Steroidal and nonsteroidal sulfamates as potent inhibitors of steroid sulfatase

Article abstract of DOI:10.1021/jm970527v

Synthetic routes to potent steroidal and nonsteroidal sulfamate-based active site-directed inhibitors of the enzyme steroid sulfatase, a topical target in the treatment of postmenopausal women with hormone-dependent breast cancer, are described. Novel compounds were examined for estrone sulfatase (E1-STS) inhibition in intact MCF-7 breast cancer cells and placental microsomes. Reaction of the sodium salt of estrone with sulfamoyl chloride gave estrone 3-O-sulfamate (EMATE, 2) which inhibits E1-STS activity potently (>99% at 0.1 mM in intact MCF-7 cells, IC₅₀ = 65 pM) in a time- and concentration-dependent manner, suggesting that EMATE is an active site- directed inhibitor. EMATE is also active in vivo orally. 5,6,7,8- Tetrahydronaphthalene 2-O-sulfamate(7) and its N-methylated derivatives (8 and 9) were synthesized, and 7 inhibits the E1-STS activity in intact MCF-7 cells by 79% at 10 μM. 4-Methylcoumarin 7-O-sulfamate (COUMATE) and its derivatives (14, 16, and 18) wee prepared to extend this series of nonsteroidal inhibitors, and COUMATE educes the E1-STS activity in placental microsomes by >90% at 10 μM. Although the orally active COUMATE is less potent than EMATE as an active site-directed inhibitor, it has the important advantage of being nonestrogenic. Analogues (20, 22, 24, 26, 27, 31, 33, 39, and 44) of COUMATE were synthesized to study its structure-activity relationships, and sulfamates of tetralones (46 and 48) and indanones (49, 51, and 53) wee also prepared. While most of these compounds were found to inhibit E1-STS activity less effectively than COUMATE, one analogue, 3,4- dimethylcoumarin 3-O-sulfamate (24), was found to be some 12-fold more potent than COUMATE as an E1-STS inhibitor in intact MCF-7 cells (IC₅₀ = 30 nM for 24, cf. 380 mM for COUMATE). Hence, highly potent sulfamate-based inhibitors of steroid sulfatase, such as EMATE, COUMATE, and 24, possess therapeutic potential and will allow the importance of estrogen formation in breast tumors via the E1-STS pathway to be assessed. A pharmacophore for active site-directed sulfatase inhibition is proposed.

Full text of DOI:10.1021/jm970527v

1068
J . Med. Chem. 1998, 41, 1068-1083
Ster oid a l a n d Non ster oid a l Su lfa m a tes a s P oten t In h ibitor s of Ster oid Su lfa ta se
L. W. Lawrence Woo,† Nicola M. Howarth,†,‡ Atul Purohit,§ Hatem A. M. Hejaz,† Michael J . Reed,§ and
Barry V. L. Potter*,†
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, St. Mary’s Hospital,
Norfolk Place, London W2 1PG, U.K.
Received August 8, 1997
Synthetic routes to potent steroidal and nonsteroidal sulfamate-based active site-directed
inhibitors of the enzyme steroid sulfatase, a topical target in the treatment of postmenopausal
women with hormone-dependent breast cancer, are described. Novel compounds were examined
for estrone sulfatase (E1-STS) inhibition in intact MCF-7 breast cancer cells and placental
microsomes. Reaction of the sodium salt of estrone with sulfamoyl chloride gave estrone 3-O-
sulfamate (EMATE, 2) which inhibits E1-STS activity potently (>99% at 0.1 µM in intact MCF-7
cells, IC50 ) 65 pM) in a time- and concentration-dependent manner, suggesting that EMATE
is an active site-directed inhibitor. EMATE is also active in vivo orally. 5,6,7,8-Tetrahy-
dronaphthalene 2-O-sulfamate (7) and its N-methylated derivatives (8 and 9) were synthesized,
and 7 inhibits the E1-STS activity in intact MCF-7 cells by 79% at 10 µM. 4-Methylcoumarin
7-O-sulfamate (COUMATE) and its derivatives (14, 16, and 18) were prepared to extend this
series of nonsteroidal inhibitors, and COUMATE reduces the E1-STS activity in placental
microsomes by >90% at 10 µM. Although the orally active COUMATE is less potent than
EMATE as an active site-directed inhibitor, it has the important advantage of being
nonestrogenic. Analogues (20, 22, 24, 26, 27, 31, 33, 39, and 44) of COUMATE were synthesized
to study its structure-activity relationships, and sulfamates of tetralones (46 and 48) and
indanones (49, 51, and 53) were also prepared. While most of these compounds were found to
inhibit E1-STS activity less effectively than COUMATE, one analogue, 3,4-dimethylcoumarin
3-O-sulfamate (24), was found to be some 12-fold more potent than COUMATE as an E1-STS
inhibitor in intact MCF-7 cells (IC50 ) 30 nM for 24, cf. 380 nM for COUMATE). Hence, highly
potent sulfamate-based inhibitors of steroid sulfatase, such as EMATE, COUMATE, and 24,
possess therapeutic potential and will allow the importance of estrogen formation in breast
tumors via the E1-STS pathway to be assessed. A pharmacophore for active site-directed
sulfatase inhibition is proposed.
In tr od u ction
is the major contribution to the estrogen content in
breast tumors.
Breast cancer causes more death in women than any
other neoplasm. This disease is particularly prevalent
in Western countries, and a recent survey shows that
the U.K. heads the incidence rate within Europe.1 In
women who develop breast cancer, about one-third to
one-half will eventually die of it. Although much
emphasis recently has been placed on the prevention
and an earlier detection of the disease by mammography
screening, there is a demand for novel therapeutic
interventions in the management of breast cancer.
The close association of estrogens with the promotion
of the growth and development of breast cancers has
long been recognized.2-4 In postmenopausal women, in
whom breast cancers most frequently occur, approxi-
mately one-third of breast tumors are hormone-depend-
ent.5 There is now substantial evidence6-9 to imply that
in situ formation of estrogen from estrogen precursors
In patients with hormone-dependent breast cancer,
the inhibition of the aromatization of androstenedione
to estrone (E1, Figure 1) in the last step of the biosyn-
thesis of estrogens has been a target for ablating
estrogens. Many potent, well-tolerated and highly
selective aromatase inhibitors are now available for
clinical use. However, the response rates of patients
with estrogen receptor positive (ER+) tumors to anas-
trozole in a recent randomized clinical trial were found
to be quite low.10,11 These findings have thus raised one
particular question as to what other mechanisms may
be responsible for the lack of improved response rates
despite the use of highly potent aromatase inhibitor
such as anastrozole.
The most likely explanation for the relative lack of
clinical efficacy of aromatase inhibitors is that the
estrone sulfatase (E1-STS) pathway (Figure 1) [i.e. the
hydrolysis of E1S to estrone], as opposed to the aro-
matase pathway, is the major route of estrogen forma-
tion in breast tumors.12,13 Evidence to support this
hypothesis includes (i) a million-fold higher steroid
sulfatase activity than aromatase activity in liver and
normal and malignant breast tissues14 and (ii) the origin
* Address correspondence to: Professor B. V. L. Potter, School of
Pharmacy and Pharmacology, University of Bath, Claverton Down,
Bath, BA2 7AY, U.K. Tel: 01225-826639. Fax: 01225-826114. E-
mail: B.V.L.Potter@bath.ac.uk.
† University of Bath.
‡ Present address: Chemistry Department, Heriot-Watt University,
Edinburgh, EH1 1HX, Scotland EH14 4AS.
§ Imperial College School of Medicine.
S0022-2623(97)00527-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/28/1998

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1069
F igu r e 1. The origin of estrogenic steroids in postmenopausal women: AR, aromatase; ST, sulfotransferase; STS, sulfatase;
17â-HSD, 17â-hydroxysteroid dehydrogenase; ER, estrogen receptor.
series of sulfamate derivatives of estrone (2 - 4, Figure
2), of which estrone 3-O-sulfamate (EMATE, 2) is the
most potent active site-directed steroidal inhibitor
synthesized to date.30,31
Unexpectedly, a recent report has shown that EMATE
and its estradiol congener possess potent estrogenic
activity.32 These findings, together with the likelihood
of E1 being released during sulfatase inhibition, as
F igu r e 2. Steroidal inhibitors of steroid sulfatase: (a) MeP-
suggested by its proposed mechanisms of action (Figure
3),33,34 have rendered EMATE unsuitable for use in the
treatment of hormone-dependent tumors. Recently,
EMATE has also been shown to have memory-enhanc-
ing effects in rats.35 In addition, studies in mice have
suggested a role of DHA-STS activity in regulation of
part of the immune response,36,37 and such a relation-
ship may also be found in humans. Since it is likely
that some of these newly discovered potential uses of
EMATE in other therapeutic areas may require drugs
to be administered from an early age, it is thus
anticipated that a potent orally active irreversible
nonsteroidal E1-STS inhibitor, which is nonestrogenic
and unlikely to be metabolized to compounds with
hormonal activity, will have much wider clinical ap-
plications than their steroidal counterparts.
Various structurally diversified nonsteroidal E1-STS
inhibitors have been reported, such as a sulfate deriva-
tive of 2-phenylindoles (e.g. 5, Figure 4)38 and (p-O-
sulfamoyl)-N-alkanoyltyramines.39,40 Our first non-
steroidal E1-STS inhibitors were 5,6,7,8-tetrahydro-
naphthalene 2-O-sulfamate (7, Figure 4) and its N-
alkylated derivatives (8 and 9). The best candidate in
this series was 7, but it is a much weaker inhibitor than
EMATE.30 We then reasoned that sulfamates of alter-
native two-ring systems such as monohydroxylated
coumarins were also likely to act as E1-STS inhibitors.
After establishing that 7-sulfooxy-4-methylcoumarin
(11, Figure 5) was a substrate for E1-STS as expected,
we synthesized and examined coumarin sulfamates (12,
14, 16, and 18), of which 4-methylcoumarin 7-O-sulfa-
mate (COUMATE, 12) was found to be the most effec-
tive inhibitor.41
SCl2/pyridine/24 h, HOCH2CH2CN/24 h, NH4OH/65 °C/5 h; (b)
NaH/DMF, H2NSO2Cl; (c) NaH/DMF, MeNHSO2Cl; (d) NaH/
DMF, Me2NSO2Cl.
of E1 from E1S in breast cancer tissue is about 10 times
more than that from androstenedione.12
In addition to the E1-STS pathway, there is a growing
awareness that another potent estrogen, androstenediol
(Adiol, Figure 1), may be of even greater importance in
the support of growth and development of hormone-
dependent breast tumors.15-17 Nearly 90% of Adiol
originates from dehydroepiandrosterone sulfate (DHA-
S) once it has been hydrolyzed to DHA by DHA-sulfatase
(DHA-STS)18 (Figure 1). Although it is still uncon-
firmed if E1-STS and DHA-STS are isoenzymes or not,
transient transfection of a placental steroid sulfatase
into COS-1 cells showed that the expressed protein was
able to hydrolyze both E1S and DHA-S.19 Hence,
steroid sulfatase inhibitors, when used alone or in
conjunction with an aromatase inhibitor, may enhance
the response of hormone-dependent tumors, such as of
the breast, to this type of endocrine therapy by reducing
not only the formation of E1 from E1S but also the
synthesis of Adiol from DHA-S.
The emergence of the biological significance of the E1-
STS pathway has prompted ourselves and several other
groups20-26 to examine a variety of compounds for the
inhibition of E1-STS activity. We found that E1S
analogues possessing sulfate surrogates such as 3-O-
phosphonates and 3-O-thiophosphonates [e.g. estrone
3-O-methylthiophosphonate (E1-3-MTP, 1, Figure 2)]27,28
and 3-O-sulfonates29 were able to inhibit the hydrolysis
of E1-STS efficiently in intact MCF-7 breast cancer cells
and also in placental and breast tumor preparations.
These findings subsequently led to our synthesis of a
To study the structure-activity relationships for
coumarin sulfamates, we have prepared and examined

