Carbon monoxide releasing therapeutic compounds and methods, products and uses related thereto

Abstract

The invention relates to carbon monoxide releasing molecules, and methods, compositions and uses relating thereto. Provided is a compound representing a carbon monoxide (CO) releasing variant of Mesalazine (MesaCORM) having a structure according to Formula Ia or Ib; or a pharmaceutically acceptable salt thereof.

Description

[0001] P136509PC00 Title: Carbon monoxide releasing therapeutic compounds and methods, products and uses related thereto. The invention relates to carbon monoxide releasing molecules (“CORMs”), and methods of synthesizing and applying the molecules. For a long time, carbon monoxide (CO) was considered to be merely a toxic gas and air pollutant. However, in recent years, the gaseous molecule was identified as an essential endogenously generated signalling molecule. While high CO concentrations are toxic, low concentrations are generally implicated in response to stress conditions. The potential of CO as a therapeutic agent was evidenced by its pronounced involvement in anti-inflammatory, cell-protective, and anti-hypertensive biological processes. These promising therapeutic effects are specifically clear for intestinal inflammation, as showcased by studies in numerous disease models. Since CO was identified as a potential disease modulating entity, growing efforts have been made to exploit the beneficial effects of CO. Small scale clinical trials in humans with CO inhalation have been carried out, but the potentially toxic characteristics of unwanted formation of carboxyhaemoglobin (COHb) in relation to systemic exposure remains a major disadvantage. Additionally, systemic administration routes, such as inhalation, require high levels of CO exposure to obtain therapeutic CO levels in the intestinal target tissue, since COHb formation functions as a sink for CO. To establish local CO delivery, Carbon Monoxide Releasing Molecules (CORMs) were introduced as a concept of using a chemically bound form of CO as a pro- drug for physiological CO release. CORMs usually contain a transition metal core surrounded by carbonyl groups as a coordinated ligand. CORM lead structures release CO immediately upon dissolution in physiological medium. Whereas different types of CORMs have been created, a number of obstacles have halted the clinical translation of current generations of CORMs to studies with human subjects. Although targeted CO release with limited systemic CO is possible by using current generation of CORMs, the lack of controlled and tissue-specific CO release patterns remain a major disadvantage. Additionally, current generation CORMs (i.e. CORM-2 and CORM-3) were reported to release more CO2 than CO. Furthermore, certain byproducts of the scaffolds of molecule- centered transition metals that are used for binding of CO show cytotoxic characteristics, raising important concerns of the druggability of these CORMs. Byrne et al. (SCIENCE TRANSLATIONAL MEDICINE 29 Jun 2022, Vol 14, Issue 651) designed gas-entrapping materials (GEMs) using components generally recognized as safe, including xanthan gum, methylcellulose, maltodextrin, and corn syrup. Solid, hydrogel, and foam GEMs containing CO could deliver different concentrations of the gas to healthy rodents and pigs through noninhaled routes. Rectally administered foam GEMs reduced tissue injury and inflammation in rodent models of colitis, acetaminophen overdose, and radiation-induced proctitis. Recent efforts have been made to tightly control the manner by which CO is released through CORMs, by taking advantage of endogenous or exogenous stimuli, such as enzyme-triggered and photo-activatable CORMs. See for example Stamellou et al. (Redox Biol.2014 Jun 5;2:739-48), Li et al.( Chembiochem.2022 Jan 5;23(1):e202100452). Animal models of intestinal inflammation have been used to investigate effects of CO therapy. See for example Takagi et al. (Nitric Oxide. 2021 Feb 1;107:19-30), Fukuda et al. (Dig Dis Sci. 2014 Jun;59(6):1142-51), Takagi et al. Dig Dis Sci. 2011 Jun;56(6):1663-71. Whereas many newly synthesized CORMs were reported in the literature to date, they lack the drug-like properties that are required for further translation into the clinic. Hence, there is a need for novel approaches for the delivery of CO directly and safely to the gastrointestinal (GI) tract, which would allow for a transforming the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, the present inventors aimed at providing novel CORMs for tissue-specific delivery of CO, in particular to the GI tract. Ideally, the CORMs exhibit a localized CO release pattern in order to overcome acute toxicity related to systemic CO exposure. Furthermore, to fully exploit the beneficial biological activity of CO specifically on diseased intestinal tissue while limiting potential negative off-target side-effects, the biodistribution of CORMs and the release kinetics have to be controlled. At least some of these goals were met by the design and synthesis of unique CORMs coupled to a ‘carrier’-prodrug, which allows for bioactive CO release triggered through local, environmental factors to establish a tightly controlled CO release, both temporally and spatially. For this purpose, η4-acyloxy-cyclohexadiene-Fe(CO)3 complexes as enzyme-triggered CORMs (ET-CORMs) were designed, in which the CO-releasing unit is conjugated to the acid moiety of the anti-inflammatory drug mesalazine (mesalamine or 5-aminosalicylic acid (5-ASA)), which functions as a pro-drug carrier. This results in unique