Supplementary Materials
Supplementary Figures
Supplementary Fig. 1
Model of the substrate binding region of caspase-2/Ac-VDVAD-CHO using ball and
stick representations.
The atoms of Ac-VDVAD-CHO are represented as follow: green = carbon, white = hydrogen,
blue = nitrogen, red = oxygen. The electric interactions (hydrogen bonds and salt bridges) are
represented by the dashed, yellow lines. Table shows the minimal energy (Kcal/mol) (Emin) of
the Casp2-inhibitor complex resulting from electric or coulombic component (Ec) together
with repulsion - attraction Van der Waals component (EVW). The interaction energy of the
Casp2-Ac-VDVAD-CHO and the Casp2-Ac-LDESD-CHO are -71.64 and -85.40 Kcal/mol
respectively. This indicates that the VDVAD sequence would lead to a better inhibition of the
Casp2 enzyme than LDESD.
Supplementary Fig. 2
Structure and in vitro effects on caspases of TRP901 and TRP801
A. Structure of TRP901. B. Structure of TRP801. Of note, TRP801 was also tested in vivo in
a well established experimental model fulminant liver injury. To proceed, 5 mg/kg of TRP801
(10% DMSO, 90% 0.9% NaCl) were administered (i.p.) to C57bl6 mice (Charles River) 30
min before injection of the JO2 anti-Fas antibody (0.3 µg/g; Pharmingen). Mice were
sacrificed at 3 h post JO2 treatment and ex vivo (TUNEL) staining was performed. TRP801
prevent liver cell death (decrease of TUNEL staining by at least 68±10%) and >70% of
TRP801-treated mice have pronounced protection.
Supplementary Fig. 3
TRP601 reduces GFAP immunoreactivity at 21 days post-stroke
Seven-day-old rats underwent electrocoagulation of the left middle cerebral artery and
transient homolateral common carotid artery occlusion (as in Fig. 3). Vehicle or 0.1 mg/kg
TRP601 was i.p. injected 1 h after the ischemic onset. After 21 days of recovery animals were
sacrificed. GFAP immunoreactivity (Cy3-anti GFAP antibody) in cortical layer VI. Vehicle
(A) and TRP601-treated (B) ischemic rats where sacrificed after 3 weeks of recovery. Note in
ischemic animals (A) a dense astrogliosis around the cavity and numerous clasmatodendritic
astrocytes (white arrows and enlarged panel), whereas astrocytes exhibiting fine processes in
TRP601-treated ischemic animals appeared to be normal. Bar represents 50 (, ) and 25
(enlarged panels) µm.
Supplementary Fig. 4
Caspase-2 triggers a TRP601 sensitive and Bid-mediated cytochrome c release from
neonatal brain mitochondria.
Immunodetection of cytochrome c (cyt. c) (A: upper panel, B) and Bid (A: lower panel) in the
supernatant of mitochondria isolated from the cortex of 7-day-old rat brain (See
Supplementary methods). Isolated mitochondria (150 g/ml protein) were incubated 30 min
at 37°C with recombinant Casp2 (rC2; 100 ng), recombinant full length Bid (60 ng; kindly
provided by Dr Xuejun Jiang, Memorial Sloan-Kettering Cancer Center, New York, NY), a
mixture of Bid plus rCasp2, or ad hoc proportions of buffers.
Supplementary Fig. 5
Kinetics of caspase-like activities during neonatal ischemic stroke
Seven-day-old rats were subjected to electrocoagulation of the left middle cerebral artery and
transient homolateral common carotid artery occlusion (as in Fig. 2). Ipsilateral penumbra
zone (IL) and contralateral counterpart (CL) of hemispheres were dissected and homogenized
at the indicated times prior to biochemical analysis. Kinetics of Ac-VDVAD-AMC, AcDEVD-AMC, and Ac-LEHD-AMC cleavage are presented. R, reperfusion. Co., control
brains (without ischemia). Data are mean ± s.e.m. (bars) values (n ≥ 3 per time point). **, P <
0.0001 (Two-way ANOVA). Asterisks indicate significance (at given time points) using
Bonferroni post hoc. Note that the earliest and quantitatively major detected caspase-like
activity is a VDVADase activity that reaches 14 pmol/min/mg at t=5h post-ischemia whereas
neither DEVDase nor LEHDase activities are detectable.
Many groups have demonstrated the difficulty in the interpretation of using peptidebased substrate or inhibitors having preferential but not strict specificity for particular
members of the caspase family. As a consequence it is well known that when used in a
biological sample which contains more than one activated caspase, it is generally impossible
to attribute the cleavage of one given aspartate-based peptide probe to one particular caspase
(see “Additional readings”). However, kinetics combining multiple probes and very careful
interpretation might deliver some information. For instance, it has been shown that DEVDbased fluorescent substrates are mainly cleaved by caspase-3 and caspase-7 and to a lesser
extent by caspase-6, -8, -9, and -10, but not (or poorly) by caspase-2 (nor by caspase-1, -4, 5). Second, It was also shown that LEHD-based fluorescent substrates are mainly cleaved by
caspase-4,-8,-9,-10, and to a lesser by caspase-6, but not (or poorly) by Caspase-2, -3,-7.
