New dispiropyrrolidine derivatives as inhibitors of the mptp mitochondrial transition pore in the treatment of ischemic reperfusion injury

Abstract

The present invention concerns a dispiropyrrolidine compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein: R1 is hydrogen or a halogen; n is 0 or 1, provided that: when n is equal to 1, R2 is a (C1-C3) alkyl, R3 and R4 are H or R2 is H and R3 and R4 together with the carbon atoms to which they are bonded form a condensed benzene ring, when n is equal to 0, R2, R3 and R4 together with the carbon atoms to which they are bonded form a condensed naphthalene structure. The compound is described herein for use as a selective inhibitor of the C subunit of the F1 / F0-ATP synthase complex in the treatment of reperfusion injury diseases.

Description

[0001] New dispiropyrrolidine derivatives as inhibitors of the mPTP mitochondrial transition pore in the treatment of ischemic reperfusion injury

[0002] DESCRIPTION

[0003] FIELD OF INVENTION

[0004] The present invention relates to differently modified compounds with a dispiropyrrolidine structure for the treatment of diseases resulting from cardiac reperfusion injury.

[0005] Specifically, the invention provides strictly selective inhibitors of the C subunit of the F1 / F0-ATP synthase complex.

[0006] The compounds of the invention, as selective inhibitors, have shown to be able to modulate the opening of the mitochondrial permeability transition pore (mPTP) both in mammalian cells and tissues and can be used to treat all diseases resulting from reperfusion injury, such as cardiac, neurological and nephrological diseases.

[0007] STATE OF THE ART

[0008] Programmed cell death is an evolutionarily conserved physiological event, essential for intrauterine and post-embryonic development, and for tissue homeostasis through the elimination of damaged cells that would otherwise lead to numerous pathological events (P. Meier, et al. Nature 2000, 407, 796). Conversely, in humans, excessive apoptosis may lead to a long list of pathological dysfunctions, such as cardiovascular, neurological and nephrological disorders. There are several types of programmed cell death, including apoptosis mediated by mitochondrial permeability transition (MPT, V. Izzo et al. Trends Cell Biol 2016, 26, 655) which results in an increase in the permeability of the inner mitochondrial membrane (IMM), usually highly impermeable, resulting in osmotic influx of solutes into the mitochondrial matrix and loss of the structural and functional characteristics of the affected mitochondria (M. Bonora et al. Oncogene 2015, 34, 1608). The MPT state is mediated by the opening of a channel called the mitochondrial permeability transition pore (mPTP), a multiprotein platform consisting of proteins that physically form a pore inside the IMM and negative and positive modulators that contribute to the functional state and formation mechanism thereof (A. Halestrap et al. Biochem Soc Trans 2010, 38, 841 ). Conditions that favor the mPTP opening include the increase in the concentration of calcium ion ([Ca2+]) in the mitochondrial matrix, restoration of pH levels, oxidative stress and presence of phosphates, all events that occur in relation to lesions due to an ischemia-reperfusion condition (G. Morciano et al. J Mol Cell Cardiol 2015, 78, 142).

[0009] Although many of the components and modulators of mPTP have been discovered in recent years, the proteins that border the channel are still the subject of intense study. Among the modulators of mPTP we include the well-known cyclophilin D (CypD), a soluble protein of the mitochondrial matrix (V. Giorgio et al. J Biol Chem 2009, 284, 33982) whose depletion was demonstrated to lead to a protective and desensitizing behavior of MPT. In two of our recent studies (M. Bonora et al. Cell Cycle 2013, 12, 674; M. Bonora et al. EMBO Rep. 2017,18, 1077), we provided experimental evidence of the fact that the C subunit of F1 / F0-ATP synthase, plays a fundamental role in the activity and formation of mPTP, demonstrating in particular a strong correlation between the expression of the C subunit and the functional state of mPTP; the depletion of this protein leads, in fact, to a reduction in the opening of the channel in response to oxidative or calcium-induced stress, while its overexpression stimulates the opening thereof (M. Bonora et al. Cell Cycle 2013, 12, 674). Furthermore, we demonstrated that the mPTP opening is a multi-step process involving i) the dissociation of F1 / F0-ATP synthase dimers and ii) an appropriate conformation of the protein ring consisting of multiple C subunits once these dimers dissociated. Other groups such as those of Alavian, Azarashvili and collaborators have independently confirmed our first observations by adding important base notions on the channel properties (K.N. Alavian et al. Proc Natl Acad Sci U S A 2014, 111 , 10580) and relative phosphorylation state (T. Azarashvili et al. Cell Calcium 2014, 55, 69). The molecular organization of the C subunit in a ringlike structure (C ring) in the IMM makes it identified as a key component in the pore assembly process (P.A. et al. Cell Death Discov 2016, 2, 16070; G. Morciano et al. Cell Death Dis 2017, 8, e2698) as well as a promising therapeutic target for the pharmacological treatment of programmed cell death (G. Morciano et al. J Mol Cell Cardiol 2015, 78, 142; R. Ferrari et al. Circ J 2017, 81 , 131 ).

