Spiro-isatin derivatives as inhibitors of the MPTP mitochondrial permeability transition PORE in the treatment of myocardial reperfusion injury

Abstract

The present invention relates to new spiro-isatin derivatives for the treatment of myocardial reperfusion injury. 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.

Description

[0001] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0002] TITLE:

[0003] SPIRO-ISATIN DERIVATIVES AS INHIBITORS OF THE MPTP MITOCHONDRIAL PERMEABILITY TRANSITION PORE IN THE TREATMENT OF MYOCARDIAL REPERFUSION INJURY

[0004] ***** ***** *****

[0005] DESCRIPTION FIELD OF INVENTION

[0006] The present invention relates to new spiro-isatin derivatives for the treatment of myocardial reperfusion injury.

[0007] 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.

[0008] STATE OF THE ART

[0009] Acute myocardial infarction (MI) is one of the leading causes of death and disability worldwide. The standard treatment of patients with ST-segment-elevation Ml (STEMI) for reducing ischemic injury is the prompt restoration of blood flow via either primary percutaneous coronary intervention (PCI) or thrombolytic therapy. Despite timely reperfusion strategies, STEMI is associated with 6-12% mortality and 14-36% prevalence of heart failure within 1 year. In particular, the so-called reperfusion injury (Rl) contributes to limit the efficacy of PCI being responsible for up to 50% of the final infarct size (A. Rout et al. Expert Opin Pharmacother 2020:21:1851). Unfortunately, there is a clear unmet need with respect to Rl therapy and this is mainly due to the incomplete understanding of the complex pathophysiology of this condition.

[0010] The so-called mitochondrial permeability transition pore (mPTP) is recognized as the main responsible for cardiomyocyte death in the final step of Rl, which makes it an emerging therapeutic target for cardioprotection (G. Morciano et al. Adv Exp Med Biol. 2017:982:169). mPTP opening mediates mitochondrial permeability transition (MPT)-driven apoptosis, during which the inner mitochondrial membrane, usuallyP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0011] highly impermeable, undergoes to a sudden and irreversible increase of permeability allowing the free entry of all molecules <1.5 kDa into the matrix which translates into ATP depletion, mitochondrial swelling, and release of cytochrome c and other apoptotic proteins ultimately leading to cell death. The increase of intra-mitochondrial calcium levels and oxygen species by-products, both typical conditions occurring when the blood flow is restored in a tissue after an ischemic insult, have been identified as triggers of mPTP opening.

[0012] Several mPTP inhibitors have been identified, among which, cyclosporin A (CsA) and TRO40303, have reached clinical studies. However, inconsistent results have been achieved as far as their cardioprotective action. It has been suggested that these disappointing results may in part be due to the indirect mechanism of action of such drug candidates. mPTP is indeed defined as a multiprotein platform including pore-forming elements and modulators that contribute to its open / closed conformational state. (T. Briston et al. Trends Pharmacol Sci. 2019:40:50) CsA is a Cyclophilin D (CypD)-dependent inhibitor while TRO40303 binds the mitochondrial translocator protein (TSPO) located in the outer mitochondrial membrane. However, both CypD and TSPO are recognized as regulators of mPTP function more than structural components of the pore.

[0013] Of note, recent data point to a role for ATP synthase in mPTP formation. (N. Mnatsakanyan et al. Nat Commun. 2019:10:5823) According to this model, the increase of mitochondrial calcium during reperfusion can convert the energy-supplying ATP synthase machine into an energy-dissipating supramolecular entity, mPTP. Specifically, independent studies provided evidence that the c subunits of F1 / F0-ATP synthase play an essential role in mPTP formation: depletion of the c subunit reduces channel opening in response to calcium- and oxidative stress-induced stimuli, whereas its over expression enhances mPTP opening. (M. Bonora et al. Cell Cycle. 2013:12:674) The recent disclosure of the Cryo-EM structure of the mammalian F-type ATP synthase provided solid confirmation of the c-ring model. (G. Pinke et al. Nat Struct Mol Biol. 2020:27:1077)

[0014] Recent studies of the inventors reported the discovery of the first mPTP inhibitorsP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0015] that target the c subunit of F1 / F0-ATP synthase. (G. Morciano et al. J Med Chem 2018:61:7131) These compounds were designed from the structure of Oligomycin A (OA), a known mPTP inhibitor that binds the c subunit of F1 / F0-ATP synthase however preventing at the same time the synthesis of ATP. (A. Pagliarini et al. Mini Rev Med Chem. 2016:16:815) The crystal structure of the OA-ATP synthase complex indicated that the spirochetal portion of OA is closely engaged by the binding cavity of the c ring. In the attempt to simplify the structure of OA to the putative pharmacophore portion, the inventors screened a small internal library of spirocyclic compounds among which PP11 was selected as a valuable HIT-compound with good activity in vitro. Further structure-activity relationship (SAR) optimization led to compound IB13 with improved potency in vitro and beneficial effects in preserving cardiac functions in an ex vivo model of Rl (Scheme 1). Noteworthy, unlike what was found for OA, the inhibition of mPTP by IB 13 was not associated with a depletion of the mitochondrial ATP content.

[0016] Scheme 1. Reference compounds for the design of novel mPTP inhibitors.

[0017] OligomycinA PP11 IB13

[0018]

[0019] % Inhibition = 30 (10 μM) % Inhibition = 31 (1 μM) % Inhibition = 67 (1 μM)

[0020] 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.

[0021] However, the need for a new pharmacological and / or therapeutic approach for the treatment of diseases resulting from reperfusion-related damage is still felt.

[0022] SUMMARY OF THE INVENTION

[0023] The inventors surprisingly identified novel spiro derivatives as potential cardioprotective agents.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0024] Surprisingly the inventors found out novel compounds by replacing the piperidine ring of the previous compounds PP11 and IB13 with an isatin bicycle.

[0025] Thus, the inventors surprisingly found new molecules which are 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.

[0026] The invention therefore allowed the identification of potent inhibitors of the mitochondrial permeability transition pore opening in living mammalian cells.

[0027] Therefore, the invention concerns a spiro-isatin derivative compound of Formula (I):

[0028] R

[0029] /

[0030]

[0031] or a pharmaceutically acceptable salt thereof,

[0032] wherein:

[0033] - R is a substituent selected from the group consisting of (C₁-C₃)alkyl and (C₁-C₃)alkyl phenyl, wherein the phenyl moiety is optionally substituted with a halogen; - R₁ is a substituent selected from the group consisting of hydrogen, halogen and (C₁-C₃)alkoxy;

[0034] - R₂ is a substituent selected from the group consisting of hydrogen, halogen and trifluoromethyl (-CF₃).

[0035] In the present invention, when the following definitions are used:

[0036] - “(Ci-Csjalkyl” means linear or branched hydrocarbon chains comprising 1 to 3 carbon atoms;

[0037] - “(Ci-Csjalkoxy” means a substituent having an (alkyl chain)-O- structure with 1 to 3 carbon atoms;

[0038] - “(Ci-Csjalkyl phenyl” or “(Ci-Csjalkyl-Ph” means a substituent having an (alkyl chain)-phenyl- structure with 1 to 3 carbon atoms, specifically it indicates a benzylP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0039] group, a ethyl benzyl group and a propyl benzyl group, and

[0040] - “halogen” means fluorine, chlorine, bromine and iodine.

[0041] In a preferred and advantageous embodiment, the invention concerns a spiro-isatin derivative compound of Formula (I):

[0042] N

[0043] o

[0044]

[0045] HN compound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione.

[0046] In another aspect, the invention therefore provided a compound of Formula (I) for use as a modulator of the mitochondrial permeability transition pore opening in mammalian cells and tissues.

[0047] Therefore, the invention concerns a compound of Formula (I) or a pharmaceutically acceptable salt thereof for use as a medicament.

[0048] 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 opening of the mitochondrial permeability transition pore (mPTP) both in mammalian cells and tissues in the treatment of reperfusion injury diseases. 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.

[0049] BRIEF DESCRIPTION OF THE FIGURES

[0050] Figure 1 reports the preparation scheme of compounds of Formula (I).

[0051] Figure 2 reports the mPTP inhibiton in AC16 living cells evaluated by calcein-cobalt assay in novel spiro-isatin derivatives.

[0052] Figure 3 reports the effects of 15j in living cardiomyocytes following H / R. A) Cellviability assay. B) Western blot. (*) p value < 0.05. (**) p value < 0.01. Figure 4 reports the effect of 15j on ATP production.

[0053] Representative kinetics of mitochondrial ATP content in living cells at restingP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0054] conditions (normoxia), in H / R of untreated cells and in H / R of 15j treated cells. Histogram with statistics of mitochondrial ATP content in living cells. (*) p value < 0.05. (****) p value < 0.0001.

[0055] DETAILED DESCRIPTION OF THE INVENTION

[0056] The invention therefore concerns a spiro-isatin derivative compound of Formula (I):

[0057] R

[0058] /

[0059]

[0060] or a pharmaceutically acceptable salt thereof,

[0061] wherein:

[0062] - R is a substituent selected from the group consisting of (C₁-C₃)alkyl and (C₁-C₃)alkyl phenyl, wherein the phenyl moiety is optionally substituted with a halogen; - R₁ is a substituent selected from the group consisting of hydrogen, halogen and (C₁-C₃)alkoxy;

[0063] - R₂ is a substituent selected from the group consisting of hydrogen, halogen and trifluoromethyl (-CF₃).

[0064] In the compound of Formula (I), R is a substituent selected from (Ci-C3)alkyl and (Ci-C3)alkyl phenyl, wherein said phenyl moiety is optionally substituted with a halogen.

[0065] Preferably, when R is (Ci-C3)alkyl, it is preferably a methyl group.

[0066] When R is (Ci-C3)alkyl phenyl, it is preferably a benzyl group.

[0067] When R is (C₁-C₃)alkyl phenyl and said phenyl moiety is optionally substituted with a halogen, preferably it is a benzyl substituted with a halogen selected from the group consisting of fluorine, chlorine, bromine and iodine, even more preferably R is a benzyl substituted with fluorine.

[0068] When R is a benzyl substituted with a F atom, it is preferably substituted in para position.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0069] In a preferred and advantageous embodiment of the invention, R is a benzyl not substituted.

[0070] In the compound of Formula (I), R1 is a substituent selected from the group consisting of hydrogen, halogen and (Ci-C3)alkoxy. Preferably when R1 is halogen, it is selected from the group consisting of fluorine, chlorine, bromine and iodine, more preferably R1 is fluorine.

[0071] Preferably when R1 is (Ci-C3)alkoxy, it is methoxy. In a preferred and advantageous embodiment of the invention, R1 is fluorine.

[0072] In the compound of Formula (I), R2 is a substituent selected from the group consisting of hydrogen, halogen and trifluoromethyl (-CF3).

[0073] Preferably when R2 is halogen, it is in para position, more preferably it is selected from fluorine, chlorine, bromine and iodine, even more preferably R2 is fluorine or chlorine.

[0074] In a preferred and advantageous embodiment of the invention, R2 is a hydrogen atom.

[0075] In a preferred and advantageous embodiment, the invention concerns a spiro-isatin derivative compound of Formula (I) wherein R is a benzyl group, R₁ is methoxy, and R₂ is a hydrogen atom.

[0076] In an even more preferred and advantageous embodiment, the invention concerns a spiro-isatin derivative compound of Formula (I) wherein R is a benzyl group, R₁ is fluorine, and R₂ is a hydrogen atom.

