Synthesis, in vitro and in vivo activity of benzophenone-based inhibitors of steroid sulfatase

Article abstract of DOI:10.1016/j.bmc.2004.02.040

Steroid sulfatase (STS) is an important new therapeutic target in oncology. Attempts to design nonsteroidal STS inhibitors, because of the oestrogenicity of the original lead oestrone 3-O-sulfamate in rodents, have led to the discovery of benzophenone-4,4′-O,O-bis-sulfamate (BENZOMATE, 3). The nonfused bicyclic BENZOMATE is a highly potent STS inhibitor in vitro, inhibiting STS activity in intact MCF-7 breast cancer cells by >70% at 0.1μM and in placental microsomes by >98% at 10μM. When MCF-7 cells were pre-treated with 3 at 1μM and then washed to remove unbound inhibitor, the initial 94% inhibition was reduced to 89% suggesting that 3, like other sulfamate-based STS inhibitors, inhibits the enzyme irreversibly. This agent also inhibits rat liver STS activity by 84% and 93% respectively 24h after a single dose of 1 or 10mg/kg, demonstrating that BENZOMATE possesses similar in vivo potency to the established potent nonsteroidal inhibitor 667COUMATE. Several modifications were made to BENZOMATE structurally and effects on in vitro activity were examined. These structure-activity relationship studies show that its carbonyl and bis-sulfamate groups are pivotal for activity, although conformational flexibility is not required. Two rigid anthraquinone-based sulfamate derivatives however showed inhibitory activity significantly better than BENZOMATE in the MCF-7 cell assay. BENZOMATE and related analogues therefore represent an important class of non-steroidal STS inhibitor and lead compounds for future drug design.

Full text of DOI:10.1016/j.bmc.2004.02.040

Bioorganic & Medicinal Chemistry 12 (2004) 2759–2772
Synthesis, in vitro and in vivo activity of benzophenone-based
inhibitors of steroid sulfatase
Hatem A. M. Hejaz,a L. W. Lawrence Woo,a Atul Purohit,b Michael J. Reedb
and Barry V. L. Pottera,*
aMedicinal Chemistry, Department of Pharmacy and Pharmacology and Sterix Ltd., University of Bath, Bath BA2 7AY, UK
bEndocrinology and Metabolic Medicine and Sterix Ltd., Faculty of Medicine, Imperial College, St. Mary’s Hospital,
London W2 1NY, UK
Received 1 October 2003; accepted 27 February 2004
Available online 15 April 2004
Abstract—Steroid sulfatase (STS) is an important new therapeutic target in oncology. Attempts to design nonsteroidal STS
inhibitors, because of the oestrogenicity of the original lead oestrone 3-O-sulfamate in rodents, have led to the discovery of ben-
zophenone-4,40-O,O-bis-sulfamate (BENZOMATE, 3). The nonfused bicyclic BENZOMATE is a highly potent STS inhibitor in
vitro, inhibiting STS activity in intact MCF-7 breast cancer cells by >70% at 0.1 lM and in placental microsomes by >98% at
10 lM. When MCF-7 cells were pre-treated with 3 at 1 lM and then washed to remove unbound inhibitor, the initial 94% inhibition
was reduced to 89% suggesting that 3, like other sulfamate-based STS inhibitors, inhibits the enzyme irreversibly. This agent also
inhibits rat liver STS activity by 84% and 93% respectively 24 h after a single dose of 1 or 10 mg/kg, demonstrating that BEN-
ZOMATE possesses similar in vivo potency to the established potent nonsteroidal inhibitor 667COUMATE. Several modifications
were made to BENZOMATE structurally and effects on in vitro activity were examined. These structure–activity relationship
studies show that its carbonyl and bis-sulfamate groups are pivotal for activity, although conformational flexibility is not required.
Two rigid anthraquinone-based sulfamate derivatives however showed inhibitory activity significantly better than BENZOMATE in
the MCF-7 cell assay. BENZOMATE and related analogues therefore represent an important class of non-steroidal STS inhibitor
and lead compounds for future drug design.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
cancer (HDBC) as a stand-alone agent or in conjunction
with an aromatase inhibitor. A recent development in
this area has seen the validation of the concept of dual
aromatase and sulfatase inhibition by a single agent.6 In
addition to its role as an anti-endocrine agent, the
therapeutic potential of a STS inhibitor has now been
widened, as recent evidence has suggested that inhibition
of STS might have a clinical role in androgen-dependent
skin diseases,7 cognitive dysfunction8;9 and also immune
function.10 For this reason, emphasis has been placed on
the development of nonsteroidal inhibitors that either
themselves, or through their metabolites, are unlikely to
exert unwanted endocrinological effects.
Steroid sulfatase (STS) is a new therapeutic target in
oncology. Since the discovery of oestrone 3-O-sulfamate
(EMATE) as a highly potent irreversible inhibitor of
STS, considerable progress has been made in developing
a number of potent nonsteroidal/steroidal STS inhibi-
tors. Highly potent steroidal analogues reported to date
include some A-ring1 and D-ring modified2–5 derivatives
of EMATE. Apart from the N-propyl- and N-(10-pyri-
din-30-ylmethyl)piperidinedione
derivatives
of
EMATE,5 as is common with other steroidal com-
pounds, these EMATE derivatives are potentially oes-
trogenic in vivo in rodents.
Some impressive inhibitory activities have been
observed with coumarin sulfamates, especially those
compounds from the tricyclic series, with 667COU-
MATE11 being shown to be some 3-fold more potent
than EMATE against STS in a placental microsomes
preparation. 667COUMATE retains all the essential
features of EMATE such as oral activity and an
The primary aim for developing an STS inhibitor has
been for the treatment of hormone-dependent breast
* Corresponding author. Tel.: +44-1225-386-639; fax: +44-1225-386-
114; e-mail: prsbvlp@bath.ac.uk
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.040

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H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
irreversible mechanism of enzyme inactivation, except
that it lacks oestrogenic activity. Since it is expected that
only a non-oestrogenic STS inhibitor will be of use in
endocrine therapy, 667COUMATE has been selected as
the first Phase I trial candidate for treating HDBC.
X
OH
X
X
(2)
(1)
Because of the structural resemblance of the naturally
occurring flavonoids to oestrogens, sulfamates of flav-
ones, isoflavones and flavanones were prepared and
shown to be moderate inhibitors of STS.12 Further
exploitation of this class of compound by others led to a
series of chromenone- and thiochromenone-based sul-
famates, of which 2-(1-adamantyl)-4H-thiochromen-
4-on-6-O-sulfamate was found to be about 170-fold
more potent than EMATE in vitro.13
Figure 1. Structures of diethylstilboestrol mono-sulfamate (1) and
diethylstilboestrol bis-sulfamate (2). X ¼ OSO2NH2.
were considerably more potent inhibitors than the fused
bicyclic tetrahydronaphthol (THN) sulfamates.18
DES-bis-sulfamate (2) was found to have an IC50 value
of 10 nM as assessed in intact MCF-7 breast cancer
cells.21 Despite these encouraging results, DES is a
known potent oestrogen and toxic compound, which
would restrict the use of its bis-sulfamoylated derivative
in treating HDBC. However, the finding that it was not
necessary to have a fused ring system for STS inhibition
encouraged us to synthesise a series of sulfamate ana-
logues with a non-fused ring structure. A prime objec-
tive of our design strategy is to introduce functionalities
into such a structural system that would increase the
sulfamoyl group transfer ability of the inhibitors. We
now report that benzophenone sulfamates, and their
analogues, are potent inhibitors of STS with in vivo
activity. Structural modifications to the lead inhibitor
have also been made in order to understand more fully
the structure–activity relationships (SARs) for this class
of nonsteroidal STS inhibitor. While this work was
being prepared for publication, in vitro activities
of some related sulfamates also reported here were
published by other groups.17;22
Recent work by Poirier and co-workers14 has shown
that 1-(p-sulfamoyloxyphenyl)-5-(p-tert-butylbenzyl)-5-
undecanol and related congeners are highly potent STS
inhibitors in vitro. Simple p-sulfamoylated benzoic acid
esters,15 substituted phenyl sulfamates16 and 4-sulfa-
moylated phenyl ketones17 have been pursued as STS
inhibitors, although none of these agents showed potent
in vitro inhibitory activity. The IC50 values for the best
two inhibitors in these series of compounds were both
reported to be 3.4 lM, some 7-fold higher than that
(0.5 lM) reported for EMATE in the same assay.17 It is
interesting to note that this IC50 value for EMATE
reported by Ahmed et al. is much higher than those
values (18–80 nM) obtained by other groups from sim-
ilar placental microsomal preparations.11;18;19
Despite the structural diversity of irreversible STS
inhibitors reported to date, they all share and support
our proposed pharmacophore, that the minimal struc-
tural requirement for STS inhibition is a phenol sulfa-
mate ester and that substituents, which exploit
favourable hydrophobic interactions with the enzyme
active site will confer higher potency to the inhibitors.20
In addition, our work on coumarin sulfamates11;20 and
on the 4-nitro analogue of EMATE1 demonstrated that
sulfamoylated inhibitors that presumably are able to
transfer more effectively their sulfamoyl group to an
essential amino acid residue in the enzyme active site
during the inactivation process are more potent STS
inhibitors. It has been reasoned that the sulfamoyl
group transfer ability of an aryl sulfamate, and hence its
inhibitory activity of STS, is related to the leaving group
ability of the parent phenol. A substantial decrease in
the inhibitory activities of analogues was observed in
our structure–activity relationship (SAR) studies on
coumarin sulfamates20 when the conjugated coumarin
ring system was disrupted such as via the saturation of
their C3–C4 double bond. The overall effect of these
disruptions is an increase in the pKa value of the
resulting phenols, rendering the parent coumarins
poorer leaving groups and hence their sulfamates
weaker inhibitors of STS.
2. Chemistry
The sulfamate derivatives of 4,40-dihydroxybenzophe-
none (3–5, Scheme 1) were synthesised as follows. When
4,40-dihydroxybenzophenone in N,N-dimethylform-
amide (DMF) was treated with 3 equiv of sodium hy-
dride followed by an excess of sulfamoyl chloride,
O
O
+
OSO2N=CHNMe2
X
X
X
(3, major)
(4)
i
O
OH
HO
O
ii
O
+
OH
X
X
X
(5, major)
(3)
Our initial studies to develop a non-steroidal STS inhib-
itor involved the sulfamoylation of diethylstilboestrol
(DES), which has two nonfused aryl rings. The mono-(1,
Fig. 1) and bis-sulfamate (2, Fig. 1) derivatives of DES21
Scheme 1. Synthesis of benzophenone derivatives 3–5. Reagents and
conditions: (i) 3NaH/DMF, excess H2NSO2Cl; (ii) NaH/DMF, excess
H2NSO2Cl. X ¼ OSO2NH2.

