Supplementary
Novel benzodiazepine-like ligands with various anxiolytic, antidepressant or pro-
cognitive profiles
Thomas D. Prevot1,2, Guanguan Li3, Aleksandra Vidojevic4, Keith A. Misquitta1,5, Corey Fee1,5
Anja Santrac4, Daniel E. Knutson3, Michael R. Stephen3, Revathi Kodali3, Nicolas M. Zahn3,
Leggy A. Arnold3, Petra Scholze6, Janet L. Fisher7, Bojan D. Marković8, Mounira Banasr1,2,5,
James M. Cook3, Miroslav Savic4* and Etienne Sibille1, 2,5*. 1Campbell Family Mental Health Research Institute of CAMH, Toronto, Canada2Department of Psychiatry, University of Toronto, Toronto, Canada3Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, Milwaukee,
USA4Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia5Department of Pharmacology and Toxicology, University of Toronto, Toronto, Canada6Department of Pathobiology of the Nervous System, Center for Brain Research, Medical
University of Vienna, Vienna, Austria7Department of Pharmacology, Physiology and Neuroscience, University of South Carolina
School of Medicine, Columbia South Carolina, USA8Department of Pharmaceutical Pharmacy, Faculty of Pharmacy, University of Belgrade,
Belgrade, Serbia
*Corresponding authors
Etienne Sibille, Ph.D., CAMH, 250 College street, room 134, Toronto, ON M5T 1R8, Canada
Tel: 416-535-8501, ext 36571; E-mail: [email protected]
Miroslav Savic, Ph.D. Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221
Belgrade. Tel: +3816427551447; E-mail: [email protected]
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ContentsSupplementary Methods..........................................................................................................................4
Chemistry...............................................................................................................................................4
Electrophysiological recordings..........................................................................................................7
Binding studies......................................................................................................................................8
Animals.................................................................................................................................................10
Pharmacokinetic characterization.....................................................................................................11
Behavioral Assessment......................................................................................................................13
Supplementary Tables...........................................................................................................................15
Supplementary Table S1: Statistical analysis for potentiation at α1/2/3/4/5/6, β1/3,γ/δ GABAA-receptors...........................................................................................................................15
Supplementary Table S2: Preferential potentiation at α5-GABAA-R....................................16
Supplementary Table S3: Statistical comparison between α1β1γ2 and α1β3γ2 potentiation at 100nM and 1µM..........................................................................................................................17
Supplementary Table S4: Half-life and percentage of compound remaining after incubation with human or mouse liver microsomes....................................................................18
Supplementary Table S5: Electrophysiological records of the GL series for α1β3γ2 and α5β3γ2 GABAA receptor subtypes...............................................................................................19
Supplementary Table S6: Ki values for all three compounds at α1/2/3/5β3γ2 receptor....20
Supplementary Table S7: The approximated % of GABA potentiation and the values of electrophysiological potentiation obtained at α2β3γ2 and α3β3γ2 receptors (presented in Figure 1), for the estimated brain free concentrations of GL-II-73, GL-II-74 and GL-II-75, administered at the doses of 1 mg/kg and 10 mg/kg, 30 minutes after administration.........21
Supplementary Table S8: Statistical analysis for each behavioral test assessing emotionality in mice........................................................................................................................22
Supplementary Table S9: Statistical analysis for the Y-maze alternation rate after acute administration in young mice (stressed and non-stressed).......................................................23
Supplementary Table S10: Statistical analysis for the Y-maze alternation rate after acute administration in old mice..............................................................................................................24
Supplementary Table S11: Statistical analysis for the Y-maze alternation rate after sub-chronic administration in young and old mice.............................................................................25
Supplementary Figures..........................................................................................................................26
Supplementary Figure S1. General synthetic route to amides..............................................26
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Supplementary Figure S2. Concentration of GL-II-73, GL-II-74 and GL-II-75 in the brain of male C57BL/6 mice after administration of a compound at doses of 1, 5 or 10mg/kg..........27
Supplementary Figure S3. Locomotor activity changes induced by DZP injection.............28
Supplementary Figure S4. Locomotor activity changes induced by αPAM injection..........29
Supplementary Figure S5. Effect of GL-II-73, GL-II-74, GL-II-75 and DZP on alternation rate in a Y-maze alternation task assessing working memory in non-stressed adult mice...30
Supplementary Figure S6. Scheme of repeated assessment of cognitive performances in the Y-Maze in function of stress exposure and α5-PAM administration...................................31
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Supplementary Methods
ChemistryBased on previous compounds (1) synthetized and screened which proved their potency at the
α5-subtype of the Bz/GABAAA-ergic receptors, a new series of metabolically more stable
compounds were prepared following the steps described below (2).
(R)-8-Ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylic acid SH-053-2'F-R-CH3-acid (2)
The ethyl ester SH-053-2'F-R-CH3 1 (20.0 g, 51.6 mmol) was dissolved in DCM (200 mL) and
EtOH (500 mL), after which solid NaOH (16.6 g, 413 mmol) was added to the solution. This
reaction mixture was heated to 55 ˚C for 0.5 h and the EtOH was removed under reduced
pressure. The remaining aq solution which remained was stirred at 0 ˚C for 10 min and then aq
HCl (1M) was added dropwise to the solution until the pH was 5 (pH paper). A pale white
precipitate which formed was left in the solution for 10 min and was then collected by filtration,
and washed with cold water after which the aq layer was also allowed to stand at rt for 10 h to
yield additional acid. The combined solids were dried in a vacuum oven at 80 ˚C for 7 h to get
pure acid 2 as a white powder (18.4 g, 51.2 mmol, 99.2%): mp 196-198 °C; []D25 = +4.00 (c
0.80, CHCl3); 1H NMR (300 MHz, DMSO-d6): δ 8.42 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.81 (d, J
= 7.8 Hz, 1H), 7.65 – 7.49 (m, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 6.51 (q, J =
6.7 Hz, 1H), 4.37 (s, 1H), 1.14 (d, J = 6.8 Hz, 3H); 13C NMR (75 MHz, DMSO-d6): δ 165.03 (s),
162.63 (s), 159.82 (d, J = 248.4 Hz), 140.49 (s), 136.40 (s), 135.52 (s), 134.78 (d, J = 1.0 Hz),
133.18 (s), 133.14 (s), 132.59 (d, J = 5.8 Hz), 131.88 (s), 129.35 (s), 128.95 (d, J = 12.6 Hz),
125.15 (d, J = 1.8 Hz), 123.97 (s), 121.05 (s), 116.37 (d, J = 20.9 Hz), 83.37 (s), 82.01 (s),
49.74 (s), 15.10 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for C21H15FN3O2 360.1143; found
360.1140.
