3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1 adenosine receptor allosteric modulators and antagonists

Article abstract of DOI:10.1016/j.bmc.2009.08.024

A series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were prepared and evaluated as potential allosteric modulators at the A₁ adenosine receptor. The structure-activity relationships of the 3- and 6-positions of a series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were explored. Despite finding that 3- and 6-substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines possess the ability to recognize an allosteric site on the agonist-occupied A₁AR at relatively high concentrations, the structural modifications we have performed on this scaffold favor the expression of orthosteric antagonist properties over allosteric properties. This research has identified 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as novel class of orthosteric antagonist of the A₁AR and highlighted the close relationship between structural elements governing allosteric modulation and orthosteric antagonism of agonist function at the A₁AR.

Full text of DOI:10.1016/j.bmc.2009.08.024

Bioorganic & Medicinal Chemistry 17 (2009) 7353–7361
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1
adenosine receptor allosteric modulators and antagonists
Luigi Aurelio a, Celine Valant b, Heidi Figler c, Bernard L. Flynn a, Joel Linden c, Patrick M. Sexton b,
a,
Arthur Christopoulos b, , Peter J. Scammells
*
*
a Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville VIC 3052, Australia
b Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Clayton, VIC 3800, Australia
c Departments of Medicine and Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were prepared and evaluated as potential
allosteric modulators at the A1 adenosine receptor. The structure–activity relationships of the 3- and
6-positions of a series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were explored. Despite find-
ing that 3- and 6-substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines possess the ability to rec-
ognize an allosteric site on the agonist-occupied A1AR at relatively high concentrations, the structural
modifications we have performed on this scaffold favor the expression of orthosteric antagonist proper-
ties over allosteric properties. This research has identified 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dines as novel class of orthosteric antagonist of the A1AR and highlighted the close relationship
between structural elements governing allosteric modulation and orthosteric antagonism of agonist
function at the A1AR.
Received 20 May 2009
Revised 14 August 2009
Accepted 15 August 2009
Available online 20 August 2009
Keywords:
Allosteric modulator
A1 adenosine receptor (A1AR)
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
revealed that the same changes in the 3-position provided compar-
ative enhancement of agonist action, yet, a marked difference was
The initial studies of Bruns et al.1,2 on the allosteric modulation
of the A1 adenosine receptor (A1AR), described four compounds of
interest, including the prototypical modulator PD 81,7231 (Fig. 1).
Improving the properties of such leads has received much subse-
quent attention and, in particular, analogues of the related PD
71,605 led to the discovery of two allosteric modulators, LUF
5484 and T-62 with improved potency over PD 81,723.3–5 Despite
selectively potentiating agonist action at the A1AR via interaction
with an allosteric site, however, a characteristic feature of all these
modulators is a propensity to also cause antagonism at higher con-
centrations,3 which is most commonly interpreted as a lower affin-
ity interaction of the modulator with the A1AR’s orthosteric site.1–3
Thus, there is an ongoing need to gain a better understanding of
the structure–activity relationships that govern the allosteric ef-
fects of these compounds, on the one hand, and the possible
orthosteric effects, on the other. There have also been additional
studies with respect to the leads PD 117,975 and PD 78,416, in
which the main focus has been variation of the benzoyl substituent
in the 3-position.4,6–8 Testing of analogs of the two lead com-
pounds PD 117,975 and PD 78,416 conducted by Bruns et al.
noted if the 6-position was N-methylated, making these com-
pounds virtually inactive.2 Bruns et al.’s reasoning for this was that
* Corresponding authors. Tel.: +61 (0)3 9905 3817 (A.C.); tel.: +61 (0)3 9903
9542; fax: +61 (0)3 9903 9582 (P.J.S.).
E-mail addresses: Arthur.Christopoulos@med.monash.edu.au (A. Christopoulos),
Peter.Scammells@pharm.monash.edu.au (P.J. Scammells).
Figure 1. The PD series, discovered as allosteric enhancers of the A1AR in the study
conducted by Bruns et al., and the second generation allosteric enhancers, LUF 5484
and T-62.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.024

7354
L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
the N-methyl derivative is more basic than the N-benzyl and there-
fore protonation causes deleterious effects; this is why the N-eth-
oxycarbonyl series are potent enhancers, an observation confirmed
by Baraldi et al.6
In a recent study conducted in our group, it was shown that
amides in the 3-position of 2-aminothiophenes analogous to PD
71,605 supported allosteric enhancement at the A1AR.9 This was
the first report in which a detailed SAR study of various carboxam-
ides in the 3-position was performed for the allosteric enhancers at
the A1AR. In the current study, we report on synthesis and biolog-
ical evaluation of analogues of PD 117,975 and PD 78,416, in which
we focus on varying the substituent in the 3- and 6-positions of 2-
amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines.
Scheme 3. Reagents: (i) R = Bn, Me, Boc, EtOH, morpholine, S8 NCCH2CN; (ii) R = Bn,
Me, EtOH, morpholine, S8, H2NCOCH2CN, then 2 M ethanolic HCl.
therefore these intermediates were used in their crude form. Ami-
dations of amino acids 3a and 3b provided the amides 6, yet vary-
ing degrees of by-products and diminished yields were apparent
and it was more expedient to Boc-protect the 2-amino group.
Saponification of the ethyl ester proceeded smoothly, provided
the acids 4a and 4b, which were far more stable than the amino
acids 3a and 3b. Amidation with the common peptide coupling re-
agents, BOP or TBTU, afforded good yields of amides 5 that were
chromatographed on silica gel. The purified carbamates were trea-
ted with excess 6 M HCl to remove the Boc group providing the
amides 6a–i as hydrochloride salts.
The synthesis of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carboxamides 7a and 7b (Scheme 3) could be achieved by
directly amidating the corresponding 3-carboxylic acids. However,
a more direct approach was pursued by performing the Gewald
synthesis with 2-cyanoacetamide. The isolated 3-carboxamides
were converted to their hydrochloride salts which greatly im-
proved their solubility in water. The corresponding 2-amino-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitriles 8a–c were
also prepared directly via a Gewald reaction between malononitri-
le and the appropriate N-substituted 4-piperidone.
2. Results and discussion
The series of compounds listed in Table 1 were synthesised by
the methods depicted in Schemes 1–3. Target 2-amino-4,5,6,7-
tetrahydrothieno[2,3-c]pyridines with different 6-substitutents
were prepared by the Gewald synthesis.10 Ethyl cyanoacetate
was reacted with the commercially available N-substituted pip-
eridones 1a–c and elemental sulfur providing 2-aminothiophenes
2a–c (Scheme 1). In the case of 2d–f, the appropriate N-substi-
tuted piperidone was first prepared from piperidone via a BOP
or TBTU mediated coupled with an N-Boc protected amino acid.
In the subsequent Gewald reaction, thiophenes 2d and 2e precip-
itated during the course of the reaction whereas 2f was isolated
following an aqueous work-up. The 2-amino-4,5,6,7-tetrahydro-
thieno[2,3-c]pyridine analog with no substituent in the 6-position
(2g), was prepared by treating 2c with 6 M HCl to cleave the Boc
group.
