Phosphate and thiophosphate biphenyl analogs as steroid sulfatase inhibitors

Article abstract of DOI:10.1002/ddr.21245

Preclinical Research In the present work, we report convenient methods for the synthesis and biological evaluation of phosphate and thiophosphate biphenyl derivatives exhibiting steroid sulfatase (STS) activity. The described synthesis is based on straigh

Full text of DOI:10.1002/ddr.21245

DRUG DEVELOPMENT RESEARCH 76 : 94–104 (2015)
Research Article
Phosphate and Thiophosphate Biphenyl Analogs as
Steroid Sulfatase Inhibitors
1
1
1
2
Sebastian Demkowicz, * Witold Kozak, Mateusz Da ꢀs ko, Maciej Masłyk,
2
1
Konrad Kubi nꢀ ski, and Janusz Rachon
1
2
Department of Organic Chemistry, Chemical Faculty, Gdansk University of Technology,
Narutowicza 11/12, 80-233, Gdansk, Poland
Department of Molecular Biology, Faculty of Biotechnology and Environment Sciences,
The John Paul II Catholic University of Lublin, Konstantyn oꢀ w 1i, 20-708 Lublin, Poland
Strategy, Management and Health Policy
Enabling
Technology, Preclinical
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Clinical Development
Phases I-III
Postmarketing
Phase IV
Genomics,
Proteomics
Research
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
In the present work, we report convenient methods for the synthesis and biological
evaluation of phosphate and thiophosphate biphenyl derivatives exhibiting steroid sulfatase (STS) activ-
0
ity. The described synthesis is based on straightforward preparation of biphenyl-4-ol and 4 -hydroxy-
biphenyl-4-carboxylic acid ethyl ester modified with various phosphate or thiophosphate moieties. The
inhibitory effects of these compounds were tested on STS isolated from human placenta and led to two
compounds of interest, 5a and 5d with IC50 values of 28.0 and 22.1 mM, respectively and that had
interesting new binding modes in the STS active site. Drug Dev Res 76 : 94–104, 2015.
V
C
2015 Wiley
Periodicals, Inc.
Key words: steroid sulfatase; STS inhibitors; breast cancer; biphenyls
INTRODUCTION
et al., 2010], antipsychotic and anxiolytic (biphenylin-
danone A) [Ruggero et al., 2006] and antimicrobial
Biphenyl, an aromatic hydrocarbon occurs natu-
rally in coal tar, crude oil, and natural gas. It forms an
incomplete combustion of mineral oil and coal. Histor-
ically, some biphenyls were widely used as intermedi-
ates in chemical syntheses that resulted in a wide
range of pesticides including the polychlorinated
biphenyls (PCBs). Emerging trends in synthetic chem-
istry have led a number of substituted biphenyl deriva-
tives prepared by various coupling reactions that
therapeutic potential [Jain et al., 2013]. These deriva-
tives have angiotensin receptor (Losartan, Telmisartan)
(biphenyl hydrazide-hydrazone) [Deep et al., 2010]
activities. Of particular interest is the use of biphenyl
scaffolds in designing new steroid sulfatase (STS)
inhibitors based on phosphate and thiophosphate
derivatives that may be potential new therapeutics for
the treatment of estrogen- and androgen-dependent
Contract grant sponsor: National Science Centre (Poland) for
financial support; contract grant number: 2011/03/D/NZ7/03985
*Correspondence to: Sebastian Demkowicz, Department
[
Zhu et al., 2004], antihypertensive (biphenyl carbox- of Organic Chemistry, Chemical Faculty, Gdansk University of
Technology, Narutowicza 11/12, 80-233 Gdansk, Poland.
ylic benzimidazole derivatives) [Kumar et al., 2006],
diuretic
E-mail: sebdemko@pg.gda.pl
(N-{(substituted)1,3-benzothiazol-2-yl}-1,1-
biphenyl-4-carboxamides) [Yar and Ansari, 2009], anti-
inflammatory (biphenyl-4-carboxylic acid 5-(aryli-
Received 5 February 2015; Accepted 14 February 2015
Published online in Wiley Online Library (wileyonlinelibrary.
dene)-2-(aryl)-4-oxothiazolidin-3-yl amides) [Deep com). DOI: 10.1002/ddr.21245
V
C
2015 Wiley Periodicals, Inc.

STEROID SULFATASE INHIBITION
95
disorders (especially breast cancer) [Nussbaumer and 806; PMR spectrum (500 MHz, DMSO, d, ppm, J/
Billich, 2004]. Among females inindustrialized coun- Hz): 3.67 (3H, d, J = 11.7, CH ), 5.11 (2H, d, JP-N
=
3
tries, breast cancer is one of the most frequently diag- 6.8, NH ), 7.28 (2H, d, J 5 7.8, Ar-H), 7.34 (1H, t,
2
nosed diseases with estimates for 2014 (according to J 5 7.3, Ar-H), 7.44 (2H, t, J 5 7.3, Ar-H), 7.61–7.67
13
National Cancer Institute data) of more than 230,000 (4H, m, Ar-H); C NMR spectrum (125 MHz,
new cases and more than 40,000 deaths from this dis- DMSO, d, ppm): 151.4 (d, JP-C 5 6.1), 140.2, 137.0,
ease in the United States. For this reason, research 129.6, 128.5, 128.0, 127.2, 121.4 (d, J 5 4.8), 53.5
P-C
31
work on the synthesis of efficient and selective STS (d, JP-C 5 5.7);
P NMR spectrum (202 MHz,
inhibitors is of particular importance.
DMSO, d, ppm): 9.50. Anal. Calcd for: C H NO P:
13
14
3
C, 59.32; H, 5.36; N, 5.32. Found: C, 59.40; H, 5.41;
N, 5.38.
METHODS AND MATERIALS
Melting points (uncorrected) were determined
with a Stuart Scientific SMP30 apparatus. NMR spectra
were recorded on a Varian Gemini 500 MHz spectrom-
0
4
-(ethoxycarbonyl)biphenyl-4-yl methyl
phosphoroamidate 3b
ꢀ
21
Yield 25%, mp 134–136 C; IR (neat, m, cm ):
331, 3251, 1703, 1601, 1568, 1527, 1493, 1272,
233, 1186, 1110, 921, 806; PMR spectrum (500
MHz, DMSO, d, ppm, J/Hz): 1.32 (3H, t, J 5 6.8,
CH ), 3.67 (3H, d, J 5 11.2, CH ), 4.32 (2H, q,
J 5 6.8, CH ), 5.14 (2H, d, J 5 6.8, NH ), 7.31
2H, d, J 5 8.3, Ar-H), 7.71–7.80 (4H, m, Ar-H), 8.01
2H, d, J 5 8.3, Ar-H); C NMR spectrum (125
MHz, DMSO, d, ppm): 166.2, 152.2 (d, JP-C 5 6.1),
eter. Chemical shifts are reported in ppm relative to the
1
3
1
residual solvent peak (CDCl 5 7.26 ppm for H, 77.0
3
13
1
ppm for C, DMSO-d 5 2.49 ppm for H, 39.5 ppm
6
13
for C) or to an external standard (85% H PO 5 0 for
3
4
31
P). Coupling constants are given in Hertz. IR spectra
3
3
were measured on a Nicolet 8700. Elemental analysis
was performed using CHNS-Carlo Erba EA-1108. Col-
umn chromatography was performed using silica gel 60
2
P-N
2
(
(
13
(230–400 mesh, Merck). Preparative thin-layer chroma-
1
44.6, 135.5, 130.5, 129.2, 128.9, 127.4, 121.5 (d, J
5
spectrum (202 MHz, DMSO, d, ppm): 9.45. Anal.
tography was performed with Polygram SIL G/UV254
silica gel (Macherey-Nagel).
P-
31
4.4), 61.4, 53.5 (d, JP-C 5 5.7), 14.9; P NMR
C
Calcd for: C H NO P: C, 57.31; H, 5.41; N, 4.18.
Found: C, 57.37; H, 5.46; N, 4.26.
16
18
5
Synthesis
General method for the synthesis of biphenyl
derivatives modified by ethyl or methyl
phosphoroamidate groups
Biphenyl-4-yl ethyl phosphoroamidate 3c
ꢀ
21
Yield 15%, mp 126–128 C; IR (neat, m, cm ):
328, 3248, 1604, 1572, 1520, 1486, 1221, 1191,
036, 954, 841, 770; PMR spectrum (500 MHz,
To an ice-cooled solution of phosphorus oxy-
chloride (0.900 g, 5.87 mmol) in dry tetrahydrofuran
3
1
(
THF; 20 mL) was added a solution of the corre-
DMSO, d, ppm, J/Hz): 1.25 (3H, t, J 5 7.3, CH3)
.01–4.08 (2H, m, CH ), 5.07 (2H, d, J 5 6.3,
sponding biphenyl-4-ol (5.87 mmol) in THF drop-
wise, followed by triethylamine (0.594 g, 5.87 mmol).
