Active site directed inhibition of estrone sulfatase by nonsteroidal coumarin sulfamates [2]

Full text of DOI:10.1021/jm950931z

J . Med. Chem. 1996, 39, 1349-1351
1349
Active Site Dir ected In h ibition of
Estr on e Su lfa ta se by Non ster oid a l
Cou m a r in Su lfa m a tes
†
‡
L. W. Lawrence Woo, Atul Purohit,
‡
,†
Michael J . Reed, and Barry V. L. Potter*
F igu r e 1. Structures of estrone (1), estrone sulfate (2),
EMATE (3), and steroid sulfamates 4 and 5.
Department of Medicinal Chemistry, School of Pharmacy
and Pharmacology, University of Bath, Claverton Down,
Bath BA2 7AY, U.K., and Unit of Metabolic Medicine,
Imperial College School of Medicine at St. Mary’s,
Norfolk Place, London W2 1PG, U.K.
Received December 21, 1995
Estrogens are the major mitogens involved in promot-
ing the growth of tumors in endocrine-dependent tis-
sues, such as the breast and endometrium. Although
plasma estrogen concentrations are similar in women
with or without breast cancer, breast tumor estrone (E1,
1
, Figure 1) and estradiol levels are significantly higher
than in normal breast tissue or blood. As in situ
synthesis of estrogen is thought to make an important
contribution to the high levels of estrogens in tumors,
specific inhibitors of estrogen biosynthesis should be of
potential value for the treatment of endocrine-depend-
ent tumors.
F igu r e 2. Structures of a phenylindole sulfate steroid sulfa-
tase inhibitor (6), THN (7), and THN sulfamates 8-10.
inhibition of rat liver E1-STS (99%) and DHA-STS (99%)
activities resulted when it was administered either
In the past two decades, much effort has been devoted
to the development of inhibitors of the aromatase
pathway which converts the androgen precursor an-
drostenedione to estrone. However, there is now con-
vincing evidence that the estrone sulfatase (E1-STS)
pathway, i.e. the hydrolysis of estrone sulfate (E1S, 2,
Figure 1) to E1, as opposed to the aromatase pathway
is the major source of estrogen in breast and endome-
trial tumors.1 This theory is supported by the only
modest reduction of plasma estrogen concentration in
postmenopausal women with breast cancer treated by
1
1
orally or subcutaneously. The bridging O atom of the
sulfamate moiety in EMATE is crucial for activity.
Thus, when the 3-O atom is replaced by other hetero-
atoms (Figure 1) as in estrone 3-N-sulfamate (4) and
estrone 3-S-sulfamate (5), these analogues are only
1
2
weak non-time-dependent inactivators.
Although optimal potency for inhibition of E1-STS
may have been attained in EMATE, a steroid nucleus
nevertheless possesses several disadvantages: first, as
a consequence of the mechanism of action of this agent,
there is a strong likelihood of estrone being released
,2
aromatase inhibitors, such as aminoglutethimide and
8
,12
during sulfatase inhibition,
and second, a recent
,4
4
-hydroxyandrostenedione3 and the relatively high
report demonstrated that, unexpectedly, EMATE and
its estradiol congener possess potent estrogenic activ-
plasma E1S concentration in these aromatase inhibitor-
treated patients. The long half-life of E1S in blood (10-
1
3
ity.
Moreover, other recently discovered potential
1
2 h) compared with the unconjugated estrogens (20
therapeutic applications of this remarkable compound
mean that it will be highly advantageous to develop
potent and orally active irreversible nonsteroidal coun-
terparts, which are nonestrogenic and do not possess
the capability of being metabolized to compounds with
hormonal activity. Thus, EMATE has been shown to
5
min), and high levels of steroid sulfatase activity in
liver and normal and malignant breast tissues, also lend
support to this theory.6
Estrone 3-O-sulfamate (EMATE, 3, Figure 1) is the
most potent E1-STS inhibitor developed to date (>99%
inhibition of E1-STS activity in intact MCF-7 cells at
1
4
have a memory-enhancing effect in rats. Studies in
mice have suggested an association between DHA-STS
0
.1 µM) and inhibits the enzyme in a time- and
concentration-dependent manner, indicating that it acts
activity and the regulation of part of the immune
as an active site directed inactivator.7
,8
Although
response which may also occur in humans.1
5,16
If a
EMATE was originally designed for the inhibition of E1-
STS, it also inhibits dehydroepiandrosterone sulfatase
nonsteroidal E1-STS inhibitor could also inhibit DHA-
STS, it not only would be more suitable for therapeutic
use in breast cancer but also would be more appropriate
for other nonmalignant conditions, such as the preven-
tion of autoimmune diseases, when drugs may need to
be administered from an early age.
(DHA-STS), the enzyme which has a pivotal role in
regulating the biosynthesis of the estrogenic steroid
8
,9
androstenediol.
