Triazole ring-opening leads to the discovery of potent nonsteroidal 17β-hydroxysteroid dehydrogenase type 2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.10.010

17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the oxidation of the highly potent steroids: the estrogen estradiol (E2) and the androgen testosterone (T) to the less active estrone and androstenedione, respectively. Inhibition of this enzyme may help maintain the local E2 level in bone tissue when the circulating E2 level drops and is therefore a novel and promising approach for the treatment of osteoporosis. In this work, a series of new nonsteroidal and achiral 17β-HSD2 inhibitors, namely N-benzyl-diphenyl-3(or 4)-carboxamide and N-benzyl-5-phenyl-thiophene-2- carboxamide was designed and the compounds were synthesized in a two to three steps reaction. A small library was built applying parallel synthesis. Highly potent 17β-HSD2 inhibitors could be identified in the thiophene-2- carboxamide class with IC₅₀ in the low nanomolar range. These compounds also showed a good selectivity profile toward 17β-HSD1 and toward the estrogen receptors α and β. The most interesting 17β-HSD2 inhibitor identified in this study is the 5-(2-fluoro-3-methoxyphenyl)-N-(3- hydroxybenzyl)-N-methylthiophene-2-carboxamide 6w displaying an IC₅₀ of 61 nM and a selectivity factor of 73 toward 17β-HSD1.

Full text of DOI:10.1016/j.ejmech.2011.10.010

European Journal of Medicinal Chemistry 46 (2011) 5978e5990
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Triazole ring-opening leads to the discovery of potent nonsteroidal
17
b-hydroxysteroid dehydrogenase type 2 inhibitors
,
,b, ,2
*
Kuiying Xu a, Yaseen A. Al-Soud a 1, Marie Wetzel a, Rolf W. Hartmann a
,
,
,2
**
Sandrine Marchais-Oberwinkler a
a Pharmaceutical and Medicinal Chemistry, Saarland University, Germany
b Helmholtz Institute for Pharmaceutical Science Saarland (HIPS), Campus C23, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
17b-Hydroxysteroid dehydrogenase type 2 (17b-HSD2) catalyzes the oxidation of the highly potent
Received 11 July 2011
Received in revised form
30 September 2011
Accepted 4 October 2011
Available online 10 October 2011
steroids: the estrogen estradiol (E2) and the androgen testosterone (T) to the less active estrone and
androstenedione, respectively. Inhibition of this enzyme may help maintain the local E2 level in bone
tissue when the circulating E2 level drops and is therefore a novel and promising approach for the
treatment of osteoporosis.
In this work, a series of new nonsteroidal and achiral 17b-HSD2 inhibitors, namely N-benzyl-diphenyl-
3(or 4)-carboxamide and N-benzyl-5-phenyl-thiophene-2-carboxamide was designed and the
compounds were synthesized in a two to three steps reaction. A small library was built applying parallel
Keywords:
17b-HSD2
Osteoporosis
Achiral inhibitors
Steroidomimetics
Selectivity
synthesis. Highly potent 17
with IC50 in the low nanomolar range. These compounds also showed a good selectivity profile toward
17 -HSD1 and toward the estrogen receptors and . The most interesting 17 -HSD2 inhibitor identified
in this study is the 5-(2-fluoro-3-methoxyphenyl)-N-(3-hydroxybenzyl)-N-methylthiophene-2-
carboxamide 6w displaying an IC50 of 61 nM and a selectivity factor of 73 toward 17 -HSD1.
b-HSD2 inhibitors could be identified in the thiophene-2-carboxamide class
b
a
b
b
b
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
osteoprotective action of estrogens is known [3]: they control bone
remodeling during reproductive life in both women and men [4,5]
and they inhibit osteoclastogenesis leading to a decrease in bone
resorption. In addition, an estrogen replacement therapy is efficient
in the treatment of bone loss and osteoporotic fractures [6e8].
Furthermore, there is substantial evidence that active androgens
like testosterone (T, Fig. 1) and dihydrotestosterone (DHT) are
involved in bone formation and might protect bones from osteo-
porosis [9,10]. Therefore, a controlled increase in active estrogen
estradiol (E2) and androgen testosterone (T) in bones of osteopo-
rotic patients will certainly slow down bone loss by lowering bone
resorption and raising bone formation and should thus reduce
osteoporotic fractures. However, the increase in E2 and T must be
restricted to the bones to avoid unwanted side-effect such as
induction of breast cancer [11].
Estrogen deficiency is believed to be responsible for the rapid
progression of osteoporosis [1]: e.g. high occurrence of this disease
has been observed in women after menopause when the estrogen
levels drop or after treatment with aromatase inhibitors, which
radically prevent estrogen biosynthesis [2]. In addition the
Abbreviations: 17
b
-HSD2, 17
b
-hydroxysteroid dehydrogenase type 2; 17
b-HSD1,
17
b-hydroxysteroid dehydrogenase type 1; E1, estrone; E2, 17b
-estradiol; T,
testosterone; A-dione, 4-androstene-3,17-dione; DHT, dihydrotestosterone;
NADP(H), nicotinamide adenine dinucleotide phosphate; NAD(H), nicotinamide
adenine dinucleotide; FC, flash chromatography; SF, selectivity factor; RBA, relative
binding affinity; ER, estrogen receptor.
* Corresponding author. Helmholtz Institute for Pharmaceutical Science Saarland
(HIPS), Campus C23, D-66123 Saarbrücken, Germany. Tel.: þ49 681 302 70300;
fax: þ49 681 302 70308.
17b-Hydroxysteroid dehydrogenase type 2 (17b-HSD2) [12] is
a trans-membrane protein localized in the endoplasmic reticulum
[13]. It is present in bones [14,15] and catalyzes the transformation
of both estrogens and androgens. It shows oxidative as well as
reductive activity in vitro, depending on the redox state of the
cofactor present (NADþ or NADH, respectively). However, in vivo it
has a predominant oxidative activity converting the highly active
** Corresponding author. Tel.: þ49 681 302 70300; fax: þ49 681 302 70308.
E-mail addresses: rwh@mx.uni-saarland.de (R.W. Hartmann), s.marchais@mx.
uni-saarland.de (S. Marchais-Oberwinkler).
URL: http://www.pharmmedchem.de
Permanent address: Department of Chemistry, Faculty of Science, Al al-Bayt
1
University, P.O. Box 130090 Al-Mafraq, Jordan.
2
RWH and SMO contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.010

K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5979
identified after selectivity screening, including compounds 7 and 8
(Fig. 3) [58]. The latter are weak inhibitors of 17 -HSD2; 7 shows
42% inhibition when tested at a concentration of 1 M and 8 30%
b
m
inhibition. Compounds 7 and 8 share some similarities with the
highly potent 2: they have a biaromatic moiety in common (rings
A þ B: pyridinethiophene in 2, biphenyl in 7 and phenylthiophene
in 8 (Fig. 3), a phenyl ring (ring C, Fig. 3) as well as a polar group
linking rings B and C. Compounds 7, 8 and 2 differ in the nature of
the linker between the B- and C-rings:
a
flexible
hydroxymethylenepyrrolidinone in 2 has been exchanged by a rigid
methylated triazole in 7 and 8, resulting in an overall linear scaffold
whereas in case of 2 there is a bent shape. The ring C in 2 and in 7, 8
covers a different area in the enzyme active site, its position might
be critical for inhibitory activity. We hypothesized that the rigidity
induced by the triazole moiety in 7 and 8 might be responsible for
their weak activity. As an amide (present in the pyrrolidinone of 2)
is a bioisostere of triazole and offers higher flexibility, it was
decided to exchange the triazole by an amide (triazole ring-
opening). In addition, as the N-methyl function present in 2, 7
and 8 might be important for the activity, N-methylated amides 4, 5
Fig. 1. 17b-HSD1, 2, 3 in sex steroid metabolism.
17
estrone (E1) and androstenedione (A-dione), using the cofactor
NADþ (Fig. 1). Thus, 17
-HSD2 is responsible for the inactivation of
E2 and T, while the biological counterparts 17 -HSD1 and 17
HSD3 activate E1 and A-dione respectively (Fig. 1). As intracellular
increase of E2 and T in bones can be achieved by inhibition of 17
b-hydroxysteroids such as E2 and T to the less active forms
b
b
b-
and 6 were designed as potential inhibitors of 17b-HSD2. Varying
the nature of the substituents R1 and R2 present on the rings A and
C, a small library of different N-benzyl-N-methylbiphenyl-4(or 3)-
carboxamides (4, 5) and N-benzyl-N-methyl-5-phenylthiophene-2-
carboxamides (6) was synthesized.
b-
HSD2, inhibition of this enzyme provides a novel and promising
approach for the treatment of osteoporosis.
There are very few potent and specific 17
described [16,17]. They can be divided into three classes (Fig. 2): 1.
steroidal spiro- -lactone 1 [18e21] (inhibition of 62% at 1 M [19]).
2. nonsteroidal chiral 4,5-disubstituted cis-pyrrolidinone
(IC50 ¼ 50 nM [22e24]). 3. nonsteroidal hydroxyphenyl naphthol 3
which like shows only moderate inhibitory activity
b-HSD2 inhibitors
3. Results
d
m
2
3.1. Chemistry
1
The synthesis pathway is depicted in Scheme 1. Amidation was
first carried out by reaction of the carbonyl chlorides 9e11 with the
corresponding benzylamines 12 under standard conditions
providing the bromophenyl 13, 14 and bromothiophene 15 deriv-
atives in very good yields (93e99%). In case of the thiophene
derivatives 15a, ether cleavage with borontrifluoride dimethyl
sulfide complex afforded the hydroxylated key intermediate 15c.
Subsequently Suzuki cross-coupling was conducted in parallel on
the hydroxyphenyl 15c or methoxyphenyl derivatives 13, 14, 15a or
the unsubstituted compound 15b with the appropriate substituted
phenyl boronic acid and afforded the final products 4e6 (yield
71e98%). Final hydroxy compounds were obtained after ether
cleavage with borontrifluoride dimethyl sulfide complex from the
methoxy analogues (yield 82%e98%).
(IC50 ¼ 302 nM [25]). A serious drawback of the steroidal inhibitors
is the risk to interact with steroid hormone receptors which could
result in unwanted severe side effects. The nonsteroidal chiral 2
was evaluated in vivo in an osteoporosis model using ovariecto-
mized cynomolgus monkeys [26]. A decrease of bone resorption
and maintenance of bone formation was observed. Despite high
experimental variations and the little efficacy, this in vivo experi-
ment validates the approach of 17
b-HSD2 inhibition. However, new
nonsteroidal 17 -HSD2 inhibitors with a good pharmacokinetic
b
profile are required for further drug development.
In the present study, we aim at the design, synthesis and in vitro
evaluation of a novel class of 17b-HSD2 inhibitors: N-benzyl-
biphenyl-3(or 4)-carboxamides (4, 5, Fig. 3) and N-benzyl-5-
phenyl-thiophene-2-carboxamides (6, Fig. 3) that, in contrast to
the existing 17b-HSD2 inhibitors, are easily accessible because they
do not contain asymmetric carbons.