Third, the VDVAD-based fluorescent substrates were found to be mainly cleaved by caspase2 and by caspase-3, and to lesser extent -7, but not (or poorly) by caspase-1,-4,-5,-6,-8,-9,-10.
Consequently, the most simple interpretation for our results is that, at early time points, there
is no (or very little) caspase-1,-3,-4,-5,-6,-7,-8,-9,-10 activities (no DEVDase and LEHDase
activities), suggesting that the early VDVADase activity we detected can be mainly attributed
to caspase-2. A contrario, at later time point like 24h post-ischemia the co-detection of high
levels of VDVADase, DEVDase, and LEHDase activities renders impossible any attribution
of these activities to a specific caspase.
Supplementary Tables
Supplementary Table 1
Lesion volumes and statistical analysis of brain lesion volumes presented in Fig. 1K.
Five-day-old ibotenate-treated mice were subjected to combined administration of TRP601
and a caspase-2 siRNA. P values for the combined treatment were calculated either relatively
to control siRNA (si2Co) or to Casp2-specific siRNA (si2-a).
Supplementary Table 2
TRP601 does not modify developmental cell death in the rat neonatal brain
A. Developmental cell death in the brain in 8-day-old (P8) and 10-day-old (P10) rat treated or
not with TRP601. White columns: rat pups were given a single injection of TRP601 (1 mg/kg;
i.p.) at 24 or 72h prior sacrifice (at P8 or P10). Grey columns are vehicle-treated conditions.
TUNEL positive nuclei within brain sections were counted in the indicated regions (%
TUNEL positive cells ± s.e.m. are indicated) using fluorescence microscopy (n = 3 pups per
group). No significant variations were observed in all the brain area except for the
mediodorsal nucleus thalamus (Kruskall-Wallis P = 0.0238 between the groups at P8). B.
Seven-day-old rat pups (P7) were given a single injection (i.p. or i.v) of TRP601 (1 mg/kg)
and were sacrificed at 24 or 72h (white rows). Brain sections from 8-day-old (P8) or 10-dayold (P10) rats were processed for TUNEL. Grey rows are vehicle-treated conditions indicative
of basal cell death at the indicated time. Only plate 12 according to Paxino’s atlas (level of
anterior commissure) was scored for TUNEL staining. Number of TUNEL positive cells (±
s.e.m.) is indicated for total hemisphere versus cortical areas. No significant cell death
variations were observed between TRP601-treated (white rows) and untreated (grey rows)
pups, either at P8 or P10.
Supplementary Table 3
Full list of proteins used in TRP601 in vitro profiling
Full list of enzymes, peptide and non-peptide receptors, nuclear receptors, ion channels or
amine transporters. Binding (Hill coefficients) or enzyme activities (IC50 values) were
determined and found not to be significantly affected by 10 µM TRP601 (Cerep, Seattle, WA,
USA). b = baboon; c = cat; d = dog; h = human; ham = hamster; m = mouse; p = pig; r = rat.
Supplementary material and methods
Chemistry
Scheme for synthesis of TRP601
O
O
O
N
a
OH
N
N
H
O
100%
1
OMe
BocHN
98%
O
BocHN
N2
BocHN
86%
O
4
O
H
N
BocHN
97%
O
Br
BocHN
95%
OMe
F
O
f
O
O
TFA.H2N
99%
F
H
N
TFA.H2N
95%
O
F
8
O
O
i
BocHN
OBn
100%
O
10
O
11
O
H
N
N
H
OBn
O
12
OMe
j
O
N
N
H
H
N
N
H
O
O
14
l
O
H
N
O
O
k
TFA.H2N
OBn
68%
O
F
O
7
h
OBn
O
9
O
e
6
g
OH
OMe
O
5
BocHN
3
O
d
O
100%
OMe
O
c
OH
OH
N
H
2
OMe
O
N
b
OtBu
N
H
O
OMe
88%
O
H
N
OBn
O
OMe
13
63%
O
O
N
N
H
H
N
O
O
N
H
O
OMe
15
a
H
N
O
O
m
OH
51%
O
N
N
H
H
N
O
N
H
O
O
H
N
O
O
O
N
H
F
O
O
F
O
16 (TRP601)
(a) H-Val-OH.HCl, BOP, HOBt, DIEA, MeCN, r.t., 3 h; (b) TFA,/DCM, reflux, 3 h; (c) NMM, ClCO2Et, THF, 0°C, 30 min, then
N2/Et2O, overnight; (d) THF/Et2O, HBr/AcOH, 0°C, 30 min; (e) 2,6-F2-C6H3OH, DMF, KF, 0°C, 3 h; (f) TFA/DCM, r.t., 90 min;
(g)H-Ala-OBn.HCl, BOP, HOBt, DIEA, MeCN, r.t., 2 h; (h) TFA/DCM, r.t., 2.5 h; (i) Boc-Asp(OMe)-OH, BOP, HOBt, DCM, r.t., 3 h;
(j) TFA/DCM, r.t., 5 h; (k) 3, BOP, HOBt, DIEA, DCM, r.t., 3 h; (l) Pd/C, MeOH/MeCN, H2, r.t., 3 days; (m) 8, HBTU, HOBt, DMF,
r.t., 2 h.
