A-ring substituted 17β-arylsulfonamides of 17β-aminoestra-1,3,5(10)-trien-3-ol as highly potent reversible inhibitors of steroid sulfatase

Article abstract of DOI:10.1016/j.bmc.2015.07.019

Steroid sulfatase (STS) catalyzes the hydrolysis of the sulfate ester group in biologically inactive sulfated steroids to give biologically active steroids. Inhibitors of STS are considered to be potential therapeutics for treating hormone-dependent cancers such as ER⁺ breast cancer. A series of 4-substituted 17β-arylsulfonamides of 17β-aminoestra-1,3,5(10)-trien-3-ol were prepared and examined as STS inhibitors. The presence of a NO₂ or Br at the 2-position of the A-ring resulted in a decrease in potency compared to their A-ring-unsubstituted counterparts. However the presence of a nitro group or fluorine atom at the 4-position of the A-ring resulted in an increase in potency and one of these compounds exhibited a K_i^app value of 1 nM. Modeling studies provided insight into how these compounds interact with active site residues. The anti-proliferative activity of the 3′-Br, 3′-CF₃, 4-NO₂-3′-Br and 4-NO₂-3′-CF₃ derivatives were examined using the NCI 60-cell-line panel and found to have mean graph midpoint values of 1.9-3.4 μM.

Full text of DOI:10.1016/j.bmc.2015.07.019

Accepted Manuscript
A-ring Substituted 17β-Arylsulfonamides of 17β-Aminoestra-1,3,5(10)-tri-
en-3-ol as Highly Potent Reversible Inhibitors of Steroid Sulfatase
Yaser A. Mostafa, Braden Kralt, Praveen P.N. Rao, Scott D. Taylor
PII:
S0968-0896(15)00602-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.019
BMC 12448
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 May 2015
29 June 2015
9 July 2015
Please cite this article as: Mostafa, Y.A., Kralt, B., Rao, P.P.N., Taylor, S.D., A-ring Substituted 17β-
Arylsulfonamides of 17β-Aminoestra-1,3,5(10)-trien-3-ol as Highly Potent Reversible Inhibitors of Steroid
Sulfatase, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.019
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A-ring Substituted 17-Arylsulfonamides of 17-Aminoestra-1,3,5(10)-trien-
-ol as Highly Potent Reversible Inhibitors of Steroid Sulfatase.
3
δ

λ
δ*
Yaser A. Mostafa, Braden Kralt, Praveen P. N. Rao and Scott D. Taylor
δ
Department of Chemistry, University of Waterloo, 200 University Ave. West, Waterloo, Ontario,
N2L 3G1, Canada
λ
School of Pharmacy, Health Sciences Campus, 200 University Ave. West, Waterloo, Ontario,
N2L 3G1.
*
Corresponding author:
Prof. Scott D. Taylor
Department of Chemistry,
University of Waterloo
2
00 University Ave. West
Waterloo, ON Canada,
N2L 3G1
Phone: 519-888-4567
Fax: 519-746-0435
Email: s5taylor@uwaterloo.ca
Keywords: steroid sulfatase, hormone therapy, enzyme inhibitors
1

Abstract
Steroid sulfatase (STS) catalyzes the hydrolysis of the sulfate ester group in biologically
inactive sulfated steroids to give biologically active steroids. Inhibitors of STS are considered to
be potential therapeutics for treating hormone-dependent cancers such as ER+ breast cancer. A
series of 4-substituted 17-arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol were
prepared and examined as STS inhibitors. The presence of a NO or Br at the 2-position of the
2
A-ring resulted in a decrease in potency compared to their A-ring-unsubstituted counterparts.
However the presence of a nitro group or fluorine atom at the 4-position of the A-ring resulted in
app
i
an increase in potency and one of these compounds exhibited a K
value of 1 nM. Modeling
studies provided insight into how these compounds interact with active site residues. The anti-
proliferative activity of the 3´-Br, 3´-CF , 4-NO -3´-Br and 4-NO -3´-CF derivatives were
examined using the NCI 60-cell-line panel and found to have mean graph midpoint values of 1.9-
.4 M.
3
2
2
3
3
2

Introduction
It is now well-established that the sex hormones are involved in the development of
1
certain cancers such as breast, prostate and endometrial cancer. Consequently, the development
of anticancer drugs that function by blocking the action or limiting the production of these
hormones has been the focus of intensive research for many years. This approach to cancer
therapy has been used extensively for the treatment of estrogen receptor positive (ER+) breast
cancer where drugs such as tamoxifen, which acts as an estrogen receptor antagonist, or letrozole
which blocks the production of estrogens by inhibiting the enzyme aromatase, have enjoyed
2
some clinical success.
However, many breast cancer patients experience relapse after
2
undergoing these treatments especially those with metastatic ER+ breast cancer. Thus, the
pursuit of alternative drugs/therapies for treating ER+ breast cancer has continued unabated.
Steroid sulfatase (STS) catalyzes the hydrolysis of the sulfate ester group in biologically
inactive sulfated steroids such as estrone sulfate (E1S) and dehydroepiandrosterone sulfate
(
(
DHEAS) to give biologically active steroids such as estrone (E1) and dehydroepiandrosterone
DHEA) (Figure 1). Several studies have suggested that STS plays an important role in the
progression of ER+ breast cancer. The production of E1 from E1S in breast cancer tissue is
approximately 10 times greater than from androstenedione, which is converted to E1 by
3
aromatase. About 90% of Adiol in post-menopausal women originates from DHEAS via
desulfation of DHEAS by STS to give DHEA which is converted into Adiol by a
4
dehydrogenase. There is approximately 50-200 times greater STS activity than aromatase
5
-7
activity in malignant breast tissues. Sulfatase activity in breast cancer cells is higher than that
8
of normal breast cells. Finally, STS expression in breast tissue is significantly higher than in
3

normal tissue and STS expression is now used as a prognostic factor in human breast
9
-11
carcinoma.
Figure 1. Biosynthesis of estrogenic steroids.
Due the role of STS in promoting ER+ breast cancer and possibly other steroid-
dependent cancers such as prostate and endometrial cancer, inhibitors of STS have been pursued
1
2a-c
as potential anticancer agents for over 20 years.
By far the most common class of STS
inhibitors are the sulfamates. Since the discovery, in 1994, that estrone sulfamate (EMATE, 1,
1
3
Figure 2) is a potent irreversible STS inhibitor, a large number of sulfamates have been
12a-c,13
prepared and examined as inhibitors of STS.
However, after over two decades of research,
1
4
only two sulfamate-based STS inhibitors have entered clinical trials. E2MATE, the estradiol
1
5
analog of EMATE, is presently in Phase II clinical trials for the treatment of endometriosis.
®
6
67-COUMATE (compound 2 in Figure 2), also known as Irusosat, (2 in Figure 2) has
4

undergone clinical trials (Phase I with postmenopausal women having ER+ breast cancer and
1
6,17
Phase II with women having advanced endometrial cancer) with mixed results.
Hence, it is
crucial to ascertain if alternative pharmacophores can be developed to broaden the potential for a
successful clinical candidate.
O
O
S
O
HN
HN
R
O
O
S
O
S
CF3
H2N
O
H2N
O
O
O
HO
O
O
HO
EMATE (1)
3, R = 3'-Br
7
667-COUMATE (2)
4
5
6
, R = 3'-CF3
, R = 4'-t-Bu
, R = 4'-Ph
Figure 2. Structures of STS inhibitors 1-7.
In contrast to irreversible sulfamate-based inhibitors, relatively few reversible, non-
12-14
sulfamate inhibitors of STS have been reported.
We previously reported that certain 17-
arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol, such as compounds 3-6 (Figure 2),
were very good reversible inhibitors of STS (IC50 values of 3-6 = 9-25 nM) when assayed with
1
8
purified STS using 4-methylumbelliferyl sulfate (4-MUS) as the substrate.
Replacing the
sulfonamide group in 4 with an amide group (compound 7) yielded a much poorer inhibitor
indicating that the sulfonamide group is key to potent inhibition.
We previously demonstrated that the affinity of E1 or E2 for STS can be increased by
introducing a small electron-withdrawing group (EWG) or atom, such as fluorine, bromine,
1
9
cyano, and nitro, at the 4-position. The reason for this increase in potency is not known though
studies showed that it was not due to the decrease in pK of the 3-OH group as there was no
correlation between the pK of the steroid and IC50 values. This observation presented to us a
a
a
potential strategy for further increasing the potency of the 17-arylsulfonamides. Here we report
5