Compounds 2a–c were subjected to sodium hydroxide medi-
ated hydrolysis providing the amino acids 3a–c which were ini-
tially used as intermediates in the synthesis of the amides 6a–i.
Attempted recrystallisation of 3a and 3b caused degradation and
The potential of the compounds listed in Table 1 to interact
allosterically with the A1AR was initially screened by evaluating
their ability to stabilize an orthosteric agonist–A1AR–G protein ter-
nary complex in an in vitro dissociation kinetic binding assay,
using 125I-ABA as the orthosteric radioligand and promoting disso-
ciation by addition of 25
lM GTPcS and the orthosteric antagonist,
100 M 8-CPT. The resulting K-score (‘kinetic score’) reported in
l
Table 1 denotes the percentage of the ternary complex remaining
after 10 min of radioligand dissociation, and is a measure of the
strength of the allosteric interaction between a given concentra-
tion of test ligand and the pre-equilibrated 125I-ABA-A1 receptor
complex; the higher this K-score percentage, the greater the ability
of the test ligand to allosterically stabilize the binding of the
orthosteric radioligand.11 Because the dissociation kinetic assays
were performed on a receptor that had been pre-equilibrated with
orthosteric radioligand, any effect of the test ligand on the dissoci-
Scheme 1. Reagents: (i) S8, EtOCOCH2CN, morpholine, EtOH; (ii) 6 M HCl, dioxane.
Scheme 2. Reagents: (i) EtOH, H2O, NaOH; (ii) Boc2O, DMAP, dioxane; (iii) BOP, DMF, DIPEA amine; (iv) 6 M HCl, EtOH.

L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
7355
Table 1
Subsequently, we determined the functional effects of the novel
compounds using an AlphaScreen plate-based assay of A1AR-med-
iated phosphorylation of ERK1/2 (pERK1/2) in intact CHO cells.12
AE activity of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines
For each compound, two concentrations (3 lM and 10 lM) were
tested alone and against an EC50 concentration of the orthosteric
agonist, R-PIA. Under these conditions an increase in functional re-
sponse represents either an allosteric enhancement of R-PIA func-
tion or intrinsic agonism of the test compound (or both). In
contrast, a reduction in R-PIA response indicates an antagonistic
effect of the compounds.
With the exception of PD81,723, which displayed some allosteric
agonism in the absence of R-PIA (yielding approx. 40% of the maxi-
mum R-PIA response at 10 lM), none of the test compounds (includ-
R1
K-scorea
(%)
EC50
M)
b
No.
R
(
l
PD 81,723
PD117,915 Bn
—
—
28.0 1.1
32.6 3.8
44.9 3.4
14.9 7.1
32.8 3.5
13.6 2.1
>20
10.3 0.10
>20
C(O)4-ClPh
C(O)OEt
C(O)OEt
C(O)OEt
C(O)OEt
C(O)OEt
2a
2b
2c
2d
2e
Bn
Me
Boc
Boc-
13.2 4.4
L
-Phe
82.5 12.4 7.2 3.9
Boc-NMe-
D-
92.9 2.0
9.1 0.6
ing PD117,915) displayed any significant effects on basal ERK1/2
signaling in the absence of agonist (not shown). Interestingly, when
tested against a concentration of R-PIA that yields approximately
50% of the maximal response, it was also noted that none of the com-
pounds, other than PD81,723 and PD 117,975, caused any apprecia-
ble potentiation of agonist responsiveness (Fig. 2). Instead, we
observed either no appreciable effect on agonist function for the
two concentrations of test compound utilized or an antagonistic
effect on R-PIA-mediated ERK1/2 phosphorylation, which was par-
ticularly striking for compounds 2c, 2d and 2e (Fig. 3). To confirm
this finding, therefore, we constructed complete R-PIA concentra-
tion–response curves in the absence or presence of increasing con-
centrations of each of these three test compounds to ascertain and
quantify the nature of their interaction with the agonist at the
A1AR (Fig. 2). Analysis of these data according to a simple model of
antagonism13 yielded the following pA2 values: 2c: 6.08 05; 2d:
6.43 0.09; 2e: 5.74 0.07; (n = 3), which are empirical measures
of antagonist potency.14 These pA2 values indicate that the
compounds are actually more potent as orthosteric antagonists of
R-PIA function than allosteric modulators of agonist dissociation,
that is, their antagonistic effects are expressed at lower concentra-
tions than any potential allosteric effects.
Phe(4-Cl)
2f
Boc-
H
L
-Cys(Trt)
C(O)OEt
C(O)OEt
C(O)OH
C(O)OH
C(O)OH
C(O)NHBn
C(O)NH(3-
ClBn)
93.2 0.4
58.5 1.8
68.3 4.7
77.1 9.9
66.1 1.8
37.6 7.3
36.0 4.1
7.9 5.5
11.7 1.1
8.0 2.9
11.6 6.1
12.1 6.6
13.1 5.8
>20
2g
3a
3b
3c
6a
6b
Bn
Me
Boc
Bn
Bn
6c
Bn
C(O)NH(3-
CF3Bn)
46.9 3.3
15.5 1.1
6d
6e
6f
Bn
Bn
Me
Me
C(O)NHNH2
C(O)NHNHPh
C(O)NHBn
C(O)NH(3-
ClBn)
C(O)NHNH2
C(O)NHNHPh
CONH2
CONH2
CN
CN
CN
82.9 13.0 8.9 3.9
55.4 11.1 7.8 3.4
87.2 7.7
88.0 6.9
12.0 6.3
13.2 4.5
6g
6h
6i
Me
Me
Bn
Me
Bn
85.7 9.2
83.1 11.9 11.5 2.8
19.1 11.3 >20
19.8 6.6
52.0 0.15 15.2 6.0
14.8 0.70 >20
4.4 3.4
7a
7b
8a
8b
8c
>20
Me
Boc
22.9 7.3
>20
a
Percentage of specific binding remaining after 10 min of dissociation of agonist,
125I]-ABA, binding to A1AR-G protein ternary complex in CHO-K1 cells stably
expressing the hA1AR (n = 3) followed pre-treatment with the candidate AE
(50 M).
Concentration of modulator producing half-maximal allosteric effect on [125I]-
[
l
b
3. Conclusions
ABA dissociation.
