Endoperoxide-based drug delivery systems useful in diagnosis and therapy and methods thereof - Patents.com

Abstract

The present invention relates to a novel drug delivery system based on an endoperoxide moiety, such as 1,2,4,5-tetraoxane, i.e., a compound of formula I, suitably modified to release an active pharmaceutical ingredient (API) in the presence of higher levels of Fe(II) in a subject, where iron metabolism disorders of formula I occur in diseases such as malaria and cancer. Thus, the present invention also relates to a biomarker comprising said compound of formula I. In another aspect, the present invention relates to a process for the synthesis of the compound of formula I and the respective intermediates. Furthermore, the present invention also relates to a process for the labeling, detection and identification of tumors in tissue samples. Thus, the present invention is applicable in the medical and pharmacological fields, in particular in the fields related to the detection and treatment of cancer, as well as the treatment of malaria.

Description

[Technical Field]

[0001] The present invention provides an endoperoxide moiety, such as 1,2,4,5-tetraoxane, suitably modified to release an active pharmaceutical ingredient (API) in the presence of higher levels of Fe(II) in a subject, i.e., a compound of formula I: [ka] Novel drug delivery systems based on the compounds death, In particular, derivative compounds include enantiomers and racemic mixtures thereof having the following formulae IIa, IIb, IIc, IId, IIe: [ka] During the ceremony, R 1 is adamantyl, R 2 is adamantyl, X is -OO-, R 3 is -CH(R 4 )-CH(R 5 )- and R 4 is hydrogen, R 5 is (4-((7-methoxyquinolin-5-yl)amino)pentyl)carbamoyloxy, R 6 is hydrogen; [ka] During the ceremony, R 1 is adamantyl, R 2 is adamantyl, X is -OO-, R3 is -CH(R 4 )-CH(R 5 )- and R 4 is hydrogen, R 5 is 1-(tert-butyl)piperazine-1,4-dicarboxylate, R 6 is hydrogen;

change

change

[0002] Iron metabolism disorders occur in diseases such as malaria and cancer. Therefore, the present invention also relates to biomarkers comprising said compounds of formula I.

[0003] In another aspect, the present invention relates to processes for the synthesis of compounds of formula I and their respective intermediates.

[0004] Additionally, the present invention also relates to a process for the labeling, detection and identification of tumors in tissue samples.

[0005] The present invention is therefore applicable in the medical and pharmacological fields, particularly those related to the detection and treatment of cancer and the treatment of malaria.

[0006] The present invention discloses drug delivery systems with two different vias based on a 1,2,4,5-tetraoxane scaffold that selectively reacts with Fe(II): the first drug delivery system spontaneously releases the active pharmaceutical ingredient (API) after reaction with Fe(II), whereas the second drug delivery system, instead of the slightly modified first system, needs to react with Fe(II) and glutathione to release the active pharmaceutical ingredient (API). [Background technology]

[0007] Iron is a cofactor for enzymes involved in important biological processes such as mitochondrial respiration and cell cycle control. Its role depends on the redox cycle between ferric iron (Fe(III)) and ferrous iron (Fe(II)). However, it can also lead to reactive oxygen species (ROS) generation through the reaction of Fe(II) with hydrogen peroxide. Despite being crucial for several biological processes, iron can also be potentially harmful.

[0008] Iron homeostasis in cells strictly maintains sufficient iron levels to ensure cell function while reducing the availability of iron in the redox-active ferrous form. The balance between iron uptake, storage, and excretion ensures iron homeostasis in cells. The mechanism of iron metabolism is primarily related to the labile iron pool, called ferrous iron (redox-active iron), present in the cytosol.

[0009] Iron metabolism disorders occur in diseases such as inflammation, infection, cancer, cardiovascular disease, and neurodegenerative diseases.

[0010] Endoperoxides, such as artemisinin and its derivatives, are used clinically for the treatment of malaria. Endoperoxides, such as 1,2,4-trioxane, 1,2,4-trioxolane, and 1,2,4,5-tetraoxane, are selectively activated by Fe(II)-rich parasite-infected erythrocytes to form carbon-centered radicals. Infected erythrocytes appear to contain elevated levels of Fe(II) compared with those detected in healthy tissues.

[0011] Antimalarial drugs have adverse side effects and increasing resistance to first-line treatments for malaria has been reported.

[0012] Several studies have reported a link between iron and cancer, showing that cancer cells have increased levels of labile iron pools to support their biological needs. Furthermore, many cancers alter the iron uptake and excretion processes, resulting in increased levels of labile iron pools compared to normal cells, a phenomenon known as "iron toxicity."

[0013] Currently, chemotherapy drugs are not selective for their site of action, and as a result, they produce severe and harmful side effects.

[0014] Iron metabolism disorders represent a novel strategy for selective drug delivery by preparing two tetraoxane-based systems. The drug delivery systems described in this invention can be administered inactively and selectively activated against a selected target, in this case, ferrous iron, thereby altering drug efficacy and drug resistance profiles.

[0015] US Pat. No. 5,629,493 describes compounds based on the 1,2,4-trioxane or 1,2,4-trioxolane ring system for the treatment of parasitic and neoplastic diseases associated with conditions presenting with elevated levels of ferrous iron.

[0016] However, Patent Document 1 makes no mention of delivery systems containing 1,2,4,5-tetraoxolane-based compounds. Furthermore, as widely recognized in the literature, the 1,2,4-trioxolane ring system does not exhibit good metabolic stability, which remains a major obstacle to clinical development. The 1,2,4,5-tetraoxolane ring system is thermodynamically and metabolically more stable than the 1,2,4-trioxolane ring system. Furthermore, these compounds are highly toxic to living organisms. Finally, the second drug delivery system disclosed is not suitable for containing the 1,2,4-trioxolane ring system due to the inherent limitations in the preparation of the 1,2,4-trioxolane scaffold.

[0017] Patent Document 2 discloses tetraoxane compounds having biological activity against the genus Plasmodium, such as adamantane-2-spiro-3'-8'-hydroxymethyl-1',2',4'-trioxaspiro[4,5]decane-1,2-benzisothiazole-1,1-dioxide (LC129), adamantane-2-spiro-3'-1',2',4',5'-tetraoxane-6'-spiro-1"-cyclohexanone (LC140), or adamantane-2-spiro-3'-1',2',4',5'-tetraoxane-6'-spiro-1"-cyclohexane (LC137). However, the compounds disclosed therein cannot selectively react with abnormally high concentrations of Fe(II) that occur in pathological conditions of abnormal iron metabolism. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] International Publication No. 2015 / 123595 [Patent Document 2] International Publication No. 2020 / 240266 Summary of the Invention [Problem to be solved by the invention]

[0019] The present invention is selective for target tissues and can contribute to the treatment of malaria and cancer, and can also be successfully used as a biomarker. Formula IIa, IIb, IIc, IId and IIe The present invention aims to overcome the problems of the prior art by providing novel 1,2,4,5-tetraoxane compounds and two drug delivery systems based on said compounds. [Means for solving the problem]

[0020] The present invention provides an endoperoxide moiety, such as 1,2,4,5-tetraoxane, suitably modified to release an active pharmaceutical ingredient (API) in the presence of higher levels of Fe(II) in a subject, i.e. Formulas IIa, IIb, IIc, IId and IIe This invention relates to a novel drug delivery system based on the compound of formula (I).

[0021] 1. New tetraoxane compounds The novel tetraoxane of the present invention teeth , Compounds of the following formulae IIa, IIb, IIc, IId and IIe, including enantiomers and racemic mixtures thereof: : [ka] During the ceremony, R 1 is adamantyl, R 2 is adamantyl, X is -OO-, R 3 is -CH(R 4 )-CH(R 5 )- and R 4 is hydrogen, R 5 is (4-((7-methoxyquinolin-5-yl)amino)pentyl)carbamoyloxy, R 6 is hydrogen; [ka] During the ceremony, R 1 is adamantyl, R 2 is adamantyl, X is -OO-, R 3 is -CH(R 4 )-CH(R5 )- and R 4 is hydrogen, R 5 is 1-(tert-butyl)piperazine-1,4-dicarboxylate, R 6 is hydrogen;

change

change

[0022] 1.1. Characteristics and Properties of the Compounds of the Invention for the Treatment of Malaria (Formulas IIa and IIb) [ka]

[0023] Compounds IIa and IIb are coupled to drugs for the treatment of malaria, defining drug delivery systems IIa and IIb. In a preferred embodiment, compound IIa is coupled to primaquine, a clinically used antimalarial drug. In another embodiment, compound IIb is coupled to boc-piperazine. These compounds exhibit half maximal inhibitory concentrations (IC) in the nanomolar range against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. 50 ) Therefore, these compounds can be applied to the treatment of malaria. Compounds IIa and IIb also include their respective racemic mixtures or their respective enantiomers.

[0024] 1.2. Characteristics and Properties of Compounds of the Invention for Cancer Treatment (Formulas IIc, IId and IIe) Further compounds are within the scope of the invention, for example, compounds of formulae IIc, IId and IIe. [ka]

[0025] Compounds IIc, IId, and IIe are coupled to anticancer drugs typically used in clinical practice, thus defining drug delivery systems IIc, IId, and IIe. In a preferred embodiment, the compounds of the present invention are coupled to doxorubicin. These compounds can induce cell death in vitro in the micromolar range in at least four types of cancer with unstable iron metabolism, where the specific cell lines are identified in parentheses: glioblastoma (U251), breast adenocarcinoma (MCF7), prostate cancer (PC3), and colorectal cancer (HCT116). Therefore, these compounds can be applied to cancer treatment. Compounds IIc, IId, and IIe also include their respective racemic mixtures or their respective enantiomers.

[0026] 1.3. Characteristics and Properties of the Compounds of the Invention (Formulas IIc, IId and IIe) for Cancer Diagnosis In addition to their ability to induce cell death in cancer cells, some other compounds with inherent fluorescence, such as doxorubicin, are useful as tumor markers when coupled with the compounds of the present invention. This fluorescence is lost when the compounds are bound to drug compounds IIc, IId, and IIe. When these fluorescent compounds are activated in cancer cells, they are released from IIc, IId, and IIe by reaction with ferrous iron, thus restoring their fluorescent properties and allowing them to be detected by fluorescence microscopy. Therefore, these compounds can also be used as drug delivery systems for tumor markers. In a preferred embodiment, doxorubicin is bound to compounds IIc, IId, and IIe used as tumor markers.

[0027] 2. Methods for preparing compounds of formula I In one embodiment of the present invention, the method for preparing compounds of formula I (see Example 1) and compounds of formula IIa, IIb, IIc and IId (see Example 2) described in the previous section is represented by the following Scheme 1: [ka] The following steps are used: (i) Sodium dichromate dihydrate, water, sulfuric acid; (ii) rhenium(VII) oxide, 50% hydrogen peroxide, acetonitrile; (iii) 2-adamantanone or 5-hydroxy-2-adamantanone, rhenium(VII) oxide, dichloromethane:acetonitrile (1:1), inert atmosphere; (iv) 4-nitrophenyl chloroformate, N,N-diisopropylethylamine, 4-dimethylaminopyridine, dichloromethane; (v) API, 1-hydroxybenzotriazole hydrate, triethylamine, N,N-dimethylformamide.