the synthesis of a series of 17-arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol bearing
a nitro group, or bromine or fluorine atom at the 2- or 4-position and their evaluation as
inhibitors of STS. Some of these compounds are highly potent reversible inhibitors of STS with
app
i
K
values as low as 1 nM. Selected inhibitors were evaluated for antiproliferative activity in
the NCI 60-cancer cell-line panel. Molecular docking studies into the STS crystal structure were
also performed to gain some insight into how they interact with active site residues.
Chemistry
2
- and 4-Nitro derivatives 20-27 and 2- and 4-bromo derivatives 28 and 29 were prepared
2
0
via the route outlined in Scheme 1. Reductive amination of 2- or 4-nitroestrone or 2- or 4-
2
1
bromo estrone with allylamine and sodium triacetoxyborohydride (STAB) gave derivatives 12-
5 in reasonable to good yields. Deallylation of 12-15 with dimethylbarbituric acid (DMBA)
and Pd(PPh gave amines 16-19 in low to good yields. Reaction of these amines with the
1
)
3 4
appropriate sulfonyl chlorides gave target compounds 20-29 in low to moderate yields though
these reactions were not optimized.
a
Scheme 1. Synthesis of 2- and 4-nitro and 2- and 4-bromo derivatives 20-29
a
Reagents and conditions: (i) allylamine, STAB, THF, rt, 2 days (63-78%); (ii) cat. Pd(PPh ) ,
3
4
o
o
DMBA, 35 C, 1h (21-71%); (iii) ArSO
2
Cl, pyr CH
2
Cl
2
, 0 C then rt O/N (26-67%).
For the evaluation of derivatives bearing a fluorine atom on the A-ring we focussed our
efforts on the preparation of the 4-fluoro-3'-CF
3
derivative 36 (Scheme 2). The synthesis of 36
6

required the preparation of 4-fluoroestrone (31) a compound that has been prepared by several
groups by a variety of routes. Compound 31 has been prepared via a multistep procedure in an
2
2-24
overall yield of 11% starting from 19-nortestosterone.
The key step in this procedure was the
regiospecific fluorination of the pyrrolidyl enamine of expensive 19-nortestosterone with toxic
perchloroylfluoride to give 4-fluoro-3-methoxyoestr-5-en-3, 17-diol. The 3-O-methyl ether
of 31 has been prepared by thermal decomposition of estrone 3-O-methyl ether 4-diazonium
2
3,25
22
fluoroborate.
Others have reported that the overall yield of this route is very low (6%).
2
6
22
Uemoto and coworkers as well as Page and coworkers have reported the synthesis of 31 and
its 2-isomer by subjecting E1 to N-fluoropyridinium triflate (NFPT), an electrophilic fluorinating
agent. Uemoto obtained the two isomers in a 45% yield in an approximately 1:1 ratio but did not
separate them. Page and coworkers reported that their 3-O-acetates could be separated by
fractional recrystallization from pet. ether-EtOH which gave the 2- and 4-isomers in yields of
5
3% and 20% respectively. We attempted to obtain 31 using this approach but were unable to
separate the two isomers either as their acetates or having a free 3-OH. To avoid formation of
the two isomers we first protected the 2-position with a tert-butyl group by subjecting estrone to
tert-butyl alcohol in the presence of cat. boron trifluoride diethyletherate which gave 2-tert-
2
7
butylestrone (30) in almost quantitative yield (Scheme 2). Reaction of 30 with NFPT followed
by removal of the tert-butyl protecting group using AlCl in nitromethane gave 31 in a 16% yield
two steps). Although the yield of 31 was low, its synthesis involved only three steps from E1
3
(
and no tedious separation of isomers was required. The 3-OH in 31 was protected with a TBS
group (84% yield) and the resulting compound, 32, was subjected to reductive amination with
BnNH and STAB which gave benzyl protected 17β-amine derivative 33 in an 82% yield.
2
Hydrogenolysis of the benzyl amine moiety in 33 gave 17β-amino derivative 34 in 97% yield.
7

Reacting 34 with excess of 3'-CF -benzenesulfonyl chloride gave sulfonamide 35 in a 79% yield.
3
Removal of the TBS group in 35 gave compound 36 in an 83% yield.
a
Scheme 2. Synthesis of 4-fluoro-subsituted derivative 36.
a
o
Reagents and conditions: (i) t-BuOH, cat. BF
3
OEt
, 0 C, 5h (16%, two steps); (iii) TBDMSCl, imidazole, DMF, 2
, STAB, AcOH, DCE, rt, 2 d (83%); (v) H , 15 wt % of Pd(OH)
MeOH/EtOAc (5:1), 12 h (97%); (vi) 3’-CF SO Cl, Et N, DMAP, CH Cl , rt, 48 h (79%);
2
, 12 h (96%); (ii) (a) NFPT, TCE, 135 C,
o
1
4 h; (b) AlCl
3
, MeNO
2
, CH
2
Cl
2
h, (84%); (iv) BnNH
2
2
2
,
3
C
6
H
4
2
3
2
2
(
vii) TBAF, THF, 3h (83%).
The X-ray crystal structure of 2-nitro derivative 25 was obtained and is shown in Figure
. The structure confirms the  stereochemistry at C-17. The nitro group is coplanar with the
3
aromatic A-ring and forms an intramolecular H-bond with the 3-OH. We were unable to obtain
diffraction quality crystals of the corresponding 4-nitro derivative 21; however, we were able to
obtain the crystal structure of 4-nitroestrone (see the supporting information). In 4-nitroestrone,
the nitro group is rotated out of the plane of the aromatic ring to avoid steric interactions with the
C-6. It is likely that the nitro group in 4-nitro derivative 21 adopts a similar conformation.
8

Figure 3. Asymmetric unit in the X-ray crystal structure of compound 25.
Results and Discussion
To ascertain the effect of C-4 substitution on the STS inhibitory activity of 17-
arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ols we initially prepared and evaluated the
2
- and 4-nitro derivatives 20-27. These and subsequent compounds were assayed using purified
STS at pH 7.0 in 20 mM Tris-HCl buffer containing 0.1 % triton X-100 and using 4-MUS as the
2
8
app
i
substrate. Their IC50 values or apparent K
i
values (K
) along with their non 4-substituted
analogs (3-6) are shown in Table 1.
9

Table 1. STS Inhibitory Activity of Compounds
-6, 20-29 and 36 with purified STS.
3
1
2
3
a
Compound R
R
R
IC50 (nM)
3
4
5
6
H
H
H
3'-Br
25
23
18
9
H
H
3'-CF
4'-t-Bu
4'-Ph
3
H
H
H
2
0
1
2
3
4
5
6
7
8
9
6
H
NO
NO
NO
NO
H
2
2
2
2
3'-Br
12
11
8
2
2
2
2
2
2
2
2
2
3
H
3'-CF
3
H
4'-t-Bu
4'-Ph
b
H
1
NO
NO
NO
NO
H
2
2
2
2
3'-Br
50
67
81
33
49
95
H
3'-CF
3
H
4'-t-Bu
H
4'-Ph
Br 3'-CF
3
3
3
Br
H
H
F
3'-CF
3'-CF
b
2.5
a
b
Errors are within ±5%; Apparent K
i
2
The introduction of a nitro group at the 4-position (R ) resulted in an increase in potency.
1
9
This is consistent with our previous finding that 4-NO E1 is more potent inhibitor than E1.
2
The effect of the 4-nitro group was most pronounced with the 4'-phenyl derivative 23 which
app
i
exhibited a K
value of 1 nM. The affinity of 23 for STS was such that its IC50 depended on the
concentration of enzyme (Figure 4) which is expected when the IC50 of an inhibitor is lower than
1
0

