Chlorophyll derivatives compounds and uses thereof

Abstract

The present disclosure relates to a compound of general forma (I) or (II) or a pharmaceutically acceptable salt thereof Furthermore, the present disclosure relates to the use of said compound in medicine, in particular in photodynamic therapy (PDT) of cancer, as well as in diagnostic. (Formula I) (Formula II)

Description

D E S C R I P T I O NCHLOROPHYLL DERIVATIVES COMPOUN DS AN D USES THEREOFTECHN ICAL FI ELD

[0001] The present disclosure relates to a series of novel chlorophyll derivatives bearing different uracilalditols moieties, their synthesis and their use in medicine, in particular in photodynamic therapy (PDT) of cancer, as well as diagnostic agents.BACKGROUND

[0002] Photosensitizers (PSs) are dyes that can absorb visible light, and in the presence of dioxygen (3O2) can generate reactive oxygen species (ROS), that can trigger cell death [1], These features have been used to treat cancerous conditions through a process denominated Photodynamic Therapy (PDT), which is a minimally invasive therapeutic modality used in the treatment of cancer and bacterial infections [2-4], The PSs already approved and utilized in clinical PDT, for the cancer treatment include Photofrin® and Photosan® - which are employed in the treatment of bladder and oesophageal cancer; Temoporfin / Foscan® are used for the treatment of head and neck cancer, while Tookad® is considered for low-risk prostate cancer. However approved, these PSs present some limitations; for instance, Photofrin® and Photosan® (first-generation PSs) are constituted by a complex mixture of monomers, dimers, and oligomers of porphyrins which make the formulations vary from batch to batch; also, these formulations present low absorption in the red region of the electromagnetic spectrum (where the light penetrates better in tissues) limiting their application to small tumors. Foscan®, a chlorin derivative in its turn, presents a better absorption in the red region, however, this dye persists in the organism for several weeks and months, causing high skin photosensitivity. In this sense, there is a need of new PS molecules with better photodynamic properties, stability and efficiency; these PSs should be obtained by high chemical purity, good absorptions in the therapeutic window (600-800 nm), high singlet oxygen ^Ch) quantum yield and minimal side-effects.

[0003] These facts are disclosed to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0004] The present disclosure relates to novel chlorophyll derivatives bearing uracil-alditols moieties, which may be used in the treatment of cancer, preferably breast cancer and pancreatic cancer, as well as imaging diagnostic agents.

[0005] The present disclosure relates to a compound family based on chlorophyll derivatives functionalized with different uracil-alditols moieties, which may be used in photodynamic therapy (PDT), in particular of cancer, and as diagnostics agents (PDD).

[0006] Surprisingly, the compounds of the present disclosure showed improved efficacy with reduced toxicity effects. In particular, the compounds of the present disclosure showed high phototoxicity in breast and pancreatic cancer cell lines and no dark toxicity in the treatment dosages. The compounds also showed strong fluorescence in red region of electromagnetic spectrum, allowing their use in diagnostic imaging.

[0007] The compounds of the present disclosure showed to be able to reduce tumoral cell number, and also to destroy cancer-associated fibroblasts (CAFs), helping to reduce the stroma barrier around the tumor cells. These results supports that the compounds of the present disclosure, are able to kill cancer cells and at the same time improve the accessibility of the therapies to the tumor.

[0008] It was also found that PDT with the compounds of the present disclosure effectively reduces the population of cancer stem cells (CSCs) / tumor initiating cells. This result supports that PDT with the compounds can be used to reduce the burden of tumor initiating cells remaining after therapy.

[0009] Photodynamic therapy (PDT) with the compounds of the present disclosure has been shown to act synergistically with chemotherapy. It was surprisingly found that when gemcitabine was administered following PDT with a compound of the present disclosure, cell viability decreased to 60% and 70% at the respective gemcitabine concentrations, demonstrating a substantially enhanced cytotoxic response in comparison with the same dose of gemcitabine alone. This clearly indicates that PDT with the compounds of the present disclosure provides a synergistic therapeutic benefit. Such synergy enables the use of lower chemotherapeutic doses while achieving superior anticancer efficacy, constituting a therapeutic effect that is advantageous over conventional therapies.

[0010] An aspect of the present disclosure relates to a compound of general formula (I) or a pharmaceutically acceptable salt thereof:(Formula I) wherein:M, R, Ri and R2 are independently selected from each other;M is selected from the list consisting of: Zn, 2H, Pd, Ru, Ni, Cu, Pt, Ir, In, Ga, Gd, Mn, Fe, Al, Si, Eu;Ri is a substituted or non-substituted alkyl C1-C10;R2 is a substituted or non-substituted alkyl C1-C10;R is selected from the list consisting of:

[0011] Another aspect of the present disclosure relates to a compound of general formula (I) or (II) or a pharmaceutically acceptable salt thereof:for use in medicine, wherein:M, R, Ri and R2 are independently selected from each other;M is selected from the list consisting of: Zn, 2H, Pd, Ru, N i, Cu, Pt, Ir, In, Ga, Gd, Mn, Fe, Al, Si, Eu;Ri is a substituted or non-substituted alkyl C1-C10;R2 is a substituted or non-substituted alkyl C1-C10;R is selected from the list consisting of:

[0012] In an embodiment for better results, M is selected from Zn, Pd, Ru, Ir, Mn. Preferably, M is Zn(ll) or 2H.

[0013] In an embodiment for better results, Ri is a non-substituted alkyl C1-C10. Preferably, Ri is a nonsubstituted alkyl Ci-C5; more preferably Ri is a non-substituted alkyl C1-C3; even more preferably Ri is CH3.

[0014] In an embodiment for better results, R2 is a non-substituted alkyl C1-C10. Preferably R2 is a nonsubstituted alkyl Ci-C5; more preferably R2 is a non-substituted alkyl C1-C3; even more preferably R2 is CH3.

[0015] In an embodiment for better results, R is selected from the list consisting of:

[0016] In an embodiment for better results, the compound is:

[0017] In an embodiment for better results, the compound is:

[0018] In an embodiment for better results, the compound is:

[0019] In an embodiment for better results, the compound is:

[0020] In an embodiment, the compounds of the present disclosure may be used in photodynamic therapy.

[0021] In an embodiment, the compounds of the present disclosure may be used in the prevention, treatment or diagnostic of cancer. Preferably, said cancer is selected from a list consisting of: skin cancer, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, lung cancer, non-small cell lung cancer, esophageal cancer, bladder cancer, head and neck cancers, oral cavity cancer, laryngeal cancer, nasopharyngeal cancer, barrett's esophagus, cholangiocarcinoma, bile duct cancer, prostate cancer, Paget's diseases of breast and vulva, pancreatic cancer, breast cancer, colon cancer, liver cancer, osteosarcoma, glioblastoma, ovarian cancer, testicular cancer, gastric cancer

[0022] More preferably, the compounds of the present disclosure may be used in the prevention, treatment or diagnostic of breast cancer or pancreatic cancer.

[0023] In an embodiment for better results, the compounds of the present disclosure may be used in the prevention, treatment or diagnostic of hyperproliferation or neoplasia.

[0024] In an embodiment, the compounds of the present disclosure may be used in the prevention, treatment or diagnostic of a disease characterized by benign or malignant cellular hyperproliferation or by areas of neovascularisation or a cancer.

[0025] In an embodiment, the compounds of the present disclosure may be used in the prevention, treatment or diagnostic of hyperproliferative tissue, in particular hyperproliferative tissue associated to cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, germ-cell tumors, blastoma, and central nervous system tumors.

[0026] In an embodiment, the compounds of the present disclosure may be used in the prevention, treatment or diagnostic of precancerous conditions and / or macular degeneration.

[0027] In an embodiment, the compounds of the present disclosure may be used in the prevention or treatment of cancer in which cancer-associated fibroblasts (CAFs) are involved.

[0028] In an embodiment, the compounds of the present disclosure may be used in combination with at least an agent selected from a list consisting of: chemotherapy agent; radiotherapy agent; thermal therapy agent; hormonal therapy agent; immunotherapy agent; targeted therapy agent; or mixtures thereof; preferably chemotherapy agent.

[0029] In an embodiment, the chemotherapy agent is selected from the list consisting of: doxorubicin, cisplatin, paclitaxel, 5-fluorouracil, mitomycin c, carboplatin, docetaxel, irinotecan, cyclophosphamide, methotrexate, vincristine, epirubicin, bleomycin, oxaliplatin, gemcitabine, or mixtures thereof; preferably the chemotherapy agent is gemcitabine.

[0030] In an embodiment, the immunotherapy agent is selected from the group consisting of: durvalumab, tremelimumab, or mixtures thereof.

[0031] In an embodiment, the targeted therapy agent is selected from the group consisting of: imatinib, alectinib, ibrutinib, palbociclib, bevacizumab, or mixtures thereof.

[0032] In an embodiment, the hormonal therapy agent is selected from the list consisting of: anastrazole, letrozoloe, exemestane, fulvestrant, elacestrant, tamoxifen, raloxifene, toremifene, leuprolide, goserelin, triptorelin, histrelin, enzalutamide, apalutamide, darolutamide, bicalutamide, flutamide, nilutamide, or mixtures thereof.

[0033] In an embodiment, the compound is administrated by oral, parenteral, intravenous, intramuscular, intranasal, intratumoral, sublingual or intratracheal route.

[0034] In an embodiment, the compound is administrated 1 to 100 h before photodynamic therapy procedure; preferably 3 to 60 h before photodynamic therapy; more preferably 10-50 h before photodynamic therapy.

[0035] Another aspect of the present disclosure relates to a substance delivery carrier comprising a compound of the present disclosure.

[0036] Another aspect of the present disclosure relates to a composition for use in treating cancer in which cancer- associated fibroblasts are involved, wherein said composition comprises a substance delivery carrier that promotes specific substance delivery to a cancer- associated fibroblast; wherein the substance delivery carrier is a compound of the present disclosure.

[0037] Another aspect of the present disclosure relates to a pharmaceutical composition comprising at least one of the compounds of the present disclosure.

[0038] In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0039] In an embodiment, the pharmaceutical composition comprises a combination of at least a compound herein described and at least a chemotherapy agent, an immunotherapy agent, or a targeted therapy agent. Preferably, the pharmaceutical composition comprises a combination of at least a compound herein described and a chemotherapy agent; more preferably the chemotherapy agent is gemcitabine.

[0040] In an embodiment, the pharmaceutical composition further comprises a surface penetration enhancer.

[0041] Another aspect of the present disclosure relates to a kit comprising a compound of the present disclosure and / or a pharmaceutical composition of the present disclosure.

[0042] Another aspect of the present disclosure relates to the use of the compounds of the present disclosure for the manufacture of a medicament for the prevention, treatment or diagnostic of cancer.

[0043] Another aspect of the present disclosure relates to a method for treating or preventing cancer in a subject, the method comprising administering a compound of the present disclosure to the subject.