H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
2761
benzophenone-4,40-O,O-bis-sulfamate (3, BENZO-
O
O
OH
MATE), was obtained as the major product. From the
same reaction, a minor product was also isolated (7%)
and it was subsequently identified as the azomethine
adduct 4, which was formed between the bis-sulfamate 3
and the reaction medium DMF. A similar reaction be-
tween a sulfamate and DMF in the presence of a base
and the possible mechanism involved has already been
reported in the synthesis of 2-nitrophenol-O-sulfamate
under related conditions.20 When 4,40-dihydroxybenzo-
phenone was sulfamoylated after treating with only
1 equiv of sodium hydride, the mono-sulfamate (40-hy-
droxybenzophenone-4-O-sulfamate 5) was obtained as
the major product and its separation from the bis-sul-
famate (3) and the starting material, was achieved by
preparative TLC.
Cl
Cl
i
+
O
(12)
HO
OH
O
O
O
ii
X
O
(13)
O
Scheme 2. Synthesis of benzo[b]naphtho[2,3-d]-furan-6,11-dione-3-O-
sulfamate (13). Reagents and conditions: (i) NaOEt/ethanol, 12 h; (ii)
NaH/DMF, H2NSO2Cl. X ¼ OSO2NH2.
4,40-dihydroxybenzophenone in ethanol in the presence
of Pd–C (10%) at room temperature under balloon
pressure for 6 h.24 Sulfamoylation of compound 17
(Scheme 3) in the usual manner gave a mixture of
compounds 18 and 19, which were separated by flash
chromatography.
A number of structural variants were designed to
examine SAR parameters. The synthesis of compounds
6–11, 14–16 and 20–21 (Fig. 2) was achieved by sulfa-
moylating the corresponding parent phenols in the usual
manner and, where applicable, the mono- and bis-sul-
famates were separated by flash chromatography.
For the preparation of compound 25 (Scheme 4), 4,40-
dihydroxybenzophenone was first protected by two tert-
butyldimethylsilyl (TBDMS) groups to give the disilyl-
ated 22, which was reacted with cyclohexyl magnesium
Benzo[b]naphtho[2,3-d]furan-6,11-dione-3-O-sulfamate
(13, Scheme 2) was prepared from the parent compound
12, which was obtained by reacting 2,3-dichloro-1,4-
naphthoquinone with resorcinol in ethanolic sodium
ethoxide solution.23
The 4,40-dihydroxydiphenylmethane (17, Scheme 3) was
prepared by catalytic hydrogenation of a solution of
O
i
HO
OH
HO
OH
(17)
O
ii
(6)
+
X
X
X
X
OH
O
O
(18)
(19)
(8)
(7)
Scheme 3. Synthesis of diphenylmethane sulfamates (18 and 19).
Reagents and conditions: (i) Pd–C/96% ethanol, 6 h; (ii) NaH/DMF,
H2NSO2Cl. X ¼ OSO2NH2.
X
X
O
(9)
X
X
O
O
O
O
X
O
(11)
(10)
i
OH
OH
X
Y
Y
OH
HO
O
(22)
ii
OH
O2
S
(14)
(15)
Y
Y
X
X
(23)
X
X
iii
S
S
OH
OH
(16)
iv
X
OH
HO
X
X
(24)
(25)
Scheme 4. Synthesis of 1-cyclohexyl-1,1-(4,40-O,O-bis-sulfamoyl-
phenyl)methanol (25). Reagents and conditions: (i) TBDMS–Cl/THF,
imidazole, 3 h; (ii) cyclohexylmagnesium chloride/ether, 12 h; (iii)
TBAF/THF, rt, 10 min; (iv) DBMP/CH2Cl2, H2NSO2Cl, 2 h.
X ¼ OSO2NH2, Y ¼ OTBDMS.
X
X
X
OH
(21)
(20)
Figure 2. Structures of sulfamates 6–11, 14–16 and 20–21.
X ¼ OSO2NH2.

2762
H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
replaced instead the alkene moiety of this compound
O
O
NO2
ii
O2N
with a carbonyl group to produce the sulfamate deriv-
atives of 4,40-dihydroxybenzophenone. The carbonyl
group is chosen on the basis that the mesomeric effect of
the benzophenone system, which is expected to lower the
pKa values of the bis-phenol, will result in a more
effective sulfamoyl group transferring agent. Indeed, the
pKa1 and pKa2 values of 4,40-dihydroxybenzophenone
in a solution of 50% v/v aqueous methanol as deter-
mined by potentiometric titration were found to be 8.32
and 9.72, respectively. In contrast, the pKa1 and pKa2
values of its unconjugated congener, bis-(4-hydroxy-
phenyl)methane were found to be 10.44 and 11.38,
respectively, under the same conditions [lit.26 (48% v/v
aq ethanol), pKa2 ¼ 11:22]. These observations promp-
ted us to investigate if sulfamate derivatives of 4,40-
dihydroxybenzophenone will also show high potency
against STS.
i
(26)
O
OH
HO
iii
(27)
O
O
X
X
X
HO
+
(28)
(29)
Scheme 5. Synthesis of benzophenone-30,3-O,O-bis-sulfamate (28) and
30-hydroxybenzophenone 3-O-sulfamate (29). Reagents and condi-
tions: (i) H2SO4/HNO3, 75 ꢁC, 30 min; (ii) (a) SnCl2/HCl, 70 ꢁC,
6 h; (b) NaNO2/H2SO4, 0 ꢁC to D; (iii) NaH/DMF, H2NSO2Cl.
X ¼ OSO2NH2.
The inhibition of STS activity in intact MCF-7 breast
cancer cells and in a placental microsomes preparation
by sulfamates 3–5 are tabulated in Tables 1 and 2,
respectively. The best compound in this series was
BENZOMATE (3), which showed >70% inhibition of
STS activity in intact MCF-7 breast cancer cells at
0.1 lM and >98% inhibition of placental microsomes
STS at 10 lM. In one experiment, when MCF-7 cells
were pre-treated with 3 at 1 lM for 2 h and then washed
to remove unbound inhibitor, the initial 94% inhibition
was reduced to 89% suggesting that 3, like other sulfa-
mate-based STS inhibitors, inhibited the enzyme irre-
chloride to give the substituted benzhydrol 23. The
TBDMS groups of compound 23 were cleaved by tetra-
butylammonium fluoride (TBAF) in THF to give the tri-
hydroxylated parent compound 24, which, as a solution
in dichloromethane, was sulfamoylated by reacting with
sulfamoyl chloride in the presence of 2,6-di-tert-butyl-4-
1
methylpyridine (DBMP) to give compound 25. The H
NMR spectrum of compound 25 showed a downfield
shift for the C3, C30, C5 and C50 protons in comparison
with those of compound 24, which is indicative of the
presence of sulfamate group at both the 4- and 40-posi-
tion. The fact that compound 25 was isolated as the
main product suggested that the tertiary alcohol was
not sulfamoylated, as anticipated in the conditions
adopted, because of steric hindrance and the use of a
weak base.
Table 1. Inhibition of STS activity in intact MCF-7 breast cancer cells
by sulfamates 3–16, 18–21, 25, 28–29
Compound
% Inhibition of STS activity in MCF-7 cells
Nitration of benzophenone with a mixture of nitric acid
and sulfuric acid gave 3,30-dinitrobenzophenone in 53%
yield (26, Scheme 5). Reduction of compound 26 with
stannous chloride in HCl followed by hydroxydeamin-
ation of the crude salt by sodium nitrite in H2SO4 gave
3,30-dihydroxy-benzophenone (27, Scheme 5).25 Sulfa-
moylation of compound 27 gave a mixture of the cor-
responding bis-(28) and mono-(29) sulfamate (Scheme
5).
0.1 lM
1 lM
10 lM
3
71.4 ꢀ 6.9
46.3 ꢀ 13.6
34.8 ꢀ 1.7
<10
95.6 ꢀ 2.1
83.5 ꢀ 1.9
83.8 ꢀ 0.5
33.0 ꢀ 10.7
24.3 ꢀ 11.9
33.0 ꢀ 10.7
67.9 ꢀ 1.9
98.6 ꢀ 1.0
94.5 ꢀ 1.6
93.9 ꢀ 0.8
50.6 ꢀ 3.3
96.6 ꢀ 1.4
83.9 ꢀ 1.2
50.01 ꢀ 0.1
39.3 ꢀ 0.1
32.7 ꢀ 2.3
16.3 ꢀ 1.5
27.4 ꢀ 0.8
98.4 ꢀ 0.3
30.7 ꢀ 5.6
98.7 ꢀ 2.8
93.9 ꢀ 1.2
96.7 ꢀ 1.0
87.9 ꢀ 2.0
83.8 ꢀ 0.2
87.9 ꢀ 2.0
97.1 ꢀ 0.8
99.1 ꢀ 0.1
90.1 ꢀ 0.8
98.6 ꢀ 0.3
87.4 ꢀ 2.7
99.7 ꢀ 0.1
97.7 ꢀ 0.4
93.3 ꢀ 0.1
92.4 ꢀ 0.1
90.1 ꢀ 0.7
64.1 ꢀ 1.1
75.1 ꢀ 2.8
99.9 ꢀ 0.1
87.2 ꢀ 1.8
4
5
6
7
<10
<10
8
9
21.3 ꢀ 1.5
93.1 ꢀ 2.1
99.3 ꢀ 0.6
46.6 ꢀ 4.5
<10
10
11
13
14
15
16
18
19
20
21
25
28
29
67.2 ꢀ 1.1
26.6 ꢀ 4.3
22.2 ꢀ 0.1
<10
3. Results and discussion
Our earlier programme of developing new classes of
nonsteroidal STS inhibitors identified that the mono-(1,
Fig. 1) and bis-sulfamate (2, Fig. 1) derivatives of di-
ethylstilboestrol (DES) are moderate STS inhibitors.21
These findings indicated that it is not necessary to have a
fused ring system for STS inhibition. Although the
reason for the inhibitory activities observed for the
sulfamate derivatives of DES is not clear, it is possible
that their conjugated stilbene system facilitates the
inactivation of STS via sulfamoylation. Because of the
toxicities associated with DES, which rule out further
modifications to the structure for drug development, we
16.1 ꢀ 4.0
<10
<10
75.3 ꢀ 0.8
<10
Monolayers of intact MCF-7 cells in 25 cm3 flasks were incubated for
20 h at 37 ꢁC with [3H]oestrone sulfate (2 nM) and sulfamates at vari-
ous concentrations. STS activity was determined by measuring the
total amount of 3H-labelled oestrone and oestradiol formed. STS
activity in untreated cells was 100–120 fmol/20 h/106 cells. Each value
represents the mean ꢀ SD of triplicate measurements. Both EMATE
and 667COUMATE showed >99% inhibitions at all concentrations.

