Neomycin-based compounds and their pharmaceutical uses

Abstract

The present invention relates to the field of medicine, particularly in the field of oncology. In particular, the present invention provides novel compounds that can be used for the treatment of various cancers such as glioblastoma, colorectal cancer or breast cancer. The present disclosure also relates to pharmaceutical compositions containing the disclosed compounds.

Description

Field of the Invention

[0001] The present invention relates to the field of medicine, in particular to the field of oncology. In particular, the present invention provides novel compounds that can be used to treat various cancers such as glioblastoma, colorectal cancer or breast cancer. The present disclosure also relates to pharmaceutical compositions containing the disclosed compounds. Background Art

[0002] Invasive gliomas are the most common and most severe primary brain tumors. The incidence of gliomas has increased over the past 20 years and has now reached 5 / 100,000. The classification of these tumors remains difficult. The WHO classification provides tumor grades (from I to IV) related to tumor invasiveness. The most severe gliomas, and also the most common, are glioblastomas (GBM) (WHO grade IV), with a median overall survival of no more than 15 months. These tumors show extensive cellular infiltration and high vascularization in the brain parenchyma. When the tumor is accessible, the standard first-line treatment is currently based on maximal surgical resection followed by concomitant chemoradiotherapy (60 Gy in 30 fractions, temozolomide 75 mg / m 2 / d for 6 weeks). Despite the observed improvement in survival in this trial, most patients survive less than 2 years after diagnosis, and less than 5% survive at 5 years, and their quality of life experiences significant deterioration and multiple debilitating symptoms.

[0003] Therefore, there is a need for more effective treatment of gliomas or glioblastomas and for the introduction of new agents in clinical trials.

[0004] GBM follows the cancer stem cell (CSC) model. This concept posits that a minority of cells within the tumor mass have long-term self-renewal and differentiation properties, which are responsible not only for tumor initiation and growth but also for the heterogeneity within the tumor. CSCs contribute to all cell subtypes that make up the tumor, including endothelial cells. Their functional properties are associated with molecular tags that combine neural and / or embryonic stem cell markers and mesenchymal cell markers. Increasing evidence supports that these self-renewing tumor cells determine tumor behavior, including proliferation, progression, invasion, and most importantly, a large part of the resistance to therapies. Thus, it is clear that the failure of current treatments to eliminate glioma-initiating cells (GICs) is a factor in tumor recurrence. In addition, GICs are not limited to adult GBM but can also be isolated from pediatric gliomas with poor prognoses, such as diffuse intrinsic pontine gliomas. Therefore, targeting GICs and their stem-like properties constitutes one of the major therapeutic challenges for significantly improving anticancer treatment. A related solution for targeting GICs is to force them to exit the stemness program and adopt a more differentiated phenotype. In this non-stem-like state, the cells lose their tumorigenicity and become vulnerable to treatment. In this context, the miR-302-367 cluster has been shown to effectively trigger a cascade of inhibitory events that disrupt the stem-like and tumorigenic properties of GiCs. In other microRNA profiling studies searching for regulators of stem cell plasticity, miR-18a* was identified as a potential candidate, and its expression is associated with the stemness state. Forcing miR-18a* expression in GiCs was found to increase clonal proliferation in vitro and tumorigenicity in vivo.

[0005] The present inventors propose to develop compounds that inhibit the stemness characteristics of cancer stem cells, such as stemness markers and self-renewal, to reduce their growth while increasing their sensitivity to anticancer treatments such as chemotherapy or radiotherapy. In the context of oncogenic and tumor-suppressive miRNAs, the present inventors developed neomycin-based compounds that are capable of replicating the inhibitory effect of the miR-302-367 cluster on stemness properties (i.e., loss of self-renewal, proliferation, and stemness markers).

[0006] The compounds developed and described herein are designed based on the conjugation of two different RNA-binding domains: (i) the aminoglycoside neomycin and (ii) the side chain of the anticancer agent bleomycin. Regarding the first domain, neomycin is a well-known and versatile RNA ligand that can bind to various structured RNAs mainly when forming non-specific interactions (electrostatic interactions). The second domain is represented by the side chain of a group of natural compounds widely used in cancer chemotherapy and known as bleomycin. These compounds act through multiple mechanisms of action, and their main mechanism of action is to inhibit the action of topoisomerase II on DNA. The side chain of bleomycin is a variable domain that is thought to be responsible for the specific interaction of these compounds with DNA targets.

[0007] Surprisingly, it has been found that the compounds can reproduce the biological effects of the miR302-367 cluster, which makes the compounds suitable for the treatment of cancer. The compounds are effective inhibitors of GiC stem cell-like and tumorigenic properties and thus are effective inhibitors of temozolomide resistance. Summary of the Invention

[0008] Accordingly, the present invention relates to compounds having the following formula (I):

[0009]

[0010] wherein X is selected from the group consisting of:

[0011]

[0012] wherein R is as follows:

[0013]

[0014] wherein n is an integer from 1 to 6, preferably 2, 3, and 4;

[0015] R1 is -NHR2, -NR3R4, or a guanidyl group;

[0016] R2 is a hydrogen atom, an amine protecting group, or an aminoalkyl group;

[0017] R3 and R4 are the same or different and independently are a hydrogen atom, an amine protecting group, or an aminoalkyl group;

[0018] wherein any amine group is optionally protected by an amine protecting group;

[0019] or a salt, stereoisomer (diastereomer, enantiomer), racemic mixture, geometric isomer, or mixture thereof.

[0020] The present disclosure also relates to pharmaceutical compositions containing the disclosed compounds and their use as medicaments, in particular for the treatment of cancers such as glioma, glioblastoma, colorectal cancer or breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Phase contrast images of GB5 morphology untreated (CTL DMSO) or treated with the indicated compounds at the indicated concentrations (compound is denoted as "Cpd"). Control cells grow as non-adherent spheres composed of numerous GSCs. When differentiated, the cells become adherent and spread on the culture dish.

[0022] Figure 2 : Effects of compounds 9a-b and 18a-b on the expression of stemness markers, clonal expansion, and mitosis in GB5 cells. (A) Nestin expression in GB5 treated with increasing amounts of compounds 9a-b and 18a was evaluated by immunofluorescence. The number of nestin-positive cells for each treatment was counted and expressed as a percentage of the control (100%). The results are the average of at least two independent experiments. (B) Immunofluorescence shows the downregulation of pluripotency markers OCT4, NANOG, and SOX2 in GB5 treated with 20 μM compound 9a compared to its control counterpart. (C) Clonal expansion assays were performed using GB5 cells as described in the Materials and Methods section. After two and three weeks, the number of spheres was counted and reported as a percentage of the initial number of cells in the wells. The results are the average of at least two independent experiments. (D) The number of mitoses in the culture was determined by detecting the number of cells positive for phosphorylated histone H3 (H3-ser10) at ser-10. H3-ser10 expression was evaluated by immunofluorescence in GB5 treated with increasing amounts of compounds 9a-b and 18a. The number of positive cells for each treatment was counted and expressed as a percentage of the control (100%). The results are the average of at least two independent experiments.

[0023] Figure 3:Orthotopic xenotransplantation of luciferase-expressing patient-derived GSCs into nude mice. (A - B) Two weeks after injection, 10 mg / kg (n = 4), 7.5 mg / kg (n = 4), or 5 mg / kg (n = 4) of compound 9a or vehicle alone as a control (ctl n = 10) were injected intraperitoneally into the mice. A) Graph showing the average tumor growth in the control group (n = 10) and each compound 9a-treated group. B) Survival rates of the entire mouse population in the untreated and treated groups were compared using the log-rank test according to the Kaplan Meier method (R command, https: / / biostatgv.sentiweb.fr / ?module=tests / surv). C) Mice were treated with compound 9a when tumors had formed. Tumor growth profiles in mice treated with DMSO (control: -▲-) or compound 9a (-■-). Tumor growth was monitored weekly by live imaging.

[0024] Figure 4 :Determination of the toxicity of the compounds to GSCs and normal human cells. (A) GB5 was incubated with increasing concentrations of compounds 9a - b and 18a. (B) Human normal neural stem cells (NNSC), human normal kidney cells (NHEK), human normal hepatocytes (HEPRG), and (C) human endothelial cells (HUVEC) were treated with increasing concentrations of compound 9a. After 7 days, XTT assays were performed on the cells as described in the methods section. The results are the average of at least two independent experiments. Cell death was confirmed by trypan blue staining (not shown).

[0025] Figure 5 :Sensitivity to TMZ was evaluated by comparing the percentage of dead cells between TMZ-treated GB5 and TMZ-treated GB5 in combination with compound 9a. GB5 cells were treated with 400 μM TMZ alone or in combination with increasing concentrations of compounds 9a - b and 18b. The results are the average of at least two independent experiments. Detailed Description of the Invention

[0027] Thus, and in a first aspect of the present invention, compounds of general formula (I) are disclosed herein:

[0028]

[0029] Wherein X is selected from the group consisting of:

[0030]

[0031] And wherein R is as follows:

[0032]

[0033] wherein n is an integer from 1 to 6, preferably 2, 3 and 4;

[0034] and R1 is -NHR2, -NR3R4 or a guanidyl group;

[0035] R2 is a hydrogen atom, an amine protecting group or an aminoalkyl group;

[0036] R3 and R4 are the same or different and are independently a hydrogen atom, an amine protecting group or an aminoalkyl group;

[0037] wherein any amine group is optionally protected by any amine protecting group;

[0038] or a salt, stereoisomer (diastereoisomer and / or enantiomer), racemic mixture, geometric isomer or mixture thereof.

[0039] According to the present invention, any compound of formula (I) may be positively charged, for example, under neutral (physiological) and acidic conditions. For example, any nitrogen atom may be an ammonium ion (N + ), including any nitrogen atom of R1.

[0040] According to the present invention, the term “(C1-C 10 ) alkyl” means a straight-chain or branched-chain saturated hydrocarbon group having 1 to 10, preferably 1 to 6 carbon atoms. Examples of straight-chain alkyl groups include, but are not limited to, those having 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl and n-decyl groups. Examples of branched-chain alkyl groups include, but are not limited to, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl and 2,2-dimethylpropyl groups. The alkyl group may be unsubstituted or substituted by an amino group (-NH2) or a protected amino group.

[0041] According to the present invention, the term “aminoalkyl” means a (C1-C 10 ) alkyl group as defined above, which is terminated by an amine group, and the amine group is optionally protected by an amine protecting group. More preferably, the aminoalkyl group is represented by the formula: -(CH2) m NH2, where m is an integer from 1 to 10, preferably 1 to 6, more preferably 4.

[0042] Amine protecting groups are well known in the art. For example, an amine protecting group forms one of the following protected amino groups with the amino group to which it is attached: 9-fluorenylmethyl carbamate (FMOC), tert-butyl carbamate (Boc), benzyl carbamate (Cbz), acetamide, trifluoroacetamide, phthalimide, benzamide, triphenylmethylamine (Tr), benzylideneamine, or p-toluenesulfonamide (Ts). Preferably, the protected amino group is tert-butyl carbamate or acetamide. Deprotection of the protected amino group is well known in the art. For example, deprotection of the Boc group can be carried out in CH2Cl2 in the presence of trifluoroacetic acid (TFA).

