Effects of conformational restriction of 2-amino-3-benzoylthiophenes on A1 adenosine receptor modulation

Article abstract of DOI:10.1021/jm1008538

2-Amino-3-benzoylthiophenes (2A3BTs) have been widely reported to act as allosteric enhancers (AEs) at the A₁ adenosine receptor (A ₁AR). Herein we describe the synthesis of a series of 1-aminoindeno[1,2-c]thiophen-8-ones and a series of (2-aminoindeno[2,1-b] thiophen-3-yl)(phenyl)methanones as conformationally rigid analogues of the 2A3BTs. These compounds were screened using a functional assay of A ₁AR-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact Chinese hamster ovary (CHO) cells to identify both potential agonistic effects as well as the ability to allosterically modulate the activity of the orthosteric agonist, N⁶-(R- phenylisopropyl)adenosine (R-PIA). All of the 1-aminoindeno[1,2-c]thiophen-8- ones (14a-c and 17a-f) proved either to be inactive or behaved as antagonists in the functional assay. However, the (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl) methanones with para-chloro substitution (compounds 25b, 25d, and 25f) did significantly augment the R-PIA response, indicating a positive allosteric effect.

Full text of DOI:10.1021/jm1008538

6550 J. Med. Chem. 2010, 53, 6550–6559
DOI: 10.1021/jm1008538
Effects of Conformational Restriction of 2-Amino-3-benzoylthiophenes on A1 Adenosine
Receptor Modulation
Luigi Aurelio,†, Celine Valant,‡, Bernard L. Flynn,† Patrick M. Sexton,‡ Jonathan M. White,§ Arthur Christopoulos,*,‡ and
Peter J. Scammells*,†
†Medicinal Chemistry and Drug Action, and ‡Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical
Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia, and School of Chemistry and the Bio21 Molecular Science and
§
Biotechnology Institute, The University of Melbourne, VIC 3010, Australia. These authors contributed equally to this work.
Received July 8, 2010
2-Amino-3-benzoylthiophenes (2A3BTs) have been widely reported to act as allosteric enhancers (AEs) at
the A1 adenosine receptor (A1AR). Herein we describe the synthesis of a series of 1-aminoindeno[1,2-c]-
thiophen-8-ones and a series of (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)methanones as conformation-
ally rigid analogues of the 2A3BTs. These compounds were screened using a functional assay of A1AR-
mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact Chinese
hamster ovary (CHO) cells to identify both potential agonistic effects as well as the ability to allosterically
modulate the activity of the orthosteric agonist, N6-(R-phenylisopropyl)adenosine (R-PIA). All of the
1-aminoindeno[1,2-c]thiophen-8-ones (14a-c and 17a-f) proved either to be inactive or behaved as
antagonists in the functional assay. However, the (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)methanones
with para-chloro substitution (compounds 25b, 25d, and 25f) did significantly augment the R-PIA response,
indicating a positive allosteric effect.
Introduction
The A1AR is one of four subtypes of G protein-coupled
receptor (GPCRa) that mediate many of the actions of the
purine nucleoside, adenosine, via coupling to multiple intracel-
lular pathways, including the inhibition of adenylate cyclase
and the phosphorylation ERK1/2.1,2 It is well documented that
there are numerous therapeutic applications for selective ade-
nosine receptor ligands, particularly in the treatment of cardi-
ovascular, inflammatory, and neurodegenerative diseases.3
To date, a number of adenosine ligands have been reported
aspotent and relativelyselectiveorthosteric agonists,4 yet they
have not been pursued as therapeutic agents due to the
associated side effects of nondiscriminately activating multi-
ple adenosine receptor subtypes and/or their tendency to
cause receptor desensitization upon prolonged use. An alter-
native approach toward selective A1AR activation that has
Figure 1. 2A3BT allosteric enhancers reported by Bruns and
co-workers.6,7
been pursued is one of allosteric modulation; ligands that can
bind to the A1AR at an allosteric site and augment the action
of endogenous adenosine are highly desirable because they
can theoretically achievehighselectivityvia receptor subtype-,
tissue-, and event-specific mechanisms.5
these compounds led to subsequent inhibition of receptor
responsiveness.7 In that study, the 2-amino and 3-keto groups
were revealed as important functionalities and that their omis-
sion or derivatization resulted in loss of activity. Interestingly,
the replacement of the thiophene with a benzene ring retained
activity, with 2-aminobenzophenone 2 showing a little over
half the degree of enhancement compared to the 2-amino-3-
benzoylthiophene (general structure 4, X = H) at 100 μM
when assayed using a radioligand dissociation kinetic para-
digm.7 This activity was improved by conformationally con-
straining the 2-aminobenzophenone 2, and therefore 1-amino-
fluoren-9-one 3 was approximately three times as active as
2-aminobenzophenone 2 at 100 μM. Collectively, these results
implied that a planar conformation of the aromatic rings of
Bruns et al.6,7 discovered that 2A3BTs, typified by the
benchmark compound, PD 81,723 (1) (Figure 1), acted as
A1AR-selective AEs, although higher concentrations of
*To whom correspondence should be addressed. For A.C.: phone,
þ61 (0)3 9903 9067; E-mail, Arthur.Christopoulos@monash.edu.
For P.J.S.: phone, þ61 (0)3 9903 9542; fax, 61 3 9903 9582; E-mail,
Peter.Scammells@monash.edu.
a Abbreviations: AE, allosteric enhancer; AR, adenosine receptor;
CHO, Chinese hamster ovary; 2A3BT, 2-amino-3-benzoylthiophene;
GPCR, G protein-coupled receptor; ERK1/2, extracellular signal-regu-
lated kinases 1 and 2; DMEM, Dulbecco’s Modified Eagle Medium;
FBS, fetal bovine serum; R-PIA, N6-(R-phenylisopropyl)adenosine.
r
pubs.acs.org/jmc
Published on Web 08/31/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6551
first forming the Knoevenagel intermediate and then cycliz-
ing with elemental sulfur in the presence of base.9 We found
that forming the Knoevenagel intermediate under Lewis
acid-mediated condensation with titanium(IV) chloride10
provided near-quantitative yields of olefin (not shown). The
olefin was then cyclized to the thiophene 10 at room tem-
perature in high yield, which was then treated with phthalic
anhydride to protect the 2-amino group. Phthalimide was
considered to be a feasible protecting group for the synthesis
of the 1-amino-8H-indeno[1,2-c]thiophen-8-one analogues
for several reasons. First, the phthalimide is not entirely
removed in the saponification step and, although it is ring-
opened in this step, it is subsequently ring-closed during the
chlorination step. Second, both valences of the 2-amino
group are removed and associated complications with the
cyclization step are eliminated. Third, phthalimide tolerates
the acidic condition of the Friedel-Crafts acylation. The
crucial step in the synthesis involved the cyclization of the
intermediate acid chloride of 12 and has been shown to be a
poor-to-moderate yielding step, as was also the case in our
hands.9 We found that replacement of thionyl chloride with
oxalyl chloride and the use of a catalytic amount of DMF to
form the corresponding acid chloride provided quantitative
yields of indenothiophenes 13 after the Friedel-Crafts acy-
lation. The phthalimide was removed by hydrazinolysis to
afford the initial conformationally restricted targets 14a-c.
Compounds 14a and 14b were elaborated further toward the
corresponding 5-aryl substituted derivatives. This initially
involved acetyl protection of the 2-amino group since the
phthalimide created problems in the subsequent Suzuki
cross-coupling reactions due to ring-opening under the basic
conditions. The acetylated derivatives 15a and 15b were
subsequently brominated with NBS/AcOH but were found
to be quite sluggish in the Suzuki cross-coupling reactions.
Figure 2. 4-Phenyl substituted 2A3BT allosteric enhancers and
proposed conformationally restricted analogues.
the 2-aminobenzophenone 2 was preferred. Therefore, one aim
of the current study was to synthesize and evaluate a correspond-
ing series of conformationally restricted 2A3BTs (Figure 1,
general structure 5) to determine if fixing the thiophene and
benzoyl group in a planar conformation imparts similar im-
provements in AE activity.
Morerecently, wealso found that certain 4-phenyl substitu-
ents on the 2-amino-3-benzoylthiophene scaffold promoted
allosteric activity (Figure 2, general structure 6), with com-
pound 7 producing a strong functional potentiation of orthos-
teric agonist activity in an assay of A1AR-mediated ERK1/2
phosphorylation.8 Thus, a second aim of the current study was
to assess the activity of conformationally restricted analogues
of 7 in which the 4-phenyl group is effectively locked in a
coplanar conformation relative to the thiophene ring via the
incorporation of a methylene tether to the 5-position (Figure 2,
general structure 8).
