5-Substituted 2-aminothiophenes as A1 adenosine receptor allosteric enhancers

Article abstract of DOI:10.1016/j.bmc.2007.10.065

Two series of 5-substituted 2-amino-4-(3-trifluoromethylphenyl)thiophenes were prepared and evaluated as allosteric enhancers at the A₁ adenosine receptor (A₁AR). In the 3-benzoyl series, a 5-phenyl group was found to confer the greatest potency (9a: ED₅₀ = 2.1 μM, AE score = 18%). However, the analogue with no 5-substituent (6b: ED₅₀ = 15.8 μM, AE score = 77%) proved to be the most efficacious. In the 3-ethoxycarbonyl series, the 5-(4-chlorophenyl) analogue was clearly the most potent and efficacious (9l: ED₅₀ = 6.6 μM, AE score = 57%). The antagonist activity of all compounds was measured using a [³H]CPX competitive binding assay.

Full text of DOI:10.1016/j.bmc.2007.10.065

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1319–1327
5-Substituted 2-aminothiophenes as A1 adenosine receptor
allosteric enhancers
Luigi Aurelio,a Heidi Figler,b Bernard L. Flynn,a Joel Lindenb and Peter J. Scammellsa,*
aDepartment of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, 381 Royal Parade,
Parkville VIC 3052, Australia
bDepartments of Medicine and Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
Received 2 May 2007; revised 17 October 2007; accepted 18 October 2007
Available online 23 October 2007
Abstract—Two series of 5-substituted 2-amino-4-(3-trifluoromethylphenyl)thiophenes were prepared and evaluated as allosteric
enhancers at the A1 adenosine receptor (A1AR). In the 3-benzoyl series, a 5-phenyl group was found to confer the greatest potency
(9a: ED50 = 2.1 lM, AE score = 18%). However, the analogue with no 5-substituent (6b: ED50 = 15.8 lM, AE score = 77%) proved
to be the most efficacious. In the 3-ethoxycarbonyl series, the 5-(4-chlorophenyl) analogue was clearly the most potent and effica-
cious (9l: ED50 = 6.6 lM, AE score = 57%). The antagonist activity of all compounds was measured using a [3H]CPX competitive
binding assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
therapeutic potential of the adenosine receptor system.
In this respect, allosteric modulators may prove clini-
cally valuable, with improved therapeutic indices owing
to their site- and event-selective actions.5 Allosteric
enhancers bind to an allosteric site, potentiating re-
sponses to agonist binding at the principle binding do-
main (or orthosteric site). Since adenosine production
is highest in ischaemic or hypoxic tissue, enhancers
which selectively amplify the actions of endogenous
adenosine would be selective for ischaemic tissue. Since
extracellular adenosine is very rapidly degraded to inac-
tive metabolites, within the circulation and interstitial
compartments, amplifying the effects of endogenous
adenosine would localize its actions to those cells under-
going ischaemic stress. Agents inducing such site-
and event-specific effects clearly offer a therapeutic
advantage.
Adenosine is an important endogenous tissue-protective
compound released during ischaemia, hypoxia or
inflammation which interacts with extracellular G-pro-
tein coupled receptors to regulate adenylyl cyclase, and
potassium and calcium ion channels.1 Four receptor
subtypes (A1, A2A, A2B and A3) have been defined based
on pharmacological properties and molecular cloning.
Considerable effort has been directed towards develop-
ing therapeutic agents targeting these receptors. Adeno-
sine (marketed as AdenocardTM) was approved for use in
the treatment of supraventricular tachycardia in the
early 1990s.2 A new synthetic A1 adenosine receptor
(A1AR) agonist, TecadenosonTM, is in clinical develop-
ment as an anti-arrhythmic agent,3 while the mixed
A1/A2AR agonist, AMP-579, reached phase II clinical
trials as a cardioprotective agent.4 Despite these ad-
vances, development of adenosine receptor agonists as
therapeutic agents has been limited by side-effects asso-
ciated with global adenosine receptor modulation and
the propensity of agonists to cause receptor desensitiza-
tion upon prolonged exposure. Such problems need to
be addressed in order to take advantage of the enormous
Routine screening for adenosine antagonists by Parke-
Davis identified a series of 2-amino-3-benzoylthiophenes
that enhanced agonist radioligand binding at A1ARs.6,7
The most effective enhancer in this series was PD81,723
(1) which proved highly selective for A1ARs, having no
major effect on agonist binding at the other adenosine
receptor subtypes or at the other G-protein coupled
receptors that were investigated (M2 muscarinic, a2
adrenergic or c-opiate receptors). The initial structure–
activity study performed by Parke-Davis established
that the amino and ketone groups were important in
Keywords: Allosteric enhancer (AE); A1 adenosine receptor (A1AR).
*
Corresponding author. Tel.: +61 3 9903 9542; fax: +61 3 9903
9582; e-mail: peter.scammells@vcp.monash.edu.au
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.065

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L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
maintaining good activity. More detailed structure–
activity relationships of 2-aminothiophene, 2-amino-
4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-6-
ing the preparation of a series of analogues. The 5-sub-
stituent was installed in the very first step of this
synthesis which is less than ideal for an investigation
of the SAR of the 5-position. For this study, an alterna-
tive synthetic approach was developed in which the two
protected 2-amino-5-bromo-4-phenylthiophenes 8a and
8b were employed as key intermediates for the incorpo-
ration of substituents in the 5-position via Suzuki–Miya-
benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine
have subsequently been reported.8–14
cores
ura cross-coupling reactions15 prior to
deprotection step (Scheme 1).