[0010] Although numerous efforts have been made for the synthesis and testing of new inhibitors of the mPTP opening (D. Fancelli et al. J Med Chem 2014, 57, 5333; L.J. Martin et al. Front Cell Neurosci 2014, 8, 433), to the best of our knowledge, none of the molecules identified to date have demonstrated specific interaction with the C subunit or have been developed for cardioprotective purposes for the treatment of myocardial infarction (Ml). It is estimated that about 50% of the final infarcted myocardial area is due to reperfusion injury (Rl), which consists of cell death following the restoration of blood flow in the artery affected by ischemia (R. Ferrari et al. Circ J 2017, 81 , 131 ). To date, only the use of broad molecular spectrum therapies has demonstrated useful efficacy in protecting the heart following Rl, while randomized clinical trials focused on more specific pharmacological targets have failed.

[0011] WO2020 / 21378 describes a family of 1 ,3,8-triazaspiro compounds for use as selective inhibitors of the C subunit of the F1 / F0-ATP synthase complex in the treatment of reperfusion injury diseases.

[0012] However, the need for a new pharmacological and / or therapeutic approach for the treatment of diseases resulting from reperfusion-related damage is still felt.

[0013] SUMMARY OF THE INVENTION

[0014] The inventors have surprisingly found that some low molecular weight molecules are able to inhibit the mPTP opening by specifically interacting with the C subunit of the F1 / F0-ATP synthase complex. The invention therefore allowed the identification of potent inhibitors of the mitochondrial permeability transition pore opening in living mammalian cells that selectively target the C subunit of the F1 / F0-ATP synthase complex. The invention therefore concerns a dispiropyrrolidine compound of Formula (I): or a pharmaceutically acceptable salt thereof wherein:

[0015] Ri is hydrogen or a halogen; n is 0 or 1 , provided that: when n is equal to 1 , R2 is a (C1-C3) alkyl, R3 and R4 are H, or R2 is H and R3 and R4 together with the carbon atoms to which they are bonded form a condensed benzene ring. when n is equal to 0, R2, R3 and R4 together with the carbon atoms to which they are bonded form a condensed naphthalene structure.

[0016] In another aspect, the invention therefore provided a compound of Formula (I) as a modulator of the mitochondrial permeability transition pore opening in mammalian cells and tissues.

[0017] Therefore, the invention concerns a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use as a medicament.

[0018] In yet a further aspect, the invention concerns a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use as a selective inhibitor of the C subunit of the F1 / F0-ATP synthase complex in the treatment of reperfusion injury diseases.

[0019] In the present invention, when the following definitions are used:

[0020] - “(C1-C3) alkyl” means linear or branched chains of hydrocarbon nature comprising from 1 to 3 carbon atoms;

[0021] - “halogen” means fluorine, chlorine, bromine and iodine;

[0022] - “a condensed naphthalene structure” means two condensed benzene rings, i.e. sharing two adjacent carbon atoms.

[0023] In another aspect, the invention concerns a composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use as a medicinal product and pharmaceutically acceptable additives.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1 reports the preparation scheme of compounds of Formula (I).

[0026] Figure 2 reports the biological activity of compounds 4a-d and 6b-c in terms of in vitro inhibition of mPTP in human ventricular cardiomyocytes. In particular, the vehicle condition (black bar) represents the mPTP opening in cells that have not undergone any treatment. The white bars, named after the wordings of the various compounds, highlight the biological activity of the compounds in inhibiting the channel opening. In fact, they correspond to lower percentages (Y axis) of mPTP opening.