[0044] Surprisingly, the compounds of the present disclosure showed low or insignificant toxicity in the dark, are photocytotoxic at nanomolar concentrations, have applicability as diagnostic agents and can be used combined with other therapeutic agents.

[0045] The use of the compounds of the present disclosure in the treatment include, but is not limited to, the use in anticancer treatment, neoadjuvant and / or adjuvant, during surgery, alone or combined with other treatments.

[0046] The use of the compounds of the present disclosure in the diagnostic include, but is not limited to, the use in imaging, molecular, and histopathological methods. In particular, the compounds of the present disclosure may be used in the following diagnostic techniques: MRI (Magnetic Resonance Imaging), CT Scan (Computed Tomography), PET Scan (Positron Emission Tomography), Fluorescence Imaging, among others.BRI EF DESCRI PTION OF THE DRAWINGS

[0047] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0048] Figure 1: Dark cytotoxicity and phototoxicity effect of molecules 1-8 in MDA-MB-231 cell line. The cells were incubated with the molecules for 5 h, washed and growth medium replaced with no molecule, and thereafter treated as follows: graphs A and B represent the phototoxicity after cells exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) followed by 48 h incubation in the dark; graphs C and Drepresent the cytotoxicity resulting from 48 h incubation in the dark. Values are the mean of three independent assays and the error bars represent the standard deviation; treatment vs control (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0049] Figure 2: Dark cytotoxicity and phototoxicity effect of molecules 1-8 in MCF-7 cell line. The cells were incubated with the molecules for 5 h, washed and growth medium replaced with no molecule, and thereafter treated as follows: graphs A and B represent the phototoxicity after cells exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) followed by 48 h incubation in the dark; graphs C and D represent the cytotoxicity resulting from 48 h incubation in the dark. Values are the mean of three independent assays and the error bars represent the standard deviation; treatment vs control (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0050] Figure 3: Dark cytotoxicity and phototoxicity effect of molecules 1-8 in PANC-1 cells. The cells were incubated with the molecules for 5 h, washed and growth medium replaced with no molecule, and thereafter treated as follows: graphs A and B represent the phototoxicity after cells exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) follows by 48 h incubation in the dark; graphs C and D represent the cytotoxicity resulting from 48 h incubation in the dark. Values are the mean of three independent assays and the error bars represent the standard deviation; treatment vs control (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0051] Figure 4: Dark cytotoxicity and phototoxicity effect of molecules 1-8 in primary human dermal fibroblast (HDF) cells. The cells were incubated with the molecules for 5 h, washed and growth medium replaced with no molecule, and thereafter treated as follows: graphs A and B represent the phototoxicity after cells exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) followed by 48 h incubation in the dark; graphs C and D represent the cytotoxicity resulting from 48 h incubation in the dark. Values are the mean of three independent assays and the error bars represent the standard deviation; treatment vs control (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0052] Figure 5: Dark cytotoxicity and phototoxicity effect of molecules 1-8 in primary human mammary epithelial cells (HMEpC). The cells were incubated with the molecules for 5 h, washed and growth medium replaced with no molecule, and thereafter treated as follows: graphs A and B represent the phototoxicity after cells exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) followed by 48 h incubation in the dark; graphs C and D represent the cytotoxicity resulting from 48 h incubation in the dark. Values are the mean of three independent assays and the error bars represent the standard deviation; treatment vs control (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0053] Figure 6: Representative images of MCF-7 cells treated with molecule 6 or DMSO (control). The cells were incubated with the molecule for 5 h, washed and growth medium replaced, followed byirradiation with red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) and thereafter incubated 24 h in the dark. Molecule 6 at IC5o (left) vs control (untreated and irradiated cells) (right).

[0054] Figure 7: Emission signal of zinc(ll) chlorin complexes (1-4) located inside the cells. Compounds 1- 4 were used at 10 pM. Measurement was taken after 5 h incubation in (A) MDA-MB-231, (B) MCF-7, (C) PANC-1, (D) HDF and (E) HMEpC cells. Control group represents untreated cells. Values are the mean fluorescence of three to five images; error bars represent the standard deviation.

[0055] Figure 8: Emission signal of free-base chlorins (5-8) located inside the cells. Compounds 5-8 were used at 10 pM. Measurement was taken after 5 h incubation in (A) MDA-MB-231, (B) MCF-7, (C) PANC-1, (D) HDF and (E) HMEpC cells. Control group represents untreated cells. Values are the mean fluorescence of three to five images; error bars represent the standard deviation.

[0056] Figure 9: Phototoxicity effect of molecule 6 in 3D micro-spheroids of MDA-MB-231 cells. The cells were incubated with molecule 6 for 5 h in the dark, washed and growth medium replaced, then exposed to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm), followed by 48 h in the dark (left). Cytotoxicity of molecule 6 (same treatment as for phototoxicity but not irradiated) in 3D micro-spheroids of MDA-MB- 231 cells in the dark (right). Control group represents untreated micro-spheroids. Data is presented as mean ± SD (10 000 cells / spheroids, 81 spheroids analysed in each experiment, n=3); treatment vs control (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0057] Figure 10: Confocal microscopy analysis of molecules 5 and 6 distribution in 3D micro-spheroids of MDA-MB-231 cells after 5 h incubation in the dark at 10 pM. Control group represents DMSO-treated micro-spheroids. (Dark Grey: Hoechst stained the nucleus; White: red channel is the fluorescence of the molecules).

[0058] Figure 11: Emission intensity of molecule 5 and 6 (10 pM) in 3D PANC-1 micro-tumors (5 h, 24 h and 48 h incubation in the dark). Values are the fluorescence intensity mean of four images, error bars represent the standard deviation; **p < 0.01, ***p < 0.001 versus control group (untreated cells) using one-way ANOVA followed by Bonferroni's post-hoc test.

[0059] Figure 12: Confocal microscopy analysis of molecules 5 and 6 distribution in 3D tumor-spheroids of PANC-1 cells after 48 h incubation at 10 pM in the dark. (Dark Grey: Hoechst stained the nucleus; White: red channel is the fluorescence of molecule 6).

[0060] Figure 13: (A) Phototoxicity effect of molecules 5 or 6 in heterotypic PDAC stratified microenvironment spheroid models (STAMs). The STAMs were incubated with 0.5 pM of either molecule for 5 h in the dark, or untreated control. Then, they were washed and growth medium replaced, followed by exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm), and incubation in the dark for 48 h. Control group is untreated and irradiated STAMs, and (B) internalization of molecules 5 and 6 in PDAC STAMs (5 h incubation at 10 pM); control group represents untreated STAMs. Data is presented as mean± SD (l.OxlO4PANC-1 cells / STAM and 4.0xl04Cancer associated fibroblasts (CAFs) / STAM, n=4 independent STAM); multiple comparisons using one-way ANOVA followed by Bonferroni's post-hoc test.

[0061] Figure 14: Morphologic changes in PDAC STAMs, treated with molecules 5 or 6 at 0.5 pM for 5 h in the dark. Before and 48 h after exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) (PDT) (ampliation 4x (left) and lOx (right)). Control group is untreated cells.

[0062] Figure 15: Phototoxicity effect of molecules 5 or 6 in PANC-l-luciferase cells used to construct the heterotypic PDAC STAMs. The STAMs were incubated with 0.5 pM of either molecule for 5 h in the dark, or untreated control. Then, they were washed and growth medium replaced, followed by exposure to red light (15 min, 6.17 mW cm'2, X = 652 ± 20 nm), and incubation in the dark for 48 h. Control is untreated and irradiated STAMs. Data is presented as mean ± SD (l.OxlO4PANC-1 cells / STAM and 4.0xl04CAFs / STAM, n=4 independent STAM); multiple comparisons using one-way ANOVA followed by Bonferroni's post-hoc test.

[0063] Figure 16: Combined effect of PDT and chemotherapy (Gemcitabine - GEM) in PANC-1 cells. The cells were treated with molecule 6 at 8.0 nM during 5 h in the dark, then washed and growth medium replaced, followed by red light irradiation for 15 min (652 ± 20 nm, 6.17 mW cm'2) and immediately after they were treated with GEM at 1.0 and 120 pM for 48 h. Control group was not treated with molecule 6, neither GEM, but the cells were treated with vehicle and irradiated with same dose of red light. Values are the mean of one independent experiment, representative of two experiments, and the error bars represent the standard deviation; ***p < 0.001 multiple comparisons using one-way ANOVA followed by Bonferroni's post-hoc test.

[0064] Figure 17. Tumorspheres microscopy analysis. The MDA-MB-231 and PANC-1 cells were treated with molecule 6 at 50 nM and 107.6 nM, respectively, during 5 h in the dark, then washed and growth medium replaced, followed by red light irradiation for 15 min (652 ± 20 nm, 6.17 mW cm'2) and incubated for another 48 h. Then, cells were trypsinized, then seeded (lxl04cells / well) and incubated for 72 h.

[0065] Figure 18. MDA-MB-231 mammospheres quantification and size analysis. Data is presented as mean ± SD (n= 3 independent wells). Treatment vs control (untreated cells) using t-test followed by Mann Whitney test.

[0066] Figure 19. Microscopic observation of skin sections 24 h post-intraperitoneal injection with molecule 6 at 0.75 mg / kg. Tissues were stained with H&E. a. adipose tissue, d. dermis, e. epidermis, h. hair follicle, m. melanocytes, k. keratin layer. Scale bar is 50 pm. Arrows indicate the structures labelled with letters.

[0067] Figure 20. Microscopic observation of pancreas, spleen, and liver sections 24 h post- intraperitoneal injection with molecule 6 at 0.75 mg / kg. Tissues were stained with H&E. Pancreas: a.acinar cells, i. islets of Langerhans, d. interlobular duct; Spleen: r. red pulp, t. trabecula, w. white pulp;Liver: h. hepatocytes, v. vein. Scale bar is 10 m.

[0068] Figure 21. Microscopic observation of skin, liver, pancreas, spleen, kidney and intestine sections 31 days post-intraperitoneal injection with molecule 6 at 0.75 mg / kg. Tissues were stained with H&E. Skin: a. adipose tissue, d. dermis, e. epidermis, f. hair follicle, m. melanocytes; Liver: h. hepatocytes, k. kupffer cells, v. vein; Pancreas: ac. acinar cells; d. interlobular duct, i. islet of Langerhans; Spleen: r. red pulp, t. trabecula, w. white pulp; Kidneys: b. Bowman's capsule, g. glomerulus; Intestine: c. crypts, p. Peyer's patch, me. muscularis externa, sm. submucosa, vi. Villus. Scale bar is 20 pm. Arrows indicate the structures labelled with letters.

