Optimization of hydroxybenzothiazoles as novel potent and selective inhibitors of 17β-HSD1

Article abstract of DOI:10.1021/jm201711b

17β-HSD1 is a novel target for the treatment of estrogen-dependent diseases, as it catalyzes intracellular estradiol formation. Starting from two recently described compounds, highly active and selective inhibitors were developed. Benzoyl 6 and benzamide 17 are the most selective compounds toward 17β-HSD2 described so far. They also showed a promising profile regarding activity in T47-D cells, selectivity toward ERα and ERβ, inhibition of hepatic CYP enzymes, metabolic stability, and inhibition of marmoset 17β-HSD1 and 17β-HSD2.

Full text of DOI:10.1021/jm201711b

Brief Article
pubs.acs.org/jmc
Optimization of Hydroxybenzothiazoles as Novel Potent and
Selective Inhibitors of 17β-HSD1
Alessandro Spadaro,†,‡ Martin Frotscher,† and Rolf W. Hartmann*,†,§
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2 , D-66123 Saarbrucken, Germany
̈
3
‡ElexoPharm GmbH, Campus A1 , D-66123 Saarbrucken, Germany
̈
1
§Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2 , D-66123 Saarbrucken, Germany
̈
3
S
* Supporting Information
ABSTRACT: 17β-HSD1 is a novel target for the treatment of estrogen-dependent diseases, as it catalyzes intracellular estradiol
formation. Starting from two recently described compounds, highly active and selective inhibitors were developed. Benzoyl 6 and
benzamide 17 are the most selective compounds toward 17β-HSD2 described so far. They also showed a promising profile
regarding activity in T47-D cells, selectivity toward ERα and ERβ, inhibition of hepatic CYP enzymes, metabolic stability, and
inhibition of marmoset 17β-HSD1 and 17β-HSD2.
Chart 1. Compounds A and B and Synthesized Compounds
INTRODUCTION
■
For the treatment of steroid hormone-dependent diseases
biosynthesis inhibitors blocking hormone formation in the
corresponding endocrine gland provide an excellent strategy as
demonstrated by the use of CYP19 inhibitors for the treatment
of breast cancer (BC).1 Three further cytochrome P450 (CYP)
enzymes are presently in the focus of drug development:
CYP17,2 CYP11B2,3 and CYP11B14 for the treatment of
prostate cancer, congestive heart failure and myocardial fibrosis,
and Cushing's syndrome, respectively. As these therapeutic
options result in systemic reductions of steroid hormone levels
that often are associated with side effects, a softer option could
be the inhibition of hormone activation in the diseased cell. For
the treatment of benign prostatic hyperplasia, this strategy has
already been proven to be successful with inhibitors of 5α-
reductase.5 More recently 17β-hydroxysteroid dehydrogenase 1
(17β-HSD1) came into the focus of interest as a novel
therapeutic target for the treatment of estrogen dependent
diseases like BC and endometriosis.6 17β-HSD1 catalyzes the
conversion of the weakly active estrone (E1) to the highly
active estradiol (E2) and is overexpressed in some BC tissues.6
Efficacy of 17β-HSD1 inhibitors against human breast cancer
cell lines has already been reported in vitro and in nude mice.7
Besides some approaches to develop steroidal inhibitors,8 there
have also been attempts by us and other groups to develop
nonsteroidal inhibitors that should be advantageous with
respect to side effects.9 Recently we reported on two new
17β-HSD1 inhibitors with different activity/selectivity profiles,
the 1,3-benzothiazol-2-ylphenylmethanone A and the N-(1,3-
benzothiazol-2-yl)benzamide B (Chart 1).10 Here we report on
the structure optimization of these two lead compounds with
regard to 17β-HSD1 inhibitory activity and selectivity toward
17β-HSD2 (the enzyme catalyzing the reverse reaction) and
the estrogen receptors (ERs) α and β. For selected compounds,
17β-HSD1 inhibition in human T47-D breast cancer cells,
inhibition of 17β-HSD1 and 17β-HSD2 from Callithrix jacchus
(marmoset, animal model for endometriosis11), of human
hepatic CYP enzymes, and metabolic stability were determined.