2
9,30
app
the concentration of enzyme used in the assay.
intercept the plot of IC50 vs. the total enzyme concentration ([E]
nitro group at the 2-position resulted in a 2-4.5-fold decrease in potency. This is in contrast to 2-
NO E1 which was approximately 2-fold more potent than E1.
The K
i
for 23 was obtained from the Y-
2
9
T
) (Figure 4). Introduction of a
2
50
40
30
20
10
0
0
20
40
60
80
[
E] (nM)
t
Figure 4. Plot of IC50 values for compound 23 as a function of [E]
T
.
Interestingly, nitrated EMATE derivatives exhibit trends, in terms of their ability to
inhibit STS, that are similar to those reported here for the 17-arylsulfonamide-based inhibitors
in that 4-nitroEMATE (37, Figure 5) is a 5-fold more potent inhibitor than EMATE while the 2-
nitro analog of EMATE is 18-fold less potent than EMATE when assayed with crude STS from
3
1
placental microsomes. The reason for these differences is not known though it was suggested
that intramolecular hydrogen bonding between the 2-nitro group and the sulfamoyl moiety in
EMATE may occur which could potentially affect its ability to interact with STS and that this
3
1
type of intramolecular interaction does not occur with the 4-nitro derivative. The enhanced
affinity of 4-nitroEMATE compared to EMATE was attributed to favorable interactions between
the nitro group and residues in the STS active site. The similar trends in inhibitory potency
between the nitrated 17-arylsulfonamide-based inhibitors presented here and the nitrated
EMATES suggest that 17-arylsulfonamide-based inhibitors in Table 1 might initially interact
1
1

with STS in a manner that is similar to EMATE and its derivatives even though their
mechanisms of action are completely different.
For studies on compounds bearing a Br or F at the 2- or 4-positions we focussed on
preparing and evaluating the 3'-CF
a Br at the 4-position was 2-fold less potent than its A-ring unsubstituted analog, compound 3
Table 1). This is in contrast to our results with E1 in that 2-BrE1 is a 10-fold more potent
3
derivatives (28, 29 and 36). Compound 28 which contained
(
1
9
inhibitor than E1. The 2-Br analog 29 was 4-fold less potent than 3. Introduction of a fluorine
app
i
to the 4-position, compound 36, resulted in a 9-fold increase in potency with a K
of 2.5 nM
(
obtained from the Y-intercept of a plot of IC50 vs. [E] - see the supporting information).
T
Figure 5. Structures of STS inhibitors 37-42 and 2-MeOE2 (43).
The development of future STS inhibitors based on the scaffold described here would be
greatly assisted by knowing how these inhibitors interact with STS. Poirier and coworkers have
reported that 17-benzylE2 inhibitors of type 38 are potent reversible inhibitors of STS (IC50
values as low as 21 nM when assayed using crude STS obtained from homogenates of HEK or
3
32-36
JEG-3 cells at pH 7.0 and [ H]E1S as substrate).
They suggested that the benzyl group
extends into the hydrophobic tunnel between the two alpha helices of STS that insert into the
1
2

membrane of the ER and this results in an increase in potency compared to E2. We reported
previously that compound 40, which has a fluorine at the 4-position is a 15-fold more potent
1
9
inhibitor (IC50 = 40 nM) than 39 under our assay conditions.
Moreover, under our assay
conditions, we have shown that 17-benzylE2 inhibitors and 17-arylsulfonamides of 17-
aminoestra-1,3,5(10)-trien-3-ol exhibit mixed inhibition suggesting that these compounds are
1
8,19,37
capable of binding at both the active site and at a secondary site outside the active site.
To study possible active site binding interactions the 3'-CF series of inhibitors were
3
docked into the crystal structure of STS (PDB ID: 1P49), after conversion of the sulfated
formylglycine75 (FGly75) hydrate to an FGly75 hydrate, using the build tool in Discovery
Studio (DS) – Structure-Based Design (SBD) software. The LibDock docking algorithm in DS
was used to investigate active site binding modes of the STS inhibitors. Figures 6A-C show the
docking poses obtained for compounds 4 (Figure 6A), 21 (Figure 6B) and the corresponding 3'-
trifluoromethyl-17-benzylE2 inhibitor 41 (Figure 6C) which has been reported by Poirier and
coworkers to have an IC50 of 126 nM when assayed using crude STS obtained homogenates of
3
35
JEG-3 cells at pH 7.0 and [ H]E1S as substrate. We also show the docking pose for compound
(Figure 6D) which is the amide analog of 4 which we had previously shown to have an IC50
value 31-times greater than 4.
The A-, B-, C- and D-rings of compounds 4, 21 and 41 share common hydrophobic
7
interactions with a variety of hydrophobic amino acids such as Val101, Leu74, Val486, Phe488
and Val177. These hydrophobic interactions are shown for compound 21 (Figure 6B) but are not
shown for compounds 4 and 41 (Figures 6A and 6C ) so that other interactions can be visualized
more clearly. When bound in the active site in this fashion the aryl sulfonamide groups in 4 and
1
3

2
1 and the benzyl portion in 41 do not extend to any significant extent down the hydrophobic
tunnel.
A (compound 4)
B (compound 21)
C (compound 41)
D (compound 7)
Figure 6. The binding poses of compounds 4 (A), 21 (B), 41 (C) and 7 (D). All hydrogens,
with the exception of the hydrogens bonded to the nitrogen of the sulfonamide groups in 4 and
2
1, the amide group in 7 and the 17-O of compound 41, have been removed for reasons of
clarity. H-bonds are shown as dashed green lines and are 2.4-3.5 Å. Hydrophobic interactions
pi-alkyl, alkyl-alkyl) are indicated by dashed purple lines and are 3.9-5.5 Å. The pi-cation
(
interaction involving Trp550 and the benzene sulfonamide moiety in 21 shown in panel B is
indicated by a dashed dark purple line (3.95 Å). The pi-donor H-bond interaction between
His346 and the 3-OH of 7 shown in panel D is indicated by a dashed light green line (4.05 Å).
The electrostatic interaction between the nitro group in 21 and Lys368 shown in panel B is
+
2
indicated by a dashed orange line (3.81 Å). The Ca ion is shown as a green sphere.
1
4

The 3-OH in compounds 4, 21 and 41 forms H-bonds with the OH and/or C=O of the
FGly hydrate. It would be expected that this interaction would be affected by the presence of an
EWG attached to the A-ring. However, there does not appear to be a relationship between
inhibitor potency (i.e. the potency of 4-nitro inhibitor 21, 4-bromo inhibitor 28 and 4-fluoro
3
8
inhibitor 36) and the pK ’s of the corresponding ortho-substituted phenols. This result suggests
a
that the effect of these EWG’s on the potency of the inhibitors may also be attributed to the
EWG’s being involved in specific interactions (H-bonding, electrostatic interaction etc.) with
active site residues. The docking pose for 21 shows the 4-NO group rotated out of the plane of
2
the aromatic A-ring, similar to the nitro group in 4-nitroestrone (see the supporting information),
and is involved in electrostatic interactions with the side chain of Lys 368. This results in a
slight rotation and shifting of the steroid backbone in 21 in relation to the steroid backbone in
compounds 4 and 41 whose A-D rings almost completely overlap as seen in Figures 7A and 7B.
The sulfonamide N-H in compounds 4 and 21 as well as the 17-OH in compound 41 form
an H-bond with the carbonyl of Val177. One of the S=O oxygens in compound 4 H-bonds with
the N-H of Gly181.
o
Although the phenyl portion of the aryl sulfonamide group of 4 is at almost a 90 angle to
the phenyl group of the benzyl moiety in 41 (see Figure 7B), a fluorine atom in both 4 and 41
forms an H-bond with the side chain of Arg98. In contrast, the CF group in 21 is rotated
3
slightly away from Arg98 and is involved in a hydrophobic interaction with side chain of Trp550
and the side chain of Arg98 is involved in a pi-cation interaction with the phenyl group of the
sulfonamide moiety.
Amide 7, which is a much poorer inhibitor than its sulfonamide analog 4, exhibits a
docking pose that is quite different from compounds 4, 21 or 41 (Figures 6D and 7C). In
1
5