[0027] Figure 3 reports the functional cardiac performance data measured in rat heart. In particular, a parameter is represented that describes the functionality of the left ventricle during the entire duration of the experiment. From the figure it can be seen that the left ventricle functions correctly and efficiently in all experimental groups before the induction of ischemia. During the 30 minutes of ischemia, in all cases, the ventricle temporarily loses its functionality causing the LVPdp parameter to fall to zero. At the time of reperfusion, if the heart is not treated with the molecules that inhibit mPTP (black trace), the ventricle only partially recovers (by approximately 39%) its functionality and this will lead to a great damage in terms of final infarction area; while if the heart is treated with compounds 4c, 6c and 4d at the time of reperfusion, the ventricle function is preserved.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] The invention therefore concerns a dispiropyrrolidine compound of Formula (I): or a pharmaceutically acceptable salt thereof wherein:

[0030] Ri is hydrogen or a halogen; n is 0 or 1 , provided that: when n is equal to 1 , R2 is a (C1-C3) alkyl, R3 and R4 are H, or R2 is H and R3 and R4 together with the carbon atoms to which they are bonded form a condensed benzene ring. when n is equal to 0, R2, R3 and R4 together with the carbon atoms to which they are bonded form a condensed naphthalene structure.

[0031] The compound of Formula (I) has R1 selected from hydrogen and halogen. Preferably when R1 is halogen, it is selected from fluorine, chlorine, bromine and iodine, more preferably R1 is fluorine.

[0032] In the compound of Formula (I) n is 0 or 1 .

[0033] When n is equal to 1 , according to the invention R2 is a (C1-C3) alkyl and R3 and R4 are H, or R2 is H and R3 and R4 together with the carbon atoms to which they are bonded form a condensed benzene ring.

[0034] R2 is a (C1-C3) alkyl, preferably it is methyl. More preferably, when n is equal to 1 , according to the invention R2 is H and R3 and

[0035] R4 together with the carbon atoms to which they are bonded form a condensed benzene ring.

[0036] When n is equal to 0, R2, R3 and R4 together with the carbon atoms to which they are bonded form a condensed naphthalene structure.

[0037] In this first aspect, the invention concerns a dispiropyrrol idine compound of Formula

[0038] (I) selected from the group consisting of:

[0039] Compound 4a: 1 ',5-dimethyldispiro[cyclohexan-1 ,3'-pyrrolidin- 2',3"-indoline]-2,2"-dione

[0040] Compound 4b: 1 '-methyldispiro[cyclohexan-1 ,3'-pyrrolidin- 2',3"-indoline]-2,2"-dione

[0041] Compound 4c: 1 '-methyl-3",4"-dihydro-1"H-dispiro[indolin- 3, 2'-pirrolidine-3',2"naphthalene]-1 ",2-dione

[0042] Compound 4d: 1'-methyl-2 / - / -dispiro[acenaphthylen-1 ,3'- pyrrolidin-2',3"-indoline]-2,2"-dione

[0043] Compound 6b: 5"-fluoro-1 '-methyldispiro[cyclohexan-1 ,3'- pyrrolidin-2',3"-indoline]-2,2"-dione

[0044] Compound 6c: 5-fluoro-1 '-methyl-3",4"-dihydro-1" / - / - dispiro[indolin-3,2'-pyrrolidin-3',2"-naphthalene]-1 ",2-dione Compound 6d: 5"-fluoro-1'-methyl-2H- dispiro[acenaphthylen-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"- dione

[0045] More preferably, the compound of the invention is selected from the group consisting of 1 '-methyl-3",4"-dihydro-1 " / - / -dispiro[indolin-3,2'-pirrolidine-3',2"naphthalene]- 1 ",2-dione (Compound 4c); 1 '-methyl-2H-dispiro[acenaphthylen-1 ,3'-pyrrolidin- 2',3"-indoline]-2,2"-dione (Compound 4d); 5-fluoro-1 '-methyl-3",4"-dihydro-1"H- dispiro[indolin-3,2'-pyrrolidin-3',2"-naphthalene]-1 ",2-dione (Compound 6c).

[0046] The invention further comprises a pharmaceutically acceptable salt of the compound of Formula (I) selected from the group consisting of hydrochloride, hydrobromide, sulfate, phosphate, acetate, succinate, oxalate, ascorbate, tartrate, gluconate, benzoate, maleate, fumarate and stearate.