H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
2763
Table 2. Inhibition of STS activity in placental microsomes by sulfa-
mates 3–16, 18–21, 25, 28–29
(20, Fig. 2) and mono-(21) sulfamate, and 1-cyclohexyl-
1,1-(4,40-bis-sulfamoyloxy-phenyl)methanol (25, Scheme
4), where extra aliphatic ring(s) are introduced; benzo-
phenone 3,30-O,O-bis-sulfamate (28, Scheme 5) and 30-
hydroxybenzophenone-3-O-sulfamate (29, Scheme 5),
where the sulfamate group(s) are relocated.
Compound
% Inhibition of STS activity in placental
microsomes
10 lM
25 lM
50 lM
100 lM
3
98.2 ꢀ 0.1
85.3 ꢀ 1.4
62.1 ꢀ 1.4
76.7 ꢀ 2.5
47.4 ꢀ 0.1
71.3 ꢀ 2.4
28.8 ꢀ 3.2
91.3 ꢀ 0.7
86.1 ꢀ 0.5
ND
ND
99.0 ꢀ 0.1
94.7 ꢀ 0.4
88.1 ꢀ 0.3
88.0 ꢀ 0.8
75.1 ꢀ 1.4
75.8 ꢀ 2.1
62.0 ꢀ 2.8
97.2 ꢀ 0.3
94.4 ꢀ 0.5
ND
99.2 ꢀ 0.0
96.6 ꢀ 0.1
92.8 ꢀ 0.2
92.3 ꢀ 0.4
82.8 ꢀ 0.2
80.5 ꢀ 1.9
75.8 ꢀ 1.2
97.7 ꢀ 0.1
95.6 ꢀ 0.1
ND
4
92.1 ꢀ 0.0
82.0 ꢀ 0.4
ND
Upon examination of the abilities of these BENZO-
MATE analogues (6–11, 13–16, 18–21, 25 and 28–29 to
inhibit STS activity (Tables 1 and 2), almost all of
them were found to be less effective STS inhibitors
than BENZOMATE, with the exception of the sulfa-
mate derivatives (10 and 11, Fig. 2) of 2,6-anthraqui-
none and benzophenone-3,30-bis-sulfamate 28 (Scheme
5), which were found to inhibit STS stronger than or to
a similar extent to BENZOMATE in MCF-7 breast
cancer cells. Although compounds 10 and 11 were
again found to be similar in potency to BENZOMATE
as STS inhibitors in placental microsomes, compound
28 was clearly less potent than BENZOMATE in this
assay. This latter finding, which is in agreement with
that of Nussbaumer et al.,22 further provides evidence
to support our reasoning that BENZOMATE is
a highly potent STS inhibitor because its sulfamate
group(s) are more efficiently transferred in the presence
of the carbonyl group in a position para to them.
Such an effect would be diminished by the relocation
of its sulfamate groups to the 3,30-position as in
compound 28 rendering the compound a weaker STS
inhibitor.
5
6
7
ND
ND
8
9
48.5 ꢀ 0.5
95.7 ꢀ 0.5
91.2 ꢀ 0.2
ND
10
11
13
14
15
16
18
19
20
21
25
28
29
65.0 ꢀ 1.8
65.3 ꢀ 0.9
33.1 ꢀ 1.7
22.1 ꢀ 0.6
11.7 ꢀ 2.3
31.4 ꢀ 1.6
<10
85.5 ꢀ 0.6
79.4 ꢀ 0.9
49.5 ꢀ 0.1
29.4 ꢀ 0.1
17.3 ꢀ 2.7
41.9 ꢀ 2.3
15.9 ꢀ 0.9
20.0 ꢀ 1.6
73.6 ꢀ 1.9
30.7 ꢀ 3.4
92.3 ꢀ 0.3
87.4 ꢀ 0.9
64.7 ꢀ 0.9
38.7 ꢀ 4.9
22.8 ꢀ 1.4
62.5 ꢀ 7.5
32.5 ꢀ 1.1
33.4 ꢀ 2.2
84.1 ꢀ 1.9
43.7 ꢀ 2.6
95.4 ꢀ 0.2
91.9 ꢀ 0.4
76.8 ꢀ 0.4
53.1 ꢀ 0.1
34.3 ꢀ 1.0
69.0 ꢀ 4.8
60.1 ꢀ 3.4
44.7 ꢀ 0.4
ND
<10
51.1 ꢀ 3.7
18.8 ꢀ 1.3
58.3 ꢀ 0.1
[3H]Oestrone sulfate (4 · 105 dpm), adjusted to 20 lM with unlabelled
substrate, with or without the inhibitor at various concentrations, was
incubated with placental microsomes (125 lg of protein/mL) for
30 min. The product formed was isolated by extraction into toluene
with [4-14C]oestrone (7 · 103 dpm) being used to monitor procedural
losses. Each value represents the mean ꢀ SD of triplicate measure-
ments. ND ¼ not determined. 667COUMATE and EMATE showed
92% and 91% inhibitions at 1 lM, respectively; and >99% at 10 lM.
The in vitro results here also show that most com-
pounds, which poorly inhibit STS in intact MCF-7 cells
(Table 1) also perform poorly when examined in pla-
cental microsomes (Table 2). In addition, bis-sulfamates
are in general more potent than their corresponding
mono-sulfamates, for example, 3 versus 5, 3 versus 6 (a
similar observation has been made22), 15 versus 16 and
18 versus 19. Although it is still unclear why such a
pattern exists, this further demonstrates that the sulfa-
mate group is the key chemical structural requirement
for potent STS inhibition.
versibly. The mono-sulfamate derivative 5 was slightly
less potent than 3 suggesting that a sulfamate group at
both the 4- and 40-positions of benzophenone is required
for maximising the inhibitory activity for this type of
STS inhibitor. We observed in related work that the
azomethine adduct of 667COUMATE (cf. 4) is inactive
as an STS inhibitor (unpublished result). The STS
inhibitory activity observed for compound 4 is thus
anticipated to be the result of its mono-sulfamate group
inactivating the enzyme via a sulfamoyl group transfer.
The sulfamate derivatives (7 and 8, Fig. 2) of trans-
chalcone are analogues of compound 6 (Fig. 2) where
the distance between the two nonfused phenyl rings has
been extended without disrupting the conjugation
between the ring system bearing the sulfamate and the
carbonyl group. Both sulfamates 7 and 8 show similar
activities in both enzyme systems at higher concentra-
tions, although compound 8 inhibits placental micro-
somes STS more strongly at 10 lM suggesting that the
positioning of the mono-sulfamate group para to the
carbonyl group might be preferred to a larger separation
between the two moieties, as in compound 7. Although
compound 8 shares similar potency to compound 6 in
our studies, Ahmed et al.17 reported an IC50 value for
compound 8 in placental microsomes being some 4-fold
lower than that of compound 6. The much weaker
inhibition than BENZOMATE observed for the chal-
cone sulfamates 7 and 8 show the importance of the
benzophenone scaffold for the biological activities of
BENZOMATE.
Having identified BENZOMATE as a potent STS
inhibitor, we prepared a range of compounds in order to
establish the SARs for this compound. These analogues
include benzophenone-4-O-sulfamate, where one of the
two phenyl rings is not substituted (6, Fig. 2); trans-
chalcone sulfamates (7 and 8, Fig. 2), where the distance
between the two nonfused-rings is extended; dibenzo-
furan-2-O-sulfamate (9, Fig. 2), 2,6-anthraquinone
bis-(10, Fig. 2) and mono-(11) sulfamate and benzo-
[b]-naphtho[2,3-d]-furan-6,11-dione-3-O-sulfamate (13,
Scheme 2), where the molecules are conformationally
restricted; sulfonyl-diphenyl-4,40-O,O-bis-sulfamate (14,
Fig. 2), and thiodiphenyl bis-(15, Fig. 2) and mono-(16)
sulfamate, where the carbonyl group is replaced by
other functionalities; diphenylmethane 4,40-O,O-bis-
sulfamate (18, Scheme 3) and 40-hydroxydiphenylme-
thane 4-O-sulfamate (19, Scheme 3), where the carbonyl
group is removed; (1,3-adamantanediyl)diphenyl bis-

2764
H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
Replacement of the carbonyl group of BENZOMATE
by a methylene unit (18, Scheme 3), or a divalent sulfur
(15, Fig. 2) or a sulfonyl group (14, Fig. 2) or aliphatic
rings (20, Fig. 2) or substitution with a cyclohexyl ring
(25, Scheme 4) significantly reduces inhibitory activity.
The lower potency observed for bis-sulfamates 15 and
18 can be attributed to their weaker sulfamoyl group
transferring potential as a result of the higher pKa val-
ues of their parent bis-phenol (pKa1 and pKa2 values of
bis-(4-hydroxyphenyl)methane were found to be 10.44
and 11.38, respectively, vide supra; pKa2 value of 4,40-
thiodiphenol was reported to be 10.8026). However, it is
surprising to find that bis-sulfamate 14 is a much weaker
STS inhibitor than BENZOMATE since the pKa2 value
of 4,40-sulfonyldiphenol was reported to be 9.41,26 which
is lower than that of 4,40-dihydroxybenzophenone (9.72,
vide supra) determined in similar conditions. The fact
that the carbonyl group of BENZOMATE is required
for potent activity suggests that this moiety might be
involved in extra binding to the enzyme active site in
addition to conjugation with the phenyl ring with acti-
vation of its sulfamate groups. Substituting the carbonyl
group with a bulky ring (adamantanediyl, 20, Fig. 2;
cyclohexyl, 25, Scheme 4) attenuates the inhibitory
activities of the BENZOMATE analogue. These results
thus highlight the presumed limited tolerance of the
enzyme to these bulky rings. It is possible that these
rings may shift the sulfamate groups at the 4- and 40-
positions away from the position occupied by the sul-
famate groups of BENZOMATE in the binding site,
and hence the analogue might not be activated effec-
tively for the sulfamoylation of the enzyme. These rings
may of course simply be too large, so that the molecule
cannot fit into the enzyme active site.
STS activity in MCF-7 cells potently and more strongly
than the tricyclic dibenzofuran-2-O-sulfamate (9, Fig.
2), it is less potent than BENZOMATE at 0.1 lM (Table
1). However, compound 13 also kills MCF-7 cells,
indicating that it might be cytotoxic and have additional
biological activities. The question obviously arises
whether their inhibition of STS results from the sulfa-
moyl group alone or is due to their other properties.
This remains to be elucidated precisely, but the follow-
ing observation was noted: as expected, the corre-
sponding starting material of 13 (i.e., 12, Scheme 2)
shows no significant inhibitory activity against STS
when examined in MCF-7 cells at 10 lM (data not
shown) while sulfamate 13 inhibited STS by 99% at the
same concentration (Table 1). When the percentage
growth inhibition of MCF-7 breast cancer cells by
compound 12 was examined, it was found to be 12%,
20% and 56% at 0.1, 1 and 10 lM, respectively, com-
pared to sulfamate 13, which inhibited the growth of
these cells by 41% and 44% at 1 and 10 lM, respectively,
and was inactive at 0.1 lM, indicating that both the
phenol 12 and the corresponding sulfamate 13 have the
same property of inhibition of the growth of MCF-7
breast cancer cells.
Having identified that benzophenone-4,40-O,O-bis-sul-
famate (BENZOMATE, Scheme 1, 3) is a potent
inhibitor, this compound was tested in vivo in rats. The
liver STS activity was found to be inhibited by 84% and
93% 24 h after a respective single oral dose of the agent
at 1 or 10 mg/kg (Fig. 3). In comparison, 667COU-
MATE inhibited rat liver STS activity by 91% and 93%
at 1 and 10 mg/kg, respectively.27 These results demon-
strate that BENZOMATE is orally active and a highly
potent nonsteroidal STS inhibitor with comparable
potency to 667COUMATE in vivo. The oestrogenicity
of BENZOMATE has not been studied in this work.
However, it has been reported that 4,40-dihydroxy-
benzophenone binds to oestrogen receptor with a rela-
Anthraquinone derivatives and 3-hydroxybenzo[b]-
naphtha-[2,3-d]furan-6,11-dione (12, Scheme 2) are
known intercalating cytostatic agents and have anti-
tumour properties. However, we considered that intro-
duction of a sulfamate group into the structure of these
compounds, might significantly engender STS inhibitory
activity and, might provide a new structural class of STS
inhibitor. Such anthraquinone derivatives are of course
conformationally restricted analogues of BENZO-
MATE, and could be used to explore details of the
potential active site binding mode. The bis-(10) and
mono-(11) sulfamates (Fig. 2) of 2,7-dihydroxyanthra-
quinone were prepared and were examined in MCF-7
cells and placental microsomes for STS inhibition. They
are potent STS inhibitors (Tables 1 and 2) with potency
comparable to or better than BENZOMATE. Thus,
compounds 10 and 11 inhibit STS activity in MCF-7
cells by 93% and >99% at 0.1 lM, respectively (Table 1)
and in placental microsomes by 91% and 86% at 10 lM,
respectively (Table 2). These findings suggest that the
conformational flexibility of BENZOMATE might not
be a prerequisite for its potent biological activity. For
comparison, 667COUMATE inhibited the STS activity
in placental microsomes by 91% and >99% at 1 and
10 lM, respectively,11 showing that compounds 10 and
11 are weaker STS inhibitors than 667COUMATE in
vitro. While the tetracyclic 3-O-sulfamoylbenzo[b]-
naphtha[2,3-d]furan-6,11-dione (13, Scheme 2) inhibits
Figure 3. In vivo effects of BENZOMATE (3) on STS activity in rat
liver tissue preparations. Female Wistar rats (Harlan Olac, Bicester,
Oxon, United Kingdom) were treated with vehicle (propylene glycol)
or 3 at 1 or 10 mg/kg with animals receiving a single dose p.o. Samples
of liver tissue were collected for assay of STS activity 24 h after
administration of the dose. Results are expressed as the percentage of
STS inhibition compared with that in control animals (mean ꢀ SD,
n ¼ 3). a, P < 0:001.