The reaction mixture was stirred under a nitrogen
atmosphere for 1 h. the triethylamine hydrochloride
precipitate removed by filtration, and sodium alkox-
ide (5.87 mmol) (freshly prepared by the addition of
absolute ethanol or methanol to 60% NaH dispersed
in mineral oil) was added. The reaction mixture was
stirred for 0.5 h, and a precipitate of NaCl formed.
4
2
P-N
NH ), 7.27 (2H, d, J 5 7.8, Ar-H), 7.34 (1H, t,
2
J 5 7.3, Ar-H), 7.44 (2H, t, J 5 7.8, Ar-H), 7.61–7.66
1
3
(
4H, m, Ar-H); C NMR spectrum (125 MHz,
DMSO, d, ppm): 151.5 (d, JP-C 5 6.6), 140.2, 136.8,
29.6, 128.5, 127.9, 127.2, 121.4 (d, J 5 4.4), 62.6
1
(
P-C
31
d, JP-C 5 5.7), 16.8 (d, JP-C 5 7.0); P NMR spec-
trum (202 MHz, DMSO, d, ppm): 8.20. Anal. Calcd
for: C H NO P: C, 60.65; H, 5.82; N, 5.05. Found:
14
16
3
After 30 min, a solution of NH in methanol (5 mL)
3
C, 60.58; H, 5.89; N, 5.09.
was added and the reaction mixture concentrated
under vacuum. The resulting residue was dispersed
in cold methanol and filtered, and solvent was evapo-
rated under vacuum. The crude product was crystal-
lized from ethyl acetate.
0
4
-(ethoxycarbonyl)biphenyl-4-yl ethyl
phosphoroamidate 3d
ꢀ
21
Yield 25%, mp 143–145 C; IR (neat, m, cm ):
343, 3253, 1707, 1606, 1564, 1525, 1492, 1396,
277, 1224, 1184, 1032, 947, 837, 773; PMR spec-
3
1
Biphenyl-4-yl methyl phosphoroamidate 3a
trum (500 MHz, DMSO, d, ppm, J/Hz): 1.23–1.34
ꢀ
21
Yield 44%, mp 173–175 C; IR (neat, m, cm ): (6H, m, CH ), 4.02–4.08 (2H, m, CH ), 4.32 (2H, q,
3
2
3
325, 3247, 1603, 1573, 1520, 1485, 1225, 1189, 922, J 5 7.3, CH ), 5.10 (2H, d, J 5 5.8, NH ), 7.31
2
P-N
2
Drug Dev. Res.

96
DEMKOWICZ ET AL.
3
1
(
(
2H, d, J 5 8.3, Ar-H), 7.72–7.80 (4H, m, Ar-H), 8.01
5 4.8), 61.5, 55.2 (d, JP-C 5 6.1), 14.6; P NMR
C
1
3
2H, d, J 5 7.8, Ar-H); C NMR spectrum (125 spectrum (202 MHz, CDCl , d, ppm): -2.95. Anal.
3
MHz, DMSO, d, ppm): 166.2, 152.2 (d, JP-C 5 6.1), Calcd for: C H O P: C, 58.29; H, 5.47. Found: C,
17
19
6
1
44.6, 135.4, 130.4, 129.2, 128.9, 127.4, 121.5 (d, JP- 58.34; H, 5.51.
5
4.4), 62.6 (d, J 5 5.3), 61.4, 16.8 (d, JP-C 5 7.0),
C
P-C
31
1
4.9; P NMR spectrum (202 MHz, DMSO, d,
Biphenyl-4-yl diethyl phosphate 3g
ppm): 8.14. Anal. Calcd for: C H NO P: C, 58.45;
H, 5.77; N, 4.01. Found: C, 58.55; H, 5.83; N, 4.11.
21
Yield 40%, oil; IR (neat, m, cm ): 1604, 1516,
1
7
20
5
1
485, 1272, 1219, 1018, 927, 842, 764, 696; PMR
spectrum (500 MHz, CDCl , d, ppm, J/Hz): 1.36–
3
General method for the synthesis of biphenyl
derivatives modified by phosphoric acid diethyl or
dimethyl ester groups
1
.40 (6H, m, CH ) 4.20–4.30 (4H, m, CH ), 7.30
3 2
(
(
2H, d, J 5 7.3, Ar-H), 7.32–7.37 (1H, m, Ar-H), 7.44
13
2H, t, J 5 7.3, Ar-H), 7.54–7.57 (4H, m, Ar-H);
C
To an ice-cooled solution of phosphorus oxy-
chloride (0.900 g, 5.87 mmol) in dry THF (20 mL)
was added a solution of the corresponding biphenyl-
NMR spectrum (125 MHz, CDCl , d, ppm): 150.5
3
(d, JP-C 5 7.0), 140.5, 138.3, 129.0, 128.6, 127.6,
1
27.2, 120.5 (d, J 5 4.8), 64.9 (d, JP-C 5 5.7), 16.4
P-C
3
1
4
-ol (5.87 mmol) in THF dropwise, followed by trie-
(d, JP-C 5 6.6);
P NMR spectrum (202 MHz,
thylamine (0.594 g, 5.87 mmol). The reaction mixture
was stirred under a nitrogen atmosphere for 1 h. The
triethylamine hydrochloride precipitate was removed
by filtration, and sodium alkoxide (11.74 mmol)
CDCl , d, ppm): -5.07. Anal. Calcd for: C H O P:
C, 62.74; H, 6.25. Found: C, 62.70; H, 6.29.
3
16 19
4
0
4
3
-(ethoxycarbonyl)biphenyl-4-yl diethyl phosphate
(
freshly prepared by the addition of absolute ethanol
h
or methanol to 60% NaH dispersed in mineral oil)
was added. The reaction mixture was stirred for 1 h,
and a precipitate of NaCl formed. The solution was
filtered, and the solvent was evaporated. The result-
ing residue was purified by column chromatography
using hexane:ethyl acetate 3:2 as an eluent to give
the desired products.
ꢀ
21
Yield 46%, mp 36–37 C; IR (neat, m, cm ):
701, 1604, 1523, 1494, 1276, 1221, 1187, 1101,
1
1
019, 931, 838, 769, 698; PMR spectrum (500 MHz,
CDCl , d, ppm, J/Hz): 1.35–1.42 (9H, m, CH ),
3
3
4
7
8
.20–4.28 (4H, m, CH ), 4.39 (2H, q, J 5 6.8, CH ),
2 2
.31 (2H, d, J 5 7.8, Ar-H), 7.57–7.62 (4H, m, Ar-H),
13
.10 (2H, d, J 5 8.3, Ar-H); C NMR spectrum (125
MHz, CDCl , d, ppm): 166.7, 151.1 (d, J 5 6.6),
3
P-C
Biphenyl-4-yl dimethyl phosphate 3e
1
44.7, 137.1, 130.3, 129.5, 128.8, 127.1, 120.7 (d, JP-
ꢀ
21
Yield 45%, mp 44–45 C; IR (neat, m, cm ):
5
4.8), 64.9 (d, J 5 5.7), 61.2, 16.4 (d, JP-C 5 6.6),
C
P-C
1
7
603, 1519, 1485, 1281, 1221, 1194, 1009, 928, 841,
65; PMR spectrum (500 MHz, CDCl , d, ppm, J/
31
1
4.6; P NMR spectrum (202 MHz, CDCl , d,
3
3
ppm): -5.18. Anal. Calcd for: C H O P: C, 60.31;
19
23
6
Hz): 3.89 (6H, d, J 5 11.2, CH ), 7.29 (2H, d,
3
H, 6.13. Found: C, 60.40; H, 6.16.
J 5 7.8, Ar-H), 7.33–7.46 (3H, m, Ar-H), 7.53–7.57
1
3
(
4H, m, Ar-H); C NMR spectrum (125 MHz,
General method for the synthesis of biphenyl
derivatives modified by phosphorodiamidate group
CDCl , d, ppm): 150.3 (d, J 5 7.0), 140.4, 138.6,
3
P-C
1
29.1, 128.7, 127.6, 127.3, 120.4 (d, J 5 4.8), 55.2
P-C
3
1
To an ice-cooled solution of phosphorus oxy-
chloride (0.900 g, 5.87 mmol) in dry THF (20 mL)
was added a solution of the corresponding biphenyl-
(
d, JP-C 5 5.7);
P NMR spectrum (202 MHz,
CDCl , d, ppm): -2.87. Anal. Calcd for: C H O P:
3
14 15
4
C, 60.43; H, 5.43. Found: C, 60.49; H, 5.50.