a
final
The thiophene core was formed via a two-step Gewald
synthesis16 in which 30-trifluoromethylacetophenone (4)
was initially reacted with either benzoylacetonitrile or
ethyl cyanoacetate, in the presence of titanium(IV)chlo-
ride17 to afford the corresponding Knoevenagel prod-
ucts 5a and 5b, respectively (Scheme 1). These olefins
were subsequently cyclised with sulfur under basic con-
ditions to yield the desired 2-aminothiophenes 6a and 6b
in 91% and 26% yield (over two steps), respectively. At-
tempts to brominate the 5-position of 6a or 6b directly,
without first protecting the 2-amino group, resulted in
degradation of the starting material to complex mixtures
(as evidenced by TLC). Accordingly, compounds 6a and
6b were conveniently Boc protected using Boc2O and
catalytic DMAP in dioxane. Heating was required to
complete the carbamoylation since no reaction occurred
at room temperature. The Boc protected 2-aminothi-
ophenes 7a and 7b were able to be smoothly converted
to the corresponding 5-bromo analogues 8a and 8b
using N-bromosuccinimide in yields of 43% and 57%
(over two steps), respectively. Derivatives 8a and 8b
were subjected to Suzuki–Miyaura cross-coupling con-
ditions in DMF/H2O mixtures using tribasic potassium
phosphate and catalytic Pd(PPh3)4 in an inert atmo-
sphere (N2) with heating (70–80 ꢁC) in a 24-h period
or alternatively under microwave irradiation. These con-
ditions provided adequate yields of the Boc protected
cross-coupled products after workup. In most cases
the cross-coupling reactions did not go to completion
even with prolonged reaction times and required the
We recently investigated the activity of a series of
2-amino-4,5-diphenylthiophenes and 2-amino-5-bromo-
4-phenylthiophenes as A1AR allosteric enhancers. This
work identified compounds that were effective AEs in
an in vitro assay that measured the test compounds’
ability to stabilize the agonist/A1AR/G-protein ternary
complex, such as [2-amino-5-bromo-4-(3-trifluorometh-
ylphenyl)thiophen-3-yl]phenyl methanone (2).12 2-Ami-
no-5-bromo-4-(3-nitrophenyl)thiophene-3-carboxylate
(3) was also found to slow the kinetics of A1AR agonist
dissociation indicating that an ethyl ester in the 3-posi-
tion can also support AE activity.12 Herein, we report
further investigation of the structure–activity relation-
ships of these classes of interesting lead compounds
which focus on the role of the 5-substituent.
2. Results and discussion
2.1. Chemistry
Our previous research on the AE activity of 2-amino-
4,5-diphenylthiophenes focused on the structure–activ-
ity relationships of the substituent in the 3-position. This
work employed a synthetic strategy in which this group
was incorporated late in the synthesis, thereby facilitat-
O
S
O
NC
R1
NH2
O
(i)
(ii)
F3C
F3C
F3C
R1
5a R1 = OEt
5b R1 = Ph
6a R1 = OEt
4
6b R1 = Ph
(iii)
S
R2
X
S
S
(v)-(vi)
(iv)
NHBoc
O
NH2
O
NHBoc
O
F3C
F3C
F3C
R1
R1
R1
7a R1 = OEt
7b R1 = Ph
8a X = Br, R1 = OEt
8b X = Br, R1 = Ph
9
Scheme 1. Reagents and conditions: (i) R1C(O)CH2CN, TiCl4, pyridine, CH2Cl2; (ii) sulfur, Et2NH, EtOH or THF; (iii) Boc2O, DMAP, dioxane;
(iv) NBS, AcOH/CH2Cl2; (v) method A: R2B(OH)2, 3 mol% Pd[P(Ph)3]4, K3PO4, DMF/H2O. Method B: R2B(OH)2, 3 mol% Pd(PPh3)2Cl2, K3PO4,
toluene/H2O, boronic acid, 150 ꢁC MW; (vi) TFA, CH2Cl2 or 6 M HCl, EtOH.

L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
1321
addition of more boronic acid and catalyst. The residues
obtained were filtered through a silica plug and the
resulting crude products were crystallised from MeOH
and MeOH/H2O. Boc deprotection was accomplished
by treating cross-coupled products with TFA, CH2Cl2
or 6 M HCl, EtOH mixtures. The crude isolated prod-
ucts 9a–n were purified by recrystallisation or column
chromatography. Overall yields for the coupling and
deprotection sequence ranged from 15% to 67%.
Although the final products 9a–n proved to be stable
upon standing for long periods when crystalline, varying
degrees of degradation were observed in CDCl3 and
DMSO-d6 solutions.
site that is independent of their allosteric activity. As a
result, the antagonist activity of the test compounds
was measured relative to [3H]CPX in a competitive bind-
ing assay since there is no allosteric effect on antagonist
radioligand binding.
Table 1 summarizes the effects of 5-substituted amino-
thiophenes. The influence of the substituent in the 5-po-
sition on AE activity was evaluated in two series, where
R1 is phenyl and ethoxy. In the first series (where
R1 = Ph), the analogue with no 5-substitutent (com-
pound 6b) proved to be the most effective allosteric en-
hancer with an AE score of 77%. A range of
substituted phenyl groups in the 5-position were also
evaluated and all of these compounds had modest max-
imal AE scores ranging from 5% to 24%. As observed
previously, efficacious compounds were not always
highly potent. In this series the 5-phenyl analogue 9a
has an ED50 value of 2.3 0.8 lM compared with
15.8 2.8 lM for the more efficacious compound 6b.
A slightly different route was used for the preparation of
compounds 2 and 11, as the acidic deprotection of the
intermediate 8b resulted in simultaneous removal of bro-
mine in the 5-position (providing 6b). Phthaloyl protec-
tion,18 bromination and then careful deprotection with
hydrazine hydrate at ambient temperature provided the
analogues 2 and 11 (Scheme 2). In the case of 5-chloro
analogue 11, there was no observed dehalogenation
when treating the intermediate phthaloylated thiophene
with hydrazine in excess in a single addition. Yet the
5-bromo derivative 2 could only ever be isolated cleanly
when careful addition of hydrazine was employed.
A contrasting situation was observed for the second
series comprised of ethyl 2-amino-4-(3-trifluoromethyl-
phenyl)thiophene-3-carboxylates (R1 = OEt). In this
series, the analogue with no substituent in the 5-position
(compound 6a) was less effective than all of the 5-phe-
nylthiophenes that were evaluated (compounds 9h–m).
The most efficacious and potent AE in this series was
compound 9k which possessed a 4-chlorophenyl group
in the 5-position (AE score = 57%, ED50 = 6.6 lM).
The other 5-phenyl compounds (9h–m) had only modest
AE scores ranging from 10% to 29%. Ethyl 2-amino-4-
(3-(trifluoromethyl)phenyl)thiophene-3-carboxylate (6a)
had a AE score of only 8%, while the 5-pyridyl analogue
9n was inactive at the test concentration.
2.2. Pharmacology
The activity of the target compounds as allosteric
enhancers of the human A1AR was evaluated in a ki-
netic assay measuring agonist dissociation. In this assay,
the A1AR agonist [125I]4-amino-3-iodo-N6-benzylade-
nosine ([125I]IABA) is incubated with membranes from
CHO-K1 cells stably expressing the hA1AR and the
ability of the candidate AE to stabilize the agonist–
A1AR–G-protein ternary complex is scored from 0 to
100 as the percentage of the ternary complex remaining
after 10 min of dissociation, initiated by the addition of
A1AR antagonist, CPX and GTPcS.14 A score of 0 cor-
responds to near complete dissociation of agonist radio-
ligand binding corresponding to the case when no AE is
added. A score a 100 would be assigned to an AE that
completely blocks radioligand dissociation. Dose–re-
sponse curves relating this score to enhancer concentra-
tion (0.1–50 lM) were fit by hyperbolic equations from
which the maximal score, or AE efficacy, was calculated
from the fit curve. In cases where the ED50 of the AE
was >10 lM, the AE efficacy could not be calculated
by curve fitting, and the maximum score was estimated
by the score at 10 lM AE. Some 2-aminothiophenes
have competitive antagonist activity at the orthosteric
Bromo substitution of the 5-position was more effective
than chloro (compare 2 and 11), though [2-amino-5-bro-
mo-4-(3-trifluoromethylphenyl)thiophen-3-yl]phenyl
methanone (2) had a lower AE score than observed in
our previous study. This compound was found to de-
grade relatively rapidly when in solution and the vari-
able results obtained in the AE assay may have
resulted from differing extents of degradation.
