Novel, potent and selective 17β-hydroxysteroid dehydrogenase type 2 inhibitors as potential therapeutics for osteoporosis with dual human and mouse activities

Article abstract of DOI:10.1016/j.ejmech.2014.06.036

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) is responsible for the oxidation of the highly active estradiol (E2) and testosterone (T) into the less potent estrone (E1) and Δ⁴-androstene-3,17-dione (Δ⁴-AD), respectively. As 17β-HSD2 is present in bones and as estradiol and testosterone are able to induce bone formation and repress bone resorption, inhibition of this enzyme could be a new promising approach for the treatment of osteoporosis. Herein, we describe the design, the synthesis and the biological evaluation of 24 new 17β-HSD2 inhibitors in the 5-substituted thiophene-2-carboxamide class. Structure-activity and structure-selectivity relationships have been explored by variation of the sulfur atom position in the central core, exchange of the thiophene by a thiazole, substitution of the amide group with a larger moiety, exchange of the N-methylamide group with bioisosteres like N-methylsulfonamide, N-methylthioamide and ketone, and substitutions at positions 2 and 3 of the thiophene core with alkyl and phenyl groups leading to 2,3,5-trisubstituted thiophene derivatives. The compounds were evaluated on human and mouse enzymes. From this study, a novel highly potent and selective compound in both human and mouse 17β-HSD2 enzymes was identified, compound 21 (IC ₅₀(h17β-HSD2) = 235 nM, selectivity factor toward h17β-HSD1 = 95, IC₅₀ (m17β-HSD2) = 54 nM). This new compound 21 could be used for an in vivo proof of principle to demonstrate the true therapeutic efficacy of 17β-HSD2 inhibitors in osteoporosis. New structural insights into the active sites of the human and mouse enzymes were gained.

Full text of DOI:10.1016/j.ejmech.2014.06.036

European Journal of Medicinal Chemistry 83 (2014) 317e337
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel, potent and selective 17b-hydroxysteroid dehydrogenase type 2
inhibitors as potential therapeutics for osteoporosis with dual human
and mouse activities
,
Enrico Perspicace a, Liliana Cozzoli a d, Emanuele M. Gargano a, Nina Hanke c,
,
b
,
, *
Angelo Carotti d, Rolf W. Hartmann a *, Sandrine Marchais-Oberwinkler a
a Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C23, D-66123 Saarbrücken, Germany
b Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C23, D-66123 Saarbrücken, Germany
c ElexoPharm GmbH, Campus A12, D-66123 Saarbrücken, Germany
d
ꢀ
Dipartimento Farmaco-Chimico, Universita degli Studi di Bari, V. Orabona 4, I-70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
17
active estradiol (E2) and testosterone (T) into the less potent estrone (E1) and
4-AD), respectively. As 17
-HSD2 is present in bones and as estradiol and testosterone are able to
induce bone formation and repress bone resorption, inhibition of this enzyme could be a new promising
approach for the treatment of osteoporosis. Herein, we describe the design, the synthesis and the bio-
b
-Hydroxysteroid dehydrogenase type 2 (17
b
-HSD2) is responsible for the oxidation of the highly
D
4-androstene-3,17-dione
Received 5 March 2014
Received in revised form
12 June 2014
Accepted 17 June 2014
Available online 17 June 2014
(
D
b
logical evaluation of 24 new 17b-HSD2 inhibitors in the 5-substituted thiophene-2-carboxamide class.
Structureeactivity and structureeselectivity relationships have been explored by variation of the sulfur
atom position in the central core, exchange of the thiophene by a thiazole, substitution of the amide
group with a larger moiety, exchange of the N-methylamide group with bioisosteres like N-methyl-
sulfonamide, N-methylthioamide and ketone, and substitutions at positions 2 and 3 of the thiophene
core with alkyl and phenyl groups leading to 2,3,5-trisubstituted thiophene derivatives. The compounds
were evaluated on human and mouse enzymes. From this study, a novel highly potent and selective
Keywords:
17b-hydroxysteroid dehydrogenase type 2
Inhibitor
Estrogen receptor
Osteoporosis
Structureeactivity relationships
compound in both human and mouse 17
HSD2) ¼ 235 nM, selectivity factor toward h17
compound 21 could be used for an in vivo proof of principle to demonstrate the true therapeutic efficacy
of 17 -HSD2 inhibitors in osteoporosis. New structural insights into the active sites of the human and
mouse enzymes were gained.
b-HSD2 enzymes was identified, compound 21 (IC50(h17
b-
b
-HSD1 ¼ 95, IC50 (m17 -HSD2) ¼ 54 nM). This new
b
b
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
activities of bone-resorbing osteoclasts (OC) and bone-forming
osteoblasts (OB).
A healthy skeleton is maintained through life by the constant
process of bone remodeling, which is regulated by the balanced
Osteoporosis is a silent bone disease often appearing after the
age of 50 in both men and women. It is characterized by a change in
bone microarchitecture, leading to a reduction in bone mass den-
sity and an increase in the risk of bone fracture [1]. Osteoporosis is
caused by the uncoupling of bone resorption from bone formation
in a sense that the OC activities by far outweigh those of the OB [2].
There are many factors responsible for the development of
osteoporosis such as: vitamin D deficiency, low calcium intake,
smoking, alcohol absorption, but also premature ovarian failure,
adrenal insufficiency, etc. Furthermore, a decrease in the active sex
steroids levels (estradiol, e.g. in women during menopause, or
testosterone in aging men) [3,4] is also considered as risk factor.
Among the different medications available, two first-line classes of
List of abbreviations: OB, osteoblast; OC, osteoclast; 17
b-HSD2, 17b-hydrox-
ysteroid dehydrogenase type 2; 17 -HSD1, 17 -hydroxysteroid dehydrogenase type
b
b
1; E1, estrone; E2, estradiol; ER, estrogen receptor; SAR, structureeactivity rela-
tionship; SSR, structureeselectivity relationship; RBA, relative binding affinity; IR,
infrared; DMF, dimethylformamide; DME, dimethoxyethane; THF, tetrahydrofur-
ane; SF, selectivity factor.
* Corresponding authors. Pharmaceutical and Medicinal Chemistry, Saarland
University, Campus C23, D-66123 Saarbrücken, Germany.
E-mail addresses: rolf.hartmann@helmholtz-hzi.de, rwh@mx.uni-saarland.de
(R.W. Hartmann), s.marchais@mx.uni-saarland.de (S. Marchais-Oberwinkler).
http://dx.doi.org/10.1016/j.ejmech.2014.06.036
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

318
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
drugs are administered to osteoporotic patients: 1. bisphospho-
nates (e.g. alendronate), which show good effects on bones in both
postmenopausal women [5] and men [6,7] but only lead to a 50%
reduction of the fracture risk and are often associated with jaw
osteonecrosis; 2. Selective estrogen receptor modulators (SERMs),
such as raloxifene [8], that are associated with an increased risk of
venous thromboembolism [9], hot flushes [10] and leg cramps [10].
Osteoporosis is an aged-related disease and as the population is
getting older, this disease is considered a serious public health
problem. Therefore, there is a need to develop new therapeutic
strategies to combat this disease.
1 (Fig. 1) resulted in a slight decrease in bone resorption and an
increase in bone formation [33-35].
We were interested in gaining deeper insight into the struc-
tureeactivity and structureeselectivity relationships (SAR and SSR,
respectively) of the 2,5-thiophene carboxamide class, which looked
very promising, to establish a basis for the rational design of even
more potent and selective inhibitors of the human 17
newly designed compounds should not inhibit 17
b
-HSD2. The
b
-HSD1, the
enzyme catalyzing the conversion of E1 into E2, as this would be
contra-productive for the increase of estradiol level in bones.
Furthermore the new 17
finity to the estrogen receptors alpha and beta (ER
keep the receptors free for E2 binding. In addition we also wanted
to develop 17 -HSD2 inhibitors able to block both the human and
b-HSD2 inhibitors should not present af-
17b
-Hydroxysteroid dehydrogenase type
2
(17
b-HSD2,
a
and ER ) to
b
(EC1.1.1.51) [11,12] catalyzes the oxidation of the highly active sex
steroids estradiol (E2), as well as testosterone (T), into their less
b
active forms estrone (E1) and
respectively, using NADþ as cofactor. 17
reverse reaction and is regarded as a target for the treatment of
breast cancer and endometriosis [13]. 17 -HSD2 and 17 -HSD1 are
D
4-androstene-3,17-dione (
D
4-AD),
rodent enzymes in order to evaluate, later on, the efficacy of the
compounds in vivo in an appropriate animal model. As the mouse is
very well characterized, small and easily available, it was decided to
b
-HSD1 catalyzes the
b
b
look for compounds able to inhibit both the mouse m17
the human h17 -HSD2.
b-HSD2 and
considered molecular switches, regulating the levels of active E2
and T in the cells. Further activation of T to the most potent
b
Our work is divided into two main parts: 1. Structural modifi-
cations in the thiophene carboxamide class to increase our
knowledge on the SAR in these series of compounds regarding the
androgen DHT is catalyzed by 5
used for treating benign prostatic hyperplasia [14e16].
17 -HSD2 is expressed in a number of tissues such as placenta,
a-reductase, inhibitors of which are
b
human enzyme (h17b-HSD2); 2. identification of compounds with
liver, small intestine, endometrium, urinary tract and to a lesser
extent in kidney, pancreas, colon, uterus, breast and prostate. This
enzyme is also present in osteoblastic cells (OB) [17e20].
good inhibitory activity on the mouse 17
comparison.
b
-HSD2 for species
In this study, we synthesized new 17
b-HSD2 thiophene car-
Estrogens are known to play a positive key role in bone physi-
ology, decreasing bone resorption. Their mode of action on bone
boxamide inhibitors derived from lead compounds 2aec and 3aec,
which showed very good potency on the mouse enzyme, moderate
density is not well understood so far. However, as 17
b
-HSD2 is
activity on the human enzyme and a good selectivity toward h17b-
expressed in OB, inhibition of this enzyme will lead to an intra-
cellular increase of E2 and T levels in bones, which should be
beneficial for osteoporosis patients.
Besides ours [21e29] there are only few non-steroidal inhibitors
of the human 17b-HSD2 (h17b-HSD2) described in literature
[30,31]. We recently reported [22,28] about substituted 2,5-
thiophene carboxamide derivatives (compounds 2aec and 3aec,
Fig. 1) as new h17b-HSD2 inhibitors. We also assessed the h17b-
HSD1. Starting from 2aec and 3aec, several structural modifica-
tions were performed such as: variations of the substitution pat-
terns on the central thiophene core, replacement of the central
thiophene core by a thiazole ring, change of the amide by a sul-
fonamide group (as successfully done in a previous study [27]), by a
thioamide and a ketone group with a methylene linker, and ex-
change of the N-methyl group on the amide by a bulkier group. In
order to investigate the electronic properties role on the activities, a
short study using infrared spectroscopy was performed on selected
compounds. The synthesized compounds, together with a selection
of previously described inhibitors as references (2aec, 3aec and
4aec) were tested for their inhibitory activity on both the human
HSD2 inhibitory activity of bishydroxyphenyl-2,5-thiophene de-
rivatives 4aec [32]. Bagi and his colleagues developed the cis-
pyrrolidinone class [4]. They validated the concept of improving
bone quality by inhibiting 17b-HSD2 using a monkey osteoporosis
model [4]. They could demonstrate that treatment with compound
and the mouse 17b-HSD2. The SAR obtained was discussed and a
Fig. 1. Described h17b-HSD2 inhibitors with inhibitory activities expressed as IC50 or percent inhibition values when tested at a concentration of 1 mM and selectivity factors (SF)
toward h17 -HSD1.
b

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
319
compound with a high inhibitory potency toward m17
identified.
b
-HSD2 was
applying a SuzukieMiyaura cross-coupling reaction with m-tolyl
boronic acid using microwave irradiation. The acidic hydrogen in
position 2 of the thiazole 17b was easily removed using n-butyl
lithium as strong base, which reacted immediately with a dry flow
of carbon dioxide leading to the corresponding carboxylate 17a. The
isolation of the protonated carboxylic acid derivative failed due to
its chemical instability (rapid decarboxylation), while the lithium
carboxylate salt could be isolated and stored at room temperature.
Consequently, in the last step, the carboxylate 17a itself was acti-
vated as acid chloride using oxalyl chloride at 0 ꢀC and reacted with
3-methoxy-N-methylaniline to afford the desired compound 17.
The synthesis of the 2,3,5-trisubstituted thiophenes 19a and 19
was performed as shown in Scheme 4 starting from the commer-
cially available thiophene-3-carboxylic acid 20. First, the dibromi-
nation of the thiophene-3-carboxylic acid 20 was performed giving
the 2,5-dibromothiophene-3-carboxylic acid 20b. The acyl chlo-
ride, freshly prepared from the corresponding carboxylic acid, was
condensed with the 3-methoxy-N-methylaniline derivative to give
the amide 20a. Double SuzukieMiyaura cross-coupling reaction
was performed with 3-methoxyphenylboronic acid affording
compound 19a in good yield. Ether cleavage using BF3.SMe2
resulted in the hydroxy derivative 19.
The 2,3,5-trisubstituted thiophene 21 was prepared as depicted
on Scheme 5 starting from the 5-bromo-3-methylthiophene-2-
carboxylic acid 22 which was obtained following a described pro-
cedure [37]. The freshly prepared acyl chloride was condensed with
the 3-methoxy-N-methylbenzylamine to give compound 22b.
Ether cleavage was performed using BF3.SMe2 affording the hy-
droxy derivative 22a followed by SuzukieMiyaura cross-coupling
reaction to afford the 2,3,5-trisubstituted thiophene 21.
The synthesis of N-methylthioamide derivatives 23a and 23 was
carried out by the reaction of the N-methylamide derivative 23b
with Lawesson reagent under dry conditions. Ether cleavage using
BF3.SMe2 afforded the hydroxylated compound 23 as depicted in
Scheme 6.
2. Results
2.1. Chemistry
The synthesis of the thiophene derivatives 5ae8a and 5e8 was
achieved following a two- or three-step reaction pathway starting
from the 5-bromo-thiophene-3-carboxylic acid 9a or 4-bromo-
thiophene-2-carboxylic acid 9b as shown in Scheme 1. Carbonyl
chloride derivatives were freshly prepared from the corresponding
carboxylic acid with thionyl chloride. The amide coupling was
carried out by reaction of the carbonyl chloride with 3-methoxy-N-
methylaniline or 3-methoxy-N-methylbenzylamine under inert
atmosphere and in dry conditions providing the corresponding
bromothiophene derivatives 5be8b in very good yields. Suzu-
kieMiyaura cross-coupling reaction was performed with 3-
methoxyphenylboronic acid and led to the compounds 5ae8a in
very good yields. Ether cleavage using boron trifluoride dimethyl
sulfide complex BF3.SMe2 afforded the hydroxy derivatives 5e8.
Thiazoles 10 and 11 were synthesized using the above procedure
described for the synthesis of compounds 5e8, starting from 2-
bromo-1,3-thiazole-5-carboxylic acid 16a as shown in Scheme 2.
In the case of thiazole derivative 11, ether cleavage on 11b, affording
the hydroxylated key intermediate 11a, was performed before
SuzukieMiyaura cross-coupling reaction.
Thiophenes 12 and 13 were prepared as depicted in Scheme 2
following a similar pathway as described above and using the key
intermediates 15a and 15b, which were synthesized as reported
[36]. The amide formation between compounds 15a and 15b and
freshly prepared 5-bromo-thiophene-2-carbonyl chloride led to
the access of N-cyclopropyl derivatives 12a and 13a. The final
compounds 12 and 13 were obtained after SuzukieMiyaura cross-
coupling reaction. For synthesis of compound 13, ether cleavage
on 13b, leading to the key intermediate 13a, was performed before
the Suzuki reaction.
The synthesis of the sulfonamide derivatives 24 and 25 was
performed as shown in Scheme 7 starting from the commercially
available 5-bromothiophene-2-sulfonyl chloride 28. It was
condensed with 3-methoxy-N-methylaniline or 3-methoxy-N-
For the synthesis of compound 17 the key intermediate 17b was
synthesized starting from the 5-bromo-1,3-thiazole 18 (Scheme 3)
Scheme 1. Synthesis of compounds 5ae8a and 5e8. Reagents and conditions: (i) SOCl2, DMF cat., toluene, reflux 4 h; (ii) Et3N, CH2Cl2, room temperature, overnight; (iii) DME/
EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave (150 ꢀC, 150 W, 20 min); (iv) BF3$S(Me)2, CH2Cl2, room temperature, 3e14 h.

