Steroid Sulfatase Inhibitors Based on Phosphate and Thiophosphate Flavone Analogs

Article abstract of DOI:10.1002/ddr.21281

Preclinical Research A series of phosphate and thiophosphate flavone derivatives were synthesized and biologically evaluated in vitro for inhibition of steroid sulfatase (STS) activity. The described synthesis includes the straightforward preparation of 7-hydroxy-2-phenyl-4H-chromen-4-one 3a, 2-(4-fluorophenyl)-7-hydroxy-4H-chromen-4-one 3b, 7-hydroxy-2-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one 3c, 7-hydroxy-2-(p-tolyl)-4H-chromen-4-one 3d modified with different phosphate or thiophosphate moieties. The inhibitory properties of the synthesized compounds were tested against human placenta STS. Some of the novel STS inhibitors had good activities against STS. In particular, the bis-(4-oxo-2-(p-tolyl)-4H-chromen-7-yl) hydrogenthiophosphate, 6i had the most potent inhibitory effect with an IC₅₀ value of 3.25 μM as compared to an IC₅₀ value of 8.50 μM for the 2-(4-trifluoromethylphenyl)-chromen-4-one-7-O-sulfamate used as a reference. Drug Dev Res 76: 450-462, 2015.

Full text of DOI:10.1002/ddr.21281

DRUG DEVELOPMENT RESEARCH 76 : 450–462 (2015)
Research Article
Steroid Sulfatase Inhibitors Based on Phosphate and
Thiophosphate Flavone Analogs
1
1
2
2
1
ꢀ
ꢀ
Witold Kozak, Mateusz Dasko, Maciej Masłyk, Konrad Kubinski, Janusz Rachon, and
Sebastian Demkowicz1*
1Department of Organic Chemistry, Chemical Faculty, Gdansk University of Technology,
Narutowicza 11/12, 80-233 Gdansk, Poland
2Department of Molecular Biology, Faculty of Biotechnology and Environment Sciences, The
ꢀ
John Paul II Catholic University of Lublin, Konstantynow 1i, 20-708 Lublin, Poland
Strategy, Management and Health Policy
Enabling
Preclinical Development
Toxicology, Formulation
Drug Delivery,
Clinical Development
Phases I-III
Preclinical
Research
Postmarketing
Phase IV
Technology,
Genomics,
Proteomics
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
A series of phosphate and thiophosphate flavone derivatives were synthesized and biologi-
cally evaluated in vitro for inhibition of steroid sulfatase (STS) activity. The described synthesis includes the
straightforward preparation of 7-hydroxy-2-phenyl-4H-chromen-4-one 3a, 2-(4-fluorophenyl)-7-hydroxy-
4H-chromen-4-one 3b, 7-hydroxy-2-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one 3c, 7-hydroxy-2-(p-
tolyl)-4H-chromen-4-one 3d modified with different phosphate or thiophosphate moieties. The inhibitory
properties of the synthesized compounds were tested against human placenta STS. Some of the novel STS
inhibitors had good activities against STS. In particular, the bis-(4-oxo-2-(p-tolyl)-4H-chromen-7-yl) hydro-
genthiophosphate, 6i had the most potent inhibitory effect with an IC50 value of 3.25 mM as compared to
an IC50 value of 8.50 mM for the 2-(4-trifluoromethylphenyl)-chromen-4-one-7-O-sulfamate used as a refer-
C
V
ence. Drug Dev Res 76 : 450–462, 2015.
2015 Wiley Periodicals, Inc.
Key words: steroid sulfatase; molecular modeling; STS inhibitors; breast cancer; flavones
et al., 2000; Yang et al., 2001], antiaggregatory [Violi
INTRODUCTION
et al., 2002] and anti-atherosclerotic [Steffen et al.,
2005] properties. The broad spectrum of activity of fla-
vonoids make them attractive as potential therapeutics.
Natural flavonoid derivatives like quercetin,
kaempferol or naringenin exhibit steroid sulfatase (STS)
inhibitory activity [Huang et al., 1997] and enzyme
responsible for the hydrolysis of steroid sulfates into
their active forms that plays a crucial role in the
Flavones are a class of flavonoids based on the
backbone of 2-phenylchromen-4-one. They are a large
family of compounds that are present in fruit, vegeta-
bles, wine, tea, and cocoa mostly as glycosides and
polymers [Harborne and Williams, 2000]. They accu-
mulated in plant tissues and can participate in the pho-
tosynthetic process [Mukohata et al., 1978]. Flavonoids
can bind to biological polymers, chelate transition
metal ions like Fe21, Cu21, Zn21, and Mg21, catalyze
electron transport and scavenge free radicals [Brandi
*Correspondence to: Sebastian Demkowicz, Department
et al., 1992]. Increased consumption of flavonoids is of Organic Chemistry, Chemical Faculty, Gdansk University of
Technology, Narutowicza 11/12, 80-233 Gdansk, Poland,
E-mail: sebdemko@pg.gda.pl
protective for circulatory system diseases [Rimm et al.,
1996; Woodman and Chan, 2004] and diabetes
Received 31 July 2015; Accepted 7 September 2015
[Jachak, 2002]. Flavonoids also have anti-inflammatory
[Yao et al., 2004], antiallergic [Nakajima et al., 2001],
Published online in Wiley Online Library (wileyonlinelibrary.
anticancer [Lamson and Brignall, 1999; Middleton com). DOI: 10.1002/ddr.21281
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2015 Wiley Periodicals, Inc.

STEROID SULFATASE INHIBITION
451
formation of biologically active hormones, including
General method for the synthesis of chalcone
androgens and estrogens. These play an important role
in the development of hormone-dependent breast can-
cer (HDBC) by promoting tumor cell growth. One
approach for the treatment of HDBC is based on STS
inhibitors.
Our research has focused on the use of differ-
ent phosphate or thiophosphate moieties in the
design of novel STS inhibitors resulted in flavone
derivatives that are stable and bioavailable and may
undergo a nucleophilic substitution reaction on the
phosphorus atom or create many interactions (e.g.,
hydrogen bonds) with a variety of amino acid resi-
dues found within the active site of the STS.
derivatives 1a-1d
2-hydroxy-4-methoxyacetophenone (5.00 g, 30.11
mmol) and the corresponding aldehyde (30.11 mmol)
were dissolved in methanol (50 mL) and potassium
hydroxide (90.33 mmol) was added in portions. The
reaction mixture was stirred for 4 h and then poured
into water. 3N HCl was added until the solution
became acidic. The resulting precipitate was filtered,
washed with water and crystallized from methanol to
give the corresponding product as crystals.
1-(2-Hydroxy-4-methoxyphenyl)-3-phenylprop-2-en-
1-one 1a
Yield 76%, mp 108–1108C; IR (neat, m, cm21
)
METHODS AND MATERIALS
790, 828, 860, 965, 1100, 1214, 1297, 1325, 1498, 1553,
1585, 2962, 3012, 3048; PMR spectrum (200 MHz,
CDCl3, d, ppm, J/Hz) 3.86 (3H, s, CH3), 6.47-6.52
(2H, m, Ar-H), 7.42–7.45 (3H, m, Ar-H), 7.58 (1H, d, J
5 15.6, CH), 7.64–7.67 (2H, m, Ar-H), 7.83 (1H, d, J
5 8.8, Ar-H), 7.89 (1H, d, J 5 15.1, CH), 13.47 (1H, s,
OH); 13C NMR spectrum (125 MHz, CDCl3, d, ppm)
55.9, 101.3, 108.0, 114.3, 120.5, 128.8, 129.3, 130.9,
131.5, 135.0, 144.7, 166.5, 167.0, 192.1.
