Structural optimization of 2,5-thiophene amides as highly potent and selective 17β-hydroxysteroid dehydrogenase type 2 inhibitors for the treatment of osteoporosis

Article abstract of DOI:10.1021/jm3014053

Inhibition of 17β-HSD2 is an attractive mechanism for the treatment of osteoporosis. We report here the optimization of human 17β-HSD2 inhibitors in the 2,5-thiophene amide class by varying the size of the linker (n equals 0 and 2) between the amide moiety and the phenyl group. While none of the phenethylamides (n = 2) were active, most of the anilides (n = 0) turned out to moderately or strongly inhibit 17β-HSD2. The four most active compounds showed an IC₅₀ of around 60 nM and a very good selectivity toward 17β-HSD1, 17β-HSD4, 17β-HSD5, 11β-HSD1, 11β-HSD2 and the estrogen receptors α and β. The investigated compounds inhibited monkey 17β-HSD2 moderately, and one of them showed good inhibitory activity on mouse 17β-HSD2. SAR studies allowed a first characterization of the human 17β-HSD2 active site, which is predicted to be considerably larger than that of 17β-HSD1.

Full text of DOI:10.1021/jm3014053

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Article
Structural Optimization of 2,5-Thiophene Amides as Highly
Potent and Selective 17#-Hydroxysteroid Dehydrogenase
Type 2 Inhibitors for the Treatment of Osteoporosis
Sandrine Marchais-Oberwinkler, Kuiying Xu, Marie Wetzel, Enrico Perspicace, Matthias
Negri, Arne Meyer, Alex Odermatt, Gabriele Möller, Jerzy Adamski, and Rolf W. Hartmann
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Nov 2012
Downloaded from http://pubs.acs.org on November 18, 2012
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Structural Optimization of 2,5ꢀThiophene Amides as Highly Potent
and Selective 17βꢀHydroxysteroid Dehydrogenase Type 2 Inhibitors
for the Treatment of Osteoporosis
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†
♦
†♦
†
†
Sandrine MarchaisꢀOberwinkler , Kuiying Xu , Marie Wetzel , Enrico Perspicace ,
‡
⊥
⊥,
§
§$
Matthias Negri , Arne Meyer , Alex Odermatt Gabriele Möller , Jerzy Adamski ,
†‡*
and Rolf W. Hartmann
†
Pharmaceutical and Medicinal Chemistry, Saarland University,
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
‡
3
Campus C2 , Dꢀ66123 Saarbrücken, Germany
⊥Division of Molecular and Systems Toxicology, Department of Pharmaceutical
Sciences, University of Basel, Klingelbergstraße 50, CHꢀ4056 Basel, Switzerland
§
Genome Analysis Center, Institute of Experimental Genetic, Helmholtz Zentrum
München, Dꢀ85764 Neuherberg, Germany
$ Lehrstuhl für Experimentelle Genetik, Technische Universität Mu nchen, Dꢀ85350
FreisingꢀWeihenstephan, Germany
♦
SMO and KX contributed equally to this work
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Abstract
Inhibition of 17βꢀHSD2 is an attractive mechanism for the treatment of osteoporosis.
We report here the optimization of human 17βꢀHSD2 inhibitors in the 2,5ꢀthiophene
amide class varying the size of the linker (n equal 0 and 2) between the amide
moiety and the phenyl group. While none of the phenethylamides (n equal 2) were
active, most of the anilides (n equal 0) turned out to either moderately or strongly
inhibit 17βꢀHSD2. The four most active compounds showed an IC50 value around 60
nM and a very good selectivity toward 17βꢀHSD1, 4, 5, 11βꢀHSD1, 2 and the
estrogen receptors α and β. The investigated compounds inhibited monkey 17βꢀ
HSD2 moderately and one of them showed a good inhibitory activity on mouse 17βꢀ
HSD2. SAR studies allowed a first characterization of the human 17βꢀHSD2 active
site, which is predicted to be considerably larger than that of 17βꢀHSD1.
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Introduction
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17βꢀhydroxysteroid dehydrogenase type 2 (17βꢀHSD2) catalyzes the conversion of
the highly active 17βꢀhydroxysteroids into the inactive 17ꢀketosteroids, i.e. the
estrogen estradiol (E2), as well as the androgens testosterone (T) and 5αꢀ
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dihydrotestosterone (DHT) into their inactive forms estrone (E1), ꢀ ꢀandrosteneꢀ3,17ꢀ
4
dione (ꢀ ꢀAD) and 5αꢀandrostanedione, respectively (Chart 1). In addition, it has
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been described to exhibit a 20αꢀdehydrogenase activity transforming
20αꢀ
2
dihydroprogesterone in progesterone and a 3βꢀdehydrogenase activity converting
4
pregnenolone into progesterone and dehydroepiandrosterone (DHEA) in ꢀ ꢀAD.
Chart 1
E2 is known to play an important role in the growth, development and maintenance of
a diverse range of tissues (e.g. reproductive tissues, brain). It is also involved in the
maintenance of bone balance, inducing bone formation and repressing bone
3
resorption by action on the osteoblasts . There is also evidence that T has beneficial
4,5
effects on bone formation .
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Osteoporosis is a systemic disease where rigidity and mechanical stability of the
bone decline. Balance between bone formation and bone resorption is disrupted,
leading to an increased risk of fractures. High incidence of this disease is observed in
women after menopause when the E2 levels drop or following treatment with
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aromatase inhibitors , which block estrogen biosynthesis. Nowadays two firstꢀline
therapies are administered to osteoporotic patients: 1. Bisphosphonates
8
9,10
(alendronate) are effective in both postmenopausal women and men . However,
they lead to reduction of only 50% of fracture risk and are often associated to
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osteonecrosis of the jaw. 2. Selective estrogen receptor modulators , also called
SERMs (raloxifene), are efficient too but are often associated with an increased risk
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of venous thromboembolism. As the reduction of circulating estrogens induces
accelerated bone loss, estrogen replacement therapy (ERT) was given to
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postmenopausal osteoporosis patients
. It reduced the risk of fractures but
increased the incidence of cardiovascular diseases and breast cancer, which
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prevented the further use of this therapy
. All therapies currently available for the
treatment of osteoporosis have limitations and none of them offers a complete cure
for the condition. Osteoporosis is an ageꢀdependent disease and due to the
increasing lifeꢀexpectancy and aging population in the industrialized countries, there
is a need for developing improved drugs to combat this disease. As 17βꢀHSD2 is
16–18
expressed in osteoblastic cells
, inhibition of 17βꢀHSD2, which will lead to an
increase in E2 and T levels locally in the bones, offers therefore the potential as a
novel therapy for osteoporosis.
Ideally, 17βꢀHSD2 inhibitors should be highly potent and selective. They should not
exhibit inhibitory activities on functionally related 17βꢀHSD subtypes like type 1, 3, 4,
5 (Chart 1). Inhibition of 17βꢀHSD type 4, which catalyzes the same reaction as type
2
is not desirable as it is ubiquitously expressed and its dysfunction leads to severe
19
human disorders e.g. Zellweger syndrome like Dꢀbifunctional protein deficiency.
Activity suppression of 17βꢀHSD1, 3 or 5, which catalyze the reverse reaction –
reduction of estrogens or androgens – will be thus counterproductive as it would
decrease E2 and T levels in bone and might lead to systemic sideꢀeffects.
17βꢀHSD2 inhibitors should not bind to the estrogen receptors (ER) α and β as it is
expected that the E2 effects are ER mediated. In addition, activation upon binding to
these receptors might lead in steroidogenic tissues to proliferative or antiꢀproliferative
effects, which should be avoided.