Despite finding that 3- and 6-substituted 2-amino-4,5,6,7-tetra-
hydrothieno[2,3-c]pyridines possess the ability to recognize an
allosteric site on the agonist-occupied A1AR at relatively high con-
centrations, we conclude that the structural modifications we have
performed on the 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridine
scaffold actually favor the expression of orthosteric antagonist
properties over allosteric properties. This finding is similar to that
made in a recent study by our group on 2-aminothienopyrida-
zines.12 Thus, in addition to identifying 2-amino-4,5,6,7-tetrahy-
drothieno[2,3-c]pyridines as novel class of orthosteric antagonist
of the A1AR, our results indicate that the close relationship be-
tween structural elements governing allosteric modulation and
orthosteric antagonism of agonist function at the A1AR may imply
that A1AR allosteric site on this GPCR is in very close proximity to
the orthosteric site.
ation of the radioligand must arise from a purely allosteric mech-
anism because the orthosteric site is already occupied by the
125I-ABA. Thus, this kinetic assay is biased towards detecting
purely allosteric effects; it should be noted, however, that this
biased assay design cannot differentiate modulators that solely
recognize the allosteric site from those that can bind to both the
orthosteric and allosteric sites. The determination of a K-score for
multiple concentrations of test ligand, up to a concentration of
50 lM, also allowed for the determination of the potency (EC50)
range for many of the compounds to mediate their half-maximal
allosteric effects on radioligand dissociation; these latter values
provide a semi-quantitative estimate of the apparent affinity of
the modulators for the allosteric site on the agonist-occupied
receptor. Where incomplete curves were attained due to low
potency, the EC50 value is reported as >20
reported as the percent change in radioligand dissociation at
50 M of modulator (Table 1).
From the results obtained in the dissociation kinetic assays and
lM and the K-score
l
4. Experimental
the resultant K-scores shown in Table 1, it was evident that the
greatest degree of allosteric stabilization of agonist–receptor com-
plexes was achieved by compounds that contained an ethyl ester in
the 3-position together with N-Boc amino acid substitution in the
6-position (e.g., compounds 2d–2f), or a methyl substitution of the
6-position together with an amide or hydrazide substitution in the
3-position (e.g., compounds 6f–6i). The potency of these com-
pounds to retard radioligand dissociation was similar to, or slightly
better than, that displayed by PD 81,723.
Melting points were determined with an Electrothermal melt-
ing point apparatus and are uncorrected. All 1H NMR spectra were
recorded on
a Bruker Avance DPX 300 spectrometer at
300.13 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 sta-
ted otherwise, samples were dissolved in CDCl3. High resolution
mass spectra were obtained on a Waters LCT Premier XE (TOF)
mass spectrometer fitted with an ESI ion source. Thin-layer

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L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
Figure 2. Effect of two different concentrations (3 lM, left bar; 10 lM, right bar) of test ligands on A1AR-mediated stimulation of ERK1/2 phosphorylation in intact CHO FlpIn
cells, in the presence of an EC50 concentration of R-PIA (determined on the same day as each assay); dashed line denotes 50% response level. The effects of PD81,723 and
PD117,915 are also shown for comparison. Data represent the mean standard deviation of two experiments conducted in triplicate.
chromatography was conducted on 0.2 mm plates using Merck Sil-
ica Gel 60 F254. Column chromatography was achieved using Merck
4.3. 6-tert-Butyl 3-ethyl 2-amino-4,5-dihydrothieno[2,3-c
]pyridine-3,6(7H)-dicarboxylate (2c)
Silica Gel 60 (particle size 0.063–0.200 lm, 70–230 mesh).
N-Boc-4-piperidone 1c (8.93 g, 44.89 mmol), ethyl cyanoacetate
(5.25 mL, 49.38 mmol) and sulfur (1.73 g, 53.87 mmol) were sus-
pended in EtOH (90 mL). Morpholine (7.85 mL, 89.79 mmol) was
added and the mixture was refluxed gently with stirring for 3 h
then allowed to stir at room temperature overnight. The resultant
precipitate was collected by suction filtration and washed with ice
cold EtOH until the filtrate was colorless (9.71 g of white powder,
66% yield).
4.1. Ethyl 2-amino-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxylate (2a)
N-Benzyl-4-piperidone 1a (5 g, 26.42 mmol), ethyl cyanoace-
tate (3.09 mL, 29.06 mmol) and sulfur (1.02 g, 31.70 mmol) were
suspended in EtOH (55 mL). Morpholine (4.62 mL, 52.84 mmol)
was added and the mixture was refluxed gently with stirring for
2 h. The cooled solution was diluted with water and extracted with
CH2Cl2 (3 ꢀ 50 mL). The combined organic layers were washed
with water, then brine, dried (MgSO4), filtered and concentrated.
The resultant residue was triturated with hot MeOH (20 mL) and
washed with ice cold MeOH to afford the desired product as an
off white powder (7.1 g, 85% yield).
Mp 140–144 °C. 1H NMR d 6.23 (br s, 2H, NH2), 4.37 (s, 2H, 7-
CH2), 4.29 (q, J = 7.2 Hz, 2H, CH2CH3), 3.64 (t, 2H, J = 5.8 Hz, 2H,
5-CH2), 2.83 (t, 2H, J = 5.7 Hz, 2H, 4-CH2), 1.50 (s, 9H, C(CH3)3),
1.36 (t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (75.4 MHz) d 165.8,
162.7, 154.7, 131.2, 113.7, 104.8, 79.9, 59.5, 42.8–40.5, 28.5, 27.1,
14.5. HR-ESMS calcd for C15H23N2O4S+ (M+1) 327.1373, found
327.1363.
Mp 110–111 °C. 1H NMR d 7.41–7.26 (m, 5H, aromatic), 6.09 (br
s, 2H, NH2), 4.26 (q, J = 7.2 Hz, 2H, CH2CH3), 3.69 (s, 2H, NCH2Ph),
3.42 (s, 2H, 7-CH2), 2.89–2.71 (m, 4H, 5-CH2 and 4-CH2), 1.33 (t,
J = 7.2 Hz, 3H, CH2CH3). 13C NMR (75.4 MHz) d 166.0, 162.3,
138.2, 131.0, 129.2, 128.35, 127.2, 114.7, 105.2, 62.0, 59.5, 51.3,
50.3, 27.3, 14.5. HR-ESMS calcd for C17H21N2O2S+ (M+1)
317.1318, found 317.1292.
4.4. (S)-Ethyl 2-amino-6-(2-(tert-butoxycarbonylamino)-3-
phenylpropanoyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxylate (2d)
N-Boc-L-Phe-OH (0.5 g, 1.89 mmol) was dissolved in DMF
(6 mL) and 4-piperidone hydrochloride monohydrate 9 (347 mg,
2.26 mmol) was added, followed by BOP reagent (1.0 g, 2.26 mmol)
and DIPEA (1.3 mL, 7.54 mmol). After 3 h stirring at room temper-
ature, the solution was diluted with EtOAc and washed with water
followed by 10% citric acid, water, saturated bicarbonate solution,
water and finally brine. The organic layer was dried (MgSO4), fil-
tered and concentrated to yield a clear colorless resin (645 mg,
99% yield). This compound (240 mg, 0.693 mmol) was dissolved
4.2. Ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxylate (2b)
N-Methyl-4-piperidone (1b) (10 mL, 86.60 mmol), ethyl cya-
noacetate (9.22 mL, 86.60 mmol) and sulfur (3.05 g, 95.26 mmol)
were suspended in EtOH (150 mL). Morpholine (15.15 mL,
173.21 mmol) was added and the mixture was refluxed gently
with stirring for 2 h. The cooled solution was diluted with water
(250 mL). The resultant yellow precipitate was collected by suction
filtration and washed with water until the filtrate was colorless
(13.4 g of yellow powder, 64% yield). A small portion was recrystal-
lised from EtOH providing an off white powder.
in EtOH (2 mL) and ethyl cyanoacetate (81
added followed by sulfur (23 mg, 0.693 mmol). Morpholine
(200 L, 2.29 mmol) was added and the mixture was stirred at
lL, 0.762 mmol) was
l
ꢁ40 °C for 2 h. After cooling on an ice bath, the resultant precipi-
tate was collected by suction filtration and washed with ice cold
EtOH to afford the desired product as a white solid (240 mg, 73%
yield).
