2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist allosteric enhancers at human a1 adenosine receptors

Article abstract of DOI:10.1021/jm010081p

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A₁ adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A₁AR (hA₁AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A₁AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA₁AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A₁AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH₂)_n- bridge, n = 3 < n = 4 < n = 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA₁AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.

Full text of DOI:10.1021/jm010081p

382
J . Med. Chem. 2002, 45, 382-389
2-Am in o-3-a r oyl-4,5-a lk ylth iop h en es: Agon ist Alloster ic En h a n cer s a t Hu m a n A1
Ad en osin e Recep tor s
C. Elisabet Tranberg,† Andrea Zickgraf,‡ Brian N. Giunta,‡ Henning Luetjens,† Heidi Figler,§
Lauren J . Murphree,§ Ruediger Falke,‡ Holger Fleischer,| J oel Linden,§ Peter J . Scammells,† and
Ray. A. Olsson*,†,‡
Centre for Chiral and Molecular Technologies, Deakin University, Geelong, Victoria 3217, Australia, Suncoast AHA
Cardiovascular Research Laboratory, Department of Internal Medicine, University of South Florida, Tampa, Florida 33612,
Departments of Medicine (Cardiology) and Molecular Physiology and Biological Physics, University of Virginia,
Charlottesville, Virginia, and Institute for Analytical Chemistry, J ohannes Gutenberg University, Mainz, Germany
Received February 21, 2001
2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A1
adenosine receptor. The present report describes syntheses and assays of the AE activity at
the human A1AR (hA1AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes
(3a -i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a -h ), three 3-aroyl-2-carboxy-4,5-
dimethylthiophenes (15a -c), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes
(17a -j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a -n ), and 15 2-amino-
3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a -o). An in vitro assay employing
the A1AR agonist [125I]ABA and membranes from CHO-K1 cells stably expressing the hA1AR
measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-
A1AR-G protein ternary complex. Compounds 3a -i had little or no AE activity, and compounds
12a -h had only modest activity, evidence that AE activity depended absolutely on the presence
of at least a methyl group at C-4 and C-5. Compounds 17a -c lacked AE activity, suggesting
the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of
compounds 17a -j, 18a -n , and 19a -o. AE activity increased with the size of the -(CH2)n- bridge,
n ) 3 < n ) 4 < n ) 5. The 3-carbethoxy substituents of 17a , 18a , and 19a did not support AE
activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para
positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus,
the hA1AR contains an allosteric binding site able to accommodate 3-aroyl substituents that
are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric
binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.
An allosteric enhancer (AE) is a compound that binds
to a receptor at a site different from the ligand binding
or orthosteric site. Such drugs amplify the activity of
an agonist or antagonist wherever and whenever the
ligand occupies its receptor. Since the actions of AEs
depend on the presence of the natural ligand, they are
event- and tissue-specific. The benzodiazepines, which
act at GABA receptors, and dihydropyridine calcium
channel blockers are familiar examples of drugs that
act at allosteric sites.
In 1990 Bruns et al.1 discovered that 2-amino-3-
benzoylthiophenes were weak antagonists but also
agonist AEs at the A1 adenosine receptor (A1AR) but
not at the A2A adenosine receptor (A2AAR). Structure-
activity correlations2 indicated that activity depended
on the 2-amino group and the carbonyl oxygen of the
benzoyl moiety, suggesting that an intramolecular
hydrogen bond making the two aromatic residues
coplanar contributed to activity. Certain electron-
withdrawing substituents such as chloro or trifluoro-
methyl at either the meta or para position of the benzoyl
moiety greatly augmented activity. Alkyl or aryl groups
at thiophene C-4, but not at C-5, promoted activity, as
did polymethylene or polymethylenamino bridges link-
ing C-4 and C-5. The most potent compound identified
by Bruns was (2-amino-4,5-dimethyl-3-thienyl)[(3-tri-
fluoromethyl)phenyl]methanone, PD81,723, which is
compound 12f in this report. Bruns was not able to
further refine the structure-activity rules because the
panel of thiophenes available to him was limited.
Allosteric enhancement of A1AR-mediated responses
might have therapeutic potential. Adenosine, acting
through A1ARs, impedes propagation of the cardiac
impulse through the atrioventricular node, the “negative
dromotropic” effect exploited in the treatment of su-
praventricular tachyarrythmias with adenosine.3,4 Sev-
eral groups have shown that 12f potentiates the nega-
tive dromotropic effect of both exogenous adenosine and
of endogenous adenosine released during hypoxia.5-10
Thus, AEs acting at the A1AR might be useful in the
prophylaxis of paroxysmal supraventricular tachyarryth-
mias. Similarly, 12f potentiates the “preconditioning”
effect of adenosine, thereby reducing the size of the
myocardial infarct produced by a coronary occlusion.11
Accordingly, AEs for the A1AR might be useful in the
* Address for correspondence: Ray A. Olsson, M.D., Dept. Internal
Medicine, MDC Box 19, University of South Florida, 12901 Bruce B.
Downs Blvd., Tampa, FL 33612-4745. Tel: (813) 974-4067. Fax: (813)
974-2189. E-mail: rolsson@hsc.usf.edu.
† Deakin University.
‡ University of South Florida.
§ University of Virginia.
| J ohannes Gutenberg University.
