Metal complex compounds as photosensitizers for photodynamic therapy

Abstract

Electrically neutral complex compounds of formula I including any stereoisomer or E / Z isomer thereof wherein: M is Fe < 2 + >, Ru < 2 + >, Os < 2 + >, Co < 3 + >, Rh < 2 + >, Rh < 3 + >, Ir < 3 + >, Ni < 2 + >, Pd < 2 + >, Pt < 2 + > or Pt < 4 + >, in particular Ru < 2 + >; t and T'are H or (C1-C3)-alkyl; lig is a bidentate ligand, in particular 2, 2 '-dipyridyl (bpy) or 4, 7-diphenyl-1, 10-phenanthroline (BPhen); and A is an anion from a pharmaceutically acceptable acid, the electrically neutral complex compounds of formula I are useful in human therapy, in particular as photosensitizers (PS) in photodynamic therapy (PDT) of cancer, skin diseases, fungal infections or microbial infections. They are almost non-toxic under dark conditions at therapeutic doses and exhibit good to excellent photoactivity under hypoxic conditions. They can be used for PDT treatment of deep hypoxia tumors by using light within a far-infrared to near-infrared light treatment window.

Description

Technical Field

[0001] The present invention relates to a novel metal complex compound which can be used as a photosensitizer (PS) for photodynamic therapy (PDT), and a novel intermediate ligand compound which can be used to prepare the complex compound. Background Art

[0002] Photodynamic therapy has been around for over 100 years, albeit in different forms. More recently, PDT has emerged as a medical intervention for treating certain types of cancer that are intractable with traditional methods. PDT is also used to treat some skin conditions (such as acne or port-wine stains), some fungal and microbial infections, and some forms of age-related macular degeneration (see SAMcFarland et al.; “Metal-based photosensitizers for photodynamic therapy: the future of multimodaloncology?”; Current Opinion in Chemical Biology, 2020, Vol. 56, pp. 23-27; and references therein).

[0003] PDT is a well-established, non-invasive modality for destroying tumors and / or tumor vasculature that is based on a combination of three components: a photosensitizer (PS), light of an appropriate wavelength (λ), and oxygen. PDT is delivered in a two-step process involving local or systemic administration of a non-toxic dose of PS, followed by photoactivation. Compared to other conventional treatment options, PDT offers several advantages in cancer treatment due to its non-invasive nature and the spatiotemporal selectivity afforded by light. Indeed, a key attribute of PDT is that toxicity is limited to regions where the three components (PS, light, and oxygen) overlap in both time and space.

[0004] The PDT effect originates from the excitation of PS to generate a singlet excited state, which undergoes intersystem crossing to form a reactive triplet state. The triplet state then converts cytotoxic singlet oxygen ( 1 O2) or participate in type I electron transfer reactions to produce other reactive oxygen species, such as superoxide or hydroxyl radicals.

[0005] Both protoporphyrin IX (PpIX) and protoporphyrin IX (PpIX) have a tetrapyrrole ring system and have been approved worldwide for use in certain cancers (see SAMcFarland et al., supra, p. 24). While these two compounds set the standard for PDT, there is ongoing interest in developing new PSs to address some of the limitations of current PSs in clinical use.

[0006] It is highly desirable to prepare water-soluble PS of high purity by direct synthesis. Certain metal complex compounds play a great role in this respect. It is known (see S. A. McFarland et al., supra, p. 25) that some PS containing metal ions have already entered clinical studies, the most promising being padeliporfin (also known as Soluble and WST 11) and PAD-1433. Padeliporfin is a tetrapyrrole Pd(II) complex compound which is used as a PS in PDT to treat low-risk prostate cancer using a vascular-targeting PDT. TLD-1433 is the first Ru(II) complex compound used as a PS in PDT clinical studies to treat non-muscle-invasive bladder cancer. TLD-1433 is a thiophene-containing complex compound, disclosed in the patent family of S. A. McFarland with priority date 2012. This patent family includes some granted patents, such as EP 2854948 B1 and US 9,676,806 B2, both of which claim PS of a variety of metal (Mn, Mo, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pt and Cu) containing thiophene complex compounds.

[0007] PDT is less efficient in treating deep-seated hypoxic tumors (i.e. tumor microenvironments that lack sufficient oxygen supply at the tissue level) because it is an oxygen-dependent process. On the other hand, while excitation with long-wavelength light is generally preferred to promote tissue penetration, in some known PS it is generally less effective in promoting the generation of type II reactive oxygen species (i.e. singlet oxygen). There is a continued interest in developing new metal complex compound-based PS for PDT that solve some of the limitations of known PS, particularly operability in the far-red and near-infrared region (NIR) of the electromagnetic spectrum and under hypoxic conditions. SUMMARY

[0008] One aspect of the present disclosure relates to providing a compound of Formula I or Formula IV, including stereoisomers and E / Z isomers,

[0009] wherein:

[0010] M is a metal cation selected from the group consisting of Fe 2+ , Ru 2+ , Os 2+ , Co 3+ , Rh 2+ , Rh 3+ , Ir 3+ , Ni 2+ , Pd 2+ , Pt 2+ , and Pt 4+ ;

[0011] y = 1, 2 or 3; and z = 0, 1 or 2; with the proviso that: when M is Ni 2+ , Pd 2+ or Pt 2+ , y + z = 2, and when M is Fe 2+ , Ru 2+ , Os 2+ , Co 3+ , Rh 2+ , Rh 2+ , Ir 2+ or Pt 2+ , y + z = 3;

[0012] m = 0, 1, 2, 3 or 4;

[0013] the groups T and T' are each, at each occurrence, independently a group selected from the group consisting of H and (C3-C6)-alkyl;

[0014]

[0015] the groups P3, P5, P6, Q3, Q4, Q5, Q6, R3 (the definition of R3 applies only to formula I, as formula IV does not contain this particular group), R5, R6 and R8 are each, at each occurrence, independently selected from the group consisting of H, (C1-C3)-alkyl, (C3-C6)-cycloalkyl, CH=CHR, CF3, CHF2, CH2F, CF2CF3, F, Cl, Br, I, OR, C(=O)OR, O(C=O)R, C(=O)NR, NRC(=O)R', NRR', phenyl, mono-, di- and tri-substituted phenyl; wherein the substituents on the phenyl ring are attached to any possible substitution position and they are independently selected from the group consisting of F, Cl, Br, I, NO2, (C1-C3)-alkyl, OH, O[(C1-C3)-alkyl], NH2, NH[(C1-C3)-alkyl] and N[(C1-C3)-alkyl]2;

[0016] the group R7 is H, NO2, OR, NRR', N(CH2COOH)2, N(CH2CH2SO3H)2, N(CH2CONHCH2CH2NMe2)2 or an unsubstituted, mono(R)-substituted, di(R,R')-substituted or tri(R,R',R") -substituted group of each of the heterocyclic groups 1-aziridinyl, 1-azetidinyl, 1-pyrrolidinyl, 1-piperidinyl, 4-morpholinyl or 1-piperazinyl, alternatively R6, R7 and R8 together form a bicyclic ring system of the formulae attached, wherein V1, V2, V3, V4, V5, V6, V7, V8, V9, V 10 , V 11 and V 12each independently a group selected from H and (Ci-C3)-alkyl;

[0017]

[0018] R4is H, (Ci-C3)-alkyl, (C3-C6)-cycloalkyl, CF3, CHF2, CH2F, CF2CF3, F, Cl, Br, I, OR, C(=0)OR, 0(C=0)R, C(=0)NR, NRC(=0)R', NRR', phenyl, mono(R)-substituted phenyl, di(R,R')-substituted phenyl, tri(R,R',R")-substituted phenyl, or a group CE1E2E3; wherein: E1and E2are independently selected from the group consisting of H, F, Cl, Br, I, OR, NRR', N02, (Ci-C3)-alkyl, phenyl, and mono(R)-substituted phenyl; and E3is OH, 0[(Ci-C3)-alkyl], or a group of the formula appended, wherein p = 0, 1, 2, 3, or 4; and D1and D2are independently at each occurrence a group selected from H, 0[CH2] q - X, S[CH2] q - X and N[[CH2] q - X]2, q = 1 or 2, and X = F, Cl, Br, or I.

[0019]

[0020] or a group of the formula appended, wherein p = 1, 2, 3, or 4; and D3is a group independently selected from H, H(C=0), [(Ci-C3)-alkyl](C=0), (OH)2P(=0)0-CH2-0(C=0), (PhO)(OH)P(=0),

[0021]

[0022] Lig is at each occurrence a bidentate ligand independently selected from the group consisting of ligands of the formulae Lig 1, Lig 2, Lig 3, Lig 4, Lig 5, Lig 6, Lig 7, Lig 8, Lig 9, Lig 10, Lig 11, Lig 12, and Lig 13 appended; wherein G is a N atom or a C atom having one negative charge which is formed in situ upon loss of H from C-H + ; and wherein R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R18 , R 19 , R 20 and R 21 are each, at each occurrence, independently a group selected from H, (Ci-C3)-alkyl, phenyl and mono-, di- and tri-substituted phenyl, the substituents attached to the phenyl ring being independently selected from F, Cl, Br, I, OR and NRR'; and

[0023] A is an anion from a pharmaceutically acceptable acid; n is an integer or fraction, whereby the compound of formula I is electrically neutral;

[0024] wherein R, R' and R" are each, at each occurrence, independently a group selected from H, F, Cl, Br, I, N02, (Ci-C3)-alkyl, OH, 0[(Ci-C3)-alkyl], NH2, NH[(Ci-C3)-alkyl] and N[(Ci-C3)-alkyl]2.

[0025]

[0026]

[0027] As used herein, the term "stereoisomers" is a general term for all isomers of a single molecule that differ only in the orientation of atoms in space, and includes enantiomeric and diastereomeric isomers. When a compound has a chiral center, it can exist in different stereoisomeric forms, such as enantiomeric or diastereomeric forms. Thus, any given compound, as mentioned herein, is intended to represent any one of the individual enantiomeric or diastereomeric forms as well as mixtures of one or more of the enantiomeric or diastereomeric forms. All stereoisomers, including enantiomers and diastereomers, of a compound described herein are contemplated within the scope of the disclosure. The disclosure also contemplates any possible E / Z isomers and mixtures thereof.