320
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
Scheme 2. Synthesis of compounds 10e13. Reagents and conditions: (i) SOCl2, DMF cat., toluene, reflux 4 h; (ii) Et3N, CH2Cl2, room temperature, overnight; (iii) BF3$S(Me)2, CH2Cl2,
room temperature, 3e14 h; (iv) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave (150 ꢀC, 150 W, 20 min).
methylbenzylamine using tetrabutylammonium hydrogen sulfate
as phase transfer reagent in a non-miscible solvents mixture
affording the brominated intermediates 26 and 27b. Suzu-
kieMiyaura cross-coupling reaction led to the final compound 24.
For compound 25, ether cleavage of 27b into 27a was performed
before the palladium-catalyzed reaction as described above.
The synthesis of the dimethylketone derivatives 29a and 29 was
performed as depicted in Scheme 8. SuzukieMiyaura cross-
coupling reaction led to the key intermediate 30a. A Friedele-
Crafts acylation was performed between the key intermediate 30a,
the freshly prepared acyl chloride 31a and aluminum chloride used
as Lewis acid to afford the methoxylated derivative 29a. Ether
cleavage using BF3.SMe2 led to the hydroxylated compound 29 as
depicted in Scheme 8. Intermediate 31b was synthesized as
described in literature [38] starting from (3-methoxyphenyl)
acetonitrile 31.
Compounds 32a and 32 were synthesized as shown in Scheme
9. 3-Methoxyacetophenone 33 was reacted with Vilsmeier-Haack
reagent to give the expected b-chloroacrolein 33b. This key in-
termediate was used for the preparation of the 2-
methylthiophenecarboxylate derivative 33a as described in the
literature [39]. Saponification of 33a led to the carboxylic acid
Scheme 3. Synthesis of compound 17. Reagents and conditions: (i) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave (150 ꢀC, 150 W, 20 min); (ii) a) dry THF, n-
BuLi, ꢁ78 ꢀCe0 ꢀC, 4 h, b) dry CO2 gas, ꢁ78 ꢀC, 5 h, room temperature, overnight; (iii) a) oxalyl chloride, DMF cat., CH2Cl2, 3 h, b) Et3N, CH2Cl2, room temperature, overnight.

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
321
Scheme 4. Synthesis of compounds 19a and 19. Reagents and conditions: (i) Br2, AcOH, room temperature, 1 h; (ii) SOCl2, DMF cat., toluene, reflux 4 h; (iii) Et3N, CH2Cl2, room
temperature, overnight; (iv) DME/EtOH/H2O (1:1:1), Cs2CO3, 3-methoxyphenylboronic acid, Pd(PPh3)4, microwave (150 ꢀC, 150 W, 20 min); (v) BF3$S(Me)2, CH2Cl2, room tem-
perature, overnight.
derivative 34. Chlorination with thionyl chloride followed by the
addition of N,O-dimethylhydroxylamine hydrochloride in pres-
ence of triethylamine afforded the intermediate 34a. Substitution
of the Weinreb amide by 3-methoxybenzylmagnesium chloride
led to the methoxylated derivative 32a. Of note, the direct Frie-
deleCrafts acylation of 30a with 2-(3-methoxyphenyl)acetyl
chloride) did not succeed in this case so the synthesis of deriv-
ative 32a was performed via the Weinreb amide instead. Ether
cleavage was performed using BF3.SMe2 affording the hydroxy
derivative 32.
2.2. Biology
2.2.1. SAR study on h17b-HSD2
The synthesized compounds were tested for their inhibitory
activity on h17 -HSD2. The inhibition values of the tested com-
b
pounds are shown in Table 1.
2.2.1.1. Influence of the substitution pattern of the thiophene core.
In both the disubstituted 3,5-thiophene and 2,4-thiophene classes
the methoxylated derivatives showed a decreased h17b-HSD2
Scheme 5. Synthesis of compound 21. Reagents and conditions: (i) SOCl2, DMF cat., toluene, reflux 4 h, (ii) Et3N, CH2Cl2, room temperature, overnight; (iii) BF3$S(Me)2, CH2Cl2, room
temperature, overnight; (iv) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave (150 ꢀC, 150 W, 20 min).

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E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
Scheme 6. Synthesis of compounds 23a and 23. Reagents and conditions: (i) Lawesson reagent, dry toluene, reflux, 4 h; (ii) BF3$S(Me)2, CH2Cl2, room temperature, overnight.
Scheme 7. Synthesis of compounds 24 and 25. Reagents and conditions: (i) NBu4HSO4, NaOH 50%, CH2Cl2, room temperature, 5 h; (ii) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4,
microwave (150 ꢀC, 150 W, 20 min); (iii) BF3$S(Me)2, CH2Cl2, room temperature, 3e14 h.
inhibitory activity compared to the 2,5-thiophene. This was
observed for compounds without linker between the amide and
the phenyl C ring, 5a and 7a (621 nM and 487 nM, respectively to be
compared to the 2,5-thiophene 2a: 68 nM) or with linker, 6a and 8a
and 8, compared to the 2,5-thiophene 3b (829 nM, 748 nM and
394 nM for compounds 6, 8 and 3b, respectively). As the 3,5- and
2,4-thiophene derivatives differ from the 2,5-thiophenes in the
position of the S atom only, these results suggest that this S atom,
depending on its place in the ring, might either be able to achieve
specific interaction with the protein thereby modulating strongly
the inhibitory potency or to modify the angles between the thio-
phene ring and the two substituents resulting in a slightly different
position/orientation of the A and C ring, which impacts the in-
teractions with amino acid side chains of the enzyme.
(34% and 27% inhibition at 1 mM, respectively to be compared to the
2,5-thiophene 3a: 63% inhibition). The hydroxylated disubstituted
3,5-thiophene and 2,4-thiophene without linker, 5 and 7, showed a
similar activity compared to the 2,5-thiophene 2b (for n ¼ 0, 44%,
39% and 34% inhibition at 1
mM for compounds 5, 7 and 2b,
respectively) or a decrease in activity for compounds with linker, 6
Scheme 8. Synthesis of compounds 29a and 29. Reagents and conditions: (i) DME/EtOH/H2O (1:1:1), Cs2CO3, Pd(PPh3)4, microwave (150 ꢀC, 150 W, 20 min); (ii) (a) MeI, NaH, dry
THF, 0 ꢀC to room temperature, overnight, (b) KOH, ethylene glycol, 150 ꢀC, overnight; (iii) SOCl2, DMF cat., toluene, reflux, 4 h; (iv) AlCl3, dry CH2Cl2, room temperature, overnight;
(v) BF3$S(Me)2, CH2Cl2, room temperature, overnight.

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
323
Scheme 9. Synthesis of compounds 32a and 32. Reagents and conditions: (i) POCl3/DMF, DMF, 0 ꢀC to room temperature, overnight; (ii) methyl thioglycolate, dry K2CO3, dry DMF,
60 ꢀC, overnight; (iii) KOH, MeOH, H2O, reflux, 4 h; (iv) SOCl2, DMF cat., toluene, reflux, 4 h; (v) N,O-dimethylhydroxylamine hydrochloride, Et3N, CH2Cl2, 0 ꢀC to room temperature,
overnight; (vi) THF, 0 ꢀC to room temperature, overnight; (vii) BF3$S(Me)2, CH2Cl2, room temperature, overnight.
2.2.1.2. Influence of the central heterocyclic core. In order to eval-
uate the importance of the central core, we investigated the ex-
change of the 2,5-thiophene by a 2,5-thiazole ring. Keeping the S
atom constant in its most favorable position between the 2 sub-
stituents, the position of the nitrogen atom on the thiazole core was
varied resulting in two symmetrical compounds 10 and 17 with the
aim to see if it can achieve an additional H-bond interaction in this
area of the binding site. Three thiazole derivatives 10, 11 and 17
a different binding mode or might be slightly shifted as the intro-
duction of the cyclopropyl group does not lead to the same decrease
in activity.
2.2.1.4. Influence of the exchange of the carboxamido group with
sulfonamide, thioamide and ketone groups. We recently described
[27] novel substituted biphenyl-N-methylsulfonamide derivatives
as novel inhibitors of h17b-HSD2. Herein, we wanted to exchange
were synthesized and all showed a loss in h17
activity compared to their 2,5-thiophene analogues 2c and 3c
(621 nM, 38% inhibition at 1 M and 296 nM for the thiazoles 10, 11
b-HSD2 inhibitory
the planar N-methylcarboxamide group by the tetrahedral N-
methylsulfonamide moiety with the aim to investigate the enzyme
binding site and increase the number of potential H-bond in-
teractions between the inhibitor and the protein. However, ex-
change of the N-methylcarboxamide by the N-methylsulfonamide
m
and 17 respectively, in comparison to 58 nM and 61 nM for 2c and
3c, respectively). Given the hypothesis that the H-bond interactions
made by the OH and OMe groups of phenyl A and C ring are impor-
tant for the stabilization of the compounds within its binding site,
introduction of the nitrogen atom in the central ring might establish
either unfavorable electronic and/or hydrophilic interactions.
was detrimental for the 17
without linker (24, 21% vs 2c, 95% inhibition at 1
linker (25, 48% vs 3c, 89% inhibition at 1 M).
b
-HSD2 inhibitory activity in both series
mM) and with
m
Exchange of the amide by a thioamide was then considered to
evaluate whether the oxygen of the carbonyl moiety establishes
any important interaction with the protein. As S is less electro-
negative than O, it will be a better lone-pair donor than the oxygen,
which means that S in C]S will be a weaker H-bond acceptor
compared to the O of the C]O amide, moreover the C]S group is
more lipophilic than the C]O group. For the methoxylated com-
pound without linker, replacement of the C]O by C]S induced a
2.2.1.3. Influence of the substituent on the amide moiety. We pre-
viously described [22] that 2,5-disubstituted thiophene carbox-
amide inhibitors must have an N-methylamide group to be active.
The primary amide and the amide substituted by a phenyl moiety
lose their activity completely. This might suggest that lipophilic
interactions, in a limited space, might play a critical role stabilizing
the inhibitor in the binding site. In this study a cyclopropyl group
was introduced in order to evaluate the volume of this lipophilic
cavity. Two N-cyclopropylamide analogues 12 and 13 were syn-
thesized and turned out to be less active than the reference N-
methylamides 2c and 3c. The N-cyclopropylamide without linker
12 was totally inactive compared to the N-methylamide analogue
2c (IC50 ¼ 58 nM) and the N-cyclopropylamide with a linker 13
showed a decreased activity compared to the N-methylamide
analogue 3c (13, IC50 ¼ 599 nM vs 3c, IC50 ¼ 61 nM). These results
point out two things: 1. The cyclopropyl group in 12 might be too
bulky to fit into the lipophilic pocket. 2. Compound 12 without
linker (inactive) and 13 with linker (moderately active) might have
dramatic loss in activity (23a, 25% vs 2a, 90% inhibition at 1
while in case of the hydroxylated derivative a slight increase in
activity was observed (23, 64% vs 2b, 34% inhibition at 1 M).
mM),
m
Therefore, from these biological results it can be concluded that the
oxygen atom of the C]O in the methoxylated 2,5-thiophene car-
boxamide derivative establishes a stronger H-bond interaction
compared to the one of the hydroxylated compound. Taken
together these results can be explained by a different binding mode
for the methoxylated and the hydroxylated compounds. Moreover,
the different sizes of the covalent radii of the S (104 pm) and the O
(66 pm), the longer C]S bond compared to the C]O bond (1.6 Å
for C]S vs 1.25 Å for C]O) and the higher lipophilicity of the C]S

324
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
Table 1
In vitro h17b-HSD2 and h17b-HSD1 inhibitory potencies of compounds 2ae32.
Entry
Central core
n
R1
R2
R3
% Inhibition at 1
h17
-HSD2a,b
m
M (IC50
)
SFd
b
h17b
-HSD1a,c
2a
0
3-OMe
e
3-OMe
90% (68 nM)
n.i. (7593 nM)
112
2b
2c
3a
3b
3c
0
0
1
1
1
3-OH
3-Me
3-OMe
3-OH
3-OMe
e
3-OH
34%
33%
e
e
3-OMe
3-OMe
3-OH
95% (58 nM)
63% (370 nM)
70% (394 nM)
89% (61 nM)
26% (6752 nM)
n.i. (>10000 nM)
21% (5449 nM)
n.i. (4452 nM)
116
>250
14
e
e
2-F
3-OH
73
5a
0
3-OMe
e
3-OMe
67% (621 nM)
43% (1641 nM)
3
5
6a
6
0
1
1
3-OH
3-OMe
3-OH
e
e
e
3-OH
3-OMe
3-OH
44%
34%
62% (829 nM)
28%
25%
50% (984 nM)
e
e
1
7a
0
3-OMe
e
3-OMe
68% (487 nM)
16% (3071 nM)
6
7
8a
8
0
1
1
3-OH
3-OMe
3-OH
e
e
e
3-OH
3-OMe
3-OH
39%
27%
70% (748 nM)
28%
15%
21% (3096 nM)
e
e
4
10
11
17
0
1
0
3-Me
3-OMe
3-Me
e
3-OMe
3-OH
54% (621 nM)
38%
18% (4821 nM)
17%
8
2-F
e
e
3-OMe
76% (296 nM)
12% (18663 nM)
63
12
13
19a
19
21
0
1
0
0
1
3-Me
3-OMe
OMe
e
3-OMe
3-OH
OMe
OH
n.i.
21
e
2-F
OMe
OH
2-F
59% (599 nM)
n.i.
n.i. (85343 nM)
n.i.
142
e
OH
74% (362 nM)
77% (235 nM)
29% (2485 nM)
n.i. (22298 nM)
7
3-OMe
3-OH
95
23a
23
0
0
0
3-OMe
3-OH
3-Me
e
e
e
3-OMe
3-OH
25%
12%
e
64% (461 nM)
21%
55% (432 nM)
n.i.
1
24
3-OMe
e