[0043] According to one specific embodiment, the compound of the present invention has the formula (I), wherein X is wherein R is as defined above, more preferably wherein n is 3 or 4, and R1 is a guanidyl group, or R1 is -NR3R4, wherein R3 is a hydrogen atom or an amine protecting group, and R4 is an aminoalkyl group;

[0044] wherein any of the amine groups is optionally protected by any amine protecting group;

[0045] or a salt, stereoisomer (diastereoisomer and / or enantiomer), racemic mixture, geometric isomer or mixture thereof.

[0046] According to another specific embodiment, the compound of the present invention has the formula (I), wherein X is wherein R is as defined above, more preferably wherein n is 3 or 4, and R1 is -NH2, or R1 is -NR3R4, wherein R3 is a hydrogen atom or an amine protecting group, and R4 is an aminoalkyl group;

[0047] wherein any of the amine groups is optionally protected by any amine protecting group;

[0048] or a salt, stereoisomer (diastereoisomer and / or enantiomer), racemic mixture, geometric isomer or mixture thereof.

[0049] According to one specific embodiment, the compound of the present invention has the formula (I), wherein R1 is -NHR2, -NR3R4 or a guanidyl group;

[0050] wherein R2 is a hydrogen atom, an amine protecting group or -(CH2) m NH2, wherein m is an integer from 1 to 6; R3 and R4 are the same or different and independently are a hydrogen atom, an amine protecting group or -(CH2) m NH2, wherein m is an integer from 1 to 6,

[0051] wherein any amine group is optionally protected by an amine protecting group.

[0052] According to a specific embodiment, m is 2, 3, 4, 5 or 6, and more preferably m is 4.

[0053] The compounds discussed herein also encompass their pure or mixed stereoisomers (diastereoisomers, enantiomers), racemic mixtures, geometric isomers, tautomers, salts, hydrates, solvates, solid forms, and mixtures thereof. Some of the compounds and their salts according to the invention can be stable in several solid forms. The invention includes all solid forms of the compounds according to the invention, which include amorphous, polymorphic, single crystal and polycrystalline forms.

[0054] The compounds according to the invention can exist in unsolvated or solvated forms, for example in solvated forms with pharmaceutically acceptable solvents such as water (hydrates) or ethanol.

[0055] More specifically, the invention relates to compounds of formula (I) wherein at least one or all of the following definitions are met:

[0056] - n is 3 or 4;

[0057] - R1 is selected from the group consisting of: -NH2, guanidyl group or -NH(CH2) m NH2 (where m is as defined above, preferably m is 3 or 4 or 5).

[0058] More specifically, the compounds of the invention are compounds of formula (I) wherein R is one of the following formulas:

[0059]

[0060] According to a specific embodiment, the compound has formula (I) wherein X is

[0061] and R is Rb or Rc;

[0062] wherein any amine group is optionally protected by any amine protecting group, preferably a Boc group;

[0063] or a salt, stereoisomer (diastereoisomer and / or enantiomer), racemic mixture, geometric isomer or mixture thereof.

[0064] According to another specific embodiment, the compound has formula (I) wherein X is

[0065] and R is Ra or Rb;

[0066] wherein any amine group is optionally protected by any amine protecting group, preferably a Boc group;

[0067] or a salt, stereoisomer (diastereoisomer and / or enantiomer), racemic mixture, geometric isomer or mixture thereof.

[0068] According to one specific embodiment, the compound of formula (I) is selected from the group consisting of:

[0069]

[0070] (Compound 9a),

[0071]

[0072] (Compound 9b),

[0073]

[0074] (Compound 18a),

[0075]

[0076] (Compound 18b),

[0077]

[0078] (Compound 8a) (which corresponds to Compound 9a with an amino protecting group), and

[0079] (Compound 8b) (which corresponds to Compound 9b with an amino protecting group).

[0080] According to a more specific embodiment, the compound of formula (I) is selected from the group consisting of:

[0081]

[0082] (Compound 9a),

[0083]

[0084] (Compound 9b),

[0085] More preferably, the compound of formula (I) is Compound 9a.

[0086] The compounds according to the present invention can be prepared by various methods known to those skilled in the art. The present invention also relates to a method for preparing the compounds of the present invention.

[0087] The compounds of the present invention are conjugates based on two different RNA-binding domains: (i) the aminoglycoside neomycin and (ii) the side chain of the anticancer agent bleomycin. According to one embodiment, the present invention relates to a method for preparing the compounds as defined above, wherein a 1,3-dipolar cycloaddition reaction is carried out to generate a triazole linker arm between the following two RNA-binding domains: (i) the aminoglycoside neomycin and (ii) the side chain of bleomycin. This means that two partners for the cycloaddition reaction are to be prepared, namely the azide derivative of neomycin and the alkyne derivatives of the side chains of bleomycin A5, B2 and A2. The preparation of the azide derivative of neomycin, Neo-N3, has been reported in the literature (Vo D.D., Staedel C., Zehnacker L., Benhida R., Darfeuille F., Duca M. Targeting the production of oncogenic microRNAs with multimodal synthetic small molecules. ACS Chem. Biol. 2014, 9, 711-721). The method for preparing the alkyne derivatives of the side chains of bleomycin A5, B2 and A2 is described in the examples. The three side chains of bleomycin carry the same bis-thiazole skeleton, and its synthesis is also illustrated in the following examples.

[0088] The compounds of the present invention have been found to limit the self-renewal of cancer stem cells and reduce the growth of such cells and render tumor cells sensitive to anticancer treatment. The compounds of the present invention seem to be able to eliminate or reduce glioma-initiating cells (GICs) or force them to acquire a non-self-renewing state or force them to lose their tumorigenicity or become susceptible to treatments such as chemotherapy and / or radiotherapy. In addition, the compounds of the present invention at effective concentrations are non-toxic to normal cells such as normal human liver, kidney or nerve cells.

[0089] Therefore, the compounds of formula (I) can be used for therapeutic purposes, particularly as medicaments.

[0090] The compounds of the present invention are more particularly useful for the treatment of cancer.

[0091] The terms "cancer", "cancerous" or "malignant" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancers include, for example, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More specific examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL), squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, osteosarcoma, pancreatic cancer, glioma, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, esophageal cancer, colon cancer, and head and neck cancer, gastric cancer, germ cell tumors, pediatric sarcomas, nasal natural killer, multiple myeloma, acute myeloid leukemia (AML), mastocytosis and any symptoms associated with mastocytosis. In one specific embodiment, the cancer is a solid tumor cancer. In another specific embodiment, the cancer is a non-solid tumor cancer. In another specific embodiment, the cancer is an epithelial tumor cancer having cancer stem cells.

[0092] The compounds of the present invention are particularly useful for treating glioma, glioblastoma multiforme or epithelial tumor cancers having cancer stem cells, including colorectal cancer or breast cancer, more preferably for treating glioma or glioblastoma multiforme, and even more preferably glioblastoma multiforme.

[0093] Accordingly, there is disclosed herein a method of treating cancer, the method comprising administering to a subject in need of such treatment an effective amount of at least one compound as defined herein or a medicament comprising the same.

[0094] The subject can be a human or any animal, preferably a human or a mammal, including cows, sheep, horses, dogs, cats, goats, etc. Preferably the subject is a human patient, regardless of his / her age or gender. This includes neonates, infants, children, adults.

[0095] As disclosed herein, the term "treatment" refers to the amelioration, prevention, or at least one symptom distinguishable therefrom, of a disease or disorder. This also means the amelioration, prevention of at least one measurable physical parameter associated with the disease or disorder being treated, which may not necessarily be distinguishable in the subject. "Treatment" further refers to inhibiting or slowing the progression of a disease or disorder, physically stabilizing a distinguishable symptom, physiologically stabilizing, for example, a physical parameter, or both. "Treatment" also refers to delaying the onset of a disease or disorder. In some specific embodiments, the target compound is administered as a prophylactic measure. In context, "prevention" refers to reducing the risk of developing a designated disease or disorder.

[0096] In a therapeutic setting, the compounds of the invention can be administered to a subject by any suitable route, including orally, topically, sublingually, parenterally (preferably intravenously), transdermally, rectally, etc. For a brief review of current methods of drug delivery, see Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference.

[0097] The invention also relates to pharmaceutical compositions comprising a compound of the invention, in particular a compound of formula (I) as described above, and a pharmaceutically acceptable carrier and / or excipient. This particular aspect also relates to preferred embodiments of the compounds of the invention disclosed above. In a particular embodiment, the pharmaceutical composition comprises a compound according to any one of the above embodiments.

[0098] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0099] The pharmaceutical compositions of the present invention are formulated according to standard pharmaceutical practices known to those skilled in the art (see, e.g., Remington: The Science and Practice of Pharmacy (20th Edition), A.R. Gennaro, ed., Lippincott Williams & Wilkins, 2000, and Encyclopedia of Pharmaceutical Technology, J. Swarbrick and J.C. Boylan, eds., 1988 - 1999, Marcel Dekker, New York). The excipients of the composition can be any pharmaceutically acceptable excipients, including specific carriers capable of targeting specific cells, cell compartments or tissues. As previously mentioned, possible pharmaceutical compositions include those suitable for oral, rectal, topical, transdermal, buccal, sublingual or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. For these formulations, conventional excipients can be used according to techniques well known to those skilled in the art. Compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions, which can optionally be prepared immediately before use from solid or lyophilized forms. For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs and concentrated drops. Non-toxic solid carriers or diluents can be used, including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For compressed tablets, a binder is also necessary as a reagent to impart cohesiveness to the powdered material. For example, starch, gelatin, sugars such as lactose or dextran, and natural or synthetic gums can be used as binders. Disintegrants are also required in tablets to facilitate the breakdown of the tablets. Disintegrants include starch, clay, cellulose, alginates, gums and cross-linked polymers. In addition, lubricants and glidants are included in tablets to prevent the tablet material from adhering to the surface during manufacture and to improve the flow characteristics of the powdered material during manufacture. Colloidal silica is most commonly used as a glidant, and compounds such as talc or stearic acid are most commonly used as lubricants. For transdermal administration, the composition can be formulated in the form of an ointment, cream or gel, and appropriate penetrants or detergents can be used to facilitate penetration, such as dimethyl sulfoxide, dimethylacetamide and dimethylformamide. For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any known suppository base by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycol (carbowax), polyoxyethylene sorbitan monostearate, and mixtures of these with other compatible materials for altering the melting point or dissolution rate. In a preferred embodiment, the pharmaceutical compositions of the present invention are suitable for parenteral administration.

[0100] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable preparation. These can be, in particular, isotonic, sterile saline solutions (monosodium or disodium phosphate, chlorides of sodium, potassium, calcium or magnesium, etc., or mixtures of such salts), or dry, especially lyophilized, compositions which, depending on the case, permit the preparation of an injectable solution when sterile water or physiological saline is added.

[0101] Drug forms suitable for injection use include sterile aqueous solutions or dispersions; preparations including sesame oil, peanut oil or aqueous solutions of propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0102] Solutions containing the compounds of the invention as the free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compounds according to the invention can be formulated in compositions in the neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid or organic acids such as, for example, acetic, oxalic, tartaric, mandelic acid, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as, for example, isopropylamine, trimethylamine, histidine, procaine, etc. The carrier can also be a solvent or dispersion medium which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal. In many cases, it is preferred to include isotonic agents such as, for example, sugars or sodium chloride. The absorption of injectable compositions can be prolonged by the use of agents delaying absorption such as, for example, aluminum monostearate and gelatin in the composition.