2,4-Dihydroxyacetophenone, benzaldehyde, 4-
fluorobenzaldehyde, 4-(trifluoromethyl)benzaldehyde,
p-tolualdehyde, phosphoryl chloride, thiophosphoryl
chloride, triethylamine, bromotrimethylsilane, potas-
sium hydroxide, anhydrous dimethyl sulfoxide, pyri-
dine hydrochloride and iodine were purchased from
Sigma-Aldrich (St. Louis, MO). Tetrahydrofuran and
methanol were dried and distilled using standard pro-
cedures. Melting points (uncorrected) were deter-
mined with a Stuart Scientific SMP30 apparatus. NMR
spectra were recorded on Varian Unity Plus 500 MHz
or Varian Gemini 200 MHz spectrometers. Chemical
shifts are reported in ppm relative to the residual sol-
3-(4-Fluorophenyl)-1-(2-hydroxy-4-methoxyphenyl)-
prop-2-en-1-one 1b
Yield 67%, mp 112-1148C; IR (neat, m, cm21
)
784, 822, 837, 958, 1123, 1210, 1280, 1364, 1503, 1570,
1634, 2851, 3047, 3080; PMR spectrum (200 MHz,
CDCl3, d, ppm, J/Hz) 3.87 (3H, s, CH3), 6.47–6.52
(2H, m, Ar-H), 7.13 (2H, t, J 5 8.3, Ar-H), 7.51 (1H, d,
J 5 15.6, CH), 7.63–7.67 (2H, m, Ar-H), 7.83 (1H, d, J
5 2.4, Ar-H), 7.85 (1H, d, J 5 24.4, CH), 13.42 (1H, s,
OH); 13C NMR spectrum (125 MHz, CDCl3, d, ppm)
55.9, 101.3, 108.1, 114.2, 116.4 (d, JF-C 5 22.0), 120.2,
130.7 (d, JF-C 5 8.8), 131.3, 131.4, 143.3, 165.4 (d, JF-C
5 252.0), 166.5, 167.0, 191.8.
1
vent peak (CDCl3 5 7.26 ppm for H, 77.0 ppm for
13C, DMSO-d6 2.49 ppm for 1H, 39.5 ppm for 13C) or
to an external standard (85% H3PO4 5 0 for 31P). Cou-
pling constants are given in Hertz. IR spectra were
measured on a Nicolet 8700. CHNS elemental analysis
was performed on a Carlo Erba EA-1108. Column
chromatography was performed using silica gel 60
(230–400 mesh, Merck). Preparative thin-layer chro-
matography was performed with Polygram SIL G/
UV254 silica gel (Macherey-Nagel).
1-(2-Hydroxy-4-methoxyphenyl)-3-(4-trifluorome-
Synthesis
thylphenyl)prop-2-en-1-one 1c
General method for the synthesis of 2-hydroxy-4-
methoxyacetophenone
Yield 71%, mp 130–1338C; IR (neat, m, cm21
)
803, 837, 958, 1015, 1108, 1225, 1320, 1370, 1507,
1578, 1640, 2851, 2938, 2969; PMR spectrum (200
MHz, CDCl3, d, ppm, J/Hz) 3.88 (3H, s, CH3), 6.48–
6.53 (2H, m, Ar-H), 7.64 (1H, d, J 5 15.6, CH),
7.67–7.77 (4H, m, Ar-H), 7.83 (1H, d, J 5 8.8, Ar-
H), 7.88 (1H, d, J 5 15.1, CH), 13.31 (1H, s, OH);
13C NMR spectrum (125 MHz, CDCl3, d, ppm) 55.9,
101.3, 108.3, 114.2, 122.9, 125.1 (q, JF-C 5 272.7),
Dimethyl sulfate (6.59 mL, 72.46 mmol) was
added to a mixture of 2,4-dihydroxyacetophenone
(11.02 g, 72.46 mmol) and anhydrous potassium car-
bonate (10.01 g, 72.46 mmol) in dry acetone (250 mL)
in a period of 15 min and refluxed for additional 5 h.
The mixture was then cooled to room temperature, fil-
tered and concentrated under vacuum. The obtained
residue was purified by silica gel column chromatogra- 126.2 (q, JF-C 5 4.0), 128.8, 131.5, 132.2 (q, JF-C
phy with CHCl3 as an eluent to give a white solid. 32.5), 138.4, 142.5, 166.8, 167.1, 191.5.
5
Drug Dev. Res.

452
KOZAK ET AL.
1-(2-Hydroxy-4-methoxyphenyl)-3-(p-tolyl)prop-2-
7-Methoxy-2-(4-trifluoromethylphenyl)-4H-
chromen-4-one 2c
en-1-one 1d
Yield 84%, mp 162–1648C; IR (neat, m, cm21
)
Yield 79%, mp 118–1198C; IR (neat, m, cm21
)
818, 850, 1070, 1110, 1280, 1320, 1330, 1416, 1600,
1624, 1651, 2842, 3011, 3074; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 3.92 (3H, s, CH3), 7.09
(1H, d, J 5 8.8, Ar-H), 7.12 (1H, s, CH), 7.35 (1H,
s, Ar-H), 7.92–7.97 (3H, m, Ar-H), 8.31 (2H, d, J 5
8.3, Ar-H); 13C NMR spectrum (125 MHz, DMSO,
d, ppm) 56.8, 101.6, 109.0, 115.6, 117.8, 125.6 (q, JF-
C 5 272.2), 126.6 (q, JF-C 5 3.5), 126.9, 127.7, 132.0
(q, JF-C 5 32.0), 135.8, 158.2, 161.0, 164.7, 177.0.
791, 828, 870, 987, 1118, 1204, 1277, 1355, 1504,
1562, 1579, 2912, 2981, 3015; PMR spectrum (200
MHz, CDCl3, d, ppm, J/Hz) 2.40 (3H, s, CH3), 3.85
(3H, s, CH3), 6.45–6.52 (2H, m, Ar-H), 7.21-7.28
(2H, m, CH), 7.50–7.57 (3H, m, Ar-H), 7.80–7.90
(2H, m, Ar-H, CH), 13.52 (1H, s, OH); 13C NMR
spectrum (125 MHz, CDCl3, d, ppm) 21.8, 55.8,
101.3, 107.9, 114.4, 119.4, 128.8, 130.0, 131.5, 132.3,
141.5, 144.8, 166.4, 166.9, 192.2.
7-Methoxy-2-(p-tolyl)-4H-chromen-4-one 2d
Yield 80%, mp 130–1328C; IR (neat, m, cm21
)
General Method for the Synthesis of 7-Methoxy-
Flavone Derivatives 2a-2d
821, 851, 1090, 1168, 1204, 1251, 1352, 1436, 1570,
1595, 1622, 2842, 2972, 3054; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 2.38 (3H, s, CH3), 3.90
(3H, s, CH3), 6.91 (1H, s, CH), 7.03–7.07 (1H, m,
Ar-H), 7.31 (1H, s, Ar-H), 7.37 (2H, d, J 5 6.3, Ar-
H), 7.90–8.10 (3H, m, Ar-H); 13C NMR spectrum
(125 MHz, DMSO, d, ppm) 21.7, 56.8, 101.6, 106.8,
115.4, 117.8, 126.8, 129.0, 130.4, 142.6, 158.2, 163.0,
164.5, 177.1.
To a solution of the corresponding chalcone
derivative (30 mmol) (1a-1d) in DMSO (20 mL) was
added a catalytic amount of iodine (0.76 g, 3 mmol)
and the reaction mixture was heated in 1408C for 1 h.
The reaction mixture was then cooled to room temper-
ature and poured into saturated aqueous sodium thio-
sulfate solution. The resulting precipitate was filtered,
washed with cold water and crystallized from methanol
to give the corresponding product as crystals.
General Method for the Synthesis of 7-Hydroxy-
Flavone Derivatives 3a-3d
7-Methoxy-2-phenyl-4H-chromen-4-one 2a
To a corresponding 7-methoxy-flavone deriva-
tive (30 mmol) (2a-2d) was added pyridine hydro-
chloride (17.33 g, 150 mmol). The mixture was
heated until solids were melted and then kept at
1808C for 5 h. The solution was then poured into
water, acidified, filtered, washed with cold water and
crystallized from methanol to give the corresponding
7-hydroxy-flavone derivative as crystals.
Yield 62%, mp 99–1028C; IR (neat, m, cm21) 667,
768, 818, 1012, 1354, 1450, 1604, 1625, 1648, 2844,
2945, 3024; PMR spectrum (200 MHz, DMSO, d,
ppm, J/Hz) 3.89 (3H, s, CH3), 6.94 (1H, s, CH), 7.04
(1H, d, J 5 8.8, Ar-H), 7.29 (1H, s, Ar-H), 7.54-7.59
(3H, m, Ar-H), 7.92 (1H, d, J 5 8.8, Ar-H), 8.07 (2H,
d, J 5 6.3, Ar-H); 13C NMR spectrum (125 MHz,
DMSO, d, ppm) 56.8, 101.6, 107.4, 115.4, 117.8, 126.9,
129.8, 131.8, 132.3, 158.2, 162.8, 164.6, 177.1.