compounds herein referred to as esterase-triggered CO-releasing mesalazine (MesaCORMs or MesET-CORMs). The MesaCORMs are cleaved by (intracellular) esterases to give highly labile intermediates which disintegrate already under slightly oxidative conditions to distribute up to 3 molecules of CO. Interestingly, it was found that either slow- or fast- CO-releasing variants could be obtained depending on the site of attachment of the acyloxy-function relative to the double bond position of the Fe(CO)3- complexed cyclohexadiene unit. Accordingly, in one embodiment the invention provides a (MesaCORM) compound representing a carbon monoxide (CO) releasing variant of Mesalazine, the MesaCORM compound having a structure according to Formula Ia or Ib: or a pharmaceutically acceptable salt thereof, wherein: Fe(CO)3 is a tricarbonyl-iron(0) group, attached in a ƞ4coordination mode to all 4 carbon atoms of the 1,3-butadiene unit; n is 0 or 1; L is selected from the group consisting of -O-CH2-X-C(O)- with X = CH2, CH2-CH2, 1,4- phenylene, or C(alkyl)2-CH2; R1, R2, R3, R4, R5, R6and R7are independently selected from the group consisting of H, alkyl (linear or branched), aryl, alkyloxy, aryloxy, and acyloxy, or wherein two or more of R1, R2, R3, R4, R5, R6and R7are connected to form a cyclic or polycyclic structure with an overall ring size of 5 to 20, and wherein each of the alkyl and aryl can be substituted by at least one of alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, hydroxy, amino, alkylamino, arylamino, halogeno, azido, oxo, imino, cyano and sulfanyl. The applicants have thus synthesized Esterase Triggered CO-releasing mesalazine (ET- CORMesa) by coupling mesalazine via an ester bond, optionally involving a linker, to the iron-carbonyl -based complex unit. Exploiting the concept of Esterase-Triggered CO- Releasing Molecules, the Mesalazine-CORM unit is selectively carried to the intestinal tissue, where ubiquitous intestinal esterases will cleave the CORM-unit from the Mesalazine. CO will be produced by the ET-CORM acting together with “liberated” Mesalazine to suppress intestinal inflammation. According to the invention, the ET- CORM is covalently coupled to mesalazine, which functions as a pro-drug carrier, to result in compounds herein referred to as Esterase triggered CO-releasing mesalazine (MesaCORMs or MesET-CORMs). CO-releasing mesalazine derivatives of the present invention are not taught or suggested in the art. Sitnikov et al. (Angew. Chem. Int. Ed. 2015, 54, 12314-12318) reported on the development of protease-triggered CORMs. The viability of the approach was demonstrated through the synthesis of compounds consisting of an η4-oxydiene–Fe(CO)3moiety connected to a penicillin G amidase (PGA)-cleavable unit through a self-immolative linker. In contrast to the present invention, the CORMs of Sitnikov only release CO and do not liberate a second bioactive molecule, let alone mesalazine. Moreover, CO release only occurs in the presence of PGA, which unlike esterase is a specific bacterial protease not present in the human body. US2011 / 0038955 relates to CORMs based on metal carbonyl complexes that are conjugated to NSAID drugs carrying a carboxylate functionality. Whereas mesalazine is mentioned among the possible NSAID in the generic structure, US2011 / 0038955 fails to teach a MesaCORM of the present invention wherein the acyloxy substituent is at either the 1- or 2- position relative to the diene unit. This structural difference is considered a key element in the mechanism of controlled, enzyme triggered CO-release. In contrast, the 5-oxy substitution in the compounds according to US2011 / 0038955 are expected to be very unstable because of a rapid, untriggered decay to a η5-pentadienyl-Fe(CO)3 cation. US 8,927,750 provides acyloxy- and phosphoryloxy-butadiene-Fe(CO)3 complexes which can deliver carbon monoxide to a physiological target, wherein release of carbon monoxide can be enzymatically-triggered. However, it fails to specifically teach or suggest a MesaCORM compound as herein disclosed. Moreover, the synthetic approach in US 8,927,750 is not suitable for the conjugation of mesalazine. In one embodiment, the invention provides a compound wherein the mesalazine moiety is coupled directly (without a Linker) via an ester bond to the iron-carbonyl -based complex unit, i.e. a structure according to Formula Ia or Ib wherein n is 0. Such compounds are suitably used to liberate CO and mesalazine upon exposure to (intestinal) esterase. See scheme 1. Scheme 1: Esterase-induced CO release from MesaCORM wherein mesalazine and CORM are directly connected via an ester bond. In another embodiment, the invention provides a compound wherein the mesalazine moiety is coupled indirectly via a Linker to the iron-carbonyl -based complex unit, i.e. a structure of according to Formula Ia or Ib wherein n is 1. See Scheme 2. are connected via a Linker. The linker L is preferably of the formula -O-CH2-X-C(=O)-, wherein X is (CH2)n, (CH2)n-O, (CH2)n-NH-, (CH2)n-N-alkyl-, wherein n = 1-3), 1,4-phenylene, 1,4-phenylene-O, C(alkyl)2- CH2- or C(alkyl)2-CH2-O-, preferably wherein alkyl is C1-4 alkyl. In one aspect, L is of the formula -O-CH2-CH2-O-C(=O)-. In another aspect, L is of the formula -O-CH2-1,4-phenylene-O-C(=O)- or -O-1,4-phenylene-CH2-O-C(=O)-. In yet another aspect, L is of the formula O-CH2-C(alkyl)2-CH2-O-C(=O)-. In some embodiments, L is of the formula -O-CH2-X-C(O)-, wherein X is CH2, CH2-CH2, 1,4-phenylene, or C(alkyl)2-CH2, preferably wherein alkyl is C1-4 alkyl. In one aspect, L is of the formula -O-CH2- CH2 -C(O)-. In another aspect, L is of the formula O-CH2- C(alkyl)2-CH2-C(O)-. In one aspect, the MesaCORM comprises an indirect trigger unit, also referred to in the art of a self-immolative linker. See scheme 3. According to the invention, substituents R1, R2, R3, R4R5, R6, and R7on the Fe(CO)3 - complexed cyclohexadiene unit are independently selected from the group consisting of H, alkyl (linear or branched), aryl, alkyloxy, aryloxy, and acyloxy, and wherein each of the alkyl and aryl can be substituted by at least one of alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, hydroxy, amino, alkylamino, arylamino, halogeno, azido, oxo, imino, cyano and sulfanyl. Alternatively, two or more of substituents R1, R2, R3, R4R5, R6, and R7are connected to form a cyclic or polycyclic structure with an overall ring size of 5 to 20, for example 5 to 10, or 6 to 7. Exemplary compounds have a structure according to Formula IIa or IIb: In one embodiment, the invention provides a compound wherein substituents R1, R2, R3, R4R5, R6, and R7are independently selected from the group consisting of H and alkyl, preferably H and unsubstituted alkyl. In a specific aspect, at least five, preferably at least six, of substituents R1, R2, R3, R4, R5, R6and R7are H. For example, provided are compounds having the structure IIIa or IIIb Formula IIIa (ANGO564) Formula IIIb (ANGO565) Synthetic methods The invention also relates to methods for the manufacture of a MesaCORM compound herein disclosed and to compounds obtainable thereby. More specifically, it provides a method for providing a compound according to the invention wherein 5-nitro-salicylic acid (1) is used as starting compound. The method comprises (i) esterification of 5-nitro-salicylic acid to attach a sidechain containing the η4-cyclohexadienyl-Fe(CO)3 complex moiety, followed by (ii) reduction of the nitro group to provide the corresponding final Esterase Triggered CO-releasing Mesalazine derivative (Mesa-CORM). Step (i) can be performed either by direct esterification or stepwise by first converting 5-nitro-salicylic acid into an activated carboxylic acid derivative and subsequent reaction with a ƞ4-cyclohexadienol-Fe(CO)3 unit. The required ƞ4-cyclohexadienol-Fe(CO)3 moiety may be provided (generated in situ) from a stable protected precursor molecule in which the dienol OH group is protected, preferentially as a silyl ether, such as a triisopropylsilyloxy (O-TIPS) group. Optionally, a derivative of 5-nitro-salicylic acid (1) in which the phenolic OH group is protected (for instance by a benzyl ether) can be employed, which additional protecting group is then cleaved off at the end of the reaction scheme. For example, the method comprises a synthetic sequence as shown in the following schemes. Suitably, to perform the esterification step (i), pyridine is added to a solution of 1 in THF and the mixture is stirred at room temp. Subsequently, 4-N,N-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) are successively added. In a second flask, the complex rac-2a or rac-2b is added to a suspension of NaH in THF and the mixture is cooled to about 0ºC before adding an agent, such as tetrabutylammonium fluoride (TBAF), to remove the silyl ether protecting group.Then, the mixture containing 1 (first flask) is transferred to the solution of thedeprotected complex (second flask) and the resulting mixture is allowed to react at room temp. for about 12-36 h to yield the nitro-intermediate. The reduction of the nitro-intermediate, step (ii), to provide the corresponding final Esterase Triggered CO-releasing Mesalazine (Mesa-CORM) rac-IIa or rac-IIb is readily achieved by subjecting the η4-acyloxy-cyclohexadiene-Fe(CO)3 complex intermediate (rac- 3a or rac-3b) to catalytic hydrogenation, e.g. using palladium on carbon (Pd / C), preferably using ethyl acetate as a solvent. For example, the reduction is carried out for about 60- 100 h under about 40 bar of an hydrogen atmosphere at room temp. The resulting crude product is suitably isolated and purified by methods known in the art, for example using filtration and column chromatography. A method for synthesis of compounds comprising a linker L may comprise four steps: (i) attaching a moiety comprising the linker to 5-nitro-salicylic acid by esterification, (ii) Methyl ester cleavage, (iii) esterification as described above for the synthesis of the non- linked MesaCORMs, and (iv) reduction. For example, for compounds comprising linker L of the formula -O-CH2-X-C(O)-, the following scheme can be used: Therapeutic applications Further aspects of the invention relate to the therapeutic uses and to pharmaceutical compositions of one or more CO-releasing compounds herein disclosed. In one embodiment, the invention provides a MesaCORM compound as herein disclosed for use as medicament. Mesalazine is the first-line treatment for ulcerative colitis, a chronic inflammatory condition involving the intestines. However, mesalazine treatment is frustrated by the lack or loss of an anti-inflammatory response. Furthermore, mesalazine has no proven anti-inflammatory effects on other organs or even in closely-related Crohn’s disease. Mesalazine is of interest as the drug is well-tolerated, safe for long-term use and side effects are rare, making it an ideal “mother compound” to develop derivative-drugs with a stronger and wider therapeutic spectrum. A CO-releasing mesalazine-derivative as provided in the present invention is capable of releasing therapeutic levels of CO and will thus be more potent than Mesalazine alone to suppress intestinal inflammation. The newly