Removal of chemical protecting groups using ionizing radiation

Abstract

The present invention is in the field of a method of preparing a pharmaceutical containing organic active ingredients, which pharmaceutical is protected by a chemical moiety, which chemical moiety can be removed for controlled release of the active ingredient of the pharmaceutical, in particular for cancer treatment. The present invention in particular is concerned with the removal of the protecting chemical moiety.

Description

[0001] P100950PC00

[0002] REMOVAL OF CHEMICAL PROTECTING GROUPS USING IONIZING RADIATION

[0003] FIELD OF THE INVENTION

[0004] The present invention is in the field of a method of preparing a pharmaceutical containing organic active ingredients, which pharmaceutical is protected by a chemical moiety, which chemical moiety can be removed for controlled release of the active ingredient of the pharmaceutical, in particular for cancer treatment. The present invention in particular is concerned with the removal of the protecting chemical moiety.

[0005] RELATED APPLICATIONS

[0006] The present application claims the benefit of priority from Dutch Patent Application NL2038549, filed on August 30, 2024, in the name of Technische Universiteit Delft, The Netherlands.

[0007] The entire contents of the above-referenced applications and of all priority documents referenced in the Application Data Sheet filed herewith are hereby incorporated by reference for all purposes.

[0008] BACKGROUND OF THE INVENTION

[0009] Pharmaceuticals, or drugs, can be administered in various forms and dosages. Typically the intention is to deliver the pharmaceutical at a targeted site in a human or animal body. However, administration and a route towards the targeted site may involve loss of effectiveness of the pharmaceutical, degradation thereof, toxic effects on not intended parts of a body, etc. In particular in view of the toxic (side-) effects the pharmaceutical may be protected, e.g. until reaching it’s destiny, such that the effectiveness is not hampered too much.

[0010] In physics, radiation relates to emission or transmission of energy in the form of waves or particles through space or a material medium. Examples are electromagnetic radiation consisting e.g. of photons, and gamma radiation (y), particle radiation consisting e.g. of particles, such as alpha radiation (a), beta radiation (P), proton radiation and neutron radiation, and further. Radiation may be ionizing or non-ionizing. Such depends typically on the energy of the radiated particles. Ionizing radiation has 10 electron volts (eV) or more. Such energies are sufficient to ionize atoms and molecules, and in particular to break chemical bonds.

[0011] In order to protect pharmaceuticals during their trajectory in the body from intake to a desired location of application they may be incorporated in another molecule or molecules, such as in micelles, e.g. is an aggregate of surfactant molecules dispersed in a liquid. Depending on the liquid these surfactant molecules may have a hydrophilic part and a hydrophobic part. Controlled release of drugs using local ionizing radiation is considered a promising approach for targeted treatment, particularly when applied in concurrent radio-chemotherapy. Therein radiation-generated reactive species often play an important role. However, the reactive species that can be used to trigger release typically have a low yield and lack selectivity. Highly oxidative species may be used, in particular when aqueous solutions containing low concentrations of organochlorides are irradiated with ionizing radiation at therapeutically relevant doses. Examples of reactive species are peroxyl radicals. Upon exposure to ionizing radiation, the oxidation may lead to cleavage of the molecule involved, and the release of loaded drugs.

[0012] Some documents may be recited relating to removal of protecting groups, in particular Ashman et al. (D01:10.1039 / D2CC06677G), Monteiro et al. (DOI:10.1055 / a-2132- 1356), and Matsushita et al. (DOI: 10.1002 / CMDC.201900324). Ashman recites Antibody-drug conjugates containing peroxide-cleavable arylboronic acid linkers are described, which target the high levels of reactive oxygen species (ROS) in cancer. The arylboronic acid linkers rapidly release a payload in the presence of hydrogen peroxide, but remain stable in plasma. Anti-HER2 and PD-L1 peroxide-cleavable ADCs exhibited potent cytotoxicity in vitro. Monteiro recites chemotherapy, which is frequently one of the first-line treatments, which frequently suffers from low selectivity to cancer cells, leading to the appearance of important side effects. Thus, it was imperative to develop a new generation of targeted alternatives that spare the healthy tissues by delivering the cytotoxic payloads safely and selectively to cancer cells. In this respect, prodrugs that are activated by tumor-specific stimuli have attracted significant attention. Despite being a hallmark of cancer and present in high concentrations in cancer cells, reactive oxygen species (ROS) have been rather underexplored as a stimulus for the preparation of targeted prodrugs, particularly when compared with an acidic pH or glutathione. Despite their lower expression, ROS have recently been gaining substantial consideration, with various ROS-re- sponsive prodrugs already reported with meaningful performances both in vitro and in vivo. This review aims to provide critical insights into this strategy by discussing the various available functional groups (with an important focus on boronic acids and their esters), their mechanisms of action, examples of their applications, advantages, limitations, and future challenges. Matsushita reports on the development of a new gemcitabine prodrug (A-GEM), which has an arylboronate moiety at the N4-position of the cytosine nu- cleobase. A-GEM was activated in the presence of hydrogen peroxide, which is found in cancer cells, and it showed less myelosuppression in an immunodeficient mouse model than gemcitabine while maintaining antitumor activity.