Mp 101–103 °C. 1H NMR d 6.03 (br s, 2H, NH2), 4.27 (q,
J = 7.2 Hz, 2H, CH2CH3), 3.39 (s, 2H, 7-CH2), 2.85 (t, 2H, J = 5.7 Hz,
2H, 5-CH2), 2.68 (t, 2H, J = 5.8 Hz, 2H, 4-CH2), 2.45 (s, 3H, NCH3),
1.34 (t, J = 7.2 Hz, 3H, CH2CH3). 13C NMR (75.4 MHz) d 166.0,
162.5, 130.5, 114.1, 104.9, 59.4, 53.2, 52.3, 45.3, 27.2, 14.4. HR-
ESMS calcd for C11H17N2O2S+ (M+1) 241.1005, found 241.1001.
Mp 143–145 °C. 1H NMR (complex rotamers) d 7.25–7.13 (m,
5H, aromatic), 5.50–4.20 (m, 6H, NH, NH2, CH2CH3 and NCHCO),
3.84–2.25 (m, 8H, 7-CH2, 5-CH2, 4-CH2 and PhCH2), 1.41 (s, 9H,
C(CH3)3), 1.37–1.29 (m, 3H, CH2CH3). HR-ESMS calcd for
C24H32N3O5S+ (M+1) 474.2057, found 474.2052.

L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
7357
provide a solid that was recrystallised from ether (455 mg, 72%
yield). This amide (400 mg, 1.01 mmol) was dissolved in EtOH
(1 mL) and ethyl cyanoacetate (118
lL, 1.11 mmol) was added fol-
lowed by sulfur (34 mg, 1.06 mmol). Morpholine (211
l
L,
2.41 mmol) was added and the mixture was stirred at ꢁ60 °C for
3 h and then at room temperature overnight. The resultant precip-
itate was collected by suction filtration and washed with ice cold
EtOH to afforded the desired product as a white solid (228 mg,
43% yield).
Mp 148–150 °C. 1H NMR (complex rotamers) d 7.26–7.09 (m,
4H, aromatic), 5.50–2.60 (m, 16H, NH2, NCH3, CH2CH3, NCHCO, 7-
CH2, 5-CH2, 4-CH2 and PhCH2,), 1.42–1.22 (m, 12H, C(CH3)3 and
CH2CH3). HR-ESMS calcd for C25H33ClN3O5S+ (M+1) 522.1824,
found 522.1822.
4.6. (R)-Ethyl 2-amino-6-(2-(tert-butoxycarbonylamino)-3-
(tritylthio)propanoyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carboxylate (2f)
L-Cys(Trt)-OH (0.5 g, 1.38 mmol) was suspended in DMF (6 mL)
and triethylamine (400 L, 2.87 mmol) was added followed by
l
Boc2O (315 mg, 1.44 mmol). After stirring at room temperature
for 10 min all of the solids had dissolved. After stirring a further
hour the mixture was diluted with water, acidified with 10% citric
acid and extracted with CHCl3 (ꢀ3). The combined organics were
washed with water, dried (MgSO4), filtered and concentrated to
provide clear colorless oil. The oil was dissolved in DMF (6 mL)
and 4-piperidone hydrochloride monohydrate (9) (254 mg,
1.65 mmol) was added followed by HOBt.H2O (253 mg, 1.65 mmol)
and TBTU (663 mg, 2.06 mmol). Finally DIPEA (1.2 mL, 6.88 mmol)
was added and the mixture was stirred at room temperature over-
night. A precipitate formed upon dilution with water. This precip-
itate was collected by suction filtration and washed with copious
amounts of water and air dried to yield a white/cream solid
(741 mg, 99% yield). This amide (300 mg, 0.551 mmol) was dis-
solved in EtOH (1.6 mL) and ethyl cyanoacetate (65
0.606 mmol) was added, followed by sulfur (18 mg, 0.551 mmol)
and morpholine (160 L, 1.83 mmol). The mixture was stirred at
lL,
l
ꢁ60 °C for 3 h then at room temperature overnight. The mixture
was concentrated to a resin and taken up in 2-propanol, filtered
and then diluted with water while stirring. The resultant precipi-
tate was collected by suction filtration and washed with copious
amounts of water and air dried to afford a pale yellow solid
(328 mg, 88% yield).
Mp 92–95 °C. 1H NMR (complex rotamers) d 7.41–7.17 (m, 15H,
aromatic), 5.30–2.35 (m, 14H, NH2, NH, CH2CH3, NCHCO, 7-CH2, 5-
CH2, 4-CH2 and TrtSCH2,), 1.48–1.30 (m, 12H, C(CH3)3 and CH2CH3).
þ
HR-ESMS calcd for C37H42N3O5S2
672.2549.
(M+1) 672.2560, found
Figure 3. R-PIA mediated stimulation of ERK1/2 phosphorylation in the absence
(d) or presence of 0.3 M (h), 1 M ( ), 10 M (s) of the
indicated test compound in intact CHO FlpIn A1AR cells. Data points represent the
mean s.e.m. of three experiments conducted in triplicate. Curves drawn through
the points represent the best global fit of a competitive model of agonist–antagonist
interaction.
l
lM (4), 3
l
r
lM (}) or 30 l
4.7. Ethyl 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxylate hydrochloride (2g)
Compound 2c (1.0 g, 3.06 mmol) was dissolved in dioxane
(10 mL) and 6 M HCl (1 mL) was added dropwise with stirring.
The mixture was heated to 80 °C for 5 min when a precipitate
developed. After stirring a further 5 min at this temperature, the
mixture was allowed to cool to room temperature and the precip-
itate was collected by suction filtration. The filter cake was washed
with dioxane and finally EtOAc to afford the desired product as a
hydrochloride salt (717 mg, 89% yield).
4.5. (R)-Ethyl 2-amino-6-(2-(tert-butoxycarbonyl(methyl)-
amino)-3-(4-chlorophenyl)propanoyl)-4,5,6,7-tetrahydrothi-
eno[2,3-c]pyridine-3-carboxylate (2e)
N-Boc-N-Me-D-Phe(4-Cl)-OH (0.5 g, 1.59 mmol) was dissolved
in DMF (5 mL) and 4-piperidone hydrochloride monohydrate (9)
(293 mg, 1.91 mmol) was added, followed by BOP (846 mg,
1.91 mmol) and DIPEA (1.11 mL, 6.37 mmol). The mixture was stir-
red at room temperature overnight and the solution was diluted
with EtOAc and washed with water followed by 10% citric acid,
water, saturated bicarbonate solution, water and finally brine.
The organic layer was dried (MgSO4), filtered and concentrated to
Mp 255 °C dec. 1H NMR (D2O) d 4.27 (q, J = 7.2 Hz, 2H, CH2CH3),
4.17 (s, 2H, 7-CH2), 3.47 (t, J = 6.2 Hz, 2H, 5-CH2), 3.05 (t, J = 6.2 Hz,
2H, 4-CH2), 1.33 (t, J = 7.2 Hz, 2H, CH2CH3). 13C NMR (D2O) d 166.4,
164.1, 129.8, 107.9, 103.5, 60.7, 41.4, 23.2, 13.4. HR-ESMS calcd for
C10H15N2O2S+ (M+1) 227.0849, found 227.0850.