[0047] In another aspect, the invention also includes a compound of Formula (I) labelled with at least one radioisotope such as, for example, tritium (3H), carbon (14C), iodine (1251) or with fluorescent probes, PET (Positron Emission Tomography) or SPECT (Single Photon Emission Tomography).

[0048] In a further aspect, the invention concerns a compound of Formula (I) for use as a medicament and pharmacologically acceptable excipients.

[0049] All preferred and inventive aspects relating to the compound of Formula (I) can be repeated herein for the compound of Formula (I) for use as a medicament.

[0050] In another aspect, the invention therefore provided a compound of Formula (I) as a modulator of the mitochondrial permeability transition pore opening in mammalian cells and tissues.

[0051] In yet a further aspect, the invention concerns a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use as a selective inhibitor of the C subunit of the F1 / F0-ATP synthase complex in the treatment of reperfusion injury diseases.

[0052] All the preferred and inventive aspects relating to the compound of Formula (I) can be repeated herein for the compound of Formula (I) for use in the treatment of reperfusion injury diseases. The compound for use according to the invention is related to the treatment of reperfusion injury diseases selected from cardiac, neurological and nephrological diseases.

[0053] According to the invention, reperfusion damage diseases are cardiac and nephrological diseases deriving from ischemic events related to heart attack and stroke.

[0054] For therapeutic application purposes, the compounds described herein can therefore be suitably formulated for the purposes of administration to mammals (in particular to human subjects), as such or associated in an appropriate pharmaceutical composition with one or more pharmaceutically acceptable excipients and / or vehicles. The compositions of the invention comprise those intended for oral, nasal, sublingual and particularly parenteral (subcutaneous, intramuscular, intravenous and intradermal) administration in the form of aqueous and non-aqueous sterile injectable preparations (solutions or suspensions).

[0055] According to the invention, the therapeutic dosage for the modulation of the mitochondrial permeability transition pore opening by the molecules object of the invention is extrapolated in vitro by using the same on cell lines expressing the cellular target. In consideration of the biological activity profile shown by the compounds of Formula (I) of the present invention, the pharmaceutical compositions comprising the same can be used for the treatment of diseases and disorders or conditions associated with the need to induce cardioprotection including, but not limited to, infarction and ischemia-reperfusion injury. In general, the invention claims a potentially useful method for the treatment of diseases mediated by alteration of the function of mPTP.

[0056] The invention will now be exemplified with reference to preparation examples of the compounds of Formula (I) of the invention and assessment of the therapeutic / medical effects of the compounds by way of example and without limitations.

[0057] EXPERIMENTAL PART

[0058] The compounds of the invention were prepared according to the scheme shown in Figure 1 .

[0059] Isatin-derived dispiropyrrolidine compounds (Compounds 4 a-d, Figure 2a) were synthesized via a one-pot reaction, in which isatin 1 , the cyclic ketone 3 a-d and sarcosine 2 are reacted together in a single reaction environment in refluxing methanol. The reaction mechanism involves the reaction between isatin and sarcosine to form an iminium ion, generated by the immediate nucleophilic addition reaction of sarcosine, which attacks the carbonyl function of isatin via the nitrogen electron pair. The iminium ion presumably undergoes two reactions: (a) decarboxylation to give azomethine ylide and (b) nucleophilic substitution to give glycolic acid, which, in turn, undergoes decarboxylation upon reaction with isatin, giving formaldehyde. The ylide reacts with formaldehyde to give the imino-alcohol, which reacts with the enol of the ketone to obtain the adduct which subsequently cyclizes with elimination of water, resulting in dispiropyrrolidine (Kumar R. S., et al., Tetrahedron Letters, 2007, 48, 7164-7168).

[0060] The dispiropyrrolidine compounds derived from 5-fluoro isatin (Compounds 6 b-d, Figure 2b) were obtained by the same procedure as the isatin derivatives (Compounds 4 a-d) but using a one-pot reaction involving 5-fluoro isatin which undergoes nucleophilic addition by the sarcosine nitrogen doublet, and the subsequent rearrangements described above, to then react in the same way with the cyclic ketone in the enolic form.