[0077] In this first aspect, the invention concerns a spiro-isatin derivative compound of Formula (I) selected from the group consisting of:

[0078] I

[0079]

[0080] HN-^ compound 15a: 1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0081]

[0082] compound 15b: 3-(4-chlorophenyl)-1'-methylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0083]

[0084] compound 15c: 5'-fluoro-1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0085]

[0086] compound 15d: 5'-methoxy-1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0087]

[0088] compound 15e: 1'-benzyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0089] 1'-benzyl-3-(4-

[0090] 1'-benzyl-3-(4-

[0091]

[0092] P024165WO-01 Notarbartolo & Gervasi S.p. A. chlorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0093]

[0094] phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0095] 1'-benzyl-5'-fluoro-3-(4-

[0096]

[0097] 1 '-benzyl-5'-methoxy-3- phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0098]

[0099] compound 15m: 1 '-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0100]

[0101] compound 15n: 1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0102]

[0103] compound 15o: 5'-fluoro-1'-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione; and

[0104]

[0105] compound 15p: 5'-fluoro-1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione.

[0106] More preferably, the compound of the invention is selected from the group consisting

[0107] of:

[0108]

[0109] - compound 15a: 1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0110]

[0111] compound 15b: 3-(4-chlorophenyl)-1'-methylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0112]

[0113] compound 15c: 5'-fluoro-1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0114]

[0115] compound 15d: 5'-methoxy-1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0116]

[0117] compound 15e: 1'-benzyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0118] 1'-benzyl-3-(4-

[0119] 1'-benzyl-3-(3-

[0120]

[0121] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0122] (trifluoromethyl)phenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0123]

[0124] phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0125]

[0126] fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0127] N

[0128]

[0129] compound 15l: 1'-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0130] N

[0131] o ^N^

[0132]

[0133] HN compound 15m: 1 '-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione.

[0134] Even more preferably, the invention concerns a spiro-isatin derivative compound of Formula (I) selected from the group consisting of:

[0135] N

[0136] o

[0137]

[0138] HN compound 15e: 1'-benzyl-3-phenylspiro[imidazolidine-4,3'-P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0139] indoline]-2', 5-dione;

[0140]

[0141] HN compound 1 chlorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;

[0142]

[0143] phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione; and

[0144] N

[0145]

[0146] compound 15l: 1'-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione.

[0147] Even more preferably, the invention concerns a spiro-isatin derivative compound of Formula (I) selected from the group consisting of:

[0148] N

[0149]

[0150] compound 15l: 1'-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione, andP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0151]

[0152] compound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione.

[0153] In an even more preferred and advantageous embodiment, the invention concerns a spiro-isatin derivative compound of Formula (I):

[0154]

[0155] compound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione. The invention further comprises a pharmaceutically acceptable salt of the compound of Formula (I) selected from the group consisting of hydrochloride, hydrobromide, sulphate, citrate, phosphate or acid phosphate, acetate, maleate, fumarate, stearate, lactate, tartrate, gluconate, salicylate, ascorbate, aspartate, succinate, oxalate and benzoate.

[0156] In a further aspect, the invention concerns a compound of Formula (I) for use as a medicament.

[0157] In another aspect, the invention provided a compound of Formula (I) for use as a modulator of the mitochondrial permeability transition pore opening in mammalian cells and tissues.

[0158] 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 opening of the mitochondrial permeability transition pore (mPTP) both in mammalian cells and tissues in the treatment of reperfusion injury diseases.

[0159] 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,P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0160] neurological and nephrological diseases.

[0161] According to the invention, reperfusion damage diseases are cardiac and nephrological diseases deriving from ischemic events related to heart attack and stroke.

[0162] 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).

[0163] 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 cardio protection including, but not limited to, infarction and ischemia-reperfusion injury. In general, the invention claims a method for the treatment of diseases mediated by alteration of the function of mPTP.

[0164] The compounds of the invention will be normally, but not necessarily, formulated into a pharmaceutical composition prior to administration to a patient. The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art.

[0165] The pharmaceutical compositions of the invention may be prepared and packaged in bulk form wherein an effective amount of a compound of the invention can be extracted and then given to the patient such as with powders, syrups, and solutions for injection. Alternatively, the pharmaceutical compositions of the invention may beP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0166] prepared and packaged in unit dosage form. A dose of the pharmaceutical composition contains at least a therapeutically effective amount of a compound of this invention (i.e., a compound of Formula (I), or a salt, particularly a pharmaceutically acceptable salt, thereof). When prepared in unit dosage form, the pharmaceutical compositions may contain from 1 pg to 1000 mg, preferably 1 mg to 100 mg, of a compound of this invention.

[0167] The compounds of the invention and the pharmaceutically acceptable excipient or excipients will typically be formulated into a dosage form adapted for administration to the patient by the desired route of administration.

[0168] Conventional dosage forms include those adapted for (1 ) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) trans-dermal administration such as trans-dermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols and solutions; and (6) topical administration such as creams, ointments, lotions, pastes, sprays and gels.

[0169] Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, granulating agents, coating agents, wetting agents, suspending agents, emulsifiers, sweeteners,, flavour masking agents, colouring agents, anti-caking agents, humectants, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch, cellulose, calcium sulphate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch, gelatine, sodium alginate, alginic acid, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calciumP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0170] stearate, and talc. Suitable carriers for oral dosage forms include but are not limited to magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, methylcellulose, sodium carboxymethyl cellulose, and the like.

[0171] Techniques used to prepare oral formulations are the conventional mixing, granulation and compression or capsules filling.

[0172] The compounds of the present invention may be also formulated for parenteral administration with suitable carriers including aqueous vehicles solutions (i.e.: saline, dextrose) or and / or oily emulsions.

[0173] 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.

[0174] EXPERIMENTAL PART

[0175] Example 1: Materials and methods

[0176] Reagents and solvents were provided by BLD, Fluorochem and Sigma-Aldrich. Reaction progress and product mixtures were monitored by thin-layer chromatography (TLC) on silica gel (precoated F254 Macherey-Nagel plates) and visualized with a UV lamp (254-nm light source). Compounds were purified through silica gel flash chromatography (silica gel 60, 40-63 pm) using appropriate eluent mixtures or on a reverse-phase Waters Prep 600 HPLC system equipped with a Jupiter column C18 (250 x 30 mm, 300 A, 15 pm spherical particle size). Reversephase purification of crude compounds was carried out using a gradient of CH3CN / H2O (with 0.1% TFA) programmed time by time, a flow rate of 20 mL / min and a UV detector with a wavelength of 220 nm. Analytical HPLC analyses were performed on a Beckman 116 liquid chromatograph equipped with a Beckman 166 diode array detector. Analytical purity of the final compounds was assessed using a XBridge® C18 column (4.6 x 150 mm, 5pm particle size) at a flow rate of 0.7 mL / min with a linear gradient from 100% of solvent A (H2O + 0.1% TFA) to 100% of solvent B (CH3CN + 0.1% TFA) over 25 mins. Analytical determinations were reported as column retention time (TR) in minutes and the purity of final compounds was > 95%P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0177] as determined by HPLC analysis carried out at a wavelength of 220 nm. Mass spectra were recorded with a Waters ESI Micromass ZQ dissolving the samples in a solution of H2O / CH3CN / TFA (40:60:0.1). Melting points were determined using glass capillaries on a Stuart Scientific electrothermal apparatus SMP3 and are uncorrected. NMR analyses were performed in CDCI3 or DMSO-d6 at ambient temperature using a Varian 400 MHz spectrometer. Chemical shifts (5) are reported in parts per million (ppm) using the peak of tetramethylsilan as an internal standard in deuterated solvents and coupling constants (J) are reported in Hertz. Splitting patterns are designed as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and b, broad.

[0178] Example 2: General synthetic procedures for the preparation of spiro-isatin derivative compounds (Compounds 15 a-p) according to the invention The inventors synthesised a series of compounds in which the general 3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione core was variously substituted at the T-, 5'-, and 3-positions (see R, R1 and R2 in Figure 1, respectively).

[0179] The compounds of the invention were prepared according to the scheme shown in Figure 1. Spiro-isatin derivative compounds (Compounds 15a-p, Figure 1) were synthesised as depicted in Scheme 1 starting from commercially available 5-substituted isatins (1-3) whose nitrogen was firstly alkylated in standard conditions with methyl iodide (Mel), benzyl bromide (BnBr) or 4-F-BnBr to give 4-11. A standard multicomponent Strecker reaction with different anilines in the presence of trimethylsilyl cyanide (TMSCN) provided the a-amino nitrile derivatives 12a-g and 12j-p. Whereas, in the case of anilines bearing the electron withdrawing CF3 group reducing their nucleophilicity, a two-step approach was required for improving reaction yields. Thus compound 7 was firstly converted into the ketimines 12'h-i that were then treated with trimethylsilyl cyanide, TMSCN affording compounds 12h-i. The subsequent base-catalysed conversion of nitriles 12a-p to the corresponding amides with hydrogen peroxide furnished 13a-p then the spyrocyclization was performed following a telescopic two-step one-pot reaction with a first treatment withP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0180] dimethylformamide dimethyl acetal (DMF-DMA), followed by the addition of acetic acid only upon the formation of the N-acylformamidine intermediates 14'a-p. The structure of the latter intermediates was assumed basing on the known reactivity of DMF-DMA with amides (https: / / doi.org / 10.1021 / jo00131a023) and on the 1H-NMR spectrum of 14'a that was isolated for analytical purposes (Figure 1). Lastly, the imidazolinone derivatives 14a-p were efficiently reduced with NaBFk to yield the final compounds 15a-p.

[0181] Example 3: Preparation of Compounds 4-6

[0182] NaH (60% dispersion in mineral oil, 1.1 eq) was added to a cooled solution of derivatives 1-3 (1 eq, 6.8 mmol) in DMF (25 mL), and the mixture was stirred at 0°C for 15 min. Mel (1.1 eq) was successively added dropwise and the reaction mixture was stirred at 0°C for 2 h and at room temperature for 1 h. Upon completion, as indicated by TLC and MS (ESI), the mixture was concentrated under vacuum. The resulting residue was then extracted with EtOAc (3x15 mL), and the combined organic layers were washed with water (1x15 mL), brine (1x10 mL) and dried over Na2SO4. The solvent was then evaporated to obtain a solid, which was triturated (Et20) and filtered to yield derivatives 4-6 as orange-red solids, used in the next step without further purification. (10.1002 / anie.201712456)

[0183]

[0184] \

[0185] 1 -methylindoline-2, 3-dione (4): Orange solid (yield 81%). 1H NMR (400 MHz, DMSO-d6) 5 7.68-7.62 (m, 1H), 7.52 (dd, J = 8.0, 2.0 Hz, 1H), 7.13-7.08 (m, 2H), 3.12 (s, 3H). MS (ESI): calculated for C9H8NO2[M+H]+ 162.06; found 162.45.