[0103] A sterile injectable solution is prepared by incorporating the required amount of the active polypeptide, along with several other ingredients (as needed) listed above, into a suitable solvent, followed by filtration sterilization. Generally, the dispersion is prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional required ingredients from its previously sterile-filtered solution.

[0104] Upon formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described above, but drug-release capsules and the like can also be used.

[0105] For example, for parenteral administration in an aqueous solution, if necessary, the solution should be appropriately buffered and first made isotonic with sufficient saline or glucose to dilute the liquid diluent. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, those skilled in the art will know the sterile aqueous media that can be used according to the present disclosure.

[0106] In a particular embodiment, the pharmaceutical composition according to the present invention comprises from 0.001 mg to 1 g of the compound of the present invention. Preferably, the pharmaceutical composition according to the present invention comprises from 0.01 mg to 800 mg of the compound of the present invention.

[0107] The pharmaceutical composition according to the present invention may comprise one or more compounds of the present invention and pharmaceutically acceptable excipients and / or carriers. These excipients and / or carriers are selected according to the form of administration as described above.

[0108] The compounds according to the present invention can be combined with pharmaceutically acceptable excipients and an optional sustained-release matrix such as a biodegradable polymer to form a therapeutic composition.

[0109] The pharmaceutical composition according to the present invention can be formulated to release the active drug substantially immediately after administration or at any predetermined time or period after administration.

[0110] Generally, the compounds according to the present invention are usually administered in a therapeutically effective amount.

[0111] "Therapeutically effective amount" means an amount of the compound according to the present invention sufficient to treat and / or prevent a disease with a reasonable benefit / risk ratio applicable to any medical treatment.

[0112] It should be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the specific compound used; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, the route of administration and the excretion rate of the specific compound used; the duration of the treatment; drugs used in combination with or concurrently with the specific compound employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art that the starting dosage level of a compound begins with a dosage of the compound that is less than the level required to achieve the desired therapeutic effect and is gradually increased until the desired effect is reached. However, the daily dosage of the product can vary within a broad range of from 0.01 to 1,000 mg per adult per day. Preferably, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for symptom adjustment in accordance with the dosage for the patient to be treated. The medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the medicament is generally provided at a dosage level of from 0.0002 mg / kg to about 20 mg / kg body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg body weight per day.

[0113] Another object of the present invention relates to a pharmaceutical composition comprising a compound according to the present invention, which is used for treating cancer, more specifically for treating cancer as detailed above, or more specifically for treating glioma or glioblastoma.

[0114] Another object of the present invention relates to the use of the compounds of the present invention and as detailed herein for preparing a pharmaceutical composition for treating cancer, more specifically for treating cancer as detailed above, or more specifically for treating glioma or glioblastoma.

[0115] In some embodiments, the compounds or compositions of the present invention are used in combination with chemotherapeutic agents or radiotherapy.

[0116] Chemotherapeutic agents include but are not limited to DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, vinca alkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, tyrosine kinase inhibitors, boron radiosensitizers (i.e., velcade) and chemotherapeutic combination therapies.

[0117] DNA alkylating agents are well known in the art and are used to treat a variety of tumors. Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as mechlorethamine, cyclophosphamide (ifosfamide, trofosfamide), chlorambucil (melphalan, prednimustine), bendamustine, uramustine, and estramustine; nitrosoureas, such as carmustine (BCNU), lomustine (semustine), fotemustine, nimustine, ranimustine, and streptozocin; alkyl sulfonates, such as busulfan (mannosulfan, treosulfan); aziridines, such as carboquone, thiotepa, triaziquone, triethylenemelamine; hydrazines (procarbazine); triazenes, such as dacarbazine and temozolomide; altretamine, and mitobronitol.

[0118] Non-limiting examples of topoisomerase I inhibitors include camptothecin derivatives, including CPT-11 (irinotecan), SN-38, APC, NPC, campothecin, topotecan, exatecan-mesylate, 9-nitro-camptothecin, 9-amino-camptothecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT, as described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and U.S. Patent Publication No. 200510250854; protoberberine alkaloids and their derivatives, including berberrubine and coralyne, as described in Li et al., (2000) Biochemistry 39(24):7107-7116 and Gatto et al., (1996) Cancer Res. 15(12):2795-2800; phenanthroline derivatives, including benzo[i]phenanthridine, Nitidine, and fagaronine, as described in Makhey et al., (2003) Bioorg. Med. Chem. 11(8):1809-1820; terbenzimidazole and its derivatives, as described in Xu (1998) Biochemistry 37(10):3558-3566; and anthracycline derivatives, including Doxorubicin, Daunorubicin, and Mitoxantrone, as described in Foglesong et al., (1992) Cancer Chemother. Pharmacol. 30(2):123-125, Crow et al., (1994) J. Med. Chem. 37(19):3191-3194, and Crespi et al., (1986) Biochem. Biophys. Res. Commun. 136(2):521-8. Topoisomerase II inhibitors include, but are not limited to, Etoposide and Teniposide.Dual topoisomerase I and II inhibitors include, but are not limited to, Saintopin and other naphthecenediones, DACA and other acridine-4-carboxamides, Intoplicine and other benzopyridoindoles, TAS-I03 and other 7H-indeno[2,1-c]quinolin-7-ones, pyrazoloacridine, XR11576 and other benzophenazines, XR 5944 and other dimeric compounds, 7-oxo-7H-dibenzo[f,ij]isoquinoline and 7-oxo-7H-benzo[e]phenanthroline and anthryl-amino acid conjugates, as described by Denny and Baguley (2003) Curr. Top. Med. Chem. 3(3):339-353. Some agents inhibit topoisomerase II and have DNA intercalating activity, such as, but not limited to, anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and anthraquinones (Mitoxantrone and Pixantrone). Examples of endoplasmic reticulum stress inducers include, but are not limited to, dimethyl-celecoxib (DMC), nelfmavir, celecoxib, and boron radiosensitizers (i.e., bortezomib (Velcade)).

[0119] Platinum-based compounds, which are a subclass of DNA alkylating agents. Non-limiting examples of such agents include Carboplatin, Cisplatin, Nedaplatin, Oxaliplatin, triplatinum tetranitrate, Satraplatin, Aroplatin, Lobaplatin, and JM-216.

[0120] Non-limiting examples of antimetabolites include folic acid-based, i.e., dihydrofolate reductase inhibitors such as aminopterin, methotrexate, and pemetrexed; thymidylate synthase inhibitors such as raltitrexed, pemetrexed; purine-based, i.e., adenosine deaminase inhibitors such as pentostatin, thiopurines such as thioguanine and mercaptopurine, halogenated / ribonucleotide reductase inhibitors such as cladribine, clofarabine, fludarabine, or guanine / guanosine: thiopurines such as thioguanine; or pyrimidine-based, i.e., cytosine / cytidine: demethylating agents such as azacitidine and decitabine, DNA polymerase inhibitors such as cytarabine, ribonucleotide reductase inhibitors such as gemcitabine, or thymine / thymidine: thymidylate synthase inhibitors such as fluorouracil (5-FU). Equivalents of 5-FU include its prodrugs, analogs, and derivatives such as 5'-deoxy-5-fluorouridine (doxifluroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur), capecitabine (Xeloda), S-I (MBMS-247616, consisting of ftorafur and two modulators 5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514, and ZD9331, as described, for example, in Papamicheal (1999) The Oncologist 4:478-487.

[0121] Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, vinflunine, vindesine, and vinorelbine.

[0122] Examples of taxanes include, but are not limited to, docetaxel, Larotaxel, Ortataxel, Paclitaxel, and Tesetaxel. An example of an epothilone is iabepilone. Examples of enzyme inhibitors include, but are not limited to, farnesyl transferase inhibitors (tipifamib); CDK inhibitors (Alvocidib, Seliciclib); proteasome inhibitors (bortezomib); phosphodiesterase inhibitors (Anagrelide; rolipram); IMP dehydrogenase inhibitors (Tiazofurine); and lipoxygenase inhibitors (Masoprocol). Examples of receptor antagonists include, but are not limited to, ERAs (Atrasentan); retinoid X receptors (Bexarotene); and sex steroids (testosterone).

[0123] Examples of tyrosine kinase inhibitors include, but are not limited to, ErbB inhibitors: HER1 / EGFR (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2 / neu (Lapatinib, Neratinib); RTK class TTI: C-kit (Axitinib, Sunitinib, Sorafenib), FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and VEGFR (Vandetanib, Semaxanib, Cediranib, Axitinib, Sorafenib); bcr-abl (Imatinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus kinase 2 (Lestaurtinib).

[0124] In a specific embodiment, the present invention relates to the compounds or compositions of the present invention for the treatment of glioblastoma. In a more specific embodiment, the compounds or compositions of the present invention are used to treat glioblastoma by eliminating or reducing glioma initiating cells (GICs) or forcing them to acquire a non-self-renewing state or lose their tumorigenicity or become susceptible to treatment.

[0125] In a specific embodiment, the present invention relates to the compounds or compositions of the present invention, which are combined with chemotherapeutic agents as defined above, more specifically with temozolomide, for the treatment of cancer, more specifically glioblastoma.

[0126] The pharmaceutical compositions of the present invention are more specifically for the simultaneous, separate or sequential administration of the compounds of the present invention and at least one chemotherapeutic agent as defined above.

[0127] The present invention will be further illustrated by the following examples. However, these examples should not be construed in any way as limiting the scope of the present invention. Detailed Description

[0128] Examples

[0129] Example 1:

[0130] Synthesis of the active compounds 9a and 9b.

[0131] The preparation of the alkyne derivatives of bleomycin A5, B2 and A2 side chains is described below. The three side chains carry the same bis-thiazole skeleton, and its synthesis is shown in Scheme 1.

[0132]

[0133] Scheme 1. Synthesis of the bis-thiazole compound 3. Reagents: a) EDC, HOBt, Et3N, CH2Cl2 / DMF, room temperature, overnight; b) LiOH, H2O / THF, room temperature (r.t.), overnight.

[0134] The introduction of the alkyne group necessary for the cycloaddition reaction was obtained by reacting compound 1 prepared following a previously published procedure (Quada JC Jr, Boturyn D, Hecht SM. Bioorg Med Chem. September 2001; 9(9):2303-14) with 4-propanoic acid in CH2Cl2 in the presence of EDC, HOBt and Et3N, thus giving the desired alkyne 2 in 50% yield. The final hydrolysis of the ester group with LiOH produced alkyne 3 for use in the coupling of the variable part of the bleomycin side chain.

[0135] Then, the alkyne derivative of the bleomycin A5 side chain was prepared as shown in Scheme 2.

[0136]

[0137] Scheme 2. Synthesis of the alkyne derivative 5 of the bleomycin A5 side chain. Reagents: a) 3, EDC, HOBt, Et3N, CH2Cl2 / DMF, room temperature, overnight.

[0138] Couple the amino group of compound 4 prepared following a previously reported procedure (Kross J, Henner WD, Haseltine WA, Rodriguez L, Levin MD, Hecht SM. Biochemistry. July 20, 1982; 21(15): 3711 - 21.) with the carboxyl group of 3 in CH2Cl2 in the presence of EDC, HOBt, and Et3N to obtain the desired A5 side chain 5 in 77% yield.

[0139] Secondly, similarly to the preparation of compound 5, synthesize the alkyne derivative of the bleomycin B2 side chain as shown in Scheme 3.