General procedure for amides
A mixture of the acid SH-053-2'F-R-CH3-acid 2 (2 g, 5.56 mmol), thionyl chloride (55.6 mmol)
and dry DCM (50 mL) was placed in an oven dried round bottom flask under argon. This
suspension was allowed to reflux at 60 ˚C for 2 h under argon. The absence of the starting
material was confirmed by TLC (silica gel). The organic solvent and excess thionyl chloride
were evaporated under reduced pressure, which was repeated 5 times with dry DCM (20 mL).
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The yellow residue, which was obtained, was dissolved in dry DCM (50 mL) and cooled to 0 ˚C
for 10 min under argon. Then the appropriate amine was added (11.12 mmol), followed by
administration of Et3N (5.56 mmol) to the reaction mixture at 0˚C and the mixture was then
allowed to warm to rt and stirred for 2-7 h depending on the amine. After the completion of the
reaction the solvent was removed under reduced pressure. The residue was treated with ice
cold water and extracted with DCM (3 X 50 mL). The combined organic layer was washed with
brine (20 mL). The solvent was removed under reduced pressure and the residue was purified
by column chromatography on silica gel to yield the corresponding pure amides.
(R)-8-Ethynyl-6-(2-fluorophenyl)-N,N,4-trimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxam GL-II-73 (3a)
The amide 3a was prepared from 2 following the general procedure with dry dimethylamine as
the nucleophile. The crude residue was purified by column chromatography (silica gel, EtOAc
and 1% MeOH) to yield pure dimethyl amide 3a as a light yellow powder (1.5 g, 3.9 mmol,
70%): mp 189-190 °C; []D25 = +40.98 (c 0.61, CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.92 (s,
1H), 7.68 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.45 – 7.35 (m,
2H), 7.21 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 9.3 Hz, 1H), 4.28 (q, J = 6.0 Hz, 1H), 3.14 (s, 1H), 3.09
(s, 3H), 2.96 (s, 3H), 1.89 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 166.10 (s), 162.69
(s), 160.19 (d, J = 250.4 Hz), 135.34 (s), 134.62 (d, J = 9.0 Hz), 133.90 (s), 133.71 (s), 132.94
(d, J = 8.4 Hz), 132.47 (s), 132.12 (d, J = 9.1 Hz), 131.29 (s), 129.18 (s), 127.57 (d, J = 11.6
Hz), 124.51 (d, J = 2.2 Hz), 122.87 (s), 121.52 (s), 116.17 (d, J = 21.3 Hz), 81.52 (s), 79.63 (s),
52.13 (s), 39.09 (s), 35.04 (s), 18.37 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for
C23H20FN4O 387.1616; found 387.1626.
(R)-N-Ethyl-8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide GL-II-74 (3b)
The amide 3b was prepared from 2 following the general procedure with dry ethylamine as the
nucleophile. The crude residue was purified by column chromatography (silica gel, 3:2 EtOAc
and hexane) to yield pure ethyl amide 3b as a white powder (1.6 g, 4.3 mmol, 78%): mp 215-
216 °C; []D25 = +44.07 (c 0.59, CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.83 (s, 1H), 7.65 (dd, J =
16.8, 7.8 Hz, 2H), 7.53 (d, J = 8.3 Hz, 1H), 7.47 – 7.39 (m, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.17 (s,
1H), 7.01 (t, J = 9.3 Hz, 1H), 6.90 (q, J = 6.3 Hz, 1H), 3.61 – 3.34 (m, 2H), 3.15 (s, 1H), 1.26 (d,
J = 9.0 Hz, 3H), 1.22 (t, J = 7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 162.81 (s), 162.51 (s),
160.13 (d, J = 250.7 Hz), 138.81 (s), 134.96 (s), 134.71 (s), 133.90 (s), 133.37 (s), 131.84 (s),
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131.76 (s), 131.37 (s), 129.78 (s), 128.86 (d, J = 10.6 Hz), 124.44 (d, J = 3.4 Hz), 122.04 (s),
121.38 (s), 116.06 (d, J = 21.5 Hz), 81.55 (s), 79.52 (s), 49.89 (s), 33.70 (s), 15.02 (s), 15.01(s);
HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for C23H20FN4O 387.1616; found 387.1618.
(R)-N-Cyclopropyl-8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide GL-II-75 (3c)
The amide 3c was prepared from 2 following the general procedure with dry cyclopropylamine
as the nucleophile. The crude residue was purified by a column chromatography (silica gel, 1:1
EtOAc and hexane) to yield pure cyclopropyl amide 3c as a white powder (1.8 g, 4.5 mmol,
82%): mp 231-232 °C; []D25 = +3.81 (c 0.46, CHCl3); 1H NMR (300 MHz, CDCl3): δ 7.80 (s, 1H),
7.64 (dd, J = 15.9, 7.6 Hz, 2H), 7.52 (d, J = 8.3 Hz, 1H), 7.47 – 7.37 (m, 2H), 7.30 – 7.19 (m,
2H), 7.01 (t, J = 9.3 Hz, 1H), 6.89 (q, J = 7.0 Hz, 1H), 3.15 (s, 1H), 2.93 – 2.67 (m, 1H), 1.26 (d,
J = 6.8 Hz, 3H), 0.87 – 0.76 (m, 2H), 0.65 – 0.51 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 164.02
(s), 162.82 (s), 160.13 (d, J = 249.9 Hz), 138.91 (s), 134.97 (s), 134.65 (s), 133.89 (s), 133.37
(s), 131.80 (d, J = 9.0 Hz), 131.58 (s), 131.36 (s), 129.78 (s), 128.81 (d, J = 14.6 Hz), 124.45 (d,
J = 3.4 Hz), 122.04 (s), 121.42 (s), 116.06 (d, J = 21.5 Hz), 81.53 (s), 79.55 (s), 49.88 (s), 22.10
(s), 14.99 (s), 6.54 (s), 6.50 (s); HRMS (ESI/IT-TOF) m/z: [M + H]+ Calcd for C24H20FN4O
399.1616; found 399.1621.