Results and Discussion
Chemistry. To the best of our knowledge, there is only one
reference describing the synthesis of 1-amino-8H-indeno-
[1,2-c]thiophen-8-one, in a patent by Moorman et al., for
the treatment of neuropathic pain.9 The synthetic route
utilized in that patent was also adopted by us in the synthesis
of our analogues, with slight modification (Scheme 1). The
synthesis began with the 3-ethoxycarbonyl thiophenes 10,
which were synthesized by the classical two-step procedure,
11
Alternatively, iodination of 15a and 15b with AgNO3/I2
provided the corresponding iodides 16a and 16b. The iodide
16a was far superior to the bromide in cross-coupling reac-
tions, which were quantitatively converted within 30 min.
Scheme 1a
a (i) TiCl4, pyridine, EtOCOCH2CN; (ii) S8, Et2NH, THF; (iii) phthalic anhydride, AcOH reflux; (iv) NaOH, H2O, EtOH; (v) Cl(CO)2Cl, CH2Cl2,
cat. DMF then AlCl3, CH2Cl2 reflux; (vi) N2H4, DMF/dioxane; (vii) Ac2O, AcOH reflux; (viii) I2, AgNO3, DMSO/CH3CN; (ix) cat Pd[PPh3]2Cl2,
2M K3PO4, DMF, boronic acid; (x) NaOH, EtOH MW.

6552 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Aurelio et al.
Several attempts to remove the acetyl group by classical
methods such as basic, acidic, and nucleophilic conditions
proved unfruitful due to the insolubility of these derivatives.
Ultimately, a suspension of the intermediates in absolute
ethanol and sodium hydroxide pellets and microwave irra-
diation removed the acetyl group. This method was advan-
tageous since, after the reaction, the final compounds 17a-f
were soluble in the ethanolic sodium hydroxide mixture. By
filtering this mixture, the insoluble impurities were removed
and the 1-aminoindenothiophen-8-ones 17a-f were recov-
ered by acidifying with acetic acid. These compounds were
triturated with boiling ethanol and in most cases provided
final compounds 17a-f in a pure state; otherwise, recrystal-
lization from DMF or DMF/MeOH provided pure com-
pound. In the case of cross-coupled products of 16b, removal
of the acetyl group resulted in intractable mixtures. Even re-
placement of the acetyl group with Boc protection resulted
in mixtures when removed under the acidic conditions. This
result was not further investigated, and the pursuit of com-
pounds of this type was abandoned.
Scheme 2a
The corresponding “unconstrained” thiophenes, which
possess a 3-benzoyl group and no substituent in the 4-position,
were prepared using the approach outlined in Scheme 2.
Briefly, this involved use of the Gewald synthesis to con-
struct the 2A3BT 20a and 20b,12 which we subsequently
acetyl protected and iodinated under standard conditions to
afford the corresponding 5-iodothiophenes 21a and 21b.
Aryl substituents were subsequently incorporated in the
5-position via Suzuki coupling with the appropriate boronic
acid. These substituents were identical to those employed
previously in the corresponding conformationally restricted
series (compounds 17a-f). Finally, a base mediated cleavage
of the acetyl protecting group afforded the desired target
compounds 22a-f and 23a-f.
a (i) Et2NH, EtOH; (ii) Ac2O, reflux; (iii) I2, AgNO3, CH3CN, DMF;
(iv) cat. Pd[PPh3]2Cl2, 2M K3PO4, DMF, boronic acid; (v) NaOH, EtOH,
H2O.
Scheme 3a
The final series of compounds, in which the 4-phenyl
is effectively locked in a coplanar conformation relative to
the thiophene ring, were prepared as described in Scheme 3.
Once again, a Gewald synthesis was used to prepare the
thiophene ring. This involved a reaction of a benzoylnitrile
18 with an appropriately substituted indanone 24. Although
these reactions were very low yielding, sufficient quantities of
the desired product could be isolated for pharmacological
evaluation in one step.
An X-ray structure of the (2-aminoindeno[2,1-b]thiophen-
3-yl)(phenyl)methanone 25c was obtained (Figure 3). This
structure highlights the expected planar nature of the mole-
cule and also supports Bruns’ original suggestion7 that a
a (i) TiCl4, pyridine; (ii) S8, Et2NH, THF, or (i) β-ala, PhCO2H
110-120 ꢀC; (ii) S8, morpholine, EtOH.
Figure 3. Ortep diagrams of the X-ray crystal structure of (2-amino-7-(trifluoromethyl)-8H-indeno[2,1-b]thiophen-3-yl)(phenyl)metha-
none (25c).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6553
Figure 4. Effect of two different concentrations (3 μM, left bar; 10 μM, right bar) of test ligands on A1AR-mediated stimulation of ERK1/2
phosphorylation in intact CHO FlpIn cells, in the absence (A) or in the presence (B) of an EC50 concentration (0.3 nM) of R-PIA (determined
on the same day as each assay); dashed line denotes 50% response level. 0% is defined as basal ERK1/2 phosphorylation in the absence
of R-PIA, whereas 100% is defined as the maximal response to a saturating concentration (100 nM) of R-PIA. Data represent the mean (
standard deviation of two experiments conducted in triplicate.
hydrogen bond between the 2-amino group and the ketone
may create an additional ring coplanar with the thiophene
ring. The “resonance-enhanced” intramolecular hydrogen
the carbonyl group, a dihedral angle C2-C13-C14-C15
42.9(2)ꢀ which minimizes steric interactions with the
indane ring.
˚
bond is characterized by the parameters; N1-H 0.87 A, N1-
Biological Activity. To assess the biological activity of the
compounds, we utilized a functional assay of A1AR-media-
ted phosphorylation of ERK1/2 in intact CHO cells.8 For each
compound, two concentrations (3 and 10 μM) were initially
tested alone (Figure 4A), to assess potential for direct agonist
effects, and against an EC50 concentration of the orthosteric
agonist, R-PIA (Figure 4B), to assess the potential for enhance-
ment or inhibition of orthosteric agonist-mediated function.
With the exception of 14b and 25b (each yielding approxi-
mately 20% of the maximum R-PIA response; Figure 4A),
none of the compounds displayed any substantial ability to
activate the receptor on their own, as evidenced by the fact
that compound-mediated phosphorylation of ERK1/2 was
within (5% of the maximum response mediated by R-PIA.
When tested in the presence of an EC50 concentration of
R-PIA, a number of the compounds were found to be inactive
in modulating the response of the orthosteric agonist when
˚
H
gen of the amino group also forms an intermolecular, hydro-
O1 130.1ꢀ, and N1 O1 2.682(2) A, the second hydro-
3 3 3
3 3 3
˚
gen bond with O1; N1-H 0.88 A, N1-H O1(-xþ3/2,
3 3 3
y-1/2, -zþ1/2) 151.5ꢀ, N1 O1(-xþ3/2, y-1/2, -zþ1/2)
3 3 3
˚
2.800 A. Delocalization of the amino group lone pair onto the
ketone carbonyl, which is enhanced due to the intramolecular
hydrogen bond between O1 and N1, is clearly apparent from
inspection of the relevant bond distances; thus the C13-O1
˚
bond distance 1.248(2) A is significantly longer than a typical
˚
ketone CdO distance of 1.20 A, the C13-C2 bond distance
˚
1.433(2) A has partial double-bond character, the C1-C2
distance 1.410(2) A is significantly longer than the second
˚
˚
thiophene double bond (C3-C4), which is 1.359(2) A,
˚
and the C1-N1 distance 1.359(2) A has some double-
bond character. There is significant twisting of the phenyl
group defined by atoms C14-C19 from coplanarity with

6554 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Aurelio et al.
Table 1. Effect of Test Compounds on A1AR-Mediated Stimulation of
ERK1/2 Phosphorylation in Intact CHO FlpIn Cells in the Presence of
an EC50 Concentration of R-PIAa
Conclusions
The major finding of this study was that, in general, con-
formational restriction in which the 3-benzoyl group of 2A3BTs
is tethered to the 4-position (general structure 5), locking the
benzoyl and thiophene ring in a planar conformation, does not
lead to improved allosteric potentiation of agonist function
at the A1AR. All of the 1-aminoindeno[1,2-c]thiophen-8-ones
(14a-c and 17a-f) that we prepared as conformationally
restricted analogues of the corresponding 2A3BTs proved either
to be inactive or behaved as antagonists in a functional assay
of A1AR-mediated phosphorylation of ERK1/2 in intact CHO
cells. Even the corresponding 2A3BTs (20a-b, 22a-f, and
23a-f) appeared to exhibit this behavior. Given that com-
pounds 20a and 22a were previously reported to be allosteric
enhancers based on their ability to slow the dissociation of [3H]
N6-cyclohexyladenosine at 100 μM concentrations,7 our find-
ings suggest that the ability of an allosteric ligand to stabilize a
pre-equilibrated agonist-receptor-G protein complex in mem-
brane preparations is not necessarily predictive of a positive
allosteric effect in whole cell functional assays.