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
325
Table 1 (continued )
Entry
Central core
n
R1
R2
R3
% Inhibition at 1
m
M (IC50
)
SFd
h17b
-HSD2a,b
h17b
-HSD1a,c
25
1
0
0
0
0
3-OMe
3-OMe
3-OH
2-F
e
3-OH
48%
28%
23%
21%
e
e
29a
29
3-OMe
3-OH
e
73% (357 nM)
54% (336 nM)
1
32a
32
3-OMe
3-OH
e
3-OMe
3-OH
17%
37%
n.i.
e
e
e
43%
n.i. no inhibition.
a
Mean value of three determinations, standard deviation less than 20% except for 8 h17
Human placental microsomal fraction, substrate E2 [500 nM], cofactor NADþ [1500
M].
M].
b-HSD2: 70% 23%.
b
c
m
Human placental cytosolic fraction, substrate E1 [500 nM], cofactor NADH [1500
m
d
SF: selectivity factor, ratio IC50(h17b-HSD1)/IC50(h17b-HSD2).
group certainly will contribute to the binding affinities of the tested
compounds.
inhibitory activity was completely lost for the methoxylated de-
rivative 19a compared to the unsubstituted 3,5-derivative 5a while
the hydroxylated derivative 19 showed a better activity compared
to the unsubstituted 3,5-derivative 5 (19, 74% (IC50 ¼ 362 nM) vs 5,
Exchange of N-methylamide by a dimethylketo group and by a
N-methylsulfonamide was undertaken to investigate whether the
planarity of the amide is important for binding of the inhibitors to
the enzyme. In the case of the methoxylated derivatives both the
dimethylketone 29a and the N-methylsulfonamides 24 and 25
resulted in a loss of inhibitory activity (29a, 28%, 24, 21% vs 2a, 90%
inhibition at 1 mM, 25, 48% vs 3c, 89% inhibition at 1 mM), indicating
that only a rather planar structure is well tolerated by the enzyme.
The hydroxylated dimethylketone derivative 29 showed a higher
44% inhibition at 1
mM). These results suggest that the 2-
hydroxyphenyl group achieves additional interactions with the
enzyme either via pi-stacking or H-bond interactions or both,
which might partially compensate the unfavorable position of the S
atom (see above). The differences in activities between the
methoxy and the hydroxy compounds (19a and 19) can be
explained by assuming that the compounds bind either in a
different or even in the same area in the enzyme, either way, the
binding pocket seems to accommodate the OH functional groups of
19 better than the methoxy functional groups of 19a.
activity (29, 73% vs 2b, 34% inhibition at 1
mM), implying that
planarity is not so critical for hydroxylated compounds. These re-
sults are also indicative of a different binding mode for the
methoxylated and the hydroxylated compounds.
Furthermore, introduction of the methyl group in position 3
(compound 21) of the 2,5-thiophene 3c slightly decreases its
The loss of activity observed for the methoxylated methylene
ketone derivative 32a (32a, 17% vs 2a, 90% inhibition at 1
m
M) re-
inhibitory activity on h17
b
-HSD2 (21, IC50 ¼ 235 nM vs 3c,
veals the importance of the methyl group in alpha position of the
carbonyl moiety (already highlighted during the investigation of
the amide substituent).
IC50 ¼ 61 nM). This suggests that insertion of a small substituent
such as a methyl group at the position 3 of the central core might
induce a partial steric clash with the enzyme. This result differs
from the one observed after introducing a 3-methyl group into
compound 4a leading to 4b without significant change in inhibitory
activity. These observations highlight the different binding modes
of compounds 4b and 21.
2.2.1.5. 2,3,5-Trisubstituted thiophene vs 2,5-disubstituted thiophene.
Three 2,3,5-trisubstituted thiophene derivatives 19a, 19 and 21
were synthesized in order to further investigate the space available
in the binding site and also with the aim to enable additional in-
teractions. In our previous work, we already described 2,3,5-
trisubstituted inhibitors in the bis(hydroxyphenyl)thiophene
class, compounds 4b and 4c, bearing an additional methyl or
hydroxyphenyl group, respectively. They showed moderate to good
2.2.2. Selectivity: inhibition of h17
estrogen receptors and
As 17 -HSD1 is involved in sex steroid metabolism and more
b-HSD1 and affinities to the
a
b
b
precisely in the reduction of E1 to E2 [41], inhibition of this enzyme
would decrease the concentration of E2 formed in the target cell
inhibitory activity on h17b-HSD2 (856 nM and 188 nM respectively,
Fig. 1) [32,40]. The introduction of a methyl group into position 3 of
the thiophene ring did not improve the inhibitory activity toward
and would be detrimental to the effect of 17
consequence 17 -HSD1 should not interact with the 17
inhibitors. Moreover, 17 -HSD2 inhibitors should not show binding
affinity for the estrogen receptors and (ER and ER ), as it is
expected that E2 effects in the treatment of osteoporosis are ER
mediated. In addition, agonistic effects would induce undesired
side-effects (induction of cell proliferation). Therefore, all com-
b
-HSD2 inhibition. As a
b
b
-HSD2
h17
b-HSD2 compared to the unsubstituted 4a (4b, IC50 ¼ 856 nM vs
b
4a, IC50 ¼ 745 nM) suggesting that the small methyl is not hin-
dering nor is it gaining any specific interaction. With the bulky
hydroxyphenyl group an increase in activity was observed
compared to the unsubstituted derivative 4a (4c, IC50 ¼ 188 nM vs
4a, IC50 ¼ 745 nM). This compound might be stabilized in the
binding site either via pi-stacking interactions (phenyl ring) or H-
bond interactions (OH) or both. We wanted to translate this infor-
mation into the thiophene carboxamide class, starting from the
bishydroxyphenyl thiophene 4a and adding the N-methylamide
chain in order to target additional interactions. Interestingly, the
a
b
a
b
pounds were tested on h17
hibitors were selected and tested for their binding affinity to ER
and ER . The disubstituted 2,4- and 3,5-thiophenes, 2,5-thiophene
thioamides and 2,5-thiophene dimethylketones were rather un-
selective exhibiting selectivity factors toward h17 -HSD1 between
1 and 6 (Table 1). However, in addition to the reference compounds
b-HSD1 and the most interesting in-
a
b
b

326
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
Table 2 (continued)
Table 2
In vitro inhibitory activities of compounds 2ae32 on m17b-HSD2/E1-formation.
Entry Central core
n
R1
R2
R3
% Inh. at 1
mM (IC50
)
m17
b
-HSD2a,b
19a
19
0
0
1
OMe
OH
OMe OMe
13%
42%
OH
OH
21
3-OMe 2-F
3-OH
95% (54 nM)
Entry Central core
n
R1
R2
R3
% Inh. at 1
mM (IC50)
m17
b
-HSD2a,b
2a
0
3-OMe
e
3-OMe 29%
4a
4b
e
e
e
e
e
e
e
e
n.i.
n.i.
2b
2c
3a
3b
3c
0
0
1
1
1
3-OH
3-Me
3-OMe
3-OH
e
e
e
e
3-OH
n.i.
3-OMe 30%
3-OMe 26%
3-OH
3-OH
16%
65%
3-OMe 2-F
4c
e
e
e
e
13%
5a
0
3-OMe
e
3-OMe 33%
5
6a
6
0
1
1
3-OH
3-OMe
3-OH
e
e
e
3-OH
3-OMe 17%
3-OH 58%
20%
n.i. no inhibition.
a
Mean value of three determinations, standard deviation less than 25%.
b
Mouse liver microsomal fraction, substrate E2 [500 nM], cofactor NADþ
[1500 M].
m
7a
0
3-OMe
e
3-OMe 16%
2a, 2c, 3a and 3c, three new h17b-HSD2 inhibitors were highly
7
8a
8
0
1
1
3-OH
3-OMe
3-OH
e
e
e
3-OH
3-OMe 17%
3-OH 38%
12%
selective: the thiazole derivative 17 (SF: 63), the 2,5-thiophene
cyclopropylamide 13 (SF: 142) and the 2,3,5-trisubstituted thio-
phene 21 (SF: 95). The most potent inhibitors 7a, 17, 19, 21 and 4c
were tested for estrogen receptor binding and showed low binding
10
11
17
0
1
0
3-Me
e
3-OMe 14%
affinity to both ER
a and ERb (RBA < 0.1% compared to the affinity of
E2, which was arbitrarily set to 100%).
3-OMe 2-F
3-OH
27%
2.2.3. SAR study on mouse m17
In previous studies [23,28], we evaluated several compounds for
their 17 -HSD2 inhibition of rat, mouse and monkey enzymes with
b-HSD2
3-Me
3-Me
e
e
3-OMe 13%
3-OMe n.i.
b
the aim to identify an animal model suitable for in vivo studies.
Based on the obtained results we hypothesized that the mouse
might be the animal model of choice and we decided to extend our
SAR knowledge on the mouse enzyme in order to design inhibitors
appropriate for mouse in vivo experiments. The inhibitory activities
of the newly synthesized compounds together with the ones of
12
0
1
0
1
0
0
0
0
0
0
13
3-OMe 2-F
3-OH
16%
24
3-Me
e
3-OMe n.i.
their reference analogues (2ae3c) on m17
are reported in Table 2 (expressed as percent inhibition values
tested at a concentration of 1 M).
b-HSD2/E1-formation
25
3-OMe 2-F
3-OH
15%
m
In the disubstituted 2,5-thiophene class with or without linker,
the methoxylated compounds 2a and 3a showed a weak inhibition
(29% and 26% at 1 mM, respectively), while the hydroxylated de-
rivatives 2b and 3b were inactive. Exchange of the methoxy group
on the A-ring by a methyl group leads to an equipotent compound
(2a vs 2c with percent inhibition values of 29% and 30% at 1 mM,
respectively), suggesting that the O of the methoxy moiety is not
involved in H-bond interaction. The addition of a fluorine atom next
to the 3-OMe in the A-ring (compound 3c) notably increases the
activity on the mouse enzyme (3c, 65% of inhibition at 1 mM) as
already observed with the human enzyme [28].
Switching the position of the S atom on the thiophene ring (for
both hydroxy and methoxy derivatives) in compounds without
linker, compounds 5a, 5, 7a and 7, or with linker, compounds 6a, 8a
29a
29
3-OMe
3-OH
e
e
e
e
e
e
3-OMe 17%
3-OH
42%
23a
23
3-OMe
3-OH
3-OMe n.i.
3-OH
26%
32a
32
3-OMe
3-OH
3-OMe 12%
3-OH
n.i.

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
327
Table 3
the N-methylamide was exchanged by a N-methylsulfonamide (2c vs
24: 30% inhibition and no inhibition at 1 M, respectively, and 3c vs
25: 65% and 15% inhibition at 1 M, respectively). Concerning the
Infrared absorption bands of the carbonyl moieties and inhibitory activities of
selected isomeric disubstituted thiophene derivatives.
m
m
2,3,5-trisubstituted derivatives, the compounds can be divided into
two groups: the ones with a 2,5-biphenylthiophene core (4be4c,19a
and 19) and the others with a 2,5-thiophene carboxamide moiety
(21). In the group with the 2,5-biphenylthiophene core, all the com-
pounds are inactive independently of the addition in position 3 of a
methyl 4b, a hydroxyphenyl 4c or a methoxylated carboxamide 19a.
However, for the hydroxylated derivative 19, a moderate activity was
observed (42% inhibition at 1 mM), indicating that the hydroxy groups
certainly participate in the stabilization of the enzymeeligand com-
plex. In the group with the 2,5-thiophene carboxamide core, intro-
ductionofamethylgroup inposition3 of thethiophenecoreresults in
Entry
Substitution
pattern on
thiophene
n
R
n
(C]O) cmꢁ1
IC50 in nM or % inh.
at 1
mM
h17
b-HSD2
a highly active compound (21, IC50 ¼ 54 nM, 95% inhibition at 1
more active than the corresponding compound without methyl (3c,
65% inhibition at 1 M). The comparison of these 2,3,5-trisubstituted
mM),
2a
5a
7a
3a
6a
8a
2b
5
2,5
3,5
2,4
2,5
3,5
2,4
2,5
3,5
2,4
2,5
3,5
2,4
0
0
0
1
1
1
0
0
0
1
1
1
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OMe
3-OH
3-OH
3-OH
3-OH
3-OH
1589
1597
1597
1591
1599
1600
1575
1584
1582
1572
1580
1580
68 nM
621 nM
487 nM
63%
34%
27%
34%
44%
39%
m
thiophenes shows that the amide group is necessary for a good
inhibitory activity, preferably at position 2 in combination with a
methyl group at position 3.
From this study it is clear that compounds of the 2,5-
disubstituted thiophene class are, in general, less potent on the
mouse than on the human enzyme except for the compound with a
linker and an additional fluoro substituent on the phenyl A-ring, 3c,
which shows the best inhibitory activity on mouse E1-formation,
better than compounds of the thiophene class without linker and
the thiazole derivatives. Furthermore, the insertion of a small alkyl
group such as methyl in position 3 of the thiophene, 21, increases
7
3b
6
394 nM
829 nM
748 nM
8
3-OH
and 8, led to molecules with similarly weak mouse enzyme
inhibitory potency as observed in the thiophene 2,5-disubstituted
the inhibitory activity on m17b-HSD2 suggesting that in position 3
series (2a and 3b): 7a vs 2a with 16% and 29% inhibition at 1
respectively, 8 vs 3b with 16% and 38% inhibition at 1 M, respec-
tively. Only for the hydroxylated compound with linker 6, a mod-
erate inhibitory activity was observed (58% inhibition at 1 M). The
mM,
of the central core lipophilic interaction is better tolerated in mouse
than in human enzyme.
m
m
introduction of additional nitrogen on the central heterocyclic core
(compounds 10, 11, 17) is detrimental for the inhibitory activity
independently of the presence or absence of the linker as seen for
2.2.4. Infrared spectroscopy analysis for selected compounds
A close analysis of the infrared spectra measured for the
methoxylated and hydroxylated 2,5-, 3,5- and 2,4-disubstituted
thiophene derivatives bearing the same substituents on the A and
C rings (compounds 2aeb, 3aeb, 5ae8, Table 3), was undertaken
focusing on the frequency of the carbonyl band, with the aim to
understand the influence of the electronic properties of these
compounds on their activity. A small difference (about 8 cmꢁ1) in
the frequency of the C]O moiety was observed comparing com-
pounds having different substitution pattern: 3,5- (5a, 6a, 6) and
2,4- (7a, 8a, 8) compared to the corresponding 2,5-disubstituted
thiophene (2a, 3a,3b, respectively). The IR spectra of the respec-
tive compounds reveal a correlation between the frequency of the
carbonyl peak of some compounds with their inhibitory activities:
the lower the frequency of the carbonyl peak the more active the
h17
hibition at 1
27% inhibition at 1
cyclopropyl group on the amide moiety diminishes the m17
inhibitory potency (without linker: 2a vs 12: 29% and no inhibition
at 1 M, respectively, and with linker: 3c vs 13; 65% and 16% in-
hibition at 1 M, respectively).
b
-HSD2 (without linker: 2c vs 10 and 17: 30%, 14% and 13% in-
M, respectively, and with linker: 3c and 11: 65% and
M, respectively). The exchange of a methyl by a
-HSD2
m
m
b
m
m
The exchange of the N-methylamide (2a and 2c) by a N-methyl-
thioamide (23a and 23) or a keto group (29a, 32a, 29 and 32) did not
improve the potency, neither in the methoxy class nor in the hydroxy
series (around 30% inhibition at 1
mM, except for the hydroxy deriv-
ative 29, 42% inhibition). Similar observations could be made when
Fig. 2. Superimposition 2,4-thiophene 7a (white) and 2,5-thiophene 2a (gray) derivatives.

