1. No category

Supplementary Data
Identification of 4-(4-Nitro-2phenethoxyphenyl)pyridine as a promising new
Lead for Discovering Inhibitors of both Human
and Rat 11β-Hydroxylase
Qingzhong Hu, a,b Jessica Kunde,a Nina Hanke,c and Rolf W. Hartmanna,b,*
a
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123
Saarbrücken, Germany
b
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-
66123 Saarbrücken, Germany
c
Elexopharm GmbH, Campus A1, 66123 Saarbrücken, Germany
* Author to whom correspondence should be addressed: Rolf W. Hartmann,
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2 3, D-66123
Saarbrücken, Germany, Phone: +49 681 302 70300. Fax: +49 681 302 70308. E-mail:
[email protected].
S1
Contents
1. Figure S1. Alignment of the sequences of hCYP11B1, hCYP11B2 and the crystal
of hCYP11B2 in complex with deoxycorticosterone (PDB ID: 4DVQ)
2. Figure S2. Ramachandran scatter plot of the hCYP11B1 homology model.
3. Experimental details and spectroscopic characterization of all intermediates.
4. HPLC Purity Control of Final Compounds.
5. 1H- and 13C-NMR spectra of the most potent and selective compounds 4–9, 18, 19
and 25.
S2
ure S1. Aliignment of the sequennces of hCY
YP11B1, hC
CYP11B2 aand the crysstal of
Figu
hCY
YP11B2 in complex
c
witth deoxycorrticosterone (PDB ID: 4DVQ)
4
S3
Figure S2. Ramachandran scatter plot of the hCYP11B1 homology model.
S4
3. Experimental details and spectroscopic characterization of all intermediates.
2-Bromo-5-fluorophenol (12b). The title compound was synthesized from 1-bromo-4fluoro-2-methoxybenzene (200 µl, 320 mg, 1.56 mmol) according to method A. The
aqueous layer was extracted three times with DCM. After drying over Na2SO4, the
solvent was removed at 700 mbar and the liquid residue was used without further
purification. 1H-NMR (CDCl3, 500 MHz): δH = 7.41 (dd, JHF = 9.0, J = 5.8 Hz, 1H), 6.78
(dd, J = 3.0, 9.6 Hz, 1H), 6.59 (ddd, J = 2.8, 7.9, 8.8 Hz, 1H), 5.63 (s, 1H); 13C-NMR
(CDCl3, 125 MHz): δC = 103.9 (d, JCF = 26.6 Hz), 104.7 (d, JCF = 3.7 Hz), 109.1 (d, JCF =
22.9 Hz), 132.4 (d, JCF = 9.2 Hz), 153.3 (d, JCF = 11.9 Hz), 163.0 (d, JCF = 245.6 Hz).
4-Bromo-3-hydroxybenzonitrile (18b). The title compound was synthesized from 4bromo-3-methoxybenzonitrile (330 mg, 1.56 mmol) according to method A. The crude
product was purified by flash chromatography on silica gel using a mixture of petroleum
ether / ethyl acetate (8:1) as eluent. Yield: 131 mg (42 %), white crystals. 1H-NMR
((CD3)2CO, 500 MHz): δH = 9.76 (br, s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 1.9 Hz,
1H), 7.19 (dd, J = 1.9, 8.2 Hz, 1H); 13C-NMR ((CD3)2CO, 125 MHz) δC = 155.7, 135.4,
125.3, 120.0, 118.7, 116.7, 113.2.
4-Bromo-3-hydroxy-N,N-dimethylbenzamide (20b). The title compound was
synthesized from 4-bromo-3-methoxy-N,N-dimethylbenzamide (300 mg, 1.16 mmol)
according to method A. Purification of the crude product was not necessary. Yield: 278
mg (98 %). 1H-NMR ((CD3)2CO, 500 MHz): δH = 9.13 (br, s, 1H), 7.54 (d, J = 8.2 Hz,
1H), 7.04 (d, J = 1.9 Hz, 1H), 6.83 (dd, J = 1.9, 8.2 Hz, 1H), 2.99 (s, 6H).
2-Bromo-3-hydroxypyridine (22b). 2-bromo-3-methoxypyridine (295 mg, 1.57 mmol)
was heated in hydrobromide acid (5 ml) to 140°C and stirred overnight. The solution was
neutralized with a saturated sodium bicarbonate solution and extracted three times with
ethyl acetate. The combined organic layers were dried over Na2SO4 and the solvent was
removed under reduced pressure. Yield: 258 mg (94 %), white crystals. 1H-NMR
((CD3)2CO, 500 MHz): δH = 9.26 (s, 1H), 7.89 (dd, J = 1.6, 4.4 Hz, 1H), 7.32–7.36 (m,
1H), 7.23–7.27 (m, 1H); 13C-NMR ((CD3)2CO, 125 MHz): δC = 152.4, 142.1, 131.9,
125.3, 124.5.
