Supporting Information Towards Functional Selectivity for GABAA Receptors: A Series of Novel Pyrazoloquinolinones
Marco Treven# (1), David C. B. Siebert# (2), Raphael Holzinger (1), Konstantina Bampali (1), Jure Fabjan (1), Zdravko Varagic (1), Laurin Wimmer (2), Friederike Steudle (3), Petra Scholze (3), Michael Schnürch (2), Marko D. Mihovilovic (2) and Margot Ernst* (1)
(1) Department of Molecular Neurosciences, Center for Brain Research, Medical University Vienna, Spitalgasse 4 – 1090 Vienna, Austria. (2) Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/163 – 1060 Vienna, Austria.
(3) Department of Pathobiology of the Nervous System, Center for Brain Research, Medical University Vienna, Spitalgasse 4 – 1090 Vienna, Austria.
*Contact:
[email protected] #
These authors contributed equally to this study
Supplementary Table S1: Calculated number of conformations and their corresponding dihedral angles (φ) of p-, m- or o-methoxy substituted pyrazoloquinolinones.
Conformation
φ of p-methoxy
φ of m-methoxy
φ of o-methoxy
1
0.0045°
0.0338°
0.8610°
2
0.0027°
0.0318°
0.8239°
3
-
0.0257°
20.4538°
4
-
-
20.3784°
The Conformation Search application in MOE generates various rotatable bond dihedral angle combinations by rotation of non-ring bonds in discrete increments followed by energy minimization which results in several reasonable conformations. The dihedral angle rules are shown in Supplementary Table S2. Conformations with dE < 7 compared to the energetic most favourable pose are shown.
Supplementary Table S2: Increments of Conformation Search application.
Bond
Description
Angles
–X
Terminal atom
{0}
X=X
Double bond
{0,180}
Triple bond / Allene
{0}
Amide / CNN resonance
{0,180}
Peroxide / Disulfide
{90,-90}
sp3-sp3
E.g. –CH2–CH2–
{180,60,-60}
aro*–X
Ortho substituted
{45,-45,135,-135}
No ortho substituents
{0,180}
aro–X
No ortho substituents
{45,-45,135,-135}
X–X
E.g. sp2–sp3
{0,60,120,180,-60,-120}
X#X
X=X=X
N–C=O
N–C=S
–O–O–
–S–S–
aro–CO2
N–C=N+
aro–OMe aro–NH2
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Supplementary Figure S3: “Syn” und “Anti” conformation of compound 4.
a
b
anti syn
Anti (panel a) and syn (panel b) conformation of compound 4. The steric hindrance in the syn conformation leads to a strong rotation of ring D up to φ=~20°. Furthermore, the anti conformation shows a 20 to 200 times stronger tilt (φ=~0.85°) compared to the m- and psubstituted compounds (φ=~0.003° and φ=~0.03°), namely compounds 3 and 2.
Supplementary Table S4: Radio displacement assay data
Compound
Ki (nM)
(n)
Ki (nM)
(n)
2
0.018 ± 0.004
3
0.55 ± 0.04
3
3
0.17 ± 0.007
4
15.4 ± 0.2
4
4
7.8 ± 0.6
4
329 ± 76
3
Ki values of compounds 2, 3 and 4 determined by displacement of [3H]flunitrazepam (α1+/γ2; KD = 4.8 ± 0.3 nM (n=3)) and [3H]Ro15-4513 in the presence of 50 mM diazepam (α6+/γ2-; KD = 1.4 ± 0.1 nM (n=3)) binding to rat cerebellar membranes (mean ± SEM).
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Supplementary Table S5: Functional data in containing receptor subtypes α1β3δ
α4β3δ
α6β3δ
n.d.
15.7
7.7
1 nM
99.5±0.4
100.0±0
102.8±4.7
10 nM
100.0±0
99.0±1.4
101.1±1.4
100 nM
100.0±0
102.1±1.7
102.1±3.8
300 nM
-
-
98.1±3.2
1 µM
100.0±0
109.0±3.3
118.4±14.3
3 µM
-
-
118.3±0.2
10 µM
108.9±0.2 106.5±0.5 2
121.4±4.5 129.8±4.0 3-4
158.9±13.4 179.5±8.7 4
Compound 3 EC50 [µM]
30 µM n
EC50 values and efficacy of compound 3 at increasing concentrations in α1β3δ, α4β3δ and α6β3δ receptors, given in % of control current (mean ± SEM). Due to low efficacy, the EC 50 value in α1β3δ could not be obtained (n.d.=not determined). Control current = 100% (GABA EC3-10).