Diazomethane was prepared in a Mini-Diazald apparatus (Aldrich) using the reported procedure of H.B. Hopps.
Mass spectra (positive mode) were recorded using a linear MALDI-TOF instrument (Bruker Bremen) using cyano-4-hydroxycinnamic acid as matrix. 1D NMR spectra were recorded in CDCl 3 or DMSO-d6 on a 300 MHz
spectrometer. Analytical HPLC was recorded at 30°C on a RP-C18 column (8 m, 3.9 x 150 mm) using linear
gradients (HPLC[A] or HPLC[B]) at a flow rate of 1.2 mL/min for 20 min with UV detection at 214 nm. These
gradient conditions were follow: HPLC[A] = 0-100% A/B; HPLC[B] = 30-100% A/B (where solvents A and B
are: A = 0.1% TFA in H2O and B = 0.08% TFA in MeCN). Semi-preparative RP-HPLC purification was
performed on a Nucleosil 250/10 300-7 C18 column. Flash chromatography purifications were performed in
silica gel 60 F254 (63-200 m mesh) using the indicated eluents. The spots were detected with UV lamp at 254
nm and/or with the Ninhydrine test.
Synthesis of quinolin-2-carbonyl-Val-OtBu (2). A solution of quinaldic acid 1 (4.33 g, 25 mmol), H-ValOtBu.HCl (5.51 g, 26.25 mmol), BOP (11.06 g, 25.01 mmol), HOBt (3.83 g, 25.02 mmol), DIEA (21.8 ml,
125.16 mmol) in MeCN (100 ml) was stirred at room temperature for 3 h, then evaporated. The residue was
dissolved in EtOAc (100 ml) and the solution was successively washed with a saturated solution of NaHCO3 (3 x
75 ml), a solution of KHSO4 1N (2 x 75 ml), brine (1x 75 ml) and water (1 x 75 ml). The organic phase was
dried over Na2SO4, then filtered, and finally evaporated under reduced pressure to afford a pale-yellow paste
which solidified to white solid on standing at 4°C (8.2 g, 100%). HPLC[B] Rt = 16.7 min. 1H NMR (300 MHz,
CDCl3)  1.05 (d, J = 7.0 Hz, 6H), 1.51 (s, 9H), 2.34 (m, 1H), 4.70 (dd, J = 4.6, 9.3 Hz, 1H), 7.60 (m, 1H), 7.76
(m, 1H), 7.86 (m, 1H), 8.16 (m, 1H), 8.29 (m, 2H), 8.77 (d, J = 8.8 Hz, 1H). 13C NMR (75 MHz, CDCl3)  17.9,
19.2, 28.1, 31.9, 57.8, 82.0, 118.9, 127.7, 127.9, 129.4, 130.0, 130.1, 137.4, 146.6, 149.5, 164.3, 171.0.
Synthesis of quinolin-2-carbonyl-Val-OH (3). A solution of quinolin-2-carbonyl-Val-OtBu 2 (7.76 g, 23.63
mmol) in DCM (50 ml) and TFA (25 ml) was refluxed for 3 h, and then evaporated. More DCM (50 ml) was
added, and evaporation was continued. This operation was repeated twice in order to get rid of the TFA. Pentane
and i-Pr2O was added to the residue and the mixture was allowed to solidify at –20°C. The excess solvent was
removed, and the residue was then evaporated to afford a white solid (6.41 g, 100%). HPLC[B] Rt = 10.1 min. 1H
NMR (300 MHz, DMSO-d6)  0.98 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.7 Hz, 3H), 2.29 (m, 1H), 4.49 (m, 1H),
7.73 (m, 1H), 7.88 (m, 1H), 8.09 (m, 1H), 8.17 (m, 2H), 8.60 (d, J = 8.2 Hz, 1H), 8.68 (d, J = 8.8 Hz, 1H), 13.03
(bs, 1H).
13
C NMR (75 MHz, DMSO-d6)  18.4, 19.6, 30.9, 57.7, 118.9, 128.6, 128.8, 129.5, 129.8, 131.1,
138.7, 146.3, 149.7, 164.1, 173.1.
Synthesis of Boc-Asp(OMe)-CHN2 (5). NMM (4.4 ml, 40.02 mmol) was added to an ice-cold solution of BocAsp(OMe)-OH (9.89 g, 40 mmol) in THF (200 ml). After stirring for 5 min, ClCO2Et (3.9 ml, 40.02 mmol) was
added and stirring was continued for 30 min at this temperature. The insoluble salt was filtered out, and the
filtrate was maintained at 0°C. Freshly prepared diazomethane (50 mmol) in Et 2O (140 ml) was added to the
unstirred solution of Boc-Asp(OMe)-OCO2Et. The mixture was allowed to rise up to room temperature over 1 h,
and then slowly stirred overnight. The excess diazomethane was destroyed by addition of AcOH (some drops).
