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Journal of Medicinal Chemistry
Article
worldwide increase in postmenopausal osteoporosis-related
bone fractures, and the growing elderly population. 17β-HSD2
in bone tissue converts the biologically active steroid
hormones, estradiol (E2) and T, into the much less active
estrone and androstenedione. The blockade of 17β-HSD2 in
bone thus increases intracellular E2 and T, and, through
estrogen and androgen receptor stimulation, inhibits bone
resorption by osteoclasts and stimulates bone formation by
osteoblasts,9−11 respectively. The expression and activity of
17β-HSD2 in human bone tissue are superior over that of 17β-
HSD1, 17β-HSD3, and 17β-HSD4,12−14 the enzymes that
catalyze the synthesis and degradation of E2 and T. A previous
study in ovariectomized cynomolgus monkeys showed that a
17β-HSD2 inhibitor increases the so-called ultimate bone
strength, that is, the maximum stress that an intact bone
specimen can sustain.15 Here, we developed a nanomolar
potent, metabolically sufficiently stable, nontoxic 17β-HSD2
inhibitor and tested this compound for activity in an
established mouse bone fracture healing model16 during a
proof-of-principle study of 28 days. Mouse bone cells express
active 17β-HSD2 as demonstrated by 17β-HSD2 immunohis-
tochemistry, using a validated antimouse 17β-HSD2 antibody
(Figure S1, Supporting Information), and shown by enzymatic
determination,17 together with androgen and estrogen
receptors (ERs).12−14
human and murine 17β-HSD2 (h + m17β-HSD2), resulting in
a small library of 16 compounds. As a starting point, the
nonsubstituted scaffold structure (compound A) was chosen.
Previous investigations revealed that the low metabolic
stability of the BSHs was primarily due to phase II
biotransformation of the phenolic OH group, which is essential
for activity.19 Therefore, stage 1 of the design strategy, aiming
at the improvement of metabolic stability, consisted in the
introduction of small substituents on the hydroxyphenyl
moiety (ring A) of compound A. These groups could protect
the OH functionality from biotransformation by the electron
withdrawal effect and/or steric hindrance (Chart 1, com-
pounds 1−8).
Compound 8 showed an enhanced metabolic stability and
was chosen as a starting point for the optimization of the
substitution pattern of the D-ring (stage 2 of the design
strategy). Only such groups were selected to be introduced
that were likely to maintain potency toward both human and
mouse 17β-HSD2.18 We aimed for a slight (three- to
fourfold) selectivity over the h17β-HSD1 enzyme (Chart 2, 9−
16). On the one hand, a highly selective 17β-HSD2 inhibitor
would induce an undesirable increase in intracellular E2 in
tissues that express similar levels of 17β-HSD2 and 17β-HSD1
and prone to E2-dependent proliferation (i.e., breast20,21 and
endometrium22), whereas on the other hand, a nonselective
17β-HSD2/1 inhibitor would likely affect the role of 17β-
HSD1 in regulating the endometrium cyclicity in women of
childbearing age.22
RESULTS AND DISCUSSION
■
As bicyclic substituted hydroxyphenylmethanones (BSHs)
bearing a sulfonamide moiety were originally designed as
inhibitors of human 17β-HSD1, most members show
selectivity toward 17β-HSD1 over 17β-HSD2.18 In addition,
BSHs display low metabolic stability, precluding their use in an
in vivo proof-of-principle study. Thus, a rational two-stage drug
design strategy focusing on rings A and D (Charts 1 and 2, see
The starting point for the syntheses of compounds 1−16
was a Friedel−Crafts reaction of 2-bromothiophene with the
appropriate benzoyl chloride, which in case of the chlorinated
3b and 4b had to be prepared from the corresponding benzoic
acids. The obtained intermediates 1b−8b were subjected to
Suzuki cross-coupling reactions with 3-aminophenylboronic
acid to afford anilines 1a−8a. The latter were reacted with the
sulfonamides using the appropriately substituted sulfonic acid
chloride, giving direct access to compound 1. Ether cleavage
using BBr3 in dichloromethane yielded the final compounds
2−16 (Scheme 1).
Chart 1. Lead Compound A and Designed Compounds 1−8
The introduction of an electron-donating methyl group on
ring A (Table 1, 1) led to a twofold decrease in inhibitory
potency toward h17β-HSD2 compared to lead A. In contrast,
the presence of an electron-withdrawing fluorine or chlorine
atom in the same position strongly increased the activity; see 1
versus 2 and 3.
The beneficial effect of electron-withdrawing substituents on
the inhibitory activity was also apparent for 4−8, in agreement
with the observations made recently for the BSHs lacking the
sulfonamide moiety.22 Metabolic stability was determined for
3, 7, and 8 as these compounds displayed selectivity over 17β-
HSD1 and nanomolar potency toward m17β-HSD2. The
trifluoro substitution pattern of 8 resulted in an improved
metabolic stability and was therefore maintained in the
subsequent optimization of ring D. All eight compounds of
this second series (9−16) showed a strong inhibition of human
and mouse 17β-HSD2 as well as a low activity toward m17β-
HSD1. Compounds 14 and 15 were especially interesting as
they displayed moderate h17β-HSD2 selectivity, which was
aimed at, as well as improved metabolic stability.
Chart 2. Lead Compound 8 and Designed Compounds 9−
16
Because of their favorable potency, selectivity, and metabolic
stability properties, 14 and 15 were selected for testing for
potential cytotoxicity. Both 14 and 15 were not toxic in the
MTT assay with HEK293 cells (i.e., <20% reduction in cell
also Supporting Information for details) was applied that, on
the one hand, aimed at improving the metabolic stability and,
on the other, at enhancing the potency and selectivity for
B
DOI: 10.1021/acs.jmedchem.8b01493
J. Med. Chem. XXXX, XXX, XXX−XXX