3.2. In vitro biological evaluation
3.2.1. Activity: inhibition of human 17
The synthesized compounds were tested for inhibitory activity
on human 17 -HSD2 in a cell-free assay using human placental
microsomal enzyme as described earlier on with minor modifica-
tions [59]. Briefly, for determination of 17 -HSD2 inhibition the
enzyme was incubated with [3H]-E2 (500 nM, 0.135 Ci) and NADþ
[1500 M] in presence of potential inhibitors. The amount of
labelled E1 formed from tritiated substrate was determined by
HPLC. The spiro- -lactone 1 described by Poirier et al. [19] was used
as reference compound and gave similar values in our test as
described in the literature [19] (68% inhibition at 1 M). IC50 values
were determined for compounds showing more than 50% inhibi-
tion at 1 M and are shown in Tables 1 and 2. For the less potent
compounds, percent inhibition values at are given.
M were
b-HSD2
2. Inhibitor design
b
As the 3D-structure of 17b-HSD2 is unknown and there is no
homology model available, drug design was based on a ligand-
based approach. This strategy was already successfully applied in
our group for CYP19 [27e29], CYP11B2 [30e33], CYP11B1 [34,35],
b
m
m
CYP17 [36e39] and 5
During the development of 17
our group [44e57], new nonsteroidal 17
a
-reductase inhibitors [40e43].
-HSD1 inhibitors performed in
-HSD2 inhibitors were
b
d
b
m
m
1
mM
Compounds showing less than 10% inhibition at 1
considered to be inactive.
m
Three compound classes have been investigated differing in the
Fig. 2. Structures of known 17
b
-HSD2 inhibitors.
nature of the central B-ring: 1,4-disubstituted benzenes (class 4);

5980
K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
Fig. 3. Designed structures.
Scheme 1. Synthesis of compounds 4e6.

K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5981
Table 1
Table 2
In vitro inhibitory potencies toward 17
1, 4, 5 and A.
b
-HSD2 and 17
b
-HSD1 of biphenyl derivatives
In vitro inhibitory potencies toward 17
derivatives 6.
b
-HSD2 and 17
b
-HSD1 of phenylthiophene
Cmpd
R2
R1
IC50 (nM)a
17
-HSD2b
Selectivity
factorsd
b
17
b
-HSD1c
Cmpd
R2
R1
IC50 (nM)a
17
-HSD2b
Selectivity
factorsd
1
e
e
65%e
494
594
n.d.
b
17
b
-HSD1c
4a
4b
4c
4d
5a
5b
5c
5d
A
3-OMe
3-OH
3-Me
3-Me
4-OMe
4-OH
3-OMe
3-OH
e
OMe
OH
OMe
OH
OMe
OH
OMe
OH
e
>100,000
13,009
>100,000
64,532
n.i.
9099
n.i.
3801
n.i.
>202
22
>375
246
n.d.
14
n. d.
8
n.d.
1
e
e
65%e
371
394
496
335
49%e
149
50%e
186
222
42%e
371
36%e
1127
520
n.i.
790
161
278
246
164
326
430
61
n.d.
>100,000
5449
>120,000
5105
n.i.
2982
n.i.
3915
265
6a
6b
6c
6d
6e
6f
6g
6h
6i
3-OMe
3-OH
4-OMe
4-OH
2-OMe
2-OH
H
OMe
OH
OMe
OH
OMe
OH
OMe
OH
H
H
H
H
H
>250
14
>240
15
n.d.
20
n.d.
21
>450
n.d.
>265
n.d.
49
>190
n.d.
55
>250
84
75
65
10
42
73
5
262
11%e
639
13%e
482
39%e
H
n.i.: no inhibition; n.d.: not determined.
a
3-OMe
3-OH
4-OMe
4-OH
2-OMe
2-OH
4-CN
>100,000
Mean value of three determinations, standard deviation less than 15%.
6j
6k
6l
10%e
b
Human placental, microsomal fraction, substrate E2 [500 nM], cofactor NADþ
>100,000
n.i.
55,741
>100,000
n.i.
43,398
>40,000
23,345
18,597
10,645
3151
[1500
m
M].
Human placental, cytosolic fraction, substrate E1 [500 nM], cofactor NADH
[500 M].
IC50 (17
c
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
m
d
H
b
-HSD1)/IC50 (17
% inhibition at 1 M.
b-HSD2).
e
OMe
OH
OH
OH
OH
OH
OH
OH
OH
OH
m
4-CN
4-OMe
3-OMe
3-N(Me)2
3-Me
3-F
4-F
2-F,3-OMe
2-F,3-OH
1,3-disubstituted benzenes (class 5, Table 1) and 2,5-thiophenes
(class 6, Table 2).
18,040
4452
202
In the first class, the 1,4-disubstituted benzenes (4aed,
Table 1), the dimethoxy (4a) and dihydroxy (4b) derivatives
showed similar moderate activities with IC50 between 500 and
600 nM. The methylated derivatives 4c (monomethoxy) and 4d
(monohydroxy) are slightly more active with the same IC50 of
260 nM. These results point out the fact that only one polar
moiety is important, the one present in the C-ring and it should
be an H-bond acceptor.
In the class of 1,3-disubstituted benzenes (5aed, Table 1), it
becomes apparent that the dihydroxy derivatives (5b, 5d) show
a better inhibition than the methoxy analogs (5a, 5c), although
their potency is still moderate (639 and 482 nM for 5b and 5d,
respectively). Shifting the hydroxy on the A-ring from the 4- (5b) to
the 3-position (5d) improves the activity slightly. Furthermore, as
the exchange of the amide function of 5b for the retroamide A [25]
is detrimental for the inhibitory activity, it can be concluded that
the polar atoms (O and N) play a role in the binding of the molecule
by tight H-bond interactions.
40
n.i.: no inhibition; n.d.: not determined.
a
Mean value of three determinations, standard deviation less than 15%.
b
Human placental, microsomal fraction, substrate E2 [500 nM], cofactor NADþ
[1500
m
M].
Human placental, cytosolic fraction, substrate E1 [500 nM], cofactor NADH
[500 M].
IC50 (17
c
m
d
b-HSD1)/IC50 (17
b-HSD2).
e
% inhibition at 1 M.
m
On the other hand, compounds monosubstituted on the
A-ring (6ie6l) show higher inhibition of the enzyme when R2 is
a methoxy group (6i, IC50 ¼ 222 nM; 6k, IC50 ¼ 371 nM)
compared to the hydroxy compounds analogues (42% for 6j and
36% for 6l at 1 mM). The A-ring 2-substituted compounds 6m and
6n again behave differently from the 3-and 4-substitued coun-
terparts, the OH derivative 6n being more potent than the
methoxy 6m with IC50 values of 1127 nM for 6m and 520 nM for
6n. However, from this study it is clear that the electron donating
3-OMe group leads to the best A-ring monosubstituted
derivative.
The disubstituted compound 6p with an OH group on the C-ring
and a cyano group in 4-position on the A-ring shows low inhibition
of the enzyme, indicating that an electron withdrawing group as R2
decreases the activity.
Thus, further compounds with a 3-hydroxy group on the C-ring
and electron donating groups as R2 on the A-ring (compounds
6qet) were synthesized. They show good to moderate inhibition of
the enzyme with IC50 values between 161 (6q) and 278 nM (6r).
Substituent like 3-Me or 4-OMe on the A-ring improve the activity
slightly compared to the unsubstituted A-ring compound (6h). As it
is known that a fluorine often enhances inhibitory potency and as
Concerning the 2,5-thiophene compound class (6), both
dimethoxy and dihydroxy derivatives (6aed, Table 2) are
moderately potent with IC50 values in the same range (between
335 and 496 nM). Exceptions are compounds 6e and 6f,
substituted with a methoxy (6e) and a hydroxy (6f) in position 2
on the A-ring: the methoxy derivative 6e exhibits low 17
inhibition (48% at 1 M) while the corresponding hydroxy 6f is
b-HSD2
m
highly potent (IC50: 148 nM). Very similar activity profile and
potencies can be observed for compounds 6g and 6h unsub-
stituted on the A-ring. This indicates that the presence of the 2-
OH group on the A-ring in 6f does not achieve specific interaction
with the protein.
The high potency of the C ring monosubstituted compound 6h
indicates that an OH group should be present in the 3 position of
the C-ring and not a methoxy.

5982
K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
there is space available in position 2 of the A-ring for substitution,
a fluorine was also introduced as R2 instead of the OH group and led
to more potent compounds 6u and 6v than the OH analogues 6j and
benzene (5a and 5c) which are inactive and for dihydroxylated
thiophene 6f which shows a good inhibitoryactivity. It is striking that
the substitutionpattern (1,4-,1,3-benzene or 2,5-thiophene) does not
have a great influence on the activity as it can be expected that
depending on the substitution pattern the A- or C-ring will cover
a completely different region of the enzyme active site.
Because of the interesting activity identified for 6f and due to the
fact that the thiophene moiety was discovered to be avery promising
heteroaromatic ring in the design of 17b-HSD1 inhibitors [47], it was
decided to focus on this class of compounds for further optimization.
In a step by step optimization process, we wanted to identify
whether OH or OMe was optimal for ring A and for ring C. Mono-
substituted compounds 6gen with only one OMe or one OH on
either ring A or ring C were evaluated. Concerning substitution on
the C-ring, R1 should be an OH group rather than an OMe group
6l, IC50 ¼ 326 nM for 6u (3-F) vs. 42% inhibition at 1
mM for 6j (3-
OH) and 430 nM for 6v (4-F) vs. 36% inhibition at 1 M for 6l (4-OH).
m
Interestingly, a significant increase in activity is observed when
the fluorine is combined with a 3-methoxy or a 3-hydroxy group on
the A-ring, resulting in the highly potent 6w (IC50 ¼ 61 nM) and 6x
(IC50 ¼ 40 nM), respectively.
3.2.2. Selectivity: inhibition of human 17
affinities for ER and ER
Since 17 -HSD1 is the major enzyme catalyzing the reverse
reaction compared to 17 -HSD2, activating E1 to E2, inhibitory
activity toward this enzyme must be avoided. 17 -HSD1 inhibition
was determined using a similar assay as described for 17 -HSD2
[47]. Briefly, human placental cytosolic 17 -HSD1 was incubated
b-HSD1 and binding
a
b
b
b
b
b
(IC50 ¼ 186 nM for the OH 6h vs. around 1
mM for the OMe 6g). Since
b
OH is superior to OMe (6g vs. 6h), a hydrogen bond acceptor
obviously exists around the C-ring in the enzyme active site.
Regarding substitution on the A-ring, the biological data of
compounds 6iel demonstrate that R2 should be an OCH3 group
rather than an OH group and it should be localized in 3-position.
In a second step, the disubstituted compound 6r with the best
R1 and R2 (a 3-OMe on the A-ring and a 3-OH on the C-ring) was
prepared and turned out to have a good inhibitory activity,
however, slightly less potent than both corresponding mono-
substituted analogues.
with tritiated E1 in the presence of NADH and the inhibitor.
Inhibitory activity was determined as % inhibition or IC50 values for
the most potent HSD2 inhibitors. A selectivity factor (SF) was
calculated as the quotient of the IC50 values for 17b-HSD1 and 17b-
HSD2 (Tables 1 and 2).