[0028] It is noted that in one preferred embodiment of the present application, formula IV can be obtained from formula I, when in formula I m > 1 (e.g. 1, 2, 3 or more) the group R3 is CH2, forming a 6-membered ring with the T' group closest to the coumarin backbone, with the proviso that T' = CH2. It is also noted that in another preferred embodiment of the present application, formula V can be obtained from formula II, when in formula II m > 1 (e.g. 1, 2, 3 or more) the group R3 is CH2, forming a 6-membered ring with the T' group closest to the coumarin backbone, with the proviso that T' = CH2.

[0029] In general, the complex compounds of formula I or IV can be prepared by reacting the corresponding intermediate compounds of formula II or V (including their corresponding stereoisomers or E / Z isomers) with the corresponding reactant compounds of formula III in a solvent or mixture of solvents, respectively, wherein m, n, y, z, P3, P5, P6, Q3, Q4, Q5, Q6, R3, R4, R5, R6, R7, R8, T, T’, M, Lig and A are as defined above. Since the intermediate compounds of formula II or V are novel, another aspect of the present disclosure relates to the provision of these compounds, which can be used for the preparation of the complex compounds of formula I or IV.

[0030]

[0031] [M(Lig) z ]A n

[0032] III

[0033]

[0034] In general, the reactant compounds of formula III and the intermediate compounds of formula II or V are reacted in molar amounts that essentially correspond to the stoichiometry determined by the values of z and y, although in some cases an excess of III can be recommended to increase the yield. In some cases, the intermediate compounds are obtained with a different anion A’ and a final anion exchange reaction is performed (e.g. with ion exchange resins).

[0035] As shown by the results in the description of the Examples section, the metal complex compounds of formula I or IV can be used as PS in anticancer PDT. For example, the complex compounds of formula la and Ic show very good phototoxicity results against the CT-26 mouse colon carcinoma cancer cell line.

[0036] Thus, another aspect of the present disclosure relates to the provision of pharmaceutical compositions comprising a therapeutically effective amount of the complex compounds of formula I or IV together with an appropriate amount of excipients, carriers or vehicles.

[0037] In dark conditions, the complex compounds of formula I are practically non-toxic at therapeutic doses. For example, the complex compound la and the complex compound Ic are both non-toxic in dark conditions, with IC 50 values exceeding 250 mM, but become highly cytotoxic upon irradiation with monochromatic visible light. As shown in Table 1, the IC 50Values in the low nanomolar range (8.2 nM for compound la at 540 nm and 7.4 nM for compound Ic at 645 nm) with very good PI values (>30487 and >33783, respectively). For comparison purposes, the results reported for TLD-1433 (PS) in clinical development are as follows: IC 50 Values in the low micromolar range (2.3 mM) and it shows moderate PI values (see S. Monro et al.; “Transition metal complexes and photodynamic therapy from a tumor-centered approach: challenges, opportunities, and highlights from the development of TLD-1433”; Chem. Rev. 2019, vol. 119, pp. 797-828).

[0038] In the CT-26 carcinoma cell line (IC 50 = 0.76 mM, PI >329) and in its analogous human HT-29 carcinoma cell line (IC 50 = 0.35 mM, PI >714), the compound of formula Ic shows a good phototoxic profile under high penetrating NIR light (740 nm); these results are superior to the corresponding results of protoporphyrin IX used as a control drug. Similar results were obtained in other human carcinoma cell lines (A-549 and A-2780) as shown in the results in Table 6. Similarly, as shown in Tables 5 and 8, compounds of formula Im, In, Io and IVa were found to be phototoxic when irradiated with NIR light (740 nm and 770 nm) in the CT-26 carcinoma cell line (for example, Im at 740 nm IC 50 = 0.17 mM, PI >588).

[0039] The complex compounds of formula I or IV show good to excellent photoactivity under hypoxic conditions. For example, compound la and compound Ic exhibit excellent photoactivity under hypoxic conditions, compound la has a specific toxicity with an excellent PI exceeding 7143 when irradiated at 540 nm. Compound Ic shows PI values of about 2900 to 3300 in the light window from 540 nm to 670 nm. Compound Im shows sub-micromolar IC 50Values (PI > 168). Such results indicate that compound I or compound IV can be used for PDT treatment of deep-seated hypoxic tumors by using light in the window of light therapy from far-red to NIR.

[0040] Aspects of the present disclosure relate to the use of complex compounds of formula I or IV for human therapy, in particular for use as photosensitizers in the photodynamic therapy of human conditions; more specifically, the human conditions are cancer, skin diseases, fungal infections or microbial infections. These aspects also relate to a method for the manufacture of a medicament for the photodynamic therapy of human cancer, skin diseases, fungal infections or microbial infections, comprising the use of complex compounds of formula I or IV, preferably in the form of a nanoformulation. In other words, it can be said that complex compounds of formula I or IV are useful in a method for the treatment of human cancer, skin diseases, fungal infections or microbial infections by photodynamic therapy.

[0041] Throughout the present disclosure and claims, the word "comprise" and variations of the word, is not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case where the word "consist" is used. Additional objects, advantages and features of the disclosure will become apparent to those skilled in the art upon examination of the disclosure or can be learned by practice of the disclosure. The following examples are provided by way of illustration and they are not intended to be limiting. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Figure 1 : 3D CT-26 multicellular tumor spheroids (MCTS) monitored growth over 7 days at increasing concentrations of Ic compared to initial size. Data are presented as mean ± SD of three independent measurements. Control: untreated MCTS, irradiated with 645 nm light (2.50 mW-cm -2 , 9.00 J-cm -2 ) for 1 h. Treated MCTS were incubated with Ic for 36 h and irradiated with 645 nm light (2.50 mW-cm -2 , 9.00 J-cm -2 ) for 1 h. Lower panel: micrographs of 3D CT-26 MCTS taken over 7 days (scale bar = 1000 pm).

[0043] Figure 2Cellular uptake of complex Compound Ic. Single confocal planes of HeLa cells incubated with the compound (10 μΜ) for 30 min at 37°C were imaged after the first observation at t=0 and 2 min. Excitation was performed with three different laser lines (405 nm, 458 nm and 514 nm). White arrows indicate mitochondria and white arrowheads vesicle staining. Black arrowheads in the right column indicate cellular bubbles. Scale bar: 20 μιη. LUT for compound images: flame color. Left and right columns: merged compound images and brightfield images.

[0044] Figure 3 Co-localization studies of Ic with Mitoview 650. Single confocal planes of HeLa cells incubated with the compound (10 μΜ, green) and Mitoview (0.1 μΜ, red). Left panel: overlay of both stains. Center: Ic' signal. Right panel: Mitoview signal. White arrows and arrowheads indicate positive and negative (vesicle staining in compound images) co-localization, respectively. Scale bar: 20 μιη.

[0045] Figure 4 Mean values of Ic concentration in plasma of Swiss Albino adult male CD1 mice (left) and pharmacokinetic parameters of Ic in mice plasma (right) after administration of 5 mg / Kg IP at different times.

[0046] Figure 5 Body weight (g) (left) and food intake (g) / animal (right) during the experimental protocol of 5 days. CD1 mice were intraperitoneally treated with vehicle and complex Compound Ic (10 mg / kg or 30 mg / kg) on day 1 and sacrificed on day 5. Results are expressed as mean ± SD (n=3 males; n=3 females).

[0047] Figure 6 Organ weight (mg) / body weight (g) ratio of animals intraperitoneally treated with vehicle and complex Compound Ic (10 mg / kg or 30 mg / kg) on day 1 and sacrificed on day 5. Results are expressed as mean ± SD (n=3 males; n=3 females). Compared to the male or female vehicle group, * p<0.05 (1-way ANOVA and Bonferroni post-hoc test).

[0048] Figure 7 In vivo PDT efficacy studies of Ic in female BALB / c mice bearing subcutaneous CT-26 syngeneic colon tumors were performed after IT administration. Irradiation was performed with a 660 nm LED light (15 min, 100 mW / cm2). The mice were treated with the compound (10 mg / kg) 24 h before irradiation. The mice were sacrificed 24 h after irradiation. The results are expressed as mean ± SD (n=5 mice per group). 2). Tumor growth inhibition curves (left) and mean tumor weights (right) of treated tumor-bearing mice (D: dark group; L: light group; x n : number of irradiations). Data are expressed as mean ± SEM of five parallel mice (n = 5). Mean tumor weight data were analyzed using one-way ANOVA followed by Bonferroni’s multiple comparison test (asterisks indicate: * p < 0.05, ** p < 0.001). DETAILED DESCRIPTION

[0049] In a particular embodiment of the complex compound of formula I or IV, the anion A is Cl - , Br - , PF6 - , PF4 - , BF4 - , CIO4 - , CF3SO3 - , SO4 2- , CF3COO - , acetate, formate and oxalate. In a particular embodiment, A is Cl - .

[0050] In a particular embodiment of the complex compound of formula I or IV, y = 1, z = 2 and the metal cation M is Fe 2+ , Ru 2+ , Os 2+ , Co 3+ , Rh 2+ , Rh 3+ , Ir 3+ or Pt 4+ . In a particular embodiment, the metal cation M is Ru 2+ .

[0051] In a particular embodiment of the complex compound of formula I or IV, the bidentate ligand Lig is Lig 1 or Lig 2; and the groups R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21each of P3, P5, P6, Q3, Q4, Q5, Q6, R3, R5, R6, and R8 is, at each occurrence, H, methyl, or phenyl. In a specific embodiment, G is N. In a specific embodiment, the bidentate ligand Lig is 2,2'-bipyridine (bpy) or 4,7-diphenyl-1,10-phenanthroline (BPhen).

[0052] In a specific embodiment, some complex compounds of formula I or IV are obtained by a reaction between an intermediate compound of formula II or V and a reactant compound of formula III, wherein M = Ru 2+ , A = Cl - , z = 2, y = 1, n = 2, Lig = 2,2'-bipyridine (bpy) or 4,7-diphenyl-1,10-phenanthroline (BPhen), the solvent mixture is a mixture of ethanol and water, and the molar ratio of compound III to compound II or V is 1.0 or slightly higher.

[0053] In a specific embodiment of the complex compounds of formula I or IV, the groups P3, P5, P6, Q3, Q4, Q5, Q6, R3 (the definition of R3 only relates to formula I, as formula IV does not contain this group), R5, R6, and R8 are, at each occurrence, H or (Ci-C3)-alkyl; the group R7 is H, N[(Ci-C3)-alkyl)]2, or NO2; alternatively, the groups R6, R7, and R8 together form a bicyclic system of the formula as shown in claim 1, wherein each of V1, V2, V3, V4, V5, V6, V7, V8, V9, V 10 , V 11 , and V 12 are H.

