WO2005105829A2 - Caspase-2 inhibitors and their biological applications - Google Patents

Abstract

The invention relates to selective new caspase-2 inhibitors that recognize caspase-2 and prevent and block its activity based on the following backbone : 2-Quinolinylcarbonyl-L-Valinyl-L-Aspartyl (methyl ester)-L-Vanilyl-L-Alaninyl-L-Aspartyl (methyl ester) 2,6-difluorophenyl ester (SEQ1) and derivatives thereof, SEQ 1 corresponding to formulae (Ia) or (Ib). Application of said inhibitors for preventing and treating diseases involving caspase-2.

Description

CASPASE-2 INHIBITORS AND THEIR BIOLOGICAL APPLICATIONS The invention relates to caspase-2 inhibitors and their biological applications, particularly for the treatment of focal and global cerebral ischemia. I / FIELD OF THE INVENTION The invention is in the field of medicinal biology and chemistry and relates to novel compounds, and pharmaceutical compositions, that inhibit pro-apoptotic caspase-2 (Nedd-2; Ich-1), to treat diseases and injuries where caspase-2 activity is implicated. More precisely : (a) The invention relates to means, methods and products, for blocking, preventing or treating cell death, particularly in vivo cerebral cell death. (b) The invention relates to the discovery that caspase-2 is an upstream major checkpoint for inhibition of neuronal cell death (especially apoptosis) in vitro and in in vivo pathological situation including cerebral hypoxia-ischemia (H-I) injuries and strokelike situations that result in brain damages (in neurons or other non-neuronal cells) : for example, MCAO (Middle Cerebral Artery Occlusion), global (as a consequence of blood flow reduction or hypoxia during cardiac arrest or cardiovascular injuries) or focal, transient or permanent, adult or neonatal H-I, ischemia with or without hypoxia and/or hypoglycaemia, H-I with or without reperfusion. (c) The invention relates to the discovery that selective caspase-2 inhibitors (for example, Qco-VDVAD-dfp) prevents or decreases in vivo cerebral cell death (d) The invention relates to the discovery that selective caspase-2 inhibitors (for example, Qco-VDVAD-dfp) prevents or decreases cerebral cell death in in vivo pathological situations including ischemic injuries following focal transient neonatal H-I (e) The invention relates to the discovery that selective caspase-2 inhibitors (for example, Qco-VDVAD-dfp) prevents or decreases cerebral cell death in in vivo pathological situations including cerebral global adult ischemia following blood flow reduction hypoxia during cardiac arrest or cardiovascular injuries.

(f) The invention relates to new applications for specific new caspase-2 inhibitors including local (intracerebroventricular, intrahippocampal, for example) delivery or systemic (intraperitoneal, intravenous....) administration to reduce cerebral cell death during pathological situations in which blood flow and oxygen pressure are disturbed, i.e. cerebral (transient or permanent) focal or global ischemia.

II / DATA SUPPORTING THE INVENTION The invention relates to means, methods and products, for blocking preventing or treating cell death, particularly neuronal cell death. Neuronal cell death occurs during embryogenesis to remove excess of neurons to ensure appropriate pre- and post-synaptic connections and to allow formation of a functional adult brain.Besides post-mitotic death related to normal ageing, environmental or genetic mutational factors may induce neuronal death in the adult human during acute injuries (for instance, hypoxia-ischemia, stroke, spinal cord injury, trauma) or chronic neurodegenerative diseases. Cell death associated with these disorders may occur by three distinct mechanisms, exhibiting morphological and biochemical features of necrosis, autophagy or apoptosis. Both physiological and pathological neuronal deaths are often associated with defective apoptosis regulation and signalling pathways that lead to this active cell suicide mechanism may be divided in cysteinyl aspartate-specific protease (caspase)-dependent versus caspase- independent pathways in mammalian cells. Neuronal apoptosis is an active cell suicide mechanism that can be divided into sequential phases, including initiation, decision, execution, and degradation. This cascade of events is driven by the activation of a specific machinery, that involve both the activation of cysteine-dependent aspartate-specific proteases (caspases) and the mitochondrion which may act as a decisive (or amplifier) regulatory organelle. Indeed, mitochondrial alterations include loss of mitochondrial inner membrane electrochemical gradient (ΔΨ_m) and release of apoptogenic factors such as cytochrome c, Smac/Diablo and Apoptosis Inducing Factor. Once released from mitochondria, these effectors trigger caspase-dependent and/or caspase- independent cytoplasmic and nuclear dismantling. Hence, mitochondrial factors combined with caspases contribute to the degradation phase of apoptosis, resulting in cell shrinkage, nuclear condensation, emission of apoptotic bodies and appearence of "eat-me" signals such as phosphatidyl-serines translocation to the outer leaflet of the plasma membrane before phagocytosis.

The respective contribution of mitochondria, caspases and other events during in vitro or in vivo neuronal apoptosis is still not elucidated, particularly with respect to a ischemic or stroke-likeinjuries. As illustrated, the inventors provide useful means and molecules enabling to inhibit caspase-2 activity in vitro and in cellula, by using specific in vitro caspases assays and serum deprivation in neuronal culture as an experimental model relevant to in vivo ischemia. The selective new caspase-2 inhibitors of the invention, that recognize caspase-2 and prevent and block its activity, are based on the following backbone : 2-Quinolinylcarbonyl-i- Valinyl-J-Aspartyl (methyl ester)-Z-Vanilyl-Z-Alaninyl-E-Aspartyl (methyl ester) 2,6- difluorophenyl ester (SEQ1) One preferred molecule corresponds to SEQ 1. Other preferred molecules comprise structures II two XIX as indicated, particularly, in

Tables 1 to 14. Another object of the invention is to provide novel products (molecules or formulations) that inhibit pro-apoptotic caspase-2 activity and induce cytprotection. Another object of the invention is to provide products and pharmaceutical compositions and methods of treatments of diseases and injuries where caspase-2 is involved. The inventors have developped pharmacological specific peptides, preferentially but not exclusively pentapeptide-based molecules, for direct inhibition of caspase-2 activity in order to attenuate in vitro and in vivo cell death mediated by caspase-2. Such inhibitors are capable of preventing or blocking caspase-2 activity in cell death. Said cells are neurons, or neuronal cells, or non neuronal cells. The above defined inhibitors are useful for in vivo inhibition of caspase-2 activity to provide neuroprotective or cerebroprotective effect. They are also useful for in vivo inhibition of caspase-2 activity to provide cytoprotective effect. According to one aspect, the invention relates to method for preventing cell death in the brain following (global of focal) ischemic injuries. As illustrated by the examples, the invention provides means, methods and products, for blocking or preventing or treating cell death, particularly in the injured (ischemic) brain. As illustrated, the inventors demonstrate that specific caspase-2 inhibitor (single intraperitoneal injected dose) induces important infarct reduction in neonatal transient hypoxia-ischemia brain injury. Infarct volume reduction was obtained when caspase-2 inliibitor administration before or after ischemic onset. Cytoprotective effects provided by the inhibitor are not mediated by temperature (hypo or hypertheπnic) regulation. But in vivo caspase-2 activity or caspase-2 processing is abolished after injection of the inhibitor. As illustrated, the inventors demonstrate that specific intracerebroventricular (ICV) administration of caspase-2 inliibitor (single dose) results in partial increase of Cresyl-Violet intensity staining, absence of Fluoro Jade B intake in damaged cells, an improvement or preservation of nuclear morphology (Hoechst 33342) as well as a considerable improvement in the behaviour of inhibitor-treated rats (refeeding post-ischemia; higher score in tests regarding spontaneous activity and reactivity). Cytoprotective and behavioural effects provided by the inhibitor are not mediated by temperature (hypo or hyperthermic) regulation. According to another aspect, the invention also relates to molecules capable of preventing caspase-2 activity and pharmaceutical compositions useful for treating diseases and injuries where caspase-2 is involved. The synthetized molecules are introduced in human or animal, under conditions for caspase-2 inhibition. The introduction step comprises chemical modifications of molecules as suitable earners or is performed by injection. Accordingly, this is another object of the invention to provide pharmaceutical compositions containing specific caspase-2 inhibitors. The pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one caspase-2 inhibitor as above defined, in association with a pharmaceutically acceptable carrier. Administration of inhibitors is performed (but is not restricted to) by oral, nasal, local (intracerebroventricular, intrahippocampal, or other intracerebral delivery, intracerebral implantation of instrumentation for mechanical delivery such as of Gelfoam® impregnated with compounds or pharmaceutical compositions, for example) or systemic (intraperitoneal, intravenous...) route, or intracerebral delivery or as aerosol to reduce in vivo cerebral cell death. The invention also relates to the use of said caspase-2-inhibitors for making drugs for preventing or treating pathologies involving caspase-2 activity. The pharmaceutical compositions of the invention are particularly useful for preventing, reducing and treating pathologies with cell death, particularly in H-I injuries and stroke-like situations brain injuries: for example, global or focal, transient or permanent, adult or neonatal H-I (ischemia with or without hypoxia / hypoglycaemia) with origin at cerebral or heart level, with or without reperfusion, or MCAO (Middle Cerebral Artery Occlusion). The pharmaceutical compositions of the invention are particularly useful for: preventing and/or treating apoptosis during chronic degenerative diseases e.g. neurodegenerative disease including Alzheimer's disease, Huntingtons' disease, Parkinsons' disease, Multiple sclerosis, amyotrophic lateral sclerosis, spinobulbar atrophy, prion disease, dementia, or preventing and/or treating retinal pericyte apoptosis, retinal neurons apoptosis glaucoma, retinal damages resulting from local ischemia, diabetic retinopaty, or preventing and/or treating epilepsy, or preventing and/or treating apoptosis during spinal cord injury, or to prevent and/or treat apoptosis resulting from traumatic brain injury, retinal ischemia or preventing and/or treating apoptosis during pathological situations of focal cerebral ischemia or providing cerebroprotective effect, or preventing and/or treating cytotoxic T cell and natural killer cell-mediated apoptosis associated with autoimmune disease and transplant rejection, or preventing and/or treating cell death of cardiac cells including heart failure, cardiomyopathy, viral infection or bacterial infection of heart, myocardial ischemia, myocardial infarct, and myocardial ischemia, coronary artery by-pass graft, or preventing and/or treating mitochondrial drug toxicity e.g. as a result of chemotherapy or HIV therapy, preventing and/or treating cell death during viral infection or bacterial infection, or preventing and/or treating inflammation or inflammatory diseases, inflammatory bowel disease, sepsis and septic shock, or preventing cell death from follicule to ovocyte stages, from ovocyte to mature egg stages and sperm (for example, methods of freezing and transplanting ovarian tissue, artificial fecondation), or preserving fertility in women and men after chemotherapy, or preserving fertility in females and males animals, or to prevent and/or treat, macular degenerescence and glaucoma, or to prevent and/or treat acute hepatitis, chronic active hepatitis, hepatitis-B, and hepatitis-C, or preventing and/or treating hair loss, and said hair loss due-to male-pattern baldness, radiation, chemotherapy or emotional stress, or treating or ameliorating skin damage (due to exposure to high level of radiation, heat, burns, chemicals, sun, and autoimmune diseases), or preventing cell death of bone marrow cells in myelodysplastic symdromes (MDS), or preventing and/or treating pancreatisis, or preventing and/or treating respiratory symdrome, or preventing and/or treating osteoarthitis, rheumatoid arthritis, psoriasis, glomerulonephritis, atheroscerosis, and graft versus host disease, or preventing and/or treating disease states associated with an increase of apoptosis, or preventing cell death in vegetals (for example: plants, flowers, thallophytes (mushrooms, seaweed) ... ) The term "treatment or treat" as used herein means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treat" can also mean prolonging survival as compared to expected survival if not receiving treatment. The invention also relates to method of treatment of pathologies with cell death, particularly ischemia and stroke injuries, comprising the administration of a therapeutically effective dose of a pharmaceutical composition such as above defined. Precised characteristics and advantages of the invention are given in the following data with reference to the figures, which represent: III / PEPTIDES AND PSEUDOPEPTIDES INHIBITORS OF CASPASE-2 1. Qco-VDVAD-dfp Qco-VDVAD-dfp (SEQ1) is a pseudopeptide (MW:827) mat inhibits selectively human recombinant caspase-2 in vitro (IC50= 80 nM) but not other cysteines-proteases. It combines pentapeptide VDVAD ((Z-Valinyl-J-Aspartyl-J-Vamlyl-Z-Alaninyl-Z-Aspartyl substituted on aspastate residues by O-methyl groups (OMe)) moieties, quinolinylcarbonyl and 2,6-difluorophenyl ester backbones. Its formula is la / lb according to the position of the substituted VD(OMe)VAD(OMe)-Oph on the quinoline nucleus: for example the 2- ■ Quinolinylcarbonyl-Z-Valinyl-Z-Aspartyl (methyl ester)-Z-Vanilyl-Z-Alaninyl-Z-Aspartyl (methyl ester) 2,6-difluorophenyl ester (II, n = 0).