[0028] In step i), oxidation of 1,3-cyclohexanediol (compound 0) produces rac-3-hydroxycyclohexanone (compound 1).

[0029] In step ii), compound 1 reacts with hydrogen peroxide in the presence of rhenium(VII) oxide to produce a dihydroperoxide (compound 2).

[0030] In step iii), the dihydroperoxide (compound 2) is reacted with a carbonyl reagent, again using a catalytic amount of rhenium(VII) oxide. 2-Adamantanone (compound 3) and 5-hydroxy-2-adamantanone (compound 4) can be used as carbonyls for the formation of the 1,2,4,5-tetraoxane scaffold of the first drug delivery system. The alcohol group at the 3" position of the cyclohexane ring generates asymmetric tetraoxanes (compounds 3 and 4) with a stereocenter, yielding two enantiomers. Therefore, this step can also be used with a racemic mixture of tetraoxanes to be applied as drug delivery systems, instead of performing a stereocontrolled synthesis of the 1,2,4,5-tetraoxane system.

[0031] Next, in step iv), compounds (3) and (4) were activated with 4-nitrophenyl chloroformate together with an excess of amine base (N,N-diisopropylethylamine and 4-dimethylaminopyridine) to produce compounds (5) and (6), respectively.

[0032] Finally, compound 5 was coupled with primaquine diphosphate (IIa) and boc-piperazine (IIb) in 55% and 33% yields, respectively. Compounds 5 and 6 were coupled with doxorubicin to give the carbamates (IIc) and (IId) in 60% and 75% yields, respectively, to generate the corresponding drug delivery systems.

[0033] Compounds 1-6, having the above formula, and the enantiomers of compounds 3-6 are included in the present invention as intermediates in the process for preparing compounds IIa, IIb, IIc, and IId, and the respective drug delivery systems.

[0034] Finally, considering that intermediate compounds 3-6 are used in the method for preparing compounds IIa, IIb, IIc, and IId, this results in the compounds and their respective drug delivery systems containing racemic mixtures or their respective enantiomers. Thus, the present invention also relates to racemic mixtures or their respective enantiomers of compounds IIa, IIb, IIc, and IId and the corresponding drug delivery systems.

[0035] In another embodiment of the present invention, the preparation of compounds of formula I (see Example 1) and compounds of formula IIe (see Example 2) is described in Scheme 2 below: [ka] The following steps are used: (i) Formaldehyde, N-methylpyrrolidine, barium hydroxide octahydrate, water:methanol (5:1); (ii) rhenium(VII) oxide, 50% hydrogen peroxide, acetonitrile; (iii) 5-hydroxy-2-adamantanone, rhenium(VII) oxide, dichloromethane:acetonitrile (1:1), inert atmosphere; (iv) 4-nitrophenyl chloroformate, N,N-diisopropylethylamine, 4-dimethylaminopyridine, dichloromethane; (v) API, 1-hydroxybenzotriazole hydrate, triethylamine, N,N-dimethylformamide.

[0036] In step i), a hydroxymethyl group was introduced at the 2-position of 2-cyclohexen-1-one (compound 7) by Morita-Baylis-Hillman reaction to give compound 8.

[0037] In step ii), compound 8 reacts with hydrogen peroxide in the presence of rhenium(VII) oxide to produce the dihydroperoxide (compound 9).

[0038] In step iii), the dihydroperoxide (compound 9) is reacted with a carbonyl reagent, again using a catalytic amount of rhenium(VII) oxide. 5-Hydroxy-2-adamantanone (compound 10) can be used as the carbonyl for the formation of the 1,2,4,5-tetraoxane scaffold of the second drug delivery system. The alcohol group at the 5-position of the adamantyl group generates an asymmetric tetraoxane (compound 10) with a stereogenic center, yielding two enantiomers. This step can also be used with a racemic mixture of tetraoxanes to be applied as a drug delivery system, instead of performing a stereocontrolled synthesis of this drug delivery system.

[0039] Next, in step iv), compound (10) was activated with 4-nitrophenyl chloroformate together with an excess of an amine base (N,N-diisopropylethylamine and 4-dimethylaminopyridine) to produce compound (11).

[0040] Finally, compound (11) was coupled with doxorubicin (IIe) in 73% yield.

[0041] Compounds 8-11 having the above formula, and the enantiomers of compounds 10 and 11, are included in the present invention as intermediates in the process for preparing compounds of formula I and formula IIe.

[0042] Finally, considering that intermediate compounds 10 and 11 are used in the process for preparing the above-mentioned compounds, this results in the compounds containing racemic mixtures or the respective enantiomers. Thus, the present invention also relates to racemic mixtures or the respective enantiomers of compounds I and IIe.

[0043] 3. Mechanism of the first drug delivery system according to the present invention The first drug delivery system contains a 1,2,4,5-tetraoxane scaffold that reacts with Fe(II) to form a cyclohexanone intermediate (Scheme 3), which spontaneously releases the API via a retro-Michael reaction (β-elimination).

[0044] Scheme 3 illustrates the mechanism of drug delivery via Fe(II) activation of the tetraoxane compounds of the present invention and subsequent β-elimination, where A represents one or more APIs that can be coupled to the tetraoxane. [ka]

[0045] Reaction of the tetraoxane present in compounds IIa, IIb, IIc, and IId with a ferrous source produces a cyclohexanone derivative, which spontaneously releases the API by β-elimination (Scheme 3).

[0046] Within the scope of the present invention, there are several suitable APIs that can be used alone or in combination for different therapeutic and / or diagnostic purposes, some of which are described below.

[0047] In this scheme, compounds IIa and IIb can be coupled to one or more APIs against Plasmodium falciparum, such as primaquine and boc-piperazine. Other compounds suitable for coupling with this system against malaria are the following and their derivatives: atovaquone, quinine sulfate, sulfonamides (sulfadoxine and sulfamethoxypyridazine), and falcipain inhibitors (dipeptidyl vinyl sulfones and peptidomimetic pyrimidine nitriles).

[0048] Compounds IIc and IId can preferably be coupled to doxorubicin, an API for cancer. Other compounds suitable for coupling with this system for cancer, alone or in combination, are the following and their derivatives: alectinib, afatinib, abiraterone acetate, azacitidine, axitinib, bendamustine, brigatinib, bosutinib, bicalutamide, calicheamicin, capecitabine, carfilzomib, combretastatin, ceritinib, cladribine, clofarabine, cobimetinib, crizotin ... Tofisin, cytarabine, dabrafenib, dasatinib, daunorubicin, decitabine, duocarmycin, enasidenib, epirubicin, eribulin mesylate, erlotinib, everolimus, etoposide, exatecan, fludarabine, fulvestrant, gemcitabine, hemiasterlin, histone deacetylase inhibitors (avexinostat, belinstat, entinostat, divinostat, mocetinostat, oxamflatin, Panobinostat, piroxamide, xinostat, vorinostat, trichostatin A, tasecinarin, tushidinostat, tubastatin A), hydroxyurea, ibrutinib, idelalisib, imatinib, irinotecan, ixabepilone, lapatinib, lenalidomide, maytansine, mitomycin C, monomethyl auristatin (MMA-E, MMA-F, dolastatin 10 and dolastatinol), methotrexate, neratinib, nilotinib, niraparib, olaparib, octreotide, osimertinib, paclitaxel, palbociclib, pazopanib, pyrrolobenzodiazepines, pemetrexed, pomalidomide, ponatinib, ribociclib, rucaparib, sorafenib, tamoxifen, triazene, trifluridine, topotecan, tublysin, vandetanib.

[0049] Other compounds suitable for coupling with this system as tumor markers include fluorophores, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), cyanines, dicyanomethines, doxorubicin, fluoresceins, nitrobenzofurazans, rhodamines, and their derivatives.

[0050] 3.1. Mechanistic proof-of-concept of the first drug delivery system Activation of the 1,2,4,5-tetraoxane scaffold after reaction with Fe(II) generates a carbon-centered radical. This reaction mechanism involves Fe(II) coordination with two available oxygens on the tetraoxane ring. The first drug delivery system contains a tetraoxane scaffold that, in the presence of Fe(II), is expected to generate a carbon-centered radical and a cyclohexanone derivative. This derivative spontaneously releases the API by β-elimination. To confirm the proposed drug delivery mechanism, compound IIc was exposed to an inorganic salt of Fe(II) and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) as described in O'Neill et al., J. Med. Chem. 2011, 54, 6443. TEMPO was used in this experiment to verify the formation of a carbon-centered radical by the drug delivery system. TEMPO reacts with the radical generated in this reaction, allowing detection by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS).

[0051] Upon Fe(II)-triggered tetraoxane activation and fragmentation of compound IIc, a TEMPO adduct was detected by UHPLC-MS, consistent with the formation of a carbon-centered radical (see Example 3). Furthermore, cyclohexanone derivatives and doxorubicin were also identified by UHPLC-MS (see Example 3), confirming API release via the proposed drug delivery mechanism.

[0052] 4. Mechanism of the second drug delivery system disclosed in the present invention An alternative second drug delivery system, which is a specific embodiment of the first system described above, also contains a 1,2,4,5-tetraoxane scaffold, which generates a cyclohex-2-enone derivative after reaction with Fe(II). This derivative does not spontaneously release the API. This derivative has an alkene group that allows for release of the API by elimination after reaction with glutathione (Michael addition) (Scheme 4). Considering that API release occurs only after reaction with Fe(II) and glutathione, which are elevated in many types of cancer, this may result in improved selectivity compared to the first drug delivery system.

[0053] Scheme 4 illustrates the mechanism of drug delivery via Fe(II)-activation, Michael addition of glutathione, and elimination of the tetraoxane compounds of the present invention, where A represents an API that can be coupled to the tetraoxane. [ka]

[0054] Reaction of the tetraoxane present in compound IIe with a ferrous source produces a cyclohex-2-enone derivative, which releases the API by elimination after reaction with glutathione (Scheme 4).

[0055] Within the scope of the present invention, there are several suitable APIs that can be used alone or in combination for different therapeutic and / or diagnostic purposes, some of which are described below.

[0056] In this scheme, compound IIe is preferably coupled to doxorubicin, an API for cancer. Other compounds suitable for coupling to this system for cancer, alone or in combination, are the following and their derivatives: alectinib, afatinib, abiraterone acetate, azacitidine, axitinib, bendamustine, brigatinib, bosutinib, bicalutamide, calicheamicin, capecitabine, carfilzomib, combretastatin, ceritinib, cladribine, clofarabine, cobimetinib, crizotin ... Tofisin, cytarabine, dabrafenib, dasatinib, daunorubicin, decitabine, duocarmycin, enasidenib, epirubicin, eribulin mesylate, erlotinib, everolimus, etoposide, exatecan, fludarabine, fulvestrant, gemcitabine, hemiasterlin, histone deacetylase inhibitors (avexinostat, belinstat, entinostat, divinostat, mocetinostat, oxamflatin, Panobinostat, piroxamide, xinostat, vorinostat, trichostatin A, tasecinarin, tushidinostat, tubastatin A), hydroxyurea, ibrutinib, idelalisib, imatinib, irinotecan, ixabepilone, lapatinib, lenalidomide, maytansine, mitomycin C, monomethyl auristatin (MMA-E, MMA-F, dolastatin 10 and dolastatinol), methotrexate, neratinib, nilotinib, niraparib, olaparib, octreotide, osimertinib, paclitaxel, palbociclib, pazopanib, pyrrolobenzodiazepines, pemetrexed, pomalidomide, ponatinib, ribociclib, rucaparib, sorafenib, tamoxifen, triazene, trifluridine, topotecan, tublysin, vandetanib.