RESULTS
■
Chemistry. The synthesis of 1−9 started from 1,3-
benzothiazol-2-ylamine or 1,3-benzothiazole. 1−3 and 5−7
were prepared in a four-step synthesis previously described10
(steps a−d in Scheme 1). 4 was synthesized in three steps
starting with the nucleophilic addition of 4iii to 4-fluoro-3-
methoxybenzaldehyde. For 8, 8iiii and 8iii were synthesized
and coupled via nucleophilic addition. Oxidation of the
resulting alcohol and ether cleavage of the silanyl ether using
tetra-n-butylammonium fluoride (TBAF) gave the final
compound. 9 was synthesized by coupling 1iii and 9iiiii,
followed by oxidation, cleavage with TBAF, boronic acid−
phenol cross-coupling reaction mediated by copper(II) acetate,
and cleavage of the two methoxy groups with boron tribromide
(BBr3). 10−18 were prepared via two steps as described10
(steps a and b in Scheme 2): (a) amide coupling between 6-
methoxy-1,3-benzothiazol-2-ylamine and the corresponding
benzoyl chloride and (b) ether cleavage. 19 was synthesized
by N-methylation of 11i with methyl iodide and sodium
hydride in DMF and subsequent ether cleavage.
Received: August 18, 2011
Published: January 26, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of Compounds 1−9
a
Reagents and conditions: (a) (1) NaNO2, H3PO4 (85%), −10 °C, 20 min, (2) H3PO2, H3PO4 (85%), −10 °C to room temp, 20 h. (b) Method A:
(1) n-BuLi, anhydrous THF, −70 to −20 °C, 1 h, (2) methoxybenzaldehyde, anhydrous THF, −15 °C, 90 min. (c) Method C: SIBX, anhydrous
THF, 0−60 °C, 20 h. (d) For 1−6, method D: pyridinium hydrochloride, 220 °C, 4 h. For 7, 8iiiii and 9, method E: BBr3, CH2Cl2, −78 °C to room
temp, 20 h. (e) Benzyl bromide, K2CO3, acetone, 100 °C, 20 h. For 9ii and 8: TBAF, THF, room temp, 2 h. (f) TBDMSiCl, imidazole, DMF, room
temp, 20 h; (g) benzeneboronic acid, Cu(AcO)2, Et3N, anhydrous CH2Cl2, room temp, 18 h.
a
Scheme 2. Synthesis of Compounds 10−19
enzymes. Compounds 2, 6, and 17 were further investigated for
inhibition of the human hepatic CYP enzymes: 3A4, 2D6, 2C9,
2C19, 1A2, and 2B6. They showed very little inhibition (54 μM
> IC50 > 3 μM) except for 1A2 which was inhibited by 2 and 6
moderately (IC50 = 1.8 and 0.57 μM). Furthermore, metabolic
stability of 6, 7, 10, and 17 was determined using human liver
microsomes. The two ketones 6 and 7 showed high intrinsic
clearance (Clint) of 94 and 108 (μL/min)/mg protein,
respectively. On the other hand, the amides 10 and 17 were
metabolically more stable, as they displayed Clint of 38 and 28,
respectively.
a
Reagents and conditions. (a) Method B: pyridine, 100 °C, 4 h. (b)
Method E: BBr3, CH2Cl2, −78 °C to room temp, 20 h. (c) 11i, NaH,
CH3I, DMF, 0 °C to room temp, 3 h.