In contrast, conformational restriction in which a 4-phenyl
substituent is effectively constrained in a planar conformation
relative to the thiophene ring via a methylene tether to the
5-position (general structure 8) gave mixed results. Three
members of the (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)-
methanone series (compounds 25b, 25d, and 25f) did signifi-
cantly augment the R-PIA response, indicating a positive
allosteric effect. Given that the other three members of this
series (compounds 25a, 25c, and 25e) did not show any
evidence of allosteric activity and that the only difference
between the two subsets of compounds was the absence or
presence of a para-chloro substituent, this result highlights the
delicate balance between allosteric enhancement and antag-
onism (or lack of activity) in terms of A1AR modulator
structure-activity relationships. The presence of the para-
chloro would not be expected to have a major influence on the
conformation of 25b, 25d, and 25f, and its marked effect on
allosteric activity presumably results from favorable interac-
tions with the allosteric binding site. Nonetheless, evaluation
of the effects of (2-amino-8H-indeno[2,1-b]thiophen-3-yl)(4-
chlorophenyl)methanone (25b) as a positive allosteric modu-
lator of R-PIA revealed that the affinity of this modulator
and its cooperativity with the agonist was comparable with the
best of the 4-phenyl substituted 2A3BTs that we reported
recently.8 For instance, in that series, (2-amino-4-(3-(trifluoro-
methyl)phenyl)thiophen-3-yl)(phenyl)methanone (7, Figure 2)
had an estimated pKB of 6.37 ( 0.16 and log Rβ of 0.38 ( 0.07
(i.e., Rβ = 2.4-fold positive cooperativity) for the stimulation
of ERK1/2 phosphorylation.
a §Effect of two different concentrations (3 and 10 μM) of the test
compound on A1AR-mediated stimulation of ERK1/2 phosphoryla-
tion in intact CHO FlpIn cells, in the presence of an EC50 concentration
(0.3 nM) of R-PIA (determined on the same day as each assay). Data
represent the mean ( standard deviation of two experiments conducted
in triplicate. 0% is defined as basal ERK1/2 phosphorylation in the
absence of R-PIA, whereas 100% is defined as the maximal response to a
saturating concentration (100 nM) of R-PIA
utilized at 3 μM; this is evidenced in Table 1 by those com-
pounds whose standard deviation includes 50% (the re-
sponse mediated by R-PIA alone). With the exception
of 25c, all compounds displayed some activity (either in-
hibitory or enhancing) when tested at 10 μM (Table 1;
Figure 4B). For compounds, 17a, 17b, 17d, 17e, 22a,
22d, 22e, 23f, and 25a, inhibition of orthosteric agonist-
mediated ERK1/2 phosphorylation was noted in the pre-
sence of both tested concentrations, whereas potentiation
of the R-PIA-mediated response was noted with 25b, 25d,
and 25f (Figure 4; Table 1).
To further assess this finding, we constructed complete
R-PIA concentration-response curves in the absence or
presence of increasing concentrations four of the compounds
that appeared to have the most striking effects, i.e., 17d, 22a,
23d, and 25b. As shown in Figure 5, the inhibitory effect
of 17d, 22a, or 23d on R-PIA-mediated ERK1/2 phosphor-
ylation was consistent with simple competitive (orthosteric)
antagonism, and application of a competitive model of inter-
action yielded the following pA2 values: 17d, 6.19 ( 0.06;
22a, 6.68 ( 10; 23d, 6.84 ( 0.08 (n = 3). In contrast, 25b be-
haved as expected of an allosteric enhancer, yielding a
concentration-dependent increase in the potency of the
orthosteric agonist. Application of an operational model of
allosterism5 to the data yielded a pKB estimate of the affinity
of the compound for the allosteric site of 6.01 ( 0.16, and
an estimate of the positive cooperativity of the interaction of
log Rβ = 0.44 ( 0.07 (i.e., Rβ = 2.7); n = 3.
In summary, we have found that certain 2A3BTs, pre-
viously thought to be allosteric enhancers, as well as the corre-
sponding conformationally restricted (1-aminoindeno[1,2-c]-
thiophen-8-one) derivatives are either inactive or behave as
orthosteric antagonists in a functional assay of A1AR-medi-
ated phosphorylation of ERK1/2. This highlights the value of
conducting functional assays rather than relying solely on
bindings assays when evaluating novel allosteric ligands of the
A1AR. In addition, we have identified a new class of A1AR allo-
steric enhancer with a indeno[2,1-b]thiophene heterocyclic core.
Experimental Section
Chemistry. Melting points were determined with an electro-
thermal melting point apparatus and are uncorrected. All H
1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6555
Figure 5. R-PIA mediated stimulation of ERK1/2 phosphorylation in the absence or presence of the indicated concentrations of test
compounds in intact CHO FlpIn A1AR cells. Data points represent the mean ( SEM of three experiments conducted in triplicate. Curves
drawn through the points represent the best global fit of a competitive (A-C) or allosteric (D) model of ligand-receptor interaction.
NMR spectra were recorded on a Bruker Avance DPX 300
spectrometer at 300.13 MHz, unless stated otherwise, or on a
Varian Unity Inova 600 spectrometer at 599.8 MHz. All 13C
NMR spectra were recorded on a Varian Unity Inova 600
spectrometer at 150.8 MHz, unless stated otherwise, or on a
Bruker Avance DPX 300 spectrometer at 75.4 MHz. Unless
stated otherwise, samples were dissolved in CDCl3. Thin-layer
chromatography was conducted on 0.2 mm plates using Merck
silica gel 60 F254. Column chromatography was achieved
using Merck silica gel 60 (particle size 0.063-0.200 μm, 70-
230 mesh). High resolution mass spectra (HR-ESI) were ob-
tained on a Waters LCT Premier XE (TOF) using electrospray
ionization. Compound purity was analyzed via LCMS (Agilent
1200 series LC coupled directly to a photodiode array detector
and an Agilent 6120 Quadrupole MS) using a Phenomenex
column (Luna 5 μm C8, 50 mm ꢀ 4.60 mm ID). All compounds
were of >95% purity.
filtered, and concentrated to a viscous amber oil. The oil is dis-
solved in THF (40 mL) and elemental sulfur (2.0 g, 62.38 mmol)
was added followed by Et2NH (10 mL) dropwise, and the
solution was stirred at room temperature for 2 h. The mixture
was diluted with ether (200 mL) and washed with water (2 ꢀ
100 mL) and finally with brine. The organic layer was separated,
dried (MgSO4), filtered, and evaporated in vacuo.
Ethyl 2-Amino-4-phenylthiophene-3-carboxylate (10a).13 Yield
97%. 1H NMR δ 7.30 (bs, 5H, ArH), 6.08 (s, 1H, H5), 5.46 (bs,
2H, NH2), 4.05 (q, J = 7.1 Hz, 2H, OCH2CH3), 0.94 (t, J = 7.2
Hz, 3H, OCH2CH3).
Ethyl 2-Amino-4-(3-chlorophenyl)thiophene-3-carboxylate (10b).13
Yield 93%. 1H NMR δ 7.30-7.16 (m, 4H, ArH), 6.21 (bs, 2H,
NH2), 6.06 (s, 1H, H5), 4.06 (q, J = 7.1 Hz, 2H, OCH2CH3), 0.98
(t, J = 7.2 Hz, 3H, OCH2CH3).
General Procedure for the Synthesis of 12a,b. To the appro-
priate thiophene 10a or 10b (30.86 mmol) in glacial acetic acid
(120 mL) was added phthalic anhydride (5.48 g, 37.03 mmol),
and the mixture was refluxed for 3 h. The mixture was concen-
trated to a solid and stirred in petroleum ether (150 mL) for
0.5 h. The solid was filtered on a Buchner funnel/flask and
washed with two portions of petroleum ether and dried (quanti-
tative recovery). The crude phthalimide 11a or 11b was sus-
pended in EtOH (120 mL) and water (120 mL) and NaOH
pellets (175 mmol) was added. The heterogeneous mixture was
heated to approximately 90 ꢀC on an oil bath for 3-4 h (moni-
tored by TLC), allowed to cool to room temperature, and
diluted with water (200 mL). The mixture was washed with
diethyl ether, and the aqueous layer was separated and cooled
on an ice bath. HCl (6M) was added dropwise with stirring to the
cooled mixture until pH ≈ 2 was obtained. A precipitate formed
General Procedure for the Synthesis of 10a,b.13 The appro-
priate acetophenone 9a or 9b (50.24 mmol) and ethyl cyanoa-
cetate (6.42 mL, 60.29 mmol) were dissolved in dry CH2Cl2
(200 mL) and cooled to 0 ꢀC in an ice bath. Neat TiCl4
(11.02 mL, 100.48 mmol) was added dropwise. After completion
of the addition, the mixture was stirred for 0.5 h and then dry
pyridine (3.40 mL) was added dropwise. The ice bath was
subsequently removed, and the mixture was stirred at room
temperature for 1 h. A further aliquot of dry pyridine (10 mL)
was added dropwise and the mixture was allowed to stir over-
night. The mixture was poured into 3 M HCl (200 mL) and the
organic layer separated. The aqueous layer was further ex-
tracted with CH2Cl2 (2 ꢀ50 mL), and the combined organics
were washed with water and finally brine, dried (MgSO4),

6556 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Aurelio et al.
and was collected on a Buchner funnel/flask and washed with
copious amounts of water.