All of these compounds have significant activity as
orthosteric antagonists, although there was a wide vari-
ation in the ratio of AE activity to antagonist activity.
For example, compound 9n had an AE score of 0%
and showed 91% inhibition of [3H]CPX binding, while
compound 6b had an AE score of 77% and at 10 lM
showed 61% inhibition of [3H]CPX binding.
Scheme 2. Reagents and conditions: (i) phthalic anhydride, AcOH, reflux; (ii) X = Cl:NCS, AcOH/CH2Cl2; X = Br:NBS, AcOH/CH2Cl2; (iii)
NH2NH2ÆH2O, EtOH, rt.

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L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
Table 1.
R2
NH2
O
S
F3C
R1
Compound
R1
R2
AE scorea SEM
Enhancer ED50 (lM)
% Inhibitionb
6a
6b
9a
9b
9c
OEt
Ph
H
7.8 0.7
76.8 8.5
18.1 0.4
18.5 1.7
4.8 0.5
23.7 0.7
22.5 2.9
7.4 0.8
13.2 0.7
14.4 1.5
28.5 2.9
10.3 1.7
57.0 5.8
17.5 0.2
14.8 1.0
0
9.4 1.5
15.8 2.8
2.3 0.8
>10
93.0 3.2
61.1 2.0
82.4 2.2
33.1 2.0
25.6 6.8
34.8 1.4
59.1 0.4
65.2 1.6
83.6 1.7
68.9 0.5
64.6 1.8
71.4 21.1
58.4 2.3
81.5 3.8
48.5 4.6
91.0 0.1
87.1 2.6
47.5 0.9
18.8 2.5
H
Ph
Ph
Ph
3-AcPh
4-AcPh
4-NO2Ph
4-ClPh
4-MeOPh
Ph
Ph
Ph
>10
>10
9d
9e
Ph
Ph
>10
>10
9f
9g
9h
9i
OEt
OEt
OEt
OEt
OEt
OEt
OEt
OEt
Ph
>10
>10
3-AcPh
4-AcPh
4-NO2Ph
4-ClPh
4-MeOPh
4-CF3Ph
4-pyridyl
Cl
>10
>10
9j
9k
9l
6.6 1.9
>10
>10
9m
9n
11
2
>10
>10
22.2 1.8
53.5 2.2
28 1.1
Ph
Br
>10
13.6 2.1
PD (1)
a For compounds with an ED50 < 10 lM this value is based on curve fitting relating AE score to AE concentration using the equation
Score = ScoreMax [AE]/(ED50 + [AE]). Data points are means SEM, N = 2–3. For compounds with ED50 values >10 lM the score at 50 lM is
*
recorded.
b Orthosteric antagonist activity, % inhibition of specific [3H]CPX binding by 10 lM allosteric enhancer, N = 3.
In summary, two series of 5-substituted 2-amino-4-(3-
trifluoromethylphenyl)thiophenes were prepared and
evaluated as allosteric enhancers at the A1AR. The first
series of compounds possessed a benzoyl group in the 3-
position. In this series, a 5-phenyl group was found to
confer the greatest potency (9a: EC50 = 2.1 lM, AE
score = 18%). However, the analogue with no 5-substi-
tuent (6b: EC50 = 15.8 lM, AE score = 77%) proved to
be the most efficacious. The second series of compounds
possessed an ethyl ester in the 3-position. In this series,
the 5-(4-chlorophenyl) analogue was clearly the most
potent and efficacious (9k: EC50 = 6.6 lM, AE
score = 57%). These values compare favourably with
PD81,723 (1, EC50 of 13.6 lM, AE score = 28%), which
has commonly been used for benchmarking new alloste-
ric enhancers. All of the compounds that were evaluated
in this study showed greater antagonist activity than
PD81,723 in a [3H]CPX competitive binding assay.
3.1. Ethyl 2-amino-4-(3-(trifluoromethyl)phenyl)thio-
phene-3-carboxylate (6a)
Compound 4 (0.8 mL, 5.32 mmol) and ethyl cyanoace-
tate (0.77 mL, 6.42 mmol) were dissolved in dry CH2Cl2
(25 mL) in a three-necked flask, fitted with a rubber sep-
tum and flushed with N2 gas. The solution was cooled to
0 ꢁC with an ice bath and neat TiCl4 (1.17 mL,
10.64 mmol) was added in a dropwise fashion. Pyridine
(360 lL) was added after 10 min. After the addition was
complete, the ice bath was removed and the solution left
to stir at room temperature. After 30 min, another ali-
quot of pyridine (1.08 mL) was added dropwise and stir-
ring was continued overnight. The mixture was diluted
with EtOAc (150 mL) and washed with 2 M HCl (2·
100 mL), H2O (200 mL) and finally with brine. The or-
ganic layer was dried (MgSO4), filtered and evaporated
to provide 1.49 g of yellow resin that slowly solidified.
The resin was taken up in dry THF (10 mL) and elemen-
tal sulfur (200 mg) was added followed by N,N-diethyl-
amine (2 mL) and the solution was stirred at room
temperature for 1.5 h. The reaction mixture was then di-
luted with water and extracted with CH2Cl2 (3· 50 mL).
The combined organic extracts were washed with water
and then brine, dried (MgSO4), filtered and evaporated
to dryness. The crude product was chromatographed
on silica gel eluting with EtOAc/pet ether (15:85), pro-
viding a pale yellow resin that solidified upon standing
3. Experimental
Melting points were determined with an Electrothermal
melting point apparatus and are uncorrected. All NMR
spectra were recorded on a Bruker Avance DPX 300
1
spectrometer. H and 13C NMR spectra were recorded
at 300.13 and 75.4 MHz, respectively, and, 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 lm, 70–230 mesh).