(R)-(8-Ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)(pyrrolidin-1-yl)methanone GL-II-76 (3d)
The amide 3d was prepared from 2 following the general procedure with dry pyrrolidine as the
nucleophile. The crude residue was purified by column chromatography (silica gel, 6:4 EtOAc
and hexane) to yield a mixture of rotamers 3d as a white powder (3:2 ratio by 1H NMR, 1.8 g,
4.4 mmol, 80%): mp 173-174 °C; []D25 = -23.91 (c 0.70, CHCl3); 1H NMR Major rotamer (300
MHz, CDCl3): δ 7.90 (s, 1H), 7.73 – 7.58 (m, 2H), 7.51 (t, J = 9.0 Hz, 1H), 7.47 – 7.35 (m, 2H),
7.23 (t, J = 7.5 Hz, 1H), 7.01 (t, J = 9.3 Hz, 1H), 4.31 (q, J = 6.1 Hz, 1H), 3.66 – 3.56 (m, 4H),
3.14 (s, 1H), 1.95 (d, J = 6.4 Hz, 3H), 1.93 – 1.69 (m, 4H); 1H NMR Minor rotamer (300 MHz,
CDCl3): δ 7.85 (s, 1H), 7.74 – 7.57 (m, 2H), 7.51 (t, J = 9.0 Hz, 1H), 7.47 – 7.35 (m, 2H), 7.23 (t,
J = 7.5 Hz, 1H), 7.01 (t, J = 9.3 Hz, 1H), 6.49 (q, J = 6.6 Hz, 1H), 3.82 (dd, J = 58.0, 32.3 Hz,
4H), 3.46 (d, J = 26.4 Hz, 4H), 3.14 (s, 1H), 1.28 (d, J = 6.4 Hz, 3H); HRMS (ESI/IT-TOF) m/z:
[M + H]+ Calcd for C25H22FN4O 413.1772; found 413.1772.
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Electrophysiological recordingsElectrophysiological recordings were performed following the methods described in Alexeev et
al (3)(2)(2)(2). Full-length cDNAs for GABAA receptor subtypes (generously provided by Dr.
Robert Macdonald, Vanderbilt University and Dr. David Weiss, University of Texas Health
Science Center, San Antonio, TX) in mammalian expression vectors were transiently
transfected into the HEK-293T cell line (GenHunter, Nashville, TN). All subtypes were rat clones
except for α2, which was a human clone. Cells were transiently transfected using calcium
phosphate precipitation. Plasmids encoding GABAA receptor subtype cDNAs were added to the
cells in 1:1:1 ratios (α:β:γ) of 2 μg each (4)(3)(3)(3). Cells were patch-clamped at −50 mV in the
whole-cell recording configuration. GABA was diluted into the bath solution from freshly made or
frozen stocks in water. Compounds were dissolved in DMSO and diluted into the bath solution
with the highest DMSO level applied to cells of 0.01%. Solutions containing GABA or GABA +
compounds were applied to cells for 5s using a 3-barrelled solution delivery device controlled by
a computer-driven stepper motor (SF-77B, Harvard Apparatus, Holliston, MA, open tip
exchange time of <50ms). There was a continuous flow of external solution through the
chamber. Currents were recorded with an Axon 200B (Foster City, CA) patch clamp amplifier.
Whole-cell currents were analyzed using the programs Clampfit (pClamp9 suite, Axon
Instruments, Foster City, CA) and Prism (Graphpad, San Diego, CA). Concentration−response
data was fit with a four-parameter logistic equation
current=minimumcurrent+(maximumcurrent−minimumcurrent)1+(10 (logEC 50−log (modulator ) )n) where n represents the Hill
number. All fits were made to normalized data with current expressed as a percentage of the
response to GABA alone for each cell.
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Binding studiesBinding studies were performed on HEK-293 cells following the methods described in (5)Human
embryonic kidney (HEK) 293 cells (AmericanType Culture Collection ATCCs CRL-1574™) were
maintained in Dulbecco's modified Eagle medium (DMEM, high glucose, GlutaMAX™
supplement, Gibco61965-059, ThermoFisher, Waltham, Massachusetts, USA) supplemented
with 10% fetal calf serum (Sigma-AldrichF7524, St. Louis, Missouri, USA), 100U/ml Penicillin-
Streptomycin (Gibco 15140-122, ThermoFisher, Waltham, Massachusetts, USA) and MEM
(Non-Essential Amino Acids Gibco 11140-035, ThermoFisher, Waltham, Massachusetts, USA)
on 10cm Cell culture dishes (Cell+, Sarstedt, Nürnbrecht, Germany) at 37 °C and 5% CO2.
HEK293 cells were transfected with cDNAs encoding rat GABAA-receptor subunits subcloned
into pCI expression vectors. The ratio of plasmids used for transfection with the calcium
phosphate precipitation method (Chen and Okayama,1987) were 3 mg α(1,2,3or5): 3 mg β3
and 15 mg γ2 per 10cm dish. Medium was changed 4–6 h after transfection. Cells were
harvested 72 days after transfection by scraping into phosphate buffered saline. After
centrifugation (10 min,12000 g, 4°C) cells were resuspended in TC50 (50 mM Tris-
CitratepH¼7.1), homogenized with an Ultra-Turraxs (IKA, Staufen, Germany) and centrifuged
(20 min,50000 g). Membranes were washed three times in TC50 as described above and frozen
at -20°C until use. Frozen membranes were thawed, re-suspended in TC50 and incubated for
90 min at 4°C in a total of 500 ml of a solution containing 50 mM Tris/citrate buffer, pH=7.1, 150
mM NaCl and 2 nM [3H]-Flunitrazepam (Perkin Elmer New England Nuclear, Waltham,
Massachusetts, USA) in the absence or presence of either 5 mM diazepam (Nycomed, Opfikon,
Switzerland) (to determine unspecific binding) or various concentrations of receptor ligands
(dissolved in DMSO, final DMSO- concentration 0.5%). Membranes were filtered through
Whatman GF/B filters and the filters were rinsed twice with 4ml of ice-cold 50 mM Tris/citrate
buffer. Filters were transferred to scintillation vials and subjected to scintillation counting after
the addition of 3 ml Rotiszint Eco plus liquid scintillation cocktail. Non-specific binding
determined in the presence of 5mM Diazepam was subtracted from total [3H]-Flunitrazepam
binding to result in specific binding. In order to determine the equilibrium binding constant KD of
3H-Flunitrazepam for the various receptor-subtypes, membranes were incubated with various
concentrations of 3H-Flunitrazepam in the absence or presence of 5 mM Diazepam. Saturation
binding experiments were analyzed using the equation Y=Bmax*X/(KD+X). Non-linear
regression analysis of the displacement curves used the equation: log(inhibitor) vs. response-
variable slope with Top=100% and Bottom=0% Y=100/(1+10^((logIC50-x)*Hill-slope)). Both
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analyses were performed using GraphPad Prism (LaJolla California USA). Drug concentrations
resulting in half maximal inhibition of specific 3H-Flunitrazepam binding (IC50) were converted to
Ki values by using the Cheng-Prusoff relationship (Cheng andPrusoff, 1973) Ki=IC50/(1+(S/KD))
with S being the concentration of the radioligand (2 nM) and the KD values listed in
Supplementary Table 6.