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L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
4.8. 2-Amino-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxylic acid (3a)
DMAP (200 mg, 1.64 mmol) and the mixture was heated with stir-
ring at ꢁ70–80 °C for 3 h then at room temperature overnight. The
solution was concentrated and then partitioned between water
and CHCl3. The organic layer was set aside and the aqueous layer
was extracted with CHCl3 (ꢀ2). The combined organic portions
were washed with water then brine, dried (MgSO4), filtered and
concentrated to a residue. This residue was suspended in EtOH
(50 mL) and water (50 mL) and KOH (3.65 g, 65.10 mmol) were
added. The mixture was at ꢁ70 °C for 3 h and then concentrated
to remove EtOH. The aqueous residue was diluted with water until
all solids had dissolved and then washed with ether (ꢀ3). The
aqueous layer was chilled on ice and acidified with 10% citric acid
to pH ꢂ 7. After extraction with EtOAc (4 ꢀ 250 mL), the combined
organics were dried (MgSO4), filtered and concentrated to yield a
yellow powder. This powder was triturated with ether and filtered
on a Buchner funnel and washed with ether to afford a yellow solid
(4.62 g, 73%).
Compound 2a (4.0 g, 12.64 mmol) was suspended in EtOH
(40 mL) and water (20 mL), dioxane (15 mL) and NaOH (2.02 g,
50.56 mmol) were added. The mixture was gently refluxed for
4 h. The solution was concentrated to a residue and partitioned be-
tween water (30 mL) and ether. The ether layer was discarded and
the aqueous phase chilled in an ice bath and slowly acidified with
6 M HCl with stirring to pH ꢂ 7. The resultant precipitate was col-
lected by suction filtration and washed with ice cold water
(10 mL). This solid was further triturated in ice cold EtOH
(15 mL) to afford the desired product as a gray solid (2.16 g, 59%
yield).
Mp 198 °C dec. 1H NMR (DMSO-d6) d 12.0 (br s, 1H, CO2H),
7.62–7.29 (m, 7H, NH2 and aromatic), 4.40–3.72 (m, 4H, 7-CH2
and NCH2Ph), 3.25 (m, 2H, 5-CH2), 2.95 (m, 2H, 4-CH2). 13C NMR
(DMSO-d6) d 166.4, 164.3, 131.4, 130.3, 130.0, 129.4, 128.8,
106.5, 102.2, 57.8, 48.6, 48.0, 23.9. HR-ESMS calcd for C15H17N2-
O2S+ (M+1) 289.1005, found 289.0997.
Mp 187 °C dec. 1H NMR (DMSO-d6) d 12.5 (br s, 1H, CO2H), 10.5
(br s, 1H, NH), 7.36–7.28 (m, 5H, aromatic), 3.69 (s, 2H, 7-CH2), 3.50
(s, 2H, NCH2Ph), 2.66–2.83 (m, 4H, 5-CH2 and 4-CH2), 1.49 (s, 9H,
C(CH3)3). 13C NMR (75.4 MHz, DMSO-d6) d 167.2, 151.2, 148.2,
137.5, 130.0, 128.9, 128.3, 127.2, 121.5, 110.9, 81.6, 60.6, 50.3,
49.3, 27.7, 26.1.
4.9. 2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxylic acid (3b)
4.12. 2-(tert-Butoxycarbonylamino)-6-methyl-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid (4b)
Compound 2b (6.4 g, 26.63 mmol) was suspended in EtOH
(40 mL) and water (30 mL) and NaOH (4.26 g, 106.52 mmol) were
added. The mixture stirred at ꢁ80 °C for 5.5 h. The solution was
concentrated to a residue and partitioned between water (30 mL)
and ether. The ether layer was discarded and the aqueous phase
chilled in an ice bath and slowly acidified with 6 M HCl with stir-
ring to pH ꢂ 7. The resultant precipitate was collected by suction
filtration and washed with ice cold water (20 mL) and 1:1 ether/
ethanol (10 mL) to afford the product as a light brown solid
(4.58 g, 81% yield).
Compound 2b (5.0 g, 20.81 mmol) was dissolved in dioxane
(38 mL) and Boc2O (9.31 g, 42.65 mmol) was added followed by
DMAP (254 mg, 2.08 mmol). The mixture was stirred at ꢁ70–
80 °C for 3 h then at room temperature overnight. The solution
was concentrated and then partitioned between water and CHCl3.
The organic layer was set aside and the aqueous layer extracted
with CHCl3 (ꢀ2). The combined organic layers were then washed
with water then brine, dried (MgSO4), filtered and concentrated
to a residue. This residue was suspended in EtOH (30 mL) and
water (15 mL) and NaOH (3.33 g, 83.25 mmol) was added. The
mixture was stirred at ꢁ70 °C for 3 h and then concentrated to re-
move EtOH. The aqueous residue was diluted with water until all
solids dissolved and then washed with ether (ꢀ3). The aqueous
layer was chilled on ice and acidified with 10% citric acid to
pH ꢂ 7. The resultant precipitate was collected by suction filtration
and washed with water. Drying under high vacuum afforded the
product as a yellow powder (4.09 g, 63% yield).
Mp 185 °C dec. 1H NMR (DMSO-d6) d 10.9 (br s, 1H, CO2H), 7.43
(br s, 2H, NH2), 4.08 (s, 2H, 7-CH2), 3.39–3.29 (m, 2H, 5-CH2), 3.01–
2.91 (m, 2H, 4-CH2), 2.81 (s, 3H, NCH3). 13C NMR (75.4 MHz,
DMSO-d6) d 166.4, 164.2, 129.5, 106.7, 102.3, 50.2, 41.8, 24.0.
HR-ESMS calcd for C9H13N2O2S+ (M+1) 213.0692, found 213.0685.
4.10. 2-Amino-6-(tert-butoxycarbonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid (3c)
Compound 2c (1.0 g, 3.06 mmol) was suspended in EtOH (5 mL)
and water (4 mL), dioxane (2 mL) and KOH (687 mg, 12.25 mmol)
were added. The mixture was stirred with gentle reflux for 2.5 h.
The cooled solution was diluted with water (100 mL) and washed
with ether. The aqueous phase was cooled in an ice bath and care-
fully acidified to pH ꢂ 5 with 10% citric acid and extracted with
CHCl3 (ꢀ2) and EtOAc (ꢀ1). The combined organic portions were
washed with brine dried (MgSO4), filtered and concentrated to a
solid (0.6 g). Trituration with MeOH afforded the desired product
as a white powder (200 mg, 22% yield).
Mp 171 °C dec. 1H NMR (DMSO-d6) d 11.21 (br s, 1H, NH), 4.45
(br s, 1H, CO2H), 3.77 (br s, 2H, 7-CH2), 2.91 (br s, 2H, 5-CH2), 2.56
(br s, 2H, 4-CH2), 1.48 (s, 9H, C(CH3)3). 13C NMR (75.4 MHz, DMSO-
d6) d 168.2, 151.4, 146.6, 130.0, 117.8, 113.7, 81.1, 51.1, 50.7, 42.9,
27.8, 24.8.