There is now substantial evidence to
suggest that androstenediol may be of even greater
1
0
importance as a promotor of breast tumor growth.
EMATE is also active in vivo as almost complete
The first attempt to develop such inhibitors was made
with the synthesis of sulfate derivatives of 2-phenylin-
doles, of which one example (6, Figure 2) had an IC50 of
80 µM, albeit as a competitive inhibitor. Such com-
pounds are expected to be substrates for steroid sulfa-
tase, and one of these sulfates was indeed hydrolyzed
*
Address correspondence to: Professor B. V. L. Potter, School of
Pharmacy and Pharmacology, University of Bath, Claverton Down,
Bath BA2 7AY, U.K. Tel. No. 1225-826639. Fax: 1225-826114.
E-mail: B.V.L.Potter@bath.ac.uk.
†
University of Bath.
Imperial College School of Medicine at St. Mary’s.
‡
17
to the free phenol upon incubation with the enzyme.
0
022-2623/96/1839-1349$12.00/0 © 1996 American Chemical Society

1
350 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Communications to the Editor
F igu r e 3. Structures of 7-hydroxycoumarin (11), 7-(sulfooxy)-
-methylcoumarin (12), and coumarin sulfamates 13-16.
4
F igu r e 4. Route a: Sulfation of 7-hydroxy-4-methylcoumarin;
pyridine/SO -pyridine complex, NaOH in MeOH. Route b:
Sulfamoylation of 7-hydroxy-4-methylcoumarin; NaH/DMF,
NSO Cl in toluene.
3
H
2
2
F igu r e 5. Dose-dependent inhibition of estrone sulfatase in
intact MCF-7 breast cancer cells by coumarin 7-O-sulfamate
(13), 4-methylcoumarin 7-O-sulfamate (14), 3,4,8-trimethyl-
coumarin 7-O-sulfamate (15), and 4-(trifluoromethyl)coumarin
Our first series of nonsteroidal inhibitors was based
upon tetrahydronaphth-2-ol (7, THN) derivatives (8-
0, Figure 2) which were A/B ring mimics of EMATE.
In intact MCF-7 breast cancer cells, it was found that
showed E1-STS inhibition activity, although much
weaker than EMATE, and the methylated derivatives
and 10 were much weaker still.7 We have now
7
-O-sulfamate (16). Assays were performed essentially as
1
7
,8
previously described. Monolayers of intact MCF-7 cells in
2
2
3
5 cm flasks were incubated for 20 h at 37 °C with [ H]estrone
8
sulfate (2 nM) and coumarin sulfamates at 0.1-10 µM. Estrone
sulfatase activity was determined by measuring the total
3
9
amount of H-labeled estrone and estradiol formed. Sulfatase
6
activity in untreated cells was 100-200 fmol/20 h/10 cells.
established that 8 only acts as a weak time- and
concentration-dependent inhibitor (data not shown).
Hence, preincubation of placental microsomes with 8 at
Each point represents the mean ( sd of triplicate measure-
ments.
Having demonstrated that 7-(sulfooxy)-4-methylcou-
marin (12) was a substrate for E1-STS, it was reasoned
that sulfamate derivatives of coumarin were likely to
act as sulfatase inhibitors. Coumarin 7-O-sulfamate
1
0 µM for 60 min resulted in less than 10% inhibition
of E1-STS activity.
In pursuit of alternative nonsteroidal mimics of
EMATE, we chose the monohydroxylated coumarin
structure, whose ring system is ubiquitous in Nature
and in many pharmaceuticals. Our core coumarin,
(
13), 4-methylcoumarin 7-O-sulfamate (14), 3,4,8-tri-
methylcoumarin 7-O-sulfamate (15), and 4-(trifluoro-
methyl)coumarin 7-O-sulfamate (16) were then synthe-
sized and evaluated for E1-STS inhibition. Compounds
7
-hydroxycoumarin (11, Figure 3), should mimic the A
and B rings of EMATE and differs from THN at the B
ring by possessing an R,â-unsaturated lactone in place
of a saturated cyclic hydrocarbon. We anticipated that
the extended conjugation of such coumarin structures
over THN should enhance the overall activity by virtue
of lowering the relative pKa of the leaving phenol
released during enzyme inactivation. Thus, we first
synthesized 7-(sulfooxy)-4-methylcoumarin (12) to ex-
amine if coumarin sulfates are substrates for E1-STS.