[0057] Other compounds suitable for coupling with this system as tumor markers include the fluorophore 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), cyanine, dicyanomethine, doxorubicin, fluorescein, nitrobenzofurazan, rhodamine, and their derivatives.

[0058] 4.1. Mechanistic proof-of-concept of a second drug delivery system As described for the first drug delivery system, the tetraoxane reaction of the second drug delivery system with a ferrous iron source also generates a carbon-centered radical through Fe(II) coordination with two oxygens of the tetraoxane scaffold. The second drug delivery system generates a cyclohex-2-enone derivative through activation by Fe(II), which then undergoes Michael addition with intracellular glutathione and subsequent elimination to release the API. To confirm the first step of the proposed drug delivery mechanism, compound IIe was exposed to an inorganic salt of Fe(II) and TEMPO as described in O'Neill et al., J. Med. Chem. 2011, 54, 6443. TEMPO was used in this experiment to verify the formation of a carbon-centered radical by the drug delivery system. TEMPO reacts with the radicals generated in this reaction, allowing their detection by UHPLC-MS. Furthermore, this experiment was performed to confirm the tetraoxane fragmentation of compound IIe to generate the cyclohex-2-enone derivative. Finally, doxorubicin is not released under these conditions due to the absence of glutathione or similar agents, and therefore Michael addition to the cyclohex-2-enone derivative cannot occur.

[0059] Fe(II)-triggered tetraoxane activation and fragmentation of compound IIe revealed the detection of TEMPO adducts by UHPLC-MS, consistent with the formation of carbon-centered radicals for both possible coordination with Fe(II) (see Example 4). Furthermore, cyclohex-2-enone derivatives were also identified by UHPLC-MS, in accordance with the proposed mechanism (see Example 4). Finally, doxorubicin was not detected in this experiment.

[0060] This drug delivery system generates a cyclohex-2-enone derivative that can undergo Michael addition with intracellular glutathione after activation with Fe(II) and subsequent release of the API. To confirm this mechanism of drug delivery, compound IIe was reacted with a ferrous iron source and N-acetylcysteine ​​(NAC) to simulate the thiol (-SH)-dependent Michael addition of glutathione. In this example, a pH above 8 is required for the thiol group to be deprotonated (-S-) to ensure this reaction occurs.

[0061] Following Fe(II)-triggered tetraoxane activation and fragmentation of compound IIe, a carbon-centered radical was detected by UHPLC-MS (see Example 4). Furthermore, following the first step of activation of this drug delivery system, a cyclohex-2-enone derivative was also detected (see Example 4). Finally, the release of doxorubicin was detected (see Example 4), confirming the mechanism of action of this drug delivery system, due to the Michael addition of NAC to the cyclohex-2-enone derivative.

[0062] 5. Biological Activity Assessment The biological activity of the compounds of the present invention and the corresponding drug delivery systems can be accessed by measuring different parameters according to known processes by those skilled in the art.

[0063] 5.1. Evaluation of antimalarial efficacy The antimalarial activity of compounds IIa and IIb can be assessed by exposing P. falciparum strains, such as the 3D7 strain, which is susceptible to chloroquine and mefloquine (see Example 5), to the compounds and using a SYBR Green I assay as described in Machado et al., Ann. Clin. Med. Microbio. 2016, 2, 1010.

[0064] Asynchronous parasites were cultured in the presence of each compound for 72 hours. Fluorescence intensity was measured using a multi-mode microplate reader (Triad, Dynex Technologies). Compounds IIa and IIb were active against malaria parasites (see Example 5, Table 1).

[0065] 5.2. Evaluation of antitumor effects Typically, antitumor effects on cancer cell proliferation are measured as the half maximal inhibitory concentration (IC) in cell cultures exposed to the compound being evaluated, as described by Florindo et. al, Dalton Trans 2016, 45, 11926. 50 ) is evaluated by measuring

[0066] I C 50 Values ​​can be calculated by dose-response curves, for example, using GraphPad Prism 8.0.2 software, followed by statistical analysis.

[0067] To this end, tumor cell lines were exposed to a range of concentrations of the appropriate compounds IIc, IId, and IIe and the corresponding drug delivery systems. After 72 hours, cell metabolic activity was assessed using the Cell Titer96® Aqueous Non-Radioactive Cell Proliferation Assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium-MTS; Promega, Madison, WI, USA), and absorbance signals were recorded using the GloMax®-MultiDetection System (Promega). Compounds IIc, IId, and IIe were able to induce cell death in all tumor cell lines tested (see Example 6, Table 2). Furthermore, compounds IIc, IId, and IIe were less potent than doxorubicin, the cancer API to which these compounds are coupled.

[0068] 5.3. Evaluating the Effects of Compounds on Toxicity Compounds IIc, IId and IIe and doxorubicin were tested in a non-tumorous cell line (AML12) to evaluate the toxicity of the compounds described in this invention. Toxicity was measured by the IC 50 The assay conditions and software can be the same as those described above for compounds IIc, IId, and IIe against the four tumor cell lines. Compounds IIc, IId, and IIe can ameliorate the toxicity associated with doxorubicin (see Example 7).

[0069] 5.4. Assessment of iron-dependent biological activity Compounds IId and IIe were incubated alone and with the iron chelator desferoxamine mesylate (DFO) in colorectal cancer cells (HCT116) as described in Mariani et al., Chem. Commun. 2016, 52, 1358 (see Example 8). Cell metabolic activity can again be determined using the MTS assay and absorbance signal detection, as described above for the evaluation of antitumor effects. IC 50 Calculations and statistical analyses can also be performed by GraphPad Prism 8.0.2.

[0070] The induction of cell death by compound IId is iron-dependent, as the cytotoxicity of this compound was reduced in the presence of the iron chelator DFO (Figure 1). Thus, the presence of Fe(II) is essential for the activity of the first drug delivery system in cells.

[0071] The second drug delivery system contains an alkene group as an additional element of selectivity compared to the first drug delivery system. Compound IIe is activated intracellularly by iron, but the primary contributor to cytotoxicity is related to Michael addition with intracellular glutathione. Induction of cell death by compound IIe showed a slight decrease in the cytotoxicity of this compound in the presence of the iron chelator DFO (Figure 2). Thus, in this example, the presence of Fe(II) activates the second drug delivery system in cells. This activation generates a cyclohex-2-enone derivative containing an alkene group, which allows for release of the API after reaction with intracellular glutathione, as further described in Example 4.

[0072] 5.5. Compounds of formula (II) as tumor markers The evaluation of the drug delivery system IIc, IId and IIe of the present invention as tumor marker is carried out by fluorescence microscopy to detect their cellular localization.Compound IId and doxorubicin are incubated in colorectal cancer cells (HCT116), and after 24 hours of incubation, images are acquired by Zeiss AxioScope.A1 microscope connected with AxioCam HR R3 camera, and analyzed by Zen Software 2012 Blue Edition.Compared to doxorubicin, compound IId exists in different cellular localization (see Example 9 and Figure 3).

[0073] 5.6. In Vivo Validation of the First Drug Delivery System Compound IId and doxorubicin were tested in a xenograft mouse model of colorectal cancer using HT-29 cells. Tumor size was analyzed during treatment and used to determine tumor volume and relative tumor volume. 20 immunodeficient Foxn1 mice were treated with HT-29 cells. nu / nuMice were divided into four groups: negative control, vehicle control, compound IId, and doxorubicin. The negative control group consisted of five mice that received no treatment. The vehicle control group consisted of five mice that received the solvent used to dissolve compound IId. Finally, five mice were treated with compound IId and five mice were treated with doxorubicin. Compound IId and doxorubicin were more efficient in reducing relative tumor mass compared to the control group (Figure 4). The reduction in relative tumor mass was greater in mice treated with compound IId compared to mice treated with doxorubicin (Figure 4). [Brief explanation of the drawings]

