CIP3m, a sn38 / gemcitabine conjugate and uses thereof in the treatment of cancers

Abstract

The present invention provides CIP3M, a SN38 / gemcitabine conjugate that is useful as an anti-cancer agent. The present invention also provides pharmaceutical composition comprising CIP3M, and the use of CIP3M and pharmaceutical compositions thereof, as therapeutic agents, in particular in the treatment of cancers, including metastatic cancers and chemoresistant cancers.

Description

[0001] CIP3M, a SN38 / Gemcitabine Conjugate and Uses thereof in the Treatment of Cancers

[0002] Related Application

[0003] The present application claims priority to European Patent Application No. EP 24 186 833.0 filed on July 5, 2024, which is incorporated herein by reference in its entirety.

[0004] Background of the Invention

[0005] Cancer is among the leading causes of mortality in developed countries. In 2022, there were an estimated 20 million new cancer cases and 9.7 million deaths worldwide (^Global Cancer Observatory", website: geo. iarc.fr / today / en, International Agency for Research on Cancer (IARC)). The IARC estimates, which use data covering 185 countries and 36 cancers, show that 10 types of cancer collectively comprised around two-thirds of new cases and deaths globally in 2022. Lung cancer was the most commonly occurring cancer worldwide accounting for 12.4% of the total new cases, followed by female breast cancer (11.6%), colorectal cancer (9.6%), prostate cancer (7.3%), and stomach cancer (4.9%). Lung cancer was also the leading cause of cancer death (18.7% of the total cancer deaths) followed by colorectal cancer (9.3%), liver cancer (7.8%), breast cancer (6.9%), and stomach cancer (6.8%).

[0006] Despite the endeavors and achievements made in treating cancers during the past decades, disease recurrence and progression remain a major obstacle to therapy. One of the main clinical issues is the development of drug resistance. Many tumors (e.g., colon cancer, pancreatic cancer, glioblastoma, and prostate cancer) are intrinsically resistant to many of the more potent cytotoxic agents used in cancer therapy. Other tumors (e.g., breast cancer), initially sensitive, recur and become resistant to the initial therapeutic agents and, very often, also resistant to other cancer drugs which were not used in the initial treatment. In addition to reducing clinical effectiveness, chemoresistance results in early termination of treatment, reduced relapse-free interval and survival. Resistance also represents a major hurdle to eradicating metastatic cancer, as metastatic tumors have higher levels of resistance to systemic conventional chemotherapies compared with primary tumors. Many strategies have been designed to combat drug resistance, either by combining currently available chemotherapeutic agents or by developing novel therapies.

[0007] Traditional chemotherapy mostly relies on the use of cytotoxic agents, which are typically unable to preferentially localize at the tumor microenvironment resulting in off- target toxicity. Improving the selectivity of the drug distribution between tumor / normal cells is challenging because of the close similarity between cancer cells and normal cells. The situation is still more complicated when dealing with a cocktail of several anticancer drugs. Two different drugs exhibit two different pharmacokinetics and two different tissue biodistributions - it is therefore very difficult to anticipate the ratio of these two drugs in tumor and normal cells.

[0008] Targeted therapy, which improves selectivity, has become an effective strategy of precision medicine for improving cancer treatment. The use of Antibody-Drug Conjugates (ADCs), a robust strategy for targeted therapy, applies to selectively deliver a potent cytotoxic compound to tumor cells and thus improve the therapeutic efficacy of the chemotherapeutic agents. Three ADC products (trastuzumab emtansine, brentuximab vedotin and inotuzumab ozogamicin) are already on the market, and several other ADC compounds are in clinical trials. Compared to ADCs, Small Molecule-Drug Conjugates (SMDCs), which are built with three modules: a targeting ligand, a linker and a drug payload, provide a new, less established perspective for targeted delivery. Nevertheless, SMDCs have several strengths: in particular they are of a non-immunogenic nature, they have lower molecular weights to support good penetration into solid tumors, and their synthesis is more manageable and cost effective (Patel et al., New J. Chem., 2021, 45: 5291; Hasan et al., Pharmacol. Res. Perspect., 2018, 6(4): e00417 ). The development of SMDCs has resulted in a number of compounds entering clinical trials, most of which exploit folate as a targeting small molecule. However, none has been approved for clinical use so far.

[0009] Although promising therapeutic approaches for cancer treatment are being developed and tested in clinical trials, there is still a need in the art for improved chemotherapeutic agents.

[0010] Summary of the Invention

[0011] The present Inventors have developed a new compound called CIP3M, which exists as an equimolar mixture of two diastereoisomers and is the first drug candidate of a new class of Small Molecule Drug Conjugates (SMDCs). CIP3M is a conjugate consisting of gemcitabine and SN-38 (which is the active metabolite of the prodrug irinotecan (CPT- 11)) covalently linked via an azlactone-based heterofunctional linker. CIP3M displays a unique mechanism of action inhibiting simultaneously DNA replication and DNA synthesis, two validated anticancer targets, resulting in antiproliferative activity in a large range of cancer cell subtypes. Indeed, it was found to exhibit in vitro activities at low nanomolar concentrations against a wide variety of tumor cell lines originating from lymphoma and from colon, pancreas, ovaries, brain, lung, breast, kidney, pharynx, prostate cancers, etc. CIP3M was observed to be particularly active on tumor cells that are sensitive and resistant to irinotecan, SN38, or gemcitabine. In addition, irrespective of the human tumor cell line tested, the potency of each of the diastereoisomers of CIP3M was found to be equivalent to that of the equimolar mixture of two diastereoisomers. The in vivo plasma stability of CIP3M has been validated in vivo using an intravenous administration in mice, showing a plasma half-life of more than 2.2 hours. The absence of toxicity of acute and repeated intravenous administration of CIP3M has been verified in mice. CIP3M has demonstrated in vivo antitumor activity in a xenograft model of human small cell lung cancer in immunodeficient mice using repeated intravenous administration.

[0012] Consequently, the present invention provides a SN38 / gemcitabine conjugate, CIP3M, having chemical formula (I), or a physiologically acceptable salt thereof.

[0013] In certain embodiments, CIP3M is an equimolar mixture of two diastereoisomers.

[0014] In other embodiments, CIP3M is CIP3M diastereoisomer of formula (II), or a physiologically acceptable salt thereof; or is CIP3M diastereoisomer of formula (III), or a physiologically acceptable salt thereof.

[0015] In yet other embodiments, CIP3M is a non-equimolar mixture of CIP3M diastereoisomer of formula (II) and CIP3M diastereoisomer of formula (III), or of physiologically acceptable salts thereof.

[0016] The present invention further relates to a pharmaceutical composition comprising an effective amount of a conjugate as defined above, and at least one pharmaceutically acceptable carrier or excipient.

[0017] In certain embodiments, the pharmaceutical composition further comprises at least one additional biologically active agent. The present invention also relates to a conjugate as defined above or a pharmaceutical composition as defined above for use as a therapeutic agent.

[0018] The present invention also relates to a conjugate as defined above or a pharmaceutical composition as defined above for use in the treatment of a proliferative cell disorder in a subject.

[0019] The present invention also relates to a conjugate as defined above or a pharmaceutical composition as defined above for use in the treatment of a cancer in a subject.

[0020] In certain embodiments, the cancer is a carcinoma, a lymphoma, a blastoma, a sarcoma, a myeloma, or a leukemia.

[0021] In certain embodiments, the cancer is selected from the group consisting of brain cancer, colon / colorectal cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, leukemia, lymphoma, myeloma, kidney cancer, head and neck cancer, urinary cancer, gastric cancer, soft tissue cancer, prostate cancer, and liver cancer.

[0022] In certain embodiments, the cancer is a small-cell lung cancer or a meningioma.

[0023] In certain embodiments, the cancer is leukemia, lymphoma or myeloma.

[0024] In certain embodiments, the cancer is a metastatic cancer.

[0025] In certain embodiments, the cancer is a chemoresistant cancer.

[0026] These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

[0027] Brief Description of the Drawing

[0028] Figure 1. Chemical formula of CIP3M (I), CIP3M diastereoisomer 1 (II), CIP3M diastereoisomer 2 (III), camptothecin / gemcitabine conjugate (IV) and the azlactone- based heterofunctional linker (V) present in CIP3M and in the camptothecin / gemcitabine conjugate.

[0029] Figure 2. (A) Scheme for the optimized chemical synthesis of CIP3M using a click reaction between alkyne 3 and Azl-N3 -Gemcitabine. (B) Scheme of the preparation of alkyne 3 from SN38 (Equation 1) and of the preparation of Azl-N3 -Gemcitabine by aminolysis of the azlactone-N3 (Equation 2). Figure 3. (A) Scheme of the preparation of the two diastereoisomers of Azl-N3- Gemcitabine by chiral HPLC. (B) Scheme of the preparation of two diastereoisomers of CIP3M by coupling of the two separate diastereoisomers of Azl-N3 -Gemcitabine with alkyne 3.

[0030] Figure 4. Microsomal stability of CIP3M in human plasma and in C57BM / 6 mouse plasma over time.

[0031] Figure 5. Concentration of CIP3M in C57BL / 6 plasma per time point after iv administration at a concentration of 3 mg / kg.

[0032] Figure 6. Antiproliferative activity of CIP3M on a large panel of cancer cell lines exemplifying 18 different indications (leukemia, lymphoma, myeloma, kidney, head and neck, breast, urinary, gastric, pancreas, lung, soft tissue, prostate, ovary, colorectal, liver, skin, esophagus, and endometrium). Each point indicates the sensitivity of one cell line. Higher is the value, higher is the sensitivity of the cell line to CIP3M.

[0033] Figure 7. Reduction of the individual volume of human small cell lung cancer NCI- H1048 tumors xenografted in immunocompromised mice (n=5-7) following intravenous administration of (A) the vehicle alone, (B) 10 mg / kg of one of the CIP3M diasteroisomers, and (C) 10 mg / kg of CIP3M. The treatment schedule was as follows: 5 days on - 2 days off, repeated twice.

[0034] Definitions

[0035] Throughout the specification, several terms are employed that are defined in the following paragraphs.

[0036] As used herein, the term “subject’ refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, cow, goat, mouse, rat, rabbit, and the like), that can develop a disease or disorder, in particular a cancer, but may or may not be suffering from the disease or disorder. Non-human subjects may be transgenic or otherwise modified animals. In most embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual” or a “patient” . The terms “individual” and “patient” do not denote a particular age. The term “patient” more specifically refers to an individual suffering from a disease or disorder, in particular a cancer. Thus, the term “cancer patient” refers to an individual suffering from a cancer. A cancer patient may or may not have been diagnosed with cancer. The term also includes individuals that have previously undergone therapy for cancer.

[0037] As used herein, the term “cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth, lack of differentiation and ability to invade local tissues and metastasize. Cancer can develop in any tissue of any organ. Examples of cancers include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia. More particularly, examples of such cancers include bone cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin’ s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

[0038] The terms “aggressive” and “invasive” are used herein interchangeably. When used herein to characterize a cancer, they refer to the proclivity of a tumor for expanding beyond its boundaries into adjacent tissue. Invasive cancer can be contrasted with organ- confined cancer wherein the tumor is confined to a particular organ. The invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, which degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the capsule, and beyond confines of the particular tissue in which that tumor is located.

[0039] The term “metastasis” , as used herein, refers to the spread of tumor cells from one organ or tissue to another location. The term also refers to tumor tissue that forms in a new location as a result of metastasis. A “metastatic cancer” is a cancer that spreads from its original, or primary, location, and may also be referred to as a “secondary cancer” or “secondary tumor”. Generally, metastatic tumors are named for the tissue of the primary tumor from which they originate. The process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location. The term “ chemoresistant cancer refers to a cancer that exhibits chemotherapy resistance or chemoresistance. The terms chemotherapy resistance and “ chemoresistance are used herein interchangeably. They refer to the insensitivity of cancer cells to chemotherapy, i.e. to the ability of cancer cells to avoid the intended therapeutic cytotoxic effects of a chemotherapeutic agent. Chemoresistance can be intrinsic or can develop over time, for example after repeated exposures to anticancer therapy. In contrast, chemosensitivity' refers to the susceptibility of cancer cells to the cytotoxic effects of a chemotherapeutic agent. Changes in chemosensitivity and chemoresistance can be measured by comparing the toxic effects of chemotherapy after a single treatment (e.g., in different patients) or by assessing changes in toxicity over the course of repeated treatments (e.g., in the same patient or different patients). An increase in chemosensitivity or decrease in chemoresistance of a cancer cell means an improvement in the therapeutic efficacy of a chemotherapeutic agent.