1
(1.52 g, 91%). Mp 65–66 ꢁC. H NMR d 7.58–7.40 (m,
4H, ArH), 6.14 (br s, 2H, NH2), 6.09 (s, 1H, 5-H),
4.04 (q, J = 7.2 Hz, 2H, CH2CH3), 0.89 (t, J = 7.2 Hz,
3H, CH2CH3). MS (APCI): m/z = 316.2 (100%)

L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
1323
(M+H)+. HR-MS (ESI) Calcd for C14H13F3NO2S+:
(M+1) 316.0614. Found: 316.0611.
trated to a solid, that was recrystallised from MeOH/
H2O (95:5) to afford a white solid (2.2 g, 96%). 1H
NMR d 10.43 (br s, 1H, NH), 7.63–7.61 (m, 1H,
ArH), 7.53–7.48 (m, 2H, ArH), 7.41–7.39 (m, 1H,
ArH), 3.95 (q, J = 7.2 Hz, 2H, CH2CH3), 1.49 (br s,
9H, C(CH3)3), 0.78 (t, J = 7.2 Hz, 3H, CH2CH3). MS
(APCI): m/z = 492.0 (48%), 494.0 (100%) (MꢁH)ꢁ.
HR-MS (ESI) Calcd for C19H19BrF3NNaO4S+:
(M+Na) 516.0062. Found: 516.0070.
3.2. [2-Amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-
yl]phenyl methanone (6b)
Compound 4 (3 mL, 3.71 g, 19.69 mmol) and benzoyl-
acetonitrile (3.00 g, 20.67 mmol) were dissolved in dry
CH2Cl2 (75 mL) in a three-necked flask, fitted with a
rubber septum and flushed with N2 gas. The solution
was cooled to 0 ꢁC with an ice bath and neat TiCl4
(4.6 mL, 42.27 mmol) was added in a dropwise fashion.
Pyridine (1.41 mL) was added after 10 min. After the
addition was complete, the ice bath was removed and
the solution left to stir at room temperature. A further
aliquot of pyridine (4.23 mL) was added after 1 h and
the reaction mixture was stirred overnight. The reaction
mixture was diluted with EtOAc (250 mL) and washed
with 2 M HCl (2· 200 mL), H2O (300 mL) and finally
with brine. The organic layer was dried (MgSO4), fil-
tered and evaporated to provide an orange resin
(7.42 g). This resin was dissolved in dry THF (40 mL),
elemental sulfur (667 mg) and N,N-diethylamine
(4 mL) were added and the reaction mixture was stirred
at room temperature overnight. The solution was di-
luted with water and extracted with CH2Cl2 (3·
100 mL). The combined organic extracts were washed
with water and then brine, dried (MgSO4), filtered and
concentration to a red/brown residue (8.03 g). The crude
product was chromatographed on silica gel eluting with
EtOAc/pet ether (20:80), supplying an orange resin that
was triturated with sec-butanol/H2O (80:20) to afford
orange crystals (1.81 g, 26%). Mp 135–138 ꢁC. 1H
NMR d 7.29–7.26 (m, 2H, ArH), 7.21–7.18 (m, 3H,
ArH), 7.13–7.06 (m, 2H, ArH), 7.00–6.95 (m, 2H,
ArH), 6.6 (br s, 2H, NH2), 6.21 (br s, 1H, 5-H). MS
(APCI): m/z = 348.1 (100%) (M+H)+. HR-MS (ESI)
Calcd for C18H13F3NOS+: (M+1) 348.0664. Found:
348.0660.
3.4. [5-Bromo-2-(tert-butoxycarbonylamino)-4-(3-(tri-
fluoromethyl)phenyl)thiophen-3-yl]phenyl methanone (8b)
Compound 6b (1.00 g, 2.88 mmol) was dissolved in diox-
ane (20 mL) and Boc2O (0.66 g, 3.02 mmol) was added to
the solution followed by DMAP (35 mg, 10 mol%). The
homogeneous solution was heated on an oil bath at 70–
80 ꢁC for 2 h. After cooling, the solution was diluted with
ether and washed with water (2·) and then brine. The or-
ganic layer was dried (MgSO4), filtered from insoluble
material and evaporated to give an orange oil (1.84 g).
This oil was chromatographed on silica gel column using
EtOAc/pet ether (5:95) as an eluent to afford a pale yel-
low resin (0.75 g, 58%). The resin (494 mg, 1.10 mmol)
was dissolved in 7 mL of CH2Cl2/AcOH (1:1) and cooled
in an ice salt bath (ꢀꢁ16 to ꢁ18 ꢁC). NBS (216 mg,
1.21 mmol) was added and the reaction mixture was stir-
red for 0.5 h on an ice/salt bath. The solution was diluted
with water (100 mL) and extracted with ether. The or-
ganic phase was washed with saturated NaHCO3 solu-
tion (until gas evolution ceases) and finally with brine.
The organic layer was dried (MgSO4), filtered from insol-
uble material and concentrated to afford a pale yellow
solid. This solid was recrystallised from MeOH/H2O
1
(95:5) resulting in a white solid (0.58 g, 99%). H NMR
d 10.55 (br s, 1H, NH), 7.26–7.22 (m, 5H, ArH),
7.18–7.12 (m, 2H, ArH), 7.02–6.97 (m, 2H, ArH), 1.53
(br s, 9H, C(CH3)3). HR-MS (ESI) Calcd for
C23H19BrF3NNaO3S+: (M+Na) 548.0113. Found:
548.0120.
3.3. Ethyl 5-bromo-2-(tert-butoxycarbonylamino)-4-(3-
(trifluoromethyl)phenyl)thiophene-3-carboxylate (8a)
3.5. General procedure for the preparation of compounds
(9a–n)
Compound 6a (3.84 g, 12.18 mmol) was dissolved in
dioxane (45 mL) and Boc2O (2.79 g, 12.79 mmol) was
added to the solution followed by DMAP (148 mg,
10 mol%). The homogeneous solution was heated on
an oil bath at 70–80 ꢁC for 2 h. After cooling, the solu-
tion was diluted with ether and washed with water (2·)
and then brine. The organic layer was dried (MgSO4),
filtered and evaporated to give a reddish brown oil
(4.9 g). This oil was chromatographed on silica gel col-
umn using EtOAc/pet ether (5:95) as an eluent to afford
a clear colourless resin (2.27 g, 45%). The resin (1.93 g,
4.65 mmol) was dissolved in 25 mL of CH2Cl2/AcOH
(1:1) and cooled in an ice salt bath (ꢀꢁ16 to ꢁ18 ꢁC).
NBS (0.91 g, 5.11 mmol) was added and the reaction
mixture was stirred for 0.5 h on an ice/salt bath. The
solution was diluted with water (200 mL) and extracted
with ether. The organic phase was washed with satu-
rated NaHCO3 solution (until gas evolution ceased)
and finally with brine. The organic layer was dried
(MgSO4), filtered from insoluble material and concen-
3.5.1. Method A. Compound 8a or 8b (100 mg) was dis-
solved in 4:1 DMF/2 M K3PO4 (1 mL, previously soni-
cated for 30 min) under an atmosphere of N2. The
appropriate boronic acid (1.2 equiv) and Pd(PPh3)4
(6.6. mg, 5.70 lmol, 3 mol%) were added and the reac-
tion mixture was heated at 70–80 ꢁC for 24 h. The solu-
tion was cooled and diluted with EtOAc (20 mL) and
washed with water. The aqueous layer was extracted
with EtOAc and the combined organics were washed
with water (3·) and finally with brine. The organic layer
was dried (MgSO4), filtered from insoluble material and
concentrated to a residue. The residue was filtered
through a silica gel plug eluting with CHCl3 and concen-
trated to a residue which was subjected to silica gel
chromatography.