la

lb

II 2-Quinolinylcarbonyl-Z-Valmyl-Z-Aspartyl (methyl ester)-L- anilyl--L-Alanmyl-Z-Aspartyl (methyl ester) 2,6-difluorophenyl ester = SEQ1

2. Modification of amino acid side chains and functional groups Other selective new caspase-2 inhibitors of the invention are based on structure III below :

III

wherein (i) the absolute configuration of each amino acid is either L or D ; (ii) R¹ and R² are a hydrogen atom, deuterium atom, C₁-20 aliphatic, substituted or unsubstituted aryl, cycloalkyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2- oxazolyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nSubstituted phenyl, (CH₂)_n(l- or 2- naphthyl). (CH₂)_nheteroaryl, CH₂N₂, CH₂Y, OH, OR, NH₂, NHR, NR₂, SR, COR, C0₂R, CONR₂₎ CH₂OCOR, CH2O-CO aryl, CH₂0-C(0) substituted aryl (ex: 2,6- dimethylbenzoyloxymethyl), CH₂0-C(0) substituted aryl, CH₂0-C(0) heteroaryl, CH₂0- C(O) substituted heteroaryl or CH₂OPOR₂ ; o

(iii) R³, R⁴, R⁵ and R⁶ are the side-chains of one of the twenty amino acids (excluding cysteine). For example, the pentapeptides can also be analogues of Leu-Asp-Glu-Ser-Asp. R³, R⁴, R⁵ and R⁶ are also C|.₂₀ aliphatic, cycloalkyl, naphthyl, substituted naphthyl, 2- benzoxazolyl, substituted 2-oxazolyl, (CH₂)_ncycloalkyl, (CH₂)nphenyl, (CH₂)_nsubstituted phenyl, (CH₂)„(1- or 2- naphthyl), (CH₂)_nheteroaryl, CH₂N₂, (CH₂)_nZ, CH₂OCOR, CH₂OCO (aryl), CH₂OCO (substituted aryl), CH₂OCO (heteroaryl), CH₂OCO (substituted heteroaryl) or CH₂OPOR₂ ; (iv) R⁷ is a hydrogen atom and R⁸ is R, U, CO(CH₂)_nNH(U), CO(CH₂)_nS(U) (v) U is (un)substituted (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, C₁.₂₀ straight chain or branched alkyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl, (CH₂)_n(l- or 2- naphthyl), (CH₂)_nheteroaryl, or a marker group such as fluorophore markers or markers useful in electronic microscopy; particularly, biotin, dinitrophenyl (DNP), iodoacetamides, DTNB, COR (ex. 2-quinolinylcarbonyl), COOR, CO(CH₂)_nNH(Z), Acridine derivatives (Red, yellow, orange ....), Fluorescein derivatives (fluorescein, FITC, FAM (carboxyfluorescein), 5-(and- 6)-carboxynaphthofluorescein, carboxyfluorescein, BCECF, naptofluorescein ), Oregon

Green® (2^,,7'-difluorofluorescein) dyes (Oregon Green® 488, Oregon Green® 514...), BODIPY® (4,4-difluoro-5,7-dimethyl-4-bora-3a.4a- diaza-^-indacene-3 -propionic acid) dyes