[0040] The term “treatment" is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease, disorder or condition (here: a cancer); (2) slowing down or stopping the progression, aggravation or deterioration of the disease, disorder or condition; (3) bringing about amelioration of the symptoms of the disease, disorder or condition; or (4) curing the disease, disorder or condition. A treatment may be administered after initiation of the disease, disorder or condition, for a therapeutic action. Alternatively, a treatment may be administered prior to the onset of the disease, disorder or condition, for a prophylactic or preventive action. In this case, the term prevention is used.

[0041] The term “combination therapy refers to a treatment of a disease (in particular here: a cancer), or a symptom thereof, or to a method for achieving a desired physiological change, which includes administering an effective amount of two or more chemical agents to treat a disease, or a symptom thereof, or to produce a physiological change, wherein the chemical agents are administered together, such as part of the same composition, or administered separately and independently at the same time or at different times (z.e., administration of each agent is separated by a finite period of time from each other). The term “combination therapy" also refers to a treatment of a disease (in particular here: a cancer), or a symptom thereof, or to a method for achieving a desired physiological change, including administering an effective amount of at least one chemical agent and at least one therapeutic procedure (e.g., surgery, radiotherapy, etc...), wherein the chemical agent and the therapeutic procedure are administered together, or separately and independently.

[0042] As used herein, the term “dosage regimen" refers to the schedule of drug administration, including formulation, route of administration, drug dose, dosing interval and treatment duration.

[0043] A “pharmaceutical composition" is defined herein as comprising an effective amount of at least one of: CIP3M (the SN38 / gemcitabine conjugate described herein), the CIP3M diastereoisomer of formula (II), the CIP3M diastereoisomer of formula (III), a physiologically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0044] The term “physiologically acceptable salt" refers to any acid addition or base addition salt that retains the biological activity and properties of the free base or free acid, respectively, and that is not biologically or otherwise undesirable. Acid addition salts are formed with inorganic acids (e.g., hydrochloric, hydrobromic, sulfuric, nitric, phosphoric acids, and the like); or organic acids (e.g., acetic, propionic, pyruvic, maleic, malonic, succinic, fumaric, tartaric, citric, benzoic, mandelic, methanesulfonic, ethanesulfonic, p- toluenesulfonic, salicylic acids, and the like). Base addition salts can be formed with inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium, magnesium, zinc, aluminum salts, and the like) or organic bases (e.g., salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethyl-aminoethanol, 2- diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, poly amine resins, and the like).

[0045] As used herein, the term “effective amount' refers to any amount of a compound (e.g., CIP3M), agent, or composition that is sufficient to fulfil its intended purpose(s), e.g., a desired biological, diagnostic, or medicinal response in a cell, tissue, system or subject. For example, an effective amount of CIP3M is an amount that can elicit a measurable amount of a desirable outcome, e.g., in a method of treatment, an amount that can reduce or ameliorate by a measurable amount, a symptom of the disease or condition that is being treated.

[0046] As used herein, the term pharmaceutically acceptable carrier or excipient refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s), and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion media, coatings, encapsulating material, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example “Remington ’s Pharmaceutical Sciences’", E.W. Martin, 18thEd., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety). Preferably, the pharmaceutical carrier or excipient is acceptable in human medicine. In certain embodiments, the pharmaceutically acceptable carrier or excipient is a veterinary acceptable carrier or excipient.

[0047] The terms approximately and “about", as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

[0048] Detailed Description of Certain Preferred Embodiments

[0049] As mentioned above, the present invention provides CIP3M, a SN38 / gemcitabine conjugate that is useful as an anti-cancer agent. The present invention also provides pharmaceutical compositions comprising CIP3M and the use of CIP3M, and pharmaceutical compositions thereof, as therapeutic agents, in particular in the treatment of cancers.

[0050] I - CIP3M

[0051] The present invention provides a new molecule, CIP3M of formula (I), which is a conjugate consisting of gemcitabine and SN38 covalently linked to each other via an azlactone-based heterofunctional linker.

[0052] As described in Example 1, CIP3M has been designed based on results of a project aimed at the development of anticancer molecules, and more particularly of “twin-drugs” or “multivalent drugs”, i.e., new drug candidates containing two or more pharmacophores covalently bound together via linkers. The linkers used have been developed by Prof. Fontaine, and are described in international patent application publication No. WO 2014 / 060357 Al ^Multifunctional Coupling Reagents Having an Azlactone Function''’, published on April 24, 2014). Within the framework of this project, interesting physicochemical and biological properties, in particular high antiproliferative activity, were only demonstrated for 5 of the hundred of homodimeric, heterodimeric and multimeric molecules tested. The molecule with the most potent antiproliferative activity on a wide panel of cells from different organs was found to be the camptothecin / gemcitabine conjugate of formula (IV) (see Figure 1). In order to increase the overall safety of the conjugate, the Inventors replaced the camptothecin part of the molecule with SN-38, an active metabolite of the chemotherapeutic agent irinotecan (or CPT-11), which is itself a derivative of camptothecin. The antiproliferation properties of CIP3M are all the more surprising that antagonistic effects have been reported for a combination of gemcitabine and SN-38 administered simultaneously to certain cancer cells (Shanks et al., Clin. Cancer Res., 2005, 11(11): 4225-4233; Peralta et al., Mol. Pharmacol., 2008, 74(3): 724-735).

[0053] 1 - The CIP3M Molecule

[0054] A. Gemcitabine

[0055] Gemcitabine (2 ’, 2 ’ -di fl uoro-2’ -deoxy cytidine, or dFdC) is a cytidine analogue active against several solid tumor types. It is sold under the brand name GEMZAR® among others. Gemcitabine is an antimetabolic specific of the S phase of the cell cycle, the DNA synthesis phase: it acts by blocking chain elongation and creation of new DNA, which results in cell death. Clinically, gemcitabine is used to treat various carcinomas: it is used as first-line treatment alone for pancreatic cancer, and in combination with cisplatin for advanced or metastatic bladder cancer and advanced or metastatic non-small cell lung cancer. It is used as a second-line treatment in combination with carboplatin for ovarian cancer and in combination with paclitaxel for breast cancer that is metastatic or cannot be surgically removed. Gemcitabine is on the World Health Organization’ s Model List of Essential Medicines.

[0056] B. SN-38, CPT-11

[0057] SN-38 ((45)-4,l l-diethyl-4,9-dihydroxy-l,4-dihydro-3H,14H-pyrano[3’,4’:6,7] indolizino[l,2-b]quinoline-3, 14-dione or 7-ethyl-10-hydroxycamptothecin) is an antineoplastic drug. It is the active metabolite of the prodrug irinotecan (an analog of camptothecin - a topoisomerase-I inhibitor). SN-38 has 1000 times more activity than irinotecan (CPT-11) itself. CPT-11 ((S)-4,l l-diethyl-3,4,12,14-tetrahydro-4-hydroxy- 3,14-dioxol / / -pyrano[3’,4’:6,7]-indolizino[l,2-b]quinolin-9-yl-[l,4’bipiperidine]-l’- carboxylate) is a derivative of camptothecin. It is sold under the brand name CAMPTOSAR® and in a pegylated liposomal form under the brand name ONIVYDE® among others. Its main use is in colon cancer (either alone or in combination with fluorouracil) and for small-cell lung cancer (in combination with cisplatin). It may also be used together with fluorouracil and folinic acid for pancreatic cancer following failure of initial treatment. CPT-11 is on the World Health Organization’s Model List of Essential Medicines.

[0058] C. Azlactone-Based Heterofunctional Linker

[0059] Within CIP3M, SN-38 and gemcitabine are covalently bound together via an azlactone-based heterofunctional linker of formula (V) (see Figure 1) developed by Prof. Fontaine and described in WO 2014 / 060347 Al. The linker is capable of playing the role of a coupling agent between different molecules of interest. The presence of the azlactone ring in the linker allows a click chemistry reaction, which takes place in mild conditions and does not require by-products elimination.

[0060] D. CIP3M

[0061] CIP3M ((S)-4,l l-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-lH- pyrano[3 ’ ,4’ :6,7]indolizino[ 1 ,2-b]quinolin-4-yl 4-( 1 -( 1 -(( 1 -(( 1 -((2R,4R,5R)-3,3-di- fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimidin- 4-yl)amino)-2-methyl-l-oxopropan-2-yl)amino)-l-oxopropan-2-yl)-lH-l,2,3-triazol-4- yl) butanoate; C44H47F2N9O12; MW = 931.9) is an equimolar mixture of two diastereoisomers. The present invention encompasses CIP3M as an equimolar mixture of two diastereoisomers, as a single (separate) diastereoisomer thereof, and as a non- equimolar mixture of diastereoisomers thereof. The two CIP3M diastereoisomers are: CIP3M diastereoisomer of formula (II) and CIP3M diastereoisomer of formula (III) (see Figure 1). The synthesis of CIP3M according to the reaction scheme developed by the present Inventors (see Example 2 and Figure 2) leads to CIP3M in the form of an equimolar mixture of two diastereoisomers due to an uncontrolled stereocenter on the linker. The synthesis of both pure diastereoisomers has been achieved (see Example 2 and Figure 3), and as shown in Example 6, the antiproliferative properties of each of the CIP3M diastereoisomers were found to be essentially identical to the antiproliferative properties of the equimolar mixture.

[0062] The terms diastereoisomers and diastereomers are used herein interchangeably. They refer to stereoisomers with two or more centers of chirality and whose molecules are not mirror images of one another. The term ' stereoisomers refers to isomeric molecules that have the same molecular formula and sequences of bonded atoms, but different three-dimensional orientations of their atoms in space. The term “non-equimolar mixture of two diastereoisomers refers to a mixture of the two diastereoisomers that has non-equal molar amounts of the two diastereoisomers (7.c., any relative molar quantities except for a 1 : 1 molar ratio), such as for example, about a molar ratio of about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85: 15, about 90:10, about 95:5.

[0063] 2 - Preparation of CIP3M and Diastereoisomers thereof

[0064] CIP3M may be prepared using any of a variety of methods known in the art. An illustrative synthesis reaction scheme is shown in Figure 2, and described in Example 2 of the Experimental Section below. The synthetic scheme is merely illustrative of some methods by which CIP3M can be synthesized, and various modifications of the synthetic reaction scheme can easily be made by one skilled in the art. In particular, where specific acids, bases, reagents, solvents, etc. are mentioned, it is understood that other acids, bases, reagents, solvents, etc. may also be used and therefore included within the scope of the present invention. Variations in reaction conditions and parameters like temperature, pressure, duration of reaction, etc., which may be used as known in the art, are also within the scope of the present invention. The starting materials and reagents used in the synthesis of CIP3M are either available from commercial suppliers or are prepared by following routine procedures.

[0065] The starting materials and the intermediates of the synthetic reaction scheme can be isolated and purified if desired using conventional techniques, including, but not limited to, filtration, distillation, crystallization, chromatography, and the like. The purified materials can be characterized using conventional means, including by measuring physical constants and recording spectra data.

[0066] CIP3M may be prepared as individual diastereoisomers by resolution from the equimolar mixture. On account of differences in their physical properties, diastereoisomers can be separated from one another through techniques like fractional crystallization, fractional distillation, chromatography, etc. Alternatively, CIP3M may be prepared as individual diastereoisomers using a separated diastereoisomer of a starting material. An illustrative synthetic reaction scheme is shown in Figure 3, and described in Example 2 of the Experimental Section below. The synthetic scheme is merely illustrative of some methods by which individual CIP3M diastereoisomers can be synthesized, and various modifications of the synthetic reaction scheme can easily be made by one skilled in the art.

[0067] Following synthesis, CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, may be of insufficient purity. It can be purified by using any of the methods known in the art for purifying organic compounds, for example, crystallization, chromatography, and the like. It is recognized that “purity” is a relative term, and not to be necessarily construed as absolute purity or absolute enrichment or absolute selection. In some embodiments, the purity of the retrieved CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, is at least or about 80%, at least or about 90% (e.g., at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, or at least or about 99%) or is approximately 100%. In some preferred embodiments, CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, is retrieved in “substantially pure form”. As used herein, the term ''substantially pure” refers to a purity that allows for the effective use of CIP3M in the applications to which it is intended. In therapeutic applications in humans, substantially pure preferably refers to at least or about 97%, at least or about 98%, at least or about 99%, or approximately 100% pure. The purity may be determined using HPLC analysis. Chemical authenticity of CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, may be established by any method well-known to those of skill in the art, including NMR.