4.13. General method for the synthesis of compounds 6a–i
Compound 4a/4b (200 mg) and the appropriate amine
(1.2 equiv) were dissolved in DMF (3 mL). BOP (1.2 equiv) and DI-
PEA (3 equiv) were added and the reaction was stirred at room
temperature overnight. Following dilution EtOAc, the solution
was washed with water (ꢀ4), brine, dried (MgSO4), filtered and
concentrated to a foam (60–80% yield). The crude product was sub-
sequently chromatographed on silica gel (30–50% EtOAc/petro-
leum ether). The resultant amide was dissolved in EtOH (1 mL)
Mp 167–170 °C. 1H NMR d 11.9 (br s, 1H, CO2H), 7.29 (br s, 2H,
NH2), 4.25 (s, 2H, 7-CH2), 3.51 (t, J = 5.8 Hz, 2H, 5-CH2), 2.66 (t, 2H,
J = 5.7 Hz, 2H, 4-CH2), 1.42 (s, 9H, C(CH3)3). 13C NMR (75.4 MHz,
DMSO-d6) d 166.5, 163.4, 153.9, 130.8, 111.8, 102.7, 79.0, 41.9,
41.4, 28.0, 26.6. HR-ESMS calcd for C13H17N2O4S- (M-1) 297.0915,
found 297.0915.
and stirred with 6 M HCl (300 lL) at room temperature overnight.
4.11. 6-Benzyl-2-(tert-butoxycarbonylamino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid (4a)
Evaporation of the solvents afforded a residue which was triturated
with dioxane. The solid was collected by suction filtration and
washed with dioxane then ether and the crude products were
recrystallised from EtOH/ether to afford compounds 6a–i as the
hydrochloride salts.
Compound 2a (5.15 g, 16.28 mmol) was dissolved in dioxane
(30 mL) and Boc2O (7.82 g, 35.81 mmol) was added followed by

L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
7359
4.14. 2-Amino-N,6-dibenzyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide hydrochloride (6a)
(m, 2H, 7-CH2), 3.56 (m, 2H, 5-CH2), 3.10–3.01 (m, 2H, 4-CH2),
2.85 (s, 3H, NCH3).). 13C NMR (75.4 MHz, DMSO-d6) d 164.7,
156.9, 140.0, 128.2, 128.0, 127.2, 126.6, 109.6, 108.7, 50.0, 49.6,
42.3, 41.3, 22.9. HR-ESMS calcd for C16H20N3OS+ (M+1) 302.1322,
found 302.1318.
The title compound 6a was synthesized as per the general
method from 4a to afford 6a as a yellow solid in 31% yield. Mp
135–140 °C. 1H NMR (DMSO-d6) d 11.15 (br s, 1H, (CH2)3N+H),
7.65–7.31 (m, 13H, aromatic, NH and NH2), 4.49–4.36 (m, 4H,
2 ꢀ NCH2Ph), 4.09 (s, 2H, 7-CH2), 3.60–3.56 (m, 2H, 5-CH2), 3.09–
3.06 (m, 2H, 4-CH2). HR-ESMS calcd for C22H24N3OS+ (M+1)
378.1635, found 378.1646.
4.20. 2-Amino-N-(3-chlorobenzyl)-6-methyl-4,5,6,7-tetrahydro-
thieno[2,3-c]pyridine-3-carboxamide hydrochloride (6g)
The title compound 6f was synthesized as per the general meth-
od from 4b to afford 6g as a yellow solid in 25% yield. Mp 190–
195 °C. 1H NMR (DMSO-d6) d 10.66 (br s, 1H, (CH2)2MeN+H),
7.64–7.56 (m, 1H, NH), 7.41–7.23 (m, 4H, aromatic), 7.02 (br s,
2H, NH2), 4.40–4.38 (m, 2H, NCH2Ph), 4.32–4.27 and 4.11–3.98
(m, 2H, 7-CH2), 3.62–3.50 (m, 2H, 5-CH2), 3.10–3.05 (m, 2H, 4-
CH2), 2.87 (s, 3H, NCH3). 13C NMR (75.4 MHz, DMSO-d6) d 165.1,
159.8, 142.8, 132.8, 130.0, 127.4, 127.0, 126.4, 125.9, 107.1, 50.1,
49.7, 41.8, 41.3, 22.9. HR-ESMS calcd for C16H19ClN3OS+ (M+1)
336.0932, found 336.0931.
4.15. 2-Amino-6-benzyl-N-(3-chlorobenzyl)-4,5,6,7-tetrahydro-
thieno[2,3-c]pyridine-3-carboxamide hydrochloride (6b)
The title compound 6b was synthesized as per the general
method from 4a to afford 6b as a yellow solid in 35% yield. Mp
210 °C dec. 1H NMR (DMSO-d6) d 10.93 (br s, 1H, (CH2)3N+H),
7.65–7.31 (m, 10H, aromatic and NH), 7.00 (br s, 2H, NH2), 4.47–
4.40 (m, 4H, 2 ꢀ NCH2Ph), 4.09 (s, 2H, 7-CH2), 3.65–3.55 (m, 2H,
5-CH2), 3.05–3.10 (m, 2H, 4-CH2). HR-ESMS calcd for
C22H23ClN3OS+ (M+1) 412.1245, found 412.1245.
4.21. 2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carbohydrazide hydrochloride (6h)
4.16. 2-Amino-6-benzyl-N-(3-(trifluoromethyl)benzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide hydrochloride
(6c)
The title compound 6h was synthesized as per the general
method from 4b to afford 6h as a yellow solid in 46% yield. Mp
195 °C dec. 1H NMR (D2O) d 4.39–4.34 and 4.14–4.09 (m, 2H, 7-
CH2), 3.72–3.66 and 3.42–3.33 (m, 2H, 5-CH2), 3.07–2.97 (m, 5H,
4-CH2 and NCH3). 13C NMR (D2O) d 166.1, 162.4, 127.5, 109.4,
105.1, 51.2, 51.1, 41.9, 22.8. HR-ESMS calcd for C9H15N4OS+
(M+1) 227.0961, found 227.0948.
The title compound 6c was synthesized as per the general
method from 4a to afford 6c as a yellow solid in 36% yield. Mp
155–160 °C. 1H NMR (DMSO-d6) d 11.19 (br s, 1H, (CH2)3N+H),
7.66–7.43 (m, 12H, aromatic, NH and NH2), 4.50–4.42 (m, 4H,
2 ꢀ NCH2Ph), 4.08 (s, 2H, 7-CH2), 3.66–3.56 (m, 2H, 5-CH2), 3.07–
3.13 (m, 2H, 4-CH2). HR-ESMS calcd for C23H23F3N3OS+ (M+1)
446.1508, found 446.1468.