10.1021/jm010081p CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/19/2001

2-Amino-3-aroyl-4,5-alkylthiophenes
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 383
alternative synthesis proceeding from 3-methyl-2-
thiophenecarboxaldehyde, 4, to generate 2-amino-3-
benzoyl-4,5-dimethylthiophenes 12a -h (Scheme 2). The
Huang-Minlon modification14 of the Wolf-Kishner
reduction of 4 gave 2,3-dimethylthiophene, 5. The
original plan called for introducing an amino function
at C-2 by nitration of 5 and then reduction of the 4,5-
dimethyl-2-nitrothiophene, 6. Unfortunately, 6 proved
difficult to purify, and the subsequent reduction gave a
tar. Such an outcome is not surprising, since the
instability of 2-amino-4,5-dialkylthiophenes is well
known.15 The alternative synthesis consisted of the tin-
(IV) chloride-catalyzed Friedel-Crafts acylation of 5
with acetyl chloride to yield 2-acetyl-4,5-dimethylthio-
phene, 7. The oxime, 8, was formed, and PCl5-catalyzed
Beckmann rearrangement of that oxime gave a mixture
of 2-acetamido-4,5-dimethylthiophene, 9, a key inter-
mediate for the synthesis of 12a -h , as well as N-methyl
2-carboxamido-4,5-dimethylthiophene, 10. Friedel-
Crafts acylation of 9 by benzoyl chlorides gave 2-aceta-
mido-3-benzoylthiophenes 11a -h . Solvent importantly
affected yield; in the case of acylation with benzoyl
chloride, replacing benzene with 1,2-dichloroethane
improved the yield from 47% to 82%. Base-catalyzed
deprotection of 11a -h gave the target thio-
phenes, 12a -h . Deprotection with acid catalyzed the
formation of dimers such as 13 that lacked AE activity.
The dimerization was not readily apparent in NMR
spectra, but it was evident in high-resolution mass
spectrometry. Deprotection of the 2,4,6-trimethylbenzoyl
compound 11h with acid did not lead to dimerization,
perhaps a result of the electronic or steric effects of the
three methyl groups.
F igu r e 1. Generic structures of the six panels of thiophenes
included in this study.
Sch em e 1
prophylaxis of coronary artery disease. Despite these
in vivo activities, the 2-aminothiophenes are not ideal
drug candidates. First, they are aromatic amines and
thus have carcinogenic potential. Second, they tend to
be unstable, though perhaps formulation could overcome
that drawback. The purpose of the present study was
not to develop aminothiophenes as drugs but, rather,
to better define their structure-activity rules in order
to design new, safer, and more stable AEs.
The present study aims at extending the work of
Bruns by examining the AE activity, at a cloned and
expressed human A1AR, of six groups of thiophenes
designed to describe those rules in further detail (Figure
1). In terms of the C-4 and C-5 substituents, those
groups are as follows: (a) no C-4 or C-5 substituents
(3a -i), (b) 4,5-dimethylthiophenes (12a -h ), and three
groups of cycloalkyl[b]thiophenes having polymethylene
bridges between C-4 and C-5 consisting of (c) three
(17a -j), (d) four (18a -n ), or (e) five (19a -o) carbon
atoms. Within each group, variations in the 3-substitu-
ent defined the structure-activity rules governing the
contribution of this substituent to AE activity. The sixth
group consisted of thiophene-2-carboxylic acids 15a -c,
included to test the hypothesis that the 2-amino group
is essential for AE activity.
Compound 10 is a side product in the pathway leading
to 12a -h , but its derivatives offered the chance to test
another inference from the Bruns study, namely, that
the 2-amino group is important for activity. That study
found that, with the exception of the 2-iodoacetamide,
derivatization of the 2-amino group destroyed AE activ-
ity. The present study built on that observation by
replacing the 2-amino group with a carboxyl group,
prepared by the hydrolysis of the amide group of 10.
Friedel-Crafts acylation at C-3 failed, probably because
the electron-withdrawing effect of the 2-substituent
made the thiophene resistant to acylation. However,
lithiation of 10 permitted acylation with benzoyl chlo-
rides, forming the amides, 14a -c. Alkaline hydrolysis
then gave compounds 15 a -c.
The method of Gewald13 served for the syntheses of
5,6-dihydro-4H-cyclopenta[b]thiophenes 17a -j, 4,5,6,7-
tetrahydrobenzo[b]thiophenes 18a -n , and 5,6,7,8-tet-
rahydro-4H-cyclohepta[b]thiophenes 19a -o (Scheme 3).
That method consists of the base-catalyzed condensation
(Knoevenagel) of a cycloalkanone 16a -c with an aryl
â-ketonitrile to form an olefin. Subsequently, that olefin
undergoes cyclization with sulfur to form a 2-amino-3-
aroylthiophene. Most of the present syntheses followed
the “one pot” variant, which consists of adding all the
reactants and catalyst at once, thereby avoiding the
necessity of isolating the intermediate olefin before the
reaction with sulfur. The two-step variant served for
making multigram quantities of 20l and 20n . Diethyl-
amine was usually the catalyst; however, a solid-phase
catalyst16 gave results similar to those using diethyl-
Ch em istr y
The reaction12 of 2,5-dihydroxy-1,4-dithiane (thioace-
taldehyde dimer, 2) with an aroylacetonitrile gave
2-amino-3-aroylthiophenes 3a -i (Scheme 1).
The base-catalyzed condensation of an aryl â-ketoni-
trile with 2-butanone to form a mixture of the E- and
Z-isomers of 2-benzoyl-3-ethylcrotonitrile, followed by
cyclization with sulfur, is a general method for the
synthesis of 2-amino-3-aroyl-4,5-dimethylthiophenes.13
However, because only the E-isomer can react with
sulfur, low yields are an inherent disadvantage of that
approach. We therefore developed a more efficient

384 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Tranberg et al.