[0068] If desired, after preparation and / or purification, CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, may be sterilized. Sterilization may be carried out using any of a wide variety of sterilization techniques known in the art, for example, by filtration through a bacteria-retaining filter, by gamma irradiation, by electron-beam irradiation, or by incorporating sterilizing agents in the form of a sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Alternatively, or additionally CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, may be lyophilized prior to use.

[0069] CIP3M, as an equimolar mixture of two diastereoisomers or as a single diastereoisomer, may be stored under appropriate conditions, either in a solution or in a solid form. For example, after preparation / purification or upon receipt from a provider, CIP3M may be kept at room temperature, away from bright light. The formulated CIP3M is stable at -20°C and can be stored for long periods of time in such conditions.

[0070] II - Therapeutic Applications of CIP3M

[0071] As already mentioned above, due to its biological activity, CIP3M may be used as a therapeutic agent. In particular, C1P3M may be used in the treatment of a cancer. Due to its activity in killing tumor cells, CIP3M represents a novel hope to treat advanced metastatic cancers. CIP3M may also find application in the treatment of chemoresistant cancers as it displays an unprecedented activity profile, i.e., high potency on an unusually large variety of tumor cells combining inhibition of two fundamental mechanisms exacerbated in multiple forms of drug resistance (DNA synthesis and DNA reparation).

[0072] In the above paragraph as well as in the following paragraphs which deal with therapeutic applications, the term CIP3M is used. However, as already mentioned above, it is to be understood that in all the therapeutic applications, CIP3M may be used in the form of an equimolar mixture of the two diastereoisomers or as a single diastereoisomer (i.e., CIP3M diastereoisomer of formula (II) or CIP3M diastereoisomer of formula (III)) or yet as a non-equimolar mixture of the two diastereoisomers.

[0073] 1 - Indications

[0074] The present invention relates to CIP3M described herein (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) for use in the treatment of a cancer in a subject. The present invention further relates to a method for the treatment of a cancer in a subject, the method comprising a step of administering to the subject in need thereof a therapeutically effective amount of CIP3M, or a pharmaceutical composition thereof. The present invention also relates to the use of CIP3M for the manufacture of a medicament for the treatment of cancer in a subject. CIP3M may be used in the form of an equimolar mixture of two diastereoisomers or as a single diastereoisomer (i.e., CIP3M diastereoisomer of formula (II) or CIP3M diastereoisomer of formula (III)) or as a non-equimolar mixture of diastereoisomers.

[0075] In the practice of the present invention, the cancer to be treated using CIP3M may be any cancer developed in any tissue or organ. Thus, the cancer may be a carcinoma, lymphoma, blastoma, sarcoma, myeloma or leukemia.

[0076] Examples of cancers that may be treated using a method according to the present invention include, but are not limited to, cancer of the bone, lung, liver, pancreas, skin, head or neck, brain, gum, tongue, skin, eye, uterus, ovary, anus / rectum, stomach, colon, nasopharynx, breast, sexual and reproductive organs, Hodgkin’s Disease, esophagus, gastrointestinal tract, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, bone marrow, bladder, kidney, pelvis, central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

[0077] In some embodiments, the subject to be treated CIP3M suffers from a cancer selected from the group consisting of acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, alveolar soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B- cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone Cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary site, carcinosarcoma, Castleman's Disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear-cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, esthesioneuroblastoma, Ewing family of tumor, Ewing family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, gliomatosis cerebri, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, hem angiosarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, Lentigo maligna melanoma, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant Glioma, mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid cancer, medulloblastoma, medulloepithelioma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, mouth cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic disease, myelodysplasia, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasm, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, Non-Hodgkin lymphoma, nonmelanoma skin cancer, non-small cell lung cancer, non-small cell lung cancer (NSCLC) which coexists with chronic obstructive pulmonary disease (COPD), ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget’s disease of the breast, pancoast tumor, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, pi neoblastoma, pituicytoma, pituitary adenoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastema, polyembryoma, precursor T- lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular cancer, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract carcinoma involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Ley dig cell tumor, sex cord-stromal tumor, Sezary syndrome, Signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, soot wart, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stomach cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial-stromal tumor, synovial sarcoma, T-cell acute, lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphatic cancer, testicular cancer, thecoma, throat cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell cancer of renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom’s macroglobulinemia, Warthin’s tumor, Wilms’ tumor, and any combination thereof.

[0078] In certain embodiments, the cancer is a solid malignant tumor. In particular, the solid cancer may be selected from the group consisting of lung cancer (such as small-cell lung cancer), meningioma, pancreatic cancer, liver cancer, cholangiocarcinoma, breast cancer, bladder cancer, and bone cancer. In particular, these cancers may be advanced metastatic cancers.

[0079] About 10% to 15% of all lung cancers are small-cell lung cancers (SCLCs). This type of lung cancer tends to grow and spread faster than non-small-cell lung cancer (NSCLC). In most patients with SCLC, the cancer has already spread beyond the lungs at the time it is diagnosed. Since this cancer grows quickly, it tends to respond well to chemotherapy and radiation therapy. Unfortunately, for most patients, the cancer will return at some point. While all lung cancers are associated with tobacco smoking, SCLC is very strongly associated with tobacco smoking.

[0080] Meningioma, also known as a meningeal tumor, is the most common type of primary brain tumor, accounting for approximately 30% of all brain tumors. It is typically a slow-growing tumor that forms from the meninges, the membranous layers surrounding the brain and spinal cord. Risks factors include exposure to ionizing radiation such as during radiation therapy, a family history of the condition, and neurofibromatosis type 2.

[0081] In certain embodiments, the cancer to be treated using CIP3M according to the present invention is a metastatic cancer, in particular an advanced metastatic cancer. In certain embodiments, the cancer to be treated using CIP3M according to the present invention is chemoresistant. Resistance to chemotherapeutic regimens has been reported for almost all the drugs used to treat the most lethal cancers. Resistance to doxorubicin, paclitaxel, 5-fluorouracil, cyclophosphamide, and carboplatin has been shown to cause cancer recurrence in breast cancer associated with poorer prognosis and shorter survival. Resistance to 5-fluorouracil, cisplatin, docetaxel and oxaliplatin result in the same outcomes in gastric cancer patients. Moreover, resistance to 5-fluorouracil, irinotecan, and oxaliplatin in colorectal cancer; to cisplatin, carboplatin, paclitaxel, and docetaxel in lung cancer; and to gemcitabine, oxaliplatin, cisplatin, and doxorubicin strongly indicate that the chemoresistance phenomenon intensively threatens the health and survival of cancer patients. In certain embodiments, the chemoresistant cancer to be treated using CIP3M is a cancer that is resistant to irinotecan or to gemcitabine.

[0082] 2 - Administration

[0083] CIP3M (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) can be administered to a subject in need thereof, in a desired dosage, by any suitable route. Various delivery systems are known and can be used to administer CIP3M, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, nanoparticles, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. CIP3M, or a pharmaceutical composition thereof, may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by adsorption through epithelial or mucocutaneous linings (e.g., oral, mucosa, rectal and intestinal mucosa, etc). Administration can be systemic or local. Parenteral administration may be directed to a given tissue of the patient, such as by catheterization. As will be appreciated by those of ordinary skill in the art, in embodiments where CIP3M is administered along with an additional therapeutic agent, CIP3M and the therapeutic agent may be administered by the same route (e.g., orally) or by different routes e.g., orally and intravenously).

[0084] CIP3M (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) may alternatively be administered incorporated in catheters, needles, or implants. 3 - Dosage

[0085] Administration of CIP3M (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) will be in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend upon the therapeutic effect desired, the severity of the disease being treated, the age, sex, weight and general health condition of the patient as well as upon the potency, bioavailability and in vivo half-life of CIP3M, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models. Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using CIP3M, further information will emerge regarding the appropriate dosage levels and duration of treatment. For example, in certain embodiments, an effective amount is one that delays or prevents the onset of cancer, and / or one that slows down or stops the progression, aggravation, or deterioration of the symptoms of cancer, and / or one that brings about amelioration of the symptoms of cancer, and / or one that prevents, delays and / or reduces the likelihood of occurrence of metastases formation and / or one that reduces the number, growth rate, size, etc... of metastases if metastases are already present in the subject. The effects of a treatment according to the invention may be monitored using any of the diagnostic assays, tests and procedures known in the art.

[0086] A treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of CIP3M, or a pharmaceutical composition thereof, may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval), monthly, yearly (e.g., in a time release form). Alternatively, the delivery may occur at multiple times during a given time period, e.g., two or more times per week, two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g., intravenous delivery.

[0087] In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg / kg body weight of the recipient per day, e.g., from about 0.01 to about 100 mg / kg of body weight per day, such as above about 0.1 mg / kg body weight per day, or in a range of from about 1 to about 10 mg / kg of body weight per day. For example, a suitable dose can be about 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, or 30 mg / kg of body weight per day.

[0088] In certain embodiments, CIP3M may be conveniently administered in unit dosage form, for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form. In other embodiments, CIP3M may be conveniently administered in unit dosage form, for example, containing about 0.0001 to 1 mg, or about 0.001 to 0.1 mg, or about 0. 1 to 1 mg or even about 10 mg per dose of the active ingredient per unit dosage form. In yet other embodiments, the dosage unit contains about 0.1 mg, about 0.5 mg, about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg, of active ingredient.

[0089] 4 - Concomitant Therapies

[0090] A treatment according to the present invention may be administered alone or in combination with another therapy (z'.e., a therapeutic agent and / or a therapeutic procedure), in particular a therapy known to be beneficial to a patient suffering from the disease to be treated. CIP3M (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients) may be administered prior to administration of the additional therapeutic agent or procedure, concurrently with the therapeutic agent or procedure, and / or following administration of the additional therapeutic agent or procedure.

[0091] Therapeutic agents that may be administered in combination with CIP3M, or a pharmaceutical composition thereof, may be selected among a large variety of biologically active compounds that are known to have a beneficial effect in the treatment of the disease to be treated (z.e., a cancer) or to have a beneficial effect to a patient in general (e.g., anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof).

[0092] Anti-cancer agents that can be used in combination with CIP3M include agents used in chemotherapy, hormonal therapy, immunotherapy, targeted therapy including smallmolecule drugs and monoclonal antibodies, and PARP inhibitors.

[0093] Anti-cancer agents that may be administered in combination with CIP3M, or pharmaceutical composition thereof, include drugs conventionally classified into one of the following groups: alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, anti-metabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones and anti-androgens. Examples of such anti-cancer agents include, but are not limited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane and amifostine.

[0094] Other examples of such anti-cancer agents include therapeutic antibodies used in the treatment of cancer, including, but not limited to, anti-CD52 antibodies such as alemtuzumab (CAMPATH™), which is used in the treatment of chronic lymphocytic leukemia; anti-VEGF antibodies including bevacizumab (AVASTIN™) used in the treatment of colorectal cancer, lung cancer and breast cancer; anti-CD33 antibodies, including gemtuzumab ozogamicin (MYLOTARG™) used in the treatment of acute myeloid leukemia; anti-CD20 antibodies including ibritumomab (ZEVALIN™) used in the treatment of lymphoma, rituximab (RITUXAN™) used in the treatment of Hodgkin lymphoma, tositumomab (BEXXAR™) used in the treatment of Hodgkin lymphoma and of atumumab (ARZERRA™) used in the treatment of chronic lymphocytic leukemia; anti-EGFR antibodies such as cetuximab (ERBITUX™) used in the treatment of colorectal cancer, head and neck cancer, and squamous cell carcinoma, and panitumumab (VECTIBEX™) used in the treatment of colorectal cancer; anti-Her2 antibodies, including trastuzumab (HERCEPTIN™) used in the treatment of breast cancer and stomach cancer; anti-CTLA4 antibodies including Ipilimumab (YERVOY™) used in the treatment of melanoma; adnectins; and domain antibodies. Active fragments and fusions of these antibodies will also find use herein.

[0095] CIP3M may be administered in combination with immunotherapy. As used herein, the term “immunotherapy” has its art understood meaning and refers to the treatment that consists in administering an immunogenic agent, i.e., an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. The immunotherapy may consist in the administration of an immune checkpoint inhibitor. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known in the art. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitors includes PD- 1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonist, and BTLA antagonists.

[0096] In certain embodiments, the immunotherapy administered in combination with CIP3M is CAR-T cell therapy. CAR-T cell therapy refers to the use of genetic engineering techniques to activate T-cells collected from peripheral blood and produce Chimeric Antigen Receptor (CAR) on the T cells, followed by mass culture and amplification in vitro and then reinfusion into patients.