[0054] In a specific embodiment of the complex compounds of formula I or IV, the group R4 is H, (Ci-C3)-alkyl, CF3, CH(CH3)OH, CH(CH3)0(C=0)-[CH2]3-Ph, CH(CH3)0(C=0)-[CH2]2-(C=0)-CH2-NH2, or a group selected from the following:

[0055]

[0056] It is noted that, in a preferred embodiment of the present application, formula IV can be obtained from formula I when, in formula I, m > 1 (e.g. 1, 2, 3, or more) and the group R3 is CH2, forming a 6-membered ring with the T' group closest to the coumarin backbone, with the proviso that T' = CH2. It is also noted that, in another preferred embodiment of the present application, formula V can be obtained from formula II when, in formula II, m > 1 (e.g. 1, 2, 3, or more) and the group R3 is CH2, forming a 6-membered ring with the T' group closest to the coumarin backbone, with the proviso that T' = CH2.

[0057] In particular embodiments, m = 0, 1, or 2. In specific embodiments, the complex compound of Formula I or Formula IV has one of the following formulae Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Im, In, Io, Ip, Iq, IVa, or IVb:

[0058]

[0059]

[0060] In specific embodiments, the intermediate compound of Formula II (IIa, IIb, IIc, IId, IIe, IIf, IIm, IIn, IIo, IIp, IIq, IIr) or Formula V (Va or Vb) has one of the following formulae IIa, IIb, IIc, IId, IIe, IIf, IIm, IIn, IIo, IIp, IIq, IIr, Va, or Vb:

[0061]

[0062]

[0063] Scheme 1 : Preparation of complex compounds of formula la, formula lb and formula Ic

[0064]

[0065] Complex compound of Formula Ia. Ligand compound of Formula IIa (31 mg, 0.073 mmol) and [Ru(bpy)2Cl2] (37 mg, 0.076 mmol) were dissolved in 3 mL of 3:1 (v / v) EtOH / H2O solution. The reaction mixture was stirred at 80 °C overnight and analyzed by HPLC-MS to confirm the formation of the product. The solution mixture was evaporated to dryness, and the resulting aqueous solution was lyophilized, and the product was isolated by silica gel column chromatography starting with hexanes, first with DCM (0 to 100%) and then MeOH (0 to 20%) increasing the eluent polarity. 41 mg of a red-brown solid was obtained (yield: 89%) and identified as the complex compound of Formula Ia. TLC: Rf (35% MeOH in DCM) 0.4. HR-ESIMS (ESI): m / z 418.1265, [C 47 H 42 N8ORu] 2+ Calcd: 418.1257.

[0066] Complex compound of formula Ib. The ligand compound of formula lib (34 mg, 0.076 mmol) and [Ru(bpy)2Cl2] (38 mg, 0.078 mmol) were dissolved in 3 mL of 3:1 (v / v) EtOH / H2O solution. The reaction mixture was stirred at 80 °C overnight and analyzed by HPLC-MS to confirm the formation of the product. The solvent was evaporated to dryness and the product was isolated by silica gel column chromatography starting with hexanes, first with DCM (0 to 100%) and then increasing the polarity of the eluent with MeOH (0 to 20%). 46 mg of dark chestnut solid was obtained (yield: 65%) and identified as the complex compound of formula Ib. TLC: Rf (1 :9 MeOH / DCM) 0.5. HR-ESIMS (ESI): m / z 430.1258, [C 49 H 42 N8ORu] 2+ Calcd: 430.1257.

[0067] Complex compound of formula Ic. The ligand compound of formula lie (31.6 mg, 0.063 mmol) and [Ru(bpy)2Cl2] (37.4 mg, 0.077 mmol) were dissolved in 2 mL of 3:1 (v / v) EtOH / H2O solution. The reaction was stirred at 80 °C overnight and analyzed by HPLC-MS to confirm the formation of the product. The solvent was evaporated to dryness and the product was isolated by silica gel column chromatography starting with hexanes, first with DCM (0 to 100%) and then increasing the polarity of the eluent with MeOH (0 to 16%). 51.4 mg of dark purple solid was obtained and identified as the complex compound of formula Ic (yield: 87%). TLC: Rf (30% MeOH in DCM) 0.4. HR-ESIMS (ESI): m / z 457.1102, [C 49 H 39 N8OF3Ru] 2+ Calcd: 457.1115.

[0068] Scheme 2: Preparation of complex compounds of formula Id, formula Ie and formula If

[0069]

[0070] Complex compound of formula Id. The ligand compound of formula lid (57.7 mg, 0.096 mmol.) and [Ru(bpy)2Cl2] (55.7 mg, 0.115 mmol) were dissolved in 6 mL of a 3:1 (v / v) EtOH / H2O solution. A few drops of DCM (0.6 mL) were used to completely dissolve the reagents. The reaction mixture was heated to 80 °C and kept under reflux overnight. The solvent was evaporated to dryness and the product was purified by flash chromatography (Puriflash system; first 0 to 100% of DCM in hexane, then 0 to 44% of MeOH in DCM, silica gel column - PF-30SIHP-F0012) to obtain 34 mg of a red-brown solid (yield: 34%) identified as the complex compound of formula Id. TLC: Rf(30% MeOH in DCM) 0.43. HR-ESIMS (ESI) m / z 506.1689, [C 58 H 54 N8O3Ru] 2+ Calculated: 506.1681.

[0071] Complex compound of formula Id. The ligand compound of formula lid (57.7 mg, 0.096 mmol.) and [Ru(bpy)2Cl2] (55.7 mg, 0.115 mmol) were dissolved in 6 mL of a 3:1 (v / v) EtOH / H2O solution. A few drops of DCM (0.6 mL) were used to completely dissolve the reagents. The reaction mixture was heated to 80 °C and kept under reflux overnight. The solvent was evaporated to dryness and the product was purified by flash chromatography (Puriflash system; first 0 to 100% of DCM in hexane, then 0 to 44% of MeOH in DCM, silica gel column - PF-30SIHP-F0012) to obtain 34 mg of a red-brown solid (yield: 34%) identified as the complex compound of formula Id. TLC: Rf(30% MeOH in DCM) 0.43. HR-ESIMS (ESI) m / z 506.1689, [C 48 H 44 N8O2Ru] 2+ Calculated: 433.1315.

[0072] Complex compound of formula If. [RuCI2(bpy)2] (18.4 mg, 0.038 mmol) and AgN03(6.5 mg, 0.038 mmol) were dissolved in a 1 : 1 (v / v) mixture of MeOH and Milli-Q water (2.4 mL) and stirred at 90 °C for 4 h in the dark. Then, a solution of ligand compound II f (28 mg, 0.038 mmol) in THF (1.2 ml) was added and the reaction mixture was allowed to react at room temperature for 72 h. Finally, KPF6(69.9 mg, 0.38 mmol) was added and allowed to react overnight. After evaporation of the solvent under reduced pressure, the product was isolated by semi-preparative RP-HPLC (gradient from 50% to 100% B in 30 min; A, 0.05% TFA in H20; B, 0.1% TFA in ACN; flow rate, 3 mL). After lyophilization, 18.8 mg of a red solid was obtained (yield: 36%). TLC: Rf(10% MeOH in DCM) = 0.48. The chloride salt of this compound was obtained by using Amberlite IRA 402 chloride resin and identified as complex compound of formula If. LR-ESIMS (ESI): m / z 575.81, [C f (30% MeOH in DCM) = 0.48. The chloride salt of this compound was obtained by using Amberlite IRA 402 chloride resin and identified as complex compound of formula If. LR-ESIMS (ESI): m / z 575.81, [C 62 H 61 Cl2N9O3Ru] 2+ Calculated: 575.6659.

[0073] Scheme 3: Preparation of complex compounds of formula Ig and formula Ih

[0074]

[0075] Complex compound of formula Ig. Ligand compound of formula Ila (20.4 mg, 0.048 mmol) and [Ru(BPhen)2CI2] (49.5 mg, 0.059 mmol) were dissolved in 3 mL of a 3: 1 (v / v) EtOH / H20 solution. The reaction mixture was stirred at 80 °C overnight and analyzed by HPLC-MS to confirm the formation of the product. The solution mixture was evaporated to dryness and the product was isolated by silica gel column chromatography starting from 100% DCM increasing the eluent polarity with MeOH (0 to 6%). 37.3 mg of a red solid was obtained (yield: 63%) and identified as complex compound of formula Ig. TLC: Rf(10% MeOH in DCM) 0.4. HR-ESIMS (positive mode): m / z 594.1892, [C 75 H 58 N8ORu] 2+ Calculated: 594.1888.

[0076] Complex compound of formula Ih. The ligand compound of formula IIc (11.0 mg, 0.022 mmol) and [Ru(BPhen)2Cl2] (41.9 mg, 0.050 mmol) were dissolved in 8 mL of 3:1 (v / v) EtOH / H2O solution. The reaction mixture was stirred at 80°C overnight and analyzed by HPLC-MS to confirm the formation of the product. The solution mixture was evaporated to dryness, and the product was separated by silica gel column chromatography starting from hexane, first with DCM (0 to 100%) and then with MeOH (0 to 26%) to increase the eluent polarity. 23.7 mg of a reddish-maroon solid was obtained (yield: 81%), which was identified as a complex compound of formula Ih. TLC: Rf (10% MeOH in DCM) 0.3. HR-ESIMS (positive ion mode): m / z 633.1740, [C 77 H 55 F3N8ORu] 2+ Calculated value: 633.1741.

[0077] Scheme 4: Preparation of complex compounds of formula Ii and formula Ij

[0078]

[0079] Mode Ii complex compound. To an ethylene glycol solution of [Os(bpy)2Cl2] (10 mg, 0.017 mmol) was added a ligand compound of formula IIa (8 mg, 0.019 mmol). The mixture was heated at 85°C under N2 for 18 hours. The solution was cooled to room temperature. A saturated aqueous solution of ammonium hexafluorophosphate was added, and the resulting dark purple precipitate was separated by filtration, washed with water, ether, and dried under vacuum. The crude product was then purified by column chromatography (silica gel, 0 to 15% MeOH in CH2Cl2). After converting the PF6 salt into a chloride salt using Amberlite IRA 410 chloride resin, 8 mg of a dark purple solid was obtained (42% yield). TLC: Rf (10% MeOH in CH2Cl2) 0.45. HR-ESIMS (positive ion mode): m / z 463.1542, [C 47 H 42 N8OOs] 2+ Calculated value: 463.1542.