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AnimalsYoung (2-3 months) or old (21-22 months) C57BL/6 mice were were obtained from Jackson
Laboratories (US), or the Military Medical Academy (Serbia) and kept in normal housing
conditions (21±2°C, relative humidity 40-45%) with a 12hr light-dark cycle (7am ON), water and
food ad libitum. Prior to behavioral assessment, animals were handled daily for 5 min, over 5
consecutive days, to reduce acute anxiety-like responses (6). Testing took place during the light
phase, and was conducted in accordance with the Canadian or U.S. institutional animal care
committee and the Ethical Commission on Animal Experimentation of the Faculty of Pharmacy
in Belgrade (carried out in accordance with the EEC Directive 86/609).
Compound preparation and administrationFor pharmacokinetic assays and behavioral testing, compounds were diluted in a vehicle
solution containing 85% distilled H2O, 14% propylene glycol (Sigma Aldrich) and 1% Tween 80
(Sigma Aldrich) to be administered intraperitoneally (i.p.) at a volume of 10 ml/kg. Working
solutions were prepared at 1, 5 or 10 mg/kg and adjusted to body weight before injection. DZP
was used as a non-selective GABAA-R PAM with known anxiolytic and no cognitive efficacies.
DZP was administered i.p. at 1.5 mg/kg in a 10ml/kg volume, based on previous studies.
For sub-chronic administration in the drinking water for 10 consecutive days, compounds
were diluted in tap-water at 30mg/kg, stirred over night at room temperature and given to the
animals in glass bottles. Bottles were changed every other day to provide freshly prepared
solutions.
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Pharmacokinetic characterization
Metabolic stability assessmentThe method used was previously described in (7). The test compounds were incubated at 10µM
with active or heat-inactivated human liver microsomes and cofactors. Aliquots were removed at
0, 10, 20, 30, 40, 50, 60 and 120 min and mixed with acetonitrile containing internal standard for
analysis. Samples were extracted and assayed using a liquid chromatography/tandem mass
spectrometry (LC-MS/MS) analytical method. On the day of the experiment, the test
compounds, prepared in DMSO, were diluted in the 100mM phosphate buffer (pH=7.4) to
achieve appropriate final concentrations. The test compounds were incubated with human or
mouse liver microsomes (0.5mg protein/mL; BD Gentest and Life Technologies) and
appropriate cofactors (2.5mM NADPH and 3.3 nM magnesium chloride) in 100mM phosphate
buffer, pH 7.4 (0.1% final DMSO), in a 37°C water bath. At selected time points, a single 100µL
aliquot was removed from each sample and mixed with 200µL of chilled acetonitrile containing
internal standard. Following brief vortexing and centrifugation, the samples were further diluted
for subsequent LC-MS/MS analysis, in triplicate. Experimental controls consisted of: (a)
incubation of all components except test compound for 0 and 60 min, (b) incubation of
Verapamil (positive control) at 10 lM for 0, 10, 20, 30, 40, 50, 60 and 120 min, and (c) incubation
of 1 and 10 µM test compound and 10 µM Verapamil with heat-inactivated microsomes (0.5 mg
protein/mL) for 0 and 60 min. Samples were analyzed by LC–MS/MS in multiple reaction
monitoring mode using positive-ion electrospray ionization. The details of the LC–MS/MS
method can be provided upon request. To determine metabolic stability, the percent remaining
at each time point was calculated:
%remaining at timeT=¿¿¿.
Pharmacokinetic profiles
Aiming to obtain the respective pharmacokinetic profiles, young mice (n = 15 per compound)
were divided into five groups of animals; each group contained three animals and corresponded
to predetermined time intervals (5, 20, 60, 180, and 720 min). The mice were treated
intraperitoneally (i.p.) at the 10 mg/kg dose and in a volume of 10 ml/kg. Also, brain
concentrations were measured 20 min after i.p. administration of the 1 mg/kg dose.
In order to parallel the design of behavioral studies, additional experiments were performed with
compounds dosed at 1, 5 and 10 mg/kg, where brain concentrations were determined 30 min
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after single i.p. injection, as well as after three i.p. injections, administered 24 h, 20 h and 1 h
prior to the concentration measurement.
At the appropriate time intervals, the blood samples were collected via cardiac puncture of mice
anesthetized with ketamine solution (10% Ketamidor, Richter Pharma Ag, Wels, Austria, dosed
i.p. at 100 mg/kg) in heparinized syringes and centrifuged at 800 rcf for 10 min to obtain plasma
samples. Mice were decapitated and brains were weighed, homogenized in 1.25 ml of methanol
and centrifuged at 3400 rcf for 20 min. Compounds were extracted from plasma and
supernatants of brain tissue homogenates by solid phase extraction, using Oasis HLB
cartridges (Waters Corporation, Milford, Massachusetts). The procedure of sample preparation
and determination of concentration by ultraperformance liquid chromatography–tandem mass
spectrometry (UPLC–MS/MS) with Thermo Scientific Accela 600 UPLC system connected to a
Thermo Scientific TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo
Fisher Scientific, San Jose, California), equipped with electrospray ionization (ESI) source, has
been already described in detail. Non-compartmental pharmacokinetic analysis was performed
using PK Functions for Microsoft Excel software (by Joel Usansky, Atul Desai, and Diane Tang-
Liuwere), while graphs were constructed in commercial statistical software Sigma Plot 12
(Systat Software Inc., USA).
In vitro hydrolytic stability study in plasmaThe compounds were tested for their hydrolytic stability in vitro at 37 °C, utilizing blank mouse
plasma spiked with the respective compound and internal standard (SH-I-048A; synthesized at
the Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, USA), as
detailed in (5).