4
-ol (5.87 mmol) in THF dropwise, followed by trie-
0
thylamine (0.594 g, 5.87 mmol). The reaction mixture
was stirred under a nitrogen atmosphere for 1 h. The
triethylamine hydrochloride precipitate was removed
by filtration and a solution of NH3 in methanol
4
-(ethoxycarbonyl)biphenyl-4-yl dimethyl
phosphate 3f
ꢀ
21
Yield 65%, mp 59–60 C; IR (neat, m, cm ):
1
8
705, 1608, 1523, 1493, 1279, 1217, 1183, 1023, 937,
42, 774; PMR spectrum (500 MHz, CDCl , d, ppm,
(5 mL) was added. After concentration under vac-
3
uum, the crude product was crystallized from metha-
nol to give the desired product.
J/Hz): 1.38–1.42 (3H, m, CH ), 3.89 (6H, dd,
3
J 5 11.2, CH ), 4.36–4.41 (2H, m, CH ), 7.31 (2H, d,
3
2
J 5 8.3, Ar-H), 7.57–7.61 (4H, m, Ar-H), 8.09 (2H, d,
1
3
Biphenyl-4-yl phosphorodiamidate 3i
J 5 8.3, Ar-H);
C NMR spectrum (125 MHz,
ꢀ
CDCl , d, ppm): 166.6, 150.9 (d, J 5 6.6), 144.6,
Yield 34%, mp 233–235 C; IR (neat, m,
3
P-C
2
1
1
37.4, 130.3, 129.6, 128.9, 127.1, 120.6 (d, JP- cm ): 3341, 3223, 1597, 1560, 1518, 1483, 1404,
Drug Dev. Res.

STEROID SULFATASE INHIBITION
97
0
1
241, 1167, 1019. 946, 839, 758; PMR spectrum 4 -(ethoxycarbonyl)biphenyl-4-yl dihydrogen
(500 MHz, DMSO, d, ppm, J/Hz): 4.32 (4H, d, JP- phosphate 3l
5
4.4, NH ), 7.26 (2H, d, J 5 7.8, Ar-H), 7.30–
ꢀ
21
N
2
Yield 53%, mp 154–158 C; IR (neat, m, cm ):
762, 1710, 1600, 1490, 1399, 1272, 1165, 974, 836,
67; PMR spectrum (500 MHz, DMSO, d, ppm, J/
7
7
.36 (1H, m, Ar-H), 7.43 (2H, t, J 5 7.8, Ar-H),
.58–7.63 (4H, m, Ar-H); C NMR spectrum (125
MHz, DMSO, d, ppm): 152.4 (d, JP-C 5 6.6), 140.4,
36.0, 129.6, 128.1, 127.8, 127.2, 121.9 (d, J
2
7
1
3
Hz): 1.33 (3H, t, J 5 7.0, CH ), 4.33 (2H, q, J 5 7.0,
CH ), 5.00–7.40 (2H, br s, OH), 7.28 (2H, d, J 5 8.7,
Ar-H), 7.70–7.82 (4H, m, Ar-H), 8.01 (2H, d, J 5 8.3,
Ar-H); C NMR spectrum (125 MHz, DMSO, d,
ppm): 166.2, 152.6, 144.6, 135.2, 130.5, 129.1, 128.9,
27.4, 121.3 (d, JP-C 5 4.8), 61.4, 14.9; P NMR
3
1
P-
31
2
5
4.4); P NMR spectrum (202 MHz, DMSO, d,
C
ppm): 15.70. Anal. Calcd for: C H N O P: C,
8.07; H, 5.28; N, 11.29. Found: C, 58.01; H, 5.34;
N, 11.37.
13
1
2
13
2
2
5
31
1
spectrum (202 MHz, DMSO, d, ppm): -5.12. Anal.
0
4
-(ethoxycarbonyl)biphenyl-4-yl phosphorodiami-
Calcd for: C H O P: C, 55.91; H, 4.69. Found: C,
15
15
6
date 3j
55.99; H, 4.74.
ꢀ
Yield 20%, mp 240–242 C; IR (neat, m,
21
cm ): 3331, 3229, 1712, 1599, 1558 1525, 1492, General method for the synthesis of biphenyl
396, 1271, 1242, 1170, 1110, 1019, 948, 833, 765; derivatives modified by ethyl or methyl
PMR spectrum (500 MHz, DMSO, d, ppm, J/Hz): chlorothiophosphate groups
1
1
4
.31–1.34 (3H, m, CH ), 4.32 (2H, q, J 5 6.8, CH ),
3 2
To an ice-cooled solution of thiophosphoryl
chloride (0.994 g, 5.87 mmol) in dry THF (20 mL)
was added a solution of the corresponding biphenyl-
.41 (4H, d, JP-N 5 3.9, NH ), 7.30 (2H, d, J 5 8.3,
2
Ar-H), 7.68–7.80 (4H, m, Ar-H), 7.99–8.02 (2H, m,
Ar-H); C NMR spectrum (125 MHz, DMSO, d,
ppm): 166.2, 153.2 (d, JP-C 5 6.6), 144.9, 134.6,
1
1
13
4
-ol (5.87 mmol) in THF dropwise, followed by trie-
thylamine (0.594 g, 5.87 mmol). The reaction mixture
was stirred under a nitrogen atmosphere for 1 h. The
triethylamine hydrochloride precipitate was removed
by filtration, and sodium alkoxide (5.87 mmol)
30.4, 129.0, 128.6, 127.3, 122.1 (d, J 5 4.4), 61.4,
P-C
31
4.9; P NMR spectrum (202 MHz, DMSO, d,
ppm): 15.75. Anal. Calcd for: C H N O P: C,
1
5
17
2
4
5
6.25; H, 5.35; N, 8.75. Found: C, 56.32; H, 5.39;
(freshly prepared by the addition of absolute ethanol
N, 8.83.
or methanol to 60% NaH dispersed in mineral oil)
was added. The reaction mixture was stirred for 1 h,
and a precipitate of NaCl formed. The solution was
filtered, and the solvent was evaporated. The result-
ing residue was purified by column chromatography
General method for the synthesis of phosphoric
acid derivatives
To an ice-cooled solution of derivative (3e) or
using petroleum ether:CHCl 3:1 as an eluent to give
3
(3f) (1 mmol) in dry DCM (7 mL) was added
the desired products.
TMSBr (4 mmol) dropwise. The reaction mixture was
stirred under a nitrogen atmosphere for 1 h. After
concentration under vacuum, 5 mL of methanol was
added. All solvents were then evaporated, and the
0
Biphenyl-4-yl methyl chlorothiophosphate 4a
ꢀ
21
Yield 60%, mp 70–73 C; IR (neat, m, cm ):
crude product was crystallized from ethyl acetate to 1600, 1519, 1483, 1218, 1186, 1016, 931, 844, 760;
give the desired product.
PMR spectrum (500 MHz, CDCl , d, ppm, J/Hz):
3
4
.05 (3H, dd, J 5 16.1, CH ), 7.26–7.49 (5H, m, Ar-
3
13
H), 7.56–7.64 (4H, m, Ar-H); C NMR spectrum
Biphenyl-4-yl dihydrogen phosphate 3k
(
1
125 MHz, CDCl , d, ppm): 149.8 (d, J 5 9.7),
ꢀ
21
Yield 37%, mp 185–187 C; IR (neat, m, cm ):
3 P-C
40.2, 139.7, 129.1, 128.8, 127.8, 127.3, 121.7 (d, J
P-
2
770, 1600, 1483, 1403, 1165, 971, 842, 757; PMR
31
5
5.3), 56.3 (d, JP-C 5 7.5); P NMR spectrum
C
spectrum (500 MHz, DMSO, d, ppm, J/Hz): 7.25
2H, d, J 5 8.3, Ar-H), 7.32 (1H, t, J 5 7.3, Ar-H),
(202 MHz, CDCl , d, ppm): 66.94. Anal. Calcd for:
3
(
C H ClO PS: C, 52.27; H, 4.05; S, 10.73. Found:
C, 52.33; H, 4.12; S, 10.79.