developed class of compounds can improve the response and remission rates of the first line treatment of UC with mesalazine by enhancing the anti-inflammatory effect of mesalazine combined with the anti-inflammatory mechanisms of the gaseous signalling molecule CO. This can lead to a reduction of the disease burden, a burden which is amplified by the side-effects of following treatment options if first line treatment fails. Such a novel compound will therefore not only be more effective in treating ulcerative colitis, but it may also enlarge the therapeutic range of mesalazine, making the drug applicable for, among others, Crohn’s disease, microscopic / collagenous colitis, celiac disease, ischemia reperfusion injury and in intestinal transplantation and other inflammatory diseases of the gut, as well as inflammatory conditions of other organs. Therefore, a pharmaceutical composition is provided comprising one or more compounds according to the invention and a pharmaceutically acceptable excipient. The pharmaceutical composition may be formulated for administering via the oral, intravenous, subcutaneous, transdermal, nasal, inhalatory, intramuscular, intraperitoneal route and / or rectal as in an enema / suppository. The pharmaceutical composition may comprise a carrier, a buffer, a stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere unduly with the efficacy of the inventive complex. The precise nature of the carrier or other material may depend on the route of administration (for example, oral, intravenous, subcutaneous, transdermal, nasal, inhalatory, intramuscular, intraperitoneal and / or rectal as in an enema or suppository). The pharmaceutical composition for oral administration may, for example, be provided in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant or slow-release polymer. In a specific aspect, the MesaCORM is formulated as an oral composition for site-specific delivery in the ileocolonic region. For example, provided is a pH-controlled pulsatile release system (PPRS) comprising a core surrounded by a coating layer, wherein said core comprises at least one MesaCORM, and wherein said coating layer comprises a pH-sensitive enteric coating material wherein a swellable agent is embedded in the form of particles and in an amount which in relation to its particle size and the structure of the coating results in a coating with a non-percolating system. For example, the swellable agent is capable of taking up at least 5 times its weight in water. Preferred swellable agents include sodium starch glycolate and cross-linked carboxymethylcellulose sodium. Such systems are known in the art as ColoPulse technology. The pharmaceutical composition in liquid form can, for example, include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Pharmaceutically acceptable amounts of other solvents may also be included, for example, where they are required for dissolving the particular inventive complex contained in the pharmaceutical composition. For intravenous or subcutaneous injection, or injection of the pharmaceutical composition at the site of affliction, the one or more compound(s) of the invention may be in the form of a parenterally acceptable solution which is pyrogen-free and has a suitable pH, isotonicity and stability. Those skilled in the art will be able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, and Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included as required. Delivery systems for needle-free injection are also known, and compositions for use with such systems may be prepared accordingly. For a pharmaceutical composition intended for delivery by any route including but not limited to oral, intravenous, subcutaneous, transdermal, nasal, inhalatory, intramuscular, intraperitoneal and / or rectal as in an enema or suppository, the compound(s) may be microencapsulated within polymeric spheres such that exposure to body fluids and subsequent carbon monoxide release is delayed. The composition may further comprise at least one additional (anti-inflammatory) active ingredient, preferably selected from the group consisting of NSAIDs, steroids such as budesonideor a bile acid such as Ursodeoxycholic acid (UDCA). In a specific aspect, the invention provides a pharmaceutical composition comprising compound IIIa (ANGO564), IIIb (ANGO565), or an admixture thereof. An admixture of a slow and a fast CO-releasing compound is particularly preferred since it allows for rapid release of a high dose in a specific area (e.g. terminal ileum) of the gut and a slower release of a lower dose in a larger area (e.g. ileocolonic) of the gut. Provided is a MesaCORM compound as herein disclosed for use in a method for treating an inflammatory disease or condition, the method comprising administering to a subject an effective amount of one or more of said compounds, preferably wherein the disease or condition is selected from the group consisting of an inflammatory bowel disease such as ulcerative colitis or Crohn's disease, microscopic / collagenous colitis, celiac disease, ischemia reperfusion injury and in intestinal transplantation. In another aspect, the invention provides a method for delivering carbon monoxide to a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition as defined above. The subject is preferably a mammalian subject, more preferably a human subject. In an alternative embodiment, the method includes providing a pharmaceutical composition for delivery of carbon monoxide to a physiological target comprising as an active ingredient at least one of the MesaCORM as defined