Plasma protein and brain tissue binding studies The rapid equilibrium dialysis assay used to determine free fraction of GL-II-73, GL-II-74 and
GL-II-75 in mouse plasma and brain tissue was the same as in (8). Free concentrations in the
brain were calculated by multiplying the obtained total brain concentrations with the appropriate
free fractions determined by rapid equilibrium dialysis (12.14%, 4.49% and 1.34% for GL-II-73,
GL-II-74 and GL-II-75, respectively).
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Behavioral Assessment Elevated plus MazeThe elevated plus maze (EPM) is designed to assess anxiety-like behaviors in rodents. The
maze is made of four white Plexiglas arms, two open arms (29x7 cm), and two enclosed arms
(29x7x17 cm) that formed a cross shape with the two open arms opposite each other. The maze
is 90 cm above the floor and illuminated at 100 lux. Young male and female (50%) mice
(Jackson Laboratories) were injected i.p. with vehicle, 5 or 10mg/kg dose of test compound or
1.5mg/kg of DZP, 30 minutes prior to testing. They were placed individually on the central
platform, facing an open arm, and allowed to explore the apparatus for 10 min. Behavior was
recorded with a digital camera and analyzed using ANY-maze Video Tracking System software
(Stoelting Co, Wood Dale, IL, USA). The time in the open arm was used to calculate the
percentage of time in the open arm presented in the results section using the following formula:
PercentageTime∈openarm=Time∈the openarmTotalTime
x 100.
Forced Swim Test Young male mice were tested in the forced swim test (FST), assessing for antidepressant
efficacy. Mice (Jackson Laboratories) were sub-chronically injected via i.p. with vehicle or 1, 5 or
10 mg/kg of test compounds 24 h, 20 h and 1 h before testing, as per standard methods in the
field for testing potential antidepressant compounds. Following standard protocol for DZP,
animals were injected only once at the dose of 1.5 mg/kg, 1 h prior testing. The protocol used
was initially described by Porsolt (9), where mice were placed in an inescapable transparent
tank filled with water (25 cm, 26±1oC). Animals were recorded for a period of 6min and a manual
count of the immobile time in the tank was measured. Immobility is defined as the minimum
amount of movement to stay afloat, between the second and the sixth minute of testing.
Compounds that reduced immobility in the FST are considered to have potential antidepressant
actions.
Spontaneous alternation in the Y-MazeYoung male and female (50%) mice (Jackson Laboratories, Bar Harbor, USA) and old males
(22 months-old) were single-housed. Young animals were subjected to a chronic stress protocol
(CS) to induce a working memory deficit. They were placed in a 50 ml Falcon® tube, twice a
day, for 1 hr during their diurnal cycle. CS was not applied on testing days. The apparatus was a
black plastic Y-maze with 3 arms, 26cm long, 8cm wide with sidewalls 13cm high and all
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separated by 120°; each arm having a sliding door. The protocol used was a modified version of
the one described in Vandesquille et al (10). Briefly, mice were first habituated to the apparatus
and to distal cues during 2 consecutive days over a 10 min free exploration session. The
following day, animals performed a training session consisting of seven successive trials where
they have to alternate between the 2 goal arms with an inter trial interval (ITI) of 30 sec. The
same general procedure used in the training session was implemented 24h later, except that
the ITI was lengthened to 90s or 60s, for young or old animals respectively. Animals were
acutely injected i.p. with vehicle, 1, 5 or 10 mg/kg dose of the test compounds or 1.5mg/kg DZP,
30 min before the beginning of the test. To dissociate memory deficits from an eventual
progressive loss of motivation, an 8th trial was added to the series which was separated from the
7th trial by a shorter ITI (5s). All animals failing to alternate at the 8 th trial were excluded from the
analysis. The alternation rate was calculated and was expressed in percentage:
Alternationrate= AlternationMaximumalternation possible
x100. The percentage of alternation during the
entire task was considered as an index of working memory performance (50% of alternation
corresponding to a random alternation rate). This test can be repeated weekly, with no
requirement for habituating the animals again. This allows us to reduce the number of animals
and test multiple doses or compounds in the same animals, with a week of wash-out between
experiments. We confirmed that the compounds were washed out of the organism of the mouse
by conducting a pilot experiment that validated our design (for more details, see
Supplementary Figure 6).
Spontaneous locomotor activityBased on previous studies from our team, we analyzed the influence of each compound on
spontaneous locomotor activity (SLA) of young mice (n=27). The apparatus was a white and
opaque Plexiglas chamber (40×25×35 cm) under dim red light (20 lux). A digital camera
mounted above the apparatus recorded the animal activity, which was tracked and analyzed
using ANY-maze Video Tracking System software (Stoelting Co, Wood Dale, IL, USA).
Compounds or vehicle was applied i.p. and a single mouse was immediately placed in the
center of the chamber. The activity was tracked for a total of 60min. Chambers were cleaned
with 70% ethanol after every trial.