1-Amino-5-chloro-8H-indeno[1,2-c]thiophen-8-one (14b). Yield
55%; mp 194-196 ꢀC. H NMR (DMSO-d6) δ 7.82 (bs, 2H,
1
NH2), 7.70 (m, 1H, aromatic), 7.51-7.48 (m, 1H, aromatic),
7.33-7.31 (m, 1H, aromatic), 6.72 (s, 1H, 3H-aromatic). 13C
NMR (DMSO-d6) δ 181.2, 158.7, 141.2, 140.1, 140.0, 137.0,
127.5, 124.4, 121.3, 114.1, 104.3. LCMS Rf (min) = 7.10. HR-
ESI calcd for C11H7ClNOSþ (M þ H) 235.9931, found 235.9940.
1-Amino-7-chloro-8H-indeno[1,2-c]thiophen-8-one (14c). Yield
22%; mp 262 ꢀC dec. 1H NMR (DMSO-d6) δ 7.79 (bs, 2H,
NH2), 7.54-7.52 (m, 1H, ArH), 7.47-7.42 (m, 1H, ArH), 7.27-
7.24 (m, 1H, aromatic), 6.70 (s, 1H, 3H-aromatic). 13C NMR
(DMSO-d6) δ 180.0, 158.8, 140.8, 139.7, 137.0, 133.5, 129.7, 129.2,
120.0, 114.4, 103.5. LCMS Rf (min) = 6.82. HR-ESI calcd for
C11H7ClNOSþ (M þ H) 235.9931, found 235.9942.
2-(2-Carboxybenzamido)-4-phenylthiophene-3-carboxylic Acid
(12a). Yield 87%; mp 196 ꢀC dec. 1H NMR (DMSO-d6) δ 13.13
(bs, 2H, 2 ꢀ CO2H), 11.64 (bs, 1H, NH), 7.93-7.90 (m, 1H,
phthalyl), 7.76-7.65 (m, 3H, phthalyl), 7.38-7.30 (m, 5H, ArH),
6.94 (s, 1H, H5). 13C NMR (DMSO-d6) δ 167.8, 167.3, 166.1,
149.2, 140.1, 137.7, 136.3, 132.6, 131.3, 131.2, 130.4, 129.4, 128.1,
127.9, 127.4, 116.2, 113.1.
2-(2-Carboxybenzamido)-4-(3-chlorophenyl)thiophene-3-carbo-
xylic Acid (12b). Yield 85%; mp 192 ꢀC dec. 1H NMR (DMSO-
d6) δ 13.18 (bs, 2H, 2 ꢀ CO2H), 11.68 (bs, 1H, NH), 7.94-7.92
(m, 1H, phthalyl), 7.75-7.67 (m, 3H, phthalyl), 7.40-7.31 (m,
4H, ArH), 7.03 (s, 1H, H5). 13C NMR (DMSO-d6) δ 167.5, 166.6,
165.8, 149.0, 139.4, 138.0, 135.9, 132.3, 132.2, 131.0, 130.7, 130.0,
129.4, 128.8, 127.9, 127.7, 127.0, 116.6, 112.6.
N-(8-Oxo-8H-indeno[1,2-c]thiophen-1-yl)acetamide (15a). Yield
1
85%; mp 212-214 ꢀC. H NMR (DMSO-d6) δ 11.42 (bs, 1H,
NH), 7.63-7.53 (m, 3H, ArH), 7.35-7.30 (m, 1H, ArH), 7.13 (s,
1H, H3), 2.25 (s, 3H, Ac). 13C NMR (DMSO-d6) δ 184.4, 169.6,
142.8, 141.2, 140.6, 140.5, 134.5, 128.6, 124.3, 122.1, 121.9, 110.6,
23.1.
General Procedure for the Synthesis of 13a,b. The bis-car-
boxylic acid 12a or 12b (22.41 mmol) was dissolved in CH2Cl2
(100 mL) under an atmosphere of N2 and DMF (200 μL) was
added. Neat oxalyl chloride (18.96 mL, 224.10 mmol) was sub-
sequently added dropwise [CAUTION: vigorous gas evolution
occurs!] over a period of an hour. The resultant mixture was
stirred overnight. The volatiles were evaporated and the residue
was redissolved in CH2Cl2 (160 mL) [NOTE: all operations
performed under an inert atmosphere]. The two neck flask was
fitted with reflux condenser, and AlCl3 (17.93 g, 134.46 mmol)
was added and the mixture was refluxed for 2 h. The mixture was
allowed to cool to room temperature and then slowly poured
with stirring into a mixture of 3 M HCl (200 mL), crushed ice
(400-600 g), and CH2Cl2 (300 mL). The organic layer was sepa-
rated and the aqueous layer extracted with CH2Cl2 (3 ꢀ 100
mL). The combined organic extracts were washed with water,
saturated bicarbonate, and finally with brine, dried (MgSO4),
filtered, and concentrated to provide a solid.
2-(8-Oxo-8H-indeno[1,2-c]thiophen-1-yl)isoindoline-1,3-dione
(13a). Yield 90%; mp 225-229 ꢀC. 1H NMR (DMSO-d6) δ
8.06-8.02 (m, 2H, phthalyl), 7.99-7.95 (m, 2H, phthalyl), 7.77
(s, 1H, H3), 7.73 (d, J = 7.5 Hz, 1H, ArH), 7.63 (t, J = 7.5 Hz,
2H, ArH), 7.58 (d, J = 7.5 Hz, 2H, ArH), 7.37 (t, J = 7.5 Hz,
2H, ArH). 13C NMR (DMSO-d6) δ 183.7, 164.9, 143.2, 140.9,
140.6, 135.8, 135.6, 134.6, 132.1, 131.7, 129.2, 125.0, 124.6,
122.2, 117.7.
N-(5-Chloro-8-oxo-8H-indeno[1,2-c]thiophen-1-yl)acetamide
(15b). Yield 75%; mp 298-300 ꢀC. 1H NMR (DMSO-d6) δ 11.53
(bs, 1H, NH), 7.76 (m, 1H, ArH), 7.58-7.55 (m, 1H, ArH),
7.36-7.33 (m, 1H, ArH), 7.21 (s, 1H, H3), 2.25 (s, 3H, Ac). 13
C
NMR (DMSO-d6) δ 183.0, 169.7, 143.2, 142.1, 139.8, 139.3,
139.2, 128.4, 125.9, 122.3, 121.9, 112.2, 23.0.
General Procedure for the Synthesis of 16a,b. Acetamide 15d
or 15e (20.55 mmol) was dissolved in DMSO (100 mL) and
CH3CN (400 mL). To the mixture was added I2 (5.74 g, 22.61
mmol) and stirred until all solids had dissolved. AgNO3 (4.19 g,
24.67 mmol) was added, and a precipitate immediately formed
with concomitant decolouration of the solution. After stirring
for 1 h, the mixture was filtered through Celite and the Celite pad
washed with ethyl acetate (500 mL). The organic mixture was
washed with water (ꢀ5), then brine, dried (MgSO4), filtered, and
concentrated to a solid. This material was triturated with ether,
collected by suction filtration, and washed with diethyl ether to
afford 16a and 16b, as bright-yellow solids.
N-(3-Iodo-8-oxo-8H-indeno[1,2-c]thiophen-1-yl)acetamide (16a).
Yield 71%; mp 250 ꢀC dec. 1H NMR (DMSO-d6) δ 11.73 (bs, 1H,
NH), 7.97 (d, J= 7.2 Hz, 1H, ArH), 7.67-7.61 (m, 2H, ArH), 7.40
(t, J= 7.5 Hz, 1H, ArH), 2.26 (s, 3H, Ac). 13C NMR (DMSO-d6) δ
182.9, 170.1, 146.3, 143.5, 141.1, 140.2, 134.4, 129.2, 124.7, 123.2,
119.7, 64.7, 22.8.