DISCUSSION AND CONCLUSION
■
Inhibition of Human 17β-HSD1 and 17β-HSD2. For the
determination of 17β-HSD1 and 17β-HSD2 inhibition, tritiated
substrates E1 or E2 and the placental enzymes 17β-HSD1
(cytosolic fraction) or 17β-HSD2 (microsomal fraction) were
used. The labeled products formed were quantified by HPLC
using radiodetection. The inhibition values (IC50) and the
selectivity factors (SFs) are shown in Table 1. A and B were
used as references. In the series of the ketones, 1−3 and 5−7
strongly inhibited 17β-HSD1. Also, in the series of the amide
derivatives stronger 17β-HSD1 inhibition than the one shown
by B was observed, with 10 and 17 exhibiting IC50 in the low
nanomolar range. Furthermore, 6 and 17 displayed strong
selectivity for 17β-HSD1 over 17β-HSD2, exhibiting the
highest selectivity factors (>130) reported in the literature.
Affinities to ERα and ERβ. The relative binding affinities
(RBA) of the most selective compounds of this study were
determined using recombinant human ERα and ERβ in a
competition assay12 with [3H]E2 (E2 was used as reference,
and its RBA value was set to 100%). Only 2 showed a slight
affinity to the ERs, while all other compounds were more than
1000-fold less potent compared to E2.
Further Biological Evaluation. Additionally, the inhib-
ition of intracellular E2 formation for selected compounds was
determined in T47-D cells (Table 2). The high cellular
activities of 2, 10, and 17 indicate that they easily permeate the
cell membrane and are not metabolized quickly. Furthermore,
2, 6, 10, and 17 were tested for inhibition of Callithrix jacchus
17β-HSD1 and 17β-HSD2 (Table 2). They showed com-
parable (6) or even stronger (2, 10, and 17) inhibition of 17β-
HSD1 but apparently lower selectivity compared to the human
Recently we discovered two new 17β-HSD1 inhibitors, A and B
(Chart 1).10 In the present study they should be optimized
regarding activity and selectivity to obtain compounds suitable
for in vivo use. Introduction of various substituents with
different electronic, lipophilic, steric, and H-bonding properties
into the benzoyl moiety of A and B led to the discovery of two
new lead compounds, 6 and 17. As suggested in our previous
study,10 where A and B were hypothesized to interact
differently with the binding site, the introduction of a variety
of substituents into the benzoyl moiety of A and B had different
influence on inhibitory activity: comparing 1 and 10, 2 and 11,
5 and 12, 6 and 13, and 7 and 15 with their parent compounds
shows that only the fluorine increased inhibitory activity in both
classes. The introduction of methyl in the 4-position and OH in
the 6-position turned out to be beneficial in the case of the
ketones (6 and 7) but was deleterious for the amides (13 and
15). The introduction of a bulky substituent decreased
inhibition (8, 9). Interestingly, methylation of the OH group
of 2 (3) or replacement by hydrogen (4) on the benzothiazole
resulted only in a slight decrease of 17β-HSD1 inhibition but in
a complete loss of selectivity toward 17β-HSD2. Changing the
F position and adding a second fluorine in the amide series
increased 17β-HSD1 inhibitory activity: 17 showed the same
IC50 of 13 nM as the best ketone 2. This may be due to
favorable electronic effects exerted by the fluorines on the H-
bonding property of the OH, to the increased lipophilicity, or
to the change in π-interacting properties of the aromatic ring as
suggested by previous SAR studies.10 Regarding selectivity in
the ketone series, introduction of the methyl group in the 4-
position (6) not only increased activity but also decreased
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Journal of Medicinal Chemistry
Brief Article
Table 1. 17β-HSD1 Inhibitory Activities and Selectivities of Compounds 1−19
cf
,
IC50 (nM)
a
b
df
,
compd
X
R1
R2
R3
HSD1
HSD2
SF
A
CO
H
H
OH
OH
OH
OCH3
H
44
1035
83
24
17
9
1
2-F
H
5
2
4-F
H
13
121
59
3
4-F
H
38
2
4
4-F
H
136
13
104
1416
4003
1538
ni
1
5
6-F
H
OH
OH
OH
OBn
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
109
148
20
nd
2
6
4-CH3
6-OH
4-CH3
4-OPh
H
H
27
7
H
78
8
H
ni
9
H
863
243
29
1457
9264
1224
1480
3804
nd
B
CONH
H
38
42
9
10
11
12
13
14
15
16
17
18
19
2-F
H
4-F
H
171
112
ni
6-F
H
34
nd
nd
nd
2
4-CH3
5-OH
6-OH
2-Cl
2-F
H
e
H
3000
ni
nd
H
nd
6-F
6-F
4-F
H
221
13
469
1774
541
136
5
2-F
99
e
e
CONCH3
4-F
667
2704
4
a
b
Human placenta, cytosolic fraction, substrate [3H]E1 + E1 [500 nM], cofactor NADH [500 μM]. Human placenta, microsomal fraction, substrate
c
d
[3H]E2 + E2 [500 nM], cofactor NAD+ [1500 μM]. Mean values of three determinations, standard deviation less than 10%. Selectivity factor:
IC50(17β-HSD2)/IC50(17β-HSD1). Calculated with LOGIT transformation. nd: not determined. ni: no inhibition (less than 10% inhibition at 1
μM).