13
12
2
7
7
.43 (2H, t, J 5 7.3, Ar-H), 7.60–7.64 (4H, m, Ar-H),
.8–9.8 (2H, br s, OH); C NMR spectrum (125
1
3
MHz, DMSO, d, ppm): 151.9 (d, JP-C 5 6.6), 140.2,
0
4
-(ethoxycarbonyl)biphenyl-4-yl methyl
1
36.6, 129.6, 128.5, 127.9, 127.2, 121.2 (d, J
P-
0
31
chlorothiophosphate 4b
5
4.8); P NMR spectrum (202 MHz, DMSO, d,
C
ꢀ
21
Yield 40%, mp 49–51 C; IR (neat, m, cm ):
ppm): -5.04. Anal. Calcd for: C H O P: C, 57.61;
1
2
11
4
H, 4.43. Found: 57.71; H, 4.50.
1701, 1608, 1523, 1491, 1275, 1210, 1182, 1106,
Drug Dev. Res.

98
DEMKOWICZ ET AL.
0
023, 921, 834, 771; PMR spectrum (500 MHz, Biphenyl-4-yl methyl thiophosphoroamidate 3a
1
CDCl , d, ppm, J/Hz): 1.42 (3H, t, J 5 6.8, CH ),
ꢀ
21
3
3
Yield 59%, mp 125–127 C; IR (neat, m, cm ):
387, 3296, 1600, 1516, 1483, 1221, 1187, 912, 763,
95; PMR spectrum (500 MHz, DMSO, d, ppm, J/
4
.04 (3H, d, J 5 16.6, CH ), 4.41 (2H, q, J 5 7.3,
3
3
6
CH ), 7.35–7.38 (2H, m, Ar-H), 7.62–7.65 (4H, m,
2
1
3
Ar-H), 8.12 (2H, d, J 5 8.3, Ar-H); C NMR spec-
trum (125 MHz, CDCl , d, ppm): 166.6, 150.4 (d, J
Hz): 3.70 (3H, d, J 5 13.7, CH ), 5.73 (2H, d, J
3
P-
3
P-
5
6.8, NH ), 7.28–7.36 (3H, m, Ar-H), 7.45 (2H, t,
J 5 7.8, Ar-H), 7.62–7.67 (4H, m, Ar-H); C NMR
spectrum (125 MHz, DMSO, d, ppm): 151.2 (d, JP-
N
2
5
9.7), 144.4, 138.5, 130.4, 129.8, 128.9, 127.2,
13
C
1
21.9 (d, JP-C 5 5.3), 61.3, 56.4 (d, JP-C 5 7.5), 14.6;
3
1
P NMR spectrum (202 MHz, CDCl , d, ppm):
3
5 7.0), 140.1, 137.3, 129.6, 128.4 128.0, 127.3,
C
31
6
6.81. Anal. Calcd for: C H ClO PS: C, 51.83; H,
1
6
16
4
122.3 (d, JP-C 5 4.8), 53.8 (d, JP-C 5 5.3); P NMR
spectrum (202 MHz, DMSO, d, ppm): 72.93. Anal.
Calcd for: C H NO PS: C, 55.90; H, 5.05; N, 5.02;
4
.35; S, 8.65. Found: C, 51.92; H, 4.40; S, 8.72.
13
14
2
0
S, 11.48. Found: C, 55.98; H, 5.11; N, 5.10; S, 11.54.
Biphenyl-4-yl ethyl chlorothiophosphate 4c
ꢀ
21
Yield 62%, mp 61–64 C; IR (neat, m, cm ):
601, 1518, 1482, 1215, 1159, 1032, 930, 839, 762;
0
4
-(ethoxycarbonyl)biphenyl-4-yl methyl
1
0
thiophosphoroamidate 3b
PMR spectrum (500 MHz, CDCl , d, ppm, J/Hz):
3
ꢀ
21
Yield 25%, mp 78–80 C; IR (neat, m, cm ):
366, 3282, 1716, 1602, 1523, 1493, 1281, 1219,
185, 1108, 908, 769, 709; PMR spectrum (500 MHz,
CDCl , d, ppm, J/Hz): 1.41 (3H, t, J 5 6.8, CH ),
.47 (2H, s, NH ), 3.86 (3H, d, J 5 14.2, CH ), 4.40
1
7
7
.48–1.53 (3H, m, CH ) 4.41–4.51 (2H, m, CH ),
3 2
3
1
.34–7.39 (3H, m, Ar-H), 7.43–7.48 (2H, m, Ar-H),
1
3
.57–7.64 (4H, m, Ar-H); C NMR spectrum (125
MHz, CDCl , d, ppm): 149.8 (d, J 5 9.7), 140.2,
3
3
3
P-C
3
(
1
39.6, 129.1, 128.7 (d, JP-C 5 1.8), 127.8, 127.4, 121.7
2 3
2H, q, J 5 6.8, CH ), 7.32–7.35 (2H, m, Ar-H),
(
d, J 5 4.8), 67.2 (d, JP-C 5 7.5), 15.9 (d, JP-C 5 8.8);
2
P-C
3
1
7
.58–7.63 (4H, m, Ar-H), 8.10 (2H, d, J 5 8.3, Ar-H);
P NMR spectrum (202 MHz, CDCl , d, ppm): 64.86.
3
1
3
C NMR spectrum (125 MHz, CDCl , d, ppm):
Anal. Calcd for: C H ClO PS: C, 53.76; H, 4.51; S,
3
1
4
14
2
1
1
(
66.7, 151.2 (d, JP-C 5 7.0), 144.8, 137.3, 130.3,
29.5, 128.6, 127.1, 121.8 (d, J 5 4.8), 61.3, 54.4
d, JP-C 5 5.3), 14.6; P NMR spectrum (202 MHz,
CDCl3, d, ppm): 71.13. Anal. Calcd for:
1
0.25. Found: C, 53.70; H, 4.57; S, 10.33.
P-C
31
0
4
-(ethoxycarbonyl)biphenyl-4-yl ethyl
0
chlorothiophosphate 4d
C H NO PS: C, 54.69; H, 5.16; N, 3.99; S, 9,13.
1
6
18
4
ꢀ
21
Yield 58%, mp 58–60 C; IR (neat, m, cm ): Found: C, 54.77; H, 5.23; N, 4.02; S, 9,18.
1
9
709, 1608, 1519, 1491, 1276, 1208, 1166, 1101, 1025,
0
32, 837, 772; PMR spectrum (500 MHz, CDCl , d, Biphenyl-4-yl ethyl thiophosphoroamidate 3c
3
ppm, J/Hz): 1.42 (3H, t, J 5 7.3, CH ), 1.50 (3H, t,
ꢀ
21
3
Yield 70%, mp 82–83 C; IR (neat, m, cm ):
425, 3285, 1600, 1541, 1514, 1483, 1389, 1217,
165, 1036, 942, 905, 840, 762, 626; PMR spectrum
J 5 7.3, CH ), 4.38–4.50 (4H, m, CH ), 7.36–7.39 (2H,
3
2
3
1
(
m, Ar-H), 7.63 (4H, d, J 5 7.3, Ar-H), 8.12 (2H, d,
1
3
J 5 8.3, Ar-H); C NMR spectrum (125 MHz, CDCl3,
d, ppm): 166.6, 150.4 (d, JP-C 5 9.7), 144.4, 138.4,
500 MHz, DMSO, d, ppm, J/Hz): 1.40 (3H, t,
J 5 6.8, CH ), 3.44 (2H, s, NH ), 4.21–4.29 (2H, m,
CH ), 7.30–7.37 (3H, m, Ar-H), 7.41–7.46 (2H, m,
Ar-H), 7.56 (4H, d, J 5 8.8, Ar-H); C NMR spec-
trum (125 MHz, DMSO, d, ppm): 150.6 (d, JP-
7.5), 140.6, 138.4, 129.0, 128.4, 127.5, 127.3,
21.7 (d, J 5 4.4), 64.3 (d, JP-C 5.3), 16.2 (d, JP-
3
2
1
30.4, 129.7, 128.9, 127.2, 121.9 (d, JP-C 5 4.8), 67.2
2
31
(
d, JP-C 5 7.0), 61.3, 15.9 (d, JP-C=8.3), 14.6; P NMR
13
spectrum (202 MHz, CDCl , d, ppm): 64.72. Anal.
3
Calcd for: C H ClO PS: C, 53.06; H, 4.71; S, 8.33.
17
18
4
5
C
Found: C, 53.13; H, 4.80; S, 8.36.