(BODIPY FL, BODIPY TMR, BODIPY TR, BODIPY 630/650, BODIPY 630/665 ),

Bimane, Coumarin derivatives (aminomethylcoumarin (AMC), AMCA, aminocoumarin, diethylaminocoumarin hydroxymethylcoυmarin; hydroxycoumarin, methoxycoumarin, AFC, ...), Cyanin derivatives (phycocyanin, allophycocyanin (APC), CY3.18, CY5.18, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7...), Erythiin/Phycoerytl rin derivatives (R-Phycoerythrin (PE), B- Phycoerythrin ...), APC PE-Cy conjugates (PE-Cy5 conjugates, PE-Cy7 conjugates, APC- Cy7 conjugates....), Calcein derivatives (calcein, SNAFL calcein....), DANS, DANSA, Cascade Blue, Lucifer yellow, NBD, Red 613, FluorX, Rhodamine derivatives(Rhodamine 123, Rhodamine 110, Rhodamine B, Rhodamine 6A, Rhodamine 6G, TRITC, X-Rhodamine, sulphorhodamin, Rliodamine Red-X, Lissamine™ rhodamine B, DHR, Rhodamine Green....), PerCP, Texas Red, TruRed, Alexa Fluo ® (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750....), Q-DOTs ™ derivatives (655,605, 585,525,...), SNARF, Zenon™ derivatives (Zenon™ Alexa Fluor® 350, Zenon™ Alexa Fluor® 488, Zenon™ Alexa Fluor® 555, Zenon™ Alexa Fluor® 594, Zenon™ Alexa Fluor® 647, Zenon™ Allophycocyanin, Zenon™ Biotin-XX, Zenon™ R-Phycoerythrin, ); NBD, Texas Red ®, QSY® dyes (QSY® 7, QSY® 9, QSY® 35, QSY® 21), Hoechst (33342, 33258), DAPI, Chromomycin A3, Mithramycin, , SYTOX (Blue, Green, Orange), Ethdium, Ethidium Bromide, 7-AAD, TOTO dyes, YOYO dyes, TO-PRO dyes, BOBO dyes, JO-PRO dyes, LO-PRO dyes, PO- PRO dyes, YO-PRO dyes, Thiazole Orange, Propidium Iodide (PI), LDS 751, Indo® dyes (Indo-1...), Fluo® dyes (Fluo-3...), DCFH, pNA, SYBR green II, SyPro (Orange, Ruby), EDANS, IR800, Dil, DiO, DiD, SNARF® derivatives, Fura dyes, QUIN dyes, NANOGOLD particules, NANOGOLD maleimide, AlexaFluor FluoroNANOGOLD, AlexaFluor FluoroNANOGOLD streptavidin, malachite green, Dabcyl, Dabsyl, Cascade yellow, dansyl, Dapoxyl, PyMPO, Pyrene, benzoxadiazole derivatives, strepavidin-/neutravidin-)biotin- labeled fluorescent microspheres, CMNB-caged fluorescein conjugate of streptavidin, calcofluor white, nile • red, Y66F, Y66H, EBFP, GFP wild type, QFP mutants H9/P4/P9/P1 1/W, GFPuv, ECFP, Y66W, S65A, S65C, S65L, S65T, EGFP, EYFP, ECFP, DsRedl, DsRed2, NANOGOLD^® particules, streptavidin-Nanogold^®, Monomaleido Nanogold®, Mono-Sulfo-NHS-Nanogold®, Monoamino Nanogold®, positively/negatively charged Nanogold® (NN, NHSN, NHSNA, NHSNS), Non-Functionalized Nanogold®, Monomaleido Nanogold®, Mono-Sulfo-NHS-Nanogold®, Monoamino Nanogold®, Nonfunctional Nanogold®, Nanogold®-conjugates, Nanogold®-streptavidin, lipide-Nanogold (Palmitoyl Nanogold®, DPPE Nanogold®, Palmitoyl Undecagold, DPPE Undecagold), Ni- NTA-Nanogold®, Alexa Fluor® 488 FluoroNanogold, Alexa Fluor® 594 FluoroNanogold, Fluorescein FluoroNanogold, HRP substrate-Nanogold. 7 R (vi) R and R are also taken together with the intervening nitrogen to form a heterocyclic ring such as substituted or unsubstituted tetrahydroquinoline, tetrahydroisoquinoline, dihydroacridine, benzazepine, pyrrolidine, morpholine, thiomortholine, piperazine, piperidine, dihydropyridine, benzimidazole, imidazole, imidazoline, pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole, piperidine, morpholine, thiomorpholine, piperazine, carbazole, phenothiazine, phenoxazine, dihydrophenazine, dihydrocinnoline, dihydroquinoxaline, dihydronaphthyridine, tetrahydronaphthyridine, dihydroacridine, indole, isoindole, dihydroindole, indoline, indazole, purine, 9,10-dihydrophenanthridine, 5H-dibenzo[b,f]azepine. 10,l l-dihydro-5H- dibenzo[b,f]azepine, β-carboline, pyrido[4,3-b]indole, 2,3,9-triazofluorene, 9-thia-2,10- diazaanthracene, thieno[3,2b]pyιτole, dihydrophenanthrine. (vii) R is a hydrogen atom, Cι-₂o aliphatic group, aryl, substituted aryl (ex: 4- nitrophenyl or coumarine derivatives), hetetoaryl (ex. 2-pyridine), substituted heteroaryl, cycloalkyl, naphthyl, substituted naphthyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl (ex: 2,6-dihalophenyl), (CH₂)_n(l- or 2- naphthyl), (CH₂)_nheteroaryl or (un)substituted (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, fluorenmethyl ; (viii) Y is an electronegative leaving group including halogens such as F, Cl, Br or I, aryl or alkylsulfonyloxy groups, trifluoromethanesulfonyloxy, OR, SR, COOR, OP(0)R₂ wherein R is an aliphatic group, an aryl group, an aralkyl group, a carbocyclic group, an alkyl carbocyclic group, or a heterocyclic group ; (ix) Z is a halogen (F, Cl, Br or I), CN, OH, OR, NH₂, NHR, NR₂, SR, COR, C0₂R, CONR₂, (x) n is 0 to 20 ; As used herein, the following definitions shall apply unless otherwise indicated. The abbreviations Q and OPH stand for quinolinylcarbonyl and 2,6-difluorophenoxy respectively. The term "aliphatic" herein means straight chained or branched C1-₂₀ hydrocarbons which are completely saturated or which contain one or more units of unsaturation. The term "alkyl" used alone or as part of a larger moiety refers to both straight or branched chains containing one to twenty carbon atoms. The term "aryl" refers to mono cyclic or polycyclic aromatic ring groups having five to fourteen atoms, such as phenyl, naphthyl or anthryl. The term "heterocyclic group" refers to saturated or unsaturated polycyclic or monocyclic ring systems containing one or more heteroatoms and a ring size of three to nine such as furanyl, thienyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, dioxolanyl, oxazolyl, thiazolyl, imidazolyl, imidazolinyl imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxalolyl, isothiazolyl, oxadiazolyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl, triazinyl, trithianyl, indolizinyl, indolyl, isoindolyl, indolinyl, benzofuranyl, benzothiophenyl, indazolyl, benzamidazolyl, benzthiazolyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1 ,8-naphthyridinyl, pteridinyl, quinuclidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, or phenoxazinyl. "Heteroaryl" refers to a heterocyclic ring that is aromatic. The term "carbocyclic group" refers to unsaturated monocyclic or polycyclic carbon ring systems of three to fourteen carbons which may be fused to aryl or heterocyclic groups. An aliphatic, alkyl, aryl, heteroaryl (ex: quinoline), heterocyclyl, or carbocyclyl, used alone or as part of a larger moiety, refers to substituted or unsubstituted groups. When substituted, these groups may contain one or more substituents. These substituents can be halogen (F, Cl, Br, I), OH, U, CO(CH₂)_nNH(U), CO(CH₂)_nS(U), OR, SR, NH₂, NHR, NR₂, OCOR, OP(0)R₂ wherein R is an aliphatic group, an aryl or substituted group, an aralkyl group, a carbocyclic group, an 1 ^*7 ^ "X 1 -1 T "30 alkyl carbocyclic group, a heterocyclic group or a radio-isotope (ex: I , H , S , C , P , P , Cr⁵¹, Ca⁴⁵, Fe^5y, Ni⁶³, Ba^{l 3J}, Cs^lj7, Eu^li2, Ra ²²⁶, Xe¹³³, technetium 99, thallium 201). FITC stands for fluorescein isothiocyanate.

3. Peptide isosteres or bioisosteres

(a) This invention also relates to carba analogues of formula IV.1 to IV.7 (Table 1) wherein (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. Table 1. Carba analogues of the pentapeptide VDVAD (compounds IV.1 to IV.7) No. Structure

(b) The invention also relates to ketomethylene analogues of formula V.l to V.7 (Table 2) in which (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously. Table 2. Ketomethylene analogues of the pentapeptide VDVAD (compounds V.l to V.7) No. Structure

(c) The invention also relates to depsi-peptides analogues of the general foraiula VI and VII wherein (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) X is O or NH and at least one X an oxygen atom (iii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above (iv) R⁹ is a hydrogen atom, (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, Cι_₂o straight chain or branched alkyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl, (CH₂)_n(l- or 2- naphthyl), (CH₂)_nheteroaryl, OCOR or OCNHR.

VI

VII (d) The invention also relates to hydroxyethylene analogues of formula VIII.1 to VIII.7 (Table 3), wherein (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. Table 3. Hydroxyethylene analogues of VDVAD ^"(compounds VIII.1 to VIII.7) No. Structure

(e) The invention also relates to reduced pentapeptide analogues of formula IX.l to IX.6 (Table 4), wherein (i) the absolute configuration of each amino acid or its isostere is either Z or D ; (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. Table 4. Reduced analogues of VDVAD (compounds IX.l to IX.6) No. Structure

(f) The invention also relates to unsaturated analogues of the formula X.l to X.7 (Table 5) wherein (i) the absolute configm-ation of each amino acid or its isostere is either Z or D ; (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. Table 5. Unsaturated analogues of VDVAD (compounds X.l to X.7) No. Structure

(g) The invention also relates to β-peptides, γ-peptides, urea, and carbamates analogues of the formula XI.1 to XI.9 (Table 6) in which (i) the absolute configuration of each amino acid or its isostere is either Z or D ; (ii) X is CH₂, NH, 0 (iii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. Table 6. Unsaturated analogues XL 1 to XI.9 No. Structure

4. Constrained analogues of VDVAD pentapeptide The invention also relates to constrained analogues of VDVAD pentapeptide shown in tables 7 to 14. In theses compounds the aryl, heteroaryl or heterocyclyl refers to substituted or unsubstituted groups. When substituted, these groups may contain one or more substituents.

These substituents can be halogen (F, Cl, Br, I), OH, U, CO(CH₂)_nNH(U), CO(CH₂)_nS(U),

OR, SR, NH₂, NHR, NR₂, OCOR, OP(0)R₂ wherein R and U are as described above. (a) The invention also relates to 3-aminopyridin-2(lH)-ones, 3~aminopyrazin-2(lH)- ones, 5-aminopyrimidin-4(3H)-ones, pyridazin-3(2H)-ones, 4-aminopyridazin-3(2H)-ones, 5- amino- l,2,4-triazin-6(lH)-ones, 5-amino-l,2,3-triazin-4(3H)-ones and 6-amino-l,2,3,4- tetrazin-5(4H)-ones XII.1 to XII.15 (Table 7) as constrained analogues of the pentapeptide

VDVAD in which

(i) the absolute configuration of each amino acid or its isostere is either Z or D ; (ii) W, X and Y are CH, C or N ; (iii) n, R, R¹, R², R³, R⁴, R⁵, R^ό, R⁷, R⁸, U, Y and Z are as described previously. Table 7. Constrained analogues of VDVAD (compounds XII.l to XII.15) No. Structure

(b) The invention also relates to 3-aminopiperidin-2-ones, 3-aminopiperazin-2-ones, 5-amino- tetrahydropyrimidin-4(lH -ones, 4-aminopiperazin-3-ones, 6-amino-l ,2,4-triazinan-5-ones, 5-amino- l,2,4-triazinan-6-ones, 5-amino-l,2,3-triazinan-4-ones and 6-amino-l,2,3,4- tetrazinan-5-ones XIII.l to XIII.15 (Table 8) in which (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) W, X and Y are CH₂, NH, O or S ; (iii) n, R, R¹ , R², R³, R⁴, R⁵, R^δ, R⁷, R⁸, U, Y and Z are as described above.

Table 8. Constrained analogues of VDVAD (compounds XIII.l to XIII.15) No. Structure

(c) Compounds of this invention also include 3-amino-3,4-dihydroquinolin-2(lH)-ones XIV.1 to XIV.15 (Table 9) in which (i) the absolute configuration of each amino acid or its isostere is either Z or D ; (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously.