7-Hydroxy-2-phenyl-4H-chromen-4-one 3a
Yield 90%, mp 247–2488C; IR (neat, m, cm21
)
1092, 1249, 1381, 1473, 1540, 1574, 1623, 2785,
3060; PMR spectrum (200 MHz, DMSO, d, ppm, J/
Hz) 6.89 (1H, s, CH), 6.92 (1H, dd, J 5 8.5, Ar-H),
6.99 (1H, d, J 5 2.0, Ar-H), 7.52–7.59 (3H, m, Ar-
H), 7.87 (1H, d, J 5 8.8, Ar-H), 8.02–8.06 (2H, m,
Ar-H), 10.83 (1H, s, OH); 13C NMR spectrum (125
MHz, DMSO, d, ppm) 103.2, 107.3, 115.8, 116.8,
126.8, 127.2, 130.0, 132.0, 132.2, 158.2, 162.6, 163.5,
177.1.
2-(4-Fluorophenyl)-7-methoxy-4H-chromen-
4-one 2b
Yield 69%, mp 161–1648C; IR (neat, m, cm21
)
673, 810, 828, 1164, 1354, 1416, 1436, 1508, 1585,
1604, 1626, 2849, 2994, 3080; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 3.90 (3H, s, CH3), 6.96
(1H, s, CH), 7.05 (1H, dd, J 5 8.8, Ar-H), 7.31 (1H,
s, Ar-H), 7.41 (2H, t, J 5 8.8, Ar-H), 7.92 (1H, d, J
5 8.8, Ar-H), 8.13-8.18 (2H, m, Ar-H); 13C NMR
spectrum (125 MHz, DMSO, d, ppm) 56.7, 101.6,
107.3, 115.4, 116.8 (d, JF-C 5 22.0), 117.7, 126.8,
128.4, 129.5 (d, JF-C 5 8.8), 158.1, 161.8, 164.5,
165.7 (d, JF-C 5 249.8), 177.0.
2-(4-Fluorophenyl)-7-hydroxy-4H-chromen-
4-one 3b
Yield 85%, mp 244–2478C; IR (neat, m, cm21
1094, 1223, 1330, 1416, 1506, 1566, 1625, 2834,
)
Drug Dev. Res.

STEROID SULFATASE INHIBITION
453
3079; PMR spectrum (200 MHz, DMSO, d, ppm, J/
4-Oxo-2-phenyl-4H-chromen-7-yl
dimethylphosphate 4e
Yield 68%, mp 97–998C; IR (neat, m, cm21
Hz) 6.88 (1H, s, CH), 6.91 (1H, d, J 5 8.8, Ar-H),
6.98 (1H, s, Ar-H), 7.38 (2H, t, J 5 8.5, Ar-H), 7.86
(1H, d, J 5 8.8, Ar-H), 8.09–8.14 (2H, m, Ar-H),
10.84 (1H, s, OH); 13C NMR spectrum (125 MHz,
DMSO, d, ppm) 56.7, 103.2, 107.2, 115.8, 116.8 (d,
JF-C 5 22.4), 127.2, 128.5, 129.5 (d, JF-C 5 9.2),
158.1, 161.6, 163.5, 165.7 (d, JF-C 5 249.8), 177.0.
)
670, 767, 861, 889, 966, 1039, 1148, 1278, 1570,
1613, 1637, 2856, 2960, 3074; PMR spectrum (200
MHz, CDCl3, d, ppm, J/Hz) 3.91 (6H, d, J 5 11.2,
CH3), 6.82 (1H, s, CH), 7.26 (1H, d, J 5 8.3, Ar-H),
7.51 (4H, d, J 5 6.3, Ar-H), 7.90 (2H, d, J 5 6.3, Ar-
H), 8.20 (1H, d, J 5 8.8, Ar-H); 13C NMR spectrum
(125 MHz, CDCl3, d, ppm) 55.5 (d, JP-C 5 6.1),
107.7, 109.2 (d, JP-C 5 4.4), 118.2 (d, JP-C 5 5.7),
121.3, 126.6, 127.9, 129.3, 131.6, 132.1, 154.8 (d, JP-
C 5 6.6), 157.2, 164.1, 177.8; 31P NMR spectrum
(202 MHz, CDCl3, d, ppm) 23.78. Anal. Calcd for:
C17H15O6P: C, 58.97; H, 4.37. Found: C, 59.01; H,
4.39.
7-Hydroxy-2-(4-trifluoromethylphenyl)-4H-chromen-
4-one 3c
Yield 88%, mp 249–2518C; IR (neat, m, cm21
)
1097, 1224, 1320, 1416, 1507, 1559, 1623, 2830,
3062; PMR spectrum (200 MHz, DMSO, d, ppm, J/
Hz) 6.92–6.96 (1H, m, Ar-H), 7.00–7.05 (2H, m, Ar-
H, CH), 7.86-7.94 (3H, m, Ar-H), 8.27 (2H, d, J 5
8.3, Ar-H), 10.90 (1H, s, OH); 13C NMR spectrum
(125 MHz, DMSO, d, ppm) 103.3, 108.8, 116.0,
116.8, 125.6 (d, JF-C 5 272.2), 126.6 (q, JF-C 5 4.0),
127.3, 127.7, 131.9 (q, JF-C 5 32.0), 135.9, 158.2,
160.9, 163.7, 177.0.
2-(4-Fluorophenyl)-4-oxo-4H-chromen-7-yl
dimethylphosphate 4f
Yield 54%, mp 145–1478C; IR (neat, m, cm21
)
670, 768, 836, 890, 968, 1029, 1153, 1236, 1285,
1511, 1612, 1636, 2856, 2920, 2962, 3081; PMR spec-
trum (200 MHz, CDCl3, d, ppm, J/Hz) 3.93 (6H, d,
J 5 11.7, CH3), 6.81 (1H, d, J 5 6.8, CH), 7.21–7.28
(3H, m, Ar-H), 7.54 (1H, s, Ar-H), 7.93 (2H, dd,
J 5 8.8, Ar-H), 8.23 (1H, d, J 5 8.8, Ar-H); 13C NMR
spectrum (125 MHz, CDCl3, d, ppm) 55.5 (d, JP-
C 5 6.1), 107.5, 109.1 (d, JP-C 5 4.8), 116.6 (d, JF-
C 5 22.4), 118.3 (d, JP-C 5 6.1), 121.2, 127.8, 128.0,
128.8 (d, JF-C 5 8.8), 154.8 (d, JP-C 5 6.6), 157.1,
163.2, 166.1 (d, JF-C 5 253.8), 177.7; 31P NMR spec-
trum (202 MHz, CDCl3, d, ppm) 23.68. Anal. Calcd
for: C17H14FO6P: C, 56.05; H, 3.87. Found: C, 56.09;
H, 3.91.
7-Hydroxy-2-(p-tolyl)-4H-chromen-4-one 3d
Yield 89%, mp 282–2858C; IR (neat, m, cm21
)
1093, 1223, 1389, 1414, 1509, 1562, 1622, 2835,
3065; PMR spectrum (200 MHz, DMSO, d, ppm, J/
Hz) 2.38 (3H, s, CH3), 6.85 (1H, s, CH), 6.92 (1H,
dd, J 5 8.8, Ar-H), 6.99 (1H, d, J 5 2.0, Ar-H), 7.36
(2H, d, J 5 8.3, Ar-H), 7.86 (1H, d, J 5 8.8, Ar-H),
7.95 (1H, d, J 5 8.3, Ar-H), 10.82 (1H, s, OH); 13C
NMR spectrum (125 MHz, DMSO, d, ppm) 21.7,
103.2, 106.6, 115.6, 116.8, 126.8, 127.2, 129.1, 130.3,
142.4, 158.1, 162.7, 163.4, 177.1.