[0140]

[0141] Scheme 3. Synthesis of the alkyne derivative 7 of the bleomycin B2 side chain. Reagents: a) 3, EDC, HOBt, Et3N, CH2Cl2 / DMF, room temperature (r.t.), overnight; b) TFA, CH2Cl2, room temperature, 1 hour; c) 1,3 - di - Boc - 2 - (trifluoromethylsulfonyl)guanidine, CH2Cl2 / MeOH, room temperature, overnight.

[0142] Briefly, couple N - Boc - 1,4 - butylamine with carboxylic acid 3 in CH2Cl2 in the presence of EDC, HOBt, and Et3N to obtain intermediate 6 in 50% yield. Deprotect the amino group in CH2Cl2 in the presence of trifluoroacetic acid, followed by guanidination in the presence of 1,3 - di - Boc - 2 - (trifluoromethylsulfonyl)guanidine to obtain the desired B2 side chain analogue 7 in 76% yield in two steps.

[0143] Then conjugate the alkynes 5 and 7 with NeoN3 using a 1,3 - dipolar cycloaddition reaction in CH3CN in the presence of a catalytic amount of CuI to produce the Boc - protected conjugates 8a - b in 75 - 92% yield (Scheme 4).

[0144]

[0145] Scheme 4. Synthesis of conjugates 9a-b. Reagents. a) 5 (for the synthesis of compound 8a) and 7 (for the synthesis of compound 8b), CuI, DIPEA, CH3CN, overnight at room temperature; b) TFA, CH2Cl2, overnight at room temperature.

[0146] After deprotection of the Boc groups of compounds 8a and 8b in the presence of trifluoroacetic acid in CH2Cl2, the final compounds 9a and 9b were obtained in 100% and 85% yields, respectively.

[0147] Example 2:

[0148] Synthesis of active compounds 18a and 18b.

[0149] As shown in Scheme 5, the preparation of analogs containing an aliphatic linker instead of a triazole linker starts with the preparation of side chain 13. First, compound 4 was coupled with compound 10 in the presence of HBTU and Et3N in CH2Cl2 to give the desired compound 11 in 51% yield. The latter was deprotected in DMF in the presence of piperidine to give compound 12 in 87% yield. Finally, 12 was coupled with succinic anhydride in CH2Cl2 to give compound 13 in 84% yield.

[0150]

[0151] Scheme 5. Synthesis of side chain intermediate 13. Reagents: a) HBTU, Et3N, CH2Cl2, overnight at room temperature; b) piperidine, DMF, 2 hours at room temperature; c) succinic anhydride, CH2Cl2, overnight at room temperature.

[0152] The synthesis of the second side chain intermediate 16 is shown in Scheme 6. As carried out previously for the preparation of 13, compound 10 was coupled with commercially available N-Boc-1,3-propanediamine in the presence of HOSu, EDC and Et3N in CH2Cl2 to give compound 14 in 56% yield. Deprotection in DMF in the presence of piperidine gave compound 15 in 89% yield. The final coupling of 15 with succinic anhydride in CH2Cl2 gave the desired compound 16 in 91% yield.

[0153]

[0154] Scheme 6. Synthesis of side chain intermediate 16. Reagents: a) HOSu, EDC, CH2Cl2, overnight at room temperature; b) piperidine, DMF, 2 hours at room temperature; c) succinic anhydride, CH2Cl2, overnight at room temperature.

[0155] The synthesis of the required analogs 18a and 18b is shown in Scheme 7. First, the modified compound neomycin containing an amino group at the 5” position was prepared following the reported procedure, and then coupled with the side chain intermediates 13 and 16 in the presence of HOSu and EDC in CH2Cl2 to afford compounds 17a and 17b in 50% and 39% yields, respectively. After deprotection of the Boc protecting group in the presence of TFA in CH2Cl2, the required compounds 18a and 18b were obtained in 86% and 100% yields, respectively.

[0156]

[0157] Scheme 7. Synthesis of the active compounds 18a and 18b. Reagents: a) 13 (for the preparation of 17a) and 16 (for the preparation of 17b), HOSu, EDC, CH2Cl2, room temperature, overnight; b) TFA, CH2Cl2, room temperature, overnight.

[0158] Experimental Section

[0159] General Methods

[0160] Reagents and solvents were purchased from Aldrich or Alfa and used without further purification. All reactions involving air- or moisture-sensitive reagents or intermediates were carried out under an argon atmosphere. Flash column chromatography was performed on silica gel (Merck, SDS 40 - 63 μm, VWR). Analytical thin-layer chromatography (TLC) was performed on Fluka analytical pre-coated silica gel on TLC Al foil with a fluorescent indicator, and the compounds were visualized by irradiation (254 nm) or by staining with ninhydrin stain or anisaldehyde stain. 1H and 1 1H 13 13C NMR spectra were recorded on a Bruker AC 200 MHz or a Bruker AC 500 MHz spectrometer. Residual 11H NMR (CDCl3, δ 7.26; CD3OD δ 3.31; DMSO-d6 δ 2.50), chemical shifts are reported in parts per million (ppm, δ). Splitting patterns are designated as follows: s (singlet), d (doublet), t (triplet), and m (multiplet), br (broad). Coupling constants (J values) are listed in Hertz (Hz). Low-resolution mass spectrometry (MS) was obtained on a ThermoFinnigan (San Jose, CA) ion trap mass spectrometer equipped with an electrospray source operating in positive and negative modes. The mass spectrometer was interfaced with an Agilent (Palo Alto, Ca) 1100 HPLC system, which included a degasser, quaternary pump, autosampler, column oven, and diode array detector. HPLC was performed using a Waters Alliance 2695 pump coupled to a Waters 996 photodiode array detector and Thermo Scientific RP-C 18 columns (250 × 4.6 mm, 5 μm, for analytical HPLC; and 250 × 10 mm, 5 μm, for semi-preparative HPLC). All HPLC analyses were carried out at room temperature. A 30-minute gradient of 5% to 60% CH3CN containing 0.1% TFA in water containing 0.1% TFA (eluent A) was used, with a flow rate of 1 mL / min for analytical HPLC and 3.5 mL / min for semi-preparative HPLC.

[0161] General procedure for 1,3-dipolar cycloaddition (General Procedure A). At room temperature, copper(I) iodide (3.20 mg, 0.0322 mmol, 0.4 eq.) and N,N-diisopropylethylamine (42.0 μL, 0.484 mmol, 6 eq.) were added to a solution of NeoN3 (Vo D.D., Staedel C., Zehnacker L., Benhida R., Darfeuille F., Duca M. Targeting the production of oncogenic microRNAs with multimodal synthetic small molecules. ACS Chem. Biol. 2014, 9, 711 - 721) (100 mg, 0.0806 mmol) and alkynes 5 and 7 (0.0887 mmol, 1.1 eq.) in CH3CN (4 mL), and the reaction mixture was stirred overnight. The solvent was then removed under reduced pressure, and the crude residue was purified by flash silica gel column chromatography using a mixture of CH2Cl2 / MeOH 95:5 as the eluent to afford the desired compounds 8a - b as colorless solids.

[0162] General procedure for Boc group deprotection (General Procedure B). Deprotection of tert-butoxycarbonyl (Boc) was achieved by treating compounds 8a-b and 17a-b (0.06 mmol) with TFA (0.6 mmol, 10 eq.) in CH2Cl2 (2 mL). After stirring the reaction mixture overnight, the solvent and residual TFA were removed under reduced pressure. The pure compounds 9a-b and 18a-b (TFA salts) were obtained as colorless solids by final precipitation in a 49:1 Et2O / MeOH mixture.

[0163] Ethyl 2'-(2-(pent-4-ynamido)ethyl)-[2,4'-bithiazole]-4-carboxylate (2). To a solution of commercially available 4-pentynoic acid (346 mg, 3.53 mmol, 1 eq.) in a 1:1 mixture of CH2Cl2 / DMF (20 mL) at room temperature were added EDC (743 mg, 3.88 mmol, 1.1 eq.), HOBt (586 mg, 3.88 mmol, 1.1 eq.) and Et3N (1.48 mL, 10.6 mmol, 3 eq.). After stirring the reaction mixture at room temperature for 15 minutes, compound 1 (1 g, 3.53 mmol) was added and the resulting reaction mixture was stirred overnight at room temperature. After evaporation of the solvent under reduced pressure, CH2Cl2 was added and the organic layer was washed with saturated NH4Cl (50 mL), H2O (50 mL) and brine (50 mL). The organic phase was concentrated under reduced pressure and the resulting product was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 98.5:1.5 as the eluent. This gave the pure desired compound 2 as a colorless solid: yield 650 mg (50%); R f = 0.10 (cyclohexane / ethyl acetate 1:1); 1 1H NMR (200 MHz, CDCl3) δ (ppm): 8.16 (s, 1H), 8.02 (s, 1H), 6.52 (br, 1H), 4.43 (q, J = 7.1 Hz, 2H), 3.76 (q, J = 6.1 Hz, 2H), 3.23 (t, J = 6.1 Hz, 2H), 2.58 - 2.33 (m, 4H), 1.92 (t, J = 2.5 Hz, 1H), 1.41 (t, J = 7.1 Hz, 3H); MS (ESI), m / z 364.6 (M+H) + (theoretical m / z 364.1).

[0164] 2'-(2-(Pent-4-ynamido)ethyl)-[2,4'-bithiazole]-4-carboxylic acid (3). To a solution of compound 2 (650 mg, 1.79 mmol) in a 1:1 mixture of H2O / THF (60 mL) was added 2N LiOH solution (1.79 mL, 3.58 mmol, 2 eq.). The reaction mixture was stirred overnight at room temperature, cooled to 0 °C and neutralized with 1M HCl solution (6 mL). During evaporation of THF under reduced pressure, a precipitate appeared. The residue was filtered and evaporated to dryness to give the desired compound 3 as a colorless solid: yield 578 mg (96%); R f = 0.1 (CH2Cl2 / MeOH 9:1); 1 1H NMR (200 MHz, CD3OD) δ (ppm): 8.35 (s, 1H), 8.20 (s, 1H), 3.64 (t, J = 6.8 Hz, 2H), 3.26 (t, J = 6.8 Hz, 2H), 2.51 - 2.33 (m, 4H), 2.24 (t, J = 2.4 Hz, 1H); 13 13C NMR (50 MHz, CD3OD) δ (ppm): 174.3, 170.7, 164.9, 164.1, 149.3, 149.2, 83.5, 70.4, 40.1, 36.1, 33.7, 15.7. MS (ESI), m / z 336.6 (M+H) + (Theoretical m / z 336.0).

[0165] (4-((tert-Butoxycarbonyl)amino)butyl)(3-(2'-(2-(pent-4-ynamido)ethyl)-[2,4'-bithiazole]-4-carboxamido)propyl)carbamate (5). Compound 3 (100 mg, 0.298 mmol) was dissolved in a 1:1 mixture of CH2Cl2 / DMF (6 mL) and EDC (69 mg, 0.358 mmol, 1.2 eq.), HOBt (54.4 mg, 0.358 mmol, 1.2 eq.) and Et3N (83.4 μL, 0.596 mmol, 2 eq.) were added. After stirring the reaction mixture at room temperature for 15 minutes, compound 4 (155 mg, 0.447 mmol, 1.5 eq.) was added. After stirring overnight at room temperature, the solvent was evaporated under reduced pressure and CH2Cl2 (60 mL) was added. The organic phase was washed with saturated NH4Cl (10 mL), H2O (30 mL) and brine (30 mL), and then concentrated under reduced pressure. Finally, the resulting product was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 97:3 as the eluent to give compound 5 as a pale yellow solid: yield 153 mg (77%); R f= 0.17 (CH2Cl2 / MeOH 97:3); 1 H NMR (200 MHz, CDCl3) δ (ppm): 8.12 (br, 1H), 8.08 (s, 1H), 7.95 (s, 1H), 6.50 (br, 1H), 4.63 (br, 1H), 3.77 (t, J = 6.1 Hz, 2H), 3.57 - 3.00 (m, 10H), 2.61 - 2.31 (m, 4H), 2.00 - 1.70 (m, 3H), 1.65 - 1.24 (m, 22H); 13 C NMR (50 MHz, CDCl3) δ (ppm): 171.0, 169.0, 161.1, 156.0, 150.9, 148.5, 123.1, 116.5, 82.8, 79.6, 69.3, 46.7, 43.9, 40.1, 38.4, 35.3, 32.7, 28.5, 28.4, 27.4, 14.8; MS (ESI), m / z 686.2 (M+Na) + (Theoretical m / z 685.3).