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Supplementary Tables
Supplementary Table S1: Statistical analysis for potentiation at α1/2/3/4/5/6, β1/3,γ/δ GABAA-
receptors
N t-test p N t-test p N t-test p N t-test p N t-test p N t-test p N t-test p N t-test pα1ß3γ2 5 2.9 0.04 5 8.05 0.0013 5 3.37 0.02 5 3.17 0.033 6 6.08 0.0017 5 6.9 0.0023 5 15.9 0.0001 5 6.68 0.0026α2ß3γ2 5 2.1 0.1 5 4.31 0.012 6 3.8 0.011 5 7.61 0.0016 6 4.1 0.009 5 3.4 0.026 5 34.04 0.0001 5 11.67 0.0003α3ß3γ2 6 7.6 0.0006 6 8.03 0.0005 5 10.2 0.0005 5 6.22 0.003 6 5.91 0.002 5 6.05 0.0038 6 5.33 0.0031 6 4.34 0.0074α4ß3γ2 5 0.35 0.73 5 2.12 0.1 5 -0.38 0.7 5 -0.44 0.68 5 0.99 0.37 5 2.01 0.11 6 1.41 0.2169 6 1.29 0.25α5ß3γ2 5 5.1 0.006 5 9.07 0.0008 5 2.9 0.04 5 7.07 0.0021 5 3.29 0.03 5 4.67 0.0095 6 4.04 0.0099 6 5.02 0.004α6ß3γ2 5 1.8 0.14 5 0.73 0.5 5 1.8 0.14 5 1.59 0.18 5 2.21 0.09 6 0.46 0.67 4 2.04 0.13 4 0.13 0.9
α1ß1γ2 4 2.35 0.077 4 4.67 0.0095 4 2.7 0.054 4 5.09 0.007 5 10.99 0.0001 5 9.97 0.0002 4 2.09 0.1046 4 8.7 0.0009
α1ß3δ 4 0.151 0.887 3 -0.47 0.669 4 2.27 0.0852 4 -0.77 0.4866
GL-II-74 GL-II-75 GL-II-76GL-II-731µM 100 nM 1µM100 nM 1µM 100 nM 1µM 100 nM
175.380 40 5.962 <.0001116.500 4 2.945 .0422162.420 4 8.052 .0013117.320 4 3.373 .0280189.680 4 3.173 .0338203.183 5 6.087 .0017346.600 4 6.926 .0023117.280 4 15.945 <.0001144.500 4 6.683 .0026
Moy. DDL t pa1, Totala1, Gl-II-73 (100nM)a1, Gl-II-73 (1uM)a1, Gl-II-74 (100nM)a1, Gl-II-74 (1uM)a1, Gl-II-75 (100nM)a1, Gl-II-75 (1uM)a1, Gl-II-76 (100nM)a1, Gl-II-76 (1uM)
Test-t univarié Eclaté par : GLMoy. théorique = 100
171.336 41 5.562 <.0001115.020 4 2.102 .1034134.100 4 4.315 .0125119.333 5 3.874 .0117199.580 4 7.612 .0016194.217 5 4.106 .0093324.820 4 3.425 .0267126.620 4 34.044 <.0001162.820 4 11.670 .0003
Moy. DDL t pa2, Totala2, Gl-II-73 (100nM)a2, Gl-II-73 (1uM)a2, Gl-II-74 (100nM)a2, Gl-II-74 (1uM)a2, Gl-II-75 (100nM)a2, Gl-II-75 (1uM)a2, Gl-II-76 (100nM)a2, Gl-II-76 (1uM)
Test-t univarié Eclaté par : GLMoy. théorique = 100
169.693 44 6.785 <.0001123.017 5 7.625 .0006136.567 5 8.036 .0005126.640 4 10.296 .0005199.220 4 6.221 .0034183.617 5 5.917 .0020321.740 4 6.055 .0038124.267 5 5.333 .0031165.567 5 4.347 .0074
Moy. DDL t pa3, Totala3, Gl-II-73 (100nM)a3, Gl-II-73 (1uM)a3, Gl-II-74 (100nM)a3, Gl-II-74 (1uM)a3, Gl-II-75 (100nM)a3, Gl-II-75 (1uM)a3, Gl-II-76 (100nM)a3, Gl-II-76 (1uM)
Test-t univarié Eclaté par : GLMoy. théorique = 100t-test comparison to 100%.
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Supplementary Table S2: Preferential potentiation at α5-GABAA-R
Subunit N
ANOVA values
p-value NANOVA values
p-value NANOVA values
p-value NANOVA values
p-value
α1 5 0.6 5 0.0015 5 0.21 5 0.0007α2 5 0.04 5 0.0017 5 0.38 5 0.059α3 6 0.44 6 0.0068 6 0.84 6 0.007α4 5 0.0002 5 <0.0001 5 0.0029 5 <0.0001α5 5 * 5 * 5 * 5 *α6 5 0.0007 5 0.0002 5 0.006 5 <0.0001α1 5 <0.0001 5 <0.0001 5 0.12 5 0.008α2 5 <0.0001 5 0.0002 5 0.19 5 0.04α3 6 <0.0001 6 0.0002 6 0.25 6 0.001α4 5 <0.0001 5 <0.0001 5 0.02 5 <0.0001α5 5 * 5 * 5 * 5 *α6 5 <0.0001 5 <0.0001 5 0.01 5 <0.0001
F(5,20)=8.2;
p=0.0002
F(5,15)=7.7;
p=0.0009
F(5,15)=11.8;
p<0.0001
F(5,15)=9.6;
p=0.0003
GL-II-73 GL-II-74 GL-II-75 GL-II-76
100n
M
1µM
F(5,20)=6.1;
p=0.0013
F(5,20)=44.3;
p<0.0001
F(5,20)=6.1;
p=0.0013
F(5,20)=16.9;
p=0.002
* refers to the group used as reference for post-hoc statistical analysis.
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Supplementary Table S3: Statistical comparison between α1β1γ2 and α1β3γ2 potentiation at
100nM and 1µM
N ANOVA p-value N ANOVA p-value N ANOVA p-value N ANOVA p-valueß3 5 * 5 * 6 * 5 *ß1 5 0.59 5 n/a 6 n/a 5 n/aß3 5 * 5 * 5 * 5 *ß1 5 0.033 5 n/a 5 n/a 5 n/a
100nM
1µM
GL-II-73 GL-II-74 GL-II-75 GL-II-76
F(1,16)=5.7 ; p=0.02
F(1,16)=0.2 ; p=0.6
F(1.19)=0.14 ; p=0.7
F(1,16)=0.19 ; p=0.66
* refers to the group used as reference for post-hoc statistical analysis.
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Supplementary Table S4: Half-life and percentage of compound remaining after incubation
with human or mouse liver microsomes
Compound Name
Half-life (min) (HLM)
% left after 2 hr. (HLM)
Half-life (min)(MLM)
% left after 2 hr. (MLM)
GL-II-73 280 ± 31 73.34 ± 0.6 472.7 ± 56 81.6 ± 0.27
GL-II-74 403 ± 40 82 ± 0.52 72 ± 6.7 32.1 ± 0.54
GL-II-75 163 ± 12 61.9 ± 0.9 106 ± 8 50 ± 0.55
GL-II-76 154 ± 5 75 ± 0.1 23 ± 1 18.6 ± 0.3
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Supplementary Table S5: Electrophysiological records of the GL series for α1β3γ2 and
α5β3γ2 GABAA receptor subtypes
α1β3γ2 α5β3γ2
Compound#
cellsAverage EC50
(±SEM)
Average max potentiation
(±SEM)
# cell
s
Average EC50
(±SEM)
Average max potentiation
(±SEM)
GL-II-73 3 892.3 ± 287.0 nM 219.9 ± 42.7% 3 890.8 ± 287.0 nM 382.8 ± 36.2%
GL-II-74 4 1136.2 ± 170.7 nM 367.3 ± 17.3% 3 206.9 ± 58.5 nM 371.2 ± 26.6%
GL-II-75 3 215.3 ± 46.2 nM 409.5 ± 16.8% 3 190.8 ± 58.8 nM 326.4 ± 19.3%
Diazepam 3 48.5 ± 6.3 nM 335.7 ± 13.0% 4 78.2 ± 8.5 nM 266.0 ± 10.8%
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Supplementary Table S6: Ki values for all three compounds at α1/2/3/5β3γ2 receptor
132 232 332 532
GL-II-73 55 ± 4µM 30 ± 5 µM 63 ± 7µM 5 ± 2 µM***
GL-II-74 1060 ± 100 nM 809 ± 130nM 1250 ± 51nM 83 ± 12nM***
GL-II-75 542 ± 88 nM 789 ± 130 nM 480 ± 62 nM 79 ± 2 nM**
;DZP 22.4 ± 5.4 nM 13.4 ±1.1 nM 20.4 ± 2.8 nM 12.1 ± 1.2 nM
Significance towards 132 ; *:p<0.05,**:p<0.01,***:p<0.001
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Supplementary Table S7: The approximated % of GABA potentiation and the values of
electrophysiological potentiation obtained at α2β3γ2 and α3β3γ2 receptors (presented in Figure 1), for the estimated brain free concentrations of GL-II-73, GL-II-74 and GL-II-75, administered
at the doses of 1 mg/kg and 10 mg/kg, 30 minutes after administration.