Table 9. Constrained analogues of VDVAD (compounds XIV.1 to XIV.15) Structure

(d) The invention also relates to 4-amino-l,2-dihydroisoquinolin-3(4H)-ones XV.1 to XV.15 (Table 10) in which (i) the absolute configuration of each amino acid or its isostere is either L ox D ; (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously. Table 10. Constrained analogues of VDVAD (compounds XV.1 to XV.15) No. Structure

(e) The invention also relates to l,2,3,4-tetrahydroisoquinoline-3-carboxamides XVI.1 to XVI.15 (Table 11) in which (i) the absolute configuration of each amino acid or its isostere is either Z or D ; (ii) n, R, R¹, R², R³, R⁴, R⁵, R⁵, R⁷, R⁸, U, Y and Z are as described previously. Table 11. Constrained VDVAD analogues (compounds XVI.1 to XVI.15) No. Structure

(f) The invention also relates to 4-amino-l,2,4,5-tetrahydrobenzo[c]azepin-3-ones XVII.1 to XVII.15 wherein (i) the absolute configuration of each amino acid or its isostere is either Z or D ; (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously. Table 12. Constrained VDVAD analogues (compounds XVII.1 to XVII.15) No. Structure

(g) The invention relates to 1,2,3,4-tetrahydronaphthalenes XVIII.1 to XVIII.15 in which (i) the absolute configuration of each amino acid or its isostere is either L ox D ; (ii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously. Table 13. Constrained amino acids XVIII.1 to XVIII.15 No. Structure

(h) The invention also relates to 2,3-dihydro-lH-indenes XIX.l to XIX.15 (Table 14) in which (i) the absolute configuration of each amino acid or its isostere is either L ox D ; (ii) n, R, R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. Table 14. Constrained VDVAD analogues (compounds XIX.l to XIX.15) Structure

Said caspase-2-inhibitors are advantageously obtained by a synthetic route as illustrated in the examples. The derivatives of the structure given on schemes 1-3 will be obtained by the one skilled in the art by using chemical synthesis classical methods.

Other characteristics and advantages of the invention will be given in the following with reference to Figures 1 to ll,which represent, respectively:

- Figure 1: the in vitro specificity and efficiency of Qco-VDVAD-dpf;

- Figure 2: the stability of formulation: HPLC determation. Qco-VDVAD-dfp; - Figure 3: the selective caspase-2 inhibition by Qco-VDVAD-dfp strongly prevents cell death and cerebral infarct of rat neonates subjected to ischemic brain injury;

- Figure 4: the in vivo inhibition of caspase-2 activity and caspase-2 processing by Q- VDVAD-OPH during neonatal ischemic brain injury;

- Figure 5: the cell death in hippocampus and cortex of ichemic rats (4VO) model; Figure 6: the impact of ischemia on CA3, Dentus gyms (DG) and cortex (COR);

Figure 7: the quantitation of cell death at 72 h post-ischemia; Figure 8: the non-toxicity of Qco-VDVAD-dfp in non-ischemic rats;

Figure 9: the neuroprotection by Qco-VDVAD-dfp at 72 h post-ischemia (4VO) in the hippocampus of the young-adult rat; Figure 10: the quantitative effect of Qco-VDVAD-dfp at 72 h post-ischemia; and Figure 11: the physiological and behaveioural tests

5. Qco-VDVAD-dfp is a selective caspase-2 inhibitor 5.1 experimental section 5.1.1 In vitro caspase activity assays. Human recombinant caspases 1 to 10 (25 U;

QuantiZyme™ Assay System, BIOMOL, Plymouth, Pennsylvania, USA) were pre-incubated 30 min at 37°C with Qco-VDVAD-dfp in assay buffer (50 mM HEPES, pH 7.4, lOOmM NaCl, 0.1% CHAPS, 10 mM DTT, ImM EDTA, 10% glycerol) and mixed with corresponding fluorogenic caspase substrates (500 μM; BIOMOL) : Ac-YVAD-AMC (caspase- 1), Ac-NDVAD-AMC (Caspase-2), Ac-DEVD-AMC (caspase-3/7), Ac-WEHD- AMC (caspase-4/5), Ac-VEID-AMC (caspase-6), Ac-IETD-AMC (caspase-8), Ac-LEHD- AMC (caspase-9), or Ac-IETD-AMC (caspase- 10). Substrate cleavage was assessed on a fluorescence microplate reader (TECAΝ, Genios; emission 510 nm, excitation 405 nm) and IC₅₀ value was determined from the dose-response sigmoid curves. Specific activity of each caspase was internally controlled with the corresponding specific inhibitors (1-2 μM, BIOMOL and MPBioMedicals). Other invtro assays were performed at CEREP or with CEREP protocoles for calpains and granzyme-besed tests). IC₅₀ determination was determined by incubating increasing concentration of Qco- VDVAD-dfp with recombinant caspase-2 in 100 μl assay buffer (50 mM HEPES, pH 7.4, 100 mM ΝaCl, 0.1% CHAPS, 10 mM DTT, ImM EDTA, 10% glycerol).. The cleavage of 50 μM VDVAD-AMC by recombinant caspase-2 (125 U) was measured after 30 min at 37°C on a fluorescence microplate reader by monitoring the fluorescence emission at 510 nm upon excitation at 405 nm. 5.1.2 Isolation, culture of primary cortical neurons and apoptosis induction. Primary cortical neurons were cultured from E14 SWISS mice embryos (Janvier). Mice were sacrificed by cervical dislocation and embryos were removed by caesarean. Cerebral cortices were extracted and tissues mechanically triturated 15 times in LI 5 medium (Gibco BRL) by using 1000 μl tips (Eppendorf), then debris were removed, and the cell suspension was centrifuged at 850 rpm for 10 min. Neurons were plated for 2 days at a high density (7.10 5 live cells per cm" -1) in Eagle's Basal Medium (Eurobio) supplemented with 1% glutamine, 5% horse serum (HS, Eurobio) and 2.5% fetal calf serum (FCS, Eurobio) onto 6 or

24 well-plates (Sarstedt), or 4-well-Lab-Tek® chambered coverglasses (Nalge Nunc

Internationnal), previously coated with lmg/ml polyethyl- enimine (Sigma). At DIV3, medium was changed daily and neurons were maintained in N5 complete medium containing 180 mg/1 glucose, 5% HS and 1% FCS, and 3 μM cytosine β-D-arabinofuranoside (Sigma) and 1 μM 5-methyl-10,l l-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801, Sigma). Purity of culture (> 95%) was controlled with an anti-Microtubule Associated Protein 2 monoclonal antibody (MAP -2, Sigma) and anti-Glial Fibrillary Acidic Protein polyclonal antibody (GFAP, Dako). Neurons were cultured used until 6 DIV on 24-well plate and subjected to serum deprivation. Briefly, neurons cultured in N5 complete medium were rapidly washed 3 times in N5 devoid of serum, and incubated for 24 hrs in N5 medium without serum, in absence or presence of Qco-VDVAD-dfp. 5.1.3 Apoptosis assays. Analysis was performed on previously stained adherent neurons by fluorescence microscopy (FM) (DM IRB inverted fluorescence microscope, Leica, Rueil-malmaison, France) equipped with a 100 W mercury short arc lamp and a X 40 N PLAN L objective or a water immersion X 100 N PLAN objective. Usually, quantitative studies were performed by both FM on approximatively 200-600 cells / field by scoring 5-10 random-selected fields per experiment and flow cytometry (FC) for higher sample throughput. FC analysis of apoptosis was performed after trypsinization of stained neurons by using a 3- color FACSCalibur cytometer equipped with a 15 mW air-cooled 488 nm argon laser (Becton Dickinson). Activated caspase-2 was detected using specific FAM-conjugated peptides (called FLICA: CaspaTag ™ fluorescein Caspase Activity Kits, Q-Biogen, lllkirch, France; ApoFluor ™ Caspase Detection Kits, MP BioMedicals): FAM-VDVAD-fmk. Viability loss was assessed by 7-AAD (Sigma) incorporation. 5.1.4 Formulation studies. Compound Qco-VDVAD-dfp (SEQ1) is soluble in Tween 20 or Pluronc F-68 excipients. Stabililty was followed throughout 7 days at RT or after freeze/thawing of acquous solution comprising various 10-100 % of excipients by using HPLC (Varian ProStar 410 equipped of Nucleosile Cjg (8 μm, 3.9 x 150 mm) with detection at 214 nm). 5.2 Results Qco-VDVAD-dfp is a selective caspase-2 inhibitor (IC50 = 65-80 nM) with no cross reactivity against other caspases or at least 32 others related-proteases or enzymes involved in signal transduction and neuronal metabolism, nitric oxide pathway or prostanglandin metabolism (Figure 1). Qco-VDVAD-dfp is cell-permeant because it prevents caspase-2 activity during serum-deprivatiion in cortical neuron cultures. In addition, Qco-VDVAD-dfp prevents neuronal death at 24 hrs in this experimental caspase-2 dependent cell death paradigm. (Figure 1). Moreover Qco-VDVAD-dfp is suitable for animal or human administration because of its high solubility in Tween 20 or Pluronic-F68 based-mixtures. No degradation of Qco-VDVAD-dfp was found when left at room temperature until 7 days or after freeze/thawing (Figure 2). These data are illustrated by the following figures : Figure 1. Figure 1:. In vitro specificity and efficiency of Qco-VDVAD-dpf.(a) In vitro specificity of Qco-VDVAD-dfp on a panel of human recombinant caspases (1 to 10; 50U). In vitro cleavage assays are performed -with 0.1 μM of Qco-VDVAD-dfp (n=3; histograms indicate % of caspase activity ± SD). (b) IC₅₀ (concentrtion that prevents 50% of Ac-VDVAD-AMC cleavage by human recombinant C2 (125 U)) determination range against recombinant human caspase-2: 65-80 nM (n=3 with different batches), (c) Cytoprotective effect of Qco-VDVAD-dfp on 24h-serum(SD)-deprived neurons. Qco-VDVAD-dfp prevents caspase-2 activity and cell death at 24 hrs (n = 3). (d) Inhibitory profile of Qco-VDVAD-dfp (1 μM) determined on 32 other in vitro pharmacology asssays related to proteases, signal transduction and neuronal metabolism, nitric oxide pathway or prostanglandin metabolism (n=2). Figure 2 . Stability of formulation: HPLC determation. Qco-VDVAD-dfp (5.10-3 M in phylological buffer containing 20 % of Tween 20) was not degraded at RT after 7 days.