[0013] The present invention relates to an improved method of removing protecting groups in organic molecules, in particular organic molecules that function as drugs, which over- comes one or more of the above disadvantages, without jeopardizing functionality and advantages.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention relates in a first aspect to a method of removing a chemical protecting group (2) from an inactive chemical compound (1) comprising providing the inactive chemical compound (1) in a solution, in an amount of 0.1 - 100 pmol / L solution, in particular 1 - 80 pmol / L, more in particular 5 - 50 pmol / L, wherein the solution comprises water, in an amount of 90 - 100 wt.% water, wherein the wt.% is relative to the total weight of the solution, and wherein the inactive chemical compound (1) comprises (i) at least one active chemical moiety (3) covalently bound to (ii) at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety, providing radiation (Sr) selected from photons and particle radiation, in particular at least one selected from gamma radiation, alpha-radiation, X-ray radiation, beta-minus radiation, beta-plus radiation, and proton radiation, wherein the radiation interacts with the water, therewith forming at least one electron, in particular at least one hydrated electron, providing an organochloride compound, wherein the organochlo- ride compound is added to the solution, such that an organochloride radical is formed, resulted from a reaction between the organochloride compound and the at least one electron is formed, preferably an organochloride peroxyradical is formed, providing chemical interaction between the organochloride radical and the at least one chemical protecting group, in particular oxidation thereof, resulting in the removal of the at least one chemical protecting group (2) from the inactive chemical compound (1), and providing separation of the active chemical moiety (3) from the at least one chemical protecting group (2) by releasing the active chemical moiety from the inactive chemical compound, the active chemical moiety (3) thereby becoming an active chemical compound (5) (see e.g. fig. 6). The chemical compound 1 is inactive, that is, in an aqueous environment, does not show significant chemical activity. By removing the chemical protecting group the chemical compound becomes active. The inactive chemical compound comprising the at least one active chemical moiety covalently bound to the at least one chemical protecting group can typically be synthesized by reacting the at least one active chemical moiety with the at least one chemical protecting group under suitable conditions; the reactants can typically be purchased from commercial suppliers. The term “protecting group” is therefore used to indicate the change in biological activity, where modification with the protecting group of a functional group on a bioactive molecule reduces or removes the biological activity. Such a protecting group in the context of the invention may also be referred to as a “cage”, and in view of the function, it may also be referred to as “self-immolated”. Equivalently in chemistry the present inactive group is protected, and later deprotected, such that the active group can not perform its chemical action. In chemistry and biology, activation is the process whereby something is prepared or excited for a subsequent reaction. The subsequent (chemical) reaction leads to a (bio-)chemical transformation of one set of (bio-)chemical substances to another. Chemical reactions typically involve forming and breaking of chemical bonds between atoms, and can often be described by a chemical equation. Substance involved are called reactants. Chemical reactions typically result in one or more products formed, which have properties different from the reactants. The presence of a protecting group on one of the reactants changes its chemical reactivity such that it can no longer participate in the reaction. Removal of the protecting group reinstates the original reactivity, allowing the reaction to occur. The present active chemical moiety may become active after release of the protecting group, e.g. in a body, and perform a biological activity there. The protecting group prevents said biological activity, and thus less side-effects are observed. The present inactive compound typically is present in relatively low concentrations, typically in an aqueous solution, comprising mainly or fully water as a solvent. Co-solvents may be present. The present chemical protecting group is ’’introduced” into a molecule typically by chemical modification of a functional group (the present inactive chemical compound) to obtain chemo selectivity in a subsequent chemical reaction, or to prevent such a reaction altogether. It often plays an important role in multistep organic synthesis. In many preparations of delicate organic compounds, specific parts of the molecules cannot survive the required reagents or chemical environments. These parts (functional groups) must then be protected. The term “compound” is typically considered to relate to a “moiety”, or distinguishable unit. The present radiation interacts with water, forming an electron (see fig. la). The electron in the present aqueous solution is typically a hydrated electron. The present organochloro compound, that is chlorinated organic compounds, whereas organochloride is also a common phrase of such organochloro compounds, compounds such as chlorinated hydrocarbons, such as chloroform, relates to a molecule having an organic backbone, and chlorine atoms. The organochloro compound may be relatively small, such as e.g. chloroform, or may be larger, such as oligomers and (small) polymers comprising chlorine atoms. At least one chlorine atom is split from the organochloro compound, forming a chloride ion and a carbon (organochloride) radical comprising optional remaining chlorine atoms. In the presence of trace amount of oxygen being present in the solution, the carbon radical, e.g. CHCh, reacts with oxygen O2 typically at a diffusion-controlled rate, leading to the formation of peroxyl radicals that are able to oxidize further species; for example:

[0016] H2O + y-rays - >H + HO + e’aq+ H+

[0017] By the provision of the organochloride peroxyradical and the subsequent reaction thereof with the chemical protecting group the chemical protecting group is separated from the inactive chemical compound. The present organochloride peroxyradical is found to be particularly effective when the present inactive chemical compound comprises at least one of a to be cleaved functional group selected from amides (R1-(C=O)-N-(R2,R3), wherein one of R2,R3 may be absent), such a carbamates, and sulfone amides, from C=C, from carbonates, from esters, and from ethers; typically a boric acid and / or carbon dioxide may be released. In comparison of the present organochloro compound to H2O2 it is noted that first of all H2O2 can not be relied on, e.g. in the body, as it is not produced through water irradiation by ionizing radiation, or in too low concentrations. Further, the reactivity of the present organochloro compound is much higher, e.g. typically two times higher, more typically at least one order of magnitude higher.