It is apparent that there is a good correlation between the
number of methoxy/hydroxy groups and the selectivity toward
17b-HSD1. Derivatives with an OH moiety at both A- and C-rings
(4b, 5b, 5d, 6b, 6d, 6f, 6x) show a low SF between 5 and 22.
However, most compounds with only one OH group at the C-ring
(4d, 6h, 6pew) exhibit a better SF between 21 and 84 except for the
fluorinated compound 6u which has a SF of 10 and the 3-methyl 4d
and the 4-methoxy derivative 6q with an extraordinarily high SF of
246 or more. Furthermore, most of the compounds with an OMe
group on the A-ring (4a, 6a, 6c, 6i, 6k) have SFs of more than 200,
with the highest value of more than 450 (6i). In summary, most of
In the next step, an additional fluorine atom was introduced in
6r in position 2 of the A-ring because of the positive ortho effect
observed for 6f. The fluorine induces an increase in activity, leading
to the highly active 6w and 6x (IC50 values of 61 nM and 40 nM for
6w and 6x, respectively). It has been shown in several examples
that fluorine may enhance binding efficacy and selectivity in
pharmaceuticals because of a variety of multipolar CeF/HeN,
CeF/C¼O, and CeF/HeC interactions between a fluorinated
ligand and protein binding sites [60,61].
the compounds are very selective toward 17
As it is expected that the biological effects of E2 in osteoporotic
cells are estrogen receptor (ER) mediated, 17 -HSD2 inhibitors
should not bind to the ERs. Binding affinities to ER and ER were
b-HSD1.
b
As selectivity toward 17b-HSD1 is an important issue for the
a
b
therapeutic concept, inhibitory activity of this enzyme should be
avoided. It was evaluated for the most potent compounds. Inter-
estingly, it can be observed that in all three classes higher selec-
tivity toward HSD1 is always observed for the compounds with an
OMe group on the A-ring (compounds 4a, 6a, 6c, 6i, 6k, 6q with
a selectivity factor >200) compared to their hydroxy analogues.
The biological results indicate that the hydroxy derivatives with
low SF are able to bind in both HSD1 and HSD2 active site while
the methoxy compounds with high SF cannot bind in the one of
HSD1. Providing that the hypothesis that the architecture of HSD1
and HSD2 active sites is very similar, is correct (the product of one
catalytic reaction is the substrate of the other), it must be
concluded that the methoxy derivatives have a different binding
mode compared to the hydroxy analogues. The binding mode
investigation of this class of compounds will be the focus of
another study.
determined for the most potent (IC50 < 500 nM) and selective
compounds (SF > 10). The ER assays were performed using
recombinant human protein. A competition assay using tritium
labelled E2, the protein and the inhibitor was applied. Separation of
bound and free E2 was carried out using hydroxyapatite. The
relative binding affinity (RBA) of E2 was set up to 100%. All tested
compounds showed no binding affinity to the ERa and ERb with
RBA values smaller than 0.1%.
4. Discussion and conclusion
In our design concept we hypothesized that the rigidity induced
by the triazole ring in 7 and 8 was responsible for the weak 17
b
-
HSD2 inhibitory activity observed. Replacement of this triazole by
the bioisostere amide moiety successfully led to the discovery of
new and highly potent 17
b-HSD2 inhibitors, especially in the
We developed a novel class of potent 17b-HSD2 inhibitors: N-
thiophene- 2-carboxamine class (compound 6). The gain in activity
observed might be caused by the fact that the amide derivatives can
adopt an energetically more favourable bent geometry similar to
the one observed in the pyrrolidinone 2 (while the triazoles 7 and 8
were linear). It seems that to reach a good inhibitory activity, the
inhibitors require some flexibility to adopt a “sandwich-like form”.
Three different classes of compounds were investigated: 1,4-
disubstituted benzenes (class 4), 1,3-disubstituted benzenes (class
5) and the 2,5-thiophenes (class 6). When substituted with dihydroxy
or dimethoxy groups (4ae4b, 5ae6d), the 5- and the 6-membered
rings all appear to be moderately potent (IC50 values between
335 nM for 6d and 639 nM for 5b) except for the dimethoxylated 1,3-
benzyl-5-phenyl-thiophene-2-carboxamides. These nonsteroidal
compounds, without asymmetric carbons are the first easily
accessible 17b-HSD2 inhibitors (2e3 steps synthesis pathway and
an overall yield between 54% and 95%). Parallel synthesis can be
applied and allows the fast production of many derivatives in
a short time. The inhibitors show a good selectivity profile toward
17b-HSD1 and the estrogen receptors a and b. The 5-(2-fluoro-3-
methoxyphenyl)-N-(3-hydroxybenzyl)-N-methylthiophene-2-
carboxamide 6w is the most promising compound identified in this
study (IC50 (HSD2) ¼ 61 nM, SF (HSD1) ¼ 73, no binding affinity to
the ERs). It will be a good tool for in vivo evaluation in a disease-
oriented model to validate the concept of 17b-HSD2 inhibition.

K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5983
5. Experimental section
dried over sodium sulfate, filtered and concentrated to dryness. The
product was purified by FC with n-hexane/ethyl acetate (6:1-3:1) as
eluant.
5.1. Chemical methods
Chemical names follow IUPAC nomenclature. Starting materials
were purchased from Aldrich, Acros, Lancaster, Roth, Merck or
Fluka and were used without purification.
5.1.1.3. General procedure for ether cleavage. MethodC. Toa solution
of methoxyphenyl compounds (1 eq) in dry dichloromethane (5 ml/
mmol of reactant), borontrifluoride dimethyl sulfide complex (6 eq/
methoxyfunction)wasadded dropwiseat0 ꢀC and stirred for4e14 h.
After the reactionwas finished, the reaction mixture was diluted with
dichloromethane and 5% aqueous NaHCO3 was added until neutral
pH. The aqueous layer was extracted with dichloromethane. The
combined organic layers were washed with brine, dried over sodium
sulfate, evaporated to dryness under reduced pressure. The product
was purified by FC, with dichloromethane/methanol (100:1e50:1) as
eluant.
Flash column chromatography (FC) was performed on silica gel
(70e200
mm), and reaction progress was monitored by TLC on
Alugram SIL G/UV254 (MachereyeNagel). Vizualisation 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 in CDCl3 and CD3COCD3. Chemical shifts are reported in
d
(parts per million: ppm), by reference to the hydrogenated resi-
dues of deuteriated solvent as internal standard, CDCl3: 7.24 ppm
d
(1H NMR) and 77 ppm (13C NMR); CD3COCD3: 2.05 ppm (1H NMR)
and 30.8 ppm (13C NMR). 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).
MS (ESI) measurements were executed using a TSQ Quantum
(Thermo Finnigan) instrument.
5.1.2. Detailed synthesis procedures
5.1.2.1. 4-Bromo-N-(3-methoxybenzyl)-N-methylbenzamide
(13).
The title compound was prepared by reaction of 4-bromo-
benzoylchloride (329 mg, 1.5 mmol) and 3-methoxybenzylamine
(227 mg, 1.5 mmol) in presence of triethylamine (0.24 ml,
1.73 mmol) according to method A. Purification by FC (n-hexane/
ethyl acetate 6:1) afforded the desired product (468 mg, yield:
93%); C16H16BrNO2; MW: 334; MS (ESI): 334e336 (M þ H)þ; 1H
NMR (CD3COCD3): 2.80e2.92 (m, 3H), 3.80 (s, 3H), 4.52e4.69 (m,
2H), 6.79 (br s, 1H), 6.86 (dd, J ¼ 8.5 Hz, 2.2 Hz, 1H), 6.95 (br s, 1H),
7.28 (t, J ¼ 7.9 Hz, 1H), 7.44 (d, J ¼ 7.9 Hz, 2H), 7.62 (br s, 2H); 13C
NMR (CD3COCD3): 55.3, 112.9, 113.6, 120.0, 123.1, 129.1, 129.4, 130.0,
131.7, 135.7, 139.1, 159.8; IR (cmꢁ1): 2931, 2835, 1630, 1600, 1586,
1488, 1398, 1257, 1070, 1011, 834, 755, 693.
IR spectra were recorded on a Spectrum 100 FTeIR spectrometer
(PerkinElmer) as neat sample.
Melting points (mp) were measured in open capillaries on
a Stuart Scientific SMP3 apparatus and are uncorrected.
The purity of the compounds was evaluated by LC/MS. The
SurveyorÒ-LC-system consisted of a pump, an autosampler, and
a PDA detector. Mass spectrometry was performed on a TSQÒ
Quantum (ThermoFisher, Dreieich, Germany). The triple quadru-
pole mass spectrometer was equipped with an electrospray
interface (ESI). The system was operated by the standard software
5.1.2.2. 3-Bromo-N-(3-methoxybenzyl)-N-methylbenzamide
(14).
The title compound was prepared by reaction of 3-bromo-
benzoylchloride (217 mg, 1 mmol) and N-methyl-3-methoxy-
benzylamine (151 mg, 1 mmol) in presence of triethylamine
(0.16 ml, 1.15 mmol) according to method A. Purification by FC (n-
hexane/ethyl acetate 6:1) afforded the desired product (320 mg,
yield: 98%); C16H16BrNO2; MW: 334; MS (ESI): 334e336 (M þ H)þ;
1H NMR (CDCl3): 2.86e3.03 (m, 3H), 3.81 (s, 3H), 4.45e4.71 (m, 2H),
6.69e6.74 (m, 1H), 6.85 (d, J ¼ 8.0 Hz, 1H), 6.89e6.92 (m, 1H),
7.24e7.26 (m,1H), 7.28 (t, J ¼ 7.9 Hz,1H), 7.37 (d, J ¼ 7.9 Hz,1H), 7.53
(s, 1H), 7.60 (s, 1H); 13C NMR (CDCl3): 50.8, 55.2, 112.5, 112.9, 118.9,
120.5, 122.6, 125.2, 125.5, 129.7, 130.0, 132.7, 138.2; IR (cmꢁ1): 2934,
1630, 1600, 1562, 1488, 1396, 1255, 1048, 746, 691.
XcaliburÒ.
A RP C18 NUCLEODURÒ 100-5 (3 mm) column
(MachereyeNagel GmbH, Dühren, Germany) was used as
stationary phase. All solvents were HPLC grade. In a gradient run
the percentage of acetonitrile (containing 0.1% trifluoroacetic acid)
in 0.1% trifluoroacetic acid in was increased from an initial
concentration of 5% at 0 min to 100% at 15 min and kept at 100%
for 5 min. The injection volume was 20
mL and flow rate was set to
800 L/min. MS analysis was carried out at a needle voltage of
m
3000 V and a 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 tested compounds have >95% chemical purity except
compound 6q which has a purity of 93%.