The solution was successively washed with a solution saturated NaHCO3 (100 ml), a solution of KHSO4 1N (100
ml) and brine (100 ml), and then dried over Na 2SO4 and filtered. The organic was then evaporated under reduced
pressure to afford a pale-yellow solid (10.63 g, 98%). HPLC[A] Rt = 13.4 min. 1H NMR (300 MHz, CDCl3) 
1.43 (s, 9H), 2.72 (m, 1H), 2.98 (m, 1H), 3.66 (s, 3H), 3.73 (s, 1H), 4.51 (m, 1H), 5.65 (m, 1H).
13
C NMR (75
MHz, CDCl3)  28.3, 35.5, 52.0, 52.7, 53.8, 80.5, 155.2, 172.0, 193.1.
Synthesis of Boc-Asp(OMe)-CH2Br (6). A solution of diazoketone 5 (7.45 g, 27.46 mmol) in Et2O (75 ml) and
THF (75 ml) was cooled down to 0°C and a solution HBr 48% (25 ml) and AcOH (25 ml) in Et 2O (50 ml) and
THF (50 ml) was added over 30 min under stirring. The mixture was then diluted with EtOAc (100 ml) and then
successively washed with brine (2 x 100 ml), saturated NaHCO3 (2 x 100 ml) and water (2 x 100 ml). The
organic phase was dried over Na2SO4 and evaporated under reduced pressure to afford a pale-yellow oil (8.08 g,
86%). 1H NMR (300 MHz, CDCl3)  1.42 (s, 9H), 2.75-3.01 (m, 2H), 3.25 (s, 3H), 4.18 (s, 2H), 4.68 (m, 1H),
5.64 (m, 1H). 13C NMR (75 MHz, CDCl3)  28.3, 32.5, 35.6, 52.2, 54.1, 80.8, 155.3, 171.9, 200.1.
Synthesis of Boc-Asp(OMe)-CH2O-2,6-F2C6H4 (7). A stirring solution of compound 6 (3.59 g, 11.07 mmol),
and 2,6-difluorophenol (1.51 g, 11.61 mmol) in DMF (30 ml) was cooled down to 0°C, and then treated with KF
(1.61 g, 27.71 mmol). After stirring for 3 h at this temperature, it was diluted with EtOAc (150 ml) and then
successively washed with saturated NaHCO3 (2 x 75 ml) and brine (2 x 75 ml). The organic phase was dried
over Na2SO4, filtered, and then evaporated under reduced pressure to afford a brown oil (3.93 g, 95%). HPLC[A]
Rt = 16.5 min. 1H NMR (300 MHz, CDCl3)  1.4 (s, 9H), 2.74-3.09 (m, 2H), 3.64 (s, 3H), 4.68 (m, 1H), 4.98 (m,
2H), 5.62 (m, 1H), 6.48-6.94 (m, 3H).
C NMR (75 MHz, CDCl3)  28.2, 36.6, 51.9, 52.6, 75.9, 80.5, 112.3,
13
123.2, 134.7, 153.7, 155.3, 171.5, 202.9.
Synthesis of TFA.H-Asp(OMe)-CH2O-2,6-F2C6H4 (8). TFA (5 ml) was added to a solution of compound 7
(0.4 g, 1.07 mmol) in DCM (5 ml). The mixture was stirred at room temperature for 90 min, and then
evaporated. The residue was redissolved in DCM and evaporated again. This operation was repeated twice to
afford a pale-yellow paste (0.41 g, 99%). HPLC[A] Rt = 11.7 min.
Synthesis of Boc-Val-Ala-OBn (10). DIEA (21.8 mL, 125.16 mmol) was added to a suspension of Boc-Val-OH
(5.43 g, 25 mmol), H-Ala-OBn.HCl (5.50 g, 25.5 mmol), HOBt (3.90 g, 25.5 mmol) and BOP (11.28 g, 25.5
mmol) in MeCN (100 ml). The mixture was stirred at room temperature for 2 h, and then evaporated. The
residue was dissolved in EtOAc (250 ml). The resulting solution was then successively washed with a saturated
solution of NaHCO3 (3 x 100 ml), brine (2 x 100 ml), a solution of KHSO 4 1N (2 x 100 ml), and water (1 x 100
ml). The organic phase was then dried over Na 2SO4, and finally evaporated under reduced pressure to afford a
white solid (9.19 g, 97%). HPLC[A] Rt = 16.1 min. 1H NMR (300 MHz, CDCl3)  0.90 (d, J = 6.9 Hz, 3H), 0.94
(d, J = 6.9 Hz, 3H), 1.41 (d, J = 7.3 Hz, 3H), 1.43 (s, 9H), 2.10 (m, 1H), 3.93 (m, 1H), 4.63 (m, 1H), 5.16 (m,
2H), 6.50 (d, J = 6.8 Hz, 1H), 7.34 (m, 5H).