DoseCompound
Estimated brain free concentrations (ng/g)
Estimatedbrain free concentrations (nmol/kg)
Approximated % of GABA potentiation
α1β3γ2
α2β3γ2
α3β3γ2
α5β3γ2
1
mg/k
g
GL-II-73 5.67 14.67 100.96<120
%
<120
%103.93
GL-II-74 4.07 10.53 100.71<120
%
<120
%118.68
GL-II-75 2.29 5.76 100.34<120
%
<120
%99.97
10
mg/k
g
GL-II-73 117.29 303.53 131.68 124.25 129.08 179.62
GL-II-74 94.93 245.67 146.18 150.02 154.85 256.67
GL-II-75 18.85 47.32 162.90 153.24 139.49 136.76
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Supplementary Table S8: Statistical analysis for each behavioral test assessing emotionality in
mice
AssayDose
(mg/kg) NANOVA values
p-valueDose
(mg/kg) NANOVA values
p-valueDose
(mg/kg) NANOVA values
p-valueDose
(mg/kg) NANOVA values
p-value
0 (i.p.) 13 - 0 (i.p.) 14 - 0 (i.p.) 13 - 0 (i.p.) 11 -5 (i.p.) 13 0.24 5 (i.p.) 13 0.32 5 (i.p.) 14 - 1.5 (i.p.) 10 0.02
10 (i.p.) 14 0.009 10 (i.p.) 14 0.005 10 (i.p.) 13 -0 (i.p.) 8 - 0 (i.p.) 8 - 0 (i.p.) 8 - 0 (i.p.) 12 -1 (i.p.) 8 0.53 1 (i.p.) 8 0.08 1 (i.p.) 8 0.96 1.5 (i.p.) 12 0.0045 (i.p.) 6 0.58 5 (i.p.) 8 0.02 5 (i.p.) 8 0.031
10 (i.p.) 8 0.0003 10 (i.p.) 8 0.0004 10 (i.p.) 9 0.0031
F(2,37)=2.8;
p=0.07
F(3,29)=5.4 ;
p=0.004
F(2,37)=3.7 ;
p=0.03
F(3,26)=7.2 ;
p=0.001
Forced Swim test
Elevated Plus Maze
GL-II-73 GL-II-74 GL-II-75 DZP
F(1,22)=9.9 ; p=0.004
F(1,19)=6.6;p=0.02
F(2,37)=4.45 ;
p=0.02
F(3,28)=5.5 ;
p=0.0042
* refers to the group used as reference for post-hoc statistical analysis.
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Supplementary Table S9: Statistical analysis for the Y-maze alternation rate after acute
administration in young mice (stressed and non-stressed)
Assay Dose (mg/kg) NANOVA values
p-value Dose (mg/kg) NANOVA values
p-value Dose (mg/kg) NANOVA values
p-valueDose
(mg/kg) NANOVA values
p-value
0 (i.p.) - NS 10 0,0002 0 (i.p.) - NS 8 0,0023 0 (i.p.) - NS 8 0,0007 0 (i.p.) - NS 6 0,030 (i.p.) -CS 10 - 0 (i.p.) 8 - 0 (i.p.) 8 - 0 (i.p.) 6 -1 (i.p.)- CS 5 0,99 1 (i.p.) 4 0,98 1 (i.p.) 6 0,01 1.5 (i.p.) 6 0,945 (i.p.)-CS 10 0,74 5 (i.p.) 10 0,97 5 (i.p.) 4 0,04510 (i.p.)-CS 12 0,01 10 (i.p.) 4 0,98 10 (i.p.) 9 0,0370 (i.p.) - NS 10 - 0 (i.p.) - NS 8 - 0 (i.p.) - NS 8 - 0 (i.p.) - NS 6 -5 (i.p.) - NS 5 n/a 5 (i.p.) - NS 9 0,49 1.5(i.p) 6 0,00510 (i.p.) - NS 10 n/a 10 (i.p.) - NS 5 0,002 10 (i.p.) - NS 7 n/a
F(1,10)=12.41 ;
p=0.0055
F(2,15)=5.2 ; p=0.01
GL-II-75 DZP
F(1,13)=1.126 ;
p=0.31
GL-II-73 GL-II-74
F(4,29)=9.5 ; p<0.0001
F(4,30)=7 ;p=0.0004
F(5,46)=9.3 ;
p<0.0001
Acut
e
Y-Maze Spontaneous Alternation (Young)
Y-Maze Spontaneous Alternation (Young non exposed to CS)
F(2,22)=2.41 ;
p=0.11
F(2,19)=8.87 ;
p=0.002
* refers to the group used as reference for post-hoc statistical analysis.
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Supplementary Table S10: Statistical analysis for the Y-maze alternation rate after acute
administration in old mice
Assay Dose (mg/kg) NANOVA values
p-value Dose (mg/kg) NANOVA values
p-value
0 (i.p.) - Young 5 0,0005 0 (i.p.) - Young 5 0,0020 (i.p.) - Old 5 - 0 (i.p.) - Old 5 -
5 (i.p.) 6 0,03 5 (i.p.) 4 0,013
F(2,11)=12.3 ;
p=0.0015
F(2,13)=14.7 ;
p=0.0005
Acut
e
GL-II-73 GL-II-75
Y-Maze Spontaneous Alternation (Ageing)
* refers to the group used as reference for post-hoc statistical analysis.