H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
2765
tive binding affinity 3 · 10ꢁ5-fold of that of 17b-oestra-
diol.28 This finding clearly suggests that neither BEN-
ZOMATE nor its putative metabolite(s) formed after
enzyme inactivation is likely to present oestrogenicity
complications.
We have observed from our experience in the handling
of many structurally diversified phenol sulfamate esters
that while these sulfamoylated compounds are perfectly
stable as solids, some of them when in solution degrade
over time to their corresponding phenolic compounds as
the result of the hydrolysis of the sulfamate group. Like
their inhibitory activity against STS in vitro, the insta-
bility of some phenol sulfamate esters in solution
apparently, and not surprisingly, also relates to the pKa
value of their corresponding phenols. Hence, highly
potent STS inhibitors such as 2-nitro- and 4-nitro-
estrone 3-O-sulfamates,1 whose respective phenolic
parent compound has a pKa value of 8.85 and 7.77 in
ethanol/water as measured spectrophotometrically,29
exhibit a much higher degree of degradation in solutions
than EMATE over the same period of time, particularly
when they are in solution in highly polar solvents such
as DMSO. To assess the stability of BENZOMATE in
solution, the disappearance of BENZOMATE, as
analysed by UV spectroscopy, over time under condi-
tions similar to those used for the in vitro assay was
performed. Hence, a freshly prepared methanolic solu-
tion of BENZOMATE was added to phosphate buffer
saline (PBS) containing 0.25 M sucrose to give a final
concentration of methanol of either 1% or 10%. The
half-life of BENZOMATE at both methanolic concen-
trations incubated at 37 ꢁC was found to be about
60 min (Fig. 4),12b demonstrating the instability of this
compound in solution.
Figure 5. Degradation of BENZOMATE in PBS containing 0.25 M
sucrose and methanol (10%) at 37 ꢁC after 70 min of incubation as
monitored by LC/MS. The peaks at tR ¼ 10:11, 9.16 and 8.34 min were
assigned by mass spectrometry as BENZOMATE (top), the mono-
sulfamate 5 (middle) and 4,40-dihydroxybenzophenone (bottom spec-
trum), respectively.
When the above degradation was studied by LC/MS,
BENZOMATE was found to break down to its mono-
sulfamate derivative, that is compound 5 and 4,40-di-
hydroxybenzophenone (Fig. 5). It appears that this
degradation is sequential with the hydrolysis of one
sulfamate group at a time. There was still some BEN-
ZOMATE detected after 130 min into the experiment
but complete disappearance of BENZOMATE was
observed after 240 min (data not shown). While such
instability of BENZOMATE may present problems in
the pharmaceutical development of this class of STS
inhibitor, the fact that it inhibits so potently the STS
activity in MCF-7 cells after an incubation period of
20 h suggests that the inactivation of STS by BENZO-
MATE is rapid and highly effective. The high potency
against liver STS activity observed for BENZOMATE
in vivo when administered orally to rats suggests also
that this apparent instability of BENZOMATE in
solution might not be as problematic as it seems. In
particular, it is thought that aryl sulfamates in vivo are
transported in red blood cells and avoid first pass
metabolism.30 The carrier protein that mediates this is
apparently carbonic anhydrase II (hCAII). Aryl sulfa-
mates bind effectively to hCAII using a coordination of
the sulfamate anion to the active site zinc atom.31;32 We
have recently determined the X-ray crystal structure of
EMATE bound to hCAII.33 It seems highly likely that
BENZOMATE would also be transported in vivo via
hCAII and any intrinsic chemical instability of this
agent could be markedly attenuated in vivo by this
effect.
Figure 4. Stability study of BENZOMATE (3) in PBS containing
0.25 M sucrose and 1% or 10% of methanol at 37 ꢁC as monitored by
UV spectroscopy. Points are mean of triplicate determinations.

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H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
4. Conclusions
spectrometer and PDA detector under the following
conditions, Ionisation technique: electrospray; Column:
Waters ‘Symmetry’ C18 (packing: 3.5 lm), 4.6 · 100 mm;
elution: gradient (flow rate): 5:95 MeOH/H2O (0.5 mL/
min) to 95:5 MeOH/H2O (1.0 mL/min) over 22 min.
Attempts to design and synthesise non-steroidal STS
inhibitors, in the light of the potent oestrogenicity of
EMATE in rodents, have led to the discovery of BEN-
ZOMATE (3), a significant improvement over initial
lead non-steroidal candidates such as THN-2-O-sulfa-
mate and bicyclic coumarin sulfamates. BENZOMATE,
a non-fused bicyclic bis-sulfamate, is a highly potent
inhibitor of STS in vitro. This agent inhibits in vivo rat
liver STS activity by 84% and 93%, respectively, at a
single oral dose (1 and 10 mg/kg), showing potency of a
similar order of magnitude to 667COUMATE, the
established potent non-steroidal STS inhibitor, which is
currently in Phase I trial. Several modifications were
made to the structure of BENZOMATE. These SAR
studies conducted on BENZOMATE show that its
carbonyl and bis-sulfamate groups are pivotal for inhi-
bition of STS although conformational flexibility is
apparently not required. BENZOMATE represents an
important lead compound and new class of STS inhib-
itor. Despite the apparent instability of BENZOMATE
in solution, it is anticipated that BENZOMATE or its
optimised derivatives might subsequently be developed
for therapeutic use in the treatment of HDBC.
5.2. Determination of the acidity constant (pKa) of 4,40-
dihydroxybenzophenone and bis-(4-hydroxyphenyl)meth-
ane
The general procedure of Albert and Serjeant34 was used
for the study. A 5 mM solution of 4,40-dihydroxybenzo-
phenone or bis-(4-hydroxyphenyl)methane in methanol/
water (1:1, 50 mL) was prepared and its pH at room
temperature read (WPA Linton Cambridge UK,
CCMD625 pH meter). The titrant (aqueous NaOH,
100 mM) was then added in equal portions. The pH was
recorded after each addition of titrant when equilibrium
was reached (after stirring). Titrations were carried out
in duplicate and the average pKa value was presented.
The pKa1 and pKa2 values of the compound were taken
as the respective pH of the mixture after 1.25 and
3.75 mL of titrant was added and were uncorrected.
5.3. Stability study of BENZOMATE
5.3.1. As monitored by UV spectroscopy. To select the
optimum wavelength for monitoring the degradation of
BENZOMATE, the UV spectrum of BENZOMATE
and its putative degradation products, that is compound
5 (Scheme 1) and 4,40-dihydroxybenzophenone, in PBS
(freshly prepared by dissolving one tablet of PBS in
100 mL of distilled water) containing 0.25 M sucrose was
individually determined. The kmax for BENZOMATE,
compound 5, 4,40-dihydroxybenzophenone and PBS
containing 0.25 sucrose was found to be 260, 295, 296
and 374 nm, respectively. The UV spectra of compound
5 and 4,40-dihydroxybenzophenone are similar.
5. Experimental
5.1. Materials and methods
All reagents and solvents employed were of general
purpose or analytical grade unless otherwise stated, and
purchased from either Aldrich or Sigma Chemicals or
Lancaster Synthesis. They were stored away from
moisture and light and dried before use where necessary.
Silica gel refers to Merck silica gel grade 60. Product(s)
and starting material were detected by either viewing
under UV light or treating with a methanolic solution of
phosphomolybic acid followed by heating. NMR spec-
tra were determined using acetone-d6, CDCl3 or DMSO-
d6 as solvent and TMS as internal standard, unless
Approximately 5 mg of BENZOMATE was dissolved in
1 mL of HPLC grade methanol. Of this freshly prepared
methanolic solution, 30 and 300 lL were, respectively,
added to 2970 and 2700 lL of PBS containing 0.25 M
sucrose in a cuvette. The mixtures were kept in a water
bath at 37 ꢁC during the course of the experiment. Each
cuvette was removed from the water bath and stirred
magnetically before absorbance was determined at
intervals between 0 and 8 h. The absorbance of the
mixture at 325 nm was determined, since at this wave-
length the absorbance of BENZOMATE is minimal in
contrast to its putative degradation products, which still
absorb UV strongly. Blanks were prepared without the
sample. The decomposition percentage versus time was
plotted (Fig. 4).
1
otherwise stated. The H NMR spectra were recorded
on a Jeol GX 270 or on a Jeol EX 400 NMR spec-
trometer. Chemical shifts are given in d units. The fol-
lowing abbreviations are used to describe resonances in
1H NMR spectra: s, singlet; d, doublet; br, broad; t,
triplet; m, multiplet and combination such as dd, dou-
blet of doublets. IR spectra were determined as KBr
discs, using a Perkin–Elmer 782 Infrared Spectropho-
tometer. UV spectra were determined using a Perkin–
Elmer Lambda 3B (UV–vis) spectrophotomer. Melting
points were determined on a Reichert-Jung Kofler Block
and were uncorrected. Mass spectra were recorded on
VG 7070 and VG Autospec instruments at the Mass
Spectrometry Service at the University of Bath.
FABꢁmass spectra were carried out using m-nitrobenz-
yl alcohol (m-NBA) as the matrix. CHN analysis was
determined using gas chromatography at the Micro-
analysis Service at the University of Bath. LC/MS was
carried out using Waters 2790 Alliance, ZQ MicroMass
5.3.2. As monitored by LC/MS. To follow the course of
the breakdown of BENZOMATE, a mixture of BEN-
ZOMATE in PBS containing 0.25 M sucrose and 10%
methanol was prepared as described above and was kept
at 37 ꢁC in a water bath throughout the experiment.