1070 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Woo et al.
F igu r e 3. Proposed mechanisms of action of EMATE in the inhibition of estrone sulfatase (E1-STS): (A) via an aminosulfene
intermediate and (B) via a nucleophilic attack by an amino acid residue in the active site. (i) Attacks by a nucleophilic amino acid
residue in the active site-selective or random sulfamoylation. (ii) No hydrolysis of the sulfamoylated E1-STS by water to regenerate
the active form of the enzyme. X, Y and Z: amino acid residue. (- - -) Hydrogen bonding.
their ability to inhibit E1-STS was not explored. In most
of our earlier works (e.g., for EMATE, 2), the sulfamoy-
lation of phenolic compounds was carried out directly
with crude residue/crystalline sulfamoyl chloride, pre-
pared according to the method of Appel and Berger.50
However, the continual handling of sulfamoyl chloride
in the solid state proved to be cumbersome since this
reagent is extremely hygroscopic and decomposes readily
even on storage at low temperature. Nevertheless, it
was found subsequently that these drawbacks could
largely be surmounted if the freshly prepared crude
sulfamoyl chloride was stored instead as a solution in
anhydrous and sulfur impurities-free toluene under
nitrogen.33
To verify that sulfamoyl chloride is stable when stored
as a solution in toluene under the conditions described
F igu r e 4. Structures of nonsteroidal inhibitors of steroid
sulfatase (5, 7-9) and 5,6,7,8-tetrahydronaphth-2-ol (6): (a)
NaH/DMF, H2NSO2Cl; (b) NaH/DMF, MeNHSO2Cl; (c) NaH/
DMF, Me2NSO2Cl.
above, its reactivity was examined by monitoring the
yield of 2-nitrophenol O-sulfamate (54, Figure 9) in the
sulfamoylation of 2-nitrophenol. 2-Nitrophenol was
initially chosen for this study because of its obvious
advantages of being structurally simple and also chro-
mophoric. However, the yield of 54 was consistently
poor. This was subsequently attributed to the instabil-
ity of 54 in the reaction mixture. Nonetheless, some
interesting results emerged during the course of this
study as outlined below.
When a solution of 54 in ethyl acetate was shaken
with 5% sodium bicarbonate solution, a bright orange
aqueous portion resulted upon separation of layers,
suggesting that the sulfamate group of 54 is cleaved in
the presence of the base and sodium 2-nitrophenolate
is generated. The pKas of the N-protons in 54 are
expected to be lower than those of EMATE by virtue of
the powerful electron-withdrawing 2-nitro group, and
although the mechanism has not yet been fully estab-
lished, it is likely that decomposition of 54 involves the
removal of the N-proton(s) of the sulfamate group by
the base. The resulting anion of 54 then collapses,
generating the highly chromophoric 2-nitrophenolate as
a consequence.
several analogues of COUMATE and its congener, 14,
in which the sulfamate group is relocated, the coumarin
ring is substituted, and the conjugation of the coumarin
ring system is disrupted. The mono- and bis-sulfamates
of a catechol-like coumarin are also examined. In
addition, we have also explored the potential of other
two-ring sulfamates such as those of tetralones and
indanones as effective inhibitors of E1-STS.
EMATE, COUMATE, and compounds 3, 4, 7-9, 11,
14, 16, and 18 have been reported previously in com-
munication form in this journal.30,41 We now describe
full details of their synthesis and review their biological
activities in conjunction with an SAR study on the
COUMATE class of compounds and related entities.
Resu lts a n d Discu ssion
The sulfamoyl group has been widely utilized as an
activity-modifying substituent in several different classes
of drugs. For example, N-substituted steroidal sulfa-
mates have been synthesized,42-46 including sulfamates
of estradiol,45,47-49 to block metabolic conjugation, but

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1071
removed (presumably by the presence of an excess
sodium hydride) and the resulting anion of 54 attacked
a molecule of DMF at the carbonyl carbon followed by
dehydration of the product upon acidification during the
aqueous workup (Figure 9). In our usual small-scale
synthesis of EMATE, such an adduct of EMATE and
DMF, which is less chromophoric than 55, was not
easily detectable on TLC. However, we did isolate a
fraction in our subsequent large-scale synthesis of
1
EMATE whose H NMR suggested the presence of the
adduct (data not shown).
The dose-response curve for the inhibition of E1-STS
in intact MCF-7 breast cancer cells by EMATE is shown
in Figure 10A. EMATE inhibits E1-STS activity by
>99% at 10, 1, and 0.1 µM with an IC50 of 65 pM.
Moreover, the IC50 for inhibition of E1-STS activity by
EMATE, measured in a placental microsome prepara-
tion (100 000 g pellet)12 using a saturating substrate
concentration (20 µM), was found to be 80 nM.30 This
demonstrates the dramatic increase in potency of
EMATE, compared to E1-3-MTP, since under the same
conditions in placental microsomes, the latter had an
IC50 of 43 µM.27,28 Unlike E1-3-MTP, which is only a
reversible inhibitor,27 EMATE inhibits E1-STS irrevers-
ibly in a time- and concentration-dependent manner in
placental microsome preparation (Figure 11A),51 indi-
cating that it acts as an active site-directed inhibitor.
Subsequent studies have also shown that EMATE
inhibits DHA-STS,19,51 the enzyme which regulates the
biosynthesis of the estrogenic androstenediol. More-
over, EMATE is active in vivo, inhibiting rat liver E1-
STS and DHA-STS activities almost completely when
given either orally or subcutaneously.52
The structure-activity relationships for EMATE have
not been extensively studied. However, from the poorer
inhibitory activities of many other E1S surrogates
available to date (e.g., E1-3-MTP), it is evident that the
sulfamate group of EMATE is a crucial motif for its
potency as an active site-directed E1-STS inhibitor. It
has also been shown that the bridging oxygen atom
between the steroid nucleus and the sulfamoyl group
(H2NSO2O-) is indispensable for the inhibitory activity
of EMATE. Hence, analogues of EMATE, where the
bridging atom is either a sulfur or nitrogen atom, are
only weak non-time-dependent inactivators.33
F igu r e 5. (A) Synthesis of coumarin sulfamates [12 (COU-
MATE), 14, 16, 18, 20, 22, 24, 26, and 27] from their
corresponding starting alcohols and 7-sulfooxy-4-methylcou-
marin (11). 4-Methyl-8-nitrocoumarin 7-O-sulfamate (29) could
not be prepared from 28 by method a: (a) NaH/DMF, H2NSO2-
Cl; (b) pyridine-SO3 complex/pyridine, NaOH in MeOH. (B)
Pechmann synthesis of coumarin 23.
In addition, Figure 10A clearly shows that a signifi-
cant reduction in the inhibitory activity of EMATE
resulted when the N atom of the sulfamate group is
increasingly methylated. Hence, estrone 3-O-(N-meth-
yl)sulfamate (3, Figure 2) and estrone 3-O-(N,N-dim-
ethyl)sulfamate (4) inhibit E1-STS activity in intact
MCF-7 breast cancer cells by only 80 and 50%, respec-
tively, at 0.1 µM, in contrast to the almost complete
inhibition by EMATE at the same concentration. More-
over, both 3 and 4 were found not to be irreversible
inhibitors since E1-STS activity recovered when reas-
saying washed intact MCF-7 cells pretreated with these
analogues.30 It has yet to be established, however, if
the reduction in potency and the apparent change in
the nature of inhibition observed for 3 and 4 are due to
an ineffectual operation of the proposed mechanisms of
action depicted in Figure 3. Recently, we have further
explored the effects of modifying the sulfamate group
of EMATE on inhibition of E1-STS activity by acylating
F igu r e 6. Hydrogenation of coumarins 10 and 13 to give the
corresponding lactonic derivatives 30 and 32 which were
sulfamoylated to 31 and 33, respectively: (a) Pd-C (10%), H2,
75 psi, 3 days; (b) Pd-C (10%), H2, 40 psi, 24 h; (c) NaH/DMF,
H2NSO2Cl.
Although 54 was the major product obtained from the
above sulfamoylation reaction, a chromophoric fraction
of higher polarity (i.e., lower Rf value on silica TLC
plate) was also afforded. This fraction was isolated and
subsequently identified as azomethine 55 (Figure 9),
which is an adduct of 54 and dimethylformamide
(DMF). A retrosynthetic analysis suggests that 55 could
have been formed when one of the N-protons of 54 was

1072 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Woo et al.
F igu r e 7. Synthesis of sulfamates 39 and 44: (a) imidazole/DMF, TBDMSCl; (b) LiAlH4/ether/-78 °C; (c) Et3N/CH2Cl2, MeSO2-
Cl; (d) (Bu)4N+F-; (e) NaH/DMF, H2NSO2Cl. TBDMS: tBu-C(CH3)2Si.
F igu r e 8. Synthesis of tetralone sulfamates (46 and 48) and
indanone sulfamates (49, 51, and 53): (a) AlCl3/toluene/reflux;
(b) NaH/DMF, H2NSO2Cl.
F igu r e 10. Dose-response curves for inhibition of estrone
sulfatase activity in intact MCF-7 breast cancer cells by (A)
estrone 3-O-sulfamate (EMATE, 2, 9), estrone 3-O-(N-methyl)-
sulfamate (3, 2), and estrone 3-O-(N,N-dimethyl)sulfamate (4,
b); and (B) 4-methylcoumarin 7-O-sulfamate (COUMATE, 12,
O) and 3,4-dimethylcoumarin 7-O-sulfamate (24, 2). Assays
were performed essentially as described previously.29,32 Mono-
layers of intact MCF-7 cells in 25 cm3 flasks were incubated
for 20 h at 37 °C with [3H]estrone sulfate (2 nM) and synthetic
analogues at various concentrations. Estrone sulfatase activity
was determined by measuring the total amount of 3H-labeled
estrone and estradiol formed. Sulfatase activity in untreated
cells was 100-120 fmol/20 h/106 cells. Each point represents
F igu r e 9. Synthesis of sulfamate 54 and azomethine 55 from
2-nitrophenol and the proposed mechanism for the formation
the mean ( sd of triplicate measurements. IC50s of EMATE,
COUMATE, and 24 are 65 pM, 380 nM, and 30 nM, respec-
of 55: (a) NaH/DMF, H2NSO2Cl.
tively.
the N atom and replacing its protons entirely with
larger alkyl groups.53 Hence, the N-acetylated deriva-
tive of EMATE inhibits the E1-STS activity irreversibly,
but less potently than EMATE while the N-benzoylated,
3-O-(piperidino)sulfamate and N,N-dibenzyl derivatives
of EMATE are only weak reversible inhibitors.
In any form of endocrine therapy, it is often desirable
to treat patients with nonsteroidal agents since the

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1073
inhibitor (data not shown). Hence, preincubation of
placental microsomes with 7 at 10 µM for 60 min
resulted in less than 10% inhibition of E1-STS activity.
With the limited success of achieving inhibition of E1-
STS with 7, we chose the monohydroxylated coumarin
structure as an alternative template whose ring system
is ubiquitous in Nature and commonly incorporated in
many pharmaceuticals. The core coumarin, 7-hydroxy-
coumarin (13, Figure 5), differs structurally from THN
by possessing a cyclic R,â-unsaturated lactone in place
of a saturated cyclic hydrocarbon. It is important,
however, to first demonstrate that coumarin sulfates
themselves are substrates for E1-STS, and hence we
synthesized 7-(sulfooxy)-4-methylcoumarin (11, Figure
5) to examine if this sulfate is hydrolyzed by the enzyme.
When 11 was incubated with placental microsomes in
the absence of EMATE, only the free coumarin 10 was
detected. However, inclusion of EMATE in the reaction
mixture completely abolished the hydrolysis of 11 by
E1-STS. These results therefore indicate that the
coumarin sulfate 11 is a substrate for this enzyme.
It was then reasoned that sulfamates of coumarin
derivatives are likely to act as steroid sulfatase inhibi-
tors. 4-Methylcoumarin 7-O-sulfamate (COUMATE, 12,
Figure 5), coumarin 7-O-sulfamate (14), 4-(trifluorom-
ethyl)coumarin 7-O-sulfamate (16) and 3,4,8-trimeth-
ylcoumarin 7-O-sulfamate (18) were hence synthesized
and evaluated for E1-STS inhibition using intact MCF-7
breast cancer cells or placental microsomes as previ-
ously described.27,30,51 The two enzyme systems adopted
for the in vitro screening of potential inhibitors are com-
plementary. The intact MCF-7 breast cancer cell sys-
tem essentially shows the ability of a potential inhibitor
to inhibit E1-STS in a live cell. However, to establish
that a potential inhibitor does compete strongly with
the natural substrate for the enzyme active site, we also
screen a given compound for inhibition of E1-STS in
placental microsomes at concentrations between 10 and
100 µM in the presence of a much higher concentration
of E1S (20 µM) [cf. in the intact MCF-7 cell system, the
E1S present is only at the physiological concentration
(2 nM) whereas the concentrations of the synthetic
agent used are in the range 0.1-10 µM]. Our experi-
ence has demonstrated that a prospective E1-STS inhib-
itor warranting further development will in general
show good inhibitory activities in both enzyme systems.
F igu r e 11. Time- and concentration-dependent inactivation
of estrone sulfatase by (A) estrone 3-O-sulfamate (EMATE, 2)51
and (B) 4-methylcoumarin-7-O-sulfamate (COUMATE, 12).
Placental microsomes (200 µg of protein) were preincubated
with 2 at various concentrations for 0-60 min and 12 at 0.5
and 10 µM for 0-30 min at 37 °C followed by incubation with
dextran-charcoal for 10 min at 4 °C. Dextran-charcoal was
sedimented by centrifugation, and portions of the supernatants
were then incubated with [3H]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 different times in each experi-
ment.
The free parent coumarins (10, 13, 15, and 17, Figure
5) were all found to be devoid of E1-STS inhibitory
activity in intact MCF-7 breast cancer cells when tested
at concentrations of up to 10 µM. In contrast, the
corresponding coumarin sulfamates (12, 14, 16, and 18,
Figure 5) inhibit E1-STS activity in the same prepara-
tion in a dose-dependent manner (Table 1). The inhibi-
tion at 10 µM ranges from 71.5% for 16 to 93.1% for
COUMATE. However, when screened in placental
microsomes, only COUMATE achieves >90% inhibition
at all three of the concentrations tested whereas the
inhibition of E1-STS by 14 and 16 is in the region of
78% at 10 µM (Table 1). Although sulfamate 18 shows
inhibitory activity comparable to that of 16 in intact
MCF-7 cells, it inhibits the enzyme very poorly in
placental microsomes with an inhibition of less than
20% at all three concentrations. The reason for these
differences in inhibitory activities of 18 is still unclear.
steroidal counterparts may themselves, or through their
metabolites, exert unwanted endocrinological effects.
Indeed, despite its high potency as an E1-STS inhibitor,
the use of EMATE in treating hormone-dependent
breast cancers is restricted since it was found to be five
times more estrogenic than ethinylestradiol orally in the
rat.32 Our first series of nonsteroidal inhibitors was
based upon tetrahydronaphth-2-ol (THN, 6, Figure 4)
derivatives 7 - 9, which are presumably A/B ring
mimics of EMATE. The best candidate in this series
was 7, albeit it was much less potent than EMATE.30
Its N-methylated derivatives, 8 and 9, are even weaker
inhibitors,30 and thus it again appears that, like their
steroidal counterparts 3 and 4 (Figure 2), the extent of
inhibition decreases with increasing methylation of the
sulfamate group. We have also established that 7 acts
only as a weak time- and concentration-dependent