[0069] Figure 22. Antitumor effect of molecule 6 in C57BL / 6 mice bearing E0771 tumors following intratumoral injection (molecule 6 = 0.75 mg / kg or CTRL = 1% DMSO, 5 h incubation) and tumor illumination (2 min, 20 mW cm-2, 400-800 nm) (left); Photograph of the tumors 5 days after PDT (end of the experiment) (bold numbers indicate relative growth compared to initial tumor size) (right). The dotted line shows the tumor borders. The numbers above each picture refer to the relative change in tumor area compared with the area prior to the treatment. The numbers below indicate the identity of the mouse.

[0070] Figure 23. Animal weight at the beginning of treatment (t0=0 h) and at the end of the experiment (t=120 h). The experiment consisted in treating C57BL / 6 mice bearing E0771 tumors following intratumoral injection (molecule 6 = 0.75 mg / kg or CTRL = 1% DMSO, 5 h incubation) and tumor illumination (2 min, 20 mW cm-2, 400-800 nm). to is the time prior treatment and tl20 is 5 days after treatment.

[0071] Figure 24. Representative H&E staining sections of the tumors treated with molecule 6 (0.75 mg / kg) or 1% DMSO (control) and tumor illumination (2 min, 20 mW cm-2, 400-800 nm). The bar graph shows the quantification of number of mitoses / HPF (high-power field) [mitoses were quantified in 10 randomly selected HPFs (40X objective)]. Treatment vs control (untreated cells) using t-test followed by Mann Whitney test.DETAILED DESCRIPTION

[0072] The present disclosure relates to a compound of general forma (I) or (II) or a pharmaceutically acceptable salt thereof. Furthermore, the present disclosure relates to the use of said compounds in medicine, in particular in photodynamic therapy (PDT) of cancer, as well as in diagnostic.

[0073] The present disclosure relates to a compound of general formula (I) or (II), or their pharmaceutically acceptable salts. Surprisingly, these compounds exhibit enhanced efficacy with significantly reduced cytotoxicity. Specifically, the compounds of the present disclosure demonstrate highphototoxicity against cell lines, in particular breast and pancreatic cancer cell lines and human primary fibroblasts, while showing no dark cytotoxicity at therapeutic doses. Additionally, these compounds exhibit fluorescence in the red region of the electromagnetic spectrum, facilitating their use in diagnostic imaging. These compounds may be used in photodynamic therapy (PDT) for cancer treatment and as diagnostic agents (PDD).

[0074] Photodynamic therapy (PDT) is a treatment that uses special drugs, called photosensitizing agents, along with light to kill cancer cells. The drugs only work after they have been activated or "turned on" by certain kinds of light (specific wavelengths). Photodynamic therapy may also be called photochemotherapy.

[0075] Based on the International Union of Pure and Applied Chemistry (IUPAC) definitions, an alkyl group is defined as a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom -Cnbhn+i. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R * H), and R3C (R * H) are primary, secondary and tertiary alkyl groups, respectively.

[0076] Alkyl chain comprises branched and unbranched chains, substituted or non-substituted. Preferably, in the present disclosure, alkyl chain relates to unbranched and non-substituted alkyl chain.

[0077] The present disclosure relates to chlorophyll derivatives bearing different uracil-alditols moieties with general formula I - or general formula II; the molecules may be used as photosensitizers (PS) for PhotoDynamic Therapy of cancer (PDT) and imaging diagnostic.

[0078] The acetoxy group (abbr. AcO or OAc; IUPAC name: acetyloxy), is a functional group with the formula -OCOCH3 and the structure -O-C(=O)-CH3.

[0079] The following table summarizes compounds 1-8 of the present disclosure.

[0080] The preparation of the chlorophyll derivatives of the present disclosure is given below.The preparation of the chlorophyll derivative, zinc(ll) complex of chlorin eg trimethyl ester (ZnChlegMe, 1) was performed and the characterization of the obtained product (1) is reported below.

[0081] ZnChlegMe (1) -XH NMR (300 MHz, CDCI3): <5 9.53 (s, 1H, H-meso), 9.48 (s, 1H, H-meso), 8.54 (s, 1H, H-meso), 8.02 (dd, 1H, J = 17.8 and 11.5 Hz, H-31), 6.17 (dd, 1H, J = 11.5 and 1.8 Hz, H-32), 6.02 (dd, 1H, J = 11.5 and 1.8 Hz, H-32), 5.24 (d,lH, H-151), 5.11 (d, 1H, H-151), 4.39-4.23 (m, 2H, H-17 and H-18), 4.19 (s, 3H, -CO2CH3), 3.83 (s, 3H, -CO2CH3), 3.80-3.71 (m, 2H, H-81), 3.58 (s, 3H, -CO2CH3), 3.44 (s, 3H, - CH3), 3.35 (s, 3H, -CH3), 3.30 (s, 3H, -CH3), 2.60-1.66 (m, 10H, H-82,-171,-172,-181) ppm. ESI-MS(+): m / z 701.3 [M+H]+and 723.4 [M+Na]+. UV-Vis (Xmax(log(e), DMF): 415 (4.89), 517 (3.65), 561 (3.57), 597 (3.82), 639 (4.48) nm.

[0082] The functionalization of the chlorophyll derivative (1) with different uracil-alditols [-Xyl (xylose), - Gal (galactose) and -Glc (glucose)] derivatives to obtain compounds ZnChlegMe-Xyl (2), ZnChlegMe-Gal (3) and ZnChlegMe-GIc (4) were performed adapting the procedure reported by Palasz et al. [5].The characterization of the obtained products (2, 3 and 4) is now reported.

[0083] ZnChlegMe-Xyl (2) - HMRS (ESI): m / z calcd for C56H64N6Oi7Zn: 1157.3698 [M+H]+; 1157.3688 found; UV-Vis (Xmax(log(e), DMF): 412 (5.04), 516, (3.77), 560 (3.67), 593 (3.88), 634 (4.64) nm.

[0084] ZnChlegMe-Gal (3) - HMRS (ESI): m / z calcd for C5gH68N60igNaZn: 1251.3728 [M+Na]+; 1251.3703 found; UV-Vis (Xmax(log(e), DMF): 412 (5.00), 516 (3.68), 559 (3.61), 593 (3.86), 634 (4.59) nm.

[0085] ZnChlegMe-GIc (4) - HMRS (ESI): m / z calcd for C5gH68N60igNaZn: 1251.3728 [M+Na]+; 1251.3688 found; UV-Vis (Xmax(log(e), DMF): 412 (5.02), 515 (3.68), 559 (3.69), 592 (3.86), 634 (4.62) nm.

[0086] The synthesis of compounds ChlegMe (5), ChlegMe-Xyl (6), ChlegMe-Gal (7) and ChlegMe-GIc (8) were performed and the characterization of the obtained products (6,7 and 8) is now reported bellow.

[0087] ChlegMe-Xyl (6) -XH NMR (500 MHz, CDCI3): 9.76 (s, 1H, H-meso), 9.49 (s, 1H, H-meso), 8.83 (s, 1H, H-meso), 7.12 (dd, 1H, J = 12.3 and 3.2 Hz, H-2'), 6.11 (dd, 1H, J = 8.2 and 2.9 Hz, H-2), 5.70 (dd, 1H, J = 8.2 and 3.3 Hz, H-3), 5.48 (ddd, 1H, J = 7.0, 5.2 and 3.3 Hz, H-4), 5.38 (d, 1H, J = 18.9 Hz, H-151), 5.27 (d, 1H, J = 18.9 Hz, H-151), 4.52-4.41 (m, 2H, H-17 and H-18), 4.33 (dd, 1H, J = 11.7 and 5.2 Hz, H-5), 4.27 (s, 3H, -CO2CH3), 4.10 (dd, 1H, J = 11.7 and 7.0 Hz, H-5), 3.83-3.79 (m, 2H, H-81) 3.78 (s, 3H, -CO2CH3), 3.64 (s, 3H, -CO2CH3), 3.60 (s, 3H, -CH3), 3.51-3.47 (m, 9H, -CH3, N-CH3), 3.44-3.42 (m, 1H, H-l), 3.26 (s, 3H, N-CH3), 2.92-2.87 (m, 1H, H-l'), 2.81 (dd, 1H, J = 14.9 and 3.3 Hz, H-l'), 2.61-2.55 (m, 1H, H-172), 2.37 (s, 3H, -OAc), 2.33 (s, 3H, -OAc), 2.23-2.19 (m, 1H, H-172), 2.14 (s, 3H, -OAc), 1.92 (s, 3H, -OAc), 1.80-1.52 (m, 8H, H-82,- 171,-181), -1.44 (s, 1H, -A / H), - 1.75 (s, 1H, -A / H) ppm. HMRS (ESI): m / z calcd for CssHssNgO : 1095.4563 [M+H]+; 1095.4532 found; UV-Vis (Xmax(log(e), DMF): 397 (5.06), 498 (4.06), 524 (3.68), 554 (3.45), 604 (3.66), 659 (4.62) nm.

[0088] ChlegMe-Gal (7) -XH NMR (500 MHz, CDCI3): 9.76 (s, 1H, H-meso), 9.52 (s, 1H, H-meso), 8.83 (s, 1H, H-meso), 6.96 (dd, 1H, J = 12.6 and 3.0 Hz, H-2'), 6.05 (t, 1H, J = 3.0 Hz, H-2), 5.62 (dd, 1H, J = 8.9 and 3.0 Hz, H-3), 5.54 (dd, 1H, J = 8.9 and 2.5 Hz, H-4), 5.43-5.34 (m, 2H, H-5 and H-151), 5.31-5.28 (m, 1H, H- 151), 4.51-4.38 (m, 3H, H-6, -17, -18), 4.27 (s, 3H, -CO2CH3), 3.98-3.93 (m, 1H, H-6), 3.84-3.80 (m, 2H, H-81) ,3.78 (s, 3H, -CO2CH3), 3.64 (s, 3H, -CO2CH3), 3.60 (s, 3H, -CH3), 3.51-3.47 (m, 9H, -CH3and N-CH3), 3.44- 3.42 (m, 1H, H-l), 3.30 (s, 3H, N-CH3), 2.93 (dd, 1H, J = 15.1 and 3.1 Hz, H-l'), 2.82-2.73 (m, 1H, H-l'), 2.60- 2.52 (m, 1H, H-172), 2.43 (s, 3H, -OAc), 2.23 (s, 3H, -OAc), 2.21-2.16 (m, 1H, H-172), 2.07 (s, 3H, -OAc), 1.80 (s, 3H, -OAc), 1.78-1.51 (m, 11H, -OAc, H-82,-171,-181), -1.74 (s, 2H, -A / H) ppm. HMRS (ESI): m / z calcd for C59H70N6O19: 1167.4774 [M+H]+; 1167.4772 found; UV-Vis (Xmax(log(e), DMF): 397 (5.03), 498 (4.08), 524 (3.75), 555 (3.56), 604 (3.72), 659 (4.58) nm.