5.1.2.3. 5-Bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-car-
boxamide (15a). The title compound was prepared by reaction of
5-bromothiophene-2-carbonyl chloride (225 mg, 1 mmol) and N-
methyl-3-methoxybenzylamine (151 mg, 1 mmol) in presence of
triethylamine (0.16 ml, 1.15 mmol) according to method A. Purifi-
cation by FC (n-hexane/ethyl acetate 6:1) afforded the desired
product (335 mg, yield: 99%) as oil; C14H14BrNO2S; MW: 340; 1H
NMR (CDCl3): 3.11 (br s, 3H), 3.80 (s, 3H), 4.72 (br s, 2H), 6.80 (s, 1H),
6.83e6.86 (dd, J ¼ 8.0 Hz, 2.0 Hz, 2H), 6.96 (br s, 1H), 7.07 (br s, 1H),
7.29 (t, J ¼ 8.0 Hz, 1H); 13C NMR (CDCl3): 55.2, 112.9, 117.2, 129.4,
129.8, 138.1, 139.5, 153.1, 160.1, 163.3; IR (cmꢁ1): 2938, 2834, 1602,
1530, 1486, 1418, 1396, 1254, 1039, 737, 693.
Compound
A was prepared according to the previously
described procedure [25].
5.1.1. General procedures
5.1.1.1. General procedure for amidation. Method A. At 0 ꢀC, a solu-
tion of 3(4)-bromobenzoyl chloride or 5-bromothiophene-2-
carbonyl chloride (1 eq) in CH2Cl2 (2 ml/eq) was added drop wise
to a solution of N-methylbenzylamine (1 eq) and triethylamine
(1.15 eq) in CH2Cl2 (2 ml/eq). The mixture was kept stirred at 0 ꢀC
for 3 h and evaporated under reduced pressure. The residue was
purified by FC with n-hexane/ethyl acetate as eluant.
5.1.1.2. General procedure for Suzuki coupling. Method B. A mixture
of aryl bromide (1 eq), substituted phenyl boronic acid (1.2 eq),
sodium carbonate (2 eq) and tetrakis(triphenylphosphine) palla-
dium (0.1 eq) in an oxygen free DME/water (1:1) solution was
stirred at 80 ꢀC for 4e14 h under nitrogen. The reaction mixture
was cooled to rt. The aqueous layer was extracted with dichloro-
methane. The combined organic layers were washed with brine,
5.1.2.4. N-benzyl-5-bromo-N-methylthiophene-2-carboxamide (15b).
The title compound was prepared by reaction of 5-bromothiophene-
2-carbonyl chloride (225 mg, 1 mmol) and N-methylbenzylamine
(121 mg, 1 mmol) in presence of triethylamine (0.16 ml, 1.15 mmol)
according to method A. Purification by FC (n-hexane/ethyl acetate
6:1) afforded the desired compound as oil (298 mg, yield: 96%).
C13H12BrNOS; MW: 310; MS (ESI): 310e312 (M þ H)þ; 1H NMR

5984
K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
(CD3COCD3): 3.14 (br s, 3H), 4.78 (br s, 2H), 7.14 (d, J ¼ 3.8 Hz, 1H),
7.25e7.40 (m, 6H); 13C NMR (CD3COCD3): 117.1, 128.3, 129.6, 131.4,
138.2, 141.9; IR (cmꢁ1): 2923, 1606, 1530, 1483, 1417, 1397, 732, 700.
(0.06 ml, 0.6 mmol) according to method C for 6 h. Purification by
FC (CH2Cl2/CH3OH 100:1/50:1) yielded the title compound as
amorph solid (30 mg, yield: 89%). C22H21NO2; MW: 331; mp:
140e141 ꢀC; MS (ESI): 332 (M þ H)þ; 1H NMR (CD3COCD3): 2.40 (s,
3H), 2.96 (s, 3H), 4.55e4.70 (m, 2H), 6.69e6.89 (m, 3H), 7.20 (t,
J ¼ 7.9 Hz, 2H), 7.35 (t, J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 7.9 Hz, 1H), 7.51 (s,
1H), 7.56 (d, J ¼ 8.2 Hz, 2H), 7.70e7.71 (m, 2H), 8.36 (br s, 1H); 13C
NMR (CD3COCD3): 21.5, 115.2, 124.9, 127.6, 128.5, 129.3, 129.7, 136.7,
139.3, 141.0, 143.0; IR (cmꢁ1): 3225, 2977, 1593, 1448, 754, 692.
5.1.2.5. 5-Bromo-N-(3-hydroxybenzyl)-N-methylthiophene-2-carbox-
amide (15c). The title compound was prepared by reaction of 5-
bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-carboxamide
15a (340 mg, 1 mmol) and borontrifluoride dimethyl sulfide
complex (0.63 ml, 6 mmol) according to method C for 5 h. Purifi-
cation by FC (CH2Cl2/CH3OH 100:1) afforded the desired compound
as amorph solid (300 mg, yield: 93%). C13H12BrNO2S; MW: 326; 1H
NMR (CD3COCD3): 3.13 (br s, 3H), 4.71 (br s, 2H), 6.76e6.79 (m, 3H),
7.13e7.14 (m, 1H), 7.20 (t, J ¼ 7.7 Hz, 1H), 7.25 (br s, 1H), 8.69 (s, 1H).
5.1.2.10. 40-Methoxy-N-(3-methoxybenzyl)-N-methyl[1,10-biphenyl]-
3-carboxamide (5a). The title compound was prepared by reaction
of 3-bromo-N-(3-methoxybenzyl)-N-methylbenzamide 14 (67 mg,
2 mmol) and 4-methoxyphenylboronic acid (36 mg, 2.4 mmol)
with tetrakis(triphenylphosphine) palladium (17 mg, 0.015 mmol)
as catalyst according to method B for 4 h. Purification by FC (n-
hexane/ethyl acetate 6:1) afforded the desired compound as oil
(70 mg, yield: 97%). C23H23NO3; MW: 361; MS (ESI): 362 (M þ H)þ;
1H NMR (CD3COCD3): 2.97 (br s, 3H), 3.79 (s, 3H), 3.84 (s, 3H),
4.57e4.73 (m, 2H), 6.83e6.88 (m, 5H), 7.30 (t, J ¼ 7.9 Hz, 1H), 7.40
(dt, J ¼ 7.9 Hz, 1.3 Hz, 1H), 7.48e7.68 (m, 5H); 13C NMR (CD3COCD3):
55.5, 55.6, 113.6, 114.5, 115.2, 125.7, 126.0, 128.1, 128.9, 129.7, 130.7,
133.4, 138.5, 141.6, 160.6; IR (cmꢁ1): 2936, 2836, 1630, 1601, 1584,
1488, 1395, 1246, 1044, 835, 802, 753.
5.1.2.6. 30-Methoxy-N-(3-methoxybenzyl)-N-methyl[1,10-biphenyl]-
4-carboxamide (4a). The title compound was prepared by reaction
of 4-bromo-N-(3-methoxybenzyl)-N-methylbenzamide 13 (67 mg,
0.2 mmol) and 3-methoxyphenylboronic acid (36 mg, 0.24 mmol)
with tetrakis(triphenylphosphine) palladium (23 mg, 0.02 mmol)
according to method B for 4 h. Purification by FC (n-hexane/ethyl
acetate 6:1/3:1) afforded the desired compound as oil (65 mg,
yield: 90%). C23H23NO3; MW: 361; MS (ESI): 362 (M þ H)þ; 1H NMR
(CD3COCD3): 2.97 (s, 3H), 3.81 (s, 3H), 3.87 (s, 3H), 4.61e4.72 (m,
2H), 6.85 (dd, J ¼ 8.2 Hz, 2.0 Hz, 2H), 6.96 (dd, J ¼ 8.2 Hz, 2.0 Hz, 2H),
7.23e7.26 (m, 2H), 7.30 (t, J ¼ 7.9 Hz,1H), 7.38 (t, J ¼ 8.0 Hz,1H), 7.57
(d, J ¼ 8.5 Hz, 2H), 7.72e7.73 (m, 2H); 13C NMR (CD3COCD3): 55.5,
55.6, 113.3, 113.6, 114.2, 120.1, 127.7, 128.5, 130.6, 130.8, 136.8, 142.5,
142.8, 161.1, 161.2; IR (cmꢁ1): 2938, 2835, 1629, 1601, 1583, 1479,
1396, 1259, 1015, 841, 764, 694.
5.1.2.11. 40-Hydroxy-N-(3-hydroxybenzyl)-N-methyl[1,10-biphenyl]-
3-carboxamide (5b). The title compound was prepared by reaction
of 40-methoxy-N-(3-methoxybenzyl)-N-methyl[1,10-biphenyl]-3-
carboxamide 5a (40 mg, 0.11 mmol) with borontrifluoride
dimethyl sulfide complex (0.14 ml, 1.32 mmol) according to
method C for 4 h. Purification by FC (CH2Cl2/CH3OH 100:1/50:1)
yielded the title compound as amorph solid (30 mg, yield: 82%).
C21H19NO3; MW: 333; mp: 128e129 ꢀC; MS (ESI): 334 (M þ H)þ;
1H NMR (CD3COCD3): 2.93e3.01 (m, 3H), 4.53e4.71 (m, 2H),
6.71e6.92 (m, 5H), 7.19 (t, J ¼ 7.9 Hz, 1H), 7.36e7.51 (m, 4H), 7.63
(br s, 1H), 7.66 (s, 1H), 8.51e8.52 (m, 1H), 8.62 (br s, 1H); 13C NMR
(CD3COCD3): 115.2, 116.7, 125.5, 125.7, 128.1, 128.9, 129.7, 130.7,
132.2, 138.1, 139.9, 141.9, 158.3, 158.7; IR (cmꢁ1): 3060, 1602, 1589,
1460, 752, 705, 687.
5.1.2.7. 30-Hydroxy-N-(3-hydroxybenzyl)-N-methyl[1,10-biphenyl]-4-
carboxamide (4b). The title compound was prepared by reaction
of 30-methoxy-N-(3-methoxybenzyl)-N-methyl[1,10-biphenyl]-4-
carboxamide 4a (36 mg, 0.1 mmol) with borontrifluoride
dimethyl sulfide complex (0.13 ml, 1.2 mmol) according to method
C for 14 h. Purification by FC (CH2Cl2/CH3OH 100:1/50:1) yielded
the title compound as amorph solid (28 mg, yield: 84%). C21H19NO3;
MW: 333; mp: 134e135 ꢀC; MS (ESI): 334 (M þ H)þ; 1H NMR
(CD3COCD3): 2.95 (s, 3H), 4.57e4.60 (m, 2H), 6.75 (dd, J ¼ 8.2 Hz,
2.0 Hz, 2H), 6.85 (dd, J ¼ 8.2 Hz, 2.0 Hz, 2H), 7.14e7.15 (m, 2H), 7.20
(t, J ¼ 8.0 Hz, 1H), 7.29 (t, J ¼ 8.0 Hz, 1H), 7.55 (d, J ¼ 8.2 Hz, 2H), 7.67
(br s, 2H), 8.38 (br s, 1H), 8.48 (s, 1H); 13C NMR (CD3COCD3): 114.7,
115.2,115.7,119.0,127.6,128.4,130.4,130.9,136.7,142.5,142.9,158.8,
158.9; IR (cmꢁ1): 3284, 2976, 1603, 1574, 1486, 1456, 764, 743, 690.