2-(Benzyloxy)-1-bromo-4-nitrobenzene (4a). The title compound was synthesized from
2-bromo-5-nitrophenol (545 mg, 2.50 mmol) according to method B. The crude product
was used directly in the next step without further purification.
1-Bromo-4-nitro-2-phenethoxybenzene (5a). The title compound was synthesized from
2-bromo-5-nitrophenol (545 mg, 2.50 mmol) according to method B. The crude product
was used directly in the next step without further purification.
1-Bromo-4-nitro-2-(3-phenylpropoxy)benzene (6a). The title compound was
synthesized from 2-bromo-5-nitrophenol (545 mg, 2.50 mmol) according to method B.
The crude product was used directly in the next step without further purification.
S5
4-Bromo-1-nitro-2-phenethoxybenzene (10a). To a solution of 5-bromo-2-nitrophenol
(120 mg, 0.55 mmol) in DMF (3 ml) Cs2CO3 (197 mg, 0.60 mmol), (2bromoethyl)benzene (83 µl, 0.60 mmol) and tetrabutylammonium iodide (5 mol%) were
added. The stirred mixture was heated to 140°C under a nitrogen atmosphere for 3 h.
After cooling, the mixture was diluted with water and extracted three times with ethyl
acetate. The combined organic layers were dried over Na2SO4 and the solvent was
removed in vacuum. The crude product was purified by flash chromatography using
petroleum ether / ethyl acetate (50:1) as eluent. Yield: 161 mg (91 %), yellow oil. 1HNMR (CDCl3, 500 MHz): δH = 7.67 (d, J = 8.5 Hz, 1H), 7.24–7.30 (m, 4H), 7.19–7.23
(m, 1H), 7.12 (d, J = 1.9 Hz, 1H), 7.09 (dd, J = 8.5, 1.9 Hz, 1H), 4.22 (t, J = 6.8 Hz, 2H),
3.11 (t, J = 6.8 Hz, 2H); 13C-NMR (CDCl3, 125 MHz): δC = 152.8, 138.6, 137.2, 129.2,
128.6, 128.3, 126.8, 126.8, 123.5, 117.9, 70.9, 35.5.
1-Bromo-4-fluoro-2-phenethoxybenzene (12a). The title compound was synthesised
from 2-bromo-5-fluorophenol 12b (3.90 mmol) according to method B. The crude
product was purified by flash chromatography on silica gel using a mixture of n-hexane /
ethyl acetate (40:1) as eluent. Yield: 836 mg (73 %), white solid. 1H-NMR (CDCl3, 500
MHz): δH = 7.39 (dd, J = 6.1, 8.7 Hz, 1H), 7.23–7.29 (m, 4H), 7.16–7.21 (m, 1H), 6.46–
6.55 (m, 2H), 4.11 (t, J = 6.9 Hz, 2H), 3.09 (t, J = 6.9 Hz, 2H); 13C-NMR (CDCl3, 125
MHz): δC = 162.7 (d, JCF = 246.5 Hz), 156.1 (d, JCF = 10.1 Hz), 137.7, 133.5 (d, JCF = 9.2
Hz) 129.2, 128.5, 126.7, 108.4 (d, JCF = 22.0 Hz), 106.4 (d, JCF = 3.7 Hz), 101.3 (d, JCF =
26.6 Hz), 70.1, 35.6.
1-Bromo-4-methoxy-2-phenethoxybenzene (14a). The title compound was synthesized
from 2-bromo-5-methoxyphenol (330 mg, 1.63 mmol) according to method B. The crude
product was purified by flash chromatography on silica gel using a mixture of petroleum
ether / ethyl acetate (20:1) as eluent. Yield: 421 mg (84 %), white solid. 1H-NMR
(CDCl3, 500 MHz): δH = 7.33 (d, J = 8.7 Hz, 1H), 7.23–7.30 (m, 4H), 7.16–7.20 (m, 1H),
6.37 (d, J = 2.7 Hz, 1H), 6.32 (dd, J = 2.7, 8.7 Hz, 1H), 4.11 (t, J = 7.0 Hz, 2H), 3.69 (s,
2H), 3.09 (t, J = 7.0 Hz, 2H); 13C-NMR (CDCl3, 125 MHz): δC = 160.3, 156.1, 138.3,
133.4, 129.5, 128.7, 126.8, 106.5, 103.2, 101.1, 70.1, 55.8, 35.9.