1
P-C
31
5
8.3); P NMR spectrum (202 MHz, DMSO, d,
C
General method for the synthesis of biphenyl
derivatives modified by ethyl or methyl
thiophosphoroamidate groups
ppm): 69.23. Anal. Calcd for: C H NO PS: C,
57.33; H, 5.50; N, 4.78; S, 10.93. Found: C, 57.29;
H, 5.53; N, 4.73; S, 10.98.
14 16 2
To an ice-cooled solution of corresponding
monochloride derivatives (4a , 4b , 4c or 4d ) (2.94
0
0
0
0
0
4 -(ethoxycarbonyl)biphenyl-4-yl ethyl
0
thiophosphoroamidate 3d
mmol) in dry THF (5 mL) a solution of NH in
3
ꢀ
21
Yield 46%, mp 72–75 C; IR (neat, m, cm ):
methanol (2 mL) was added. After concentration
under vacuum, the resulting residue was purified by 3415, 3311, 1706, 1600, 1537, 1523, 1493, 1386,
column chromatography using CHCl :petroleum 1279, 1215, 1170, 1035, 950, 910, 837, 769, 637;
3
ether 3:1 as an eluent to give the desired products.
PMR spectrum (500 MHz, CDCl , d, ppm, J/Hz):
3
Drug Dev. Res.

STEROID SULFATASE INHIBITION
99
1
4
.37–1.43 (6H, m, CH ), 3.48 (2H, s, NH ), 4.21– spectrum (202 MHz, CDCl , d, ppm): 67.65. Anal.
3 2 3
.28 (2H, m, CH ), 4.37–4.42 (2H, m, CH ), 7.33 Calcd for: C H O PS: C, 55.73; H, 5.23; S, 8.75.
2
2
17 19
5
(
(
2H, d, J 5 8.3, Ar-H), 7.57–7.63 (4H, m, Ar-H), 8.01 Found: C, 55.79; H, 5.28; S, 8.82.
2H, d, J 5 7.3, Ar-H); C NMR spectrum (125
1
3
0
MHz, CDCl , d, ppm): 166.7, 151.3 (d, JP-C 5 7.5), Biphenyl-4-yl diethyl thiophosphate 3g
3
1
44.8, 137.2, 130.3, 129.4, 128.6, 127.1, 121.9 (d, JP-
21
Yield 58%, oil; IR (neat, m, cm ): 1604, 1514,
483, 1213, 1165, 1017, 921, 842, 762, 696; PMR spec-
5
4.8), 64.3 (d, J 5 4.8), 61.3, 16.2 (d, JP-C 5 8.3),
C
P-C
1
31
1
4.6; P NMR spectrum (202 MHz, CDCl , d,
3
trum (500 MHz, CDCl , d, ppm, J/Hz): 1.38–1.41 (6H,
3
ppm): 69.21. Anal. Calcd for: C H NO PS: C,
1
7
20
4
m, CH ) 4.24–4.31 (4H, m, CH ), 7.25–7.28 (2H, m,
3
2
5
5
5.88; H, 5.52; N, 3.83; S, 8.78. Found: C, 55.95; H,
.60; N, 3.92; S, 8.85.
Ar-H), 7.33–7.37 (1H, m, Ar-H), 7.42–7.46 (2H, m,
13
Ar-H) 7.56 (2H, d, J 5 8.3, Ar-H); C NMR spectrum
(
1
125 MHz, CDCl , d, ppm): 150.5 (d, J 5 7.9),
3 P-C
General method for the synthesis of biphenyl
derivatives modified by thiophosphoric acid
diethyl or dimethyl ester groups
40.5, 138.5, 129.0, 128.5, 127.6, 127.3, 121.4 (d, J
P-
31
5
4.8), 65.4 (d, JP-C 5 5.7), 16.2 (d, JP-C 5 7.5);
P
C
NMR spectrum (202 MHz, CDCl , d, ppm): 64.16.
3
To an ice-cooled solution of corresponding Anal. Calcd for: C16
H O PS: C, 59.61; H, 5.94; S,
19 3
0
0
0
0
9
.95. Found: C, 59.69; H, 5.98; S, 10.01.
monochloride derivatives (4a , 4b , 4c or 4d ) (2.94
mmol) in dry THF (5 mL) sodium alkoxide (2.94
mmol) (freshly prepared by the addition of absolute 4 -(ethoxycarbonyl)biphenyl-4-yl diethyl
ethanol or methanol to 60% NaH dispersed in min- thiophosphate 3h
0
0
ꢀ
2
1
eral oil) was added. The reaction mixture was stirred
for 1 h, and a precipitate of NaCl formed. The solu-
tion was filtered, and the solvent was evaporated.
The resulting residue was purified by column chro-
matography using hexane:ethyl acetate 2:1 as an elu-
ent to give the desired products.
Yield 48%, mp 44–47 C; IR (neat, m, cm ):
701, 1605, 1521, 1493, 1276, 1215, 1182, 1103,
016, 928, 836, 770, 703; PMR spectrum (500 MHz,
1
1
CDCl , d, ppm, J/Hz): 1.37–1.43 (9H, m, CH ),
3
3
4
7
8
.23–4.31 (4H, m, CH ), 4.40 (2H, q, J 5 7.3, CH ),
2 2
.26–7.30 (2H, m, Ar-H), 7.58–7.64 (4H, m, Ar-H),
.10 (2H, d, J 5 8.3, Ar-H); C NMR spectrum (125
MHz, CDCl , d, ppm): 166.7, 151.1 (d, J 5 7.5),
P-C
144.7, 137.3, 130.3, 129.5, 128.7, 127.1, 121.7 (d, JP-
13
0
Biphenyl-4-yl dimethyl thiophosphate 3e
3
ꢀ
21
Yield 30%, mp 36–37 C; IR (neat, m, cm ):
5
4.8), 65.4 (d, J 5 5.7), 61.2, 16.2 (d, JP-C 5 7.5),
P-C
31
1
6
601, 1518, 1481, 1219, 1182, 1014, 932, 827, 762,
C
1
4.6; P NMR spectrum (202 MHz, CDCl , d,
93; PMR spectrum (500 MHz, CDCl , d, ppm, J/
3
3
ppm): 64.11. Anal. Calcd for: C H O PS: C, 57.86;
H, 5.88; S, 8.13. Found: C, 57.93; H, 5.92; S, 8.20.
Hz): 3.89 (6H, d, J 5 14.2, CH ), 7.25–7.28 (2H, m,
19 23
5
3
Ar-H), 7.33–7.38 (1H, m, Ar-H), 7.44 (2H, t, J 5 8.3,
1
3
Ar-H) 7.55–7.59 (4H, m, Ar-H); C NMR spectrum
General method for the synthesis of biphenyl
(
125 MHz, CDCl , d, ppm): 150.3 (d, J 5 7.5),
3 P-C
derivatives modified by thiophosphorodiamidate group
1
40.4, 138.7, 129.0, 128.6, 127.6, 127.3, 121.3 (d, J
P-
3
1
5
4.8), 55.5 (d, JP-C 5 5.7); P NMR spectrum
To an ice-cooled solution of thiophosphoryl
C
(
202 MHz, CDCl , d, ppm): 67.70. Anal. Calcd for: chloride (0.994 g, 5.87 mmol) in dry THF (20 mL)
3
C H O PS: C, 57.13; H, 5.14; S, 10.90. Found: C, was added a solution of the corresponding biphenyl-
14
15
3
5
7.21; H, 5.20; S, 10.98.
4-ol (5.87 mmol) in THF dropwise, followed by trie-
thylamine (0.594 g, 5.87 mmol). The reaction mixture
was stirred under a nitrogen atmosphere for 3 h. The
triethylamine hydrochloride precipitate was removed
by filtration and a solution of NH3 in methanol
0
4
-(ethoxycarbonyl)biphenyl-4-yl dimethyl
0
thiophosphate 3f
ꢀ
21
Yield 48%, mp 54–56 C; IR (neat, m, cm ):
710, 1602, 1523, 1492, 1279, 1219, 1179, 1103,
022, 931, 829, 771, 700; PMR spectrum (500 MHz,
(5 mL) was added. After concentration under vac-
1
1
uum, the crude product was crystallized from metha-
nol to give the desired product.