above, administering the pharmaceutical composition to an area of the subject requiring treatment, and triggering a release of carbon monoxide by hydrolysis in the area so as to treat the subject. Also provided is a method for treating an inflammatory disease or condition, the method comprising administering to a subject having said disease or condition an effective amount of a compound according to the invention. Preferably, the disease or condition is selected from the group consisting of an inflammatory bowel disease such as ulcerative colitis or Crohn's disease, microscopic / collagenous colitis, celiac disease, ischemia reperfusion injury and in intestinal transplantation. LEGEND TO THE FIGURES Figure 1: (A)1H-NMR (500 MHz, CDCl3) and (B)13C-NMR analysis (125 MHz, CDCl3) of representative MesaCORM compound ANGO564 (rac-IIIa). Figure 2: (A)1H-NMR (500 MHz, CDCl3) and (B)13C-NMR analysis (125 MHz, CDCl3) of representative MesaCORM compound ANGO565 (rac-IIIb). Figure 3: In vitro CO release profiles of slow CO releasing MesaCORM ANGO564 (♦) and fast CO releasing MesaCORM ANGO565 (▲) induced by pig liver esterase (PLE) inphosphate buffer (0.1 M; pH 7.4) / DMSO=5 : 1 as detected by headspace GC.Figure 4: In vitro toxicity tests using DLD-1 cells (human intestinal cell line) demonstrating that representative MesaCORMs are non-toxic up to concentrations of to 50 μM as evidenced by SYTOX® green staining for detecting necrosis. Figure 5: In vitro toxicity tests using DLD-1 cells demonstrating that representative MesaCORMs are non-toxic up to concentrations of up to at least 50 μM (8 and 12 hr exposure) as evidenced by measuring caspase-3 activity for detecting apoptosis. Anti-FAS was used as positive control. Figure 6: MesaCORMs dose-dependently (0.5-50 µM) induce HO-1 expression (as indicator of in vivo CO release) in intestinal DLD-1 cells under control conditions and under cytokine treatment to mimic inflammation. Non-esterase-triggered CORM3 (Ru(CO)3Cl-glycinate), inactive CORM3 (which does not release CO, unconjugated CORMs (ANGO210 and ANGO460) and Mesalazin were included as comparative compounds. For details, see Example 3. Figure 7: MesaCORMs dose-dependently (0.5-50 µM) reduce cytokine-induced iNOS expression (marker of inflammation) in intestinal DLD-1 cells under control conditions and under cytokine treatment to mimic inflammation. Non-esterase-triggered CORM3 (Ru(CO)3Cl-glycinate), inactive CORM3 (which does not release CO, unconjugated CORMs (ANGO210 and ANGO460) and Mesalazin were included as comparative compounds. For details, see Example 3. Figure 8: Fast CO-releasing MesaCORM (ANGO565) shows a strong induction of CO release (evidenced by HO-1 gene expression) in human intestinal organoids. EXPERIMENTAL SECTION Example 1: Synthesis of slow and fast releasing MesaCORMs SYNTHESIS of ANGO564 (rac-IIIa) All operations were performed under exclusion of air using an atmosphere of argon. 5- nitro-salicylic acid (1) was purchased from a commercial supplier, and the TIPS- protected cyclohexadienol-Fe(CO)3 complexes rac-2a’ and rac-2b’ were prepared aspreviously reported (see: S. Romanski et al. Dalton Trans., 2012, 41, 13862). To a solution of 1 (281 mg, 1.53 mmol, 2.4 eq.) in THF (3.3 mL) were added DMAP (3.0 mg, 0.03 mmol, 0.05 eq.) and pyridine (0.37 mL, 4.58 mmol, 7.2 eq.). A yellow solid precipitated which dissolved upon addition of TMSCl (0.40 mL, 3.14 mmol, 4.9 eq.). In a second flask, NaH (75 mg, 60% in paraffin, 1.91 mmol, 3.0 eq.) was suspended in THF (4.0 mL). Complex rac-2a’ (250 mg, 0.64 mmol, 1.0 eq.) was added and the mixture was cooled to 0 °C before TBAF (1.0 M in THF, 0.75 mL, 0.75 mmol, 1.2 eq.) was added. The mixture was stirred for 15 min at 0 °C before pyridine (0.10 ml, 1.24 mmol, 2.0 eq) was added dropwise. Subsequently, DMAP (39 mg, 0.32 mmol, 0.5 eq.) and EDC·HCl (185 mg, 0.97 mmol, 1.5 eq.) were added successively. The mixture of 1 in THF (flask) was added to the deprotected complex and the resulting mixture was stirred for 24 h at room temp. The solvent was removed under reduced pressure and the crude product (rac-3a’) was purified by filtration through a short pad of silica (SiO2, CyHex / EtOAc = 10:1). Subsequently, rac-3a’ was immediately dissolved in EtOAc (7.5 ml) and Pd / C (20 w%, 42 mg) was added. The mixture was stirred for 92 h under 40 bar of an hydrogen atmosphere at r.t. The crude product was filtered over a short pad of celite with EtOAc to removed residues of Pd / C. Then, the product rac-IIIa (90 mg, 0.24 mmol, 38%) was obtained by column chromatography (SiO2, CyHex / EtOAc = 5:1) as a yellow solid. 1H-NMR: (500 MHz, CDCl3) δ = 9.83 (s, 1H, OH), 7.12 (d, J = 2.9 Hz, 1H, H-7), 6.94 (dd, J = 8.8, 2.9 Hz, 1H, H-5), 6.87 (d, J = 8.8 Hz, 1H, H-4), 5.62 (dd, J = 6.7, 2.1 Hz, 1H, H-6’), 3.57 – 3.37 (m, 3H, NH2, H-2’), 2.93 (dt, J = 6.3, 3.0 Hz, 1H, H-5’),1.94 – 1.86 (m, 1H, H-3’a), 1.86 – 1.78 (m, 1H, H-3’b), 1.67 – 1.59 (m, 2H, H-4’).13C-NMR: (125 MHz, CDCl3): δ = 210.7 (Fe(CO)3), 169.7 (C-1), 155.6 (C-3), 138.9 (C-6), 128.5 (C-1’), 125.6 (C-5), 118.7 (C-4), 114.5 (C-7), 111.1 (C-2), 80.3 (C-6’), 59.4 (C-2’), 52.3 (C-5’), 24.8 (C-3’), 23.7 (C-4’). HR-MS (ESI): Calc.: [M+H]+= 372.0171 amu, Obs. = 372.0208 amu. FT-IR: (ATR) ῦ [cm-1] = 3451 (w), 3366 (w), 3266 (w), 3015 (w), 2933 (w), 2855 (w), 2044 (s), 1959 (s), 1687 (m), 1628 (m), 1592 (w), 1490 (m), 1457 (m), 1430 (w), 1381 (w), 1343 (m), 1300 (m), 1266 (w), 1219 (m), 1195 (m), 1165 (s), 1119 (m), 1075 (m), 1053 (m), 1023 (w), 995 (w), 924 (w), 874 (w), 829 (m), 780 (m), 761 (w), 738 (w), 703 (w), 657 (m), 609 (m), 574 (m), 515 (m).