Sch em e 2
Sch em e 3
in proportion to the size of the C4-C5 polymethylene
bridge, -(CH2)3- < -(CH2)4- < -(CH2)5-. However, that
comparison was limited to three compounds. Between-
group comparisons of members of 17, 18, and 19 having
the same C-3 substituents showed that AE activity
increased according to the size of the C4-C5 polymeth-
ylene bridge (Figure 2).
The 3-aroyl moieties contributed importantly to AE
activity. None of the cycloalkylthiophenes having a
3-carboxyethyl substituent, namely, 17a , 18a , and 19a ,
were active. An unsubstituted benzoyl group supported
a low level of AE activity, and both 3- and 4-fluoroben-
zoyl groups generally did likewise. Other benzoyl sub-
stituents increased AE activity, the rank order for all
substituents being H ) F , Cl < Br < I ) Ph ) cHex.
Both the 1- and the 2-isomers of 3-naphthoylthiophenes
had substantial AE activity. QSAR analysis24 showed
that neither of the electronic parameters, σm or σp, of
the 3-benzoyl substituent accounted for differences in
AE activity (r2 for the regressions of AE activity on
either Hammett parameter were <0.1 and were not
significant; data not shown). However, the hydrophobic
and steric parameters, π and molar refractivity, respec-
tively, better accounted for the effect of the 3-aroyl
substituents on AE activity (Figure 3). That the analysis
could not distinguish between hydrophobicity and steric
bulk is not surprising, since those substituent param-
eters tend to be covariant.24 For the substituent groups
studied here, the regression of π on molar refractivity
had r2 ) 0.83. Although most of the 3-aroyl substituents
were planar, thiophenes having 4-phenylbenzoyl (17i,
18k , 19m ) or 4-cyclohexylbenzoyl (18l, 19n ) substitu-
ents had excellent activity. Because the phenyl group
can rotate around the axis of the bond joining it to the
benzoyl moiety and a cyclohexane group is not planar,
the high AE activity of those compounds suggests that
planarity in that portion of the molecule is not critical.
amine. Neither LiCl,17 nor zeolites,18 which suffice for
Knoevenagel condensations of aldehydes, catalyzed the
condensation of cycloalkanones 16a -c.
Bromoacetylarenes were the starting materials for the
preparation of the â-ketonitriles used to synthesize the
thiophenes. Since only a few were commercially avail-
able, we prepared them by reacting acetoarenes with
elemental bromine in glacial acetic acid,19 1,4-dioxane
dibromide,20 copper(II) bromide,21 or tetrabutylammo-
nium tribromide.22 Brominations by means of Cu(II)Br
or tetrabutylammonium tribromide were rapid, clean,
and nearly quantitative. By contrast, brominations with
either Br2/acetic acid or dioxane dibromide required over
2 equiv of brominating agent to drive the reaction to
completion. Reacting the bromoacetylarenes with NaCN
in cooled ethanol-water generated the â-ketonitriles.23
Resu lts a n d Discu ssion
Table 1 lists the chemical characteristics of the novel
compounds.
Five panels of compounds provided information about
the structure-activity rules governing modifications at
thiophene C-3, C-4, and C-5 (Table 2 and Figure 2). The
very low to absent activities of 3a -i are evidence that
substituents at C-4 and/or C-5 are essential for activity.
Consistent with that interpretation, the 4,5-dimethyl-
thiophenes 12a -h have somewhat better AE activity.
The AE activities of those cycloalkylthiophenes 17-19
having the same C-3 substituents as 12a -h were much
higher, evidence that while the dimethyl substituents
could support activity, their contribution was less than
optimal. In the study by Bruns,1 AE activity increased
Thiophene-2-carboxylic acids 17a -c lacked activity,
support for the idea2 that AE activity depends on the
2-amino group.
In summary, analysis of the results by QSAR supports
a model of the hA1AR that contains an allosteric binding
site able to accommodate 3-aroyl substituents that are
bulky and hydrophobic but not necessarily planar. A
second region in the A1AR interacts additively with
alkyl substituents at thiophene C-4 and/or C-5. The lack

2-Amino-3-aroyl-4,5-alkylthiophenes
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 385
Ta ble 1. Characteristics of Novel 2-Aminothiophenes
no.
3b
3d
3e
3g
3h
3i
R3, R4, R5
3-FPh, H, H
3-BrPh, H, H
4-FPh, H, H
yield, %
purificationa
mp, °C
formula
anal.
