Synthesis and structure-activity relationships of thieno[2,3-b]pyrroles as antagonists of the GnRH receptor

Article abstract of DOI:10.1016/j.bmcl.2007.09.099

A new class of small-molecule GnRH antagonists, the thieno[2,3-b]pyrroles, was designed. Herein, the synthesis and structure-activity relationships are described. Substitution at the C4 position was investigated; during this study, it was observed that in

Full text of DOI:10.1016/j.bmcl.2007.09.099

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Bioorganic & Medicinal Chemistry Letters 17 (2007) 6448–6454
Synthesis and structure–activity relationships
of thieno[2,3-b]pyrroles as antagonists of the GnRH receptor
a
a
b
Jean Claude Arnould,a, Benedicte Delouvrie, Pascal Boutron, Al G. Dossetter,
*
´ ´
´
Kevin M. Foote,b Annie Hamon,a Urs Hancox,b Craig S. Harris,a Mike Hutton,b
Maryannick Lamorlettea and Zbigniew Matusiakb
aAstraZeneca, Centre de Recherches, PI Pompelle, BP 1050, 51689 Reims Cedex 2, France
bAstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Received 4 September 2007; revised 28 September 2007; accepted 29 September 2007
Available online 4 October 2007
Abstract—A new class of small-molecule GnRH antagonists, the thieno[2,3-b]pyrroles, was designed. Herein, the synthesis and
structure–activity relationships are described. Substitution at the C4 position was investigated; during this study, it was observed
that introducing piperazines and piperidines improved the physical properties of the compounds while retaining good in vitro
potency. This exploration led to the discovery of amidopiperidines with improved pharmacokinetic properties.
Ó 2007 Elsevier Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH), also
known as LHRH, is a decapeptide hormone secreted
and produced by the hypothalamus. It binds to
GnRH receptors on the pituitary and stimulates the
release of follicle stimulating hormone (FSH) and
luteinising hormone (LH) which regulate gonadal ste-
roid hormone production regulating in turn progester-
one, oestrogens and androgens.1,2 Several hormone
dependent diseases such as endometriosis, breast and
prostate cancer, caused by over-stimulation of the go-
nadal steroids, can be treated by GnRH peptide-based
agonists. Antagonism of the GnRH receptor consti-
tutes a valid alternative to agonists. They compete
with endogenous GnRH at the receptor level and pro-
voke a rapid suppression of the formation of LH
without the concomitant ‘flare’ effect which is induced
by peptide agonists in reaction to the initial over-stim-
ulation of the receptors.3a Several peptide antagonists
have been evaluated in the clinic for the treatment
of hormone-sensitive diseases.3 Orally administered
small-molecule GnRH antagonists would present an
advantage over the peptide antagonists and efforts to-
wards orally active GnRH receptor antagonists have
been reported in the literature (Fig. 1).4 T-98475 (1)
and analogues were the first orally active small mole-
cules to be disclosed.5 Indole derivatives (2) were re-
ported to be potent and orally active GnRH
antagonists.6 Uracil derivatives (3) with potent antag-
onist activity have also been disclosed.7
In this paper, we describe our efforts towards potent or-
ally bioavailable small-molecule GnRH antagonists and
disclose the SAR study of thieno[2,3-b]pyrroles 4
(Fig. 2).8
The thienopyrrole core 5 with the functional amino
group on the C4 side-chain was constructed by a Fischer
indole approach according to Scheme 1.9
Commercially available ethyl thiophen-2-ylacetic acid
ester was dimethylated with an excess of CH3I in the
presence of NaH. The resulting thiophene 7 was selec-
tively nitrated with NO2BF4 to form 8 which was re-
duced by catalytic hydrogenation over palladium on
charcoal to give the desired 2-aminothiophene. Boc pro-
tection of the amine gave carbamate 9 which was depro-
tonated with NaH and aminated with O-
(diphenylphosphinyl)hydroxylamine, to afford hydra-
zine 10.10 Ketone 11 was condensed with hydrazine 10
under tailored Fischer indole conditions to give pro-
tected thieno[2,3-b]pyrrole 12.8 Deprotection and Boc
reprotection provided intermediate 13. Introduction of
Keywords: GnRH; Receptor; Antagonist; Thieno[2,3-b]pyrrole;
LHRH.
*
Corresponding author. Tel.: +33 326616848; fax: +33 326616842;
e-mail: jean-claude.arnould@astrazeneca.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.099