2-(5-Chloro-8-oxo-8H-indeno[1,2-c]thiophen-1-yl)isoindoline-
1,3-dione and 2-(7-chloro-8-oxo-8H-indeno[1,2-c]thiophen-1-yl)-
isoindoline-1,3-dione (13b). Yield 81% of a mixture of isomers in
an approximate 7:3 ratio of the 5-chloro to 7-chloro and used in
the next step without purification.
N-(5-Chloro-3-iodo-8-oxo-8H-indeno[1,2-c]thiophen-1-yl)acet-
amide (16b). Yield 86%; mp 275-278 ꢀC. 1H NMR (DMSO-d6)
δ 11.89 (bs, 1H, NH), 7.90 (d, J = 1.5 Hz, 1H, ArH), 7.64 (d, J =
8.1 Hz, 1H, ArH), 7.47 (dd, J = 1.8 Hz, 8.1 Hz, 1H, ArH). 13
C
General Procedure for the Synthesis of 14a-c. The phthali-
mide 13a or 13b (12.07 mmol) was suspended in DMF:dioxane
(1:1, 20 mL) at room temperature. After the dropwise addition
of hydrazine hydrate (1 mL), all of the solids had dissolved. The
reaction mixture was stirred for 1 h, diluted with ether (100 mL),
and stirred for a further 10 min. The mixture was filtered through
Celite, and the organics were washed thoroughly with water, then
brine, dried (MgSO4), and concentrated. The residue was chro-
matographed on silica gel eluting with 30% ethyl acetate/petro-
leum ether to yield the desired product (14a-c) as a solid. A small
portion of each were recrystallized from MeOH for analytical
purposes. Compounds 14a and 14b were subsequently refluxed
for 10 min in a mixture of Ac2O:AcOH (1:1, 10 mL). The mixture
was concentrated and triturated with ether. The resultant solid
was collected by suction filtration and washed with ether to
provide a yellow solid 15a and 15b.
NMR (DMSO-d6) δ 180.8, 169.6, 146.0, 141.5, 141.1, 139.1, 138.3,
128.4, 125.7, 122.5, 118.7, 66.2, 22.3.
General Procedure for the Synthesis of 17a-f. The iodide 16a
(650 mg, 1.76 mmol) in DMF (11 mL, degassed) was added 2 M
K3PO4 (3.25 mL, degassed) and the appropriate boronic acid
(3.52 mmol) followed by Pd[PPh3]2Cl2 (124 mg, 10 mol %).
After heating for 10-15 min at 75-80 ꢀC under an atmosphere
of N2, a precipitate formed. The mixture was stirred for a further
30-45 min at 75-80 ꢀC. The mixture was then diluted with
water (30 mL), and the precipitate was collected by suction
filtration. This solid was washed with water (50 mL) and diethyl
ether (50 mL) to provide quantitative yields of crude cross-
coupled products. The crude cross-coupled product was sus-
pended in EtOH (12 mL) and NaOH (4 pellets ≈ 800 mg)
was added and the mixture was capped in a sealed vessel
and heated (microwave) at 150 ꢀC for 20 min. The cooled
solution was filtered to remove insoluble material, concentra-
ted to a residue that was dissolved in water (50 mL), and
acidified with AcOH until no more precipitate formed. The
solid was collected on a Buchner funnel/flask and washed
with water (100 mL), dried, and then suspended in EtOH
(5 mL) and refluxed for 10 min. The cooled mixture was
1-Amino-8H-indeno[1,2-c]thiophen-8-one (14a). Yield 73%,
1
mp 128-130 ꢀC. H NMR (DMSO-d6) δ 7.66 (bs, 2H, NH2),
7.55-7.44 (m, 3H, ArH), 7.31-7.26 (m, 1H, ArH), 6.60 (s, 1H,
H3). 13C NMR (DMSO-d6) δ 183.1, 158.8, 143.2, 142.0, 139.1,
132.8, 128.1, 123.3, 121.5, 114.8, 103.0. LCMS Rf (min) =
6.58. HR-ESI calcd for C11H8NOSþ (M þ H) 202.0321, found
202.0318.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6557
(2-Aminothiophen-3-yl)(4-chlorophenyl)methanone (20b).12
collected by suction filtration and washed with EtOH and then
ether. If required, the resultant solid was recrystallized from
DMF or DMF/MeOH.
1
Yield 68%; mp 168-170 ꢀC. H NMR (599.8 MHz) δ 7.62 (d,
J = 7.2 Hz, 1H, ArH), 7.42 (d, J = 7.2 Hz, 1H, ArH), 6.98 (bs,
2H, NH2), 6.83 (d, J = 4.8 Hz, 1H, H4), 6.14 (d, J = 4.8 Hz, 1H,
H5). 13C NMR δ 189.8, 166.5, 139.1, 136.9, 129.6, 128.4, 127.3,
114.8, 106.4. LCMS Rf (min) = 7.12. HR-ESI calcd for C11H9-
ClNOSþ (M þ H) 238.0088, found 238.0091.
1-Amino-3-phenyl-8H-indeno[1,2-c]thiophen-8-one (17a). Yield
40%; mp 224-226 ꢀC dec. 1H NMR (DMSO-d6) δ 7.86 (bs, 2H,
NH2), 7.60-7.47 (m, 5H, ArH), 7.44-7.38 (m, 3H, ArH),
7.33-7.29 (m, 1H, ArH). 13C NMR (DMSO-d6) δ 182.4, 157.8,
142.8, 138.3, 135.6, 132.7, 132.4, 129.1, 128.1, 128.0, 127.9, 123.1,
120.9, 120.3, 115.9. LCMS Rf (min) = 7.59. HR-ESI calcd for
C17H12NOSþ (M þ H) 278.0634, found 278.0647.
General Procedure for the Synthesis of 21a and 21b. The
appropriate thiophene 20a or 20b (12.62 mmol) was refluxed
for 10 min in acetic anhydride (15 mL). The cooled mixture was
quenched with bicarbonate solution and extracted with diethyl
ether. The ether layer was dried (MgSO4), filtered, and concen-
trated to a solid that was dissolved in CH3CN (50 mL) and DMF
(50 mL). Iodine (3.52 g, 13.88 mmol) was added, and the mixture
was stirred until it had dissolved. Neat AgNO3 (2.57 g, 15.14
mmol) was added, and the iodine color dissapeared after 1 min
with precipitation of AgI. After stirring a further 20 min, the
mixture was diluted with EtOAc (∼100 mL) and filtered through
Celite. The mixture was washed with dilute Na2S2O3 and then
H2O (ꢀ4) and finally brine, dried (MgSO4), filtered, and con-
centrated to a solid. The products were recrystallized from
MeOH (21a) and iPrOH (21b).
1-Amino-3-(4-methoxyphenyl)-8H-indeno[1,2-c]thiophen-8-one
(17b). Yield 27%; mp 215-218 ꢀC. 1H NMR δ 7.68 (d, J = 7.5
Hz, 1H, ArH), 7.53 (d, J = 8.4 Hz, 3H, ArH), 7.33 (t, J = 7.4 Hz,
1H, ArH), 7.25 (t, J = 6.9 Hz, 1H, ArH), 6.99 (d, J = 8.4 Hz, 1H,
ArH), 5.52(bs, 2H, NH2), 3.88(s, 3H, OMe). 13C NMR (DMSO-
d6) δ 182.3, 159.2, 157.5, 142.8, 138.4, 134.6, 132.3, 129.3, 127.7,
124.8, 123.1, 120.7, 120.5, 115.7, 114.5, 55.3. LCMS Rf (min) =
7.63. HR-ESI calcd for C18H14NO2Sþ (M þ H) 308.0740, found
308.0736.
1-Amino-3-(4-(dimethylamino)phenyl)-8H-indeno[1,2-c]thiophen-
8-one (17c). Yield 56%; mp 241 ꢀC dec. 1H NMR (DMSO-d6) δ
7.71 (bs, 2H, NH2), 7.54 (d, J = 7.5 Hz, 2H, ArH), 7.43-7.38 (m,
3H, ArH), 7.27 (t, J = 7.5 Hz, 1H, ArH), 6.82 (d, J = 9.0 Hz, 2H,
ArH), 2.97 (s, 6H, NMe2). 13C NMR (DMSO-d6) δ 182.6, 157.7,
150.5, 143.2, 139.1, 133.6, 132.6, 129.1, 127.8, 123.5, 122.6, 121.2,
120.2, 116.3, 112.7, 40.3. LCMS Rf (min) = 7.63. HR-ESI calcd
for C19H17N2OSþ (M þ H) 321.1056, found 321.1059.
