73 as a yellow solid.
To begin an experiment, growth medium was removed from
the culture plates and cells were washed once with Hanks
buffered saline solution. The wash solution was then removed
and replaced with fresh Hanks solution containing forskolin
(1 µM), rolipram (20 µM), CPA (0.01 nM), adenosine deaminase
(2 U/mL), and the allosteric enhancer to be tested. Forskolin
was used to stimulate the activity of adenylyl cyclase, rolipram
to inhibit cAMP phosphodiesterase, adenosine deaminase to
degrade endogenous adenosine, and CPA to cause a small
increase of the number of activated adenosine receptors.
Yield: 77%; mp 132-134 C (petroleum ether). IR (KBr) cm-1
:
3412, 1607, 1578, 1509, 1420, 1303, 1254, 1176, 1112, 1029,
778; 1H NMR (CDCl3): δ 2.17 (s, 2H), 3.77 (s, 3H), 3.81 (s,
1H), 6.78 (d, 2H, J ) 8.6 Hz), 7.28 (bs, 2H), 7.29 (m, 2H), 7.51
(m, 5 H), 7.93 (m, 2H).
P r ep a r a tion of (2-Acetyla m in o-4,5,6,7-tetr a h yd r oben -
zo[b]th iop h en -3-yl)n a p h th a len -1-ylm eth a n on e (75). Py-
ridine (10 drops) was added to a stirred solution of 34 (1.6 g,
5.2 mmol) in Ac2O (12 mL) at room temperature. The solution
was refluxed for 2 h, poured in water, and extracted with
EtOAc (50 mL). The extract was washed successively with
saturated aqueous NaHCO3, water, and brine, dried over Na2-
SO4, and concentrated under vacuo. Recrystallization from
petroleum ether afforded 75 as a yellow solid. Yield ) 88%,
After 6 min of incubation at 36 °C in the presence of drugs,
the incubation solution was removed, and hydrochloric acid
(final concentration, 50 mM) was added to cells to terminate
drug action. The content of cAMP in acidified extracts of cells
was determined by radioimmunoassay as previously de-
scribed.14c Because the magnitude of the effects of allosteric
enhancers on CHO:hA1 cells changed subtly with passage
number and differed slightly among different aliquots of cells,
the action of tested compounds and the action of the reference
compound PD 81,723 were assayed in each experiment.
1
mp 234 °C. H NMR (CDCl3): δ 1.34 (m, 4H), 1.61 (t, 2H, J )
6.0 Hz), 2.36 (s, 3H), 2.62 (t, 2H, J ) 6.2 Hz), 7.51 (m, 4H),
7.92 (m, 4H), 12.3 (s, 1H).
Allosteric enhancement was measured as the action of a test
compound at different concentrations (0.01, 0.1, 1, and 10 µM)
to reduce the cAMP content of CHO:hA1 cells in the presence
of 0.05-0.1 nM CPA. CPA (0.05-0.1 nM) alone causes a slight
reduction of cAMP content of cells by activation of A1-adenosine
receptors. Allosteric enhancement of the action of CPA causes
a further reduction of the cAMP content of CHO:hA1 cells.
Because the spontaneous activity of adenosine receptors in
CHO:hA1 cells causes an inhibition of adenylyl cyclase activity
even in the absence of an agonist,30 antagonists of adenosine
receptors increase cAMP content of cells. Therefore, com-
pounds that increased cAMP content of cells in this study were
provisionally identified as A1-adenosine receptor antagonists.
(2-Acet yla m in ob en zo[b]t h iop h en -3-yl)n a p h t h a len -1-
ylm eth a n on e (76). A mixture of 75 (621 mg, 1.8 mmol), 10%
Pd-C (50% wet, 1.4 g), and CHCl3 (20 mL) was stirred at room
temperature for 10 min, and then the solvent was evaporated.
The resulting powder was heated at 130 °C for 20 h, cooled to
rt, and extracted with EtOAc. The insoluble solids were filtered
out, and the filtrate was concentrated under vacuo. Recrys-
tallization from petroleum ether afforded 76 as a yellow solid.
Yield ) 60%, mp 134 °C. 1H NMR (CDCl3) δ: 2.44 (s, 3H),
6.30 (d, 1H, J ) 8.4 Hz), 6.86 (t, 1H, J ) 7.4 Hz), 7.14 (t, 1H,
J ) 7.4 Hz), 7.51 (m, 4H), 7.71 (d, 1H, J ) 7.8 Hz), 7.95 (t,
1H, J ) 6.6 Hz), 7.95 (d, 1H, J ) 7.8 Hz), 12.8 (s, 1H).