[0097] Anti-cancer therapeutic procedures that may be performed in combination with administration of CIP3M, or a pharmaceutical composition thereof, include, but are not limited to, surgery, radiation therapy, radio-immunotherapy, and the like.

[0098] Ill - Pharmaceutical Compositions, Packs and Kits of CIP3M

[0099] 1 - Pharmaceutical Compositions

[0100] As mentioned above, in therapeutic applications, CIP3M described herein, may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition comprising an effective amount of CIP3M described herein, either as an equimolar mixture of two diastereoisomers or as a single diastereoisomer (CIP3M diastereoisomer of formula (II) or CIP3M diastereoisomer of formula (III)) or as a non-equimolar mixture of the two diastereoisomers, and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises one or more additional biologically active agents.

[0101] The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “unit dosage form”, as used herein, refers to a physically discrete unit of CIP3M for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

[0102] A. Formulation

[0103] A pharmaceutical composition described herein may be administered in any amount and using any route of administration effective for achieving the desired prophylactic and / or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration (orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes, or by injection into tissue), and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.

[0104] Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3 -butanediol or in beta-cyclodextrin. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

[0105] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection.

[0106] Tn order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

[0107] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to the CIP3M molecule described herein, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or another solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, beta-cyclodextrins, fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0108] Solid dosage forms for oral administration include, for example, lozenges, troches, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches and sublingual tablets. In such solid dosage forms, CIP3M may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as, for example, carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as nonionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, magnesium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0109] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. Examples of embedding compositions which can be used include polymeric substances and waxes.

[0110] In certain embodiments, it may be desirable to administer an inventive composition locally. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, by injection, by means of a catheter, by means of suppository, or by means of a skin patch or stent or other implant.

[0111] For topical administration, the composition is preferably formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingredient by either passive or active release mechanisms. Transdermal administrations include all administrations across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, solutions, sprays, and suppositories (rectal and vaginal).

[0112] Transdermal administration may be accomplished through the use of a transdermal patch containing an active ingredient i.e., CIP3M described herein) and a carrier that is non-toxic to the skin and allows the delivery of the ingredient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi- permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

[0113] Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository’s melting point, and glycerine. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

[0114] Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations can be found, for example, in “ Remington ’s Pharmaceutical Sciences’’’, E.W. Martin, 18thEd., 1990, Mack Publishing Co.: Easton, PA.

[0115] B. A dditional Biologically A ctive Agents

[0116] In certain embodiments, CIP3M is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, but are not limited to, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof. Other examples of suitable biologically active agents include, but are not limited to, the therapeutic agents described above, in particular the anti-cancer agents.

[0117] In such pharmaceutical compositions, CIP3M and the at least one additional biologically active agent may be combined in one or more preparations for simultaneous, separate or sequential administration of CIP3M and the biologically active agent(s). More specifically, an inventive composition may be formulated in such a way that CIP3M and the biologically active agent(s) can be administered together or independently from each other. For example, CIP3M and the biologically active agent can be formulated together in a single pharmaceutical composition. Alternatively, they may be maintained (e.g., in different compositions and / or containers) and administered separately, thereby constituting a pharmaceutical kit or pack.

[0118] 2 - Pharmaceutical Packs or Kits

[0119] In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of CIP3M, to a subject in need thereof, for therapeutic purpose.

[0120] Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Packs or kits according to the invention may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.

[0121] In certain embodiments, a pharmaceutical pack or kit can include a device for administering CIP3M, or a composition thereof, e.g., syringe needle, pen device, jet injector or another needle-free injector.

[0122] In certain embodiments, a pharmaceutical pack or kit includes one or more additional therapeutic agent(s), as described above.

[0123] Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.

[0124] An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

[0125] The invention will be further illustrated by the following examples. However, these examples and associated figures should not be interpreted in any way as limiting the scope of the present invention.

[0126] Examples

[0127] The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually carried out or data were actually obtained.

[0128] Example 1: Design of CIP3M

[0129] The present Inventors have first conducted a project with the aim of associating molecules with proven biological properties via a binding molecule (or linker). In particular, the aim was to covalently associate anticancer products (registered or not) and measure the effectiveness of the resulting molecule on cancer cells resistant to current therapies and compare it to the effectiveness of the separate components.

[0130] This work was based on the linkers developed by the team of Professor Laurent Fontaine (PCT publication No. WO 2014 / 060347 Al, “Multifunctional Coupling Reagents Having an Azlactone Function”, published on April 24, 2014). The presence of an azlactone ring in these linkers has the advantage of allowing a click chemistry reaction with the primary amines present in numerous (bio)molecules of interest. This reaction can in fact be carried out under mild conditions (organic or aqueous medium, ambient temperature) and without elimination of by-products, which is of considerable interest for applications in the medical sector. This family of new linkers is of particular interest in therapeutic chemistry for the formation of “multivalent” or “twin-drugs”, that is to say new drug candidates containing two (or more) pharmacophores covalently bound via a linker.

[0131] The anti-cancer compounds used in this study were: cytidine, cytarabine, gemcitabine hydrochloride, zidovudine (AZT), camptothecin (CPT), aminoglutethimide, and chlorambucil. Around a hundred homodimeric, heterodimeric and multimeric molecules have been synthesized.

[0132] The biological activity of the different molecules synthesized during this project was evaluated by the company CIPREVO (Antony, France) on three distinct cell lines:

[0133] HCT-116 (human colorectal carcinoma cell line);

[0134] FIT -29 (human colorectal carcinoma cell line); and MiaPaCa (human pancreas carcinoma cell line.

[0135] The different compounds were solubilized at a concentration of 10'2M in DMSO. On day 0 (DO), the cells were seeded in 96-well plates at the following densities: HT-29: 500 cells / well; HT-116: 1000 cells / well; MiaPaCa2: 2000 cells / well. On day 1 (DI), 10 pL of solution of the different compounds were added to wells. The concentration tested was 10 pM. Each compound was tested in 6 wells. On day 4 (D4), cell survival (WST) was measured, and the percentage of inhibition was calculated for each dose of product tested and compared to the control wells.

[0136] Among the different molecules tested, only 5 demonstrated interesting physicochemical and biological properties. The molecule with the most potent antiproliferative activity was found to be the camptothecin / gemcitabine conjugate of formula (IV) (see Figure 1). In order to increase the overall safety of the conjugate, the Inventors replaced the camptothecin moiety of the molecule, which is known to exhibit adverse side effects, with SN-38, an active metabolite of the chemotherapeutic agent irinotecan (or CPT-11), which is itself a derivative of camptothecin approved by the health regulatory agencies.

[0137] Example 2: Synthesis of CIP3M and of the two Diastereoisomers Thereof

[0138] 1. Synthesis of CIP3M

[0139] A robust synthetic process to prepare CIP3M has been established by the Inventors. The reaction conditions for each step have been optimized to be suitable for industrial production. CIP3M was synthesized by a key step of click-chemistry between alkyne 3 and Azl-N3 -Gemcitabine (See Figure 2(A)), which leads to CIP3M as a mixture of two diastereoisomers in a 1 : 1 molar ratio. The alkyne 3 was prepared from SN38 in three steps (See Figure 2(B), Equation 1: Boc protection, esterification and Boc deprotection), and Azl-N3 -Gemcitabine was obtained by aminolysis of the azlactone-N3 (See Figure 2(B), Equation 2)).

[0140] A - Synthesis of Azl-N 3 -Gemcitabine

[0141] To a suspension of Gemcitabine hydrochloride (602 mg, 2.01 mmol, 1 eq.) in dry DMF (0.625 M) was added freshly distilled diisopropylethylamine (DIPEA, 384 pL, 2.41 mmol, 1.2 eq.). The resulting mixture was stirred at 50°C for 15 minutes. Then the excess of DIPEA and half of the volume of DMF were removed under reduced pressure (vacuum pump, 50°C). A solution of Azlactone (366 mg, 2.01 mmol, 1 eq.) in DMF (1.25 M) was then added at room temperature and the resulting solution was stirred overnight at 50°C. The solvent was removed under reduced pressure and the product was purified on silica gel chromatography (CFECh / MeOH 95 / 5 to 90 / 10) affording Azl-N3-Gemcitabine (as a mixture of two diastereoisomers) (768 mg, 86%) as a white solid.

[0142] H NMR (400 MHz, CD3OD): 5 (ppm) 8.35 (d, 1H, J= 7.6 Hz, =CH), 7.51 (d, 1H, . / =

[0143] 7.6 Hz, =CH), 6.25 (dd, 1H, J= 7.2 Hz, J= 7.2 Hz, =CH), 4.31 (dt, 1H, f = 12.2 Hz, Jd= 8.5 Hz, CH), 4.00 - 3.92 (m, 3H, CH and CH2), 3.84 - 3.78 (m, 3.81H, CH2), 1.52 (s, 3H, CH3), 1.51 (s, 3H, CH3), 1.45 (d, 3H, J= 6.9 Hz, CH3).

[0144] 13C NMR (100 MHz, CD3OD): 5 (ppm) 175.9 (C=O), 172.8 (C=O), 165.2 (C=N), 157.7 (C=O), 146.0 (=CH), 123.9 (t, J= 259 Hz, CF2), 98.2 (=CH), 86.5 (m, CH), 82.8 (m, CH), 70.1 (dd, J = 23.6 Hz, J = 22.3 Hz, CH), 60.2 (CH2), 59.0 (CH), 58.8 (C), 24.6 (CH3),

[0145] 24.6 (CH3), 16.9 (CH3).

[0146] HRMS (Q-TOF ESI+): [M+Na]+: Ci6H2iF2N7NaO6, calculated: 468.1414, experimental: 468.1406.

[0147] FT-TR: u (cm'1) 3305, 21 12, 1727, 1657, 1554, 1484, 1388, 1316, 1196, 1123, 1073, 810, 787.

[0148] B - Synthesis of Alkyne 3

[0149] 1) Synthesis of compound 1: (S)-tert- Butyl (4,ll-diethyl-4-hydroxy-3,14- dioxo-3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-9-yl) carbonate. To a solution of SN38 (100 mg, 0.255 mmol) in anhydrous CH2CI2 under Argon (10 mL) were added BOC2O (75 mg, 0.34 mmol, 1.3 eq.) and pyridine (0.6 mL) at room temperature. After being stirred overnight, the reaction mixture was washed with HC1 (0.5 M) three times, with an aqueous saturated solution of NaHCCE and with brine. The organic phase was dried over MgSC , filtered and concentrated under reduced pressure affording compound 1 (C27H28N2O7, Mw: 492.1897) as a white solid (117 mg, 0.238 mmol, 93%).

[0150] RMN ’H (200 MHz, CDCI3) : 8 8.24 (d, J= 9.2 Hz, 1H), 7.90 (d, J= 2.4 Hz, 1H), 7.67 (dd, J = 9.2, 2.4 Hz, 1H), 7.65 (s, 1H), 5.76 (d, J= 16.4 Hz, 1H), 5.31 (d, J = 16.4 Hz, 1H), 5.27 (s, 2H), 3.73 (s, 1H), 3.22-3.11 (2H), 1.92-1.87 (2H), 1.61 (s, 9H), 1.40 (t, J = 7.6 Hz, 3H), 1.04 (t, J= 7.4 Hz, 3H). NMR data in accordance with literature (Yuqin Yao et al., Anti-Cancer Drugs, 2013, 24: 270-277).

[0151] 2) Synthesis of compound 2: (N)-9-((tert-butoxycarbonyl)oxy)-4,ll-diethyl-

[0152] 3.14-dioxo-3,4,12,14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-4-yl hex-5-ynoate A mixture of 5-Hexynoic acid (40 pL, 0.366 mmol, 3.1 eq.), DMAP (6 mg, 0.049 mmol, 0.4 eq.) andEDCI (86 mg, 0.448 mmol, 3.8 eq.) in anhydrous CH2CI2 (1 mL) was stirred 15 minutes at 25°C. Then compound 1 (57 mg, 0.116 mmol, 1 eq.) in DCM (0.8 mL) was added. The resulting mixture was stirred at 25°C for 4 hours. The reaction mixture was hydrolyzed, and chloroform was added. The aqueous phase was extracted three times with chloroform and the combined organic phases were washed with water, saturated Na2CO3aqueous solution, brine, dried over MgSCh, filtered and concentrated under reduced pressure. The crude product was purified by preparative TLC (Heptane / AcOEt 4:6) affording compound 2 (C33H34N2O8, Mw: 586,2315) as a yellow solid (57 mg, 0.097 mmol, 84%).