C NMR (75 MHz, CDCl3)  17.7, 18.3, 19.2, 28.3, 31.0, 48.1,
13
59.8, 67.2, 79.9, 128.2, 128.5, 128.6, 135.3, 155.8, 171.1, 172.5.
Synthesis of TFA.H-Val-Ala-OBn (11). TFA (25 ml) was added to a solution of compound 10 (10 g, 26.42
mmol) in 25 ml of DCM. The mixture was stirred for 2.5 h, and then evaporated. The residue was redissolved in
DCM and evaporated again. This operation was repeated three more times to afford a white solid (9.85 g, 95%).
HPLC[A] Rt = 11.5 min. 1H NMR (300 MHz, DMSO-d6)  0.90 (d, J = 7.0 Hz, 3H), 0.92 (d, J = 7.0 Hz, 3H), 1.35
(d, J = 7.3 Hz, 3H), 2.05 (m, 1H), 3.61 (m, 1H), 4.43 (m, 1H), 5.13 (s, 2H), 7.36 (m, 5H), 8.19 (s, 3H), 9.92 (d, J
= 6.6 Hz, 1H).
C NMR (75 MHz, DMSO-d6)  17.2, 18.0, 18.6, 30.2, 43.3, 57.6, 66.6, 128.4, 128.6, 128.9,
13
136.2, 168.3, 172.3.
Synthesis of Boc-Asp(OMe)-Val-Ala-OBn (12). DIEA (13.1 ml, 75.2 mmol), was added to a solution of BocAsp(OMe)-OH (3.71 g, 15 mmol), compound 11 (5.93 g, 15.1 mmol), BOP (6.68 g, 15.1 mmol), , HOBt (2.31 g,
15.1 mmol) in DCM (150 ml). The mixture was stirred at room temperature for 3 h, and then successively
washed with a saturated solution of NaHCO3 (4 x 100 ml), a solution of KHSO4 1N, brine (1 x 100 ml) and
water (2 x 100 ml). The organic phase was dried over Na2SO4, filtered and evaporated to afford a white solid
(7.61 g, 100%). HPLC[A] Rt = 15.9 min. 1H NMR (300 MHz, DMSO-d6)  0.77 (d, J = 6.6 Hz, 3H), 0.82 (d, J =
6.6 Hz, 3H), 1.30 (d, J = 7.3 Hz, 3H), 1.38 (s, 9H), 1.94 (m, 1H), 2.70-2.83 (m, 2H), 3.57 (s, 3H), 4.19-4.35 (m,
3H), 5.11 (s, 2H), 7.32-7.37 (m, 6H), 7.45 (d, J =9.0 Hz, 1H), 8.50 (d, J = 6.6 Hz, 1H).
Synthesis of TFA.H-Asp(OMe)-Val-Ala-OBn (13). TFA (20 ml) was added to a solution of compound 12 (6.0
g, 11.82 mmol) in DCM (20 ml). The mixture was stirred at room temperature for 5 h, and then evaporated. The
residue was redissolved in DCM and evaporated again. This operation was repeated three times. The resulting
residue was triturated in iPr20 (50 ml), filtered, and dried under reduced pressure to afford a white solid (5.43 g,
88%). HPLC[A] Rt = 12.6 min. 1H NMR (300 MHz, DMSO-d6)  0.85 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.9 Hz,
3H), 1.31 (d, J = 7.2 Hz, 3H), 1.99 (m, 1H), 2.83 (m, 2H), 3.64 (s, 3H), 4.20-4.34 (m, 3H), 5.11 (s, 2H), 7.35 (m,
5H), 8.35 (s, 3H), 8.51-8.54 (m, 2H).
Synthesis of quinolin-2-carbonyl-Val-Asp(OMe)-Val-Ala-OBn (14). Prepared from compound 3 (2.09, 7.67
mmol), compound 13 (4.0 g, 7.67 mmol), BOP (3.41 g, 7.7 mmol), HOBt (1.18 g, 7.7 mmol), DIEA (6.5 ml,
37.3 mmol) in DCM (100 ml) using the procedure described for the synthesis of compound 12. The desired
product was obtained as a white solid (3.45 g, 68%). HPLC[A] Rt = 17.3 min. 1H NMR (300 MHz, DMSO-d6) 
0.74 (d, J = 7.0 Hz, 3H), 0.80 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 7.1 Hz, 3H), 0.95 (d, J = 7.0 Hz, 3H), 1.30 (d, J =
7.3 Hz, 3H), 1.92 (m, 1H), 2.15 (m, 1H), 2.56-2.82 (m, 2H), 3.54 (s, 3H), 4.19 (m, 1H), 4.33 (m, 1H), 4.51 (m,
1H), 4.71 (m, 1H), 5.10 (s, 2H), 7.35 (m, 5H), 7.66 (d, J = 8.8 Hz, 1H), 7.74 (m, 1H), 7.88 (m, 1H), 8.10 (d, J =
8.1 Hz, 1H), 8.16-8.20 (m, 2H), 8.41 (d, J = 6.5 Hz, 1H), 8.60 (d, J= 8.4 Hz, 1H), 8.68-8.72 (m, 2H).
Synthesis of quinolin-2-carbonyl-Val-Asp(OMe)-Val-Ala-OH (15). 70 mg of 10% Pd/C was added to a
solution of compound 14 (3.45 g, 5.22 mmol) in a solution of MeOH (100 ml) and MeCN (50 ml). The mixture
was degassed, stirred under H2 atmosphere for three days, filtered over celite, and then evaporated. The residue
was then chromatographed on silica gel in cyclohexane/EtOAc/AcOH (30/70/5) to afford a white solid (1.88 g,
63%). HPLC[A] Rt = 14.5 min.