[0074] [Figure 1] Figure 1 shows the effect of cell death (Y-axis) in colorectal cancer cells (HCT116) in the presence of: Compound IId alone (represented by column #3); Compound IId and an iron chelator (deferoxamine mesylate - DFO) (represented by column #4); DFO alone (represented by column #2); and dimethyl sulfoxide (DMSO) as a control compound (represented by column #1). On the Y-axis, cell viability is shown, as determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-)2H-tetrazolium (MTS) metabolic assay. Metabolically active (viable) cells metabolize MTS to formazan, which is quantified by absorbance. A higher amount of formazan indicates that the cells can survive when exposed to the compound, whereas a lower amount of formazan indicates that the compound can induce cell death. From this figure, it can be observed that compound IId induces cell death, and co-treatment of compound IId with DFO promotes a lower induction of cell death. Thus, the cell death conferred by compound IId is iron-dependent. Data represent the mean ± standard error of the mean (SEM) of three independent experiments relative to the untreated control (DMSO). Ordinary one-way ANOVA and Tukey's multiple comparison test. Vs. DMSO: δ p<0.0001; Vs. compound IId without DFO: **p<0.01. [Figure 2]FIG. 2 shows the effect of cell death (Y-axis) in colorectal cancer cells (HCT116) in the presence of: Compound IIe alone represented by column #3, Compound IIe and an iron chelator (deferoxamine mesylate - DFO) represented by column #4, DFO alone represented by column #2, and DMSO as a control compound represented by column #1. On the y-axis, cell viability is shown, as determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) metabolic assay. Metabolic active (viable) cells metabolize MTS to formazan, which is quantified by absorbance. A higher amount of formazan indicates that the cells are viable when exposed to the compound, whereas a lower amount of formazan indicates that the compound is capable of inducing cell death. From this figure, it can be observed that compound IIe induces cell death, and that co-treatment of compound IIe with DFO promotes a slight reduction in the induction of cell death by compound IIe. Therefore, the cell death conferred by compound IIe may be iron-dependent, since ferrous iron (Fe(II)) activates this compound in cells. Data represent the mean ± SEM of three independent experiments normalized to the untreated control (DMSO). Ordinary one-way ANOVA. vs. DMSO: δ p<0.0001. [Figure 3]Figure 3 shows images of HCT116 cells after 24 hours of incubation with system control (DMSO), doxorubicin, and Compound IId. Fluorescence emission was detected using a 460 nm emission filter (blue channel) and a 560 nm emission filter (red channel). Hoechst dye 33258 is a fluorescent dye used to stain cell nuclei, and its fluorescence emission can be detected by the blue channel. Doxorubicin has inherent fluorescence, and its emission wavelength is detected by the red channel. The merge is the overlap of the fluorescence detected by the blue and red channels imparted by Hoechst dye 33258 and doxorubicin, respectively. AA represents the system control (DMSO) and the fluorescence emission in the blue channel, AB represents the system control (DMSO) and the fluorescence emission in the red channel, AC represents the system control (DMSO) and the merge of the fluorescence emission from both channels (blue and red), AD represents the zoomed-in field for the system control (DMSO) and the merge of the fluorescence emission from both channels (blue and red), BA represents doxorubicin (1 μM) and the fluorescence emission in the blue channel, BB represents doxorubicin (1 μM) and the fluorescence emission in the red channel, and BC represents doxorubicin. (1 µM) and the merge of fluorescence emission from both channels (blue and red). BD represents a zoomed-in field of doxorubicin (1 µM) and the merge of fluorescence emission from both channels (blue and red). CA represents compound IId (1 µM) and the fluorescence emission in the blue channel. CB represents compound IId (1 µM) and the fluorescence emission in the red channel. CC represents compound IId (1 µM) and the merge of fluorescence emission from both channels (blue and red). CD represents a zoomed-in field of compound IId (1 µM) and the merge of fluorescence emission from both channels (blue and red). Objective: 63x magnification. Scale bar = 50 µm. From these images, it can be observed that fluorescence in the system control (DMSO) is only present in the blue channel, meaning that only nuclei can be detected (images AA, AC, and AD).Doxorubicin can be observed to accumulate in the nucleus through colocalization of fluorescence in the blue and red channels (images BC and BD). Meanwhile, compound IId exhibits fluorescence around the nucleus (images CC and CD). Because compound IId has no intrinsic fluorescence, this means that compound IId is activated in the cytosol, where ferrous iron (Fe(II)) levels rise, releasing doxorubicin. [Figure 4] Figure 4 shows the relative tumor volume (Y-axis) for four groups of mice treated with colorectal cancer cells (HT-29) over the treatment days (X-axis): mice treated with compound IId, represented by open circles, and mice treated with doxorubicin, represented by open squares. Mice without treatment (negative control group) are represented by open inverted triangles, and mice receiving cremophor in phosphate-buffered saline, the solvent in which compound IId is dissolved for administration (vehicle control), are represented by filled circles. On the Y-axis, the relative tumor volume in mm3 is shown for each group of mice, determined by the ratio between the volume on the indicated day and the volume at the start of treatment. From this figure, it is possible to observe a reduction in tumor mass progression in mice treated with compound IId and doxorubicin compared to the negative control group and vehicle control. Furthermore, compound IId was more effective in reducing tumor mass than doxorubicin. Mixed-effects analysis (e.g., two-way ANOVA) and Tukey's multiple comparison test were used. On day 12, mice treated with compound IId showed a significant reduction in tumor volume compared to vehicle controls: *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0075] Example 1. Synthesis of 1,2,4,5-tetraoxane compounds of formula I Cyclocondensation of dihydroperoxide (2) in the presence of a carbonyl (2-adamantanone or 5-hydroxy-2-adamantanone) produced the 1,2,4,5-tetraoxane scaffolds (3) and (4). Alcohol activation at the 3-position with 4-nitrophenyl chloroformate produced compounds (5) and (6). Finally, API coupling with compounds (5) and (6) produced the four compounds (IIa-IId) described in this invention for the first drug delivery system.

[0076] Cyclocondensation of dihydroperoxide 9 in the presence of a carbonyl reagent, in this case 5-hydroxy-2-adamantanone, produced the 1,2,4,5-tetraoxane scaffold 10. Alcohol activation of 10 with 4-nitrophenyl chloroformate produced 11. Finally, API coupling with 11 produced compound IIe, described in this invention for the second drug delivery system.

[0077] To this end, several intermediate compounds (1-6 and 8-11) were prepared according to the following methods.

[0078] 1.1. Synthesis of Compound (1): rac-3-hydroxycyclohexanone [ka]

[0079] A solution of sodium dichromate (3.01 g, 10.3 mmol) in concentrated sulfuric acid / water (1.7 mL / 12 mL) was added dropwise to a solution of 1,3-cyclohexanediol (3.45 g, 29.7 mmol) in diethyl ether (12 mL). After the addition was complete, the reaction was allowed to stir at 25 °C for 3.5 hours. The reaction mixture was extracted with ethyl acetate (3 × 30 mL), and the organic fractions were combined, dried over anhydrous sodium sulfate, filtered, and evaporated. Purification by flash chromatography using hexane:ethyl acetate (2:8) as the eluent isolated compound 1 as a colorless oil (1.35 g, 11.8 mmol) and 1,3-cyclohexanediol (1.26 g, 10.8 mmol).

[0080] Yield: 63%; 1 H NMR (300MHz, CDCl3): δ(ppm)4.18~4.10(m,1H), 2.93(br s,1H), 2.60(dd,J=14.1,4.2Hz,1H), 2.37(dd,J=13.8,7.5Hz,1H), 2.27(t,J=6.6Hz,2H), 2.09~1.88(m,2H), 1.80~1.55(m,2H); 13 C NMR (75MHz, CDCl3): δ(ppm)210.4,69.8,50.5,41.0,32.8,20.8;MS(ES + ):[M+H] + 114.9(12).

[0081] 1.2. Synthesis of Compound (2): 3,3-Dihydroperoxycyclohexan-1-ol [ka]

[0082] A 25 mL round-bottom flask was charged with rac-3-hydroxycyclohexanone (0.219 g, 1.92 mmol) and rhenium(VII) oxide (0.044 g, 0.092 mmol) in acetonitrile (10.5 mL). 50% hydrogen peroxide (0.40 mL, 7.0 mmol) was then added at room temperature and stirred for 40 minutes. The mixture was filtered under vacuum through a silica plug and washed with ethyl acetate. The solution was concentrated, and compound 2 was isolated as a colorless oil (0.313 g, 1.91 mmol).

[0083] Yield: 100%; 1 H NMR (300MHz, (CD3)2CO): δ(ppm)δ9.99(s,1H), 9.67(s,1H), 3.91~3.63(m,2H), 2.46~2. 36(m,1H), 2.13~2.07(m,1H), 1.91~1.82(m,1H), 1.69~1.59(m,1H), 1.53~1.14(m,4H); 13 C NMR (75MHz, (CD3)2CO): δ(ppm) 110.7, 67.5, 39.6, 39.4, 35.6, 20.4;

[0084] 1.3. Synthesis of Compound (3): Dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-ol [ka]

[0085] A 25 mL round-bottom flask was charged with 2-adamantanone (0.433 g, 2.88 mmol) and rhenium(VII) oxide (0.020 g, 0.041 mmol) in dichloromethane (5.7 mL) under an inert atmosphere. 3,3-Dihydroperoxycyclohexan-1-ol (0.313 g, 1.91 mmol) in acetonitrile (5.7 mL) was then added at room temperature and stirred for 45 minutes. The mixture was filtered under vacuum through a silica plug and washed with ethyl acetate. The solution was concentrated and purified by flash chromatography in hexane:ethyl acetate (7:3) to isolate compound 3 as a white solid (0.315 g, 1.06 mmol).

[0086] Yield: 56%; 1 H NMR (300MHz, CDCl3): δ(ppm)4.52~4.45(m,1H), 3.11~3.03(m,1H), 2.16~1.67(m,21H); 13 C NMR (75MHz, CDCl3): δ(ppm) 110.7, 73.2, 41.3, 37.0, 35.8, 33.8, 33.2, 31.0, 27.1, 25.9; MS(ES + ):[M+H] + 297.2(10);[M-tetraoxane cleavage] + 167.0(100).

[0087] 1.4. Synthesis of Compound (4): Dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3",5-diol [ka]

[0088] The same method was used to synthesize compound 4 using 3,3-dihydroperoxycyclohexan-1-ol (0.141 g, 0.861 mmol) as the starting material; however, 2-adamantanone was replaced with 5-hydroxy-2-adamantanone (0.216 g, 1.30 mmol). Purification by flash chromatography with hexane:ethyl acetate (2:8) afforded compound 4 as a white solid (0.145 g, 0.464 mmol).

[0089] Yield: 54%; 1 H NMR (300MHz, CDCl3): δ(ppm)4.63~4.59(m,1H), 3.18~3.12(m,1H), 2.59~2.39(m,2H), 2.16~1.50(m,18H); 13 C NMR (100MHz, CDCl3): δ(ppm) 108.6, 74.6, 66.7, 43.7, 42.1, 41.5, 38.8, 34.7, 30.4, 29.9; MS(ES + ): [M-Tetraoxane cleavage] + 183.0 (100). Elemental analysis: Found: C, 61.46; H, 7.60. 16 H 24 O6 requires C, 61.52; H, 7.74.

[0090] 1.5. Synthesis of Compound (5): Dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl(4-nitrophenyl)carbonate [ka]

[0091] Compound 3 (0.120 g, 0.404 mmol) was dissolved in dichloromethane (1.6 mL), followed by the addition of N,N-diisopropylethylamine (0.21 mL, 1.21 mmol) and 4-dimethylaminopyridine (0.0520 g, 0.421 mmol). At 0 °C, 4-nitrophenyl chloroformate (0.168 g, 0.814 mmol) was added, and the reaction was stirred under an inert atmosphere for 15 min, 10 min at 0 °C to room temperature, and 20 min at room temperature. The reaction mixture was diluted with diethyl ether (30 mL) and washed with 5% potassium bisulfate (10 mL) and saturated sodium bicarbonate solution (5 × 10 mL). The organic phase was washed with brine (1 × 25 mL), dried over anhydrous sodium sulfate, then filtered and concentrated. Compound 5 was isolated as a white solid (0.0416 g, 0.090 mmol) by purification by flash chromatography with hexane:ethyl acetate (9:1).

[0092] Yield: 22%; 1 H NMR (300MHz, CDCl3): δ(ppm)8.28(dd,J=7.2,2.4Hz,2H), 7.39(dd,J=6.9,2.1Hz,2H), 4.93(s,1H), 3.32(br s,1H), 3.13(br s,1H), 2.23~1.57(m,20H); 13 C NMR (75MHz, CDCl3): δ(ppm)155.7, 151.7, 145.5, 125.4, 122.0, 111.0, 75.6, 37.0, 33.2, 30.6, 27.1; MS(ES + ):[M-NO2] + 415.2(20).

[0093] 1.6. Synthesis of Compound (6): 5-Hydroxydispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl(4-nitrophenyl)carbonate [ka]

[0094] Compound 6 was prepared using the same method, except that the starting material was compound 4 (0.151 g, 0.483 mmol). Purification by flash chromatography with hexane:ethyl acetate (1:1) afforded compound 6 as a white solid (0.0996 g, 0.208 mmol).