5.1.2.12. 30-Methoxy-N-(3-methoxybenzyl)-N-methyl[1,10-biphenyl]-
3-carboxamide (5c). The title compound was prepared by reaction
of 3-bromo-N-(3-methoxybenzyl)-N-methylbenzamide 14 (67 mg,
0.2 mmol) and 3-methoxyphenylboronic acid (36 mg, 0.24 mmol)
with tetrakis(triphenylphosphine) palladium (23 mg, 0.02 mmol)
as catalyst according to method B for 4 h. Purification by FC (n-
hexane/ethyl acetate 6:1) afforded the desired compound as oil
(71 mg, yield: 98%). C23H23NO3; MW: 361; MS (ESI): 362 (M þ H)þ;
1H NMR (CD3COCD3): 2.98 (br s, 3H), 3.79 (s, 3H), 3.85 (s, 3H),
4.58e4.73 (m, 2H), 6.81 (br s, 1H), 6.86 (dd, J ¼ 7.9 Hz, 2.2 Hz, 1H),
6.93e6.99 (m, 2H), 7.13 (br s, 1H), 7.22 (br s, 1H), 7.29 (t, J ¼ 7.9 Hz,
1H), 7.36 (br s, 1H), 7.46 (dt, J ¼ 7.6 Hz, 1.6 Hz, 1H), 7.52 (br s, 1H),
7.71 (br s, 1H), 7.73 (s, 1H); 13C NMR (CD3COCD3): 54.9, 55.5, 55.6,
88.6, 113.2, 113.6, 114.2, 120.1, 126.2, 126.8, 128.7, 129.8, 130.5, 130.8,
138.5, 140.1, 141.8, 142.5, 161.1, 161.2; IR (cmꢁ1): 2947, 2836, 1629,
1600, 1585, 1489, 1458, 1257, 1042, 782, 752, 696.
5.1.2.8. N-(3-methoxybenzyl)-N,30-dimethyl[1,10-biphenyl]-4-carbo-
xamide (4c). The title compound was prepared by reaction of 4-
bromo-N-(3-methoxybenzyl)-N-methylbenzamide 13 (53 mg,
0.15 mmol) and 3-methylphenylboronic acid (25 mg, 0.23 mmol)
with tetrakis(triphenylphosphine) palladium (17 mg, 0.015 mmol)
according to method B for 6 h. Purification by FC (n-hexane/ethyl
acetate 6:1/3:1) afforded the desired compound as oil (50 mg,
yield: 95%). C23H23NO2; MW: 345; MS (ESI): 346 (M þ H)þ; 1H NMR
(CD3COCD3): 2.40 (s, 3H), 2.97 (s, 3H), 3.80 (s, 3H), 4.60e4.72 (m,
2H), 6.81e6.98 (m, 3H), 7.20 (d, J ¼ 7.3 Hz, 1H), 7.30 (t, J ¼ 8.0 Hz,
1H), 7.35 (t, J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 7.6 Hz, 1H), 7.51 (s, 1H), 7.56
(d, J ¼ 8.5 Hz, 2H), 7.71 (d, J ¼ 7.3 Hz, 2H); 13C NMR (CD3COCD3):
21.5, 55.5, 113.6, 124.9, 127.6, 128.5, 129.3, 129.7, 130.6, 136.6, 139.3,
141.0, 143.0, 161.1; IR (cmꢁ1): 2934, 1630, 1606, 1586, 1485, 1396,
1259, 845, 763, 696.
5.1.2.13. 30-Hydroxy-N-(3-hydroxybenzyl)-N-methyl[1,10-biphenyl]-
3-carboxamide (5d). The title compound was prepared by reaction
of 30-methoxy-N-(3-methoxybenzyl)-N-methyl[1,10-biphenyl]-3-
carboxamide 5c (54 mg, 0.15 mmol) with borontrifluoride
dimethyl sulfide complex (0.19 ml, 1.8 mmol) according to
method C for 5 h. Purification by FC (CH2Cl2/CH3OH 100:1/50:1)
yielded the title compound as oil (46 mg, yield: 92%). C21H19NO3;
MW: 333; MS (ESI): 334 (M þ H)þ; 1H NMR (CD3COCD3): 2.96 (br
5.1.2.9. N-(3-hydroxybenzyl)-N,30-dimethyl[1,10-biphenyl]-4-carbo-
xamide (4d). The title compound was prepared by reaction of N-(3-
methoxybenzyl)-N,30-dimethyl[1,10-biphenyl]-4-carboxamide 4c
(35 mg, 0.1 mmol) with borontrifluoride dimethyl sulfide complex

K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5985
s, 3H), 4.54e4.70 (m, 2H), 6.74e6.91 (m, 4H), 7.02e7.27 (m, 4H),
7.44 (d, J ¼ 7.6 Hz, 1H), 7.51 (br s, 1H), 7.67 (br s, 1H), 7.69 (s, 1H),
8.37 (br s, 1H), 8.45 (s, 1H); 13C NMR (CD3COCD3): 114.7, 115.2,
115.6, 119.0, 119.9, 126.2, 126.7, 128.6, 129.7, 130.9, 138.4, 139.9,
142.0, 142.5, 158.7, 158.8; IR (cmꢁ1): 3208, 2985, 1587, 1574, 1454,
752, 693.
116.8, 122.5, 126.1, 128.2, 130.6, 137.1, 140.1, 149.0, 158.8, 158.9; IR
(cmꢁ1): 3316, 2971, 1609, 1581, 1442, 1160, 815, 690.
5.1.2.18. N-(3-methoxybenzyl)-5-(2-methoxyphenyl)-N-methylthio-
phene-2-carboxamide (6e). The title compound was prepared by
reaction of 5-bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-
carboxamide 15a (90 mg, 0.26 mmol) and 2-methoxyphenylboronic
acid (48 mg, 0.32 mmol) with tetrakis(triphenylphosphine) palla-
dium (30 mg, 0.026 mmol) as catalyst according to method B for 4 h.
Purification by FC (n-hexane/ethyl acetate 6:1) afforded the desired
compound as amorph solid (92 mg, yield: 93%). C21H21NO3S; MW:
367; mp: 101e102 ꢀC; MS (ESI): 368 (M þ H)þ; 1H NMR (CD3COCD3):
3.16 (br s, 3H), 3.80 (s, 3H), 3.97 (s, 3H), 4.80 (br s, 2H), 6.87 (dd,
J ¼ 8.0 Hz, 2.0 Hz, 1H), 6.92e6.94 (m, 2H), 7.03 (td, J ¼ 7.0 Hz, 1.0 Hz,
1H), 7.15 (dd, J ¼ 8.0 Hz, 1.0 Hz, 1H), 7.29e7.36 (m, 2H), 7.39 (br s,1H),
7.52 (d, J ¼ 4.0 Hz, 1H), 7.76 (dd, J ¼ 8.0 Hz, 1.5 Hz, 1H); 13C NMR
(CD3COCD3): 55.5, 56.0, 112.9, 113.5, 121.8, 123.1, 125.5, 128.8, 129.5,
130.2, 130.6, 132.0, 138.6, 140.3, 143.5, 156.7, 161.1; IR (cmꢁ1): 2948,
1594, 1480, 1255, 1161, 1019, 754, 672.
5.1.2.14. N-(3-methoxybenzyl)-5-(3-methoxyphenyl)-N-methylthio-
phene-2-carboxamide (6a). The title compound was prepared by
reaction of 5-bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-
carboxamide 15a (124 mg, 0.37 mmol) and 3-methoxyphenylbor-
onic acid (67 mg, 0.44 mmol) with tetrakis(triphenylphosphine)
palladium (43 mg, 0.04 mmol) as catalyst according to method B for
6 h. Purification by FC (n-hexane/ethyl acetate 6:1) afforded the
desired compound as oil (130 mg, yield: 96%). C21H21NO3S; MW: 367;
MS (ESI): 368 (M þ H)þ; 1H NMR (CD3COCD3): 3.16 (br s, 3H), 3.80 (s,
3H), 3.86 (s, 3H), 4.79 (br s, 2H), 6.86e6.88 (m,1H), 6.91e6.95 (m, 3H),
7.23 (t, J ¼ 2.0 Hz,1H), 7.25e7.28 (m,1H), 7.30 (t, J ¼ 8.0 Hz,1H), 7.35 (t,
J ¼ 8.0 Hz,1H), 7.41 (br s, 2H); 13C NMR (CD3COCD3): 55.5, 55.7, 112.1,
113.5, 114.9, 119.1, 124.3, 130.6, 131.1, 135.7, 138.7, 140.1, 148.1, 161.1,
161.3; IR (cmꢁ1): 2964, 2838,1593,1578,1480,1400,1277,1253,1158,
1033, 752, 731, 682.
5.1.2.19. N-(3-hydroxybenzyl)-5-(2-hydroxyphenyl)-N-methylthio-
phene-2-carboxamide (6f). The title compound was prepared by
reaction of N-(3-methoxybenzyl)-5-(2-methoxyphenyl)-N-meth-
ylthiophene-2-carboxamide 6e (48 mg, 0.13 mmol) with borontri-
fluoride dimethyl sulfide complex (0.16 ml,1.56 mmol) according to
method C for 14 h. Purification by FC (CH2Cl2/CH3OH 100:1/50:1)
yielded the title compound as amorph solid (40 mg, yield: 90%).
C19H17NO3S; MW: 339; mp: 177e178 ꢀC; MS (ESI): 340 (M þ H)þ;
1H NMR (CD3COCD3): 3.15 (br s, 3H), 4.76 (br s, 2H), 6.77 (dd,
J ¼ 8.0 Hz, 2.0 Hz,1H), 6.81e6.84 (m, 2H), 6.92 (td, J ¼ 7.6 Hz, 0.9 Hz,
1H), 7.04 (dd, J ¼ 8.0 Hz, 0.9 Hz, 1H), 7.16e7.22 (m, 2H), 7.39 (br s,
1H), 7.55 (d, J ¼ 3.4 Hz, 1H), 7.70 (dd, J ¼ 8.0 Hz, 0.9 Hz, 1H), 8.40 (s,
1H), 9.34 (s, 1H); 13C NMR (CD3COCD3): 115.2, 117.3, 120.9, 121.4,
125.3, 128.9, 130.0, 130.6, 138.1, 140.1, 144.5, 154.6, 158.8; IR (cmꢁ1):
3300, 2941, 1617, 1564, 1477, 749, 693.
5.1.2.15. N-(3-hydroxybenzyl)-5-(3-hydroxyphenyl)-N-methylthio-
phene-2-carboxamide (6b). The title compound was prepared by
reaction of N-(3-methoxybenzyl)-5-(3-methoxyphenyl)-N-meth-
ylthiophene-2-carboxamide 6a (70 mg, 0.19 mmol) with borontri-
fluoride dimethyl sulfide complex (0.24 ml, 2.28 mmol) according to
method C for 4 h. Purification by FC (CH2Cl2/CH3OH 100:1/50:1)
yielded the title compound as amorph solid (59 mg, yield: 91%).