1074 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Woo et al.
Ta ble 1. Inhibition of Estrone Sulfatase Activity in Intact
MCF-7 Breast Cancer Cells by Sulfamates 7, 12, 14, 16, 18, 20,
22, 24, 26, 27, 31, 33, 39, 44, 46, 48, 49, 51, and 53 at Various
Concentrationsa
activity exhibited by the coumarin sulfamates may be
due, in part, to a facilitated sulfamoylation of the
enzyme. It is likely that the breaking of the S-O bond
in the sulfamate group of 12, 14, 16, and 18 during
sulfamoylation of E1-STS is assisted by the extended
conjugation present in the coumarin motif which im-
proves the leaving group ability of coumarins (10, 13,
15, and 17, Figure 5).
% inhibition ( SD of E1-STS activity in
intact MCF-7 cells at various concentrations
compd
0.1 µM
1 µM
10 µM
7
12*
14
16
18
20
22
24†
26
27
31
33
39
44
46
48
49
51
53
51.7 ( 2.5
42.9 ( 0.7
23.9 ( 4.1
9.9 ( 3.9
9.3 ( 4.2
56.7 ( 0.4
85.6 ( 2.7
66.0 ( 7.2
28.3 ( 5.3
46.4 ( 5.0
17.4 ( 3.4
34.1 ( 1.8
94.9 ( 0.3
78.8 ( 1.9
93.1 ( 2.2
90.4 ( 2.3
71.5 ( 3.6
75.1 ( 5.5
39.1 ( 5.3
72.1 ( 1.3
98 ( 0.4
In light of the encouraging inhibitory activities of
COUMATE, it is important to have a better understand-
ing of its structure-activity relationships since this
inhibitor represents a good lead template for the design
of more potent nonsteroidal E1-STS inhibitors. Hence,
we have synthesized several analogues of COUMATE
and its congener, 14, in which (i) the sulfamate group
has been relocated (20, Figure 5), ii) the aromatic ring
has been substituted (22, Figure 5), (iii) both the 3- and
4-positions have been substituted with methyl groups
(24, Figure 5), and (iv) the coumarin ring system has
been disrupted (31 and 33, Figure 6; 39 and 44, Figure
7). The mono- (26) and bis-sulfamates (27) (Figure 5)
of the catechol-like coumarin, 25, have also been pre-
pared.
<10
33.8 ( 5.3
85.4 ( 1.1
<10
<10
<10
<10
<10
<10
<10
30.2 ( 3.9
68.2 ( 2.4
60.6 ( 2.4
11.7 ( 8.2
47.8 ( 1.7
15.5 ( 9.9
22.6 ( 5.8
91 ( 0.6
31.2 ( 2.6
32.3 ( 7.4
28.5 ( 5.2
99.3 ( 1.1
76.2 ( 10.2
78.1 ( 2.9
93.5 ( 0.8
74.1 ( 2.8
71.3 ( 1
12.0 ( 8.8
11.4 ( 3.1
49.0 ( 4.8
<10
10.4 ( 3
<10
13.5 ( 3
33 ( 2
99 ( 2
a
For details of the assay, refer to the legend of Figure 10.
*<10% at 1 and 10 nM. †<10% at 1 nM and 22.7% ( 4.7 at 10
nM.
4-Methylcoumarin 6-O-sulfamate (20, Figure 5) and
6-methoxycoumarin 7-O-sulfamate (22) were prepared
by direct sulfamoylation of their corresponding starting
coumarins (19 and 21). 3,4-Dimethyl-7-hydroxycou-
marin (23, Figure 5), required for the synthesis of
sulfamate 24, was prepared in 25% yield by a Pechmann
reaction involving the condensation of resorcinol with
ethyl 2-methylacetoacetate in the presence of concen-
trated sulfuric acid. When 6,7-dihydroxy-4-methylcou-
marin, 25, was treated with 2 equiv of sodium hydride
followed by an excess of sulfamoyl chloride (5 equiv), a
mixture of the catechol-like coumarin sulfamates, 26
and 27, was formed. The separation of the mono-
sulfamate, 26 from the bis-sulfamate, 27, was achieved
by preparative TLC. An attempt to sulfamoylate 7-hy-
droxy-4-methyl-8-nitrocoumarin (28, Figure 5) in DMF
using sulfamoyl chloride failed to give the desired
4-methyl-8-nitrocoumarin 7-O-sulfamate (29, Figure 5).
It is evident that the most effective inhibitor in this
series is COUMATE. Its IC50 for inhibition of E1-STS,
measured using intact MCF-7 cells, was found to be 380
nM (Figure 10B). As with EMATE, COUMATE inhibits
placental microsomes E1-STS activity in a time- and
concentration-dependent manner and in a biphasic
fashion (Figure 11B), indicating a similar mechanism
of action to that proposed for EMATE, as depicted in
Figure 3. At 10 µM, COUMATE reduces the original
E1-STS activity by 95% after preincubation of the
enzyme with the inhibitor for 20 min (Figure 11B).
Also, at the same concentration, COUMATE inhibits
placental microsomal DHA-STS activity by 94%.54 In
contrast to EMATE, however, COUMATE is devoid of
estrogenic activity since it did not stimulate the growth
of intact MCF-7 breast cancer cells or the uteri of
ovariectomized rats.54 It has also been shown that
COUMATE is able to block completely the ability of E1S
to stimulate uterine growth in ovariectomized rats.54
Moreover, COUMATE is orally active in vivo and, after
multiple dosing (10 mg/kg/day for 7 days), inhibits E1-
STS activity in rat liver by 85%.54 However, the
duration of this inhibition is limited since liver E1-STS
activity is almost fully restored 7 days after single or
multiple dosing with COUMATE.54 When EMATE was
examined in vivo in a similar manner, the rat liver E1-
STS activity remained inhibited (>95%) for over a week
following the cessation of dosing.52
The conjugation in 7-hydroxycoumarin (13, Figure 5)
was disrupted by hydrogenation in the presence of 10%
palladium-charcoal. While the disubstituted double
bond of 13 was completely reduced in 24 h at 40 psi,
the sterically more hindered trisubstituted double bond
in 4-methyl-7-hydroxycoumarin (10, Figure 5) remained
unaffected under this hydrogenation condition. (()-3,4-
Dihydro-7-hydroxy-4-methylcoumarin (30, Figure 6) was
subsequently obtained in 71% yield when 10 was
hydrogenated at 75 psi for 3 days. Direct hydrogenation
of COUMATE and 14 (Figure 5) to 31 and 33, respec-
tively, has not yet been attempted. However, from our
experience with other sulfamates, it is likely that the
sulfamate groups of COUMATE and 14 will be stable
to hydrogenation conditions used in the above experi-
ments, and hence this may be a more direct route to 31
and 33.
The significant increase in the inhibition of E1-STS
activity by coumarin sulfamates (12, 14, 16, and 18) in
comparison to 7 can presumably be attributed to their
different ring systems. The initial E1-STS/coumarin
sulfamate complex may form more readily than E1-
STS/7 since there may be more favorable interactions
between the amino acid residues in the active site and
the R,â-unsaturated lactone motif of the coumarin
derivative. Subsequent inactivation of the enzyme may
thus be accomplished more efficiently with the coumarin
sulfamates. Alternatively, the enhanced inhibitory
The conjugation of the coumarin motif can also be
disrupted by replacing the carbonyl group in the lactone
with a methylene group. An attempt to convert a model
coumarin to the corresponding R,â-unsaturated ether
by the method of Kraus et al.55 failed. Since there is
no apparent working direct synthetic route to com-

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1075
Ta ble 2. Inhibition of Estrone Sulfatase Activity in Placental
Microsomes by Sulfamates 7, 12, 14, 16, 18, 20, 22, 24, 26, 27,
31, 33, 39, 44, 46, 48, 49, 51, and 53 at Various
Concentrationsa
activity poorly in intact MCF-7 breast cancer cells (Table
1) also perform poorly when examined in placental
microsomes (Table 2). The only exception is sulfamate
31 where the situation is apparently reversed.
% inhibition ( SD of E1-STS activity in
placental microsomes at various concentrations
The dose-response curves for the inhibition of E1-
STS in intact MCF-7 breast cancer cells by COUMATE
and 24 are compared in Figure 10B and the IC50 of 24
is found to be 30 nM, some 12-fold lower than that of
COUMATE. It is likely that the higher potency ob-
served for 24 is the result of its extra methyl group at
the 3-position which might have rendered a tighter
binding of the inhibitor to the enzyme active site
through hydrophobic interactions with neighboring
amino acid residues. Although no time- and concentra-
tion-dependent inactivation studies have been per-
formed, it is reasonable to expect that 24, like COU-
MATE, is also an active site-directed inhibitor.
compd
10 µM
<10
50 µM
<10
97.8 ( 0.1
91.9 ( 0.8
94.3 ( 0.3
11.5 ( 1.8
<10
42.9 ( 1.9
98.8 ( 1.3
<10
<10
70.6 ( 2.8
15.0 ( 0.9
36.9 ( 1.8
33.2 ( 1.5
54.3 ( 4.0
16.5 ( 2.1
100 µM
7
12*
14
16
18
20
22
24†
26
27
31
33
39
44
46
48
49
51
53
12.5 ( 3.0
99 ( 0.4
94.4 ( 0.1
96.3 ( 0.3
12.5 ( 1.6
<10
69.9 ( 1.3
99.2 ( 1.6
<10
<10
82.1 ( 1.0
34.4 ( 0.3
47.2 ( 2.3
34.5 ( 6.0
56.1 ( 4.6
27.0 ( 1.1
93.0 ( 0.8
78.2 ( 0.1
79.4 ( 0.1
17.4 ( 3.1
<10
<10
96.9 ( 4.1
<10
<10
53.4 ( 1.6
<10
19.9 ( 2.1
<10
16.1 ( 5.8
12.3 ( 1.0
<10
19.6 ( 3.1
<10
An introduction of a substituent at either the C-6 (22
cf. 14, and 27 cf. COUMATE, Tables 1 and 2) or the
C-8 position (18 cf. 24, Tables 1 and 2) of the coumarin
ring significantly reduces the inhibitory activity of the
sulfamate. Since coumarin sulfamates are anticipated
to be A/B ring mimics of EMATE, our results have
therefore highlighted the limited tolerance of the en-
zyme to substituents at these positions. It is possible
that the methoxy group in 22, the C-8 methyl group in
18, and the second sulfamate group in 27 may have
caused the sulfamate group at C-7 to be shifted in the
binding site from the usual position occupied by the
sulfamate group of EMATE/COUMATE, and hence it
could not be activated effectively for the sulfamoylation
of the enzyme. These substituents may also conceivably
shield the sulfamate group in such a manner that the
putative proton abstraction, the first vital step proposed
for the mechanism of E1-STS inhibition by EMATE, is
prevented from occurring.
<10
37.7 ( 0.9
<10
<10
50.3 ( 1.2
<10
a
[3H]Estrone sulfate (4 × 105 dpm), adjusted to 20 µM with
unlabeled substrate, with or without the inhibitor at various
concentrations, was incubated with placental microsomes (125 µg
of protein/mL) for 30 min. The product formed was isolated by
extraction into toluene with [4-14C]estrone (7 × 103 dpm) being
used to monitor procedural losses. Each value represents the mean
( s.d. of triplicate measurements. *<10% at 0.1 µM and 62.7% (
0.5 at 1 µM. †34.7% ( 0.1 at 0.1 µM and 88.2% ( 1.9 at 1 µM.
pounds of this type, a multistep approach is required
for the synthesis of 2H-1-benzopyran-7-ol (38, Figure
7), the starting material for the corresponding sulfamate
(39, Figure 7). Hence, 2,3-dihydro-7-hydroxy-4H-1-
benzopyran-4-one (34, Figure 7) was synthesized from
resorcinol and acrylnitrile according to the method of
Breytenbach and Rall56 and then silylated. The result-
ing protected pyranone (35, Figure 7) was reduced and
the resulting mixture of the enantiomeric alcohols (36,
Figure 7) dehydrated to give the silylated pyranol (37,
Figure 7). Finally, deprotection of 37 gave crude 2H-
1-benzopyran-7-ol, 38, which was then sulfamoylated
immediately, without further purification, because of
noticeable decomposition. The sulfamate 39 obtained
was also unstable and had to be purified by preparative
TLC before analysis and testing. It appears that this
class of compounds is intrinsically unstable as the
structurally related analogue, 2,7,8-trimethyl-2-(4,8,12-
trimethyltrideca-3-(E),7(E),11-trienyl)-2-H-benzopyran-
6-ol, has also been reported as showing noticeable
oxidative decomposition upon storage.57
The above multistep approach to 38 was also applied
to the synthesis of 7,8-dihydronaphth-2-ol (43, Figure
7). Although alcohol 43 was found to be unstable in a
manner similar to its analogue 38, 7,8-dihydronaph-
thalene 2-O-sulfamate (44, Figure 7), in contrast to the
benzopyran sulfamate 39, is a stable crystalline product.
Upon examination of the abilities of these COUMATE
analogues (20, 22, 24, 26, 27, 31, 33, 39, and 44) to
inhibit E1-STS activity (Tables 1 and 2), almost all of
them were found to be less effective inhibitors of E1-
STS than COUMATE with the exception of 3,4-dimeth-
ylcoumarin 7-O-sulfamate (24), which inhibits the en-
zyme to a greater extent. These results have also shown
a general pattern that analogues which inhibit E1-STS
Relocation of the sulfamate group from the usual C-7
to the C-6 position virtually abolishes the inhibitory
activities of the coumarin sulfamates in both of the
biological systems (20 and 26, cf. COUMATE, Tables 1
and 2). It is likely that on binding to the enzyme active
site, the sulfamate groups in 20 and 26, which are at
the C-6 position of the coumarin ring, as opposed to the
C-7 position in COUMATE, are not in close proximity
to the essential amino acid residues responsible for their
subsequent activation. The poor inhibition shown by
sulfamates 20 and 26 could also be a consequence of
the relatively poor leaving group abilities of their
respective starting coumarins (19 and 25, Figure 5). It
is reasonable to expect that the pKas of 19 and 25 will
be similar to that of a simple phenol (ca. 10) since their
hydroxy group at the C-6 position is not conjugated to
the R,â-unsaturated lactone moiety of the coumarin
ring.
The significance of the coumarin moiety to the inhibi-
tory activities of COUMATE and 14 is further exempli-
fied by the poor inhibition observed for their analogues
where the conjugation of the ring system is disrupted.
When the R,â-olefinic double bond was saturated (31
and 33, Figure 6) or the lactone disrupted, either by
replacement of the carbonyl group with a methylene
group (39, Figure 7) or by removal of the entire moiety
(44, Figure 7), the analogues so afforded were found to
be less effective inhibitors of E1-STS than COUMATE