[0153] 1H NMR (400 MHz, CDC13): 8 8.22 (d, J= 9.2 Hz, 1H), 7.90 (d, J= 2.5 Hz, 1H), 7.68(dd, , , , , , , 17.2 Hz, 1H), 5.41 (d, 17.2 Hz, 1H),

[0154] 5.23 (d, J= 2.1 Hz, 2H), 3.15 (q, J= 7.6 Hz, 2H), 2.73-2.58 (m, 2H), 2.33 - 2.24 (m, 3H), 2.16 (m, 1H), 2.03 (t, J = 2.7 Hz, 1H), 1.90-1.83 (m, 2H), 1.62 (s, 9H), 1.39 (t, J = 7.6 Hz, 3H), 0.99 (t, J= 7.5 Hz, 3H).

[0155] 13C NMR (100 MHz, CDC13): 8 172.1, 167.6, 157.4, 151.9, 151.6, 150.1, 147.3, 146.8, 146.1, 145.6, 131.9, 127.6, 127.3, 125.3, 120.2, 114.3, 96.0, 83.1, 76.0, 69.6, 67.2, 49.3, 32.5, 31.9, 27.8 (3C), 23.3 (2C), 17.9, 17.7, 14.0, 7.7.

[0156] HRMS-ESI calculated for CssH^NaOs: m / z 609.2207 ([M+Na]+), found: 609.2195.

[0157] 3) Synthesis of compound 3: (N)-4,ll-Diethyl-9-hydroxy-3,14-dioxo-

[0158] 3.4.12.14-tetrahydro-lH-pyrano[3',4':6,7]indolizino[l,2-b]quinolin-4-yl hex-5- ynoate. To a solution of compound 2 (57 mg, 0.097 mmol) in CH2CI2 (1 mL) was added a solution of TFA (0.6 mL) in CH2CI2 (2 mL). The reaction mixture was stirred at room temperature for 1 h30 and then concentrated under reduced pressure. The crude product was purified by preparative TLC (CH2Cl2 / MeOH 98:2) affording compound 3 (C28H26N2O6, Mw: 486,1791) as a yellowish solid (33 mg, 0.068 mmol, 70%).

[0159] H NMR (400 MHz, CDC13): 5 8.08 (d, J= 9.4 Hz, 1H), 7.44 (dd, J= 9.4, 2.5 Hz, 1H), 7.24 (d, J = 2.5 Hz, 1H), 7.15 (s, 1H), 5.70 (d, J = 17.1 Hz, 1H), 5.41 (d, J = 17.1 Hz, 1H), 5.23 (d, J= 2.9 Hz, 2H), 2.98 (q, J= 7.6 Hz, 2H), 2.74-2.59 (m, 2H), 2.33-2.26 (m, 3H), 2.16 (m, 1H), 2.03 (t, J= 2.6 Hz, 1H), 1.93-1.84 (m, 2H), 1.31 (t, J= 7.6 Hz, 3H), 1.01 (t, J= 7.5 Hz, 3H).

[0160] 13C NMR (100 MHz, CDC13): 8 172.5, 167.7, 157.6, 155.8, 149.3, 147.1, 146.4, 144.9, 143.6, 132.1, 128.5, 127.1, 122.6, 119.5, 105.4, 95.7, 83.1, 76.3, 69.7, 67.2, 49.3, 32.7, 32.0, 23.4, 23.2, 17.8, 13.7, 7.7.

[0161] HRMS-ESI calculated for C28H26N2NaO6: m / z 509.1683 ([M+Na]+), found: 509.1669.

[0162] C - Synthesis of CIP3M as an Equimolar Mixture of Two Diastereoisomers

[0163] To a solution of Azl-N3-Gemcitabine (31 mg, 0.070 mmol, mixture of diastereoisomers), alkyne 3 (33 mg, 0.067 mmol) and TBTA (1.8 mg, 0.05 eq.) in THF (1 mL) and H2O (1 mL) was added a solution of CuSO4-5H2O in water (220 pL, 0.05 equiv, 4 mg / mL) and a solution of sodium ascorbate in H2O (220 pL, 0.1 equiv, 6 mg / mL). The resulting solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure. TLC preparative (CH2Cl2 / MeOH / H2O 9 : 1 :0.1) gave compound CIP3M (C44H47F2N9O12,) (25 mg, 0.027 mmol, 40%) isolated as a yellow solid.

[0164] H NMR (500 MHz, DMSO): 6 10.6 (bs, 1H), 10.3 (bs, 1H), 8.64 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.99 (d, J= 9.8 Hz, 1H), 7.90 (d, J= 3.9 Hz, 1H), 7.41-7.38 (m, 2H), 7.25 (d, J= 7.6 Hz, 1H), 6.94 (s, 1H), 6.30 (m, 1H), 6.15 (m, 1H), 5.48 (s, 2H), 5.37 (q, J = 7.4 Hz, 1H), 5.28 (s, 2H), 5.27 (m, 1H), 4.17 (m, 1H), 3.88 (m, 1H), 3.79 (d, J= 12.8 Hz, 1H), 3.64 (d, J= 12.8 Hz, 1H), 3.11-3.05 (m, 2H), 2.68-2.64 (m, 2H), 2.58-2.55 (m, 2H), 2.17-2.11 (m, 2H), 1.91-1.84 (m, 2H), 1.65 (d, J= 8.6 Hz, 3H), 1.41-1.36 (m, 6H), 1.28 (t, J= 7.4 Hz, 3H), 0.91 (t, J= 13 Hz, 3H).

[0165] 13C NMR (125 MHz, DMSO): 174.5, 171.7, 168.3, 167.3, 163.3, 156.7, 156.5, 154.0, 148.5, 146.9, 145.7, 145.4, 144.5, 143.5, 142.7, 131.4, 128.2, 127.9, 122.8 (t, J= 259 Hz), 122.3, 121.1, 117.7, 104.7, 95.9, 93.7, 80.9, 75.7, 68.3 (t, J = 22.5 Hz), 66.2, 58.7, 57.7, 56.8, 49.4, 32.5, 30.1, 28.9, 24.1, 24.0, 23.9, 22.2, 22.0, 17.8, 13.2, 7.4.

[0166] HRMS-ESI calculated for C44H47F2N9NaOi2: m / z 954.3204 ([M+Na]+), found: 954.3200.

[0167] CIP3M in powder form was found to be chemically stable. Stored at room temperature, in powder form for several months, CIP3M was found to display essentially identical effects on the proliferation of cells of the human colorectal carcinoma HCT-116 cell line in vitro as freshly prepared CIP3M.

[0168] 2. Synthesis of the Two Diastereoisomers of CIP3M

[0169] Chiral HPLC was performed on the Azl-N3-Gemcitabine to separate the two diastereoisomers (dia 1 and dia 2) (see Figure 3(A)). The configuration of the indicated stereocenter has not yet been confirmed. Each of the two separate diastereoisomers of Azl-N3-Gemcitabine was then coupled with alkyne 3 using the same conditions of clickchemistry as previously described (see Figure 3(B)).

[0170] A - Separation of the two Diastereoisomers of Azl-N3-Gemcitabine

[0171] The separation of the two diastereoisomers of Azl-N3-Gemcitabine was performed by chiral chromatography.

[0172] 1) Analytical Separation of the Diastereoisomers

[0173] The chiral HPLC analyses were performed using an Agilent 1260 Infinity unit (pump G1311B, autosampler G1329B, DAD G1315D), with Agilent OpenLAB CDS Chemstation LC and CE Drivers (A.02.08SP1) and Agilent OpenLAB Intelligent reporting (A.01.06.111). The analytical column (250x4.6 mm) used was a Chiral Art Cellulose SZ from YMC Europe, cellulose tris (3-chloro-4-methylphenylcarbamate) immobilised on silica. Heptane, hexane and ethanol, HPLC grade, were degassed and filtered on a 0.45 pm millipore membrane before use. Retention times Rt in minutes, retention factors ki = (Rti-Rto) / Rto and enantioselectivity factor a = k2 / ki and resolution Rs = 1.18 (Rt2- Rti) / (wi + W2) are given. Rto was determined by injection of tri-tertio- butyl benzene and Wi was the peak width at half-height.

[0174] The sample was dissolved in a mixture of ethanol and heptane, injected on the chiral column, and detected with an UV detector at 254 nm. The flowrate was 1 mL / min. The data obtained are presented in Table 1 below.

[0175] Table 1. Results of the analytical separation of the two diastereoisomers of Azl-N3- Gemcitabine. 2) Preparative Separation of the Diastereoisomers

[0176] The preparative chiral HPLC separation was performed using an Agilent 1260 Infinity unit (pump G1311C, autosampler G1329B, DAD G1365D and fraction collector G1364C), monitored by Agilent OpenLAB CDS Chemstation LC. The preparative column (250 x 10 mm, 5 pm) used was a Chiral Art Cellulose SZ from YMC Europe, cellulose tris (3-chloro-4-methylphenylcarbamate) immobilized on silica.

[0177] About 212 mg of Azl-N3-Gemcitabine were dissolved in 30 mb of the mobile phase. The chromatographic conditions used were: Chiral Art Cellulose SZ (250 x 10 mm), hexane / ethanol (70 / 30) as mobile phase, flow-rate = 5 mL / min, UV detection at 254 nm. Injections of 50, 100, 150, 200, 250 and 300 pL were performed. The maximum volume that could be injected before peaks overlap was 300 pL (about 2 mg). 96 additional injections of 300 pL were performed every 11 minutes. The diastereoisomers were collected, solvents were evaporated, and the purity of each fraction was checked by analytical chromatography. The data obtained are presented in Table 2 below.

[0178] Table 2. Results of the preparative separation of the two diastereoisomers of Azl-N3- Gemcitabine.

[0179] On Chiral Art Cellulose SZ (250 x 10 mm, 5 pm), 20 hours and 6 liters of mobile phase were needed for 100 injections and to obtain 100 mg of each of the diastereomers of Azl-N3-Gemcitabine.

[0180] B - Preparation of the First Diastereoisomer of CIP3M— CIP3M dia 1

[0181] To a solution of azide (18 mg, 0.040 mmol, dia 1), alkyne 3 (19.5 mg, 0.040 mmol) and TBTA (1.1 mg, 0.05 eq.) in THF (0.6 mL) and H2O (0.6 mL) was added a solution of CUSO4’5H2O in water (120 pL, 0.05 eq., 4 mg / mL) and a solution of sodium ascorbate in H2O (130 pL, 0.1 eq., 6 mg / mL). The resulting solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure. TLC preparative (CH2C12 / MeOH / H2O 9 :1 :0.1) gave compound CIP3M - dia 1 (15 mg, 0.016 mmol, 40%) isolated as a yellow solid. H NMR (500 MHz, DMSO): 8 10.60 (bs, 1H), 10.30 (bs, 1H), 8.64 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.99 (d, J= 9.8 Hz, 1H), 7.91 (s, 1H), 7.41-7.38 (m, 2H), 7.26 (d, J= 7.6 Hz, 1H), 6.94 (s, 1H), 6.30 (d, J= 6.2 Hz, 1H), 6.15 (t, J = 7.5 Hz, 1H), 5.48 (s, 2H), 5.37 (q, J= 7.1 Hz, 1H), 5.30-5.25 (m, 3H), 4.17 (m, 1H), 3.87 (dt, J= 8.5, 3.1 Hz, 1H), 3.79 (d, J= 12.3 Hz, 1H), 3.66 (m, 1H), 3.11-3.05 (m, 2H), 2.68-2.64 (m, 2H), 2.58-2.55 (m, 2H), 2.18-2.10 (m, 2H), 1.90-1.83 (m, 2H), 1.67 (d, 7.2 Hz, 3H), 1.41 (s, 3H), 1.36

[0182] (s, 3H), 1.28 (t, J= 7.5 Hz, 3H), 0.90 (t, J= 7.4 Hz, 3H).

[0183] 13C NMR (125 MHz, DMSO): 174.5, 171.7, 168.3, 167.3, 163.3, 156.7, 156.5, 154.0, 148.5, 146.9, 145.7, 145.4, 144.5, 143.5, 142.7, 131.4, 128.2, 127.9, 122.8 (t, J= 259 Hz), 122.3, 121.1, 117.7, 104.7, 95.9, 93.7, 80.9, 75.7, 68.3 (t, J = 22.5 Hz), 66.2, 58.7, 57.7, 56.8, 49.4, 32.5, 30.1, 28.9, 24.1, 24.0, 23.9, 22.2, 22.0, 17.8, 13.2, 7.4.