20
Although 17βꢀHSD2 was already revealed in 1985 by Blomquist and characterized
1
by Wu in 1993 , very few 17βꢀHSD2 inhibitor classes have been reported to date.
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Among the steroidal inhibitors, Poirier and colleagues described a series of steroidal
21–24
spirolactone derivatives
, the most potent compound is the C17ꢀspiroꢀδꢀlactone 1
22
25–27
(
Chart 2, IC50 = 34 nM ). Wood et al.
reported about a novel class of cisꢀ
pyrrolidinones as active and selective nonsteroidal 17βꢀHSD2 inhibitors, with 2 (Chart
) one of the most potent compound (IC50 = 50 nM in a cellꢀfree assay). Three further
classes of nonsteroidal potent and selective 17βꢀHSD2 inhibitors were recently
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2
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published by our group (Chart 2): the hydroxyphenylnaphthꢀ1ꢀol 3a and 3b
, the
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hydroxyphenylmethanones and derived from the triazole 4 , the amides 5a, 6b, 6c,
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and 7 . These amide derivatives are all substituted by a benzyl group which is linked
to a biphenylamide 5a or a phenylthiophene 6b, 6c, 7 moiety.
Chart 2
At the time we started this work, a proof of concept for therapeutic efficacy of 17βꢀ
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HSD2 inhibition had been described using compound 2 in vivo in a monkey model ,
showing a decrease of bone resorption and maintenance of bone formation. Despite
high variations and the moderate effects observed this in vivo experiment validates
this approach and underlines the need for new optimized 17βꢀHSD2 inhibitors.
In the current report, we describe the optimization of 17βꢀHSD2 inhibitors in the
biphenylamide and phenylthiophene amide classes focusing on the suppression of
the methylene from the benzyl group (anilide derivatives) or its replacement by an
ethylene linker (phenethylamide derivatives). The synthesis of a small library of
achiral derivatives, the biological evaluation and the structure activity relationship
(SAR) of the new 17βꢀHSD2 inhibitors will be presented and compared to the benzyl
32
analogues . The selectivity toward further HSD enzymes and the cytotoxicity profile
of the best candidates were investigated. Selectivity toward 17βꢀHSD1 was achieved
based on the expertise from the group developing potent and selective inhibitors of
34–45
17βꢀHSD1
. In order to identify which species could be more suitable to perform a
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proof of concept in a preclinical model, the most potent and selective compounds (at
the human enzymes) identified in this study were further tested for their ability to
inhibit 17βꢀHSD2 from different species (rodents and monkey).
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Design
In a previous study, it was shown that, starting from the weakly active disubstituted
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triazole 4 , opening of the triazole central moiety led to the discovery of a new
class of biphenylamideꢀ 5a and phenylthiophene amides 6b, 6c and 7 as 17βꢀHSD2
inhibitors (Chart 2). All the compounds discovered in this class share a methylated
amide and two hydroxy/methoxyphenyl moieties differentiated in this study as ring A
and C (Chart 2). In addition ring C is attached to the nitrogen of the amide via a
methylene linker (n = 1, benzyl group). The compounds differ in their central ring B,
which is either a 1,4ꢀphenyl, 1,3ꢀphenyl or 2,5ꢀthiophene group. Moderately active
compounds were identified in the class of the 1,3ꢀphenyl derivatives 5a, showing IC50
around 500 nM. Moderate to good active molecules were discovered in the 2,5ꢀ
thiophene class 6b and 6c, with IC50 around 380 nM with the exception of 7, the most
active and promising 17βꢀHSD2 inhibitor (IC50 = 61 nM and selectivity factor of 73
toward 17βꢀHSD1).
Having the hypothesis that the central core B and the hydroxy/methoxyphenyl A bind
at the same position in the enzyme, variation of the linker size n (n = 0, 1 and 2) will
bring ring C into different areas of the binding cavity as seen on Figure 1. Variation of
the size of the linker will help mapping the enzyme’s active site, which is unknown,
providing information on the space available there and the global size of the inhibitor
accepted by the enzyme as well as on inhibitor rigidity (n = 0) / flexibility (n = 2)
tolerated by the enzyme.
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In order to investigate deeper the enzyme’s active site and in an attempt to optimize
this class of compounds, a small library of 17βꢀHSD2 inhibitors was synthesized
keeping the phenyl C unchanged and varying the size of the linker (n = 0 and n = 2,
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to be compared with n = 1 previously described ) as well as the substituents at the
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ring A in both biphenyl and phenylthiophene amides classes (Chart 3, compounds 8ꢀ
41).
Figure 1
Chart 3
Results
Chemistry
The synthesis of the 1,3ꢀphenyl derivatives 8 to 11, depicted in Scheme 1 and the
one of the 2,5ꢀthiophene derivatives 12 to 41, depicted in Scheme 2, was performed
following a two to three steps reaction pathway. First, amidation was carried out by
reaction of the commercially available 5ꢀbromothiophene 12c or the 3(4)ꢀ
bromobenzoyl chloride 8c with substituted anilines 8d or with the 2ꢀ(3ꢀ
methoxyphenyl)ꢀNꢀmethylethanamine 27c under standard conditions (Method A:
triethylamine, dichloromethane at 0°C for 3h) providing the brominated intermediates
8b, 12b, 16b, 22b, 24bꢀ27b, 33bꢀ36b in isolated yields between 57% and 99%.
Subsequently, Suzuki coupling using tetrakis triphenylphosphine palladium and
sodium carbonate in a mixture DME/water 1:1 (Method B) afforded the biphenyl
amides 8aꢀ11a or the phenylthiophene amides derivatives 12, 13aꢀ16a, 16, 17, 18a,
19ꢀ21, 22a, 23ꢀ26, 27aꢀ29a, 30ꢀ41 with good yields. Methoxy compounds were
submitted to ether cleavage using boron trifluoride dimethyl sulphide complex and
yielded the hydroxy molecules 8ꢀ11, 13ꢀ16, 18, 22, 27ꢀ29.
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Scheme 1
Scheme 1
Scheme 2
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Biological Results
1. Inhibition of human 17βꢀHSD2 in cellꢀfree assay and cellular assay
7βꢀHSD2 inhibitory activities of the synthesized compounds were first evaluated
in a cellꢀfree assay. Human placental enzyme was obtained and used according to
1
23,46,47
described methods
. Briefly, incubations were run with the enzyme microsomal
fraction, tritiated E2, cofactor and inhibitor. The separation of substrate and product
was accomplished by HPLC. The percent inhibition values of compounds 8ꢀ41 are
shown in Table 1ꢀ3. The IC50 values determined for selected compounds are
reported in Table 4. Compounds showing less than 10% inhibition when tested at a
concentration of 1 ꢁM were considered to be inactive. The spiroꢀδꢀlactone 1,
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described by Poirier et al. , was taken as external reference (68% at 1ꢁM in our test;
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2ꢀ66% at 1ꢁM in their assay).
In the 1,3ꢀphenyl class (Table 1), comparison of the biological results indicates that
the best 17βꢀHSD2 inhibitory activities are obtained either when n equal 0 and the
substituents R1 and R2 are methoxy groups (compounds 8a, 9a, 10a) or when n
equal 1 and R1 and R2 are hydroxy moieties (compounds 5a, 5c). Taking away R2
(
R2: H, 11 and 11a) is detrimental for the activity, independent on the nature of R1. It
indicates that R2 is important for the stabilization of the molecule in the active site.
R1 and R2 therefore interact with amino acids from the binding cavity and behave as
Hꢀbond acceptors when n equal 0 or Hꢀbond donors when n equal 1.