General Method for the Synthesis of Dimethyl
Phosphate Derivatives 4e-4h
4-Oxo-2-(4-trifluoromethylphenyl)-4H-chromen-7-yl
dimethylphosphate 4g
To an ice-cooled solution of phosphoryl chloride
(92 mg, 0.6 mmol) in dry THF (10 mL) was added
dropwise a solution of the corresponding 7-hydroxy-
flavone derivative (3a-3d) (0.6 mmol) in THF, fol-
lowed by triethylamine (61 mg, 0.6 mmol). The reac-
tion mixture was stirred under nitrogen for 1 h. The
triethylamine hydrochloride precipitate was removed
Yield 48%, mp 161–1648C; IR (neat, m, cm21
)
770, 830, 844, 873, 1014, 1034, 1115, 1319, 1440,
1644, 2864, 2966, 3082; PMR spectrum (200 MHz,
DMSO, d, ppm, J/Hz) 3.86 (6H, d, J 5 11.7, CH3),
7.21 (1H, s, CH), 7.39 (1H, dd, J 5 8.8, Ar-H), 7.72
(1H, d, J 5 1.0, Ar-H), 7.94 (2H, d, J 5 8.3, Ar-H),
by filtration and sodium methoxide (81 mg, 1.5 8.10 (1H, d, J 5 8.8, Ar-H), 8.35 (2H, d, J 5 8.3, Ar-
H); 13C NMR spectrum (125 MHz, DMSO, d, ppm)
mmol) (freshly prepared by the addition of absolute
methanol to 60% NaH dispersed in mineral oil) was 56.0 (d, JP-C 5 6.1), 109.2, 110.1 (d, JP-C 5 4.8), 119.0
added. The reaction mixture was stirred for 1 h, and (d, JP-C 5 4.8), 121.4, 124.5 (q, JF-C 5 272.2), 126.6
a precipitate of NaCl was formed. The solution was (q, JF-C 5 3.5), 127.8, 128.0, 132.1 (q, JF-C 5 32.0),
filtered, and the solvent evaporated. The resulting 135.5, 154.9 (d, JP-C 5 6.1), 157.1, 161.8, 177.0; 31P
residue was purified by column chromatography NMR spectrum (202 MHz, DMSO, d, ppm) 23.64.
using CHCl3 as an eluent to give the desired Anal. Calcd for: C18H14F3O6P: C, 52.19; H, 3.41.
products.
Found: C, 52.22; H, 3.39.
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KOZAK ET AL.
t, J 5 8.8, Ar-H), 7.57 (1H, d, J 5 1.5, Ar-H), 8.03
4-Oxo-2-(p-tolyl)-4H-chromen-7-yl
dimethylphosphate 4h
Yield 50%, mp 138–1408C; IR (neat, m, cm21
(1H, d, J 5 8.8, Ar-H), 8.18 (2H, dd, J 5 8.8, Ar-H);
13C NMR spectrum (125 MHz, DMSO, d, ppm)
107.5, 109.3 (d, JP-C 5 5.3), 117.0 (d, JF-C 5 22.0),
119.1 (d, JP-C 5 5.7), 120.2, 127.2, 128.3, 129.7 (d, JF-
C 5 9.2), 156.6 (d, JP-C 5 5.7), 157.1, 162.4, 165.8 (d,
)
816, 854, 869, 973, 1035, 1153, 1242, 1270, 1370,
1615, 1640, 2854, 2922, 2956, 3075; PMR spectrum
(200 MHz, CDCl3, d, ppm, J/Hz) 2.46 (3H, s, CH3),
3.93 (6H, d, J 5 11.2, CH3), 6.88 (1H, s, CH), 7.26
(1H, s, Ar-H), 7.34 (2H, d, J 5 7.8, Ar-H), 7.54 (1H,
d, J 5 2.0, Ar-H), 7.83 (2H, d, J 5 8.3, Ar-H), 8.24
(1H, d, J 5 8.8, Ar-H); 13C NMR spectrum (125
MHz, CDCl3, d, ppm) 21.8, 55.5 (d, JP-C 5 6.1),
107.0, 109.2 (d, JP-C 5 4.4), 118.1 (d, JP-C 5 5.7),
121.2, 126.5, 127.9, 128.7, 130.1, 142.9, 154.7 (d, JP-
C 5 6.6), 157.2, 164.5, 177.8; 31P NMR spectrum
(202 MHz, CDCl3, d, ppm) 23.68. Anal. Calcd for:
C18H17O6P: C, 60.00; H, 4.76. Found: C, 60.08; H,
4.80.
J
F-C 5 250.7), 177.1; 31P NMR spectrum (202 MHz,
DMSO, d, ppm) 25.57. Anal. Calcd for:
C15H10FO6P: C, 53.59; H, 3.00. Found: C, 53.57; H,
3.04.
4-Oxo-2-(4-trifluoromethylphenyl)-4H-chromen-7-yl
dihydrogenphosphate 4k
Yield 73%, mp 2438C (with decomposition); IR
(neat, m, cm21) 640, 666, 842, 952, 1112, 1130, 1168,
1325, 1447, 1621, 3080, 3096; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 2.60-5.00 (2H, br s,
OH), 7.17 (1H, s, CH), 7.30 (1H, dd, J 5 8.8, Ar-H),
7.60 (1H, d, J 5 2.0, Ar-H) 7.94 (2H, d, J 5 8.3, Ar-
H), 8.05 (1H, d, J 5 8.8, Ar-H), 8.32 (2H, d, J 5 8.3,
Ar-H); 13C NMR spectrum (125 MHz, DMSO, d,
ppm) 109.1, 109.3 (d, JP-C 5 5.3), 119.3 (d, JP-
C 5 5.7), 120.2, 125.6 (q, JF-C 5 277.5), 126.6 (q, JF-
C 5 3.5), 127.2, 127.9, 132.0 (q, JF-C 5 32.0), 135.6,
156.8 (d, JP-C 5 5.7), 157.2, 161.6, 177.1; 31P NMR
spectrum (202 MHz, DMSO, d, ppm) 25.59. Anal.
Calcd for: C16H10F3O6P: C, 49.76; H, 2.61. Found:
C, 49.81; H, 2.65.
General Method for the Synthesis of Phosphoric
Acid Derivatives 4i-4l
To an ice-cooled solution of derivatives (4e-4h)
(1 mmol) in dry DCM (10 mL), TMSBr (0.66 mL, 5
mmol) was added dropwise. The reaction mixture
was stirred under nitrogen for 1.5 h. After concentra-
tion under vacuum, 5 mL of methanol was added. All
solvents were then evaporated, and the crude prod-
uct was washed with ethyl acetate to give the desired
product.
4-Oxo-2-(p-tolyl)-4H-chromen-7-yl dihydrogenphos-
phate 4l
4-Oxo-2-Phenyl-4H-chromen-7-yl
dihydrogenphosphate 4i
Yield 78%, mp 2488C (with decomposition); IR
(neat, m, cm21) 657, 818, 837, 941, 1015, 1165, 1254,
1448, 1609, 3033, 3077, 3097; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 2.49 (3H, s, CH3),
2.60–5.20 (2H, br s, OH), 6.98 (1H, s, CH), 7.28
(1H, dd, J 5 8.8, Ar-H), 7.38 (2H, d, J 5 8.3, Ar-H),
7.56 (1H, d, J 5 2.0, Ar-H), 7.99 (2H, d, J 5 8.3, Ar-
H), 8.02 (1H, d, J 5 8.8, Ar-H); 13C NMR spectrum
(125 MHz, DMSO, d, ppm) 21.7, 106.9, 109.2 (d, JP-
C 5 4.8), 119.0 (d, JP-C 5 5.3), 120.3, 126.9, 127.1,
128.9, 130.4, 142.7, 157.1, 163.5, 177.1; 31P NMR
spectrum (202 MHz, DMSO, d, ppm) 25.57. Anal.
Calcd for: C16H13O6P: C, 57.84; H, 3.94. Found: C,
57.75; H, 3.89.
Yield 74%, mp 2518C (with decomposition); IR
(neat, m, cm21) 666, 774, 830, 945, 1007, 1156, 1250,
1454, 1613, 3074, 3099; PMR spectrum (200 MHz,
DMSO, d, ppm, J/Hz) 2.80-5.80 (2H, br s, OH), 7.03
(1H, s, CH), 7.29 (1H, dd, J 5 8.8, Ar-H), 7.54-7.64
(4H, m, Ar-H), 8.03 (1H, d, J 5 8.8, Ar-H), 8.10 (2H,
d, J 5 6.8, Ar-H); 13C NMR spectrum (125 MHz,
DMSO, d, ppm) 107.6, 109.3 (d, JP-C 5 5.3), 119.1 (d,
JP-C 5 5.7), 120.3, 127.0, 127.2, 129.8, 131.7, 132.5,
156.6 (d, JP-C 5 5.7), 157.2, 163.3, 177.2; 31P NMR
spectrum (202 MHz, DMSO, d, ppm) 25.60. Anal.
Calcd for: C15H11O6P: C, 56.62; H, 3.48. Found: C,
56.60; H, 3.45.