J. C. Arnould et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6448–6454
6449
O
N
F
O
N
N
O
N
F
O
N
H
N
O
N
O
H2N
O
S
O
F
F
N
F
N
1 : T-98475
2
3: NBI-42902
Figure 1. Small-molecule GnRH receptor antagonist.
R
R
NH2
OH
N
R
O
O
R1
O
R1
R1
N
R2
N
R2
N
N
N
R2
N
S
S
S
5
6
4
Figure 2. General synthesis of thieno[2,3-b]pyrroles.
O
O
O
O
O
a
c,d
b
e
O
O
O
O
O
S
S
NO2
N
S
S
H
O
NH2
P
O
7
8
9
O
O
N
Phth
NH2
NBoc
O
N
O
O
i, j, k
g, h
11
O
O
O
O
O
N
NH2
R1
S
N
EtO
N
EtO
N
N
S
S
f
S
R2
10
O
12
13
5
OH
14
l
OH
OH
N
OH
m
n
O
O
O
R1
N
O
N
HO
N
S
S
S
R2
15
6
16
Scheme 1. Reagents and conditions: (a) NaH 60%, THF, 0 °C; CH3I, THF, 18-crown-6, 5 h; 90%; (b) NO2BF4, DME, À55 °C 5 h then À5 °C
overnight, 88%; (c) 10% Pd/C, EtOH/EtOAC, H2, 9 h, 100%; (d) Boc-O-Boc, THF, reflux, 16 h, 68%; (e) NaH 60%, DMF, 5 °C, 2 h, overnight, 95%;
(f) 11, 2-butanol, ZnCl2 120 °C, 6 h; (g) EtOH, NH2–NH2, H2O, 75%, 4 h; (h) MeOH, TFA, Boc-O-Boc, 60 °C, 4 h, quant; (i) NaOH 1 N, EtOH,
60 °C, 4 h, quant; (j) 7-azabicyclo[2.2.1]heptane, HCl, DIPEA, HATU, CH2Cl2, 0.5 h; quant; (k) CH2Cl2, TFA, 15 min, 57%; (l) 14, 2-butanol,
ZnCl2, 110 °C, 5 h, 60%; (m) EtOH, NaOH 1 N, 60 °C, 4 h, 95%; (n) 7-azabicyclo[2.2.1]heptane, HCl, DIPEA, HATU, CH2Cl2, 0.5 h, 70%.
the amide group at the C2 position was achieved by
saponification and a subsequent amide coupling under
basic conditions using HATU.11 Deprotection with
TFA afforded the key intermediate 5. A similar ap-
proach gave the hydroxy thienopyrrole core 6.
anates to core 5 and subsequent reaction of the interme-
diate 18 with secondary amines. Ureas 23–25 were
prepared by reaction with p-nitrophenylchloroformate
and further reaction with amines.
The synthesized compounds12 were evaluated for their
ability to compete for [125I]-D-Trp6 GnRH radioligand
binding to rat and human receptors.13 Their functional
antagonism activity was established by inhibition of
GnRH stimulated LH release.14
A series of thienopyrroles incorporating non-basic
N-acylguanidino and urea groups was prepared accord-
ing to Scheme 2. Synthesis of acylguanidines 19–22 was
achieved by the addition of N-alkoxycarbonylisothiocy-

6450
J. C. Arnould et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6448–6454
R3
R3
O
O
O
R4
N
NH2
N
O
H
N
N
HN
N
S
R4
HN
R5
R5
O
R1
N
R2
a
N
b
S
O
R1
O
N
R1
S
R2
N
R2
N
S
5
18
19-22
O
R4
N
c,d
HN
R5
O
R1
N
N
S
R2
23-25
R
OH
R'
Cl
N
e
f
O
O
O
N
N
N
N
N
N
S
S
S
6
26
27-45
Scheme 2. Reagents and conditions: (a) alkoxycarbonylisothiocyanate, CH2Cl2, TFA, 2 h, quant; (b) CH2Cl2, EDCI, DIEA, NR4R5, overnight,
63%; (c) 4-nitrophenylchloroformate, Et3N, CH2Cl2, 0 °C, 10 min, 0.5 h, quant; (d) R4R5NH, 2 h 30, 50%; (e) SOCl2, DMF (cat), CH2Cl2, 0 °C,
10 min; 25 °C, 2 h, 85%; (f) K2CO3, NR1R2, CH3CN, DMF, 80 °C.
Table 1. Binding and functional affinities of thienopyrroles 19–25 towards the rat and human GnRH receptors
Compound
R1R2N
R4R5N
R3
r IC50, nMa
h IC50, nMa
r cell IC50, nMa
Sol. lMb
N
19
Me2N
i-Pr
100
244
452
—
N
N
N
20
21
22
i-Pr
Me
i-Pr
15
8
55
35
79
3
0.8
4.6
1.4
N
N
N
N
N
N
3
27
12
N
N
N
23
24
25
—
—
—
64
<1
16
96
2
445
13
1.5
—
N
N
N
N
N
N
56
29
0.6
N
r, rat; h, human; sol., solubility.
a Values are geometric means of several experiments.
b Phosphate buffer 0.1 M, pH 7.4, 25 °C.