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Synthesis Commercially available reagents were used without further purification. Reactions were monitored by thin layer chromatography with silica gel 60 F254 plates (E. Merck, Darmstadt, Germany). HPLC chromatography was carried out with the Autopurification system by Waters using fluoro-phenyl columns. 1H and 13C NMR spectra were recorded on Bruker AC 200 (1H: 200MHz,
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C: 50 MHz), Bruker Avance Ultrashield 400 (1H: 400 MHz,
13
C: 101
MHz) or Bruker Avance IIIHD 600 spectrometer equipped with a Prodigy BBO cryo probe (1H: 600 MHz,
13
C: 151MHz). Chemical shifts are reported in parts per million (ppm) and
were calibrated using DMSO-d6 as internal standard. Multiplicities are denoted by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), ddd (doublet of doublet of doublet), t (triplet), dt (doublet of triplet) and m (multiplet). Melting points were determined with a Büchi Melting Point B-545 apparatus. HR-MS was measured on an Aglient 6230 LC TOFMS mass spectrometer equipped with an Aglient Dual AJS ESI-Source.
The Synthesis of compounds 3 (LAU159), 4 (LAU165), 5 (DCBS142), 6 (DCBS32), 7 (DCBS146), 8 (DCBS120) and 9 (DCBS152A) was conducted in analogy to previously outlined synthetic routes (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013).
Compound
3:
8‐Chloro‐2‐(3‐methoxyphenyl)‐2H,3H,5H‐pyrazolo[4,3‐c]quinolin‐3‐one
(LAU159)
LAU159 was synthesized according to the literature (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013) in 66% yield (yellow solid, 108 mg, 0.33 mmol). 1H NMR (200 MHz, DMSO-d6) δ = 3.80 (s, 3H), 6.76 (d, J = 8.3 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H), 7.51 - 7.90 (m, 4H), 8.16 (s, 1H), 8.72 (d, J = 5.5 Hz, 1H), 12.96 (br s, 1H). 13C NMR (50 MHz, DMSO-d6) δ = 55.1, 104.4, 106.3, 109.5, 110.9, 119.9, 121.1, 121.6, 129.5, 130.2, 130.6, 134.2, 139.5, 5
141.0, 141.9, 159.5, 161.5. HR-MS: calculated [C17H13ClN3O2+]: 326.0691; found [C17H13ClN3O2+]: 326.0688 (diff.: 0.92 ppm). M.p.: decomposes > 300 °C.
Compound
4:
8‐Chloro‐2‐(2‐methoxyphenyl)‐2H,3H,5H‐pyrazolo[4,3‐c]quinolin‐3‐one
(LAU165)
LAU165 was synthesized according to the literature (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013) in 28% yield (yellow solid, 46 mg, 0.14 mmol). 1H NMR (200 MHz, DMSO-d6) δ = 3.74 (s, 3H), 7.04 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H) 7.29-7.45 (m, 2H), 7.62-7.73 (m, 2H), 8.02 (d, J = 2.0 Hz, 1H) 8.66 (s, 1H), 12.78 (br s, 1H,). 13C NMR (50 MHz, DMSO-d6) δ = 55.6, 105.3, 112.5, 120.2, 120.3,. 120.9, 121.4, 127.7, 129.3, 129.4, 129.7, 130.3, 134.0, 139.1, 141.4, 155.1, 161.6. HR-MS: calculated [C17H13ClN3O2+]: 326.0691; found [C17H13ClN3O2+]: 326.0678 (diff.: 3.99 ppm). TLC (10% EtOAc in MeOH). M.p.: decomposes > 300 °C.
Compound
5:
8-Chloro-2-(m-tolyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one
(DCBS142)
DCBS142 was synthesized according to the literature (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013) in 63% yield (yellow solid, 36 mg, 0.12 mmol). 1H NMR (400 MHz, DMSO-d6) δ 2.37 (s, 3H), 7.00 (d, J = 7.5 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.68 – 7.77 (m, 2H), 7.99 – 8.07 (m, 2H), 8.14 – 8.22 (m, 1H), 8.76 (s, 1H), 12.91 (br s, 1H).
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C NMR
(101 MHz, DMSO-d6) δ 21.4, 106.4, 115.9, 119.2, 120.0, 121.2, 121.7, 124.9, 128.5, 130.2, 130.7, 134.3, 138.0, 139.7, 139.9, 141.9, 161.4. HR-MS: calculated [C17H13ClN3O+]:
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310.0742; found [C17H13ClN3O+]: 310.1249 (diff.: 4.77 ppm). TLC (5% MeOH in CH2Cl2): Rf=0.24. M.p.: decomposes > 300 °C.