H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
2767
Aliquots were removed at intervals between 0 and 5 h
and assessed by LC/MS. The retention time of 4,40-di-
hydroxybenzophenone, compound 5 and BENZO-
MATE was found to be around 8–9, 9–10 and 10–
11 min, respectively. The results at t ¼ 70 min are shown
in Figure 5.
The band at Rf 0.57 (chloroform/acetone, 2:1) gave a
beige residue (183 mg), which was further purified by
recrystallisation from acetone/hexane (1:2) to give 4 as
white crystals (130 mg, 7%); mp 158–160 ꢁC; mmax (KBr)
1
3340–3240 (NH2), 1650 (C@O), 1370 (SO2) cmꢁ1; H
NMR (270 MHz, acetone-d6) 3.09 (3H, s, NCH3), 3.3
(3H, s, NCH3), 7.36 (2H, br s, ex. with D2O,
OSO2NH2), 7.48 (2H, d, J 8.79 Hz, ArH), 7.52 (2H, d, J
8.8 Hz, ArH) 7.82 (2H, d, J 8.8 Hz, ArH), 7.84 (2H, d, J
8.8 Hz, ArH) and 8.14 (1H, s, SO2N@CH); MS m=z
5.4. Biological assay of sulfamates
(FABþ)
427.9
[100,
(MþH)þ],
349.0
[20,
In vitro studies in placental microsomal (100,000 g)
preparations or in intact MCF-7 breast cancer cells, and
in vivo studies in rats were performed essentially as
described previously.35 For details, see legends of indi-
vidual figure and tables.
(MþHꢁSO2NH)þ], 294.1 [10, (Mþ2HꢁSO2N@CH
ꢁN(Me)2)þ]; MS m=z (FABꢁ) 425.9 [100, (MꢁH)ꢁ],
347.0 [40, (MꢁSO2NH2)ꢁ], 293.0 [10, (Mþ
HꢁSO2N@CHꢁN(Me)2)ꢁ]; Acc. MS (FABþ) 428.0582,
C16H18N3O7S2 requires 428.0586.
5.5. General method for sulfamoylation
5.6.2. 40-Hydroxybenzophenone-4-O-sulfamate (5). Upon
sulfamoylation using 1 equiv of NaH, 4,40-dihydroxy-
benzophenone (300 mg, 1.40 mmol) gave a crude prod-
uct (420 mg) of which 100 mg was fractionated on
preparative TLC (chloroform/acetone, gradient). The
white residue that isolated (46 mg) was further purified
by recrystallisation from ethyl acetate/hexane (1:2) to
give 5 as white crystals (32 mg, 32%); mp > 152 ꢁC (dec);
TLC (chloroform/acetone, 4:1 and 2:1): Rf s 0.27 and
0.41, respectively; mmax (KBr) 3500–3000 (NH2 and OH),
Starting with the parent compound, the sulfamate
derivatives were prepared essentially as previously
described.36 In this regard, a solution of the appropriate
parent compound in anhydrous DMF (or another sol-
vent as specified) was treated with sodium hydride [60%
dispersion; 1.2 and 2.5 equiv. for monohydroxyl and
dihydroxyl compounds, respectively, unless stated
otherwise] at 0 ꢁC under an atmosphere of N2. After
evolution of hydrogen had ceased, a freshly concen-
trated solution of sulfamoyl chloride in toluene36 [ex-
cess, ca. 5–6 equiv] was added and the reaction mixture
poured into brine after warming to room temperature
overnight. Ethyl acetate was added and the organic
fraction that separated was washed exhaustively with
brine, dried (MgSO4), filtered and evaporated under
reduced pressure. In general, the crude product obtained
was purified by flash chromatography or preparative
TLC followed by recrystallisation to give the corre-
sponding sulfamate. All new compounds were charac-
terised by spectroscopic and combustion analysis.
1630 (C@O), 1590, 1380 (SO2) cmꢁ1
,
1H NMR
(270 MHz, acetone-d6) 7.0 (2H, d, J 8.8 Hz, C30-H, and
C50-H), 7.3 (2H, br s, ex. with D2O, OSO2NH2), 7.48
(2H, d, J 8.4 Hz, C3-H, and C5-H), 7.77 (2H, d, J
8.4 Hz, ArH), 7.8 (2H, d, J 8.42 Hz, ArH) and 8.1 (1H,
br s, ex. with D2O, OH); MS m=z (FABþ) 447.1 [10,
(MþHþNBA)þ], 294.0 [100, (MþH)þ], 215.1 [10,
(MþHꢁSO2NH)þ]; MS m=z (FABꢁ) 446.1 [20, (Mþ
NBA)ꢁ], 292.1 [100, (MꢁH)ꢁ], 213.1 [30, (MꢁSO2-
NH2)ꢁ]. Found C, 53.1; H, 3.9; N, 4.55; C13H11NO5S
requires C, 53.24; H, 3.78; N, 4.78.
5.6. Synthesis of sulfamates
5.6.3. Benzophenone-4-O-sulfamate (6). Upon sulfa-
moylation, 4-hydroxybenzophenone (1.0 g, 5.05 mmol)
gave a crude product (1.45 g), which was fractionated by
flash chromatography (chloroform/acetone, 8:1). The
creamy residue that isolated (716 mg) was further puri-
fied by recrystallisation from ethyl acetate/hexane (1:2)
to give 6 as white crystals (495 mg, 35%); mp 134–136 ꢁC
[lit.17 140–142 ꢁC]; TLC (chloroform/acetone, 4:1 and
8:1): Rf s 0.68 and 0.35, respectively; mmax (KBr) 3360
5.6.1. Benzophenone-4,40-O,O-bis-sulfamate (3) and
azomethine derivative (4). Upon sulfamoylation, 4,40-di-
hydroxybenzophenone (1.0 g, 4.67 mmol) gave a crude
product (1.63 g), which was fractionated by flash chro-
matography (chloroform/acetone gradient). The band at
Rf 0.46 (chloroform/acetone, 2:1) gave a creamy residue
(1.17 g), which was further purified by recrystallisation
from acetone/chloroform (1:2) to give 3 as white crystals
(730 mg, 43%); mp 182–184 ꢁC; mmax (KBr) 3340 (NH2),
1
(NH2), 1390 (SO2) cmꢁ1; H NMR (270 MHz, acetone-
1660 (C@O), 1370 (SO2) cmꢁ1 1H NMR (270 MHz,
;
d6) 7.35 (2H, br s, ex. with D2O, SO2NH2), 7.5 (2H, d, J
8.8 Hz, C3-H and C5-H), 7.55–7.75 (3H, m, C30-H, C40-
H and C50-H), 7.84 (2H, m, C20-H and C60-H), 7.88 (2H,
d, J 8.8 Hz, C2-H and C6-H); MS m=z (FABþ) 278.0
[100, (MþH)þ], 198.0 [10, (MþHꢁSO2NH2)þ]; MS m=z
(FABꢁ) 430.0 [15, (MþNBA)ꢁ], 276.0 [100, (MꢁH)ꢁ],
197.0 [25, (MꢁSO2NH2)ꢁ]; Acc. MS m=z (FABþ)
278.0503, C13H12NO4S requires 278.0487. Found C,
56.1; H, 4.01; N, 5.12; C13H11NO4S requires C, 56.31; H,
4.0; N, 5.05.
acetone-d6) 7.36 (4H, br s, ex. with D2O, 2 · OSO2NH2),
7.51 (4H, d, J 8.8 Hz, ArH) and 7.9 (4H, d, J 8.4 Hz,
ArH); MS m=z (FABþ) 372.9 [100, (MþH)þ], 293.0 [30,
(MþHꢁSO2NH2)þ], 213.1 [10, (MþHꢁ2 · SO2NH2)þ];
MS m=z (FABꢁ) 370.9 [100, (MꢁH)ꢁ], 291.9 [82,
(MꢁSO2NH2)ꢁ], 213.0 [20, (MþHꢁ2 · SO2NH2)ꢁ];
Acc. MS (FABþ) 373.0177, C13H13N2O7S2 requires
373.0164. Found C, 41.9; H, 3.24; N, 7.50;
C13H12N2O7S2 requires C, 41.93; H, 3.25; N, 7.52.