[0184] HRMS-ESI calculated for C44H47F2N9NaOi2: m / z 954.3204 ([M+Na]+), found: 954.3188.

[0185] C - Preparation of the Second Diastereoisomer of CIP3M- CIP3M dia 2

[0186] To a solution of azide (18 mg, 0.040 mmol, dia 2), alkyne 3 (19.5 mg, 0.040 mmol) and TBTA (1.1 mg, 0.05 eq.) in THF (0.6 mL) and H2O (0.6 mL) was added a solution of CuSO4-5H2O in water (120 qL, 0.05 eq., 4 mg / mL) and a solution of sodium ascorbate in H2O (130 qL, 0.1 equiv, 6 mg / mL). The resulting solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure. TLC preparative (CH2C12 / MeOH / H2O 9 : 1 :0.1) gave compound CIP3M- dia 2 (13 mg, 0.014 mmol, 35%) isolated as a yellow solid.

[0187] H NMR (500 MHz, DMSO): 8 10.60 (bs, 1H), 10.31 (bs, 1H), 8.64 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 9.8 Hz, 1H), 7.90 (s, 1H), 7.41-7.38 (m, 2H), 7.26 (d, J = 7.4 Hz, 1H), 6.94 (s, 1H), 6.30 (d, J= 6.3 Hz, 1H), 6.17 (t, J= 7.5 Hz, 1H), 5.48 (s, 2H), 5.37 (q, J= 7.1 Hz, 1H), 5.30-5.27 (m, 3H), 4.18 (m, 1H), 3.88 (dt, J= 8.5, 3.0 Hz, 1H), 3.79 (d, J= 12.5 Hz, 1H), 3.64 (m, 1H), 3.08 (q, J= 7.5 Hz, 2H), 2.67-2.64 (m, 2H), 2.58-2.55 (m, 2H), 2.18-2.10 (m, 2H), 1.90-1.84 (m, 2H), 1.63 (d, J = 7.2 Hz, 3H), 1.41 (s, 3H), 1.37 (s, 3H), 1.28 (t, J= 7.5 Hz, 3H), 0.90 (t, J= 7.4 Hz, 3H).

[0188] 13C NMR (125 MHz, DMSO): 174.6, 171.7, 168.3, 167.3, 163.3, 156.7, 156.5, 154.0, 148.5, 146.9, 145.7, 145.4, 144.4, 143.5, 142.7, 131.4, 128.2, 127.9, 122.8 (t, J= 259 Hz), 122.3, 121.1, 117.7, 104.7, 95.9, 93.7, 80.9, 75.7, 68.3 (t, J = 223 Hz), 66.2, 58.7, 57.7, 56.8, 49.4, 32.5, 30.1, 28.9, 24.1, 24.0, 23.9, 22.2, 22.0, 17.6, 13.2, 7.4.

[0189] HRMS-ESI calculated for C44H47F2N9NaOi2: m / z 954.3204 ([M+Na]+), found: 954.3190. Example 3: Physico-Chemical and Biophysical Properties of CIP3M

[0190] 1. Solubility and Stability of CIP3M in Aqueous Medium

[0191] Drug solubility is one of the core factors which affect the movement of a drug from the site of administration into the bloodstream to the tumor site and the rest of the body. The solubility and stability of CIP3M in solution in aqueous vehicles were studied by the CRO DRUGABILIS (Les Ulis, France) in the presence or absence of excipients. The excipients tested were: Tween 80 (5%), Cremophor EL (5%), Solutol HS15 (5%), Lutrol F68 (5%) and HPpCD (30%), all at pH 7.4. The vehicle containing 30% HPpCD in phosphate buffer pH7.4 achieved the highest solubility with approximately 3-4 mg / mL of CIP3M (measured 24 hours after preparation of the solution), while a solubility of around 0.05 mg / mL or less were obtained with the other excipients.

[0192] The manufacturing feasibility of the 30% HPPCD formulation was verified, and its stability evaluated: no decrease in titer was observed after a freeze / thaw cycle.

[0193] The CIP3M formulation in 30% HPpCD / tp pH7.4 vehicle was manufactured in a sterile manner for in vivo studies. With this formulation, CIP3M was found to be stable in different conditions (after one cycle of freeze / thaw; after 8 days at 4°C; and after 8 days at 20°C). This allows for the convenient use of CIP3M solutions stored by freezing. Protection of CIP3M from light is strictly recommended to avoid CIP3M degradation.

[0194] 2. Stability of CIP3M in Human Plasma and Mouse Plasma

[0195] In vitro microsomal stability assays are widely used in drug discovery as an indicator of in vivo stability, which affects pharmacokinetics. This approach supports the strategy used by drug discovery teams of applying the in vitro data to triage compounds for in vivo pharmacokinetic and efficacy studies and may guide structural modifications to improve metabolic stability.

[0196] Consequently, the stability of CIP3M has been studied by the CRO ONCODESIGN (Les Ulis, France). Figure 4 shows the relation time / concentration of CIP3M analyzed by LC / MS. In the conditions of the experiments, CIP3M clearance from microsomal stability assay was found to be 34.9 pL / min / mg proteins in C57BM / 6 mice. Based on the results obtained in mice, CIP3M appears as an excellent candidate for in vivo pharmacokinetic studies. Example 4: Pharmacokinetic Parameters of CIP3M in Mice

[0197] The role of pharmacokinetics (PK) in drug discovery is to support the optimization of the absorption, distribution, metabolism, and excretion (ADME) properties of compounds with the ultimate goal to achieve a concentration-time profile in the body that is adequate for the desired efficacy and safety profile. The pharmacokinetic parameters of CIP3M have been studied in mice by the CRO ONCODESIGN. CIP3M concentrations in plasma have been determined by LC-MS / MS. The concentrations of CIP3M obtained in C57BL / 6 mouse plasma samples per time point after intravenous administration of 3mg / kg are presented in Figure 5.

[0198] Based on the results obtained in these experiments, the pharmacokinetic parameters for CIP3M were calculated and are presented in Table 3 below. Such values characterized for CIP3M with a half-life superior to 2.2 hours allow the early generation of PK / PD relationships linking in vitro potency and pharmacological activity (mode-of-action) and antitumoral efficacy in animal models.

[0199] Table 3. Pharmacokinetic parameters of CIP3M in mice.

[0200] Example 5: In vitro Evidence of CIP3M Properties

[0201] 1. Antiproliferative Activity of CIP3M on Human Tumor Cell Lines in 2D

[0202] The study has been conducted at the CRO GENEMARIN (Palaiseau, France). The antiproliferative activity of CIP3M has been evidenced by several experiments.

[0203] In a first set of experiments, the effects of CIP3M and of ACC486PP (CIP3M without the gemcitabine payload) on the proliferation of cells of four human tumor cell lines: A2780 (ovarian cancer), Caco2 (colorectal cancer), MDA-MB-231 (breast cancer) and MiaPaCa2 (pancreas cancer) were assessed at 96 hours and compared to the parent molecules, i.e., SN38 and gemcitabine. As shown in Table 4 below, which reports the results of 3 sets of experiments, CIP3M was found to be active on all four cell lines tested with IC50 values ranging from 23 nM to 135 nM, meaning less than a 6-fold difference between the most sensitive and the less sensitive cell lines. By comparison, ACC486PP (CIP3M without the gemcitabine payload) was found to be active but with a higher range of IC50 values (from 84 nM to 1054 nM). For the parent compounds, the range of efficacies was 1 nM to 23 nM for Gemcitabine (23 -fold) and 1 nM to 69 nM (69-fold) for SN38. It is noteworthy that ACC486PP (CIP3M without the gemcitabine payload) was observed to be particularly poorly active on the Caco2 cell line which was found to be the most sensitive to the gemcitabine treatment.

[0204] Table 4. Antiproliferative activity of CIP3M on 4 different human tumor cell lines compared to controls.

[0205] 2. Antiproliferative Activity of CIP3M on Human Tumor Cell Lines Depends on the Exposure Time The study has been conducted at the CRO GENEMARIN (Palaiseau, France). It is of great importance to assess the time of contact between a drug and tumor cells needed to trigger an antiproliferative effect. The value of this time of contact will impact the manner in which the drug may be used in patients. Briefly, a long time of required exposure is usually associated with a protracted treatment administrated by oral route whereas a short time of contact usually allows an acute treatment by intravenous route or by oral route.

[0206] In the case of CIP3M, such a parameter was evaluated using the wash-out period approach. Increasing times of exposure (3, 6, 24 hours) were compared to a long-term exposure (48 hours). Table 5 below summarizes the data generated with the human colon cancer cell line HCT116 / H3E5. In both experiments performed, it is noteworthy that CIP3M as well as the other tested controls (SN38 and gemcitabine) were all active after just a couple of hours. However, the compound displaying the lowest shift of IC50 values between 3 and 48 hours (5.1 and 6.9) was CIP3M. This indicates that a short exposure of tumor cells with CIP3M is sufficient to trigger cell death. This duration of 3 hours is highly compatible with the current plasma half-life observed in the in vivo mouse experiment with a value of over 2 hours.

[0207] Table 5. Wash-out induced IC50 shifts in HCT116 / H3E5 cells. 3. Large Spectrum of CIP3M Antiproliferative Activity against Human Tumor Cell

[0208] Lines in 2D

[0209] The studies have been conducted at the CRO GENEMARIN (Palaiseau, France). The in vitro antiproliferative activity of CIP3M has been screened across a diverse, clinically relevant panel of cell lines. Such data of drug response, which identify sensitive and resistant cells, can help with patient stratification for CIP3M and / or with identification of potential drug combinations.

[0210] Table 6 below shows a large spectrum of sensitivity of tumor cell lines for CIP3M from low nanomolar range (HCT116) to low micromolar range (H-MESO-1). Table 6. Antiproliferative activity of CIP3M on a large panel of human tumor cell lines.

[0211] 4. CIP3M Activity against 60 Human Tumor Cell Lines in 2D

[0212] The data obtained in the previous paragraph has encouraged to perform a larger screening to identify specific signatures of sensitive and resistant cells using the CRO REVITY (United Kingdom) and evaluate the sensitivity of CIP3M to 60 cell lines exemplifying 18 different cancer indications, z.e., leukemia, lymphoma, myeloma, kidney, head and neck, breast, urinary, gastric, pancreas, lung, soft tissue, prostate, ovary, colorectal, liver, skin, esophagus, and endometrium.

[0213] A study was conducted for assessing single agent activity of CIP3M in 384-well viability assay using 60 cell lines grown in vendor recommended media. After one day of subculture in 384-well plate, treatment was initiated for 4 days (96 hours). Cell viability was measured using CellTiterGlo2.0 (Promego). Three replicates were performed to account for assay variability. At the time of treatment, a “time zero” set of assay plates (which did not receive any treatment) were collected to enable calculating of the Growth Inhibition and Doubling Time.

[0214] The results obtained are presented in Table 7 below and in Figure 6. Table 7. Antiproliferative activity of CIP3M on a large panel of human tumor cell lines.

[0215] Table 7 and Figure 6 clearly demonstrate that CIP3M exhibits an antiproliferative activity on a very large spectrum of cancer cell lines.

[0216] Example 6: Effects of CIP3M Diastereoisomers Compared to CIP3M as an Equimolar Mixture of Two Diastereoisomers on HCT116-H3E5, MiaPaCal and NCI-H1048 Tumor Cell Lines Proliferation

[0217] All the experiments presented in this section were performed by the CRO GENEMARIN SAS.

[0218] CIP3M exists as an equimolar mixture of two diastereoisomers on the gemcitabine side of the molecule. Therefore, it was of interest to test the effects of the two diastereoisomers separately and to compare their activities to that of the equimolar mixture. The two stereoisomers and the equimolar mixture were tested on the human colorectal cancer HCT116 cell line (H3E5 SN38 sensitive variant), on the human pancreatic cancer MiaPaCa2 cell line and on the human small cell lung cancer NCI-H1048 cell line. Materials and Methods

[0219] Chemotherapy Agents. CIP3M, as an equimolar mixture of two diastereoisomers, CIP3M diastereoisomer 1 and CIP3M diastereoisomer 2 were used in the present study.

[0220] Cell Culture and Proliferation Measurement Reagents. DMSO (molecular biology grade) was purchased from Sigma Aldrich; RMPI-1640 and DMEM were purchased from PanBiotechn (D Dutscher); DMEM / F12 and Insulin-Transferin- Selenium lOOx (ITS) were purchased from Gibco (Thermo Fisher); (3 estradiol and hydrocortisone were purchased from Thermofisher; Foetal calf serum (South Africa origin), Trypsin-EDTA lx in Sodium, and PBS without Ca2+ / Mg2+were purchased from Dominique Dutscher; L-glutamine 200 mM and Penicillin / Streptomycin were purchased from Gibco; and Resazurin was purchased from Acros Organics.