[0080] Complex compound of formula Ij. To a solution of [Os(bpy)2Cl2] (14 mg, 0.024 mmol) in ethylene glycol was added the ligand compound of formula lie (12 mg, 0.028 mmol). The mixture was heated at 85 °C under N2for 18 h. The solution was cooled to room temperature. A saturated aqueous solution of ammonium hexafluorophosphate was added and the resulting dark purple precipitate was isolated by filtration, washed with water, diethyl ether and dried under vacuum. The crude product was then purified by column chromatography (silica gel, 0 to 15% MeOH in CH2Cl2). After conversion of the PF6salt into the chloride salt using Amberlite IRA 410 chloride resin, 7 mg of a dark purple solid was obtained (28% yield). Characterization: TLC: Rf(10% MeOH in CH2Cl2) 0.40. HR-ESIMS (positive mode): m / z 502.1402, [C 51 H 45 F3N8OOs] 2+ Calculated: 502.1401.

[0081] Scheme 5: Preparation of complex compounds of formula Ik

[0082]

[0083] Complex compound of formula Ik. To a degassed solution of [Os(BPhen)2Cl2] (30 mg, 0.032 mmol) in ethylene glycol was added the ligand compound of formula Ha (14.8 mg, 0.035 mmol). The mixture was heated at 110 °C under N2for 18 h. The solution was cooled to room temperature. A saturated aqueous solution of ammonium hexafluorophosphate was added and the resulting dark purple precipitate was isolated by filtration, washed with water, diethyl ether and dried under vacuum. The crude product was first purified by column chromatography (silica gel, 0 to 15% MeOH in CH2Cl2) and then further purified by preparative HPLC (gradient from 0 to 100% B in 30 min, flow rate: 10 mL min -1 ,R t = 15.4 min). After conversion of the PF6salt into the chloride salt using Amberlite IRA 410 chloride resin, 9 mg of a dark purple solid was obtained (22% yield). TLC: Rf(10% MeOH in CH2Cl2) 0.51. HR-ESIMS (positive mode): m / z 639.2181, [C 75 H 58 N8OOs] 2+ Calculated: 639.2169.

[0084] Scheme 6: Preparation of complex compounds of formula Im

[0085]

[0086] Complex compound of formula Im. The ligand compound of formula IIm (31.6 mg, 0.060 mmol) and [Ru(bpy)2Cl2] (37.4 mg, 0.077 mmol) were dissolved in a 3:1 (v / v) EtOH / H2O (2 mL) solution and the reaction mixture was stirred at 90 °C overnight. The solvent was evaporated to dryness and the product was purified by silica gel column chromatography starting with hexanes, first with DCM (0 to 100%) and then increasing the eluent polarity with MeOH (0 to 11%). 10 mg of blue solid was obtained (yield: 17%) identified as the complex compound of formula Im. TLC: Rf (30% MeOH in DCM) 0.3. HR-ESIMS (ESI): m / z 470.1193, [C 51 H 41 F3N8ORu] 2+ Calculated: 470.1199

[0087] Scheme 7: Preparation of ligand compounds of formula Ila, formula lib and formula lie

[0088]

[0089] 4-Methyl-4'-((trimethylsilyl)methyl)-2,2'-bipyridine of formula 1. A solution of 4,4'-dimethyl-2,2'-bipyridine (2 g, 10.86 mmol) in anhydrous THF (80 mL) was added via a cannula to a cooled (-78 °C) solution of LDA in THF (12 mL, 11.95 mmol) under an argon atmosphere. The resulting chestnut mixture was stirred at -78 °C for 1 h. Then, trimethylsilyl chloride (2 mL, 11.95 mmol) was added to the crude product, which turned blue, just 10 seconds later, 10 mL of anhydrous ethanol was added carefully, which caused the solution to turn yellow. The crude product was transferred to a separatory funnel containing a saturated NaHC03solution (200 mL) and extracted with DCM (3 x 150 mL). The combined organic phases were washed with brine (150 mL), dried over anhydrous Na2S04, filtered and evaporated to dryness to obtain 2.12 g of the title compound as a yellow solid (yield: 76%) which was used without further purification.

[0090] 4-(chloromethyl)-4'-methyl-2,2'-bipyridine of formula 2. 4-methyl-4'-((trimethylsilyl)methyl)-2,2'-bipyridine (2.12 g, 7.80 mmol), hexachloroethane (7.39 g, 31.2 mmol) and cesium fluoride (4.76 g, 31.2 mmol) were dissolved in anhydrous ACN (120 mL) under argon atmosphere and the resulting solution was stirred at 60 °C for 3.5 h. The reaction mixture was partitioned between 50 mL H2O and 50 mL AcOEt and transferred to a separatory funnel. The aqueous phase was extracted with ethyl acetate (3 x 50 mL) and the combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and evaporated to dryness. The product was purified by column chromatography (silica gel, 0 to 10% MeOH in DCM) to obtain 1.01 g of the title compound as a yellow solid (yield: 57%). TLC: Rf(1 :9 MeOH / DCM) 0.6. LR-ESIMS (ESI): m / z 218.8, [C 12 H 11 N2Cl + H] + Calculated: 218.1.

[0091] 2-(4'-methyl-[2,2'-bipyridin]-4-yl)acetonitrile of formula 3. 4-(chloromethyl)-4'-methyl-2,2'-bipyridine (1 g, 4.57 mmol), 18-crown-6 (26.8 mg, 0.09 mmol) and KCN (3.39 g, 36.5 mmol) were dissolved in 100 mL of ACN and stirred at room temperature overnight. HPLC-MS analysis of the crude product showed almost no product formation. Then, 214 mg of 18-crown-6 (0.81 mmol) were added and the solution was stirred at 50 °C overnight. After confirming by HPLC-MS that the reaction had been completed, the reaction mixture was evaporated to dryness, dissolved in deionized water (100 mL) and extracted with DCM (3 x 100 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and evaporated to dryness. After purification by column chromatography (silica gel, 50% to 100% AcOEt in hexanes), 615 mg of the title compound were obtained as a white solid (yield: 64%). TLC: Rf(AcOEt 100%) 0.7. HR-ESIMS (ESI): m / z 210.1026, [C 13 H 11 N3+H] + Calculated: 210.1025.

[0092] Thiocoumarin of formula 4. 7-Diethylamino-4-methylcoumarin (1.72 g, 7.42 mmol) and Lawesson's reagent (1.65 g, 4.08 mmol) were dissolved in toluene (40 mL) and heated at 100 °C for 12 h. After evaporation under reduced pressure, the dark residue was purified by column chromatography (silica gel, DCM) to obtain the thiocoumarin of formula 4 as an orange solid (1.70 g, 92%): TLC Rf(DCM) 0.50. HRMS (ESI-TOF) m / z [M+H] + , C 14 H 18 NOS 248.1109, found 248.1099.

[0093] Thiocoumarin of formula 5. 2,3,6,7-Tetrahydro-9-methyl-1H,5H-quinolizino[9,1- gh]coumarin (2 g, 7.8 mmol) and Lawesson's reagent (1.89 g, 4.7 mmol) were dissolved in 100 mL of toluene and stirred at 100 °C overnight. The dark green crude product was evaporated to dryness and the product was isolated by column chromatography (silica gel, 0 to 70% DCM in hexanes). 1.44 g of the thiocoumarin of formula 5 was obtained as an orange solid (yield: 68%). TLC: Rf(100% DCM) 0.8. HR-ESIMS (ESI): m / z 272.1104, [C 16 H 17 NOS + H] + Calculated for: 272.1102.

[0094] Thiocoumarin of formula 6. 2,3,6,7-Tetrahydro-9-trifluoromethyl-1H,5H-quinolizino[9,1- gh]coumarin (1 g, 3.23 mmol) and Lawesson's reagent (791 mg, 1.94 mmol) were dissolved in toluene (50 mL). The yellow solution was stirred at 100 °C overnight. The dark red solution was evaporated to dryness and the thiocoumarin of formula 6 was isolated by column chromatography (silica gel, 0 to 40% DCM in hexanes). 1.14 g of the thiocoumarin of formula 6 was obtained as a golden-orange solid (yield: 95%). Golden-orange solid. TLC: Rf(100% DCM) 0.9. HR-ESIMS (ESI): m / z 326.0821, [C 16 H 14 F3NOS + H] + Calculated for: 326.0816.

[0095] Ligand compound of Formula IIa. To a solution of sodium hydride (568 mg of a 60% dispersion in mineral oil, 14.15 mmol) and compound 3 (712 mg, 3.40 mmol) in anhydrous acetonitrile (300 mL) was added 7-(N,N-diethylamino-4-methyl-2-thioxycoumarin (703 mg, 5.66 mmol). The orange solution was stirred for 3 h. Then, silver nitrate (962 mg, 4.98 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The dark-brown solution was analyzed by HPLC-MS to confirm the formation of the desired product in the crude product and the solution was evaporated under reduced pressure. The product was isolated by silica gel column chromatography starting with hexanes, first increasing the eluent polarity with DCM (0 to 100%) and then with MeOH (0 to 10%). 451 mg of an orange / red solid was obtained (yield: 37%) identified as the ligand compound of Formula IIa. TLC: Rf (1 :9 MeOH / DCM) 0.5. HR-ESIMS (ESI): m / z 423.2181, [C 27 H 26 N4O+H] + Calculated: 423.2179

[0096] Ligand compound of Formula IIb. To a solution of NaH (38 mg of a 60% dispersion in mineral oil, 1.58 mmol) and compound 3 (46 mg, 0.22 mmol) in anhydrous ACN (12 mL) was added the thioxycoumarin of Formula 5 (50 mg, 0.18 mmol). The brown solution was stirred at 50 °C for 3 h. Then, silver nitrate (71 mg, 0.41 mmol) was added and the crude product was stirred at room temperature for 2 h. The product was isolated by silica gel column chromatography starting with hexanes, first increasing the eluent polarity with DCM (0 to 100%) and then with MeOH (0 to 10%). 20 mg of an orange / brown solid was obtained (yield: 20%) identified as the ligand compound of Formula IIb. TLC: Rf (5% MeOH / DCM) 0.5. HR-ESIMS (ESI): m / z 447.2186, [C 29 H 26 N4O+H] + Calculated: 447.2179.