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5.6.4. trans-Chalcone-4-O-sulfamate (7). Upon sulfa-
moylation, trans-4-hydroxychalcone (1.0 g, 4.46 mmol)
gave a crude product (1.44 g), which was fractionated by
flash chromatography (chloroform/acetone, 8:1). The
creamy residue that obtained (719 mg) was further
purified by recrystallisation from ethyl acetate/hexane
(1:2) to give 7 as white crystals (464 mg, 34%); mp 136–
138 ꢁC; TLC (chloroform/acetone, 8:1, 4:1 and 2:1): Rf s
0.24, 0.48 and 0.66, respectively; mmax (KBr) 3500–3220
5.6.7. Anthraquinone-2,6-O,O-bis-sulfamate (10) and 2-
hydroxyanthraquinone-6-O-sulfamate (11). Upon sulfa-
moylation,
2,6-dihydroxyanthraquinone
(1.0 g,
4.16 mmol) gave crude products (1.41 g) of which a
100 mg sample was fractionated on preparative TLC
(chloroform/acetone, gradient). The fraction at Rf 0.39
(chloroform/acetone, 2:1) gave a yellow residue (32 mg),
which was further purified by recrystallisation from
acetone/hexane (1:2) to give 10 as yellow crystals (24 mg,
20%); mp > 220 ꢁC (dec); TLC (chloroform/acetone,
4:1): Rf 0.26; mmax (KBr) 3380–3260 (NH2), 1680 (C@O),
(NH2), 1670 (C@O), 1390 (SO2) cmꢁ1
;
1H NMR
(270 MHz, acetone-d6) 7.27 (2H, s, ex. with D2O,
OSO2NH2), 7.42 (2H, d, J 8.79 Hz, C3-H and C5-H),
7.62 (2H, m, C30-H, C40-H and C50-H), 7.85 (2H, d, J
14.7 Hz, CH@CH), 7.95 (5H, d, J 6.2 Hz, C2-H and C6-
H) and 8.18 (2H, d, J 7.7 Hz, C20-H and C60-H); MS m=z
1
1390 (SO2) cmꢁ1; H NMR (270 MHz, acetone-d6) 7.57
(4H, br s, ex. with D2O, 2 · SO2NH2), 7.84 (2H, dd, J
2.4 and 8.4 Hz, C3-H and C7-H), 8.17 (2H, d, J 2.6 Hz,
C1-H and C5-H) and 8.39 (2H, d, J 8.4 Hz, C4-H and
C8-H); MS m=z (FABþ) 398.0 [40, (M)þ], 240.1 [30,
(Mꢁ2 · SO2NH)þ]; MS m=z (FABꢁ) 551.1 [25,
(MþNBA)ꢁ], 398.1 [100, (M)ꢁ], 239.1 [70, (MþHꢁ
2 · SO2NH2)ꢁ]; Acc. MS m=z (FABþ) 397.9887,
C14H10N2O8S2 requires 397.9879.
(FABþ)
304.0
[100,
(MþH)þ],
224.1
[10,
(MþHꢁSO2NH2)þ]; MS m=z (FABꢁ) 302.0 [100,
(MꢁH)ꢁ], 223.0 [40, (MꢁSO2NH2)ꢁ]; Acc. MS (FABþ)
304.0646, C15H14NO4S requires 304.0644. Found C,
59.1; H, 4.29; N, 4.64 C15H13NO4S requires C, 59.40; H,
4.32; N, 4.62.
The fraction at Rf 0.46 (chloroform/acetone, 2:1) gave a
white residue (46 mg), which was further purified by
recrystallisation from ethyl acetate/hexane (1:2) to give
11 as white crystals (31 mg, 21%); mp > 285 ꢁC (dec);
TLC (chloroform/acetone, 4:1): Rf 0.3; mmax (KBr) 3500–
3000 (OH, NH2), 1670 (C@O) and 1390 (SO2) cmꢁ1; 1H
NMR (270 MHz, acetone-d6) 7.34 (1H, dd, J 2.6 and
8.4 Hz, C3-H), 7.53 (2H, br s, ex. with D2O, SO2NH2),
7.65 (1H, d, J 2.6 Hz, C1-H), 7.77 (1H, dd, J 2.4 and
8.4 Hz, C7-H), 8.13 (1H, d, J 2.6 Hz, C5-H), 8.2 (1H, d,
J 8.4 Hz, C4-H), 8.33 (1H, d, J 8.4 Hz, C8-H) and 9.98
(1H, s, ex. with D2O, OH); MS m=z (FABþ) 320.0 [100,
(MþH)þ], 240.1 [30, (MþHꢁSO2NH2)ꢁ], 225.1 (15);
MS m=z (FABꢁ) 473.2 [20, (MþHþNBA)ꢁ], 318.1 [100,
(MꢁH)ꢁ], 239.1 [90, (MꢁSO2NH2)ꢁ]; Acc. MS (FABþ)
320.0224, C14H10NO6S requires 320.0229. Found C,
52.5; H, 2.84; N, 4.25; C14H9NO6S requires C, 52.67; H,
2.84; N, 4.39.
5.6.5. trans-Chalcone-40-O-sulfamate (8). Upon sulfa-
moylation, trans-40-hydroxychalcone (1.0 g, 4.46 mmol)
gave a crude product (1.5 g), which was fractionated by
flash chromatography (chloroform/acetone, 8:1). The
creamy residue that obtained (723 mg) was further
purified by recrystallisation from ethyl acetate/hexane
(1:2) to give 8 as white crystals (425 mg, 31%); mp 187–
189 ꢁC [lit.17 190–193 ꢁC]; TLC (chloroform/acetone,
8:1, 4:1 and 2:1): Rf s 0.17, 0.26 and 0.62, respectively; 1H
NMR (270 MHz, acetone-d6) 7.34 (2H, s, ex. with D2O,
OSO2NH2), 7.49 (5H, m, C-30, 50, 3, 4 and C5-H), 7.88
(4H, m, CH@CH and C2-H, C6-H), 8.25 (2H, d, J
8.8 Hz, C20-H and C60-H); MS m=z (FABþ) 304.0 [100,
(MþH)þ], 225.1 [10, (Mþ2HꢁSO2NH2)þ]; MS m=z
(FABꢁ) 302.0 [100, (MꢁH)ꢁ], 223.1 [35, (MꢁSO2-
NH2)ꢁ]; Acc. MS (FABþ) 304.0654, C15H14NO4S
requires 304.0644. Found C, 59.1; H, 4.29; N, 4.65;
C15H13NO4S requires C, 59.40; H, 4.32; N, 4.62.
5.6.8. 3-Hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione
(12). Resorcinol (6.6 g, 60 mmol) in ethanol (80 mL)
was added dropwise to a stirred ethanolic sodium eth-
oxide solution (prepared by dissolving 3.8 g of sodium
metal in 120 mL of absolute ethanol) containing 2,3-di-
chloro-1,4-naphtho-quinone (6.9 g, 30 mmol) at 0 ꢁC.
After stirring at room temperature overnight, the black
reaction mixture was acidified with 5 N HCl at 0 ꢁC. The
yellow solid that precipitated was collected by filtration,
washed successively with water, methanol, diethyl ether
and air dried to give crude 12 as a deep yellow solid
(7.1 g, 88%); mp > 310 ꢁC [lit.23 >300 ꢁC]; TLC (chlo-
roform/acetone, 8:1): Rf 0.47; mmax (KBr) 3400 (OH),
5.6.6. Dibenzofuran-2-O-sulfamate (9). Upon sulfamoyl-
ation, 2-hydroxydibenzofuran (1.0 g, 5.43 mmol) gave a
crude product (1.31 g), which was fractionated by flash
chromatography (chloroform/acetone, 8:1). The beige
residue that obtained (561 mg) was further purified by
recrystallisation from ethyl acetate/hexane (1:2) to give 9
as white crystals (319 mg, 23%); mp > 120 ꢁC (dec); TLC
(chloroform/acetone, 8:1, 4:1 and 2:1): Rf s 0.31, 0.55 and
0.70, respectively; mmax (KBr) 3400 (NH2), 1600 (C@O),
1
1370 (SO2) cmꢁ1; H NMR (270 MHz, acetone-d6) 7.18
(2H, br s, ex. with D2O, SO2NH2), 7.46 (2H, m, C4-H
and C5-H), 7.56 (1H, t, J 7.7 Hz, C7-H), 7.7 (2H, t, J
7.3 Hz, C3-H and C6-H), 8.07 (1H, d, J 2.2 Hz, C1-H)
and 8.16 (1H, d, J 7.7 Hz, C8-H); MS m=z (FABþ) 417.0
[13, (MþHþNBA)þ], 263.1 [100, (M)þ], 183.1 [60,
(MꢁSO2NH2)þ]; MS m=z (FABꢁ) 416.0 [15,
(MþNBA)ꢁ], 262.0 [100, (MꢁH)ꢁ]. Found C, 54.6; H,
3.46; N, 5.22; C12H9NO4S requires C, 54.75; H, 3.42; N,
5.32.
1670 and 1620 (C@O) cmꢁ1 1H NMR (400 MHz,
;
DMSO-d6) 7.05 (1H, d, J 8.5 Hz, C4-H), 7.16 (1H, s, C2-
H), 7.2–7.7 (2H, m, C8-H and C9-H), 7.95 (1H, d, J
8.5 Hz, C5-H), 8.2–8.7 (2H, m, C7-H and C10-H) and
10.53 (1H, br s, ex. with D2O, C3-OH); MS m=z (FABþ)
265.1 [70, (MþH)þ], 255.3 (75), 243.2 (30), 173.2 (100);
MS m=z (FABꢁ) 263.1 [100, (MꢁH)ꢁ], 242.1 (20), 210.1
(25), 198.1 (45), 181.1 (35); Acc. MS (FABþ) 265.0502,
C16H9O4 requires 265.0501.

H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
2769
5.6.9. Benzo[b]naphtho[2,3-d]furan-6,11-dione-3-O-sulfa-
mate (13). Upon sulfamoylation, 12 (500 mg, 1.89 mmol)
gave a crude product (655 mg), which was fractionated
by flash chromatography (chloroform/acetone, gradi-
ent). The yellow residue that obtained (510 mg) was
further purified by recrystallisation from acetone/hexane
(1:3) to give 13 as white crystals (450 mg, 69%);
mp > 270 ꢁC (dec); TLC (chloroform/acetone, 8:1):
Rf 0.28; mmax (KBr) 3280, 3380 (NH2), 1680 (C@O), 1380
(FABþ) 376.9905, C12H13N2O6S3 requires 376.9936.
Found C, 38.8; H, 3.24; N, 7.37; C12H12N2O6S3 requires
C, 38.29; H, 3.21; N, 7.44.
The fraction at Rf 0.57 (chloroform/acetone, 2:1) gave a
white residue (123 mg), which was further purified by
recrystallisation from ethyl acetone/hexane (1:4) to give
16 as white crystals (110 mg, 10%); mp 170–172 ꢁC (dec);
TLC (chloroform/acetone, 4:1): Rf 0.35; mmax (KBr)
1
(SO2) cmꢁ1
;
1H NMR (270 MHz, DMSO-d6) 7.52
3500–3000 (NH2 and OH), 1390 (SO2) cmꢁ1; H NMR
(1H, dd, J 2.0 and 8.4 Hz, C4-H), 7.85–8.0 (3H, m,
ArH), 8.1–8.2 (2H, m, ArH), 8.25 (2H, br s, ex. with
D2O, SO2NH2) and 8.27 (1H, d, J 8.4 Hz, ArH);
MS m=z (FABþ) 344.1 [100, (MþH)þ], 263.1
[30, (MꢁSO2NH2)þ]; MS m=z (FABꢁ) 242.2 [100,
(MꢁH)ꢁ]; 264.2 [20, (MꢁSO2NH)ꢁ]; Acc. MS (FABþ)
344.0218, C16H10NO6S requires 344.0229. Found C,
55.83; H, 2.68; N, 3.96; C16H9NO6S requires C, 55.98;
H, 2.64; N, 4.08.
(400 MHz, acetone-d6) 6.73 (2H, br s, ex. with D2O,
OSO2NH2), 6.95 (2H, d, J 8.5 Hz, C30-H and C50-H),
7.18 (2H, d, J 8.8 Hz, ArH), 7.25 (2H, d, J 8.8 Hz, ArH),
7.42 (2H, d, J 8.5 Hz, ArH) and 8.97 (1H, br s, ex. with
D2O, OH); MS m=z (FABþ) 297.0 [100, (M)þ], 217.1
[20, (MꢁSO2NH2)þ]; MS m=z (FABꢁ) 450.1 [20,
(MþNBA)ꢁ], 296.1 [100, (MꢁH)ꢁ]; 216.1 [20,
(MꢁHꢁSO2NH2)ꢁ]; Acc. MS (FABþ) 297.0129,
C12H11NO4S2 requires 297.0129. Found C, 48.2; H,
3.89; N, 4.57; C12H11NO4S2 requires C, 48.47; H, 3.73;
N, 4.71.
5.6.10. Sulfonyldiphenyl-4,40-O,O-bis-sulfamate (14).
Upon sulfamoylation, 4,40-sulfonyldiphenol (1.0 g,
4.00 mmol) gave a brown residue (1.56 g). A sample of
this crude material (150 mg) was fractionated on pre-
parative TLC (chloroform/acetone, gradient). The pale
white residue that obtained (117 mg) was further puri-
fied by recrystallisation from acetone/hexane (1:2) to
give 14 as white crystals (94 mg, 67%); mp 174–176 ꢁC;
TLC (chloroform/acetone, 4:1 and 2:1): Rf s 0.22 and
0.43, respectively; mmax (KBr) 3380–3240 (NH2), 1380
5.6.12. 4,40-Dihydroxydiphenylmethane (17). To a solu-
tion of 4,40-dihydroxybenzophenone (1.0 g, 4.67 mmol)
in ethanol (25 mL) Pd–C (10%, 200 mg) was added and
the resulting suspension was subjected to hydrogenation
at balloon pressure at room temperature for 6 h. Upon
removal of the supported catalyst by filtration, the fil-
trate was evaporated to give a white residue (1.02 g),
which was recrystallised from ethyl acetate/hexane (1:2)
to give 17 as white crystals (854 mg, 91%); mp 158–
160 ꢁC [lit.24 161–162 ꢁC]; TLC (chloroform/acetone, 8:1
and 4:1): Rf s 0.28 and 0.63, respectively; mmax (KBr)
1
(SO2) cmꢁ1; H NMR (270 MHz, acetone-d6) 7.41 (4H,
br s, ex. with D2O, 2 · OSO2NH2) 7.57 (4H, d, J 8.8 Hz,
C3-H, C5-H, C30-H and C50-H) and 8.12 (4H, d, J
8.8 Hz, C2-H, C6-H, C20-H and C60-H); MS m=z
(FABþ) 562.1 [10, (MþHþNBA)þ], 409.0 [60,
(MþH)þ], 330.1 [50, (MþHꢁSO2NH)]; MS m=z
1
3500–3000 (OH), 1600 cmꢁ1; H NMR (270 MHz, ace-
tone-d6) 3.8 (2H, s, CH2), 6.75 (4H, m, ArH), 7.03 (4H,
m, ArH) and 8.1 (2H, br s, ex. with D2O, 2 · OH); MS
m=z (FABþ) 200.1 [100, (M)þ]; Acc. MS (FABþ)
200.0842, C13H12O2 requires 200.0915.
(FABꢁ)
407.1
[100,
(MꢁH)ꢁ],
328.1
[25,
(MꢁSO2NH2)ꢁ], 249.1 [25, (MþHꢁ2 · SO2NH2)ꢁ];
Acc. MS m=z (FABꢁ) 406.9675, C12H11N2O8S3 requires
406.9678. Found C, 35.4; H, 3.0; N, 6.71; C12H12N2O8S3
requires C, 35.29; H, 2.96; N, 6.86.
5.6.13. 4,40-Bis-sulfamoyloxydiphenylmethane (18) and 4-
sulfamoyloxy-40-hydroxydiphenylmethane (19). Upon
sulfamoylation, 17 (825 mg, 4.12 mmol) gave a beige
crude product (1.3 g), which was fractionated by flash
chromatography (chloroform/acetone, gradient). The
fraction at Rf 0.43 (chloroform/acetone, 4:1) gave a
white residue (471 mg), which was further purified by
recrystallisation from acetone/chloroform (1:2) to give
18 as white crystals (389 mg, 26%); mp 175–177 ꢁC; mmax
5.6.11. Thiodiphenyl-4,40-O,O-bis-sulfamate (15) and 40-
hydroxythiodiphenyl-4-O-sulfamate (16). Upon sulfa-
moylation, 4,40-thiodiphenol (760 mg, 3.48 mmol) gave a
crude product (1.09 g), which was fractionated by flash
chromatography (chloroform/acetone, gradient). The
fraction at Rf 0.42 (chloroform/acetone, 2:1) gave a
beige residue that (889 mg), which was further purified
by recrystallisation from acetone/hexane (1:2) to give 15
as white crystals (665 mg, 50%); mp 142–144 ꢁC; TLC
(chloroform/acetone, 4:1): Rf 0.25; mmax (KBr) 3400–3220
1
(KBr) 3500–3000 (NH2), 1390 (SO2) cmꢁ1; H NMR
(270 MHz, acetone-d6) 4.03 (2H, s, CH2), 7.10 (4H, br s,
ex. with D2O, 2 · OSO2NH2), 7.25 (4H, d, J 8.8 Hz, C2-
H, C20-H, C6-H and C60-H) and 7.34 (4H, d, J 8.8 Hz,
C3-H, C30-H, C5-H and C50-H); MS m=z (FABþ) 512.0
[40, (MþHþNBA)þ], 358.0 [90, (M)þ], 279.0 [50,
(MþHꢁSO2NH2)þ]; MS m=z (FABꢁ) 511.1 [40,
(MþNBA)þ], 357.1 [100, (MꢁH)þ], 278.0 [30,
(MꢁSO2NH2)þ]. Found C, 43.5; H, 3.89; N, 7.64;
C13H14O6N2S2 requires C, 43.57; H, 3.94; N, 7.82.
(NH2), 1590, 1390 (SO2) cmꢁ1 1H NMR (270 MHz,
;
acetone-d6) 7.22 (4H, br s, ex. with D2O, 2 · OSO2NH2),
7.34 (4H, d, J 8.8 Hz, C3-H, C5-H, C30-H and C50-H)
7.44 (4H, d, J 6.6 Hz, C2-H, C6-H, C20-H and C60-H);
MS m=z (FABþ) 530.1 [10, (MþHþNBA)] 376.0 [100,
(M)þ], 297.0 [40, (MþHꢁSO2NH2)þ]; 217.1 [20,
(MþHꢁ2 · SO2NH2)þ]; MS m=z (FABꢁ) 529.2 [10,
(MþNBA)ꢁ], 375.1 [100, (MꢁH)ꢁ], 296.1 [40,
(MꢁSO2NH2)ꢁ]; 216.1 [10, (Mꢁ2SO2NH2)ꢁ]; Acc. MS
The fraction at Rf 0.52 (chloroform/acetone, 4:1) gave a
beige residue (150 mg), which was recrystallised from