[0186] o

[0187] Y L >o

[0188]

[0189] \

[0190] 5-fluoro-1-methylindoline-2, 3-dione (5): Dark orange solid (yield 85%). 1H NMR (400 MHz, DMSO-d6) 5 7.58-7.52 (m, 1H), 7.47-7.43 (m, 1H), 7.20-7.14 (m, 1H), 3.14 (s, 3H). MS (ESI): calculated for C9H7FNO2[M+H]+ 180.05; found 180.12.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0191] o

[0192]

[0193] 5-methoxy-1 -methylindoline-2, 3-dione (6): Red solid (yield 71%). 1H NMR (400 MHz, DMSO-d6) 5 7.26-7.21 (m, 1H), 7.11-7.09 (m, 1H), 7.06 (d, J = 8.0 Hz, 1H), 3.75 (s, 3H), 3.09 (s, 3H). MS (ESI): calculated for C10H10NO3 [M+H]+ 192.07; found 192.14.

[0194] Example 4: Preparation of Compounds 7-11

[0195] K2CO3 (3 eq) and the appropriate benzyl bromide (1.5 eq) were added to a solution of derivatives 1-3 (1 eq, 6.8 mmol) in MeCN (0.10 M) at room temperature. The resulting mixture was then heated at reflux overnight. Upon completion, as indicated by TLC and MS (ESI), the reaction mixture was cooled, filtered and concentrated. The crude residue was then triturated (Et20) and filtered to yield derivatives 7-11 as orange-red solids, which were used in the next step without further purification. (10.1002 / ange.201707531)

[0196]

[0197] 1 -benzylindoline-2, 3-dione (7): Orange solid (yield 82%). 1H NMR (400 MHz, DMSO-d6) 5 7.62-7.54 (m, 2H), 7.43 (d, J = 7.2 Hz, 2H), 7.39-7.24 (m, 3H), 7.14-7.08 (m, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.91 (s, 2H). MS (ESI): calculated for C15H12NO2 [M+H]+ 238.09; found 238.30.

[0198]

[0199] 1-benzyl-5-fluoroindoline-2, 3-dione (8): Dark orange solid (yield 89%). 1H NMR (400 MHz, DMSO-d6) 5 7.53-7.40 (m, 4H), 7.38-7.24 (m, 3H), 6.98-6.93 (m, 1H),P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0200] 4.91 (s, 2H). MS (ESI): calculated for C15H11FNO2 [M+H]+ 256.08; found 256.37.

[0201] 1-benzyl-5-methoxyindoline-2, 3-dione (9): Red solid (yield 93%). 1H NMR (400 MHz, DMSO-d6) 5 7.44-7.39 (m, 2H), 7.37-7.24 (m, 3H) 7.19-7.14 (m, 2H), 6.93-6.87 (m, 1 H), 4.88 (s, 2H), 3.74 (s, 3H). MS (ESI): calculated for C16H14NO3 [M+H]+ 268.10; found 268.25.

[0202] 1-(4-fluorobenzyl)indoline-2, 3-dione (10): Dark orange solid (yield 79%). 1H NMR (400 MHz, DMSO-d6) 57.62-7.44 (m, 4H), 7.24 -7.04 (m, 3H), 6.98 (d, J = 7.7 Hz, 1H), 4.90 (s, 2H). MS (ESI): calculated for C15H11FNO2 [M+H]+ 256.08; found 256.17.

[0203]

[0204] 5-fluoro-1-(4-fluorobenzyl)indoline-2, 3-dione (11): Red solid (yield 78%). 1H NMR (400 MHz, CDCI3) 57.37-7.29 (m, 3H), 7.26-7.19 (m, 1H), 7.10-7.03 (m, 2H), 6.76-6.71 (m, 1H), 4.91 (s, 2H). MS (ESI): calculated for C15H10F2NO2 [M+H]+ 274.07; found 274.30.

[0205] Example 5. Preparation of compounds 12a-g and 12j-p

[0206] To a solution of derivatives 4-11 (1 eq, 4.2 mmol) in glacial AcOH (15 mL) was added the appropriate aniline (1 eq) at 0°C. Trimethylsilyl cyanide (TMSCN) (3 eq) was then added at 0°C and the reaction mixture was stirred at room temperatureP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0207] overnight. Upon completion, as indicated by TLC and MS (ESI), the reaction mixture was basified to pH 10 using 2M NaOH and suspended with Ac2OEt (20 mL). The organic layers were then washed with water (2x15 mL), brine (1x10 mL) and dried over Na2SO4. The solvent was evaporated under reduced pressure to give a crude solid residue, which was triturated (Et20) and filtered. Derivatives 12a-n were obtained as pale orange solids and used in the next step without further purification.

[0208] N

[0209] N

[0210] \ 1-methyl-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12a): Pale orange solid (yield 59%). 1 H NMR (400 MHz, DMSO-d6) 57.59-7.49 (m, 2H), 7.26-7.18 (m, 2H), 7.15 (s, 1H), 7.13-7-07 (m, 2H), 6.77-6.71 (m, 3H), 3.25 (s, 3H). MS (ESI): calculated for C16H14N3O [M+H]+ 264.11; found 264.26.

[0211] Cl

[0212] N

[0213] \ 3-((4-chlorophenyl)amino)-1-methyl-2-oxoindoline-3-carbonitrile (12b): Pale orange solid (yield 48%). 1 H NMR (400 MHz, DMSO-d6) 57.62-7.50 (m, 2H), 7.29 (s, 1 H), 7.26-7.14 (m, 4H), 6.83-6.80 (m, 2H), 3.24 (s, 3H). MS (ESI): calculated for C16H13CIN3O [M+H]+ 298.07; found 298.27.

[0214] N

[0215] N

[0216] \ 5-fluoro-1-methyl-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12c): Pale orange solid (yield 52%). 1H NMR (400 MHz, DMSO-d6) 5 7.54 (dd, J = 7.7, 2.7 Hz, 1H), 7.46-7.38 (m, 1H), 7.29-7.23 (m, 1H), 7.18 (s, 1H), 7.15-7.08 (m, 2H), 6.76-6.73 (m,

[0217]

[0218] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0219] 3H), 3.25 (s, 3H). MS (ESI): calculated for C16H13FN3O [M+H]+ 282.10; found 282.34.

[0220]

[0221] 5-methoxy-1-methyl-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12d): Pale orange solid (yield 54%). 1H NMR (400 MHz, DMSO-d6) 57.17-7.11 (m, 2H), 7.12-7.04 (m, 4H), 6.73-6.68 (m, 3H), 3.73 (s, 3H), 3.21 (s, 3H). MS (ESI): calculated for C17H16N3O2 [M+H]+ 294.12; found 294.04.

[0222]

[0223] 1-benzyl-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12e): Pale orange solid (yield 75%). 1 H NMR (400 MHz, DMSO-d6) 57.62-7.56 (m, 1 H), 7.47-7.43 (m, 1 H), 7.39-7.25 (m, 6H), 7.21-7.16 (m, 2H), 7.06-7.00 (m, 2H), 6.74 (t, J = 7.3 Hz, 1H), 6.71-6.63 (m, 2H), 5.07 (d, J = 15.7 Hz, 1H), 4.94 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H18N3O [M+H]+ 340.14; found 340.35.

[0224]

[0225] 1-benzyl-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carbonitrile (12f): Pale orange solid (yield 69%). 1 H NMR (400 MHz, DMSO-d6) 57.61 -7.57 (m, 1 H), 7.48-7.06 (m, 9H), 6.94-6.86 (m, 2H), 6.79-6.71 (m, 2H), 5.02 (d, J = 15.7 Hz, 1H), 4.89 (d, J = 15.7 Hz, 1 H). MS (ESI): calculated for C22H17FN3O [M+H]+ = 358.14; found 358.56.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0226]

[0227] 1-benzyl-3-((4-chlorophenyl)amino)-2-oxoindoline-3-carbonitrile (12g): Pale orange solid (yield 71 %). 1 H NMR (400 MHz, DMSO-d6) 57.63-7.59 (m, 1 H), 7.48-7.44 (m, 1H), 7.40 (s, 1H), 7.37-7.25 (m, 5H), 7.22-7.16 (m, 2H), 7.11-7.05 (m, 2H), 6.77-6.69 (m, 2H), 5.03 (d, J = 15.7 Hz, 1H), 4.92 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H17CIN3O [M+H]+ 374.11; found 374.21.

[0228] 1-benzyl-5-fluoro-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12j): Pale orange solid (yield 72%). 1H NMR (400 MHz, DMSO-d6) 5 7.58 (dd, J = 7.6, 2.7 Hz, 1H), 7.39-7.27 (m, 7H), 7.24-7.18 (m, 1H), 7.10-6.99 (m, 2H), 6.81-6.71 (m, 1H), 6.67 (dd, J = 8.6, 1.0 Hz, 2H), 5.08 (d, J = 15.7 Hz, 1H), 4.94 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H17FN3O [M+H]+ 358.14; found 358.30.

[0229] 1-benzyl-5-fluoro-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carbonitrile (12k): Pale orange solid (yield 71 %). 1 H NMR (400 MHz, DMSO-d6) 57.59 (dd, J = 7.7, 2.7 Hz, 1H), 7.35-7.26 (m, 6H), 7.17-7.12 (m, 1H), 7.11 (s, 1H), 6.91 (t, J = 8.9 Hz, 2H),

[0230]

[0231] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0232] 6.79-6.72 (m, 2H), 5.02 (d, J = 15.8 Hz, 1H), 4.88 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C22H16F2N3O [M+H]+ 376.13; found 376.24.

[0233] 1-benzyl-5-methoxy-2-oxo-3-(phenylamino)indoline-3-carbonitrile (121): Pale orange solid (yield 81 %). 1 H NMR (400 MHz, DMSO-d6) 57.38-7.26 (m, 6H), 7.21 -7.18 (m, 1H), 7.13-7.09 (m, 1H), 7.06-6.99 (m, 3H), 6.74 (t, J = 7.3 Hz, 1H), 6.67-6-64 (m, 2H), 5.05 (d, J = 15.6 Hz, 1H), 4.94 (d, J = 15.6 Hz, 1H), 3.73 (s, 3H). MS (ESI): calculated for C23H20N3O2[M+H]+ 370.16; found 370.24.

[0234] 1-(4-fluorobenzyl)-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12m): Pale orange solid (yield 84%). 1 H NMR (400 MHz, DMSO-d6) 57.60-7.56 (m, 1 H), 7.48-7.42 (m, 1H), 7.38-7.32 (m, 2H), 7.27 (s, 1H), 7.23-7.14 (m, 4H), 7.04-6.99 (m, 2H), 6.75-6.70 (m, 1H), 6.66-6.60 (m, 2H), 5.04 (d, J = 15.6 Hz, 1H), 4.91 (d, J = 15.6 Hz, 1H). MS (ESI): calculated for C22H17FN3O [M+H]+ 358.14; found 358.71.

[0235] 1-(4-fluorobenzyl)-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carbonitrile (12n): Pale orange solid (yield 89%). 1H NMR (400 MHz, DMSO-d6) 57.61-7.57 (m, 1H),

[0236]

[0237] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0238] 7.48-7.42 (m, 1 H), 7.30 (dd, J = 8.80, 5.40 Hz, 2H), 7.22-7.13 (m, 5H), 7.10 (s, 1H), 6.90 (t, J = 8.9 Hz, 2H), 6.76-6.71 (m, 2H), 5.01 (d, J = 15.7 Hz, 1H), 4.88 (d, J = 15.7 Hz, 1 H). MS (ESI): calculated for C22H16F2N3O [M+H]+ 376.13; found 376.68.