[0097] Ligand compound of Formula IIc. To a solution of NaH (18 mg of a 60% dispersion in mineral oil, 0.77 mmol) and compound 3 (41 mg, 0.18 mmol) in anhydrous ACN (35 mL) was added a thio-coumarin of Formula 6 (50 mg, 0.15 mmol). The chestnut-colored solution was stirred for 3 h. Then, silver nitrate (56 mg, 0.32 mmol) was added and the crude product was stirred at room temperature for 2 h. The product was isolated by silica gel column chromatography starting with hexanes, first with DCM (0 to 100%) and then increasing the eluent polarity with MeOH (0 to 20%). 58 mg of a bright red solid was obtained (yield: 75%) identified as the ligand compound of Formula IIc. Bright red solid. TLC: Rf(5% MeOH / DCM) 0.5. HR-ESIMS (ESI): m / z 501.1889, [C 29 H 23 F3N4O+H] + Calculated: 501.1887.

[0098] Scheme 8: Preparation of ligand compounds of formula IId, formula lie and formula IIf

[0099]

[0100] Compound of Formula 11. A mixture of coumarin of Formula 10 (1.60 g, 6.12 mmol), phenylbutyric acid (1.50 g, 9.15 mmol), EDC (1.75 g, 9.15 mmol) and 4-dimethylaminopyridine (DMAP) (1.12 g, 9.15 mmol) was cooled at 0 °C under an argon atmosphere and then dissolved in DCM (100 mL). The mixture was stirred at 0 °C for 15 min and then at room temperature for 17 h. Then, the solution was washed with saturated NH4Cl (2 x 100 mL), 5% aqueous NaHCO3(1 x 100 mL, 2 x 50 mL) and deionized water (100 mL). The organic layer was dried over anhydrous MgSO4, filtered and evaporated under reduced pressure. The product was isolated by silica gel column chromatography (silica gel, 50% to 100% DCM in hexanes, 1% to 3% MeOH in DCM) to obtain 2 g of the title compound as a yellow / orange solid (yield: 80%). TLC: Rf(DCM) 0.6. HR-ESIMS (ESI): m / z 408.2169, [C 25 H 29 NO4+H] + Calculated: 408.2175.

[0101] Compound of formula 12. Under an argon atmosphere, Lawesson's reagent (1.54 g, 3.81 mmol) was added to a toluene solution (60 mL) of the compound of formula 11 (1.94 g, 4.75 mmol). The mixture was stirred in the dark at 105 °C overnight. A color change from dark brown to light yellow was observed. After removal of the solvent under reduced pressure, the product was isolated by column chromatography (silica gel, 100% to 30% hexanes in DCM) to obtain 1.67 g of the title compound as an orange solid (yield: 83%). TLC: Rf (10% MeOH in DCM) = 0.4. HR-ESIMS (ESI): m / z 424.1936, [C f (DCM) = 0.78. HR-ESIMS (ESI): m / z 424.1936, [C 25 H 29 NO3S+H] + Calculated: 424.1946.

[0102] Ligand compound of formula lid. First, sodium hydride (68.3 mg of a 60% dispersion in mineral oil, 1.71 mmol) and 2-(4'-methyl-[2,2'-bipyridinyl]-4-yl)acetonitrile (formula 3) (357 mg, 1.71 mmol) were dissolved in anhydrous ACN (30 mL) and stirred at 35 °C under an argon atmosphere for 15 min. Then, a solution of the coumarin of formula 12 (362 mg, 0.85 mmol) in anhydrous ACN (15 mL) was added to the previous flask and the resulting reaction mixture was stirred at 35 °C under an argon atmosphere in the dark for another 4 h. After 4 h, AgN03(363 mg, 2.14 mmol, 2.5 eq.) was added and stirred for another 2 h. A color change from brown to dark red / crimson was observed. After evaporation of the solvent under reduced pressure, the product was isolated by silica column chromatography starting from a 1 : 1 mixture of DCM and hexanes, increasing the eluent polarity with 10% to 100% DCM and then MeOH (0 to 1%) in DCM. 155 mg of an orange / red solid were obtained (yield: 30%) identified as the ligand compound of formula lid. TLC: Rf (10% MeOH in DCM) = 0.4. HR-ESIMS (ESI): m / z 599.3014, [C f (10% MeOH in DCM) = 0.4. HR-ESIMS (ESI): m / z 599.3014, [C 38 H 38 N4O3+H] + Calculated: 599.3022.

[0103] Ligand compound of Formula IIe. To a solution of ligand compound of Formula IId (41.7 mg, 0.070 mmol) in 2:1 (v / v) ACN / H20 (7 mL) was added 10% NaOH (6.65 mL, 0.070 mmol) and the reaction mixture was stirred at room temperature in the dark overnight. The color of the reaction mixture changed from orange / yellow to bright orange with solid particles. After evaporation to dryness under reduced pressure, the crude product was purified by flash chromatography (hexane solution of DCM (50% to 100%), then MeOH in DCM (0 to 10%), Puriflash system, silica gel column PF-DLE-F0012) to obtain 26.9 mg of orange / red solid (yield: 85%) identified as ligand compound of Formula IIe. TLC: Rf (10% MeOH in DCM) 0.57. HR-ESIMS (ESI): m / z 452.56, [C 29 H 28 N4O2+H] + Calculated: 453.2291.

[0104] Ligand compound of Formula IIf. Under an argon atmosphere, chlorambucil (46.54 mg, 0.153 mmol), 4-dimethylaminopyridine (DMAP) (18.69 mg, 0.153 mmol) and l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HC1) (29.33 mg, 0.153 mmol) were added to a round bottom flask containing the compound of Formula 14 (46.10 mg, 0.102 mmol), which was placed in an ice bath. The reagents were dissolved in DCM (13 mL) and the reaction mixture was stirred for 15 min. Then, the flask was removed from the ice bath and allowed to react at room temperature in the dark overnight. Once the reaction was complete (confirmed by TLC and HPLC), the DCM was evaporated under reduced pressure and then purified by column chromatography (silica gel, 0 to 100% DCM in hexane, then 0 to 1% MeOH in DCM) to obtain 49.9 mg of orange / red solid (yield: 66%) identified as ligand compound of Formula IIf. TLC: Rf (10% MeOH in DCM) 0.75. HR-ESIMS (ESI): m / z 739.85, [C 42 H 45 Cl2N5O3+H] + Calculated: 738.2978.

[0105] Scheme 9: Preparation of ligand compounds of formula IIm

[0106]

[0107] Cinnamycin of formula 16. To a cooled (-78°C) solution of CH3CN (0.7 mL, 13.4 mmol) in dry THF (40 mL) under argon was added n-butyllithium (5.2 mL of a 2.5 M solution in hexane, 13 mmol). The resulting clear colorless solution was stirred at -78°C for 15 min, after which the appearance of a white suspension was observed. Then, a solution of 2,3,6,7-tetrahydro-9-trifluoromethyl-1 H,5H-quinolizino[9,1 -gh]cinnoline (1.023 g, 3.31 mmol) in dry THF (35 mL) was slowly added and the reaction mixture was stirred at -78°C under argon for 30 min. The resulting orange mixture was quenched at -78°C by the addition of a saturated aqueous NH4CI solution (35 mL), while a white solid precipitated. The mixture was allowed to warm to room temperature and extracted with AcOEt (3 x 50 mL). The combined organic phases were evaporated to dryness. To the resulting crude oil was added a 0.5 M aqueous HCI solution (200 mL) and the mixture was stirred vigorously for 3 h. The solution color changed from orange to brown. This solution was extracted with AcOEt (3 x 80 mL), dried over anhydrous MgS04, filtered and evaporated to dryness under reduced pressure. The crude mixture was purified by flash chromatography (silica gel, 0 to 15% AcOEt in hexanes). 760 mg of the title compound were obtained as a bright orange solid (yield: 70%). Orange solid TLC: Rf(25% AcOEt in hexanes) 0.38. HR-MS (ESI): m / z 332.1209, [C 18 H 15 F3N2O+H] + Calculated: 333.1215.

[0108] Coumarin aldehyde of formula 17. The nitrile coumarin of formula 16 (300 mg, 0.90 mmol) was dissolved in dry toluene (35 mL) under argon atmosphere and the mixture was stirred for 10 min (orange solution). Then, 1 M DIBALH solution in toluene (1.35 mL, 1.35 mmol) was added and the reaction mixture was stirred at room temperature under argon atmosphere for 30 min (color changed to chestnut). The crude product was cooled in an ice bath and acetone (4 mL) was added to decompose the excess reagent (color changed from chestnut to dark red). After the addition of saturated potassium sodium tartrate (25 mL), the crude product was extracted with AcOEt (3 x 25 mL) and the organic phase was washed with water (25 mL) and brine (25 mL), dried over anhydrous MgS04, filtered and evaporated to dryness under reduced pressure. The compound was purified by column chromatography on silica gel with hexane, first with DCM (0 to 100%) and then with MeOH (0 to 0.5%) increasing polarity. 150 mg of the title compound were obtained as a red solid (yield: 50%). TLC: Rf (5% MeOH in DCM) 0.74. HR-MS (ESI): m / z 336.1206, [C 18 H 16 F3NO2+H] + Calculated: 336.1211.

[0109] Ligand compound of formula III m. To a solution of coumarin aldehyde of formula 17 (50 mg, 0.15 mmol) in dry ethanol (7 mL) was added piperidine (70 mg, 0.83 mmol) and 2-(4'-methyl-[2,2'-bipyridin]-4-yl)acetonitrile (46 mg, 0.22 mmol). The reaction mixture (dark chestnut) was stirred at 80 °C overnight. After evaporation to dryness under reduced pressure, the compound was purified by column chromatography on silica gel with ethane, first with DCM (0 to 100%) and then with MeOH (0 to 2%) increasing polarity. 66 mg of a purple solid were obtained, identified as the ligand compound of formula III m (yield: 83%). TLC: Rf (5% MeOH in DCM) 0.4. HR-ESI MS (ESI): m / z 527.2077, [C 31 H 25 F3N4O+H] + Calculated: 527.2053.

[0110] Scheme 10: Preparation of ligand compounds of formula IIo

[0111]

[0112] Nitrile coumarin of formula 18. To a toluene solution (15 mL) of coumarin aldehyde (207 mg, 0.62 mmol) of formula 17 was added (triphenylphosphoranylidene)acetonitrile (850 mg, 2.82 mmol). The solution was degassed with argon, the vial was sealed, and the mixture was stirred at 60 ° C for 7 days. Once the starting aldehyde was completely consumed, deionized water (H2O) was added to the crude product, which was then extracted with AcOEt (3x30 mL). The combined organic phases were dried over anhydrous MgSO4, filtered, and evaporated to dryness under reduced pressure, and the crude product was purified by silica gel column chromatography with hexane, with AcOEt (0 to 12%) increasing the polarity. 184 mg of the title compound was obtained as a red solid (yield: 84%). TLC: Rf (50% AcOEt in hexane) 0.90. HR-MS(ESI): m / z 359.1366, [C 20 H 17 F3N2O+H] + Calculated value: 359.1357.