Table 1
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In the 2,5ꢀthiophene class (Table 2), the highest inhibition data are observed with the
compounds having the linker n equal 0 and the substitutents R1 and R2 methoxys
(13a) or when the linker n is 1 and R1/R2 are hydroxys (6e) as observed in the 1,3ꢀ
phenyl class. The compounds with the linker n equal 2 are either inactive (28, 29a) or
weakly active (27a, 27) independent on the substituents at rings A and C. With the
ethylene linker the compounds might be too long and/or too flexible to fit in the
enzyme active site. They were not further investigated.
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Table 2
Focusing on compounds with n equal 0, the influence of the central core can also be
evaluated. Comparing the 1,3ꢀphenyl to the 2,5ꢀthiophene derivatives: 9a to 13a: 64
and 90% inh. @ 1 ꢁM, respectively; 10a to 14a: 40 and 66% inh. @ 1 ꢁM,
respectively and 11a to 15a: 37 and 82% inh. @ 1 ꢁM, respectively, it is obvious that
the 2,5ꢀthiophene is better than the 1,3ꢀphenyl moiety. This preference is difficult to
explain as both aromatic moieties can establish a πꢀstacking interaction with aromatic
amino acids from the active site. However the overall electronic density and the
molecular electrostatic potential (MEP) differ depending on the nature of the central
scaffold. It is likely that the MEP induced by the 2,5ꢀthiophene leads to a better
recognition with the corresponding region in the binding cavity. This property has
39,48
already been evidence in the discovery of 17βꢀHSD1 inhibitors
thiophene class only was further investigated in the rest of the study.
. Thus, the 2,5ꢀ
Furthermore the influence of the A and C ring substituents on the activity can be
deduced in the 2,5ꢀthiophene class with the linker n equal 0. Taking away the
methoxy group on the A ring (R2: H 15a/ R2: OMe 13a, 82/90% inh. @ 1 ꢁM,
respectively) does not influence the potency of the compound, indicating that this
group does not play a critical role for stabilization of the inhibitor in the active site.
Deleting the same group on the C ring (R1: H 16a/ R1: OMe 13a, 67/90% inh. @ 1
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methoxy is involved in specific interactions that stabilize the inhibitor in the binding
cavity or that it affects the electrostatic potential of the C ring which again favors the
binding.
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The importance of electronic effects is evidenced also by the fact that replacement of
the methoxy moiety at the A ring (13a, 90% inh. @ 1 ꢁM) by other electron donating
groups (3ꢀMe 19, 3ꢀNMe
is well accepted whereas replacement by electron withdrawing groups (4ꢀCN 18a or
ꢀF 23: 48, 67% inh. @ 1 ꢁM, respectively) leads to a decrease in activity. Same is
valid also for the C ring, where exchange of the methoxy moiety 13a (90% inh. @ 1
M) by electron withdrawing groups like 3ꢀCF 24 (75% inh. @ 1 ꢁM) or 2ꢀF 25 (68%
1 ꢁM) slightly reduces the 17βꢀHSD2 inhibitory activity while in presence of the
2
20, 3ꢀSMe 21: 95, 82 and 88% inh. @ 1 ꢁM, respectively)
3
ꢁ
3
@
electron donating 3ꢀMe 22a the percentage inhibition does not change. Introduction
of the large 3ꢀPh 26 (83% inh. @ 1 ꢁM) is also well tolerated by the enzyme
indicating that there is space in the area of the binding site for introduction of bulky
substituents.
A fluorine has been introduced at the 3ꢀmethoxyphenyl A ring as second substituent
in this ring (compounds 30ꢀ32, 36ꢀ39, Table 3) in the 2,5ꢀthiophene class with the
linker n equal 0. Introduction at the 2 position led to 31 and 32 (93% and 85% inh. @
1
ꢁM, respectively), which have similar activity as the corresponding compounds 13a
and 22a. This substituent does not achieve any specific interaction with the active
site but is also not disturbing the stabilization. Addition of the fluorine at position 4
induces a slight loss in activity 39 (66% inh. @ 1 ꢁM). This decrease in activity is
consistent with the electronic effect described previously (replacement of a methoxy
for an electron withdrawing group, compounds 18a and 23).
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Addition of a third substituent on the A ring, a 6ꢀF (37 94% inh. @ 1 ꢁM) does not
increase the potency of 31.
Compound 31 (93% inh. @ 1 ꢁM) differing from 35 (49% inh. @ 1 ꢁM) in the
displacement of the methoxy group on the C ring from the 3 to the 4 position, leads to
a decrease in activity and reveals the importance of the interaction achieved by this
group, which must have the right orientation.
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It was then investigated if the methyl group on the amide function of 31 is necessary
for activity: exchange with a hydrogen 33 or a phenyl 34 led to two inactive
compounds. The methyl group might be located in a small lipophilic cavity and
participate actively in the stabilization of the compound. Loss of this group prevents
this interaction and the phenyl group might be too big to fit into this lipophilic cavity.
Table 3
For the most active compounds showing more than 70% inhibition @ 1 ꢁM, IC50 were
determined in the cellꢀfree assay and are shown in Table 4. Four highly active
compounds with the linker n equal 0 (13a, 19, 31 and 32) were identified displaying
IC values around 60 nM. They are equipotent to the previously described 7 carrying
50
a methylene linker. Five other interesting compounds (12, 20, 26, 36 and 37) were
discovered with an IC50 values between 100 and 200 nM.
Table 4
The inhibitory activity of the most potent compounds on 17βꢀHSD2 was also
evaluated in a cellular model system, using the MDAꢀMBꢀ231 cell line. The
compounds’ efficiency is either expressed as IC50 for the most potent compounds or
as percentage of inhibition for the others (Table 4). The data obtained lie in the same
range as the one from the cellꢀfree inhibition, with IC50 values around 100 nM or
below. The results indicate that the compounds can permeate the cell membrane, are
stable in the cell, and are not quickly metabolized.
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. Selectivity aspect
As 17βꢀHSD1 catalyzes the reduction of E1 to E2, the reversed 17βꢀHSD2 reaction,
it should not be affected by 17βꢀHSD2 inhibitors. In the 1,3ꢀphenyl class (compounds
0
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aꢀ11), the selectivity observed toward this enzyme (Table 1) is very good: no or a
very weak inhibition of the enzyme was measured at a concentration of 1 ꢁM. In the
series of the 2,5ꢀthiophenes, independent of the linker size (n equal 0, 1 or 2,
compounds 12ꢀ29), the same results were observed except for the middle active
17βꢀHSD2 inhibitors 14, 15 and 30 (69%, 60% and 67% 17βꢀHSD2 inh. @ 1 ꢁM,
respectively), which showed around 50% 17βꢀHSD1 inhibition at 1 ꢁM. For the most
potent 17βꢀHSD2 inhibitors (Table 4), the selectivity was expressed as selectivity
factor (SF) calculated as the ratio of IC50 (17βꢀHSD2) over IC50 (17βꢀHSD1). For the
compounds with an IC50 (17βꢀHSD2) below 200 nM, the SF varied between 26 and
above 800, except for 26 with a SF of 8. The selectivity toward 17βꢀHSD1 is good to
very good for most of the new 17βꢀHSD2 inhibitors described. It is even better for the
compounds without linker (SF of 112, 116, above 800 and 132 for 13a, 19, 31 and
32) compared to the one with a methylene linker (SF of 73 for 7).