1076 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Woo et al.
(Tables 1 and 2). For sulfamates 39 and 44, their poor
inhibitory activities observed might be attributed to a
weaker binding to the enzyme active site since a site of
potential hydrogen bonding with neighboring amino acid
residues has been deleted. However, the differences in
the inhibitory activities of 31 and 33 when compared
to those of their R,â-unsaturated lactone counterparts
are unlikely to be explained simply by the structural
modification to COUMATE and 14. It is anticipated
that these sulfamates (COUMATE, 14, 31, and 33) will
all bind in a similar manner and tightness to the
enzyme active site. We therefore attribute the superior
inhibition shown by COUMATE and 14 to the better
leaving group abilities of their starting coumarins, 10
and 13, respectively, as a result of the conjugation in
their coumarin rings. It is now clear that a study on
the pKa values of 6, 10, 13, 15, 17, 19, 21, 23, 25, 30,
32, 38, and 43 is warranted in order to establish if a
correlation between the leaving group abilities of these
phenols and the inhibitory activities of their correspond-
ing sulfamates exists.
F igu r e 12. Proposed pharmacophore for an EMATE-like
steroid sulfatase inhibitor. X = H or a substituent; Y =
additional functionalities (including fused or adjacent/remote
ring structures).
mental to its biological activity. Replacement of the
entire coumarin ring of COUMATE with other simple
two-ring systems having similar leaving group poten-
tials have also resulted in analogues which are less
effective inhibitors of E1-STS. However, when the
3-position of COUMATE is substituted with a methyl
group, the analogue 3,4-dimethylcoumarin 3-O-sulfa-
mate, 24, is some 12-fold more potent than COUMATE
as an E1-STS inhibitor in intact MCF-7 breast cancer
cells. These structure-activity relationship studies
conducted on COUMATE clearly show that its coumarin
ring is pivotal for inhibition of E1-STS and that highly
potent, irreversible, and nonestrogenic E1-STS inhibi-
tors, which are structurally related to COUMATE, may
subsequently be designed and developed for therapeutic
use in the treatment of hormone-dependent breast
cancers.
All of the studies reported here, and by ourselves and
others since the publication of our initial report,30 have
demonstrated the absolute requirement for at least a
phenol sulfamate ester for activity. It seems clear that
this must be the active pharmacophore required for
steroid sulfatase inhibition, but that other groups, not
necessarily a ring structure (see refs 39 and 40),
presumably provide the extra motifs required for effec-
tive binding to the active site and/or activation of the
sulfamoyl group and can modulate activity and estro-
genicity.61 We therefore propose the pharmacophore
illustrated in Figure 12 as the definitive molecular motif
required in an EMATE-like steroid sulfatase inhibitor.
No compound has yet been synthesized, acting like
EMATE, that does not possess this functionality which
we believe will be a crucial factor in the design of further
active site-directed inhibitors of steroid sulfatase.
Finally, to explore the potential of other two-ring
sulfamates as effective inhibitors of E1-STS, we have
synthesized and examined tetralone 6-O-sulfamate (46,
Figure 8), indanone 5-O-sulfamate (51, Figure 8), and
the sulfamates of their analogues (48, 49, and 53, Figure
8). Although some of these sulfamates show encourag-
ing inhibition in intact MCF-7 breast cancer cells (48
and 53, Table 1), all of the sulfamates inhibit E1-STS
activity in placental microsomes to a lesser extent than
COUMATE (Table 2). For sulfamates 46 and 51,
preliminary results from our studies in progress have
indicated that the pKa values of the starting alcohols,
6-hydroxy-1-tetralone (45, Figure 8), and 5-hydroxy-1-
indanone (50, Figure 8) respectively, are in the same
range as that of 7-hydroxy-4-methylcoumarin (10, Fig-
ure 5) [ca. 8.1 (unpublished results)]. It is therefore
interesting to note that the inhibitory activity of COU-
MATE cannot be reproduced by simply replacing the
coumarin ring with other two-ring systems having
leaving group abilities similar to that of 10. The
relatively disappointing inhibitory activities observed
for this series of tetralone and indanone sulfamates
could well be the result of less effective binding of these
compounds to the enzyme active site.
Exp er im en ta l Section
All chemicals were either purchased from Aldrich Chemical
Co. (Gillingham, Dorset, U.K.) or Lancaster Synthesis (More-
cambe, Lancashire, U.K.). Crystalline starting alcohols were
dried in vacuo at 60 °C for 3 h prior to use. All organic
solvents, of A.R. grade, were supplied by Fison plc (Lough-
borough, U.K.) and stored over 4 Å molecular sieves. Anhy-
drous dimethylformamide (DMF), used for all the sulfamoy-
lation reactions, was purchased from Aldrich and was stored
under a positive pressure of N2 after use. Sulfamoyl chloride
was prepared by an adaptation of the method of Appel and
Berger50 and was stored as a solution in toluene as described
by Woo et al.33 No titration was attempted on this sulfamoyl
chloride solution whose molarity was estimated according to
the weight of the original crude sulfamoyl chloride obtained
after workup. An appropriate volume of this solution was
freshly concentrated in vacuo immediately before use. For the
synthesis of EMATE, the crude sulfamoyl chloride solid was
used instead. N-Methylsulfamoyl chloride was prepared from
N-methylsulfamic acid by treatment with phosphorus pen-
tachloride;58 N,N-dimethylsulfamoyl chloride was purchased
from Aldrich. Pyridine was dried by refluxing over sodium
hydroxide pellets followed by distillation and stored over 4 Å
molecular sieves.
In conclusion, EMATE (2, Figure 2) is the most potent
active site-directed steroidal E1-STS inhibitor synthe-
sized to date. However, a significant reduction in the
inhibitory activity of EMATE with an apparent change
in the nature of inhibition results when the N atom of
its sulfamate group is increasingly methylated. An
attempt to design and synthesize nonsteroidal E1-STS
inhibitors, in the light of the potent estrogenicity of
EMATE, has led to the development of COUMATE (12,
Figure 5). This is a significant improvement over our
first lead nonsteroidal candidate, 5,6,7,8-tetrahydronaph-
thalene 7-O-sulfamate (7, Figure 4). COUMATE is an
orally active and nonestrogenic active site-directed
steroid sulfatase inhibitor, albeit less potent than
EMATE. Modifications made to the structure of COU-
MATE by (i) the relocation of its C-7 sulfamate group
to the C-6 position, (ii) substitutions of the aromatic ring
at the C-6 or C-8 positions, and (iii) the disruption of
the conjugated coumarin ring all proved to be detri-
E1S and E1 were purchased from Sigma Chemical Co.
(Poole, U.K.). [6,7-3H]E1S (specific activity, 50 Ci/mmol) and

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1077
[4-14C]E1 (specific activity, 52 mCi/mmol) were purchased from
New England Nuclear (Boston, MA). [6,7-3H]E1 (specific
activity, 97 Ci/mmol) was obtained from the Amersham
International Radiochemical Centre (Amersham, U.K.).
2.25 mmol), N-methylsulfamoyl chloride (280 mg, 2.14 mmol),
and anhydrous DMF (5 mL) were used. Purification was
effected using flash chromatography (chloroform/methanol, 98:
2) to afford 3 as a cream-colored powder (90 mg, 0.247 mmol,
24%): 1H NMR (270 MHz, CDCl3) δ 0.91 (3H, s, CH3), 1.40-
1.68 (6H, m) 1.97-2.57 (7H, series of m), 2.91 (2H, m), 2.94
(3H, d, J ) 5.13 Hz, CH3NH), 4.70 (1H, br q, exchanged with
D2O, J ) 5.13 Hz, MeNH), 7.04 (2H, m, C2-H and C4-H), and
7.30 (1H, d, J ) 8.06 Hz, C1-H); 13C NMR (100.4 MHz, CDCl3)
δ 13.78 (q, C18) 21.54 (t), 25.72 (t), 26.21 (t), 29.39 (t), 30.23
(q, CH3NH), 31.46 (t), 35.81 (t), 37.88 (d), 44.09 (d), 47.87 (s,
C13), 50.34 (d), 118.84 (d),121.79 (d), 126.72 (d), 138.60 (s),
138.66 (s), 147.97 (s), 220.82 (s, CO); MS (CI, isobutane) m/z
(rel intensity) 364 [13, (M + H)+], 271 [70, (M + H-MeNSO2)+],
69 (100). Anal. (C19H25NO4S‚1/4H2O) C, H, N.
Thin layer chromatography (TLC) was performed on pre-
coated plates (Merck TLC aluminum sheets silica gel 60 F254
,
Art. No. 5554). Product(s) and starting material (SM) were
detected by either viewing under UV light or treating with a
methanolic solution of phosphomolybdic acid followed by
heating. Preparative TLC was performed on precoated plates
(Merck TLC silica gel 60 F254, 20 × 20 cm, layer thickness 2
mm, art. no. 5717) and bands were visualized under UV light.
Flash column chromatography was performed on silica gel
(Sorbsil C60). IR spectra were determined by a Perkin-Elmer
782 infrared spectrophotometer, and peak positions are ex-
pressed in cm-1 1H and DEPT-edited 13C NMR spectra were
.
Estr on e 3-O-(N,N-Dim eth yl)su lfa m a te (4). Sodium hy-
dride (60% dispersion, 74 mg, 1.85 mmol) was added to a
stirred solution of estrone (500 mg, 1.85 mmol) in anhydrous
DMF (5 mL) at 0 °C. After the evolution of hydrogen had
ceased, N,N-dimethylsulfamoyl chloride (531 mg, 3.70 mmol)
was added and the resulting mixture was left to stir overnight
at room temperature. The reaction mixture was then quenched
with brine (100 mL) and the aqueous portion extracted with
ethyl acetate (100 mL). The organic fraction that separated
was further washed with brine (5 × 50 mL), dried (MgSO4),
filtered, and evaporated in vacuo to give a white residue which
was fractionated by flash chromatography (chloroform/ethyl
acetate, 15:1 to 9:1, gradient). The crude product obtained was
further purified by recrystallization from ethyl acetate/hexane
(1:1) to afford 4 as white crystals (170 mg, 450 µmol, 24%):
mp 176-177 °C; 1H NMR (270 MHz, CDCl3) δ 0.92 (3H, s,
CH3), 1.4-2.6 (13H, m), 2.92 (2H, m, C6-H2), 2.99 [6H, s,
N(CH3)2], 7.03 (2H, m, C2-H and C4-H), and 7.29 (1H, d,
J C2-H,C1-H ≈ 8 Hz, C1-H); 13C NMR (100.4 MHz, CDCl3) δ 13.75
(q, C18), 21.50 (d), 25.66 (t), 26.17 (t), 29.32 (t), 31.43 (t), 35.78
(t), 37.82 (d), 38.08 (q, CH3N), 38.73 (q, CH3N), 44.05 (d), 47.84
(s, C13), 50.31 (d), 118.71 (d) 121.63 (d), 126.59 (d), 138.30 (s),
138.43 (s), 148.00 (s), 220.69 (s, CO); MS (EI, 70 eV) m/z (rel
intensity) 377 (100, M+), 269 [20, (M - Me2NSO2)+]. Anal.
(C20H27NO4S) C, H, N.
5,6,7,8-Tetr ah ydr on aph th alen e 2-O-Su lfam ate (7). This
was prepared in a manner similar to the preparation of 4.
5,6,7,8-Tetrahydronaphth-2-ol (500 mg, 3.306 mmol), sodium
hydride (60% dispersion, 132 mg, 3.306 mmol), sulfamoyl
chloride (2 equiv), and DMF (5 mL) were used. Purification
of the syrupy light brown crude was effected using flash
chromatography (ethyl acetate/hexane, 1:4 to 1:2, gradient) to
give 7 as a clear pale yellow syrup which solidified to a cream-
colored waxy solid when kept under a stream of nitrogen over
the weekend (624 mg, 2.745 mmol, 83%): TLC (ethyl acetate/
hexane, 1:1) Rf 0.58, cf. Rf 0.66 (SM); IR (KBr) 3440, 3260,
3100, 2920, 2840, 1610, 1500, 1370, 1180; 1H NMR (400 MHz,
CDCl3) δ 1.79 (4H, m, C6-H2 and C7-H2), 2.75 (4H, m, C5-H2
and C8-H2), 4.90 (2H, br s, exchanged with D2O, OSO2NH2),
7.03 (2H, m, C1-H and C3-H) and 7.08 (1H, d, J ) 9.2 Hz,
C4-H); 13C NMR (100.4 MHz, CDCl3) δ 22.64 (t), 22.87 (t), 28.83
(t), 29.35 (t), 118.94 (d), 122.09 (d), 130.23 (d), 136.32 (s), 138.98
(s), 147.61 (s); MS (FAB+) m/z (rel intensity) 381.0 [14, (M +
H + NBA)+], 227.1 (100, M+), 147.1 [45, (M - H2NSO2)+]; MS
(FAB-) m/z (rel intensity) 380.0 [26, (M + NBA)-], 226.0 [100,
(M - H)-]; HRMS (FAB+) m/z 227.061 74 (M+), calcd for
C10H13NO3S 227.061 62. Anal. (C10H13NO3S) C, H, N.
recorded with a J EOL J MN-GX270 and J MN-GX400 NMR
spectrometers, and chemical shifts are reported in parts per
million (ppm, δ) relative to tetramethylsilane (TMS) as an
internal standard. For compounds 35, 37, 40, and 42, where
no TMS was included, the chemical shifts are reported relative
to CDCl3 at 7.26 ppm. Mass spectra were recorded at the Mass
Spectrometry Service Centre, University of Bath, and the
EPSRC National Mass Spectrometry Service Centre, Swansea.
FAB-mass spectra were carried out using m-nitrobenzyl
alcohol (NBA) as the matrix unless stated otherwise. Elemen-
tal analyses were performed by the Microanalysis Service,
University of Bath, and are within ( 0.4% of theory unless
otherwise stated. Melting points were determined using a
Reichert-J ung Thermo Galen Kofler block and are uncorrected.
Biological studies were performed essentially as described
previously.27,30,51 For more details, see legends of individual
figures or tables. To examine whether the sodium salt of
7-(sulfooxy)-4-methylcoumarin (11, Figure 5) 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 product formed at the end of the
incubation was extracted into diethyl ether. After evaporation
of solvent, the residue obtained was examined by TLC using
ethyl acetate/methanol (4:1) as eluent. In this mobile phase,
the coumarin sulfate, 11, and the parent 7-hydroxy-4-meth-
ylcoumarin (10, Figure 5) have Rf values of 0.79 and 0.95,
respectively.
Estr on e 3-O-Su lfa m a te (2). Sodium hydride (60% disper-
sion, 70 mg, 1.73 mmol) was added to a stirred solution of
estrone (230 mg, 850 µmol) in anhydrous DMF (5 mL) at 0
°C. Subsequently, sulfamoyl chloride (200 mg, 1.73 mmol) was
added carefully, and the resulting mixture was allowed to
warm to room temperature whereupon stirring was continued
for a further 96 h. The reaction mixture was then poured onto
a cold, saturated solution of sodium bicarbonate (20 mL). The
aqueous solution was extracted with dichloromethane (5 × 15
mL), and the combined organic extracts were dried over
anhydrous MgSO4. Removal of the solvent in vacuo followed
by repeated coevaporation with toluene (3 × 30 mL) to remove
the final traces of DMF gave a viscous pale brown oil which
solidified on standing. Purification was effected using flash
chromatography (chloroform/methanol, 96:4). The final prod-
uct was further purified by precipitation from chloroform by
addition of pentane, and 2 was afforded as a white crystalline
solid (230 mg, 659 µmol, 76%): mp 195-197 °C; TLC (chlo-
roform/methanol, 96:4) Rf 0.36; 1H NMR (270 MHz, methanol-
d4) δ 0.91 (3H, s, CH3), 1.46-1.67 (6H, m), 1.89-2.54 (7H,
series of m), 2.92 (2H, m), 7.04 (2H, br d, J ) 10.44 Hz, C2-H
and C4-H), and 7.33 (1H, br d, J ) 8.42 Hz, C1-H); 13C NMR
(100.4 MHz, methanol-d4) δ 14.53 (q, C18), 22.80 (t), 27.24 (t),
27.73 (t), 30.68 (t), 33.05 (t), 37.01 (t), 39.76 (d), 45.73 (d), 50.27
(s, C13), 51.86 (d), 120.76 (d), 123.54 (d), 127.89 (d), 139.83
(s), 150.27 (s), 223.87 (s, CO); MS (EI, 70 eV) m/z (rel intensity)
349 (9, M+), 270 [100, (M - HNSO2)+]. Anal. (C18H23NO4S)
C, H, N.
5,6,7,8-Tetr a h yd r on a p h th a len e 2-O-(N-Meth yl)su lfa -
m a te (8). This was prepared according to the procedure used
for the preparation of 2. 5,6,7,8-Tetrahydronaphth-2-ol (150
mg, 1.01 mmol), sodium hydride (60% dispersion, 80 mg, 2.02
mmol), N-methylsulfamoyl chloride (260 mg, 2.02 mmol), and
DMF (5 mL) were used. Purification was effected using
preparative TLC (chloroform/methanol, 96:4) to give 8 as a
yellow/green viscous oil (90 mg, 0.373 mmol, 37%): 1H NMR
(270 MHz, CDCl3) δ 1.77 (4H, quintet, J ) 3.25 Hz, C6-H2 and
C7-H2), 2.74 (4H, br d, J ) 4.22 Hz, C5-H2 and C8-H2), 2.87
(3H, d, J ) 5.13 Hz, CH3NH), 4.85 (1H, br q, exchanged with
D2O, J ) 4.94 Hz, CH3NH), 6.95-7.25 (3H, m, C1-H, C3-H,
Estr on e 3-O-(N-Meth yl)su lfa m a te (3). This was pre-
pared in a manner similar to the preparation of 2. Estrone
(290 mg, 1.07 mmol), sodium hydride (60% dispersion, 90 mg,