[0166] (4-(2'-(2-(Pent-4-ynamido)ethyl)-[2,4'-bithiazole]-4-carboxamido)butyl)carbamic acid tert-butyl ester (6). Compound 3 (200 mg, 0.597 mmol) was dissolved in the mixture CH2Cl2 / DMF 1:1 (12 mL) and EDC (127 μL, 0.716 mmol, 1.2 eq.), HOBt (109 mg, 0.716 mmol, 1.2 eq.) and Et3N (167 μL, 1.19 mmol, 2 eq.) were added. After stirring for 15 minutes at room temperature, (4-aminobutyl)carbamic acid tert-butyl ester (169 mg, 0.896 mmol, 1.5 eq.) was added and the reaction mixture was stirred overnight at room temperature. After evaporation of the solvent under reduced pressure, CH2Cl2 (100 mL) was added and the mixture was washed with saturated NH4Cl (10 mL), H2O (30 mL) and brine (30 mL). The organic layer was concentrated to dryness and the resulting residue was purified by flash column chromatography using the mixture CH2Cl2 / MeOH 97:3 as the eluent to give the desired compound 7 as a pale yellow solid: yield 151 mg (50%); R f = 0.17 (CH2Cl2 / MeOH 97:3); 11H NMR (200 MHz, CDCl3) δ (ppm): 8.09 (s, 1H), 7.88 (s, 1H), 7.45 (t, J = 5.6 Hz, 1H), 6.48 (br, 1H), 4.63 (br, 1H), 3.77 (q, J = 6.1 Hz, 2H), 3.48 (q, J = 6.4 Hz, 2H), 3.25 (t, J = 6.1 Hz, 2H), 3.16 (q, J = 6.1 Hz, 2H), 2.59 - 2.34 (m, 4H), 1.93 (t, J = 2.5 Hz, 1H), 1.75 - 1.50 (m, 4H), 1.43 (s, 9H); MS (ESI), m / z 528.8 (M+Na) + (Theoretical m / z 528.2).

[0167] (tert-Butyl (4-(2'-(2-(pent-4-ynamido)ethyl)-[2,4'-bithiazole]-4-N,N'-di-Boc-guanidino)butyl)carbamate (7). Compound 6 (151 mg, 0.298 mmol) was dissolved in CH2Cl2 (4 mL) and TFA (0.46 mL, 5.97 mmol, 20 eq.) was added. After stirring the reaction mixture at room temperature for 1 h, the solvent was evaporated under reduced pressure and the crude product was used in the next step without further purification. The crude product was dissolved in a CH2Cl2 / MeOH 9:1 mixture (2.2 mL) and 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine (176 mg, 0.799 mmol, 1.5 eq.) and Et3N (125 μL, 0.894 mmol, 3 eq.) were added. After stirring the reaction mixture overnight, the solvent was evaporated under reduced pressure and the resulting residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 97:3 as the eluent to give the desired compound 7 as a pale yellow solid: yield 148 mg (76%, over 2 steps); R f f = 0.29 (CH2Cl2 / MeOH 95:5); 1 1H NMR (200 MHz, CDCl3) δ (ppm): 8.36 (br, 1H), 8.11 (s, 1H), 7.89 (s, 1H), 7.51 (t, J = 6.1 Hz, 1H), 6.54 (br, 1H), 4.63 (br, 1H), 3.77 (q, J = 6.1 Hz, 2H), 3.59 - 3.36 (m, 4H), 3.25 (t, J = 6.2 Hz, 2H), 2.59 - 2.35 (m, 4H), 1.93 (t, J = 2.5 Hz, 1H), 1.77 - 1.62 (m, 4H), 1.48 (s, 18H); 1313C NMR (50 MHz, CDCl3) δ (ppm): 171.3, 169.0, 163.4, 162.2, 161.2, 156.2, 153.3, 150.6, 148.3, 123.7, 116.5, 83.2, 82.7, 79.5, 69.5, 40.4, 38.9, 38.4, 35.3, 32.6, 28.2, 28.0, 26.9, 26.4, 14.8; MS (ESI), m / z 649.0 (M+H) + (Theoretical m / z 648.3).

[0168] (4-((tert-Butoxycarbonyl)amino)butyl)(3-(2'-(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)propanamido)ethyl)-[2,4'-bithiazole]-4-carboxamido)propyl)carbamic acid tert-butyl ester - Boc-Neo(8a). General procedure A was used for the reaction between NeoN3 and alkyne 5 to give compound 8a as a colorless solid: yield 116 mg (76%); R f = 0.2 (CH2Cl2 / MeOH 95 / 5); 1 1H NMR (500 MHz, CD3OD) δ (ppm): 8.20 (s, 1H), 7.98 (s, 1H), 5.41 (s, 1H), 5.11 (s, 1H), 4.94 (s, 1H), 4.68 - 4.51 (m, 1H), 4.42 - 4.11 (m, 3H), 3.82 - 3.73 (m, 2H), 3.70 - 3.32 (m, 18H), 3.28 - 3.11 (m, 4H), 3.10 - 2.97 (m, 4H), 2.75 - 2.52 (m, 2H), 2.05 - 1.78 (m, 3H), 1.62 - 1.29 (m, 77H); 13 13C NMR (125 MHz, CD3OD) δ (ppm): 159.2, 158.9, 158.6, 158.5, 158.2, 111.5, 100.5, 98.9, 86.1, 81.0, 80.8, 80.7, 80.4, 80.3, 79.9, 75.5, 74.7, 74.5, 73.3, 72.9, 72.8, 71.7, 69.1, 56.7, 54.8, 53.6, 51.1, 49.2, 42.5, 42.1, 41.1, 40.4, 35.9, 33.9, 29.0, 28.9, 28.8, 28.4; MS (ESI), m / z 1903.9 (M+H) + (Theoretical m / z 1902.9).

[0169] tert-Butyl-N-(4-((diaminomethylene)amino)butyl)-2'-(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)propanamido)ethyl)-[2,4'-bithiazole]-4-carboxamide-Boc-Neo(8b). General procedure A was used for the reaction between Neo-N3 and alkyne 7 to afford compound 8b as a colorless solid: yield 129 mg (85%); R f = 0.46 (CH2Cl2 / MeOH 92:8); 1 1H NMR (500 MHz, CD3OD) δ (ppm): 8.19 (s, 1H), 8.17 (s, 1H), 7.92 (s, 1H), 5.42 (s, 1H), 5.11 (s, 1H), 4.94 (s, 1H), 4.84 - 4.78 (m, 1H), 4.65 - 4.58 (m, 1H), 4.41 - 4.10 (m, 3H), 3.96 (t, J = 6.3 Hz, 1H), 3.95 - 3.86 (m, 1H), 3.76 - 3.71 (m, 2H), 3.67 - 3.34 (m, 16H), 3.30 - 2.97 (m, 6H), 2.69 - 2.53 (m, 2H), 2.02 - 1.89 (m, 3H), 1.76 - 1.64 (m, 4H), 1.62 - 1.19 (m, 75H); 13 13C NMR (125 MHz, CD3OD) δ (ppm): 174.9, 170.9, 164.6, 164.0, 163.4, 159.3, 158.9, 158.5, 158.3, 158.1, 158.0, 157.6, 154.2, 151.6, 149.4, 125.6, 125.2, 119.0, 111.4, 100.5, 98.8, 86.1, 84.5, 81.0, 80.8, 80.7, 80.6, 80.5, 80.4, 80.3, 75.5, 74.6, 74.5, 73.2, 72.9, 72.8, 71.6, 69.0, 56.7, 55.8, 53.6, 53.3, 52.6, 51.1, 49.9, 43.8, 42.5, 42.1, 41.5, 40.3, 40.1, 36.6, 35.9, 33.9, 33.1, 28.9, 28.8, 28.6, 28.3, 28.0, 27.7, 22.7, 13.3; MS (ESI), m / z 1687.8 (M + 3H - 2Boc) + (theoretical m / z 1687.8).

[0170] N-(3-((4-Aminobutyl)amino)propyl)-2'-(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)propanamido)ethyl)-[2,4'-bithiazole]-4-carboxamide-neomycin (9a). The general procedure B was applied to compound 8a to give the desired compound 9a as a colorless solid: yield 66 mg (100%); retention time 14.5 min; 1 H NMR (500 MHz, CD3OD) δ (ppm): 8.25 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 5.95 (d, J = 3.6 Hz, 1H), 5.42 (d, J = 3.8 Hz, 1H), 5.34 (d, J = 1.4 Hz, 1H), 4.94 - 4.86 (m, 1H), 4.71 (dd, J = 14.9, 5.6 Hz, 1H), 4.52 - 4.44 (m, 2H), 4.35 - 4.31 (m, 1H), 4.17 - 3.97 (m, 5H), 3.84 (t, J = 9.0 Hz, 1H), 3.77 (t, J = 4.1 Hz, 1H), 3.72 - 3.38 (m, 13H), 3.31 - 3.20 (m, 4H), 3.15 - 2.95 (m, 9H), 2.65 - 2.56 (m, 2H), 2.49 - 2.40 (m, 1H), 2.10 - 1.98 (m, 3H), 1.87 - 1.72 (m, 4H). 13 C NMR (125 MHz, CD3OD) δ (ppm): 174.8, 162.1, 161.9, 161.8, 161.7, 111.3, 97.0, 96.7, 86.7, 81.7, 77.8, 76.7, 74.8, 74.1, 72.8, 72.2, 71.9, 69.4, 69.3, 69.2, 55.1, 54.8, 53.0, 52.1, 50.2, 48.3, 46.6, 41.7, 40.2, 40.0, 37.1, 36.2, 33.9, 33.8, 29.5, 28.0, 27.8, 25.6, 24.4, 22.6; HRMS (ESI), m / z 1102.5138 (M + H) + (C 44 H 76 N 15 O 14 S2 requires 1102.5132).