[0113] Formula 19 coumarin aldehyde. Under an argon atmosphere, the nitrile coumarin of formula 18 (103mg, 0.29mmol) was dissolved in anhydrous THF (30mL), and the mixture was cooled in an ice bath. Then, a toluene solution of 1M DIBALH (3.0mL, 2.95mmol) was added, and the reaction mixture was stirred at room temperature for 30min under an argon atmosphere. The crude product was cooled in an ice bath and acetone (10mL) was added to decompose the excess reagent. After adding saturated potassium sodium tartrate (25mL) and stirring for 30min, the crude product was extracted with DCM (3x50mL), the organic phase was washed with brine (50mL), dried over anhydrous MgSO4, filtered and evaporated to dryness under reduced pressure. The compound was purified by silica gel column chromatography with hexane, first with DCM (0 to 100%), then with MeOH (0 to 0.5%) to increase polarity. 78mg of the title compound was obtained as a yellow solid (yield: 69%). TLC: Rf (5% MeOH in DCM) 0.52. HR-MS (ESI): m / z 362.1362, [C 20 H 17 F3N2O+H] + Calculated value: 362.1359.

[0114] Ligand compound of Formula IIo. To a solution of coumarin aldehyde of Formula 19 (32 mg, 0.088 mmol) in absolute ethanol (10 mL) was added piperidine (45 mg, 0.531 mmol) and 2-(4'-methyl-[2,2'-bipyridinyl]-4-yl)acetonitrile (23 mg, 0.11 mmol). The reaction mixture was stirred at 80 °C overnight. After evaporation to dryness under reduced pressure, the compound was purified by column chromatography on silica gel with hexane first with DCM (0 to 100%) and then increasing polarity with MeOH (0 to 1%). 44 mg of purple solid was obtained, identified as ligand compound of Formula IIo (yield: 89%). TLC: Rf (5% MeOH in DCM) 0.59. LR-MS (ESI): m / z 553.25, [C 33 H 27 F3N4O+H] + Calculated: 553.2210.

[0115] Scheme 11 : Preparation of ligand compounds of formula IIn

[0116]

[0117] Cinnamonic coumarin of Formula 20. Under an argon atmosphere, to a cooled (-78 °C) solution of CH3CN (2.42 mL, 34.6 mmol) in dry THF (40 mL) was added n-butyllithium (13.8 mL, 2.5 M in hexanes, 34.6 mmol). The resulting clear colorless solution was stirred at -78 °C for 15 min, after which the appearance of a white suspension was observed. Then, a solution of 7-(diethylamino)-4-methyl-2H-chromen-2-one coumarin (2.008 g, 8.65 mmol) in dry THF (30 mL) was slowly added and the reaction mixture was stirred at -78 °C under argon for 30 min. The resulting white mixture was quenched still at -78 °C by the addition of saturated aqueous NH4C1 (35 mL), precipitating a white solid. The mixture was allowed to warm to room temperature and extracted with AcOEt (3 x 50 mL). The combined organic phases were evaporated to dryness. To the resulting orange crude oil was added 0.5 M aqueous HC1 (200 mL) and the mixture was stirred vigorously for 16 h. The solution turned brown and a yellow solid precipitated. 50 mL of DCM were added to dissolve the precipitate and the mixture was extracted with DCM (3 x 100 mL), dried over anhydrous MgS04, filtered and evaporated to dryness under reduced pressure, affording a brown solid. The crude mixture was purified by column chromatography on silica gel (0 to 22% AcOEt in hexanes). 2.01 g of the title compound was obtained as a bright orange solid (yield: 92%). Orange solid TLC: Rf (50% AcOEt in hexanes) 0.61. LR-ESI MS (ESI): m / z 255.2, [C16 H 18 N2O+H] + Calculated: 255.1492.

[0118] Coumarin aldehyde of formula 21. The nitrile coumarin of formula 20 (500 mg, 1.97 mmol) was dissolved in dry toluene (25 mL) under argon atmosphere and the mixture was stirred for 10 min (orange solution). Then, 1 M DIBALH in toluene (3.0 mL, 2.95 mmol) was added and the reaction mixture was stirred at room temperature under argon atmosphere for 30 min. The crude product was cooled in an ice bath and acetone (10 mL) was added to decompose the excess reagent. After the addition of saturated potassium sodium tartrate (40 mL), the crude product was extracted with DCM (3 x 50 mL) and the organic phase was washed with brine (50 mL), dried over anhydrous MgS04, filtered and evaporated to dryness under reduced pressure. The compound was purified by silica gel column chromatography with hexane, first with DCM (0 to 100%) and then MeOH (0 to 0.5%) increasing polarity. 78 mg of the title compound was obtained as a yellow solid (yield: 15%). TLC: Rf (5% MeOH in DCM) 0.52. LR-ESIMS (ESI): [C 16 H 19 NO2+H] + m / z 258.2, calculated: 258.1489.

[0119] Ligand compound of formula III n. To a solution of coumarin aldehyde of formula 21 (47 mg, 0.18 mmol) in dry ethanol (10 mL) was added piperidine (84 mg, 1.0 mmol) and 2-(4'-methyl-[2,2'-bipyridin]-4-yl)acetonitrile (44 mg, 0.20 mmol). The reaction mixture was stirred at 80 °C overnight. After evaporation to dryness under reduced pressure, the compound was purified by silica gel column chromatography with hexane, first with DCM (0 to 100%) and then MeOH (0 to 2%) increasing polarity. 79 mg of a purple solid was obtained, identified as ligand compound of formula III n (yield: 96%). TLC: Rf (5% MeOH in DCM) 0.38. HR-ESIMS (ESI): m / z 449.2336, [C 29 H 28 N4O+H] + Calculated: 449.2336.

[0120] Scheme 12: Preparation of ligand compounds of formula IIp

[0121]

[0122] Ligand compound of formula IIp. Sodium hydride (66 mg of a 60% dispersion in mineral oil, 1.64 mmol) and 2-(4'-methyl-[2,2'-bipyridinyl]-4-yl)acetonitrile (106 mg, 0.506 mmol) were first dissolved in anhydrous THF (15 mL) and stirred at 40 °C under argon atmosphere for 15 min. Then, a solution of 4-methyl-2H-chromen-2-one (85 mg, 0.482 mmol) in anhydrous THF (15 mL) was added to the previous flask and the resulting reaction mixture was stirred in the dark at 40 °C under argon atmosphere for further 4 h. After 4 h, AgN03(163 mg, 0.964 mmol) was added and stirred at 35 °C for further 2 h. After evaporation of the solvent under reduced pressure, the compound was purified by column chromatography on silica gel with hexane, first with DCM (0 to 100%) and then increasing the polarity with MeOH (0 to 1%). 92 mg of an orange solid were obtained (yield: 54%) identified as the ligand compound of formula IIp. TLC: Rf (2.5% MeOH in DCM) 0.25. HR-MS (ESI): m / z 352.1451, [C 23 H 17 N3O+H] + Calculated for C20H20N3O+H 352.1444.

[0123] Scheme 13: Preparation of ligand compounds of formula IIq and formula IIr and complex compounds of formula Iq

[0124]

[0125] Ligand compound of formula IIq. 4-Dimethylaminopyridine (DMAP) (20 mg, 0.164 mmol) and l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI) (38 mg, 0.198 mmol) were added sequentially to an ice-cooled DCM solution (10 mL) of N-Boc-5-amino levulinic acid (38 mg, 0.164 mmol). After stirring the mixture at 0 °C for 15 min, a DCM solution (5 mL) of the compound of formula lie (61 mg, 0.135 mmol) was added dropwise at the same temperature. Then, the ice bath was removed and the resulting mixture was further stirred at room temperature for 16 h. After completion of the reaction (TLC, HPLC), the volatiles were concentrated under reduced pressure and the resulting crude product was purified by flash column chromatography (silica gel, 0 to 5% MeOH in DCM) to obtain 31 mg of an orange / red solid (yield: 34%) identified as the ligand compound of formula IIq. LR-MS (ESI): m / z [M+H] + , C 38 H 43 Calculated for C26H30N5O6 666.33, found 666.57.

[0126] Ligand compound of Formula IIr. To an ice-cooled solution of ligand compound of Formula IIq (15 mg, 0.022 mmol) in anhydrous THF (2 mL) was added 4 N HC1 in dioxane (675 μL, 2.70 mmol) and the resulting mixture was warmed to room temperature and further stirred for 5 h. After completion of the reaction (HPLC-MS), the reaction mixture was neutralized with 10% (w / v) aqueous NaHC03solution (10 mL), diluted with water (20 mL), extracted with DCM (3 x 20 mL) and the combined organic extracts were dried over anhydrous MgS04, filtered and concentrated under reduced pressure to obtain crude ligand compound of Formula IIr (12 mg, yield: 94%) as an orange / red solid which was used in the next step without any further purification. LR-MS (ESI) m / z [M+H] + , C 33 H 35 Calcd for C26H28N5O4, 566.28, Found, 566.45.

[0127] Complex compound of formula Iq. Method A: Under an argon atmosphere, the ligand compound of formula IIq (16 mg, 0.024 mmol) and [Ru(bpy)2Cl2] (10 mg, 0.021 mmol) were dissolved in 4 mL of a 3:1 (v / v) EtOH / H2O solution. The reaction mixture was stirred at 80°C overnight and analyzed by HPLC-MS to confirm product formation. The reaction mixture was evaporated to dryness, and the residue was taken up in anhydrous THF (2 mL) and treated with 4N HCl in dioxane (600 μL, 2.37 mmol) at 0°C, allowed to warm to room temperature, and further stirred for 5 h. After completion of the reaction (HPLC-MS), the reaction mixture was neutralized with a 10% (w / v) aqueous solution of NaHCO3 (5 mL), diluted with water (20 mL), extracted with DCM (3 x 20 mL), and the combined organic extracts were dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to obtain the crude product. Method B: Under an argon atmosphere, the ligand compound of Formula IIr (11 mg, 0.019 mmol) and [Ru(bpy)2Cl2] (9 mg, 0.019 mmol) were dissolved in 4 mL of a 3:1 (v / v) EtOH / H2O solution. The reaction mixture was stirred at 80°C overnight and analyzed by HPLC-MS to confirm the formation of the product. The reaction mixture was evaporated to dryness to obtain a crude product. In both methods, the desired complex compound of Formula Iq was obtained as a maroon solid by preparative HPLC (C18 column; mobile phase, A: 0.1% HCOOH in water, B: 0.1% HCOOH in ACN; gradient: 10% to 80% B in 30 min). Analytical HPLC (C18 column; mobile phase, A: 0.1% HCOOH in water, B: 0.1% HCOOH in ACN; gradient: 5% to 100% B in 5 minutes): Rt = 2.14 min. LR-MS (ESI): m / z [M] 2+ , C 53 H 51 N9O4Ru 2+ The calculated value is 489.65 and the actual value is 489.60.