compound 7, no H-bond is formed between the 3-OH and FGly hydrate as the entire inhibitor is
shifted away from the FGly hydrate towards the entrance of the active site. There is a weak pi-
donor H-bond interaction between the 3-OH and the imidazole ring of His346. The carbonyl of
the amide is involved in two H-bonds with the side chain of Arg98. The CF
3
group is involved
in weak hydrophobic interactions with Phe104 and Phe553.
A
B
C
Figure 7. Overlay of the binding poses of (A); 4 (grey) and 21 (gold), (B); 4 (grey) and 41
(
gold) and, (C); 4 (grey) and 7 (gold).
These differences in binding modes may, at least in part, explain the differences in
potency between these compounds; however, as 17-benzylE2 inhibitors and 17-
1
9
arylsulfonamides of 17-aminoestra-1,3,5(10)-trien-3-ol exhibit mixed inhibition, which
indicates that these inhibitors bind at both the active site and a site outside the active site, then
these modeling studies do not provide a complete picture of all inhibitor-protein interactions. In
the absence of an x-ray structure of an STS-inhibitor complex, the location of the additional
binding site would be entirely speculative.
Many sulfamate-based STS inhibitors have been examined for antiproliferative activity in
cell-based assays. Previous reports have demonstrated that the antiproliferative activity
displayed in in vitro assays using MCF-7 cells were predictive of the spectrum of desirable
biological properties obtained with certain sulfamate-based STS inhibitors such as 2-
3
9-41
MeOEMATE (42, Figure 5).
It is believed that the antitumor activity of compounds such as
1
6

4
0
4
2 is due to their ability to disrupt the tubulin-microtubule equilibrium in cells. In contrast,
very few reversible STS inhibitors have been evaluated for their antiproliferative activity in cell-
based assays. Potter and coworkers reported 51% inhibition of growth of MCF-7 cells with 10

M compound 39 which had been previously shown by Poirier and coworkers to be a
moderately good STS inhibitor (IC50 = 310 nM) when assayed using STS obtained from
3
39
homogenates of JEG-3 cells and [ H]-E1S as substrate.
Compounds 3, 4, 20, and 21 were screened for their antiproliferative activity in the NCI
+
human 60-cancer cell line panel which included two ER breast cancer cell lines (MCF-7 and T-
-
4
7D) and four ER breast cancer cell lines (HS578T, BT-549, MDA-MB-468, MDA-MB-231).
The GI50 values with these breast cancer cell lines are shown in Table 2. The GI50’s of these
compounds with these breast cancer cell lines ranged from for 1.8-5.0 M and there was little
-
difference in cytotoxicity between the ER+ and ER lines. There was no correlation between
STS inhibitory activity and GI50 values though this was not unexpected. In comparison, 2-
methoxyestradiol (MeOE2, 43, Figure 5) an experimental drug candidate (under trade name of
®
Panzem ) undergoing clinical trials for treating breast cancer and ovarian cancer, exhibits a GI50
3
9-41
value of 2.4 M with MCF-7 cells.
The GI50 values of 4-substituted EMATE derivatives
such as 4-nitroEMATE with MCF-7 cells have not been reported. In contrast, many 2-
substituted EMATE derivatives have been examined for their ability to inhibit the growth of
3
9-41
MCF-7 cells.
The GI50 values of these compounds depended greatly on the appended
substituents and the presence of a sulfamate group at the 3-position was found to be important
for antiproliferative activity. Some of these compounds exhibited GI50 values with MCF-7 and
other cancer cell lines in the low nanomolar range. The GI50 values for compounds 3, 4, 20, and
2
1 with the other 54 cell lines evaluated in the screen were between 1-10 M. The mean graph
1
7

midpoint (MGM) data in Table 2 which represent the average of the GI50 values with all 60 cell
lines, were between 1.9-3.4 M for these four compounds. In comparison, MeOE2 exhibited an
3
9-41
MGM value of 1.3 M in this screen.
Table 2. GI50 values (M) and MGM (M) values of compounds 3, 4, 20, 21 obtained from the
NCI screening.
Compound MCF-7
T-47D
5.0
HS578T BT-549 MDA-MB-468 MDA-MB-231
MGM
3.3
3
3.7
2.7
1.9
1.8
4.0
3.2
2.1
2.2
3.1
2.7
1.6
1.9
2.4
4.1
2.2
2.7
3.9
2.4
1.5
1.9
4
3.7
2.5
1.8
3.4
1.9
2.2
2
0
1
2
Conclusions
In this study we set out to further refine STS inhibitory activity of 17-arylsulfonamides
of 17-aminoestra-1,3,5(10)-trien-3-ol by examining the effect of small EWGs at the 2- and or
4
- position of the A-ring. The presence of a NO , F or Br at the 2-position of the A-ring resulted
2
in a decrease in potency compared to their A-ring-unsubstituted counterparts. In contrast, the
presence of a nitro group or fluorine atom at the 4-position of the A-ring resulted in an increase
app
in potency. Compounds 23 and 36 were found to be tight-binding inhibitors with K
and 2.5 nM respectively. Modeling studies performed on the 3´-CF
reasons for differences in binding affinity amongst this series of compounds. The anti-
proliferative activity of the 3´-Br, 3´-CF , 4-NO -3´-Br and 4-NO -3´-CF derivatives were
i
values of
1
3
series revealed possible
3
2
2
3
examined using the NCI 60 cell-line panel. The mean graph midpoint (MGM) values of these
compounds, 1.9-3.4 M, were similar to that previously reported for 2-methoxyestradiol (1.3

M) an experimental drug candidate undergoing clinical trials for treating breast cancer and
ovarian cancer. Further studies to determine the estrogenic properties of these compounds and if
1
8

these compounds interact with STS at more than one site are in progress.
Experimental Section
General Procedures. All buffers, starting materials and reagents were obtained from
commercial suppliers. THF was distilled from sodium-benzophenone, pyridine was distilled
from KOH pellets, 1,2-DCE and 1,1,2-TCE were dried by standing over activated type 4A
molecular sieves, CH
2
Cl was distilled from calcium hydride under nitrogen. Benzylamine and
2
allylamine were dried by distillation from KOH pellets and stored in the dark over type 4 Å
molecular sieves. Dioxane was distilled from Na and stored over type 4 Å molecular sieves.
Silica gel chromatography was performed using silica gel (60Å, 230-400 mesh) obtained from
1
13
19
Silicycle (Laval, Quebec, Canada). H-, C-, and F-NMR spectra were recorded on a Bruker
Avance 300 spectrometer. For NMR spectra obtained using CDCl as the solvent, chemical
3
1
shifts () for H NMR spectra are reported relative to internal Me
4
Si ( 0.0 ppm), chemical shifts
1
3
for C spectra are relative to the residual solvent peak (77.0 ppm, central peak), and chemical
1
9
shifts for F-NMR are relative to a CFCl
3
( 0.0 ppm) external standard. High-resolution
(
HRMS) electrospray ionization (ESI) mass spectra were obtained on a Waters/Micromass
QTOF Ultima Global mass spectrometer or a Thermo Q-Exactive Orbitrap mass spectrometer.
Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected.
Cellular studies were done using human tumor cell lines of the cancer screening panel (NCI60)
at the U.S. National Cancer Institute (Bethesda, USA). STS was purified as previously
4
2
®
described.
All fluorescent measurements were carried out on a SpectraMax GeminiXS
o
fluorimeter (Molecular Devices, Sunnyvale, CA, USA) at 24 C in black microtiter plates from
Corning (Corning, MA, USA). All determinations were carried out in triplicate and errors
reported as  5% of obtained results.
1
9