[0018] In a second aspect the present invention relates to a method of treating tumors, the method comprising applying at least one of radiotherapy, immunotherapy and chemotherapy, wherein the method comprises providing an organochloro compound.

[0019] In a third aspect the present invention relates to a use of an organochloro compound in a method of removing a chemical protecting group (2) from an inactive chemical compound (1), wherein the inactive chemical compound comprises an active chemical moiety (3) covalently bound to at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety.

[0020] In a fourth aspect the present invention relates to an organochloro compound for use in treatment of cancer, in particular in treatment of tumors, more in particular in treatment of hypoxic tumors. These hypoxic tumors can be treated under low oxygen concentrations, in particular under a partial oxygen pressure of <5% relative, more in particular <2% relative, even more in particular < 0.5% relative, in particular as measured in the tissue.

[0021] In a fifth aspect the present invention relates to an inactive chemical compound (1) comprising (i) at least one active chemical moiety (3) covalently bound to (ii) at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety, for use as a pharmaceutical in the treatment of cancer, in particular in treatment of tumors, more in particular in treatment of hypoxic tumors, wherein the chemical protecting group (2) is selected from at least one of a chemical protecting group comprising boron, preferably comprising at least one of a boronic acid group and an ester derivative of a boronic acid, and a chemical protecting group comprising sulfur, preferably comprising a thioether, in particular wherein the treatment further comprises providing an organochloro compound and providing radiation (Sr), selected from photons and particle radiation.

[0022] In a sixth aspect the present invention relates to a pharmaceutical dosage comprising an inactive chemical compound (1), the inactive chemical compound comprising (i) at least one active chemical moiety (3) covalently bound to (ii) at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety, wherein the chemical protecting group (2) is selected from at least one of a chemical protecting group comprising boron, preferably comprising at least one of a boronic acid group and an ester derivative of a boronic acid, and a chemical protecting group comprising sulphur, preferably comprising a thioether.

[0023] Thereby the present invention provides a solution to one or more of the above mentioned problems.

[0024] Advantages of the present description are detailed throughout the description. References to the figures are not limiting, and are only intended to guide the person skilled in the art through details of the present invention.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates in a first aspect to the method of removing a chemical protecting group.

[0027] In an exemplary embodiment of the present method of removing a chemical protecting group the solution is a polar solution, in particular an aqueous solution, more in particular a solution comprising at least one selected from deionized water, phosphate buffer saline solution, and serum.

[0028] In an exemplary embodiment of the present method of removing a chemical protecting group an amine compound (7) is additionally provided to the solution, in particular a tertiary amine compound, in particular the amine compound is provided in a concentration of 1 - 1000 pmol / L solution, more in particular in a concentration of 5 - 800 pmol / L, more in particular in a concentration of 10 - 650 pmol / L. Such in particular supports the deprotection of boronic acid and esters.

[0029] In an exemplary embodiment of the present method of removing a chemical protecting group the chemical protecting group (2) is selected from at least one of a chemical protecting group comprising boron, preferably comprising at least one of a boronic acid group and an ester derivative of a boronic acid, comprising selenium, in particular a selenoether, and a chemical protecting group comprising sulfur, preferably comprising a thioether.

[0030] In an exemplary embodiment of the present method of removing a chemical protecting group the method is performed under condition wherein oxygen is present, in particular under condition of low oxygen concentration, in particular wherein the oxygen concentration of the solution is between 0.1 and 1250 pmol / L, more in particular between 0.5 and 500 pmol / L, more in particular between 1 and 62,5 pmol / L.

[0031] In an exemplary embodiment of the present method of removing a chemical protecting group the radiation is applied in a clinical dose, and / or wherein the radiation is selected from gamma radiation, alpha radiation, X-ray radiation, beta-minus radiation, beta- plus radiation and proton radiation, in particular with a radiation dose between 0.1 and 100 Gy, in particular between 0.5 and 80 Gy, more in particular between 1 and 60 Gy.

[0032] In an exemplary embodiment of the present method of removing a chemical protecting group the active chemical compound (5) is a pharmaceutical compound, in particular a drug, more in particular an anti-cancer drug, even more in particular an anti-cancer drug that is incorporated in newly formed DNA when a cell replicates, such as a drug that is catalyzed by the enzyme deoxy cytidine kinase (DCK).

[0033] In an exemplary embodiment of the present method of removing a chemical protecting group the organochloro compound is selected from di- and trichloro compounds, typically small molecular organochloros, in particular from organochloros with a size of 4 - 15 atoms, more in particular with a size of 4 - 10 atoms, preferably the organochloro is selected from at least one of chloroform, methyl trichloro acetate, dichloromethane, 2,2,2- trichloroethanol, and 2,2-dichloroethanol, and from polymer comprising organochloro compounds, in particular poly (meth)acrylic polymer comprising compounds, in particular relatively small polymers with an average number molecular weight (according to GPC) of < 10 kDa (e.g. on a Prominence- 1 GPC of Shimadzu against polystyrene standards, e.g. ASTM D5296-19). Such polymers may be synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. The present chemicals in general were typically purchased from commercial suppliers, and typically used without further purification.