Compound 6: 2-(3-Bromophenyl)-8-chloro-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (DCBS32)
DCBS32 was synthesized according to the literature (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013) and purified by HPLC to give a yellow solid (104 mg, 0.28 mmol, 75%). 1
H NMR (600 MHz, DMSO-d6) δ 7.37 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H), 7.43 (t, J = 8.0 Hz,
1H), 7.69 – 7.80 (m, 2H), 8.21 (t, J = 1.4 Hz, 1H), 8.27 (ddd, J = 8.2, 2.1, 1.1 Hz, 1H), 8.47 (t, J = 2.0 Hz, 1H), 8.82 (s, 1H), 13.03 (br s, 1H).
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C NMR (151 MHz, DMSO-d6) δ 106.0,
117.1, 119.9, 120.5, 121.3, 121.7, 121.8, 126.6, 130.5, 130.88, 130.92, 134.4, 140.1, 141.2, 142.6, 161.7. TLC (5% MeOH in CH2Cl2): Rf=0.43. M.p.: decomposes > 300 °C.
Compound 7: 3-(8-Chloro-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (DCBS146)
DCBS146 was synthesized according to the literature (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013) in 71% yield (yellow solid, 67 mg, 0.21 mmol). 1H NMR (600 MHz, DMSO-d6) δ 7.62 (dt, J = 7.6, 1.4 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.71 (dd, J = 8.8, 2.3 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 2.3 Hz, 1H), 8.57 – 8.61 (m, 1H), 8.66 (t, J = 1.9 Hz, 1H), 8.80 (s, 1H), 13.07 (br s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 105.6, 111.7, 118.8, 120.2, 120.9, 121.3, 122.6, 123.0, 127.2, 130.2, 130.3, 130.5, 135.9, 140.7, 141.4, 143.3, 161.9. HR-MS: calculated [C17H10ClN4O+]: 321.0538; found [C17H10ClN4O+]:
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321.0546 (diff.: 2.54 ppm). TLC (10% MeOH in CH2Cl2): Rf=0.62. M.p.: decomposes > 300 °C.
Precursor of Compound 8: 8-Chloro-2-(3-nitrophenyl)-2,5-dihydro-3H-pyrazolo[4,3c]quinolin-3-one (DCBS119)
DCBS119 was synthesized according to the literature (Fryer et al., 1993; Savini et al., 2001; Varagic et al., 2013) in 92% yield (yellow solid, 112 mg, 0.33 mmol). 1H NMR (600 MHz, DMSO-d6) δ 7.72 – 7.78 (m, 3H), 8.02 (ddd, J = 8.1, 2.3, 1.0 Hz, 1H), 8.20 (t, J = 1.4 Hz, 1H), 8.65 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 8.85 (s, 1H), 9.11 (t, J = 2.2 Hz, 1H), 13.12 (s, 1H).
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C NMR (151 MHz, DMSO-d6) δ 105.8, 112.2, 118.4, 119.8, 121.4, 121.8, 124.0,
130.4, 130.7, 131.0, 134.4, 140.4, 140.6, 143.0, 148.1, 162.0. HR-MS: calculated [C16H10ClN4O3+]: 341.0436; found [C16H10ClN4O3+]: 341.0433 (diff.: 0.84 ppm). TLC (2% MeOH in CH2Cl2): Rf=0.25. M.p.: decomposes > 300 °C.
Compound 8: 2-(3-Aminophenyl)-8-chloro-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (DCBS120)
8-Chloro-2-(3-nitrophenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (100 mg, 0.293 mmol) was dissolved in 6 mL EtOH, Na2S·9H2O (423 mg, 1.76 mmol) was added and the reaction mixture was heated to reflux. After 2 h water (10 mL) and 2 N HCl were added to adjust pH 5-6. The precipitate was collected by filtration, washed with satd. NaHCO3, water (3 x 20 mL) and was dried under reduced pressure to give 2-(3-aminophenyl)-8-chloro-2,5dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (yellow solid, 75 mg, 0.24 mmol, 82%). 1H NMR (600 MHz, DMSO-d6) δ 5.22 (s, 2H), 6.39 (dd, J = 7.9, 2.1 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 8.0, 1.9 Hz, 1H), 7.44 – 7.47 (m, 1H), 7.68 – 7.74 (m, 2H), 8.11 (d, J = 2.0 Hz, 8
1H), 8.73 (s, 1H), 12.86 (s, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 104.6, 106.6, 106.9,
110.3, 120.1, 121.0, 121.7, 128.9, 130.1, 130.5, 134.2, 139.4, 140.7, 141.5, 149.0, 161.3. HRMS: calculated [C16H12ClN4O+]: 311.0700; found [C16H12ClN4O+]: 311.0702 (diff.: 2.04 ppm). TLC (10% MeOH in CH2Cl2): Rf=0.52. M.p.: decomposes > 300 °C.