[0239]

[0240] 5-fluoro-1 -(4-fluorobenzyl)-2-oxo-3-(phenylamino)indoline-3-carbonitrile (12o): Pale orange solid (yield 87%). 1 H NMR (400 MHz, DMSO-d6) 57.56 (dd, J = 7.6, 2.6 Hz, 1H), 7.37-7.32 (m, 3H), 7.28 (s, 1H), 7.26-7.22 (m, 1H), 7.21-7.13 (m, 2H), 7.05-7.00 (m, 2H), 6.77-6.72 (m, 1H), 6.66-6.61 (m, 2H), 5.04 (d, J = 15.6 Hz, 1H), 4.91 (d, J = 15.6 Hz, 1H). MS (ESI): calculated for C22H16F2N3O [M+H]+ 376.13; found 376.42.

[0241]

[0242] 5-fluoro-1-(4-fluorobenzyl)-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carbonitrile (12p): Pale orange solid (yield 83%). 1H NMR (400 MHz, DMSO-d6) 57.60 (dd, J = 7.7, 2.7 Hz, 1 H), 7.37-7.26 (m, 3H), 7.23-7.12 (m, 3H), 7.11 (s, 1 H), 6.96-6.88 (m, 2H), 6.77-6.72 (m, 2H), 5.00 (d, J = 15.7 Hz, 1H), 4.87 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H15F3N3O [M+H]+ 394.12, found 394.41.

[0243] Example 6: Preparation of compounds 12h-i

[0244] To a solution of derivative 7 (1 eq, 4.2 mmol) in glacial AcOH (15 mL) was added the appropriate aniline (1 eq) at 0°C, and the mixture was stirred at room temperature for 3 h. Upon completion, as indicated by TLC, the solution was basified to pH 10 using 2M NaOH and suspended with Ac2OEt (20 mL). The organic layers were washed with water (2x15 mL), brine (1x10 mL) and dried over Na2SO4. TheP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0245] solvent was then evaporated under reduced pressure to give imine intermediates, as orange solids, which were used in the next step without further purification. To a solution of the obtained imine intermediates (1 eq, 4.0 mmol) in MeOH (15 mL) was added TMSCN, and the mixture was stirring at 50°C for 18 h. Upon completion, as indicated by TLC and MS (ESI), the solution was basified to pH 10 using a solution NaHCOs 5%. The resulting mixture was then suspended with Ac2OEt (20 mL), and then washed with water (2x15 mL), brine (1x10 mL) and dried over Na2SO4. The organic layers were evaporated under reduced pressure to give a solid residue which was purified by flash chromatography (EtOAc / PE as the eluent). Derivatives 12o-p were obtained as orange pale solids.

[0246] N

[0247] 2=0

[0248] N

[0249]

[0250] 1-benzyl-2-oxo-3-((3-(trifluoromethyl)phenyl)amino)indoline-3-carbonitrile (12h): Orange solid (yield 49%). 1 H NMR (400 MHz, DMSO-d6) 57.69-7.62 (m, 2H), 7.52-7.45 (m, 1 H), 7.34-7.25 (m, 6H), 7.25-7.18 (m, 3H), 7.10-7.05 (m, 1H), 7.03-6.98 (m, 1H), 5.03 (d, J = 15.8 Hz, 1H), 4.94 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C23H17F3N3O [M+H]+ 408.13; found 408.68.

[0251] F

[0252] F-J

[0253] N

[0254] 2=0

[0255] N

[0256]

[0257] 1-benzyl-2-oxo-3-((4-(trifluoromethyl)phenyl)amino)indoline-3-carbonitrile (12i): Orange solid (yield 53%). 1H NMR (400 MHz, DMSO-d6) 57.94 (s, 1H), 7.65-7.61 (m, 1H), 7.52-7.45 (m, 1H), 7.41-7.18 (m, 9H), 6.84 (d, J = 8.5 Hz, 2H), 5.04 (d, J = 15.6 Hz, 1H), 4.96 (d, J = 15.6 Hz, 1H). MS (ESI): calculated for C23H17F3N3OP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0258] [M+H]+ 408.13; found 408.37.

[0259] Example 7: Preparation of compounds 13a-p

[0260] Carbonitrile derivatives 12a-m (1 eq, 3.0 mmol) was dissolved in acetone (25 mL) and stirred for 10 min. A solution 1 N Na2COs (3eq) was then added to the reaction solution, followed by the addition of H2O2 (30% w / w, 27 eq). The resulting mixture was stirred at room temperature for 2 h. Upon completion, as indicated by TLC and MS (ESI), the volatile organic solvent was evaporated. The residue was suspended with CH2CI2 (3x15 mL), and the organic layers were washed with water (2x15 mL), brine (1x10 mL) and dried over Na2SO4. The solvent was then evaporated under reduced pressure and the crude material was purified by flash chromatography (EtOAc / PE as the eluent) to yield derivatives 13a-p as pale-yellow solids. (10.1002 / adsc.201200630)

[0261] CX? zx JZ' NH2

[0262] \ 1-methyl-2-oxo-3-(phenylamino)indoline-3-carboxamide (13a): Pale yellow solid (yield 70%). 1H NMR (400 MHz, DMSO-d6) 57.53 (s, 1H), 7.42-7.36 (m, 1H), 7.32-7.24 (m, 2H), 7.13 (d, J = 7.8 Hz, 1H), 7.08-7.02 (m, 1H), 6.94-6.91 (m, 2H), 6.54 (t, J = 7.3 Hz, 1 H), 6.38-6.31 (m, 3H), 3.20 (s, 3H). MS (ESI): calculated for C16H16N3O2 [M+H]+ 282.12; found 282.26.

[0263] 3-((4-chlorophenyl)amino)-1-methyl-2-oxoindoline-3-carboxamide (13b): Pale yellow solid (yield 71%). 1H NMR (400 MHz, DMSO-d6) 5 7.52 (s, 1H), 7.41-7.34 (m, 2H), 7.32-7.26 (m, 1 H), 7.14-7.02 (m, 2H), 7.00-6.93 (m, 2H), 6.56 (s, 1 H), 6.43-6.37 (m, 2H), 3.17 (s, 3H). MS (ESI): calculated for C16H15CIN3O2 [M+H]+ 316.08;

[0264]

[0265] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0266] found 316.30.

[0267] NH2

[0268] o

[0269] 5-fluoro-1-methyl-2-oxo-3-(phenylamino)indoline-3-carboxamide (13c): Pale yellow solid (yield 46%). 1H NMR (400 MHz, DMSO-d6) 57.58 (s, 1H), 7.37 (s, 1H), 7.26-7.10 (m, 3H), 6.99-6.89 (m, 2H), 6.59-6.49 (m, 1H), 6.40 (s, 1H), 6.35-6.31 (m, 2H), 3.17 (s, 3H). MS (ESI): calculated for C16H15FN3O2 [M+H]+ 300.11; found 300.30.

[0270] o

[0271] NH\\

[0272] o 12

[0273] N

[0274] \

[0275] 5-methoxy-1-methyl-2-oxo-3-(phenylamino)indoline-3-carboxamide (13d): Pale yellow solid (yield 65%). 1 H NMR (400 MHz, DMSO-d6) 57.50 (s, 1 H), 7.26 (s, 1 H), 7.08-7.01 (m, 1 H), 6.96-6.86 (m, 4H), 6.60-6.45 (m, 1 H), 6.38-6.27 (m, 3H), 3.67 (s, 3H), 3.15 (s, 3H). MS (ESI): calculated for C17H18N3O3 [M+H]+ 312.13; found 312.40.

[0276] 'NH2

[0277] O

[0278]

[0279] 1-benzyl-2-oxo-3-(phenylamino)indoline-3-carboxamide (13e): Pale yellow solid (yield 87%). 1 H NMR (400 MHz, DMSO-d6) 57.58 (s, 1 H), 7.49 (s, 1 H), 7.45-7.20 (m, 7H), 7.07-6.94 (m, 2H), 6.93-6.84 (m, 2H), 6.59-6.54 (m, 1 H), 6.49 (s, 1 H), 6.42-6.37 (m, 2H), 5.02 (d, J = 15.7 Hz, 1H), 4.84 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H20N3O2[M+H]+ 358.16; found 358.74.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0280] 1-benzyl-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carboxamide (13f): Pale yellow solid (yield 63%). 1 H NMR (400 MHz, DMSO-d6) 57.58-7.53 (m, 2H), 7.42-7.38 (m, 1 H), 7.35-7.22 (m, 6H), 7.05-6.98 (m, 1 H), 6.95 (d, J = 7.8 Hz, 1 H), 6.76-6.68 (m, 2H), 6.47 (s, 1 H), 6.46-6.39 (m, 2H), 4.95 (d, J = 15.8 Hz, 1 H), 4.82 (d, J = 15.8 Hz, 1H). MS (ESI): C22H19FN3O2 [M+H]+ 376.15; found 376.29

[0281] 1 -benzyl-3-((4-chlorophenyl)amino)-2-oxoindoline-3-carboxamide (13g): Pale yellow solid (yield 45%). 1H NMR (400 MHz, DMSO-d6) 5 7.59 (s, 2H), 7.45-7.24 (m, 7H), 7.07-6.98 (m, 2H), 6.95-6.89 (m, 2H), 6.73 (s, 1 H), 6.52-6.43 (m, 2H), 4.97 (d, J = 15.7 Hz, 1 H), 4.86 (d, J = 15.7 Hz, 1 H). MS (ESI): calculated for C22H19CIN3O2 [M+H]+ 392.12; found 392.33.

[0282] 1-benzyl-2-oxo-3-((3-(trifluoromethyl)phenyl)amino)indoline-3-carboxamide (13h): Pale yellow solid (yield 52%). 1H NMR (400 MHz, DMSO-d6) 5 7.69 (s, 1H), 7.60 (s, 1H), 7.44 (dd, J = 7.5, 0.9 Hz, 1H), 7.41-7.34 (m, 2H), 7.34-7.23 (m, 4H), 7.12-7.01 (m, 3H), 7.01-6.95 (m, 2H), 6.89-6.84 (m, 1H), 6.69-6.64 (m, 1H), 4.93 (d, J =

[0283]

[0284] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0285] 15.8 Hz, 1H), 4.88 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C23H19F3N3O2 [M+H]+ 426.14; found 426.57.

[0286] 1 -benzyl-2-oxo-3-((4-(trifluoromethyl)phenyl)amino)indoline-3-carboxamide (13i): Pale yellow solid (yield 78%). 1H NMR (400 MHz, DMSO-d6) 57.65-7.58 (m, 2H), 7.40 (dd, J = 6.4, 1.5 Hz, 3H), 7.35-7.26 (m, 4H), 7.22-7.16 (m, 3H), 7.07-7.01 (m, 2H), 6.58 (d, J = 8.6 Hz, 2H), 4.96 (d, J = 15.7 Hz, 1H), 4.87 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C23H19F3N3O2 [M+H]+ 426.14; found 426.30.

[0287] 1-benzyl-5-fluoro-2-oxo-3-(phenylamino)indoline-3-carboxamide (13j): Pale yellow solid (yield 73%). 1 H NMR (400 MHz, DMSO-d6) 57.68-7.53 (m, 2H), 7.43-7.21 (m, 6H), 7.21-7.05 (m, 1H), 7.01-6.95 (m, 1H), 6.92-6.85 (m, 2H) 6.57 (t, J = 7.3 Hz, 2H), 6.38 (d, J = 7.7 Hz, 1H), 4.99 (d, J = 15.7 Hz, 1H), 4.84 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H19FN3O2 [M+H]+ 376.15; found 376.23.