IV.Effets of Qco-DVAD-dfp in rats following focal cerebral ischemia Intro Stroke is the third most common cause of death in adults in the developed. world, and an important cause of mortality and chronic neurological morbidity in children. Many strokes in children happen in the perinatal period, soon before birth or within the month after. Risk of ischaemic stroke in the mother also increases near the time of birth, and is 34 times more common in the 2 days before and 1 day after delivery than earlier in pregnancy or in the non- pregnant state. The heightened vulnerability to ischaemic stroke in both mother and child, and also to thromboses in non-cerebral sites is probably related to activation of coagulant mechanisms by parturition presumably an evolutionary adaptation to lessen the risk of haemorrhage at this crucial time. Perinatal ischaemic stroke is a cerebrovascular event around the time of birth with pathological or radiological evidence of focal arterial infarction. Most perinatal strokes occur in the territory of the middle cerebral artery (MCA). There is a predominance of left hemisphere lesions, which may be caused by haemodynamic differences from a patent ductus arteriosus, or a more direct route involving the left common carotid. The distribution of cerebral infarction differs somewhat with gestational age-preterm infants tend to have multifocal lesions involving the cortical or lenticulostriate branches of the MCA, whereas full-term infants tend to have occlusions of the main branch.

1. Experimental model 1.1 Transient unilateral focal ischemia model

Newborn Wistar rats (dam plus 9 pups per litter) were obtained from Janvier (Le Genest-St- Isle, France) when the pups were 3-4 days of age. The pups were housed with their dam under a 12:12 h light-dark cycle with food and water freely available. Animal experimentation was conducted according to the French and European Community guidelines for the care and use of experimental animals. Ischemia was performed in 7 day-old rats (17-21 g), as previously described (Renolleau et al, 1998). Rat pups were anesthetized with an intraperitoneal injection of chloral hydrate (350 mg/kg). Anesthetized rats were positioned on their back and a median incision was made in the neck to expose the left common carotid artery. Rats were then placed on the right side and an oblique skin incision was made between the ear and the eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. Then, the left middle cerebral artery, exposed just after its appearance over the rhinal fissure, was coagulated at the inferior level of the cerebral vein. After this procedure, a clip was placed to occlude the left common carotid artery. Rats were then placed in an incubator to avoid hypothermia. After 50 min, the clip was removed. Carotid blood flow restoration was verified with the aid of a microscope. Neck and cranial skin incisions were then closed. During the surgical procedure, body temperature was maintained at 37-38°C. Pups were transferred in an incubator (32°C) until recovery then after to their dams. Qco-VDVAD-dfp was administered intraperitoneally at a dose ranging from 0.001 to 10 mg/kg 5-15 min before the ischemic onset or 1 h after ischemia. Control animals received an equivalent volume of 0.9 % saline containing 10 % DMSO (n=15), the vehicle required to solubilize the caspase inhibitors (vehicle-treated group). The mortality rate during ischemia or before killing did not differ between Qco-VDVAD-dfp and vehicle-treated groups (< 4%). Rats were killed 48 hours after reperfusion and brains were removed. The infarct lesion (pale zone) was visually scored by an observer blinded to the treatment of animals. Brains without a clear ischemic pale zone were observed under a magnifying glass. Those exhibiting no clear MCA occlusion were discarded. Sixteen sections from anterior striatum to posterior hippocampus (corresponding to plates 9 to 27 in Paxinos' rat brain atlas) were selected, taken at equally spaced 0.5-mm intervals. The lesion areas were measured on cresyl violet-stained sections using an image analyzer (NIH image software), and the distances between respective coronal sections were used to calculate the infarct volume. Brain sections were processed for DNA strand breaks (TUNEL assay) using the in situ Fluorescein Cell Death Detection Kit (Roche, Meylan, France) according to manufacturer's instructions. For Tunnel analysis, brains were then fixed 2 days in 4 % buffered formaldehyde. Fifty- micrometer coronal brain sections were cut on a cryostat and collected on gelatin-coated slides. Statistical analysis was performed as followed. Assuming a beta risk of 0.2 and an alpha risk of 0.05, it was estimated that 15-16 animals in each group were needed to detect a 50% infarct volume reduction between two groups. Because three groups of animals are compared in the experiments, these values are only informative. A predetermined list with blocks of six animals was used to randomized the animals among the three groups. An investigator blind to the treatment condition did all measurements. The difference between the means was assessed by the non-parametric multiple comparison test of Kruskall-WaHis, followed by the Newman- Keul's test for non-parametric values. We consider differences to be significant at the 5 % level (P<0 05).

1.2 In vitro VDVAD-AMC cleavage in brain l sates and caspase-2 cleavage by Western blot analysis

Protein extraction and Western Blot analysis Brain hemispheres were lysed in 10 M Hepes, 5mM MgC12, 42 M KC1, ImM DTT, 0.5 % CHAPS supplemented by complete protease inhibitors cocktail (Roche, Meylan, France) by using a manual potter on ice. Homogenates were centrifuged at 10000 g / 4°C for 10 min before keeping surnageant. Protein concentration was determined using BCA test.

Proteins (50 μg) were separated on 12.5%> polyacrylamide gels and transferred to PVDF membranes (Amersham). Immunostaining was revealed using ECL (Amersham Pharmacia Biotech). The monoclonal anti-mouse caspase-2 antibody (52 kDa; 11B4, Alexis Biochemicals) was used at a 1 : 1000 dilution; actin (42kDa; Sigma; antibody diluted 1 :5000) is used as an equal loading control. Caspase-2 activity (100 μg protein brain sample) was assessed in 100 μl assay buffer (50 mM HEPES, pH 7.4, lOOrnM NaCl, 0.1% CHAPS, 10 mM DTT, ImM EDTA, 10% glycerol). The cleavage of 50 μM VDVAD-AMC by recombinant caspase-2 was measured after 2h at 37°C on a fluorescence microplate reader by monitoring the fluorescence emission at 510 nm upon excitation at 405 nm. For inhibition of VDVADase activity, inhibitors (2 μM) were pre-incubated 30 min at 37°C in presence of caspase-2 prior to subsequent incubation with 50 μM VDVAD-AMC (30 min, 37°C). No noticeable fluorescence background was observed with VDVAD-AMC alone. 2. Results In this transient unilateral focal ischemia model, rat pups underwent permanent left middle cerebral artery occlusion in association with transient occlusion of the left common carotid artery with reperfusion. Brains were then analyzed 48 hours later, a time point at which the infarct was stabilized without significant edema (no more than 1.5 %). Ischemia induced an infarct volume of 55.0 ± 3,4 mm , which represents a 22.1 ± 1.4 % damage in the lesioned ipsilateral hemisphere. Infarct volumes appeared normally distributed (between 15 and 26 %) (Figure 3). A single dose of Qco-VDVAD-dfp (5 mg/kg; i.p.) was administrated to rat pups before the ischemic onset. This single dose induced a highly significant 74 % reduction in infarct volume (5.7 ± 2.3 %, pO.Ol ((median = 0.5) compared to the control group (V=22.1 ± 1.4 %, median = 21) in the Newman-Keul's test) (Figure 3). Interestingly, On the 12 studied animals, four Qco-VDVAD-dfp-treated animals display intermediate protection (V=16.5 ± 1.32 %) and eight animals display either complete protection or a very marked smaller infarct (median = 0.5%), visible only at the level of the middle cerebral artery (levels corresponding to plates 12 and 13) but not at that of the dorsal) and hippocampus (plate 21) compared to the ischemic control animals (Figure 3). A single dose of Qco-VDVAD-dfp (0.1 mg/kg; i.p.) was administrated 1 h after MCAO (corresponding to the beginning of reperfusion) to rat pups. This single dose induced a highly significant 44 % reduction in infarct volume (9.95 ± 2.8 %, pO.Ol compared to the control group (V=22.64 ± 1.6 %) in the Newman-Keul' s test) (Figure 3).

In addition, terminal transferase dUTP nick end labeling (TUNEL) is significantly decreased in Qco-VDVAD-dfp-treated animals (Figure 3).