47
56
69
41
71
63
54
38
94
63
93
84
71
90
86
88
59
52
74
55
73
52
37
79
32
14
23
18
75
21
28
38
28
33
26
64
30
84
42
31
20
34
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
H
H
E
E
E
E
H
E
E
E
E
H
H
E
E
E
E
E
E
E
E
155-6
135
C
C
C
C
11H8FNOS
11H8BrNOS
11H8FNOS
11H8BrNOS
C11H7Cl2NOS
15H11NOS
13H12FNOS
13H12ClNOS
13H12BrNOS
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
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
142-4
153-4
138-40
143-5
106
4-BrPh, H, H
3,4-Cl2Ph, H, H
2-naphth, H, H
3-FPh, Me, Me
3-ClPh, Me, Me
3-BrPh, Me, Me
3-CH3Ph, Me, Me
3-PhPh, Me, Me
mesityl, Me, Me
Ph, 2-COOH
3-CF3Ph, 2-COOH
4-PhPh, 2-COOH
CO2Et, -(CH2)3-
3ClPh, -(CH2)3-
3BrPh, -(CH2)3-
4F-Ph, -(CH2)3-
2-naphth, -(CH2)3-
CO2Et, -(CH2)4-
3-FPh, -(CH2)4-
3-BrPh, -(CH2)4-
4-CH3Ph, -(CH2)4-
4-CNPh, -(CH2)4-
4-PhPh, -(CH2)4-
4-cHexPh, -(CH2)4-
2-naphth, -(CH2)4-
CO2Et, -(CH2)5-
3ClPh, -(CH2)5-
3-BrPh, -(CH2)5-
3-IPh, -(CH2)5-
4FPh, -(CH2)5-
C
C
C
C
12b
12c
12d
12e
12g
12h
15a
15b
15c
17a
17c
17d
17e
17i
18a
18c
18e
18j
18k
18l
18m
18n
19a
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
19n
19o
114-7
128-30
118-20
161-3
156-9
212-4
187-9
218-19
95-7
C14H15NOS
C
C
C
19H17NOS
16H19NOS
13H13OS
C15H11F3O3S
20H16O3S
C
C10H13NO2S
C14H12ClNOS
C14H12BrNOS
C14H12FNOS
C15H11NOS
C11H15NO2S
C15H14FNOS
C15H14BrNOS
C16H17NOS
C16H14N2OS
C21H19NOS
C21H25NOS
C19H17NOS
C12H17NO2S
C16H16ClNOS
C16H16BrNOS
C16H16INOS
C16H16FNOS
C16H16ClNOS
C16H16BrNOS
C16H16INOS
C17H19NO2S
C17H19NO2S
C21H21NOS
C21H27NOS
C19H19NOS
C19H19NOS
185
200-3
151-3
154-6
95-7
112-4
118-21
138-40
208-10
109-11
114-6
104-6
116-9
74-6
80-2
100-1
78-80
97-9
4ClPh, -(CH2)5-
4BrPh, -(CH2)5-
4-IPh, -(CH2)5-
3-CH3OPh, -(CH2)5-
4-CH3OPh, -(CH2)5-
4-PhPh, -(CH2)5-
4-cHxPh, -(CH2)5-
1-naphth, -(CH2)5-
2-naphth, -(CH2)5-
153-5
176-8
71-3
124-6
55-7
131-3
92-4
120-2
a
Solvents for crystallization: E, ethanol-water; H, hexane.
of activity of thiophenes 3a -i and the thiophene-2-
carboxylic acids 17a -c suggests that the 2-amino group
is necessary but not sufficient for AE activity at the A1-
AR.
basis of an agonist activity profile may give ambiguous
results. Potentiation by an allosteric enhancer could be
an additional criterion for deciding that the A1AR rather
than the A3AR initiates a response.
The original design of the study included measure-
ments of the A1AR antagonistic activity of candidate
AEs, measured as their ability to compete with the
binding of [3H]CPX. Table 2 includes those results.
Several of the candidate AEs had substantial antago-
nistic activity. However, when it became clear part of
the way through the study that the two activities were
unrelated (r2 ) 0.057, n ) 28), assays of antagonism
were discontinued.
All of the compounds underwent screening for AE
activity at both the hA2AAR and the hA3AR, but none
were active. Likewise, the AEs did not affect the rate
of dissociation of antagonists from the hA1AR, evidence
that there was no allosteric effect on antagonist binding.
Differential activity at the A1AR and A3AR may have a
practical application. Since N6-substituted adenosines
are agonists at both the A1AR and A3AR, assigning a
biological response to one or the other receptor on the
This study included seven compounds previously
studied by Bruns et al.2 (12a , 12f, 17b, 18b, 18d , 18f,
19b), seven by van der Klein et al.25 (3a , 3c, 18d , 18f,
18g, 18h , 18i), and 15 by Baraldi et al.26 (3a , 3f, 17b,f-
h , 18b,f-h ,l, and 19b,f,g,k ). The studies of Bruns et
al. and of van der Klein et al. assayed A1AR AE activity
under different conditions and examined the rat rather
than the human A1AR. The study of Baraldi et al.
employed a functional assay in cultured cells expressing
the hA1AR. Accordingly, direct comparisons of the
results of the present study with the earlier studies are
unwarranted. However, the data reported by Bruns et
al. and van der Klein et al. support the idea that the
size/hydrophobicity of the 3-aroyl group influences AE
activity as much as the kinds of alkyl groups at C-4 and
C-5. The present study identified several compounds
substantially more active than 12f, which was the most
active compound identified by Bruns. In that regard our

386 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Ta ble 2. Summary of Allosteric Enhancer Activity
Tranberg et al.
F igu r e 2. Influence of the size of the thiophene C-4 and C-5
substituents on AE activity. Legend: 4,5-H, compounds 3a -
i; 4,5-Me, compounds 12a -h ; cPENT, compounds 17b-i;
cHEX, compounds 18b,d -h ,l,m ; and cHEP, compounds 19b-
d ,f-h ,l,o. Note that methyl groups are the smallest substit-
uents that support activity and that activity of the cycloalka-
[b]thiophenes parallels the size of the cycloalkyl moiety.
AE score,
inhibition of
a
no.