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Supplementary Table S11: Statistical analysis for the Y-maze alternation rate after sub-chronic
administration in young and old mice
Assay Dose (mg/kg) NANOVA values
p-value Dose (mg/kg) NANOVA values
p-value
0 (p.o.) - NS 6 0,001 0 (p.o.) - NS 6 0,00510 (p.o.) -CS 5 - 0 (p.o.) 5 -30(p.o.)-CS 6 0,005 30(p.o.) 6 0,33
0 (p.o.) - Young 5 0,0001 0 (p.o.) - Young 6 0,00120 (p.o.) - Old 6 - 0 (p.o.) - Old 8 -
30(p.o.) 4 0,0001 30(p.o.) 5 0,46
Chro
nic
Y-Maze Spontaneous Alternation (Young)
F(2,14)=28.1 ;
p=0.0001
F(2,14)=8.1 ; p=0.0045
Y-Maze Spontaneous Alternation (Ageing)
F(2,12)=73.83 ;
p=0.0001
F(2,16)=10.8 ;
p=0.001
GL-II-73 GL-II-75
* refers to the group used as reference for post-hoc statistical analysis.
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Supplementary Figures
Supplementary Figure S1. General synthetic route to amides
Summarized in these schemes are the principal steps for the synthesis of the GL-II
series compounds from the parent compound SH-053-2'F-R-CH3 (1). A detailed
synthesis of SH-053-2'F-R-CH3 (1) is summarized in the US Patent 7,618,958.
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Supplementary Figure S2. Concentration of GL-II-73, GL-II-74 and GL-II-75 in plasma and
brain of male C57BL/6 mice after administration of a compound at doses of 1, 5 or 10 mg/kg, 30
min after single i.p. injection (A), as well as after three i.p. injections, administered 24 h, 20 h
and 1 h (B) prior to concentration measurement.
In order to parallel the design of behavioral studies (elevated plus maze, Y maze and forced
swim test), plasma and brain concentrations were determined 30 min after single i.p. injection
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(A), as well as after three i.p. injections, administered 24 h, 20 h and 1 h (B) prior to
concentration measurement with compounds dosed at 1, 5 and 10 mg/kg. In the appropriate
time intervals, the samples were collected from mice anesthetized with ketamine solution (10%
Ketamidor, dosed i.p. at 100 mg/kg). Compounds were extracted from plasma and supernatants
of brain tissue homogenates by solid phase extraction. The procedure of sample preparation
and determination of concentration by ultraperformance liquid chromatography–tandem mass
spectrometry (UPLC–MS/MS) has been already described in Obradovitch et al.
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Supplementary Figure S3. Locomotor activity changes induced by DZP injection
The effect of the referent non-selective PAM DZP dosed i.p. at 10 mg/kg was assessed on
distance traveled in 5-min bins during recording in the locomotor activity test in male mice.
Animals were injected and immediately placed in an opaque Plexiglas chamber under dim red
light (20 lux) for 1hr. Chambers were cleaned with 70% ethanol after every trial. Distance
travelled per 5-min bin was quantified a posteriori using ANYmaze Video Tracking System. A
two-way repeated measures ANOVA showed a significant effect of factor Treatment (Treatment:
F(1,132)=17.376, p=0.001; Time: F(11,132)=1.822, p=0.056; Interaction: F(11,253)=0.809,
p=0.631). Post-hoc comparisons demonstrated that DZP induced a hypolocomotor response in
time intervals 10-35 min and 40-55 min suggesting a sedative effect in the spontaneous
locomotor activity test in mice, in accordance with the approximated substantial potentiation of
α1-containing GABAA receptors.
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Supplementary Figure S4. Locomotor activity changes induced by αPAM injection
The effect of GL-II-73, GL-II-74 and GL-II-75 all dosed i.p. at 10 mg/kg were assessed on
distance traveled in 5-min bins during recording in the locomotor activity test in male mice.
Animals were injected with vehicle (VEH) or the test compounds and immediately placed in an
opaque Plexiglas chamber under dim red light (20 lux) for 1 hr. Chambers were cleaned with
70% ethanol after every trial. Distance travelled per 5-min bin was quantified a posteriori using
ANYmaze Video Tracking System. A two-way repeated measures ANOVA revealed a
significant effect of all factors (Treatment: F(3,253)=6.52, P=0.002; Time: F(11,253)=14.59,
P<0.001; Interaction: F(33,253)=3.25, P<0.001). Post-hoc comparisons of 5-min bins
demonstrated that GL-II-74 induced a hyperlocomotor response in time intervals 0-5 min and
50-55 min, while GL-II-75 elicited a similar stimulant-like effect in the first 5 min, but a consistent
hypolocomotion in the whole period 15-40 min.
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Supplementary Figure S5. Effect of GL-II-73, GL-II-74, GL-II-75 and DZP on alternation rate in
a Y-maze alternation task assessing working memory in non-stressed adult mice
The effect of each compound and the non-selective PAM DZP was assessed on alternation
performance in the Y-maze to evaluate potential cognitive deficit induced by the compound
under baseline conditions. Animals received 5 or 10mg/kg of the compounds, or 1.5mg/kg of
DZP, 30 min prior to testing. GL-II-74 was the only compound to show decreased alternation
rate after 10 mg/kg administration, similarly comparable to DZP. **p<0.001 compared to “No
Stress-Vehicle” group.
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469
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471
472
473
No drug -No stress
No Drug -Stress
Drug - Stress
No Drug -Stress
0102030405060708090
100
ControlStress
Alte
rnati
on R
ate
(%)
** ***
Supplementary Figure S6. Scheme of repeated assessment of cognitive performances in the
Y-Maze in function of stress exposure and α5-PAM administration
The same animals (N=10 per condition: Control or Stress) were evaluated weekly in the
Y-Maze task, assessing working memory abilities. The first week, after habituation to
the maze, the animals were tested without receiving the compound and without being
exposed to chronic stress yet. After that first assessment, animals belonging to the
Stress group were exposed to 1 week of chronic stress (CS), and re-evaluated. Results
showed that 1 week of CS induced alternation deficit in that task. The following week,
while the stress group was still exposed to CS, animals were injected with GL-II-73, 10
mg/kg. Results showed that the compound administration restored the alternation rate
that was altered the week before due to CS exposure. However, if we keep exposing
the animal to CS, without injecting them, the alternation rate drops down again,
suggesting that the single drug administration does not have long term effect, and that
the compound is washed out from the organism.
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