328
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
inhibitor (Table 3). As the frequency depends on the
p
-electron
methoxylated compounds (the exchange of amide for sulfonamide
or dimethylketone as well as the replacement of the methylamide
by a cyclopropylamide are detrimental for the activity), which is
indicative of a rather planar surface binding area of the human
enzyme. The space in the human enzyme active site seems larger
than that of the mouse enzyme, as indicated by the inactivity or low
activity of the tri-substituted bishydroxyphenylthiophenes on
conjugation state, the IR spectra show that the conjugation state of
the compounds influences their activities. In consequence, the
different electronic properties, which correlate with different fre-
quencies of the C]O stretch for these compounds, are determinant
for the biological activity.
However, the difference in activity between the 2,5- and the 3,5-
or 2,4-disubstituted thiophene is certainly mainly coming from
their different 3D structures, which induce different positions of
the phenyl C ring and therefore different interactions between the
ligand functional groups and amino acids in the active site (as seen
of the superimposition picture of the 2,4-thiophene 7a with the
2,5-thiophene 2a, Fig. 2).
mouse versus moderate to good activity on human 17b-HSD2.
However, the small lipophilic methyl group in position 3 of the
central core of 21 fits better into the mouse enzyme binding site
than into the human one. The presence of a small lipophilic pocket
in the mouse enzyme binding site might explain this result.
In general the 2,5-disubstituted thiophene derivatives are less
active on the mouse than on the human enzyme, except for com-
In case of the hydroxylated 2b, 5 and 7 no correlation between
the IR C]O frequency and the biological activity can be found. This
result might be explained by a different binding mode as already
highlighted in the SAR study.
pound 21, which is four times more potent on m17
b-HSD2
(IC50 ¼ 54 nM) than on h17 -HSD2 (IC50 ¼ 235 nM) indicating
b
clearly different structures of both enzymes. None of the variations
undertaken either at the central ring or at the amide function
improved this low potency. The geometry of the compounds
induced by the substitution pattern at the 5-membered ring might
not be optimal for the stabilization of the compounds in the mouse
enzyme. Compounds bearing a 6-membered ring as central core or
sharing a bulkier lipophilic group on the central ring will be
investigated in the future to verify this hypothesis. The SAR in the
mouse enzyme did not allow to gain many information on the
mouse enzyme active site. However, compounds with a hydroxy
group on the C ring and a methylene linker exhibited slightly su-
perior activity. From this study, it is also clear that the human
binding site is larger than the one of the mouse.
3. Discussion
The main aim of this study was the development of novel active
andselective17b-HSD2inhibitorsinthethiophenecarboxamideclass
with a good potencyon both the human and mouse enzymes and also
a good selectivity in both species. Different modifications were made
such as variation of the sulfur atom position on the thiophene ring,
exchange of the thiophene by a thiazole, substitution of the amide
group by a larger moiety (cyclopropyl), exchange of the N-methyl-
amide group by bioisosteres such as N-methylthioamide, N-methyl-
sulfonamide and methylene ketone, and exploration of the positions
2 and 3 of the thiophene ring with alkyl and phenyl substituents
leading to the 2,3,5-trisubstituted thiophene derivatives.
4. Conclusion
In the optimization process, the 2,5-, 3,5- and 2,4-disubstituted
thiophene carboxamide regioisomers were explored. From the bio-
logical results it became apparent that the h17b-HSD2 inhibitory ac-
In this study, we described the optimization of h17
b-HSD2 and
m17 -HSD2 inhibitors in the 5-substituted thiophene-2-
b
tivity was strongly influenced by the substitution pattern. The
position of the S atom with respect to the two substituents has a
strong impact on the biological activity due to the modification of the
overall geometry conformation of the 2,5-, 3,5- and 2,4-thiophene
derivatives. It also has an influence on the electronic distribution all
over the molecule, which might modulate biological activity as
already described [40]. The higher potency in the 2,5-thiophene class
might also indicate that no intramolecular coulombic attraction be-
tween the S(thiophene)-O(carbonyl) occurs as already described in
thiophene GABA receptorligands [42]and other thiophenes actingon
different targets [43]. All the changes undertaken to modify the
centralcore have beenineffective forthe improvementof the potency
of thecompoundson the humanand mouseenzymes(change of theS
position in the ring with respect to the two substituents and intro-
duction of an N atom, exchanging the thiophene by a thiazole).
However, the binding modes of the differently substituted thiophene
rings deserve further investigation. The IR spectra revealed that
electronic effects might also participate to the stabilization of the
compoundsinthe enzymebinding site along withhydrogen bonding,
pi-stacking and van der Waals interactions.
carboxamide class by variation of the sulfur atom position in the
central core, the exchange of the thiophene by a thiazole, the sub-
stitution of the amide group with a larger moiety (cyclopropyl),
exchange of the N-methylamide group with bioisosteres such as N-
methylsulfonamide, N-methylthioamide and methylene ketone and
exploration of the positions 2 and 3 of the thiophene core with alkyl
and phenyl moieties leading to the 2,3,5-trisubstituted thiophene
derivatives. The biological activities obtained indicate a very sharp
SAR in this class of compounds. The geometry of the compounds is
also quite restricted allowing only little variation around the thio-
phene core, the 2,5-disubstituted thiophene derivatives being the
best substitution pattern. Our SAR studies lead us to conclude that
the active site of the human 17b-HSD2 is quite large, larger than the
one of the mouse 17 -HSD2. Furthermore our investigations result
b
in the discovery of the highly active compound 21, a 2,3,5-
trisubstituted thiophene containing a methyl group in position 3
of the thiophene. This compound shows a reasonable IC50 of 235 nM
in the human enzyme with a good selectivity factor toward h17
HSD1 (SF ¼ 95), and a very good IC50 of 54 nM in mouse 17 -HSD2.
Furthermore, as this compound shows very low binding to the ER
and ER , it could be used for an in vivo proof of principle to
b-
b
a
The structural modifications performed led to a loss in activity
on the human enzyme, indicating a very sharp SAR in this class
of compounds.
b
demonstrate efficacy of 17b-HSD2 inhibitors in osteoporosis.
The SAR study deduced from the human enzyme in the 2,5-
thiophene class, highlights contradictory biological results for the
methoxy- and hydroxy substituted derivatives (by exchanging
amide for thioamide or dimethylketone). These results indicate that
the methoxylated compounds do not bind exactly in the same area
in the binding site as the hydroxylated corresponding analogues. In
addition, it is also obvious that the planarity around the amide
group is very important for a good potency, at least for the
5. Experimental section
5.1. Chemical methods
Chemical names follow IUPAC nomenclature, starting materials
were purchased from Aldrich, Acros, Combi-Blocks or Fluka and
were used without purification.

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
329
Column chromatography was performed on silica gel
(70e200 m) and reaction progress was monitored by TLC on
MgSO4, filtered and the solution was concentrated under reduced
pressure. The residue was purified by silica gel column chroma-
tography using hexanes and EtOAc as eluant or by trituration in a
mixture of diethyl ether/petroleum ether to afford the desired
compound.
m
Alugram SIL G/UV254 (MachereyeNagel). Visualization was
accomplished with UV light.
1H NMR and 13C NMR spectra were measured on a Bruker
AM500 spectrometer (at 500 MHz and 125 MHz, respectively) at
300 K. Chemical shifts are reported in d (parts per million: ppm), by
5.2.2. General procedure for SuzukieMiyaura coupling (method B)
In a sealed tube the previously prepared bromo-N-hetero-
arylcarboxamide derivative (1 eq.) was introduced followed by the
corresponding boronic acid (1.5 eq.), cesium carbonate (3 eq.),
tetrakis(triphenylphosphine)palladium (0.02 eq.) and a mixture of
DME/EtOH/H2O (1:1:1, v:v:v, 3 mL) as solvent. The reactor was
flushed with N2 and submitted to microwave irradiation (150 ꢀC,
150 W) for 20 min. After cooling to room temperature, a mixture of
EtOAc/H2O (1:1, v:v, 2 mL) was added to stop the reaction. The
aqueous layer was extracted with EtOAc (3 ꢃ 10 mL). The organic
layer was washed once with brine and once with water, dried over
MgSO4, filtered and the solution was concentrated under reduced
pressure. The residue was purified by column chromatography
using hexanes and EtOAc as eluant to afford the desired compound.
reference to the hydrogenated residues of deuteriated solvent as
internal standard: 2.05 ppm (1H NMR) and 29.80 and 206.30 ppm
(
13C NMR) for CD3COCD3, 2.50 ppm (1H NMR) and 39.50 ppm (13
C
C
NMR) for (CD3SOCD3), 7.26 ppm (1H NMR) and 77.16 ppm (13
NMR) for CDCl3. Signals are described as br (broad), s (singlet),
d (doublet), t (triplet), dd (doublet of doublets), ddd (doublet of
doublet of doublets), dt (doublet of triplets) and m (multiplet). All
coupling constants (J) are given in Hertz (Hz).
Melting points (mp) were measured in open capillaries on a
Stuart Scientific SMP3 apparatus and are uncorrected.
IR spectra were recorded neat on a Perkin Elmer Spectrum 100
FTIR spectrophotometer or on a Bruker Tensor 27 (indicated in
text).
Mass spectrometry was performed on a TSQ® Quantum (Ther-
moFisher, Dreieich, Germany). The triple quadrupole mass spec-
trometer was equipped with an electrospray interface (ESI). The
purity of the compounds was assessed by LC/MS. The Surveyor®-
LC-system consisted of a pump, an auto sampler, and a PDA de-
tector. The system was operated by the standard software Xcali-
bur®. An RP C18 NUCLEODUR® 100-5 (3 mm) column
(MachereyeNagel GmbH, Dühren, Germany) was used as station-
ary phase. All solvents were HPLC grade. In a gradient run the
percentage of acetonitrile (containing 0.1% trifluoroacetic acid) was
increased from an initial concentration of 0% at 0 min to 100% at
5.2.3. General procedure for ether cleavage (method C)
To a solution of methoxy heteroaryl derivative (1 eq.) in CH2Cl2
(10 mL) at 0 ꢀC boron trifluoride methyl sulfide complex (6 eq. per
methoxy group) was added. The reaction mixture was warmed up
to room temperature and stirred overnight. Methanol was added to
quench the reaction at 0 ꢀC. After warming up to room temperature
for 1 h, the solvent was carefully removed under reduced pressure
(temperature of bath was 25 ꢀC). Cold water was added to the
residue and the aqueous layer was extracted with CH2Cl2
(3 ꢃ 15 mL). The organic layer was washed once with water, dried
over MgSO4, filtered and evaporated to dryness under reduced
pressure. The residue was purified by column chromatography
using hexanes and EtOAc as eluant or triturated in the mixture
diethyl ether/petroleum ether and filtered off to afford the desired
compound.
15 min and kept at 100% for 5 min. The injection volume was 15
mL
and the flow rate was set to 800 L/min. MS analysis was carried
m
out at the needle voltage of 3000 V and the capillary temperature of
350 ꢀC. Mass spectra were acquired in positive mode from 100 to
1000 m/z and UV spectra were recorded at the wave length of
254 nm and in some cases at 360 nm.
All microwave irradiation experiments were carried out in a
CEM-Discover microwave apparatus.
5.3. Detailed synthesis procedure for the preparation of compounds
MPLC purifications were performed on Teledyne Isco Combi-
Flash Companion.
5.3.1. 2-Bromo-N-(3-methoxyphenyl)-N-methyl-1,3-thiazole-5-
carboxamide (10a)
Synthesized according to method A using 2-bromo-1,3-thiazole-
5-carboxylic acid (416 mg, 2 mmol) and 3-methoxy-N-methylani-
All tested compounds exhibited ꢂ95% chemical purity as
measured by HPLC/MS.
The following compounds were prepared according to previ-
ously described procedures: N-cyclopropyl-3-methoxyaniline 15a
[36]; N-(3-methoxybenzyl)cyclopropanamine 15b [36]; 2,5-
dibromothiophene-3-carboxylic acid 20b [44]; 5-bromo-3-
line (262 ml, 2 mmol). The residue was purified by silica gel column
chromatography (hexanes/EtOAc 70:30) to afford 10a as pale
brown solid (393 mg, 60%). Mp: 106e107 ꢀC. IR (neat): 3098, 3059,
3014, 2966, 2943, 2838, 1633, 1582 cmꢁ1 1H NMR (CD3COCD3)
.
methylthiophene-2-carboxylic
acid
22
[37];
N,5-bis(3-
d
3.67 (s, 3H), 3.85 (s, 3H), 6.99 (ddd, J ¼ 0.9, 2.0, 7.7 Hz, 1H), 7.05 (t,
methoxyphenyl)-N-methylthiophene-2-carboxamide 23b [28]; 2-
(3-methoxyphenyl)-2-methylpropanoic acid 31b [38]; 3-chloro-3-
(3-methoxyphenyl)prop-2-enal 33b [45].
J ¼ 2.3 Hz, 1H), 7.09 (ddd, J ¼ 0.9, 2.5, 8.4 Hz, 1H), 7.18 (s, 1H), 7.44 (t,
J ¼ 8.1 Hz, 1H); 13C NMR (CD3COCD3)
d 38.6, 56.0, 114.9, 115.8, 121.3,
131.8, 136.8, 144.9, 145.7, 155.6, 159.9, 162.1; LCeMS (ESI): 325.77,
327.45 [M]þ.
5.2. General procedures
5.2.1. General procedure for amide formation (method A)
5.3.2. 2-Bromo-N-(3-methoxybenzyl)-N-methyl-1,3-thiazole-5-
carboxamide (11b)
Synthesized according to method A using 2-bromo-1,3-thiazole-
5-carboxylic acid (418 mg, 2 mmol) and 3-methoxy-N-methyl-
A solution of bromo heteroaryl carboxylic acid (2 mmol), thionyl
chloride (4 mmol) and DMF (5 drops) in toluene (10 mL) was
refluxed at 110 ꢀC for 4 h. The reaction mixture was cooled to room
temperature and the solvent and the excess of thionyl chloride
removed under reduced pressure. To the residue was added at 0 ꢀC
the corresponding N-methyl amine (2 mmol) and Et3N (2 mmol) in
CH2Cl2 (10 mL) under N2 atmosphere. After 30 min at 0 ꢀC, the ice
bath was removed and the solution was warmed up and stirred at
room temperature overnight. The reaction mixture was extracted
twice with CH2Cl2 (2 ꢃ 15 mL) and the organic layer dried over
benzylamin (298 mL, 2 mmol). The residue was purified by silica gel
column chromatography (hexanes/EtOAc 70:30) to afford 11b as
yellow oil (530 mg, 78%). IR (neat): 2971, 2934, 2835, 1611 cmꢁ1. 1H
NMR (CD3COCD3)
2H), 6.86e6.93 (m, 3H), 7.26e7.34 (m, 1H), 7.63e8.14 (m, 1H); 13C
NMR (CD3COCD3) 37.1, 52.4, 55.5, 113.7, 114.5, 119.4, 120.9, 130.7,
139.5, 143.2, 154.7, 161.1, 161.5; LCeMS (ESI): 339.70, 341.52 [M]þ.
d 2.98e3.34 (m, 3H), 3.79 (s, 3H), 4.68e4.82 (m,
d