3a
3b
3c
3d
3e
R
%
[3H]CPX binding, %a
Ph
0.2 ( 0.01
0
0
0.2 ( 0.03
0
15 ( 6
3-FPh
3-ClPh
3-BrPh
4-FPh
3f
3g
3h
3i
4-ClPh
4-BrPh
3,4-Cl2Ph
2-naphth
Ph
0
0.8 ( 0.3
1.7 ( 0.8
3.2 ( 1.8
9
12a
12b
12c
12d
12e
12f
12g
12h
15a
15b
15c
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
18m
18n
19a
19b
19c
19d
19e
19f
19g
19h
19i
19j
19k
19l
19m
19n
19o
3-FPh
0
3-ClPh
3-BrPh
3-CH3Ph
3-CF3Ph
4-PhPh
mesityl
Ph
3-CF3Ph
4-PhPh
OEt
14 ( 1
16 ( 4
18 ( 0.5
19 ( 2.9
12 ( 1.9
0
0
0
0
42 ( 7
0.4 ( 0.1
20 ( 3.6
16 ( 2
13 ( 3
68 ( 1
22 ( 4
70 ( 7
62 ( 5
38 ( 3
31 ( 4
3.5 ( 1.8
19 ( 5
22 ( 5
70 ( 9
49 ( 1
17 ( 3
68 ( 10
83 ( 5
86 ( 13
19 ( 7
19 ( 0.9
33 ( 2
99 ( 6
86 ( 1
0.3 ( 0.01
13 ( 3.8
65 ( 8
78 ( 2.6
86 ( 9
22 ( 2.7
65 ( 6.7
86 ( 3.8
96 ( 4.7
99 ( 3.1
85 ( 10
88 ( 4.6
77 ( 11
81 ( 7.2
75 ( 9.6
58 ( 9
56 ( 7
Ph
3-FPh
3-ClPh
3-BrPh
4-FPh
4-ClPh
4-BrPh
4-PhPh
2-naphth
OEt
56 ( 15
36 ( 7
40 ( 4
20 ( 6
21 ( 3
53 ( 5
42 ( 9
35 ( 4
78 ( 8
49 ( 13
56 ( 6
69 ( 9
61 ( 7
62 ( 11
36 ( 4
21 ( 6
Ph
3-FPh
3-ClPh
3-BrPh
4-FPh
4-ClPh
4-BrPh
4-IPh
4-CH3Ph
4-CNPh
4-PhPh
4-cHxPh
2-naphth
OEt
F igu r e 3. QSAR analysis of the effect of 3-aroyl substituents
on the AE activity of 2-amino-3-aroylthiophenes. Top panel:
dependence of AE activity on log P, an index of hydrophobicity.
Bottom panel: dependence of AE activity on molar refractivity,
an index of steric bulk. See text for additional discussion.
observations support those of van der Klein et al.25 but
not Baraldi et al., who found that 12f was the most
potent compound tested.26
47 ( 7
38 ( 3
68 ( 6
10 ( 2
7 ( 3
Assa y of Alloster ic En h a n cer Activity. The assay
devised to screen candidates for AE activity measures
the ability of an AE to stabilize the agonist-receptor-G
protein ternary complex,7,27 manifested as a decrease
in the rate of agonist dissociation (Figure 4). The first
phase of the assay achieved agonist binding equilibrium.
The second phase, a 5 min exposure to a candidate AE,
was sufficiently long for binding of the AE to the ternary
complex without discernible (<5%) loss of bound agonist
due to any coexisting antagonist activity of the AE. The
third phase measured the rate at which bound agonist
dissociated, driven both by competition at the ortho-
steric site with a large excess of antagonist as well as
by displacement of GDP from the G protein by GTPγS.
Ph
3-ClPh
3-BrPh
3-IPh
4-FPh
4-ClPh
4-BrPh
4-IPh
3-OCH3Ph
4-OCH3Ph
4-PhPh
4-cHxPh
1-naphth
2-naphth
7 ( 4
9 ( 4
13 ( 3
a
Concentration of allosteric enhancer was 100 µM. All data are
mean ( SEM of three separate assays, each in triplicate.

2-Amino-3-aroyl-4,5-alkylthiophenes
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 387
d, J ) 5.2 Hz, 1H, H-5. 13C NMR (CDCl3) δ: 13.0, 13.6, 120.6,
129.9, 132.6, 133.0.
(4,5-Dim eth yl-2-th ien yl)(m eth yl)m eth a n on e (7). A solu-
tion of 5 (15.16 g, 135 mmol) and acetyl chloride (9.6 mL, 135
mmol) in 60 mL of benzene dried over Na was cooled to - 5
°C and vigorously stirred during the addition of a solution of
tin(IV) chloride in 50 mL of benzene over a period of 1 h. The
reaction mixture was removed from the cold bath and stirred
for an additional hour at room temperature. The slow addition
of 4 mL of concentrated HCl in 28 mL of water quenched the
reaction. The organic layer was separated, washed with 2 ×
10 mL water, dried over Na2SO4, and evaporated to give 20.8
g of crude product. Chromatography on a column of silica
eluted with petroleum ether:ethyl acetate (10:1) and evapora-
tion of relevant fractions gave a viscous yellow oil, 16.42 g,
79%. 1H NMR (CDCl3) δ: 2.08, s, 3H, COCH3; 2.31, s, 3H, CH3;
2.41, s, 3H, CH3; 7.33, s, 1H, H-3. 13C NMR (CDCl3) δ: 13.3,
13.7, 26.1, 134.7, 135.3, 139.1, 143.4, 190.0.