2770
H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
acetone/hexane (1:2) to give 19 as white crystals (120 mg,
10%); mp 128–130 ꢁC; TLC (chloroform/acetone, 8:1):
Rf 0.27; mmax (KBr) 3500–3300 (NH2), 3240 (OH) 1390
(C@O) cmꢁ1; 1H NMR (400 MHz, CDCl3) 0.24 (12H, s,
2 · Si(CH3)2), 1.0 (18H, s, 2 · (CH3)3), 6.9 (4H, AA0BB0,
C3-H, C5-H, C30-H and C50-H) and 7.73 (4H, AA0BB0,
C2-H, C6-H, C20-H and C60-H); MS m=z (FABþ) 443.4
[60, (MþH)þ], 385.3 [10, (MꢁC(CH3)3)þ], 235 (90), 73.0
(100); MS m=z (FABꢁ) 441.3 [10, (MꢁH)ꢁ], 401.2 (10),
327.2 [100, (MꢁHꢁ2 · C(CH3)3)þ], 255.1 (10). Found
C, 67.6; H, 8.64; C25H38O3Si2 requires C, 67.82; H, 8.65.
1
(SO2) cmꢁ1; H NMR (270 MHz, acetone-d6) 3.90 (2H,
s, CH2), 6.77 (2H, d, J 8.8 Hz, C30-H and C50-H), 7.07
(4H, d, J 8.1 Hz, 2H ex. with D2O, ArH and OSO2NH2),
7.21 (2H, d, J 8.8 Hz, ArH), 7.28 (2H, d, J 8.8 Hz, ArH)
and 8.18 (1H, br s, ex. with D2O, OH); MS m=z (FABþ)
279.0 [100, (M)þ], 200.1 [30, (MþHꢁSO2NH2)þ]; MS
m=z (FABꢁ) 432.2 [40, (MþNBA)ꢁ], 278.1 [100,
(MꢁH)ꢁ]; Acc. MS (FABþ) 279.0584, C13H13O4NS
requires 279.0643. Found C, 56.0; H, 5.16; N, 4.74;
C13H13O4NS requires C, 55.90; H, 4.69; N, 5.01.
5.6.16. 1-Cyclohexyl-1,1-di-[(4-(tert-butyldimethylsilyl-
oxy)-phenyl]methanol (23). To a solution of 22 (500 mg,
1.13 mmol) in dry ether (50 mL), cyclohexyl magnesium
chloride (1.2 mL, 2.4 mmol) was added dropwise with
stirring at 0 ꢁC under N2. The reaction mixture after
being stirred overnight at room temperature was diluted
with dilute HCl and extracted with ether. The combined
ethereal extracts were washed with water, brine, dried
(MgSO4), filtered and evaporated to give crude 23 as a
pale white oil (550 mg, 92%). TLC analysis of this crude
5.6.14. (1,3-Adamantanediyl)diphenyl-4,40-O,O-bis-sulfa-
mate (20) and 40-hydroxy-(1,3-adamantanediyl)diphenyl-
4-O-sulfamate (21). Upon sulfamoylation, 4,40-(1,3-ada-
mantanediyl)diphenol (210 mg, 655 lmol) gave a crude
product (273 mg), which was fractionated by flash
chromatography (chloroform/acetone, gradient). The
fraction at Rf 0.25 (chloroform/acetone, 4:1) gave a
white residue (159 mg), which was further purified by
recrystallisation from acetone/hexane (1:2) to give 20 as
white crystals (112 mg, 36%); mp 197–199 ꢁC; mmax (KBr)
1
has shown a single spot although H NMR has indi-
cated the presence of starting material (ca. 10–20%);
TLC (chloroform): Rf 0.61; mmax (film) 3420 (OH),
1
1600 cmꢁ1; H NMR (400 MHz, DMSO-d6) 0.14 (12H,
s, 2 · Si(CH3)2), 0.92 (18H, s, 2 · (CH3)3), 0.96–2.32
(11H, m), 4.95 (1H, s, ex. with D2O, OH), 6.7 (4H, d, J
8.5 Hz, C3-H, C5-H, C30-H and C50-H) and 7.31 (4H, d,
J 8.8 Hz, C2-H, C6-H, C20-H and C60-H); MS m=z
(FABþ) 525.5 [30, (MꢁH)þ], 443.4 [100, (Mꢁcyclo-
hexyl)þ], 427.4 (50), 319 (20), 73.0 (60); MS m=z (FABꢁ)
679.0 [10, (MþNBA)ꢁ], 525.5 [40, (MꢁH)ꢁ], 441.3 [20,
(MꢁHꢁcyclohexyl)ꢁ], 411.4 (100), 327.3 [50,
(MꢁcyclohexaneꢁSi(CH3)2C(CH3)3]ꢁ, 317.3 (40); Acc.
MS (FABþ) 526.3214, C31H50O3Si2 requires 526.3298.
3420–3240 (NH2), 1390 (SO2) cmꢁ1
;
1H NMR
(270 MHz, acetone-d6) 1.8–2.25 (14H, m), 7.12 (4H, br s,
ex. with D2O, 2 · SO2NH2), 7.24–7.32 (4H, AA0BB0,
ArH), 7.48–7.56 (4H, AA0BB0, ArH); MS m=z (FABþ)
478.1 [80, (M)þ], 399.2 [30, (MþHꢁSO2NH2)þ]; MS m/z
(FABꢁ) 631.2 [30, (MþNBA)ꢁ], 477.2 [100, (MꢁH)ꢁ],
398.2 [20, (MꢁSO2NH2)ꢁ]; Acc. MS (FABþ) 478.1227,
C22H26N2O6S2 requires 478.1232. Found C, 55.1; H,
5.63; N, 5.85; C22H26N2O6S2 requires C, 55.21; H, 5.48;
N, 5.85.
The fraction at Rf 0.39 (chloroform/acetone, 4:1) gave a
white residue (38 mg), which was further purified by
recrystallisation from ethyl acetate/hexane (1:2) to give
21 as white crystals (20 mg, 8%); mp 140–142 ꢁC; mmax
5.6.17. 1-Cyclohexyl-1,1-di-(4-hydroxyphenyl)methanol
(24). To a solution of 23 (500 mg, 950 lmol) in dry
THF (10 mL), tetra-n-butylammonium fluoride (5.6 mL
of 1 M solution in THF, 5.68 mmol) was added drop-
wise. The resulting mixture was stirred for about 5–
10 min and then diluted with ethyl acetate (100 mL). The
organic layer was washed with water (100 mL), dried
(MgSO4), filtered and evaporated to give a white solid
(280 mg), which was recrystallised from acetone/hexane
(1:2) to give 24 as white crystals (260 mg, 92%); mp 115–
117 ꢁC; TLC (chloroform and chloroform/acetone, 8:1):
Rf s 0.21 and 0.52; mmax (KBr) 3500–3140 (OH),
1
(KBr) 3500–3000 (NH2 and OH), 1370 (SO2) cmꢁ1; H
NMR (270 MHz, acetone-d6) 1.8–2.29 (14H, m), 6.78
(2H, d, J 8.8 Hz, C30-H and C50-H), 7.08 (2H, br s, ex.
with D2O, SO2NH2), 7.26 (4H, br d, J 8.79 Hz, ArH),
7.5 (2H, d,J 8.8 Hz, ArH) and 8.15 (1H, br s, ex. with
D2O, OH); MS m=z (FABþ) 399.1 [100, (M)þ], 320.2
[15, (MþHꢁSO2NH2)ꢁ], 306.1 (45), 288.1 (15); MS m=z
(FABꢁ) 552.3 [30, (MþNBA)ꢁ], 398.2 [100, (MꢁH)ꢁ];
Acc. MS (FABþ) 400.1533, C22H26NO4S requires
400.1583.
1
1600 cmꢁ1; H NMR (400 MHz, DMSO-d6) 0.96–2.26
(11H, m), 4.74 (1H, s, ex. with D2O, OH), 6.66 (4H, d, J
8.8 Hz, C3-H, C5-H, C30-H and C50-H), 7.21 (4H, d, J
8.8 Hz, C2-H, C6-H, C20-H and C60-H) and 9.1 (2H, s,
ex. with D2O, 2 · OH); MS m=z (FABþ) 298.2 [10,
(M)þ], 215.1 [100, Mꢁcyclohexyl]þ, 205.2 (15); MS m=z
(FABꢁ) 451.3 [40, (MþNBA)ꢁ], 297.3 [100, (MꢁH)ꢁ],
279.3 (30), 213.2 [30, (MꢁHꢁcyclohexane)ꢁ]; Acc. MS
(FABþ) 298.1558, C19H22O3 requires 298.1569.
5.6.15. 4,40-(Di-tert-butyldimethylsilyloxy)benzophenone
(22). To a solution of 4,40-dihydroxybenzophenone
(5.0 g, 23.3 mmol) in anhydrous DMF (15 mL), tert-
butyldimethyl-chlorosilane (4.22 g, 28 mmol) and imid-
azole (4.0 g, 58.8 mmol) were added and the reaction
mixture stirred under N2 for 3 h. After dilution with 5%
aq NaHCO3 (100 mL) and ethyl acetate (200 mL), the
organic layer that separated was washed with water,
brine, dried (MgSO4), filtered and evaporated to give 22
as a pale white oil, which solidified on standing (10 g,
97%); TLC (chloroform): Rf 0.69; mmax (KBr) 1620
5.6.18. 1-Cyclohexyl-1,1-di-(4-sulfamoyloxyphenyl)meth-
anol (25). To a solution of 24 (150 mg, 503 lmol) and
2,6-di-tert-butyl-4-methylpyridine (930 mg, 4.53 mmol)
in dichloromethane (25 mL) was added dropwise with