e
f
Table 2. Binding Affinities for the ERα and ERβ, 17β-HSD1 Inhibitory Activity in T47-D Cells, and Inhibition of Callithrix
jacchus 17β-HSD1 and 17β-HSD2 by Selected Compounds
% inhibition
b
d
e
RBA (%)
a
calHSD1
calHSD2,
50 nM
a
c
compd
X
R1
R2
ERα
ERβ
IC50 (nM), T47-D
5 nM 50 nM
A
1
CO
H
H
H
H
H
H
H
H
H
H
6-F
<10
0.1
<1
<1
2-F
<0.1
<1
2
4-F
11
80
34
40
5
6-F
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.1
6
4-CH3
6-OH
H
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
258
83
f
7
365
B
10
12
17
CONH
245
g
2-F
73
67
87
48
51
g
6-F
152
g
2-F
37
a
b
Human recombinant protein, incubation with 10 nM [3H]E2 and inhibitor for 1 h. RBA: relative binding affinity in percent (E2 = 100%), mean
c
d
value of three determinations, standard deviation less than 10%. Mean value of three determinations, standard deviation less than 10%; Marmoset
e
placenta, cytosolic fraction, substrate [3H]E1 + E1 [500 nM], cofactor NADH [500 μM]. Marmoset placenta, microsomal fraction, substrate
f
g
[3H]E2 + E2 [500 nM], cofactor NAD+ [1500 μM]. Standard deviation less than 15%. Calculated with LOGIT transformation at 50 nM inhibitor.
inhibition of 17β-HSD2 and affinity to the ERs. Thus, 6 shows
the highest selectivity toward 17β-HSD2 described so far (SF =
148). In the amide series introduction of fluorine in the 4-
position (11) led to a decrease in selectivity toward 17β-HSD2,
as observed for 2 in the ketone series, while introducing a
fluorine in the 2-position (10) and the 6-position (12) did not
alter selectivity. The difluoro-substituted 17 showed the best
selectivity in this series toward 17β-HSD2 and the ERs with
values similar to those of 6. To summarize, we have discovered
two highly potent 17β-HSD1 inhibitors that are the most
selective compounds described so far. Compounds 6 and 17
also show very good intracellular activity and high potency
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Journal of Medicinal Chemistry
Brief Article
toward the marmoset target enzyme (IC50 < 50 nM in the case
of 6 and IC50 < 5 nM in the case of 17). Recently published
sequence alignment studies13 showed that human and
marmoset 17β-HSD1 share high homology (80% identity and
85% similarity). Interestingly, among the seven human 17β-
HSD1 amino acid residues (S142, N152, Y155, Y218, H221,
R258, and E282) described10 as potential interaction partners
of A and B, only one amino acid variation was observed in the
Callithrix jacchus enzyme: E282N. Similar binding modes in the
marmoset enzyme as described for the two classes in human
17β-HSD1 can be postulated. In summary 6 and 17 should be
suitable candidates that after optimization could be evaluated in
a marmoset model of endometriosis.