1078 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Woo et al.
and C4-H); 13C NMR (100.4 MHz, CDCl3) δ 22.61 (t), 22.80
(t), 28.74 (t), 29.29 (t), 30.10 (q, CH3NH), 118.61 (d), 121.76
(d), 130.16 (d), 135.97 (s), 138.89 (s), 147.51 (s); MS (CI,
isobutane) m/z (rel intensity) 242 [100, (M + H)+].
NH)+]; MS (FAB-) m/z (rel intensity) 407.1 [15, (M - H +
NBA)-], 254 [100, (M - H)-], 175.1 [32, (M - SO2NH2)-].
Anal. (C10H9NO5S) C, H, N.
Cou m a r in 7-O-Su lfa m a te (14). This was prepared from
7-hydroxycoumarin (500 mg, 3.082 mmol) in a manner similar
to the preparation of 12. Purification by flash chromatography
(chloroform/acetone, 8:1 to 2:1, gradient) gave the crude
product which was further purified by recrystallization from
ethyl acetate/hexane (1:1) to give 14 as dull white crystals (239
mg, 991.0 µmol, 32%): mp 170.0-171.5 °C; 1H NMR (270 MHz,
DMSO-d6/CDCl3, ca. 1:25) δ 6.42 (1H, d, J ) 9.7 Hz, C3-H),
7.29 (1H, dd, J ) 2.3 and 8.5 Hz, C6-H), 7.38 (1H, d, J ) 2.2
Hz, C8-H), 7.51 (2H, br s, exchanged with D2O, OSO2NH2),
7.54 (1H, d, J ) 8.4 Hz, C5-H), and 7.76 (1H, d, J ) 9.7 Hz,
C4-H); MS (EI, 70 eV) m/z (rel intensity) 241.0 (10, M+), 162.0
[97, (M - SO2NH)+], 134.0 (100); HRMS (EI, 70 eV) m/z
241.006 84 (M+), calcd for C9H7NO5S 241.004 49. Anal. (C9H7-
NO5S) C, H, N.
5,6,7,8-Tet r a h yd r on a p h t h a len e 2-O-(N,N-d im et h yl)-
su lfa m a te (9). This was prepared in a manner similar to the
preparation of 2. 5,6,7,8-Tetrahydronaphth-2-ol (300 mg, 2.02
mmol), sodium hydride (60% dispersion, 160 mg, 4.05 mmol),
N,N-dimethylsulfamoyl chloride (580 mg, 4.05 mmol), and
DMF (5 mL) were used. Purification of the crude material
was effected using flash chromatography (chloroform) to yield
9 as a colorless oil (370 mg, 1.45 mmol, 72%): 1H NMR (270
MHz, CDCl3) δ 1.78 (4H, quintet, J ) 3.25 Hz, C6-H2 and C7-
H2), 2.75 (4H, br d, J ) 6.04 Hz, C5-H2 and C8-H2), 2.958 (3H,
s, CH3N), 2.960 (3H, s, CH3N), 6.98-7.07 (3H, m, C1-H, C3-
H, and C4-H); 13C NMR (100.4 MHz, CDCl3) δ 22.70 (t), 22.93
(t), 28.80 (t), 29.39 (t), 38.66 (q, CH3N), 38.73 (q, CH3N), 118.61
(d), 121.76 (d), 130.10 (d), 135.68 (s), 138.79 (s), 147.71 (s); MS
(EI, 70 eV) m/z (rel intensity) 255 (59, M+), 147 [100, (M -
Me2NSO2)+].
4-(Tr iflu or om eth yl)cou m a r in 7-O-Su lfa m a te (16). This
was prepared from 7-hydroxy-4-(trifluoromethyl)coumarin (900
mg, 3.832 mmol) in a manner similar to the preparation of
12. Purification by flash chromatography (diethyl ether/
chloroform, 1:4) gave the crude product which was further
purified by recrystallization from ethyl acetate/hexane (1:3)
to give 16 as white needle-shaped crystals (160 mg, 517.4 µmol,
14%): mp 165-168 °C; 1H NMR (270 MHz, acetone-d6) δ 6.99
7-(Su lfooxy)-4-m eth ylcou m a r in , Sod iu m Sa lt (11). To
a stirred solution of 7-hydroxy-4-methylcoumarin (1.0 g, 5.676
mmol) in anhydrous pyridine (20 mL), under an atmosphere
of N2, was added sulfur trioxide-pyridine complex (1.8 g, 11.35
mmol), and the reaction mixture was stirred overnight. After
removal of pyridine, methanol (20 mL) was added to the
creamy syrup obtained, and the resulting light yellow solution
was basified (pH ∼ 8) by dropwise addition of sodium hydrox-
ide in methanol (1 M). The bright yellow precipitate formed
was collected by filtration and washed with fresh methanol.
The combined methanolic filtrates were then concentrated to
30-40 mL, and diethyl ether (ca. 120 mL in total) was added
in portions until complete precipitation had occurred. The
light beige precipitate was collected by filtration and recrystal-
lized from methanol/diethyl ether (1:1) to give 11 as light
creamy yellow crystals (409 mg, 1.604 mmol, 28%): mp 172-
175 °C dec; TLC (methanol/diethyl ether, 1:2) Rf 0.68, cf. Rf
0.83 (SM); 1H NMR (270 MHz, DMSO-d6) δ 2.41 (3H, s, CH3),
6.27 (1H, s, C3-H), 7.20 (2H, m, C6-H and C8-H), and 7.70
(1H, d, J ) 8.8 Hz, C5-H); MS (FAB-) m/z (rel intensity) 431.0
[15, (M + NBA)-], 408.0 [8, M - Na + NBA)-], 255.0 [100, (M
- Na)-], 175.1 [14, (M - Na - SO3)-]; HRMS (FAB-, glycerol)
m/z 254.998 23 [(M - Na)-], calcd for C10H7O6S 254.996 33.
Anal. (C10H7O6NaS‚H2O) C, H, N. HPLC [Spherisorb ODS5
column (25 × 4.6 mm); mobile phase, MeOH/H2O (70:30); flow
rate, 1 mL/min; λmax, 316 nm]; tR ) 1.5 min (cf. 7-hydroxy-4-
methylcoumarin ) 3.6 min).
4-Meth ylcou m a r in 7-O-Su lfa m a te (12). A stirred solu-
tion of 4-methyl-7-hydroxycoumarin (500 mg, 2.753 mmol) in
anhydrous DMF (5 mL) was treated with sodium hydride (60%
dispersion, 110 mg, 2.753 mmol) at 0 °C under an atmosphere
of N2. After evolution of hydrogen had ceased, sulfamoyl
chloride (2 equiv) was added. The reaction mixture was stirred
at room temperature overnight and then poured into water
(150 mL). The resulting mixture was extracted with ethyl
acetate (150 mL) and the organic portion that separated was
washed with brine (5 × 100 mL), dried (MgSO4), filtered, and
evaporated in vacuo at 40 °C. Purification by flash chroma-
tography (chloroform/acetone, 8:1 to 2:1, gradient) gave the
crude product which was further purified by recrystallization
from acetone/chloroform (3:5) to give 12 as colorless rhombic
crystals (281 mg, 1.101 mmol, 40%): mp 165-167 °C; TLC
(chloroform/acetone, 4:1) Rf 0.26, cf. Rf 0.44 (SM); IR (KBr)
3320, 3180, 3080, 1700, 1620, 1560, 1380, 1195, 1125; 1H NMR
(400 MHz, acetone-d6) δ 2.50 (3H, d, 4J C3-H,Me ) 1.22 Hz, CH3),
6.33 (1H, d, 4J Me,C3-H ) 1.22 Hz, C3-H), 7.30 (1H, d, J C6-H,C8-H
) 2.14 Hz, C8-H), 7.31 (1H, dd, J C8-H,C6-H ) 2.44 Hz and
J C5-H,C6-H ) 8.54 Hz, C6-H), 7.40 (2H, br s, D2O exchanged,
OSO2NH2), and 7.86 (1H, d, J C6-H,C5-H ) 8.24 Hz, C5-H); 13C
NMR (100.4 MHz, acetone-d6) δ 18.53 (q, CH3), 110.99 (d),
115.17 (d, C3), 119.12 (d), 119.25 (s), 127.27 (d, C5), 153.19
(s), 153.54 (s), 154.96 (s), 160.14 (s). (These assignments were
confirmed by 13C-1H correlation experiments); MS (FAB+) m/z
(rel intensity) 256.1 [100, (M + H)+], 177.1 [20, (M + H - SO2-
4
(1H, d, J F, H ≈ 1 Hz, C3-H), 7.46 (1H, dd, J ) 2.4 and 8.1 Hz,
C6-H), 7.48 (1H, d, J ) 2.4 Hz, C8-H), 7.53 (2H, br s,
exchanged with D2O, OSO2NH2), and 7.89 (1H, m, C5-H) [1H
NMR spectrum of 16 in DMSO-d6/CDCl3 (ca. 1:15) showed
partial decomposition to the starting coumarin]; MS (EI, 70
eV) m/z (rel intensity) 309.0 (2, M+), 230.0 [77, (M - SO2NH)+],
202.0 (100); HRMS (EI, 70 eV) m/z 308.987 43 (M+), calcd for
C
10H6F3NO5S 308.991 88. Anal. (C10H6F3NO5S) C, H, N.
3,4,8-Tr im eth ylcou m a r in 7-O-Su lfa m a te (18). This was
prepared from 7-hydroxy-3,4,8-trimethylcoumarin (1.0 g, 4.896
mmol) in a manner similar to the preparation of 12. The crude
material was recrystallized from hot ethyl acetate whereupon
238 mg of unreacted starting material was recovered. The
residue, obtained from the evaporation of the mother liquor,
was fractionated by flash chromatography (diethyl ether) to
give the crude product which was further purified by recrys-
tallization from acetone/hexane (1:2) to give 18 as pale yellow
crystals (312 mg, 1.101 mmol, 22%): mp 197-202 °C; 1H NMR
(270 MHz, acetone-d6) δ 2.18 (3H, s, CH3), 2.38 (3H, s, CH3),
2.46 (3H, s, CH3), 7.37 (1H, d, J ) 8.8 Hz, C6-H), 7.39 (2H, br
s, exchanged with D2O, OSO2NH2) and 7.68 (1H, d, J ) 8.8
Hz, C5-H); MS (EI, 70 eV) m/z (rel intensity) 283.1 (10, M+),
204.1 [45, (M - SO2NH)+], 43.1 (100); HRMS (EI, 70 eV) m/z
283.049 67 (M+), calcd for C12H13NO5S 283.051 44. Anal.
(C12H13NO5S) C, H, N.
4-Meth ylcou m a r in 6-O-Su lfa m a te (20). This was pre-
pared from 6-hydroxy-4-methylcoumarin (500 mg, 2.810 mmol)
in a manner similar to the preparation of 12. The crude
material was recrystallized from warm acetone/hexane (2:1)
to yield 20 as soft creamy rod-shaped crystals (285 mg, 1.118
mmol). Further recrystallization of the residue, recovered from
the evaporation of the mother liquor, from acetone/hexane (2:
1) gave a second crop of 20 (114 mg, 447 µmol, 56% overall):
1
mp 214-223 °C; H NMR (270 MHz, acetone-d6) δ 2.50 (3H,
s, CH3), 6.41 (1H, s, C3-H), 7.24 (2H, br s, exchanged with D2O,
OSO2NH2), 7.41 (1H, d, J ) 9.0 Hz, C8-H), 7.57 (1H, dd, J )
2.0 and 9.4 Hz, C7-H) and 7.71 (1H, d, J ) 2.0 Hz, C5-H); MS
(FAB+) m/z (rel intensity) 256.1 [100, (M + H)+]; MS (FAB-)
m/z (rel intensity) 407.9 [35, (M - H + NBA)-], 253.9 [100,
(M - H)-]; HRMS (FAB+) m/z 256.026 32 [(M + H)+], calcd
for C10H10NO5S 256.027 97. Anal. (C10H9NO5S) C, H, N.
6-Meth oxycou m a r in 7-O-Su lfa m a te (22). This was pre-
pared from 7-hydroxy-6-methoxycoumarin (70 mg, 346.0 µmol)
in a manner similar to the preparation of 12. Purification by
flash chromatography (chloroform/acetone, 4:1) gave the crude
product which was further purified by recrystallization from
acetone/hexane (1:2) to give 22 as beige crystals (59 mg, 217.5
µmol, 63%): mp 174-176 °C; 1H NMR (270 MHz, acetone-d6)