C19H17NO3S; mp: 175e176 ꢀC; MW: 339; MS (ESI): 340 (M þ H)þ; 1H
NMR (CD3COCD3): 3.15 (br s, 3H), 4.76 (br s, 2H), 6.77e6.79 (m, 1H),
6.80e6.83 (m, 2H), 6.85 (ddd, J ¼ 8.0 Hz, 2.5 Hz, 0.9 Hz,1H), 7.15e7.18
(m, 2H), 7.21 (t, J ¼ 8.0 Hz, 1H), 7.26 (t, J ¼ 7.9 Hz, 1H), 7.34 (d,
J ¼ 3.5 Hz, 1H), 7.39 (br s, 1H), 8.35 (s, 1H), 8.54 (s, 1H); 13C NMR
(CD3COCD3): 113.5,115.2,116.4,118.0,124.0,130.7,131.1,135.7,138.4,
140.0,148.4,158.8,158.9; IR (cmꢁ1): 3111,1605,1570,1481, 752, 685.
5.1.2.20. N-(3-methoxybenzyl)-N-methyl-5-phenylthiophene-2-carbo-
xamide (6g). The title compound was prepared by reaction of 5-
bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-carboxamide
15a (90 mg, 0.27 mmol) and phenyl boronic acid (43 mg,
0.35 mmol) with tetrakis(triphenylphosphine) palladium (31 mg,
0.027 mmol) as catalyst according to method B for 14 h. Purification
by FC (n-hexane/ethyl acetate 6:1) afforded the desired compound
as oil (87 mg, yield: 96%). C20H19NO2S; MW: 337; MS (ESI): 338
(M þ H)þ; 1H NMR (CD3COCD3): 3.17 (br s, 3H), 3.80 (s, 3H), 4.80 (br
s, 2H), 6.88 (ddd, J ¼ 8.2 Hz, 2.5 Hz, 0.9 Hz, 1H), 6.92 (s, 1H), 6.93 (d,
J ¼ 7.9 Hz, 1H), 7.32 (t, J ¼ 8.2 Hz, 1H), 7.36 (t, J ¼ 7.6 Hz, 1H),
7.41e7.46 (m, 4H), 7.71 (d, J ¼ 7.6 Hz, 2H); 13C NMR (CD3COCD3):
55.5, 113.5, 124.1, 126.7, 129.2, 130.0, 130.6, 134.5, 140.1, 148.3, 161.1;
IR (cmꢁ1): 2968, 1592, 1481, 1394, 1264, 751, 686.
5.1.2.16. N-(3-methoxybenzyl)-5-(4-methoxyphenyl)-N-methylthio-
phene-2-carboxamide (6c). The title compound was prepared by
reaction of 5-bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-
carboxamide 15a (130 mg, 0.38 mmol) and 4-methoxyphenylbor-
onic acid (70 mg, 0.46 mmol) with tetrakis(triphenylphosphine)
palladium (44 mg, 0.04 mmol) as catalyst according to method B for
6 h. Purification by FC (n-hexane/ethyl acetate 6:1) afforded the
desired compound as amorph solid (138 mg, yield: 98%).
C21H21NO3S; MW: 367; mp: 115e116 ꢀC; MS (ESI): 368 (M þ H)þ; 1H
NMR (CD3COCD3): 3.17 (br s, 3H), 3.80 (s, 3H), 3.84 (s, 3H), 4.79 (br s,
2H), 6.87 (d, J ¼ 8.0 Hz,1H), 6.91e6.93 (m, 2H), 7.00 (d, J ¼ 8.8 Hz, 2H),
7.27e7.32 (m, 2H), 7.38 (br s, 1H), 7.63 (d, J ¼ 8.8 Hz, 2H); 13C NMR
(CD3COCD3): 55.5, 55.7, 113.5, 115.4, 122.9, 127.1, 128.1, 130.6, 137.6,
140.2, 148.5, 161.0, 161.1; IR (cmꢁ1): 2976, 1602, 1573, 1483, 1400,
1266, 736, 698.
5.1.2.21. N-(3-hydroxybenzyl)-N-methyl-5-phenylthiophene-2-carbo-
xamide (6h). The title compound was prepared by reaction of N-(3-
methoxybenzyl)-N-methyl-5-phenylthiophene-2-carboxamide 6g
with borontrifluoride dimethyl sulfide complex (0.13 ml,
1.20 mmol) according to method C for 6 h. Purification by FC
(CH2Cl2/CH3OH 100:1/50:1) yielded the title compound as
amorph solid (63 mg, yield: 98%). C19H17NO2S; MW: 323; mp:
140e141 ꢀC; MS (ESI): 346 (M þ Na)þ; 1H NMR (CD3COCD3): 3.16
(br s, 3H), 4.76 (br s, 2H), 6.78 (ddd, J ¼ 8.2 Hz, 2.5 Hz, 0.9 Hz, 1H),
6.81e6.83 (m, 2H), 7.22 (t, J ¼ 7.8 Hz, 1H), 7.36 (tt, J ¼ 8.2 Hz, 1.3 Hz,
1H), 7.40e7.46 (m, 4H), 7.71 (d, J ¼ 7.6 Hz, 2H), 8.37 (s, 1H); 13C
NMR (CD3COCD3): 115.2, 124.0, 126.7, 129.2, 130.0, 130.7, 134.5,
138.7, 140.0, 148.3, 158.8; IR (cmꢁ1): 3176, 2982, 1599, 1563, 1499,
734, 688.
5.1.2.17. N-(3-hydroxybenzyl)-5-(4-hydroxyphenyl)-N-methylthio-
phene-2-carboxamide (6d). The title compound was prepared by
reaction of N-(3-methoxybenzyl)-5-(4-methoxyphenyl)-N-meth-
ylthiophene-2-carboxamide 6c (100 mg, 0.27 mmol) with boron-
trifluoride dimethyl sulfide complex (0.34 ml, 3.24 mmol) according
to method C for 4 h. Purification by FC (CH2Cl2/CH3OH 100:1/50:1)
yielded the title compound as amorph solid (85 mg, yield: 92%).
C19H17NO3S; MW: 339; mp: 135e136 ꢀC; MS (ESI): 340 (M þ H)þ; 1H
NMR (CD3COCD3): 3.15 (br s, 3H), 4.75 (br s, 2H), 6.76e6.80 (m, 3H),
6.90 (d, J ¼ 8.5 Hz, 2H), 7.19e7.23 (m, 2H), 7.36 (br s, 1H), 7.54 (d,
J ¼ 8.5 Hz, 2H), 8.40 (s,1H), 8.70 (s,1H); 13C NMR (CD3COCD3): 115.2,

5986
K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5.1.2.22. N-benzyl-5-(3-methoxyphenyl)-N-methylthiophene-2-carbo-
xamide (6i). The title compound was prepared by reaction of N-
benzyl-5-bromo-N-methylthiophene-2-carboxamide 15b (80 mg,
0.26 mmol) and 3-methoxyphenylboronic acid (47 mg,
0.31 mmol) with tetrakis(triphenylphosphine) palladium (30 mg,
0.026 mmol) as catalyst according to method B for 4 h. Purifi-
cation by FC (n-hexane/ethyl acetate 6:1) afforded the desired
compound as amorph solid (75 mg, yield: 86%). C20H19NO2S;
MW: 337; MS (ESI): 338 (M þ H)þ; 1H NMR (CD3COCD3): 3.17 (br
s, 3H), 3.86 (s, 3H), 4.82 (br s, 2H), 6.94 (ddd, J ¼ 8.2 Hz, 2.5 Hz,
0.9 Hz, 1H), 7.23 (t, J ¼ 2.0 Hz, 1H), 7.27 (ddd, J ¼ 7.9 Hz, 1.6 Hz,
0.9 Hz, 1H), 7.29e7.32 (m, 1H), 7.34e7.41 (m, 7H); 13C NMR
(CD3COCD3): 55.7, 112.1, 114.9, 119.1, 124.3, 128.2, 129.6, 131.1,
135.7, 138.5, 148.1, 161.3.
(76 mg, yield: 87%). C20H19NO2S; MW: 337; MS (ESI): 338 (M þ H)þ;
1H NMR (CD3COCD3): 3.16 (br s, 3H), 3.97 (s, 3H), 4.82 (br s, 2H),
7.03 (td, J ¼ 7.6 Hz, 1.3 Hz, 1H), 7.15 (dd, J ¼ 8.2 Hz, 1.3 Hz, 1H),
7.29e7.41 (m, 7H), 7.52 (d, J ¼ 4.0 Hz,1H), 7.76 (dd, J ¼ 7.8 Hz,1.6 Hz,
1H); 13C NMR (CD3COCD3): 56.0, 112.9, 121.8, 123.1, 125.5, 128.1,
128.8, 129.5, 130.2, 132.0, 138.6, 143.5, 156.7; IR (cmꢁ1): 2965, 2838,
1595, 1480, 1253, 1158, 1033, 771, 682.
5.1.2.27. N-benzyl-5-(2-hydroxyphenyl)-N-methylthiophene-2-car-
boxamide (6n). The title compound was prepared by reaction of N-
benzyl-5-(2-methoxyphenyl)-N-methylthiophene-2-carboxamide
6m (55 mg, 0.16 mmol) with borontrifluoride dimethyl sulfide
complex (0.10 ml, 0.96 mmol) according to method C for 14 h.
Purification by FC (CH2Cl2/CH3OH 100:1) yielded the title
compound as amorph solid (49 mg, yield: 93%). C19H17NO2S; MW:
323; mp: 185e186 ꢀC; MS (ESI): 324 (M þ H)þ; 1H NMR (CD3COCD3):
3.16 (br s, 3H), 4.83 (br s, 2H), 6.93 (ddd, J ¼ 8.5 Hz, 7.3 Hz,1.3 Hz,1H),
7.03 (dd, J ¼ 7.8 Hz, 1.3 Hz, 1H), 7.18 (ddd, J ¼ 8.5 Hz, 7.3 Hz, 1.3 Hz,
1H), 7.28e7.32 (m, 1H), 7.35e7.41 (m, 5H), 7.56 (d, J ¼ 4.0 Hz, 1H),
7.70 (dd, J ¼ 7.8 Hz, 1.6 Hz, 1H), 9.23 (s, 1H); 13C NMR (CD3COCD3):
117.3,121.0,121.5,125.4,128.1,129.0,129.6,130.0,138.2,138.7,154.6;
IR (cmꢁ1): 2986, 1587, 1455, 752, 697.
5.1.2.23. N-benzyl-5-(3-hydroxyphenyl)-N-methylthiophene-2-carbox-
amide (6j). The title compound was prepared by reaction of N-
benzyl-5-(3-methoxyphenyl)-N-methylthiophene-2-carboxamide
6i (65 mg, 0.19 mmol) with borontrifluoride dimethyl sulfide
complex (0.12 ml, 1.14 mmol) according to method C for 4 h. Puri-
fication by FC (CH2Cl2/CH3OH 100:1) yielded the title compound as
oil (55 mg, yield: 88%). C19H17NO2S, MW: 323; MS (ESI): 324
(M þ H)þ; 1H NMR (CD3COCD3): 3.17 (br s, 3H), 4.82 (br s, 2H), 6.85
(ddd, J ¼ 8.2 Hz, 2.2 Hz, 0.9 Hz, 1H), 7.16e7.18 (m, 2H), 7.26 (t,
J ¼ 7.8 Hz, 1H), 7.29e7.33 (m, 1H), 7.34e7.41 (m, 6H), 8.55 (s, 1H);
13C NMR (CD3COCD3): 113.5, 116.4, 118.0, 124.0, 128.2, 129.6, 131.1,
135.7, 138.5, 148.4, 158.9; IR (cmꢁ1): 3285, 2967, 1602, 1571, 1488,
744, 691.