F igu r e 4. Experimental protocol for the assay of allosteric
enhancer activity. See text for discussion.
Adding GTPγ greatly reduced the time necessary to
perform an assay. In the absence of GTPγS, dissociation
can be very slow, t1/2 ∼ 60 min. Upon addition of GTPγS,
a fraction of the receptors release the agonist relatively
quickly and the remainder release the agonist very
slowly. We have previously shown that AEs increase the
fraction of receptors coupled to G proteins, which bind
agonists with high affinity. Our data are consistent with
the possibility that in kinetic experiments the rapidly
dissociating component represents receptors not coupled
to G proteins and the slowly dissociating component
represents receptors tightly coupled to G proteins. Our
data suggest that the population of receptor-G protein
complexes increases and remains relatively stable in the
presence of enhancers, even after the addition of GTPγS.
Over the time course of the assay, dissociation can be
very slow, but in such instances it drops toward the level
of unspecific binding in a matter of hours.
1-(4,5-Dim eth yl-2-th iop h en -2-yl)-eth a n on e Oxim e (8).
A mixture of 7 (33.1 g, 215 mmol), hydroxylamine hydrochlo-
ride (32.9 g, 473 mmol), and barium carbonate (91.7 g, 495
mmol) in 500 mL of ethanol was heated at reflux for 8 h, the
salts were filtered, and the filtrate was evaporated to an off-
white solid. Crystallization from ethanol-water afforded 30.8
g (85%) of pure 8. Four recrystallizations improved the E:Z
1
ratio of isomers from 4:1 to 14:1. H NMR (CDCl3) δ: 2.11 (s,
3H, CH3CdNOH), 2.26 (s, 3H, CH3), 2.32 (s, 3H, CH3), 6.94
(s, 1H, H-3), 9.62 (br s, 1H, OH). 13C NMR (CDCl3) δ: 11.4,
12.6, 12.9, 129.2, 132.7, 133.1, 135.8, 149.4.
N-(4,5-Dim eth yl-th iop h en -2-yl)a ceta m id e (9) a n d 4,5-
Dim eth yl-th ioph en e-2-car boxylic Acid Meth ylam ide (10).
A solution of 8 (0.304 g, 1.8 mmol) in 5 mL of dry ether was
cooled to 0 °C and stirred vigorously during the addition of
PCl5 (0.4 g, 1.9 mmol) at a rate that kept the temperature at
0 °C. Stirring on ice continued for 15 min and at room
temperature for an additional 30 min. The addition of 1 mL
of water at a rate keeping the temperature at <20 °C quenched
the reaction. Under cooling, NaOH was added to bring the pH
to 5-6, and the product was extracted into ether. Evaporation
gave 0.324 g of crude product that was purified by elution from
a silica gel column with petroleum ether-ethyl acetate 1:1 to
give 9 (0.097 g, 32%). 1H NMR (CDCl3) δ: 2.03 (s, 3H, COCH3),
2.16 (s, 3H, CH3), 2.24 (s, 3H, CH3), 6.39 (s, 1H, H-3), 9.08 (br
s, 1H, NHCdO). 13C NMR (CDCl3) δ: 12.3, 13.4, 23.0, 115.6,
124.8, 129.5, 134.2, 167.3. Additional fractions contained 10
Exp er im en ta l Section
Melting points are uncorrected. Elemental analyses agreed
within (0.4% of calculated composition. 1H NMR spectra were
consistent with the putative structures. Trans-World Chemi-
cals, Rockville, MD, supplied 3′-iodoacetophenone. One recrys-
tallization from methanol removed minor impurities from
4-acetylbiphenyl. All other starting materials were from Ald-
rich and were used as received. The brominations19-22 of
acetylarenes and their conversions to aroylacetonitriles23 fol-
lowed the methods cited.
(2-Am in o-th ien -3-yl)(p h en yl)m eth a n on e (3a ) Gen er a l
Meth od A. Benzoylacetonitrile (1.45 g, 10 mmol), 2,5-dihy-
droxy-1,4-dithiane (0.76 g, 5 mmol), and diethylamine (0.73 g
) 1.04 mL, 10 mmol) in 4 mL of absolute ethanol were heated
in a Teflon-sealed pressure tube for 4 h at 50 °C with frequent
stirring on a vortex mixer. By 2 h starting materials had
dissolved, and shortly thereafter the product began to crystal-
lize. After refrigerating the tube overnight, the product was
filtered off and washed with a little methanol to give bright
yellow crystals. Yield: 1.3 g, 64%. 1H NMR (CDCl3) δ: 6.14
(d, 1H, H-5), 6.88 (d, 1H, H-4), 6.95 (br s, 2H, NH2), 7.5-7.7
(m, 5H, C6H5).
2,3-Dim eth ylth iop h en e (5). Heating a mixture of 3-meth-
yl-2-thiophene carboxaldehyde, 4 (58.6 g, 464 mmol), 80%
hydrazine hydrate (97 mL, 1.62 mol), and 200 mL of ethylene
glycol to an internal temperature of 130-160 °C caused
hydrazine and water to distil. The reaction mixture was cooled
to below 60 °C, and the water-immiscible fraction of the
distillate was returned to the flask. The addition of KOH (91.0
g, 1.62 mol) and reheating caused vigorous gas evolution when
the temperature reached 90-100 °C. Reflux continued for 15
min after gas evolution ceased; steam distillation then sepa-
rated 5. Product in the distillate was extracted into ether, the
extract washed with 6 N HCl, dried over CaCl2, and evapo-
rated. Distillation over sodium gave 5 as a colorless oil, bp
139.5-140.5, yield 39.8 g, 77%. 1H NMR (CDCl3) δ: 2.21, s,
3H, CH3; 2.41, s, 3H, CH3; 6.84, d, J ) 5.1 Hz, 1H, H-4; 7.03,
1
(0.060 g, 20%). H NMR (CDCl3) δ: 2.10 (s, 3H, CH3), 2.33 (s,
3H, CH3), 2.94 (d, 3H, NHCH3), 6.21 (br s, 1H, NH, 7.22 (1H,
ArH). 13C NMR (CDCl3) δ: 13.4, 13.5, 26.6, 131.0, 133.2, 134.0,
138.2, 162.8.
