Novel aminopyridazine derivative of minaprine modified by radiolysis presents potent anti-inflammatory effects in LPS-stimulated RAW 264.7 and DH82 macrophage cells

Radiation molecularly transforms naturally occurring products by inducing the methoxylation, hydroxylation, and alkylation of parent compounds, thereby affecting the anti-inflammatory capacities of those compounds. Minaprine (1) modified by ionizing radiation generated the novel hydroxymethylation hydropyridazine (2), and its chemical structure was determined based on NMR and HRESIMS spectra. Compared to the original minaprine, the novel generated product showed a highly enhanced anti-inflammatory capacity inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 and DH82 macrophage cells. In addition, minaprinol (2) effectively inhibited cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) at the protein level and pro-inflammatory cytokine (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10) production in macrophages.

Inflammation occur in response to injury and involves localized accumulations of body fluids, plasma proteins, and white blood cells associated with immune system action or physical injury and infection 1,2 . Inflammatory responses lead to the excessive production of inflammatory mediators such as pro-inflammatory nitric oxide (NO), prostaglandin E 2 (PGE 2 ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), or the inhibition of secretions by anti-inflammatory cytokines such as IL-10 and so on [3][4][5] . The formation of inflammatory mediators plays a major role in mediating inflammation based on their ability to convert arachidonic acid into leukotriene, thromboxane, and prostaglandins through the reaction of cyclooxygenase-2 (COX-2) 6 . In addition, NO production is regulated by inducible NO synthase (iNOS) 7 . The excessive inflammation can lead to a variety of chronic inflammation-based diseases, such as diabetes, cancer, arthritis, and cardiovascular disease 8 . These inflammatory diseases are fatal for humans as well as companion animals. Although research on drugs for inflammatory diseases in humans has been conducted, the development of similar prescription drugs for companion animals is limited. As the public adoption of companion animals has increased worldwide amidst the COVID-19 pandemic 9 , the related industries are also growing rapidly; therefore, additional prescription drugs must be developed for companion animals.
Gamma irradiation has received wide attention due to its potential to decrease the toxicity of alkaloids, steroids, and flavonoids and increase the biological capacities of these compounds 10 . Radiation transformation technology (RTT) for the development of candidate drugs has advantages of being simple, rapid, low cost, and environmentally friendly compared to organic solvent-based chemical synthesis 11 . Recently, the anti-depressant drug nomifensine (Merital®) was reported to effectively improve the anti-cancer properties in breast cancer cells after exposed to ionizing radiation 12 . Radiation induces the molecular transformation of natural products by inducing the methoxylation, hydroxylation, and alkylation of mother compounds 13,14 . However, studies on the structural modification of alkaloid-based drugs using γ-irradiation technology are still very limited. Thus, the www.nature.com/scientificreports/ exact chemical structure and anti-inflammatory potential of radiolytic molecules in aminopyridazine derivatives must be evaluated and better understood to promote their use in the treatment of inflammatory diseases. The monoamine oxidase inhibitor, minaprine (Brantur®) is used as an anti-depressant and shows inhibitory effects on acetylcholinesterase in vivo; however, it was withdrawn from the anti-depressant drug market in 1996 because it causes convulsions 14,15 . Recently, a new class of aminopyridazine derivatives exhibiting various biological activities, such as anti-cancer, anti-diabetic, and cardiovascular activities, have been developed, and their value as functional materials has been demonstrated 16 . As part of our continuing search for novel drugs for humans and companion animals, we herein report the radiolytic modification of minaprine into a novel hydroxymethylated hydropyridazine derivative (2) that exhibits significantly enhanced anti-inflammatory properties compared to the original minaprine.

Result and discussion
Determination and isolation of newly generated product. Ionizing radiation was applied as previously described. A sample solution containing pure minaprine (400 mg) in methanol (400 mL) was directly irradiated with gamma-rays, and the transformation pattern was analyzed using a reverse-phases HPLC apparatus. After irradiation with a dose of 30 kGy, minaprine (1) was nearly reduced and the newly generated peak at a t R 6.2 min corresponded to a conversion rate of 95% (Fig. 1A,B ). The reactants irradiated at a dose of 30 kGy showed enhanced inhibitory effects on NO production in lipopolysaccharide (LPS)-stimulated RAW 264.7 and DH82 macrophages compared with pure minaprine (Fig. 1C,D). Repeated column chromatographic separation of the 30 kGy-irradiated reactants led to the isolation and purification of the novel hydropyridzine derivative 2.
The new hydroxyalkylated product 2 was found to contain rare functional groups with a nitrogen moiety at the aminopyridazine of minaprine ( Fig. 2A).
Radiolysis of methanolic conditions in the presence of an atmosphere produces various free radicals, such as hydroxymethyl ( · CH 2 OH), hydroxyl ( • OH), methoxy (CH 3 O · ), and peroxyl (HOO · ), which are subsequently green-synthesis of new drug candidates 19,20 . Our results suggest that the hydroxymethyl ( · CH 2 OH) radical may also be involved in the modification of compound 2 when minaprine is exposed to ionizing radiation in methanolic solution. This is the first example of the novel radiolytic hydroxymethylation of the amionpyridazine drugs.