3.5.2. Method B. Compound 8a or 8b (200 mg) was dis-
solved in 4:1 DMF/2 M K3PO4 (2 mL, previously soni-
cated for 30 min) in a sealed vial (microwave pressure

1324
L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
tube). The appropriate boronic acid (1.2 equiv) and
Pd(PPh3)2Cl2 (8.0 mg, 11.40 lmol, 3 mol%) were added
and the mixture was heated with stirring to 150 ꢁC for
15 min. If the reaction was incomplete (by TLC), a fur-
ther equivalent of boronic acid and 3 mol% of catalyst
were added and microwave heating was continued for
10 min at 150 ꢁC. The solution was cooled and the tolu-
ene layer was filtered through a silica gel plug eluting
with EtOAc and finally chloroform and concentrated
to a residue.
(s, 3H, Ac), 1.90 (br s, 2H, NH2). HR-MS (ESI) Calcd
for C26H19F3NO2S+: (M+1) 466.1083. Found: 466.1081.
3.5.2.4. [2-Amino-5-(4-nitrophenyl)-4-(3-trifluorometh-
ylphenyl)thiophen-3-yl]phenyl methanone (9d). Method A:
The residue obtained was dissolved in a minimum of
MeOH and crystallised upon the addition of water.
The resultant solid was filtered and washed with H2O
to provide a yellow powder (75 mg, 69%). This cross-
coupled product (50 mg, 87.94 lmol) was dissolved in
CH2Cl2 (1 mL), TFA (1 mL) was added and the reaction
mixture was stirred at room temperature. After 2 h, the
mixture was diluted with CHCl3 (10 mL) and concen-
trated to a solid that was filtered and washed with water
(43 mg). The solid was chromatographed on silica gel
eluting with EtOAc/pet ether (20:80) to afford a semi-so-
lid that was triturated with 80% MeOH/H2O to give an
3.5.2.1. [2-Amino-5-phenyl-4-(3-trifluoromethylphenyl)-
thiophen-3-yl]phenyl methanone (9a). Method B: The res-
idue obtained was sufficiently pure for the subsequent
deprotection step. It was dissolved in CH2Cl2 (1 mL),
TFA (1 mL) was added and the reaction mixture was
stirred at room temperature. After 3 h the mixture was
diluted with CHCl3 (10 mL) and concentrated to give
a reddish brown residue. The residue was chromato-
graphed on a silica gel column eluting with EtOAc/pet
ether (15:85) to provide a light yellow resin which slowly
crystallised upon standing at 4 ꢁC. Complete crystallisa-
tion was induced by sonicating in a minimum of metha-
nol to afford a yellow powder (74 mg, 45%). Mp 86–
1
orange solid (22 mg, 41%). Mp 221–229 ꢁC. H NMR d
8.01 (d, J = 8.7 Hz, 2H, ArH), 7.19–7.17 (m, 3H, ArH),
7.12–7.04 (m, 6H, ArH), 6.98–6.93 (m, 2H, ArH), 2.31
(br s, 2H, NH2). HR-MS (ESI) Calcd for
C24H16F3N2O3S+: (M+1) 469.0828. Found: 469.0828.
3.5.2.5. [2-Amino-5-(4-chlorophenyl)-4-(3-trifluoromethyl-
phenyl)thiophen-3-yl]phenyl methanone (9e). Method A:
The residue obtained was crystallised from MeOH.
The solid was filtered and washed with ice-cold MeOH
to provide a tan coloured powder (51 mg, 48%). This
cross-coupled product (46 mg, 82.44 lmol) was dis-
solved in CH2Cl2 (1 mL), TFA (200 lL) was added
and the reaction mixture was stirred at room tempera-
ture. After 1.5 h, the mixture was diluted with CHCl3
(10 mL) and evaporated to give a reddish brown residue.
The residue was triturated with a minimum amount of
ice-cold MeOH and the resultant solid was filtered and
washed with MeOH/H2O (60:40) to afford a yellow pow-
der (31 mg, 82%). Mp 185–194 ꢁC. 1H NMR d 7.20–6.90
(m, 13H, ArH), 5.21 (br s, 2H, NH2). MS (APCI): m/z =
60.0 (40%), 458.0 (100%) (M+H)+. HR-MS (ESI)
Calcd for C24H16ClF3NOS+: (M+1) 458.0588. Found:
458.0591.
1
89 ꢁC. H NMR d 7.23–6.93 (m, 14H, ArH), 3.15 (br
s, 2H, NH2). HR-MS (ESI) Calcd for C24H17F3NOS+:
(M+1) 424.0983. Found: 424.0982.
3.5.2.2. [5-(3-Acetylphenyl)-2-amino-4-(3-trifluoromethyl-
phenyl)thiophen-3-yl]phenyl methanone (9b). Method A:
The crude product was chromatographed on silica gel
eluting with EtOAc/pet ether (5:95) to provide 40 mg
of clear pale yellow resin that was triturated with
MeOH/water. The resultant solid was filtered and
washed with H2O providing pale yellow powder
(38 mg, 35%). This cross-coupled product (30 mg,
53.04 lmol) was dissolved in CH2Cl2 (1 mL) and TFA
(400 lL) was added at room temperature. After 1.5 h,
the mixture was diluted with CHCl3 (10 mL), evapo-
rated and chromatographed on silica gel eluting with
CHCl3 to provide 26 mg of yellow resin. The resin was
slowly crystallised from MeOH with careful addition
of water to give a pale brown powder (15 mg, 61%).
3.5.2.6. [2-Amino-5-(4-methoxyphenyl)-4-(3-trifluoro-
methylphenyl)thiophen-3-yl]phenyl methanone (9f). Meth-
od B: The residue obtained was sufficiently pure for the
next step. It was dissolved in CH2Cl2 (1 mL), TFA
(1 mL) was added and the reaction mixture was stirred
at room temperature. After 3 h, the mixture was diluted
with CHCl3 (10 mL) and evaporated to give a reddish
brown residue. Chromatography on a silica gel column,
eluting with 15:85 EtOAc/pet ether (15:85), yielded a
light yellow resin. This material was crystallised by son-
icating in a minimum of methanol to afford a yellow
1
Mp 160–164 ꢁC. H NMR d 7.77–7.74 (m, 1H, ArH),
7.56 (m, 1H, ArH), 7.27–6.95 (m, 11H, ArH), 2.35 (s,
3H, Ac), 1.88 (br s, 2H, NH2). HR-MS (ESI) Calcd
for C26H19F3NO2S+: (M+1) 466.1083. Found: 466.1081.