J. C. Arnould et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6448–6454
6451
Table 2. Binding and functional affinities of piperazine thienopyrroles 27–35 towards the rat and human GnRH receptors
R1
N
N
O
N
X
S
N
Compound
X
NR1
r IC50, nMa
h IC50, nMa
r cell IC50, nMa
Sol. lMb
O
N
27
CH2
110
162
184
87
N
O
O
N
28
29
CH2
CH2
172
74
177
195
209
89
104
39
N
N
O
N
O
N
30
—
116
495
161
2
N
N
O
N
31
32
33
34
C@O
CH2
CH2
CH2
101
529
104
155
170
696
345
331
197
—
71
O
N
—
N
O
N
947
280
107
236
O
N
N
N
O
N
O
O
N
N
N
N
35
CH2
55
74
171
425
r, rat; h, human; sol., solubility.
a Values are geometric means of several experiments.
b Phosphate buffer 0.1 M, pH 7.4, 25 °C.
Table 1 lists the binding affinity data, functional activ-
ity and solubility for compounds 19–25. Replacement
of the diethylamide or pyrrolidine amide group at the
C2 position by more lipophilic bicyclic amides led to
a dramatic improvement in the affinity for GnRH
receptors (rat and human) which translated into the
functional assay (20 vs 19; 24 vs 23). Solubility and free
drug (not shown) were improved by replacement of the
isopropoxy group by methoxy group (21 vs 20).
Replacement of the pyrrolidinopyridine group by
piperidinopyridine led to less potent compounds (22
vs 20; 25 vs 24).
Overall, the introduction of acylguanidine or urea side-
chains at the C4 position of the thieno[2,3-b]pyrrole
gave very potent compounds. This suggests that there
is no requirement for the side-chain to be basic.6 Never-
theless, the physical properties were less than desired,

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J. C. Arnould et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6448–6454
Table 3. Binding and functional affinities of piperidine thienopyrroles 36–46 towards the rat and human LHRH receptors
R1
N
O
N
X
S
N
Compound
X
R1
r IC50, nMa
h IC50, nMa
r cell IC50, nMa
AUC lM.h
O
36
CH2
51
178
191
0.006b
N
O
37
CH2
94
275
743
0.018b
N
N
O
O
N
38
39
40
CH2
C@O
CH2
51
132
371
233
111
238
381
0.041b
0.360b
27.1d
O
O
O
108
64
N
O
N
O
O
O
41
42
43
CH2
CH2
CH2
30
62
76
169
173
184
214
210
216
39.3d
190d
358d
N
O
O
OMe
N
N
O
O
N
44c
CH2
43
88
220
0.254d
O
N
O
O
45
46
CH2
CH2
116
30
338
33
117
36
0.526d
0.002b
N
O
O
N
a Values are geometric means of several experiments.
b AUC dosed in rat at 1 mg/kg po (vehicle: propylene glycol).
c Drawing represents piperidine-R1.
d AUC dosed in rat at 10 mg/kg po (vehicle: propylene glycol).