[0288]

[0289] 1 -benzyl-5-fluoro-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carboxamide (13k): Pale yellow solid (yield 61%). 1H NMR (400 MHz, DMSO-d6) 57.63 (s, 2H), 7.37-P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0290] 7.23 (m, 6H), 7.17-7.08 (m, 1 H), 6.97-6.91 (m, 1 H), 6.79-6.71 (m, 2H), 6.55 (s, 1 H), 6.47-6.39 (m, 2H), 4.94 (d, J = 15.8 Hz, 1H), 4.84 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C22H18F2N3O2 [M+H]+ 394.14; found 394.23.

[0291]

[0292] 1 -benzyl-5-methoxy-2-oxo-3-(phenylamino)indoline-3-carboxamide (13I): Pale yellow solid (yield 60%). 1 H NMR (400 MHz, DMSO-d6) 57.55 (s, 1 H), 7.45 (s, 1 H), 7.41-7.33 (m, 2H), 7.34-7.19 (m, 3H), 7.02-6.99 (m, 1H), 6.92-6.82 (m, 4H), 6.55 (t, J = 7.3 Hz, 1 H), 6.47 (s, 1 H), 6.37 (d, J = 7.7 Hz, 2H), 4.97 (d, J = 15.7 Hz, 1 H), 4.79 (d, J = 15.7 Hz, 1H), 3.65 (s, 3H). MS (ESI): calculated for C23H22N3O3 [M+H]+ 388.17; found 388.24.

[0293]

[0294] 1 -(4-fluorobenzyl)-2-oxo-3-(phenylamino)indoline-3-carboxamide (13m): Pale yellow solid (yield 70%). 1 H NMR (400 MHz, DMSO-d6) 57.56 (s, 1 H), 7.49 (s, 1 H), 7.45-7.38 (m, 2H), 7.36 (dd, J = 7.6, 1.1 Hz, 1H), 7.32-7.25 (m, 1H), 7.19-7.08 (m, 2H), 7.03-6.98 (m, 2H), 6.90-6.80 (m, 2H), 6.57-6.51 (m, 1 H), 6.47 (s, 1 H), 6.35 (dd, J = 8.7, 1.0 Hz, 2H), 4.97 (d, J = 15.7 Hz, 1H), 4.83 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H19FN3O2 [M+H]+ 376.15; found 376.33.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0295]

[0296] 1 -(4-fluorobenzyl)-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carboxamide (13n): Pale yellow solid (yield 61%). 1H NMR (400 MHz, DMSO-d6) 57.62-7.58 (m, 2H), 7.45-7.36 (m, 3H), 7.33-7.27 (m, 1H), 7.19-7.11 (m, 2H), 7.08-6.98 (m, 2H), 6.78-6.72 (m, 2H), 6.51 (s, 1H), 6.47-6.41 (m, 2H), 4.94 (d, J = 15.7 Hz, 1H), 4.85 (d, J = 15.7 Hz, 1 H). MS (ESI): calculated for C22H18F2N3O2 [M+H]+ 394.14; found 394.05.

[0297]

[0298] 5-fluoro-1 -(4-fluorobenzyl)-2-oxo-3-(phenylamino)indoline-3-carboxamide (13o): Pale yellow solid (yield 56%). 1H NMR (400 MHz, DMSO-d6) 5 7.64 (s, 1H), 7.58 (s, 1H), 7.48-7.35 (m, 2H), 7.30 (dd, J = 8.0, 2.7 Hz, 1H), 7.19-7.08 (m, 3H), 7.04- 6.99 (m, 1 H), 6.92-6.86 (m, 2H), 6.61-6.53 (m, 2H), 6.35 (dd, J = 8.6, 1.0 Hz, 2H), 4.96 (d, J = 15.7 Hz, 1H), 4.85 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H18F2N3O2 [M+H]+ 394.14; found 394.68.

[0299]

[0300] 5-fluoro-1-(4-fluorobenzyl)-3-((4-fluorophenyl)amino)-2-oxoindoline-3-carboxamide (13p): Pale yellow solid (yield 58%). 1 H NMR (400 MHz, DMSO-d6) 57.69-7.66 (m, 2H), 7.42-7.33 (m, 3H), 7.21-7.12 (m, 3H), 7.03-6.98 (m, 1H), 6.83-6.73 (m, 2H),P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0301] 6.59 (s, 1 H), 6.48-6.41 (m, 2H), 4.93 (d, J = 15.7 Hz, 1H), 4.87 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C22H17F3N3O2 [M+H]+ 412.13; found 412.25.

[0302] Example 8: Preparation of compounds 14a-p

[0303] To a solution of carboxamide derivatives 13a-p (1 eq, 1.3 mmol) in MeOH (5 mL) was added DMF-DMA (3 eq) and the reaction mixture was then stirred at 55°C for 1-2 h. After formation of the formamidine intermediates, as indicated by TLC, few drops of AcOH were added to the mixture, which was stirred at 55°C for 2 h. Upon completion, as indicate by TLC, the volatile fraction was evaporated, and the crude residue was suspended with EtOAc (3x15 mL). The organic layers were washed with water (1x10 mL), brine (1x10 mL), and dried over Na2SO4. The solvent was then evaporated under reduced pressure and the obtained crude residue was purified by flash chromatography (AcOEt / PE as the eluent) to yield byciclic derivatives 14a-p as pale white solids. In addition, a small amount of derivatives 14a-p was then purified by semi-preparative HPLC to improve the chemical characterization performed by NMR.

[0304]

[0305] 1'-methyl-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14a): Pale white solid (yield 37%). 1 H NMR (400 MHz, DMSO-d6) 59.80 (s, 1 H), 7.48-7.42 (m, 1 H), 7.34 (t, J = 8.0 Hz, 2H), 7.28-7.16 (m, 3H), 7.09-7.04 (m, 3H), 3.29 (s, 3H). MS (ESI): calculated for C17H14N3O2 [M+H]+ 292.11; found 392.28.

[0306]

[0307] 3-(4-chlorophenyl)-1'-methylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14b): Pale white solid (yield 41%). 1H NMR (400 MHz, DMSO-d6) 5 9.76 (s, 1H), 7.51- 7.34 (m, 2H), 7.29-6.98 (m, 4H), 6.37-6.24 (m, 2H), 3.27 (s, 3H). MS (ESI): calculated for C17H13CIN3O2 [M+H]+ 326.07; found 326.26P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0308] 5'-fluoro-1'-methyl-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14c): Pale white solid (yield 32%).1 H NMR (400 MHz, DMSO- d6) 59.77 (s, 1 H), 7.38-7.24 (m, 5H), 7.23-7.16 (m, 1H), 7.06-7.02 (m, 2H), 3.27 (s, 3H). MS (ESI): calculated for C17H13FN3O2 [M+H]+ 310.10; found 310.33.

[0309] 5'-methoxy-1'-methyl-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14d): Pale white solid (yield 37%). 1H NMR (400 MHz, DMSO- d6) 5 9.74 (s, 1H), 7.36- 7.29 (m, 2H), 7.22-7.12 (m, 2H), 7.07-7.02 (m, 2H), 7.00-6.93 (m, 2H), 3.64 (s, 3H), 3.24 (s, 3H). MS (ESI): calculated for C18H16N3O3 [M+H]+ 322.19; found 322.39.

[0310] 1'-benzyl-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14e): Pale white solid (yield 31%). 1 H NMR (400 MHz, DMSO-d6) 59.78 (s, 1H), 7.42-6.97 (m, 14H), 5.05 (d, J = 15.8 Hz, 1H), 5.00 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C23H18N3O2 [M+H]+ 368.14; found 368.22.

[0311] 1'-benzyl-3-(4-fluorophenyl)spiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14f): Pale

[0312]

[0313] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0314] white solid (yield 34%). 1 H NMR (400 MHz, DMSO-d6) 59.66 (s, 1 H), 7.38-7.23 (m, 7H), 7.21-7.02 (m, 6H), 5.04 (d, J = 15.9 Hz, 1H), 4.97 (d, J = 15.9 Hz, 1H). MS (ESI): calculated for C23H17FN3O2 [M+H]+ 386.13; found 386.74.

[0315] N N

[0316] N

[0317] 1'-benzyl-3-(4-chlorophenyl)spiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14g): Pale white solid (yield 29%). 1H NMR (400 MHz, DMSO-d6) 5 9.76 (s, 1H), 7.46- 7.24 (m, 7H), 7.12-6.93 (m, 4H), 6.35-6.28 (m, 2H), 5.01 (d, J = 15.8 Hz, 1H), 4.97 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C23H17CIN3O2 [M+H]+ 402.10; found 402.28.

[0318] N N

[0319] N

[0320] 1'-benzyl-3-(3-(trifluoromethyl)phenyl)spiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14h): Pale white solid (yield 40%). 1H NMR (400 MHz, DMSO-d6) 5 9.94 (s, 1H), 7.55-7.45 (m, 1H), 7.40-7.25 (m, 7H), 7.21-7.05 (m, 5H), 5.06 (d, J = 15.9 Hz, 1H), 4.99 (d, J = 15.9 Hz, 1H). MS (ESI): calculated for C24H17F3N3O2 [M+H]+ 436.13; found 436.46.

[0321] F

[0322] N pF F N

[0323] N

[0324] 1'-benzyl-3-(4-(trifluoromethyl)phenyl)spiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14i): Pale white solid (yield 37%). 1H NMR (400 MHz, DMSO-d6) 5 10.00 (s, 1H), 7.56-7.26 (m, 7H), 7.19-7.04 (m, 3H), 6.56 (s, 1H), 6.43 (d, J = 8.7 Hz, 2H), 5.04 (d,

[0325]

[0326] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0327] J = 15.7 Hz, 1H), 4.95 (d, J = 15.7 Hz, 1H). MS (ESI): calculated for C24H17F3N3O2 [M+H]+ 436.13, found 436.39.

[0328] 1'-benzyl-5'-fluoro-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14j): Pale white solid (yield 28%). 1 H NMR (400 MHz, DMSO-d6) 59.78 (s, 1 H), 7.48-6.68 (m, 12H), 6.34-6.28 (m, 1H), 5.06 (d, J = 15.8 Hz, 2H), 4.96 (d, J = 15.8 Hz, 2H); MS (ESI): calculated for C23H17FN3O2 [M+H]+ 386.13; found 386.17.

[0329] 1'-benzyl-5'-fluoro-3-(4-fluorophenyl)spiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14k): Pale white solid (yield 48%). 1H NMR (400 MHz, DMSO-d6) 5 9.66 (s, 1H), 7.43 (dd, J = 7.9, 2.6 Hz, 1H), 7.38-7.23 (m, 6H), 7.23-7.16 (m, 3H), 7.16-7.09 (m, 2H), 5.04 (d, J = 15.9 Hz, 1H), 4.97 (d, J = 15.9 Hz, 1H). MS (ESI): calculated for C23H16F2N3O2 [M+H]+ 404.12; found 404.31.

[0330]

[0331] 1'-benzyl-5'-methoxy-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (141): Pale white solid (yield 35%). 1H NMR (400 MHz, DMSO-d6) 5 9.76 (s, 1H), 7.36-7.17 (m, 7H), 7.06-6.97 (m, 4H), 6.92-6.87 (m, 2H), 5.01 (d, J = 15.8 Hz, 1H), 4.97 (d, J = 15.8 Hz, 1H), 3.62 (s, 3H). MS (ESI): calculated for C24H20N3O3 [M+H]+ 398.15; found 398.24.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0332] 1'-(4-fluorobenzyl)-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14m): Pale white solid (yield 39%). 1H NMR (400 MHz, DMSO-d6) 5 9.79 (s, 1H), 7.39- 7.33 (m, 3H), 7.30-7.24 (m, 3H), 7.23-7.14 (m, 4H), 7.07-6.98 (m, 3H), 5.05 (d, J = 15.9 Hz, 1H), 4.99 (d, J = 15.9 Hz, 1H). MS (ESI): calculated for C23H17FN3O2 [M+H]+ 386.13; found 386.35.