H. A. M. Hejaz et al. / Bioorg. Med. Chem. 12 (2004) 2759–2772
2771
stirring a freshly concentrated solution of sulfamoyl
chloride in toluene (ca. 0.7 M, 3.0 mmol). After 2 h of
stirring, the solution was diluted with dichloromethane
(100 mL). The organic layer was washed with water,
brine, dried (MgSO4), filtered and evaporated to give a
residue (450 mg), which was fractionated by flash chro-
matography (chloroform/acetone, gradient). The white
solid that obtained (180 mg) was further purified by
recrystallisation from acetone/hexane (1:2) to give 25 as
pale white crystals (120 mg, 52%); mp 213–215 ꢁC (dec);
TLC (chloroform, and chloroform/acetone, 8:1); Rf s
0.12 and 0.31, respectively; mmax (KBr) 3500–3000 (NH2),
1600, 1380 (SO2) cmꢁ1; 1H NMR (400 MHz, DMSO-d6)
1.31 (1H, br s), 1.57 (6H, br s), 2.15 (4H, br s), 7.25 (8H,
m, ArH), 8.0 (4H, br s, ex. with D2O, SO2NH2) and 9.68
(1H, br s, ex. with D2O, OH); MS m=z (FABþ) 457.1
[10, (MþH)þ], 206.2 (100); MS m=z (FABꢁ) 608.2 [40,
(MꢁHþNBA)ꢁ], 455 [15, (MꢁH)ꢁ], 249.0 (100); Acc.
MS (FABþ) 457.1065, C19H25N2O7S2 requires 457.1103.
precipitate (tetrazonium salt) was added (cold, 0–5 ꢁC)
in small portions to boiling 1 N sulfuric acid (100 mL).
The resulting red solution was heated with charcoal for
decolourisation and the suspension was filtered hot. On
cooling the filtrate, the yellow precipitate that formed
was collected by filtration, washed with water and air
dried to give a yellow powder (1.92 g, 49%); which was
recrystallised from acetone/hexane (1:2) to give 27 as
yellow crystals (1.3 g, 33%); mp 161–163 ꢁC (lit.25 163–
164 ꢁC); TLC (chloroform/acetone, 8:1): Rf 0.63; mmax
1
(KBr) 3500–3000 (OH), 1630 (C@O) cmꢁ1; H NMR
(270 MHz, acetone-d6) 7.12 (2H, m, C4-H and C40-H),
7.23 (4H, m, C6-H, C60-H, C2-H and C20-H), 7.38 (2H,
t, J 8.1 Hz, C5-H and C50-H) and 8.74 (2H, br s, ex. with
D2O, 2 · OH); MS m=z (FABþ) 215.1 [100, (MþH)þ],
199.1 (10), 185.1 (15); MS m=z (FABꢁ) 367.2 [40,
(MþNBA)þ], 213.1 [50, (MꢁH)ꢁ], 139.1 (5); Acc. MS
(FABþ) 215.0715, C13H11O3 requires 215.0708.
5.6.21. Benzophenone-3,30-O,O-bis-sulfamate (28) and 30-
hydroxybenzophenone-3-O-sulfamate (29). Upon sulfa-
moylation, 27 (1.0 g, 4.67 mmol) gave a dark yellow
residue (1.42 g), which was fractionated by flash chro-
matography (chloroform/acetone, gradient). The band
at Rf 0.21 (chloroform/acetone, 4:1) gave a yellow resi-
due (567 mg), which was further purified by recrystalli-
sation from acetone/hexane (1:2) to give 28 as yellow
crystals (310 g, 18%); mp 141–143 ꢁC; TLC (chloroform/
acetone, 4:1): Rf 0.42; mmax (KBr) 3360–3580 (NH2), 1650
(C@O), 1380 (SO2); 1H NMR (270 MHz, acetone-d6)
7.29 (4H, br s, ex. with D2O, 2 · SO2NH2) 7.65 (4H, m,
ArH) and 7.77 (4H, m, ArH); MS m=z (FABþ) 373.0
[100, (MþH)þ], 211.1 [20, (MꢁHꢁ2 · SO2NH2)þ]; MS
m=z (FABꢁ) 371.1 [100, (MꢁH)ꢁ]; Acc. MS (FABþ)
373.0165, C13H13N2O7S2 requires 373.0164. Found C,
42.2; H, 3.23; N, 7.21; C13H12N2O7S2 requires C, 41.93;
H, 3.25; N, 7.52.
5.6.19. 3,30-Dinitrobenzophenone (26). To a stirred mix-
ture of benzophenone (10 g, 54.88 mmol) in concd sul-
furic acid (55 mL) at room temperature was added a
mixture of concd nitric acid (6 mL) and concd sulfuric
acid (14 mL). The reaction mixture was then slowly
heated to 75 ꢁC, and maintained at this temperature for
30 min. After cooling to room temperature, the reaction
mixture was poured onto crushed ice. The gummy mass
that formed hardened over time and after being grinded
into powder was washed with water until the washings
were neutral. The cake/powder that collected was air
dried and recrystallised from butanone (73 mL). The
solid that formed overnight was washed first with
butanone and then with ethanol to give crude 26 as a
yellow solid (7.9 g, 53%). An analytical sample was
further recrystallised from methanol to give 26 as pale
yellow crystals; mp 144–146 ꢁC (lit.25 148–149 ꢁC); TLC
(chloroform/acetone, 8:1): Rf 0.8; mmax (KBr) 1670
(C@O) cmꢁ1; 1H NMR (270 MHz, CDCl3) 7.78 (2H, t, J
7.9 Hz, C5-H and C50-H), 8.15 (2H, dt, J ꢂ 2 and
7.7 Hz, ArH), 8.53 (2H, ddd, J ꢂ 1–2 and 8.2 Hz, ArH)
and 8.64 (2H, t, J ꢂ 2 Hz, C2-H and C20-H); MS m=z
(FABþ) 273.0 [100, (MþH)þ], 259.1 (90), 243.1 (50);
MS m=z (FABꢁ) 425.2 [30, (MþNBA)ꢁ], 272.1 [100,
(M)ꢁ], 257.2 (10); Acc. MS (FABþ) 273.0500,
C13H9N2O5 requires 273.0512. Found C, 57.1; H, 2.89;
N, 10.30; C13H8N2O5 requires C, 57.36; H, 2.96; N,
10.29.
The fraction at Rf 0.56 (chloroform/acetone, 2:1) gave a
yellow residue (215 mg), which was further purified by
recrystallisation from ethyl acetate/hexane (1:2) to give
29 as yellow crystals (163 mg, 12%); mp 190–192 ꢁC;
TLC (chloroform/acetone, 4:1): Rf 0.31; mmax (KBr)
3220–3000 (NH2 and OH), 1640 (C@O), 1400 (SO2)
1
cmꢁ1; H NMR (270 MHz, acetone-d6) 6.64 (2H, br s,
ex. with D2O, OSO2NH2), 7.17 (1H, t, J 7.5 Hz, C50-H),
7.47 (2H, m, C20-H and C40-H), 7.58 (2H, dd, J 8.1 Hz,
C4-H and C5-H), 7.79 (1H, d, J 7.3 Hz, C60-H), 8.0 (1H,
d, J 8.8 Hz, C6-H), 8.11 (1H, s, C2-H) and 8.87 (1H, br
s, ex. with D2O, OH); MS m=z (FABþ) 447.1 [10,
(MþHþNBA)þ], 294.0 [100, (MþH)þ], 214.1 [10,
(MþHꢁSO2NH2)þ]; MS m=z (FABꢁ) 446.1 [20,
(MþNBA)ꢁ], 292.0 [100, (MꢁH)ꢁ], 213.0 [30, (MꢁSO2-
NH2)ꢁ]; Acc. MS (FABþ) 294.0411, C13H11NO5S
requires 294.0436.
5.6.20. 3,30-Dihydroxybenzophenone (27). A mixture of
26 (5.0 g, 18.37 mmol), tin chloride (25 g, 131.9 mmol)
and concentrated hydrochloric acid (35 mL) was stirred
at 70 ꢁC for 6 h. The dark yellow crystalline benzophe-
none-3,30-diammonium chlorostannate that separated
was collected by filtration. After re-suspending the solid
collected in concentrated hydrochloric acid (35 mL) at
0 ꢁC, this cold suspension was added dropwise to a cold
solution (at 0 ꢁC) of sodium nitrite (2.56 g in 10 mL
water). After the addition was completed, the suspen-
sion was stirred at 0 ꢁC for 1 h and then quickly filtered
by means of a cold glass funnel. The resulting yellow
Acknowledgements
This work was supported by Sterix Ltd. We thank Mrs.
C. M. Parker for assistance in the preparation of this
manuscript and Ms. A. C. Smith for technical assistance.

2772
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19. Jutten, P.; Schumann, W.; Hartl, A.; Heinisch, L.; Grafe,
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