[0034] In an exemplary embodiment of the present method of removing a chemical protecting group the organochloro compound comprises at least two chloride atoms, and / or wherein the concentration of the organochloro compound in the solution is between 0.01- 50 mM, preferably between 0.1-35 mM, more preferably between 1-20 mM, and / or wherein the organochloro is conjugated with at least one of a particle, a protein, and a polymer, in particular a nanoparticle, such as a nanoparticle with a core diameter of 20- 100 nm, such as 30-50 nm.

[0035] In an exemplary embodiment of the present method of removing a chemical protecting group the chemical interaction between the organochloride radical, preferably the or- ganochloride peroxyradical, and the at least one chemical protecting group (2) is a chemical reaction, preferably an oxidation reaction.

[0036] In an exemplary embodiment of the present method of removing a chemical protecting group the active chemical compound is selected from pharmacogenomic and phar- macogenetic drugs, in particular from drugs that have nucleophilic functional groups in their molecular structure, in particular from drugs that have hydroxy (ROH), amino (RR’NH or RR’R”N), thiol (RSH) or carboxyl (RCOOH) functional groups in their molecular structure, in particular from chemotherapeutics, more in particular from hydro- philic drugs, such as from pyrimidine nucleoside prodrugs, such as 2',2'-difluoro 2'deox- ycytidine (gemcitabine) (CAS 95058-81-4), from cytotoxic alkaloids, such as camptothe- cin, and doxorubicin (CAS 23214-92-8), and derivates thereof.

[0037] In an exemplary embodiment of the present method of removing a chemical protecting group the chemical interaction between the organochloride radical and the at least one chemical protecting group is one of accompanied by a hydrolysis reaction (H), and followed by a hydrolysis reaction (H), therewith releasing the active chemical moiety (3) from the inactive chemical compound (1), the active chemical moiety (3) thereby becoming an active chemical compound (5).

[0038] In an exemplary embodiment of the present method of removing a chemical protecting group the method is comprised in a method of medical treatment.

[0039] In an exemplary embodiment of the present method of removing a chemical protecting group the method of medical treatment is applied locally by providing radiation locally to a subject, in particular to one selected from a human body and an animal body.

[0040] In an exemplary embodiment of the present method of removing a chemical protecting group the method of medical treatment is a method of treating tumors, in particular hypoxic tumors.

[0041] In an exemplary embodiment of the present method of treating tumors an organochloride radical, preferably an organochloride peroxyradical, is formed resulted from a reaction between the organochloro compound and at least one electron.

[0042] In an exemplary embodiment of the present use in the method an organochloride radical, preferably an organochloride peroxyradical, is formed resulted from a reaction between the organochloro compound and at least one electron.

[0043] In an exemplary embodiment of the present use the at least one electron is formed by radiation (Sr) selected from photons and particle radiation, preferably selected from at least one of gamma radiation, alpha radiation, X-ray radiation, beta-minus radiation, betaplus radiation, and proton radiation.

[0044] In an exemplary embodiment of the present organochloro compound the treatment comprises at least one of radiotherapy, immunotherapy and chemotherapy.

[0045] In an exemplary embodiment of the present organochloro compound the organochloro compound is selected from small molecular organochlorides, in particular from or- ganochloros with a size of 4 - 15 atoms, more in particular with a size of 4 - 10 atoms, preferably the organochloro is selected from at least one of chloroform, methyl trichloro acetate, dichloromethane, 2,2,2-trichloroethanol, and 2,2-dichloroethanol, and from polymer comprising organochloro compounds, in particular poly (meth)acrylic polymer comprising compounds, and / or wherein the organochloro compound comprises at least two chloride atoms, and / or wherein the concentration of the organochloro compound in the solution is between 0.01- 50 mM, preferably between 0.1-35 mM, more preferably between 1-20 mM, and / or wherein the organochloro is conjugated with at least one of a particle, a protein, and a polymer.

[0046] In an exemplary embodiment the present pharmaceutical dosage further comprises an organochloro compound.

[0047] In an exemplary embodiment of the present pharmaceutical dosage the organochloro compound is selected from small molecular organochloros, in particular from or- ganochloros with a size of 4 - 15 atoms, more in particular with a size of 4 - 10 atoms, preferably the organochloro is selected from at least one of chloroform, methyl trichloro acetate, dichloromethane, 2,2,2-trichloroethanol, and 2,2-dichloroethanol, and from polymer comprising organochloro compounds, in particular poly (meth)acrylic polymer comprising compounds, and / or wherein the organochloro compound comprises at least two chloride atoms, and / or wherein the organochloro is conjugated with at least one of a particle, a protein, and a polymer.

[0048] In an exemplary embodiment the present pharmaceutical dosage further comprises an amine compound (7), in particular a tertiary amine compound.