330
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
5.3.3. 2-Bromo-N-(3-hydroxybenzyl)-N-methyl-1,3-thiazole-5-
carboxamide (11a)
111.4, 113.8, 113.9, 120.3, 131.0, 131.8, 132.0, 139.0, 146.8, 161.5, 163.5;
LCeMS (ESI): 325.20, 327.21 [M]þ.
Synthesized according to method
C using 11b (530 mg,
1.55 mmol) and BF3.SMe2 (980 L, 9.32 mmol). The product was
purified on MP-LC (hexanes/EtOAc 100:0 to 80:20, 15 min, 80:20 to
m
5.3.8. 5-Bromo-N-(3-methoxybenzyl)-N-methylthiophene-3-
carboxamide (7b)
60:40, 35 min) afforded the desired product as pale red oil (236 mg,
Synthesized according to method A using 5-bromothiophene-3-
carboxylic acid (414 mg, 2 mmol) and 3-methoxy-N-methyl-
benzylamine (298 mL, 2 mmol). The residue was purified by silica
47%). IR (neat): 3286, 3101, 2969, 2928, 2871, 1731, 1702, 1603 cmꢁ1
.
1H NMR (CD3COCD3)
d
2.99e3.34 (m, 3H), 4.61e4.82 (m, 2H),
6.72e6.92 (m, 3H), 7.60e8.13 (m, 1H), 8.47 (br s, 1H); 13C NMR
gel column chromatography (hexanes/EtOAc 70:30) to afford 7b as
(CD3COCD3)
d
36.6, 52.3,115.3,119.4,130.7,134.6,139.8,144.3,158.7,
yellow oil (277 mg, 41%). IR (neat, Bruker Tensor 27): 3094, 2995,
162.3, 171.3; LCeMS (ESI): 327.93, 328.94 [MþH]þ.
2918, 2833,1622 cmꢁ1. 1H NMR (CD3COCD3)
d
3.10 (br s, 3H), 3.79 (s,
3H), 4.66 (s, 2H), 6.86 (dd, J ¼ 1.9, 8.0 Hz, 1H), 6.88e6.96 (m, 2H),
7.24e7.37 (m, 2H), 7.60e7.84 (m, 1H); 13C NMR (CD3COCD3)
37.1,
5.3.4. 5-Bromo-N-cyclopropyl-N-(3-methoxyphenyl)thiophene-2-
carboxamide (12a)
d
46.7, 55.6, 112.7, 113.6, 114.4, 121.0, 128.8, 129.4, 130.6, 131.4, 138.8,
Synthesized according to method A using 5-bromothiophene-2-
carboxylic acid (414 mg, 2 mmol) and freshly synthesized N-
cyclopropyl-3-methoxyaniline 15a (2 mmol). The residue was pu-
rified by silica gel column chromatography (hexanes/EtOAc 70:30)
to afford 12a as yellow solid (343 mg, 49%). Mp: 60e61 ꢀC. IR (neat):
3086, 3014, 2946, 2841, 1740, 1630, 1597 cmꢁ1. 1H NMR (CD3COCD3)
140.0, 161.1; LCeMS (ESI): 340.21, 342.21 [M]þ.
5.3.9. 4-Bromo-N-(3-methoxyphenyl)-N-methylthiophene-2-
carboxamide (6b)
Synthesized according to method A using 4-bromothiophene-2-
carboxylic acid (414 mg, 2 mmol) and 3-methoxy-N-methylaniline
d
0.58e0.62 (m, 2H), 0.78e0.82 (m, 2H), 3.28e3.32 (m, 1H), 3.82 (s,
(262 mL, 2 mmol). The residue was purified by silica gel column
3H), 6.66 (d, J ¼ 4.1 Hz,1H), 6.86 (ddd, J ¼ 1.0, 1.9, 7.9 Hz,1H), 6.91 (t,
chromatography (hexanes/EtOAc 70:30) to afford 6b as pale brown
solid (665 mg, 99%). Mp: 79e82 ꢀC. IR (neat, Bruker Tensor 27):
3103, 2997, 2964, 2939, 2935, 2831, 1709, 1624, 1589 cmꢁ1. 1H NMR
J ¼ 2.3 Hz, 1H), 6.95 (d, J ¼ 4.1 Hz, 1H), 7.03 (ddd, J ¼ 0.9, 2.6, 8.5 Hz,
1H), 7.38 (t, J ¼ 8.2 Hz, 1H); 13C NMR (CD3COCD3)
d 7.8, 33.1, 55.9,
115.1, 116.0, 118.5, 122.5, 131.1, 132.9, 142.0, 143.3, 161.6, 162.6;
(CD3COCD3)
d
3.37 (s, 3H), 3.82 (s, 3H), 6.47 (d, J ¼ 1.5 Hz, 1H), 6.94
LCeMS (ESI): 351.72, 354.07 [MþH]þ.
(ddd, J ¼ 0.9, 2.1, 7.8 Hz, 1H), 7.00 (t, J ¼ 2.4 Hz, 1H), 7.04 (ddd,
J ¼ 0.9, 2.6, 8.3 Hz,1H), 7.40 (t, J ¼ 8.1 Hz,1H), 7.58 (d, J ¼ 1.5 Hz,1H).
5.3.5. 5-Bromo-N-cyclopropyl-N-(3-methoxybenzyl)thiophene-2-
carboxamide (13b)
13C NMR (CD3COCD3)
d 39.1, 56.0, 109.2, 114.6, 115.1, 121.1, 129.2,
131.5, 134.0, 141.0, 146.0, 161.2, 161.9; LCeMS (ESI): 325.97, 328.97
Synthesized according to method A using 5-bromothiophene-2-
carbonyl chloride (416 mg, 2 mmol) and freshly synthesized N-(3-
methoxybenzyl)cyclopropanamine 15b (2 mmol). The residue
was purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford 13b as yellow oil (302 mg, 41%). IR (neat): 3089,
[M]þ.
5.3.10. 4-Bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-
carboxamide (8b)
Synthesized according to method A using 4-bromothiophene-2-
carboxylic acid (413 mg, 2 mmol) and 3-methoxy-N-methyl-
3008, 2946, 2838, 1737, 1600 cmꢁ1 1H NMR (CD3COCD3)
.
d
0.78e0.81 (m, 2H), 0.88e0.92 (m, 2H), 2.98e3.04 (m, 1H), 3.77 (s,
benzylamine (298 mL, 2 mmol). The residue was purified by silica
3H), 4.74 (s, 2H), 6.82e6.85 (m, 1H), 6.88e6.91 (m, 2H), 7.18 (d,
gel column chromatography (hexanes/EtOAc 70:30) to afford 8b as
J ¼ 4.1 Hz, 1H), 7.23e7.27 (m, 1H), 7.61 (d, J ¼ 4.1 Hz, 1H); 13C NMR
yellow oil (680 mg, 99%). IR (neat, Bruker Tensor 27): 3109, 3009,
(CD3COCD3)
d
11.3, 32.0, 51.9, 55.4, 113.2, 114.1, 117.9, 120.5, 130.4,
2960, 2939, 2837, 1610, 1591 cmꢁ1. 1H NMR (CD3COCD3)
d 3.17 (br s,
131.5, 132.3, 141.0, 142.5, 160.9, 164.1; LCeMS (ESI): 365.66, 367.91
3H), 3.79 (s, 3H), 4.76 (br s, 2H), 6.86e6.92 (m, 3H), 7.30 (t,
[MþH]þ.
J ¼ 7.5 Hz, 1H), 7.32e7.50 (m, 1H), 7.71 (s, 1H); 13C NMR (CD3COCD3)
d
38.9, 46.3, 55.7, 109.5, 113.6, 114.0, 120.5, 128.1, 130.7, 131.7, 139.8,
5.3.6. 5-Bromo-N-cyclopropyl-N-(3-hydroxybenzyl)thiophene-2-
141.1, 161.1, 163.2; LCeMS (ESI): 340.00, 342.01 [M]þ.
carboxamide (13a)
Synthesized according to method
C
using 13b (278 mg,
5.3.11. 5-(3-Methylphenyl)-1,3-thiazole (17b)
0.76 mmol) and BF3.SMe2 (480 L, 4.56 mmol). The product as
m
Synthesized according to method B using 5-bromo-1,3-thiazole
(1 g, 6.10 mmol), m-tolyl boronic acid (1.24 g, 9.14 mmol), cesium
carbonate (5.96 g, 18.30 mmol) and tetrakis(triphenylphosphine)
palladium (140 mg, 0.02 eq) in an oxygen free DME/EtOH/water
(1:1:1) (6 mL). The residue was purified by silica gel column
chromatography (hexanes/EtOAc 70:30) to afford 17b as colorless
brown solid (270 mg, 100%) was directly obtained analytically pure
and used in the next step without further purification. Mp: 128 ꢀC.
IR (neat): 3268, 3098, 3002, 2975, 2928, 2429, 1740, 1603,
1570 cmꢁ1. 1H NMR (CD3COCD3)
d 0.77e0.81 (m, 2H), 0.87e0.92 (m,
2H), 2.96e3.02 (m, 1H), 4.70 (s, 2H), 6.73 (ddd, J ¼ 0.9, 2.7, 7.8 Hz,
1H), 6.78e6.80 (m, 2H), 7.13e7.17 (m, 1H), 7.18 (d, J ¼ 4.1 Hz, 1H),
oil (1.06 g, 99%). IR (neat): 3086, 3026, 2957, 2922, 2862, 1605 cmꢁ1
1H NMR (CD3COCD3)
2.37 (s, 3H), 7.17e7.20 (m, 1H), 7.32 (t,
J ¼ 7.7 Hz, 1H), 7.45e7.48 (m, 1H), 7.50e7.52 (m, 1H), 8.19 (s, 1H),
.
7.60 (d, J ¼ 4.1 Hz, 1H), 8.28 (br s, 1H); 13C NMR (CD3COCD3)
d
11.3,
d
31.9, 51.9, 114.9, 115.1, 117.9, 119.5, 130.4, 131.5, 132.3, 140.9, 142.5,
158.5, 164.1; LCeMS (ESI): 351.75, 353.28 [M]þ.
8.93 (s, 1H); 13C NMR (CD3COCD3)
d 21.4, 124.8, 128.3, 129.99,
130.01, 132.1, 139.8, 140.1, 153.2; LCeMS (ESI): 176.07 [MþH]þ.
5.3.7. 5-Bromo-N-(3-methoxyphenyl)-N-methylthiophene-3-
carboxamide (5b)
Synthesized according to method A using 5-bromothiophene-3-
carboxylic acid (415 mg, 2 mmol) and 3-methoxy-N-methylaniline
5.3.12. Lithium 5-(3-methylphenyl)-1,3-thiazole-2-carboxylate
(17a)
To a solution of 17b (1.05 g, 6 mmol) in dry THF (50 mL) was
added drop wise n-BuLi (2.88 mL, 7.21 mmol, 2.5 M in hexane)
at ꢁ78 ꢀC under N2 atmosphere. The reaction mixture was kept
at ꢁ78 ꢀC for 1 h and was warmed up (without removing ice bath)
to ꢁ5 ꢀC (in about 3 h). Then, the reaction mixture was cooled
at ꢁ78 ꢀC and dry carbon dioxide gas was bubbled through the
(262
chromatography (hexanes/EtOAc 70:30) to afford 5b as pale brown
oil (340 mg, 69%). 1H NMR (CD3COCD3)
3.36 (s, 3H), 3.77 (s, 3H),
mL, 2 mmol). The residue was purified by silica gel column
d
6.80 (ddd, J ¼ 0.9, 1.9, 7.9 Hz, 1H), 6.86e6.89 (m, 2H), 6.91 (d,
J ¼ 1.6 Hz,1H), 7.26e7.29 (m, 2H); 13C NMR (CD3COCD3)
d 38.4, 55.8,

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
331
solution for 3 h. The reaction mixture was warmed up to room
5.3.16. N-(3-Methoxyphenyl)-N-methyl-2-(3-methylphenyl)-1,3-
temperature and the bubbling of dry CO2 gas was continued over-
night. Solvent was removed under reduced pressure (bath tem-
perature of the rotary evaporator at 20 ꢀC). The residue was
triturated with diethyl ether and filtered to afford the desired
compound as a pale brown solid (977 mg, 72%). IR (neat): 3301,
3095, 3032, 2960, 2922, 2868, 1737, 1618 cmꢁ1. 1H NMR (CD3SOCD3)
thiazole-5-carboxamide (10)
Synthesized according to method
B
using 10a (200 mg,
0.61 mmol), m-tolyl boronic acid (105 mg, 0.77 mmol), cesium
carbonate (596 mg, 1.83 mmol) and tetrakis(triphenylphosphine)
palladium (14 mg, 0.02 eq.). The residue was purified on silica gel
(hexanes/EtOAc 70:30) to afford the desired product as beige solid
(170 mg, 82%). Mp: 87e90 ꢀC. IR (neat): 3065, 3005, 2924, 1626,
d
2.34 (s, 3H), 7.16 (d, J ¼ 7.2 Hz, 1H), 7.31 (t, J ¼ 7.5 Hz, 1H), 7.46 (d,
J ¼ 7.2 Hz, 1H), 7.50 (s, 1H), 8.12 (s, 1H); 13C NMR (CD3SOCD3)
d
20.9,
1588 cmꢁ1. 1H NMR (CD3COCD3)
d 2.36 (s, 3H), 3.40 (s, 3H), 3.83 (s,
123.6, 126.9, 128.99, 129.06, 131.2, 138.5, 141.0, 161.7, 170.3; LCeMS
3H), 6.98e7.00 (m, 1H), 7.04e7.06 (m, 2H), 7.28e7.30 (m, 2H), 7.33
(t, J ¼ 7.5 Hz, 1H), 7.42 (dt, J ¼ 0.7, 7.8 Hz, 1H), 7.64e7.66 (m, 1H),
(ESI): 219.95 [M-Li þ H]þ.
7.70e7.71 (m, 1H); 13C NMR (CD3COCD3)
d 21.2, 38.7, 55.9, 114.7,
115.2, 121.1, 124.5, 127.7, 129.9, 131.6, 132.4, 134.0, 134.7, 139.8, 145.8,
5.3.13. 5-Bromo-N-(3-methoxyphenyl)-N-methylthiophene-2-
sulfonamide (26)
147.6, 161.2, 161.9, 171.6; LCeMS (ESI): 338.68 [M]þ.
To a stirred solution of 3-methoxy-N-methylaniline (137 mg,
1 mmol) in CH2Cl2 (3 mL) was added NaOH 50% (1 mL) followed by
n-tetrabutyl ammonium hydrogen sulfate (51 mg, 0.15 mmol). After
few minutes, 5-bromothiophene-2-sulfonyl chloride (262 mg,
1 mmol) was added to the reaction mixture. The solution was
stirred at room temperature for 5 h. Water (10 mL) was added to
quench the reaction followed by EtOAc (10 mL). The aqueous layer
was extracted twice with EtOAc (2 ꢃ 15 mL). The organic layer was
dried over MgSO4, filtered and the solution was concentrated under
reduced pressure. The product was purified on silica gel (hexanes/
EtOAc 8:2) to afford the desired compound as yellow oil (277 mg,
77%). IR (neat): 3101, 2938, 2835, 1734, 1602, 1354 cmꢁ1. 1H NMR
5.3.17. 2-(2-Fluoro-3-methoxyphenyl)-N-(3-hydroxybenzyl)-N-
methyl-1,3-thiazole-5-carboxamide (11)
Synthesized according to method
B using 11a (104 mg,
0.32 mmol), 2-fluoro-3-methoxyphenylboronic acid (81 mg,
0.48 mmol), cesium carbonate (311 mg, 0.95 mmol) and tetrakis(-
triphenylphosphine) palladium (7 mg, 0.02 eq.). The residue was
purified by MP-LC (hexanes/EtOAc 100:0 to 60:40, 60 min) followed
by a trituration cold acetonitrile to afford the desired product as
beige solid (83 mg, 70%). Mp: 140e141 ꢀC. IR (neat): 3220, 3029,
2993, 2931, 1591 cmꢁ1. 1H NMR (CD3COCD3)
d 3.20 (br s, 3H), 3.97
(s, 3H), 4.76 (br s, 2H), 6.77e6.85 (m, 3H), 7.22 (t, J ¼ 7.6 Hz, 1H),
7.26e7.32 (m, 2H), 7.84 (br s, 1H), 8.36 (br s, 1H); 13C NMR
(CD3COCD3)
d 3.27 (s, 3H), 3.77 (s, 3H), 6.78e6.81 (m, 2H),
6.90e6.92 (m, 1H), 7.26e7.30 (m, 2H), 7.31 (d, J ¼ 4.1 Hz, 1H); 13C
(CD3COCD3)
d 56.9, 104.4, 115.3, 115.4, 116.12, 116.14, 120.1, 122.3,
122.4, 125.57, 125.60, 139.7, 149.3, 149.4, 150.0, 152.0, 158.8, 162.9;
NMR (CD3COCD3) d 38.7, 55.8, 113.5, 114.2, 119.3, 120.1, 130.5, 132.2,
LCeMS (ESI): 372.70 [MþH]þ.
134.3, 138.9, 143.2, 161.0; LCeMS (ESI): 361.07, 362.90 [MþH]þ.
5.3.18. N-(3-Methoxyphenyl)-N-methyl-5-(3-methylphenyl)-1,3-
thiazole-2-carboxamide (17)
To a solution of 17a (450 mg, 2 mmol) in CH2Cl2 (15 mL) was
added drop wise oxalyl chloride (340 mL, 4 mmol) followed by few
5.3.14. 5-Bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-
sulfonamide (27b)
To
a stirred solution of 3-methoxy-N-methylbenzylamine
(302 mg, 2 mmol) in CH2Cl2 (6 mL) was added NaOH 50% (2 mL)
followed by n-tetrabutyl ammonium hydrogen sulfate (102 mg,
0.30 mmol). After few minutes, 5-bromothiophene-2-sulfonyl
chloride (524 mg, 2 mmol) was added to the reaction mixture.
The solution was stirred at room temperature for 5 h. Water (10 mL)
was added to quench the reaction, followed by EtOAc (10 mL). The
aqueous layer was extracted twice with EtOAc (2 ꢃ 15 mL). The
organic layer was dried over MgSO4, filtered and the solution was
concentrated under reduced pressure. The product was purified on
silica gel (hexanes/EtOAc 80:20) to afford the desired compound as
yellow oil (620 mg, 82%). IR (neat): 3101, 2937, 2835, 1740, 1600,
drops of DMF at 0 ꢀC under N2 atmosphere. The reaction mixture
was stirred at 0 ꢀC for 10 min and at room temperature for 3 h. The
solvent was removed under reduced pressure (bath temperature of
the rotary evaporator: 20 ꢀC). The residue was diluted in dry CH2Cl2
(10 mL) and 3-methoxy-N-methylaniline (262 mL, 2 mmol) was
added followed by triethylamine (280 mL, 2 mmol). The solution
was stirred overnight at room temperature under N2 atmosphere.
The solvent was removed under reduced pressure (bath tempera-
ture of rotary evaporator: 20 ꢀC), Na2CO3 2 N (15 mL) followed by
EtOAc (15 mL) were added. The aqueous layer was extracted once
more with EtOAc (15 mL). The organic layer was washed twice with
Na2CO3 2 N (15 mL), once with water (15 mL), dried over MgSO4,
filtered and the solution was concentrated under reduced pressure.
Purification on silica gel (hexanes/EtOAc 70:30) to afford the
desired product as yellow solid (520 mg, 77%). Mp: 84e86 ꢀC. IR
1345 cmꢁ1. 1H NMR (CD3COCD3)
d 2.70 (s, 3H), 3.80 (s, 3H), 4.21 (s,
2H), 6.88e6.91 (m, 1H), 6.92e6.95 (m, 2H), 7.28e7.31 (m, 1H), 7.38
(d, J ¼ 4.1 Hz, 1H), 7.52 (d, J ¼ 4.1 Hz, 1H); 13C NMR (CD3COCD3)
d
35.0, 54.7, 55.5, 114.2, 114.7, 119.7, 121.3, 130.6, 132.5, 133.7, 138.3,
140.0, 161.0; LCeMS (ESI): 376.22, 378.65 [M]þ.
(neat): 3095, 3014, 2975, 2919, 1633, 1588 cmꢁ1
.
1H NMR
(CD3COCD3) 2.36 (s, 3H), 3.54 (br s, 3H), 3.78 (s, 3H), 6.87 (ddd,
d
5.3.15. 5-Bromo-N-(3-hydroxybenzyl)-N-methylthiophene-2-
sulfonamide (27a)
J ¼ 0.6, 2.5, 8.3 Hz, 2H), 6.92 (t, J ¼ 2.1 Hz, 1H), 7.20e7.22 (m, 1H),
7.27 (t, J ¼ 8.1 Hz, 1H), 7.32 (t, J ¼ 7.7 Hz, 1H), 7.43e7.47 (m, 1H), 7.49
Synthesized according to method
C
using 27b (528 mg,
(br s, 1H), 7.93 (br s, 1H); 13C NMR (CD3COCD3)
d 21.3, 39.5, 55.7,
1.40 mmol) and BF3.SMe2 (880 L, 8.42 mmol). The precipitate
m
113.6, 113.8, 120.1, 124.87, 124.93, 128.3, 128.4, 130.1, 130.55, 130.62,
formed was filtered to give the desired product as yellow solid
(445 mg, 88%) was used in the next step without any purification.
Mp: 105e107 ꢀC. IR (neat): 3438, 3104, 3026, 2981, 2931, 1600,
131.5, 139.7, 139.9, 144.3, 146.6, 161.2; LCeMS (ESI): 338.85 [MþH]þ.
5.3.19. N-Cyclopropyl-N-(3-methoxyphenyl)-5-(3-methylphenyl)
thiophene-2-carboxamide (12)
1337 cmꢁ1
.
1H NMR (CD3COCD3)
d
2.69 (s, 3H), 4.16 (s, 2H), 6.80
(ddd, J ¼ 0.8, 2.6, 9.0 Hz, 1H), 6.81e6.84 (m, 1H), 6.86e6.88 (m, 1H),
Synthesized according to method B using 12a (205 mg,
7.19 (t, J ¼ 7.9 Hz, 1H), 7.38 (d, J ¼ 4.0 Hz, 1H), 7.51 (d, J ¼ 4.0 Hz, 1H),
0.58 mmol), m-tolyl boronic acid (118 mg, 0.87 mmol), cesium
carbonate (570 mg, 1.75 mmol) and tetrakis(triphenylphosphine)
palladium (14 mg, 0.02 eq). The residue was purified on MP-LC
(hexanes/EtOAc 100:0 to 80:20, 10 min, 80:20 to 60:50, 45 min)
8.38 (s, 1H); 13C NMR (CD3COCD3)
d 35.0, 54.7, 115.8, 116.0, 119.6,
120.3, 130.6, 132.4, 133.6, 138.3, 140.0, 158.7; LCeMS (ESI): 361.88,
363.73 [MþH]þ.