[0089] ChlegMe-GIc (8) -XH NMR (500 MHz, CDCI3): 9.76 (s, 1H, H-meso), 9.50 (s, 1H, H-meso), 8.84 (s, 1H, H-meso), 7.12 (dd, 1H, J = 12.5 and 3.0 Hz, H-2'), 6.03 (dd, 1H, J = 9.1 and 3.0 Hz, H-2), 5.79 (dd, 1H, J = 9.1 and 2.4 Hz, H-3), 5.52 (dd, 1H, J = 8.5 and 2.4 Hz, H-4), 5.39 (d, 1H, J = 19.0 Hz, H-151), 5.26 (d, 1H, J = 19.0 Hz, H-151), 5.18-5.13 (ddd, 1H, J = 8.5, 4.9 and 2.8 Hz, H-5), 4.52-4.42 (m, 2H, H-17 and H-18), 4.27 (s, 3H, -CO2CH3), 4.19 (dd, 1H, J = 12.8 and 2.8 Hz, H-6), 4.05 (dd, 1H, J = 12.8 and 4.9 Hz, H-6), 3.84-3.80 (m, 2H, H-81), 3.80 (s, 3H, -CO2CH3), 3.64 (s, 3H, -CO2CH3), 3.60 (s, 3H, -CH3), 3.51-3.47 (m, 9H, -CH3and N- CH3), 3.46-3.44 (m, 1H, H-l), 3.27 (s, 3H, -CH3), 2.98-2.87 (m, 1H, H-l'), 2.78 (dd, 1H, J = 15.3 and 3.0 Hz, H-l'), , 2.62-2.53 (m, 1H, H-172), 2.43 (s, 3H, -OAc), 2.31 (s, 3H,-OAc), 2.15 (s, 3H, -OAc), 2.02-1.97 (m, 1H- 172), 1.94 (s, 3H, -OAc), 1.87 (s, 3H, -OAc), 1.77-1.70 (m, 8H, H-82, -171, -181), -1.43 (s, 1H, -A / H), -1.75 (s, 1H, -A / H) ppm. HMRS (ESI): m / z calcd for C59H70N6O19: 1167.4774 [M+H]+; 1167.4759 found; UV-Vis (Xmax(log(e), DMF): 397 (5.01), 497 (4.01), 523 (3.60), 556 (3.33), 605 (3.59), 659 (4.56) nm.

[0090] Photophysical and Biological Properties:

[0091] The absorption spectra of all molecules (zinc(ll) complexes and free-base derivatives) are in accordance with the typical absorption spectrum expected for a chlorin derivative; the absorption spectra of zinc(ll) complexes (1-4) present a strong Soret band at ca 412-415 nm, and the four Q. bands between 515 and 639 nm, and the last Q. band is more intense than the other three bands. In its turn, for the free- base chlorins (5-8) is observed that the last Q. band appears at ca 659-664 nm; free-base chlorins present an absorption band in a wavelength above 650 nm, where the light better penetrates in the tissues. The introduction of the uracil-alditols in 1 cause a slight hypsochromic shift (blue shift) in the bands of 2, 3, and 4 molecules; and when these conjugates are compared with the corresponding free-base molecules 6, 7, and 8 are observed a bathochromic shift (red shift) in the last Q. band and a hypsochromic shift at the Soret band. Compounds 6, 7, and 8 when compared with compound 5, showed a hypsochromic shift in all absorption bands.

[0092] The zinc(ll) complexes (1, 2, 3 and 4) revealed to be more efficient to produce1O2than the free- base molecules (5, 6, 7 and 8). The introduction of uracil-alditols moieties did not extinguish the ability of these macrocycles to generate1O2.The singlet oxygen quantum yield ((Pa) found are presented in Table 1.

[0093] Table 1. Singlet oxygen quantum yield (( a) data in DMF.

[0094] The cytotoxicity and phototoxicity of all molecules were assessed in different cancer cell lines [triple-negative breast cancer cell line (MDA-MB-231), luminal breast cancer cell line (MCF-7), human pancreatic cancer cell line (PANC-1) and in human mammary epithelial cells (HMEpC) and human dermal fibroblasts (HDF). All the molecules are soluble in DMSO; therefore, in all experiments, the stock solutions of the molecules were diluted to be added to the cells with a maximum of 0.1% (v / v) of DMSO. Additionally, the controls were prepared in the same way, never exceeding a maximum of 0.1% (v / v) of DMSO. Cell viability was assessed with crystal violet assay (Figure 1-5), and the IC5o values were determined and are shown in Table 2.

[0095] Table 2. IC5o values for molecules 1-8, after exposure to red light (PDT) (15 min, 6.17 mW cm'2, X = 652 ± 20 nm) in breast cancer lines (MDA-MB-231 and MCF-7), pancreatic cell line (PANC-1), and primary non-tumorigenic cells (HDF).

[0096] The analysis of Table 2 revealed that molecule 1 showed an IC5o of 1.08 pM in MDA-MB-231, 1.78 pM in MCF-7, 0.84 pM in PANC-1 and 0.95 pM in HDF. Molecule 1 was more efficient in MDA-MB-231, PANC-1 and HDF than in MCF-7 (p<0.001). Molecule 2 showed an IC50of 0.90 pM in MDA-MB-231, 0.58 pM in MCF-7, 0.89 pM in PANC-1 and 1.04 pM in HDF. Molecule 2 was more effective in MCF-7 than in HDF (p<0.05).

[0097] Molecule 3 showed an IC50of 1.20 pM in MDA-MB-231, 1.17 pM in MCF-7, 0.96 pM in PANC-1 and 1.06 pM in HDF. No significant differences between each cell line were observed.

[0098] Molecule 4 showed an IC50of 1.18 pM in MDA-MB-231, 0.79 pM in MCF-7, 0.93 pM in PANC-1 and 0.96 pM in HDF. Molecule 4 was more effective in MCF-7 than in MDA-MB-231 (p<0.05).

[0099] Molecule 5 showed an IC50of 103.25 nM in MDA-MB-231, 63.74 nM in MCF-7, 107.60 nM in PANC- 1 and 106.20 in HDF. No significant differences were observed between each cell line.

[0100] Molecule 6 showed an IC50of 50.69 nM in MDA-MB-231, 9.07 nM in MCF-7, 8.66 nM in PANC-1 and 11.03 nM in HDF. No significant differences were observed between each cell line.

[0101] Molecule 7 showed an IC50of 77.57 nM in MDA-MB-231, 15.89 nM in MCF-7, 11.00 nM in PANC- 1 and 14.08 nM in HDF. No significant differences were observed between each cell line.

[0102] Molecule 8 showed an IC50of 49.25 nM in MDA-MB-231, 12.70 nM in MCF-7, 11.27 nM in PANC- 1 and 11.58 in HDF. No significant differences were observed between each cell line.

[0103] Regarding free base derivatives (6, 7 and 8) it was observed that they are more effective in all cell lines than the zinc(ll) molecules (2, 3 and 4) (p<0.001). Molecule 5 was more efficient in MDA-MB-231, MCF-7, PANC-1 and HDF than molecules 1, 2, 3 and 4 (p<0.001). Molecule 1 was less effective than molecules 5, 6, 7 and 8 in all cell lines. Moreover, molecule 1 was less effective than molecules 2, 3 and 4 in MCF-7 cells (p<0.001). Also, molecule 2 was more effective in MCF-7 cells than molecule 3, and consequently molecule 3 was more effective than molecule 4 (p<0.05) in the same cell line.

[0104] The phototoxicity effect was also tested in HMEpC cells. The doses used for cancer cells (0.1 - 10 pM for molecules 1, 2, 3 and 4; and 10 - 750 nM for molecules 5, 6, 7 and 8) showed more phototoxicity in HMEpC.

[0105] Molecule 1 showed significant phototoxicity starting at 1.0 pM in MDA-MB-231 (45%), PANC-1 (70%) and HDF (75%) cells; at 0.5 pM in MCF-7 (20%); and 0.1 pM in HMEpC (60%). The same molecule in the dark showed a significant 20% toxicity at 10 pM in MDA-MB-321 and at 1.0 pM in MCF-7 cell lines; and 5.0 pM in HMEpC (30%). No cytotoxicity by molecule 1 in the dark was recorded in PANC-1 or HDF cells.

[0106] Molecule 2 showed significant phototoxicity starting at 1.0 pM in MDA-MB-231 (45%), PANC-1 (70%) and HDF (45%) cells; at 0.5 pM in MCF-7 (30%); and 0.1 pM in HMEpC (85%). This molecule in darkconditions showed significant cytotoxicity at 100 pM in MDA-MB-231 (60%), MCF-7 (50%); and some cytotoxicity at 0.1 pM in HMEpC (15%). No cytotoxicity by molecule 2 in the dark was recorded in PANC- 1, or HDF cells.

[0107] Molecule 3 showed significant photocytotoxicity at 1.0 pM in MDA-MB-231 (45%), PANC-1 (45%) and HDF (40%) cells; at 0.5 pM in MCF-7 (15%) and at 0.1 pM in HMEpC (90%). In the dark, no cytotoxicity was observed in PANC-1 and HDF cells; in MDA-MB-231 dark toxicity was found at 100 pM (70%) and in HMEpC at 5.0 pM (20%).

[0108] Molecule 4 showed significant photocytotoxicity at 1.0 pM in MDA-MB-231 (20%), PANC-1 (60%) and HDF (50%); at 0.5 pM in MCF-7 (20%) and at 0.1 pM in HMEpC (95%). In the dark molecule 4 has cytotoxicity at 100 pM in MDA-MB-231 (60%) and at 5.0 pM in HMEpC (20%) cells. No dark cytotoxicity by molecule 4 was found in MCF-7, PANC-1 and HDF.

[0109] Molecule 5 showed significant photocytotoxicity at 5.0 nM in MDA-MB-231 (20%), at 50 nM in MCF-7 (20%), at 75 nM in HMEpC (70%), at 100 nM in HDF (40%) and at 750 nM in PANC-1 (95%). In dark conditions, cytotoxicity was recorded at 100 pM in MDA-MB-231 (20%). No dark cytotoxicity was found in MCF-7 PANC-1, HDF and HMEpC.

[0110] Molecule 6 showed significant photocytotoxicity at 7.5 nM in MCF-7 (30%), at 10 nM in PANC-1 (65%), HDF (30%) and HMEpC (85%) cells; and at 50 nM in MDA-MB-231 (50%). Molecule 6 in the dark showed a 55% cytotoxicity in MDA-MB-231 at 100 pM. No dark cytotoxicity was recorded in MCF-7, PANC- 1, HDF and HMEpC.

[0111] Molecule 7 showed significant photocytotoxicity at 7.5 nM in MCF-7 (20%), at 10 nM in PANC-1 (30%), HDF (25%) and HMEpC (85%), and at 50 nM in MDA-MB-231 (25%). In the dark, the same molecule showed a 40% cytotoxicity in MDA-MB-231 at 100 pM. No dark cytotoxicity was found in MCF-7, PANC-1, HDF and HMEpC.