[0002] SYNTHESIS of ANGO565 (rac-IIIb) To a solution of 1 (187 mg, 1.02 mmol, 2.0 eq.) in THF (2.4 mL) was added pyridine (0.25 mL, 3.06 mmol, 6.0 eq.) and the mixture was stirred for 30 min at r.t. Subsequently, DMAP (20 mg, 0.15 mmol, 0.3 eq.) and EDC·HCl (149 mg, 0.77 mmol, 1.5 eq.) were added successively. In a second flask, NaH (60 mg, 60% in paraffin, 1.53 mmol, 1.5 eq.) was suspended in THF (2.4 mL). Complex rac-2b’ (199 mg, 0.51 mmol, 1.0 eq.) was added and the mixture was cooled to 0 °C before TBAF (1.0 M in THF, 0.60 mL, 0.60 mmol, 1.2 eq.) was added. The mixture was stirred for 5 min at 0 °C. The mixture of 1 in THF (flask 1) was added to the solution of the deprotected complex and the mixture was stirred for 4 h at room temp. The solvent was removed under reduced pressure and the crude product (rac-3b’) was purified by filtration through a short pad of silica (SiO2, CyHex / EtOAc = 10:1). Subsequently, rac-3b’ was immediately dissolved in EtOAc (4.5 mL) and Pd / C (10 w%, 47 mg) was added. The mixture was stirred for 48 h under 40 bar of an hydrogen atmosphere at room temp. The crude product was filtered over a short pad of celite with EtOAc to removed residues of Pd / C. Then, the product rac-IIIb (90 mg, 0.24 mmol, 47%) was obtained by column chromatography (SiO2, CyHex / EtOAc = 5:1) as a yellow solid. M(C16H13FeNO6): 373.13 g / mol TLC: Rf (CyHex / EtOAc = 5:1) = 0.11 1H-NMR: (500 MHz, CDCl3) δ = 10.10 (s, 1H, OH), 7.13 (d, J = 2.8 Hz, 1H, H7), 6.89 (dd, J = 8.8, 2.8 Hz, 1H, H-5), 6.83 (d, J = 8.7 Hz, 1H, H4), 5.52 (dq, J = 4.4, 1.1 Hz, 1H, H-2’), 5.18 (ddd, J = 5.9, 4.6, 0.9 Hz, 1H, H-3’), 3.45 (s, 2H, NH2), 3.27 – 3.03 (m, 1H, H-4’), 2.30 – 2.14 (m, 1H, H-6’), 2.03 – 1.86 (m, 2H, H-5’), 1.79 – 1.69 (m, 1H, H-6’). 13C-NMR: (125 MHz, CDCl3): δ = 211.1 (Fe(CO)3), 168.6 (C-1), 155.3 (C- 3), 138.5 (C-6), 124.9 (C-5), 118.5 (C-4), 114.7 (C-7), 112.1 (C- 2), 103.6 (C-1’), 81.1 (C-3’), 80.4 (C-2’), 60.8 (C-4’), 27.2 (C-6’), 24.4 (C-5’). HR-MS (ESI): calc.: [M+H]+= 372.0171 amu, found: = 372.0209 amu. FT-IR: (ATR) ῦ [cm-1] = 3446 (w), 3364 (w), 3225 (w), 3070 (w), 3032 (w), 2939 (w), 2858 (w), 2045 (s), 1957 (s), 1677 (m), 1625 (m), 1596 (w), 1490 (m), 1382 (w), 1343 (w), 1328 (w), 1300 (m), 1220 (m), 1195 (m), 1135 (m), 1109 (m), 1069 (m), 1056 (w), 1030 (w), 1003 (m), 977 (w), 902 (m), 866 (w), 828 (m), 785 (m), 738 (w), 687 (w), 640 (w), 610 (s), 559 (m), 519 (m). Example 2: In vitro CO release. In this example, exemplary Mesa-CORMs (ANGO564 and ANGO565) were analyzed for their CO-releasing properties using previously described methods. The headspace GC measurements were performed on a Thermo Scientific Trace 1300 headspace gas chromatograph with a TriPlus RSH autosampler. A thermal conductivity detector (TCD) and a Shincarbon ST 100 / 1201.0 mm × 2 m 1 / 16” OD silico column were used. The data was analyzed using Chromeleon® 7 software. Helium was used as the carrier gas at a flow rate of 15 ml / min. The inlet temperature and the detector temperature were 200 °C. The injection volume was 50 μl. The parameters of the standard temperature program flow15_split10_10min used are: 0-2.5 min 35 °C, then 70 °C at 20 °C / min, then 1 min at 70 °C, then 35 °C at 20 °C / min, then 1 min at 35 °C. To record the calibration curve, eight headspace vials (BGB Analytics, cat. No.200410-F, 10 ml) were filled with 0.2 ml DMSO and 1.0 ml phosphate buffer (pH = 7.4). The vials were sealed with gas-tight silicone / PTFE septa with aluminum crimp caps (BGB Analytics, cat.No.20030500) and degassed with argon for 10 min. Subsequently, a defined amount of argon was replaced by CO (0.00 ml, 0.05 ml, 0.10 ml, 0.25 ml, 0.50 ml, 1.00 ml, 1.50 ml, 2.00 ml). After equilibration of the vials for 10 min at 37 °C, the composition of the gas space was determined via headspace GC. The measurement was repeated three times and a calibration curve was generated from the values. In the case of ANGO564, CO release was observed within minutes which plateauedat about 1.7 equiv. after 14 h. For ANGO565, a slow and sustained CO release wasobserved up to nearly 2.5 equiv. after 100 h (Figure 3). Satisfactorily, both compounds showed no spontaneous CO release in the absence of PLE. Example 3: In vitro toxicity tests. In vitro necrosis and apoptosis assays were performed on intestinal DLD-1 cells to evaluate a possible cytotoxic effect of the MesaCORMs. Necrosis assay Necrosis was assessed using SYTOX® green, which is a nucleic acid stain and shows green-fluorescent when binding to nucleic acids. It is impermeant to live cells but penetrates the compromised membranes of dead cells, indicating the dead / necrotic cells in a population. -DLD-1 cells (human intestinal cell line) were plated on a separate 12-wellplate for each compound.- The tested compounds were mesalazine, unconjugated first generation ET-CORMs (ANGO210 and ANGO460), slow-releasing MesaCORM (ANGO564), fast-releasing MesaCORM (ANGO565) and H2O2. -Mesalazine and CORMs were dissolved in DMSO. On the plate with theDLD-1 cells, this mixture was diluted in a 1:1000 ratio until a DMSO level of <0.1% was reached. - SYTOX® green was diluted in a 1:40.000 ratio. First it was diluted in a 1:400 ratio in a medium (1µL sytox + 399 µL medium). After this, it was further diluted in a 1:200 ratio in the 12-well plate (3µL sytox in 600µL medium). The experiment was performed in duplicate with the following concentrations of the compound per plate. For each plate, 0.1% DMSO was used as a control to confirm that the necrosis was induced by the compound and not by the DMSO dilution. Mesalazine ANGO210 ANGO460 ANGO564 ANGO565 H2O20.