N-(3-Ben zoyl-4,5-d im et h yl-t h iop h en -2-yl)a cet a m id e
(11a ). Gen er a l Meth od B. A solution of 1.71 M tin(IV)
chloride (3.1 mL, 5.3 mmol) in 1,2-dichloroethane was added
dropwise to a suspension of 9 (0.241 g, 1.42 mmol) and benzoyl
chloride (0.31 mL, 2.66 mmol) in 1,2-dichloroethane, and the
mixture was refluxed for 10.5 h. The reaction was quenched
with ice, and the organic phase was washed sequentially with
2 N HCl, water, and 2 N NaOH. Drying over CaCl2 and
evaporation gave a solid that was purified by chromatography
on silica gel eluted with petroleum ether-ethyl acetate 5:1.
Recrystallization from ethanol-water gave 0.32 g of pure
product as yellow crystals, 82%. 1H NMR (CDCl3) δ: 1.6 (s,
3H, CH3), 2.23 (s, 3H, CH3), 2.24 (s, 3H, CH3), 7.4-7.6 (m,
5H, C6H5), 11.1 (br s, 1H, NH). 13C NMR (CDCl3) δ: 12.4, 14.8,
23.6, 122.4, 124.7, 127.6, 128.3, 128.4, 131.9, 140.3, 146.4,
167.4, 195.0.
(2-Am in o-4,5-d im et h yl-t h iop h en -3-yl)(p h en yl)m et h a -
n on e (12a ). Gen er a l Meth od C. A solution of 11a (0.3 g, 1.1
mmol) in KOH (3.5 equiv in methanol-water 1:1) was refluxed
for 45 min, evaporated, and taken up in dichloromethane. The
solution was washed three times with water, dried, and
evaporated to a solid that was recrystallized from ethanol-
water as yellow crystals. Yield: 0.25 g, 98%. 1H NMR (CDCl3)
δ: 1.5 (s, 3H, CH3), 2.1 (s, 3H, CH3), 6.4 (br s, 2H, NH2), 7.2-

388 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Tranberg et al.
7.5 (m, 5H, C6H5). 13C NMR (CDCl3) δ: 12.5, 15.2, 114.9, 117.2,
binding to equilibrium, the incubation mixture consisted of 10
mM HEPES, pH 7.2, containing 0.5 mM MgCl2, 1 U/mL
adenosine deaminase, 0.5 nM [125I] N6-(4-aminobenzyl)adeno-
sine {[125I]ABA}, and 10 µg of membrane protein (Bmax = 4
pmol/mg protein) in a final volume of 100 µL. After 90 min at
room temperature, the addition of 50 µL of a 0.3 mM solution
of a candidate AE initiated stabilization of the ternary
complex. Five minutes later, the addition of 50 µL of a solution
of 400 µM 8-cyclopentyltheophylline and 200 µM GTPγS
initiated the dissociation of the ternary complex. Ten minutes
later, filtering through Whatman GF/C membranes, washing,
drying, and counting 125I-activity measured residual agonist.
The percentage of specifically bound agonist remaining after
10 min of dissociation served as an index of AE activity
127.8, 128.0, 128.8, 130.4, 141.7, 162.8, 193.0.
4,9-Bis-(3-flu or op h en yl)-2,3,7,8-tetr a m eth yl-[2,3-b:2′,3′-
b′]-d ith ien o-1,5-d ia zocin e (13). A solution of 11a (0.54 g,
1.86 mmol) in ethanolic 0.5 N HCl was heated at reflux for 7
h, cooled, and alkalinized with NaOH. Extracting into dichlo-
romethane, drying, and evaporation gave a solid that was
purified by chromatography on silica gel eluted with petroleum
ether-ethyl acetate 10:1. Crystallization from ethanol-water
gave orange crystals, 0.254 g, 57%. 1H NMR (CDCl3) δ: 1.6
(s, 3H, CH3), 2.3 (s, 3H, CH3), 7.1-7.5 (m, 4H, C6H4F). 13C
NMR (CDCl3) δ: 13.0, 13.2, 115.4 (d, J ) 22.8 Hz), 118.1 (d, J
) 21.3 Hz), 123.5, 124.8 (d, J ) 2.6 Hz), 130.3, 130.7, 140.2
(d, J ) 7.3 Hz), 153.0, 162.8 (d, j ) 246.2 Hz), 169.1 (d, J )
2.6 Hz). ES-MS m/z 463.1 (M + 1), 485.1 (M + Na).
(4,5-Dim eth yl-2-m eth ylca r ba m oyl-th iop h en -3-yl)(p h e-
n yl)m eth a n on e (14a ). A solution of 10 (0.40 g, 2.37 mmol)
in 20 mL of dry THF was cooled to -70 °C and stirred during
the addition of tert-butyllithium (5.21 mmol). After 30 min of
stirring, benzoyl chloride (0.42 g ) 0.35 mL, 3 mmol) was
added, and the mixture was warmed to room temperature.