Synthesis of quinolinyl-2-carbonyl-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH2O-2,6-F2C6H3 (16). DIEA (871
l, 5 mmol) was added to a solution of compound 15 (0.572 g, 1 mmol), compound 8 (0.426 g, 1.1 mmol),
HBTU (0.38 g, 1 mmol) and HOBt (0.153 g, 1 mmol) in DMF (10 ml). The solution was stirred for 2h at room
temperature and then evaporated. The residue was dissolved in EtOAc (150 ml) and then successively washed
with a solution of KHSO4 1N (3 x 50 ml), saturated NaHCO3 (3 x 50 ml) and brine (1 x 50 ml). The organic
phase was dried over Na2SO4 and then evaporated to give a pale-yellow solid (0.78 g). 100 mg of this crude
product was purified by semi-preparative RP-HPLC the above-mentioned conditions to afford of a white solid
after lyophilization (54 mg, 51%). HPLC[A] Rt = 16.6 min. 1H NMR (300 MHz, DMSO-d6)  0.71-0.78 (m, 6H),
0.90 (d, J = 6.7 Hz, 3H), 0.93 (d, J = 6.1 Hz, 3H), 1.18 (d, J = 6.6 Hz, 3H), 1.89 (m, 1H), 2.12 (m, 1H), 2.57–2.66
(m, 2H), 2.74-2.84 (m, 2H), 3.53 (s, 3H), 3.56 (s, 3H), 4.14 (m, 1H), 4.21 (m, 1H), 4.48 (m, 1H), 4.56-4.71 (m,
2H), 5.01 (s, 2H), 7.06 (m, 3H), 7.62 (m, 1H), 7.71 (m, 1H), 7.86 (m, 1H), 8.07-8.16 (m, 4H), 8.46 (d, J = 7.7
Hz, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.65-8.68 (m, 2H). MS (m/z) 850 [M+Na+], 866 [M+K+].
Molecular modeling
Molecular modeling with Insight II. Inhibitors were manually modified by Biopolymer module of Accelrys. The
enzyme atoms were fixed and the inhibitor minimalized with the Discover module using 2000 steps of
conjugated gradient until the RMS was < 0.0001 Kcal/mol.Ǻ. Non covalent energy values (Van der Waals and
coulombic forces) were then calculated with Insight II module. Molecular modeling with Discovery Studio 2.0.
A binding site with radius 6 Å around the inhibitor was defined and the interaction energy (Van der Waals forces
and coulombic forces) were calculated using Discovery Studio 2 (Accelrys). Inhibitors were manually
constructed using the backbone of the original inhibitor and was minimized using a CHARMm forcefield. The
obtained inhibitor was introduced in the defined binding site and minimized. After minimization, interaction
energies were calculated.
Animal experiments
Studies were conducted in compliance with Animal Health regulations (Council Directive No. 86/609/EEC of
24th November 1986). Animals were acclimated for a period of at least 2-3 days before the beginning of the
treatment period. The required number of animals were selected according to body weight and clinical condition
and randomly allocated to groups, so that the average body weight in each group is similar. Animals were
identified individually by a color mark. Pups stayed with their dams during the study. The animal room
conditions were rigorously controlled: temperature: 21°C; light/dark cycle: 12h/12h. The animals were housed
with free access to food and water. Rat pups were weaned at 3 weeks of age. Animal housing, care and
application of experimental procedures were conducted according to the European Community guidelines for the
care and use of experimental animals.
Unilateral transient focal ischemia in rat neonates (neonatal stroke model)
7-day-old Wistar rats (Janvier, Le Genest-St-Isle, France) were anesthetized with chloral hydrate (i.p., 350
mg/kg), and subjected to focal ischemia with reperfusion as described
10,41
. Then, animal were killed at
appropriate time points from reperfusion to 48h after reperfusion or at 21 days post-reperfusion, and their brains
were removed. The infarct lesion (pale zone) was visually scored by a blinded observer. The brains were then
fixed for 2 days in 4 % PFA. 50 μm coronal brain sections were cut on a cryostat and collected on gelatin-coated
slides. Lesion areas were measured on Cresyl violet–stained sections using an image analyzer (Nikon SMZ680),
and distances between respective coronal sections were used to calculate the infarct volume
10, 41
. Alternatively,
brain sections were processed for GFAP immunoreactivity (Cy3-anti GFAP antibody) or DNA strand breaks
(TUNEL assay) using the in situ Fluorescein Cell Death Detection Kit (Roche, Meylan, France) and
counterstained with Hoechst 33342. In vivo administration of propidium iodide was done intrajugularly (100 µl;
10 mg/kg) as described by Unal Cevik and Dalkara in 2003, with slight modifications.