[0333] 1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14n): Pale white solid (yield 39%). 1H NMR (400 MHz, DMSO-d6) 5 9.66 (s, 1H), 7.60-6.77 (m, 10H), 6.58-6.14 (m, 2H), 5.03 (d, J = 15.8 Hz, 1H), 4.95 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C23H16F2N3O2 [M+H]+ 404.12; found 404.35.

[0334]

[0335] 5'-fluoro-1'-(4-fluorobenzyl)-3-phenylspiro[imidazole-4,3'-indoline]-2',5(3H)-dione (14o): Pale white solid (yield 45%).1H NMR (400 MHz, DMSO-d6) 5 9.79 (s, 1H), 7.51-7.45 (m, 1H), 7.40-7.32 (m, 2H), 7.29-7.21 (m, 1H), 7.18-7.09 (m, 5H), 7.03- 7.96 (m, 3H), 5.05 (d, J = 15.8 Hz, 1H), 4.99 (d, J = 15.8 Hz, 1H). MS (ESI): calculated for C23H16F2N3O2 [M+H]+ 404.12, found 404.18.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0336]

[0337] 5'-fluoro-1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazole-4,3'-indoline]- 2',5(3H)-dione (14p): Pale white solid (yield 40%). 1 H NMR (400 MHz, DMSO-d6) 5 9.66 (s, 1 H), 7.60-6.77 (m, 9H), 6.58-6.14 (m, 2H), 5.03 (d, J = 15.3 Hz, 1H), 4.96 (d, J = 15.3 Hz, 2H). MS (ESI): calculated for C23H15F3N3O2 [M+H]+ 422.11; found 422.40.

[0338] Example 9: Preparation of compounds 15a-pTo a cooled solution of unsaturated spiro derivatives 14a-p (1 eq, 0.2 mmol) in MeOH (10 mL) was added in small portions NaBH4 (1.3 eq), and the reaction mixture was then stirred at room temperature for 3-4 h. Upon completion, as indicate by TLC and MS (ESI), the mixture was quenched with water. The volatile fraction was then concentrated under reduced pressure and the resulting residue was suspended with EtOAc (2x15 mL). The organic layers were washed with water (1x15 mL), brine (1x10 mL), and dried over Na2SO4. The solvent was then evaporated, and the obtained crude material was purified by flash chromatography (AcOEt / PE as the eluent) to yield the final derivatives 15a-p as white solids.

[0339]

[0340] 1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15a): White solid (yield 44%). mp 230-231 °C. 1H NMR (400 MHz, DMSO-d6) 59.43 (bs, 1H), 7.43-7.36 (m, 1H), 7.21-7.16 (m, 2H), 7.09-6.99 (m, 3H), 6.66 (t, J = 7.3 Hz, 1H), 6.16 (dd, J = 8.7, 0.9 Hz, 2H), 5.07 (d, J = 4.8 Hz, 1H), 4.91 (d, J = 4.8 Hz, 1H), 3.23 (s, 3H). 13C NMR (101 MHz, DMSO-d6) 5 172.42 (C), 167.83 (C), 143.75 (C), 142.85 (C), 129.88 (CH), 129.12 (CH), 126.13 (C), 123.10 (CH), 122.90 (CH), 118.05 (CH), 111.87 (CH), 109.46 (CH), 70.50 (C), 59.76 (CH2), 26.45 (CH3). MS (ESI):P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0341] calculated for C17H16N3O2 [M+H]+ 294.1237; found 294.4462. HPLC: tR = 15.07.

[0342] I

[0343]

[0344] 3-(4-chlorophenyl)-1 '-methylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15b): White solid (yield 60%). mp 228-229°C. 1H NMR (400 MHz, DMSO-d6) 59.48 (bs, 1H), 7.46-7.38 (m, 1H), 7.26-7.17 (m, 2H), 7.14-7.00 (m, 3H), 6.22-6.15 (m, 2H), 5.08 (d, J = 4.6 Hz, 1H), 4.92 (d, J = 4.6 Hz, 1H), 3.24 (s, 3H). 13C NMR (101 MHz, DMSO-d6) 5 172.07 (C), 167.58 (C), 143.75 (C), 141.73 (C), 130.05 (CH), 128.89 (CH), 125.66 (C), 123.14 (CH), 122.99 (CH), 121.95 (C), 113.43 (CH), 109.60 (CH), 70.51 (C), 59.84 (CH2), 26.51 (CH3). MS (ESI): calculated for C17H15CIN3O2 [M+H]+ 328.0847; found 328.4014. HPLC: tR = 16.22.

[0345]

[0346] 5'-fluoro-1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15c): White solid (yield 70%). mp 212-232°C. 1H NMR (400 MHz, DMSO-d6) 5 9.52 (bs, 1H), 7.31-7.19 (m, 3H), 7.13-7.06 (m, 2H), 6.70 (d, J = 7.3 Hz, 1H), 6.17 (dd, J = 8.7, 0.8 Hz, 2H), 5.10 (d, J = 4.6 Hz, 1H), 4.92 (d, J = 4.6 Hz, 1H), 3.25 (s, 3H). 13C NMR (101 MHz, DMSO-d6) 5172.30 (C), 167.28 (C), 158.75 (C, 1JC-F = 240 Hz), 142.65 (C), 139.99 (C), 129.23 (CH), 127.94 (C, 3JC-F = 8 Hz), 118.20 (CH), 116.27-116.03 (CH, 2JC-F = 24 Hz), 111.83 (CH), 111.30 (CH, 2JC-F = 25 Hz), 110.53 (CH, 3JC- F = 8 Hz), 70.68 (C), 59.71 (CH2), 26.66 (CH3). MS (ESI): calculated for C17H15FN3O2 [M+H]+ 312.1143; found 312.4261. HPLC: tR = 16.10.

[0347]

[0348] 5'-methoxy-1 '-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15d):P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0349] White solid (yield 45%). mp 229-230°C. 1H NMR (400 MHz, DMSO-d6) δ9.40 (bs, 1 H), 7.16-7.01 (m, 3H), 6.95 (dd, 1H, J = 8.5, 2.6 Hz), 6.87-6.82 (m, 1H), 6.66 (t, J = 7.3 Hz, 1H), 6.23-6.12 (m, 2H), 5.08 (d, J = 4.7 Hz, 1H), 4.89 (d, J = 4.7 Hz, 1H), 3.66 (s, 3H), 3.20 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ172.62 (C), 168.35 (C), 156.40 (C), 143.47 (C), 137.62 (C), 129.69 (CH), 128.06 (C), 118.58 (CH), 114.75 (CH), 112.42 (CH), 110.75 (CH), 110.55 (CH), 71.34 (C), 60.28 (CH2), 56.02 (CH3), 27.06 (CH3). MS (ESI): calculated for C18H18N3O3 [M+H]+ 324.1343; found 324.3919. HPLC: tR = 16.02.

[0350]

[0351] 1'-benzyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15e): White solid (yield 48%). mp 234-235°C. 1H NMR (400 MHz, DMSO-d6) δ9.48 (bs, 1H), 7.49-7.24 (m, 6H), 7.21 (dd, J = 7.4, 0.8 Hz, 1H), 7.08 (d, J = 7.8 Hz, 1H), 7.03-6.96 (m, 3H), 6.69-6.63 (m, 1H), 6.16 (d, J = 7.9 Hz, 2H), 5.10 (d, J = 4.8 Hz, 1H), 4.99 (d, J = 2.5 Hz, 2H), 4.94 (d, J = 4.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 173.21 (C), 168.35 (C), 143.34 (C), 143.25 (C), 136.27 (C), 130.32 (CH), 129.56 (CH), 129.02 (CH), 128.01 (CH), 126.75 (C), 123.87 (CH), 123.56 (CH), 118.74 (CH), 112.60 (CH), 110.63 (CH), 71.18 (C), 60.36 (CH2), 43.66 (CH2); MS (ESI): calculated for C23H20N3O2[M+H]+ 370.1550; found 370.1786. HPLC: tR = 18.43.

[0352] 1'-benzyl-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione (15f): White solid (yield 46%). mp 228-229°C. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (bs, 1H), 7.44-7.39 (m, 2H), 7.39-7.34 (m, 2H), 7.34-7.29 (m, 2H), 7.23 (dd, J = 7.4, 0.8 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.06-7.00 (m, 1H), 6.90-6.84 (m, 2H), 6.20-6.13 (m,

[0353]

[0354] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0355] 2H), 5.10 (d, J = 4.7 Hz, 1H), 5.00 (d, J = 4.9 Hz, 2H), 4.94 (d, J = 4.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 172.50 (C), 167.76 (C), 155.37 (C, 1 JC-F = 236 Hz), 142.73 (C), 139.54 (C), 135.65 (C), 129.86 (CH), 128.48 (CH), 127.48 (CH), 127.34 (CH), 125.92 (C), 123.36 (CH), 123.06 (CH), 115.56 (CH, 2JC-F = 22 Hz), 113.07 (CH, 1 JC-F = 7 Hz), 110.12 (CH), 70.82 (C), 60.07 (CH2), 43.10 (CH2). MS (ESI): calculated for C23H18FN3O2 [M+H]+ 388.4219; found 388.1456. HPLC: tR = 21.40.

[0356]

[0357] 1'-benzyl-3-(4-chlorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione (15g): White solid (yield 49%). mp 255-256°C. 1H NMR (400 MHz, DMSO-d6) δ9.52 (bs, 1H), 7.45-7.38 (m, 2H), 7.38-7.27 (m, 6H), 7.26-7.20 (m, 1H), 7.10 (d, J = 7.8 Hz, 1H), 7.07-6.97 (m, 3H), 6.20-6.09 (m, 2H), 5.09 (d, J = 4.8 Hz, 1H), 4.99 (d, J = 9.2 Hz, 2H), 4.94 (d, J = 4.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 172.85 (C), 168.09 (C), 143.28 (C), 142.22 (C), 136.21 (C), 130.52 (CH), 129.30 (CH), 129.05 (CH), 128.07 (CH), 127.94 (CH), 126.25 (C), 123.93 (CH), 123.67 (CH), 122.60 (C), 114.10 (CH), 110.75 (CH), 71.18 (C), 60.45 (CH2), 43.72 (CH2). MS (ESI): calculated for C23H19ClN3O2 [M+H]+ 404.1160; found 404.4582. HPLC: tR = 24.08.