332
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
to afford the desired product as yellow solid (181 mg, 86%)ꢁ. 1Mp:
.
1H
5.3.23. N,5-bis(3-methoxyphenyl)-N-methylthiophene-3-
carboxamide (5a)
68e69 ꢀC. IR (neat): 3092, 3014, 2972, 2922, 1627, 1600 cm
NMR (CD3COCD3)
d
0.61e0.64 (m, 2H), 0.79e0.83 (m, 2H), 2.33 (s,
Synthesized according to method B using 5b (163 mg, 0.5 mmol)
and 3-methoxyphenylboronic acid (91 mg, 0.6 mmol), cesium
carbonate (489 mg, 1.5 mmol) and tetrakis(triphenylphosphine)
palladium (12 mg, 0.02 eq.). The residue was purified by silica gel
column chromatography (hexanes/EtOAc 70:30) to afford 5a as
yellow oil (168 mg, 95%). IR (neat): 3104, 3061, 3001, 2938, 1635,
3H), 3.31e3.35 (m, 1H), 3.81 (s, 3H), 6.73 (d, J ¼ 4.0 Hz, 1H), 6.88
(ddd, J ¼ 0.9, 2.0, 7.7 Hz, 1H), 6.92 (t, J ¼ 2.3 Hz, 1H), 6.99 (ddd,
J ¼ 0.9, 2.6, 8.5 Hz, 1H), 7.13e7.14 (m, 1H), 7.15 (d, J ¼ 4.0 Hz, 1H),
7.26 (t, J ¼ 7.7 Hz, 1H), 7.36 (t, J ¼ 8.1 Hz, 1H), 7.37e7.39 (m, 1H),
7.42e7.43 (m, 1H); 13C NMR (CD3COCD3)
d 8.1, 21.3, 33.2, 55.8, 114.5,
115.8, 122.3, 123.7, 123.8, 127.2, 129.8, 129.9, 130.9, 132.9, 134.4,
139.5, 139.6, 144.0, 149.3, 161.5, 163.7; LCeMS (ESI): 363.86
[MþH]þ.
1597 cmꢁ1. 1H NMR (CD3COCD3)
d 3.40 (s, 3H), 3.76 (s, 3H), 3.82 (s,
3H), 6.83e6.89 (m, 4H), 6.99 (t, J ¼ 1.9 Hz, 1H), 7.05 (ddd, J ¼ 0.9, 1.6,
7.6 Hz, 1H), 7.17 (d, J ¼ 1.4 Hz, 1H), 7.25e7.30 (m, 3H); 13C NMR
(CD3COCD3)
d 38.3, 55.6, 55.8, 111.7, 113.8, 113.9, 114.5, 118.8, 120.4,
125.5, 129.4, 130.9, 131.0, 135.9, 139.2, 143.5, 147.2, 161.2, 161.5,
164.6; LCeMS (ESI): 353.86 [MþH]þ.
5.3.20. N-Cyclopropyl-5-(2-fluoro-3-methoxyphenyl)-N-(3-
hydroxybenzyl)thiophene-2-carboxamide (13)
Synthesized according to method
B using 13a (104 mg,
0.32 mmol), 2-fluoro-3-methoxyphenylboronic acid (81 mg,
0.48 mmol), cesium carbonate (311 mg, 0.95 mmol) and tetrakis(-
triphenylphosphine) palladium (7 mg, 0.02 eq). The residue was
purified by preparative HPLC (acetonitrile/water 40:60 to 100:0,
solvent containing 1 mL of TFA for 1 L of solvent) to afford the
desired product as beige solid (41 mg, 37%). Mp: 149e150 ꢀC. IR
5.3.24. N-(3-methoxybenzyl)-5-(3-methoxyphenyl)-N-
methylthiophene-3-carboxamide (7a)
Synthesized according to method
B using 7b (100 mg,
0.29 mmol) and 3-methoxyphenylboronic acid (53 mg, 0.35 mmol),
cesium carbonate (283 mg, 0.87 mmol) and tetrakis(-
triphenylphosphine) palladium (7 mg, 0.02 eq.). The residue was
purified by silica gel column chromatography (hexanes/EtOAc
60:40) to afford 7a as yellow oil (71 mg, 67%). IR (neat): 3086, 3064,
(neat): 3253, 3008, 2972, 2937, 1600, 1570 cmꢁ1
.
1H NMR
(CD3COCD3) 0.77e0.80 (m, 2H), 0.87e0.92 (m, 2H), 3.00e3.06 (m,
d
1H), 3.94 (s, 3H), 4.74 (s, 2H), 6.73e6.76 (m, 1H), 6.81e6.84 (m, 2H),
7.13e7.22 (m, 3H), 7.32e7.35 (m, 1H), 7.52 (dd, J ¼ 1.0, 4.1 Hz, 1H),
3001, 2956, 2937, 1626, 1597 cmꢁ1. 1H NMR (CD3COCD3)
d
3.04 (br s,
3H), 3.79 (s, 3H), 3.84 (s, 3H), 4.70 (s, 2H), 6.84e6.97 (m, 4H),
7.16e7.34 (m, 4H), 7.53e7.77 (m, 2H); 13C NMR (CD3COCD3)
37.3,
7.78 (m, 1H), 8.31 (br s, 1H); 13C NMR (CD3COCD3)
d 11.2, 32.1, 51.9,
d
56.7, 114.07, 114.08, 114.9, 115.1, 119.5, 120.57, 120.58, 122.8, 122.9,
125.5, 125.6, 127.2, 127.3, 130.4, 131.8, 140.67, 140.71, 141.1, 141.29,
141.31, 148.9, 149.6, 149.7, 150.9, 158.5, 165.1 (additional peaks due
to coupling of carbon with fluorine); LCeMS (ESI): 397.66 [MþH]þ.
51.4, 55.5, 55.7, 112.0, 113.6, 114.6, 119.0, 119.6, 121.0, 124.8, 126.2,
126.8, 130.7, 131.1, 135.9, 139.1, 140.3, 144.9, 161.1, 161.2; LCeMS
(ESI): 367.89 [MþH]þ.
5.3.21. N-(3-Methoxyphenyl)-N-methyl-5-(3-methylphenyl)
thiophene-2-sulfonamide (24)
5.3.25. N,4-bis(3-methoxyphenyl)-N-methylthiophene-2-
carboxamide (6a)
Synthesized according to method
B using 26 (142 mg,
Synthesized according to method
B using 6b (216 mg,
0.39 mmol), m-tolyl boronic acid (80 mg, 0.59 mmol), cesium car-
bonate (385 mg, 1.18 mmol) and tetrakis(triphenylphosphine)
palladium (10 mg, 0.02 eq). The residue was purified on silica gel
(hexanes/EtOAc 8:2) to afford the desired product as yellow oil
0.66 mmol) and 3-methoxyphenylboronic acid (151 mg,
0.99 mmol), cesium carbonate (324 mg, 1.98 mmol) and tetrakis(-
triphenylphosphine) palladium (8 mg, 0.02 eq.). The residue was
purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford 6a as yellow oil (177 mg, 76%). IR (neat): 3098,
3057, 3001, 2955, 2935, 1624,1599, 1584 cmꢁ1. 1H NMR (CD3COCD3)
(147 mg, 99%). IR (neat): 3101, 3008, 2925, 1602 cmꢁ1 1H NMR
.
(CD3COCD3)
d
2.38 (s, 3H), 3.29 (s, 3H), 3.75 (s, 3H), 6.80 (dd, J ¼ 0.9,
2.2 Hz, 1H), 6.81e6.82 (m, 1H), 6.89 (ddd, J ¼ 0.8, 2.5, 8.4 Hz, 1H),
7.23e7.28 (m, 2H), 7.34 (t, J ¼ 7.7 Hz, 1H), 7.39 (d, J ¼ 4.0 Hz, 1H),
7.48e7.49 (m, 1H), 7.50 (d, J ¼ 4.0 Hz, 1H), 7.53e7.54 (m, 1H); 13C
d
3.40 (s, 3H), 3.80 (s, 3H), 3.82 (s, 3H), 6.83 (ddd, J ¼ 0.9, 2.6, 8.3 Hz,
1H), 6.90 (t, J ¼ 2.1 Hz, 1H), 6.92 (d, J ¼ 1.6 Hz, 1H), 6.97e7.00 (m,
2H), 7.03 (q, J ¼ 1.6 Hz, 1H), 7.04 (dd, J ¼ 0.9, 2.6, 1H), 7.25 (t,
J ¼ 7.8 Hz, 1H), 7.39e7.43 (m, 1H), 7.77 (d, J ¼ 1.6 Hz, 1H); 13C NMR
NMR (CD3COCD3)
d 21.3, 38.8, 55.7, 113.5, 114.0, 119.3, 124.1, 124.3,
127.6, 130.1, 130.4, 130.8, 133.5, 134.7, 135.9, 140.0, 143.6, 152.0,
(CD3COCD3)
d 38.8, 55.5, 55.9, 112.0, 114.0, 114.7, 114.8, 119.0, 121.2,
160.9; LCeMS (ESI): 373.62 [M]þ.
126.2,130.8,131.0, 131.4,137.4, 140.4,142.2,146.6,161.1, 161.9, 162.3;
LCeMS (ESI): 354.05 [MþH]þ.
5.3.22. 5-(2-Fluoro-3-methoxyphenyl)-N-(3-hydroxybenzyl)-N-
methylthiophene-2-sulfonamide (25)
Synthesized according to method
B
using 27a (200 mg,
5.3.26. N-(3-methoxybenzyl)-4-(3-methoxyphenyl)-N-
0.55 mmol), 2-fluoro-3-methoxyphenylboronic acid (141 mg,
0.83 mmol), cesium carbonate (540 mg, 1.66 mmol) and tetrakis(-
triphenylphosphine) palladium (13 mg, 0.02 eq). The residue was
purified by preparative HPLC (acetonitrile/water 40:60 to 100:0,
solvent containing 1 mL of TFA for 1 L of solvent) to afford the
desired product as colorless oil (42 mg, 19%). IR (neat): 3438, 3107,
methylthiophene-2-carboxamide (8a)
Synthesized according to method B using 8b (340 mg, 1 mmol)
and 3-methoxyphenylboronic acid (228 mg, 1.5 mmol), cesium
carbonate (977 mg, 3 mmol) and tetrakis(triphenylphosphine)
palladium (23 mg, 0.02 eq). The residue was purified by silica gel
column chromatography (hexanes/EtOAc 70:30) to afford 8a as
yellow oil (312 mg, 85%). IR (neat): 3087, 3061, 3000, 2955, 2935,
3029, 2981, 2928, 1600, 1337 cmꢁ1. 1H NMR (CD3COCD3)
d 2.72 (s,
3H), 3.95 (s, 3H), 4.19 (s, 2H), 6.80 (ddd, J ¼ 0.9, 2.5, 8.3 Hz, 1H),
1600, 1583 cmꢁ1. 1H NMR (CD3COCD3)
d 3.21 (br s, 3H), 3.80 (s, 3H),
6.83e6.85 (m, 1H), 6.89e6.90 (m, 1H), 7.17e7.27 (m, 3H), 7.37e7.40
3.82 (br s, 3H), 4.81 (s, 2H), 6.86e6.89 (m, 2H), 6.92e6.96 (m, 2H),
(m,1H), 7.67 (dd, J ¼ 0.6, 4.1 Hz,1H), 7.71 (dd, J ¼ 1.3, 4.0 Hz,1H); 13
C
7.16 (m, 2H), 7.28e7.33 (m, 2H), 7.79 (br s, 1H), 7.92 (s, 1H); 13C NMR
NMR (CD3COCD3)
d
35.1, 54.7, 56.8, 114.8, 115.7, 115.9, 120.3, 120.5,
(CD3COCD3) d 40.2, 55.5, 55.6, 112.6, 113.5, 113.9, 118.0, 119.4, 125.0,
121.9, 125.8, 127.7, 130.5, 133.3, 138.5, 143.7, 148.9, 149.6, 150.9,
127.1, 128.0, 128.5, 128.6, 130.7, 130.8, 137.4, 140.2,140.3,142.6,161.1,
158.6; LCeMS (ESI): 407.61 [M]þ.
161.2; LCeMS (ESI): 368.05 [MþH]þ.