Inhibitory effects of anti-inflammatory in LPS-stimulated macrophage cells.
The planner structure characterized for compound 2 was evaluated to determine its anti-inflammatory activity in both RAW 264.7 and DH82 macrophage cells. Treatment with compounds 1 and 2 did not affect the viability of RAW 264.7 and DH82 cells at 5% or less (up to 200 μM) (Figs. 3A and 4A). We evaluated the anti-inflammatory effects of the novel hydropyridazine derivative minaprinol (2) by detecting the production of pro-inflammatory mediators including NO and PGE 2 in LPS-stimulated RAW 264.7 and DH82 macrophages (Figs. 3 and 4).
Minaprinol (2) possesses hydroxymethyl functionality, and at concentrations of 200 μM, it showed potent enhanced inhibitory activities against NO and PGE 2 production in LPS-stimulated RAW 264.7 cells, with reductions of approximately 10.6 μM and 490.4 pg/mL relative to the parent minaprine, respectively (Fig. 3B,C). In DH82 cells, significant suppression of NO and PGE 2 was observed (Fig. 4B,C). During inflammation, the enzymes COX-2 and iNOS promote the secretion of pro-inflammatory mediators 21 . Thus, 2 was investigated by western blot analysis to determine whether it inhibited COX-2 and iNOS in LPS-induced macrophage cells. As shown in Figs. 3D and 4D, compound 2 suppressed LPS-induced COX-2 and iNOS protein expression in a dose-dependent manner, resulting in the inhibition of pro-inflammatory mediator production, such as NO and PGE 2 .
Changes in the secretion of pro-inflammatory cytokines, including TNF-α and IL-6, and anti-inflammatory cytokines, such as IL-10, by LPS-stimulated macrophages were determined. Figure 3G-I displays the inhibitory effects of the newly generated product 2 from irradiated minaprine on the secretion of inflammatory cytokines TNF-α, IL-6, and IL-10 by LPS-stimulated RAW 264.7 cells (Fig. 3). Minaprinol (2) also showed increased inhibition of pro-and anti-inflammatory cytokine production compared to minaprine (1) in DH82 cells from canine ( Fig. 4G-I). The newly generated candidate 2 did not affect the mRNA expression levels of iNOS and Arg-1 in RAW 264.7 macrophage, which suggested did not involve in macrophage polarization (Fig. 3J,K). Interestingly, the hydroxymethylation of aminopyridazine induced by ionizing radiation improved the anti-inflammatory properties of LPS-stimulated macrophages. In a recent study, chrysin and luteolin showed improved anti-inflammatory activities in vitro and in vivo induced by γ-irradiation, and the chemical structure of hydroxyalkyl-substituted compounds was identified 12,21 . In addition, radiolytic transformation of alkaloids and steroids showed potent enhanced anti-cancer effects in melanoma, breast, liver, and lung cancer 11,22-25 . Colour change and modification of anti-inflammatory agents from minaprine by radiolysis. The effects of ionizing irradiation on the colour changes and modification of new compound caused by hydroxymethylation of minaprine, which is most common type of hydroaminopyridazine, were examined. The white color of minaprine was gradually reduced in a dose-dependent manner and was effectively transformed at 30 kGy dose of gamma irradiation (Fig. 5A).  www.nature.com/scientificreports/ Contents of the isolated compound from the irradiated mianprine of 5, 10, 20, 30, and 40 kGy were quantified using the external standard method and the results are shown in Fig. 5B. Five concentrations points (n = 5) were used for the preparation of the calibration curve and the calibration curve of pure solutions of the standard compounds was completely linear (R 2 > 0.999). The retention times of newly formed minaprinol (2, t R 6.3 min) and minaprine (1, t R 8.2 min) were detected for five irradiated reactants. Quantitative analysis revealed that the absolute contents of the most potent minaprinol (2) in the irradiated samples at 5, 10, 20, 30, and 40 kGy were 98.6 ± 0.6, 202.7 ± 1.5, 554.6 ± 1.6, 912.0 ± 2.0, and 886.0 ± 2.0 mg/g, respectively, which is in accordance with The chemical characteristics of the modified minaprine were checked by UV spectrometry (Fig. 5C). The absorbance at 280 nm was attributed to the pyridazine bonds of minaprine (1). In contrast, 30 kGy-irradiated samples and decreased absorbance at 280 nm compared to that of control (0 kGy), which suggested greater transformation to dihydropyridazine backbone. These results suggest that the content of minaprinol (2) increased up to 30 kGy dose of ionizing radiation as the major product from minaprine. Our results indicate that hydroxymethylation induced by the application of ionizing radiation to minaprine may be beneficial in suppressing the generation of excessive inflammation in companion animal macrophage cells.