[0171] N-(4-((Diaminomethylene)amino)butyl)-2'-(2-(3-(1-methyl-1H-1,2,3-triazol-4-yl)propanamido)ethyl)-[2,4'-bithiazole]-4-carboxamide-neomycin (9b). The general procedure B was applied to compound 8b to give the desired product 9b as a colorless solid: yield 97 mg (100%); retention time 15.9 min; 1 H NMR (500 MHz, CD3OD) δ (ppm): 8.20 (s, 1H), 8.17 (s, 1H), 7.94 (s, 1H), 5.94 (d, J = 3.7 Hz, 1H), 5.42 (d, J = 3.9 Hz, 1H), 5.35 (d, J = 1.5 Hz, 1H), 4.86 - 4.82 (m, 1H), 4.71 (dd, J = 14.9, 5.6 Hz, 1H), 4.52 - 4.44 (m, 2H), 4.35 - 4.31 (m, 1H), 4.17 - 3.97 (m, 5H), 3.84 (t, J = 9.0 Hz, 1H), 3.74 (t, J = 4.3 Hz, 1H), 3.71 - 3.39 (m, 13H), 3.31 - 3.20 (m, 6H), 3.02 (t, J = 7.2 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H), 2.49 - 2.40 (m, 1H), 2.10 - 1.98 (m, 1H), 1.77 - 1.64 (m, 4H); 13 C NMR (125 MHz, CD3OD) δ (ppm): 174.7, 170.9, 163.6, 158.7, 151.6, 149.4, 148.6, 125.2, 125.0, 118.9, 111.3, 97.0, 96.7, 86.7, 81.7, 77.8, 76.7, 74.8, 74.1, 72.8, 72.2, 71.9, 69.4, 69.3, 69.2, 55.9, 55.1, 53.0, 52.9, 51.2, 50.2, 49.9, 43.8, 42.2, 41.7, 40.2, 39.8, 36.2, 33.8, 29.5, 28.0, 27.9, 27.3, 22.6, 18.8, 17.3; MS (ESI), m / z 1087.4770 (M + H) + (C 42 H 70 N 16 O 14 S2 requires 1087.4772).

[0172] (3-(2'-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-[2,4'-bithiazole]-4-carboxamido)propyl)(4-((tert-butoxycarbonyl)amino)butyl)carbamic acid tert-butyl ester (11). To a solution of 2'-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)-[2,4'-bithiazole]-4-carboxylic acid 10 (1.24 g, 2.60 mmol) in anhydrous CH2Cl2 (10 mL) was added HBtU (1.18 g, 3.12 mmol, 1.2 eq) and triethylamine (724 μL, 5.20 mmol, 2 eq). The reaction mixture was stirred at room temperature for 30 minutes, then spermidine 4 (0.99 g, 2.86 mmol, 11 eq) was added. After stirring the reaction mixture overnight at room temperature, saturated NH4Cl solution was added and the reaction mixture was extracted with CH2Cl2. The combined organic phases were washed with water and brine, dried over Na2SO4, and then concentrated under reduced pressure. The crude residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 90:10 as the eluent to give the pure compound 11 as a colorless oil: yield 1.06 g (51%); R f = 0.39 (CH2Cl2 / MeOH 9:1); 1 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.96 (br s, 1H), 7.74 (d, J = 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.27 (t, J = 7.4 Hz, 2H), 5.56 (NH, 1H), 4.63 (NH, 1H), 4.41 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.8 Hz, 1H), 3.72–3.64 (m, 2H), 3.47 (br s, 2H), 3.33 (br s, 2H), 3.24 (t, J = 6.1 Hz, 2H), 3.22–3.16 (m, 2H), 3.16–3.06 (m, 2H), 1.83 (br s, 2H), 1.64–1.50 (m, 2H), 1.50–1.39 (m, 20H); 1313C NMR (50 MHz, CDCl3) δ 168.94, 162.28, 161.33, 156.48, 156.29, 156.13, 151.07, 148.74, 143.98, 141.44, 127.81, 127.13, 125.15, 123.31, 120.10, 116.56, 79.72, 66.79, 47.37, 46.84, 44.10, 40.30, 40.14, 36.33, 33.24, 28.61, 28.53, 28.28, 27.55, 25.96; MS (ESI) m / z 804.87 (M+H) + 。

[0173] (3-(2'-(2-Aminoethyl)-[2,4'-bithiazole]-4-carboxamido)propyl)(4-((tert-butoxycarbonyl)amino)butyl)carbamic acid tert-butyl ester (12). To a solution of compound 11 (1.06 g, 1.32 mmol) in DMF (10 mL) was added piperidine (2.5 mL). The reaction mixture was stirred at room temperature for 3 h, concentrated under reduced pressure, and the residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 90:10 as the eluent to give pure compound 12 as a colorless solid: yield 668 mg (87%); R f = 0.29 (CH2Cl2 / MeOH 95:05); 1 1H NMR (400 MHz, CD3OD) δ 8.17 (s, 1H), 8.15 (s, 1H), 3.43 (t, J = 6.7 Hz, 2H), 3.40–3.30 (m, 2H), 3.27–3.20 (m, 4H), 3.13 (t, J = 6.52 Hz, 2H), 3.04 (t, J = 6.7 Hz, 2H), 1.87 (br s, 2H), 1.57 (br s, 2H), 1.51–1.38 (m, 20H); 13 13C NMR (50 MHz, CD3OD) δ 171.38, 164.00, 163.40, 158.51, 157.55, 151.71, 149.54, 124.85, 118.26, 80.97, 79.78, 51.06, 48.06, 45.53, 42.14, 40.97, 38.04, 36.64, 34.80, 29.60, 28.80, 28.73, 28.34, 26.63; MS (ESI) m / z 583.07 (M+H) + 。

[0174] 4-((2-(4-((3-((tert-Butoxycarbonyl)(4-((tert-butoxycarbonyl)amino)butyl)amino)propyl)carbamoyl)-[2,4'-bithiazol]-2'-yl)ethyl)amino)-4-oxobutanoic acid (13). Succinic anhydride (30.9 mg, 0.31 mmol, 1.2 eq) was added to a solution of compound 12 (150 mg, 0.26 mmol) in CH2Cl2 (1.2 mL). The reaction mixture was stirred overnight at room temperature and then the solvent was removed under reduced pressure. The crude residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 95:5 as the eluent to give pure compound 13 as a colorless solid: 148 mg (84%); R f = 0.25 (CH2Cl2 / MeOH 95:5); 1 1H NMR (400 MHz, CDCl3) δ 9.68 (br s, OH), 8.11 (br s, NH), 8.01 (s, 1H), 7.85 (s, 1H), 7.08 (br s, NH), 4.76 (br s, NH), 3.64 (dd, J = 6.1, 12.2 Hz, 2H), 3.37 (br s, 2H), 3.24 (br s, 2H), 3.17 (t, J = 6.4 Hz, 2H), 3.11 (br s, 2H), 3.04 (br s, 2H), 2.61 (t, J = 6.7 Hz, 2H), 2.46 (t, J = 6.7 Hz, 2H), 1.75 (br s, 2H), 1.55–1.42 (m, 2H), 1.38 (s, 9H), 1.35 (s, 9H); 13 13C NMR (101 MHz, CDCl 3 ) δ 175.54, 172.51, 169.00, 162.13, 161.33, 156.11, 155.52, 150.59, 148.32, 123.43, 116.71, 79.70, 79.12, 46.75, 44.01, 40.15, 38.79, 36.35, 32.69, 30.91, 29.68, 28.45, 28.39, 28.11, 27.33, 25.83; MS (ESI) m / z 682.93 (M+H) 1+ .

[0175] (2-(4-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)-[2,4'-bithiazol]-2'-yl)ethyl)carbamic acid (9H-fluoren-9-yl)methyl ester (14). To a solution of compound 10 (292.0 mg, 0.612 mmol) in anhydrous CH2Cl2 (2 mL) was added EDC (199.3 mg, 1.04 mmol, 1.7 eq) and HOSu (119.7 mg, 1.04 mmol, 1.7 eq). The reaction mixture was stirred at room temperature for 2 h, then N-Boc-1,3-propanediamine (149.9 mg, 0.86 mmol, 1.4 eq) was added. The reaction mixture was stirred overnight at room temperature, water was added and the mixture was extracted with CH2Cl2. The combined organic phases were washed with water and brine, dried over Na2SO4 and then concentrated under reduced pressure. The crude residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 98:2 as the eluent to give pure compound 14 as a colorless solid: 216 mg (56%); R f = 0.69 (CH2Cl2 / MeOH 95:05); 1 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.88 (s, 1H), 7.79–7.69 (m, 2H + 1NH), 7.56 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.28–7.21 (t, J = 7.5 Hz, 2H), 5.67 (br s, NH), 5.20 (br s, NH), 4.40 (d, J = 6.9 Hz, 2H), 4.19 (t, J = 6.8 Hz, 1H), 3.73–3.62 (m, 2H), 3.52 (dd, J = 12.7, 6.4 Hz, 2H), 3.30–3.15 (m, 4H), 1.76 (p, J = 6.3 Hz, 2H), 1.44 (s, 9H); 13 13C NMR (101 MHz, CDCl3) δ 169.01, 162.47, 161.67, 156.51, 150.81, 148.50, 143.98, 141.42, 127.81, 127.13, 125.15, 123.63, 120.10, 116.59, 79.26, 66.77, 47.37, 40.19, 37.48, 36.33, 33.27, 30.38, 28.57; MS (ESI) m / z 633.87 (M + H) + .

[0176] (3-(2'-(2-Aminoethyl)-[2,4'-bithiazole]-4-carboxamido)propyl)carbamic acid tert-butyl ester (15). To a solution of compound 14 (210.0 mg, 0.331 mmol) in DMF (2.9 mL) was added piperidine (0.75 mL). The reaction mixture was stirred at room temperature for 4 h and the solvent was removed under reduced pressure. Water was added and the mixture was extracted with CH2Cl2. The combined organic phases were washed with water and brine, dried over Na2SO4, and then concentrated under reduced pressure. The crude residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 85:15 as the eluent to give pure compound 15 as a colorless solid: 121 mg (89%); R f = 0.27 (CH2Cl2 / MeOH 91:09); 1 1H NMR (400 MHz, CD3OD) δ 8.18 (s, 1H), 8.14 (s, 1H), 3.49 (t, J = 6.8 Hz, 2H), 3.27–3.10 (m, 6H), 1.82 (p, J = 6.5 Hz, 2H), 1.47 (s, 9H); 13 13C NMR (101 MHz, CD3OD) δ 171.40, 163.92, 163.40, 158.49, 151.60, 149.42, 124.82, 118.19, 79.90, 42.24, 38.73, 37.79, 36.91, 30.94, 28.78; MS (ESI) m / z 411.87 (M+H) + .