[0095] Yield: 43%; 1 H NMR (300MHz, CDCl3): δ(ppm)8.28(dd,J=7.2,2.4Hz,2H), 7.36(dd,J=6.9,2.4Hz,2H), 4.76~4.69(m, 1H), 3.34~3.26(m, 1H), 2.68~1.74(m, 20H); 13 C NMR (75MHz, CDCl3): δ(ppm)155.4, 150.0, 145.5, 125.4, 121.9, 81.9, 73.9, 41.3, 39.8, 38.2, 34.7, 30.6, 29.9;.

[0096] 1.7. Synthesis of Compound (8): 2-(hydroxymethyl)cyclohex-1-en-1-one [ka]

[0097] A 10 mL round-bottom flask was charged with barium hydroxide octahydrate (0.0079 g, 0.0250 mmol) dissolved in water / methanol (5:1; 2:0.5 mL), and N-methylpyrrolidine (5.5 μL, 0.0520 mmol) was added. Formaldehyde (0.12 mL, 1.56 mmol) and compound 7 (0.10 mL, 1.04 mmol) were then added sequentially to the previous solution, and the reaction was stirred at 0 °C for 21 h. The reaction mixture was quenched with hydrochloric acid solution (concentration: 1 mol / L) until pH = 3. Saturated sodium bicarbonate solution was then added until pH ~ 7, and the resulting aqueous phase was extracted with dichloromethane (3 × 20 mL). The organic fractions were combined, dried over anhydrous sodium sulfate, filtered, and evaporated. Purification by flash chromatography using hexane:ethyl acetate (4:6) as the eluent resulted in the isolation of compound 8 as a colorless oil (0.0706 g, 0.560 mmol).

[0098] Yield: 54%; 1 H NMR (300MHz, CDCl3): δ(ppm)6.93(t,J=4.2Hz,1H), 4.24(q,J=1.2Hz,2H), 2.48~2.35(m,4H), 2.32~2.12(br s,1H), 2.08~1.95(m,2H); 13 C NMR (75MHz, CDCl3): δ(ppm)200.6, 147.9, 138.2, 61.3, 38.2, 25.6, 22.7; MS(ES + ):[M+H] + 17(75).

[0099] 1.8. Synthesis of Compound (9): (6,6-dihydroperoxycyclohex-1-en-1-yl)methanol [ka]

[0100] A 25 mL round-bottom flask was charged with compound 8 (0.0774 g, 0.614 mmol) and rhenium(VII) oxide (0.0171 g, 0.0353 mmol) in acetonitrile (3.7 mL). 50% hydrogen peroxide (0.14 mL, 2.46 mmol) was then added at room temperature and stirred for 50 minutes. The mixture was filtered under vacuum through a silica plug and washed with ethyl acetate. The solution was concentrated, and compound 9 was isolated as a colorless oil (0.105 g, 0.598 mmol).

[0101] Yield: 97%; 1 H NMR (300MHz, (CD3)2CO): δ(ppm)9.97(s,1H), 9.65(s,1H), 4.26~4.13(m,1H) , 4.02~3.89(m,1H), 3.71~3.62(m,1H), 2.49~1.25(m,2H), 2.01~1.62(m,4H); 13 C NMR (75MHz, (CD3)2CO): δ(ppm) 145.1, 59.5, 37.8, 26.1, 23.8.

[0102] 1.9. Synthesis of Compound (10): 2"-(hydroxymethyl)dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-2"-en-5-ol [ka]

[0103] A 25 mL round-bottom flask was charged with 5-hydroxy-2-adamantanone (0.338 g, 2.03 mmol) and rhenium(VII) oxide (0.0165 g, 0.034 mmol) in dichloromethane (4.0 mL) under an inert atmosphere. Compound 9 (0.234 g, 1.33 mmol) in acetonitrile (4.0 mL) was then added at room temperature and stirred for 1 hour. The mixture was filtered under vacuum through a silica plug and washed with ethyl acetate. The solution was concentrated and purified by flash chromatography with hexane:ethyl acetate (2:8) to isolate compound 10 as a white solid (0.132 g, 0.406 mmol).

[0104] Yield: 31%; 1 H NMR (300MHz, CDCl3): δ(ppm)4.68~4.59(m,1H), 3.24~3.15(m,1H), 2.49~2.39(m,2H), 2.16~1.68(m,18H); MS(ES + ):[M+ACN+H] + 366(30), [M+Na] + 347(30).

[0105] 1.10. Synthesis of Compound (11): 5-Hydroxydispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-2"-en-2"-yl)methyl(4-nitrophenyl)carbonate [ka]

[0106] Compound 10 (0.126 g, 0.388 mmol) was dissolved in dichloromethane (1.53 mL), followed by the addition of N,N-diisopropylethylamine (0.20 mL, 1.17 mmol) and 4-dimethylaminopyridine (0.0510 g, 0.413 mmol). At 0 °C, 4-nitrophenyl chloroformate (0.162 g, 0.788 mmol) was added, and the reaction was stirred under an inert atmosphere for 15 min, 10 min from 0 °C to room temperature, and 30 min at room temperature. The reaction mixture was diluted with diethyl ether (30 mL) and washed with saturated sodium bicarbonate solution (5 × 30 mL). The organic phase was washed with brine (1 × 25 mL), dried over anhydrous sodium sulfate, then filtered and concentrated. Compound 11 was isolated as a white solid (0.0565 g, 0.115 mmol) by flash chromatography with hexane:ethyl acetate (1:1).

[0107] Yield: 30%; 1H NMR (300MHz, CDCl3): δ(ppm)8.28(dd,J=7.0,2.4Hz,2H), 7.36(dd,J=7.0,2.4Hz,2H), 4.76~4.69(m, 1H), 3.34~3.26(m, 1H), 2.66~1.77(m, 20H). MS(ES) + ):[M+ACN+Na] + 553(15), [M+NH4] + 507(100).

[0108] Example 2. Synthesis of compounds of the present invention (IIa, IIb, IIc, IId and IIe) 2.1. Synthesis of Compound (IIa): Dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl(4-(7-methoxyquinolin-5-yl)amino)pentyl)carbamate) [ka]

[0109] To a solution of primaquine diphosphate (0.042 g, 0.0922 mmol) in N,N-dimethylformamide (0.34 mL) was added triethylamine (12.0 μL, 0.0866 mmol). After 30 min, 1-hydroxybenzotriazole hydrate (0.012 g, 0.0888 mmol) was added to the previous solution, followed by compound 5 (0.0205 g, 0.0444 mmol) in N,N-dimethylformamide (0.34 mL). The mixture was stirred overnight at room temperature. Brine (20 mL) was then added to the mixture, which was then extracted with ethyl acetate (3 × 20 mL). The organic fractions were combined, washed with distilled water (1 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by preparative thin-layer chromatography using hexane:ethyl acetate (7:3) as the eluent. Compound IIa was isolated (0.0143 g, 0.024 mmol).

[0110] Yield: 55%; 1H NMR (300MHz, CDCl3): δ(ppm)8.52(dd,J=4.2,1.5Hz,1H), 7.91(dd,J=8.4,1.5Hz,1H), 7.30(dd,J=8.4,4.2Hz,1H), 6.32(dd,J=17.4,2.4Hz,2H), 6.00(br s,1H), 4.82(br s,1H), 4.70(br s,1H), 3.89(s,3H)3.62(br s,1H), 3.18(br s,3H), 2.81(br s,1H), 2.15~1.62(m,24H), 1.30(d,J=6.3Hz,3H);MS(ES + ):[M+H] + 582(100).

[0111] 2.2. Synthesis of Compound (IIb): 1-(tert-butyl) 4-dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl)piperazine-1,4-dicarboxylate [ka]

[0112] To a solution of boc-piperazine (0.014 g, 0.0752 mmol) in N,N-dimethylformamide (0.27 mL) was added triethylamine (10.0 μL, 0.0684 mmol). After 30 min, 1-hydroxybenzotriazole hydrate (0.0098 g, 0.0704 mmol) was added to the previous solution, followed by compound 5 (0.0158 g, 0.0342 mmol) in N,N-dimethylformamide (0.27 mL). The mixture was stirred overnight at room temperature. Then, brine (20 mL) was added to the mixture, followed by extraction with ethyl acetate (3 × 20 mL). The organic fractions were combined, washed with distilled water (1 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by preparative thin-layer chromatography using hexane:ethyl acetate (8:2) as the eluent. Compound IIb was isolated (0.0057 g, 0.011 mmol).

[0113] Yield: 33%;1 H NMR (300MHz, CDCl3): δ(ppm) 4.91(br s,1H), 3.41(m,8H), 3.12(br s,1H), 3.00~2.56(m,1H), 2.31~1.55(m,20H), 1.46(s,9H).

[0114] 2.3. Synthesis of Compound (IIc): dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl (3-hydroxy-2-methyl-6-((2S,4S)-4,5,12-trihydroxy-4-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate [ka]

[0115] To a solution of doxorubicin hydrochloride (0.112 g, 0.193 mmol) in N,N-dimethylformamide (0.76 mL) was added triethylamine (27.0 μL, 0.193 mmol). After 30 minutes, 1-hydroxybenzotriazole hydrate (0.027 g, 0.194 mmol) was added to the previous solution, followed by compound 5 (0.0445 g, 0.0964 mmol) in N,N-dimethylformamide (0.76 mL). The mixture was stirred at room temperature for 3 hours. Then, brine (20 mL) was added to the mixture, followed by extraction with ethyl acetate (3 × 20 mL). The organic fractions were combined, washed with distilled water (1 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by preparative thin-layer chromatography using 4% dichloromethane:methanol as the eluent. A red powder consistent with compound IIc was isolated (0.050 g, 0.058 mmol).

[0116] Yield: 60%; 1H NMR (400MHz, CDCl3): δ(ppm)13.94(d,J=4.4Hz,1H),13.18(s,1H),8.00(d,J=7.6Hz,1H),7.76(t,J=8.0Hz,1H),7.37(d,J=8.4Hz,1H)5.49(br s,1H),5.25(br s,1H),5.13~5.04(m,1H),4.75(s,3H),4.59~4.46(m,1H),4.15-4.09(m,1H),4.06(s,3H)3.83(br s,1H),3.67(br s, 1H), 3.28~2.85 (m, 4H), 2.77 (br s,1H),2.68~2.37(m,1H),2.36~2.26(m,1H)2.24~2.08(m,2H),2.04~1.57(m,21H),1.28(d,J=6.8Hz,3H); 13 C NMR (101MHz, CDCl3): δ(ppm)214.0, 187.1, 186.7, 161.1, 156.3, 155.7, 155.1, 135.9, 135.5, 133.7, 120.9, 119.9, 118.6, 111.6, 111.5 MS(ES + ):[M+Na] + 888(90);HRMS(ESI):m / z[MH] - C 44 H 50 NO 17 Calculated value: 864.3084, measured value: 864.3062.

[0117] 2.4. Synthesis of Compound (IId): 5-hydroxydispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl (3-hydroxy-2-methyl-6-(((2S,4S)-4,5,12-trihydroxy-4-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate [ka]

[0118] Compound IId was prepared using the same method, except that the starting material was compound 6 (0.0310 g, 0.0649 mmol). Purification by preparative thin-layer chromatography in dichloromethane:methanol resulted in the isolation of 4% of compound IId as a red powder (0.0429 g, 0.049 mmol).