2-(4-Fluorophenyl)-4-oxo-4H-chromen-7-yl
dihydrogenphosphate 4j
General Method for the Synthesis of
Methylchlorothiophosphate Derivatives 5e-5h
Yield 88%, mp 3208C (with decomposition); IR
(neat, m, cm21) 660, 752, 835, 948, 1011, 1165, 1241,
1504, 1601, 3088, 3099; PMR spectrum (200 MHz,
DMSO, d, ppm, J/Hz) 2.60-4.40 (2H, br s, OH), 7.03
To an ice-cooled solution of thiophosphoryl
chloride (102 mg, 0.6 mmol) in dry THF (10 mL)
was added dropwise a solution of the corresponding
7-hydroxy-flavone (3a-3d) (0.6 mmol) in THF, fol-
(1H, s, CH), 7.29 (1H, dd, J 5 8.8, Ar-H), 7.42 (2H, lowed by triethylamine (61 mg, 0.6 mmol). The
Drug Dev. Res.

STEROID SULFATASE INHIBITION
455
reaction mixture was stirred under nitrogen for 3 h. 6.89 (1H, s, CH), 7.34 (1H, d, J 5 8.8, Ar-H), 7.56
The triethylamine hydrochloride precipitate was (1H, t, J 5 2.0, Ar-H), 7.80 (2H, d, J 5 8.3, Ar-H),
removed by filtration, and sodium methoxide (32 mg, 8.04 (2H, d, J 5 8.3, Ar-H), 8.28 (1H, d, J 5 8.8, Ar-
0.6 mmol) (freshly prepared by the addition of abso- H); 13C NMR spectrum (125 MHz, CDCl3, d, ppm)
lute methanol to 60% NaH dispersed in mineral oil) 56.6 (d, JP-C 5 7.0), 109.2, 111.0 (d, JP-C 5 5.7), 119.7
was added. The reaction mixture was stirred for 1 h. (d, JP-C 5 5.3), 122.2, 124.9 (q, JF-C 5 272.7), 126.4
The solution was filtered, and the solvent evaporated. (q, JF-C 5 4.0), 127.0, 128.1, 133.6 (q, JF-C 5 32.9),
The resulting residue was purified by column chro- 135.0, 154.1 (d, JP-C 5 9.2), 156.9, 162.4, 177.4; 31P
matography using CHCl3:hexane 1:1 as eluent to give NMR spectrum (202 MHz, CDCl3, d, ppm) 65.32.
the desired products.
Anal. Calcd for: C17H11ClF3O4PS: C, 46.96; H, 2.55;
S, 7.38. Found: C, 47.00; H, 2.51; S, 7.41.
4-Oxo-2-Phenyl-4H-chromen-7-yl methylchloro-
thiophosphate 5e
4-Oxo-2-(p-tolyl)-4H-chromen-7-yl methylchloro-
thiophosphate 5h
Yield 77%, mp 1028C (with decomposition); IR
(neat, m, cm21) 645, 728, 812, 885, 966, 1039, 1147,
Yield 52%, mp 110–1138C; IR (neat, m, cm21
)
1363, 1439, 1610, 1643, 2958, 3065; PMR spectrum 644, 717, 816, 969, 1033, 1137, 1369, 1441, 1612,
(200 MHz, CDCl3, d, ppm, J/Hz) 4.07 (3H, d, 1640, 2849, 2952, 3071; PMR spectrum (200 MHz,
J 5 16.6, CH3), 6.96 (1H, s, CH), 7.34 (1H, d, CDCl3, d, ppm, J/Hz) 2.46 (3H, s, CH3), 4.07 (3H,
J 5 8.3, Ar-H), 7.54–7.59 (4H, m, Ar-H), 7.95 (2H, d, d, J 5 16.6, CH3), 6.92 (1H, s, CH), 7.31-7.36 (3H,
J 5 7.3, Ar-H), 8.29 (1H, d, J 5 8.8, Ar-H); 13C NMR m, Ar-H), 7.55 (1H, t, J 5 2.0, Ar-H), 7.84 (2H, d,
spectrum (125 MHz, CDCl3, d, ppm) 56.6 (d, JP- J 5 8.3, Ar-H), 8.28 (1H, d, J 5 8.8, Ar-H); 13C NMR
C 5 7.0), 107.7, 110.9 (d, JP-C 5 5.7), 119.5 (d, JP- spectrum (125 MHz, CDCl3, d, ppm) 21.8, 56.6 (d,
C 5 4.8), 122.0, 126.7, 128.0, 129.4, 131.4, 132.3,
JP-C 5 7.0), 107.1, 110.9 (d, JP-C 5 5.7), 119.3, 122.1,
154.0 (d, JP-C 5 8.8), 157.0, 164.6, 177.8; 31P NMR 126.6, 127.6, 127.9, 128.6, 130.1, 143.1, 153.9, 156.9,
spectrum (202 MHz, CDCl3, d, ppm) 65.33. Anal. 164.7, 177.7; 31P NMR spectrum (202 MHz, CDCl3,
Calcd for: C16H12ClO4PS: C, 52.40; H, 3.30; S, 8.74. d, ppm) 65.34. Anal. Calcd for: C17H14ClO4PS: C,
Found: C, 52.45; H, 3.36; S, 8.72.
53.62; H, 3.71; S, 8.42. Found: C, 53.58; H, 3.70; S,
8.43.
2-(4-Fluorophenyl)-4-oxo-4H-chromen-7-yl
methylchlorothiophosphate 5f
General Method for the Synthesis of
Dimethylthiophosphate Derivatives 5i-5l
Yield 58%, mp 1228C (with decomposition); IR
(neat, m, cm21) 646, 726, 824, 890, 972, 1032, 1141,
To an ice-cooled solution of the corresponding
1371, 1439, 1613, 1637, 2958, 3065; PMR spectrum methylchlorothiophosphate derivative (5e-5h) (0.6
(200 MHz, CDCl3, d, ppm, J/Hz) 4.06 (3H, d, mmol) in dry THF (10 mL) sodium methoxide
J 5 16.1, CH3), 6.80 (1H, s, CH), 7.22 (2H, t, J 5 8.3, (32 mg, 0.6 mmol) (freshly prepared by the addition
Ar-H), 7.32 (1H, d, J 5 8.8, Ar-H), 7.52 (1H, t, of absolute methanol to 60% NaH dispersed in min-
J 5 2.0, Ar-H), 7.93 (2H, dd, J 5 8.8, Ar-H), 8.26 eral oil) was added. The reaction mixture was stirred
(1H, d, J 5 8.8, Ar-H); 13C NMR spectrum (125 for 1 h. The solution was filtered and the solvent
MHz, CDCl3, d, ppm) 56.6 (d, JP-C 5 7.0), 107.7, evaporated. The resulting residue was purified by col-
110.8 (d, JP-C 5 5.7), 116.8 (d, JF-C 5 22.4), 119.4 (d, umn chromatography using CHCl3:hexane 2:1 as an
JP-C 5 5.3), 122.1, 127.7, 128.0, 128.8 (d, JF-C 5 8.8), eluent to give the desired products.
153.9 (d, JP-C 5 9.2), 156.8 (d, JP-C 5 1.8), 163.2,
166.2 (d, JF-C 5 254.2), 177.5; 31P NMR spectrum
(202 MHz, CDCl3, d, ppm) 65.32. Anal. Calcd for:
C16H11ClFO4PS: C, 49.95; H, 2.88; S, 8.33. Found:
C, 49.99; H, 2.91; S, 8.36.
4-Oxo-2-phenyl-4H-chromen-7-yl dimethylthio-
phosphate 5i
Yield 86%, mp 126–1288C; IR (neat, m, cm21
)
766, 825, 882, 970, 1022, 1153, 1244, 1368, 1438,
1609, 1641, 2848, 2952, 3075; PMR spectrum (200
MHz, CDCl3, d, ppm, J/Hz) 3.91 (6H, d, J 5 14.2,
CH3), 6.92 (1H, s, CH), 7.23-7.27 (1H, m, Ar-H),
7.45 (1H, t, J 5 2.0, Ar-H), 7.51–7.56 (3H, m, Ar-H),
4-Oxo-2-(4-trifluoromethylphenyl)-4H-chromen-7-yl
methylchlorothiophosphate 5g
Yield 60%, mp 135-1378C; IR (neat, m, cm21
)
652, 704, 824, 890, 966, 1028, 1127, 1371, 1442, 7.91–7.94 (2H, m, Ar-H), 8.23 (1H, d, J 5 8.8, Ar-H);
1615, 1639, 2952, 3087; PMR spectrum (200 MHz, 13C NMR spectrum (125 MHz, CDCl3, d, ppm) 55.8
CDCl3, d, ppm, J/Hz) 4.07 (3H, d, J 5 16.6, CH3), (d, JP-C 5 5.7), 107.5, 110.3 (d, JP-C 5 5.3), 119.4 (d,
Drug Dev. Res.