[0049] In an exemplary embodiment of the present pharmaceutical dosage the pharmaceutical dosage is comprised in a solution, wherein the solution comprises water, in an amount of [90 - 100 wt.%] water, wherein the wt.% is relative to the total weight of the solution, and wherein the concentration of the inactive chemical compound (1) is 0.1 - 100 pmol / L solution, in particular 1 - 80 pmol / L, more in particular 5 - 50 pmol / L, and / or wherein the concentration of the organochloro compound in the solution is between 0.01-50 mM, preferably between 0.1-35 mM, more preferably between 1-20 mM, and / or wherein the concentration of the amine compound (7) is 1 - 500 pmol / L solution, in particular 5 - 400 pmol / L solution, more in particular 10 - 300 pmol / L solution, in particular wherein the dosage comprises nanoparticles, wherein the nanoparticles comprise an amphiphilic surface active compound, in particular selected from polymers, the surface active compound forming a layer, and within the layer the inactive compound, wherein a dosage of the inactive compound is 0.1-1 mg / mg surface active compound, in particular 0.2-0.5 mg / mg.

[0050] The invention is further detailed by the accompanying figures and examples, which are exemplary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

[0051] SUMMARY OF FIGURES

[0052] Figures 2-13 show details of the present invention, whereas fig. 1 shows an aspect of prior art. DETAILED DESCRIPTION OF FIGURES

[0053] In the figures:

[0054] 1. inactive chemical compound

[0055] 2. chemical protecting group

[0056] 3. active chemical moiety in its inactive form by a covalent bond to a chemical protecting group

[0057] 4. removed chemical protecting group (parts)

[0058] 5. active chemical compound

[0059] 6. rest groups of an experimental probe for investigating double bond oxidation

[0060] 7. tertiary amine compound

[0061] 8. water unstable intermediate compound, which will be hydrolyzed (H) in water Sh hydrogen peroxide stimulus

[0062] Sr radiation stimulus in the presence of organochloride iE increased efficacy of boronic acid oxidation in the presence of (tertiary) amine H hydrolysis of water unstable intermediate compounds

[0063] Figure 1 shows prior art oxidative cleavage of stilbene by radiation in water containing organochloride. (a) Proposed sequence of events; (b) chemical structure of a comparable compound and block copolymer PSP. Fig. 1c shows various reactive organo- chloro species.

[0064] Figure 2 shows prior art oxidative cleavage of PSP and the reaction products.

[0065] Figure 3 shows (a) the deprotection of a boronic acid derivative by radiation and the catalytic effect of amines, (b) fluorescence emission spectrum of 1 before and after 8 Gy of gamma-irradiation (excitation at 330 nm; 10 pM probe in PBS, pH 7.4; 0.1 vol% of organochloride if present; 100 pM of amine if present; samples were incubated for 30 min after irradiation); (b) HPLC-UV chromatograph determines the oxidation of 1 (10 pM in water; 0.1 vol% of organochloride if present; 100 pM of amine if present; samples were incubated for 30 min after irradiation).

[0066] Figure 4 shows (a) schematic illustration of the reactions of 4-(methyl Thio)phenol under irradiation in water and water / TCE; (b)1H NMR spectrum of 4-(methyl Thio)phe- nol before and after irradiation (spectrum were taken within 3 hours after irradiation).

[0067] Figure 5 shows the deprotection of anti-tumor drug, gemcitabine, by radiation.

[0068] Figure 6 shows the oxidation of a thioether derivative followed by the hydrolysis of the unstable intermediate.

[0069] Fig. 7a shows a chemical structure of gemcitabine and 7b of camptothecin (CAS 7689-03-4).

[0070] Fig. 8 shows an exemplary reaction scheme. In the first step radiation and water causes formation of a reactive species. This species and oxygen form a peroxyl radical. Next, the boronic acid is provided with an OR group. The boronic acid is subsequently released, and also the C-X bond is cleaved, releasing the active group, indicated with “cargo”.

[0071] Fig. 9 shows self-immolation of exemplary benzylboronic acids and esters thereof by the present method, under radiation and the provision of the present organochloro compound.

[0072] Fig. 10 shows hydrolysis of thioether / sulfide esters / carbamates / carbonates upon oxidation to sulfoxide, by the present method, under radiation and the provision of the present organochloro compound.

[0073] Fig. 11 shows the use of the present protecting group with gemcitabine, and the use of the present method, under radiation and the provision of the present organochloro compound. Fig. 12 shows two comparable results for camptothecin. For the first structure, Inventors synthesized a polymer construct with camptothecin. The polymer (R in the structure) contains trichloro ethanol esters as the sidechains of the polymer. These tricholoro ethanol groups were the source of organochloride in the experiment, no small molecule organochloride was added. The polymer had on average one camptothecin unit per polymer chain. Irradiation (60Gy) of a 2mg / ml polymer solution in PBS resulted in 30% release of camptothecin. For the second structure inventors synthesized a camptothecin prodrug with the camptothecin hydroxyl functional group protected as a thioether-containing ester. Radiation-induced oxidation of the thioether functionality resulted in accelerated hydrolysis of the ester, leading to formation of native camptothecin. Radiation experiments were performed on 10 pM of prodrug in PBS / 12 mM trichloro ethanol; 8Gy of gamma radiation gave statistically significant release of camptothecin whereas controls or ImM hydrogen peroxide did not.