Inhibitors of 17βꢀHSD2 should have no affinity for the estrogen receptors (ER) α and
β, as it is expected that most E2 effects are ER mediated. All the compounds with an
IC50 (17βꢀHSD2, cellꢀfree assay) below 500 nM were evaluated for their relative
binding affinity (RBA) to the ERα and ERβ in a competitive assay using a previously
47,49
described assay
and taking E2 as internal reference. All of the tested compounds
showed a RBA below 0.1%, compared to the affinity of E2, which was arbitrarily set
to 100%.
1
7βꢀHSD4 catalyzes the oxidation of E2 into E1 as 17βꢀHSD2 (Chart 1) and is
ubiquitously expressed. 17βꢀHSD5 is a reductive enzyme, it converts the inactive
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DHEA and 4ꢀandrosteneꢀ3,17ꢀdione into the potent 5ꢀandrosteneꢀ3β,17βꢀdiol and
testosterone, respectively (Chart 1). In order to avoid systemic side effects and not to
counteract the effect of 17βꢀHSD2 inhibition, inhibition of these enzymes should be
avoided.
10
11
12
13
14
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17
18
19
20
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24
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26
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The five most potent compounds 7, 13a, 19, 31 and 32 were evaluated for their
ability to inhibit these two enzymes, using recombinant human 17βꢀHSD4 and 17βꢀ
50,51
HSD5 expressed in E. Coli following the described procedure
. The compounds
did not show any inhibition of 17βꢀHSD4 when tested at a concentration of 1 ꢁM and
inhibited 17βꢀHSD5 only weakly (inhibition between 17% and 33% at 1 ꢁM, Table 5).
Table 5
11βꢀHSDs are involved in the glucocorticoid biosynthesis. 11βꢀHSD1 catalyzes the
transformation of the inactive cortisone into the potent cortisol and 11βꢀHSD2 the
52
reverse reaction. Some inhibitors of 11βꢀHSD1 have a close structural analogy to
the amides identified in this study. In addition, 17βꢀHSD2 has a relatively high
53
sequence identity with 11βꢀHSD2 (45%) . Therefore, the selectivity profile of the five
structurally most relevant compounds 7, 13a, 19, 31 and 32 was thus extended to
these two enzymes using the recombinant enzymes 11βꢀHSD1 and 11βꢀHSD2 stably
54
transfected in HEKꢀ293 cells following the described procedure . Absence to very
low 11βꢀHSD1 and 11βꢀHSD2 inhibition was observed at a concentration of 2 ꢁM
except for 7, which showed an 11βꢀHSD1 IC50 of 1 ꢁM for 11βꢀHSD1. This activity is
not negligible but compared to the 17βꢀHSD2 IC50 of 61 nM for 17βꢀHSD2, a
selectivity factor of around 16 might be acceptable, especially regarding the fact that
11βꢀHSD1 activates glucocorticoids and elevated glucocorticoids have been
55
associated with osteoporosis .
3. Further tests
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The lipophilicity profile of 7, 13a, 19, 31 and 32 was evaluated by calculation of the
logP values (Table 4). For most of the compounds it is between 4 and 5, or slightly
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above 5, which is still in a good range according to the Lipinski rule of 5 and which
should be correlated to a good permeability.
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The cytotoxicity of 7, 13a, 19, 31 and 32 was evaluated in the MDAꢀMBꢀ231 cell line
57
based on MTT conversion following the procedure described by Denisot et al.
at
three different concentrations: 2.5, 10 and 50 ꢁM. No cytotoxicity could be observed
even at the highest concentration after 3 h incubation (data not shown).
In order to identify the appropriate species for demonstration of in vivo efficacy in a
diseaseꢀoriented model, the five most potent compounds 7, 13a, 19, 31 and 32 were
tested for their ability to inhibit the enzyme responsible for E2 into E1ꢀtransformation
from three different animals: rat, mouse and monkey Callithrix Jacchus. The
compounds were evaluated in a cellꢀfree assay using the microsomal fraction of liver
preparation from rat and mouse. In case of the monkey, the microsomal enzyme was
gained from placenta. The compounds showed middle activity on the monkey
enzyme, between 45 and 53% inh. @ 1 ꢁM (Table 6). They were inactive to very low
active in the rat, the best one being 7 with 25% inh. @ 1 ꢁM. In mouse compounds
13a, 19 and 31 were also barely active (between 26 and 30% inh. @ 1 ꢁM) except
for 32 and 7 which were middle to good active with 45% and 65% inh. @ 1 ꢁM,
respectively. It is striking that such a difference in activity is observed between the rat
(Rattus norvegicus) and the mouse (Mus. Musculus) 17βꢀHSD2 inhibition data, as
the protein sequence of both species is highly similar. However from this study,
compound 7, identified as highly active and selective 17βꢀHSD2 inhibitor at the
human enzyme, exhibits the highest 17βꢀHSD2 inhibition on the mouse enzyme. This
result suggests that the mouse might be a promising species to perform an in vivo
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experiment using compound 7 and to verify that 17βꢀHSD2 inhibitors could be
effective for the treatment of osteoporosis.
Table 6
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Discussion
The aim of this study was the optimization of 17βꢀHSD2 inhibitors from the amide
class by variation of the size of the linker (n) located between the amide function and
the C ring. Introduction of an ethylene linker (n equal 2) is detrimental for the activity
independent of the central moiety 1,3ꢀphenyl or 2,5ꢀthiophene. The compounds might
be too long or too flexible. Taking out these two carbons linker (n equal 0) led to the
identification of four promising compounds 13a, 19, 31 and 32 with IC50 values
around 60 nM. Interestingly these compounds all bear a methoxy function on the C
ring while the equally active 7 with a methylene linker (n equal 1) is hydroxylated on
this ring. High activity is only achieved when n equals 1 and the C ring is
hydroxylated (7) or n equals 0 and C ring is methoxylated (13a, 19, 31 and 32). It is
striking that there is no difference in activity between these two series of compounds.
These requirements to achieve high activity are also intriguing: these data suggest
that these two series of compounds may not interact with the same amino acids in
the binding site. Thus, a different binding mode could be expected for these two
groups of inhibitors.
From this study it is clear that the 2,5ꢀthiophene central core is superior to the 1,3ꢀ
phenyl independent of the size of the linker n equal 0 or 1. This result was already
observed developing 17βꢀHSD1 inhibitors. The molecular electrostatic potential
induced by the thiophene on the whole molecule might lead to better interactions with
the enzyme active site. Further, the thiophene differs from the phenyl ring in the
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presence of dꢀorbitals present on the sulfur. They might allow the thiophene
derivatives to undergo specific interactions compared to the phenyl one.
The human 17βꢀHSD2 accepts ligands with a high structural diversity: steroidal
substrates E2, T, 20αꢀdihydroprogesterone, pregnenolone and DHEA, steroidal
inhibitor spirolactone 1, nonsteroidal inhibitors cisꢀpyrrolidinone 2 and amides 7 and
31. These ligands differ in their shape and volume but they are all very large. In order
to map the enzyme active site , which is unknown, and having the hypothesis that all
these compound bind in the enzyme active site, we compared the new nonsteroidal
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17βꢀHSD2 inhibitors with two different linker sizes (31 for n = 0 and 7 n = 1) with the
25–27
equipotent cisꢀpyrrolidinone 2 described by Wood et al.
. Visualization of the 3Dꢀ
structure of these three compounds, after energy minimization, highlights the folded
shape of compound 2 with the thiophene ring almost parallel to the pyrrolidinone
moiety (Figure 2). Thereby the two aromatic groups, phenyl and pyridine, are directed
in opposite direction while for 7 and 31 they assume an elongated shape.