456
KOZAK ET AL.
J
P-C 5 4.8), 121.2, 126.7, 127.7, 129.4, 131.5, 132.3, 111.1, 119.6, 121.7, 127.0, 127.5, 128.7, 130.4, 142.9,
155.0 (d, JP-C 5 7.0), 157.1, 164.6, 177.9; 31P NMR
spectrum (202 MHz, CDCl3, d, ppm) 66.77. Anal.
Calcd for: C17H15O5PS: C, 56.35; H, 4.17; S, 8.85.
Found: C, 56.42; H, 4.22; S, 8.79.
154.6, 156.9, 163.7, 177.0; 31P NMR spectrum (202
MHz, DMSO, d, ppm) 66.50. Anal. Calcd for:
C18H17O5PS: C, 57.44; H, 4.45; S, 8.52. Found: C,
57.47; H, 4.41; S, 8.50.
2-(4-Fluorophenyl)-4-oxo-4H-chromen-7-yl
dimethylthiophosphate 5j
Yield 83%, mp 130–1328C; IR (neat, m, cm21
General Method for the Synthesis of Biflavone
Chlorothiophosphates 6a-6d
)
757, 886, 973, 1024, 1147, 1237, 1370, 1439, 1612,
1635, 2850, 2966, 3074; PMR spectrum (200 MHz,
DMSO, d, ppm, J/Hz) 3.88 (6H, d, J 13.7, CH3),
7.08 (1H, s, CH), 7.32 (1H, dd, J 5 8.8, Ar-H), 7.44
(2H, t, J 5 8.8, Ar-H), 7.66 (1H, t, J 5 2.0, Ar-H),
8.08 (1H, d, J 5 8.8, Ar-H), 8.20–8.24 (2H, m, Ar-H);
13C NMR spectrum (125 MHz, DMSO, d, ppm) 56.3
(d, JP-C 5 6.1), 107.4, 111.1 (d, JP-C 5 4.8), 116.9 (d,
To an ice-cooled solution of thiophosphoryl
chloride (102 mg, 0.6 mmol) in dry THF (10 mL)
was added dropwise a solution of the corresponding
7-hydroxy-flavone (3a-3d) (1.2 mmol) in THF, fol-
lowed by triethylamine (122 mg, 1.2 mmol). The
reaction mixture was stirred under nitrogen for 24 h.
The triethylamine hydrochloride precipitate removed
by filtration, and the solvent evaporated. Due to their
instability, compounds (6a), (6b), and (6d) were used
as raw products for further synthesis. Analog (6c)
was purified by column chromatography using
CHCl3:AcOEt 15:1 as eluent to give the desired
products.
J
F-C 5 22.0), 119.8, 121.4, 127.5, 128.0, 129.8 (d, JF-
C 5 9.2), 154.6 (d, JP-C 5 7.0), 156.9, 162.8, 165.9 (d,
F-C 5 250.7), 177.2; 31P NMR spectrum (202 MHz,
J
DMSO, d, ppm) 66.45. Anal. Calcd for:
C17H14FO5PS: C, 53.69; H, 3.71; S, 8.43. Found: C,
53.75; H, 3.77; S, 8.46.
4-Oxo-2-(4-trifluoromethylphenyl)-4H-chromen-
7-yl dimethylthiophosphate 5k
Bis-(4-oxo-2-(4-trifluoromethylphenyl)-4H-
chromen-7-yl) chlorothiophosphate 6c
Yield 66%, mp 186–1888C; IR (neat, m, cm21
Yield 82%, mp 157–1608C; IR (neat, m, cm21
)
)
705, 835, 891, 962, 1030, 1155, 1320, 1371, 1439,
1610, 1643, 2852, 2954, 3075; PMR spectrum (200
MHz, CDCl3, d, ppm, J/Hz) 3.91 (6H, d, J 5 13.7,
CH3), 6.90 (1H, s, CH), 7.26 (1H, s, Ar-H), 7.47
(1H, t, J 5 2.0, Ar-H), 7.80 (2H, d, J 5 8.3, Ar-H),
8.05 (2H, d, J 5 8.3, Ar-H), 8.24 (1H, d, J 5 8.8, Ar-
H); 13C NMR spectrum (125 MHz, CDCl3, d, ppm)
55.8 (d, JP-C 5 5.7), 109.0, 110.3 (d, JP-C 5 5.3), 119.5
(d, JP-C 5 4.8), 121.5, 123.8 (q, JF-C 5 272.2), 126.3
(q, JF-C 5 3.5), 126.9, 127.8, 133.5 (q, JF-C 5 32.9),
135.1, 155.0 (d, JP-C 5 7.0), 157.0, 162.2, 177.6; 31P
NMR spectrum (202 MHz, CDCl3, d, ppm) 66.78.
Anal. Calcd for: C18H14F3O5PS: C, 50.24; H, 3.28; S,
7.45. Found: C, 50.21; H, 3.34; S, 7.48.
829, 959, 1113, 1320, 1417, 1440, 1509, 1609, 1647,
2926, 3066; PMR spectrum (200 MHz, CDCl3, d,
ppm, J/Hz) 6.90 (2H, s, CH), 7.43 (2H, dt, J 5 8.8,
Ar-H), 7.62 (2H, t, J 5 2.0, Ar-H), 7.80 (4H, d,
J 5 8.3, Ar-H), 8.05 (4H, d, J 5 8.3, Ar-H), 8.34 (2H,
d, J 5 8.8, Ar-H); 13C NMR spectrum (125 MHz,
CDCl3, d, ppm) 109.0, 110.9 (d, JP-C 5 5.7), 119.3,
122.4, 126.2, 126.7, 128.1, 134.0, 134.6, 153.5, 156.6,
162.2, 176.9; 31P NMR spectrum (202 MHz, CDCl3,
d, ppm) 57.00. Anal. Calcd for: Anal. Calcd for:
C32H16ClF6O6PS: C, 54.21; H, 2.27; S, 4.52. Found:
C, 54.28; H, 2.30; S, 4.55.
General Method for the synthesis of biflavone
methylthiophosphate 6e
4-Oxo-2-(p-tolyl)-4H-chromen-7-yl dimethylthio-
phosphate 5l
Yield 90%, mp 137–1408C; IR (neat, m, cm21
)
To a solution of biflavone chlorothiophosphate
(6c) (708 mg, 1.0 mmol) in dry THF (10 mL) was
added methanol (0.04 mL, 1 mmol) followed by
potassium carbonate (138 mg, 1 mmol). The reaction
mixture was stirred under nitrogen for 24 h and fil-
tered. After concentration under vacuum, the result-
ing residue was purified by column chromatography
using CHCl3:AcOEt 10:1 as eluent to give the
710, 818, 882, 962, 1019, 1152, 1328, 1367, 1437,
1603, 1637, 2849, 2950, 3068; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 2.39 (3H, s, CH3), 3.88
(6H, d, J 5 13.7, CH3), 7.02 (1H, s, CH), 7.31 (1H,
d, J 5 8.8, Ar-H), 7.39 (2H, d, J 5 8.3, Ar-H), 7.65
(1H, s, Ar-H), 8.03 (2H, d, J 5 7.8, Ar-H), 8.08 (1H,
d, J 5 8.8, Ar-H); 13C NMR spectrum (125 MHz,
DMSO, d, ppm) 21.8, 56.3 (d, JP-C 5 6.1), 107.0, desired product.
Drug Dev. Res.