[0074] Fig. 13a, b show the The diameter of spheroids after various treatments as a function of time, shown for samples that are (a) not irradiated and (b) irradiated with 6 Gy X-rays; Gemcitabine (Gem)+2-dimethylaminobenzoic acid (amine A)+tri chloro ethanol (T): addition of 81 nm. Gem, 5 pM A and 500 pM T; Benzylboronic acid-Gem (BOH- Gem)+A+T: addition of 81 nM prodrug, 5 pM A and 500 pM T; BOH-Gem+T: addition of 81 nM BOH-Gem and 500 pM T; BOH-Gem: addition of 81 nM BOH-Gem; Control: addition of 5 pM A and 500 pM T. One-way ANOVA t-test, n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001. (c) Light microscopy images of spheroids treated with indicated conditions, scale bar = 400 pm. (Gem represents addition of 81 pM gemcitabine; BOH-Gem A+T represents addition of 81 pM BOH-Gem, 5 pM A and 500 pM T).

[0075] The figures are further detailed in the description of the experiments below. EXAMPLES / EXPERIMENTS

[0076] Experimental Methods

[0077] All compounds were purchased from commercial suppliers (Sigma Aldrich (Zwijndrecht, the Netherlands), Tokyo Chemical Industry (Zwijndrecht, the Netherlands) and abcr Gute Chemie (Karlsruhe, Germany)) and used without further purification unless otherwise specified. Reactions were monitored by thin-layer chromatography on a silica gel plate and visualized by UV light (254 nm) or stained using a KMnOVsolution. Fluorescence spectra were recorded with Spex Fluorolog- 3 equipped with a standard 90° setup. Flash column chromatography was carried out on a 30 cm column loaded with 230 - 400 mesh silica gel. 'H-NMR spectra were recorded on an Agilent-400 MR DD2 (Santa Clara, United States) (399.67 MHz) at 298 K. Milli-Q water was obtained by purification of demineralized water with a Milli-Q IQ 7000 (Darmstadt, Germany) machine equipped with a Millipak 0.22 pM filter. High-performance liquid chromatography-mass spectrometry (HPLC-MS) was performed on a LTQ XL spectrometer from Thermo Scientific (Bleiswijk, The Netherlands) that was connected with a Shimadzu HPLC setup with D2 detector and Discovery C18 reverse phase column (Kyoto, Japan). Water / MeCN with 0.1% (v / v) formic acid was used as the mobile phase at a flow rate of 0.2 mL / min. The irradiations with y-rays were performed using a Nordi on 22060Co gamma cell (Ottawa, Canada). The dose rate at the experimental date was around 460 Gy / h which is calculated based on the decay law and the half-life of60Co. The delivered dose was calculated by the dose rate at the date of the experiments multiplied by the exposure time. Radiation was given in one fraction unless otherwise specified. The X-ray irradiation was carried out using an X-ray source (Philips MCN 321 variable-energy X-ray tube, Eindhoven, The Netherlands) performed in a working voltage of 240 keV and a current of 10 mA. The dose rate of the X-ray source was determined using the method as described in literature.

[0078] Results

[0079] In Figure 3, 7-amino-4-methylcoumarin (AMC, CAS 26093-31-2) is used as the active chemical compound, 2,2,2-trichloroethanol (TCE, CAS 115-20-8) is used as the organochloride and 2-(dimethylamino)benzoic acid is used as amine. The emission intensity of AMC is quenched if the amino group bonds with a carbamate group. The oxidation of boronic acid results in the hydrolysis of the carbamate and activates the fluorescence emission of AMC. As shown in Figure 3b, irradiation (8 Gy) of boronic acid protected AMC in PBS leads to a slight increase in emission intensity at 441 nm attribute to the oxidation of arylboronic acid by hydrogen peroxide and subsequently activating AMC. The introduction of 2,2,2-trichloroethanol (TCE) results in enhanced AMC activation as evidenced by a higher emission intensity, indicating that the peroxyl radical generated from PBS / TCE irradiation can oxidize boronic acid moiety. Moreover if amine is presented in PBS / TCE, more AMC is activated, suggesting the enhancing effect of tertiary amine in the irradiation process. However, irradiation in PBS / amine results in a much less AMC activation than that in PBS / TCE / amine, suggesting that the present of TCE is crucial in the oxidation process. This conclusion is corroborated by high-performance liquid chromatography (HPLC) (Figure 3c), where irradiation in water / TCE / amine results in the highest conversion to AMC in all the experimental groups.

[0080] In Figure 4, 4-(methyl Thio)phenol is used as the model thioether and 2,2,2- trichloroethan-l-ol (TCE) is used as the model organochloride. Proton nuclear magnetic resonance (XH NMR) was employed to quantify the oxidation. 4-(Methyl- thio)phenol was dissolved in water or in water / 0.1 vol% TCE with a concentration of 50 pM. After exposure to gamma-radiation (60 Gy) in water, theXH NMR spectrum remained the same as it is before irradiation (Figure 4b). Although water radiolysis generates hydrogen peroxide that can oxidise thioether, the low concentration after 60 Gy (ca. 4.2 pM) and the slow reaction kinetic cause the product undetectable using NMR. However, if 0.1 vol% TCE was present while irradiating, the methyl peak shifted from 2.30 to 2.73 ppm, which corresponded to the sulfoxide compound. This indicates that the formed peroxyl radical can oxidise thioether rapidly.

[0081] It should be appreciated that for commercial application it may be preferable to use one or more variations of the present system, which would similar be to the ones disclosed in the present application and are within the spirit of the invention.