1
3-16 were prepared by first treating a solution of the
appropriate coumarin in anhydrous DMF with sodium
hydride (1 equiv) at 0 °C under N2. Sulfamoyl chloride
(
ca. 1.5 equiv), stored as a standard solution in dry
1
2
purified toluene, was then added, and after the
mixture was warmed to room temperature overnight
and the reaction quenched, the crude product, after
workup for each coumarin sulfamate, was purified by
flash chromatography and recrystallization (yields, 24-
7
-Hydroxy-4-methylcoumarin in dry pyridine was treated
6
0%). All new compounds were fully characterized by
1
8
with sulfur trioxide-pyridine complex (2 equiv) under
N2 (Figure 4a). After removal of pyridine, basification,
precipitation, and crystallization of the residue from
methanol/ether (1:1), pure 12 was obtained. To examine
whether 12 could act as a substrate for E1-STS, 100 µg
of the compound was incubated for 1 h with placental
microsomes in the absence or presence of EMATE (10
µM). The unconjugated coumarin formed at the end of
the incubation was extracted with diethyl ether. After
evaporation of solvent, the residue was examined by
TLC using ethyl acetate/methanol (80:20) as eluent, in
which the coumarin sulfate 12 and 7-hydroxy-4-meth-
ylcoumarin had Rf values of 0.79 and 0.95, respectively.
Only unconjugated 7-hydroxy-4-methylcoumarin was
detected after incubation of 12 with placental mi-
crosomes. The inclusion of EMATE in the reaction
mixture completely abolished the hydrolysis of 12 by
E1-STS, indicating that the coumarin sulfate is indeed
a substrate for the sulfatase.
spectroscopic and combustion analysis. The synthesis
of 4-methylcoumarin 7-O-sulfamate (14) is shown in
Figure 4b.
These coumarin sulfamates were then tested for their
ability to inhibit E1-STS activity using intact MCF-7
breast cancer cells or placental microsomes (100 000 g
7,8,19
fraction) essentially as previously described.
The
free parent coumarins of all coumarin sulfamates
prepared were devoid of E1-STS inhibitory activity
when tested up to 10 µM. In contrast, all four coumarin
sulfamates 13-16 inhibited estrone sulfatase inhibitory
activity in a dose-dependent manner (Figure 5), and the
inhibition at 10 µM ranged from 71.5% for 16 to 93.3%
for 14. The IC50 for inhibition of E1-STS by 14, the most
effective inhibitor, measured using intact MCF-7 cells
was 380 nM. As with EMATE, 14 also inhibited E1-
STS activity in a time- and concentration-dependent
manner in a biphasic fashion (Figure 6), indicating a
similar mechanism of action (potential chemical modi

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1351
Ack n ow led gm en t. This work was supported by The
Cancer Research Campaign. B.V.L.P. is a Lister Insti-
tute Research Professor.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for 14 (2 pages). Ordering information is given on any
current masthead page.
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(
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(
control, b; 0.5 µM, 2; and 10 µM, f) for 0-30 min at 37 °C
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(
°
3
portions of the supernatants were then incubated with [ H]-
estrone sulfate (20 µM) for 1 h at 37 °C to assess remaining
sulfatase activity. Duplicate experiments were run at each
concentration, but assays for residual activity were taken at
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fication of two active site residues). At 10 µM, 14
reduced the original E1-STS activity by 95% after
preincubating the enzyme with the inhibitor for 20 min.
Also, 14 inhibited placental microsomal DHA-STS ac-
tivity by 93.6% at the same concentration (data not
shown).
1
987, 50, 269-279.
(
7) Howarth, N. M.; Purohit, A.; Reed, M. J .; Potter, B. V. L. Estrone
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In order to examine if 14 possessed estrogenic activity
and also to test its ability to inhibit E1-STS in vivo, it
was administered to rats (1 mg/kg subcutaneously in
propylene glycol for 5 days) 14 days after ovariectomy
had been performed. Administration of 14 did not result
in any significant increase in the uterine weight in these
rats, showing that 14 was devoid of any estrogenic
agonist properties, in contrast to EMATE. Thus, the
results, as (uterine weight × 100)/total body weight,
expressed as mean ( sd for control, EMATE and 14
were 0.04 ( 0.01, 0.13 ( 0.02, and 0.04 ( 0.02,
respectively. The E1-STS activity in the uteri obtained
3
4, 11508-11514.
(
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(
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1
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1
1
from these animals, determined as described, was
inhibited by 89.4% compared with the activity in
untreated animals. Preliminary data obtained in a
(
1
1
similar fashion to those described previously also
demonstrate potent oral activity in rats for 14, similar
to that observed for EMATE.
(
In summary, nonsteroidal inhibitors have been de-
veloped that, while potent in vivo as sulfatase inhibitors,
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coumarin sulfamates tested, 4-methylcoumarin 7-O-
sulfamate (14), together with coumarin 7-O-sulfamate
(
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(
(
(13), appear most active in vitro, with 14 being shown
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like EMATE. These coumarin sulfamates therefore
represent key lead compounds for the optimization of
nonsteroidal sulfatase inhibition. The further develop-
ment of such inhibitors should enable the therapeutic
use of sulfatase inhibitors to be broadened, not only for
endocrine-dependent cancers, but also for other condi-
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a potential therapeutic agent in
J M950931Z