Unilateral focal ischemia with hypoxia in rodent neonates (hypoxia-ischemia model)
Eight-day-old rats or alternatively 9-day-old mice were exposed to hypoxia-ischemia (HI). HI was induced by
unilateral ligation of the left carotid artery followed by hypoxia (7.8% O 2 for rats and 10% O2 for mice, 36°C)
for 50 min
32,45
. Three days after HI, the pups were killed and perfused. The brains were carefully removed,
embedded in paraffin and cut in 6 µm thick sections on a microtome. Infarction volume and tissue loss
percentage were measured after MAP2 staining at each anatomical level. Infarction was measured as the MAP2negative staining area in the ipsilateral hemisphere, and the tissue loss was measured by subtracting the area of
MAP2-positive staining in the ipsilateral hemisphere from the area of the contralateral hemisphere. The Cavalieri
principle, where V=ΣAPt, was used to calculate the infarction volume and tissue loss (V as the total volume, ΣA
as the sum of the areas measured, P as the inverse of the section sampling fraction, and t as the section
thickness).
NMDA receptor-induced excitotoxicity in newborn mice
All ibotenate injections were administered on the fifth postnatal day (P5), as described previously
. The pups
32
were decapitated at different times after intracerebral ibotenate injection, and the brains were fixed in 4%
formaldehyde. After embedding in paraffin, 15 µm sections were cut on the coronal plane, from the frontal to the
occipital pole. Every third section was stained with cresyl-violet and the total volume of the lesion was measured
using the Neurolucida software-controlled computer system (MicroBright- Field Europe, Magdeburg, Germany).
Sections adjacent to those used for cresyl violet staining were used for immunohistochemistry. Sections
containing excitotoxic lesions were reacted with anti-GFAP (Dako, Glostrup, Denmark) or biotinylated
Griffonea Simplicifolia I isolectin (Vector, Burlingame, CA). Labeled antigens were detected using avidin-biotin
horseradish peroxidase kits (Vector), as described previously 32. Quantitative RT-PCR analysis was performed as
described 32.
Isolation of brain mitochondria
Seven-day-old Wistar rats (12-18g) were decapitated. Cortical areas were microdissected and rapidly immersed
in ice-cold mitochondrial isolation buffer (0.25M sucrose, 0.5mM K +-EDTA, 10mM Tris-HCl, pH7.4). Samples
were homogenised in 12% Percoll in MIB using Dounce homogenizers with glass pestles. Mitochondria were
then isolated by differential centrifugation on Percoll gradients. For cytochrome c release assays, isolated brain
mitochondria were incubated with recombinant Bid or human recombinant Casp2 in a buffer containing: 0.2 M
sucrose, 5 mM succinate, 10 mM MOPS, 10 µM EGTA, 2 µM rotenone and 1 mM H 3PO4 (pH 7.4). For
combined rCasp2 plus Bid treatments, rCasp2 was incubated 30 min at 37°C with Bid in a buffer (containing 50
mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 1 mM EDTA and 10% glycerol) prior addition
to isolated mitochondria. Western Blot analysis was performed on concentrated supernatant after centrifugation
(10 000 g; 10 min), using mouse anti-cytochrome c antibody (Clone 7H8-2C12, BD Pharmingen, 1/500) and
rabbit anti-Bid antibody (AB1730, Chemicon International, 1/500). Bid and human recombinant Caspase-2 were
gift from Dr. Xuejun Jiang.
Brain biochemistry
Brain samples (1-2 mm3) were processed at 4 °C into a glass tube containing 800 µl of a buffer containing:
HEPES 10 mM pH 7.4, KCl 42 mM, MgCl 2 5 mM, DTT 1 mM, CHAPS 0.5%, EDTA 0.1 mM, PMSF 1 mM,
leupeptin 1 µg/ml, pepstatin A 1 µg/ml, cytochalasin B 1 µM, chymopapain 10 µg/ml, antipain 1 µg/ml. Tissue
were then crushed using a glass Potter, then frozen for 24h (-80°C), prior to thawing and elimination of debris
(10 min, 4°C, 2000g). 100 µg of supernatant was diluted in buffer A and incubated for 2-3 hours at 37°C in the
presence of the above-described caspase substrates (50 µM). Caspase activities were monitored by
spectrofluorimetry (ex= 380 nm; em= 465 nm). Alternatively, samples (50 µg of protein) were subjected to
western blot analysis with monoclonal anti-caspase-2 antibodies (1:1000, clone 11B4, Alexis Biochemicals, or
Ab-1, Lab Vision). Mitochondrial and cytosolic fractions from similar tissue samples were subjected to
cytochrome c (1:1000, clone 6H2B7, BD Pharmingen), porin (1:1000, #4866, Cell Signaling) and actin (1:5000,
clone 1B4, Sigma) detection by western blot analysis.