2
- and 4-Nitroestrone (8 and 9). To a stirred solution of E1 (1.7 g, 6.4 mmol) in glacial
o
acetic acid (90 mL) at 70-75 C was added a mixture of concentrated nitric acid (0.4 mL) and
glacial acetic acid (10 mL). Stirring was continued at room temperature for 18 h during which a
precipitate formed. The mixture was filtered and washed with hot glacial acetic acid to afford
(
200 mg, 20%) of 8 as a pale yellow powder. The filtrate was concentrated, the residue was
dissolved in benzene (30 mL), aq. NaHCO (2%, 20 mL) was added and the mixture stirred for 6
3
h. The layers were separated and the aqueous layer was acidified with conc. HCl (10 mL). The
mixture was filtered and the filtrate was concentrated and recrystallized from ethanol (95%, 10
mL) to afford 9 as bright yellow crystals (250 mg, 23%). Characterization data for 8: Mp: 277-
o
20
o
1
2
80 C (lit 273-276 C); H-NMR (CDCl , 300 MHz) δ 9.40 (brs, 1H, ArOH), 7.44 (d, J = 8.8
3
Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.20-3.14 (m, 1H), 3.02-2.94 (m, 1H), 2.55-2.46 (m, 1H),
o
2
.35-1.94 (m, 6H), 1.67-1.23 (m, 8H), 0.90 (s, 3H,). Characterization data for 9: Mp: 181-183 C
2
0
o
1
(
lit: 183-184 C); H-NMR (CDCl
H), 2.95-2.92 (m, 2H), 2.54-2.39 (m, 2H), 2.17-1.96 (m, 5H), 1.61-1.41 (m, 7H), 0.89 (s, 3H,
H-18).
3
, 300 MHz) δ 10.32 (brs, 1H, ArOH), 7.90 (s, 1H), 6.80 (s,
1
4
-Bromoestrone (10). To a stirred solution of E1 (500 mg, 1.8 mmol) in ethanol (50
mL), was added NBA (255 mg, 1.8 mmol). The mixture was stirred for 24 h during which a
precipitate formed. The mixture was filtered and washed with cold ethanol. Recrystalization
o
22
o
1
from ethanol afforded 10 as white solid (575 mg, 89%). Mp: 264-265 C (lit: 263-265 C); H-
NMR (DMSO-d , 300 MHz) δ 9.80 (s, 1H, ArOH), 7.07 (d, J = 8.8 Hz, 1H), 6.72 (d, J = 8.5 Hz,
H), 2.87-2.79 (m, 1H), 2.63-2.51 (m, 1H), 2.42-1.92 (m, 6H), 1.72-1.65 (m, 1H), 1.55-1.27 (m,
H), 0.77 (s, 3H).
6
1
5
2
0

2
2
2
-Bromoestrone (11). To a stirred solution of estratrien-1,3,5(10)-3-acetate (1 g, 3.2
o
mmol) in TFA (20 mL) at 0 C (ice bath) was added thallium trifluoroacetate (3.5 g, 6.4 mmol),
and the mixture stirred for 24 h. The TFA was removed by rotary evaporation, the residue was
washed with DCE (2 x 10 mL) then dried under high vacuum for 6 h. The residue was dissolved
in 1,4-dioxane (50 mL), CuBr
The solvent was removed, the residue was dissolved in dichloromethane (25 mL), washed with
water (2 x 20 mL), brine (2 x 20 mL), dried (Na SO ) and concentrated. Purification by flash
chromatography (ethyl acetate/hexane, 4:1) afforded 2-bromo-estratrien-1,3,5(10)-3-acetate as
2
(1.14 g, 7.9 mmol) was added and mixture was refluxed for 3 h.
2
4
o
22
o
1
white solid (0.9 g, 73%). Mp: 166-167 C (lit: 146-147 C); H-NMR (CDCl
3
, 300 MHz) δ 7.46
(
s, 1H), 6.82 (s, 1H), 2.85-2.82 (m, 2H), 2.53-2.44 (m, 1H), 2.31-1.93 (m, 9H), 1.59-1.39 (m,
6
H), 0.88 (s, 3H). To a stirred solution of 2-bromo-estratrien-1,3,5(10)-3-acetate (0.50 g, 1.3
mmol) in methanol (20 mL) was added potassium carbonate (0.8 g, 6.3 mmol). The mixture was
stirred under reflux for 3 h. The solvent was then removed and water (20 mL) and
dichloromethane (20 mL) added to the residue. The layers were separated and the aq. layer
extracted with dichloromethane (20 mL). The combined organics were washed with brine (20
mL), dried (Na
2
SO ) and then concentrated. Purification by flash chromatography (ethyl
4
o
22
o
acetate/hexane, 3:2) afforded 11 as white solid (0.45 g, 91%). Mp: 194-196 C (lit: 194-195 C);
1
H-NMR (CDCl , 300 MHz) δ 7.32 (s, 1H), 6.74 (s, 1H), 5.26 (s, 1H, ArOH), 2.83-2.80 (m, 2H),
3
2
.53-2.44 (m, 1H), 2.32-1.93 (m, 6H), 1.59-1.39 (m, 7H), 0.88 (s, 3H).
General procedure for the reductive amination of 8-11. To a stirred solution of 8-11 in
THF (for 8 and 9, 0.050 g/mL THF) or DCE (for 10 and 11, 0.020 g/mL DCE) was added 4
equiv allylamine, 4 equiv glacial acetic and 2.5 equiv sodium triacetoxyborohydride (STAB).
The mixture was stirred for 1-3 d, after which aq. saturated NaHCO (20 mL) was added and the
3
2
1

mixture stirred was for additional 10 min. The mixture was extracted with dichloromethane, the
combined organics washed with aq. saturated NaHCO
3
, water and brine, then dried (Na
2
SO
4
) and
concentrated. Purification by flash chromatography gave compounds 12-15.
1
7-Allylamino-4-nitro-estratrien-1,3,5(10)-3-ol
(12).
Purification
by
flash
chromatography (methanol/chloroform, 1:9) afforded 12 as a yellow solid (350 mg, 63%); Mp
o
1
1
5
50-151 C; H-NMR (DMSO-d
.90-5.70 (m, 1H), 5.15 (d, J = 17 Hz, 1H), 5.00 (d, J = 10 Hz, 1H), 3.18 (m, 1H, overlaps with
O peak), 3.15 (m, 1H), 2.25-2.00 (m, 2H), 2.00-1.70 (m, 3H), 1.49-1.02 (m, 7H), 0.64 (s, 3H,
6
, 300 MHz) δ 7.20 (d, J = 8.9 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H),
H
2
1
3
)
; C-NMR (DMSO-d
6
, 75 MHz) δ 147.7, 141.0, 138.2, 131.2, 128.3, 128.1, 115.7, 115.1, 68.2,
+
5
1.8, 51.2, 43.7, 43.1, 38.1, 37.8, 29.2, 26.5, 26.3, 24.1, 23.4, 12.2; HRMS (ESI ) calcd. for
+
C
21
H
29
N
2
O (M+H) 357.2178; found 357.2186.
3
1
7-Allylamino-2-nitro-estratrien-1,3,5(10)-3-ol
(13).
Purification
by
flash
chromatography (ethyl acetate/hexane, 3:7) afforded 13 as a yellow solid (415 mg, 73%); Mp
o
1
1
1
6
63-164 C; H-NMR (CDCl , 300 MHz) δ 7.95 (s, 1H), 6.81 (s, 1H), 5.90 (dddd, J = 6.0, 10.2,
3
1.9, 16.3 Hz, 1H), 5.51 (brs, 1H), 5.09 (ddd, J = 1.7, 17.2, 18.8 Hz, 2H), 3.27 (dd, J = 1.3 and
.0 Hz, 2H), 2.92-2.82 (m, 2H), 2.67-2.61 (m, 1H), 2.30-2.26 (m, 1H), 2.15-1.98 (m, 3H), 1.89
13
(
m, 1H), 1.71-1.20 (m, 8H), 0.73 (s, 3H). C-NMR (CDCl
3
, 75 MHz) δ 152.8, 149.2, 137.4,
1
1
33.9, 131.7, 121.4, 118.8, 115.5, 68.3, 52.2, , 51.1, 43.5, 42.9, 38.1, 37.6, 29.7, 26.7, 26.2, 23.4,
+
+
1.8; HRMS (ESI ) calcd. for C21
H
29
N
2
O (M+H) 357.2178; found 357.2176.
3
4
-Bromo-17-allylamino-estratrien-1,3,5(10)-3-ol (14). Purification by flash
chromatography (ethyl acetate/hexane, 1:4) afforded 14 as white solid (160 mg, 71%). Mp: 145-
o
1
1
47 C; H-NMR (CDCl , 300 MHz) δ 7.14 (d, J = 8.5 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 5.96-
3
2
2