STEROID SULFATASE INHIBITION
457
8.19 (4H, m, Ar-H); 13C NMR spectrum (125 MHz,
DMSO, d, ppm) 107.1, 109.3 (d, JP-C 5 5.3), 116.6 (d,
Bis-(4-oxo-2-(4-trifluoromethylphenyl)-4H-
chromen-7-yl) methylthiophosphate 6e
J
F-C 5 22.2), 119.2, 119.7, 126.2, 128.1, 129.4 (d, JF-
Yield 36%, mp 1918C (with decomposition); IR
(neat, m, cm21) 825, 896, 964, 1070, 1110, 1236,
1320, 1439, 1491, 1608, 1649, 2953, 3067; PMR spec-
trum (200 MHz, DMSO, d, ppm, J/Hz) 4.10 (3H, d,
J 5 14.2, CH3), 7.24 (2H, s, CH), 7.49 (2H, d,
J 5 8.3, Ar-H), 7.85 (2H, s, Ar-H), 7.93 (4H, d,
J 5 8.3, Ar-H), 8.16 (2H, d, J 5 8.8, Ar-H), 8.36 (4H,
d, J 5 7.8, Ar-H); 13C NMR spectrum (125 MHz,
DMSO, d, ppm) 57.3, 109.0, 111.3, 119.2, 121.8,
124.3 (q, JF-C 5 272.2), 126.6 (q, JF-C 5 16.7), 127.7,
132.2 (q, JF-C 5 32.0), 135.2, 156.8, 158.0, 161.6,
176.9; 31P NMR spectrum (202 MHz, DMSO, d,
ppm) 45.30. Anal. Calcd for: C33H19F6O7PS: C,
56.26; H, 2.72; S, 4.55. Found: C, 56.22; H, 2.75; S,
4.56.
C 5 8.7), 156.8, 157.9 (d, JP-C 5 7.8), 162.0, 167.0,
176.9; 31P NMR spectrum (202 MHz, DMSO, d,
ppm) 45.27. Anal. Calcd for: C30H17F2O7PS: C,
61.02; H, 2.90; S, 5.43. Found: C, 60.95; H, 2.94; S,
5.48.
Bis-(4-oxo-2-(4-trifluoromethylphenyl)-4H-
chromen-7-yl) hydrogenthiophosphate 6h
Yield 38%, mp 2538C (with decomposition); IR
(neat, m, cm21) 829, 958, 1068, 1113, 1241, 1320,
1440, 1494, 1623, 2940, 3074; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 7.13 (2H, s, CH), 7.34
(2H, dd, J 5 8.8, Ar-H), 7.70 (2H, s, Ar-H), 7.91 (4H,
d, J 5 8.3, Ar-H), 7.99 (2H, d, J 5 8.3, Ar-H), 8.32
(4H, d, J 5 8.3, Ar-H); 13C NMR spectrum (125
MHz, DMSO, d, ppm) 108.7, 109.3, 119.2, 119.9,
124.2 (q, JF-C 5 272.4), 126.3, 127.6, 131.6 (q, JF-
C 5 32.2), 135.5, 156.8, 158.1 (d, JP-C 5 7.8), 161.2,
176.9; 31P NMR spectrum (202 MHz, DMSO, d,
ppm) 45.23. Anal. Calcd for: C32H17F6O7PS: C,
55.66; H, 2.48; S, 4.64. Found: C, 55.74; H, 2.55; S,
4.67.
General method for the synthesis of biflavone
hydrogenthiophosphates 6f-6i
To a solution of corresponding biflavone chloro-
thiophosphates (6a-6d) (1.0 mmol) in THF (10 mL)
was added a water solution of potassium carbonate
(0.5 mL). The reaction mixture was stirred for 24 h.
After concentration under vacuum, the resulting resi-
due was purified by column chromatography using
AcOEt:MeOH 20:1 as eluent to give the desired
products.
Bis-(4-oxo-2-(p-tolyl)-4H-chromen-7-yl) hydrogen-
thiophosphate 6i
Yield 41%, mp 2038C (with decomposition); IR
(neat, m, cm21) 816, 957, 1091, 1145, 1243, 1370,
1412, 1436, 1617, 2922, 3069; PMR spectrum (200
MHz, DMSO, d, ppm, J/Hz) 2.48 (6H, s, CH3), 6.93
(2H, s, CH), 7.30 (2H, d, J 5 8.8, Ar-H), 7.36 (4H, d,
J 5 7.8, Ar-H), 7.64 (2H, s, Ar-H), 7.93–8.00 (6H, m,
Ar-H); 13C NMR spectrum (125 MHz, DMSO, d,
ppm) 21.5, 106.6, 109.3, 117.0, 119.4 (d, JP-C 5 13.5),
126.2, 126.6, 128.7, 130.1, 142.3, 156.8, 157.8, 163.0,
176.9; 31P NMR spectrum (202 MHz, DMSO, d,
ppm) 45.28. Anal. Calcd for: C32H23O7PS: C, 65.97;
H, 3.98; S, 5.50. Found: C, 66.05; H, 3.96; S, 5.55.
Bis-(4-oxo-2-phenyl-4H-chromen-7-yl) hydrogen-
thiophosphate 6f
Yield 46%, mp 1978C (with decomposition); IR
(neat, m, cm21) 821, 840, 958, 1092, 1145, 1241,
1373, 1438, 1492, 1620, 2952, 3065; PMR spectrum
(200 MHz, DMSO, d, ppm, J/Hz) 6.98 (2H, s, CH),
7.33 (2H, d, J 5 8.3, Ar-H), 7.55-7.58 (6H, m, Ar-H),
7.66 (2H, s, Ar-H), 7.97 (2H, d, J 5 8.3, Ar-H), 8.09
(4H, d, J 5 7.8, Ar-H); 13C NMR spectrum (125
MHz, DMSO, d, ppm) 107.2, 109.2, 119.2, 119.7,
126.2, 126.7, 130.1, 131.5, 132.1, 156.8, 158.0, 161.5,
177.0; 31P NMR spectrum (202 MHz, DMSO, d,
ppm) 45.29. Anal. Calcd for: C30H19O7PS: C, 64.98;
H, 3.45; S, 5.78. Found: C, 65.04; H, 3.49; S, 5.76.
Enzyme Purification
STS was extracted from human placenta and
purified to homogeneity following a multi-step chro-
matography protocol as previously described [Her-
nandez-Guzman et al., 2001].
Bis-(4-oxo-2-(4-fluorophenyl)-4H-chromen-7-yl)
hydrogenthiophosphate 6g
Yield 38%, mp 2388C (with decomposition); IR
(neat, m, cm21) 829, 958, 1092, 1145, 1236, 1374,
1438, 1506, 1621, 2924, 3072; PMR spectrum (200
In Vitro Activity Assay
The reaction mixture, at a final volume of 100
MHz, DMSO, d, ppm, J/Hz) 6.98 (2H, s, CH), 7.33 mL, contained 20 mM Tris-HCl pH 7.4, 1 mM NPS
(2H, d, J 5 8.3, Ar-H), 7.39 (4H, t, J 5 8.8, Ar-H), (p-nitrophenyl sulphate), various concentrations of an
7.66 (2H, s, Ar-H), 7.97 (2H, d, J 5 8.8, Ar-H), 8.14- inhibitor (0.1–500 mM) and 5 U of purified enzyme (1
Drug Dev. Res.

458
KOZAK ET AL.
Fig. 1. Docked binding mode for compounds 6i (yellow), 4j (red) and 2-(4-trifluoromethylphenyl)-chromen-4-one-7-O-sulfamate (green).
U is the amount of enzyme that hydrolyzes 100 mM of 1.1.2 software. For all of the docking studies, a grid
˚
˚
˚
NPS in 1 h at 378C). The reaction was performed at
378C for 15 min and stopped by the addition of 100
mL of 1 M NaOH. Ten samples containing various
concentration of inhibitor (0.1–500 mM) were per-
formed in triplicate. The absorbance of the released
p-nitrophenol was measured at 405 nm using a Micro-
plate Reader Biotek ELx800 (SERVA). IC50 values
were calculated using GraphPad Prism (version 4.0)
software. To calculate IC50 values, 10 data points in
triplicate were used. In the first step the concentration
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 (variable slope).
box size of 27A 3 27A 3 27A, centered on the Cb
atom of amino acid 75, was used. The best poses for
a particular ligand were visually inspected. Illustra-
tions of the 3D models were created using VMD 1.9
(University of Illinois at Urbana – Champaign).