1-Bromo-2-phenethoxy-4-(trifluoromethyl)benzene (16a). The title compound was
synthesized from 2-bromo-5-(trifluoromethyl)phenol (380 mg, 1.58 mmol) according to
method B. The crude product was purified by flash chromatography on silica gel using a
mixture of petroleum ether as eluent. Yield: 382 mg (70 %), white solid. 1H-NMR
(CDCl3, 500 MHz): δH = 7.56 (dd, J = 0.9, 8.2 Hz) 7.23–7.29 (m) 7.16–7.20 (m) 7.00
(ddd, J = 0.6, 1.9, 8.2 Hz) 6.96 (d, J = 1.9 Hz) 4.18 (t, J = 6.8 Hz) 3.11 (t, J = 6.8 Hz);
13
C-NMR (CDCl3, 125 MHz): δC = 155.6, 137.7, 133.8, 130.9 (q, JCF = 33.0 Hz), 129.2,
128.5, 126.8, 123.69 (q, JCF = 272.2 Hz), 118.4 (q, JCF = 3.7 Hz), 116.3, 109.5 (q, JCF =
3.7 Hz), 70.2, 35.6.
4-Bromo-3-phenethoxybenzonitrile (18a). The title compound was synthesized from 4bromo-3-hydroxybenzonitrile 18b (130 mg, 0.66 mmol) according to method B. The
crude product was purified by flash chromatography on silica gel using a mixture of
petroleum ether / ethyl acetate (20:1) as eluent. Yield: 140 mg (70 %), yellowish solid.
S6
1
H-NMR (CDCl3, 500 MHz): δH = 7.55 (d, J = 8.2 Hz, 1H), 7.24–7.29 (m, 4H), 7.17–
7.21 (m, 1H), 7.03 (dd, J = 1.9, 8.2 Hz, 1H), 6.97 (d, J = 1.9 Hz, 1H), 6.96–6.98 (m, 1H),
4.16 (t, J = 6.8 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H); 13C-NMR (CDCl3, 125 MHz): δC =
155.6, 137.5, 134.2, 129.2, 128.5, 126.8, 125.2, 118.3, 118.1, 115.4, 112.0, 70.3, 35.5.
4-Bromo-N,N-dimethyl-3-phenethoxybenzamide (20a). The title compound was
synthesized from 4-bromo-3-hydroxy-N,N-dimethylbenzamide 20b (290 mg, 1.19 mmol)
according to method B. The crude product was purified by flash chromatography on
silica gel using a mixture of petroleum ether / ethyl acetate (1:1) as eluent. Yield: 339 mg
(82 %). 1H-NMR (CDCl3, 500 MHz): δH = 7.48 (d, J = 8.2 Hz, 1H), 7.23–7.30 (m, 4H),
7.16–7.21 (m, 1H), 6.87 (d, J = 1.9 Hz, 1H), 6.77 (dd, J = 1.9, 8.2 Hz, 1H), 4.17 (t, J =
6.8 Hz, 2H), 3.10 (t, J = 6.8 Hz, 2H), 2.99 (br, s, 3H), 2.88 (br, s., 3H); 13C-NMR
(CDCl3, 125 MHz): δC = 170.7, 155.5, 138.1, 136.8, 133.3, 129.4, 128.7, 126.8, 120.3,
113.8, 112.1, 70.2, 35.9.
2-Bromo-3-phenethoxypyridine (22a). The title compound was synthesised from 2bromo-3-hydroxypyridine 22b (228 mg, 1.31 mmol) according to method B. The crude
product was purified by flash chromatography on silica gel using a mixture of petroleum
ether / ethyl acetate (7:1) as eluent. Yield: 232 mg (64 %). 1H-NMR (CDCl3, 500 MHz)
δH = 7.89 (dd, J = 1.6, 4.7 Hz, 1H), 7.24–7.29 (m, 4H), 7.16–7.21 (m, 1H), 7.09 (dd, J =
4.7, 8.2 Hz, 1H), 7.00 (dd, J = 1.6, 8.2Hz, 1H), 4.15 (t, J = 6.8 Hz, 2H), 3.10 (t, J = 6.8
Hz, 2H); 13C-NMR (CDCl3, 125 MHz): δC = 152.3, 141.2, 137.6, 133.1, 129.2, 128.5,
126.7, 123.3, 119.5, 70.1, 35.6.