1-Amino-3-(pyrimidin-5-yl)-8H-indeno[1,2-c]thiophen-8-one
(17d). Yield 46%; mp 294 ꢀC dec. 1H NMR (DMSO-d6) δ 9.20
(s, 1H, ArH), 9.04 (s, 2H, ArH), 7.99 (bs, 2H, NH2), 7.59-7.56
(m, 1H, ArH), 7.48-7.33 (m, 3H, ArH). 13C NMR (DMSO-d6) δ
182.8, 159.1, 157.7, 155.9, 143.1, 139.4, 138.2, 133.2, 129.1, 128.1,
123.7, 121.1, 116.5, 112.1. LCMS Rf (min) = 6.78. HR-ESI calcd
for C15H8N3OS- (M - H) 278.0394, found 278.0392.
1-Amino-3-(pyridin-4-yl)-8H-indeno[1,2-c]thiophen-8-one (17e).
Yield 59%; mp 272 ꢀC dec. 1H NMR (DMSO-d6) δ 8.67-864 (m,
2H, ArH), 7.99 (bs, 2H, NH2), 7.67-7.64 (m, 1H, ArH), 7.60-
7.55 (m, 3H, ArH), 7.51-7.42 (m, 1H, ArH), 7.40-7.34 (m, 1H,
ArH). 13C NMR (DMSO-d6) δ 182.9, 158.7, 150.8, 143.1, 140.7,
139.0, 138.2, 133.1, 129.1, 123.7, 122.3, 121.9, 117.2, 116.8. LCMS
Rf (min) = 5.87. HR-ESI calcd for C16H11N2OSþ (M þ H)
279.0587, found 279.0598.
N-(3-Benzoyl-5-iodothiophen-2-yl)acetamide (21a). Yield 71%;
1
mp 162-164 ꢀC. H NMR δ 12.06 (bs, 1H, NH), 7.69-7.67
(m, 2H, ArH), 7.61-7.48 (m, 3H, ArH), 6.96 (s, 1H, H4), 2.24
(s, 3H, Ac). 13C NMR δ 191.5, 168.1, 155.2, 139.4, 135.1, 132.1,
128.6 (ꢀ2), 121.7, 66.1, 23.4.
N-(3-(4-Chlorobenzoyl)-5-iodothiophen-2-yl)acetamide (21b).
1
Yield 84%; mp 175-177 ꢀC. H NMR δ 11.99 (bs, 1H, NH),
7.64 (d, J = 8.7 Hz, 2H, ArH), 7.49 (d, J = 8.7 Hz, 2H, ArH),
7.23 (s, 1H, 4H-aromatic), 2.24 (s, 3H, Ac). 13C NMR δ 190.0,
168.1, 155.4, 138.4, 137.6, 134.6, 130.0, 128.9, 121.4, 66.4, 23.4.
General Procedure for the Synthesis of 22a-f and 23a-f. The
appropriate iodide 21a or 21b (1.23 mmol) in DMF (7.7 mL,
degassed) was added 2 M K3PO4 (2.28 mL, degassed) and the
appropriate boronic acid (2.46 mmol) followed by Pd[PPh3]2Cl2
(10 mol %, 87 mg) and heated with stirring on an oil bath
at 75-80 ꢀC in an N2 atmosphere for 1 h. The mixture was
diluted with ether and washed with water (ꢀ4) then brine, dried
(MgSO4), filtered, and evaporated to dryness. The crude cross-
coupled product was suspended in EtOH (15 mL), dioxane
(10 mL), and water (5 mL) and NaOH (250 mg, 6.25 mmol)
was added and the mixture was stirred on an oil bath at
30-35 ꢀC for 1-3 h (monitored by TLC). The cooled solu-
tion was diluted with water, and if a solid formed, it was filtered
on a Buchner funnel/flask, otherwise it was extracted with die-
thyl ether. The crude hydrolyzed compounds were chromato-
graphed on silica gel (10-50% EtOAc/petroleum ether) and
then recystallized from MeOH. For compounds 22f and
23f, after completion of the hydrolysis, the mixture was
diluted with water (∼100 mL) and acidified to pH 3 (2 M
HCl). The resultant solid was collected by suction filtration
and washed with plenty of water before being recrystallized
from MeOH.
4-(1-Amino-8-oxo-8H-indeno[1,2-c]thiophen-3-yl)benzoic Acid
(17f). Yield 53%; mp 318 ꢀC dec. 1H NMR (DMSO-d6) δ 13.01
(bs, 1H, CO2H), 8.05 (d, J = 8.1 Hz, 2H, ArH), 7.92 (bs, 2H,
NH2), 7.71 (d, J = 8.1 Hz, 2H, ArH), 7.62-7.56 (m, 2H, ArH),
7.45 (t, J = 7.4 Hz, 1H, ArH), 7.35 (t, J = 7.4 Hz, 1H, ArH). 13
C
NMR (DMSO-d6) δ 182.9, 167.3, 158.5, 143.2, 138.5, 133.0,
130.6, 130.5, 130.2, 128.8, 128.2, 127.6, 123.6, 121.7, 119.4,
116.6. LCMS Rf (min) = 7.15. HR-ESI calcd for C18H12NO3Sþ
(M þ H) 322.0532, found 322.0543.
General Procedure Synthesis of 20a-b.12 The appropriate
nitrile 18a or 18b (34.45 mmol), 2,5-dihydroxy-1,4-dithiane 19
(2.62 g, 17.22 mmol), and diethylamine (3.58 mL) were sus-
pended in EtOH (14 mL) and stirred at 50 ꢀC for several hours,
during which time a solid precipitated. The mixture was diluted
with EtOH (∼5 mL) and chilled on an ice bath with stirring
(∼0.5 h). The fine yellow powder was filtered using a Buchner
funnel/flask and washed with ice-cold EtOH until a pale-yellow
filtrate was obtained. The solid was left to dry overnight,
providing 20a and 20b that were used in the next step without
further purification. A small portion of each were recrystallized
from MeOH.
(2-Amino-5-phenylthiophen-3-yl)(phenyl)methanone (22a). Yield
1
66%; mp 159-161 ꢀC. H NMR δ 7.73-7.71 (m, 2H, ArH),
7.55-7.45 (m, 3H, ArH), 7.41-7.38 (m, 2H, ArH), 7.34-7.29 (m,
2H, ArH), 7.23-7.18 (m, 1H, ArH), 7.12 (s, 1H, H4), 7.08 (bs, 2H,
NH2). 13C NMR δ 191.3, 166.2, 140.8, 133.8, 130.9, 128.9, 128.3,
128.2, 126.8, 124.8, 123.9, 122.8, 116.0. LCMS Rf (min) = 7.69.
HR-ESI calcd for C17H14NOSþ (M þ H) 280.0791, found
280.0800.
(2-Amino-5-(4-methoxyphenyl)thiophen-3-yl)(phenyl)methan-
one (22b). Yield 66%; mp 158-160 ꢀC. 1H NMR δ 7.73-7.70 (m,
2H, ArH), 7.54-7.45 (m, 3H, ArH), 7.32 (d, J = 7.5 Hz, 2H,
ArH), 7.03 (bs, 2H, NH2), 6.97 (s, 1H, H4), 6.86 (d, J = 7.5 Hz,
2H, ArH), 3.81 (s, 3H, OMe). 13C NMR δ 191.2, 165.6, 158.7,
140.9, 130.8, 128.3, 128.1, 126.6, 126.2, 124.0, 121.4, 116.0, 114.3,
55.4. LCMS Rf (min) = 7.58. HR-ESI calcd for C18H16NO2Sþ
(M þ H) 310.0896, found 310.0909.
(2-Aminothiophen-3-yl)(phenyl)methanone (20a).12 Yield 64%;
mp 144-146 ꢀC. 1H NMR δ 7.68-7.66 (m, 2H, ArH), 7.51-7.42
(m, 3H, ArH), 6.96 (bs, 2H, NH2), 6.88 (d, J = 5.7 Hz, 1H, H4),
6.13 (d, J = 5.4 Hz, 1H, H5). 13C NMR δ 191.3, 166.4, 140.8,
130.8, 128.2 (ꢀ2), 127.7, 115.0, 106.1. LCMS Rf (min) = 6.68.
HR-ESI calcd for C11H10NOSþ (M þ H) 204.0478, found
204.0485.

6558 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Aurelio et al.
(2-Amino-5-(4-(dimethylamino)phenyl)thiophen-3-yl)(phenyl)-
methanone (22c). Yield 42%; mp 206-208 ꢀC. 1H NMR δ
7.73-7.72 (m, 2H, ArH), 7.51-7.46 (m, 3H, ArH), 7.28-726
(m, 2H, ArH), 6.98 (bs, 2H, NH2), 6.91 (s, 1H, H4), 6.88 (d, J =
7.8 Hz, 2H, ArH), 2.96 (s, 6H, NMe2). 13C NMR δ 191.1, 165.2,
149.6, 141.0, 130.7, 128.2 (ꢀ2), 126.0, 125.0, 122.3, 120.0, 116.0,
112.7, 40.5. LCMS Rf (min) = 7.18. HR-ESI calcd for
C19H19N2OSþ (M þ H) 323.1213, found 323.1216.