Blood gas analyses
Heparinized capillary tubes were filled (~100 µl) with arterial blood collected by direct intraventricular puncture.
Arterial pH, partial pressure of oxygen and carbon dioxide (PaO2, PaCO2), and concentration of bicarbonate
(HCO3-) were measured using a blood gas analyzer (Roche OMNI, Manheim Germany).
Hemodynamic evaluations
Heart function and intracranial blood flow velocities were analyzed on anesthetized (chloral hydrate: i.p. 350
mg/kg) and heated rat pups (~37 °C) using a commercially available echocardiograph (Vivid 7, General Electric
Healthcare) with a linear 12 MHz probe. Echography and Doppler exploration were performed on anesthetized
rat pups before and one hour after TRP601 (1 mg/kg) or vehicle injection. Left ventricular dimensions of the
heart, and systolic and diastolic left ventricular functions were evaluated. Cerebral perfusion was evaluated by
measuring the resistive index (RI) of anterior cerebral arteries using transcranial color-coded duplex Doppler
sonography.
Toxicology rationale
Chromosome aberration assays in cultured human lymphocytes and bacterial reverse mutation (Ames) tests with
and without S9 fraction demonstrated no genetic toxicity. TRP601 had no hemolytic potency, no effect on
bleeding time, no impact on platelet aggregation, and low toxicities on various cultured primary cells. We then
investigated toxicology in adult animals, together with dedicated studies in rodent and non-rodent neonates (see
below).
Toxicology in adult animals
TRP601 was found to be not genotoxic when evaluated in a battery of in vitro and in vivo assays, and showed no
antigenic response in rat. In multiple-dose GLP regulatory studies conducted at the Centre International of
Toxicology (CIT, Evreux, France) in adult dogs no TRP601-related cytotoxic effects were observed following
intravenous administration for 14 days at doses up to 3 mg/kg/day (NOAEL). Using a clinically acceptable
formulation, a good vascular and perivascular local tolerance was found in rabbit ear (CIT, Evreux, France).
Toxicology in newborn dogs
Studies with multiple-dose intravenous injections (once every 3 days during a 2-week period, starting on postnatal day 1 in newborn Beagle dogs showed the no observed effect level (NOEL) at 15 mg/kg/injection once
every 3 days.
Repeated TRP601 administrations to newborn Beagles were carried out at CIT (Centre
International of Toxicology, Evreux, France) in compliance with the following Good Laboratory Practice
regulations: OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17 and
all subsequent OECD consensus documents, Directive 2004/10/EC of the European Parliament and of the
Council of 11 February 2004 on the harmonization of laws, regulations and administrative provisions relating to
the application of the principles of good laboratory practice and the verification of their applications for tests on
chemical substances (OJ No. L 50 of 20 February 2004).
Toxicology in newborn rats
Single-dose administrations of TRP601 to 7-day-old rat pups revealed low toxicity (DL50i.v.=60mg/kg;
DL30i.p.>200mg/kg). Single doses were administered to Wistar rat neonates (Janvier) at dose levels of 0
(vehicle), 1, 5, 10, 50, 100, 200 and 400 mg/kg (i.p., 25 ml/kg.) or 1, 5, 10, 20, 40, 60, 80 400 mg/kg.(i.v., 5
ml/kg, intrajugular). Offspring were evaluated after a single dose by assessment of gripping and tail reflex on
days 1 (at 1h and 2h post injection), 2, 3, 5, 8, 11 and 15. Observations were made for maturation of physical
characteristics: eye opening, incisor eruption and ear unfolding. The body weight of each animal was recorded
daily. In addition, the Irwin procedure (54 items) was used to explore the behavioural, neurological and
autonomic profiles of pups. Each parameter was checked at least once a day (at the same time) for clinical
examination, mortality or signs of morbidity. Any animals showing signs of poor clinical condition were killed
by cervical dislocation under anaesthesia. At the end of the observation period, all rats were sacrificed under
anesthesia. Brain, thymus, lungs, heart, liver, kidneys and spleen were preserved in 4% PFA. Macroscopic post-
mortem examination of organs and further microscopic analysis of hematoxylin-eosin stained paraffin-embedded
sections were performed by blinded pathologist (Biodoxis, Romainville, France).
Safety pharmacology
In vivo cardiovascular safety evaluations (telemetry, ECG in vigil canines) were done by Porsolt & Partners
Pharmacology (PORSOLT S.A.S., France) and were completed with in-vitro Ikr assays (hERG) were done by
CEREP (Seattle, WA, USA) and in line with the ICH S7B guideline (www.ich.org). In vivo safety pharmacology
studies for CNS (Irwin tests, activimetry, Rotarod and Morris maze) and for respiratory function (whole body
plethysmograpy) were performed in conscious rats (by Porsolt & Partners Pharmacology, France), in line with
the ICH guideline S7A.
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