[0112] Molecule 8 showed significant photocytotoxicity at 7.5 nM in MCF-7 (20%), at 10 nM in PANC-1 (30%), HDF (25%) and HMEpC (90%), and at 50 nM in MDA-MB-231 (55%). In dark conditions, molecule 8 showed cytotoxicity at 10 pM in MDA-MB-231 (30%). No dark cytotoxicity was noted in MCF-7, PANC-1, HDF and HMEpC.

[0113] In Figure 6, the microscopic images of MCF-7 cells 24 h after being exposed to red light are presented (15 min, 6.17 mW cm'2, X = 652 ± 20 nm). The cells treated with molecule 6 became rounded and detached from the plate, as a sign of cell death.

[0114] Live cell imaging was conducted to investigate the effect of structure on the internalization across different cell lines (Figure 7 and 8). Since all molecules are emissive in the red region ("'660 nm) of electromagnetic spectrum, they can be visualized when excited with blue-green light in the range of 400-532 nm. Therefore, internalization was imaged using a Zeiss LSM 880 with Airyscan microscope and quantified as pixel intensity in the picture / total number of cells. An average of 60 cells per treatment were analysed using confocal microscopy and ImageJ software to quantify the fluorescence intensity / # cells as a measure of molecule internalization.

[0115] In general, molecules without uracil-alditol moieties (1 and 5) appear to internalize better in the cells, except in MCF-7 for molecule 5.

[0116] Molecule 1 seems to internalize better in HMEpC cells than in the other cell lines.

[0117] Molecule 2 seems to internalize better in HMEpC cells than in the other cell lines.

[0118] Molecule 3 seems to internalize better in HMEpC and PANC-1 than in the other cell lines.

[0119] Molecule 4 seems to internalize better in HMEpC than in the other cell lines.

[0120] Molecule 5 seems to internalize better in PANC-1 than in the other cell lines.

[0121] Molecule 6 seems to internalize better in PANC-1 than in the other cell lines.

[0122] Molecule 7 seems to internalize better in PANC-1 than in the other cell lines.

[0123] Molecule 8 seems to internalize better in PANC-1 than in the other cell lines.

[0124] Considering the preceding results, molecule 6 was selected for studies in 3D micro-spheroids of MDA-MB-231 cell line. Viability was evaluated using the 3D CellTiterGlo reagent, and the results are presented in Figure 9.

[0125] As can be observed in Figure 9, molecule 6 under light conditions was very effective in 3D microspheroids at concentration starting in 50 nM. In dark conditions, no cytotoxicity effect was observed in the tested conditions.

[0126] Additionally, the internalization of molecule 5 and 6 in the 3D micro-spheroids was assessed using confocal microscopy. For the purpose of this study, molecule 5 was selected as a reference to enable the comparative analysis of molecule 6. As shown in Figure 10, after 5 h of incubation, the molecules 5 and 6 efficiently internalizes the micro-spheroids. The molecules are distributed all over the spheroids.

[0127] The studies in 3D micro-tumors were also conducted using PANC-1 cell line. For these assays, molecule 6 was chosen, and molecule 5 was selected as a reference. The internalization of molecules 5 and 6 was studied after 5 h of incubation and for longer periods (24 h and 48 h). As shown in Figure 11, after 5 h incubation, both molecules are efficiently internalized by the micro-tumor. Besides, even after 24 h and 48 h, these molecules are still internalized by the spheroids, revealing that these molecules are not excreted or metabolized by the cancer cell.

[0128] In Figure 12, is shown a representative image of the fluorescence distribution of molecules 5 (reference) and 6 in four 3D tumor-spheroids of PANC-1 cells after 48 h incubation. The molecules are distributed all over the spheroids.

[0129] We tested a more complex model mimicking the tumor microenvironment. This is the heterotypic PDAC stratified tumor spheroid model (STAM), which consist of PANC-1 cancer cells, surrounded by CAFs, mimicking the desmoplastic microenvironment of human PDAC [6], The viability of STAMs was assessed using the CellTiterGlo Assay (which assesses the viability of both CAFs and PANC-1 cells together). In this model of micro-tumors, phototoxicity was observed for both molecules. Notably, molecule 6 stood out as the more efficient of the two, reducing STAM viability by 50% (Figure 13A). The compounds were found throughout all the micro-tumor and molecule 6 was better internalized (Figure 13B).

[0130] Comparing of the micro-tumor morphology before and after irradiation (Figure 14), it is clear that molecule 6, and to a lesser extent molecule 5, are able to disrupt the outer layer of the micro-tumor. Since this consists of the CAFs, this compound can disrupt the tumor stroma to facilitate the entry of itself or other drugs.

[0131] To validate the phototoxic effect in Figure 13 occurs in the PANC-1 cancer cells, a study was performed using 3D STAMs of PANC-1 cells expressing luciferase. The viability of PANC-1 luciferase spheroids was assessed using the OneGlo Luciferase Assay. The results obtained are represented in Figure 15 and show that both molecules 5 and 6 are able to kill the pancreatic cancer cell population. As observed in Figure 15, the OneGlo analysis revealed that molecule 5 and molecule 6 reduced the luciferase signal in 48% and 37%, respectively, indicating that PANC-1 cells are dying. Thus, taking in mind the results in Figure 13A, both molecules 5 and 6 are able to reduce tumoral cells, and also to destroy the CAFs, helping to reduce the stroma barrier around the tumor cells. Given that in Figure 13A molecule 6 was more effective than molecule 5, molecule 6 is also better able to kill CAFs.Use of novel PSs in combination with chemotherapy

[0132] PDT with compound 6 was able to enter and kill PDAC cells and also the surrounding stroma cells (CAFs) as evidenced by the disaggregated STAM (Figure 13 and 14). This supports that combining chemotherapy (using for instance Gemcitabine, GEM) and PDT with compound 6 improves the accessibility of therapy to the tumor. Because PDT causes oxidative stress, the PDT sensitizes the cells to chemotherapy and allows the reduction of chemotherapeutic drug concentration needed to achieve a similar effect. In Figure 16 is represented the results supporting this claim.

[0133] As shown in Figure 16, the treatment of PANC-1 cells with GEM at 1.0 pM and 120 pM causes a decrease in cell viability of 28% and 40%, respectively; the viability of cells treated with molecule 6 at 8 nM cause a reduction in cell viability of 38%, however when GEM at 1.0 pM and 120 pM was addedimmediately after PDT it was observed a decrease in PANC-1 cells viability of 60% and 70%, respectively. Thus, a pre-treatment of PDT with molecule 6 allow to sensitize the cells and increase the toxic effect of GEM. This reduces the amount of chemotherapeutic needed to achieve an effect.Effect of PDT on Tumor Initiating Potential

[0134] Cancer stem cells (CSCs), or cancer initiating cells, are characterized by self-renewal and tumorigenic potential, are often drug-resistant, contributing to tumor recurrence and metastasis. Therefore, there is a need to find therapies that reduce the number of cells with tumor initiating potential that remain after therapy and are the cause of relapse and metastasis. To evaluate if PDT applied with molecule 6 reduces the number of tumor-initiating cells in the surviving MDA-MB-231 and PANC-1 cell population post-PDT treatment, we assessed their capacity to form tumorspheres (grow in low attachment and no serum conditions). After PDT protocol, in both MDA-MB-231 and PANC-1 cells, with molecule 6, the cells were trypsinized. A low density of 1.0 xlO4cells mL1was seeded in agarose-coated 12-well plates and cultured in serum-free medium with B-27 supplement for 72 h, allowing only CSC- like / tumor initiating cells to form tumorspheres. Tumorspheres were subsequently analysed using light microscopy. As observed in Figure 17, both MDA-MB-231 and PANC-1 cells treated with molecule 6 demonstrated a reduced ability to form tumorspheres compared to the control group (untreated cells). In fact, PDT with molecule 6 reduced the number of MDA-MB-231 mammospheres by ca. 50% (28 vs 49 in control group, p = 0.0383). Also, the mammospheres in the control group had an average area 1.5 times larger (3378.3 vs 2194.2 pm2, p = 0.0500) than those resulting from treated cells (Figure 18). These results support that PDT with molecule 6 effectively can reduce the population of CSCs / tumor initiating cells and the proliferative capacity, which can be further improved using regimens of multiple irradiation sessions.In vivo experiments

[0135] As a proof of concept, molecule 6 was used to test the toxicity, biodistribution and anti-tumoral effect.

[0136] The C57BL / 6 mouse model was used to evaluate if molecule 6 is toxic, as well as its biodistribution in tissues and organs after 5 or 24 h of intraperitoneal (i.p.) injection. Consequently, 2 males and 3 females were injected intraperitoneally with molecule 6 at 0.25 mg / kg and 0.75 mg / kg (mg of compound 6 per kg of mice body weight). After 5 or 24 h, the animals were sacrificed, and their organs and tissues were collected. Throughout the 24 h period post-injection, the animals were kept under a normal light cycle program and monitored for any signs of cytotoxicity or behavioural changes.Biodistribution

[0137] The concentration of molecule 6 accumulated in each organ and tissue was determined and the results are presented in Table 3. First, the photosensitizing molecules available in the market often accumulate in skin which is responsible for patient high photosensitivity for prolonged periods, and the patients need to protect themselves from exposure to light. Molecule 6 amounts found in skin, were insignificant (co. two orders of magnitude lower than in the organs with higher concentration), and internalization in eyes, which could potentially also represent an undesired phototoxic effect in patients was also negligible. The molecule 6 showed to be present in a varying degree in various organs, including spleen, pancreas, intestine, gonadal adipose tissue, bladder and ovaries. The biodistribution profile demonstrated differences in tissue accumulation of the molecule between 5 h and 24 h post-injection, with higher accumulation at the short period of 5 h.

[0138] Table 3. Biodistribution of molecule 6, after 24 h or 5 h of intraperitoneal injection (i.p.). The values are represented in ng of molecule 6 per mg of tissue (ng / mg). One male and one female were used as control without treatment and these values were used as blank; M2 and F2: male 2 and female 2 injected with 0.25 mg / kg of molecule 6 after 24 h; M3 and F3: male 3 and female 3 injected with 0.75 mg / kg of molecule 6 after 24 h; F4: female 4 injected with 0.75 mg / kg of molecule 6 after 5 h. (detection limit: 1X10-5ng / mg)n.d. - not-detected: below de limit of detection; Gon. adi. tis. - Gonodal adipose tissue; Subc. adip. tis. -Subcutaneous adipose tissue.Short-term toxicity

[0139] Throughout the 24-h period, all animals were observed every 2 h the first 12 h and thereafter at 24 h, and no signs of toxicity or behavioural changes were observed in mice injected i.p. with 0.25 mg / kg or 0.75 mg / kg of molecule 6. The animals didn't show any signs of edema, erythema or any damage in skin throughout this period.