4-(5-Amino-4-(4-chlorobenzoyl)thiophen-2-yl)benzoic Acid (23f).
Yield 50%; mp 274-276 ꢀC dec. 1H NMR (DMSO-d6) δ 12.88
(bs, 1H, CO2H), 8.72 (bs, 2H, NH2), 7.86 (d, J = 7.8 Hz, 2H,
ArH), 7.69 (d, J = 8.1 Hz, 2H, ArH), 7.58 (d, J = 7.5 Hz, 2H,
ArH), 7.56 (d, J = 7.5 Hz, 2H, ArH), 7.33 (s, 1H, H4). 13C NMR
δ 188.1, 167.8, 167.0, 139.2 137.8, 135.5, 130.1, 129.9, 128.5,
128.1, 124.5, 124.0, 120.8, 114.2. LCMS Rf (min) = 7.39. HR-ESI
calcd for C18H13ClNO3Sþ (M þ H) 358.0299, found 358.0316.
General Procedure for the Synthesis of 25a,b. 1-Indanone 24a
(200 mg, 1.51 mmol) and the appropriate nitrile 18a or 18b (1.51
mmol) were dissolved in dry CH2Cl2 (10 mL) and cooled to 0 ꢀC
in an ice bath. Neat TiCl4 (166 μL, 1.51 mmol) was added
dropwise, and the mixture was stirred for 0.5 h. Dry pyridine
(111 μL) was added dropwise, and the mixture was warmed to
room temperature and stirred for 1 h. A further aliquot of dry
pyridine (308 μL) was added dropwise, and stirring was con-
tinued overnight. The mixture was poured into 3 M HCl (20 mL)
and the organic layer separated. The aqueous layer was further
extracted with CH2Cl2 (2 ꢀ 10 mL), and the combined organics
were washed with water and brine before being dried (MgSO4),
filtered, and concentrated to a viscous amber oil. The oil was
dissolved in THF (3 mL) and elemental sulfur (53 mg, 1.66
mmol) and Et2NH (400 μL) were added. The reaction was
stirred at ∼60 ꢀC for 2 h. The deep blue-purple mixture was
concentrated to a residue and then chromatographed on silica
gel (10% EtOAc/petroleum ether) to afford 25a,b as solids that
were recrystallized from iPrOH.
(2-Amino-8H-indeno[2,1-b]thiophen-3-yl)(phenyl)methanone
(25a). Yield 9%; mp 183-185 ꢀC. 1H NMR δ 7.74-7.71 (m, 2H,
ArH), 7.55-7.50 (m, 1H, ArH), 7.43-7.34 (m, 3H, ArH), 7.02-
6.97 (m, 1H, ArH), 6.77 (t, J = 7.5 Hz, 1H, ArH), 6.58 (bs, 2H,
NH2), 5.67 (d, J = 7.8 Hz, 1H, ArH), 3.69 (s, 2H, CH2). 13C
NMR (DMSO-d6) δ 191.9, 168.5, 146.1, 142.2, 141.5, 139.7,
131.8, 129.1, 128.7, 126.3, 126.0, 124.0, 123.8, 121.3, 111.0, 34.6.
LCMSRf (min) = 7.38. HR-ESI calcd for C18H14NOSþ (M þ H)
292.0791, found 292.0803.
(2-Amino-8H-indeno[2,1-b]thiophen-3-yl)(4-chlorophenyl)methan-
one (25b). Yield 18%; mp 198-200 ꢀC. 1H NMR δ 7.69 (d, J =
7.8 Hz, 2H, ArH), 7.39-7.37 (m, 3H, ArH), 7.03 (t, J = 7.4 Hz,
1H, ArH), 6.86 (t, J = 7.5 Hz, 1H, ArH), 6.59 (bs, 2H, NH2), 5.80
(d, J = 7.8 Hz, 1H, ArH), 3.70 (s, 2H, CH2). 13C NMR (DMSO-
d6) δ 190.3, 168.8, 146.2, 141.9, 139.7, 139.5, 138.0, 130.7, 128.9,
126.6, 126.1, 124.1, 124.0, 121.2, 110.6, 34.6. LCMS Rf (min) =
7.74. HR-ESIcalcdfor C18H13ClNOSþ (M þ H) 326.0401, found
326.0403.
(2-Amino-5-(pyrimidin-5-yl)thiophen-3-yl)(phenyl)methanone
(22d). Yield 15%; mp 150-152 ꢀC dec. 1H NMR δ 9.03 (s, 1H,
ArH), 8.74 (s, 2H, ArH), 7.71-7.70 (m, 2H, ArH), 7.56-7.50
(m, 3H, ArH), 7.26 (s, 1H, H4), 7.20 (bs, 2H, NH2). 13C NMR δ
191.3, 166.8, 156.2, 152.2 (ꢀ2), 140.3, 131.2, 128.5, 128.1, 125.9,
116.1, 115.5. LCMS Rf (min) = 6.82. HR-ESI calcd for
C15H12N3OSþ (M þ H) 282.0696, found 282.0696.
(2-Amino-5-(pyridin-4-yl)thiophen-3-yl)(phenyl)methanone (22e).
Yield 42%; mp 218-220 ꢀC dec. 1H NMR δ 8.49 (s, 2H, ArH),
7.71-7.70 (m, 2H, ArH), 7.56-7.50 (m, 3H, ArH), 7.37 (s, 1H,
H4), 7.34 (bs, 2H, NH2), 7.23 (m, 2H, ArH). 13C NMR δ 190.5,
168.0, 149.7, 141.1, 140.5, 130.6, 128.1, 127.7, 126.0, 119.2, 118.2,
114.9. LCMS Rf (min) = 5.27. HR-ESI calcd for C16H13N2OSþ
(M þ H) 281.0743, found 281.0754.
4-(5-Amino-4-benzoylthiophen-2-yl)benzoic acid (22f). Yield
1
37%; mp 284-286 ꢀC. H NMR (DMSO-d6) δ 12.83 (bs, 1H,
CO2H), 7.86 (d, J = 8.4 Hz, 2H, ArH), 7.67-7.65 (m, 2H, ArH),
7.56-7.52 (m, 5H, ArH and NH2), 7.30 (s, 1H, H4). 13C NMR δ
189.5, 167.5, 167.0, 140.6, 137.8, 130.8, 130.1, 128.4, 128.0,
127.9, 124.6, 123.9, 120.5, 114.3. LCMS Rf (min) = 7.15. HR-
ESI calcd for C18H14NO3Sþ (M þ H) 324.0689, found 324.0688.
(2-Amino-5-phenylthiophen-3-yl)(4-chlorophenyl)methanone (23a).
Yield 37%; mp 168-170 ꢀC. 1H NMR δ 7.67 (d, J = 7.2 Hz, 2H,
ArH), 7.45 (d, J = 7.2 Hz, 2H, ArH), 7.41-7.30 (m, 4H, ArH),
7.25-7.20 (m, 1H, ArH), 7.07 (bs, 2H, NH2), 7.06 (s, 1H, H4).
13C NMR δ 189.8, 166.2, 139.1, 137.0, 133.6, 129.6, 128.9, 128.6,
126.9, 124.8, 124.3, 122.3, 115.8. LCMSRf (min) = 8.05. HR-ESI
calcd for C17H13ClNOSþ (M þ H) 314.0401, found 314.0409.
(2-Amino-5-(4-methoxyphenyl)thiophen-3-yl)(4-chlorophenyl)-
1
methanone (23b). Yield 58%; mp 139-141 ꢀC dec. H NMR δ
7.66 (d, J = 7.5 Hz, 2H, ArH), 7.45 (d, J = 7.5 Hz, 2H, ArH),
7.32 (d, J = 8.1 Hz, 2H, ArH), 7.03 (bs, 2H, NH2), 6.91 (s, 1H,
H4), 6.87 (d, J = 8.1 Hz, 2H, ArH), 3.81 (s, 3H, OMe). 13C NMR
(150.8 MHz) δ 189.7, 165.9, 158.8, 139.2, 136.9, 129.6, 128.5,
126.4, 126.2, 124.3, 120.9, 115.7, 114.3, 55.4. LCMS Rf (min) =
7.92. HR-ESI calcd for C18H15ClNO2Sþ (M þ H) 344.0507,
found 344.0493.