J. C. Arnould et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6448–6454
6453
resulting in poor bioavailabilities, therefore further
improvement was needed.
and 35%, respectively, compared to 4% for 27). Further-
more, those compounds showed an ability to inhibit the
production of LH after oral administration in both
acute and chronic in vivo rat models. For example, com-
pound 40 administered orally at 75 mg/kg bid provided
prolonged suppression of circulating LH levels in a cas-
trated rat model.16
Introduction of piperazine, pyrrolidine and piperidine
groups was then considered in an attempt to improve
the physical properties. These compounds were pre-
pared by reaction of the appropriate amines with the
chloro intermediate 26 derived from core 6, in the pres-
ence of a base (Scheme 2).
In conclusion, we have developed a route to a new class
of small-molecule antagonists of the GnRH receptor.
Introduction of substituted piperazines and piperidines
at the C4 position substantially improved the physical
properties. In these series, amidopiperidines showed im-
proved pharmacokinetic parameters while retaining
good potency.
Right-hand side-chains with substituted piperazines
contributed to improved physical properties, more spe-
cifically the solubility (Table 2). This was achieved to
the detriment of binding and functional activities that
were generally reduced. A rigid one-carbon linker be-
tween the thienopyrrole nucleus and the piperazine led
to poor solubility (30 vs 29). On the contrary, introduc-
tion of a carbonyl function on the linker generally im-
proved solubility (31 vs 29).
Acknowledgments
We acknowledge the excellent technical expertise of the
following scientists: Dawn Brison; Rob Davies; Del-
phine Duval; Mickael Maudet; Adina Hughes and Dia-
na Stafford. We thank Madeleine Brady, Geoff Bird and
Romuald Laine for helpful discussions.
Variation of R1 showed that amidomethyl substitution
on the piperazine was giving good potency associated
with overall good physical properties. Potency decreased
dramatically with amide substitution (32). We observed
during our investigation that functional activity was cor-
related with lipophilicity (33 vs 29). We also discovered
during this study that some compounds were inhibitors
of the cytochrome P450 3A4 enzyme. In fact, suscepti-
bility to Cyp 3A4 is a general feature of this class of lipo-
philic antagonists.6h,i Nevertheless, we succeeded in
diminishing the 3A4 inhibition with a variety of our
piperazine compounds, as exemplified by 34 and 35
(3A4 IC50: 6 lM and >10 lM, respectively, compared
to 0.1 lM for 27).15
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oral bioavailability as exemplified by 42 and 43 (38%

6454
J. C. Arnould et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6448–6454
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expressing human GnRH receptors. [125I]-D-Trp6 GnRH
having specific activity of 20 Ci/mmol was used as the
radiolabelled ligand. Competitive binding was measured
in a 80 mM Tris (4 mM MgCl) buffer containing 0.25%
bovine serum albumin BSA, pH 7.4, and the compound of
interest at concentrations between 0.3 and 3000 nM in a
final concentration of 0.5% DMSO with a total incubation
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[
125I]-D-Trp6 GnRH to receptors by 50%.
14. Functional assay. Primary pituitary cells were isolated
from >12-week-old female AP Han Wistar rats by
enzymatic digestion with a mixture of collagenase and
pancreatin. The suspended cells were then cultured for 4
days in 24-well tissue culture plates. On the day of the
assay, the cells were washed and then treated with medium
containing 10 nM GnRH and the compound of interest at
concentrations between 3 and 3000 nM in a final concen-
tration of 0.1% DMSO. After a 4-h incubation, the
medium was removed and centrifuged. The supernatant was
then analyzed for content of LH using a EIA kit specific for
rat LH (Amersham). The results are reported as an IC50
value, which is the antagonist concentration, required to
inhibit the GnRH stimulated LH release by 50%.
15. The microsomes were obtained from yeast expressing the
human CYP3A4 and the human cytochrome P450 reduc-
tase and were provided by AstraZeneca Biotech Labora-
tories (ABL) Sodertalje, Sweden. IC50 was determined
using 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC) as
the substrate.
16. Chronic LH suppression. 12-week-old male AP Han Wistar
rats were castrated. At least 10 days post surgery
compound (75 mg/kg) or vehicle (ICME Imwitor 988
49%, Cremaphor RH40 27%, Miglyol 12%, ethanol 13%)
alone was administered by oral gavage bid at a dose
volume of 5 ml/kg. Terminal blood samples were taken
12 h after either the first or second dose. Plasma LH levels
were measured by rat LH specific radioimmunoassay
(Amersham). Inhibition is reported as a percentage of
control plasma LH.
8. (a) Foote, K. M.; Matusiak, Z.; Dossetter, A. G.;
´
Arnould, J. C.; Lamorlette, M. A.; Delouvrie, B.; Hamon,
A. WO 2004-018480, Chem. Abstr. 2004, 182888;