[0119] Yield: 75%; 1 H NMR (400MHz, DMSO): δ(ppm)13.97(s,1H),13.21(s,1H),7.89~7.80(m,2H),7.59(d,J=7. 2Hz,1H),6.56(d,J=8.0Hz,1H),5.39(s,1H)5.17(s,1H),4.86(d,J=6.0Hz,2H),4.84(br s,1H),4.66(d,J=5.6Hz,1H),4.55(d,J=5.6Hz,2H),4.51(br s,1H)4.11(d,J=6.8Hz,2H),3.95(s,3H),3.62(br s,2H), 3.44~3.37(m,2H), 3.02~2.82(m,4H), 2.76~2.60(m,1H), 2.45~1.51(m, 19H), 1.09(d, J=6.4Hz, 3H); 13 CNMR (101MHz, DMSO): δ(ppm)213.9, 186.5, 186.4, 176.7, 160.8, 156.1, 154.5, 154.1, 136.2, 135.5, 134.6, 134.1, 119.9, 119.7, 119.0, 110. 8, 110.6, 100.4, 75.975.0,73.6,69.8,68.1,66.7,63.7,56.6,46.7,4 0.9,38.5,36.5,34.8,34.7,34.2,32.1,29.8,29.7,29.2,17.0;MS(ES + ):[M+2ACN+H] + 964(25), [M+CH3OH+H] + 914(45), [M+Na] + 904(30);MS(ES-):[MH] -880(35), [M+HCOOH-H] - 925(20).

[0120] 2.5. Synthesis of Compound (IIe): 5-hydroxydispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-2"-en-2"-yl)methyl (3-hydroxy-2-methyl-6-(((1R,3R)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamate [ka]

[0121] To a solution of doxorubicin hydrochloride (0.0743 g, 0.128 mmol) in N,N-dimethylformamide (0.50 mL) was added triethylamine (18.0 μL, 0.128 mmol). After 30 minutes, 1-hydroxybenzotriazole hydrate (0.0183 g, 0.135 mmol) was added to the previous solution, followed by the addition of compound 11 (0.0312 g, 0.064 mmol) in N,N-dimethylformamide (0.50 mL). The mixture was stirred at room temperature for 7 hours. Then, brine (20 mL) was added to the mixture, followed by extraction with ethyl acetate (3 × 20 mL). The organic fractions were combined, washed with distilled water (1 × 20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by preparative thin-layer chromatography (TLC) using 4% dichloromethane:methanol as the eluent. Compound IIe was isolated as a red powder (0.0415 g, 0.046 mmol).

[0122] Yield: 73%; 1H NMR (400MHz, DMSO): δ (ppm) 13.95 (s, 1H), 13.20 (s, 1H), 7.90~7.76 (m, 2H), 7.58 (d, J = 7.6Hz, 1H), 6.56 (d, J = 8.0Hz, 1H), 5. 37(s,1H)5.16(s,1H),4.91~4.81(m,2H),4.73~4.61(m,1H),4.60~4.45(m,4H),4.11(d,J=7.2Hz,1H),3.94(s,3H),3.62(br s,2H),3.39(br s,2H), 3.04~2.75(m,4H), 2.40~1.50(m,20H), 1.41(d,J=12Hz,1H), 1.09(d,J=6.4Hz,3H); 13 CNMR (101MHz, DMSO): δ (ppm) 213.9, 186.5, 186.4, 176.7, 160.8, 156.1, 154.5, 154.1, 136.2, 135.5, 134.6, 134.0, 119.9, 119.7, 119.0, 110. 7. 110.6, 100.4, 75.975.0,73.5,69.8,68.1,66.7,63.7,56.6,46.7,4 0.9,38.5,36.5,34.8,34.7,34.2,32.1,29.8,29.7,29.2,17.0;MS(ES + ):[M+H] + 894(35), [M+Na] + 916(70);MS(ES - ):[MH] - 892(40).

[0123] Example 3 - Demonstration of the concept of the first delivery mechanism theory A solution of compound IIc (0.009 mmol) in dichloromethane was added to a Schlenk tube under an inert atmosphere and charged with iron(II) bromide (2 equivalents) in acetonitrile and TEMPO (2 equivalents). The reaction was allowed to stir at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with distilled water (1 × 10 mL) and brine (1 × 10 mL). The organic fraction was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was analyzed by UHPLC-MS. The mobile phase consisted of Millipore water containing 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.30 mL min -1 A gradient elution was applied consisting of 10% B for 1 min, 10–95% B for 5 min, and 95% B for 4 min, finally returning to the initial conditions (10% B) for 5 min.

[0124] The tetraoxane reaction of compound IIc with an Fe(II) source and TEMPO was performed, and it was confirmed that tetraoxane fragmentation and, more importantly, spontaneous β-elimination of the cyclohexanone derivative occurred, liberating doxorubicin.

[0125] UHPLC-MS analysis of this reaction revealed two possibilities for coordination with Fe(II) in the positive mode ([M+H] + =324) and negative mode ([MH] - =322;[MH] - = 855 and [M+CN-H] - = 881), confirming the tetraoxane fragmentation with the formation of a carbon-centered radical (Scheme 5). Furthermore, in accordance with the proposed mechanism, 2-adamantanone ([M + K] + =189) and cyclohexanone derivatives ([MH] - Importantly, doxorubicin was also detected ([M+H] + =544;[MH] - =542), and API release by β-elimination of the cyclohexanone derivative was confirmed (Scheme 6).

[0126] Scheme 5 shows the Fe(II) activation and TEMPO reaction of compound IIc to form the TEMPO adduct. [ka]

[0127] Scheme 6 shows the activation of compound IIc by cyclohexanone derivatives and the release of doxorubicin. [ka]

[0128] Example 4 - Mechanistic proof of concept of a second drug delivery system To verify the ferrous activation and TEMPO trapping of the second drug delivery system, the following protocol was performed. A solution of compound IIe (0.0077 g, 0.009 mmol) in dichloromethane was added to a Schlenk tube under an inert atmosphere and charged with iron(II) bromide (2 equivalents) in tetrahydrofuran and TEMPO (2 equivalents). The reaction was allowed to stir at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate (10 mL) and washed with distilled water (1 × 10 mL) and brine (1 × 10 mL). The organic fraction was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was analyzed by UHPLC-MS. The mobile phase consisted of Millipore water containing 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.30 mL min -1 A gradient elution was applied consisting of 30% B for 1 min, 30–95% B for 5 min, and 95% B for 4 min, and finally returned to the initial conditions (30% B) for 5 min.

[0129] To verify the ferrous activation and N-acetylcysteine ​​(NAC) Michael addition of the second drug delivery system, the following protocol was performed. 250 μL of a 10 mM stock solution of compound IIe in tetrahydrofuran was added to a Schlenk flask and charged with 250 μL of iron(II) bromide (2 equivalents) and NAC (1.5 equivalents) in 250 μL of Millipore water under an inert atmosphere. 100 μL of sodium hydroxide (6.25 mol / L) was then added to ensure a basic pH. The reaction was allowed to stir at room temperature for 24 hours. The reaction mixture was diluted with distilled water (10 mL) and extracted with ethyl acetate (10 mL). The aqueous fraction was acidified with hydrochloric acid (1 mol / L) and extracted with ethyl acetate (2 × 10 mL). The aqueous phase was neutralized with sodium hydroxide (6.25 mol / L) and extracted with ethyl acetate (2 × 10 mL). The organic fractions were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was analyzed by UHPLC-MS. The mobile phase consisted of Millipore water containing 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.30 mL min -1 A gradient elution was applied consisting of 30% B for 1 min, 30–95% B for 5 min, and 95% B for 4 min, and finally returned to the initial conditions (30% B) for 5 min.

[0130] Compound IIe reacted with an iron source and TEMPO scavenger, confirming tetraoxane fragmentation, and more importantly, no doxorubicin was released under these conditions.

[0131] UHPLC-MS analysis of this reaction revealed two TEMPO adducts ([M+H]) in positive mode for two possibilities of coordination with Fe(II). + =340;[M+K] + =907) was detected, confirming tetraoxane fragmentation with the formation of a carbon-centered radical (Scheme 7).

[0132] Scheme 7 shows the Fe(II) activation and TEMPO trapping of compound IIe to form the TEMPO adduct. [ka]

[0133] Furthermore, according to the proposed mechanism, 5-hydroxy-2-adamantanone ([M+NH4] + = 184; [M + DMSO + H] + =245) and cyclohex-2-enone derivatives ([M+K] + =734; [M + DMSO + H] + =774) was also identified (Schemes 7 and 8). Finally, doxorubicin was not detected in this experiment.

[0134] Scheme 8 shows the Fe(II) activation of compound IIe to give the cyclohex-2-enone derivative. Reagents and conditions: (i) iron(II) bromide, TEMPO, tetrahydrofuran. [ka]

[0135] Compound IIe was reacted with an Fe(II) source and N-acetylcysteine ​​(NAC) to confirm the mechanism of the second drug delivery system. UHPLC-MS analysis of this reaction showed a positive mode ([M+H] + = 713 and [M+Na] + =735), we detected one carbon-centered radical (Scheme 9), confirming tetraoxane fragmentation upon reaction with Fe(II).

[0136] Scheme 9 shows the Fe(II) activation of compound IIe to generate a carbon-centered radical. [ka]

[0137] Furthermore, according to the first step of the activation of the second drug delivery system, the cyclohex-2-enone derivative ([M+Na] + =718 and [M+K] +=734) was also detected. Finally, the release of doxorubicin was detected, and the Michael addition of NAC to the cyclohex-2-enone derivative occurred, thus validating the mechanism of action of the second drug delivery system (Scheme 10).

[0138] Scheme 10 shows the drug delivery mechanism of compound IIe, which was verified after detection of doxorubicin. [ka]

[0139] Example 5. Evaluation of the biological activity of compounds of formula II against malaria Sample preparation Compounds were dissolved in RPMI-1640 containing L-glutamine (Biowest™) supplemented with AlbuMAX II (Gibco™), hereafter referred to as RPMIc, to give an intermediate solution of 100 μM.

[0140] Plasmodium falciparum in vitro culture Laboratory-adapted Plasmodium falciparum strain 3D7, a chloroquine- and mefloquine-sensitive strain, was serially cultured as described elsewhere (Machado et al., Ann. Clin. Med. Microbio. 2016, 2, 1010). Parasites were cultured at 5% hematocrit, 37°C, and in an atmosphere containing 5% CO2. 0.5% AlbuMAX II (Gibco™) was used as a substitute for human serum in the culture medium.

[0141] Antimalarial activity was determined using a whole-cell SYBR GreenI assay Staging and parasitemia were identified by light microscopy of Giemsa-stained thin blood smears. Antimalarial activity was determined using the SYBR Green I assay (Machado et al., Ann. Clin. Med. Microbio. 2016, 2, 1010 and Lobo et al., Malar. J. 2018, 17, 1). Briefly, asynchronous parasites were cultured for 72 hours in the presence of 1:3 serial dilutions of each compound. Fluorescence intensity was measured using a multimode microplate reader (Triad, Dynex Technologies) at excitation and emission wavelengths of 485 and 535 nm, respectively, and analyzed by nonlinear regression using GraphPad Prism to determine IC values. 50 value was determined.