4.22. 2-Amino-6-methyl-N’-phenyl-4,5,6,7-tetrahydrothi-
eno[2,3-c]pyridine-3-carbohydrazide hydrochloride (6i)
4.17. 2-Amino-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carbohydrazide hydrochloride (6d)
The title compound 6i was synthesized as per the general meth-
od from 4b to afford 6i as a yellow solid in 22% yield. Mp 190 °C
dec. 1H NMR (DMSO-d6) d 10.56 (br s, 1H, (CH2)3N+H), 8.99 (br s,
1H, NHNHPh), 7.17–7.12 and 6.81–6.68 (m, 5H, aromatic), 4.33–
4.28 (m, 1H, NHNHPh), 4.08–4.04 (m, 2H, 7-CH2), 3.62 (m, 2H, 5-
CH2), 3.17–3.10 (m, 2H, 4-CH2), 2.89 (s, 3H, NCH3). 13C NMR
(75.4 MHz, DMSO-d6) d 165.8, 160.1, 149.7, 128.7, 127.8, 118.5,
114.5, 112.5, 107.4, 50.1, 49.7, 41.2, 22.8. HR-ESMS calcd for
C15H19N4OS+ (M+1) 303.1274, found 303.1279.
The title compound 6d was synthesized as per the general
method from 4a to afford 6d as a yellow solid in 65% yield. Mp
191 °C dec. 1H NMR (DMSO-d6) d 11.60 (br s, 1H, (CH2)3N+H),
10.20 (br s, 1H, NHNH2), 7.73–7.68 and 7.49–7.46 (2 m, 7H, aro-
matic and NH2), 4.43 (br s, 2H, NCH2Ph), 4.07 (s, 2H, 7-CH2),
3.66–3.30 (m, 4H, 5-CH2 and NHNH2), 3.07–3.17 (m, 2H, 4-CH2).
13C NMR (75.4 MHz, DMSO-d6) d 165.7, 163.4, 131.5, 129.9,
129.5, 128.8, 127.8, 107.3, 103.1, 57.1, 48.2, 47.2, 22.6. HR-ESMS
calcd for C15H19N4OS+ (M+1) 303.1274, found 303.1279.
4.23. 2-Amino-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carboxamide hydrochloride (7a)
4.18. 2-Amino-6-benzyl-N’-phenyl-4,5,6,7-tetrahydro-
thieno[2,3-c]pyridine-3-carbohydrazide hydrochloride (6e)
N-Benzyl-4-piperidone 1a (1.67 g, 8.84 mmol), 2-cyanoacet-
amide (743 mg, 8.84 mmol) were suspended in EtOH (2.6 mL).
Sulfur (309 mg, 9.64 mmol) and morpholine (885 lL, 10.12
The title compound 6a was synthesized as per the general
method from 4a to afford 6e as a yellow solid in 33% yield. Mp
180 °C dec. 1H NMR (DMSO-d6) d 11.15 (br s, 1H, (CH2)3N+H),
8.99 (br s, 1H, NHNHPh), 7.73–7.68 and 7.49–7.46 (2 m, 12H, aro-
matic and NH2), 4.43 (br s, 2H, NCH2Ph), 4.07 (s, 2H, 7-CH2),
3.66–3.30 (m, 4H, 5-CH2 and NHNH2), 3.17–3.07 (m, 2H, 4-
CH2). HR-ESMS calcd for C21H23N4OS+ (M+1) 379.1587, found
379.1606.
mmol) were added and the mixture was heated on an oil bath
at 60 °C for 3 h during which time a solid precipitated. After
cooling, the precipitate was collected by suction filtration and
washed with ice cold EtOH (2.21 g, 87% yield) and recrystallised
from MeOH. A sample of recrystallised material (0.5 g) was
dissolved in dioxane (10 mL) and 6 M HCl was added dropwise
until no further precipitation occurred. The hydrochloride salt
was collected by suction filtration and washed with dioxane
and finally EtOAc.
4.19. 2-Amino-N-benzyl-6-methyl-4,5,6,7-tetrahydrothieno-
[2,3-c]pyridine-3-carboxamide hydrochloride (6f)
Mp 200–205 °C. 1H NMR (D2O) d 4.56-4.39 4.56–4.39 (m, 2H,
7-CH2), 4.18–4.17 (m, 2H, NCH2Ph), 3.85–3.78 and 3.46–3.36
(2 m, 2H, 5-CH2), 3.06–3.02 (m, 2H, 4-CH2). 13C NMR (D2O) d
168.9, 158.2, 131.0, 130.3, 129.3, 128.4, 128.1, 110.3, 109.6, 59.2,
49.3, 48.9, 22.8. HR-ESMS calcd for C15H18N3OS+ (M+1) 288.1165,
found 288.1144.
The title compound 6f was synthesized as per the general meth-
od from 4b to afford 6f as a yellow solid in 39% yield. Mp 200 °C
dec. 1H NMR (DMSO-d6) d 10.98 (br s, 1H, (CH2)2MeN+H), 7.57–
7.23 (m, 8H, aromatic, NH and NH2), 4.41 (m, 2H, NCH2Ph), 4.06

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L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
4.24. 2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carboxamide hydrochloride (7b)
Mp 195–197 °C. 1H NMR d 4.36 (s, 2H, 7-CH2), 3.66 (t, J = 5.7 Hz,
2H, 5-CH2), 2.59 (t, J = 5.7 Hz, 2H, 4-CH2), 1.48 (s, 9H, C(CH3)3). 13
C
NMR (rotamers) d 161.3, 154.6, 131.0, 116.7, 116.1, 115.1, 87.6,
80.4, 42.6, 42.0, 41.4, 40.0, 28.4, 24.7. HR-ESMS calcd for
C13H18N3O2S+ (M+1) 280.1114, found 280.1109.
N-Methyl-4-piperidone 1b (1.0 g, 8.84 mmol) and 2-cyano-
acetamide (743 mg, 8.84 mmol) were suspended in EtOH
(2.6 mL). Sulfur (309 mg, 9.64 mmol) and morpholine (885 lL,
10.12 mmol) were added and the mixture was heated on an oil
bath at 60 °C for 3 h during which time a solid precipitated. After
cooling, the precipitate was collected by suction filtration and
washed with ice cold EtOH (1.69 g, 90% yield) and recrystallised
from MeOH. A sample of the recrystallised material (0.5 g) was
dissolved in dioxane (10 mL) and 6 M HCl was added dropwise
until no further precipitation occurred. The hydrochloride salt
was filtered by suction filtration and washed with dioxane and
finally EtOAc.
4.28. Dissociation kinetic assays
Determination of allosteric modulation of orthosteric radioli-
gand dissociation kinetics consisted of three phases: (1) binding
to equilibrium of the agonist, [125I]-ABA, to the A1AR-G protein ter-
nary complex; (2) stabilization of that complex by adding vehicle
or increasing doses of allosteric modulator for 30 min, and (3) dis-
sociation of the complex by adding a combination of an A1AR
orthosteric antagonist, 100 lM BW-1433, and 25 lM GTPcS for
Mp 207 °C dec. 1H NMR (D2O) d 4.20-4.15 4.20–4.15 and 3.95–
3.90 (2 m, 2H, 7-CH2), 3.53–3.45 and 3.21–3.12 (2 m, 2H, 5-CH2),
2.91–2.79 (m, 5H, 4-CH2 and NCH3). 13C NMR (75.4 MHz, D2O) d
168.4, 154.8, 127.9, 112.4, 112.0, 51.3, 51.0, 42.0, 22.8. HR-ESMS
calcd for C9H14N3OS+ (M+1) 212.0852, found 212.0850.