3.5.2.3. [5-(4-Acetylphenyl)-2-amino-4-(3-trifluoromethyl-
phenyl)thiophen-3-yl]phenyl methanone (9c). Method A:
The residue obtained was chromatographed on silica
gel eluting with EtOAc/pet ether (5:95) to provide a clear
pale yellow resin that was crystallised from MeOH/H2O
(90:10). The solid obtained was filtered and washed with
H2O to give a pale yellow powder (54 mg, 50%). This
cross-coupled product (40 mg, 70.72 lmol) was dis-
solved in CH2Cl2 (1 mL), TFA (300 lL) was added
and the reaction mixture was stirred at room tempera-
ture. After 2 h the mixture was diluted with CHCl3
(10 mL), evaporated and chromatographed on silica
gel eluting with CHCl3 providing a yellow powder
(19 mg, 58%). Mp 190–196 ꢁC. 1H NMR d 7.74 (d,
J = 8.4 Hz, 2H, ArH), 7.20–6.91 (m, 11H, ArH), 2.52
1
powder (36 mg, 22%). Mp 68–71 ꢁC. H NMR d 7.21–
7.19 (m, 2H, ArH), 7.09–6.91 (m, 9H, ArH), 6.77–6.72
(m, 2H, ArH), 3.74 (br s, 3H, CH3O) 2.25 (br s, 2H,
NH2). HR-MS (ESI) Calcd for C25H19F3NO2S+:
(M+1) 454.1089. Found: 454.1090.
3.5.2.7. Ethyl 2-amino-5-phenyl-4-(3-trifluoromethyl-
phenyl)thiophene-3-carboxylate (9g). Method B: The res-
idue obtained was sufficiently pure for the next step. It
was dissolved in CH2Cl2 (1 mL), TFA (1 mL) was added

L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
1325
and the reaction was stirred at room temperature. After
3 h, the mixture was diluted with CHCl3 (10 mL) and
evaporated to give a reddish brown residue. This residue
was chromatographed on a silica gel column eluting
with EtOAc/pet ether (15:85) to provide a pink resin
(141 mg), which solidified upon standing. Complete
crystallisation was induced by sonicating in 50% aque-
ous methanol to yield a pink powder (76 mg, 48%).
Mp 113–116 ꢁC. 1H NMR d 7.53 (br s, 2H, ArH),
7.38–7.30 (m, 2H, ArH), 7.20–6.95 (m, 5H, ArH), 4.00
(br s, 2H, NH2), 3.95 (q, J = 7.2 Hz, 2H, CH2CH3),
0.80 (t, J = 7.2 Hz, 3H, CH2CH3). HR-MS (ESI) Calcd
for C20H17F3NO2S+: (M+1) 392.0932. Found: 392.0929.
solved in CH2Cl2 (1 mL), TFA (200 lL) was added
and the reaction mixture was stirred at room tempera-
ture. After 3 h, the mixture was diluted with CHCl3
(10 mL) and concentrated to a reddish brown residue.
The residue was chromatographed on a silica gel column
eluting with EtOAc/pet ether (15:85), providing a red-
dish brown resin (82 mg). The resin was crystallised
from MeOH/water to afford a reddish brown powder
(33 mg, 54%). Mp 178–184 ꢁC. 1H NMR d 7.97 (d,
J = 8.7 Hz, 2H, ArH), 7.60 (d, J = 7.8 Hz, 1H, ArH),
7.53 (s, 1H, ArH), 7.42 (t, J = 7.7 Hz, 1H, ArH), 7.32
(d, J = 7.5 Hz, 1H, ArH), 7.08 (d, J = 8.7 Hz, 2H,
ArH), 3.95 (q, J = 7.2 Hz, 2H, CH2CH3), 0.79 (t,
J = 7.2 Hz, 3H, CH2CH3). HR-MS (ESI) Calcd for
C20H16F3N2O4S+: (M+1) 437.0777. Found: 437.0781.
3.5.2.8. Ethyl 5-(3-acetylphenyl)-2-amino-4-(3-trifluo-
romethylphenyl)thiophene-3-carboxylate (9h). Method A:
The residue obtained was crystallised from MeOH. The
solid was filtered and washed with ice-cold MeOH to
give an off-white powder (75 mg, 69%). This cross-cou-
pled product (75 mg, 140.57 lmol) was dissolved in
CH2Cl2 (1 mL), TFA (200 lL) was added and the reac-
tion mixture was stirred at room temperature. After 3 h,
the mixture was diluted with CHCl3 (10 mL) and con-
centrated to a brown/red residue. The residue was chro-
matographed on a silica gel column eluting with EtOAc/
pet ether (15:85) to provide a brown semi-solid (35 mg).
The solid was dissolved in a minimum of MeOH and
precipitated by the addition of water, yielding an off-
3.5.2.11. Ethyl 2-amino-5-(4-chlorophenyl)-4-(3-tri-
fluoromethylphenyl)thiophene-3-carboxylate (9k). Meth-
od A: The residue obtained was crystallised from
MeOH. The solid was filtered and washed with ice-cold
MeOH to give a white powder (102 mg, 96%). This
cross-coupled product (100 mg, 190.13 lmol) was dis-
solved in CH2Cl2 (1 mL), TFA (1 mL) was added and
the reaction mixture was stirred at room temperature.
After 3 h, the mixture was diluted with CHCl3 (10 mL)
and concentrated to a reddish brown residue. The resi-
due was chromatographed on a silica gel column eluting
with EtOAc/pet ether (10:90) to give a pink resin
(70 mg). The resin was recrystallised from EtOH/water
to afford a pink powder (57 mg, 70%). Mp 127–
1
white powder (33 mg, 54%). Mp 164–170 ꢁC. H NMR
d 7.70 (m, 1H, ArH), 7.56–7.54 (m, 3H, ArH), 7.41–
7.31 (m, 2H, ArH), 7.26–7.23 (m, 2H, ArH), 4.12 (br
s, 2H, NH2), 3.95 (q, J = 7.2 Hz, 2H, CH2CH3), 2.32
(s, 3H, Ac), 0.79 (t, J = 7.2 Hz, 3H, CH2CH3). HR-
MS (ESI) Calcd for C22H19F3NO3S+: (M+1) 434.1032.
Found: 434.1031.