[0221] Cell Culture Consumables. 75 cm2T flasks for cell cultures and 96-well plates for cell cultures were purchased from Falcon (D Dutscher).

[0222] Cell Lines Cells of the HCT116-H3E5 cell line of human colorectal cancer (NSCLC) were provided by Dr. Philippe Pourquier (Montpellier, France), and cultured, as adherent cells, in a culture medium comprising RPMI-40, 10% FCS, 1% pen / strep, for a total of 5 to 7 passages. Cells of the MiaPaCa2 cell line (ATCC # CRL-1420) of human pancreatic cancer were cultured, as adherent cells, in a culture medium comprising DMEM, 10% FCS, 1% pen / strep for a total of 6 to 8 passages. Cells of the NCI-H1048 cell line of human small cell lung cancer were cultured, as adherent cells, in a culture medium comprising DMEME / F12, 5% heat-inactivated FCS, 1% glutamine, 1% pen / strep, 1% ITS complement, for a total of 3 to 5 passages.

[0223] Cell Cultures. Cells were maintained in culture at 37°C, 5% CO2 and humidity- saturated atmosphere in one of the cell culture media described above. Cells were maintained in 75 cm2culture flasks either for maintenance or for experiments. The cells which are all adherent cells were sub-cultured twice a week.

[0224] Cell Proliferation Assay Adherent cells were seeded in 96 flat bottomed-well cell culture treated plates at the following densities: for the HCT116 / H3E5 cell line: 500 cells / well; for the MiaPaCa2 cell line: 2000 cells / well; and for the NCI-H1048 cell line: 3000 cells / well.

[0225] The plates were kept at 37°C, 5% CO2, saturating humidity for 24 hours to allow the cells to adhere. The next day, the cells were treated with 10 uL / well of a chemotherapy agent (lOx final concentration). The cells were treated with the chemotherapy agents in a concentration-dependent manner (semi logarithmic progression, 3 wells per concentration).

[0226] Results

[0227] Cells were plated in 96-well cell culture treated plates and treated with the chemotherapy agents according to the protocol described above. CIP3M and its diastereoisomers were tested at concentrations ranging from 1 nM to 10 pM (semi log progression). The two diastereoisomers CIP3M, as an equimolar mixture of the two diastereoisomers were able to inhibit cell proliferation of the three human tumor cell lines tested in a nanomolar range (see Table 8). Irrespective of the cell line tested, the potency of each of the diastereoisomers was equivalent to that of CIP3M, as an equimolar mixture of the two diastereoisomers.

[0228] Table 8. Mean IC50 values of CIP3M (equimolar mixture) and diastereoisomers on HCT116-H3E5, MiaPaCa2 and NCI-H1048 cell lines.

[0229] Example 7: Effects of CIP3M compared to CPT-11, Gemcitabine, and Lurbinectedin on Cell Proliferation of a Panel of Human Small Cell Lung Cancers

[0230] Cell Lines

[0231] All the experiments presented in this section were performed at GENEMARIN SAS (Faculte des Sciences d’ Orsay, Orsay, France).

[0232] Introduction

[0233] Small cell lung cancer is a very aggressive cancer with a five-year survival rate of less than 10% despite the existing chemotherapies and surgery protocols. Therefore, there is still a need to find new treatments against this very aggressive cancer. The present study aims to evaluate in vitro a new strategy using CIP3M and assess its capacity to inhibit cell proliferation of a panel of seven human small cell lung cancer cells lines (NCI- H69, NCI-H82, NCI-H1018, NCI-H1105, NCI-H1417 and NCI-H1882) and one nonsmall cell lung cancer cell line (A549). Another aim of this study was to compare the antiproliferative activity of CIP3M to a benchmark molecule, Lurbinectedin, which is currently approved as a second line treatment of metastatic small cell lung cancer patients, and to the two controls: SN38 and Gemcitabine separately.

[0234] Materials and Methods

[0235] Chemotherapy Agents. SN38 and Gemcitabine were purchased from Sigma Aldrich, and Lurbinectedin was purchased from MedChem (Clinisciences).

[0236] Cell Culture and Proliferation Measurement Reagents. DMSO (molecular biology grade) was purchased from Sigma Aldrich; RMPI-1640 and DMEM were purchased from PanBiotechn (D Dutscher); DMEM / F12 and Insulin-Transferin- Selenium lOOx (ITS) were purchased from Gibco (Thermo Fisher); P estradial and hydrocortisone were purchased from Thermofisher; Foetal calf serum (South Africa origin), Trypsin-EDTA lx in Sodium, and PBS without Ca2+ / Mg2+were purchased from Dominique Dutscher; L-glutamine 200 mM and Penicillin / Streptomycin were purchased from Gibco; and Resazurin was purchased from Acros Organics.

[0237] Cell Culture Consumables. 75 cm2T flasks for cell cultures and 96-well plates for cell cultures were purchased from Falcon (D Dutscher).

[0238] Cell Lines. The A549 cell line (ATCC # CRM-CCL-185) of human non-small cell lung cancer (NSCLC) were cultured, as adherent cells, in a culture medium comprising 90% DMEM, 10% heat inactivated FCS, 1% glutamine, 1% pen / strep, for a total of 8 to 12 passages. Cells of the NCI-H69 cell line (ATCC # HTB-119) of human lung carcinoma (small cell lung cancer, SCLC) were cultured, as multicellular aggregates / suspension, in a culture medium comprising 90% RPMI-1640, 10% heat inactivated FCS, 1% glutamine, 1% pen / strep for a total of 1 to 6 passages. Cells of the NC1-H82 cell line (ATCC # HTB-175) of human small cell lung carcinoma (SCLC) were cultured, as multicellular aggregates / suspension, in a culture medium comprising 90% RPMI-1640, 10% heat inactivated FCS, 1% glutamine, 1% pen / strep for a total of 2 to 6 passages. Cells of the NCI-H345 cell line (ATCC # HTB-180) of human small cell lung carcinoma (SCLC) were cultured, as multicellular aggregates / suspension, in a culture medium comprising DMEM / F12, 5% heat inactivated FCS, 1% glutamine, 1% pen / strep, 1% ITS complement for a total of 4 to 6 passages. Cells of the NCLH1048 cell line (ATCC # CRL-5853) of human small cell lung carcinoma (SCLC) were cultured, as adherent cells, in a culture medium comprising DMEM / F12, 5% heat inactivated FCS, 1% glutamine, 1% pen / strep, 1% ITS complement for a total of 6 to 10 passages. Cells of the NCI-H1105 cell line (ATCC # CRL-5856) of human small cell lung carcinoma (SCLC stage E) were cultured, as multicellular aggregates / suspension, in a culture medium comprising DMEM / F 12, 5% heat inactivated FCS, 1% Glutamine, l% Pen / strep, 1% ITS complement for a total of 4 to 8 passages. Cells of the NCI-H1417 cell line (ATCC # CRL-5869) of human small cell lung carcinoma (SCLC stage E) were cultured, as multicellular aggregates / suspension, in a culture medium comprising 90% RPMI- 1640, 10% heat inactivated FCS, 1% glutamine, 1% pen / strep for a total of 3 to 6 passages. Cells of the NCLH1882 cell line (ATCC # CRL-5853) of human small cell lung carcinoma (SCLC stage E) were cultured, as adherent cells, in a culture medium comprising DMEM / F 12, 5% heat inactivated FCS, 1% Glutamine, 1% Pen / strep, 1% ITS complement, 10 nM P-estradiol and hydrocortisone for a total of 3 to 4 passages.

[0239] Cell Cultures. Cells were maintained in culture at 37°C, 5% CO2 and humidity- saturated atmosphere in one of the cell culture media described above. Cells were maintained in 75 cm2culture flasks either for maintenance or for experiments. Adherent cells were sub-cultured twice a week (A549 and NCI-H1048) and once a week for the NCLH1 182 cell line. The other cell lines growing in suspension and / or aggregates were diluted (1 / 2) once a week in fresh medium, or fresh medium was added thereto accordingly. For maintenance and expansion, cell aggregates were not dissociated.

[0240] Cell Proliferation Assay Adherent cells were seeded in 96 flat bottomed-well cell culture treated plates at the following densities: for the A549 cell line: 500 cells / well; for the NCI-H1048 cell line: 3000 cells / well; and for the NCLH1882 cell line: 10 000 cells / well.

[0241] For suspension / aggregate growing cells, aggregates were dissociated by passing 10 times the suspension through a 10 mL sterile syringe and a 18G x 11 / 2needle. The suspension was then diluted five times in fresh complete culture medium before being distributed in 96 well plates. These cell lines do not support higher dilutions. The plates were kept at 37°C, 5% CO2, saturating humidity for 24 hours to allow the cells to adhere or to settle. The next day, the cells were treated with 10 pL / well of a chemotherapy agent (lOx final concentration). The cells were treated with the chemotherapy agents in a concentration-dependent manner (semi logarithmic progression, 3 wells per concentration). The plates were then incubated for 96 hours at 37°C, 5% CO2, saturating humidity. After a 96 hour-treatment with chemotherapy agents, 20 pL / well of resazurin (0.15mg / ml in PBS) were added. The cell plates were incubated for 1 to 3 hours in a cell culture incubator. Cell proliferation was evaluated by measuring the reduction of resazurin in resorufin by mitochondrial activity. This reduction reaction was followed by measurement of resorufin fluorescenceeXc' 520 nm / Xem: 600 nm) using the multimode reader PHERAstar (BMG Labtech). IC50 were calculated using a three parameters regression of the survival percentages versus log(concentration) curve (GraphPad Prism 8 software) for all the cell lines except for the NCI-H345 cell line were the log(inhibitor) vs. normalized response - variable slope fitting model was used. The experiments were performed at least twice.

[0242] Results

[0243] Cells were plated in 96-well cell culture treated plates and treated with the chemotherapy agents according to the protocol described above. Lurbinectedin, the benchmark agent, was tested at concentrations ranging from 0.01 to 100 nM (semi log progression). All the other compounds were tested at concentrations ranging from 1 nM to 10 pM (semi log progression) and 0.1 to 100 nM when necessary.

[0244] Discussion

[0245] In this study, the antiproliferative effects of CIP3M were evaluated on a non-smallcell lung cancer cell line (A549) and on a panel of seven small-cell lung cancer cell lines. Its antiproliferative activity was compared to that of its individual moi eties (SN38 and Gemcitabine). Lurbinectedine, was used as a benchmark in this study.

[0246] Adherent cells (A549, NCI-H1048 and NCI-H1882) were seeded at fixed densities, as mentioned above. Cells growing in suspension and aggregates were systematically diluted 1 :5 after dissociation of the aggregates being dissociated, as described above. It was noticed that after higher dilutions (z.e., 10 times or more), the cells did not survive, or some aggregates were formed, randomly leading to extremely scattered biological replicates. Under the experimental conditions used, the cells were able to reassociate in aggregates as seen by microscopy observations in 96 wells cell culture plates.

[0247] The results obtained are presented in the following table. Table 9. Mean IC50 values of the different compounds tested measured in the different cell lines.

[0248] * indicates adherent growing cells, while the others are suspension / aggregates growing cells.

[0249] As can be seen from the results presented above in Table 9, Lubinectedin was found to be the most potent compound in terms of cell proliferation inhibition of all the cell lines tested, with an IC50 ranging from 0.1 nM (NCI-H1018) to 2.7 nM (A549). This ICsovalues range is in accordance with the data reported in the literature. SN38 alone was more potent than Gemcitabine alone. CIP3M was found to be active on all cell lines tested generally in the same range of potency as Gemcitabine.

[0250] CIP3M was able to efficiently prevent cell proliferation of a wide panel of human small-cell lung cancer cell lines. When the cell lines were ranked according to the respective IC50 values of chemotherapy agents, NCI-H1048 and NCI-H1105 were found to be the two most sensitive cell lines to all compounds and particularly to the CIP3M molecule.

[0251] Example 8: Antiproliferative Activity of CTP3M on Drug-Resistant Cell Lines

[0252] Clinically relevant drug-resistant cell lines are developed by mimicking the conditions that are experienced by cancer patients during chemotherapy and the newly established cell lines display drug-resistance compared to their parental cell line. The resistance can be induced using different ways including a protracted exposure to the drug, by specific gene mutations, by change of gene expression of drug transporter or by inhibition of a drug transporter. The antiproliferative activity of CIP3M on human drug resistant tumoral cell lines have been evidenced by different studies presented below.