[0177] 4-((2-(4-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)-[2,4'-bithiazole]-2'-yl)ethyl)amino)-4-oxobutanoic acid (16). To a solution of compound 15 (100.0 mg, 0.24 mmol) in DCM (1.0 mL) was added succinic anhydride (26.0 mg, 0.26 mmol, 1.08 eq). The reaction mixture was stirred at room temperature for 2 h and then concentrated under reduced pressure. The crude residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 95:5 as the eluent to give pure compound 16 as a colorless solid: 86.9 mg (91%); R f = 0.43 (CH2Cl2 / MeOH 91:09); 11H NMR (400 MHz, CD3OD) δ 8.10 (s, 1H), 8.06 (s, 1H), 3.57 (t, J = 6.8 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 3.20 (t, J = 6.8 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H), 2.57–2.51 (m, 2H), 2.46–2.41 (m, 2H), 1.73 (p, J = 6.5 Hz, 2H), 1.38 (s, 9H); 13 13C NMR (101 MHz, CD3OD) δ 176.09, 174.68, 170.65, 163.96, 163.47, 158.54, 151.56, 149.46, 124.86, 118.46, 79.96, 40.06, 38.73, 37.79, 33.67, 31.56, 30.92, 30.22, 28.76; MS (ESI) m / z 511.80 (M+H) + 。

[0178] 4-((2-(4-((3-((tert-Butoxycarbonyl)(4-((tert-butoxycarbonyl)amino)butyl)amino)propyl)carbamoyl)-[2,4'-bithiazol]-2'-yl)ethyl)amino)-4-oxobutanoic acid amide-Boc6-neomycin (17a). To a solution of compound 13 (34.1 mg, 0.040 mmol) in anhydrous CH2Cl2 (1.0 mL) was added EDC (14.0 mg, 0.073 mmol, 1.8 eq) and HOSu (8.0 mg, 0.070 mmol, 1.7 eq). The reaction mixture was stirred at room temperature for 30 minutes and then Neo(Boc)6-NH2 compound (63.0 mg, 0.051, 1.3 eq) was added. The reaction mixture was stirred overnight at room temperature, then water was added and the mixture was extracted with CH2Cl2. The combined organic phases were washed with water and brine, dried over Na2SO4, and then concentrated under reduced pressure. The crude residue was purified by flash column chromatography using a mixture of CH2Cl2 / MeOH 95:05 as the eluent to give the pure compound 17a as a colorless solid: 37.6 mg (50%); R f = 0.43 (CH2Cl2 / MeOH 93:07); 11H NMR (400 MHz, CD3OD) δ 8.19 (s, 1H), 8.18 (s, 1H), 5.41 (s, 1H), 5.10 (s, 1H), 4.87 (s, 1H), 4.29 (s, 1H), 4.06 (br s, 1H), 3.99–3.87 (s, 4H), 3.76 (s, 2H), 3.64 (t, J = 6.7, 1H), 3.61–3.57 (m, 3H), 3.57–3.41 (m, 7H), 3.41–3.31 (m, 6H), 3.31–3.12 (m, 7H), 3.06 (t, J = 6.7 Hz, 2H), 2.67–2.49 (m, 4H), 2.01–1.92 (s, 1H), 1.88 (br s, 2H), 1.58 (br s, 2H), 1.53–1.34 (m, 75H); 13 13C NMR (101 MHz, CD3OD) δ 173.50, 173.37, 169.36, 162.69, 162.06, 157.49, 157.14, 157.06, 156.82, 156.48, 150.29, 148.13, 123.52, 117.24, 110.53, 99.31, 97.79, 86.71, 79.81, 79.61, 79.38, 79.28, 79.06, 78.93, 78.91, 78.82, 78.42, 74.64, 74.18, 73.11, 71.96, 71.43, 71.34, 70.30, 67.68, 55.56, 52.20, 51.0, 50.08, 46.47, 44.51, 42.22, 41.22, 40.60, 39.59, 38.80, 36.49, 34.41, 32.34, 31.29, 31.02, 27.63, 27.52, 27.46, 27.42, 27.37, 26.94; HRMS (ESI), m / z 1879.9263 (M+H) + (C 83 H 141 N 13 O 31 S2 requires 1879.9292).

[0179] 4-((2-(4-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)-[2,4'-bithiazol]-2'-yl)ethyl)amino)-4-oxobutyric acid - amido - Boc6 - neomycin (17b). To a solution of compound 16 (33.0 mg, 0.065 mmol, 1.8 eq) in anhydrous CH2Cl2 (400 μL) was added EDC (21.0 mg, 0.109 mmol, 3.1 eq) and HOSu (12.5 mg, 0.109 mmol, 3.1 eq). The reaction mixture was stirred at room temperature for 6 h, then Neo(Boc)6 - NH2 compound (42.5 mg, 0.035 mmol) was added. The reaction mixture was stirred overnight at room temperature. Water was added and the mixture was extracted with CH2Cl2. The combined organic phases were washed with water and brine, dried over Na2SO4, and then concentrated under reduced pressure. The crude residue was purified by flash chromatography using a mixture of CH2Cl2 / MeOH 95:5 as the eluent to give the pure compound 17b as a colorless solid: yield 23.2 mg (39%); R f = 0.34 (CH2Cl2 / MeOH 95:05); 1 1H NMR (400 MHz, CD3OD) δ 8.19 (s, 2H), 5.41 (s, 1H), 5.07 (s, 1H), 4.87 (s, 1H), 4.29 (br s, 1H), 4.13–4.01 (m, 1H), 4.00–3.86 (m, 4H), 3.76 (br s, 2H), 3.70–3.42 (m, 10H), 3.42–3.10 (m, 12H), 2.67–2.47 (m, 4H), 2.02–1.90 (m, 1H), 1.80 (p, J = 6.4, 2H), 1.55–1.31 (m, 64H); 1313C NMR (101 MHz, CD3OD) δ 174.89, 174.76, 170.76, 164.10, 163.58, 158.89, 158.59, 158.53, 158.46, 158.21, 157.86, 151.64, 149.51, 124.94, 118.69, 111.88, 100.74, 99.15, 88.08, 81.22, 80.79, 80.68, 80.46, 80.30, 80.21, 80.00, 76.03, 75.58, 74.49, 73.37, 72.81, 72.73, 71.69, 69.07, 56.95, 53.59, 52.47, 51.46, 43.62, 42.70, 42.02, 40.19, 38.77, 37.83, 35.78, 33.73, 32.69, 32.43, 30.98, 29.01, 28.91, 28.87, 28.84, 28.79, 28.76; HRMS (ESI), m / z 1707.7983 (M+H) + (C 74 H 123 N 12 O 29 S2 requires 1707.7955).

[0180] 4-((2-(4-((3-((4-Aminobutyl)amino)propyl)carbamoyl)-[2,4'-bithiazol]-2'-yl)ethyl)amino)-4-oxobutanoic acid amide-neomycin (18a). Deprotection of compound 17a (30.0 mg, 0.016 mmol) was carried out following the general procedure B to afford pure compound 18a as a colorless solid: yield 14.8 mg (86%). Retention time 3.6 min; 11H NMR (400 MHz, D2O) δ 8.24 (s, 1H), 8.12 (s, 1H), 5.97 (d, J = 3.9 Hz, 1H), 5.41 (d, J = 3.9 Hz, 1H), 5.29 (d, J = 1.4 Hz, 1H), 4.37 (t, J = 5.1 Hz, 1H), 4.34–4.22 (m, 4H), 4.13 (t, J = 9.6 Hz, 1H), 4.06–3.91 (m, 3H), 3.84 (br s, 1H), 3.79–3.70 (m, 1H), 3.67–3.58 (m, 4H), 3.58–3.31 (m, 10H), 3.29 (t, J = 6.5, 2H), 3.19–3.07 (m, 4H), 3.04 (t, J = 6.9 Hz, 2H), 2.55 (s, 5H), 2.10–2.00 (m, 2H), 1.92 (q, J = 12.6 Hz, 1H), 1.85–1.70 (m, 4H); 13 13C NMR (101 MHz, D2O) δ 175.40, 174.70, 170.87, 163.67, 163.44, 163.09, 162.98, 162.73, 162.38, 148.79, 146.96, 125.02, 120.68, 118.86, 117.78, 114.88, 111.98, 109.41, 95.77, 94.91, 84.91, 80.56, 77.34, 75.21, 73.35, 72.28, 70.34, 70.11, 69.84, 68.08, 67.57, 67.40, 53.16, 50.79, 49.55, 48.47, 46.92, 45.13, 41.35, 40.45, 39.96, 39.01, 38.74, 36.22, 32.06, 30.78, 30.58, 25.74, 23.87, 22.73; HRMS (ESI), m / z 1078.5023 (M+H)+ (C 43 H 76 N 13 O 15 S2 requires 1078.5020).

[0181] 4-((2-(4-((3-aminopropyl)carbamoyl)-[2,4'-bithiazol]-2'-yl)ethyl)amino)-4-oxobutanoic acid - amido - neomycin (18b). Compound 17b (22.0 mg, 0.013 mmol) was deprotected following General Procedure B to afford pure compound 18b as a colorless solid: 13.1 mg (100%); retention time 2.4 min; 11H NMR (400 MHz, D2O) δ 8.23 (s, 1H), 8.11 (s, 1H), 5.96 (d, J = 3.9 Hz, 1H), 5.40 (d, J = 3.8 Hz, 1H), 5.29 (br s, 1H), 4.36 (t, J = 5.1 Hz, 1H), 4.31 (t, J = 4.8 Hz, 1H), 4.30–4.26 (m, 1H), 4.26–4.22 (m, 2H), 4.12 (t, J = 9.6 Hz, 1H), 4.06–3.91 (m, 3H), 3.83 (s, 1H), 3.74 (t, J = 9.7 Hz, 1H), 3.66–3.48 (m, 9H), 3.48–3.30 (m, 6H), 3.28 (t, J = 6.4 Hz, 2H), 3.10 (t, J = 7.6 Hz, 2H), 2.59–2.47 (m, 5H), 2.07–1.98 (m, 2H), 1.92 (q, J = 12.7 Hz, 1H); 13 13C NMR (101 MHz, D2O) δ 175.39, 174.68, 170.86, 163.60, 163.44, 163.09, 162.93, 162.74, 162.39, 148.83, 146.96, 124.95, 120.67, 118.87, 117.77, 114.87, 111.97, 109.40, 95.78, 94.91, 84.90, 80.56, 77.34, 75.21, 73.35, 72.28, 70.33, 70.11, 69.84, 68.08, 67.56, 67.39, 53.15, 50.79, 49.55, 48.46, 41.34, 40.44, 39.96, 39.00, 37.05, 36.22, 32.06, 30.78, 30.57, 27.88, 26.80; HRMS (ESI), m / z 1007.4287 (M + H) + (C 39 H 67 N 12 O 15 (S2 requires 1007.4285).

[0182] Example 2: Biological Activity of the Compounds of the Invention

[0183] A - material

[0184] The nestin (MAB5326) and H3Pser10 (AB-5176) antibodies were purchased from Millipore (Millipore S.A.S., 39 Route Industrielle de la Hardt Molsheim Alsace 67120, France) and Abcam (24 rue Louis Blanc, 75010, Paris, France), respectively. The cell proliferation kit XTT assay (reference number: 11465015001) was purchased from Roche diagnostic (2, Avenue du Vercors, BP 59, 38242 Meylan Cedex, France). HepaRG, HEK293, media and supplements were purchased from Thermo Fisher scientific. TMZ was provided by Dr Chneiweiss, CAL?

[0185] B-cell culture: Patient-derived cells GB5 were isolated from the surgical resection of human primary GBM provided by the Neurosurgery Department of the University Hospital of Nice. TG6 (patient-derived cells from primary GBM) and HNNSC25 (human normal neural stem cells) were provided by Dr Hervé Chneiweiss of the University of Pierre and Marie Curie, Paris. The resulting primary cultures (GB5, TG6, HNNSC25) were grown in NS34+ medium containing EGF and bFGF (DMEM-F12 1 / 1 ratio, 10 mM glutamine, 10 mM Hepes, 0.025% sodium bicarbonate, N2, G5 and B27).

[0186] HepaRG TM is a terminally differentiated hepatocyte derived from human hepatic progenitor cells, which retains many characteristics of primary human hepatocytes. HepaRG was grown in William's E medium supplemented with Tox medium supplement and glutamax.

[0187] HEK293 (human embryonic kidney cells) were grown in DMEM supplemented with glutamax and 10% fetal bovine serum.