1
129 ꢁC. H NMR d 7.55–7.48 (m, 2H, ArH), 7.39–7.26
(m, 2H, ArH), 7.16–6.91 (m, 4H, ArH), 3.94 (q,
J = 7.2 Hz, 2H, CH2CH3), 0.78 (t, J = 7.2 Hz, 3H,
CH2CH3). HR-MS (ESI) Calcd for C20H16ClF3NO2S+:
(M+1) 426.0537. Found: 426.0552.
3.5.2.9. Ethyl 5-(4-acetylphenyl)-2-amino-4-(3-trifluo-
romethylphenyl)thiophene-3-carboxylate (9i). Method A:
The residue obtained was crystallised from MeOH.
The solid was filtered and washed with ice-cold MeOH
to give a peach coloured powder (100 mg, 93%). This
cross-coupled product (100 mg, 187.42 lmol) was dis-
solved in CH2Cl2 (1 mL), TFA (400 lL) was added
and the reaction mixture was stirred at room tempera-
ture. After 2 h, the mixture was diluted with CHCl3
(10 mL) and concentrated to a black/green residue.
The residue was chromatographed on a silica gel column
eluting with CHCl3, providing a yellow resin (54 mg)
that solidified upon standing. The solid was recrystal-
lised from MeOH to afford a yellow powder (13.5 mg,
3.5.2.12. Ethyl 2-amino-5-(4-methoxyphenyl)-4-(3-tri-
fluoromethylphenyl)thiophene-3-carboxylate (9l). Method
B: The residue obtained was sufficiently pure for the
next step. It was dissolved in CH2Cl2 (1 mL), TFA
(1 mL) was added and the reaction mixture was stirred
at room temperature. After 3 h, the mixture was diluted
with CHCl3 (10 mL) and concentrated to a reddish
brown residue. The residue was chromatographed on a
silica gel column eluting with EtOAc/pet ether (15:85)
to provide a light purple resin which slowly solidified
upon standing. Complete crystallisation was induced
by sonication in 50% aqueous methanol, giving a light
purple powder (91 mg, 49%). Mp 112–116 ꢁC. 1H
NMR d 7.51–7.49 (m, 2H, ArH), 7.37–7.27 (m, 2H,
ArH), 7.10–6.65 (m, 4H, ArH), 3.94 (q, J = 7.2 Hz,
2H, CH2CH3), 3.73 (br s, 3H, CH3O) 0.79 (t,
J = 7.2 Hz, 3H, CH2CH3). HR-MS (ESI) Calcd for
C21H19F3NO3S+: (M+1) 422.1038. Found: 422.1031.
1
16%). Mp 167–170 ꢁC. H NMR d 7.71 (d, J = 8.4 Hz,
2H, ArH), 7.58–7.53 (m, 2H, ArH), 7.41–7.30 (m, 2H,
ArH), 7.05 (d, J = 8.4 Hz, 2H, ArH), 3.93 (q,
J = 7.2 Hz, 2H, CH2CH3), 2.51 (s, 3H, Ac), 0.79 (t,
J = 7.2 Hz, 3H, CH2CH3). HR-MS (ESI) Calcd for
C22H19F3NO3S+: (M+1) 434.1032. Found: 434.1030.
3.5.2.13. [2-Amino-4-(3-trifluoromethylphenyl)-5-(4-tri-
fluoromethylphenyl)thiophen-3-yl]phenyl methanone (9m).
Method A: The residue obtained was crystallised from
MeOH. The solid was filtered and washed with ice-cold
MeOH to give a light brown powder (100 mg, 88%).
This cross-coupled product (100 mg, 178.73 lmol) was
dissolved in CH2Cl2 (1 mL), TFA (400 lL) was added
and the reaction mixture was stirred at room tempera-
3.5.2.10. Ethyl 2-amino-5-(4-nitrophenyl)-4-(3-trifluo-
romethylphenyl)thiophene-3-carboxylate (9j). Method A:
The residue obtained was crystallised from MeOH.
The solid was filtered and washed with ice-cold MeOH
to give a brownish yellow powder (75 mg, 69%). This
cross-coupled product (75 mg, 139.79 lmol) was dis-

1326
L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
ture. After 2 h, the mixture was diluted with CHCl3
(10 mL) and concentrated to a reddish brown residue.
The residue was chromatographed on a silica gel column
eluting with CHCl3, providing a brown solid (55 mg).
The solid was triturated with a minimum of ice-cold
80% MeOH/H2O to yield a brown powder (35 mg,
was concentrated and chromatographed on silica gel
eluting with CHCl3 to afford a yellow resin (60 mg).
This material was dissolved in a minimum of MeOH
and precipitated with water (21 mg, 20%). Mp 139–
141 ꢁC. H NMR d 7.24–7.06 (m, 9H, ArH and NH2),
6.99–6.93 (m, 2H, ArH). HR-MS (ESI) Calcd for
C18H12ClF3NOS+: (M+1) 382.0275. Found: 382.0276.
1
1
43%). Mp 135–140 ꢁC. H NMR d 7.57–7.53 (m, 2H,
ArH), 7.41–7.36 (m, 3H, ArH), 7.32–7.29 (m, 1H,
ArH), 7.08 (d, J = 8.1 Hz, 2H, ArH), 3.94 (q,
J = 7.2 Hz, 2H, CH2CH3), 2.04 (br s, 2H, NH2), 0.79
(t, J = 7.2 Hz, 3H, CH2CH3). HR-MS (ESI) Calcd for
C21H16F6NO2S+: (M+1) 460.0800. Found: 460.0800.
3.7. [2-Amino-5-bromo-4-(3-(trifluoromethyl)phenyl)thio-
phen-3-yl]phenyl methanone (2)
Compound 10 (60 mg, 0.13 mmol) was dissolved in
50:50 CH2Cl2/AcOH (1 mL) and cooled to 0 ꢁC with
ice bath. NBS (27 mg, 0.15 mmol) was added and the
reaction mixture was stirred at room temperature for
1.5 h. The solution was concentrated to a residue
and then taken up in EtOAc. The EtOAc solution
was washed with 5% sodium bicarbonate solution
(2·), water and brine. The organic layer was dried
(MgSO4), filtered and concentrated to give a pale yel-
low solid (65 mg, 93%). HR-MS (ESI) Calcd for
C26H14BrF3NO3S+: (M+1) 555.9824. Found: 555.9824.
The crude product (50 mg, 0.09 mmol) was dissolved
in 0.5 mL of EtOH and vigorously stirred at room tem-
perature. Hydrazine hydrate (6.0 lL, 0.124 mmol) was
added slowly and dropwise. After stirring at room tem-
perature for 20 min, dioxane (0.5 mL) was added to sol-
ubilise all materials and was left to stir overnight. The
next day a further 5 lL of hydrazine hydrate was added
and after 1 min a precipitate formed. The solution was
immediately concentrated and the residue obtained
was filtered through a silica gel plug eluting with 50:50
EtOAc/pet ether to provide a pale yellow resin that
solidifies upon standing at 4 ꢁC. Complete crystallisa-
tion was induced by dissolving in a minimum of MeOH
and adding water to afford a green solid (37 mg, 96%).