Preliminary docking studies were carried out to
assess of capability of potential new STS inhibitors to
interact with the ligand binding domain (LBD). The
newly designed phosphate and thiophosphate flavone
analogs, theoretically possess STS-binding ability. The
docked free binding energies of these compounds (in
the range 23.2 to 24.6 kcal mol21) were compared
to a reference 2-(4-trifluoromethylphenyl)-chromen-
4-one-7-O-sulfamate (25.2 kcal mol21). As shown in
Figure 1, the phosphate groups were in close proxim-
ity to the proposed catalytic amino acid, FGly75 and
were surrounded by a hydrophobic amino acid resi-
dues in the active site of STS. Encouraged by previ-
ous work [Demkowicz et al., 2015a,b] we evaluated
biflavone thiophosphate derivatives, which led to val-
ues within the lowest energy range (26.2 to 27.0
kcal mol21). In this particular case, one nonpolar
part of the biflavone skeleton was oriented inside the
STS active site and underwent nonpolar interactions
with amino acid side chains of the hydrophobic
pocket formed by Leu74, Arg98, Arg99, Val101,
Leu103, Val177, Phe178, Thr180, Thr484, Val486,
and Phe488. Another part of flavone scaffold was sit-
uated outside the entrance to the STS active site. It
is notworthy that compound (6i) exhibited a short
RESULTS AND DISCUSSION
Molecular Modeling
All ligands were constructed using Portable
HyperChem 8.0.7 Release (Hypercube Incorpora-
tion), and were optimized using a MM1 force field
and the nonlinear Polak–Ribierꢀe conjugate gradient
algorithm using one-dimensional searches terminating
˚
at a gradient of 0.01 kcal mol21 21. The X-ray crys-
A
tal structure of human STS was taken from the Pro-
tein Data Bank (accession code 1P49). Standard
preparation procedures including removing the water
molecules from crystallization and subsequent con-
version of catalytic amino acid FGly75 (formylgly-
cine) to the gem-diol form were performed.
Molecular modeling was performed 10 times for each
investigated ligand structures using AutoDock Vina distance from its OH group to Thr484 residue (3.53
Drug Dev. Res.

STEROID SULFATASE INHIBITION
459
Fig. 2. Synthesis of flavone scaffolds (3a-3d).
˚
A) which could facilitate hydrogen bonding and may iodine in D < SO to yield 7-methoxyflavones (2a-2d)
[Kosmeder et al., 2000]. Finally, a simple method for
deprotecting methoxy groups was performed by heat-
ing with pyridine hydrochloride to give 7-
hydroxyflavones (3a-3d). The detailed synthesis of the
scaffolds is presented in Figure 2.
have significant influence for its activity.
Chemistry
Many convenient synthetic methods have been
described for the synthesis of flavones and their
derivatives e.g., Claisen–Schmidt condensation
[Sashidhara et al., 2012], Baker–Venkataraman rear-
rangement [Mahal and Venkataraman, 1934], Allan–
Robinson reaction [Allan and Robinson, 1924], Vils-
meier–Haack reaction [Su et al., 2009], Ionic Liquid
Promoted synthesis [Sarda et al., 2006], Fries rear-
rangement, Wittig reaction or modified Schotten–
Baumann reaction [Singh et al., 2014]. The Baker–
Venkataraman method is one of the most common
procedures for the preparation of flavones and their
derivatives and involves the conversion of o-hydroxya-
cetophenone into a phenolic ester, which undergoes
an intramolecular Claisen condensation to form a b-
diketone derivative under basic conditions. Finally, b-
diketone derivative is cyclized to flavone’s ring by an
acid-catalyzed cyclodehydration.
Encouraged by our previous research and the
high biological activities of flavones, we decided to
synthesize a series of phosphate and thiophosphate
flavone analogs as compounds with potential STS
inhibitory activity. In the first step of scaffolds syn-
thesis, commercially available 2,4-dihydroxyacetophe-
none was treated with dimethyl sulfate in the
presence of anhydrous potassium carbonate to give 2-
hydroxy-4-methoxyacetophenone in good yields
[Nakazawa, 1962]. Treatment of 2-hydroxy-4-
methoxyacetophenone with aryl aldehydes in methano-
lic KOH solution afforded the corresponding chalcone
derivatives (1a-1d). Next, the cyclization of the chal-
Next, functionalization of the hydroxyl group of the
synthesized flavone scaffolds was performed (see Fig. 3).
Previous studies conducted showed that the highest STS
activities exhibited dimethylphosphate, dimethylthiophos-
phate, dihydrogenphosphate, and methylchlorothiophos-
phate analogs [Kozak et al., 2014, 2015; Demkowicz et al.,
2015c]. Therefore, we decided to apply these functional
groups in the synthesis of flavone based STS inhibitors. In
the first step, stable and readily available compounds (3a-
3d) were reacted with phosphoryl or thiophosphoryl chlo-
ride in the presence of triethylamine at 08C to afford
phosphoryl or thiophosphoryl dichloride derivatives (4a-
4d) and (5a-5d). After removal of precipitated triethyl-
amine hydrochloride, raw phosphoryl or thiophosphoryl
dichlorides (4a-4d) and (5a-5d) were treated at 08C with
MeONa in different stoichiometric ratios to obtain the
corresponding phosphate and thiophosphate flavone
derivatives in good yield. Finally, the reaction of flavone
dimethylphosphates (4e-4h) with bromotrimethylsilane
(TMSBr) gave the corresponding dihydrogenphosphate
analogs (4i-4l).
To further study structure-activity relationships,
we synthesized additional thiophosphate analogs (6a-
6i), which exhibited promising binding modes in
modeling studies (Fig. 4). Thiophosphoryl dichlorides
(5a-5d) were treated with corresponding flavone
scaffold (3a-3d) in the presence of triethylamine,
obtaining the compounds (6a-6d) with good yield. In
the course of the investigation the biflavone chloro-
thiophosphate analogs (6a, 6b, 6d) were found to be
cones was accomplished with a catalytic amount of highly unstable with rapid hydrolysis being observed.
Drug Dev. Res.

460
KOZAK ET AL.
Fig. 3. Synthesis of phosphate (4e-4l) and thiophosphate (5e-5l) flavone derivatives.
Therefore, the synthesis of hydrogenthiophosphate source in the in vitro activity assays. A summary of
the results is shown in Table 1.
analogs (6f-6i) was performed using biflavone chloro-
thiophosphate analogs generated in situ (6a, 6b, 6d)
via nucleophilic substitution reactions using H2O/
K2CO3. The exception was compound (6c), that had
a high resistance to hydrolysis. In this case, biflavone
chlorothiophosphate derivative (6c) could be used to
prepare the methylthiophosphate analog (6e) by
treatment with CH3OH/K2CO3.
In the course of investigation, we found that the
compounds based on 7-hydroxy-2-phenyl-4H-chromen-
4-one (3a), 2-(4-fluorophenyl)-7-hydroxy-4H-chrom-
en-4-one (3b), 7-hydroxy-2-(4-trifluoromethylphenyl)-4
H-chromen-4-one (3c), 7-hydroxy-2-(p-tolyl)-4H-chro-
men-4-one (3d) modified by different phosphate or thio-
phosphate moieties in the 7-position, exhibited poor STS
inhibitory potency. Only the dihydrogenphosphate (4i-
4j) and the methylchlorothiophosphate (5e-5h) analogs
demonstrated good activities in the STS enzyme assays
with the best IC50 value of 29.0 mM being for 2-(4-fluoro-
phenyl)-4-oxo-4H-chromen-7-yl dihydrogen phosphate
(4j) and 38.4 mM for 4-oxo-2-(p-tolyl)-4H-chromen-7-yl
methyl chlorothiophosphate (5h). The highest STS inhi-
STS Enzyme Assays
The ability of synthesized compounds to inhibit
STS was determined using an in vitro assaymusing
STS enzyme extracted from human placenta tissue
and purified by 3-step chromatography. After purifi-
cation, the fractions were used directly as an enzyme bition (in the low micromolar range) occurred in a series
Fig. 4. Synthesis of biflavone derivatives (6a-6i).
Drug Dev. Res.

STEROID SULFATASE INHIBITION
461
ACKNOWLEDGMENT
TABLE 1. Activities of the Synthesized Compounds and a Reference
Inhibitor (2-(4-Trifluoromethylphenyl)-Chromen-4-one-7-O-Sulfa-
mate) in STS Enzyme Assays
We gratefully acknowledge the National Science
Centre (Poland) for financial support (grant no. 2011/
03/D/NZ7/03985).
No.
IC50 [lM]
Lp.
IC50 [lM]
4e
4f
>500
>500
5i
5j
>500
>500
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4g
4h
4i
4j
4k
4l
5e
5f
5g
5h
>500
>500
50.2 6 4.8
29.0 6 4.4
>500
5k
5l
6c
6e
6f
6g
6g
6i
>500
>500
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