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
333
5.3.27. N,5-bis(3-methoxyphenyl)-N-methylthiophene-2-
carbothioamide (23a)
(CD3COCD3)
(br s, 2H), 7.19e7.22 (m, 2H), 7.76 (br s, 1H), 7.82 (s, 1H); 13C NMR
(CD3COCD3) 37.1, 52.7, 114.0, 115.3, 115.0, 115.4, 118.4, 119.4, 124.9,
d 3.19 (br s, 3H), 4.77 (br s, 2H), 6.76e6.86 (m, 4H), 7.12
A solution of N,5-bis(3-methoxyphenyl)-N-methylthiophene-2-
carboxamide 23b (382 mg, 1.08 mmol) in dry toluene and the
Lawesson reagent (437 mg, 1.08 mmol) were heated at reflux under
N2 atmosphere for 3 h. After cooling, water was added to the so-
lution and the aqueous layer was extracted with toluene
(3 ꢃ 20 mL), washed once with brine and once with water, dried
over MgSO4, filtered and the solution was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (hexanes/EtOAc 80:20) to afford 23a as yellow
solid (252 mg, 63%). Mp: 101e102 ꢀC. IR (neat): 3080, 3004, 2963,
d
127.9, 128.7, 130.7, 130.8, 137.4, 139.9, 142.8, 158.7, 164.7; LCeMS
(ESI): 340.09 [MþH]þ.
5.3.32. N,5-bis(3-hydroxyphenyl)-N-methylthiophene-2-
carbothioamide (23)
Synthesized according to method
C using 23a (120 mg,
0.32 mmol) and BF3.SMe2 (402 L, 3.84 mmol). The residue was
m
purified by silica gel column chromatography (hexanes/EtOAc
40:60) to afford 23 as pale brown solid (106 mg, 97%). Mp:
174e177 ꢀC. IR (neat): 3185, 2263, 1584 cmꢁ1. 1H NMR (CD3COCD3)
2938, 1591, 1577 cmꢁ1. 1H NMR (CD3COCD3)
d 3.80 (s, 3H), 3.84 (s,
3H), 3.85 (s, 3H), 6.49 (d, J ¼ 4.1 Hz, 1H), 6.89e6.92 (m, 2H),
6.95e6.98 (m, 2H), 7.10 (d, J ¼ 4.1 Hz,1H), 7.12 (t, J ¼ 2.1 Hz,1H), 7.16
(ddd, J ¼ 0.9, 1.8, 7.7 Hz, 1H), 7.30 (t, J ¼ 8.1 Hz, 1H), 7.35 (dt, J ¼ 0.7,
d
3.82 (s, 3H), 6.53 (d, J ¼ 4.0 Hz, 1H), 6.78e6.84 (m, 3H), 6.85e6.88
(m, 1H), 7.03e7.09 (m, 3H), 7.21 (t, J ¼ 8.0 Hz, 1H), 7.28 (t, J ¼ 8.1 Hz,
1H); 13C NMR (CD3COCD3)
47.6,113.1, 114.1,116.1, 116.4, 117.8,118.1,
7.9 Hz, 1H); 13C NMR (CD3COCD3)
d
47.6, 55.7, 55.9, 111.7, 112.2,
d
114.8, 115.0, 118.8, 119.2, 124.1, 130.4, 131.1, 131.4, 135.6, 146.7, 149.4,
123.8, 130.7, 131.1, 131.5, 135.6, 146.5, 149.3, 150.3, 158.8, 159.4,
150.0, 161.2, 161.7, 191.0; LC-MS (ESI): 370.12 [MþH]þ.
190.8; LCeMS (ESI): 341.80 [M]þ.
5.3.28. N,5-bis(3-hydroxyphenyl)-N-methylthiophene-3-
carboxamide (5)
5.3.33. 2,5-Dibromo-N-(3-methoxyphenyl)-N-methylthiophene-3-
carboxamide (20a)
Synthesized according to method
C using 5a (161 mg,
0.46 mmol) and BF3.SMe2 (290 L, 2.76 mmol). The residue was
m
Synthesized according to method A using 20b (414 mg,
triturated in the mixture diethyl ether/petroleum ether to afford 5
1.45 mmol) and 3-methoxy-N-methylaniline (190 mL, 1.45 mmol).
as green solid (193 mg, 98%). Mp: 164e166 ꢀC. IR (neat): 3212, 2924,
The residue was purified by silica gel column chromatography
1584 cmꢁ1. 1H NMR (CD3COCD3)
d
3.38 (s, 3H), 6.75e6.80 (m, 4H),
(hexanes/EtOAc 80:20) to afford 20a as yellow oil (431 mg, 73%). 1H
6.94e6.96 (m, 2H), 7.16e7.25 (m, 4H); 13C NMR (CD3COCD3)
d
38.4,
NMR (CD3COCD3)
8.3 Hz, 1H), 6.82e6.85 (m, 2H), 6.95e6.98 (m, 1H), 7.23 (t, J ¼ 8.1 Hz,
1H); 13C NMR (CD3COCD3)
55.7, 56.1, 113.2, 114.1, 119.7, 129.6,
d
3.39 (s, 3H), 3.76 (s, 3H), 6.81 (ddd, J ¼ 0.8, 2.5,
113.2, 115.3, 115.9, 117.7, 119.2, 125.2, 129.4, 130.8, 131.0, 135.8, 139.0,
143.8, 147.0, 158.8, 159.2, 164.8; LC-MS (ESI): 325.87 [M]þ.
d
130.6, 130.8, 131.7, 140.8, 145.3, 161.0, 163.7; LCeMS (ESI): 402.79,
5.3.29. N-(3-hydroxybenzyl)-5-(3-hydroxyphenyl)-N-
methylthiophene-3-carboxamide (7)
406.80, 404.93 [M]þ.
Synthesized according to method C using 7a (48 mg, 0.13 mmol)
and BF3.SMe2 (82
gel column chromatography (hexanes/EtOAc 40:60) to afford 7 as
pale brown oil (44 mg, 98%). IR (neat): 3203, 2971, 2925, 1582 cmꢁ1
1H NMR (CD3COCD3)
2.09 (s, 3H), 4.67 (s, 2H), 6.76e6.83 (m, 4H),
7.12e7.25 (m, 4H), 7.50e7.74 (m, 2H); 13C NMR (CD3COCD3)
37.1,
51.1, 113.4, 115.2, 116.0, 117.9, 124.3, 125.9, 126.6, 130.55, 130.56,
130.7, 131.1, 135.8, 138.9, 140.0, 145.2, 158.8, 158.9; LCeMS (ESI):
339.87 [M]þ.
m
L, 0.78 mmol). The residue was purified by silica
5.3.34. N-2,5-tris(3-methoxyphenyl)-N-methylthiophene-3-
carboxamide (19a)
.
Synthesized according to method B using 20a (133 mg,
d
0.33 mmol) and 3-methoxyphenylboronic acid (75 mg, 0.49 mmol),
cesium carbonate (401 mg, 1.23 mmol) and tetrakis(-
triphenylphosphine) palladium (1 mg, 0.02 eq). The residue was
purified by silica gel column chromatography (hexanes/EtOAc
70:30) to afford 19a as yellow oil (143 mg, 94%). IR (neat): 3064,
d
3002, 2955, 2937, 1639, 1596, 1578 cmꢁ1. 1H NMR (CDCl3)
d 3.35 (br
5.3.30. N,4-bis(3-hydroxyphenyl)-N-methylthiophene-2-
carboxamide (6)
s, 3H), 3.52 (br s, 3H), 3.80 (s, 3H), 3.85 (s, 3H), 5.90e5.93 (m, 1H),
6.09e6.13 (m, 1H), 6.55e6.58 (m, 1H), 6.61e6.64 (m, 1H), 6.78e6.81
(m, 1H), 6.84e6.90 (m, 3H), 7.08e7.09 (m, 1H), 7.14e7.16 (m, 1H),
7.22e7.25 (m, 1H), 7.27 (d, J ¼ 8.0 Hz, 1H), 7.38 (s, 1H); 13C NMR
Synthesized according to method
C using 6a (164 mg,
0.46 mmol) and BF3.SMe2 (290 L, 2.76 mmol). The residue was
m
triturated in the mixture diethyl ether/petroleum ether to afford 6
as pale brown solid (56 mg, 37%). Mp: 187e189 ꢀC. IR (neat): 3232,
(CDCl3) d 55.0, 55.28, 55.33, 55.34, 110.7, 111.1, 112.3, 112.7, 113.5,
114.3, 118.15, 118.20, 120.4, 124.6, 128.7, 129.4, 129.9, 134.0, 134.6,
134.8, 141.1, 143.4, 144.2, 159.4, 159.7, 159.9, 167.2; LCeMS (ESI):
460.42 [MþH]þ.
3104, 2925, 1580 cmꢁ1 1H NMR (CD3COCD3)
. d 3.38 (s, 3H), 6.74
(ddd, J ¼ 0.9, 2.5, 8.2 Hz, 1H), 6.85 (t, J ¼ 2.2 Hz, 1H), 6.86e6.89 (m,
2H), 6.90 (t, J ¼ 2.2 Hz, 1H), 6.93 (ddd, J ¼ 1.0, 2.5, 8.3 Hz, 1H), 6.98
(d, J ¼ 1.5 Hz, 1H), 7.16 (t, J ¼ 7.9 Hz, 1H), 7.32 (t, J ¼ 7.9 Hz, 1H), 7.69
(d, J ¼ 1.6 Hz, 1H), 8.40 (s, 1H), 8.73 (s, 1H); 13C NMR (CD3COCD3)
5.3.35. N-2,5-tris(3-hydroxyphenyl)-N-methylthiophene-3-
carboxamide (19)
d
38.9, 113.7, 115.3, 116.1, 116.2, 118.1, 120.0, 126.1, 130.8, 131.1, 131.4,
137.4, 140.3, 142.3, 146.5, 158.7, 159.5, 162.4; LCeMS (ESI): 326.09
[MþH]þ.
Synthesized according to method
C using 19a (71 mg,
0.15 mmol) and BF3.SMe2 (283 L, 2.70 mmol). The residue was
m
triturated in the mixture diethyl ether/petroleum ether to afford 19
as pale green solid (64 mg, 97%). Mp: 139e142 ꢀC. IR (neat): 3253,
5.3.31. N-(3-hydroxybenzyl)-4-(3-hydroxyphenyl)-N-
methylthiophene-2-carboxamide (8)
2929, 1580 cmꢁ1. 1H NMR (CD3SOCD3)
d 3.25 (br s, 3H), 6.17e6.26
Synthesized according to method
C
using 8a (281 mg,
(m, 2H), 6.45e6.43 (m, 2H), 6.72e6.78 (m, 3H), 6.85e7.03 (m, 3H),
0.76 mmol) and BF3.SMe2 (480 L, 4.56 mmol). The residue was
m
7.17e7.33 (m, 3H), 9.37 (br s, 1H), 9.32 (br s, 2H); 13C NMR
purified by silica gel column chromatography (hexanes/EtOAc
40:60) to afford 8 as pale brown solid (296 mg, 97%). Mp:
174e177 ꢀC. IR (neat): 3273, 3097, 2961, 2926, 1580 cmꢁ1. 1H NMR
(CD3SOCD3) d 64.9, 111.8, 113.2, 113.5, 113.9, 115.2, 115.3, 116.0,
116.57, 116.59, 116.7, 117.9, 124.7, 124.8, 129.0, 129.9, 130.2, 133.8,
134.0, 134.3, 144.1, 157.4, 157.6, 157.8; LCeMS (ESI): 417.81 [M]þ.