3.5.2.14. Ethyl 2-amino-5-(4-pyridyl)-4-(3-trifluoro-
methylphenyl)thiophene-3-carboxylate (9n). Method A:
The residue obtained was chromatographed on silica
gel eluting with EtOAc/pet ether (15:85) to give a clear
colourless resin (32 mg). The resin was crystallised by
dissolving in MeOH and precipitating with water. The
solid was washed with water to provide a white powder
(26 mg, 26%). This cross-coupled product (25 mg,
50.76 lmol) was dissolved in EtOH (1 mL), 6 M HCl
(200 lL) was added and the reaction mixture was stirred
at room temperature. After 2 h, the mixture was heated
on an oil bath at 50–60 ꢁC for 24 h. The solution was
concentrated to a green/yellow solid. The solid was
recrystallised from EtOH/ether to afford a green solid
as the hydrochloride salt (21 mg, 96%). Mp 164–
1
170 ꢁC. H NMR (DMSO) d 8.44 (d, J = 6.0 Hz, 2H,
ArH), 8.36 (br s, 2H, NH2), 7.84 (d, J = 7.5 Hz, 1H,
ArH), 7.71–7.66 (m, 2H, ArH), 7.56 (d, J = 7.5 Hz,
1H, ArH), 7.11 (d, J = 6.3 Hz, 2H, ArH), 3.86 (m, 2H,
CH2CH3), 0.72 (t, J = 7.2 Hz, 3H, CH2CH3). HR-MS
(ESI) Calcd for C19H16F3N2O2S+: (M+1) 393.0879.
Found: 393.0868.
1
3.6. [2-Amino-5-chloro-4-(3-(trifluoromethyl)phenyl)thio-
phen-3-yl]phenyl methanone (11)
Mp 122–124 ꢁC. H NMR d 7.25–7.06 (m, 7H, ArH),
6.98–6.93 (m, 2H, ArH), 6.35 (br s, 2H, NH2). MS
(APCI): m/z = 426.0 (90%), 428.0 (100%) (M+H)+.
HR-MS (ESI) Calcd for C18H12BrF3NOS+: (M+1)
425.9775. Found: 425.9772.
Compound 6b (0.65 mg, 1.86 mmol) and phthalic anhy-
dride (0.33 g, 2.23 mmol) were dissolved in 17 mL of gla-
cial acetic and refluxed overnight. The solution was
concentrated and the resultant solid was recrystallised
from sec-butanol/H2O (80:20) to give compound 10 as
a pale yellow solid (661 mg). This crude product
(200 mg, 0.42 mmol) was dissolved in 50:50 CHCl3/
AcOH (4 mL) and NCS (67 mg, 0.50 mmol) was added.
After 10 min, DMF (0.5 mL) was also added to solubi-
lise all solids. The homogeneous solution was stirred
at room temperature for 3 h. An additional portion of
NCS (67 mg, 0.50 mmol) was added and the reaction
mixture was stirred overnight. The solution was diluted
with water and extracted with EtOAc. The organic
phase was washed with dilute sodium bicarbonate solu-
tion and then water. The organic layer was dried
(MgSO4), filtered and concentrated to a yellow resin.
The resin was filtered through a silica gel plug eluting
with diethyl ether to give a pale yellow foam that was
crystallised from MeOH (142 mg, 66%). The resultant
solid (140 mg, 0.27 mmol) was dissolved in 2 mL of
dioxane/EtOH (1:1) and vigorously stirred at room tem-
perature. Hydrazine hydrate (18.6 lL, 0.38 mmol) was
added and stirring was continued for 2.5 h. The mixture
3.8. Assay of AE activity
The assay of AE activity consisted of three phases: (1)
binding to equilibrium of the agonist, [125I]IABA, to
the A1AR–G-protein ternary complex; (2) stabilization
of that complex by adding vehicle or AE for 30 min
and (3) dissociation of the complex by adding a combi-
nation of an A1AR antagonist, 100 lM BW-1433, and
25 lM GTPcS for 10 min. Compounds were scored be-
tween 0% (no different than AE vehicle) and 100% (com-
plete abolition of [125I]IABA dissociation). The assay
employed membranes from CHO-K1 cells stably
expressing the hA1AR. For agonist binding to equilib-
rium (phase 1) the buffer consisted of 10 mM Hepes,
pH 7.2, containing 0.5 mM MgCl2, 1 U/mL adenosine
deaminase, 0.5 nM [125I]IABA and 10 lg of membrane
protein in a final volume of 100 lL applied to 96-well
Millipore GF/C glass fibre filter plates. After 90 min at
room temperature the addition of 50 lL of AE (0.1–
50 lM, final) initiated stabilization of the ternary com-
plex (phase 2). Thirty minutes later 50 lL solution con-

L. Aurelio et al. / Bioorg. Med. Chem. 16 (2008) 1319–1327
1327
taining BW-1433 and GTPcS was added to initiate the
References and notes
dissociation of the ternary complex. Ten minutes later
membranes were filtered, washed, dried and counted
for residual [125I]IABA. The percentage of specifically
bound agonist remaining after 10 min of dissociation
served as an index of AE activity:
1. Jacobson, K. A.; Gao, Z.-G. Nat. Rev. Drug Disc. 2006, 5,
247–264.
2. Lerman, B. B.; Belardinelli, L. Circulation 1991, 83, 1499–
1509.
3. Sorbera, L. A.; Castaner, J.; Martin, L.; Bayes, M. Drugs
Future 2002, 27, 846–849.
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% AE score ¼ 100 ꢂ ðB ꢁ BoÞ=ðBeq ꢁ BoÞ
where B is the residual binding (cpm) bound at the
end of 10 min of dissociation in the presence of an
AE; Bo is the residual binding (cpm) at the end of
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means no dissociation and a score of zero means com-
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3.9. Assay of A1R antagonist activity
CHO-K1 membranes expressing human A1 adenosine
receptors were resuspended at 400 lg/mL in HE buffer
containing 1 U/mL adenosine deaminase. Fifty microli-
tres of membrane solution was added to 50 lL HE buf-
fer containing [3H] CPX (2 nM). One hundred
microlitres of HE buffer with either vehicle, enhancer
or NECA (to define non-specific) was added. The final
drug concentrations were 10 lM for enhancer and
100 lM for NECA. Samples were incubated for
90 min at room temperature, filtered and counted on a
liquid scintillation counter. Binding was performed in
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group, even when CsF was employed as the base under
anhydrous conditions.
Acknowledgments
The authors wish to thank the Australian Research
Council (Discovery Grant DP0558184) and NIH R01
HL56111 for financial support.