ItaCORMs: conjugation with a CO-releasing unit greatly enhances the anti-inflammatory activity of itaconates

Endogenous itaconate as well as the gasotransmitter CO have recently been described as powerful anti-inflammatory and immunomodulating agents. However, each of the two agents comes along with a major drawback: Whereas itaconates only exert beneficial effects at high concentrations above 100 μM, the uncontrolled application of CO has strong toxic effects. To solve these problems, we designed hybrid prodrugs, i.e. itaconates that are conjugated with an esterase-triggered CO-releasing acyloxycyclohexadiene–Fe(CO)3 unit (ItaCORMs). Here, we describe the synthesis of different ItaCORMs and demonstrate their anti-inflammatory potency in cellular assays of primary murine immune cells in the low μmolar range (<10 μM). Thus, ItaCORMs represent a promising new class of hybrid compounds with high clinical potential as anti-inflammatory agents.


Inert gas
Unless otherwise stated, all reactions were run under an inert atmosphere using Schlenk technique and argon 4.6 of the company Linde (99.996%, <1 ppm H2O, <1 ppm O2). Before being used glassware was evacuated, heated out with a heat gun and then flushed with argon three times.

Solvents and reagents
Unless otherwise stated all chemicals and solvents were purchased from commercial suppliers (Acros, Alfa-Aesar, Carbolution, Merck, Rockwood-Lithium, Sigma-Aldrich, TCI and VWR) and were used without further purification. Diisopropylamine, hexamethyldisalazane and 2-cyclohexenone were distilled and stored under argon atmosphere. The concentration of n-butyllithium was determined by titration. 1 Non-absolute solvents were distilled before use.
Absolute THF and toluene were obtained by distillation under argon atmosphere over sodium with benzophenone used as indicator. CH2Cl2 was dried over CaCl2 and distilled on the day of use. Other solvents were used without further purification unless otherwise noted.

Removal of solvents
Solvents were removed under reduced pressure using a rotary evaporator of the company Büchi and a membrane pump. The temperature of the water bath was set to 40 °C. Residual solvents were then removed under oil pump vacuum.

Nuclear magnetic resonance
NMR spectra ( 1 H and 13 C) were recorded on the devices Avance II 300 ( 1 H NMR: 300 MHz, 13 C NMR: 75 MHz), Avance 400 ( 1 H NMR: 400 MHz, 13 C NMR: 100 MHz), Avance III 499 ( 1 H NMR: 500 MHz) and Avance AVIII-HD 500 ( 1 H NMR: 500 MHz, 13 C NMR: 125 MHz) of the company Bruker. Proton as well as carbon chemical shifts δ are reported in ppm (parts per million) relative to tetramethylsilane. All spectra were measured at room temperature. The multiplicity of signals is described with "s" for singlet, "d" for doublet, "t" for triplet as well as "m" for multiplet and "br" for broad signal. Coupling constants are given in Hertz. The assignment of signals was done using suitable 2D-NMR spectra (COSY, HMQC/HSQC, HMBC, NOESY).

Synthesis of ItaCORM 1a
In

Synthesis of ItaCORM 2b
In
The solvent was removed under reduced pressure and the crude product was purified by column chromatography on SiO2 (Cyhex/EtOAc; 6:1). The desired product ItaCORM 4b was

1.4
In situ quantification of CO-release

Headspace-GC-system
For the CO-release quantification a Thermo Scientific Trace 1300 headspace gas chromatograph equipped with a TriPlus RSH autosampler was used. The detector was a thermal conductivity detector (TCD). The system was equipped with a Shin carbon ST 10/120 1.0 mm x 2 m 1/16'' OD silico column. For the programming of the system Chromeleon ® 7 Data System Software was used.

Gas-chromatography conditions for GC-Headspace system
Helium was used as carrier gas with a flow of 15 mL/min. A defined gas volume of the vial was substituted for 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). The vials were then equilibrated at 37 °C for 10 min and the composition of the gas phase was subsequently determined by headspace GC analysis.
The respective measurements were repeated three times and a calibration curve was then generated.

In situ quantification of the CO-release of ItaCORMs
The respective complex (36 µmol) was dissolved in DMSO (0.      1.6 Crystal data and structure refinement

Animals
For the experiments performed, primary dendritic cells were isolated from C57BL/6 mice aged 8 weeks or older. The animals were bred and held in the facilities of the Eberhard Karls University Tübingen under specific pathogen free conditions. Mice were handled in accordance with the guidelines of the local authorities (Regierungspräsidium Tübingen, Germany).

Isolation of primary dendritic cells
The isolation of bone marrow derived dendritic cells (BMDCs) was performed according to a protocol of Madaan et al.. 4 Initially, the bone marrow was isolated from femurs and tibias of C57BL/6 mice. After erythrocyte lysis, the cell suspension was plated with 2x10 6 cells/ml on petri dishes (non-tissue culture grade). The culture medium consisted of DMEM supplemented with 10 % fetal calf serum, 1 % nonessential amino acids, 1 % sodium pyruvate, 1 % HEPES, 1 % antibiotics, 25 µM ßmercaptoethanol and 20 ng/ml recombinant granulocyte-macrophage colonystimulating factor (GM-CSF). On day 3 and day 6 after plating of the cells, replenishment of culture medium was conducted by addition of fresh medium.
Immature dendritic cells were harvested on day 7 by careful collection of non-adherent cells. Finally, immature BMDCs were plated for subsequent in vitro experiments with 1x10 6 cells/ml.

Viability
BMDCs were treated with 1 µg/ml LPS for 24 h and increasing concentrations of ItaCORMs. Then, cell viability was determined using two different methods: (i) the Cell Proliferation Kit II (XTT) from Merck KGaA and (ii) the Pierce LDH Cytotoxicity Assay Kit from Thermo Fisher Scientific. Analyses were performed as described in the manufacturer's protocols.

Western blot
For the analyses of the HO-1 and STAT1 signaling pathways, BMDCs were stimulated with 1 µg/ml LPS and ItaCORMs or their corresponding reference substances for 2 h.
Subsequently, Western blotting was performed as previously described in detail. 5 In the present study, α-tubulin (1:5000; Novus Biologicals) was applied as loading control.

Analyses of cytokines
For the analyses of inflammatory cytokine levels, BMDCs were treated with 1 µg/ml LPS and ItaCORMs or their respective reference substances. After 24 h of stimulation, the cell culture supernatant was collected. Measurements of IL-12p70 and IL-23 concentrations were performed using the commercially available kits DuoSet ELISA Mouse IL-12p70, DuoSet ELISA Mouse IL-23 in combination with the DuoSet Ancillary Reagent Kit 2 from R&D Systems. The whole procedure was performed as described in the manufacturer's protocol.