334
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
5.3.36. 5-Bromo-N-(3-methoxybenzyl)-N,3-dimethylthiophene-2-
carboxamide (22b)
5.3.40. 2-(3-Methoxyphenyl)-1-[5-(3-methoxyphenyl)thiophen-2-
yl]-2-methylpropan-1-one (29a)
Synthesized according to method
methylthiophene-2-carboxylic acid 22 (442 mg, 2 mmol) and 3-
methoxy-N-methylbenzylamine (298 L, 2 mmol). The residue
A
using 5-bromo-3-
To a solution of 2-(3-methoxyphenyl)-2-methylpropanoic acid
31b (510 mg, 2.63 mmol) in dry toluene (10 mL) was added thionyl
chloride (760 mL, 10.50 mmol) followed by 5 drops of DMF. The
m
was purified by silica gel column chromatography (hexanes/EtOAc
reaction mixture was heated at reflux for 4 h under N2 atmosphere.
After cooling the solvent was removed under reduced pressure to
afford the corresponding acyl chloride. To a solution of 2-(3-
methoxyphenyl)thiophene 30a (500 mg, 2.63 mmol) in dry
CH2Cl2 (10 mL) was added the previously freshly prepared acyl
chloride, followed by aluminum chloride (526 mg, 3.94 mmol)
at ꢁ20 ꢀC and under N2 atmosphere. After 30 min the ice bath was
removed and the reaction mixture was stirred at room temperature
overnight. Cold water (10 mL) was added to quench the reaction.
The inorganic salts were filtered on Celite and the aqueous layer
was extracted three times with CH2Cl2. The organic layer was
washed with water, dried over MgSO4, filtered and the solution was
concentrated under reduce pressure. The residue was purified by
silica gel column chromatography using hexanes and EtOAc as
eluant (95:5) to afford 29a as colorless solid (330 mg, 34%). Mp:
109e113 ꢀC. IR (neat): 3018, 2992, 2975, 2951, 2929, 1640, 1605,
70:30) to afford 22b as pale brown oil (710 mg, 99%). 1H NMR
(CD3COCD3)
6.85e6.89 (m, 3H), 6.97 (s, 1H), 7.27e7.30 (m, 1H); 13C NMR
(CD3COCD3) 13.5, 33.9, 51.6, 54.1, 111.5, 112.3, 112.6, 119.2, 129.2,
d 2.22 (s, 3H), 2.95 (s, 3H), 3.79 (s, 3H), 4.65 (s, 2H),
d
132.2, 132.4, 137.5, 138.2, 159.6, 163.1; LCeMS (ESI): 353.81, 355.88
[MþH]þ.
5.3.37. 5-Bromo-N-(3-hydroxybenzyl)-N,3-dimethylthiophene-2-
carboxamide (22a)
Synthesized according to method
C using 22b (625 mg,
1.76 mmol) and BF3.SMe2 (1.11 mL, 10.59 mmol). The desired
product obtained as pale brown oil (598 mg, 99%) was analytically
pure and was used in the next step without any purifications. 1
H
NMR (CD3COCD3)
d
2.22 (s, 3H), 2.95 (s, 3H), 4.62 (s, 2H), 6.74e6.79
14.9,
1577 cmꢁ1. 1H NMR (CD3COCD3)
d 1.63 (s, 6H), 3.79 (s, 3H), 3.84 (s,
(m, 3H), 6.97 (s, 1H), 7.16e7.20 (m, 1H); 13C NMR (CD3COCD3)
d
3H), 6.85e6.88 (m, 1H), 6.92e6.94 (m, 2H), 6.95 (dd, J ¼ 0.9, 2.6 Hz,
1H), 7.04 (d, J ¼ 4.1 Hz, 1H), 7.18 (t, J ¼ 2.1 Hz, 1H), 7.21 (ddd, J ¼ 0.9,
1.6, 7.8 Hz, 1H), 7.26 (d, J ¼ 4.1 Hz, 1H),7.28e7.34 (m, 2H); 13C NMR
35.4, 53.1, 113.2, 115.2, 115.4, 119.6, 130.7, 133.4, 133.7, 139.2, 139.4,
158.6, 164.9; LCeMS (ESI): 339.80, 341.81 [M]þ.
(CD3COCD3)
d 28.0, 52.0, 55.5, 55.7, 112.3, 112.7, 113.3, 115.4, 119.3,
119.5, 125.2, 130.8, 131.1, 135.3, 135.5, 142.1, 147.9, 151.4, 161.1, 161.2,
196.3. LCeMS (ESI): 367.11 [MþH]þ.
5.3.38. 5-(2-Fluoro-3-methoxyphenyl)-N-(3-methoxybenzyl)-N,3-
dimethylthiophene-2-carboxamide (21)
Synthesized according to method
B using 22a (326 mg,
0.96 mmol), 2-fluoro-3-methoxyphenylboronic acid (244 mg,
1.44 mmol), cesium carbonate (937 mg, 2.87 mmol) and tetrakis(-
triphenylphosphine) palladium (22 mg, 0.02 eq.). The residue was
purified by silica gel column chromatography (hexanes/EtOAc
60:40) followed by trituration in EtOAc and filtration under
reduced pressure to afford 21 as colorless solid (210 mg, 57%). Mp:
123e125 ꢀC. IR (neat): 3162, 2985, 2970, 2935, 1599 cmꢁ1. 1H NMR
5.3.41. 2-(3-Hydroxyphenyl)-1-[5-(3-hydroxyphenyl)thiophen-2-
yl]-2-methylpropan-1-one (29)
Synthesized according to method
C using 29a (250 mg,
0.68 mmol) and BF3.SMe2 (861 L, 8.19 mmol). The residue was
m
triturated in the mixture of diethyl ether/petroleum ether to afford
29 as pale green solid (170 mg, 71%). Mp: 209e213 ꢀC. IR (neat):
3363, 3230, 2974, 1621, 1594 cmꢁ1. 1H NMR (CD3COCD3)
d 1.60 (s,
(CD3COCD3)
d 2.29 (s, 3H), 2.97 (s, 3H), 3.92 (s, 3H), 4.66 (s, 2H),
6H), 6.77 (ddd, J ¼ 0.9, 2.5, 8.1 Hz, 1H), 6.82 (t, J ¼ 2.0 Hz, 1H),
6.84e6.87 (m, 2H), 7.05 (d, J ¼ 4.1 Hz,1H), 7.11 (t, J ¼ 2.0 Hz,1H), 7.13
(ddd, J ¼ 1.0,1.8, 7.6 Hz,1H), 7.21 (t, J ¼ 7.9 Hz,1H), 7.22 (d, J ¼ 4.1 Hz,
1H), 7.24 (t, J ¼ 7.9 Hz, 1H), 8.32 (s, 1H), 8.56 (s, 1H); 13C NMR
6.76e6.84 (m, 3H), 7.11 (dt, J ¼ 1.6, 8.1 Hz, 1H), 7.16 (dt, J ¼ 1.3,
8.0 Hz, 1H), 7.19 (t, J ¼ 5.8 Hz, 1H), 7.23e7.28 (m, 1H), 7.34 (s, 1H),
8.39 (s, 1H); 13C NMR (CD3COCD3)
d 14.9, 16.5, 56.7, 113.71, 113.72,
115.4, 119.7, 120.30, 120.32, 122.7, 122.8, 125.4, 125.5, 129.9, 130.0,
130.6, 132.57, 132.61, 137.1, 137.2, 138.4, 139.8, 148.8, 149.5, 149.6,
150.8, 158.7, 165.5 (additional peaks due to coupling of carbon with
fluorine); LCeMS (ESI): 385.89 [M]þ.
(CD3COCD3)
d 28.0, 51.9, 113.6, 114.2, 114.9, 116.9, 118.16, 118.25,
124.9, 130.8, 131.2, 135.3, 135.4,141.9, 147.9, 151.5, 158.7, 158.9, 196.4.
LCeMS (ESI): 339.10 [MþH]þ.
5.3.42. Methyl 5-(3-methoxyphenyl)thiophene-2-carboxylate (33a)
To a solution of 3-chloro-3-(3-methoxyphenyl)prop-2-enal 33b
(5 g, 25.43 mmol) and dry potassium carbonate (4.22 g,
30.52 mmol) in dry DMF (40 mL) was added drop wise under N2
atmosphere methyl thioglycolate (2.78 mL, 30.52 mmol) diluted in
dry DMF (10 mL). The reaction mixture was heated at 60 ꢀC over-
night. After cooling to room temperature the reaction mixture was
poured into ice water solution under stirring. The aqueous layer
was extracted three times with EtOAc (3 ꢃ 30 mL). The organic
layer was washed once with water, dried MgSO4, filtered and the
solution was concentrated under reduced pressure. The pale brown
solid obtained was pure enough to be used in the next step without
further purification (1.04 g, 82%). Mp: 38e39 ꢀC. IR (neat, Bruker
Tensor 27): 3101, 3071, 3014, 2974, 2943, 2833, 1705, 1601 cmꢁ1. 1H
5.3.39. 2-(3-Methoxyphenyl)thiophene (30a)
A
solution of 2-bromothiophene (326 mg, 8 mmol), 3-
methoxyphenylboronic acid (456 mg, 12 mmol), cesium carbon-
ate (1.96 g, 24 mmol) and tetrakis(triphenylphosphine)palla-
dium(0) (46 mg, 0.2 mmol) in O2-free DME/EtOH/H2O (3 mL, 1:1:1)
was heated under microwave irradiation at 150 ꢀC/150 W for
20 min. After cooling to room temperature water and EtOAc (2 mL,
1:1) were added to quench the reaction. The solution was extracted
three times with EtOAc (3 ꢃ 10 mL). The organic layer was dried
over MgSO4, filtered and the solution was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography using hexanes and EtOAc as eluant (95:5) to afford
30a as pale yellow oil (698 mg, 46%). 1H NMR (CD3COCD3)
d 3.85 (s,
3H), 6.88 (ddd, J ¼ 0.7, 2.5, 8.2 Hz, 1H), 7.11 (dd, J ¼ 3.7, 5.1 Hz, 1H),
7.21 (t, J ¼ 1.8 Hz, 1H), 7.23e7.25 (m, 1H), 7.32 (t, J ¼ 8.0 Hz, 1H), 7.44
(dd, J ¼ 1.1, 5.2 Hz, 1H), 7.46 (dd, J ¼ 1.2, 3.7 Hz, 1H); 13C NMR
NMR (CD3COCD3)
d
3.87 (s, 3H), 3.88 (s, 3H), 6.98 (ddd, J ¼ 0.9, 2.5,
8.2 Hz, 1H), 7.27 (t, J ¼ 2.0 Hz, 1H), 7.31 (ddd, J ¼ 1.0, 1.6, 7.6 Hz, 1H),
7.37 (t, J ¼ 8.1 Hz, 1H), 7.52 (d, J ¼ 4.0 Hz,1H), 7.76 (d, J ¼ 4.0 Hz,1H);
(CD3COCD3)
d
55.6, 112.0, 113.9, 119.0, 124.5, 126.0, 129.0, 131.0,
13C NMR (CD3COCD3)
d 52.5, 55.7, 112.3, 115.5, 119.3, 125.2, 131.2,
136.6, 144.8, 161.2. LCeMS (ESI): 190.18 [M]þ.
133.0, 135.2, 135.4, 151.6, 161.3, 162.8. LCeMS (ESI): 248.94 [M]þ.

E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
335
5.3.43. 5-(3-Methoxyphenyl)thiophene-2-carboxylic acid (34)
solution of methyl 5-(3-methoxyphenyl)thiophene-2-
5.3.46. 2-(3-Hydroxyphenyl)-1-[5-(3-hydroxyphenyl)thiophen-2-
yl]ethanone (32)
A
carboxylate 33a (5.15 g, 20.74 mmol) and potassium hydroxide
(4.65 g, 82.96 mmol) in a mixture of MeOH/water (100 mL/50 mL)
was heated to a slow reflux for 4 h. Methanol was removed under
reduced pressure. The aqueous layer was extracted three times
with diethyl ether. The aqueous layer was slowly acidified at 0 ꢀC
with concentrated hydrochloric acid until pH ¼ 2. The precipitated
formed was filtered off and washed twice with water to afford 34 as
pale brown solid (519 mg, 53%). Mp: 161e164 ꢀC. IR (neat, Bruker
Tensor 27): 2725 (br), 1659, 1601, 1576 cmꢁ1. 1H NMR (CD3SOCD3)
Synthesized according to method
C
using 32a (578 mg,
1.71 mmol) and BF3.SMe2 (2.16 mL, 20.52 mmol). The residue was
recrystallized in a mixture of ethanol/water to afford 32 as yellow
solid (251 mg, 48%). Mp: 183e187 ꢀC. IR (neat): 3426, 3151, 3092,
3024, 2971, 1621,1591 cmꢁ1. 1H NMR (CD3COCD3)
d 4.19 (s, 2H), 6.72
(ddd, J ¼ 0.9, 2.4, 8.0 Hz, 1H), 6.83e6.86 (m, 2H), 6.89 (ddd, J ¼ 1.0,
2.5, 8.1 Hz, 1H), 7.14 (t, J ¼ 7.8, 1H), 7.20 (t, J ¼ 2.1, 1H), 7.22 (ddd,
J ¼ 1.1, 1.7, 7.7 Hz,1H), 7.28 (t, J ¼ 8.1, 1H), 7.49 (d, J ¼ 4.1 Hz,1H), 7.96
(d, J ¼ 4.1 Hz, 1H), 8.32 (br s, 1H), 8.67 (br s, 1H); 13C NMR
d
3.82 (s, 3H), 6.97 (ddd, J ¼ 0.7, 2.5, 8.2 Hz, 1H), 7.26 (t, J ¼ 1.9 Hz,
(CD3COCD3) d 46.1, 113.7, 114.7, 117.2, 117.3, 118.3, 121.5, 125.4, 130.4,
1H), 7.28e7.31 (m, 1H), 7.37 (t, J ¼ 8.0 Hz, 1H), 7.59 (d, J ¼ 3.9 Hz,
131.3, 135.2, 135.5, 137.5, 143.6, 153.2, 158.4, 158.9, 190.9. LCeMS
1H), 7.72 (d, J ¼ 3.9 Hz, 1H), 13.13 (br s, 1H); 13C NMR (CD3SOCD3)
(ESI): 310.97 [M]þ.
d
55.3, 111.2, 114.6, 118.3, 124.9, 130.4, 133.3, 134.1, 134.2, 149.6,
159.8, 162.7. LCeMS (ESI): 234.97 [MþH]þ.
5.4. Biological assays
5.4.1. h17
The placental microsomal enzyme was used for the human 17
HSD2 cell free assay as described previously [46]. The enzyme
h17 -HSD2 was incubated with tritiated E2 (final concentration:
500 nM) in the presence of the cofactor NADþ (1500
M) and po-
tential inhibitor (test concentration: 1 M) as previously described
[46]. Substrate and product were separated by high performance
liquid chromatography (HPLC) and the amounts of labeled E2
(substrate) and E1 (product) were quantified. IC50 values were
determined for compounds showing more than 55% inhibition at
b-HSD2 cell free assay
5.3.44. N-methoxy-5-(3-methoxyphenyl)-N-methylthiophene-2-
carboxamide (34a)
b
-
A solution of 5-(3-methoxyphenyl)thiophene-2-carboxylic acid
34 (4.71 g, 20.12 mmol) and thionyl chloride (5.85 mL, 80.48 mmol)
in dry toluene (100 mL) was refluxed for 4 h. The reaction mixture
was concentrated to dryness under reduced pressure. CH2Cl2
(50 mL) was added and the solution was cooled to 0 ꢀC. N,O-
dimethylhydroxylamine hydrochloride (2.94 g, 30.18 mmol) was
added to the solution followed by Et3N (4.20 mL, 30.18 mmol). The
reaction mixture was warmed up to room temperature and stirred
overnight. Water (50 mL) was added to quench the reaction. The
aqueous layer was extracted three times with CH2Cl2. The organic
phase was washed once with HCl 1 N and once with water, before
being dried over MgSO4, filtered and the solution was concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography using hexanes and EtOAc as eluant
(70:30) to afford 34a as orange oil (5.95 g, 97%). IR (neat, Bruker
Tensor 27): 3080, 3007, 2974, 2941, 2839, 2372, 1715, 1630, 1607,
b
m
m
1 mM on h17b-HSD2 as shown in Table 1.
5.4.2. h17
Inhibition of h17
cedure as for the h17
b
-HSD1 cell free assay
-HSD1 was determined using a similar pro-
-HSD2 cell free assay. h17 -HSD1, gained
b
b
b
from the placental cytosolic fraction, was incubated with tritiated
E1 (final concentration: 500 nM) in the presence of the cofactor
NADH (500 mM) and potential inhibitor (test concentration: 1 mM).
Separation and quantification of labeled E1 (substrate) and E2
(product) was performed by HPLC using radio flow detection.
1578 cmꢁ1. 1H NMR (CD3COCD3)
d 3.33 (s, 3H), 3.86 (s, 3H), 3.87 (s,
3H), 6.96 (ddd, J ¼ 1.0, 2.6, 8.1 Hz, 1H), 7.28 (t, J ¼ 2.1 Hz, 1H), 7.31
Selectivity factors were calculated as follows: SF ¼ IC50(h17
b-
(ddd, J ¼ 1.0,1.6, 7.6 Hz,1H), 7.37 (t, J ¼ 8.1 Hz,1H), 7.49 (d, J ¼ 4.1 Hz,
1H), 7.86 (d, J ¼ 4.1 Hz, 1H); 13C NMR (CD3COCD3)
d
33.1, 55.7, 62.1,
HSD1)/IC50(h17 -HSD2).
b
112.3, 115.0, 119.2, 124.3, 131.1, 133.7, 135.6, 135.9, 150.9, 161.3, 162.4.
LCeMS (ESI): 278.23 [MþH]þ.
5.4.3. ER binding affinity assay
The binding affinity of selected compounds to ERa and ERb was
determined according to the recommendations of the US Envi-
ronmental Protection Agency (EPA) by their Endocrine Disruptor
Screening Program (EDSP) [47] using recombinant human proteins.
5.3.45. 2-(3-Methoxyphenyl)-1-[5-(3-methoxyphenyl)thiophen-2-
yl]ethanone (32a)
To the N-methoxy-5-(3-methoxyphenyl)-N-methylthiophene-
2-carboxamide 34a (2.53 g, 9.11 mmol) in dry THF (20 mL) was
added at 0 ꢀC drop wise a solution of 3-methoxybenzylmagnesium
chloride 0.25 M in THF (36.4 mL, 9.11 mmol). The reaction mixture
was stirred 1 h at 0 ꢀC and warmed up to room temperature
overnight. The reaction mixture was quenched at 0 ꢀC by addition
of concentrated hydrochloric acid and extracted with EtOAc
(4 ꢃ 25 mL). The organic layer was washed with water, dried over
MgSO4, filtered and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using
hexanes and EtOAc as eluant (70:30) to afford 32a as yellow solid
(578 mg, 33%). Mp: 61e64 ꢀC. IR (neat): 3076, 3014, 2988, 2957,
Briefly, 1 nM of ER
a
and 4 nM of ER
b
, respectively, were incubated
with [3H]-E2 (3 nM for ER
a
and 10 nM for ER
b
) and inhibitor. The
potential inhibitors were dissolved in DMSO (5% final concentra-
tion). Evaluation of non-specific binding was performed with un-
labeled E2 at concentrations 100-fold of [3H]-E2 (300 nM for ER
a
and 1000 nM for ERb). After incubation, ligandereceptor complexes
were selectively bound to hydroxyapatite (83.5 g/L in TE-buffer)
and measurements were performed by liquid scintillation count-
ing. From these results the percentage of [3H]-E2 displacement by
the compounds was calculated. The plot of percentage displace-
ment versus compound concentration resulted in sigmoidal bind-
ing curves. The compound concentrations to displace 50% of the
receptor bound [3H]-E2 were determined. Unlabeled E2 IC50 values
were determined in each experiment and used as reference. The E2
2937, 1652, 1578 cmꢁ1. 1H NMR (CD3COCD3)
d 3.77 (s, 3H), 3.86 (s,
3H), 4.25 (s, 2H), 6.82 (dd, J ¼ 2.4, 8.1 Hz, 1H), 6.93e6.99 (m, 3H),
7.23 (t, J ¼ 7.9, 1H), 7.27 (t, J ¼ 1.9, 1H), 7.30e7.32 (m, 1H), 7.37 (t,
J ¼ 8.0, 1H), 7.55 (d, J ¼ 3.9 Hz, 1H), 7.99 (d, J ¼ 3.9 Hz, 1H); 13C NMR
IC50 values accepted were 3 nM 20% for ER
a and 10 nM 20% for
ER . Relative Binding Affinity (RBA) was determined by applying
b
(CD3COCD3)
d
46.1, 55.5, 55.8, 112.4, 112.9, 115.7, 116.2, 119.4, 122.6,
the following equation: RBA[%] ¼ (IC50(E2)/IC50(compound)) ꢃ 100
[47]. This results in an RBA value of 100% for E2. Compounds were
tested at a concentrations of 1000 ꢃ IC50(E2). Compounds with less
125.7, 130.3, 131.2, 135.2, 135.5, 137.5, 143.7, 153.1, 160.8, 161.3, 190.8.
LCeMS (ESI): 338.99 [M]þ.

336
E. Perspicace et al. / European Journal of Medicinal Chemistry 83 (2014) 317e337
than 50% displacement of [3H]-E2 at
1000 ꢃ IC50(E2) were classified as RBA <0.1%.
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Conflict of interest
steroid-5alpha-reductase type
1479e1487.
1 and 2, Bioorg. Med. Chem. 8 (2000)
Authors declare no conflict of interest.
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