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1079
δ 3.92 (3H, s, OCH3), 6.43 (1H, d, J ) 9.7 Hz, C3-H), 7.31 (2H,
br s, exchanged with D2O, OSO2NH2), 7.36 (1H, s, C8-H), 7.44
(1H, s, C5-H), and 7.98 (1H, d, J ) 9.7 Hz, C4-H); MS (FAB+)
m/z (rel intensity) 271.9 [100, (M + H)+], 191.9 [20, (M -
HNSO2)+]; MS (FAB-) m/z (rel intensity) 269.8 [100, (M -
H)-], 190.9 [40, (M - H2NSO2)-]; HRMS (FAB+) m/z 272.022 46
[(M + H)+], calcd for C10H10NO6S 272.022 88. Anal. (C10H9-
NO6S) C, H, N.
2SO2NH)-]; HRMS (FAB+) m/z 350.996 01 [(M + H)+], calcd
for C10H11N2O8S2 350.995 68. Anal. (C10H10N2O8S2) C, H, N.
The band at Rf 0.62 gave a white residue which was further
purified by recrystallization from acetone/hexane (1:2) to 26
as white crystals (60 mg, 221.4 µmol, 24%): mp 203-205 °C;
1H NMR (DMSO-d6) δ 2.37 (3H, s, CH3), 6.22 (1H, s, C3-H),
6.89 (1H, s, C8-H), 7.59 (1H, s, C5-H), ∼8 (2H, br s, exchanged
with D2O, OSO2NH2) and ∼11 (1H, very br s, OH); MS (FAB+)
m/z (rel intensity) 272.1 [100, (M + H)+], 255.2[65, (M + H -
NH3)+], 193.2[35, (M + H - SO2NH)+]; MS (FAB-) m/z (rel
intensity) 270.0 [100, (M - H)-], 191.1[30, (M - SO2NH2)-];
7-Hyd r oxy-3,4-d im eth ylcou m a r in (23). Resorcinol (1.21
g, 11.0 mmol) was dissolved in hot ethyl 2-methylacetoacetate
(1.6 g, 10.0 mmol). The mixture was then cooled to 0 °C, and
concentrated sulfuric acid (98%, 15 mL) was added dropwise
with stirring. The resulting yellow/brown mixture was stirred
overnight at room temperature and then poured onto a mixture
of ice/water. The bright yellow precipitate was collected by
suction filtration and washed exhaustively with water. After
the precipitate was redissolved in acetone, the solution was
evaporated and drying of the mass obtained was effected
azeotropically with isopropyl alcohol. Recrystallization was
performed by first redissolving the crude product in hot
isopropyl alcohol (30 mL) followed by the dropwise addition
of hexane (30 mL). Upon cooling, 23 (369 mg, 1.942 mmol)
was separated as pale beige crystals. The residue, recovered
from the evaporation of the mother liquor, was recrystallized
in a similar manner to give a second crop of 23 (105 mg, 552.6
µmol, 25% overall); mp 245-265 °C; 1H NMR (270 MHz,
DMSO-d6) δ 2.05* (3H, s, C3-CH3), 2.33* (3H, s, C4-CH3), 6.68
(1H, d, J ) 2.6 Hz, C8-H), 6.78 (1H, dd, J ) 2.6 and 8.8 Hz,
C6-H), 7.60 (1H, d, J ) 8.8 Hz, C5-H), and 10.36 (1H, s,
exchanged with D2O, OH); MS (FAB+) m/z (rel intensity) 191.1
[100, (M + H)+]; MS (FAB-) m/z (rel intensity) 343.3 [46, (M
+ NBA)-], 189.2 [100, (M - H)-]. Anal. (C11H10O3) C, H.
*These assignments were aided by a 1H NMR spectrum of 10
in DMSO-d6.
3,4-Dim eth ylcou m a r in 7-O-Su lfa m a te (24). This was
prepared in a manner similar to the preparation of 12 from
23 (400 mg, 2.103 mmol), sodium hydride (1 equiv), but using
more sulfamoyl chloride (5 equiv). Purification by flash
chromatography (chloroform/acetone, 15:1 to 8:1, gradient)
gave the crude product which was further purified by recrys-
tallization from acetone/hexane (8:3) to give 24 as white
crystals (75 mg, 278.5 µmol). Upon recrystallization of the
residue, recovered from the evaporation of the mother liquor,
from acetone/hexane (2:5) gave a second crop of 24 (70 mg,
259.9 µmol, 25% overall); mp 194-196 °C; TLC (ethyl acetate/
chloroform, 1:1) Rf 0.46, cf. Rf 0.55 (23); IR (KBr) 3380, 3200,
3090, 1680, 1610, 1560, 1390, 1200; 1H NMR (270 MHz,
acetone-d6) δ 2.17 (3H, s, C3-CH3), 2.47 (3H, s, C4-CH3), 7.35
(4H, m, reduced to 2H on exchange with D2O, C6-H, C8-H,
and OSO2NH2), and 7.85 (1H, d, J ) 8.1 Hz, C5-H); MS (FAB+)
m/z (rel intensity) 270.1 [100, (M + H)+], 190.1 [10, (M -
HNSO2)+]; MS (FAB-) m/z (rel intensity) 268.2 [100, (M -
H)-], 189.2 [28, (M - H2NSO2)-]; HRMS (FAB+) m/z 270.044 82
[(M + H)+], calcd for C11H12NO5S 270.043 62. Found: C, 48.4;
H, 4.05; N, 5.34. C11H11NO5S requires C, 49.06; H, 4.12; N,
5.20.
HRMS (FAB+) m/z 272.023 27 [(M + H)+], calcd for C10H10
-
NO6S 272.022 88. Found: C, 43.8; H, 3.37; N, 5.11. C10H9-
NO6S requires C, 44.28; H, 3.34; N, 5.16.
(()-3,4-Dih yd r o-7-h yd r oxy-4-m eth ylcou m a r in (30). A
solution of 7-hydroxy-4-methylcoumarin (1.0 g, 5.676 mmol)
in ethanol (100 mL) was shaken with Pd-C (10%, 3.0 g) under
hydrogen at 75 psi for 3 days. After removal of the supported
catalyst by suction filtration, the filtrate was evaporated, and
the residue obtained was fractionated by flash chromatography
(diethyl ether). The yellow/green syrupy crude product was
further purified by recrystallization from chloroform/hexane
(5:3) to give 30 as white crystals (719 mg, 4.035 mmol, 71%):
1
mp 105-111 °C; H NMR (270 MHz, CDCl3) δ 1.31 (3H, d, J
) 7.0 Hz, CH3), 2.55 (1H, dd, J C4-H,C3-H ) 7.5 Hz and
J C3-H
Hz and J C3-H
) 15.8 Hz, C3-HA), 2.83 (1H, dd,AJ C4-H,C3-HB ) 5.5
,C3-H
B
A
) 15.8 Hz, C3-HB), 3.11 (1H, m, C4-H),
,C3-H
A
B
5.23 (1H, s, exchanged with D2O, OH), 6.62 (2H, m, C6-H and
C8-H), and 7.08 (1H, d, J ) 7.9 Hz, C5-H). Anal. (C10H10O3)
C, H.
(()-3,4-Dih yd r o-4-m eth ylcou m a r in 7-O-Su lfa m a te (31).
This was prepared from 30 (500 mg, 2.806 mmol) in a manner
similar to the preparation of 12. Purification by flash chro-
matography (chloroform/acetone, 20:1 to 4:1, gradient) gave
the crude product which was further purified by recrystalli-
zation from ethyl acetate/hexane (1:1) to give 31 as white
crystals (357 mg, 1.387 mmol, 49%): mp 152-155 °C; 1H NMR
(400 MHz, acetone-d6) δ 1.32 (3H, d, J ) 7.0 Hz, CH3), 2.63
(1H, dd, J C4-H,C3-H ) 7.0 Hz and J C3-H
) 15.9 Hz, C3-
,C3-H
A
HA), 2.93 (1H, dd, JAC4-H,C3-H ) 5.6 Hz aBnd J C3-H
B ) 16.0
,C3-H
A
B
Hz, C3-HB), 3.30 (1H, m, C4-H), 7.00 (1H, d, J ) 2.4 Hz, C8-
H), 7.11 (1H, dd, J ) 2.3 and 8.4 Hz, C6-H), 7.22 (2H, br s,
exchanged with D2O, OSO2NH2), and 7.44 (1H, d, J ) 8.2 Hz,
C5-H); MS (FAB+) m/z (rel intensity) 411.0 [24, (M + H +
NBA)+], 258.1 [100, (M + H)+]; MS (FAB-) m/z (rel intensity)
410.0 [22, (M + NBA)-], 256.0 [100, (M - H)-]. Anal. (C10H11
NO5S) C, H, N.
-
3,4-Dih yd r o-7-h yd r oxycou m a r in (32). A solution of
7-hydroxycoumarin (2.0 g, 12.34 mmol) in ethanol (120 mL)
was shaken with Pd-C (10%, 2.0 g) under hydrogen at 40 psi
for 24 h. After removal of the supported catalyst by suction
filtration, the filtrate was evaporated and the residue obtained
was fractionated by flash chromatography (diethyl ether). The
light brown crude product was further purified by recrystal-
lization from toluene to give 32 as white crystals (1.25 g, 7.623
mmol, 61%): mp 134-137 °C (lit.59 mp 132-133 °C); 1H NMR
(400 MHz, CDCl3) δ 2.78 (2H, m, C3-H2), 2.93 (2H, t, J ≈ 7.0
Hz, C4-H2), 5.42 (1H, s, exchanged with D2O, OH), 6.60 (2H,
m, C6-H and C8-H), and 7.04 (1H, d, J ) 8.8 Hz, C5-H); MS
(FAB+) m/z (rel intensity) 165.1 [100, (M + H)+]; HRMS
(FAB+) m/z 165.056 23 [(M + H)+], calcd for C9H9O3 165.055 17.
Anal. (C9H8O3) C, H.
7-Hyd r oxy-4-m eth ylcou m a r in 6-O-Su lfa m a te (26) a n d
4-Meth ylcou m a r in 6,7-O,O-Disu lfa m a tes (27). These were
obtained when 6,7-dihydroxy-4-methylcoumarin (500 mg, 2.550
mmol) was sulfamoylated in a manner similar to the prepara-
tion of 12 using sodium hydride (2 equiv) and sulfamoyl
chloride (5 equiv). A 120 mg sample of the crude material (330
mg) was purified by fractionating on preparative TLC (ethyl
acetate/acetone/hexane, 2:1:1).
The band at Rf 0.74, cf. SM (Rf 0.46), gave a beige residue
which was further purified by recrystallization from ethyl
acetate/hexane (1:2) to give 27 as white crystals (24 mg, 68.53
µmol, 7%): mp 169-172 °C; 1H NMR (400 MHz, DMSO-d6) δ
2.44 (3H, s, CH3), 6.50 (1H, s, C3-H), 7.55 (1H, s, C8-H), 7.81
(1H, s, C5-H), 8.21 (2H, br s, exchanged with D2O, OSO2NH2),
and 8.40 (2H, br s, exchanged with D2O, OSO2NH2); MS
(FAB+) m/z (rel intensity) 350.9 [100, (M + H)+], 272.0 [30,
(M + H - SO2NH)+]; MS (FAB-) m/z (rel intensity) 348.9 [100,
(M - H)-], 269.9 [40, (M - SO2NH2)-], 191.0 [30, (M - H -
3,4-Dih yd r ocou m a r in 7-O-Su lfa m a te (33). This was
prepared from 32 (500 mg, 3.049 mmol) in a manner similar
to the preparation of 12. Purification by flash chromatography
(chloroform/acetone, 20:1 to 4:1, gradient) gave the crude
product which was further purified by recrystallization from
ethyl acetate/hexane (2:1) to give 33 as white crystals (378 mg,
1
1.554 mmol, 51%): mp 169.5-171.5 °C; H NMR (400 MHz,
DMSO-d6/CDCl3, ca. 1:15) δ 2.80 (2H, t, J ≈ 7.3 Hz, C3-H2),
3.02 (2H, t, J ≈ 7.3 Hz, C4-H2), 7.10 (2H, m, C6-H and C8-H),
7.16 (2H, br s, exchanged with D2O, OSO2NH2), and 7.22 (1H,
d, J ) 7.6 Hz, C5-H); MS (FAB+) m/z (rel intensity) 397.0 [30,
(M + H + NBA)+], 244.1 [100, (M + H)+], 164.1 [26, (M - SO2-