[0358]

[0359] 1'-benzyl-3-(3-(trifluoromethyl)phenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione (15h): White solid (yield 58%). mp 238-239°C. 1H NMR (400 MHz, DMSO-d6) δ 9.59 (bs, 1 H), 7.44-7.38 (m, 2H), 7.37-7.23 (m, 5H), 7.18 (t, J = 7.9 Hz, 1 H), 7.11 (d, J = 7.8 Hz, 1 H), 7.05-6.97 (m, 2H), 6.51 (s, 1 H), 6.30 (dd, J = 8.3, 2.4 Hz, 1 H), 5.18 (d, J = 5.0 Hz, 1H), 5.07-4.92 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.74 (C), 167.84 (C), 143.68 (C), 143.29 (C), 136.14 (C), 130.67 (CH), 130.63 (CH),P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0360] 129.05 (CH), 128.03 (CH), 127.80 (CH), 125.86 (C), 124.43 (C) 123.99 (CH), 123.77 (CH), 116.22 (CH), 114.96 (CH), 110.74 (CH), 108.55 (CH), 71.11 (C), 60.38 (CH2), 43.75 (CH2). MS (ESI): calculated for C24H19F3N3O2 [M+H]+ 438.1424; found 438.1981. HPLC: tR = 25.42.

[0361]

[0362] 1'-benzyl-3-(4-(trifluoromethyl)phenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione (15i): White solid (yield 46%). mp 251-252°C. 1H NMR (400 MHz, DMSO-d6) δ 9.61 (bs, 1H), 7.47-7.41 (m, 2H), 7.39-7.25 (m, 7H), 7.15 (d, J = 7.9 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.29 (d, J = 8.7 Hz, 2H), 5.19 (d, J = 5.1 Hz, 1H), 5.03 (m, 3H).

[0363] 13C NMR (101 MHz, DMSO-d6) δ 171.96 (C), 167.17 (C), 145.48 (C), 142.72 (C), 135.60 (C), 130.08 (CH), 128.46 (CH), 127.50 (CH), 127.42 (CH), 126.25 (CH), 125.90 (C), 125.35 (C), 123.37 (CH), 123.18 (CH), 118.23 (C), 111.88 (CH), 110.28 (CH), 70.42 (C), 59.79 (CH2), 43.23 (CH2). MS (ESI): calculated for C24H19F3N3O2 [M+H]+ 438.1424; found 438.4498. HPLC: tR = 24.82.

[0364]

[0365] 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15j): White solid (yield 49%). mp 238-239°C. 1H NMR (400 MHz, DMSO-d6) δ 9.54 (bs, 1H), 7.46-6.96 (m, 11 H), 6.68 (t, J = 7.3 Hz, 1H), 6.15 (d, J = 7.8 Hz, 2H), 5.10 (d, J = 4.5 Hz, 1H), 5.00 (s, 2H), 4.93 (d, J = 4.5 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 173.08 (C), 167.78 (C), 159.31 (C, 1JC-F = 241 Hz), 143.13 (C), 139.43 (C), 136.07 (C), 129.64 (CH), 129.06 (CH), 128,61 (C, 3JC-F = 9Hz), 128.11 (CH), 128.03 (CH), 118.86 (CH), 116.67 (CH, 2JC-F = 23 Hz), 112.53 (CH), 112.02 (CH, 2JC-F = 26 Hz), 111.66 (CH, 3JC-F = 7Hz), 71.34 (C), 60.30 (CH2), 43.78 (CH2). MS (ESI):P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0366] calculated for C23H19FN3O2 [M+H]+ 388.1456; found 388.2196; HPLC: tR = 23.17.

[0367]

[0368] 1'-benzyl-5'-fluoro-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione (15k): White solid (yield 54%). mp 242-243°C. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (bs, 1H), 7.41-7.32 (m, 4H), 7.31-7.23 (m, 2H), 7.20-7.14 (m, 1H), 7.09 (dd, J = 8.7, 4.2 Hz, 1H), 6.87 (t, J = 8.9 Hz, 2H), 6.17-6.11 (m, 2H), 5.08 (d, J = 4.6 Hz, 1H), 4.99 (d, J = 6.7 Hz, 2H), 4.92 (d, J = 4.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ172.96 (C), 167.77 (C), 159.34 (C, 1 JC-F = 241 Hz), 156.00 (C, 1 JC-F = 236 Hz), 139.88 (C), 139.48 (C), 136.03 (C), 129.09 (CH), 128.12 (CH), 127.93 (CH), 116.80 (CH, 2JC-F = 23 Hz), 116.22 (CH, 2JC-F = 22 Hz), 113.59 (CH, 3JC-F = 7 Hz), 112.08 (CH, 2JC-F = 25 Hz), 111.75 (C, 3JC-F = 8 Hz), 111.75 (CH, 3JC-F = 8 Hz), 71.56 (C), 60.58 (CH2), 43.79(CH2). MS (ESI): calculated for C23H18F2N3O2 [M+H]+ 406.1362; found 406.3859. HPLC: tR = 23.92.

[0369]

[0370] 1 '-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15I): White solid (yield 52%). mp 240-241 °C. 1H NMR (400 MHz, DMSO-d6) δ9.45 (bs, 1H), 7.45-7.37 (m, 2H), 7.37-7.24 (m, 3H), 7.07-6.93 (m, 3H), 6.88-6.82 (m, 2H), 6.67 (t, J = 7.3 Hz, 1H), 6.16 (d, J = 7.8 Hz, 2H), 5.11 (d, J = 4.5 Hz, 1H), 4.96 (d, J = 6.6 Hz, 2H), 4.92 (d, J = 4.6 Hz, 1H), 3.63 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ172.84 (C), 168.30 (C), 156.42 (C), 143.39 (C), 136.45 (C), 136.34 (C), 129.56 (CH), 128.99 (CH), 128.13 (C), 128.01 (CH), 118.69 (CH), 114.71 (CH), 112.59 (CH), 111.21 (CH), 110.86 (CH), 71.45 (C), 60.32 (CH2), 55.99 (CH3), 43.69 (CH2). MS (ESI): calculated for C24H22N3O3 [M+H]+ 400.1656; found 400.0746; HPLC: tRP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0371] = 17.85.

[0372]

[0373] 1 '-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15m): White solid (yield 49%). mp 241-242°C. 1H NMR (400 MHz, DMSO-d6) δ9.48 (bs, 1 H), 7.51-7.40 (m, 2H), 7.32-7.28 (m, 1 H), 7.24-7.08 (m, 4H), 7.03-6.96 (m, 3H), 6.66 (t, J = 7.3 Hz, 1H), 6.14 (dd, J = 8.7, 0.9 Hz, 2H), 5.10 (d, J = 4.8 Hz, 1H), 4.99 (s, 2H), 4.94 (d, J = 4.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 173.20 (C), 168.31 (C), 162.02 (C, 1 JC-F = 244 Hz), 143.32 (C), 143.09 (C), 132.49 (C), 130.35 (CH), 130.10 (CH, 3JC-F = 8 Hz), 129.57 (CH), 126.75 (C), 123.91 (CH), 123.63 (CH), 118.76 (CH), 115.85 (CH, 2JC-F = 21 Hz), 112.54 (CH), 110.59 (CH), 71.17 (C), 60.36 (CH2), 42.92 (CH2). MS (ESI): calculated for C23H19FN3O2 [M+H]+ 388.1456; found 388.4308. HPLC: tR = 20.77.

[0374]

[0375] 1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione (15n): White solid (yield 52%). mp 237-238°C. 1H NMR (400 MHz, DMSO-d6) δ 9.49 (bs, 1H), 7.46-7.41 (m, 2H), 7.34-7.29 (m, 1H), 7.25-7.15 (m, 3H), 7.11 (d, J = 7.8 Hz, 1H), 7.05-6.99 (m, 1H), 6.86 (t, J = 8.90 Hz, 2H), 6.18-6.10 (m, 2H), 5.08 (d, J = 4.7 Hz, 1H), 5.00-4.95 (m, 2H), 4.92 (d, J = 4.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 173.08 (C), 168.29 (C), 162.02 (C, 1 JC-F = 244 Hz), 155.95 (C, 1 JC- F = 236 Hz), 143.15 (C), 140.07 (C), 132.46 (C), 130.47 (CH), 129.98 (CH, 3JC-F = 8 Hz), 126.48 (C), 123.96 (CH), 123.70 (CH), 116.17 (CH, 2JC-F = 23 Hz), 115.89 (CH, 2JC-F = 21 Hz), 113.57 (CH, 3JC-F = 7 Hz), 110.66 (CH), 71.40 (C), 60.63 (CH2), 42.93 (CH2). MS (ESI): calculated for C23H18F2N3O2 [M+H]+ 406.1362; foundP024165WO-01 Notarbartolo & Gervasi S.p. A.

[0376] 406.4291. HPLC: tR = 23.20.

[0377]

[0378] 5'-fluoro-1'-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione (15o): White solid (yield 51%). mp 249-250°C. 1H NMR (400 MHz, DMSO-d6) δ 9.54 (bs, 1H), 7.46-7.42 (m, 2H), 7.25 (dd, J = 7.7, 2.5 Hz, 1H), 7.22-7.11 (m, 4H), 7.04-6.98 (m, 2H), 6.68 (t, J = 7.3 Hz, 1H), 6.13 (dd, J = 8.7, 0.9 Hz, 2H), 5.10 (d, J = 4.7 Hz, 1H), 4.99 (s, 2H), 4.93 (d, J = 4.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 173.10 (C), 167.76 (C), 162.06 (C, 1 JC-F = 244 Hz), 159.34 (C, 1 JC-F = 241 Hz), 143.11 (C), 139.27 (C), 132.32 (C), 130.12 (CH, 3JC-F = 8 Hz), 129.65 (CH), 128.61 (C, 3JC-F = 8 Hz), 118.89 (CH), 116.70 (CH, 2JC-F = 24 Hz), 115.89 (CH, 2JC-F = 21 Hz), 112.47 (CH), 112.07 (CH, 2JC-F = 25 Hz), 111.63 (CH, 3JC-F = 8 Hz), 71.33 (C), 60.30 (CH2), 43.04 (CH2). MS (ESI): calculated for C23H18F2N3O2 [M+H]+ 406.1362; found 406.2602. HPLC: tR = 23.48.

[0379]

[0380] 5'-fluoro-1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]- 2', 5-dione (15p): White solid (yield 51%). mp 221-222°C. 1H NMR (400 MHz, DMSO-d6) δ9.57 (bs, 1 H), 7.47-7.42 (m, 2H), 7.30-7.11 (m, 5H), 6.90 (t, J = 8.8 Hz, 2H), 6.17-6.12 (m, 2H), 5.10 (d, J = 4.6 Hz, 1H), 5.03-4.97 (m, 2H), 4.93 (d, J = 4.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ172.39 (C), 167.17 (C), 161.48 (C, 1JC-F = 244 Hz), 158.80 (C, 1 JC-F = 241 Hz), 155.44 (C, 1 JC-F = 236 Hz), 139.29 (C), 138.75 (C), 131.69 (C), 129.43 (CH, 3JC-F = 8 Hz), 127.77 (C, 3JC-F = 9 Hz), 116.26 (CH, 2JC-F = 23 Hz), 115.68 (CH, 2JC-F = 22 Hz), 115.35 (CH, 2JC-F = 22 Hz), 112.95 (CH, 3JC-F = 8 Hz), 111.54 (CH, 2JC-F = 25 Hz), 111.15 (CH, 3JC-F =

[0381]

[0382] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0383] 8 Hz), 70.98 (C), 60.00 (CH2), 42.48 (CH2). MS (ESI): calculated for C23H17F3N3O2 [M+H]+ 424.1267; found 424.4342. HPLC: tR = 23.43.