Workup consisted of quenching the reaction with saturated
aqueous NH4Cl and extraction of product into ethyl acetate.
The extract was dried over MgSO4 and evaporated, and the
product was purified by chromatography on silica gel eluted
with hexanes-ethyl acetate 1:1. Yield: 0.356 g, 55%. 1H NMR
(CDCl3) δ: 1.83 (s, 3H, CH3), 2.37 (s, 3H, CH3), 2.79 (d, 3H,
NHCH3), 6.58 (br s, 1H, NH), 7.42-7.78 (m, 5H, ArH).
AE activity ) 100 × (B - Bo)/(Beq - Bo)
where B ) residual binding (cpm) bound at the end of 10 min
of dissociation in the presence of an AE, Bo ) residual binding
(cpm) at the end of 10 min of dissociation in the absence of an
AE, and Beq ) cpm bound at the end of 90 min of equilibration.
Assa y of A1AR An ta gon ist Activity. Assays of antago-
nism of equilibrium binding by allosteric enhancers used
membranes from CHO-K cells expressing the hA1AR. Assays,
in triplicate, consisted of mixing 50 µL aliquots of membrane
suspensions (15 µg of protein) in 10 mM HEPES, pH 7.4,
containing 1 mM EDTA, 1 u/mL adenosine deaminase, and 4
nM [3H]CPX with 50 µL of either 200 µM enhancer dissolved
in HEPES buffer containing 10% methyl sulfoxide or, as
controls, HEPES containing 10% DMSO. Additional aliquots
of membrane suspension mixed with 50 µL of 200 µM NECA
in HEPES-10% DMSO served for measurements of unspecific
binding. Incubation for 3 h at room temperature established
binding equilibrium. Filtration through Whatman GF/C mem-
branes separated free and bound radioligand. The membranes
were washed three times and dried, and 3H activity was
measured by liquid scintillation spectrometry. Inhibition was
expressed as percentage of control specific binding. Table 2
reports the mean ( SEM of three separate assays.
(2-Ca r boxy-4,5-d im eth ylth iop h en -3-yl)(p h en yl)m eth a -
n on e (15a ). A solution of 14a (0.281 g, 1.03 mmol) in
methanol-water 1:1 containing 10% KOH was heated at reflux
for 12 h, neutralized, and extracted with ethyl acetate. The
solid after evaporation was crystallized from ethanol. Yield:
0.19 g, 71%.
(2-Am in o-4,5-d ih yd r ocyclop en t a [b]t h iop h en -3-yl)(3-
br om op h en yl)m eth a n on e (17d ). Gen er a l Meth od D. A
mixture of sulfur (0.176 g, 5.5 mg-at), 3-bromobenzoylaceto-
nitrile (1.35 g, 5.5 mmol), and cyclopentanone (0.463 g ) 0.482
mL, 5.5 mmol) in 4 mL anhydrous ethanol was heated at 50
°C in a Teflon-capped pressure tube for 4 h. Cooling overnight
deposited crystalline product, which was filtered off, washed
with a little cold methanol, and dried. TLC showed the
Ack n ow led gm en t. This work was supported by
NIH HL-56111 (to J .L.), a grant from the Australian
Research Council (to P.J .S.), and the Ed C. Wright chair
in Cardiovascular Research, University of South Florida
(R.A.O.).
1
material was pure; yield 1.2 g, 62%. H NMR (CDCl3) δ: 2.16
(m, 4H, H-4 and H-6), 2.65 (m, 2H, H-5), 7.07 (br s, 2H, NH2),
7.3-7.6 (m, 4H, C6H4Br).
Refer en ces
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
bip h en yl)m eth a n on e (18l). A mixture of 4-phenylbenzoyl-
acetonitrile (4.42 g, 0.02 mol), cyclohexanone (1.96 g ) 2.1 mL,
0.02 mol), â-alanine (0.18 g, 0.002 mol), glacial acetic acid (2
mL), and toluene (100 mL) was heated at reflux in a flask fitted
with a Dean-Stark trap and condenser. After 18 h, TLC
(hexane:ethyl acetate 3:1) showed complete conversion of the
nitrile, Rf 0.48, to the olefin, Rf 0.67. The residue after
evaporation was taken up in ethyl acetate, washed twice with
50 mL of water, dried over MgSO4, and evaporated to a glass.
Weight: 4.6 g, 76%. Sulfur (0.673 g, 0.021 mol) was suspended
in a solution of the olefin in 50 mL of anhydrous ethanol,
diethylamine (1 mL) was added, and the dark suspension was
stirred at room temperature until the sulfur had disappeared.
Product that crystallized out on cooling in an ice bath was
filtered off, washed with a little methanol, and dried. TLC
(hexane:ethyl acetate 1:3) showed only product, Rf 0.50.
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1
Yield: 4.5 g, 67% based on starting nitrile. H NMR δ: 1.57
(m, 2H, cyclohexyl), 1.81 (m, 2H, cyclohexyl), 1.97 (q, 2H,
cyclohexyl), 2.59 (q, 2H, cyclohexyl), 6.75 (br s, 2H, NH2), 7.45-
7.75 (m, 9H, biphenyl).
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of an A1AR antagonist to compete with agonist at the ortho-
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2-Amino-3-aroyl-4,5-alkylthiophenes
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