[0061] Example 1 : Preparation of Compounds 4a-d

[0062] Isatin (500 mg, 3.4 mmol) was solubilized in MeOH (15 ml) in a 100 ml flask. Sarcosine (453.9 mg, 5.1 mmol) and methylcyclohexanone (417.26 pl, 3.4 mmol) were added to it. The reaction was left under stirring at reflux for 12h. The reaction was monitored by mass spectroscopy (ESI) and TLC (Thin Layer Chromatography). The reaction mixture was then quenched with sat NaHCOs and deionized H2O; MeOH was evaporated, and the organic phase was extracted with ethyl acetate, and subsequently the product of interest was isolated by column chromatography using an eluent mixture of the petroleum ether / AcOEt (1 :1 ) type and obtaining a yellow-orange solid.

[0063] 1 ',5-Dimethyldispiro[cyclohexan-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"-dione

[0064] (Compound 4 a)

[0065] Yield 51 %.1H NMR (400 MHz, Chloroform-d) 5 9.01 (s, 1 H), 7.29 - 7.09 (m, 1 H), 7.05 - 6.75 (m, 3H), 3.29 - 3.13 (m, 2H), 2.83 - 2.69 (m, 1 H), 2.60 (dt, J = 14.6, 3.1 Hz, 1 H), 2.26 - 2.11 (m, 1 H), 2.03 (s, 3H), 1.90 - 1.62 (m, 2H), 1.40 - 1.06 (m, 3H), 0.79 (d, J = 6.4 Hz, 3H).13C NMR (101 MHz, CDCIs) 5 211.40, 179.40, 141.51 , 129.51 , 127.51 , 126.93, 122.64, 110.30, 76.34, 62.84, 52.07, 45.37, 41.70, 35.71 ,

[0066] 34.66, 34.54, 28.66, 21.86.

[0067] 1 '-Methyldispiro[cyclohexan-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"-dione (Compound 4 b)

[0068] Yield 51 %.1H NMR (400 MHz, Chloroform-d) 5 8.86 (s, 1 H), 7.32 - 7.09 (m, 1 H), 7.09 - 6.93 (m, 2H), 6.90 (d, J = 7.8 Hz, 1 H), 3.26 (h, J = 3.9, 3.4 Hz, 2H), 2.68 (ddd, J = 12.7, 7.5, 5.3 Hz, 1 H), 2.44 (dt, J = 13.5, 3.9 Hz, 1 H), 2.22 (dd, J = 14.6, 4.4 Hz, 1 H), 2.08 (s, 3H), 2.03 - 1 .91 (m, 1 H), 1 .77 (ddd, J = 14.9, 11.5, 7.0 Hz, 1 H), 1 .67 (ddd, J = 14.7, 10.9, 3.7 Hz, 2H), 1.59 - 1.45 (m, 2H), 1.43 - 1.32 (m, 1 H).13C NMR (101 MHz, CDCI3) 5 212.01 , 179.02, 141.71 , 135.09, 129.55, 128.33, 127.09,

[0069] 123.44, 122.75, 115.29, 110.23, 62.83, 52.14, 41.30, 35.85, 35.64, 34.10, 27.70,

[0070] 25.44, 21.69.

[0071] 1 '-Methyl-3",4"-dihydro-1 "H-dispiro[indolin-3,2'-pyrrolidin-3',2"-naphthalene]-1 ",2- dione (Compound 4 c)

[0072] Yield 52%.1H NMR (400 MHz, Chloroform-d) 5 10.20 (s, 1 H), 8.35 - 8.01 (m, 4H), 7.62 (ddd, J = 8.6, 7.3, 1 .5 Hz, 1 H), 7.41 - 6.43 (m, 2H), 4.39 (s, 3H), 3.49 (s, 4H), 2.79 - 2.02 (m, 9H).13C NMR (101 MHz, CDCI3) 5 210.13, 162.80, 152.08, 143.47, 138.59, 135.13, 133.27, 131.81 , 128.45, 127.94, 126.87, 125.20, 123.52, 118.73, 115.15, 114.57, 109.84, 57.72, 51.38, 27.74, 26.04

[0073] 1 '-Methyl-2H-dispiro[acenaphthylen-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"-dione (Compound 4 d)