Superimposition of 7 and 31 to 2 (Figure 2) may indicate that the area occupied by
the different compounds varies and sustains the hypothesis that compound 2 may fit
into the enzyme’s active site with another binding mode compared to 7 and 31. In
addition, the fact that compound 2 does not require any hydroxy or methoxy groups
to achieve high activity in contrary to 7 or 31 also suggests a different positioning of
these compounds in the binding site. It could therefore be deduced that the 17βꢀ
HSD2 active site may be very large. This space could easily be used to achieve
selectivity toward other enzymes by introduction of appropriate substituents. It is
striking that compounds with a linker n equal 2 were inactive although they were
expected to fit from the steric hindrance point of view. It might indicate that the
flexibility induced by the ethylene linker is not appropriate.
Figure 2
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The selectivity profile of the 17βꢀHSD2 inhibitor is an important issue. Not to
counteract the therapeutic concept, the functionally related enzymes like 17βꢀHSD1,
4, 5 should not be inhibited and no binding affinity to the ERs should be identified.
The most potent 17βꢀHSD2 inhibitors identified in this study 13a, 19, 31 and 32 as
well as 1, 2 and 7 are all selective toward 17βꢀHSD1 and do not bind to the estrogen
receptors. It might indicate that the 17βꢀHSD2 active site is larger than that of 17βꢀ
HSD1 and the binding domains of the ERs. The size difference of the binding sites of
these proteins is an interesting property as it could facilitate the gain in selectivity of
the 17βꢀHSD2 inhibitor toward 17βꢀHSD1 and ERs. In addition it is notable that the
most active compounds with a linker equal 0, i.e. 13a, 19, 31 and 32 have a much
higher selectivity factor than the one compound with n equal 1 i.e. compound 7. In
case n equal 0, the C ring seems to adopt a conformation of higher steric hindrance
in the 17βꢀHSD1 active site than in the case of n equal 1.
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11βꢀHSD1 and 11βꢀHSD2 catalyze the oxidoreduction at position 11 of cortisone in
cortisol. These two enzymes have an important function in the glucocorticoid
biosynthesis and should not be inhibited. Based on structural similarities identified
between 11βꢀHSD1 inhibitors and our amides derivatives, it is also important to verify
the selectivity of our inhibitors toward these enzymes. None of the new amides
discovered in this study inhibit 11βꢀHSD1 or 11βꢀHSD2 to an appreciable extend,
except for compound 7 which shows an IC value of 1 ꢁM for 11βꢀHSD1 and an IC
50
50
value of 61 nM for 17βꢀHSD2, the ratio resulting in a selectivity factor (SF) of 16.
Based on the fact that elevated glucocorticoids levels have been associated with
osteoporosis, moderate 11βꢀHSD1 inhibition might be still of advantage for the
therapeutic concept.
Inhibition of 17βꢀHSD2 is a completely new approach for the treatment of
osteoporosis compared to the existing treatments. A therapeutic effect similar to the
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one observed with estrogen replacement therapy (ERT), which has already been
proven to be efficient in the treatment of osteoporosis, is expected after treatment
with 17βꢀHSD2 inhibitors. ERT is not recommended because of severe sideꢀeffects
caused by the systemic increase in E2 at the necessary high doses. Inhibition of 17βꢀ
HSD2 will allow an increase in E2 in lower doses and only in targeted organs where
the enzyme is present i.e. in organs like placenta, brain, bone, breast and ovaries. In
addition after menopause, the ovaries do not function anymore and there is atrophy
of the breast and of the uterus connected to a reduction of the metabolism/
catabolism of the tissues. The levels in androstenedione and estrone in these organs
may be reduced and therefore the amount of E2 and testosterone as well.
Consequently treatment with 17βꢀHSD2 inhibitors may be less susceptible to induce
breast cancer compared to ERT. 17βꢀHSD2 inhibition is thus not expected to induce
severe adverseꢀeffects. A targeted effect in the bones should result in a superior drug
compared to SERMs or bisphosphonates.
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The five most potent compounds 7, 13a, 19, 31 and 32 were investigated regarding
their ability to inhibit 17βꢀHSD2 from other species in order to identify an appropriate
species for conducting in vivo experiments. The compounds were tested on the rat,
mouse and monkey 17βꢀHSD2. Only inhibitor 7 showed a good inhibition on the
mouse enzyme. At this stage, the mouse can be considered as potential species to
perform the in vivo proof of concept. It has the advantage that it’s easily accessible
58,59
and is well described as it is often used for the study of bone diseases
. Metabolic
stability as well as the pharmacokinetic profile of compound 7 have to be evaluated in
the mouse to validate this species as adequate model.
In this study, we described the optimization of 17βꢀHSD2 inhibitors in the 2,5ꢀ
thiophene and 1,3ꢀphenylamide class by variation of the linker size between the C
ring and the amide moiety. It led to the discovery of four new highly active
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compounds with the C ring directly attached to the amide 13a, 19, 31 and 32 with an
IC value around 60 nM in a cellꢀfree assay, a very good cellular activity in the same
50
range as in the cellꢀfree assay and a very high selectivity factor toward 17βꢀHSD1
above 100 and even higher than 800 for 31. The compounds 13a, 19, 31 and 32 are
equipotent to the compound with the methylene linker 7 but show higher selectivity
toward 17βꢀHSD1. SAR studies allowed a first characterization of the 17βꢀHSD2
active site which must be quite large and certainly larger than the one of 17βꢀHSD1.
The mouse was identified as potential animal in order to perform an in vivo proof of
concept for the demonstration of the efficacy of 17βꢀHSD2 inhibitors in osteoporosis.
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Supporting information available
Chemical synthesis and characterization of all compounds as well as HPLC purity
determination. This material is free of charge via the Internet at http://pubs.acs.org.
Experimental Section
1. Chemical methods
Chemical names follow IUPAC nomenclature.
Starting materials were purchased from Aldrich, Acros, Lancaster, Roth, Merck,
CombiꢀBlocks or Fluka and were used without purification.
Flash column chromatography (FC) was performed on silica gel (70ꢀ200 ꢀm), and
reaction progress was monitored by TLC on Alugram SIL G/UV254 (Machereyꢀ
Nagel). Visualization was accomplished with UV light.
1
13
H NMR and C NMR spectra were measured on a Bruker AM500 spectrometer (at
500 MHz and 125 MHz, respectively) at 300 K in CD COCD . Chemical shifts are
3
3
reported in δ (parts per million: ppm), by reference to the hydrogenated residues of
1
deuteriated solvent as internal standard: 2.05 ppm ( H NMR) and 30.8 and 206.3
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ppm ( C 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 measurements were executed using a TSQ Quantum equipped with an
electrospray interface (ESI) or an atmospheric pressure chemical ionization source
(APCI) (Thermo Fischer, Dreieich, Germany) instrument. GC/MS spectra were
measured on a GCD Series G1800A (Hewlett Packard) instrument with an Optimaꢀ5ꢀ
MS (0.25 ꢂM, 30 m) column (Macherey Nagel).
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IR spectra were recorded on a Spectrum 100 FTꢀIR 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 Surveyor®ꢀLCꢀsystem
consisted of a pump, an auto sampler, and a PDA detector. Mass spectrometry was
performed on a TSQ® Quantum (ThermoFisher, Dreieich, Germany). The triple
quadrupole mass spectrometer was equipped with an electrospray interface (ESI) or
an atmospheric pressure chemical ionisation (APCI). The system was operated by
the standard software Xcalibur®. A RP C18 NUCLEODUR® 100ꢀ5 (3 mm) column
(MachereyꢀNagel 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 was increased from
an initial concentration of 0% at 0 min to 100 % at 15 min and kept at 100 % for 5
min. The injection volume was 15 ꢁL and flow rate was set to 800 ꢁL/min. MS
analysis was carried out at a needle voltage of 3000 V and a capillary temperature of
3
50 °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.