CDCl , d, ppm, J/Hz): 1.41 (3H, t, J 5 7.3, CH ),
3
3
3
.89 (6H, d, J 5 13.7, CH ), 4.40 (2H, q, J 5 7.3,
3
0
Biphenyl-4-yl thiophosphorodiamidate 3i
CH ), 7.26–7.29 (2H, m, Ar-H), 7.59–7.63 (4H, m,
Ar-H), 8.10 (2H, d, J 5 8.3, Ar-H); C NMR spec-
trum (125 MHz, CDCl , d, ppm): 166.7, 151.0, 3383, 3240, 1599, 1514, 1480, 1403, 1219, 1186,
2
1
3
ꢀ
21
Yield 51%, mp 187–190 C; IR (neat, m, cm ):
3
1
44.7, 137.5, 130.3, 129.6, 128.8, 127.1, 121.6 (d, J
1167, 1016, 885, 841, 773; PMR spectrum (500 MHz,
P-
31
5
4.8), 61.3, 55.5 (d, JP-C 5 5.7), 14.6; P NMR DMSO, d, ppm, J/Hz): 4.88 (4H, d, JP-N 5 3.4, NH2),
C
Drug Dev. Res.

100
DEMKOWICZ ET AL.
Figure 1. Docked binding modes for compounds 3l (red), 3f (green), 5d (blue) and biphenyl-4-yl sulfamate (yellow). [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
7
7
.22–7.35 (3H, m, Ar-H), 7.42 (2H, t, J 5 7.3, Ar-H), atmosphere for 3 h. The triethylamine hydrochloride
.60–7.64 (4H, m, Ar-H); C NMR spectrum (125 precipitate was removed by filtration, and the solvent
1
3
MHz, DMSO, d, ppm): 151.9 (d, JP-C 5 7.9), 140.4, was evaporated. The resulting residue was purified by
1
36.6, 129.6, 128.0, 127.8, 127.2, 123.0 (d, 5 J
column chromatography using CH Cl :hexane 1:6 as
P-C
2 2
31
4.8); P NMR spectrum (202 MHz, DMSO, d, eluent to give the desired products.
ppm): 70.44. Anal. Calcd for: C H N OPS: C,
1
2
13
2
5
4.54; H, 4.96; N, 10.60; S, 12.13. Found: C, 54.62;
0
Biphenyl-4-yl dichlorothiophosphate 2a
H, 5.03; N, 10.66; S, 12.19.
ꢀ
21
Yield 68%, mp 65–67 C; IR (neat, m, cm ):
1
7
599, 1516, 1482, 1210, 1189, 1159, 1015, 915, 838,
0
4
-(ethoxycarbonyl)biphenyl-4-yl
61; PMR spectrum (500 MHz, CDCl , d, ppm, J/
3
0
thiophosphorodiamidate 3j
13
Hz): 7.35–7.67 (9H, m, Ar-H); C NMR spectrum
ꢀ
21
Yield 44%, mp 183–185 C; IR (neat, m, cm ): (125 MHz, CDCl , d, ppm): 149.9 (d, J 5 14.0),
3
P-C
3
1
379, 3242, 1710, 1599, 1523, 1489, 1397, 1273, 1221, 140.5, 139.9, 129.2, 128.9 (d, J 5 10.6), 128.0,
186, 1168, 1116, 1022, 884, 834, 776; PMR spectrum 127.4, 121.9 (d, JP-C 5 5.7); P NMR spectrum (202
P-C
31
(
500 MHz, DMSO, d, ppm, J/Hz): 1.31–1.34 (3H, t, MHz, CDCl , d, ppm): 55.08.
3
J 5 6.8, CH ), 4.32 (2H, q, J 5 6.8, CH ), 4.90 (4H, d,
3
2
JP-N 5 3.9, NH ), 7.31–7.34 (2H, m, Ar-H), 7.70–7.81
2
General method for the synthesis of
di(biphenyl-4-yl) chlorothiophosphate 5a
13
(
4H, m, Ar-H), 8.01 (2H, d, J 5 8.8 Ar-H); C NMR
spectrum (125 MHz, DMSO, d, ppm): 166.2, 152.7 (d,
JP-C 5 7.6), 144.8, 135.1, 130.4, 129.1, 128.4, 127.4,
To a solution of biphenyl-4-yl dichlorothiophos-
0
3
1
phate (2a ) (1.0 mmol) in dry THF (15 mL) was
1
(
23.1 (d, JP-C 5 4.8), 61.4, 14.9; P NMR spectrum
202 MHz, DMSO, d, ppm): 70.46. Anal. Calcd for:
C H N O PS: C, 53.56; H, 5.09; N, 8.33; S, 9.53.
added the solution of biphenyl-4-ol (1.0 mmol) fol-
lowed by triethylamine (1.0 mmol). The reaction mix-
ture was stirred under a nitrogen atmosphere for
15
17
2
3
Found: C, 53.50; H, 5.12; N, 8.37; S, 9.62.
2
4 h. The triethylamine hydrochloride precipitate was
removed by filtration, and the solvent was evapo-
rated. The resulting residue was washed with hot
ethyl acetate to give the desired products.
General method for the synthesis of biphenyl-4-yl
dichlorothiophosphate 2a
0
ꢀ
21
To an ice-cooled solution of thiophosphoryl chlo-
Yield 55%, mp 210–212 C; IR (neat, m, cm ):
ride (0.848 g, 0.05 mol) in dry THF (20 mL) was added 1599, 1518, 1481, 1213, 1186, 1157, 1016, 945, 932,
a solution of biphenyl-4-ol (0.05 mol) in THF 841, 760; PMR spectrum (500 MHz, CDCl , d, ppm,
3
dropwise, followed by triethylamine (0.505 g, 0.05 mol). J/Hz): 7.37–7.48 (10H, m, Ar-H), 7.57–7.65 (8H, m,
13
The reaction mixture was stirred under a nitrogen Ar-H); C NMR spectrum (125 MHz, CDCl , d,
3
Drug Dev. Res.

STEROID SULFATASE INHIBITION
101
ppm): 150.0 (d, JP-C 5 10.1), 140.2, 139.9, 129.1, added a water solution of potassium carbonate
3
1
1
28.8, 127.8, 127.4, 121.8 (d, JP-C 5 5.3); P NMR (0.5 mL). The reaction mixture was stirred for 24 h.
spectrum (202 MHz, CDCl , d, ppm): 59.97. Anal. After concentration under vacuum, the resulting resi-
3
Calcd for: C H ClO PS: C, 65.98; H, 4.15; S, 7.34. due was purified by column chromatography using
24
18
2
Found: C, 65.93; H, 4.22; S, 7.40.
AcOEt:MeOH 20:1 as eluent to give the desired
products.
ꢀ
General method for the synthesis of
di(biphenyl-4-yl) methyl thiophosphate 5b
Yield 48%, mp 267 C with decomposition; IR
21
(neat, m, cm ): 3382, 1603, 1514, 1483, 1205, 1166,
105, 1009, 903, 834, 757; PMR spectrum (500 MHz,
DMSO, d, ppm, J/Hz): 7.27–7.32 (6H, m, Ar-H),
1
To a solution of di(biphenyl-4-yl) chlorothio-
phosphate (5a) (1.0 mmol) in dry THF (15 mL) was
added methanol (1.0 mmol) followed by potassium
carbonate (1.0 mmol). The reaction mixture was kept
under reflux for 24 h and filtered. After concentration
under vacuum, the resulting residue was purified by
column chromatography using CH Cl :hexane 1:2 as
7
.42 (4H, t, J 5 7.8, Ar-H), 7.55–7.63 (8H, m, Ar-H);
C NMR spectrum (125 MHz, DMSO, d, ppm):
53.4 (d, JP-C 5 7.9), 140.6, 135.3, 129.6, 127.9,
1
3
1
1
31
27.6, 127.7, 121.8 (d, JP-C 5 5.3); P NMR spec-
trum (202 MHz, DMSO, d, ppm): 45.51. Anal. Calcd
2
2
for: C H O PS: C, 68.89; H, 4.58; S, 7.66. Found:
24
19
3
eluent to give the desired products.
ꢀ
21
C, 68.94; H, 4.51; S, 7.72.
Yield 34%, mp 123–125 C; IR (neat, m, cm ):
1
7
597, 1518, 1482, 1216, 1188, 1165, 1015, 913, 844,
64; PMR spectrum (500 MHz, CDCl , d, ppm, J/
Enzyme Purification
3
Hz): 4.02 (3H, dd, J 5 11.7, CH ) 7.25–7.38 (6H, m,
3
STS was extracted from human placenta and
purified to homogeneity following a multistep chro-
matography protocol as previously described [Her-
nandez-Guzman et al., 2001].