[0142] Compounds IIa and IIb were potent against the chloroquine-sensitive 3D7 strain of Plasmodium falciparum. Compounds IIa and IIb had IC values ​​of 32.82 nM and 11.38 nM, respectively. 50 Both compounds were as potent as chloroquine (CQ), a clinically used antimalarial drug (Table 1). [Table 1]

[0143] Example 6. Evaluation of the biological activity of compounds of Formula II in cancer treatment PC3 human prostate carcinoma, MCF7 human breast carcinoma, HCT116 human colorectal carcinoma, and U251 human glioblastoma were obtained from the American Type Culture Collection (ATCC).

[0144] Cell lines were cultured under adherent conditions in Roswell Park Memorial Institute (RPMI) 1640 (PC3), Dulbecco's Modified Eagle's Medium (DMEM; MCF7 and U251), and McCoy's 5A (HCT116), all supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotic / antimycotic solution (all Gibco, ThermoFisher Scientific, Paisley, UK). MCF7 medium was supplemented with 1% (v / v) GlutaMAX (Gibco); U251 medium was supplemented with 1% (v / v) GlutaMAX and 1% (v / v) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (both from Gibco). All cell lines were cultured at 37°C in a humidified atmosphere of 5% CO (HeraCell 150i CO Incubator, ThermoFisher Scientific).

[0145] To quantitatively evaluate the inhibitory potential of a compound, the half maximal inhibitory concentration (IC 50 ) were identified by dose-response curves. Cell lines were plated in 96-well plates at a cell density of 5000 cells / well, and after 24 hours, the cells were treated with Compounds IIc, IId, IIe, doxorubicin, Compound 3, and Compound 10 (1.5 × 10 -3 After 72 hours of incubation, cells were treated with MTS (at concentrations ranging from 0.01 to 1 mM). After 72 hours of incubation, cellular metabolic activity was assessed by measuring MTS metabolism. Viable (metabolically active) cells metabolize MTS to formazan, which is quantified by absorbance. The greater the amount of formazan, the more cells survive after exposure to the compound; the less formazan, the more metabolically inactive cells.

[0146] For all assays, cell metabolic activity was measured using the Cell Titer96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS; Promega, Madison, WI, USA), and absorbance signals were recorded using the GloMax®-MultiDetection System (Promega). IC was calculated using GraphPad Prism 8.0.2 software (San Diego, CA, USA). 50 All statistical analyses were performed. All data are expressed as mean ± SEM from at least three independent experiments. Differences between sample groups were assessed according to normality of value distribution, assessed by Shapiro-Wilk test, one-way analysis of variance, or Kruskal-Wallis test, followed by Bonferroni or Dunn's multiple comparison test. A p value of less than 0.05 was considered significant.

[0147] All compounds were tested alone and at appropriate doses in a panel of cancer cell lines and the results are shown in Table 2.

[0148] As can be observed from Table 2, compound IIc was potent in all cell lines in the micromolar range, except for the U251 cell line, where it was in the submicromolar range. Furthermore, compound IIc was less potent than doxorubicin alone.

[0149] Compound IId was also tested in previously used cancer cell lines (U251, PC3, MCF7, and HCT116). Despite differences in lipophilicity, compound IId was also potent in the micromolar range, less potent than doxorubicin.

[0150] Compound 3 was tested as a control to demonstrate that the efficacy of compounds IIc and IId against cancer cells was dependent on the API coupled to compound 3. Compound 3 was chosen because it represents the core structure of compounds IIc and IId.

[0151] Compound (3) was not active against the cell lines tested. These results indicate that the potency of compounds IIc and IId depends on the API coupled in the system, rather than on the tetraoxane structure.

[0152] Compound IIe was potent in the micromolar range in all cancer cell lines. Compound IIe was less potent than doxorubicin in all cancer cell lines evaluated.

[0153] Compound 10 was tested as a control to demonstrate that the efficacy of compound IIe against cancer cells depends on the API coupled to compound 10. Compound 10 was chosen because it represents the core structure of compound IIe.

[0154] Compound 10 was not active in any of the cell lines tested (Table 2). Therefore, these results demonstrate that the tetraoxane scaffold is not cytotoxic and that the contributing factor to the activity of compound IIe is the API coupled to the system, in this case, doxorubicin. [Table 2]

[0155] Example 7. Evaluation of the effect of compounds of formula II on toxicity to non-tumor cells To evaluate the toxic effects of doxorubicin, compounds IIc, IId, IIe, and doxorubicin were tested on a non-tumor cell line (AML12), and the results are shown in Table 3. The AML12 mouse immortalized hepatocyte cell line was obtained from the European Collection of Authenticated Cell Cultures (Porton Down, UK). The AML12 cell line was cultured under adherent conditions in DMEM / nutrient mixture F-12 (Gibco, Thermo Fisher Scientific, Paisley, UK) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS; Gibco), 1% (v / v) antibiotic / antimycotic solution (Gibco), 1% (v / v) insulin-transferrin-selenium (Gibco), and 40 ng / mL dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). The cell line was cultured at 37°C in a humidified atmosphere of 5% CO (HeraCell 150i CO incubator, ThermoFisher Scientific). AML12 cells were plated in 96-well plates at a cell density of 5000 cells / well, and after 24 hours, the cells were treated with compounds IIc, IId, and IIe and doxorubicin (1.5 × 10 -3 Cells were treated with 100 μg of ... 50 All statistical analyses were performed. All data are expressed as mean ± SEM from at least three independent experiments. Differences between sample groups were assessed according to the Shapiro-Wilk test, one-way analysis of variance, or Kruskal-Wallis test, followed by Bonferroni or Dunn's multiple comparison test to determine normality of the value distribution. A p value of less than 0.05 was considered significant.

[0156] From Table 3, it can be observed that both compounds IIc and IId were able to induce lower toxicity than doxorubicin. Interestingly, compound IIc was 10-fold less toxic when compared to doxorubicin. However, AML12 cells were highly resistant to compound IId, with IC 50 The ratio was 4294.

[0157] In conclusion, the first drug delivery system according to the present invention can reduce the toxicity of doxorubicin.

[0158] From Table 3, it can be seen that compound IIe is less toxic than doxorubicin in non-tumorous cells. Interestingly, compound IIe was 180 times less toxic than doxorubicin. Therefore, the second drug delivery system can reduce the toxicity associated with doxorubicin. [Table 3]

[0159] Example 8. Evaluation of iron-dependent biological activity For the iron competition assay, HCT116 cells were incubated with 1 μM DFO (column 2 in Figure 1 ), 10 μM Compound IId (column 3 in Figure 1 ), and 10 μM Compound IId and 1 μM DFO (column 4 in Figure 1 ). Additionally, HCT116 cells were incubated with 1 μM DFO (column 2 in Figure 2 ), 10 μM Compound IIe (column 3 in Figure 2 ), and 10 μM Compound IIe and 1 μM DFO (column 4 in Figure 2 ). After 24 hours of incubation, cellular metabolic activity was measured. Cell Titer96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS; Promega, Madison, WI, USA) was used to measure the absorbance signal, and the absorbance signal was recorded using a GloMax®-MultiDetection System (Promega). IC was analyzed using GraphPad Prism 8.0.2 software (San Diego, CA, USA). 50 All statistical analyses were performed. All data are expressed as mean ± SEM from at least three independent experiments. Differences between sample groups were assessed according to the Shapiro-Wilk test, one-way analysis of variance, or Kruskal-Wallis test, followed by Bonferroni or Dunn's multiple comparison test to assess normality of value distribution. A p value of less than 0.05 was considered significant.

[0160] Compound IId was incubated with a ferrous chelator (deferoxamine mesylate - DFO) in HCT116 cells. A significant decrease in the potency of compound IId was detected (column #4 of Figure 1 ) compared with compound IId alone (column #3 of Figure 1 ). Thus, cell death caused by compound IId is iron-dependent. Compound IIe was incubated with the iron chelator DFO in HCT116 cells. A slight decrease (7%) in the potency of compound IIe was detected (column #4 of Figure 2 ) compared with compound IIe alone (column #3 of Figure 2 ). While compound IIe is likely activated by Fe(II) as described, the primary contributor to its cytotoxicity is related to the reaction of the cyclohex-2-enone derivative with glutathione (Michael addition), which allows for the release of the API by elimination (see Example 4). The cyclohex-2-enone derivatives have an alkene group that only becomes available after tetraoxane activation with Fe(II), which allows for improved selectivity compared to first drug delivery systems.

[0161] Example 9. Evaluation of the biological activity of compounds of formula II as tumor markers Determination of quantum yield Ultraviolet (UV) spectra were monitored using a Thermo Scientific Evolution 201 UV-Visible Spectrophotometer. Fluorescence measurements were performed using a SHIMADZU Spectro Fluorescence Photometer RF-6000 instrument. Fluorescence quantum yields were measured using fluorescein in 0.1 M sodium hydroxide as the standard (Φf = 0.95). All solvents were analytical reagent grade and purchased from Alfa Acer or Sigma-Aldrich. Doxorubicin possesses fluorescent properties. Diluted solutions of compounds IIc, IId, and doxorubicin in water containing 30% dimethyl sulfoxide (DMSO) were prepared. The quantum yield of doxorubicin was 2.8%, and the quantum yields of compounds IIc and IId were less than 1%.

[0162] Fluorescence imaging for compound cellular localization HCT116 cells were plated at 2.5 x 10 on a glass coverslip. 5Cells were plated in 35 mm dishes at a cell density of 1000 cells / ml. 24 hours after plating, cells were incubated with system control (DMSO) or 1 μM Compound IId or doxorubicin for an additional 24 hours. Cells were gently washed with 1x phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA), pH 7.4, at room temperature. Fixed cells were incubated with Hoechst dye 33258 (5 μg / mL) for 10 minutes in the dark. Cells were washed three times with 1x PBS and mounted on glass slides with 7 μL of Mowiol®. Images were acquired using a Zeiss Axio Scope.A1 microscope coupled to an AxioCam HR R3 camera and analyzed using Zen Software 2012 Blue Edition (Carl Zeiss Microscopy, GmbH).

[0163] The photoluminescence properties of doxorubicin are lost in the drug delivery system of Formula II (quantum yield less than 1%). Therefore, we next investigated the cellular localization of compound IId in cells. Interestingly, fluorescence imaging in HCT116 cells showed that after 24 hours of incubation, doxorubicin accumulated in the nucleus (Figures 3B-C and 3D-D), while compound IId was activated and released doxorubicin in the adipose tissue where labile iron pools increased (Figures 3C-C and 3D-D).