1080 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Woo et al.
NH)+]; MS (FAB-) m/z (rel intensity) 396.0 [22, (M + NBA)-],
242.0 [100, (M - H)-]; HRMS (FAB+) m/z 244.027 44 [(M +
H)+], calcd for C9H10NO5S 244.027 97. Anal. (C9H9NO5S) C,
H, N.
2,3-Dih yd r o-7-h yd r oxy-4H -1-b en zop yr a n -4-on e (34).
This compound was synthesized from resorcinol and acrylni-
trile as described by Breytenbach and Rall.56
decomposition after 24 h at room temperature but appeared
to be stable if stored under nitrogen at -20 °C. Sulfamate 39
was further purified by preparative TLC (ethyl acetate/hexane,
1:1) before analysis and bioassay: 1H NMR (400 MHz, CDCl3)
δ 4.84 (2H, m, C2-H2), 4.98 (2H, br s, exchanged with D2O,
OSO2NH2), 5.78 (1H, dt, J C2-H
≈ 3.5 Hz and J C4-H,C3-H
)
,C3-H
2
4
10.1 Hz, C3-H), 6.40 (1H, dt, J ≈ 1.6 Hz and J C3-H,C4-H ) 7.9
Hz, C4-H), 6.74 (1H, d, J ) 2.4 Hz, C8-H), 6.82 (1H, dd, J )
2.4 and 8.2 Hz, C6-H), and 6.95 (1H, d, J ) 8.2 Hz, C5-H); MS
(FAB+) m/z (rel intensity) 226 [47, (M - H)+], 147 [100, (M -
SO2NH2)+]; HRMS (FAB+) m/z 226.017 34 [(M - H)+], calcd
for C9H8NO4S 226.017 41.
2,3-Dih yd r o-7-[d im eth yl(1,1-d im eth yleth yl)silyl]-4H-1-
ben zop yr a n -4-on e (35). To a stirred mixture of 34 (800 mg,
4.873 mmol) and imidazole (1.35 g, 19.63 mmol) in DMF (5
mL) under nitrogen at room temperature was added dimethyl-
(1,1-dimethylethyl)silyl chloride (1.03 g, 6.628 mmol). After
being left to stir overnight, the reaction mixture was diluted
with diethyl ether (100 mL). The resulting mixture was then
washed with 1 M HCl (100 mL) and then brine (4 × 50 mL),
dried (MgSO4), filtered, and evaporated. The residue obtained
was fractionated by flash chromatography (ethyl acetate/
hexane, 1:8 to 1:2, gradient) to give 35 as a pale green/yellow
liquid (1.33 g, 4.777 mmol, 98%): 1H NMR (400 MHz, CDCl3,
no TMS) δ 0.24 [6H, s, Si(CH3)2], 0.98 [9H, s, C(CH3)3], 2.75
(2H, t, J ) 6.4 Hz, C3-H2), 4.50 (2H, t, J ) 6.4 Hz, C2-H2),
6.37 (1H, d, J ) 2.1 Hz, C8-H), 6.50 (1H, dd, J ) 2.3 and 8.7
Hz, C6-H), and 7.80 (1H, d, J ) 8.6 Hz, C5-H); MS (EI, 70 eV)
m/z (rel intensity) 278.2 (24, M+), 221.1 (100, [M - C(CH3)3]+),
179.1 (6, [M + H - Si(CH3)C(CH3)3]+), 85.1 (8, [SiC(CH3)3]+),
57.1 (23, [C(CH3)3]+).
2H-1-Ben zop yr a n -7-ol (38). Lithium aluminum hydride
(5.1 mL, 1.0 M in THF) was added to a stirred solution of 35
(1.28 g, 4.598 mmol) in diethyl ether (10 mL) at -78 °C under
nitrogen. The reaction mixture was allowed to warm to room
temperature over 2 h and then poured cautiously onto ice.
After acidification with hydrochloric acid (1 M, 20 mL), the
resulting mixture was extracted with diethyl ether (100 mL).
The organic layer that separated was further washed with
brine exhaustively, dried (MgSO4), filtered, and evaporated to
give crude 36 as a light brown syrup (1.14 g): TLC (ethyl
acetate/hexane, 1:1) Rf 0.51, cf. Rf 0.67 (35). This crude product
was used immediately in the next reaction without further
purification.
A mixture of the crude enantiomeric alcohols 36 (1.14 g),
triethylamine (2.5 mL, 17.76 mmol), and methanesulfonyl
chloride (350 µL, 4.432 mmol) in dichloromethane (20 mL) was
stirred at room temperature for 24 h. After the reaction
mixture was concentrated in vacuo, the residue afforded was
redissolved in diethyl ether (100 mL) and washed with water
(5 × 50 mL). The combined aqueous washes were then back-
extracted with chloroform (4 × 50 mL). The organic extracts
were combined, dried (MgSO4), filtered, and evaporated. The
crude residue was fractionated by flash chromatography (ethyl
acetate/hexane, 1:16 to 1:8, gradient) to give the silylated 2H-
1-benzopyran-7-ol (37) as a pale green/yellow oil (840 mg, 3.201
mmol, 70%): 1H NMR (270 MHz, CDCl3, no TMS) δ 0.19 [6H,
s, Si(CH3)2], 0.97 [9H, s, C(CH3)3], 4.77 (2H, m, C2-H2), 5.62
6-[Dim eth yl(1,1-d im eth yleth yl)silyl]-1-tetr a lon e (40).
The silylation reaction was performed in a manner similar to
35. 6-Hydroxy-1-tetralone (45) (1.8 g, 11.10 mmol), DMF (10
mL), imidazole (1.9 g, 27.74 mmol), and dimethyl(1,1-dimeth-
ylethyl)silyl chloride (2.1 g, 13.51 mmol) were used. The
residue obtained from the workup was fractionated by flash
chromatography (ethyl acetate/hexane, 1:8) to give 40 as a pale
1
yellow liquid (2.93 g, 10.60 mmol, 96%); H NMR (270 MHz,
CDCl3, no TMS) δ 0.23 [6H, s, Si(CH3)2], 0.98 [9H, s, C(CH3)3],
2.13 (2H, quintet, J ≈ 6 Hz, C3-H2), 2.60 (2H, t, J ≈ 6.5 Hz,
C4-H2), 2.89 (2H, t, J ≈ 6.0 Hz, C2-H2), 6.65 (1H, d, J ) 2.7
Hz, C5-H), 6.74 (1H, dd, J ) 2.3 and 8.7 Hz, C7-H) and 7.95
(1H, d, J ) 8.6 Hz, C8-H); MS (EI, 70 eV) m/z (rel intensity)
276.2 (35, M+), 261.3 [7, (M - CH3)+], 219.1 (100, [M -
C(CH3)3]+), 192.1 (8, [M + H - SiC(CH3)3]+), 163.1 (8, [M +
2H - Si(CH3)2C(CH3)3]+), 115.1 (12, [Si(CH3)2C(CH3)3]+), 57.1
(15, [C(CH3)3]+).
7,8-Dih yd r on a p h th -2-ol (43). The multistep synthesis to
38 from 35 was used to prepare this compound from 40. 40
(2.49 g, 9.009 mmol), diethyl ether (30 mL), and lithium
aluminum hydride (9.9 mL, 1.0 M in THF) were used, and
crude 41 was afforded as a pale yellow syrup (2.10 g).
Crude enantiomeric alcohols 41 (2.10 g), triethylamine (4.5
mL, 31.96 mmol), methanesulfonyl chloride (700 µL, 8.862
mmol), and dichloromethane (50 mL) were used. Fractionation
of the crude residue by flash chromatography (ethyl acetate/
hexane, 1:8) gave the silylated 7,8-dihydronaphth-2-ol (42) as
a pale yellow oil (1.40 g, 5.376 mmol, 60%): 1H NMR (270
MHz, CDCl3, no TMS) δ 0.19 [6H, s, Si(CH3)2], 0.98 [9H, s,
C(CH3)3], 2.28 (2H, m, C7-H2), 2.73 (2H, t, J ) 8.2 Hz, C8-H2),
5.89 (1H, dt, J C7-H
≈ 4.4 Hz and J C5-H,C6-H ) 9.5 Hz, C6-
,C6-H
H), 6.40 (1H, d, J 2 ) 9.7 Hz, C5-H), 6.62 (2H, m, C1-H and
C3-H), and 6.87 (1H, d, J ) 8.6 Hz, C4-H); MS (EI, 70 eV) m/z
(rel intensity) 260.2 (38, M+), 203.1 (100, [M - C(CH3)3]+).
Silylated 7,8-dihydronaphth-2-ol (42) (500 mg, 1.920 mmol),
diethyl ether (25 mL), and tetra-n-butylammonium fluoride
(2.0 mL, 1.0 M in THF) were used and gave 7,8-dihydronaphth-
2-ol (43) as an unstable light yellow/brown syrup (390 mg).
This crude 43 was sulfamoylated immediately.
7,8-Dih yd r on a p h th a len e 2-O-Su lfa m a te (44). This was
prepared from crude 43 (390 mg) in a manner similar to the
preparation of 12. Purification by flash chromatography (ethyl
acetate/hexane, 1:8 to 1:4, gradient) gave the crude product
which was further purified by recrystallization from ethyl
acetate/hexane (1:10) to afford 44 as fine white crystals (210
(1H, dt, J C2-H
≈ 3.5 Hz and J C4-H,C3-H ) 9.7 Hz, C3-H),
,C3-H
6.29 (1H, d, J 2) 2.9 Hz, C8-H), 6.35 (1H, dd, J ) 2.4 and 8.1
Hz, C6-H), 6.37 (1H, d, J ) 9.7 Hz, C4-H), and 6.81 (1H, d, J
) 8.1 Hz, C5-H); MS (EI, 70 eV) m/z (rel intensity) 262.2 (55,
M+), 205.1 (100, [M - C(CH3)3]+), 163.0 (13, [M + H - Si-
(CH3)C(CH3)3]+).
1
mg, 822.7 µmol, 43%): mp 100-102 °C; H NMR (270 MHz,
CDCl3) δ 2.32 (2H, m, C7-H2), 2.81 (2H, t, J ) 8.2 Hz, C8-H2),
A mixture of tetra-n-butylammonium fluoride (2.0 mL, 1 M
in THF), 37 (500 mg, 1.905 mmol), and diethyl ether (25 mL)
was stirred at room temperature for 10 min. The reaction
mixture was then diluted with diethyl ether (100 mL) and the
resulting mixture washed with brine (5 × 50 mL), dried
(MgSO4), filtered, and evaporated to give crude 2H-1-benzopy-
ran-7-ol (38) as an unstable light brick red liquid (380 mg);
TLC (ethyl acetate/hexane, 1:2) Rf 0.53, cf. Rf 0.68 (37). This
crude 38 was sulfamoylated immediately.
2H-1-Ben zop yr a n 7-O-Su lfa m a te (39). This was pre-
pared from crude 38 (380 mg) in a manner similar to the
preparation of 12. Purification of the crude material obtained
immediately by flash chromatography (ethyl acetate/hexane,
1:4 to 1:1, gradient) gave 39 as a greenish yellow syrup (240
mg, 1.056 mmol, 55%). This syrup exhibited noticeable
4.93 (2H, br s, exchanged with D2O, OSO2NH2), 6.07 (1H, dt,
J C7-H
≈ 4.6 Hz and J C5-H,C6-H ) 9.5 Hz, C6-H), 6.45 (1H,
,C6-H
2
d, J ) 9.7 Hz, C5-H), and 7.0-7.15 (3H, m, Ar-H); MS (FAB+)
m/z (rel intensity) 225.1 (100, M+), 145.1 [30, (M - SO2NH2)+];
MS (FAB-) m/z (rel intensity) 224.0 [100, (M - H)-]. Anal.
(C10H11NO3S) C, H, N.
6-Hyd r oxy-1-tetr a lon e (45). 6-Methoxy-1-tetralone (6.0
g, 34.04 mmol) was added to a stirred suspension of aluminum
chloride (11.3 g, 85.1 mmol) in anhydrous toluene (150 mL)
at room temperture, under nitrogen. The reaction mixture was
refluxed for 30 min, cooled, and cautiously quenched with
water. The resulting mixture was then extracted with ethyl
acetate (150 mL). The organic portion that separated was
washed with water exhaustively, dried (MgSO4), filtered, and
evaporated in vacuo. Recrystallization of the orange brown