[0188] C-Methods:

[0189] Clonal expansion: TG6 and GB5 were seeded at 10 cells / well in 96-well plates in NS34+ medium only or in NS34+ medium containing increasing concentrations of compounds 9a-b and 18a-b. The actual number of cells in the wells was determined by direct counting immediately after seeding. After two and three weeks, the resulting sphere numbers in each well were evaluated by direct counting. The % clonal efficiency was calculated by dividing the number of cells seeded in the well by the number of resulting spheres.

[0190] II - XTT assay: TG6 and GB5 were seeded at 2000 cells / well in 96-well plates in NS34+ only or in NS34+ containing 10, 20, 25, 50, and 100 μM of compounds 9a-b and 18a-b. The cultures were incubated for 4 days and cell proliferation was evaluated according to the manufacturer's instructions.

[0191] III - Immunofluorescence assay: TG6 and GB5 were seeded at 2000 cells / well in 24-well plates in NS34+ only or in NS34+ containing 5, 7.5, 10, 12.5, 15, 20, and 25 μM of compounds 9a-b and 18a-b. The cultures were incubated for 4 days, and the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and then washed with PBS. The cells were then incubated with 20 mM ammonium chloride for 5 minutes at room temperature and washed 5 times with PBS. Blocking and hybridization were achieved in PBS containing 10% FCS and 0.1% Triton X100. Anti-nestin and anti-H3 phospho-serine 10 antibodies were incubated for 2 hours at room temperature and then washed with PBS. Fluorescently conjugated secondary antibodies and Hoechst 33342 nuclear counterstaining reagent were hybridized for one hour at room temperature. The cells were fixed with an anti-fade reagent and then imaged, counted, and quantified using a fluorescence microscope (TiE Nikon) and NiS software (Nikon).

[0192] IV - TMZ assay: TG6 and GB5 were seeded at 2000 cells / well in 96-well plates in NS34+ or treated with medium containing only 400 μM TMZ or mixed with 10, 25, and 50 μM of compounds 9a-b and 18a-b. The cultures were incubated for 4 days, and cell viability was evaluated by counting live and dead cells by XTT assay and trypan blue staining.

[0193] V - Toxicity test: NNSC, GB5, TG6, HEK293, and HepaRG, HUVEC were seeded at 2000 cells / well in 96-well plates in their respective individual media or in media containing increasing concentrations of compounds 9a-b from 10 to 500 μM. The cultures were incubated for 4 days and cytotoxicity was evaluated by XTT assay. Cell viability was also assessed by counting live and dead cells by trypan blue staining.

[0194] Results

[0195] Differentiation of patient-derived glioma stem cells (GSC or GiC) is accompanied by profound changes in their behavior and morphology. In fact, when grown in 3D in defined medium, they become adherent and adopt the typical morphology of differentiated cells, usually exemplified by a weak nucleus / cytoplasm ratio and cytoplasmic extensions. Along with these phenotypic changes, stemness markers such as nestin, NANOG, OCT4, SOX2 are downregulated, and GSC tumorigenicity as well as its clonal expansion efficiency are inhibited.

[0196] 1) Effects of compound treatment on GSC phenotype:

[0197] Patient-derived GSC (GB5) was treated for three days with increasing concentrations of compounds 9a-b and 18a-b (10 to 100 uM). 10 uM of compound 9a-b and 18a and 25 uM of compound 18b induced changes in adhesion and GSC morphology, which are reminiscent of differentiated GBM cells in culture ( Figure 1 ). Similar results were also obtained with TG6.

[0198] 2) Effects of compound treatment on stemness properties and tumorigenicity:

[0199] Patient-derived GSC (GB5) was treated for seven days with increasing concentrations of compounds capable of inducing changes in GSC morphology. To evaluate the effect on stemness maintenance, we observed the expression of stemness and progenitor cells such as nestin by immunofluorescence. As shown in Table 1 and Figure 2 A, the results showed a decrease in nestin expression when GSC was treated with compounds 9a-b and 18a. The strongest effect was obtained with compound 9a treatment (IC50 = 10 uM). Similar effects on OCT4, NANOG, and SOX2 expression were observed after treatment with compound 9a ( Figure 2 B).

[0200] The effects of the selected compounds on GSC clonal expansion and its mitotic ability were further investigated ( Figure 2 B and Figure 2 C). Treatment with compounds 9a-b and 18a altered GSC clonal expansion and mitosis. Compound 9a treatment provided the most effective inhibition of clonal expansion (IC50 < 10 uM), while both compounds 9a-b were the most effective in inhibiting mitosis (IC50 < 10 uM) ( Figure 2 C and Figure 2 D).

[0201] Orthotopic xenografts were performed in nude mice using luminescent patient-derived GSCs. Two weeks after injection, compound 9a at 10 mg / kg (n = 4), 7.5 mg / kg (n = 4), or 5 mg / kg (n = 4) or vehicle alone as control (ctl n = 10) was injected intraperitoneally into the mice three days a week (Monday, Wednesday, Friday, suspended over the weekend). Weekly, each mouse was subjected to real-time imaging (IVIS lumina III) to detect tumor occurrence and progression. In the control group, all control mice developed tumors. Among the mice treated with compound 9a, only two mice (one in the 10 mg / kg and one in the 5 mg / kg treatment groups, respectively) out of 12 mice developed tumors. Figure 3 A is a graph showing the average tumor growth in the control group (n = 10) and each compound 9a treatment group (note that the two mice that developed tumors in the treatment groups have been excluded from the average). Figure 3 B shows the survival rates of the entire mouse population in the untreated and treated groups compared using the log-rank test according to the Kaplan Meier method (R command, https: / / biostatgv.sentiweb.fr / ?module=tests / surv). In this figure, the two mice that developed tumors in the treatment group are represented. Based on these results, it was thus observed in nude mice that had been orthotopically xenografted with luminescent patient-derived GSCs that treatment with compound 9a injected intraperitoneally at 10 mg / kg, 7.5 mg / kg, and 5 mg / kg prevented tumor occurrence and development ( Figure 3 A and Figure 3 B).

[0202] To further determine whether compound 9a might also alter the tumor growth of tumors that had already formed before treatment, 50,000 GSCs expressing the luciferase gene (GB1-luc) for luminescent in vivo imaging were injected into the brains of 12 nude mice. When the tumors reached an appropriate size including a total photon flux between 2.10 6 and 1.10 7 per second, the mice were treated with DMSO (n = 6) or compound 9a (n = 6) at a dose of 7.5 mg / kg three times a week, suspended over the weekend. Tumor growth was controlled weekly by real-time imaging. As shown in the results in Figure 3 C (control: -▲-; and compound 9a: -■-), compound 9a inhibited tumor growth. The difference between the two groups was significant at week 11, ***P value = 0.017. In summary, these results indicate that compound 9a not only inhibits tumor occurrence and development but also inhibits the growth of tumors that have already formed.

[0203] 3) Effect of compound treatment on cell survival:

[0204] XTT assay allows the determination of cytotoxicity / cytostatic effects by measuring cell metabolism. The final toxicity of compounds 9a-b and 18a was tested by XTT assay as described in the Materials and Methods section. At working concentrations of 10 to 25 μM, the compounds were not toxic to GSCs ( Figure 4 A). However, compounds 9a-b and 18a showed relative toxicity at 50 μM and higher concentrations ( Figure 4 A). These XTT results were confirmed by trypan blue staining, which directly revealed the percentage of dead cells. The toxicity of compound 9a was further tested on normal neural stem cells (NNSC), human kidney (NHEK), human hepatocytes (HEPRG), and human endothelial cells (HUVEC). When used between 10 and 50 μM, the compound was not toxic to NNSC, NHEK, HEPRG, and HUVEC cells. At 100 μM and above, compound 9a was toxic ( Figure 4 ).

[0205] 4) Effects of compounds 9a-b and 18b on the sensitivity of GSCs to temozolomide (TMZ):

[0206] Temozolomide is the reference chemotherapy for GBM treatment. To evaluate whether the compounds might sensitize GSCs to TMZ, GB5 cells pretreated with TMZ (400 μM) were incubated with compounds 9a-b and 18b at 10, 25, and 50 μM. After three days, the experiment was stopped and the cells were subjected to XTT assay. At 10 μM, the results showed that the TMZ sensitivity of compounds 9a-b and 18b increased by 2-fold, 1.8-fold, and 1.7-fold, respectively ( Figure 5 ). The maximum effects of compounds 9a-b and 18b at 50 μM were 2.9-fold, 2.8-fold, and 3-fold, respectively. Cytotoxicity was confirmed by trypan blue staining.

Claims

1. A compound of general formula (I), wherein X is selected from the group consisting of: and wherein R is as follows: where n is an integer from 1 to 6; and R1 is -NHR2, -NR3R4 or a guanidyl group; R2 is a hydrogen atom, an amine protecting group or an aminoalkyl group; R3 and R4 are the same or different and are independently a hydrogen atom, an amine protecting group or an aminoalkyl group; wherein any amine group is optionally protected by any amine protecting group; or a salt thereof.

2. The compound according to claim 1, wherein X is where R is as defined in claim 1, and R1 is a guanidyl group, or R1 is -NR3R4, wherein R3 is a hydrogen atom or an amine protecting group, and R4 is an aminoalkyl group.

3. The compound according to claim 1, wherein X is where R is as defined in claim 1, and R1 is -NH2, or R1 is -NR3R4, wherein R3 is a hydrogen atom or an amine protecting group, and R4 is an aminoalkyl group.

4. The compound according to any one of claims 1 - 3, wherein R1 is -NHR2, -NR3R4 or a guanidyl group; R2 is a hydrogen atom, an amine protecting group or -(CH2) m NH2, where m is an integer from 1 to 6; R3 and R4 are the same or different and independently are a hydrogen atom, an amine protecting group or -(CH2) m NH2, where m is an integer from 1 to 6.

5. The compound according to claim 4, wherein m is 2, 3, 4, 5 or 6.

6. The compound according to any one of claims 1 - 3, wherein at least one or all of the following definitions are satisfied: n is 3 or 4; R1 is selected from the group consisting of: -NH2, a guanidyl group, and -NH(CH2) m NH2, where m is as defined in claim 4 or 5.

7. The compound according to any one of claims 1 - 3, wherein R is one of the following formulas:

8. The compound according to claim 1, wherein the compound is selected from the group consisting of:

9. The compound according to claim 1, wherein the compound is:

10. The compound according to claim 1, wherein n is an integer selected from 2, 3 or 4.

11. The compound according to claim 2, wherein n is 3 or 4.

12. The compound according to claim 3, wherein n is 3 or 4.

13. The compound according to claim 5, wherein m is 4.

14. The compound according to claim 6, wherein m is 3, 4 or 5.

15. A pharmaceutical composition comprising a compound of formula (I) as described in any one of the foregoing claims and a pharmaceutically acceptable carrier and / or excipient.

16. Use of a compound according to any one of claims 1 - 14 in the preparation of a medicament for the treatment of cancer.

17. The use according to claim 16, wherein the cancer is selected from glioma, glioblastoma or epithelial tumor cancer having cancer stem cells.

18. The use according to claim 17, wherein the cancer is glioma or glioblastoma.

19. The use according to claim 18, wherein the cancer is glioblastoma.

20. Use of a compound according to any one of claims 1 - 14 in combination with a chemotherapeutic agent or radiotherapy in the preparation of a medicament for the treatment of cancer.

21. The use according to claim 20, wherein a compound according to any one of claims 1 - 14 is combined with a chemotherapeutic agent for the treatment of glioblastoma.

22. The use according to claim 20 or 21, wherein the chemotherapeutic agent is temozolomide.

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