[0074] Yield 90%.1H NMR (400 MHz, Chloroform-d) 5 7.90 (d, J = 8.1 Hz, 1 H), 7.85 (d, J = 7.0 Hz, 1 H), 7.70 (dd, J = 7.7, 5.8 Hz, 2H), 7.56 (dd, J = 8.1 , 7.0 Hz, 1 H), 7.47 (dd, J = 8.4, 7.0 Hz, 1 H), 6.91 (td, J = 7.7, 1.3 Hz, 1 H), 6.76 (td, J = 7.6, 1.1 Hz, 1 H), 6.46 (d, J = 7.7 Hz, 1 H), 3.84 (s, 1 H), 3.64 (td, J = 9.5, 8.6, 4.1 Hz, 1 H), 3.06 (td, J = 11.9, 5.1 Hz, 1 H), 2.46 (ddd, J = 12.8, 8.5, 4.2 Hz, 1 H), 2.30 (s, 4H).13C NMR (101 MHz, Chloroforme d 204.87, 177.95, 142.05, 141.52, 132.66, 132.53, 131.68, 131.28, 130.34, 129.92, 129.44, 128.94, 128.68, 128.22, 128.12, 126.65, 124.89,

[0075] 122.66, 122.53, 121.70, 121.58, 109.67, 109.60, 79.00, 63.48, 52.45, 35.98, 31.73. Example 2: Preparation of Compounds 6 b-d 5-Fluoroisotin (500 mg, 3.03 mmol) was solubilized in MeOH (15 ml) in a 100 ml flask. Sarcosine (404.54 mg, 4.54 mmol) and the appropriate cyclic ketone (314.04 pl, 3.03 mmol) were added to it. The reaction was left under stirring at reflux for 12 h. The reaction was monitored by mass spectroscopy (ESI) and TLC (Thin Layer Chromatography). The reaction mixture was then quenched with sat NaHCOs and deionized H2O; MeOH was evaporated, and the organic phase was extracted with ethyl acetate. The organic phase was evaporated after drying with anhydrous sodium sulfate, and the product of interest was analyzed by column chromatography, using an eluent mixture of the petroleum ether / AcOEt (1 :1 ) type. After evaporating the solvent, a yellow-orange solid was obtained.

[0076] 5"-Fluoro-1 '-methyldispiro[cyclohexan-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"-dione (Compound 6 b)

[0077] Yield 72%.1H NMR (400 MHz, Chloroform-d) 5 8.56 (s, 1 H), 6.95 (td, J = 8.7, 2.6 Hz, 1 H), 6.89 - 6.76 (m, 2H), 3.33 - 3.12 (m, 2H), 2.62 (ddd, J = 12.8, 7.4, 5.7 Hz, 1 H), 2.43 - 2.22 (m, 2H), 2.07 (s, 3H), 1 .99 (ddd, J = 12.6, 8.4, 6.8 Hz, 1 H), 1 .83 (ddd, J = 15.0, 11.1 , 7.0 Hz, 1 H), 1.75 - 1.55 (m, 2H), 1.45 - 1.37 (m, 1 H).13C NMR (101 MHz, CDCI3) 5 211.58, 179.03, 160.25, 157.86, 137.59, 116.13, 115.89, 115.21 , 114.96, 110.65, 110.57, 62.95, 52.02, 41.19, 35.79, 35.36, 34.03, 25.34, 21.67.19F NMR (376 MHz, CDCI3) 5 -119.58, -119.59, -119.61 , -119.62, -119.63, - 119.64.

[0078] 5-Fluoro-1 '-methyl-3",4"-dihydro-1"H-dispiro[indolin-3,2'-pyrrolidin-3',2"- naphthalene]-1 ",2-dione (Compound 6 c)

[0079] Yield 38%.1H NMR (400 MHz, Chloroform-d) 5 8.29 (s, 1 H), 8.09 (dd, J = 7.7, 1.7 Hz, 1 H), 7.37 - 7.16 (m, 3H), 6.94 - 6.81 (m, 1 H), 6.76 - 6.62 (m, 2H), 6.62 - 6.44 (m, 1 H), 3.53 (q, J = 8.4 Hz, 1 H), 3.25 (ddd, J = 10.6, 8.8, 4.3 Hz, 1 H), 2.58 (ddd, J = 12.7, 9.1 , 4.2 Hz, 3H), 2.42 (dt, J = 14.5, 4.1 Hz, 1 H), 2.34 - 2.19 (m, 2H), 2.10 (s, 3H).13C NMR (101 MHz, cdcl3) 5 198.74, 159.86, 157.46, 143.25, 137.47, 137.45, 133.26, 133.03, 128.14, 127.85, 126.97, 115.82, 115.58, 110.06, 109.98, 57.97, 51.89, 35.57, 33.37, 31.96, 26.07.19F NMR (376 MHz, CDCI3) 5 -120.56.