Claims

CLAIMS1. A method of removing a chemical protecting group (2) from an inactive chemical compound (1) comprising providing the inactive chemical compound (1) in a solution, in an amount of 0.1 - 100 pmol / L solution, in particular 1 - 80 pmol / L, more in particular 5 - 50 pmol / L, wherein the solution comprises water, in an amount of 90 - 100 wt.% water, wherein the wt.% is relative to the total weight of the solution, and wherein the inactive chemical compound (1) comprises (i) at least one active chemical moiety (3) covalently bound to (ii) at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety, providing radiation (Sr) selected from photons and particle radiation, in particular at least one selected from gamma radiation, alpha-radiation, X-ray radiation, beta-minus radiation, beta-plus radiation, and proton radiation, wherein the radiation interacts with the water, therewith forming at least one electron, in particular at least one hydrated electron, providing an organochloro compound, wherein the organochloro compound is added to the solution, such that an organo- chloride radical is formed, resulted from a reaction between the organochloro compound and the at least one electron is formed, preferably an organochloride peroxyradical is formed, providing chemical interaction between the organochloride radical and the at least one chemical protecting group, resulting in the removal of the at least one chemical protecting group (2) from the inactive chemical compound (1), and providing separation of the active chemical moiety (3) from the at least one chemical protecting group (2) by releasing the active chemical moiety from the inactive chemical compound, the active chemical moiety (3) thereby becoming an active chemical compound (5).

2. The method according to claim 1, wherein the solution is a polar solution, in particular an aqueous solution, more in particular a solution comprising at least one selected from deionized water, phosphate buffer saline solution, and serum.

3. The method according to claim 1 or 2, wherein an amine compound (7) is additionally provided to the solution, in particular a tertiary amine compound, in particular the amine compound is provided in a concentration of 1 - 1000 pmol / L solution, more in particular in a concentration of 5 - 800 pmol / L, more in particular in a concentration of 10 - 650 pmol / L.

4. The method according to any of claims 1-3, wherein the chemical protecting group (2) is selected from at least one ofa chemical protecting group comprising boron, preferably comprising at least one of a bo- ronic acid group and an ester derivative of a boronic acid, comprising selenium, in particular a selenoether, and a chemical protecting group comprising sulfur, preferably comprising a thioether.

5. The method according to any of claims 1-4, wherein the method is performed under condition wherein oxygen is present, in particular under condition of low oxygen concentration, in particular wherein the oxygen concentration of the solution is between 0.1 and 1250 pmol / L, more in particular between 0.5 and 500 pmol / L, more in particular between 1 and 62,5 pmol / L.

6. The method according to any of claims 1-5, wherein the radiation is applied in a clinical dose, and / or wherein the radiation is selected from gamma radiation, alpha radiation, X-ray radiation, beta-minus radiation, beta-plus radiation and proton radiation, in particular with a radiation dose between 0.1 and 100 Gy, in particular between 0.5 and 80 Gy, more in particular between 1 and 60 Gy.

7. The method according to any of claims 1-6, wherein the active chemical compound (5) is a pharmaceutical compound, in particular a drug, more in particular an anti-cancer drug.

8. The method according to any of claims 1-7, wherein the organochloro compound is selected from small molecular organochloros, in particular from organochloros with a size of 4 - 15 atoms, more in particular with a size of 4 - 10 atoms, preferably the organochloro is selected from at least one of chloroform, methyl trichloro acetate, dichloromethane, 2,2,2-trichloroethanol, and 2,2-dichloroethanol, and from polymer comprising organochloro compounds, in particular poly (meth)acrylic polymer comprising compounds, and / or wherein the organochloro compound comprises at least two chloride atoms, and / or wherein the concentration of the organochloro compound in the solution is between 0.01- 50 mM, preferably between 0.1-35 mM, more preferably between 1-20 mM, and / or wherein the organochloro compound is conjugated with at least one of a particle, a protein, and a polymer, in particular a nanoparticle, such as a nanoparticle with a core diameter of 20-100 nm.

9. The method according to any of claims 1-8, wherein the chemical interaction between the organochloride radical, preferably the organochloride peroxyradical, and the at least one chemical protecting group (2) is a chemical reaction, preferably an oxidation reaction.

10. The method according to any of claims 1-9, wherein the active chemical compound is selected from pharmacogenomic and pharmacogenetic drugs, in particular from drugs that have nucleophilic functional groups in their molecular structure, in particular from drugs that have hydroxy (ROH), amino (RR’NH or RR’R”N), thiol (RSH) or carboxyl(RCOOH) functional groups in their molecular structure, in particular from chemotherapeutics, more in particular from hydrophilic drugs, such as from pyrimidine nucleoside prodrugs, such as 2',2'-difluoro 2'deoxycytidine (gemcitabine) (CAS 95058-81-4), from cytotoxic alkaloids, such as camptothecin, and doxorubicin (CAS 23214-92-8), and deri- vates thereof.

11. The method according to any of claims 1-10, wherein the chemical interaction between the organochloride radical and the at least one chemical protecting group is one of accompanied by a hydrolysis reaction (H), and followed by a hydrolysis reaction (H), therewith releasing the active chemical moiety (3) from the inactive chemical compound (1), the active chemical moiety (3) thereby becoming an active chemical compound (5).

12. The method according to any of claims 1-11, wherein the method is comprised in a method of medical treatment.13 The method according to claim 12, wherein the method of medical treatment is applied locally by providing radiation locally to a subject, in particular to one selected from a human body and an animal body.