[0253] ,S'-(4-Nitrobenzyl)-6-thioinosine (NBTI) is a synthetic small molecule acting as a selective blocker of the High Equilibrating Nucleoside Transporter (hENTl) involved in the Gemcitabine penetration in cancer cells (Nordh el al., World J Gastroenterol., 2014, antiproliferative effect of gemcitabine. Here, the aim of the study was to evaluate the effects of NBTI on the antiproliferative activity of CIP3M and its components SN38 and Gemcitabine alone on the human colon cancer variants cell lines of HCT116: HCT116 / H3E5 (SN38 sensitive) and HCT116 / C4 (SN38 resistant). This approach allowed to evaluate CIP3M on cell lines that are: sensitive to both SN38 and gemcitabine, sensitive to SN38 only (in the presence of NBTI), sensitive to gemcitabine only (variant C4), and resistant to both SN38 and gemcitabine (variant C4 pretreated with NBTI).

[0254] The results obtained are presented in Table 10 below.

[0255] Table 10. NBTI effects on HCT116 / H3E5 sensitivity and HCT116 / C4 sensitivity to chemotherapy agents (Graphpad analysis).

[0256] NBTI was found to induce a loss of sensitivity only to Gemcitabine in HCT116 / H3E5 and in HCT116-C4. The lack of effect of NBTI on CIP3M on HCT116 / H3E5 may be explained by the high sensitivity of this cell line to SN38 (IC50 = 0.73 nM). Conversely, NBTI was able to lower the sensitivity not only to Gemcitabine, but also to CIP3M in the HCT116-C4 variant. This can be explained by the more preponderant role of Gemcitabine in this context of SN38 resistance. Such data may positively impact the safety margin of CIP3M over Irinotecan (Prodrug of SN38) or Gemcitabine with a reduced range of active doses to trigger antiproliferative effects pending the respective sensitivity of each tumor to SN38 or Gemcitabine.

[0257] Example 9: Evaluation of CIP3M in Athymic Human Xenograft Tumor Model

[0258] All the experiments presented in this section were performed at ONCODESIGN SERVICES (Villebon-sur-Yvette, France). The objective of the study was to evaluate the in vivo dose-response efficacy of CIP3M in nude athymic mice bearing subcutaneous NCI-H1048 human lung cancer cells, by assessing the tumor growth inhibition after repeated injections.

[0259] Materials and Methods

[0260] Use of Animals

[0261] Ethical Statement. Animal housing and experimental procedures were conducted according to the French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals (Decret n° 2013-118 du ler Fevrier 2013 relatif a la protection des animaux utilises a des fins scientifiques; National Research Council (US), Institute for Laboratory Animal Research (US) and National Academies Press (US), Eds., Guide for the care and use of laboratory animals, 8thEd. Washington, DC: National Academies Press, 2011). The animal facility is authorized by the French authorities (CFH: Agreement N° B 91 962 106). All animal procedures (including surgery, anesthesia and euthanasia as applicable) used in the current study were submitted to the Institutional Animal Care and Use Committee of Oncodesign Services (Oncomet) approved by French authorities [CNREEA agreement N° 91 (Oncodesign Services)].

[0262] Housing Conditions. Animals were maintained in specific-pathogen free health status according to the Federation for Laboratory Animal Science Associations guidelines. Animals were individually identified with tail tattoos. They were maintained in housing rooms under controlled environmental conditions: temperature: 22 ± 2°C; humidity 55 ± 10%; photoperiod (12h light / 12h dark); HEPA filtered air; and minimum of 15 air exchanges per hour with no recirculation. Each cage was labeled with a specific code. Animal enclosures provided sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described in Oncodesign Services standard operating procedures.

[0263] Test Substances

[0264] The test substances were CIP3M (2.9 mg / mL, stored at -20°C, and protected from ambient light). The CIP3M was diluted with HPpCD 30% (pH=7.4, stored at +4°C or - 20°C).

[0265] Experimental Design and Treatments

[0266] Cancer Cell line and Cell Culture Method. Cells from the NCI-H1048 cell line were grown as monolayers at 37°C in a humidified atmosphere (5% CO2, 95% air). The culture medium was DMEM-F12 containing 1% Glutamax supplemented with 5% fetal bovine serum (heat inactivated), 1% Penicillin-Streptomycin and 1% Insulin-Transferrin- Selenium. The cells were adherent to plastic flasks. For experimental use, tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene, in Hanks’ medium without calcium or magnesium and neutralized by addition of complete culture medium. Cells were counted and viability was assessed using a 0.25% trypan blue exclusion assay.

[0267] Animals. Healthy immunodeficient female athymic nude mice, 8-week-old at reception, were obtained from Charles River. The mice were irradiated with a gammasource (1.44 Gy),60Co, BioMep, France before reception.

[0268] Tumor Induction. Tumors were induced by subcutaneous injection of 5xl06NCI- H1048 cells in 200 pL of RPMI 1640 containing 50% (v / v) matrigel into the right flank of female athymic nude mice. NCI-H1048 tumor cell implantation was performed 24 hours after a whole-body irradiation with a gamma-source (1.44 Gy),60Co, BioMep, France).

[0269] Randomization DO. Animals were randomized based on their individual tumor volume. Randomization was performed when values reached a mean of 126 mm3. Homogeneity between groups was tested by an analysis of variance (ANOVA).

[0270] Treatment DO. The treatment was administered intravenously (IV) slowly through the caudal vein. The recommended pH formulation for intravenous administration was pH 5.0-8 0 (minimum pH 3.0). The administration volume was 5 mL / kg adjusted to the most recent individual body weight. The treatment schedule was as follows:

[0271] Group 1 : animals (7) received one daily IV injection of vehicle (HPpCD 30%) for five consecutive days (QlDx5),

[0272] Group 6: animals (7) received one daily IV injection CIP3M at 7.5 mg / kg for five consecutive days (QlDx5).

[0273] Animal Monitoring

[0274] Clinical Monitoring. All study data, including animal body weight measurements, tumor volume, clinical and mortality records, and treatment were scheduled and recorded in the Vivo Manager database (Biosystemes, France). Animal viability and behavior was observed daily. A clinical follow-up was performed if deemed necessary. Body weights were measured three times a week. The length and width of the tumor was measured three times a week with calipers. Animals were observed for four weeks after the start of treatment (three weeks post-treatment). At the end of the four-week follow-up period, the animals were euthanized.

[0275] Humane Endpoints. Humane endpoints requiring specific action were established including: providing a nutritive gel diet in case of a bodyweight loss > 15% (compared to a reference day, e.g., the first day of treatment); providing fluid or hydrogel in case of animal dehydration; and euthanasia if tumor exceeds 10% of normal body weight or exceeds 2000 mm3(xenogenic tumors in mice); if tumor interferes with ambulation or nutrition; 20% of bodyweight loss (compared to a reference day, e.g., the first day of treatment); poor body condition, emaciation, cachexia, significant dehydration, etc.

[0276] Anesthesia and Analgesia Anesthesia and analgesia were administered according to the research ministry project authorization. Non-pharmacological care, such as rehydration or gel diet, was provided, because body weight losses >10% were observed for groups treated by references.

[0277] Euthanasia and Necropsy. Euthanasia of animals was performed by over dosage on gas anesthesia (isoflurane) or CO2 induction, followed by cervical dislocation or exsanguination. If physical methods for euthanasia (cervical dislocation) were necessary, they were performed by highly skilled and trained technicians. Necropsy (macroscopic examination) was performed on all animals euthanized in the study and, if possible, on all euthanized moribund animals; and those found dead.

[0278] Data Management

[0279] Health Parameters. Individual, mean and medium body weights of animals were measured, and body weight change (BWC) of treated animals was calculated.

[0280] Efficacy Parameter. The treatment efficacy was assessed in terms of the effects of the test substance on the tumor volumes of treated animals relative to control animals. The following criteria of anti-tumor efficacy were evaluated: tumor volume, tumor growth inhibition, and time to reach target tumor volume.

[0281] Tumor Volume. Tumor volume was estimated by the formula: width2x length

[0282] Tumor Volume

[0283] Tumors which were palpable and not measurable using calipers were assigned a volume of 4 mm3, indicating the technical limit measure. Tumor volume of 1000 mm3is considered to be equal to 1 g. Individual, mean and median tumor volumes were provided. A comparison between group tumor volumes (TVs) was performed at a fixed day if necessary, or in the case of MRI.

[0284] Tumor Growth Inhibition Tumor growth inhibition (TGI), defined as the ratio of the median tumor volumes of treated versus control (T / C%), was calculated using the formula:

[0285] Median tumor volume of treated group at DX

[0286] T / C% — 100 x - > - > Median tumor volume of vehicle treated group at DX where DX: Day of measurement. The optimal value is the minimal T / C% ratio reflecting the maximal tumor growth inhibition achieved. Tumor growth inhibition was classified as follows:

[0287] <20%: high inhibition

[0288] 21 to 40%: moderately high inhibition

[0289] 41 to 80%: moderate inhibition

[0290] >81%: low inhibition.

[0291] If fewer than four animals were present in each group, the calculation of tumor growth inhibition was considered as not relevant.

[0292] Statistical Tests Statistical analysis comparisons were performed using an ANOVA. A p value < 0.05 was considered significant. If significant, pairwise tests were performed using the Bonferroni / Dunn correction. In order to control for multiple comparisons, either the threshold of significance was adjusted, or the p value was adjusted (padj). The log-Rank test was used to compare the distribution of time until the occurrence of the event of interest between the groups. A p-value < 0.05 was considered significant.

[0293] Results

[0294] Anti-tumoral Activity. The growth of the subcutaneous NCI-H1048 tumors in female athymic nude mice was monitored throughout the experiment. The parameters of tumor growth, tumor growth delay and tumor growth inhibition were used to evaluate the anti -turn oral activity of CIP3M in mice bearing subcutaneous NCI-H1048 tumors.

[0295] Animals treated with 10 mg / kg of CIP3M or one of the CIP3M diastereoisomers were found to exhibit a decrease in the tumor volume the days following the treatment. Body weights were found to be unaffected by treatment. In summary, the CIP3M treatment at the tested dose (10 mg / kg) and with the model selected herein (female athymic nude mice bearing subcutaneous NCI-H1048 tumors) was well tolerated and a strong antitumor activity was observed compared to control.

[0296] Figure 7 presents results showing the reduction of the individual volumes of human small cell lung cancer NCI-H1040 tumors xenografted in immunocompromised mice (n=5-7) following intravenous administration of 10 mg / kg of CIP3M or of one of the CIP3M diasteroisomers, the treatment schedule being as follows: 5 days on - 2 days off, repeated twice.

Claims

ClaimsWhat is claimed is:

1. A SN38 / gemcitabine conjugate, CIP3M, having chemical formula (I), or a physiologically acceptable salt thereof.of two diastereoisomers.

3. The conjugate according to claim 1, wherein CIP3M is CIP3M diastereoisomer of formula (II), or a physiologically acceptable salt thereof.

4. The conjugate according to claim 1, wherein CIP3M is CIP3M diastereoisomer of formula (III), or a physiologically acceptable salt thereof.

5. The conjugate according to claim 1, wherein CIP3M is a non-equimolar mixture of CIP3M diastereoisomer of formula (II) and CIP3M diastereoisomer of formula (III), or of physiologically acceptable salts thereof.

6. A pharmaceutical composition comprising an effective amount of a conjugate according to any one of claims 1 to 5, and at least one pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition further comprises at least one additional biologically active agent.

8. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for use as a therapeutic agent.

9. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for use in the treatment of a proliferative cell disorder in a subject.

10. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for use in the treatment of a cancer in a subject.

11. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for the use according to claim 10, wherein the cancer is a carcinoma, a lymphoma, a blastoma, a sarcoma, a myeloma, or a leukemia.

12. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for the use according to claim 10, wherein the cancer is selected from the group consisting of brain cancer, colon / colorectal cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, leukemia, lymphoma, myeloma, kidney cancer, head and neck cancer, urinary cancer, gastric cancer, soft tissue cancer, prostate cancer, and liver cancer.

13. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for the use according to claim 10, wherein the cancer is a small-cell lung cancer or a meningioma.

14. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for the use according to any one of claims 10 to 13, wherein the cancer is metastatic.

15. A conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 6 or claim 7 for the use according to claim any one of claims 10 to 14, wherein the cancer is chemoresistant.

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