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 2 µM1 µM 1 µM 1 µM 1 µM 1 µM 5 µM10 µM 10 µM 10 µM 10 µM 10 µM 10 µM20 µM 20 µM 20 µM 20 µM 20 µM50 µM 50 µM 50 µM 50 µM 50 µM100 µM 100 µM 100 µM 100 µM 100 µMThe results shown in Figure 4 demonstrate that the DMSO 0.1% solvent control, and Mesalazine and ANGO210 do not induce necrosis at any concentration tested. ANGO460, ANGO564 and ANGO565 only cause necrosis at the highest concentration of 100 µM. In conclusion, this experiment shows that both MesaCORMs (ANGO564 and ANGO565) are non-toxic (regarding induction of necrosis) up to concentrations of at least 50 µM for DLD-1 cells. Apoptosis Apoptosis was assessed by detecting caspase-3 activation using synthetic tetrapeptide fluorogenic Caspase-3 substrate Ac-DEVD-AMC after a 8h and 12 h incubation with test compound. Caspase-3 cleaves the tetrapeptide between D and AMC, thus releasing the fluorogenic AMC, which can be quantified in a spectrofluorometer at an excitation wavelength of 380 nm and an emission wavelength range of 430-460 nm. An anti-Fas (CD95) antibody was used as positive control. -DLD-1 cells (human intestinal cell line) were plated at a density of about70-80% confluency on a separate 12-well plate for each compound. -The tested compounds were mesalazine, unconjugated first generation ET-CORMs (ANGO210 and ANGO460), slow-releasing MesaCORM (ANGO564) and fast-releasing MesaCORM (ANGO565) / Each test compound was used at 1, 10, 20, 50 and 100 µM final concentration. -0.1% DMSO was used as solvent control.- Anti-FAS ( Beckman Coulter IM2387; used at 1:200 dilution) was used aspositive control. The results in Figure 5 show that 0.1% DMSO does not induce apoptosis. Mesalazine also shows to be non-toxic up to 100 µM. As expected, Anti-Fas induces apoptosis. ANGO210 and ANGO460 do not induce apoptosis up to concentrations of 100 µM. ANGO564 and ANGO565 do not induce apoptosis up to concentrations of 50 µM. At 100 µM, both ANGO564 and ANGO565 show toxicity in the form of apoptosis. This is consistent with the data from the sytox green experiments, which show necrotic toxicity at the 100 µM concentrations. In conclusion, this experiment shows that both CORMesalazines (ANGO564 and ANGO565) are non-toxic (regarding induction of apoptosis) up to concentrations of at least 50 µM. Example 4: Dose-dependent regulation of gene expression. This example demonstrates that exemplary MesaCORMs can regulate the expression of marker genes HO-1 (indicator of in vivo CO release) and iNOS (as marker for inflammation) in intestinal DLD-1 cells under control conditions and under cytokine treatment (mimics inflammation). Materials and Methods DLD-1 cells (human intestinal cell line) were plated on separate 12-well plates for each test compound. The tested compounds were CORM-3, inactive CORM-3 (which does not release carbon monoxide), mesalazine, ANGO210, ANGO460, slow-releasing MesaCORM (ANGO564) and fast-releasing MesaCORM (ANGO565). DMSO (0.1%) was used as solvent control. To mimic an inflammatory state, the DLD-1 cells were exposed to a cytokine mix (CM) comprising IL-1β, TNF-α and INFγ (at 10 ng / ml final concentration each). The concentrations of the compounds on the plates were as follows: CORM-3 Mesalazine ANGO210 ANGO460 ANGO564 ANGO565 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO 0.1% DMSO CM CM CM CM CM CM CORM3 0.5 µM 0.5 µM 0.5 µM 0.5 µM 0.5 µM 100µM CORM3 0.5 µM + CM 0.5 µM + CM 0.5 µM + CM 0.5 µM + CM 0.5 µM + CM 100µM + CM iCORM3 5 µM 5 µM 5 µM 5 µM 5 µM 100µM iCORM3 5 µM + CM 5 µM + CM 5 µM + CM 5 µM + CM 5 µM + CM 100µM + CM 50 µM 50 µM 50 µM 50 µM 50 µM 50 µM + CM 50 µM + CM 50 µM + CM 50 µM + CM 50 µM + CM The experiment was performed with a 16h incubation time. A qRT-PCR analysis was performed for gene expression of HO-1 (as indicator of in vivo CO release) and NOS (as marker for inflammation). The results are shown in Figures 6 and 7. HO-1 expression: Figure 6 shows that 0.1% DMSO and CM alone do not induce HO-1 expression. CORM-3 on its own induces HO-1 but this effect greatly diminishes when combined with the cytokine mix. Mesalazine on its own does not seem to induce HO-1 expression. ANGO210 only induces HO-1 expression in concentrations of 50 µM, this effect persists when combined with the cytokine mix. The same was observed for ANGO460, with induction of HO-1 expression only in concentrations of 50 µM. The slow releasing MesaCORM ANGO564 dose-dependently induces HO-1 expression. The fast releasing MesaCORM ANGO565 also dose-dependently induces HO-1 expression, with an even greater effect than the other compounds in the 50 µM concentration (up to a 15 fold change gene expression). NOS expression: Figure 7 shows that both MesaCORMs show dose-dependent reduction in cytokine- induced iNOS expression. The fast releasing ANGO565 shows greater reduction of iNOS expression than the slow releasing ANGO564. ANGO210 and ANGO460 also show a dose-dependent reduction in iNOS expression. Mesalazine only shows some reduction in iNOS expression in concentrations of 50 µM. CORM3 also shows some reduction in iNOS expression while inactive CORM3 does not show any reduction at all (as expected). In conclusion, both MesaCORMs show a dose-dependent reduction in cytokine induced iNOS expression. Example 5: Suppression of cytokine-induced damage in human organoids. This example demonstrates that MesaCORMs of the invention can suppress cytokine-induced damage to primary human intestinal organoids. Intestinal crypts were isolated from resection material / endoscopic biopsies. These crypts were put in EM+ medium (organoid medium.). The stem cells in these crypts will grow out to organoids, whereas the rest of the crypt will die and are washed away in time. When the organoids are growing well and the dead cells from the crypts are washed away, the organoids were used for experiments. The different MesaCORMs were tested on human colonic organoids plated on a 96- well plate. To mimic an inflammatory state, the organoids were exposed to a cytokine mix (CM) consisting of IL-1β, TNF-α and INF-γ (at 5 nm each). The organoids were then treated for 16 hours with the different compounds: 0.1% DMSO as control, CORM3, inactive CORM 3, mesalazine, ANGO460 and the fast releasing MesaCORM ANGO565. There were no visible signs of cytotoxic or apoptotic effects on the organoids (data not shown). A qRT-PCR analysis was performed for gene expression of HO-1 (normalized to 18S rRNA) as indicator of in vivo CO release. Figure 8 demonstrates that the fast releasing ANGO565 induces HO-1 mRNA expression indicative of in vivo CO release in human intestinal organoids much stronger than CORM3, mesalazine or ANGO460 separately.