[0384] Example 10: Biological evaluation of compounds of the invention Example 10.1: In vitro activity against mPTP opening

[0385] The inhibitory activity of compounds 15a-e, g,h, j-m against Ca2+-mediated mPTP opening has been investigated in human ventricular cardiomyocytes (AC16) via a calcein-cobalt (Co2+) assay. (M. Bonora etal. Nat Protoc. 2016:11:1067) Living cells were pre-treated with the potential inhibitors at 1 pM concentration then ionomycin (1pM) was added as Ca2+ionophore. The novel compounds along with PP11 and IB13 employed as internal references were evaluated in triplicate and results are reported in Table 1.

[0386] This in vitro investigation confirmed the new spiro-isatin template as potent mPTP inhibitors. Moreover, the inventors by exploring the structure-activity relationship (SAR) demonstrated that the activity was highly dependent on the substitution pattern of the bicyclic core.

[0387] As far as the substitution of the isatin nitrogen (R substituent in Table 1 ), compounds bearing a small methyl group exhibited a comparable or slightly higher activity to that of PP11 with percent inhibition ranging from 42.6 (15d) to 49.2 (15a). When the N1'-methyl group was replaced by a benzyl, a significant increase of potency was observed especially if comparing 15c (l% = 47.4) to 15j (l% = 62.6) and 15d (l% = 42,6) to 151 (1% = 59,5). While the para substitution of the N1 '-benzyl group with a fluorine atom was particularly detrimental for the activity (see compounds 151 (1% = 59.8) vs 15 m (1% = 20.6)).

[0388] The contribution of 5'-substitution (R1 substituent in Table 1 ) seems to be particularly dependent on the presence of a methyl or a benzyl moiety at the isatin nitrogen: while N1'-methyl derivatives poorly tolerate the substitution of 5'-position (compare 15a with 15c and 15d), the combination of a N1 '-benzyl moiety with a fluorine atom (15j vs 15e) or with a methoxy group (151 vs 15e) resulted in enhanced potency. The SAR study was extended to the phenyl ring of the imidazolidinone region (R2 substituent in Table 1) where few halogenated moieties have been introduced.P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0389] However, except for the 4-CI-phenyl derivatives 15b (l% = 44.8) and 15g (l% = 54.2), the substitution of the 3-phenyl ring resulted in a marked loss of activity (see compound 15k (l% = 11.37).

[0390] The preliminary in vitro assay allowed to identify compound 15j as a potent inhibitor (l% = 62.6) with the greatest effect in reducing mPTP opening among the investigated spiro-isatin derivatives (Figure 2), almost equipotent to the previously identified inhibitor IB13 (l% = 66.80) and with significant improved potency if compared to PP11 (l% = 40.19), thus this compound was selected for further biological evaluations.

[0391] Table 1: Percent inhibition of the novel spiro-isatin derivatives against mPTP opening (Me indicates a methyl group, Bn indicates a benzyl group, and OMe indicates a methoxy group).

[0392] mPTP

[0393] Compd R Ri R2inhibition Statistic (%, 1pM)a

[0394] PP11 - - - 40.19 P<0.0001 IB13 - - - 66.80 P<0.0001 15a Me H H 49.18 P<0.0001 15b Me H 4-CI 44.77 P<0.0001 15c Me F H 47.39 P<0.0001 15d Me OMe H 42.59 P<0.0001 15e Bn H H 51.90 P<0.0001 15g Bn H 4-CI 54.22 P<0.0001 15h Bn H 3-CF319.99 P<0.0001 15j Bn F H 62.60 P<0.0001 15k Bn F 4-F 11.37 P<0.01 151 Bn OMe H 59.48 P<0.0001

[0395]

[0396] P024165WO-01 Notarbartolo & Gervasi S.p. A.

[0397] 15m 4-F-Bn H H 20.64 P<0.0001

[0398]

[0399] aCalcein-Co2+assay in human ventricular cardiomyocytes (AC 16) pre-treated with compounds (1pM) then treated with ionomycin (1pM). PP11 and IB13 were evaluated as positive control.

[0400] Example 10.2: Cardioprotective effect of 15j in vitro.

[0401] In addition, to evaluate the cardioprotective effect of compound 15j, the most promising one from the screening depicted in Figure 2 and Table 1, a cell-death assay and a cell-viability assay were performed, by western blot and fluorescence flow cytometry respectively. In Figure 3A, in a context of hypoxia-reoxygenation (H / R) mimicking ischemia / reperfusion (l / R), it can be noticed that AC16 cells treated with 15j maintain cell viability similar to normoxia condition, while H / R triggered a drastic decrease in untreated living cells. This difference in viability can be due to two proteins engaged in the mechanism of cell-death, cleaved Parp and cleaved Caspase 3 (Figure 3B). Their levels are reduced after 15j treatment when compared with the untreated sample.

[0402] Example 10.3: Effect of 15j on mitochondrial ATP content

[0403] Given the key role of ATP synthase in both PTP formation and the cellular energy production, the basal mitochondrial ATP was monitored by the luciferin -luciferase method. (G Morciano et al. Nat Protoc. 2017:12:1542) Also, the continuous production of ATP, especially after ischemia, is essential for cardiomyocyte survival and for heart contraction. The most sensitive and reliable technique for measuring ATP is based on the bio luminescent luciferin-luciferase reaction. Luciferase is an enzyme derived from the North American firefly Photinus pyralis that chemically generates light as a byproduct of the oxidation of the small-molecule substrate D-luciferin. Especially, the reaction catalyzed by luciferase in the presence of magnesium ions implicates the conversion of D-luciferin into oxyluciferin, producing a flash of yellow-green light proportional to the amount of ATP present.

[0404] Especially, we aimed to assess the effects of 15j either in normoxia or following H / R conditions. As depicted in Figure 4A-B, compound 15j mostly preserves the mitochondrial ATP content becoming significantly decreased after H / R.

Claims

P024165WO-01 Notarbartolo & Gervasi S.p. A.CLAIMS1. A spiro-isatin derivative compound of Formula (I):R / or a pharmaceutically acceptable salt thereof,wherein:- R is a substituent selected from the group consisting of (C₁-C₃)alkyl and (C₁-C₃)alkyl phenyl, wherein the phenyl moiety is optionally substituted with a halogen; - R₁ is a substituent selected from the group consisting of hydrogen, halogen and (C₁-C₃)alkoxy;- R₂ is a substituent selected from the group consisting of hydrogen, halogen and trifluoromethyl (-CF₃).

2. The compound according to claim 1, wherein R is (Ci-C3)alkyl, preferably a methyl group.

3. The compound according to claim 1, wherein R is (Ci-C3)alkyl phenyl, preferably R is a benzyl group.

4. The compound according to anyone of claims 1-3, wherein R1 is a halogen, selected from the group consisting of fluorine, chlorine, bromine and iodine, preferably it is fluorine.

5. The compound according to anyone of claims 1-3, wherein R1 is (Ci-C3)alkoxy, preferably R1 is a methoxy group.

6. The compound according to anyone of claims 1-5, wherein R2 is a halogen selected from the group consisting of fluorine, chlorine, bromine and iodine, it is fluorine or chlorine.

7. The compound according to anyone of claims 1 -5, wherein R2 is a hydrogen atom.

8. The compound according to claim 1, wherein R is a benzyl group, R1 is methoxy,P024165WO-01 Notarbartolo & Gervasi S.p. A.and R2 is a hydrogen atom.

9. The compound according to claim 1, wherein R is a benzyl group, R1 is fluorine, and R2 is a hydrogen atom.

10. The compound according claim 1, wherein the spiro-isatin derivative compound of Formula (I) is selected from the group consisting of:compound 15a: 1 '-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;compound 15b: 3-(4-chlorophenyl)-1'-methylspiro[imidazolidine-4,3'-indoline]-2',5- dione;compound 15c: 5'-fluoro-1 '-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5- dione;compound 15d: 5'-methoxy-1'-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]- 2',5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.compound 15e: 1'-benzyl-3-phenylspiro[imidazolidine-4,3'- indoline]-2', 5-dione;compound 15f: 1'-benzyl-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2',5-dione;compound 15g: 1'-benzyl-3-(4-chlorophenyl)spiro[imidazolidine-4,3'-indoline]-2',5-dione;compound 15h: 1'-benzyl-3-(3-(trifluoromethyl)phenyl)spiro[imidazolidine-4,3'-indoline]-2',5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.compound 15i: 1'-benzyl-3-(4-(trifluoromethyl)phenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;compound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione;compound 15k: 1 '-benzyl-5'-fluoro-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;compound 151: 1'-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione;compound 15m: 1'-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.compound 15n: 1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2',5-dione;FO=^^N^S's^HN—compound 15o: 5'-fluoro-1'-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'- indoline]-2', 5-dione; andNNcompound 15p: 5'-fluoro-1'-(4-fluorobenzyl)-3-(4-fluorophenyl)spiro[imidazolidine- 4, 3'-indoline]-2', 5-dione.

11. The compound according to claim 9, wherein the compound of Formula (I) is selected from the group consisting ofINcompound 15a: 1 '-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;NP024165WO-01 Notarbartolo & Gervasi S.p. A.compound 15b: 3-(4-chlorophenyl)-1'-methylspiro[imidazolidine-4,3'-indoline]-2',5-dione;HN—1compound 15c: 5'-fluoro-1 '-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione;compound 15d: 5'-methoxy-1 '-methyl-3-phenylspiro[imidazolidine-4,3'-indoline]- 2', 5-dione;compound 15e: 1'-benzyl-3-phenylspiro[imidazolidine-4,3'-indoline]-2', 5-dione;compound 15g: 1'-benzyl-3-(4-chlorophenyl)spiro[imidazolidine-4,3'-indoline]-2',5-dione;P024165WO-01 Notarbartolo & Gervasi S.p. A.compound 15h: 1'-benzyl-3-(3-(trifluoromethyl)phenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;compound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione;HN— compound 15k: 1 '-benzyl-5'-fluoro-3-(4-fluorophenyl)spiro[imidazolidine-4,3'-indoline]-2', 5-dione;compound 151: 1'-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione; andcompound 15m: 1'-(4-fluorobenzyl)-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione.

12. The compound according to claim 11, wherein the compound of Formula (I) is selected from the group consisting ofP024165WO-01 Notarbartolo & Gervasi S.p. A.compound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione, andcompound 151: 1'-benzyl-5'-methoxy-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione.

13. The compound according to claim 12, wherein the compound of Formula (I) iscompound 15j: 1 '-benzyl-5'-fluoro-3-phenylspiro[imidazolidine-4,3'-indoline]-2',5-dione.

14. The compound according to anyone of claims 1 to 13, wherein the pharmaceutically acceptable salt of the compound of Formula (I) is selected from the group consisting of hydrochloride, hydrobromide, sulphate, citrate, phosphate or acid phosphate, acetate, maleate, fumarate, stearate, lactate, tartrate, gluconate, salicylate, ascorbate, aspartate, succinate, oxalate and benzoate.

15. A compound of Formula (I) according to anyone of claims 1 to 14 for use as a medicament.

16. A compound of Formula (I) according to claim 15, for use as a selective inhibitor of the opening of the mitochondrial permeability transition pore (mPTP) both inP024165WO-01 Notarbartolo & Gervasi S.p. A.mammalian cells and tissues in the treatment of reperfusion injury diseases.

17. The compound for use according to claim 16, wherein the reperfusion injury diseases are cardiac, neurological or nephrological diseases.

18. The compound for use according to claim 16 or 17, wherein the reperfusion injury diseases are cardiac or nephrological diseases resulting from ischemic events related to heart attack and stroke.

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