5.1.2.28. 5-(4-Cyanophenyl)-N-(3-methoxybenzyl)-N-methylthio-
phene-2-carboxamide (6o). The title compound was prepared by
reaction of 5-bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-
carboxamide 15a (80 mg, 0.24 mmol) and 4-cyanophenylboronic
acid (42 mg, 0.29 mmol) with tetrakis(triphenylphosphine) palla-
dium (28 mg, 0.024 mmol) as catalyst according to method B for
14 h. Purification by FC (n-hexane/ethyl acetate 6:1) afforded the
desired compound as amorph solid (80 mg, yield: 92%).
C21H18N2O2S; MW: 362; mp: 137e138 ꢀC; MS (ESI): 363 (M þ H)þ;
1H NMR (CD3COCD3): 3.18 (br s, 3H), 3.80 (s, 3H), 4.80 (br s, 2H),
6.87e6.89 (m, 1H), 6.91e6.93 (m, 2H), 7.31 (t, J ¼ 7.9 Hz, 1H), 7.46 (s,
1H), 7.60 (d, J ¼ 3.3 Hz, 1H), 7.84 (d, J ¼ 8.8 Hz, 2H), 7.92 (d,
J ¼ 8.2 Hz, 2H); 13C NMR (CD3COCD3): 55.5, 112.3, 113.6, 119.1, 126.3,
127.3, 130.7, 133.9, 138.7, 140.0, 145.7, 161.2; IR (cmꢁ1): 2941, 2223,
1604, 1583, 1486, 1397, 1253, 1041, 750, 697.
5.1.2.24. N-benzyl-5-(4-methoxyphenyl)-N-methylthiophene-2-carbox-
amide (6k). The title compound was prepared by reaction of N-
benzyl-5-bromo-N-methylthiophene-2-carboxamide 15b (72 mg,
0.23 mmol) and 4-methoxyphenylboronic acid (42 mg, 0.27 mmol)
with tetrakis(triphenylphosphine) palladium (27 mg, 0.023 mmol)
as catalyst according to method B for 5 h. Purification by FC (n-
hexane/ethyl acetate 6:1) afforded the desired compound as
amorph solid (75 mg, yield: 96%). C20H19NO2S; MW: 337; mp:
126e127 ꢀC; MS (ESI): 338 (M þ H)þ; 1H NMR (CD3COCD3): 3.16 (br
s, 3H), 3.84 (s, 3H), 4.82 (br s, 2H), 7.00 (d, J ¼ 8.8 Hz, 2H); 7.27 (d,
J ¼ 3.8 Hz, 1H), 7.29e7.32 (m, 1H), 7.35e7.41 (m, 5H), 7.63 (d,
J ¼ 8.8 Hz, 2H); 13C NMR (CD3COCD3): 55.7, 115.4, 122.9, 127.1, 128.1,
128.2, 129.6, 137.6, 138.6, 148.5, 161.0; IR (cmꢁ1): 2937, 1596, 1572,
1486, 1245, 1179, 1024, 732, 695.
5.1.2.29.
5-(4-Cyanophenyl)-N-(3-hydroxybenzyl)-N-methylthio-
phene-2-carboxamide (6p). The title compound was prepared by
reaction of 5-(4-cyanophenyl)-N-(3-methoxybenzyl)-N-methyl-
thiophene-2-carboxamide 6o (60 mg, 0.17 mmol) with borontri-
fluoride dimethyl sulfide complex (0.11 ml, 1.02 mmol) according
5.1.2.25. N-benzyl-5-(4-hydroxyphenyl)-N-methylthiophene-2-carbox-
amide (6l). The title compound was prepared by reaction of N-
benzyl-5-(4-methoxyphenyl)-N-methylthiophene-2-carboxamide
6k (56 mg, 0.17 mmol) with borontrifluoride dimethyl sulfide
complex (0.11 ml, 1.02 mmol) according to method C for 6 h.
Purification by FC (CH2Cl2/CH3OH 100:1) yielded the title
compound as amorph solid (49 mg, yield: 91%). C19H17NO2S; MW:
323; mp: 170e171 ꢀC; MS: 324 (M þ H)þ; 1H NMR (CD3COCD3): 3.16
(br s, 3H), 4.82 (br s, 2H), 6.91 (d, J ¼ 8.8 Hz, 2H), 7.23 (d, J ¼ 3.8 Hz,
1H), 7.28e7.32 (m, 1H), 7.34e7.40 (m, 5H), 7.55 (d, J ¼ 8.8 Hz, 2H),
8.64 (s, 1H); 13C NMR (CD3COCD3): 116.8, 122.5, 126.1, 128.2,
129.6, 137.2, 138.6, 149.0, 158.9; IR (cmꢁ1): 3211, 1611, 1573, 1482,
735, 698.
to method C for 14 h. Purification by FC (CH2Cl2/CH3OH
100:1/50:1) yielded the title compound as amorph solid (55 mg,
yield: 95%). C20H16N2O2S; MW: 348; mp: 166e167 ꢀC; MS (ESI):
349 (M þ H)þ; 1H NMR (CD3COCD3): 3.16 (br s, 3H), 4.76 (br s, 2H),
6.77e6.82 (m, 3H), 7.21 (t, J ¼ 7.7 Hz, 1H), 7.46 (br s, 1H), 7.60 (d,
J ¼ 2.8 Hz, 1H), 7.83 (d, J ¼ 8.5 Hz, 2H), 7.92 (d, J ¼ 8.5 Hz, 2H), 8.37
(s, 1H); 13C NMR (CD3COCD3): 112.3, 115.2, 115.3, 118.8, 119.1,
126.3, 127.3, 130.7, 133.9, 138.7, 139.9, 145.7, 158.8; IR (cmꢁ1):
3204, 2934, 2227, 1600, 1573, 1483, 751, 696.
5.1.2.30. N-(3-hydroxybenzyl)-5-(4-methoxyphenyl)-N-methylthio-
phene-2-carboxamide (6q). The title compound was prepared
by reaction of 5-bromo-N-(3-hydroxybenzyl)-N-methylthiophene-
2-carboxamide
15c
(49
mg,
0.15
mmol)
and
4-
5.1.2.26. N-benzyl-5-(2-methoxyphenyl)-N-methylthiophene-2-car-
boxamide (6m). The title compound was prepared by reaction of N-
benzyl-5-bromo-N-methylthiophene-2-carboxamide 15b (80 mg,
0.26 mmol) and 2-methoxyphenylboronic acid (47 mg, 0.31 mmol)
with tetrakis(triphenylphosphine) palladium (30 mg, 0.026 mmol)
as catalyst according to method B for 14 h. Purification by FC (n-
hexane/ethyl acetate 6:1) afforded the desired compound as oil
methoxyphenylboronic acid (27 mg, 0.18 mmol) with tetrakis(-
triphenylphosphine) palladium (17 mg, 0.015 mmol) as catalyst
according to method B for 6 h. Purification by FC (n-hexane/ethyl
acetate 6:1) afforded the desired compound as oil (48 mg, yield:
90%). C20H19NO3S; MW: 353; MS (ESI): 354 (M þ H)þ; 1H NMR
(CD3COCD3): 3.15 (br s, 3H), 3.84 (s, 3H), 4.75 (br s, 2H), 6.77 (dd,
J ¼ 8.0 Hz, 2.2 Hz, 1H), 6.80e6.83 (m, 2H), 7.00 (d, J ¼ 8.8 Hz, 2H),

K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5987
7.21 (t, J ¼ 8.0 Hz, 1H), 7.27 (d, J ¼ 3.5 Hz, 1H), 7.31 (br s, 1H), 7.63 (d,
J ¼ 8.8 Hz, 2H), 8.35 (s, 1H); 13C NMR (CD3COCD3): 55.7, 115.2, 115.4,
122.9, 127.1, 128.1, 129.6, 130.6, 132.0, 133.4, 140.1, 143.9, 158.8; IR
(cmꢁ1): 3150, 2939, 1599, 1482, 1244, 1178, 1034, 745, 699.
5.1.2.35. 5-(3-Fluorophenyl)-N-(3-hydroxybenzyl)-N-methylthio-
phene-2-carboxamide (6u). The title compound was prepared by
reaction of 5-bromo-N-(3-hydroxybenzyl)-N-methylthiophene-2-
carboxamide 15c (49 mg, 0.15 mmol) and 3-fluorophenylboronic
acid (25 mg, 0.18 mmol) with tetrakis(triphenylphosphine)
palladium (17 mg, 0.015 mmol) as catalyst according to method B
for 14 h. Purification by FC (n-hexane/ethyl acetate 6:1/3:1)
afforded the desired compound as oil (40 mg, yield: 78%).
C19H16FNO2S; MW: 341; MS (ESI): 342 (M þ H)þ; 1H NMR
(CD3COCD3): 3.16 (br s, 3H), 4.76 (br s, 2H), 6.77e6.82 (m, 3H),
7.11e7.15 (m, 1H), 7.21 (t, J ¼ 7.7 Hz, 1H), 7.42 (br s, 1H), 7.46e7.51
(m, 3H), 7.54 (d, J ¼ 8.2 Hz, 1H), 8.35 (s, 1H); 13C NMR (CD3COCD3):
113.2, 115.3, 115.7, 115.9, 122.7, 125.1, 130.7, 131.9, 132.0, 136.8,
139.2, 140.1, 144.4, 146.5, 148.3, 150.9, 158.8, 160.9, 163.1, 165.1; IR
(cmꢁ1): 3660, 2976, 1610, 1576, 1480, 1407, 1276, 1243, 1154, 786,
697.
5.1.2.31. N-(3-hydroxybenzyl)-5-(3-methoxyphenyl)-N-methylthio-
phene-2-carboxamide (6r). The title compound was prepared by
reaction of 5-bromo-N-(3-hydroxybenzyl)-N-methylthiophene-2-
carboxamide 15c (33 mg, 1 mmol) and 3-methoxyphenylboronic
acid (18 mg, 0.12 mmol) with tetrakis(triphenylphosphine) palla-
dium (12 mg, 0.01 mmol) as catalyst according to method B for 6 h.
Purification by FC (n-hexane/ethyl acetate 6:1/3:1) afforded the
desired compound as oil (25 mg, yield: 71%). C20H19NO3S; MW:
353; MS (ESI): 354 (M þ H)þ; 1H NMR (CD3COCD3): 3.16 (br s, 3H),
3.86 (s, 3H), 4.76 (br s, 2H), 6.77e6.82 (m, 3H), 6.94 (ddd, J ¼ 8.0 Hz,
2.5 Hz, 0.9 Hz,1H), 7.21 (t, J ¼ 8.0 Hz,1H), 7.23 (t, J ¼ 2.0 Hz,1H), 7.27
(d, J ¼ 8.0 Hz,1H), 7.35 (t, J ¼ 8.0 Hz,1H), 7.41 (br, s, 2H), 8.36 (s,1H);
13C NMR (CD3COCD3): 55.7, 112.1, 114.9, 115.3, 119.1, 130.6, 131.1,
135.7, 140.0, 148.1, 158.8, 161.2; IR (cmꢁ1): 3193, 2957, 1606, 1583,
1463, 1287, 1240, 732, 686.