The protective effect provided by Qco-VDVAD-dfp is not mediated by corporal temperature regulation (Figure 3). Finally, Qco-VDVAD-dfp well target in vivo caspase-2 : effectively, it inhibited caspase-2 activity as well as caspase-2 processing in brain of ischemic rat pups (Figure 4). We demonstrate that caspase-2 is a relevant target with good neuroprotective prognosis in neonatal stroke, since in vivo inactivation of caspase-2 results in massive reduction of infarct volume during transient focal ischemia. We suggest that caspase-2 may be a valid target for in vivo prevention of hypoxic-ischemic encephalopathy at birth and perinatal stroke, a major health care in human newborns. These data are illustrated by the following figures : Figure 3: Selective caspase-2 inhibition by Qco-VDVAD-dfp strongly prevents cell death and cerebral infarct of rat neonates subjected to ischemic brain injury, (a, left panel) Mean infarct volumes at 48 hrs after ischemia in vehicle- (n=17) and Qco-VDVAD-dfp- (i.p., 5 mg/kg; n=12) treated rats (mean ± SEM). Qco-VDVAD-dfp induces 74 % reduction (_*** = p<0.001 , Kruskall-Wallis test) when administrated 5-15 min before ischemic onset; (a, right panel) Qco-VDVAD-dfp treatments provide 2 groups displaying high/total (height) or low protection (four). Single infarct volume data are plotted. Bold and thin horizontal bars represent the group median and mean, respectively, (b) Representative cresyl violet-stained coronal sections from animals at 48 hrs post-reperfusion at the level of dorsal hippocampus (plate 21, Paxinos' rat brain atlas) and anterior commissure (plate 12). Dotted lines indicate infarct area. Arrow indicates the absence of infarct in Qco-VDVAD-dfp-treated animal. Bar represents 130 μm. (c, left panel) Mean infarct volumes at 48 hrs after ischemia in vehicle- (n=22) and Qco-VDVAD-dfp- (i.p., 0.1 mg/kg; n=19) treated rats (mean ± SEM). Qco- VDVAD-dfp induces 44 % reduction (_*** = p<0.001, Kruskall-Wallis test) when administrated lh after ischemic onset, (c, right panel) Single infarct volume data are plotted. Bold and thin horizontal bars represent the group median and mean, respectively, (d) Cell death in the ipsilateral cortex of vehicle- and Qco-VDVAD-dfp-treated animals. Representative fluorescence micrographs (from plate 12) after in situ 3 'OH end DNA labelling (bar: 100 μm). (e) Corporal temperature was measured before and after Qco-VDVAD-dfp admnistration (pre and post-ischemia). Figure 4: Ischemic rat pups were treated with 5 mg/kg (i.p) of Qco-VDVAD-dfp. Animals were sacrified at 24 hrs. Brain homogenates were subjected to caspase-2 activity assay and western blot analysis. IL : ipsilateral injured hemisphere, CL : contralateral unlesionned hemisphere, (a) Caspase-2 activity determination in vehicle versus Qco- VDVAD-dfp-treated rats, (b) Western blot analysis of caspase-2 processing in brain homogenates from vehicle- versus Qco-VDVAD-dfp-treated rats.

V.Effects of Qco-DVAD-dfp in rats following global cerebral ischemia Global cerebral ischemia is a condition that may be be induced after cardiac arrest or cardiovascular disturbances, and that results in both blood flow reduction and hypoxia. Lesions appear in selectively vulnerable brain regions and neurons may be damaged by apoptosis during such global cerebral ischemia. Global ischemia results also in widespread and global loss of energy metabolites combined with diffuse brain edema and global damage. Mechanisms involved in lesion growth may include cystein-proteases (caspases) activation, excitotoxicity, peri-infarct depolarizations, lactacidosis, microcirculatory disturbances, and flow-metabolism uncoupling among others. If some (executiormer) caspases may be activated during global ischemia, no one can comment on the role of caspase-2 in such pathological situations. Recently, we have developped a new selective caspase-2 inhibitor (2- Quinolinylcarnonyl-Z-Valinyl-Z-Aspartyl (methyl ester)-Z-Vanilyl-Z-Alaninyl-Z-Aspartyl (methyl ester) 2,6-difluorophenyl ester, nammed Qco-VDVAD-dfp, that provided strong in vivo neuroprotection during neonatal cerebral ischemia. Here we investigated its cytoprotective-mediated effects against cerebral lesions occuring after cardiac arrest and behevioural-related benefits. Thus we tested Qco-VDVAD-dfp's effect in an in vivo experimental model of global and transient cerebral ischemia (4VO, four vessel occlusion) in the young adult rat, based on Pulsinelli's one (1979). 1 Experimental model 1.1 VO model Global cerebral ischemia was induced by four-vessel occlusion (4VO) according a Pulsinelli's derived method (Pulsinelli et al., 1982; Pulsinelli and Buchman, 1988) in young- adult rats (males Wistar aged of 10-12 weeks, 320 g +/- 10 g; Janvier). The first day, head of anesthetised rats was positionned in stereotaxic ear bars and tilted down at approximatively 30° to the horizontal. After a midline incision at the level of cervical spine, both vertebral arteries were exposed under microscope and then coagulated by electocautery needle through the alar foramina at the level of first cervical vertebra. Both common carotides were then exposed 24 h later and clamped for 20-30 min (rats fell in the coma when electrocautherisation of both vertebral arteries have been well performed). Carotid arteries were then declamped to allow blood flow reperfusion. Vehicle and Qco-VDVAD-dfp (II, n=0) were administrated at the level of the left cerebral ventricule in the first fifth minutes of ischemia (carotides occlusion). Rats were let in their cage with waer et food ad libitum. 1.2 Estimation of cytoprotection The selective loss of vulnerable cells and Qco-VDVAD-dfp's effects

(intracerebroventricular (ICV) administrated) have been evaluated at 72 h. Rats were sacrificied and brain fixed by trans-cardiac perfusion with paraformaldehyde. Frontal brain slices (25 μm) were strained by Cresyl-Violet or co-stained by Hoechst 33342 and Fluorojade B to assess cell death in the hippocampus and in the cortex. Cresyl-Violet is a pink-red dye that labels cytoplamic body Nissl (endoplasmic reticulum structures) and nuclei in living cells thus resulting in pale or absence of staining in dying cells. Fluoro JadeB (green fluorescence) intake is possible only in cells which have permeable plasma membrane, thus more caracteristic of dying cells. Hoechst 33342 (blue fluorescence) label nuclei all all cells and allows to appreciate nuclear morphology changes during cell death. Cells were counted at the level of canula, as well as 650 μm and 780 μm after the cannula (antero-posterior axis) in the left brain hemisphere. Cells are counted in three successives slices at such levels in the hippocampus (CAl, CA3, girus dentus) and cortex. Cresyl-Violet stained slices were observed under white light (Nikon Eclipse E 800M microscope equipped with x40 et x20 objectives and LEICA IM50 software). Both Fluoro Jade B and Hoechst stained slices were observed under a Leica DMIRB inverted fluorescence microscope equiped with x40 objective and LEICA IM1000 / Qfluorobase software (BP 340-80 excitation filter combined with LP 425 emission filter for Hoechst; BP 480/40 excitation filter combined with BP 515-560 emission filter for Fluoro Jade B). 1.3 Estimation of behavioural gain

Corporal temperature was measured during churgical procedure at day 1 (from anesthesia to stepl (vertebral electrocauterisation and cannula putting up) and step 2 ( coratid isolation) of chirurgical procedure during. Corporal temperature was also measured 0, 10, 20 minutes after the beginning of ischemia at day 2. Temperature was measured 24, 48, 72H post- ischemia and before final anesthesia for perfusion at day 5. Behavioural studies (feeding, spontaneous activity and reactivity) were performed and evaluated by scoring. Feeding : observation of stomacal contains at day 5 If refeeding = 1 ; If no refeeding = 0 Spontaneous activity : general behaviour of animals in their cage (alternance of active versus inactive phases) and reaction to prehension)

Vivacity = 1; More or less vivacious = 0.5; No or poor vivacity = 0. Reactivity : animal are sensitive and reactive to noise and move to the origin of the noise. If curious = 1 ; If relatively curious = 0,5; If not very curious = 0. 2 Results Figures 5-7 show that cell death occur at the level of CAl in the hippocampus (and a few in the cortex) of 72h post-ischemic adult rats: 90-100%o of cells have lost their Cresyl- violet labelling and have abnormal cellular morphologies, 40-60 % exhibited nuclear alteration, and 40-50% retained FluoroJade B in the CAl (CAla,b and c sub-areas) of ischemic brain. No sign of cell death was found in CA3, DG (Dentus girus) (also in CA2 and CA4, data not shown).. Ischemia was also controlled by the presence of microglial cells thaht exhibit stick (resident microglia) or sickle (migrant microglia invading the brain after blood brain barrier rupture) shapes. Figure 8 shows no cell death (below basal threshold : 10 %>) in non-ischemic rats (icv) treated with 60-600 ng of Qco-VDVAD-dfp whatever the level of observation in the hippocampus or in the cortex : absence of Cresyl-Violet negative cells, absence of abnormal nuclei, no Fluoro Jade B incorporation. Thus Qco-VDVAD-dfp is not topically toxic at 60- 600 ng. Figures 9-10 show the cytoprotective effects of Qco-VDVAD-dfp at the level of CAl in ischemic brains. In sharp contrast to ischemic rats, Qco-VDVAD-dfp's treated animals have less abnormal nuclei (nuclei were bigger and less retracted) and few cells incorporated FluoroJade B (between 10-20% instead of 50-60%). Thus colore slices looked like strongly to non-ischemic' ones. Moreover Cresyl-violet staining intensity was partially restored to the level of non-ischemic animal, but without total recovery of the cellular morphology. Qco-VDVAD-dfp's effects were independent of regulatory effects on temperature (Figure 11). Qco-VDVAD-dfp 'have benefitory effects on the general behaviour of ischemic treated rat because these rats have a better scoring than untreated rats: they were more active, more reactive to noise and they fed them (Figure 11). These data are illustrated by the following figures : Figure 5 : Cell death in hippocampus and cortex of ichemic rats (4VO) model. A: Cell death in the hippocampus (CAl, CA3, DG) and cortex of 72h post-ischemic adult rats. Global and transient cerebral ischemia was induced by 4 vessels occlusion (4VO; n = 5). Rats were treated (icv) with DMSO (here, 7,255%; 0.7255%) not shown). Slices are stained by Cresyl-Violet. B: Cell death in the hippocampus (CAl) of 72h post-ischemic adult rats. Global and transient cerebral ischemia was induced by 4 vessels occlusion ((4VO; n = 5). Rats were treated (icv) with DMSO ( 7,255%). Slices are stained by Hoechst 33432 (blue nuclei) and Fluoro Jade B (green nuclei and cytoplasm). C: High magnification of Hoechst and Fluoro Jade B fluorescence micrographs (size are increased by 2 and 6 folds, respectively). D: Microglial activation as a marker of ischemia. Microglial cells exhibit stick (resident microglia) or sickle (migrant microglia invading the brain after blood brain barrier rupture) shapes. Figure 6 : Impact of ischemia on CA3, Dentus gyrus (DG) and cortex (COR). Cell death in the hippocampus (CA3, DG) and cortex (COR) at 72h post-ischemia in adult rats.