[0140] The histological analysis of tissue sections after 24 h i.p. with compound 6 is represented in Figure 19 and 20. The longitudinal skin sections from both control mice (untreated) and mice treated with 0.75 mg kg1of molecule 6, after 24 h, reveal no notable differences. In Figure 19, H&E staining highlights distinct structural features, such as the epidermis (e), adipose tissue (a), keratin layer (k) and dermis (d) that remained unchanged in the treated animals. This is in agreement with no bioaccumulation of the compound in skin and hence no damage and an improvement over current molecules.

[0141] Figure 20 shows sections corresponding to pancreas, spleen and liver from both control mice (untreated) and mice, 24 h-after being treated with 0.75 mg kg1of molecule 6. The tissues do not disclose changes in their structures. For instance, in pancreas sections in both control and treated animals exhibited normal islet of Langerhans (i) and the acinar cells (a). In the case of spleen, it is possible to observe the red pulp (r) (consisting of connective tissue and venous sinuses) and the white pulp (w) (lymphatic tissue) unchanged after treatment. In the liver, there were no signs of inflammation and the cells in treated animals had a similar appearance as the in the control.Long-term cytotoxicity

[0142] C57BL / 6 mice were used to evaluate the long-term toxicity of molecule 6 after 31 days of injection. Ten males were injected intraperitoneally with 0.75 mg / kg of molecule 6 or vehicle solution (1% DMSO in PBS). Simultaneously with the injection of the compound, the back of the animal was depilated with over-the-counter depilatory cream for humans. Throughout the period post-injection, the animals were kept ad libitum under a normal 12 h light / 12 h dark cycle program and monitored every 24 h the first 72 h and thereafter every 2 days for any signs of cytotoxicity or behavioural changes, none of which were observed. No variation in body weight was observed between treated and untreated animals.Throughout the 31 days no signs of toxicity or behavioural changes were observed, and the depilated skin had the same appearance in the treated animals compared with the controls. On day 31, the animals were sacrificed, and their organs and tissues were collected. H&E staining of the main organs (Figure 21), including the skin, liver, pancreas, spleen, kidneys and intestine, revealed no differences in their morphological structures compared with those of control (untreated) mice.In vivo anti-tumoral effect

[0143] C57BL / 6 12-month-old female mice with one E0771 tumor in the left inguinal side were used to evaluate the antitumoral effect of molecule 6. The tumors that developed over the course of 15 days showed different sizes at the beginning of the treatment, mimicking what is observed in clinical settings.

[0144] First, the uptake of the compound by the tumor and other organs was evaluated using two administration routes (intratumor or intraperitoneal). Mouse F5 was injected intraperitoneally, and mouse F6 was injected intratumorally with a dose of 0.75 mg / kg of molecule 6. The amount of molecule 6 present in the tumor was analysed after 5 h. The total amount of molecule 6 in the tumor tissue of the intratumorally injected animal was 0.142 ng / mg, that corresponds to 19% of injected compound. In contrast, no detectable molecule 6 was found in the tumor of the intraperitoneally injected animal.

[0145] PDT was performed only once, on day 15, and the experiment concluded on day 20. Six females, with E0771 tumors in the left inguinal flank, were injected intratumorally with 0.75 mg / kg of molecule 6 or vehicle solution (1% DMSO in PBS). After 5 h, the tumoral area was irradiated with LUMACARE system with white light (2 min, 20 mW cm'2, 400-800 nm). The animals were kept ad libitum under a normal light cycle program and monitored for any signs of toxicity or behavioural changes, and the tumor size was recorded for 5 days. The results are presented in Figure 22. No toxicity or behavioural changes were observed. Tumors in the control group (#1, #8, #20) continued to grow, doubling or even quadrupling in size. In contrast, the treated tumors either remained stable (#2) or decreased in size by approximately 20 to 30% (#5 and #3, respectively). The animals' weight was also monitored (Figure 23). Overall, there was no change in weight within the 5 days following treatment. Visually, it was also possible to see less blood irrigation in the tumors treated with molecule 6. Tumor sections, stained with H&E, from treated animals exhibited a reduced number of mitosis compared to control group (1.3±0.4 vs 4.6±1.2 mitoses / HPF). These findings indicate that molecule 6 significantly inhibits tumor cell proliferation in vivo.MATERIALS AND METHODSGeneral procedures

[0146] The commercial reagents used were as supplied, due to their high purity. Toluene and methanol were purified or dried according to the literature procedures. All the1H spectra were recorded on Bruker AMX 300 Advance NMR. The assignments of the protons were confirmed using two-dimensional COSY (1H / 1H). High-resolution mass spectra were recorded on a LTQ Orbitrap XL mass spectrometer (ThermoFischer Scientific, Bremen, Germany) using chloroform as solvent. UV-Vis spectra were recorded on an UV-2501-PC Shimadzu spectrophotometer, and emission spectra were recorded on a HORIBA Fluoromax Plus spectrofluorometer, using DMF as solvent and at 20 °C. Chromatographic purification was carried out with preparative thin-layer chromatography (TLC), 20x20 cm glass plates coated with silica gel 60 (Merck, 0.5 mm). Analytical TLC was carried out on precoated sheets with silica gel 60 (Merck, 0.2 mm).Synthesis of photoactive molecules:

[0147] Preparation of chlorophyll derivative, ZnChlegMe (1)

[0148] Extraction: Dried Spirulina maxima (100 g) was added to a dried methanol solution with H2SO45% (500 mL) for 48 h under stirring at room temperature and protected from light. The reaction mixture was filtered, and the obtained solid was washed with methanol and ethyl acetate. The solvent was evaporated under reduced pressure, and the crude was placed in ice and refrigerated at 0 °C. Subsequently, the residue was neutralized with sodium bicarbonate and after neutralization was transferred to a porous funnel containing silica gel, eluting with water and then with ethyl acetate. The collected fraction was washed, and the solvent evaporated.

[0149] Cyclopentanone ring opening and esterification of carboxylic acid groups: The crude was redissolved in dry methanol (15 mL) under a nitrogen atmosphere and allowed to stir for 10 min. Thereafter, a solution of sodium methoxide in methanol 0.5 M (10.29 mL) was added and the mixture was allowed to stir at 0 °C for 1 h and overnight at room temperature. After the reaction was complete, it was extracted with dichloromethane and the organic phase was evaporated under reduced pressure.

[0150] Zinc (II) complexation: The residue was re-dissolved in chloroform (90 mL) and it was added zinc(ll) acetate di-hydrate (Zn CHaCChh.ZI- O, 515 mg, 2.35 mmol, 1.5 equiv.) and methanol (30 mL). The reaction mixture was kept under stirring for 25 min between 50 °C and 70 °C, but in general at 65 °C. After the reaction was complete, the residue was washed and extracted with dichloromethane. The solvent was evaporated to dryness and the residue was purified by preparative thin layer chromatography using a mixture of dichloromethane:methanol (1%) as the eluent.

[0151] Synthesis of u racil -a Id itol s -Xyl,-Gal,-Glc

[0152] The uracil-alditols -Xyl,-Gal,-Glc were prepared in two steps according to the literature procedure [5,7], Their spectroscopic data are in accordance with the literature.

[0153] Synthesis of ZnChlegMe-Xyl,-Gal,-Glc (2, 3 and 4)

[0154] ZnChlegMe (1) (20 mg; 35.6 pmol; 1 equiv.) and uracil-alditol derivatives -Xyl,-Gal,-Glc (71.2 pmol; 2 equiv.) were dissolved in dry toluene (2 mL) and the reaction mixture was maintained under stirring for 1 h at 120 °C. After this time, the solvent was evaporated under reduced pressure. The crude was purified by thin-layer chromatography using dichloromethane-methanol (2%) mixture as eluent. The productswere obtained in 58, 56 and 57 % yield, respectively ZnChlegMe-Xyl (2), ZnChlegMe-Gal (3) and ZnChlegMe- Glc (4).

[0155] Synthesis of ChlegMe (5) and ChlegMe-Xyl,-Gal,-Glc (6, 7 and 8)

[0156] ZnChlegMe (1) and ZnChlegMe-Xyl (2), ZnChlegMe-Gal (3), ZnChlegMe-GIc (4) derivatives (20 mg; 16.3 pmol) were dissolved in chloroform (10 mL) and it was added trifluoroacetic acid (10%; 100 pL). The reaction mixture was maintained under stirring for 30 min. After this time, the reaction mixture was neutralized with sodium hydrogen carbonate and washed with water and dichloromethane. The products 5, 6, 7 and 8 were then purified by thin-layer chromatography using dichloromethane-methanol (2%) mixture as eluent.Photophysical Characterization:Spectrophotometric and spectrofluorimetric properties

[0157] The fluorescence quantum yields (<1>F) of the molecules 2-8 were obtained using a solution of ZnChlegMe (1) as standard (<t>F= 0.14, in DMF). In all measures in quartz cuvettes (lxl cm), it was recorded the absorption spectra of the compound and of the standard in DMF, and samples were excited at the wavelength of the absorbance (= 0.02-0.04) crossing point of both.Singlet Oxygen Generation

[0158] In quartz cuvettes (l x l cm), the absorbance of the solution of each molecule (1-8) in DMF was set to ~ 0.1 at 420 nm; then it was added an aliquot of a solution of DMA at 30 pM. The solutions were irradiated at 420 nm and the absorbance of the solution was monitored at 378 nm, each 60 s during 600 s.Biological Assays:Cell Culture

[0159] The MDA-MB-231 and MCF-7 cells were obtained from ATCC, the cells grow in RPMI-1640 medium (Gibco™, ThermoFisher Scientific) supplemented with 10% of fetal bovine serum (FBS) (Gibco™, ThermoFisher Scientific) and 1% penicillin / streptomycin (PEST) (Gibco™, ThermoFisher Scientific). PANC- 1 and HDF were obtained from ATCC; the cells grow in DMEM (Gibco™, ThermoFisher Scientific) high glucose medium supplemented with 10% of FBS and 1% PEST. HMEpC were purchased from CELL Application, Inc.; the cells grow in Mammary Epithelial cell medium (PromoCell) supplement with bovine pituitary extract (0.004 mL / mL), epidermal growth factor (10 ng / mL), insulin (5 pg / mL) and hydrocortisone (0.5 pg / mL) (PromoCell). Human Pancreatic CAF-Stellate Cells (CAFs) were obtained from Vitro Biopharma (USA); the cells grow in MSC-GRO™ Pancreatic Stellate CAFs Maintenance Medium. All cells were maintained in an incubator (Panasonic Healthcare Co, Ltd) at 37gC with 5% CO2 atmosphere.Photosensitizers

[0160] All the molecules (1-8) were soluble in DMSO; therefore, in all experiments, the stock solutions of the molecules were diluted to be added to the cells with a maximum of 0.1% (v / v) of DMSO. Additionally, the controls were prepared in the same way, never exceeding a maximum of 0.1% (v / v) of DMSO.Light source