[0080] 5"-Fluoro-1 '-methyl-2 / - / -dispiro[acenaphthylene-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"- dione (Compound 6 d) Yield 40%.1H NMR (400 MHz, Chloroform-d) 5 7.98 - 7.87 (m, 1 H), 7.77 - 7.66 (m, 1 H), 7.60 (ddd, J = 8.1 , 7.1 , 5.5 Hz, 1 H), 7.50 (dd, J = 8.3, 7.1 Hz, 2H), 7.02 (dd, J = 8.6, 2.6 Hz, 1 H), 6.66 - 6.57 (m, 1 H), 6.39 (dd, J = 8.4, 4.2 Hz, 2H), 3.87 - 3.78 (m, 1 H), 3.61 (ddd, J = 10.7, 8.8, 4.1 Hz, 3H), 3.05 (ddd, J = 13.0, 10.7, 5.4 Hz, 2H).13C NMR (101 MHz, Chloroform-d) 5 204.60, 160.14, 157.74, 142.01 , 132.47, 132.00, 131.43, 130.35, 128.70, 128.28, 128.06, 125.01 , 122.59, 121.83, 121.77, 116.04, 115.81 , 114.67, 114.41 , 110.15, 110.07, 63.59, 52.43, 35.89, 31.78.19F NMR (376 MHz, Chloroform-d) 5 -120.60 (d, J = 488.2 Hz).

[0081] Example 3: Assessment of mPTP in vitro inhibition

[0082] In order to determine the biological effect of the compounds described herein, a single cell fluorescence microscopy assay, named Cobalt-Calcein (for technical details refer to M. Bonora et al. Nat Protoc 2016, 11 , 1067) was carried out in viable human ventricular cardiomyocytes in which the mPTP opening induced by cytosolic and mitochondrial calcium overload is assessed in a highly specific way, i.e. same as happens in conditions of ischemia and reperfusion. In detail, the mPTP opening was stimulated by the addition of the ionophore ionomycin (500 nM) and the pore opening kinetics were evaluated in terms of the average slope of the mitochondrial fluorescent signal decrease. As reported in Figure 2a-b, the 15-minute pretreatment with Compounds 4 a-d, 6 b-c, at a concentration of 1 pM, resulted in a desensitization of the mPTP opening when compared with non-pretreated cells in which the same intracellular overload of calcium had been induced. In all cases, applying the one-way ANOVA statistical test, the Compounds 4 a-d, 6 b-c inhibit mPTP in a statistically significant manner with a p value ranging from <0.05 to < 0.0001.

[0083] Example 4: Assessment of the beneficial effects of PTP inhibition in an animal model Considering the ability of these inhibitors to desensitize the mPTP opening (Figure 2), the effects of some compounds of the invention (4c, 4d and 6c) were studied in an animal model of cardiac reperfusion injury. For this purpose, beating rat hearts were isolated and placed in an extracorporeal circulation system called Langendorff. This system made it possible to study in detail the behavior of the heart, kept alive by the mechanical circulation system.

[0084] The protocol involved stabilizing the heart for 20 min, once cannulated through the aorta, and then retrograde perfusion was then progressively interrupted to induce 30 min of global ischemia followed by a few hours of reperfusion. Following heart stabilization, the left ventricle developed a given systolic pressure (LVPdp) which was measured and stored as an index of cardiac performance. Figure 3 showed how this parameter was normal in stabilization conditions (black trace), how it progressively decreased to 0 when ischemia was induced, and how this parameter was restored to varying degrees in proportion to the damage that the heart had suffered. Following reperfusion, the control heart had undergone a strong decrease in the LVDP value due to the induced infarction and compared to the moment of stabilization. The data improves if, at the time of reperfusion, the heart underwent an in situ treatment with Compounds 4c (grey trace), 4d (light gray trace) and 6c (dark gray trace).