4,4,5,5-Tetramethyl-2-(4-nitro-2-phenethoxyphenyl)-1,3,2-dioxaborolane (24a). 1bromo-4-nitro-2-phenethoxybenzene (400 mg, 1.24 mmol) was dissolved in dioxane (20
ml) and bis(pinacolato)diboron (472 mg, 1.86 mmol) and KOAc (365 mg, 3.72 mmol)
were added. The mixture was deoxygenated and flushed with nitrogen three times before
PdCl2(PPh3)2 (5 mol%) was added. The resulting suspension was heated under reflux for
4 h. After cooling, water and ethyl acetate were added and the phases were separated. The
water phase was extracted three times with ethyl acetate and the combined organic layers
were dried over Na2SO4. The solvent was removed under reduced pressure and the
residue was dissolved in ethanol and stored overnight at 7 °C. The precipitated crystals
were separated from the solution and washed with little amounts of cooled ethanol. Yield:
319 mg (70 %). 1H-NMR (CDCl3, 500 MHz): δH = 7.78 (d, J = 1 Hz, 2H) 7.63 (t, J = 1.0
Hz, 1H) 7.38–7.42 (m, 2H) 7.32 (m, 2H) 7.23–7.28 (m, 1 H) 4.27 (t, J = 6.6 Hz, 2H) 3.18
(t, J = 6.6 Hz, 2H) 1.39 (s, 12H).
S7
4. HPLC Purity Control of Final Compounds
The Surveyor®-LC-system consisted of a pump, an autosampler, and a PDA detector. Mass
spectrometry was performed on a TSQ® Quantum (Thermo Electron Corporation, Dreieich,
Germany). The triple quadrupole mass spectrometer was equipped with an electrospray interface
(ESI). The system was operated by the standard software Xcalibur®. A RP C18 NUCLEODUR®
100-5 (125  3 mm) column (Macherey-Nagel GmbH, Duehren, Germany) was used as
stationary phase. All solvents were HPLC grade. In a gradient run the percentage of acetonitrile
(containing 0.1% triflouro-acetic acid) in water was increased from an initial concentration of 3%
at 0 min to 100% at 15 min and kept at 100% for 3 min. The injection volume was 10 µl and flow
rate was set to 350 µl/min. MS analysis was carried out at a spray voltage of 3800 V, a capillary
temperature of 350 °C and a source CID of 10 V. Spectra were acquired in positive mode from
100 to 1000 m/z and full scan UV-mode. In some cases APC ionization had to be applied.
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Rt [min]
7.25
8.67
5.63
9.07
9.13
9.28
9.02
9.21
9.33
9.80
9.57
9.57
9.32
9.07
9.20
10.30
10.13
8.85
8.71
7.56
7.51
7.73
7.91
9.70
9.55
Purity [%]
96.5
98.5
97.7
97.5
96.7
98.3
96.5
98.7
97.6
98.6
98.1
96.9
97.5
95.6
98.4
98.7
98.3
99.5
98.5
97.7
97.4
98.6
95.6
99.1
98.8
S8
200
ppm (t1)
150
100
71.073
132.226
131.048
128.758
128.350
127.148
122.989
116.488
107.870
135.307
134.194
136.758
156.003
149.963
149.310
148.558
35000
30000
N
O2 N
50
25000
O
20000
15000
10000
50000
0
0
50000
N
O2 N
2.05
8.0
6.17
1.03
ppm (t1)
1.02
2.04
1.01
1.00
9.0
7.0
6.0
5.0
4.0
3.0
40000
O
30000
20000
10000
0
2.0
1.0
0.0
5.117
7.877
7.873
7.857
7.847
7.810
7.794
7.412
7.395
7.283
7.268
7.255
7.240
7.225
8.544
8.534
8.714
8.710
200
ppm (t1)
150
100
35.408
69.887
122.889
116.144
106.969
126.738
128.995
128.535
130.854
132.185
156.248
149.931
149.179
148.549
137.559
136.919
133.810
15000
N
O2N
50
10000
O
50000
0
0
N
O2N
7.0
6.0
5.0
2.06
2.07
8.0
2.06
4.49
1.05
1.02
1.03
1.03
1.01
1.00
9.0
ppm (t1)
4.0
3.0
2.0
O
50000