[0161] For the 2D and 3D biological assays, a 12x8 LED array system with continuous light was used, with the option to also use pulsed light. This LED system emits red light with a centred peak emission at 652 nm and a bandwidth of 20 nm. The irradiance was set at 6.17 mW cm'2. For in vivo studies, a LUMACARE system with continuous light was used. LUMACARE emits white light (400-800 nm) from a halogen lamp delivered via fibre-optic bundle. The irradiance was set at 20 mw cm'2.Photodynamic Treatment in 2D culture

[0162] MDA-MB-231 (1.0 x 104cells / well), MCF-7 (1.5 x 104cells / well), PANC-1 (1.0 x 104cells / well), HDF (3.2 x 103cells / well) and HMEpC (1.0 x 104cells / well) cells were seeded in 96 well microplates and maintained until they reached 70-80% confluency; then, each chlorin derivative was added (1-8) at different concentrations, and the cells were incubated for 5 h. After that, the cells were washed with Dulbecco's phosphate buffer saline (DPBS) followed by addition of red phenol free RPMI and irradiation of the plate with red light (X = 652 ± 20 nm) at an irradiance of 6.17 mW.cm'2during 15 min. The microplate was incubated for another 48 h, and then the cells were fixed with 4% of paraformaldehyde. The cells were, then, washed twice with PBS. The viability assay was performed with crystal violet assay according to literature procedure [8], At the same time, a similar set up was used to study toxicity of the molecules in the dark, but in this case the cells were not irradiated. All incubations of cells were carried out in standard culture conditions (5% CO2 atmosphere incubator and at 37°C).2D Culture Confocal Images

[0163] MDA-MB-231 (3.1 x 104cells / well), MCF-7 (4.7 x 104cells / well), PANC-1 ((3.1 x 104cells / well), HDF (3.1 x 104cells / well) and similar number of HMEpC cells were plated onto 8-well culture microscopy slides and incubated for 24 h. Then, cells were treated with each molecule (1-8) at a concentration of 10 pM and incubated for 5 h. After that, cells were washed twice with DPBS and stained with Hoechst for 15 min; subsequently, the cells were washed twice with DPBS and stained with calcein-AM for 15 min. Finally, cells were washed with DPBS and red phenol free medium was added. To obtain the images, a Z-stack was acquired using a Zeiss LSM 880 with Airyscan microscope and the images shown are the result of maximum intensity projection. ImageJ software was used to quantify the fluorescence intensity as pixel intensity in the picture / total number of cells.3D micro-spheroids generation and characterization

[0164] Agarose gel (2%) molds with 81-wells that allows the formation of 3D spheroids in each well were placed onto 12-well plates. MDA-MB-231 cells or PANC-1 were seeded (180 pL) in the agarose molds (1.0 x 104cells / spheroid) and incubated at 37gC with 5% CO2 atmosphere for 7 days.Photodynamic treatment in 3D Micro-spheroids

[0165] MDA-MB-231 cells were seeded into agarose gel molds (1.0 x 104cells / spheroid) and incubated for 7 days. The MDA-MB-231 spheroids were treated with 180 pL of molecule 6 at different concentrations and incubated for 5 h. Then, the spheroids were washed with DPBS and red phenol free RPMI was added, then the spheroids were irradiated with red light (X = 652 ± 20 nm) at an irradiance of 6.17 mW.cm'2during 15 min. Afterwards, the spheroids were incubated for 48 h. The viability of 3D micro-spheroids was assessed using RPMI, and CellTiterGlo reagent in a ratio of 1:1. The spheroids were incubated at 37 °C for 5 min under stirring, followed for additional 25 min incubation at room temperature. The luminescence was measured in a microplate reader (Synergy HTX). At the same time, to evaluate toxicity of the molecule in the dark, a similar study was performed, but the spheroids were not irradiated.3D Micro-Spheroids Confocal images

[0166] The 3D micro-spheroids of MDA-MD-231 or PANC-1 cells were prepared as previously described and were incubated for 7 days. Subsequently, the spheroids were treated with molecule 5 and 6 at 10 pM for 5, 24 and 48 h. The spheroids were washed twice with DPBS and were fixed with 4% paraformaldehyde for 20 min. Afterwards, the spheroids were washed twice with PBS and stained with Hoechst for 15 min and finally washed twice with PBS. To obtain the images, a Z-stack was acquired using a Zeiss LSM 880 with Airyscan microscope and the images shown are the result of maximum intensity projection. ImageJ software was used to quantify the fluorescence intensity as pixel intensity in the picture.Photodynamic treatment in heterotypic PDAC stratified tumor spheroid models (STAM)

[0167] STAMs scaffold-free models were generated by using the liquid overlay technique (LOT). From this standpoint, tumor-associated cells were dispensed in a temporal-controlled manner in ultra-low adhesion (ULA) plates that promote cellular self-aggregation into a scaffold-free microtumor. For assembling heterotypic 3D STAMS, a two-step strategy was established. In a first step a 3D spheroid core comprising PANC-1 cells (1.0 x 104cells per model) was placed in each well of the plate. At day 6 of maturation, a suspension of human pancreatic cancer-associated fibroblasts (CAFs, 4.0 x 104cells) was seeded in the wells containing the tumor core spheroid composed by cancer cells, in order to establish the stratified 3D heterotypic model. The tumor-stroma model was matured for 14 days. Then, the STAMs were treated with molecule 5 and 6 at 500 nM for 5 h. After that, the STAMs were washed with DPBS and it was added red phenol free DMEM; the plate was irradiated with red light (X = 652 ± 20 nm) at an irradiance of 6.17 mW.cm'2during 15 min and incubated for 48 h. The viability of STAMs was assessed using DMEM and CellTiterGlo as explained before, and the viability of luciferase were assessed usingDMEM and OneGlo reagent in a ratio of 1:1. and then it was measured the luminescence with a multiwell plate reader (Tecan Infinite M200).STAM Confocal Images

[0168] The STAMs were prepared as previously described. Then, the STAMs were treated withjnolecule 5 and 6 at 10 pM for 5 h. Afterwards, the STAMs were washed with DPBS and fixed with PFA 4% during 20 min; then it was washed twice with PBS and stained with Hoechst for 15 min and washed twice with PBS. To obtain the images, a Z-stack was acquired using a Zeiss LSM 880 with Airyscan microscope and the images shown are the result of maximum intensity projection.Combinatory effect of Chemotherapy and PDT

[0169] PANC-1 cells were seeded (1.0 x 104cells / well) in 96 well microplates and maintained for 48 h; then it was added molecule 6 at I C5o concentration and incubated for 5 h. After that, the cells were washed with DPBS and red phenol free DMEM was added, and the plates were irradiated with red light (X = 652 ± 20 nm) at an irradiance of 6.17 mW.cm'2during 15 min. Afterwards, Gemcitabine (GEM) was added in its IC20 and IC50 concentrations. Controls of cells without molecules, and only with Gemcitabine were made. The microplate was incubated for another 48 h, and then the cells were fixed with 4% of paraformaldehyde. The cells were, then, washed twice with PBS. The viability assay was performed with crystal violet assay according to literature procedure [8], At the same time, a similar study was performed, but the cells were not treated with light.Mammospheres / Tumorsphere Formation

[0170] MDA-MB-231 or PANC-1 (2.95 x 104cells / well) were seeded in 100 mm plates and maintained in an incubator during 24 h; following that, molecule 6 was added at 50 nM for MDA-MB-231 or at 8.66 nM for PANC-1. For the control 0.1% DMSO was added. The plates were incubated for 5 h. Thereafter, the cells were washed with DPBS and phenol red-free RPMI 1640 or DMEM was added, and the plates were irradiated with red light (X = 652 ± 20 nm) at an irradiance of 6.17 mW.cm'2during 15 min. The plates were then incubated for another 48 h. The cells (MDA-MB-231 and PANC-1) were trypsinized, then seeded (1.0 x 104cells / well) into 12-well plates coated with agarose gel 2% and incubated for 72 h. At the endpoint mammospheres / tumorspheres were observed in EVOS M5000 microscope. FIJI ImageJ software was used to measure the area of the mammospheres / tumorspheres.In vivo Cytotoxicity and Biodistribution

[0171] C57BL / 6 mice (2 males and 3 females) were injected intraperitoneally with molecule 6 at 0.25 mg / kg or 0.75 mg / kg. The animals were kept, under vigilance, in a room with a normal 12 h light / 12 h dark cycle program for 5 or 24 h. At 5 or 24 h, the animals were sacrificed, and the organs and tissues were collected for subsequent analysis. Molecule 6 was extracted by mixing ~50 mg of each tissue and organ with SDS 2% (500 mL) and each sample was homogenized with a tissue homogenizer (Potter-Elvehjem). After that, each homogenate was kept 1 h under stirring in a rotary stirrer protected from light. Then, the samples were centrifuged 3 min at 10,000 rpm, and 100 pL of each sample were added to 1900 pL DMF. Following that, the samples were again centrifuged 3 min at 10 000 rpm and the supernatant was then collected, and fluorescence of molecule 6 present in the extracts was measured with a Horiba- Fluoromax Plus fluorimeter (excitation wavelength at 397 nm slit 10 nm, emission wavelength range from 600 to 750 nm, slit 5 nm). The concentration of molecule 6 accumulated in each organ and tissue was determined using a calibration curve where a known different PS concentrations were dissolved in the same extraction solvent (SDS:DMF) (plot of fluorescence intensity versus concentration).In vivo long-term cytotoxicity

[0172] C57BL / 6 mice (10 males) were injected intraperitoneally with molecule 6 at 0.75 mg / kg (n = 5) or vehicle (1% DMSO in PBS; n= 5). A small area of the skin was shaved. The animals were kept ad libitum, under vigilance for 31 days. After that, the mice were sacrificed. The organs and tissues were collected for subsequently analysis.In vivo phototoxicity

[0173] C57BL / 6 mice were injected subcutaneously with E0771 cells (1.0 x 10scells / 100 pL / mice) in the left inguinal flank. The animals were kept ad libitum, under vigilance, and the tumor grow was monitored. After 15 days, the tumoral area was depilated with over-the-counter face depilatory cream for human use, and the mice were injected intratumorally with molecule 6 at 0.75 mg / kg (n = 3) or vehicle (1% DMSO in PBS; n= 3). The animals were kept ad libitum, under vigilance for 5 h. Then, the tumors were irradiated with white light (LUMACARE system) for 2 min (20 mW cm'2). After that, the animals were kept ad libitum, under vigilance, and the tumor volume was recorded at 24 h, 48 h, 96 h and 120 h. The mice were then sacrificed, and the organs, tissues and tumors were collected for subsequently analysis.In vivo biodistribution