General Procedure for the Synthesis of 25c-f. Indanone 24b
and 24c (500 mg, 2.50 mmol), the appropriate nitrile 18a or 18b
(3.25 mmol), benzoic acid (305 mg, 2.50 mmol), and β-alanine
(45 mg, 20 mol %) was heated at 110-120 ꢀC for 3-6 h (moni-
tored by TLC). The reaction was then cooled to ∼60 ꢀC and
EtOH (1 mL) was added, followed by morpholine (655 μL, 7.49
mmol) and elemental sulfur (120 mg, 3.75 mmol). The mixture
was stirred at this temperature for 3 h and then cooled to room
temperature, diluted with ether, and washed with water. The
organic layer was dried (MgSO4), filtered, and concentrated to a
residue that was chromatographed on silica gel (CH2Cl2),
providing 25c-f as solids. Recrystallization (25c,d from ether/
pet. ether and 25e,f from MeOH) afforded the pure products.
(2-Amino-7-(trifluoromethyl)-8H-indeno[2,1-b]thiophen-3-yl)-
(phenyl)methanone (25c). Yield 21%; mp 188-190 ꢀC. 1H NMR
δ 7.71 (d, J = 7.5 Hz, 2H, ArH), 7.54 (t, J = 7.2 Hz, 1H, ArH),
7.41(t, J = 7.2 Hz, 2H, ArH), 7.23(d, J = 8.1 Hz, 1H, ArH), 6.88
(t, J = 7.8 Hz, 1H, ArH), 6.63 (bs, 2H, NH2), 5.83 (d, J = 7.8 Hz,
1H, ArH), 3.89 (s, 2H, CH2). 13C NMR (DMSO-d6) δ 191.6,
168.9, 143.4, 141.4, 141.1, 141.0, 131.9, 129.0, 128.8, 127.4, 126.5,
126.0 (q, J = 31.9 Hz), 124.5 (q, J = 273.0 Hz), 124.3, 120.5 (q,
J = 4.5 Hz), 110.5, 33.8. LCMS Rf (min) = 8.09. HR-ESI calcd
for C19H13F3NOSþ (M þ H) 360.0664, found 360.0664.
(2-Amino-5-(4-(dimethylamino)phenyl)thiophen-3-yl)(4-chloro-
1
phenyl)methanone (23c). Yield 35%; mp 170-172 ꢀC dec. H
NMR δ 7.67 (d, J = 8.4 Hz, 2H, ArH), 7.44 (d, J = 8.1 Hz, 2H,
ArH), 7.27 (d, J = 8.2 Hz, 2H, ArH), 7.00 (bs, 2H, NH2), 6.85 (s,
1H, H4), 6.68 (d, J = 8.7 Hz, 2H, ArH), 2.96 (s, 6H, NMe2). 13
C
NMR δ 189.6, 165.7, 149.7, 139.4, 136.9, 129.8, 128.6, 126.1,
125.4, 122.2, 119.5, 115.8, 112.8, 40.6. LCMS Rf (min) = 7.65.
HR-ESI calcd for C19H18ClN2OSþ (M þ H) 357.0823, found
357.0840.
(2-Amino-5-(pyrimidin-5-yl)thiophen-3-yl)(4-chlorophenyl)-
methanone (23d). Yield 18%; mp 229-231 ꢀC dec. 1H NMR δ
9.04 (s, 1H, ArH), 8.75 (s, 2H, ArH), 7.66 (d, J = 7.5 Hz, 2H,
ArH), 7.48 (d, J = 7.2 Hz, 2H, ArH), 7.26 (s, 1H, H4), 7.20 (bs,
2H, NH2). 13C NMR δ 188.7, 168.2, 155.6, 151.7 (ꢀ2), 138.9,
136.4, 129.3, 128.3, 125.0, 114.7, 114.5. LCMS Rf (min) = 7.13.
HR-ESI calcd for C15H11ClN3OSþ (M þ H) 316.0306, found
316.0313.
(2-Amino-5-(pyridin-4-yl)thiophen-3-yl)(4-chlorophenyl)methan-
one (23e). Yield 37%; mp 262-264 ꢀC dec. 1H NMR (DMSO-d6)
δ 8.77 (bs, 2H, NH2), 8.43 (d, J = 4.8 Hz, 2H, ArH), 7.69 (d,
J = 7.2 Hz, 2H, ArH), 7.58 (d, J = 7.5 Hz, 2H, ArH), 7.50 (s, 1H,
H4), 7.42 (d, J = 4.8 Hz, 2H, ArH). 13C NMR (DMSO-d6) δ
188.1, 167.9, 149.8, 140.6, 139.0, 135.6, 129.9, 128.5, 126.2, 118.7,
118.3, 114.1. LCMS Rf (min) = 5.67. HR-ESI calcd for
C16H12ClN2OSþ (M þ H) 315.0353, found 315.0366.
(2-Amino-7-(trifluoromethyl)-8H-indeno[2,1-b]thiophen-3-yl)-
(4-chlorophenyl)methanone (25d). Yield 17%; mp 152-154 ꢀC

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6559
dec. 1H NMR δ7.67 (d, J = 7.8 Hz, 2H, ArH), 7.39 (d, J = 7.5 Hz,
2H, ArH), 7.28-7.26 (m, 1H, ArH), 6.97 (t, J = 8.1 Hz, 1H, ArH),
6.63 (bs, 2H, NH2), 5.96 (d, J = 7.8 Hz, 1H, ArH), 3.90 (s, 2H,
CH2). 13C NMR (DMSO-d6) δ 190.0, 169.0, 143.4, 140.9, 140.7,
139.6, 138.2, 130.6, 129.1, 127.7, 126.7, 126.3 (q, J = 28.2 Hz),
124.5 (q, J = 273.3 Hz), 124.2, 120.7 (q, J = 4.5 Hz), 110.2, 33.8.
LCMS Rf (min) = 8.56. HR-ESI calcd for C19H12ClF3NOSþ
(M þ H) 394.0275, found 394.0280.
basal ERK1/2 phosphorylation in the absence of R-PIA and
100% representing the maximal response attained at a saturating
concentration of R-PIA (100 nM). For the full concentration-
response curves, the data were expressed as a percentage of the
ERK1/2 phosphorylation mediated after a 6 min exposure to
DMEM containing 3% FBS.
Acknowledgment. This research was supported by Discovery
grant DP0877497 of the Australian Research Council and Pro-
gram grant 519461 of the National Health and Medical Research
Council (NHMRC) of Australia. A.C. is a Senior, and P.M.S. a
Principal Research Fellow, of the NHMRC. We are grateful to
Dr. Michael Crouch, TGR Biosciences, Adelaide, for generously
providing the ERK SureFire Alphascreen kit reagents.
(2-Amino-5-(trifluoromethyl)-8H-indeno[2,1-b]thiophen-3-yl)-
(phenyl)methanone (25e). Yield 1%; mp 202-207 ꢀC dec. 1H
NMR δ 7.68 (d, J = 7.8 Hz, 2H, ArH), 7.56 (t, J = 7.4 Hz, 1H,
ArH), 7.44-7.39 (m, 3H, ArH), 7.26-7.24 (m, 1H, ArH), 6.71 (bs,
2H, NH2), 5.78 (s, 1H, ArH), 3.74 (s, 2H, CH2). LCMSRf (min) =
7.74. HR-ESI calcd for C19H13F3NOSþ (M þ H) 360.0664, found
360.0666.
(2-Amino-5-(trifluoromethyl)-8H-indeno[2,1-b]thiophen-3-yl)-
(4-chlorophenyl)methanone (25f). Yield 1%; mp 202-204 ꢀC
dec. 1H NMR δ 7.63 (d, J = 8.4 Hz, 2H, ArH), 7.45-7.39 (m,
3H, ArH), 7.30-7.26 (m, 1H, ArH), 6.74 (bs, 2H, NH2), 5.85 (s,
1H, ArH), 3.75 (s, 2H, CH2). LCMS Rf (min) = 7.99. HR-ESI
calcd for C19H12ClF3NOSþ (M þ H) 394.0275, found 394.0274.
Crystallography. Intensity data for 25c were collected with an
Oxford Diffraction Sapphire CCD diffractometer using Cu KR
radiation (graphite crystal monochromator λ = 1.54184). The
temperature during data collection was maintained at 130.0(1)
using an Oxford cooling device. The structures were solved by
direct methods and difference Fourier synthesis. Thermal ellip-
soid plots were generated using the program ORTEP-314 inte-
grated within the WINGX15 suite of programs.
Supporting Information Available: LC-MS analysis of com-
pounds 14a-c, 17a-f, 20a-b, 22a-f, 23a-f, and 25a-f. Crystal-
lographic data for 25c have been deposited with the Cambridge
Crystallographic Data Centre (CCDC code 790719). This materi-
al is available free of charge via the Internet at http://pubs.acs.org.
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