[0164] Example 10 - In vivo validation of the first drug delivery system The antitumor effect of compound IId was tested in a xenograft mouse model. nu / nu Mice were inoculated with colorectal cancer cell lines (2 × 10) suspended in PBS (phosphate-buffered saline) solution (100 μL) into the right flank. 6HT-29 cells). Treatment began when tumor masses became palpable. Four groups with five mice per group were established: negative control group—mice that did not receive any treatment; vehicle control—mice administered with cremophor in PBS (phosphate-buffered saline) solution (the solvent used to solubilize compound IId); IId—mice administered with the compound under test; doxorubicin—mice administered with doxorubicin solubilized in PBS (phosphate-buffered saline) solution. All test formulations were administered intravenously via the tail vein at a therapeutic dose of 2 mg / kg body weight for a total of eight injections. The dose administered to the mice was established by the ratio between the mass of the compound and the body weight of the animals. Tumor size was monitored regularly using a digital caliper. The volume of each tumor was calculated according to the following formula: V (mm 3 )=(L×W 2 ) / 2, where L and W represent the longest and shortest axes of the tumor, respectively. Relative tumor volume was also determined for each animal: the ratio of the volume on a given day to the volume at the start of treatment. Body weight was also assessed periodically.

[0165] Mice treated with compound IId and doxorubicin showed greater tumor mass reduction compared to the negative control and vehicle controls (Figure 4). Furthermore, tumor mass reduction was greater in mice treated with compound IId (Figure 4). The molecular weight of compound IId is 30% higher than that of doxorubicin. Therefore, the number of moles of doxorubicin released from compound IId was 30% lower than that of doxorubicin administered alone. Perhaps experiments in which animals were administered the same number of moles of both compounds could result in a greater difference in tumor mass reduction between compound IId and doxorubicin.

Claims

1. Compounds of formula I, including enantiomers and racemic mixtures thereof, 【Chemistry 1】 During the ceremony, R １ and R ２ are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; R １ and R ２ can couple to form substituted or unsubstituted cycloalkyl and substituted or unsubstituted heterocycloalkyl; X is -CH ２ O-, -OCH ２ - or -O-O-, R ３ is -C(R ４ ) (R ５ ) -, -CH(R ４ )-CH(R ５ )- or -C(R ４ ) = C(R ５ ) - and R ４ represents hydrogen, -OH, -CH ２ OH, -CH ２ O(C=O)A or -CH ２ O(C=S)A, where A is independently one or more APIs, proteins and antibodies for the diagnosis and treatment of inflammation, diabetes, cardiovascular disease, malaria, rare diseases, neurodegenerative diseases, infectious diseases and cancer; R ５ and R ６ are independently hydrogen, —OH, —O(C═O)A, or —O(C═S)A, where A is independently one or more APIs, proteins, and antibodies for the diagnosis and treatment of inflammation, diabetes, cardiovascular disease, malaria, rare diseases, neurodegenerative diseases, infectious diseases, and cancer; The compound has the following formula: Formula IIa, 【Chemistry 2】 During the ceremony, R 1 is adamantyl; R 2 is adamantyl; X is —O—O—, R 3 is —CH(R 4 )—CH(R 5 )—; R 4 is hydrogen; R 5 is (4-((7-methoxyquinolin-5-yl)amino)pentyl)carbamoyloxy; R 6 is hydrogen; Formula IIb 【Transformation 3】 During the ceremony, R 1 is adamantyl; R 2 is adamantyl; X is —O—O—, R 3 is —CH(R 4 )—CH(R 5 )—; R 4 is hydrogen; R 5 is 1-(tert-butyl)piperazine-1,4-dicarboxylate; R 6 is hydrogen; Formula IIc 【Chemistry 4】 During the ceremony, R 1 is adamantyl; R 2 is adamantyl; X is —O—O—, R 3 is —CH(R 4 )—CH(R 5 )—; R 4 is hydrogen; R 5 is (3-hydroxy-2-methyl-6-((2S,4S)-4,5,12-trihydroxy-4-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyloxy; R 6 is hydrogen; Formula IId 【Transformation 5】 During the ceremony, R 1 is 5-hydroxyadamantyl; R 2 is 5-hydroxyadamantyl; X is —O—O—, R 3 is —CH(R 4 )—CH(R 5 )—; R 4 is hydrogen; R 5 is (3-hydroxy-2-methyl-6-((2S,4S)-4,5,12-trihydroxy-4-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyloxy; R 6 is hydrogen; Formula IIe 【Transformation 6】 During the ceremony, R 1 is 5-hydroxyadamantyl; R 2 is 5-hydroxyadamantyl; X is —O—O—, R 3 is —C(R 4 )═C(R 5 )—; R 4 is (3-hydroxy-2-methyl-6-(((1R,3R)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyloxy; R 5 is R 6 is hydrogen characterized in that it is represented by any one of compound.

2. 10. A drug delivery system based on the tetraoxane compounds of formula IIa, IIb, IIc, IId and IIe according to claim 1, wherein the compounds are suitably modified to release an active pharmaceutical ingredient (API), wherein compound IIa or IIb is coupled to one or more antimalarial APIs, compound IIc, IId or IIe is coupled to one or more antitumor APIs, or compound IIc, IId or IIe is coupled to one or more tumor marker APIs.

3. The drug delivery system of claim 2, characterized in that the antimalarial API is selected from primaquine and boc-piperazine, atovaquone, quinine sulfate, sulfonamides (sulfadoxine and sulfamethoxypyridazine) and falcipain inhibitors (dipeptidyl vinyl sulfone and peptidomimetic pyrimidine nitrile), and combinations or derivatives thereof.

4. The antitumor API is doxorubicin, alectinib, afatinib, abiraterone acetate, azacitidine, axitinib, bendamustine, brigatinib, bosutinib, bicalutamide, calicheamicin, capecitabine, carfilzomib, combretastatin, ceritinib, cladribine, clofarabine, cobimetinib, crizotinib, cryptophycin, cytarabine, dabrafenib, dasati Nib, daunorubicin, decitabine, duocarmycin, enasidenib, epirubicin, eribulin mesylate, erlotinib, everolimus, etoposide, exatecan, fludarabine, fulvestrant, gemcitabine, hemiasterlin, histone deacetylase inhibitors (abexinostat, belinstat, entinostat, divinostat, mocetinostat, oxamflatin, Panobinostat, piroxamide, xinostat, vorinostat, trichostatin A, tasecinarin, tushidinostat, tubastatin A), hydroxyurea, ibrutinib, idelalisib, imatinib, irinotecan, ixabepilone, lapatinib, lenalidomide, maytansine, mitomycin C, monomethyl auristatin (MMA-E, MMA-F, dolastatin 10 and dolastatinol), methotrexate, neratinib, 3. The drug delivery system of claim 2, wherein the drug is selected from nilotinib, niraparib, olaparib, octreotide, osimertinib, paclitaxel, palbociclib, pazopanib, pyrrolobenzodiazepines, pemetrexed, pomalidomide, ponatinib, ribociclib, rucaparib, sorafenib, tamoxifen, triazenes, trifluridine, topotecan, tublysin, vandetanib, and combinations or derivatives thereof.

5. A drug delivery system based on the tetraoxane compounds of formula IIc, IId and IIe according to claim 1, characterized in that the tumor marker API is selected from 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), cyanine, dicyanomethine, doxorubicin, fluorescein, nitrobenzofurazan, rhodamine, and combinations or derivatives thereof.

6. A method for preparing a 1,2,4,5-tetraoxane-based drug delivery system having formula I according to any one of claims 2 to 5, comprising the following steps: 【Transformation 7】 Including, (i) Sodium dichromate dihydrate, water, and sulfuric acid react to oxidize 1,3-cyclohexanediol, compound 0, to produce rac-3-hydroxycyclohexanone, compound 1; (ii) rhenium(VII) oxide, 50% hydrogen peroxide, acetonitrile, wherein compound 1 reacts with hydrogen peroxide in the presence of rhenium(VII) oxide to produce the dihydroperoxide compound 2; (iii) 2-adamantanone or 5-hydroxy-2-adamantanone, rhenium(VII) oxide, dichloromethane:acetonitrile (1:1), inert atmosphere; the dihydroperoxide, compound 2, is reacted with a carbonyl reagent using a catalytic amount of rhenium(VII) oxide; 2-adamantanone (compound 3) and 5-hydroxy-2-adamantanone (compound 4) can be used as a carbonyl for the formation of tetraoxanes; (iv) 4-nitrophenyl chloroformate, N,N-diisopropylethylamine, 4-dimethylaminopyridine, dichloromethane; Compounds 3 and 4 are activated with 4-nitrophenyl chloroformate along with an excess of an amine base (N,N-diisopropylethylamine and 4-dimethylaminopyridine) to produce compounds (5) and (6), respectively; (v) API, 1-hydroxybenzotriazole hydrate, triethylamine, N,N-dimethylformamide. Compound 5 is coupled with primaquine diphosphate to give compound IIa or boc-piperazine to give compound IIb, or two carbonates are coupled with doxorubicin to give carbamates IIc and IId. A method characterized by:

7. in step (i), sodium dichromate dihydrate, water, sulfuric acid, and compound 0 are replaced with formaldehyde, N-methylpyrrolidine, barium hydroxide octahydrate, water:methanol (5:1), and compound 7; In step (ii), compound 1 is replaced by compound 8, In step (iii), compound 2 is replaced by compound 9, In step (iv), compound 3 or 4 is replaced by compound 10, In step (v), compound 5 or 6 is replaced with compound 11 7. A method for preparing the drug delivery system according to claim 6.

8. 1. An intermediate compound used in the process for preparing a 1,2,4,5-tetraoxane-based drug delivery system having formula I according to claim 6 or claim 7, the intermediate compound having the following formulas 1 to 11: Compound 1 is rac-3-hydroxycyclohexanone, Compound 2 is 3,3-dihydroperoxycyclohexan-1-ol, Compound 3 is dispiro[adamantan-2,3'-[1,2,4,5]tetraoxan-6',1"-cyclohexan]-3"-ol; Compound 4 is dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3",5-diol; Compound 5 is dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl(4-nitrophenyl)carbonate; Compound 6 is 5-hydroxydispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-3"-yl(4-nitrophenyl)carbonate; Compound 7 is 2-cyclohexen-1-one, Compound 8 is 2-(hydroxymethyl)cyclohex-1-en-1-one, Compound 9 is (6,6-dihydroperoxycyclohex-1-en-1-yl)methanol; Compound 10 is 2"-(hydroxymethyl)dispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-2"-en-5-ol; Compound 11 is 5-hydroxydispiro[adamantane-2,3'-[1,2,4,5]tetraoxane-6',1"-cyclohexane]-2"-en-2"-yl)methyl(4-nitrophenyl)carbonate. An intermediate compound, characterized in that it is represented by:

9. A drug delivery system comprising a compound of formula IIa, IIb, IIc, IId and IIe according to claim 1 or said compound according to any one of claims 2 to 4 for use as a medicament.

10. - the compounds of formula IIa and IIb according to claim 3 are used as antimalarials, The compound IIc, IId or IIe according to claim 4 is used as an antitumor agent.

10. The drug delivery system of claim 9.

11. A drug delivery system comprising a compound of formula IIc, IId and IIe as defined in claim 1, or a compound IIc, IId or IIe as defined in claim 5 for use as a tumor marker.

Images