Global and transient cerebral ischemia was induced by 4 vessels occlusion ((4VO; n = 5).

Rats were treated (icv) with DMSO (here, 7,255%>; 0.7255%) not shown). Slices are co-stained by Fluoro Jade B / Hoechst at CAl level (n = 5). Figure 7 : Quantitation of cell death at 72 h post-ischemia. Cell death in the hippocampus (CA1,CA3, DG,) and cortex (COR) at 72h post-ischemia in adult rats. Global and transient cerebral ischemia was induced by 4 vessels occlusion ((4VO; n = 5). Rats were treated (icv) with DMSO (here, 7,255%>; 0.7255% not shown) (n = 5). A: Quantitation of Cresyl-Violet positives cells at the level of injection of DMSO or TRP6 (1), 650 μm after (2) or 780 μm after (3). B: Quantitation of abnormal nuclei assessed by Hoechst staining in the hippocampus (CAl_a,b,o. CA3, DG) and cortex (COR) of ischemic and non-ischemic brains. C: Quantitation of FluoroJade B positive cells in the hippocampus (CAl_a>bj0, CA3, DG) and cortex (COR) of ischemic and non-ischemic brains. Figure 8 : Non-toxicity of Qco-VDVAD-dfp in non-ischemic rats. Cell death in the hippocampus (CA1,CA3, DG,) and cortex (COR) at 72h after icv administration of DMSO (here, 7,255%; 0.7255% not shown) or Qco-VDVAD-dfp (60 or 600 ng) (n = 5) in non- ischemic rats. A: Quantitation of Cresyl-Violet positives cells in presence of increasing doses of TRP6 at the level of injection of DMSO or Qco-VDVAD-dfp (1), 650 μm after (2) or 780 μm after (3). B: Quantitation of abnormal nuclei assessed by Hoechst staining in the hippocampus (CAl_a,_b,_c, CA3, DG) and cortex (COR) of vehicle-trated of Qco-VDVAD-dfp treated rats. C: Quantitation of FluoroJade B positive cells in the hippocampus (CAl_a,b,c, CA3, DG) and cortex (COR) of ischemic and non-ischemic brains. Figure 9 : Neuroprotection by Qco-VDVAD-dfp at 72 h post-ischemia (4VO) in the liippocampus of the young-adult rat. A: Microgaphs (x400) representative for CAl and its sub-areas (CAla,CAlb, CAlc) in vehicle-treated (DMSO 0.7255 %) or Qco-VDVAD-dfp- treated (60 ng) ischemic rats versus non-ischemic animals (vehicle treated). Slices are stained with Cresyl-Violet (upper line), Hoechst (blue nuclei in left columns) or Fluoro Jade B (green fluorescence in right columns). B : High magnification (6 folds) of Hoechst and Fluoro Jade B fluorescence micrographs. Figure 10 : Quantitative effect of Qco-VDVAD-dfp at 72 h post-ischemia. Cell death in the hippocampus (CA1 ,CA3, DG,) and cortex (COR) at 72h post-ischemia in adult rats. Global and transient cerebral ischemia was induced by 4 vessels occlusion ((4VO; n = 5). Rats were treated (icv) with DMSO (here, 7,255%; 0.7255% not shown) or Qco-VDVAD-dfp (60 or 600 ng) (n = 5). A: Quantitation of Cresyl-Violet positives cells in presence of increasing doses of TRP6 at the level of injection of DMSO or Qco-VDVAD-dfp (1), 650 μm after (2) or 780 μm after (3). B: Quantitation of abnormal nuclei assessed by Hoechst staining in the hippocampus (CAl_a,b,c, CA3, DG) and cortex (COR) of vehicle-trated of Qco-VDVAD- dfp-treated rats. C: Quantitation of Fluoro Jade B positive cells in the hippocampus (CAl_aιb,c, CA3, DG) and cortex (COR) of ischemic and non-ischemic brains. Figure 11 : Physiological and behavioural tests. A: Physiologial studies. Corporal temperature measurment from anesthesia to stepl (vertebral electrocauterisation and cannula putting up) and step 2 ( coratid isolation) of chirurgical procedure during day 1; 0, 10, 20 minutes after the beginning of ischemia at day 2; 24, 48, 72H post-ischemia and before final anesthesia for perfusion at day 5. B : Behavioural studies : mean of parameters per group. Feeding : observation of stomacal contains at day 5, If refeeding = 1, If no refeeding = 0; Spontaneous activity : general behaviour of animals in their cage (alternance of active versus inactive phases) and reaction to prehension), Vivacity = 1, More or less vivacious = 0.5, No or poor vivacity = 0; Reactivity : animal are sensitive and reactive to noise and move to the origin of the noise. If curious = 1, If relatively curious = 0.5, If not very curious = 0. C : Behavioural studies : total scoring (sum of total scores per group).

Example: Obtention of caspase-2 inhibitors of structure II The main steps used for the synthesis are summarized on schemes 1 to 3

("lOTHF >^"M\t --5^'-C iVEtOCOCl (b>CE_:H_: fc)Et_:DTEF,H3. AeOK fd)D F KF 6-cιfl .icplι-nol <e}TPA DCM

Scheme 1

7

Scheme 2

10 11

12 13

14 IS

(a)DCMDlEi-.Boc-Vsl-OH.DCC HOB- i DCMTFArt:.}. ill. (c) DCM, IEA. Boc-A.p(OM«)-OH. BOP.HOBt (d^';DCM DIEA, Qo-Val-OH. BOP. HOBt. (e) MeOH, cat. H,, ?d C. rt. i-2 jotus (f) DCM, DEA. TF.A-H-A.p(OMe)-CH_:-0-{2.6-difluoropheuyk). BOP. HOBt.

Scheme 3

Bibliographic references

Pulsinelli WA, Waldman S, Rawlinson D, Plum F, Moderate hyperglycemia augments ischemic brain damage: a neuropathologic study in the rat. Neurology. 1982, 32, :1239-1246.

Pulsinelli W.A., Buchman A.M. (1988). The Four- vessel Occlusion Rat Model: Method for Complete Occlusion of vertebral arteries and control of collateral circulation. Stroke. 19, 913- 914

Renolleau, S., D. Aggoun-Zouaoui, Y. Ben- Ari, and C. Charriaut-Marlangue. 1998. A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis. Stroke 29: 1454-1461.

Claims

1/ Selective new caspase-2 inliibitors that recognize caspase-2 and prevent and block its activity based on the following backbone : 2-Quinolinylcarbonyl-E-Valinyl-Z,-Aspartyl (methyl ester)-Z-Vanilyl-I-Alaninyl-Z.-Aspartyl (methyl ester) 2,6-difluorophenyl ester (SEQ1) and derivatives thereof, SEQ 1 corresponding to formulae la or lb:

la

lb

2/ Selective new caspase-2 inliibitors according to claim 1 based on structure II

II

3/ Selective new caspase-2 inhibitors according to claim 1 based on structure III:

III wherein (i) the absolute configuration of each amino acid is either L or D ; 1 (ii) R and R are a hydrogen atom, deuterium atom, Cι.₂₀ aliphatic, substituted or unsubstituted aryl, cycloalkyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2- oxazolyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl, (CH₂)_n(l- or 2- naphthyl), (CH₂)„heteroaryl, CH₂N₂, CH₂Y, OH, OR, NH₂, NHR, NR₂, SR, COR, C0₂R, CONR₂, CH2OCOR, CH.O-CO aryl, CH₂0-C(0) substituted aryl (ex: 2,6- dimethylbenzoyloxymethyl), CH₂0-C(0) substituted aryl, CH₂0-C(0) heteroaryl, CH 0- C(O) substituted heteroaryl or CH₂OPOR₂ ; (iii) R³, R⁴, R³ and R^δ are the side-chains of one of the twenty amino acids (excluding cysteine). For example, the pentapeptides can also be analogues of Leu-Asp-Glu-Ser-Asp. R³, R⁴, R³ and R⁶ are also C).₂₀ aliphatic, cycloalkyl, naphthyl, substituted naphthyl, 2- benzoxazolyl, substituted 2-oxazolyl, (CH2)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_πsubstituted phenyl, (CH₂)_n(l- or 2- naphthyl), (CH₂)„heteroaryl, CH₂N₂, (CH₂)_nZ, CH₂OCOR, CH₂OCO (aryl), CH₂OCO (substituted aryl), CH₂OCO (heteroaryl), CH₂OCO (substituted heteroaryl) or CH2OPOR2 ; (iv) R⁷ is a hydrogen atom and R^s is R, U, CO(CH₂)_nNH(U), CO(CH₂)„S(U) (v) U is (un)substituted (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, C₁-₂₀ straight chain or branched alkyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl, (CH₂) (l- or 2- naphthyl), (CH₂)_nheteroaryl, or a marker group such as fluorophore markers or markers useful in electronic microscopy; (vi) R⁷ and R⁸ are also taken together with the intervening nitrogen to form a heterocyclic ring such as substituted or unsubstituted tetrahydroquinoline, tetrahydroisoquinoline, dihydroacridine, benzazepine, pyrrolidine, morpholine, thiomortholine, piperazine, piperidine, dihydropyridine, benzimidazole, imidazole, imidazoline, pyrrole, pyrrolidine, pyrroline, pyrazole, pyrazoline, pyrazolidine, triazole, piperidine, morpholine, thiomorpholine, piperazine, carbazole, phenothiazine, phenoxazine, dihydrophenazine, dihydrocinnoline, dihydroquinoxaline, dihydronaphthyridine, tetrahydronaphthyridine, dihydroacridine, indole, isoindole, dihydroindole, indoline, indazole, purine, 9,10-dihydrophenanthridine, 5H-dibenzo[b,fJazepine, 10,l l-dihydro-5H- dibenzo[b,fjazepine, β-carboline, pyrido [4,3 -b] indole, 2,3,9-triazofluorene, 9-thia-2,10- diazaanthracene, thieno[3,2b]pyιτole, dihydrophenanthrine. (vii) R is a hydrogen atom, .2₀ aliphatic group, aryl, substituted aryl (ex: 4-nitrophenyl or coumarine derivatives), hetetoaryl (ex. 2-pyridine), substituted heteroaryl, cycloalkyl, naphthyl, substituted naphthyl, (CH₂)_ncycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl (ex: 2,6-dihalophenyl), (CH₂)_n(l- or 2- naphthyl), (CH₂)_nheteroaryl or (un)substituted (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, fluorenmethyl ; (viii) Y is an electronegative leaving group including halogens such as F, Cl, Br or I, aryl or alkylsulfonyloxy groups, trifluoromethanesulfonyloxy, OR, SR, COOR, OP(0)R₂ wherein R is an aliphatic group, an aryl group, an aralkyl group, a carbocyclic group, an alkyl carbocyclic group, or a heterocyclic group ; (ix) Z is a halogen (F, Cl, Br or I), CN, OH, OR, NH₂, NHR, NR₂, SR, COR, C0₂R,

(x) n is 0 to 20 ; 4/ Selective new caspase-2 inliibitors according to claim 1 , based on structure IV, comprising carba analogues of formula IV.1 to IV.7, wherein (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. No. Structure

5/ Selective new caspase-2 inhibitors according to claim 1 , based on stmcture V, comprising Ketomethylene analogues of the pentapeptide VDVAD, of formula V.l to V.7, wherein (i) the absolute configuration of each amino acid or its isostere is either L or D ; (ii) n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. No. Structure

6/ Selective new caspase-2 according to claim 1, based on structure VI, comprising depsi- peptides analogues of formula VI, : - the absolute configuration of each amino acid or its isostere is either L ox D ; - X is 0 or NH and at least one X an oxygen atom - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above - R⁹ is a hydrogen atom, (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, Ci-₂₀ straight chain or branched alkyl, (CH₂)nCycloalkyl, (CH₂)_nphenyl, (CH₂)_nsubstituted phenyl, (CH₂)_n(l- or 2- naphthyl), (CH₂)_nheteroaιyl, OCOR or OCNHR.

VI

VII

II Selective new caspase-2 inhibitors according to claim 1 , based on structure VII comprising depsi-peptides analogues of formula VI, : - the absolute configuration of each amino acid or its isostere is either L ox D - X is O or NH and at least one X an oxygen atom - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R^s, U, Y and Z are as described above - R⁹ is a hydrogen atom, (2-, 3-, 4-, 5-, 6-, 7- or 8-) quinolinyl, C].₂o straight chain or branched alkyl, (CH₂)_ncycloalkyl, (CH )_nphenyl, (CH )_nsubstituted phenyl, (CH₂)_n(l- or 2- naphthyl), (CH₂)_nheteroaryl, OCOR or OCNHR.

VII 8/ Selective new caspase-2 inliibitors according to claim 1, based on structure VIII, comprising hydroxyethylene analogues of formula VIII.l to VIII.7, wherein - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R\ R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. No. ^' Structure

9/ Selective new caspase-2 inhibitors according to claim 1, based on stmcture IX, comprising reduced pentapeptide analogues of formula IX.1 to IX. 6, wherein: - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R⁴, R^S, R⁶, R⁷, R⁸, U, Y and Z are as described above. No. Structure

10/ Selective new caspase-2 inhibitors according to claim 1, based on structure X, comprising unsaturated analogues of formula X.1 to X.7, , wherein: - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above. No. Structure

11/ Selective new caspase-2 inliibitors according to claim 1, based on structure XI, comprising beta-peptides, gamma-peptides, urea and carbamates analogues of foπnula XI.1 to XI.9, wherein: - the absolute configuration of each amino acid or its isostere is either L or D ; - X is CH₂, NH, O - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R^s, U, Y and Z are as described above. No. Structure

12/ Selective new caspase-2 inhibitors according to claim 1, based on structure XII, comprising constrained analogues of VDVAD, of fomiula XII.l to XII.15, wherein: - the absolute configuration of each amino acid or its isostere is either L or D ; - W, X and Y are CH, C or N ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously No. Structure

13/ Selective new caspase-2 inhibitors according to claim 1, based on structure XIII, comprising constrained analogues of VDVAD, of fonrmla XIII.l to XIII.15, wherein: - the absolute configuration of each amino acid or its isostere is either L ox D ; - W, X and Y are CH₂, NH, 0 or S ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described above No. Structure

14/ Selective new caspase-2 inliibitors according to claim 1, based on structure XIV, comprising constrained analogues of VDVAD, of foπnula XIV.1 to XIV.15, wherein - the absolute configuration of each amino acid or its isostere is either L ox D ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously. No. Structure

15/ Selective new caspase-2 inliibitors according to claim 1, based on structure XV, comprising constrained analogues of VDVAD, of formula XV.1 to XV.15, wherein - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously. No. Structure

16/ Selective new caspase-2 inliibitors according to claim 1, based on structure XNI, comprising constrained analogues of VDVAD, of foπnula XNI.l to XNI.15, wherein - the absolute configuration of each amino acid or its isostere is either L x D ; - n, R, R\ R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously No. Structure

17/ Selective new caspase-2 inliibitors according to claim 1, based on structure XNII, comprising constrained analogues of λHDVAD, of formula XVII. l to XNII.15, wherein - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R⁴, R\ R⁶, R⁷, R⁸, U, Y and Z are as described previously No. Structure

18/ Selective new caspase-2 inliibitors according to claim 1, based on stmcture XVIII, comprising constrained analogues of VDVAD, of fomiula XVIII.1 to XVIII.15, wherein - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, U, Y and Z are as described previously No. Structure

19/ Selective new caspase-2 inhibitors according to claim 1, based on stmcture XIX, comprising constrained analogues of VDVAD, of formula XIX.1 to XIX.15, wherein - the absolute configuration of each amino acid or its isostere is either L or D ; - n, R, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R^s, U, Y and Z are as described previously No. Structure

20/ Pharmaceutical compositions comprising an effective amount a least one compound as defined in anyone of claims 1 to 19, .in association or not with a pharmaceutically acceptable carrier.

21/ The phamiaceutical compositions according to claim 20, for administration by oral, nasal, local (intracerebroventricular, intrahippocampal,. other intracerebral delivery, intracerebral implantation of instrumentation for mechanical delivery) or systemic (for example: intraperitoneal, intravenous....) administration or intradeπnic delivery, intrathecal delivery or as aerosol to reduce in vivo cerebral cell death.

22/ The pharmaceutical compositions according to claim 20 or 21 , for preventing or blocking caspase-2 activity in cell death.

23/ The pharmaceutical compositions according to claim 22, wherein said cells are neurons, or neuronal cells or non-neuronal cells. 24/ The phamiaceutical compositions of claim 20 or 21, for providing neuroprotective or cerebroprotective effect.

25/ The pharmaceutical compositions of claim 20 or 21, for providing cytoprotective effect.

26/ The phamiaceutical compositions according to claim 20 or 21, for the treatment of pathological situation including ischemic injuries following cerebral focal ischemia

27/ The pharmaceutical compositions according to claim 20 or 21, for the treatment of pathological situation including ischemic injuries following cerebral focal ischemia as a consequence of MCAO.

28/ The pharmaceutical compositions according to claim 20 or 21, for the treatment of pathological situation including ischemic injuries following cerebral focal ischemia as a consequence of strokes at birth or in the perinatal period.

29/ The phamiaceutical compositions according to claim 20 or 21. for the treatment of pathological situation including ischemic injuries following cerebral focal ischemia as a consequence of thromboses in non-cerebral sites. 30/ The pharmaceutical compositions according to claim 20 or 21, for the treatment of focal arterial infarction.

31/ The pharmaceutical compositions according to claim 20 or 21, for the treatment of ischemic lesions in the territory of the middle cerebral artery (MCA) or left cerebral hemisphere, caused by haemodynamic differences from a patent ductus arteriosus, or a more direct route involving the left common carotid.

32/ The phamiaceutical compositions according to claim 20 or 21, for the treatment of ischemic multifocal lesions involving the cortical or lenticulostriate branches of the MCA, or occlusion of the main branch.

33/ The pharmaceutical compositions according to claim 20 or 21, for the treatment of pathological situation including ischemic injuries following cerebral global ischemia as a consequence of blood flow reduction or hypoxia during cardiac arrest or cardiovascular injuries.

34/ The phamiaceutical compositions according to claim 20 or 21, for the treatment of pathological situation including pathological situations in which blood flow and oxygen pressure are disturbed, that resulted in cerebral (transient or permanent) damages.