5
1
.83 (m, 1H), 5.18-5.04 (m, 2H), 3.46-3.27 (brs overlapped by an AB system, 3H), 2.93-2.85 (m,
H), 2.72-2.62 (m, 2H), 2.28-1.91 (m, 5H), 1.72-1.70 (m, 1H), 1.47-1.23 (m, 7H), 0.72 (s, 3H,
1
3
H-18); C-NMR (CDCl
3
, 75 MHz) δ 150.1, 137.4, 136.5, 134.4, 125.4 , 115.6, 113.7, 112.6,
+
6
8.3, 52.1, 51.4, 44.1, 42.9, 38.0, 37.9, 31.1, 29.7, 27.5, 26.7, 23.4, 11.8; HRMS (ESI ) calcd. for
+
C
21
H29NOBr (M+H) 390.1433; found 390.1443.
2
-Bromo-17-allylamino-estratrien-1,3,5(10)-3-ol (15). Purification by flash
chromatography (ethyl acetate/hexane, 1:4) afforded 15 as white solid (120 mg, 71%). Mp: 173-
o
1
1
2
5
7
3
75 C; H-NMR (CDCl
3
, 300 MHz) δ 7.30 (s, 1H), 6.65 (s, 1H), 5.94-5.87 (m, 1H), 5.2-5.05 (m,
H), 4.10 (brs, 2H), 3.86 (t, J = 5.7 Hz, 1H), 3.30-3.28 (m, 2H), 2.75-2.63 (m, 3H), 2.20-1.97 (m,
1
3
H), 1.84-1.81 (m, 1H), 1.70-1.67 (m, 1H), 1.43-1.23 (m, 7H), 0.72 (s, 3H); C-NMR (CDCl
3
,
5 MHz) δ 150.4, 137.7, 136.8, 134.0, 128.9, 116.1, 116.0, 107.4, 68.2, 52.1, 51.2, 43.7, 43.0,
-
-
8.4, 37.8, 29.2, 27.1, 26.4, 23.4, 11.7; HRMS (ESI ) calcd. for C21H27NOBr (M-H) 388.1276;
found 388.1272.
General method for deallylation of 12-15. To a stirred solution of compounds 12-15 in
dry DCM (0.020 g/mL) was added a solution of N,N-dimethylbarbituric acid (DMBA, 3 equiv)
and 15 mol% Pd(Ph
3
)
4
in dry dichloromethane (0.17 g DMBA/mL dichloromethane). The
o
mixture was stirred for 3 h at 35 C under an argon atmosphere and then diluted with
dichloromethane. The mixture was washed with sat. NaHCO
and concentrated under vacuum. Pure 16-19 was obtained using flash chromatography.
7-Amino-4-nitro-estratrien-1,3,5(10)-3-ol (16). Purification by flash chromatography
3
, water, brine then dried (Na
2
SO
4
)
1
(
methanol/chloroform/NH OH, 9:90:1) afforded 16 as a yellow solid (150 mg, 47%). Mp 167-
4
o
1
69 C; This compound exhibited very limited solubility, with or without heating, in all organic
solvents tested including CHCl
3
, MeOH, H O and DMSO. Prolonged heating in DMSO results
2
2
3

1
in some decomposition. H-NMR (CDCl
3
, 300 MHz) δ 7.44 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 9.0
Hz, 1H), 3.20 (m, 2H), 2.96-2.78 (m, 3H), 2.23-2.15 (m, 4H), 1.97-1.92 (m, 2H), 1.70 (m, 1H),
1
3
1
1
.46-1.17 (m, 8H), 0.72 (s, 3H);
C-NMR (CDCl
3
, 75 MHz)  151.9, 134.2, 134.1, 132.8,
16.4, 115.8, 68.2, 52.0, 51.4, 44.5, 43.0, 38.0, 37.5, 29.6, 27.8, 26.83, 26.80, 23.3, 11.9; HRMS
+
+
(
ESI ) calcd. for C18
7-Amino-2-nitro-estratrien-1,3,5(10)-3-ol (17). Purification by flash chromatography
methanol/chloroform/NH OH, 9:90:1) afforded 17 as an orange solid (220 mg, 71%). Mp 133-
, 300 MHz) δ 7.95 (s, 1H), 6.81 (s, 1H), 3.77 (brs, 2H), 2.92-2.70 (m,
H), 2.33-2.28 (m, 1H), 2.14-2.00 (m, 2H), 1.87 (m, 2H), 1.70-1.64 (m, 1H), 1.53-1.48 (m, 1H),
H
25
N
2
O
3
(M+H) 317.18597; found 317.18601.
1
(
4
o
1
1
3
1
1
35 C; H-NMR (CDCl
3
1
3
.38-1.15 (m, 6H), 0.65 (s, 3H). C-NMR (CDCl
3
, 75 MHz) δ 152.8, 149.2, 133.8, 131.7, 121.4,
+
18.8, 62.7, 51.9, 43.4, 42.8, 38.4, 36.3 , 30.9, 29.8, 26.7, 26.1, 23.3, 11.1; HRMS (ESI ) calcd
+
for C18
H
25
N
2
O
3
(M+H) 317.1865; found 317.1853.
4
-Bromo-17-amino-estratrien-1,3,5(10)-3-ol
OH, 9:90:1) afforded 18 as a white solid (58 mg,
3%). This compound exhibited very limited solubility, with or without heating, in all organic
solvents tested including CHCl , MeOH, H O and DMSO. Prolonged heating in DMSO results
in some decomposition. Mp: 163-165 C. H-NMR (DMSO-d
.90 Hz, 1H), 6.83 (broad d, J = 7.0 Hz, 1H), 3.4 (bs, 1H), 2.81 (broad d, J = 17.5 Hz, 1H), 2.25
m, 1H), 2.11 (broad s, 1H), 1.95-1.70 (m, 3H), 1.69-1.55 (m, 1H), 1.37-1.03 (m, 7H), 0.58 (s,
(18).
Purification
by
flash
chromatography (methanol/chloroform/NH
4
6
3
2
o
1
6
, 300 MHz) δ 7.11 (broad d, J =
7
(
1
3
3
3
H); C-NMR (DMSO-d
6
, 75 MHz)  151.7, 136.3, 132.6, 124.8, 113.1, 112.4, 51.3, 43.6, 40.3,
+
+
7.9, 36.2, 30.8, 27.1, 26.1, 22.9, 10.9; HRMS (ESI ) calcd. for C18
H
25NOBr (M+H) 350.1120;
found 350.1127.
2
4