0
1.0
0.0
3.149
4.402
7.240
7.225
7.529
7.512
7.409
7.393
7.382
7.366
7.352
7.340
7.331
7.317
7.760
7.744
8.007
7.990
7.898
8.773
8.724
8.714
O2 N
200
ppm (t1)
150
100
50
30.704
32.267
68.386
131.103
128.779
128.619
126.411
123.213
116.372
107.374
132.576
141.014
136.960
134.054
156.616
150.270
149.511
148.869
35000
30000
25000
N
O
20000
15000
10000
50000
0
0
O2 N
2.06
2.10
2.00
2.01
1.12
1.97
1.01
1.02
ppm (t1)
0.97
0.99
0.98
1.02
1.00
5.0
2.091
2.076
2.061
2.679
4.067
7.385
7.375
7.369
7.360
7.246
7.232
7.179
7.164
7.150
7.096
7.080
8.627
8.621
7.921
7.917
7.905
7.900
7.880
7.864
7.774
7.769
7.465
7.448
8.788
8.785
80000
70000
60000
N
50000
O
40000
30000
20000
10000
0
-10000
0.0
20000
15000
N
O2 N
200
ppm (t1)
150
100
50
O
10000
50000
0
0
71.350
116.670
108.219
127.355
124.261
131.241
129.024
128.652
156.222
150.087
149.140
144.347
135.478
134.923
5.215
7.963
7.943
7.517
7.501
7.483
7.474
7.369
7.356
7.334
8.685
60000
N
O2 N
50000
O
40000
30000
20000
10000
0
2.01
8.0
5.00
3.06
2.00
1.99
9.0
ppm (t1)
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
O2N
200
ppm (t1)
150
100
50
35.620
70.104
116.312
107.316
127.043
124.297
129.241
128.822
131.066
134.533
156.456
149.978
149.114
144.231
137.814
30000
25000
N
O
20000
15000
10000
50000
0
0
N
O2N
6.0
5.0
2.04
7.0
2.03
8.0
2.02
3.16
2.09
1.03
1.00
1.00
2.01
9.0
ppm (t1)
4.0
3.0
2.0
O
50000
0
1.0
0.0
3.042
4.308
7.792
7.788
7.417
7.400
7.285
7.271
7.257
7.240
7.228
7.142
7.129
7.886
7.882
7.869
7.865
8.605
8.592
O2 N
200
ppm (t1)
150
100
50
30.471
32.073
68.210
116.023
107.210
130.816
128.558
128.346
126.203
123.997
140.723
134.255
149.800
148.936
144.221
156.331
25000
20000
N
O
15000
10000
50000
0
0
2.100
2.709
4.092
7.116
7.101
7.188
7.473
7.269
7.255
7.931
7.910
7.788
8.705
40000
N
30000
O2 N
O
20000
10000
0
5.0
4.0
3.0
2.00
6.0
2.01
7.0
1.99
8.0
1.97
1.04
2.31
2.97
0.98
0.98
1.96
9.0
ppm (t1)
2.0
1.0
0.0
NC
200
ppm (f1)
150
100
50
35.429
69.666
115.102
113.030
123.142
118.452
125.035
126.734
155.974
149.166
148.187
137.628
137.539
132.761
131.806
131.218
128.956
128.534
35000
30000
25000
N
O
20000
15000
10000
5000
0
0
1500
N
NC
6.0
5.0
2.15
7.0
2.00
8.0
2.03
1.01
3.46
3.00
1.04
2.00
9.0
ppm (f1)
4.0
3.0
2.0
1000
O
500
0
1.0
0.0
3.022
4.223
7.124
7.108
7.276
7.262
7.247
7.227
7.213
7.198
7.179
7.368
7.352
7.333
7.680
8.606
8.659
70000
60000
N
NC
200
ppm (f1)
150
100
50
50000
O
40000
30000
20000
10000
0
0
35.384
69.596
113.410
115.163
118.361
124.914
124.124
126.757
131.089
128.947
128.539
155.895
149.368
144.501
137.565
132.620
7.0
6.0
5.0
2.02
8.0
2.01
1.99
1.00
5.22
1.02
1.01
2.00
9.0
ppm (f1)
4.0
3.0
3.047
4.256
8.616
8.604
7.386
7.370
7.344
7.341
7.328
7.311
7.297
7.282
7.263
7.251
7.204
7.159
7.146
3000
2500
N
2000
NC
O
1500
1000
500
0
2.0
1.0
0.0
15000
N
10000
O2 N
200
ppm (f1)
150
100
O
5000
0
50
0
36.042
35.980
70.305
107.259
136.404
131.943
129.872
129.304
127.338
125.179
116.515
139.335
150.601
149.623
157.907
6.0
5.0
4.0
2.76
7.0
2.15
2.19
8.0
2.00
1.04
4.14
1.04
2.04
2.00
9.0
ppm (f1)
3.148
4.066
4.445
7.512
7.495
7.341
7.325
7.311
7.297
7.281
7.247
7.233
7.219
7.186
7.175
7.812
7.802
7.797
8.429
8.417
1500
N
1000
O2 N
3.0
2.0
O
500
0
1.0
0.0