10 min. A score was determined at each point between 0% (no dif-
ferent than vehicle) and 100% (complete prevention of [125I]-ABA
dissociation). The data were fitted to a variable slope sigmoidal
curve and the max and EC50 values extrapolated form the curve.
The assay employed membranes from CHO-K1 cells stably express-
ing the human A1AR. For agonist binding to equilibrium (phase 1)
the buffer consisted of 10 mM HEPES, pH 7.2, containing 0.5 mM
4.25. 2-Amino-6-benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carbonitrile (8a)
MgCl2, 1 U/mL adenosine deaminase, 0.5 nM [125I]-ABA and 10
of membrane protein in a final volume of 100 L applied to 96 well
Millipore GF/C glass fiber filter plates. After 90 min at room tem-
perature the addition of 50 L of test modulator (0.1–50 M, final)
initiated stabilization of the ternary complex (phase 2). After
30 min, a solution containing BW-1433 and GTP S (50 L) was
lg
l
N-Benzyl-4-piperidone 1a (5.0 g, 26.42 mmol) and malononitri-
le (1.75 g, 26.42 mmol) were dissolved in EtOH (50 mL). Sulfur
(932 mg, 29.06 mmol) and morpholine (4.62 mL, 52.84 mmol)
were added and the mixture was refluxed for 2 h. After cooling,
the solution was diluted with water (100 mL). A precipitate formed
which was collected by suction filtration and washed with
water. This afforded the product as a dark brown solid (6.4 g,
90% yield).
l
l
c
l
added to initiate the dissociation of the ternary complex. Ten min-
utes later membranes were filtered, washed, dried and counted for
residual [125I]-ABA. The percentage of specifically bound agonist
remaining after 10 min of dissociation served as an index of mod-
ulator activity, and was calculated as follows:
Mp 138–141 °C. 1H NMR d 7.40–7.30 (m, 5H, aromatic), 4.68 (br
s, 2H, NH2), 3.76 (s, 2H, NCH2Ph), 3.45 (s, 2H, 7-CH2), 2.85 (t,
J = 5.7 Hz, 2H, 5-CH2), 2.66 (t, J = 5.7 Hz, 2H, 4-CH2). 13C NMR
(75.4 MHz) d 161.0, 137.9, 131.0, 129.2, 128.7, 128.5, 127.5,
117.8, 115.4, 61.8, 50.8, 49.61, 24.7.
K-score ¼ 100 ꢀ ðB ꢃ BoÞ=ðBeq ꢃ BoÞ
where B = residual binding (cpm) bound at the end of 10 min of dis-
sociation in the presence of an AE, Bo = residual binding (cpm) at the
end of 10 min of dissociation in the absence of an AE and Beq = cpm
bound at the end of 90 min of equilibrium binding. The percentage
of specific binding remaining after 10 min of dissociation consti-
tutes an index of modulator activity for ranking candidate com-
pounds. A score of 100% means no dissociation and a score of zero
means complete dissociation. Note, we have previously referred to
the K-score as the AE score, but this implies that a retardation of
dissociation kinetics will always result in allosteric enhancement
at equilibrium, which is not always the case.
4.26. 2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carbonitrile (8b)
N-Methyl-4-piperidone 1b (2.0 g, 17.67 mmol) and malononit-
rile (1.17 g, 17.67 mmol) were dissolved in EtOH (10 mL). Sulfur
(577 mg, 18.0 mmol) and diethylamine (2 mL) were added and
the mixture was heated to 60 °C for 1 h then left to stir at room
temperature overnight. The resultant precipitate was collected by
suction filtration and washed to EtOH to form the product as a yel-
low solid (1.75 g, 51% yield).
4.29. A1AR-mediated ERK 1/2 phosphorylation
Mp 183–185 °C. 1H NMR (DMSO-d6) d 7.00 (br s, 2H, NH2), 3.24
(s, 2H, 7-CH2), 2.62–2.58 (m, 2H, 5-CH2), 2.48–2.40 (m, 2H, 4-CH2),
2.32 (s, 3H, NCH3). 13C NMR (DMSO-d6) d 163.9, 130.0, 116.5, 114.9,
83.2, 52.7, 51.6, 45.3, 24.8. HR-ESMS calcd for C9H12N3S+ (M+1)
194.0746, found 194.0732.
Chinese hamster ovary (CHO) FlpIn cells, stably transfected
with the human A1AR (FlpIn-CHO A1 cells), were grown to 90% con-
fluence and maintained in Dulbecco’s modified eagle medium
(DMEM) containing 20 mM HEPES, 5% fetal bovine serum (FBS)
and 200 lG/mL of hygromycin at 37 °C in a humidified incubator
containing 5% CO2: 95% O2. Cells were then harvested by trypsin-
ization followed by centrifugation (300 g, 5 min). Cells were then
seeded into 96-well plates at a density of 50,000 cells/well. After
4 h, the cells were washed twice with phosphate-buffered saline
(PBS) and then maintained in DMEM containing 20 mM HEPES
for at least 4 h. Prior to agonist stimulation, cells were pretreated
for 30 min with 1 u/mL adenosine deaminase (ADA) and then for
4.27. tert-Butyl 2-amino-3-cyano-4,5-dihydrothieno[2,3-c]pyri-
dine-6(7H)-carboxylate (8c)
N-Boc-4-piperidone 1c (0.5 g, 2.51 mmol) and malononitrile
(182 mg, 2.76 mmol) were dissolved in EtOH (5 mL). Sulfur
(85 mg, 2.64 mmol) and morpholine (200 lL, 2.29 mmol) were
added and the mixture was heated to 70–80 °C for 2 h. The mixture
was cooled on an ice bath and the resultant precipitate was col-
lected by suction filtration and washed with ice cold EtOH. This
afforded the product as a pale peach solid (440 mg, 63% yield).
30 min with test compound (3 or 10 lM for initial screens; more
detailed concentration–response analyses for compounds 2c, 2d
and 2e) at 37 °C. R-PIA was then added and stimulation allowed
to proceed for 5 min before the reaction was terminated by the re-

L. Aurelio et al. / Bioorg. Med. Chem. 17 (2009) 7353–7361
7361
moval of media and the addition of 100
l
L of SureFireTM lysis buffer
References and notes
to each well. The plate was then agitated for 1–2 min. A 4:1 v/v
dilution of lysate:SureFireTM activation buffer was made in a total
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volume of 50
beads:activated lysate mixture:SureFireTM reaction buffer in
11 L total volume was then transferred to a white opaque 384-
l
L. A 1:100:120 v/v dilution of AlphaScreenTM
a
l
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Acknowledgments
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This research was supported by Discovery Grant DP0558184 of
the Australian Research Council, Project Grant 400134 of the Na-
tional Health and Medical Research Council (NHMRC) of Australia,
and National Institutes of Health Grant R01 HL56111. A.C. is a Se-
nior, and P.M.S. a Principal, Research Fellow of the NHMRC. We are
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