[0128] Scheme 14: Preparation of ligand compounds of formula Va and formula Vb

[0129]

[0130] Ligand compound of formula Va. To a solution of coumarin aldehyde (92 mg, 0.32 mmol) of formula 22 in anhydrous ethanol (10 mL) were added piperidine (166 mg, 2.0 mmol) and 2-(4'-methyl-[2,2'-bipyridine]-4-yl)acetonitrile (68 mg, 0.32 mmol). The reaction mixture was stirred at 80 ° C overnight. After evaporation to dryness under reduced pressure, the compound was purified by silica gel column chromatography with hexane, first with DCM (0 to 100%) and then with MeOH (0 to 1%) to increase polarity. 73 mg of a purple solid was obtained, which was identified as a ligand compound of formula Va (yield: 47%). TLC: Rf (5% MeOH in DCM) 0.59. HR-MS (ESI): m / z 475.2495, [C 31 H 30 N4O+H] + Calculated value: 475.2492.

[0131] Ligand compound of formula Vb. To a solution of coumarin aldehyde (40 mg, 0.155 mmol) of formula 23 in anhydrous ethanol (8 mL) were added piperidine (79 mg, 0.932 mmol) and 2-(4'-methyl-[2,2'-bipyridine]-4-yl)acetonitrile (33 mg, 0.155 mmol). The reaction mixture was stirred at 80 ° C overnight. After evaporation to dryness under reduced pressure, the compound was purified by silica gel column chromatography with hexane, first with DCM (0 to 100%) and then with MeOH (0 to 2%) to increase the polarity. 32 mg of a brown solid was obtained, which was identified as a ligand compound of formula Vb (yield: 46%). TLC: Rf (5% MeOH in DCM) 0.30. LR-MS (ESI): m / z 449.07, [C 27 H 20 N4O3+H] + Calculated value: 449.1608.

[0132] General procedure for the synthesis of complex compounds of formula In, formula Io, formula Ip, formula IVa and formula IVb.

[0133] The corresponding ligand compound of Formula IIn, Formula IIo, Formula IIp, Formula Va or Formula Vb and [Ru(bpy)2Cl2] (1.1 equivalents) were dissolved in a mixture of 1:1 (v / v) EtOH / H2O, and the reaction mixture was stirred at 80°C to 90°C under an argon atmosphere for 24 to 48 hours. The reaction was followed by HPLC-MS. Once complete conversion was achieved, the solvent was evaporated to dryness, and the product was purified by silica gel column chromatography with DCM and increasing the eluent polarity with MeOH. The compound was obtained as a racemic mixture of chloride salts and characterized by HR-ESIMS.

[0134] In: HR-ESIMS (ESI): m / z 431.1336, [C 49 H 44 N8ORu] 2+ Calcd: 431.1336. Analytical HPLC (5% to 100% B in 5 min, formic acid additive): Rt = 2.46 min.

[0135] Io: HR-ESIMS (ESI): m / z 483.1282, [C 53 H 43 F3N8ORu] 2+ Calcd: 483.1272. Analytical HPLC (5% to 100% B in 5 min, formic acid additive): Rt = 2.88 min.

[0136] Ip: HR-ESIMS (ESI): m / z 382.5898, [C 43 H 33 N7ORu] 2+ Calcd: 382.5890. Analytical HPLC (5% to 100% B in 5 min, formic acid additive): Rt = 2.25 min.

[0137] IVa: HR-ESIMS (ESI): m / z 444.1420, [C 51 H 44 N8ORu] 2+ Calcd: 444.1414. Analytical HPLC (5% to 100% B in 5 min, formic acid additive): Rt = 2.68 min.

[0138] IVb: HR-ESIMS (ESI): m / z 431.0972, [C 47 H 36 N8O3Ru] 2+ Calcd: 431.0972. Analytical HPLC (5% to 100% B in 5 min, formic acid additive): Rt = 2.39 min.

[0139] Phototoxicity evaluation of complex compounds of formula I or formula IV

[0140] Several complex compounds of formula I or IV were evaluated for phototoxicity on several (human and mouse) cancer cell lines and on human normal cells under normoxic (21% 02) and hypoxic (2% 02) conditions. The results obtained are reported in Tables 1 to 7, appended hereto. In all cases, the phototoxicity index (PI) was calculated as follows:

[0141] PI = IC 50 (dark - unirradiated cells) / IC50 (irradiated cells).

[0142] Cell lines and cell culture conditions. The following cancer cell lines were used: CT-26 (mouse, colon carcinoma); HT-29 (human, colorectal adenocarcinoma); A-549 (human, lung adenocarcinoma); A-2780 (human, ovarian carcinoma). The following human normal cells were used: RPE-1 (human retinal pigment epithelial cell-1), HEK-293 (human embryonic kidney) and MRC-5 (human fetal lung fibroblast). Cell lines were cultured in their specific medium and maintained at 37°C in a humidified atmosphere of 5% CO2. CT-26 cell line was cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin antibiotics (Gibco). HT-29 cell line was cultured in McCoy medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin antibiotics (Gibco). A-549 cell line was cultured in DMEM / F-12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin antibiotics (Gibco). A-2780 cell line was cultured in RPMI medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics (Gibco). RPE-1 cell line was cultured in DMEM / F-12 medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics (Gibco). HEK-293 cell line was cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics (Gibco). MRC-5 cell line was cultured in DMEM / F-12 medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics (Gibco).

[0143] Phototoxicity evaluation of complex compounds of Formula I or Formula IV in 2D monolayer cells under normoxia (21% O2). The phototoxicity of the test compounds under normoxia (21% O2) was assessed by using a fluorescent cell viability assay with Resazurin (Acros Organics). Briefly, cells were seeded in 96-well plates in triplicate at a density of 4x10 3cells / well in a final volume of 100 μL / well. After 24 h, cells were treated with increasing concentrations of metal complexes and control compounds. Test compounds were serially diluted from stock solutions in DMSO at 10 mM or 25 mM with cell culture medium, and the serial dilutions were added to a final concentration range of 0.001 μM to 250 μM in a final volume of 100 μL per well. Cells were incubated with the samples in the dark for 4 h, after which the medium was refreshed. To investigate the phototoxic effects of the test compounds, cells were exposed to 540 nm (spectral half-width: 32 nm, 40 min, 3.75 mW cm -2 , 9.0 J cm -2 ), 645 nm (spectral half-width: 32 nm, 60 min, 2.50 mW cm -2 , 9.0 J cm -2 ), 670 nm (spectral half-width: 32 nm, 60 min, 3.75 mW cm -2 , 13.5 J cm -2 ), 740 nm (spectral half-width: 32 nm, 60 min, 3.50 mW cm -2 , 12.6 J cm -2 ), and 770 nm (spectral half-width: 32 nm, 60 min, 6.75 mW cm -2 , 24.3 J cm -2 ) light using an Atlas Photonics LUMOS BIO irradiator. To investigate the cytotoxic effects of the samples in the dark, cells were not irradiated. A 44 h recovery period for untreated cells was then allowed, while the temperature was maintained at 37 °C throughout the experiment. After 44 h of incubation, the medium was removed by aspiration, and 100 μL of complete medium containing resazurin (final concentration 0.2 mg / mL) was added. After 4 h of incubation at 37 °C, the fluorescence signal of the resorufin product (λ ex = 540 nm, λ em = 590 nm) was measured using an Infinite 200 PRO microplate reader from TECAN. IC 50 values were then calculated based on the inhibition rate curves. All experiments were performed in triplicate in three independent studies, with three biological replicates per concentration level (n = 3).

[0144] Phototoxicity evaluation of complex compounds of formula I or formula IV in 2D monolayer cells under hypoxia (2% 02) The phototoxicity of test compounds under hypoxia (2% O2) was assessed by using a fluorescent cell viability assay with resazurin (Acros Organics). Cells were seeded in 96-well plates in triplicate at a density of 2.5 x 10 3Cells were plated at 5000 cells / well in a final volume of 100 μL / well in a 96-well plate and placed in a 2% O2incubator. Flasks containing the corresponding cell culture medium were also prepared in a 2% O2incubator. After 48 h of incubation under hypoxia, cells were treated with increasing concentrations of metal complexes and control compounds. Test compounds were serially diluted from a stock solution of 10 mM or 25 mM in DMSO with cell culture medium, and serial dilutions were added to a final concentration of 0.01 μM to 250 μM in a final volume of 100 μL per well. Cells were incubated with the samples in the dark for 4 h, after which the medium was refreshed. To investigate the phototoxic effects of the samples under hypoxic conditions, a hypoxic chamber glovebox from Plas-Labs (856 series) was used. Cells were exposed to light (see previous paragraph) using an Atlas Photonics LUMOS BIO illuminator placed inside the hypoxic chamber. To assess the cytotoxicity of the samples in the dark, cells were not illuminated. Cells were then allowed to recover for a 44 h treatment-free period inside the 2% O2incubator, and the temperature was maintained at 37 °C throughout the experiment. After 44 h of incubation, the medium was removed by aspiration, and 100 μL of complete medium containing resazurin (final concentration 0.2 mg / mL) was added. After 4 h of incubation at 37 °C in a 21% O2incubator, the fluorescence signal of the Resorufin product was measured using an Infinite 200 PRO microplate reader from TECAN (λ ex = 540 nm, λ ex = 590 nm). IC 50 values were calculated as described in the previous paragraph.

[0145] Several complex compounds of formula I or formula IV were irradiated after with light of different wavelengths (l) under normoxic and hypoxic conditions. phototoxicity of the substances on cancer cells. The IC50values obtained under different conditions 50 and the phototoxicity index (PI) are shown in Tables 1 to 8, appended Figure 1 Standard error of the mean with n = 3, “nd” means “not determined”.