ASSOCIATED CONTENT
* Supporting Information
Synthetic procedures, characterization, and HPLC purities of
intermediates and final compounds and all biological assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 681 302 70300. Fax: +49 681 302 70308. E-mail:
rwh@mx.uni-saarland.de. Web: www.helmholtz-hzi.de/hips.
■
Author Contributions
The manuscript was written with contributions of all authors.
All authors have given approval to the final version.
EXPERIMENTAL SECTION
■
Notes
Chemistry. General. Chemical names follow IUPAC nomencla-
ture. Starting materials were purchased and used without purification.
1H and 13C NMR spectra were measured on a Bruker AM500
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
spectrometer (500 MHz), and mass spectra (ESI) were recorded on a
MSQ electro mass spectrometer (ThermoFisher). The purity for final
compounds was ≥95% and was determined by HPLC-MS (see SI).
The following compounds were prepared according to previously
described procedures: 6-methoxy-1,3-benzothiazole (1iii),14 6-hy-
droxy-1,3-benzothiazole (8iiiii),14 [3-(tert-butyldimethylsilanyloxy)-4-
m e t h y l ] b e n z a l d e h y d e ( 8 i i i ) , 1 5 3 - m e t h o x y - 4 - ( t e r t -
butyldimethylsilanyloxy)benzaldehyde (9iiiii).16 Synthetic details and
characterization of compounds can be found in SI. Method A involves
formation of methanols, method B amide formation, method C
oxidation with SIBX, method D ether cleavage with PyHCl, and
method E ether cleavage with BBr3. 6ii, 17i, 6i, 6, and 17 are presented
as examples.
We thank Mrs. Henn and Ludwig for performing the biological
tests, Drs. Zapp and Boettcher for NMR and MS analytics, and
Professor Einspanier for supplying marmoset placenta.
ABBREVIATIONS USED
■
BC, breast cancer; 17β-HSD1, 17β-hydroxysteroid dehydrogen-
ase type 1; 17β-HSD2, 17β-hydroxysteroid dehydrogenase type
2; E1, estrone; E2, 17β-estradiol; EDD, estrogen-dependent
disease; ER, estrogen receptor; SF, selectivity factor; RBA,
relative binding affinity; CC, column chromatography
(6-Methoxy-1,3-benzothiazol-2-yl)(4-methyl-3-
methoxyphenyl)methanol (6ii). 6ii was prepared by reaction of 6-
methoxy-1,3-benzothiazole (1iii) (0.25 g, 1.5 mmol), n-BuLi (0.61
mL, 1.5 mmol), and 4-methyl-3-methoxybenzaldehyde (0.18 g, 1.2
mmol) according to method A. The product was purified by CC
(hexane/ethyl acetate, 60:40): yield, 89% (0.42 g).
ADDITIONAL NOTE
■
For the sake of clarity, IUPAC nomenclature is not strictly
followed except for the experimental part where the correct
IUPAC names are given.
2,6-Difluoro-3-methoxy-N-(6-methoxy-1,3-benzothiazol-2-
yl)benzamide (17i). 17i was prepared by reaction of 6-methoxy-1,3-
benzothiazol-2-ylamine (0.3 g, 1.79 mmol) and 2,6-difluoro-3-
methoxybenzoyl chloride (0.4 g, 1.79 mmol) according to method
B. The product was purified by CC (dichloromethane/methanol,
95:05): yield, 29% (0.18 g).
(4-Methyl-3-methoxyphenyl)(6-methoxy-1,3-benzothiazol-
2-yl)methanone (6i). 6i was prepared by reaction of 6ii (0.33 g, 1.03
mmol) and 2-iodoxybenzoic acid (0.58 g, 2.06 mmol) according to
method C. The product was purified by CC (hexane/ethyl acetate,
85:15): yield, quantitative (0.33 g).
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̈
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