(2-Am in oben zo[b]th iop h en -3-yl)n a p h th a len -1-ylm eth -
a n on e (77). A mixture of 76 (160 mg, 0.464 mmol), 1 N
aqueous NaOH (0.5 mL, 0.5 mmol), and EtOH (10 mL) was
refluxed for 5 h and then concentrated in vacuo. The residue
was diluted with EtOAc, washed successively with water and
brine, dried over Na2SO4, and evaporated in vacuo. The residue
was purified by flash chromatography on silica gel using
EtOAc-petroleum ether (2:8, v/v) as eluent. Recrystallization
from petroleum ether furnished 77 as a yellow solid. Yield )
78%; mp 162-164 °C. IR (KBr) cm-1: 3351, 1587, 1466, 1420,
Mem br a n e P r ep a r a tion fr om CHO-A1 Cells. For mem-
brane preparation the culture medium was removed. The cells
were washed with PBS and scraped off T75 flasks in ice-cold
hypotonic buffer (5 mM Tris HCl, 2 mM EDTA, pH 7.4). The
cell suspension was homogenized using a Polytron, and the
homogenate was spun for 10 min at 1000g. The supernatant
was then centrifuged for 30 min at 100 000g. The membrane
pellet was resuspended in 50 mM Tris HCl buffer pH 7.4 and
incubated with 2U/ml of ADA for 30 min at 37 °C. Then the
suspension was stored at -80 °C. The protein concentration

808 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 5
Baraldi et al.
was determined according to a Bio-Rad method with bovine
albumin as a standard reference.31
(1 µM) and test agents (10 µM). Specific binding was then
measured at 2-180 min after the addition of R-PIA (1 µM)
and test agents (10 µM).
Mem br a n e P r ep a r a tion fr om Ra t Cor tex a n d Hu m a n
Br a in . Cerebral cortical tissue from each species was homog-
enized using a Polytron (setting 6, 20 s) in 20 volumes of ice-
cold 50 mM Tris-HCl, pH 7.4. This crude membrane homoge-
nate was then centrifuged at 48 000g for 15 min at 4 °C. The
resulting pellet was resuspended in buffer containing 2 IU/ml
ADA to 20 mg/mL original tissue weight and incubated at 37
°C for 30 min to remove endogenous adenosine. This mem-
brane homogenate was recentrifuged at 48 000g for 15 min at
4 °C. The resulting membrane pellet was resuspended and
recentrifuged at 48 000g for 15 min at 4 °C. The final
membrane pellets were stored at -80 °C until the time of
assay.
Da ta An a lysis. All values are expressed as mean ( SEM
of three independent experiments. Inhibitory binding con-
stants, Ki, were 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.32 A weighted non linear least-squares
curve fitting program LIGAND was used for computer analysis
of saturation, competition, and kinetic experiments.33 For
experiments with two comparison groups, statistical analysis
was performed with a two-tailed t test. Differences between
group mean values were considered significant at P e 0.05.
Ad en osin e Recep tor Bin d in g. To determine the effect of
the new series of derivatives of PD 81,723 on the binding of
ligands to A1, A2A, and A3 receptors, membranes from CHO:
hA1, hA2A, hA3, rat cortex, and human brain were incubated
in a buffer solution in the absence and presence of test
compounds. Test agents were dissolved in DMSO and added
to the assay from a 100-fold concentrated solution in DMSO.
Control incubations also contained 1% DMSO. Bound and free
radioactivities were separated by filtering the assay mixture
through Whatman GF/B glass-fiber filters using a Micro-Mate
196 cell harvester (Packard Instrument Company). The filter-
bound radioactivity was counted on Top Count Microplate
Scintillation Counter (efficiency 57%) with Micro-Scint 20.
Binding of 1 nM [3H]CCPA to A1 receptors in CHO:hA1, rat,
and human brain membranes in the absence and presence of
increasing concentrations of test compounds was carried out
in triplicate at 25 °C for 90 min in 50 mM Tris-HCl, pH 7.4.
Nonspecific binding was defined as binding in the presence of
1 µM R-PIA.
Ack n ow led gm en t. We wish to thank King Phar-
maceuticals and MIUR (60% and 40%) for support of
this research. We are thankful to Prof. Karl-Norbert
Klotz (Institut fu¨r Pharmakologie und Toxikologie,
Universita¨t Wu¨rzburg, Germany) for supplying CHO-
hA3 cell lines. Maria del Carmen Nun˜ez is the recipient
of a Fundacio`n Ramo`n Areces fellowship.
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