[0146] Table 1. Phototoxicity of complex compounds of Formula la and Formula Ic on CT-26 cancer cells under normoxic (21% O2) and hypoxic (2% O2) conditions.

[0147]

[0148] Table 2. Phototoxicity of complex compounds of Formula Ig and Formula Ih on CT-26 cancer cells under normoxic (21% O2) and hypoxic (2% O2) conditions.

[0149]

[0150] Table 3. Phototoxicity of complex compounds of Formula Id on CT-26 cancer cells under normoxic (21% O2) conditions

[0151]

[0152] Table 4. Phototoxicity of complex compounds of formula If on CT-26 cancer cells under normoxic (21% 02) and hypoxic (2% 02) conditions

[0153]

[0154] Table 5. Phototoxicity of complex compounds of formula Im on CT-26 cancer cells under normoxic (21% 02) conditions.

[0155]

[0156] Table 6. Phototoxicity of complex compounds of formula Ic on human cancer cells.

[0157]

[0158] Table 7. Phototoxicity of complex compounds of formula Ic on human normal cells.

[0159]

[0160] Table 8. Phototoxicity of complex compounds of the indicated formula on CT-26 cancer cells under normoxic (21% 02) and hypoxic (2% 02).

[0161]

[0162]

[0163] In addition, it was noted that complex compound Ic was able to inhibit the growth of 3D tumor spheroids (Figure 1 1 Figure 2 ), targeting mitochondria as a specific subcellular target (Figure 1 1 Figure 3 and Figure 4 ). Ic in vivo studies showed good biodistribution profile and excellent safety profile. As shown in Figure 1 1 Figure 5 , the compound reached maximum concentration in mouse plasma 30 min after administration (5 mg / kg) and was completely eliminated 24 h later. In the toxicology maximum tolerated dose (MTD) study, all parameters of animals treated (male and female albino Swiss CD1 mice) at two study concentrations (10 mg / kg and 30 mg / kg) were comparable to the control group (Figure 1 1 Figure 6 and Figure 7 ): body weight, body weight gain, food consumption, organ weights, gross necropsy, hematology and biochemistry analyses. No deaths and clinical signs were recorded in the observation period (5 days) in vehicle and Ic treated male and female experimental groups. In addition, complex compound Ic was able to reduce tumor growth in a murine subcutaneous colorectal cancer model (Figure 1 1 ​ ).

Claims

1. A compound of formula I, including any stereoisomer or E / Z isomer thereof, in: M is selected from the following metal cations: Fe 2+ 、Ru 2+ 、Os 2+ 、Co 3+ , Rh 2+ , Rh 3+ 、Ir 3+ 、Ni 2+ 、Pd 2+ , Pt 2+ and Pt 4+ ; y=1, 2 or 3; and z=0, 1 or 2; provided that: when M is Ni 2+ 、Pd 2+ or Pt 2+ When y+z=2, and when M is Fe 2+ 、Ru 2+ 、Os 2+ 、Co 3+ , Rh 2+ , Rh 3+ 、Ir 3+ or Pt 4+ When y+z=3; m = 0, 1, 2, 3, or 4; The groups T and T' are each independently selected at each occurrence from the group consisting of: H and (C3-C6)-alkyl; The groups P3, P5, P6, Q3, Q4, Q5, Q6, R3, R5, R6 and R8 are each independently selected from the group consisting of H, (C1-C3)-alkyl, (C3-C6)-cycloalkyl, CH=CHR, CF3, CHF2, CH2F, CF2CF3, F, Cl, Br, I, OR, C(=O)OR, O(C=O)R, C(=O)NR, NRC(=O)R', NRR', phenyl, monosubstituted phenyl, disubstituted phenyl and trisubstituted phenyl wherein the substituents on the phenyl ring are attached to any possible substitution position and are independently selected from the group consisting of: F, Cl, Br, I, NO2, (C1-C3)-alkyl, OH, O[(C1-C3)-alkyl], NH2, NH[(C1-C3)-alkyl] and N[(C1-C3)-alkyl]2; wherein when m≥1 and the group R3 is CH2, a 6-membered ring can be formed between the CH2 of R3 and the T' group closest to the coumarin main chain, provided that T'=CH2, thereby providing a compound of formula IV; The group R7 is H, NO2, OR, NRR', N(CH2COOH)2, N(CH2CH2S3H)2, N(CH2CONHCH2CH2NMe2)2, or: an unsubstituted, mono(R)-substituted, di(R,R')-substituted or tri(R,R',R")-substituted group of each of the heterocyclic groups 1-aziridinyl, 1-azetidinyl, 1-pyrrolidinyl, 1-piperidinyl, 4-morpholinyl or 1-piperazinyl, alternatively, R6, R7 and R8 together form a bicyclic ring system of the attached formula, wherein V1, V2, V3, V4, V5, V6, V7, V8, V9, V 10 、V 11 and V 12 are each a group independently selected from H and (C1-C3)-alkyl; the radical R4 is H, (C1-C3)-alkyl, (C3-C6)-cycloalkyl, CF3, CHF2, CH2F, CF2CF3, F, Cl, Br, I, OR, C(═O)OR, O(C═O)R, C(═O)NR, NRC(═O)R′, NRR′, phenyl, mono-(R)-substituted phenyl, di-(R,R′)-substituted phenyl, tri-(R,R′,R″)-substituted phenyl or the radical CE1E2E3; wherein: E1 and E2 are independently selected from the group consisting of H, F, Cl, Br, I, OR, NRR', NO2, (C1-C3)-alkyl, phenyl, and mono(R)-substituted phenyl; and E3 is OH, O[(C1-C3)-alkyl], or a group of the accompanying formula, wherein p=0, 1, 2, 3, or 4; and D1 and D2 are independently selected from the group consisting of H, O[CH2], q -X, S[CH2] q -X and N[[CH2] q -X]2, q=1 or 2, and X=F, Cl, Br or I. or a group of the formula attached, wherein p=1, 2, 3 or 4; and D3 is a group independently selected from the following: H, H(C=O), [(C1-C3)-alkyl](C=O), (OH)2P(=O)O-CH2-O(C=O), or (PhO)(OH)P(=O) Lig is a bidentate ligand independently selected from the group consisting of ligands of the following formulae: Lig 1, Lig 2, Lig 3, Lig 4, Lig 5, Lig 6, Lig 7, Lig 8, Lig 9, Lig 10, Lig 11, Lig 12, and Lig 13, at each occurrence; wherein G is a nitrogen or carbon atom having a negative charge resulting from the loss of H from CH in the formation of a carbon-metal bond. + is formed in situ; and wherein R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 、R 18 、R 19 、R 20 and R 21 is, at each occurrence, a group independently selected from the group consisting of H, (C1-C3)-alkyl, phenyl, and mono-, di-, and tri-substituted phenyl, the substituents attached to the phenyl ring being independently selected from the group consisting of F, Cl, Br, I, OR, and NRR'; and A is an anion derived from a pharmaceutically acceptable acid; n is an integer or fraction such that the compound of Formula I is electrically neutral; wherein R, R' and R" at each occurrence are each independently selected from the group consisting of H, F, Cl, Br, I, NO2, (C1-C3)-alkyl, OH, O[(C1-C3)-alkyl], NH2, NH[(C1-C3)-alkyl] and N[(C1-C3)-alkyl]2.

2. The compound according to claim 1, wherein The anion A is selected from: Cl - Br - PF6 - PF4 - 、BF4 - 、ClO4 - CF3SO3 - 、SO4 2- CF3COO - , acetate, formate and oxalate.

3. The compound according to claim 2, wherein The anion A is Cl - .

4. The compound according to any one of claims 1 to 3, wherein y=1, z=2, and the metal cation M is Fe 2+ 、Ru 2+ 、Os 2+ 、Co 3+ , Rh 2+ , Rh 3+ 、Ir 3+ or Pt 4+ .

5. The compound according to any one of claims 1 to 4, wherein The bidentate ligand Lig is Lig 1 or Lig 2; and the group R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 、R 18 、R 19 、R 20 and R 21 Each of the groups is independently selected at each occurrence from the group consisting of: H, methyl, and phenyl.

6. The compound according to any one of claims 1 to 5, wherein G is N.

7. The compound according to claim 6, wherein The bidentate ligand Lig is 2,2′-bipyridine (bpy) or 4,7-diphenyl-1,10-phenanthroline (BPhen).

8. The compound according to any one of claims 1 to 7, wherein Each of the groups P3, P5, P6, Q3, Q4, Q5, Q6, R3, R5, R6 and R8 is independently selected at each occurrence from H and (C1-C3)-alkyl; and the group R7 is H, N[(C1-C3)-alkyl)]2 or NO2; alternatively, the groups R6, R7 and R8 together form a bicyclic ring system of the formula as shown in claim 1, wherein V1, V2, V3, V4, V5, V6, V7, V8, V9, V 10 、V 11 and V 12 Each of them is H.

9. The compound according to any one of claims 1 to 8, wherein The group R4 is H, (C1-C3)-alkyl, CF3, CH(CH3)OH, CH(CH3)O(C=O)-[CH2]3-Ph or a group of the following formula Or a group of the formula CH(CH3)O(C=O)-[CH2]2-(C=O)-CH2-NH2.

10. The compound according to any one of claims 1 to 9, wherein m=0, 1 or 2.

11. The compound according to any one of claims 1 to 10, wherein The metal cation M is Ru 2+ .

12. The compound according to claim 11, wherein Formula I is selected from the group consisting of the appended Formulas Ia, Ib, Ic, Id, Ie, If, Ig, Ih, Im, In, Io, Ip, Iq, IVa, and IVb:

13. A compound of formula II, including any stereoisomer or E / Z isomer thereof, wherein m, P3, P5, P6, Q3, Q4, Q5, Q6, R3, R4, R5, R6, R7, R8, T and T' are as defined in any one of claims 1, 8, 9 and 10.

14. The compound according to claim 13, wherein formula II is selected from the group consisting of the following formulae: IIa, IIb, IIc, IId, IIe, IIf, IIm, IIo, IIn, IIp, IIq, IIr, Va, Vb:

15. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as defined in any one of claims 1 to 12 and a suitable amount of an excipient, carrier or solvent.

16. A compound of formula I as defined in any one of claims 1 to 12 for use in human therapy.

17. Use of a compound of formula I as defined in any one of claims 1 to 12 as a photosensitizer in photodynamic therapy of a human condition.

18. The compound for use according to claim 17, wherein The human condition is cancer, skin disease, fungal infection or microbial infection.

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