Claims

Claims1. A compound representing a carbon monoxide (CO) releasing variant ofMesalazine (MesaCORM), the compound having a structure according to Formula Ia or Ib:or a pharmaceutically acceptable salt thereof, wherein: n is 0 or 1; Fe(CO)3 is a tricarbonyl-iron(0) group, attached in a ^4coordination mode to all 4 carbon atoms of the 1,3-butadiene unit; L is -O-CH2-X-C(O)- wherein X = CH2, CH2-CH21,4-phenylene, or C(alkyl)2- CH2; R1, R2, R3, R4,R5, R6and R7are independently selected from the group consisting of H, alkyl (linear or branched), aryl, alkyloxy, aryloxy, and acyloxy, or wherein two or more of R1, R2, R3, R4, R5, R6and R7are connected to form a cyclic or polycyclic structure with an overall ring size of 5 to 20, and wherein each of the alkyl and aryl can be substituted by at least one of alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, hydroxy, amino, alkylamino, arylamino, halogeno, azido, oxo, imino, cyano and sulfanyl.

2. The compound of claim 1, wherein R1, R2, R3, R4, R5, R6 and R7 areindependently selected from the group consisting of H and alkyl.

3. The compound of claim 1 or 2, wherein at least three of R1, R2, R3, R4,R5, R6and R7are H.

4. The compound according to any one of the preceding claims, wherein nis zero.

5. The compound of claim 1, having a structure according to Formula IIaor IIb:

6. The compound according to claim 5, having the structure IIIa or IIIb7. A pharmaceutical composition comprising one or more compoundsaccording to any one of claims 1-6 and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, furthercomprising at least one additional anti-inflammatory active ingredient.

9. The pharmaceutical composition according to claim 7 or 8, comprisingcompound IIIa (ANGO564), IIIb (ANGO565), or an admixture thereof.

10. A compound according to any one of claims 1-6 for use as medicament.

11. A compound according to any one of claims 1-6 for use in a method fortreating an inflammatory disease or condition, the method comprising administering to a subject an effective amount of one or more of said compounds.

12. The compound for use according to claim 11, wherein the disease orcondition is selected from the group consisting of an inflammatory bowel disease such as ulcerative colitis or Crohn's disease, microscopic / collagenous colitis, celiac disease and ischemia reperfusion injury in intestinal transplantation.

13. A method for delivering carbon monoxide to a subject in need thereof,the method comprising administering to the subject a pharmaceutical composition according to any one of claims 7-9.

14. A method for providing a compound according to any one of claims 1-6,comprising (i) esterification of 5-nitro-salicylic acid to either directly or stepwise attacha sidechain containing the η4-cyclohexadienyl-Fe(CO)3 complex moiety, followed by (ii) reduction of the nitro group to provide the corresponding final EsteraseTriggered CO-releasing Mesalazine (Mesa-CORM).

15. The method according to claim 14, comprising performing one or morereactions according to the following reaction scheme16. The method according to claim 14 or 15, wherein the reduction stepcomprises subjecting the η4-acyloxy-cyclohexadiene-Fe(CO)3 complex to catalytic hydrogenation with palladium on carbon (Pd / C), preferably using ethyl acetate as a solvent.

17. A method for treating an inflammatory disease or condition, the methodcomprising administering to a subject having said disease or condition an effective amount of a compound according to any one of claims 1-6.

18. The method according to claim 17, wherein the disease or condition isselected from the group consisting of an inflammatory bowel disease such as ulcerative colitis or Crohn's disease, microscopic / collagenous colitis, celiac disease and ischemia reperfusion injury in intestinal transplantation.

19. The method according to claim 17 or 18, comprising administering apharmaceutical composition comprising compound IIIa (ANGO564), IIIb (ANGO565), or an admixture thereof.

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