[0174] C57BL / 6 mice were injected subcutaneously with E0771 cells (1 x 10scells / 100 pL / mice) in the left inguinal flank. The animals were kept ad libitum, under vigilance, and the tumor grow was monitored. The mice were injected intratumorally (n=l) or intraperitoneally (n=l) with molecule 6 at 0.75 mg / kg. The animals were kept ad libitum, under vigilance for 5 h. After that, the mice were sacrificed, and the organs, tissues and tumors were collected for subsequently analysis.Haematoxylin and Eosin (H&E) Staining

[0175] Organs (skin, liver, pancreas, spleen, kidneys and intestine) and tumors were fixed using 10% formalin for at least 24 h, then dehydrated through increasing alcohols, respectively 70% (30 min, 2 changes), 80% (30 min, 2 changes), 96% (30 min, 2 changes) and 100% (45 min, 2 changes), and xylene (1 h, 2 changes). The organs were paraffin-embedded for 1 h (2 changes), following by paraffin blocksinclusion. The paraffin blocks were sectioned in a microtome (5 pm thick), and thereafter the slides containing tissue sections were deparaffinized in xylene (5 min, 2 changes) and rehydrated through decreasing alcohols: 100% (5 min, 2 changes), 96% (5 min), 70% (5 min) and 50% (5 min). After that the slides were rinsed with tap water and then stained with H&E. Briefly, slides were stained with Harris Haematoxylin for 10 min. The slides were washed with distilled water and differentiated in acid alcohol (5 dips), followed by immersion in distilled water. The nuclei were stained by placing the slides in tap water for 10 min. Thereafter, slides were rinsed with distilled water and immersed in 96% alcohol. The slides were immersed in eosin for 3 s and washed in tap water for 2 min. Finally, the slides containing the tissues sections were dehydrated though increasing alcohols: 96% (10 dips, 2 changes), 100% (10 dips, 2 changes) and then xylene (2 min, 2 changes). The slides were then mounted in DPX medium, and histopathological changes were observed using a Zeiss bright field microscope.

[0176] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0177] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[0178] The following dependent claims further set out particular embodiments of the disclosure.References1. Abrahamse, H; Hamblin, M. New photosensitizers for photodynamic therapy. Biochem J. 2016, 473, 347-364, doi:10.1042 / BJ20150942.2. Mesquita, M.Q.; Dias, C.J.; Neves, M.G.P.M.S.; Almeida, A.; Faustino, M.A.F. Revisiting current photoactive materials for antimicrobial photodynamic therapy. Molecules 2018, 23, 2424-2470, doi:10.3390 / molecules23102424.3. Mesquita, M.Q.; Dias, C.J.; Gamelas, S.; Fardilha, M.; Neves, M.G.P.M.S.; Faustino, M.A.F. An insight on the role of photosensitizer nanocarriers for Photodynamic Therapy. Ann. Brazilian Acad. Sci. 2018, 90, 1101-1130, doi:10.1590 / 0001-3765201720170800.4. Dias, C.J.; Helguero, L.; Faustino, M.A.F. Current Photoactive Molecules for Targeted Therapy of Triple-Negative Breast Cancer. Molecules 2021, 26, 7654, doi:10.3390 / molecules26247654.5. Palasz A, Kalinowska-Tluscik J, Jablonski M Application of 2,4,6-trioxo-pyrimidin-5-ylidene alditols in the synthesis of pyrano [2, 3-d] pyrimidines containing a sugar moiety by hetero-Diels-Alder reactions and by conjugate Michael addition-cyclizations. Tetrahedron 2013, 69, 8216-8227.6. Monteiro, M. V.; Gaspar, V.M.; Mendes, L.; Duarte, I.F.; Mano, J.F. Stratified 3D Microtumors as Organotypic Testing Platforms for Screening Pancreatic Cancer Therapies. Small Methods 2021, 5, 1-15, doi:10.1002 / smtd.202001207.7. Armarego, W.L.F. Purification of Laboratory Chemicals; fourth ed. Oxford: Butterworth- Heinemann, 1996; ISBN 0128054565.8. Feoktistova, M.; Geserick, P.; Leverkus, M. Crystal violet assay for determining viability of cultured cells. ColdSpring Harb. Protoc. 2016, 2016, 343-346, doi:10.1101 / pdb.prot087379.

Claims

C L A I M S1. Compound of general formula (I) or a pharmaceutically acceptable salt thereof:(Formula I) wherein:M, R, Ri and R2 are independently selected from each other;M is selected from the list consisting of: Zn, 2H, Pd, Ru, Ni, Cu, Pt, Ir, In, Ga, Gd, Mn, Fe, Al, Si, Eu;Ri is a substituted or non-substituted alkyl C1-C10;R2 is a substituted or non-substituted alkyl C1-C10;R is selected from the list consisting of:

2. Compound of general formula (I) or (II) or a pharmaceutically acceptable salt thereof:for use in medicine, wherein:M, R, Ri and R2 are independently selected from each other;M is selected from the list consisting of: Zn, 2H, Pd, Ru, N i, Cu, Pt, Ir, In, Ga, Gd, Mn, Fe, Al, Si, Eu;Ri is a substituted or non-substituted alkyl C1-C10;R2 is a substituted or non-substituted alkyl C1-C10;R is selected from the list consisting of:

3. Compound according to any of the previous claims wherein M is selected from Zn, Pd, Ru, Ir, Mn.

4. Compound according to any of the previous claims wherein M is Zn(ll) or 2H.

5. Compound according to any of the previous claims wherein Ri is a non-substituted alkyl Ci-Cio.

6. Compound according to any of the previous claims wherein Ri is a non-substituted alkyl Ci-C5; preferably a non-substituted alkyl C1-C3; more preferably Ri is CH3.

7. Compound according to any of the previous claims wherein R2 is a non-substituted alkyl C1-C10.

8. Compound according to any of the previous claims wherein R2 is a non-substituted alkyl Ci-C5; preferably a non-substituted alkyl C1-C3; more preferably R2 is CH3.

9. Compound according to any of the previous claims wherein R is selected from the list consisting of:

10. Compound according to any of the previous claims wherein the compound is:

11. Compound according to any of the previous claims wherein the compound is:

12. Compound according to the previous claim 2 wherein the compound is:

13. Compound according to any of the previous claims wherein the compound is:

14. Compound according to any of the previous claims 2-13 for use in photodynamic therapy.

15. Compound according to any of the previous claims 2-13 for use in the prevention, treatment or diagnostic of cancer.

16. Compound according to any of the previous claims 2-13 for use in the prevention, treatment or diagnostic of hyperproliferation or neoplasia.

17. Compound according to any of the previous claims 2-13 for use in the prevention, treatment or diagnostic of a disease characterized by benign or malignant cellular hyperproliferation or by areas of neovascularisation or a cancer.

18. Compound according to any of the previous claims 2-13 for use in the prevention, treatment or diagnostic of hyperproliferative tissue, in particular hyperproliferative tissue associated to cancer, carcinoma and / or myeloma.

19. Compound according to any of the previous claims 2-13 for use in the prevention, treatment or diagnostic of precancerous conditions and / or macular degeneration.

20. Compound for use according to the previous claim 15 wherein the cancer is selected from a list consisting of: skin cancer, basal cell carcinoma, squamous cell carcinoma, actinic keratosis, lung cancer, non-small cell lung cancer, esophageal cancer, bladder cancer, head and neck cancers, oral cavity cancer, laryngeal cancer, nasopharyngeal cancer, Barrett's esophagus, cholangiocarcinoma, bile duct cancer, prostate cancer, Paget's diseases of breast and vulva pancreatic cancer, breast cancer, colon cancer, liver cancer, osteosarcoma, glioblastoma, gastric cancer, ovarian cancer; preferably breast cancer or pancreatic cancer.

21. Compound according to any of the previous claims 2-20 for use in the prevention or treatment of cancer in which cancer- associated fibroblasts (CAFs) are involved.

22. Compound according to any of the previous claims 2-21 for use in combination with at least an agent selected from a list consisting of: chemotherapy agent; radiotherapy agent; thermal therapy agent; hormonal therapy agent; immunotherapy agent; targeted therapy agent; or mixtures thereof; preferably chemotherapy agent.

23. Compound for use according to the previous claim wherein the chemotherapy agent is selected from the list consisting of: doxorubicin, cisplatin, paclitaxel, 5-fluorouracil, mitomycin c, carboplatin, docetaxel, irinotecan, cyclophosphamide, methotrexate, vincristine, epirubicin, bleomycin, oxaliplatin, gemcitabine, or mixtures thereof; preferably the chemotherapy agent is gemcitabine.

24. Compound for use according to the previous claim 22 wherein the immunotherapy agent is selected from the group consisting of: durvalumab, tremelimumab, or mixtures thereof.

25. Compound for use according to the previous claim 22 wherein the targeted therapy agent is selected from the group consisting of: imatinib, alectinib, ibrutinib, palbociclib, bevacizumab, or mixtures thereof.

26. Compound for use according to the previous claim 22 wherein the hormonal therapy agent is selected from the list consisting of: anastrazole, letrozoloe, exemestane, fulvestrant, elacestrant, tamoxifen, raloxifene, toremifene, leuprolide, goserelin, triptorelin, histrelin, enzalutamide, apalutamide, darolutamide, bicalutamide, flutamide, nilutamide, or mixtures thereof.

27. Compound according to any of the previous claims 2-26wherein the compound is administrated by oral, parenteral, intravenous, intramuscular, intranasal, sublingual or intratracheal route.

28. Compound according to any of the previous claims 1-T1 wherein the compound is administrated 1 to 100 h before photodynamic therapy procedure; preferably 3 to 60 h before photodynamic therapy; more preferably 10-50 h before photodynamic therapy.

29. Substance delivery carrier comprising the compound described in any of the previous claims 2-13, wherein said carrier promotes specific substance delivery to a cancer- associated fibroblast.

30. Composition for use in treating cancer in which cancer- associated fibroblasts are involved, wherein said composition comprises a substance delivery carrier according to the previous claim 29.

31. Pharmaceutical composition comprising at least one of the compounds according to any of the previous claims 2-13.

32. Pharmaceutical composition according to the previous claim further comprising a pharmaceutically acceptable carrier.

33. Pharmaceutical composition according to any of the previous claims 31-32 comprising a combination of at least a compound according to any of the previous claims 1-13 and at least a chemotherapy agent, an immunotherapy agent, or a targeted therapy agent according to any of the previous claims 23-26; preferably a chemotherapy agent according to the previous claim 23; more preferably the chemotherapy agent is gemcitabine.

34. Pharmaceutical composition according to any of the previous claims 31-33 further comprising a surface penetration enhancer.

35. Kit comprising a compound as described in any of the previous claims 1-13 and / or a pharmaceutical composition as described in any of the previous claims 31-34.

36. The use of the compounds according to any of the previous claims 2-13 for the manufacture of a medicament for the prevention, treatment or diagnostic of cancer.

37. A method for treating or preventing cancer in a subject, the method comprising administering the compound according to any of the previous claims 2-13 to the subject.

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