14. The method according to any of claims 12 or 13, wherein the method of medical treatment is a method of treating tumors, in particular hypoxic tumors.

15. A method of treating tumors, the method comprising applying at least one of radiotherapy, immunotherapy and chemotherapy, wherein the method comprises providing an organochloro compound.

16. The method according to claim 15, wherein an organochloride radical, preferably an organochloride peroxyradical, is formed resulted from a reaction between the organochloro compound and at least one electron.

17. Use of an organochloro compound in a method of removing a chemical protecting group (2) from an inactive chemical compound (1), wherein the inactive chemical compound comprises an active chemical moiety (3) covalently bound to at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety.

18. Use according to claim 17, wherein in the method an organochloride radical, preferably an organochloride peroxyradical, is formed resulted from a reaction between the organochloro compound and at least one electron.

19. Use according to claim 18, wherein the at least one electron is formed by radiation (Sr) selected from photons and particle radiation, preferably selected from at least one of gamma radiation, alpha radiation, X-ray radiation, beta-minus radiation, beta-plus radiation, and proton radiation.

20. Organochloro compound for use in treatment of cancer, in particular in treatment of tumors, more in particular in treatment of hypoxic tumors.

21. Organochloro compound for use according to claim 20, wherein the treatment comprises at least one of radiotherapy, immunotherapy and chemotherapy.

22. Organochloro compound for use according to any of claims 20 or 21, wherein the organochloro compound is selected from small molecular organochlorides, in particular from organochloros with a size of 4 - 15 atoms, more in particular with a size of 4 - 10 atoms, preferably the organochloro is selected from at least one of chloroform, methyl trichloro acetate, dichloromethane, 2,2,2-trichloroethanol, and 2,2-dichloroethanol, and from polymer comprising organochloro compounds, in particular poly (meth)acrylic polymer comprising compounds, and / or wherein the organochloro compound comprises at least two chloride atoms, and / or wherein the concentration of the organochloro compound in the solution is between 0.01- 50 mM, preferably between 0.1-35 mM, more preferably between 1-20 mM, and / or wherein the organochloro is conjugated with at least one of a particle, a protein, and a polymer.

23. Inactive chemical compound (1) comprising (i) at least one active chemical moiety (3) covalently bound to (ii) at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety, for use as a pharmaceutical in the treatment of cancer, in particular in treatment of tumors, more in particular in treatment of hypoxic tumors, wherein the chemical protecting group (2) is selected from at least one of a chemical protecting group comprising boron, preferably comprising at least one of a bo- ronic acid group and an ester derivative of a boronic acid, comprising selenium, such as seleno ethers, and a chemical protecting group comprising sulfur, preferably comprising a thioether, wherein the treatment further comprises providing an organochloro compound.

24. Inactive chemical compound (1) for use according to claim 23, wherein the treatment further comprises providing radiation (Sr), selected from photons and particle radiation.

25. A pharmaceutical dosage, in particular a pro-drug, comprising an inactive chemical compound (1), the inactive chemical compound comprising (i) at least one active chemical moiety (3) covalently bound to (ii) at least one chemical protecting group (2), the at least one chemical protecting group thereby inactivating the active chemical moiety, wherein the chemical protecting group (2) is selected from at least one of a chemical protecting group comprising boron, preferably comprising at least one of a boronic acid group and an ester derivative of a boronic acid, and a chemical protecting group comprising sulphur, preferably comprising a thioether, further comprising an organochloro compound.

26. The pharmaceutical dosage according to claim 25, wherein the organochloro compound is selected from small molecular organochloros,.

27. The pharmaceutical dosage according to claim 26, wherein the organochloro compound is selected from organochloros with a size of 4 - 15 atoms, in particular with a size of 4 - 10 atoms, preferably the organochloro is selected from at least one of chloroform, methyl trichloro acetate, dichloromethane, 2,2,2-trichloroethanol, and 2,2-dichloroetha- nol, and / or wherein the organochloro compound comprises at least two chloride atoms, and / or wherein the organochloro is conjugated with at least one of a particle, a protein, and a polymer.

28. The pharmaceutical dosage according to any of claims 25-27, further comprising an amine compound (7), in particular a tertiary amine compound.

29. The pharmaceutical dosage according to any of claims 25-28, wherein the pharmaceutical dosage is comprised in a solution, wherein the solution comprises water, in an amount of 90 - 100 wt.% water, wherein the wt.% is relative to the total weight of the solution, and wherein the concentration of the inactive chemical compound (1) is 0.1 - 100 pmol / L solution, in particular 1 - 80 pmol / L, more in particular 5 - 50 pmol / L, and / or wherein the concentration of the organochloro compound in the solution is between 0.01- 50 mM, preferably between 0,1-35 mM, more preferably between 1-20 mM, and / or wherein the concentration of the amine compound (7) is 1 - 500 pmol / L solution, in particular 5 - 400 pmol / L solution, more in particular 10 - 300 pmol / L solution.

30. The pharmaceutical dosage according to any of claims 25-29, wherein the dosage comprises nanoparticles, wherein the nanoparticles comprise an amphiphilic surface active compound, in particular selected from polymers, the surface active compound forming a layer, and within the layer the inactive compound, wherein a dosage of the inactive compound is 0.1-1 mg / mg surface active compound, in particular 0.2-0.5 mg / mg.

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