2
-Bromo-17-amino-estratrien-1,3,5(10)-3-ol
chromatography (methanol/chloroform/NH OH, 9:90:1) afforded 4.26 as a white solid (20 mg,
, 300 MHz) δ 7.33 (s, 1H), 6.69 (s, 1H), 3.47 (s, 1H),
.73-2.78 (m, 3H), 2.62 (br s, 2H), 2.29-2.04 (m, 3H), 1.90-1.81 (m, 2H), 1.75-1.64 (m, 1H),
(19).
Purification
by
flash
4
o
1
2
2
1
1
1%). Mp: 190-191 C; H-NMR (CDCl
3
1
3
.52-1.13 (m, 7H), 0.67 (s, 3H); C-NMR (DMSO-d
6
, 75 MHz) δ 152.0, 136.6, 132.1, 129.0,
+
16.2, 106.6, 62.3, 51.3, 43.1, 42.4, 38.4, 36.1, 30.6, 28.5, 26.8, 25.8, 22.9, 10.9; HRMS (ESI )
+
calcd. for C18
H
25NOBr (M+H) 350.1120; found 350.1111.
General procedure for synthesis of sulfonamides 20-28. To a stirred solution of 16-19
o
in dry pyridine (0.16 mmol/mL) at 0 C was added a solution of the appropriate sulfonyl chloride
(
1.2 equiv) in dichloromethane (1 mL) drop-wise via a syringe pump over 10 min. The reaction
was stirred for 16 h at room temperature. The mixture was concentrated by rotary evaporation
pyridine and the residue dissolved in chloroform or ethyl acetate , washed with water and brine,
then dried with anhydrous Na
chromatography.
2
SO and concentrated. Purification was achieved using flash
4
4
-Nitro-17β-(3'-bromobenzene)sulfonamide-1,3,5(10)-estratrien-3-ol
(20).
Purification by flash chromatography (ethyl acetate/hexane, 3:7) afforded 20 as a yellow solid
o
1
(
41 mg, 48%). Mp 230-231 C; H-NMR (CDCl
J = 6.9 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.44-7.35 (m, 2H), 6.92 (d, J = 8.7 Hz, 1H), 4.55 (d, J
9.2 Hz, 1H), 3.21-3.09 (m, 2H), 2.94-2.88 (m, 1H), 2.23-2.15 (m, 2H), 1.89-1.65 (m, 4H),
3
, 300 MHz) δ 9.40 (s, 1H), 8.02 (s, 1H), 7.80 (d,
=
1
3
1
1
2
5
.44-1.19 (m, 8H), 0.72 (s, 3H, H-18); C-NMR (CDCl
3
, 75 MHz) δ 152.1, 143.1, 136.1, 135.6,
34.0, 133.6, 132.8, 130.6, 130.0, 125.5, 122.9, 121.5, 116.5, 63.3, 50.8, 44.2, 42.9, 37.6, 36.3,
+
+
9.5, 27.8, 26.5, 22.9, 11.8; HRMS (ESI ) calcd. for C24
H
28
N
2
O SBr (M+H) 535.0902; found
5
35.0909.
2
5

4
-Nitro-17β-(3'-trifluoromethylbenzene)sulfonamide-1,3,5(10)-estratrien-3-ol (21).
Purification by flash chromatography (ethyl acetate/hexane, 1:4) afforded 21 as a yellow solid
o
1
(
44 mg, 53%). Mp 263-264 C; H-NMR (CD
H), 7.88 (d, J = 7.8 Hz, 1H), 7.73 (overlapping dd, J = 8.0 Hz), 7.18 (d, J = 8.9 Hz, 1H), 6.74
d, J = 8.9 Hz, 1H), 3.20-3.08 (m, 2H), 2.73-2.51 (m, 1H), 2.24-2.12 (m, 1H), 2.08-1.94 (m, 1H),
3
OD, 300 MHz) δ 8.15 (s, 1H), 8.11 (d, J = 7.7 Hz,
1
(
1
3
1
1
1
2
C
.85-1.50 (m, 4H), 1.41-1.02 (m, 8H), 0.69 (s, 3H); C-NMR (CD OD, 75 MHz) δ 147.9, 144.9,
3
42.1, 133.4, 132.4 (q, J = 33.5 Hz), 131.7, 131.4, 129.9 (q, J = 3.2 Hz), 129.7, 129.0, 126.7,
24.9 (q, J = 271.6 Hz), 124.84 (q, J = 3.2 Hz), 115.4; 64.8, 52.0, 44.9, 44.1, 39.5, 37.7, 29.1,
19
+
7.3, 25.2, 23.9, 12.4 F-NMR (CDCl
3
, 282 MHz), δ -63.1; HRMS (ESI ) calcd for
+
25
H
28
N
2
O F
5 3
S (M+H) 525.1671; found 525.1667.
4
-Nitro-17β-(4'-t-butylbenzene)sulfonamide-1,3,5(10)-estratrien-3-ol
(22).
Purification by flash chromatography (ethyl acetate/hexane, 2.5:7.5) afforded 22 as a yellow
o
1
solid (32 mg, 40%). Mp 228-229 C; H-NMR (CDCl
3
, 300 MHz) δ 9.40 (s, 1H), 7.77 (d, J = 8.4
Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.6 Hz, 1H), 4.35 (d, J =
9
1
1
3
.3 Hz, 1H), 3.22-3.06 (m, 2H), 2.95-2.87 (m, 1H), 2.20-2.11 (m, 2H), 1.90-1.77 (m, 3H), 1.67-
1
3
.59 (m, 1H), 1.43-1.12 (m, 17 H), 0.71 (s, 3H); C-NMR (CDCl
3
, 75 MHz) δ 156.4, 152.2,
38.0, 136.1, 134.1, 133.7, 132.9, 126.9, 125.9, 116.5, 63.1, 50.9, 44.3, 42.8, 37.6, 36.2 , 35.1,
+
+
1.1, 29.5, 27.8, 26.5, 22.9, 11.8; HRMS (ESI ) calcd. for C28
H
37
N
2
O S (M+H) 513.2423;
5
found 513.2432.
4
-Nitro-17β-4'-biphenylsulfonamide-1,3,5(10)-estratrien-3-ol (23). Purification by
flash chromatography (methanol/chloroform, 1:8) afforded 23 as a yellow solid (23 mg, 27%).
o
1
Mp 270-271 C; H-NMR (CDCl
3
, 300 MHz) δ 9.40 (s, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.70 (d, J
=
8.3 Hz, 2H), 7.61 (d, J = 6.8 Hz, 2H), 7.49-7.38 (m, 4H), 6.80 (d, J = 8.6 Hz, 1H), 4.51 (d, J =
2
6

8
1
1
3
.9 Hz, 1H), 3.47-3.17 (m, 2H), 3.12-3.06 (m, 1H), 2.22-2.15 (m, 3H), 1.91-1.80 (m, 3H), 1.66-
1
3
.60 (m, 2H), 1.44-1.15 (m, 8H), 0.73 (s, 3H); C-NMR (CDCl
3
, 75 MHz) δ 152.1, 145.4,
39.7, 139.2, 136.1, 134.0, 133.7, 132.7, 129.0, 128.5, 127.6, 127.3, 116.5, 63.3, 51.1, 44.3, 42.8,
+
+
7.6, 36.3, 29.5, 27.8, 26.5, 23.0, 11.8; HRMS (ESI ) calcd. for C30
H
33
N
2
O S (M+H) 533.2110;
5
found 533.2098.
-Nitro-17β-(3'-bromobenzene)sulfonamide-1,3,5(10)-estratrien-3-ol
Purification by flash chromatography (ethyl acetate/hexane, 3:7) afforded 24 as a yellow solid
2
(24).
o
1
(
56 mg, 66%). Mp 242-243 C; H-NMR (CDCl , 300 MHz) δ 10.40 (s, 1H), 8.03 (s, 1H), 7.91
3
(
s, 1H), 7.80 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.38 (dd, J = 7.9 and 7.9 Hz, 1H), 6.80
s, 1H), 4.68 (d, J = 9.3 Hz, 1H), 3.19 (q, J = 8.7 Hz, 1H), 2.86-2.80 (m, 2H), 2.27-2.23 (m, 1H),
(
1
3
2
1
5
.11 (m, 1H), 1.91-1.65 (m, 4H), 1.46-1.14 (m, 7H), 0.70 (s, 3H); C-NMR (CDCl , 75 MHz) δ
3
52.8, 148.9, 143.0, 135.6, 133.3 (C-5), 131.7, 130.6, 130.0, 125.5, 122.9, 121.5, 118.8, 63.3,
+
1.0, 43.2, 42.8, 38.2, 35.9, 29.5, 26.5, 25.8, 23.1, 11.8; HRMS (ESI ) calcd. for C24
H
28
N
2
5
O SBr
+
(
M+H) 535.0902; found 535.0902.
2
-Nitro-17β-(3'-trifluoromethylbenzene)sulfonamide-1,3,5(10)-estratrien-3-ol (25).
Purification by flash chromatography (methanol/chloroform, 0.5:9.5) afforded 25 as a yellow
o
1
solid (55 mg, 67%). Mp 278-279 C; H-NMR (CDCl
3
, 300 MHz) δ 10.40 (s, 1H), 8.15 (s, 1H),
.07 (d, J = 8.07 Hz, 1H), 7.91 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.66 (dd, J = 7.4 and 7.9 Hz,
H), 6.80 (s, 1H), 4.59 (d, J = 9.6 Hz, 1H), 3.22 (q, J = 8.5 Hz, 1H), 2.89 (m, 2H), 2.26-2.22 (m,
8
1
1
1
3
H), 2.11 (m, 1H), 1.91-1.85 (m, 2H), 1.73-1.68 (m, 2H), 1.49-1.10 (m, 7H), 0.70 (s, 3H); C-
, 75 MHz) δ 152.8, 148.9, 142.5, 133.3, 131.7 (q, J = 33.3 Hz), 131.7, 130.2, 129.8,
29.1 (q, J = 3.6 Hz, Ar-CH), 124.1 (q, J = 3.8 Hz, Ar-CH), 123.2 (q, J = 271.3 Hz, C-CF ),
NMR (CDCl
3
1
3
2
7