5.1.2.36.
5-(4-Fluorophenyl)-N-(3-hydroxybenzyl)-N-methylthio-
phene-2-carboxamide (6v). The title compound was prepared by
reaction of 5-bromo-N-(3-hydroxybenzyl)-N-methylthiophene-2-
carboxamide 15c (49 mg, 0.15 mmol) and 4-fluorophenylboronic
acid (25 mg, 0.18 mmol) with tetrakis(triphenylphosphine)
palladium (17 mg, 0.015 mmol) as catalyst according to method B
for 14 h. Purification by FC (n-hexane/ethyl acetate 5:1/3:1)
afforded the desired compound as oil (43 mg, yield: 84%).
C19H16FNO2S; MW: 341; MS (ESI): 342 (M þ H)þ; 1H NMR
(CD3COCD3): 3.16 (br s, 3H), 4.75 (br s, 2H), 6.77e6.82 (m, 3H),
7.19e7.24 (m, 3H), 7.37 (d, J ¼ 3.7 Hz, 1H), 7.40 (br s, 1H), 7.73e7.76
(m, 2H), 8.35 (br s, 1H); 13C NMR (CD3COCD3): 115.3, 116.8, 116.9,
124.3, 127.8, 128.7, 128.8, 130.6, 131.0, 138.9, 140.0, 147.1, 158.8,
162.7, 164.2, 164.6; IR (cmꢁ1): 3168, 2956, 1598, 1561, 1479, 1228,
735, 699.
5.1.2.32.
5-(3-(Dimethylamino)phenyl)-N-(3-hydroxybenzyl)-N-
methylthiophene-2-carboxamide (6s). The title compound was
prepared by reaction of 5-bromo-N-(3-hydroxybenzyl)-N-methyl-
thiophene-2-carboxamide 15c (49 mg, 0.15 mmol) and N,N-
dimethyl-3-aminophenylboronic acid (30 mg, 0.18 mmol) with tet-
rakis(triphenylphosphine) palladium (17 mg, 0.015 mmol) as catalyst
according to method B for 14 h. Purification by FC (n-hexane/ethyl
acetate 5:1/3:1) afforded the desired compound as oil (45 mg, yield:
82%). C21H22N2O2S; MW: 366; MS (ESI): 367 (M þ H)þ; 1H NMR
(CD3COCD3): 3.00 (s, 6H), 3.15 (br s, 3H), 4.75 (br s, 2H), 6.75e6.82 (m,
4H), 6.98 (d, J ¼ 4.0 Hz,1H), 7.00 (s,1H), 7.19e7.26 (m, 2H), 7.36 (s,1H),
7.39 (br s, 1H), 8.36 (s, 1H); 13C NMR (CD3COCD3): 40.5, 110.5, 113.5,
114.9,115.2,123.7,130.5,135.0,138.1,140.1,149.6,152.1,157.8,158.8; IR
(cmꢁ1): 3649, 2977, 2808, 1594, 1571, 1484, 1440, 1232, 742, 687.
5.1.2.37. 5-(2-Fluoro-3-methoxyphenyl)-N-(3-hydroxybenzyl)-N-
methylthiophene-2-carboxamide (6w). The title compound was
prepared by reaction of 5-bromo-N-(3-hydroxybenzyl)-N-meth-
ylthiophene-2-carboxamide 15c (49 mg, 0.15 mmol) and 2-
fluoro-3-methoxyphenylboronic acid (31 mg, 0.18 mmol) with
tetrakis(triphenylphosphine) palladium (17 mg, 0.015 mmol) as
catalyst according to method B for 14 h. Purification by FC (n-
hexane/ethyl acetate 5:1/3:1) afforded the desired compound
as amorph solid (40 mg, yield: 72%). C20H18FNO3S; MW: 371; mp:
169e170 ꢀC; MS (ESI): 372 (M þ H)þ; 1H NMR (CD3COCD3): 3.16
(br s, 3H), 3.93 (s, 3H), 4.76 (br s, 2H), 6.77e6.83 (m, 3H),
7.13e7.23 (m, 3H), 7.31 (td, J ¼ 7.8 Hz, 1.6 Hz, 1H), 7.45 (br s, 1H),
7.48 (br s, 1H), 8.35 (s, 1H); 13C NMR (CD3COCD3): 56.7, 114.0,
114.1, 115.3, 120.5, 120.6, 122.7, 122.8, 125.6, 127.2, 130.7, 139.9,
140.7, 148.9, 149.6, 150.9, 158.8; IR (cmꢁ1): 2977, 1606, 1584, 1471,
1265.
5.1.2.33. N-(3-methoxybenzyl)-N-methyl-5-m-tolylthiophene-2-car-
boxamide (6ta). The title compound was prepared by reaction of
5-bromo-N-(3-methoxybenzyl)-N-methylthiophene-2-carboxamide
15a (53 mg, 0.15 mol) and 3-methylphenylboronic acid (24 mg,
0.18 mmol) with tetrakis(triphenylphosphine) palladium (17 mg,
0.015 mmol) as catalyst according to method B for 6 h. Purification by
FC (n-hexane/ethyl acetate 6:1) afforded the desired compound as
colorless oil (50 mg, yield: 95%). C21H21NO2S; MW: 351; MS (ESI):
352 (M þ H)þ; 1H NMR (CD3COCD3): 2.38 (s, 3H), 3.17 (br s, 3H), 3.80
(s, 3H), 4.80 (br s, 2H), 6.86e6.89 (m, 1H), 6.91e6.93 (m, 2H), 7.19 (d,
J ¼ 7.6Hz,1H), 7.29e7.33 (m, 2H), 7.38e7.42 (m, 2H), 7.49(d, J¼ 7.6 Hz,
1H), 7.53 (s, 1H); 13C NMR (CD3COCD3): 21.3, 55.5, 113.5, 123.8, 123.9,
127.3, 129.9, 130.0, 130.6, 134.4, 138.5, 139.7, 140.1, 148.5, 161.1; IR
(cmꢁ1): 3010, 2917, 1592, 1491, 1280, 1262, 1159, 774, 751, 689.
5.1.2.38.
5-(2-Fluoro-3-hydroxyphenyl)-N-(3-hydroxybenzyl)-N-
5.1.2.34. N-(3-hydroxybenzyl)-N-methyl-5-m-tolylthiophene-2-car-
boxamide (6t). The title compound was prepared by reaction of N-
(3-methoxybenzyl)-N-methyl-5-m-tolylthiophene-2-carboxamide
6ta (35 mg, 0.1 mmol) with borontrifluoride dimethyl sulfide
complex (0.06 ml, 0.6 mmol) according to method C for 4 h. Puri-
fication by FC (CH2Cl2/CH3OH 100:1) yielded the title compound as
amorph solid (30 mg, yield: 89%). C20H19NO2S; MW: 337; mp:
156e157 ꢀC; MS (ESI): 338 (M þ H)þ; 1H NMR (CD3COCD3): 2.37 (s,
3H), 3.15 (br s, 3H), 4.75 (br s, 2H), 6.78 (ddd, J ¼ 8.2 Hz, 2.5 Hz,
0.9 Hz, 1H), 6.80e6.83 (m, 2H), 7.17e7.22 (m, 2H), 7.32 (t, J ¼ 7.8 Hz,
1H), 7.37e7.43 (m, 2H), 7.49 (d, J ¼ 7.8 Hz, 1H), 7.53 (s, 1H), 8.46 (s,
1H); 13C NMR (CD3COCD3): 21.3, 115.2, 123.8, 123.9, 127.3, 129.9,
130.0, 130.7, 134.4, 138.5, 139.7, 140.0, 148.5, 158.8; IR (cmꢁ1): 3225,
2932, 1588, 1566, 1489, 740, 688.
methylthiophene-2-carboxamide (6x). The title compound was
prepared by reaction of 5-(2-fluoro-3-methoxyphenyl)-N-(3-
hydroxybenzyl)-N-methylthiophene-2-carboxamide 6w (30 mg,
0.08 mmol) with borontrifluoride dimethyl sulfide complex
(0.05 ml, 0.48 mmol) according to method C for 14 h. Purification
by FC (CH2Cl2/CH3OH 100:1) yielded the title compound as
amorph solid (25 mg, yield: 87%). C19H16FNO3S; MW: 357; mp:
167e168 ꢀC; MS (ESI): 358 (M þ H)þ; 1H NMR (CD3COCD3): 3.16
(br s, 3H), 4.76 (br s, 2H), 6.77e6.99 (m, 3H), 7.01 (td, J ¼ 8.0 Hz,
1.6 Hz, 1H), 7.08 (td, J ¼ 8.0 Hz, 0.9 Hz, 1H), 7.21 (t, J ¼ 8.0 Hz, 2H),
7.44 (br s, 1H), 7.46 (s, 1H), 8.36 (s, 1H), 8.86 (d, J ¼ 1.6 Hz, 1H); 13C
NMR (CD3COCD3): 115.3, 118.4, 119.7, 122.9, 125.6, 126.6, 127.0,
127.1, 130.6, 139.6, 140.0, 141.1, 146.7, 148.3, 150.3, 158.8; IR (cmꢁ1):
3113, 2924, 1599, 1571, 1493, 751, 694.

5988
K. Xu et al. / European Journal of Medicinal Chemistry 46 (2011) 5978e5990
5.2. Biological methods
added and samples were measured in a liquid scintillation counter
(Rack Beta Primo 1209, Wallac, Turku). For determination of the
relative binding affinity (RBA), inhibitor and E2 concentrations
required to displace 50% of the receptor bound labelled E2 were
determined. RBA[%] ¼ IC50(E2)/IC50(compound) ꢂ 100. The RBA
value for E2 was arbitrarily set at 100%.
[2,4,6,7-3H]-E1 and [2,4,6,7-3H]-E2 were purchased from Per-
kinElmer, Boston. Quickszint Flow 302 scintillator fluid was bought
from Zinsser Analytic, Frankfurt. Other chemicals were purchased
from Sigma, Roth or Merck.
5.3. 17
placental enzyme
b-HSD1 and 17b-HSD2 enzyme preparation from human
Acknowledgments
We thank Josef Zapp for the NMR measurement, Ms. Martina
Jankowski for the LC-Mass spectra as well as Jannine Ludwig and
Jörg Haupenthal for the biological tests. Kuiying Xu and Yaseen
Al-Soud are grateful to the Alexander von Humboldt Foundation
17b-HSD1 and 17b-HSD2 were obtained from human placenta
according to previously described procedures [62,63]. Fresh human
placenta was homogenized and the enzymes were separated by
fractional centrifugation at 1000 g, 10,000 g and 150,000 g. For the
(AvH) for
a
fellowship. We acknowledge the Deutsche
purification of 17
b-HSD1, the cytosolic fraction was precipitated
Forschungsgemeinschaft (DFG) for financial support (HA1315/12-1).
with ammonium sulfate. 17
b
-HSD2 was obtained from the micro-
somal fraction. Aliquots containing 17
stored frozen.
b-HSD1 or 17b-HSD2 were
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