Potent Inhibitors of Steroid Sulfatase
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1081
residue from hot toluene (three times) gave 45 as fine beige
crystals (5.09 g, 31.38 mmol, 92%): mp 151-155 °C (lit.60 mp
148-152 °C); 1H NMR (270 MHz, CDCl3) δ 2.11 (2H, quintet,
J ≈ 6 Hz, C3-H2), 2.63 (2H, t, J ≈ 6.0 Hz, C4-H2), 2.91 (2H, t,
J ≈ 6.0 Hz, C2-H2), 6.54 (1H, br s, exchanged with D2O, OH),
6.70 (1H, d, J ) 2.4 Hz, C5-H), 6.78 (1H, dd, J ) 2.6 and 8.6
Hz, C7-H), and 7.98 (1H, d, J ) 8.6 Hz, C8-H); MS (EI, 70 eV)
m/z (rel intensity) 162.1 (60, M+), 134.0 (100). Anal. (C10H10O2)
C, H.
1-Tetr a lon e 6-O-Su lfa m a te (46). This was prepared from
45 (1.0 g, 6.165 mmol) in a manner similar to the preparation
of 12. Purification by flash chromatography (chloroform/
acetone, 8:1 to 2:1, gradient) gave the crude product which was
further purified by recrystallization from acetone/hexane (7:
4) to afford 46 as dull white rod-shaped crystals (447 mg, 1.853
mmol, 30%): mp 174-175 °C; 1H NMR (270 MHz, acetone-
d6) δ 2.13 (2H, quintet, J ≈ 6 Hz, C3-H2), 2.62 (2H, t, J ≈ 6.6
Hz, C4-H2), 3.02 (2H, t, J ≈ 5.8 Hz, C2-H2), 7.28 (4H, m,
reduced to 2H upon exchange with D2O, C5-H, C7-H, and
OSO2NH2), and 7.99 (1H, d, J ) 8.8 Hz, C8-H); MS (FAB+)
m/z (rel intensity) 242.1 [100, (M + H)+]; MS (FAB-) m/z (rel
intensity) 240.0 [100, (M - H)-], 161.0 [10, (M - SO2NH2)-].
Anal. (C10H11NO4S) C, H, N.
7-Hyd r oxy-1-tetr a lon e (47). This was prepared from
7-methoxy-1-tetralone (4.0 g, 22.69 mmol) in a manner similar
to the preparation of 45. Recrystallization of the orange brown
residue from acetone/hexane (1:5) afforded 47 as fine white
crystals (3.49 g, 21.52 mmol, 95%): mp 165-167 °C; 1H NMR
(270 MHz, acetone-d6) δ 2.07 (2H, m, C3-H2), 2.55 (2H, t,
J ≈ 6.5 Hz, C4-H2), 2.87 (2H, t, J ) 5.9 Hz, C2-H2), 7.02
(1H, dd, J ) 2.9 and 8.2 Hz, C6-H), 7.17 (1H, d, J ) 8.1 Hz,
C5-H), 7.39 (1H, d, J ) 2.6 Hz, C8-H), and 8.55 (1H, br s,
exchanged with D2O, OH); MS (FAB+) m/z (rel intensity) 163.1
[100, (M + H)+]; MS (FAB-) m/z (rel intensity) 314.3 [65, (M
- H + NBA)-], 161.1 [100, (M - H)-]. Anal. (C10H10O2) C,
H.
1-Tetr a lon e 7-O-Su lfa m a te (48). This was prepared from
47 (1.0 g, 6.164 mmol) in a manner similar to the preparation
of 12. Purification by flash chromatography (chloroform/
acetone, 4:1) gave the crude product which was further purified
by recrystallization from acetone/hexane (1:1) to give 48 as
beige crystals (433 mg, 1.797 mmol, 29%): mp 150-152 °C;
1H NMR (270 MHz, acetone-d6) δ 2.16 (2H, m, C3-H2), 2.63
(2H, t, J ≈ 6.5 Hz, C4-H2), 3.02 (2H, t, J ≈ 6 Hz, C2-H2), 7.21
(2H, br s, exchanged with D2O, OSO2NH2), 7.47 (2H, m, C5-H
and C6-H), and 7.84 (1H, d, J ≈ 1 Hz, C8-H); MS (FAB+) m/z
(rel intensity) 242.2 [100, (M + H)+]; MS (FAB-) m/z (rel
intensity) 394.1 [43, (M + NBA)-], 240.1 [100, (M - H)-].
Anal. (C10H11NO4S) C, H, N.
HRMS (FAB+) m/z 149.058 79 [(M + H)+], calcd for C9H9O2
149.060 25. Found: C, 72.40; H, 5.46. C9H8O2 requires C,
72.96; H, 5.44.
1-In d a n on e 5-O-Su lfa m a te (51). This was prepared from
50 (250 mg, 1.687 mmol) in a manner similar to the prepara-
tion of 12. Purification by flash chromatography (chloroform/
acetone, 4:1) gave the crude product which was further purified
by recrystallization from acetone/hexane (1:2) to afford 51 as
beige crystals (121 mg, 532.9 µmol, 31%): mp 174-176 °C;
1H NMR (270 MHz, acetone-d6) δ 2.68 (2H, t, J ≈ 6 Hz, C3-
H2), 3.19 (2H, t, J ≈ 5.9 Hz, C2-H2), 7.36 (3H, m, 2H exchanged
with D2O, OSO2NH2 and C6-H), 7.51 (1H, s, C4-H) and 7.71
(1H, d, J ) 8.5 Hz, C7-H); MS (FAB+) m/z (rel intensity) 228.1
[100, (M + H)+], 147.1 [10, (M - SO2NH2)+]; MS (FAB-) m/z
(rel intensity) 226.1 [100, (M - H)-], 147.1 [20, (M -
SO2NH2)-]; HRMS (FAB+) m/z 228.032 69 [(M + H)+], calcd
for C9H10NO4S 228.033 05. Anal. (C9H9NO4S) C, H, N.
6-Hyd r oxy-1-in d a n on e (52). This was prepared from
6-methoxy-1-indanone (2.5 g, 14.95 mmol) in a manner similar
to the preparation 45. Recrystallization of the yellow/brown
residue from hot toluene afforded 52 as light yellow/brown
1
crystals (2.0 g, 13.50 mmol, 90%): mp 156-158 °C; H NMR
(270 MHz, DMSO-d6/CDCl3, ca. 1:20) δ 2.66 (2H, m, C3-H2),
3.04 (2H, t, J ≈ 5.7 Hz, C2-H2), 7.13 (2H, m, C5-H and C7-H),
7.31 (1H, d, J ) 8.9 Hz, C4-H), and 9.23 (1H, br s, exchanged
with D2O, OH). Anal. (C9H8O2) C, H.
1-In d a n on e 6-O-Su lfa m a te (53). This was prepared from
52 (545 mg, 3.683 mmol) in a manner similar to the prepara-
tion of 12. Purification by flash chromatography (chloroform/
acetone, 4:1) gave the crude product which was further purified
by recrystallization from acetone/hexane (1:2) to give 53 as
yellow crystals (315 mg, 1.388 mmol, 37%): mp 155-157 °C;
1H NMR (270 MHz, acetone-d6) δ 2.71 (2H, m, C3-H2), 3.19
(2H, t, J ≈ 5.9 Hz, C2-H2), 7.26 (2H, br s, exchanged with D2O,
OSO2NH2), 7.57 (2H, m, C5-H and C7-H) and 7.67 (1H, d, J )
9.2 Hz, C4-H); MS (FAB+) m/z (rel intensity) 228.0 [100, (M
+ H)+], 148.0 [15, (M - SO2NH)+]; MS (FAB-) m/z (rel
intensity) 225.9 [100, (M - H)-], 146.9 [8, (M - SO2NH2)-];
HRMS (FAB+) m/z 228.033 56 [(M + H)+], calcd for C9H10
NO4S 228.033 05. Anal. (C9H9NO4S) C, H, N.
-
2-Nitr op h en ol O-Su lfa m a te (54) a n d Azom eth in e (55).
2-Nitrophenol (1.391 g, 10.0 mmol) was sulfamoylated in a
manner similar to the preparation of 12. Purification by flash
chromatography (ethyl acetate/hexane, 1:1) gave the crude
2-nitrophenol O-sulfamate which was further purified by
recrystallization from hot chloroform to afford 54 as white
crystals (333 mg, 745.8 µmol). The residue recovered, from
the evaporation of the mother liquor, was recrystallized from
chloroform/hexane to give further crops of 54 (a total of 252
1-In d a n on e 4-O-Su lfa m a te (49). This was prepared from
4-hydroxy-1-indanone (500 mg, 3.375 mmol) in a manner
similar to the preparation of 12. Purification by flash chro-
matography (chloroform/acetone, 8:1) gave the crude product
which was further purified by recrystallization from ethyl
acetate/hexane (1:3) to give 49 as brown/yellow crystals (80
1
mg, 564.4 µmol, 26% overall); mp 102-103 °C; H NMR (270
MHz, CDCl3) δ 5.29 (2H, br s, exchanged with D2O, OSO2NH2),
7.48 (1H, m, C4-H or C5-H), 7.66 (1H, dd, J ) 1.5 and 8.3 Hz,
C3-H or C6-H), 7.71 (1H, m, C4-H or C5-H), and 8.05 (1H, dd,
J ) 1.5 and 8.3 Hz, C3-H or C6-H); MS (EI, 70 eV) m/z (rel
intensity) 218 (2, M+), 139 [100, (M - SO2NH)+]; MS (CI,
isobutane) m/z (rel intensity) 219 [27, (M + H)+], 202 [10, (M
+ H - NH3)+], 140 [100, (M + H - SO2NH)+], 122 [15, (M -
OSO2NH2)+]. Anal. (C6H6N2O5S) C, H, N.
1
mg, 352.4 µmol, 11%): mp 193-196 °C; H NMR (270 MHz,
acetone-d6) δ 2.67 (2H, m, C3-H2), 3.25 (2H, t, J ≈ 5.9 Hz, C2-
H2), 7.39 (2H, br s, exchanged with D2O, OSO2NH2), 7.52 (1H,
t, J ) 7.7 Hz, C6-H), 7.64 (2H, m, C5-H and C7-H); MS (FAB+)
m/z (rel intensity) 228.1 [100, (M + H)+]; MS (FAB-) m/z (rel
intensity) 226.0 [100, (M - H)-]; HRMS (FAB+) m/z 228.031 21
[(M + H)+], calcd for C9H10NO4S 228.033 05. Anal. Found:
C, 46.0; H, 3.94; N, 5.83. C9H9NO4S requires C, 47.57; H, 3.99;
N, 6.16.
A fraction of higher polarity was also collected from the flash
column which upon recrystallization from hot isopropyl alcohol
afforded 55 as white crystals (163 mg, 597.1 µmol, 6%): mp
122-123 °C; 1H NMR (270 MHz, CDCl3) δ 3.03 (3H, s,
N-CH3), 3.22 (3H, s, N-CH3), 7.39 (1H, m, Ar-H), 7.65 (2H,
m, Ar-H), 7.90 (1H, dd, J ) 1.3 and 8.2 Hz, Ar-H), and 8.09
(1H, s, NdCH); 13C NMR (100.4 MHz, CDCl3) δ 35.79 (q), 41.99
(q), 125.33 (d), 125.93 (d), 126.85 (d), 134.23 (d), 142.94 (s),
143.21 (s), 161.36 (d); MS (EI, 70 eV) m/z (rel intensity) 273
(4, M+), 135 [100, (Me2NCHdNSO2)+]; MS (CI, isobutane) m/z
(rel intensity) 274 [28, (M + H)+], 135 [100, (Me2NCHdNSO2)+].
Anal. (C9H11N3O5S) C, H, N.
5-Hyd r oxy-1-in d a n on e (50). This was prepared from
5-methoxy-1-indanone (1.0 g, 6.166 mmol) in a manner similar
to the preparation of 45. Recrystallization of the orange brown
residue from acetone/hexane (1:5) gave 50 as fine yellow/brown
crystals (787 mg, 5.312 mmol, 86%): mp 182-185 °C; 1H NMR
(270 MHz, acetone-d6) δ 2.55 (2H, m, C3-H2), 3.04 (2H, t,
J ≈ 6 Hz, C2-H2), 6.9 (2H, m, C4-H and C6-H), 7.53 (1H,
d, J ) 8.1 Hz, C7-H), and 9.37 (1H, br s, exchanged with
D2O, OH); MS (FAB+) m/z (rel intensity) 149.1 [100, (M +
H)+]; MS (FAB-) m/z (rel intensity) 147.1 [100, (M - H)-];
Ack n ow led gm en t. This work was supported by The
Cancer Research Campaign.

1082 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
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