1
9
1
21.5, 118.8, 63.4, 51.0, 43.2, 42.8, 38.2, 35.9, 29.5, 26.5, 25.8, 23.0, 11.8; F-NMR (CDCl ,
3
+
+
2
82 MHz), δ -63.1; HRMS (ESI ) calcd. for C25
H
28
N
2
O
5
F S (M+H) 525.1671; found 525.1656.
3
2
-Nitro-17β-(4'-t-butylbenzene)sulfonamide-1,3,5(10)-estratrien-3-ol
(26).
Purification by flash chromatography (ethyl acetate/hexane, 1:4) afforded 26 as a yellow solid
o
1
(
21 mg, 26%). Mp 212-213 C; H-NMR (CDCl
3
, 300 MHz) δ 10.37 (app. d, 1H), 7.91 (s, 1H),
.80-7.77 (2 overlapping d, J = 8.4 Hz, 2H), 7.50-7.47 (2 overlapping d, J = 8.4 Hz, 2H), 6.80 (s,
H), 4.57 (d, J = 9.2 Hz, 1H), 3.15 (apparent t, J = 8.6 Hz, 1H), 2.85 (m, 2H), 2.24-2.20 (m, 1H),
7
1
2
1
3
.09-2.01 (m, 1H), 1.84-1.75 (m, 3H), 1.65-1.62 (m, 1H), 1.49-1.14 (m, 17 H), 0.69 (s, 3H); C-
NMR (CDCl
3
, 75 MHz) δ 156.4,152.8, 148.9, 137.9, 133.5, 131.7, 126.9, 125.9, 121.5, 118.8,
+
6
3.1, 51.1, 43.2, 42.8, 38.2, 35.9, 35.1, 31.1, 29.5, 26.5, 25.8, 23.1, 11.7; HRMS (ESI ) calcd. for
+
C
28
H
37
N
2
O S (M+H) 513.2423; found 513.2424.
5
2
-Nitro-17β-4'-biphenylsulfonamide-1,3,5(10)-estratrien-3-ol (27). Purification by
flash chromatography (ethyl acetate/hexane, 1:9) afforded 27 as a yellow solid (36 mg, 43%).
o
1
Mp 265-266 C; H-NMR (CDCl
3
, 300 MHz) δ 10.40 (s, 1H), 7.96-7.91 (m, 3H), 7.71 (d, J = 8.3
Hz, 2H), 7.61 (d, J = 7.7 Hz, 2H), 7.49-7.38 (m, 3H), 6.80 (s, 1H), 4.67 (d, J = 9.2 Hz, 1H), 3.21
(
q, J = 8.7 Hz, 1H), 2.90-2.77 (m, 2H), 2.26-2.21 (m, 2H), 1.90-1.63 (m, 4H), 1.40-1.12 (m, 8H),
1
3
0
1
2
.71 (s, 3H); C-NMR (CDCl
3
, 75 MHz) δ 152.8, 149.2, 145.5, 139.7, 139.2, 133.8 (C-5),
31.7, 129.1, 128.5, 127.6, 127.3, 121.5, 118.9, 63.3, 51.1, 43.3, 42.8, 38.1, 36.0, 29.5, 26.5,
+
+
5.8, 23.1, 11.8; HRMS (ESI ) calcd. for C30
H
33
N
2
O S (M+H) 533.2110; found 533.2103.
5
4
-Bromo-17β-(3'-trifluoromethylbenzene)sulfonamide-1,3,5(10)-estratrien-3-ol (28).
Purification by flash chromatography (ethyl acetate/hexane, 1:9) afforded 28 as a white solid (15
o
1
mg, 48%). Mp 215-217 C; H-NMR (CDCl , 300 MHz) δ 8.15 (s, 1H), 8.07 (d, J = 7.9 Hz, 1H),
3
2
8

7
8
1
8
.82 (d, J = 7.9 Hz, 1H), 7.65 (dd, J = 7.8 and 7.8 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.81 (d, J =
.5 Hz, 1H), 5.49 (brs, 1H), 4.72 (d, J = 9.4 Hz, 1H), 3.20 (q , J = 8.6 Hz, 1H), 2.91-2.83 (m,
H), 2.69-2.60 (m, 1H), 2.24-2.15 (m, 2H), 1.92-1.83 (m, 2H), 1.72-1.62 (m, 3H), 1.37-1.08 (m,
1
3
H), 0.69 (s, 3H); C-NMR (CDCl , 75 MHz) δ 150.1, 142.5, 136.2, 133.9, 131.5 (q, J = 33.2
3
Hz) 130.2, 129.8, 129.1 (q, J = 3.4 Hz), 125.5, 124.1 (q, J = 3.7 Hz), 122.1 (q, J = 306.7 Hz),
1
9
1
2
5
13.6, 112.7, 63.5, 50.9, 43.8, 42.9, 38.0, 36.3, 30.9, 29.5, 27.2, 26.2, 11.8; F-NMR (CDCl ,
3
-
-
82 MHz), δ -63.1; HRMS (ESI ) calcd. for C25
H
26NO
3
F SBr (M-H) 556.0769; found
3
56.0756.
2
-Bromo-17β-(3'-trifluoromethylbenzene)sulfonamide-1,3,5(10)-estratrien-3-ol (29).
Purification by flash chromatography (ethyl acetate/hexane, 0.5:9.5) afforded 29 as a white solid
o
1
(
10 mg, 33%). Mp 233-234 C; H-NMR (CDCl
3
, 300 MHz) δ 8.14 (s, 1H), 8.06 (d, J = 7.7 Hz,
H), 7.82 (d, J = 8.0 Hz, 1H), 7.65 (dd, J = 7.7 and 8.0 Hz, 1H), 7.27 (s, 1H), 6.70 (s, 1H, ArH),
.23 (brs, 1H), 4.45 (d, J = 9.2 Hz, 1H), 3.20 (app. t , J = 9.0 Hz, 1H), 2.74 (m, 2H), 2.21-2.12
1
5
1
3
(
(
m, 2H), 1.90-1.79 (m, 2H), 1.70-1.62 (m, 2H), 1.40-1.12 (m, 8H), 0.69 (s, 3H); C-NMR
CDCl , 75 MHz) δ 149.9, 142.5, 138.0, 134.1, 131.8 (q, J = 33.9 Hz), 130.3, 129.9, 129.2 (q, J
3.7 Hz), 128.7, 124.2 (q, J = 3.7 Hz), 115.8, 107.4, 63.5, 51.0, 43.5, 42.9, 38.5, 36.2 , 29.6,
3
=
1
9
+
2
9.0, 26.9, 26.0, 23.1, 11.9; F-NMR (CDCl , 282 MHz), δ -63.0; HRMS (ESI ) calcd. for
3
+
C
25
H28NO
3
F
3
SBr (M+H) 558.0925; found 558.0927.
2
7
4
-Fluoroestrone (31). A stirred mixture of 30 (0.652 g, 2.00 mmol) and N-
fluoropyridinium triflate (NFPT, 0.980 g, 4.00 mmol) in dry 1,1,2-trichloroethane (TCE, 24 mL)
in a glass pressure tube (CAUTION!: This reaction should be performed in a fume hood behind a
o
blast shield) under argon atmosphere was heated at 135 C for 15 h. The TCE was then removed
in vacuo, water (100 mL) was added and reaction mixture was extracted with dichloromethane (4
2
9