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All tested compounds have ≥ 95% chemical purity except compounds 15a and 21
which have a purity of 94% and 90%, respectively.
31
32
32
32
Compounds 4 , 5aꢀd , 6aꢀl , 7 were prepared according to the previously
described procedure.
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General procedures
General procedure for amidation. Method A. At 0 °C, a solution of 3(4)ꢀ
bromobenzoyl chloride or 5ꢀbromothiopheneꢀ2ꢀcarbonyl chloride (1 eq) in CH
ml/eq) was added drop wise to a solution of the corresponding amine (1 eq) and
triethylamine (1.15 eq) in solution in CH Cl (2 ml/eq). The mixture was kept stirred at
°C for 3 h and evaporated under reduced pressure. The residue was purified by FC
2 2
Cl (2
2
2
0
with nꢀhexane/ethyl acetate or dichloromethane as eluant.
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) palladium (0.1 eq) in an oxygen free DME/water (1:1)
solution was stirred at 80 °C for 4 to 14 h under nitrogen. The reaction mixture was
cooled to room temperature. The aqueous layer was extracted with dichloromethane.
The combined organic layers were washed with brine, dried over sodium sulfate,
filtered and concentrated to dryness. The product was purified by FC with nꢀ
hexane/ethyl acetate, dichloromethane or dichloromethane/methanol as eluant.
General procedure for ether cleavage. Method C. To a solution of methoxyphenyl
compounds (1 eq) in dry dichloromethane (5 ml/mmol of reactant), borontrifluoride
dimethyl sulfide complex (6 eq/methoxy function) was added drop wise at 0 °C and
stirred for 6 to 14 h. After the reaction was finished, the reaction mixture was diluted
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with dichloromethane and 5% aqueous NaHCO
3
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
as eluant.
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Detailed synthesis procedures of the most interesting compounds
Nꢀ(3ꢀMethoxyphenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀcarboxamide
(12).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (40 mg, 0.12 mmol) and 4ꢀ
methoxyphenylboronic acid (22 mg, 0.14 mmol) with tetrakis(triphenylphosphine)
palladium (14 mg, 0.012 mmol) according to method B for 6 h. Purification by FC
(CH
2
Cl
2
/CH
3
OH 200:1) afforded the desired compound as brown solid (40 mg, yield:
+
1
92%). C20
3
H19NO S; MW: 353; mp: 119ꢀ120 °C; MS (ESI): 354 (M+H) ; H NMR
3 3
(CD COCD ): 3.37 (s, 3H), 3.81 (s, 3H), 3.82 (s, 3H), 6.55 (d, J = 4.1 Hz, 1H), 6.92ꢀ
6
.96 (m, 3H), 6.97ꢀ7.01 (m, 2H), 7.02 (d, J = 4.1 Hz, 1H), 7.37 (td, J = 7.9, 0.6 Hz,
H), 7.51 (d, J = 9.1 Hz, 2H).
1
N,5ꢀBis(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀcarboxamide (13a).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (75 mg, 0.23 mmol) and 3ꢀ
methoxyphenylboronic acid (41 mg, 0.27 mmol) with tetrakis(triphenylphosphine)
palladium (27 mg, 0.023 mmol) according to method B for 5 h. Purification by FC
2 2
(CH Cl ) afforded the desired compound as yellow solid (80 mg, yield: 98%).
+
1
20 3
C H19NO S; MW: 353; mp: 116ꢀ117 °C; MS (ESI): 354 (M+H) ; H NMR
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(CD
3
COCD
3
): 3.38 (s, 3H), 3.82 (s, 3H), 3.83 (s, 3H), 6.58 (d, J = 4.1 Hz, 1H), 6.90
(ddd, J = 8.2, 2.5, 0.9 Hz, 1H), 6.94 (ddd, J = 7.6, 1.6, 0.9 Hz, 1H), 6.98ꢀ7.01 (m,
2
H), 7.12 (t, J = 2.0 Hz, 1H), 7.15 (ddd, J = 7.6, 1.6, 0.9 Hz, 1H), 7.15 (d, J = 4.1 Hz,
H), 7.30 (t, J = 8.0 Hz, 1H), 7.38 (td, J = 8.0, 0.9 Hz, 1H).
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Nꢀ(3ꢀMethoxyphenyl)ꢀNꢀmethylꢀ5ꢀphenylthiopheneꢀ2ꢀcarboxamide (15a).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (75 mg, 0.23 mmol) and phenylboronic acid (33
mg, 0.27 mmol) with tetrakis(triphenylphosphine) palladium (27 mg, 0.023 mmol)
according to method B for 4 h. Purification by FC (nꢀhexane/ethyl acetate 10:1→6:1)
afforded the desired compound as beige solid (70 mg, yield: 94%). C19
H17NO
2
S; MW:
+
1
3
3
23; mp: 126ꢀ127 °C; MS (ESI): 324 (M+H) ; H NMR (CD COCD ): 3.38 (s, 3H),
3
3
.82 (s, 3H), 6.60 (d, J = 4.0 Hz, 1H), 6.94 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 6.99ꢀ7.01
(m, 2H), 7.16 (d, J = 4.0 Hz, 1H), 7.32 (ddt, J = 8.0, 6.3, 1.2 Hz, 1H), 7.36ꢀ7.41 (m,
3H), 7.59 (d, J = 8.0 Hz, 2H).
Nꢀ(3ꢀMethoxyphenyl)ꢀNꢀmethylꢀ5ꢀmꢀtolylthiopheneꢀ2ꢀcarboxamide (19).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (33 mg, 0.1 mmol) and 3ꢀmethylphenylboronic
acid (19 mg, 0.14 mmol) with tetrakis(triphenylphosphine) palladium (12 mg, 0.01
mmol) according to method B for 8 h. Purification by FC (nꢀhexane/ethyl acetate
10:1→6:1) afforded the desired compound as beige solid (26 mg, yield: 77%).
+
1
C
20
H
19NO
2
S; MW: 337; mp: 128ꢀ129 °C; MS (ESI): 338 (M+H) ; H NMR
COCD ): 2.34 (s, 3H), 3.38 (s, 3H), 3.81 (s, 3H), 6.57 (d, J = 4.0 Hz, 1H), 6.94
ddd, J = 7.9, 2.2, 0.9 Hz, 1H), 6.98ꢀ7.01 (m, 2H), 7.13 (d, J = 4.0 Hz, 1H), 7.14ꢀ7.16
m, 1H), 7.27 (t, J = 7.9 Hz, 1H), 7.36ꢀ7.39 (m, 2H), 7.41ꢀ7.42 (m, 1H).
(CD
3
3
(
(
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ꢀ(3ꢀ(Dimethylamino)phenyl)ꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀ
carboxamide (20).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (49 mg, 0.15 mmol) and 3ꢀ
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(dimethylamino)phenylboronic acid (30 mg, 0.18 mmol) with
tetrakis(triphenylphosphine) palladium (17 mg, 0.015 mmol) according to method B
for 14 h. Purification by FC (nꢀhexane/ethyl acetate 10:1→6:1) afforded the desired
compound as orange solid (26 mg, yield: 47%). C21
H
22
N
2
O
2
S; MW: 366; mp: 121ꢀ122
): 2.96 (s, 6H), 3.38 (s, 3H), 3.82 (s,
H), 6.56 (d, J = 4.0 Hz, 1H), 6.72 (dd, J = 8.2, 2.4 Hz, 1H), 6.86 (d, J = 7.6 Hz, 1H),
.89 (t, J = 2.0 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.98ꢀ7.01 (m, 2H), 7.10 (d, J = 4.0
+
1
3 3
°C; MS (ESI): 367 (M+H) ; H NMR (CD COCD
3
6
Hz, 1H), 7.19 (t, J = 7.9 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H).