Ar-H), 7.45 (4H, t, J 5 7.8, Ar-H), 7.54–7.63 (8H, m,
13
Ar-H); C NMR spectrum (125 MHz, CDCl , d,
3
ppm): 150.3 (d, JP-C 5 7.9), 140.4, 139.0, 129.1, 128.6,
1
27.7, 127.3, 121.5 (d, JP-C 5 4.8), 56.0 (d, JP-C 5 5.7);
3
1
P NMR spectrum (202 MHz, CDCl , d, ppm):
3
In vitro STS assay
6
4
1.08. Anal. Calcd for: C H O PS: C, 69.43; H,
.89; S, 7.41. Found: C, 69.51; H, 4.94; S, 7.47.
2
5
21
3
The reaction mixture, at a final volume of 100
mL, contained 20 mM Tris-HCl pH 7.4, 1 mM NPS
(
p-nitrocatechol sulfate), various concentrations of
General method for the synthesis of
di(biphenyl-4-yl) thiophosphoroamidate 5c
inhibitor (0.1–500 mM) and 5 U of purified enzyme
(
mM of NPS in 1 h at 37 C). The reaction was per-
formed at 37 C for 15 min and was halted by the
addition of 100 mL of 1 M NaOH. Ten samples con-
taining various concentration of inhibitor (0.1–500
mM) were performed in triplicate. The absorbance of
the released p-nitrophenol was measured at 405 nm
using a Microplate Reader Biotek ELx800 (SERVA).
IC50 values were calculated using GraphPad Prism
1 U is the amount of enzyme that hydrolyzes 100
To a solution of di(biphenyl-4-yl) chlorothio-
phosphate (5a) (1.0 mmol) in THF (15 mL) was
ꢀ
ꢀ
added a methanol solution of NH (1 mL). The reac-
3
tion mixture was stirred for 1 h. After concentration
under vacuum, the resulting residue was purified by
column chromatography using CHCl :AcOEt 10:1 as
3
eluent to give the desired products.
ꢀ
21
Yield 67%, mp 239–242 C; IR (neat, m, cm ):
404, 3230, 1601, 1535, 1513, 1482, 1217, 1184, 1162,
015, 915, 841, 760; PMR spectrum (500 MHz, DMSO,
3
1
(
version 4.0) software. In the first step the concentra-
tion values were transformed to log(concentration).
To compute the IC50 values, the options were
selected as follows: Nonlinear Regression (Curve
Fit)/Classic equation/Sigmoidal dose–response (vari-
able slope).
d, ppm, J/Hz): 6.21 (2H, t, JP-N 5 6.8, NH ) 7.34–7.39
2
(
6H, m, Ar-H), 7.46 (4H, t, J5 7.3, Ar-H), 7.65–7.72
13
(8H, m, Ar-H); C NMR spectrum (125 MHz, DMSO,
d, ppm): 151.0 (d, JP-C 5 7.0), 140.1, 137.6, 129.7, 128.5,
31
1
(
28.1, 127.3, 122.4 (d, JP-C 5 4.4); P NMR spectrum
202 MHz, DMSO, d, ppm): 67.33. Anal. Calcd for:
C H NO PS: C, 69.05; H, 4.83; N, 3.36; S, 7.68.
RESULTS AND DISCUSSION
Molecular Modeling
24
20
2
Found: C, 69.12; H, 4.89; N, 3.43; S, 7.70.
The X-ray structure of human STS was taken
from the Protein Databank (Protein Data Bank
accession code 1P49). After standard preparation of
General method for the synthesis of
di(biphenyl-4-yl) hydrogen thiophosphate 5d
To a solution of di(biphenyl-4-yl) chlorothio- enzyme structure (including conversion of the
phosphate (5a) (1.0 mmol) in THF (15 mL) was catalytic amino acid FGly75 (formylglycine) to the
Drug Dev. Res.

102
DEMKOWICZ ET AL.
Figure 2. Synthesis of biphenyl-4-yl phosphate and thiophosphate derivatives.
˚
gem – diol form) the docking analysis was carried (3.10 A) could favor the binding via hydrogen bond-
out using the Autodock Vina 1.1.2 (The Molecular ing and may have a significant impact for its
Graphics Laboratory, Scripps Research Institute) activity.
software. Docking studies were performed 10 times
for each compound with the aim of studying the
capability of the designed inhibitors to interact with
Chemistry
the ligand binding domain (LBD) of STS (Fig. 1).
Substituted biphenyls can be prepared syn-
All the newly designed compound candidates thetically by various methods include: coupling reac-
theoretically bound STS. Analysis of the docking tions (Hiyama [Cornelissen et al., 2014], Hiyama-
0
0
studies for compounds (3a–3l) and (3a –4d ) Denmark [Denmark and Tymonko, 2005], Kumada
showed that these STS inhibitors could adopt [Ackermann et al., 2010], Negishi [Liu et al., 2013],
substrate-like poses in active site of STS in a similar Suzuki [Dreher et al., 2009] or Stille coupling [Mee
manner to the mode of reported biphenyl-4-yl sulfa- et al., 2004]), Ullmann reaction [Zhang et al.,
mate (used as reference) with the phosphate or thi- 2015], reaction of Grignard reagents with bromo-
ophosphate groups directed toward the catalytic benzene [Huynh and Jothibasu, 2009] or by reac-
cavity of STS (predicted binding energies from -4.5 tions of phenyl lithium with fluorobenzene [Zhu
21
to -6.8 kcal mol ). The best docking results were and Wang, 2014] or benzyne [Burdon et al., 1979]
obtained for the di(biphenyl-4-yl) thiophosphates Figure 2.
(5a–5d) which exhibited slightly different docking
As a result of the theoretical study, we decided
modes (not previously described) and led to values to synthesize phosphate and thiophosphate deriva-
within the lowest energy range (from -7.1 to -8.0 tives based on two biphenyl scaffolds. 4-phenylphenol
2
1
kcal mol ). In this case, one of the nonpolar (1a) was commercially available, analog (1b) was
0
biphenyl skeleton was oriented in the centre of the obtained via Fisher esterification of the 4 -
active site and underwent nonpolar interaction with hydroxybiphenyl-4-carboxylic acid with ethanol in the
side chains of the hydrophobic pocket formed by presence of sulfuric acid as an acidic catalyst. In the
Leu74, Arg98, Arg99, Val101, Leu103, Val177, first step, compounds (1a) and (1b) were treated
Phe178, Thr180, Thr484, Val486, and Phe488. with phosphoryl or thiophosphoryl chloride in the
ꢀ
Another part of biphenyl scaffolds is located outside presence of triethylamine at 0 C to yield phosphoryl
the entrance to the STS active site. For compound or thiophosphoryl dichloride derivatives (2a, 2b) and
0 0
5d) a short distance of the OH group to Thr484 (2a , 2b ). The progress of reactions was monitored
(
Drug Dev. Res.

STEROID SULFATASE INHIBITION
103
TABLE 1. Activities of the Synthesized Compounds in STS Enzyme
Assays
Lp.
IC50 [lM]
Lp.
IC50 [lM]
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
98.0 6 10.0
132.4 6 16.8
86.6 6 6.4
128.2 6 15.4
134.1 6 15.3
115.9 6 11.5
95.6 6 8.6
106.7 6 13.7
101.2 6 12.3
113.5 6 15.6
69.2 6 6.4
161.0 6 20.3
103.8 6 10.8
77.5 6 8.9
3h
3i
3j
3i
3j
108.6 6 13.9
125.6 6 14.9
139.6 6 20.9
97.2 6 9.0
117.7 6 14.1
101.7 6 13.9
69.3 6 7.8
51.2 6 4.7
53.5 6 5.8
48.0 6 5.2
46.8 6 6.6
28.0 6 4.3
58.0 6 6.8
51.2 6 6.2
22.1 6 3.9
91.4 6 7.9
0
a
b
c
d
0
0
0
3k
3l
0
0
c
d
e
f
0
4a
0
0
4b
4c
0
0
0
e
f
4d
5a
5b
5c
Figure 3. Synthesis of di(biphenyl-4-yl) thiophosphates (5a-5d).