Compound 9: 3-(8-Chloro-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzoic acid (DCBS152A)
3-(8-Chloro-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile
(20
mg,
0.063 mmol) and NaOH (18 mg, 0.44 mmol) were dissolved in 1.5 mL EtOH/H2O (v/v) and the reaction mixture was heated to reflux. After 18 h the mixture was acidified with 2 M HCl and the precipitate was collected by filtration, washed with water (3 mL), light petroleum (15 mL), EtOAc (20 mL) and dried under reduced pressure to give 3-(8-chloro-3-oxo-3,5dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzoic acid (yellow solid, 15 mg, 0.044 mmol, 70%). 1H NMR (400 MHz, DMSO-d6) δ 7.56 (t, J = 8.0 Hz, 1H), 7.67 – 7.80 (m, 3H), 8.19 (d, J = 2.3 Hz, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.78 (s, 1H), 8.83 (s, 1H), 13.02 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 106.1, 119.3, 120.0, 121.3, 122.0, 122.6, 124.9, 129.1, 130.4, 130.8, 131.5, 134.7, 140.1, 140.2, 142.5, 161.7, 167.4. HR-MS: calculated [C17H11ClN3O3+]: 340.0483; found [C17H11ClN3O3+]: 340.0474 (diff.: 2.71 ppm). TLC (20% MeOH in CH2Cl2): Rf=0.58. M.p.: decomposes > 300 °C.
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Supplementary Figure S6: 1H NMR spectrum of compound 3 (LAU159)
Supplementary Figure S7: APT NMR spectrum of 3 (LAU159)
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Supplementary Figure S8: 1H NMR spectrum of compound 4 (LAU165)
Supplementary Figure S9: APT NMR spectrum of compound 4 (LAU165)
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Supplementary Figure S10: 1H NMR spectrum of compound 5 (DCBS142)
Supplementary Figure S11: 13C NMR spectrum of compound 5 (DCBS142)
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Supplementary Figure S12: 1H NMR spectrum of compound 6 (DCBS32)
Supplementary Figure S13: 13C NMR spectrum of compound 6 (DCBS32)
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Supplementary Figure S14: 1H NMR spectrum of compound 7 (DCBS146)
Supplementary Figure S15: 13C NMR spectrum of compound 7 (DCBS146)
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Supplementary Figure S16: 1H NMR spectrum of the precursor of compound 8
Supplementary Figure S17: 13C NMR spectrum of the precursor of compound 8
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Supplementary Figure S18: 1H NMR spectrum of compound 8 (DCBS120)
Supplementary Figure S19: 13C NMR spectrum of compound 8 (DCBS120)
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Supplementary Figure S20: 1H NMR spectrum of compound 9 (DCBS152A)
Supplementary Figure S21: 13C NMR spectrum of compound 9 (DCBS152A)
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References Fryer, R.I., Zhang, P., Rios, R., Gu, Z.Q., Basile, A.S., and Skolnick, P. (1993). Structureactivity relationship studies at the benzodiazepine receptor (BZR): a comparison of the substitutent effects of pyrazoloquinolinone analogs. J. Med. Chem. 36: 1669–73. Savini, L., Chiasserini, L., Pellerano, C., Biggio, G., Maciocco, E., Serra, M., et al. (2001). High affinity central benzodiazepine receptor ligands. Part 2: quantitative structure-activity relationships and comparative molecular field analysis of pyrazolo[4,3-c]quinolin-3-ones. Bioorg. Med. Chem. 9: 431–44. Sieghart, W., and Schuster, A. (1984). Affinity of various ligands for benzodiazepine receptors in rat cerebellum and hippocampus. Biochem. Pharmacol. 33: 4033–8. Simeone, X., Siebert, D.C.B., Bampali, K., Varagic, Z., Treven, M., Rehman, S., et al. (2017). Molecular tools for GABAA receptors: High affinity ligands for β1-containing subtypes. Sci. Rep. 7: 5674. Varagic, Z., Wimmer, L., Schnürch, M., Mihovilovic, M.D., Huang, S., Rallapalli, S., et al. (2013). Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β- interface. Br. J. Pharmacol. 169: 371–83.
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