Nꢀ(3ꢀMethoxyphenyl)ꢀNꢀmethylꢀ5ꢀ(3ꢀ(methylthio)phenyl)thiopheneꢀ2ꢀ
carboxamide (21).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (33 mg, 0.1 mmol) and 3ꢀ
(methylthio)phenylboronic acid (23 mg, 0.14 mmol) with tetrakis(triphenylphosphine)
palladium (12 mg, 0.01 mmol) according to method B for 8 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1→7:1) afforded the desired compound as yellowish solid
+
(
22 mg, yield: 59%). C20
H
19NO
2
S
2
; MW: 369; mp: 109ꢀ110 °C; MS (ESI): 370 (M+H) ;
): 2.52 (s, 3H), 3.38 (s, 3H), 3.82 (s, 3H), 6.59 (d, J = 4.0 Hz,
H), 6.94 (ddd, J = 8.0, 1.9, 0.9 Hz, 1H), 6.98ꢀ7.02 (m, 2H), 7.18 (d, J = 4.0 Hz, 1H),
1
3 3
H NMR (CD COCD
1
7
7
.23 (dt, J = 6.9, 1.9 Hz, 1H), 7.30ꢀ7.35 (m, 2H), 7.38 (td, J = 8.0, 0.9 Hz, 1H), 7.44ꢀ
.45 (m, 1H).
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ꢀ(2ꢀFluoroꢀ3ꢀmethoxyphenyl)ꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀ
carboxamide (31).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (40 mg, 0.12 mmol) and 2ꢀfluoroꢀ3ꢀ
methoxyphenylboronic acid (25 mg, 0.14 mmol) with tetrakis(triphenylphosphine)
palladium (14 mg, 0.012 mmol) according to method B for 14 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1→6:1) afforded the desired compound as brown solid (30
10
11
12
13
14
15
16
17
18
19
20
21
22
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48
49
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52
53
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56
57
58
59
60
+
1
mg, yield: 66%). C20
NMR (CD COCD ): 3.38 (s, 3H), 3.82 (s, 3H), 3.90 (s, 3H), 6.65 (dd, J = 4.0, 1.0 Hz,
H), 6.95 (ddd, J = 7.6, 1.8, 1.0 Hz, 1H), 6.99ꢀ7.01 (m, 2H), 7.09ꢀ7.20 (m, 3H), 7.23
dd, J = 4.0, 1.0 Hz, 1H), 7.37 (dd, J = 9.1, 7.9 Hz, 1H).
3
H18FNO S; MW: 371; mp: 159ꢀ160 °C; MS (ESI): 372 (M+H) ; H
3
3
1
(
5ꢀ(2ꢀFluoroꢀ3ꢀmethylphenyl)ꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀ
carboxamide (36).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (49 mg, 0.15 mmol) and 2ꢀfluoroꢀ3ꢀ
methylphenylboronic acid (28 mg, 0.18 mmol) with tetrakis(triphenylphosphine)
palladium (17 mg, 0.015 mmol) according to method B for 14 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1) afforded the desired compound as colorless solid (50 mg,
+
1
yield: 94%). C20
2
H18FNO S; MW: 355; mp: 142ꢀ143 °C; MS (APCI): 356 (M+H) ; H
NMR (CD COCD ): 2.29 (d, J = 2.5 Hz, 3H), 3.39 (s, 3H), 3.81 (s, 3H), 6.63 (dd, J =
3
3
4.1, 0.9 Hz, 1H), 6.94 (ddd, J = 7.9, 1.9, 0.9 Hz, 1H), 6.98ꢀ7.01 (m, 2H), 7.11 (t, J =
7.9 Hz, 1H), 7.22 (dd, J = 4.1, 0.9 Hz, 1H), 7.23ꢀ7.25 (m, 1H), 7.36ꢀ7.39 (m, 1H),
7.45ꢀ7.48 (m, 1H)..
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5ꢀ(2,6ꢀDifluoroꢀ3ꢀmethoxyphenyl)ꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀ
carboxamide (37).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (33 mg, 0.1 mmol) and 2,6ꢀdifluoroꢀ3ꢀ
methoxyphenyl boronic acid (26 mg, 0.14 mmol) with tetrakis(triphenylphosphine)
palladium (12 mg, 0.01 mmol) according to method B for 14 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1) afforded the desired compound as orange solid (10 mg,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
yield: 26%). C20
NMR (CD COCD
1H), 6.95 (ddd, J = 7.6, 1.9, 0.9 Hz, 1H), 6.99ꢀ7.01 (m, 2H), 7.05 (ddd, J = 11.4, 9.1,
.2 Hz, 1H), 7.16 (td, J = 9.1, 5.0 Hz, 1H), 7.23 (dt, J = 4.1, 1.1 Hz, 1H), 7.36ꢀ7.39
m, 1H).
H
17
F
2
NO
3
S; MW: 389; mp: 147ꢀ148 °C; MS (ESI): 390 (M+H) ; H
3
3
): 3.40 (s, 3H), 3.82 (s, 3H), 3.90 (s, 3H), 6,71 (dt, J = 4.1, 0.9 Hz,
2
(
5ꢀ(3ꢀFluoroꢀ4ꢀmethoxyphenyl)ꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀ
carboxamide (38).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (40 mg, 0.12 mmol) and 3ꢀfluoroꢀ4ꢀ
methoxyphenylboronic acid (25 mg, 0.14 mmol) with tetrakis(triphenylphosphine)
palladium (14 mg, 0.015 mmol) according to method B for 6 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1→6:1) afforded the desired compound as yellow solid (40
+
1
mg, yield: 72%). C20
3
H18FNO S; MW: 371; mp: 157ꢀ158 °C; MS (ESI): 372 (M+H) ; H
NMR (CD COCD ): 3.38 (s, 3H), 3.82 (s, 3H), 3.91 (s, 3H), 6.55 (d, J = 4.0 Hz, 1H),
3
3
6.94 (ddd, J = 7.9, 1.9, 0.9 Hz, 1H), 6.99ꢀ7.01 (m, 2H), 7.08 (d, J = 4.0 Hz, 1H), 7.15
(t, J = 8.5 Hz, 1H), 7.33ꢀ7.39 (m, 3H).
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5ꢀ(2,4ꢀDimethoxyphenyl)ꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀ
carboxamide (41).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀ(3ꢀmethoxyphenyl)ꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 12b (40 mg, 0.12 mmol) and 2,4ꢀ
dimethoxyphenylboronic acid (27 mg, 0.14 mmol) with tetrakis(triphenylphosphine)
palladium (14 mg, 0.012 mmol) according to method B for 14 h. Purification by FC (nꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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33
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hexane/ethyl acetate 10:1→6:1) afforded the desired compound as colorless solid
+
(35 mg, yield: 74%). C21
H
21NO
4
S; MW: 383; mp: 117ꢀ118 °C; MS (ESI): 384 (M+H) ;
1
3 3
H NMR (CD COCD ): 3.37 (s, 3H), 3.82 (s, 3H), 3.83 (s, 3H), 3.89 (s, 3H), 6.57
(ddd, J = 8.5, 2.4, 0.9 Hz, 1H), 6.63ꢀ6.64 (m, 2H), 6.92 (ddd, J = 7.9, 2.1, 0.9 Hz, 1H),
6
.97 (t, J = 2.1 Hz, 1H), 7.00 (ddd, J = 8.2, 2.7, 0.9 Hz, 1H), 7.15 (d, J = 4.3 Hz, 1H),
.37 (t, J = 8.1 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H).