g
h
0
g
82.3 6 7.3
5d
31
by P NMR spectra. After removal of precipitated
triethylamine hydrochloride, raw phosphoryl or thio-
Biphenyl-4-yl
sulfamate
0
0
phosphoryl dichlorides (2a, 2b) and (2a , 2b ) were
ꢀ
treated at 0 C with nucleophilic agents including
MeONa, EtONa or NH /MeOH in different stoichio- A comparison of the biphenyl chlorothiophosphates
3
0
0
metric ratios to obtain the corresponding phosphate (4a –4d ) with biphenyl dialkoxy phosphates (3e–3h)
and thiophosphate biphenyl derivatives in good yield and thiophosphates (3e –3h ) indicated that their
0
0
(
(
Figure 2). Finally, biphenyl dimethyl phosphates STS-inhibitory activity was attenuated. A simialr
3e, 3f) were transformed into the corresponding effect was observed when introducing the NH moi-
2
dihydrogenphosphate analogs (3k, 3l) by treatment ety into the phosphorus atom. Among all synthesized
with bromotrimethylsilane (TMSBr). To further study compounds the highest STS inhibition occurred in a
the structure-activity relationships, we synthesized series of di(biphenyl) thiophosphate derivatives (5a–
additional thiophosphate analogs (5a–5d), which 5d), which is in agreement with the data of molecu-
exhibited very promising binding modes in molecular lar modeling studies. Two compounds, (5a) and its
modeling studies. The detailed synthesis is shown in derivative (5d) had IC50 values of 28.0 and 22.1 lM
0
Figure 3. In this case, thiophosphoryl dichloride (2a ) respectively as compared to the IC50 value of 91.4
was treated with 4-phenylphenol (1a) in the presence lM for biphenyl-4-yl sulfamate used as reference.
of triethylamine, obtaining the compound (5a) with Furthermore, increased biological activity of (5d)
good yield. Next, methylthiophosphate (5b), thio- may result from the formation of hydrogen bonds
phosphoroamidate (5c) and hydrogenthiophosphate with Thr484 residue in the STS active site, which
analogs (5d) were prepared via nucleophilic substitu- may be critical for the stability of the enzyme-
tion reactions using MeOH/K CO , NH /MeOH or inhibitor complex and lead to a more effective inacti-
2
3
3
H O/K CO , respectively (Fig. 3).
vation of STS.
2
2
3
STS Enzyme Assays
CONCLUSIONS
A series of biphenyl phosphates and thiophos-
The structure-activity relationship of the synthe-
sized biphenyl derivatives with respect to the STS phates have been synthesized, and their affinity for
enzyme was evaluated. The affinity of all synthesized STS have been determined by means of in vitro STS
compounds for STS was determined using an in vitro assays. Docking studies on these compounds have
STS assay [Vaccaro et al., 1987; Woo et al., 2010]. demonstrated that the new STS inhibitors could
Table 1 shows a summary of the results where it is adopt substrate-like poses in active site of STS in a
clearly shown that phosphate and thiophosphate similar manner to the mode of reported biphenyl-4-yl
biphenyl derivatives (3a–3l) exhibit moderate STS sulfamate. Moreover, we found that di(biphenyl) thi-
inhibitor activities. We found good inhibitory potency ophosphates exhibited a slightly different docking
0
0
in case of chlorothiophosphate derivatives (4a –4d ) modes (not previously described) to that of the
with IC50 values in the range of 46.8 to 53.5 lM. reported sulfamate-based STS inhibitor and led to
Drug Dev. Res.

104
DEMKOWICZ ET AL.
increase of inhibitory potency. In the course of our
investigation, the greatest inhibitory effect of STS
applications in Kumada-Corriu couplings. Eur J Inorg Chem
3:1926–1931.
1
was observed with compound (5d) which had an Jain ZJ, Gide PS, Kankate RS. Biphenyls and their derivatives as
synthetically and pharmacologically important aromatic struc-
tural moieties. Arab J Chem, DOI: 10.1016/j.arabjc.2013.07.035.
IC50 value of 22.1 lM. This outcome suggests that
the identified hydrogen bond between thiophosphate
moieties and Thr484 could favor binding and may
have a significant impact on enzyme–ligand complex
stability for STS.
ACKNOWLEDGMENT
Kumar JR, Jawahar J, Pathak DP. 2006. Synthesis of benzimida-
zole derivatives as anti-hypertensive agents. Eur J Chem 3:278–
2
85.
Liu Z, Dong N, Xu M, Sun Z, Tu T. 2013. Mild Negishi Cross-
Coupling Reactions Catalyzed by Acenaphthoimidazolylidene
Palladium Complexes at Low Catalyst Loadings. J Org Chem
7
8:7436–7444.
The authors gratefully acknowledge the
National Science Centre (Poland) for financial sup-
port (grant no. 2011/03/D/NZ7/03985).
Mee SPH, Lee V, Baldwin JE. 2004. Stille coupling made easier -
the synergic effect of copper(I) salts and the fluoride ion.
Angew Chem Int Ed 43:1132–1136.
Nussbaumer P, Billich A. 2004. Steroid sulfatase inhibitors. Med
Res Rev 24:529–576.
REFERENCES
Ackermann L, Kapdi AR, Schulzke C. 2010. Air-stable secondary
phosphine oxide or chloride (pre) ligands for cross-couplings of
unactivated alkyl chlorides. Org Lett 12:2298–2301.
Ruggero G, Jones CK, Hemstapat K, Nong Y, Echemendia
NG, Williams LC, Paulis TD, Conn PJ. 2006. Biphenyl-inda-
none A, a positive allosteric modulator of the metabotropic
glutamate receptor subtype 2, has antipsychotic- and
anxiolytic-like effects in mice. J Pharmacol Exp Ther 318:
173–185.
Burdon J, Gill SH, Parsons IW, Tatlow JC. 1979. Hexafluoro-1,2-
naphthalyne. J Chem Soc Chem Commun 24:1147–1148.
Cornelissen L, Cirriez V, Vercruysse S, Riant O. 2014. Copper-
catalyzed Hiyama cross-coupling using vinylsilanes and benzylic
electrophiles. Org Lett 16:3024–3027.
Vaccaro AM, Salvioli R, Muscillo M, Renola L. 1987. Purification
and properties of arylsulfatase C from human placenta. Enzyme
Deep A, Jain S, Sharma PC, Verma P, Kumar M, Dora CP. 2010.
Design and biological evaluation of biphenyl-4-carboxylic acid
hydrazide-hydrazone for antimicrobial activity. Acta Pol Pharm
Drug Res 67:255–259.
3
7:115–126.
Woo LWL, Jackson T, Putey A, Cozier G, Leonard P, Acharya
KR, Chander SK, Purohit A, Reed MJ, Potter BVL. 2010.
Highly potent first examples of dual aromatase-steroid sulfatase
inhibitors based on a Biphenyl template. J Med Chem 53:
Deep A, Jain S, Sharma PC. 2010. Synthesis and anti-
inflammatory activity of derivatives N-[5-(arylmethyl)-2-(aryl)-4-
2
155–2170.
0
oxo-3-thiazolidinyl]-1,1 -biphenyl-4-carboxamide
Acta Pol Pharm Drug Res 67:63–67.
derivatives.
Yar MS, Ansari ZH. 2009. Synthesis and in vivo diuretic activity
of biphenyl benzothiazole-2-carboxamide derivatives. Acta Pol
Pharm Drug Res 66:387–392.
Denmark SE, Tymonko SA. 2005. Sequential cross-coupling of
,4-bissilylbutadienes: synthesis of unsymmetrical 1,4-disubsti-
1
tuted 1,3-butadienes. J Am Chem Soc 127:8004–8005.
Zhang C, Sun Q, Chen H, Tan Q, Xu W. 2015. Formation of pol-
yphenyl chains through hierarchical reactions: Ullmann cou-
pling followed by cross-dehydrogenative coupling. Chem
Commun 51:495–498.
Dreher SD, Lim SE, Sandrock DL, Molander GA. 2009. Suzuki-
Miyaura Cross-Coupling Reactions of Primary Alkyltrifluorobo-
rates with Aryl Chlorides. J Org Chem 74:3626–3631.
Hernandez-Guzman FG, Higashiyama T, Osawa Y, Ghosh D.
Zhu F, Wang ZX. 2014. Nickel-catalyzed cross-coupling of aryl
fluorides and organozinc reagents. J Org Chem 79:4285–4292.
2
001. Purification, characterization and crystallization of human
placental estrone/dehydroepiandrosterone sulfatase,
membrane-bound enzyme of the endoplasmic reticulum.
J Steroid Biochem Mol Biol 78:441–450.
a
Zhu JR, Bai J, Cai NS, Tang B, Fan WH, Guo JZ, Ke YN, Guo
JX, Sheng LH, Lu ZY, Cheng NN. 2004. Efficacy and safety of
telmisartan vs. losartan in control of mild-to-moderate hyper-
tension: A multicentre, randomised, double-blind study. Int J
Clin Pract Suppl 145:46–49.
Huynh HV, Jothibasu R. 2009. Formation of homoleptic tetracar-
bene versus cis-chelating dicarbene complexes of nickel(II) and
Drug Dev. Res.