Claims

CLAIM1. A dispiropyrrolidine compound of Formula (I):or a pharmaceutically acceptable salt thereof wherein:Ri is hydrogen or a halogen; n is 0 or 1 , provided that: when n is equal to 1 , R2 is a (C1-C3) alkyl, R3 and R4 are H, or R2 is H and R3 and R4 together with the carbon atoms to which they are bonded form a condensed benzene ring when n is equal to 0, R2, R3 and R4 together with the carbon atoms to which they are bonded form a condensed naphthalene structure.

2. The compound according to claim 1 , wherein R1 is halogen, preferably selected from fluorine, chlorine, bromine, and iodine, more preferably R1 is fluorine.

3. The compound according to claim 1 or claim 2, wherein R2 is a (C1-C3) alkyl, preferably it is methyl.

4. The compound according to any one of claims 1 to 3, wherein when n is equal to 1 , R2 is H, and R3 and R4 together with the carbon atoms to which they are bonded form a condensed benzene ring.

5. The compound according to any one of claims 1 to 3, wherein when n is equal to 0, R2, R3 and R4 together with the carbon atoms to which they are bonded form a condensed naphthalene structure.

6. The compound according to claim 1 , wherein the dispiropyrrol idine compound ofFormula (I) is selected from the group consisting of:Compound 4a1 ',5-dimethyldispiro[cyclohexan-1 , 3'-pyrrol id in-2' , 3"- indoline]-2,2"-dioneCompound 4b1 '-methyldispiro[cyclohexan-1 , 3'-pyrrol id in-2' ,3"-indoline]- 2,2"-dioneCompound 4c1 '-methyl-3",4"-dihydro-1" / - / -dispiro[indolin-3,2'-pyrrolidin-3',2"-naphthalene]-1 ",2-dioneCompound 4d1 '-methyl-2 / - / -dispiro[acenaphthylen-1 , 3'-pyrrol id in-2', 3"- indoline]-2,2"-dioneCompound 6b5"-fluoro-1 '-methyldispiro[cyclohexan-1 ,3'-pyrrolidin-2',3"- indoline]-2,2"-dioneCompound 6c5-fluoro-1 '-methyl-3",4"-dihydro-1 " / - / -dispiro[indol in-3, 2'- pyrrolidin-3',2"-naphthalene]-1 ",2-dioneCompound 6d 5"-fluoro-1 '-methyl-2 / - / -dispiro[acenaphthylen-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"-dione57. The compound according to claim 6, wherein the compound of Formula (I) is selected from the group consisting of 1 '-methyl-3",4"-dihydro-1" / - / -dispiro[indolin- 3, 2'-pyrrolidin-3',2"naphthalene]-1 ",2-dione (Compound 4c); 1 '-methyl-2 / - / - dispiro[acenaphthylen-1 ,3'-pyrrolidin-2',3"-indoline]-2,2"-dione (Compound 4d); 5- fluoro-1 '-methyl-3",4"-dihydro-1" / - / -dispiro[indolin-3,2'-pyrrolidin-3',2"-naphthalene]- 1 ",2-dione (Compound 6c).

8. The compound according to any one of claims 1 to 7, wherein the pharmaceutically acceptable salt of the compound of Formula (I) is selected from the group consisting of hydrochloride, hydrobromide, sulfate, phosphate, acetate, succinate, oxalate, ascorbate, tartrate, gluconate, benzoate, maleate, fumarate, and stearate.

9. The compound according to any one of claims 1 to 8, wherein the compound of Formula (I) is labeled with at least one radioisotope selected from tritium (3H), carbon (14C), iodine (1251) or with fluorescent probes, suitable for use in PET (Positron Emission Tomography) and SPECT (Single Photon Emission Tomography).

10. A compound of Formula (I) according to any one of claims 1 to 9, for use as a medicament.

11. A compound of Formula (I) according to any one of claims 1 to 9, for use as a selective inhibitor of the C subunit of the F1 / F0-ATP synthase complex in the treatment of reperfusion injury diseases.

12. The compound for use according to claim 11 , wherein the reperfusion injury diseases are cardiac, neurological and nephrological diseases.

13. The compound for use according to claim 11 or 12, wherein the reperfusion injury diseases are cardiac and nephrological diseases resulting from ischemic events related to heart attack and stroke.

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