7
5ꢀ(3ꢀMethoxyphenyl)ꢀNꢀmethylꢀNꢀmꢀtolylthiopheneꢀ2ꢀcarboxamide (22a).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀmethylꢀNꢀmꢀ
tolylthiopheneꢀ2ꢀcarboxamide 22b (78 mg, 0.25 mmol) and 3ꢀmethoxyphenylboronic
acid (45 mg, 0.3 mmol) with tetrakis(triphenylphosphine) palladium (29 mg, 0.025
mmol) according to method B for 4 h. Purification by FC (nꢀhexane/ethyl acetate
25:1→10:1) afforded the desired compound as colorless solid (65 mg, yield: 77%).
+
1
C
20
H
19NO
2
S; MW: 337; mp: 97ꢀ98 °C; MS (ESI): 338 (M+H) ; H NMR (CD
.36 (s, 3H), 3.37 (s, 3H), 3.83 (s, 3H), 6.51 (d, J = 4.1 Hz, 1H), 6.90 (ddd, J = 8.2,
.5, 0.9 Hz, 1H), 7.10ꢀ7.11 (m, 1H), 7.13ꢀ7.17 (m, 3H), 7.23 (s, 1H), 7.26 (d, J = 7.6
3 3
COCD ):
2
2
Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H).
5ꢀ(3ꢀHydroxyphenyl)ꢀNꢀmethylꢀNꢀmꢀtolylthiopheneꢀ2ꢀcarboxamide (22).
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The title compound was prepared by reaction of 5ꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylꢀNꢀmꢀ
tolylthiopheneꢀ2ꢀcarboxamide 22a (40 mg, 0.12 mmol) with borontrifluoride dimethyl
sulfide complex (0.08 ml, 0.72 mmol) according to method C for 14 h. Purification by
FC (CH
yield: 79%). C19
(CD COCD ): 2.36 (s, 3H), 3.37 (s, 3H), 6.52 (d, J = 4.1 Hz, 1H), 6.81 (ddd, J = 8.0,
2 2 3
Cl /CH OH 100:1→50:1) afforded the title compound as beige solid (30 mg,
0
1
2
3
4
5
6
7
8
9
0
1
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3
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7
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9
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0
+
1
H
2
17NO S; MW: 323; mp: 157ꢀ158 °C; MS (ESI): 324 (M+H) ; H NMR
3
3
2.5, 0.9 Hz, 1H), 7.03ꢀ7.06 (m, 2H), 7.08 (d, J = 4.1 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H),
7.20 (d, J = 7.6 Hz, 1H), 7.22 (s, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.36 (t, J = 8.0 Hz,
1H), 8.51 (s, 1H).
5ꢀ(2ꢀFluoroꢀ3ꢀmethoxyphenyl)ꢀNꢀmethylꢀNꢀ(mꢀtolylthiophene)ꢀ2ꢀcarboxamide
(32).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀmethylꢀNꢀmꢀ
tolylthiopheneꢀ2ꢀcarboxamide 22b (46 mg, 0.15 mmol) and 2ꢀfluoroꢀ3ꢀ
methoxyphenylboronic acid (31 mg, 0.18 mmol) with tetrakis(triphenylphosphine)
palladium (17 mg, 0.015 mmol) according to method B for 14 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1) afforded the desired compound as colorless solid (45 mg,
+
1
yield: 85%). C20
H
18FNO
2
S; MW: 355; mp: 120ꢀ121 °C; MS (ESI): 356 (M+H) ; H
NMR (CD COCD
3
3
): 2.36 (s, 3H), 3.38 (s, 3H), 3.90 (s, 3H), 6.58 (dd, J = 4.0, 1.0 Hz,
1H), 7.08ꢀ7.19 (m, 4H), 7.21 (dd, J = 4.1, 1.0 Hz, 1H), 7.23ꢀ7.27 (m, 2H), 7.36 (t, J =
7.7 Hz, 1H).
5ꢀ(3ꢀMethoxyphenyl)ꢀNꢀmethylꢀNꢀ(3ꢀ(trifluoromethyl)phenyl)thiopheneꢀ2ꢀ
carboxamide (24).
The title compound was prepared by reaction of 5ꢀbromoꢀNꢀmethylꢀNꢀ(3ꢀ
(trifluoromethyl)phenyl)thiopheneꢀ2ꢀcarboxamide 24b (36 mg, 0.1 mmol) and 3ꢀ
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methoxyphenylboronic acid (20 mg, 0.13 mmol) with tetrakis(triphenylphosphine)
palladium (12 mg, 0.01 mmol) according to method B for 6 h. Purification by FC (nꢀ
hexane/ethyl acetate 10:1→5:1) afforded the desired compound as yellow solid (35
+
1
mg, yield: 90%). C20
NMR (CD COCD ): 3.47 (s, 3H), 3.83 (s, 3H), 6.57 (d, J = 4.1 Hz, 1H), 6.91 (ddd, J =
8.2, 2.5, 0.9 Hz, 1H), 7.11 (t, J = 2.2 Hz, 1H), 7.14 (ddd, J = 7.6, 1.6, 0.9 Hz, 1H),
16 3 2
H F NO S; MW: 391; mp: 97ꢀ98 °C; MS (ESI): 392 (M+H) ; H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
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37
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44
45
46
47
48
49
50
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3
3
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.19 (d, J = 4.1 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H), 7.69ꢀ7.78 (m, 3H), 7.81ꢀ7.82 (m,
13
3 3
H); C NMR (CD COCD ): 39.0, 55.6, 112.0, 115.0, 119.0, 124.1, 125.3, 125.4,
25.8, 125.9, 131.1, 131.7, 132.2, 132.5, 133.0, 133.5, 135.5, 138.2, 146.3, 149.2,
ꢀ
1
161.2, 162.6; IR (cm ): 3046, 2963, 1608, 1438, 1330, 1117, 707 .
Nꢀ(Biphenylꢀ3ꢀyl)ꢀ5ꢀ(3ꢀmethoxyphenyl)ꢀNꢀmethylthiopheneꢀ2ꢀcarboxamide (26).
The title compound was prepared by reaction of Nꢀ(biphenylꢀ3ꢀyl)ꢀ5ꢀbromoꢀNꢀ
methylthiopheneꢀ2ꢀcarboxamide 26b (37 mg, 0.1 mmol) and 3ꢀmethoxyphenylboronic
acid (18 mg, 0.12 mmol) with tetrakis(triphenylphosphine) palladium (12 mg, 0.01
mmol) according to method B for 14 h. Purification by FC (nꢀhexane/ethyl acetate
8
:1) afforded the desired compound as yellow solid (38 mg, yield: 95%). C H NO S;
25 21 2
+
1
MW: 399; mp: 108ꢀ109 °C; MS (ESI): 400 (M+H) ; H NMR (CD
3H), 3.81 (s, 3H), 6.65 (d, J = 4.0 Hz, 1H), 6.89 (dd, J = 8.0, 2.1 Hz, 1H), 7.09 (s, 1H),
.12 (d, J = 7.6 Hz, 1H), 7.15 (d, J = 4.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.36ꢀ7.39
m, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.57 (t, J = 7.8 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H),
.71ꢀ7.74 (m, 2H).
3 3
COCD ): 3.46 (s,
7
(
7
LogP determination
The LogP values were calculated from CambridgeSoft Chem & Bio Draw 11.0 using
the ChemDrawPro 11.0 program.
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