Conclusion
In the present study, we first confirmed that minaprine is easily hydroxymethylated to the aminopyridazine compound 2. Minaprinol (2) showed more potent anti-inflammatory activity toward RAW 264.7 and DH82 macrophage cells than the original minaprine. When mianprine was induced by ionizing radiation at 30 kGy, the conversion rate to minaprinol (2) was 95% or more, and it was efficiently produced (Fig. 1A and B). It would be interesting to extend the results to the companion animal drug industry for the development of novel antiinflammatory therapeutics in dogs (as well as humans) based on hydroxymethylated minaprine derivatives. Overall, this study offers a unique approach to the production of novel aminopyridazines with greatly enhanced anti-inflammatory capacity.

Methods
Chemicals and instruments. Minaprine, acetonitrile, methanol, formic acid (HPLC grade), CD 3 OD, lipopolysaccharides (LPS), and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany). All other reagents and chemicals purchased and used in this study were of analytical grade. Spectra of 1 H and 13 C nuclear magnetic resonance (NMR) were measured on a Avance NEO-600 instrument (Bruker, Karlsruhe, Germany) operated at 600 and 150 MHz, respectively. Chemical shifts are given in δ (ppm) values relative to those of the solvent CD 3 OD (δ H 3.35; δ C 49.0) on a tetramethylsilane (TMS) scale. The ESI mass spectra were measured by a Vanquish UPLS System (Thermo Fisher Scientific, MA, USA). YMC gel ODS AQ 120-50S (particle size 50 μm; YMC Co., Kyoto, Japan) was used for the column chromatogrphy, while a microplate reader (Infinite F200, Tecan Austria GmBH, Grodig, Austria) was used to measure the absorbance. A semi-prepratative high performance liquid chromatography (HPLC), Agilent HPLC 1200 system (Agilent Technologies, Palo Alto, CA, USA) equipped with a phtodiode array detector (PDA, 1200 Infinity series, Agilent Technologies) and a series of YMC-Pack ODS A-302 column (4.6 mm i.d. × 150 mm, particle size 5 μm; YMC Co., Kyoto, Japan) were used to purify the compound.
Preparation of irradiated sample. Ionizing radiation was carried out at room temperature, using a cobalt-60 irradiator (Point source AELC, IR-79, MDS Nordion International Co. Ltd.) at the Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute (Jeongup, Korea). The source strength was approximately 320 kCi with dose rate of 10 kGy/h. Pure minaprine (400 mg) in methanol (400 mL) in chapped glass bottle was directly irradiated with 5, 10, 20, 30, and 40 kGy dose. The methanolic solution of irradiated samples was immediately evaporated to remove the solvent and dried. The dried minaprine irradiated at 30 kGy dose showed the most improved NO production inhibitory effects in LPS-induced RAW264.7 cells. www.nature.com/scientificreports/ Isolation and quantitation of newly generated product. The irradiated minaprine reactant (265.6 mg) at 30 kGy dose was directly subjected to column chromatography over a YMC GEL ODS AQ 120-50S column (1.0 cm i.d. × 43 cm, particle size 50 μm) with 25% MeOH in H 2 O, to afford pure compound 2 (206.1 mg, t R 6.3 min) (Fig. S1). HPLC analysis was conducted on a YMC-Pack ODS A-302 column (4.6 mm i.d. × 150 mm; 5 μm particle size; YMC Co., Kyoto, Japan), and the gradient solvent system initiated with 0.1% HCOOH (flow rate: 1.0 mL/min; detection: UV 280 nm; temperature: 40 °C), increased to CH 3 CN over 15 min. In addition, the purity of each individual compound isolated was also determined by HPLC with values more than 99%. The newly modified product (2) from minaprine (1) was monitored using their retention times (t R ) and compared with original mianprine. Stock solutions of minaprine (1) and minaprinol (2) were prepared in MeOH, each at 1 mg/mL. Working solutions were then obtained, as mixtures of these stock solutions after serial dilutions with methanol, to achieve five concentration levels in the range of 1 to 0.0625 mg/mL. The working solutions were filtered through a syringe filter (Fisher Scientific, Fair Lawn, MJ) prior HPLC injection. After this, the linearity was determined by linear regression analysis of the integrated peak areas (Y) vs. the concentration of each standard (X mg/mL) at five different concentrations.
Minaprinol (2) (Fig. S2-S9). Cell viability assay. The effects of the newly formed product from irradiated minaprine on cell viability in RAW 264.7 and DH82 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method 26 . Cells were seeded at a density of 5 × 10 4 cells/well into 96-well plates and incubated for 24 h at 37 °C. The cells were treated with minaprine and isolated compound 2 (50, 100, and 200 μM) in dissolved free medium, and incubated for 24 h at 37 °C. A solution of MTT (0.5 mg/mL) was added to each well and the plates were incubated for 3 h at 37 °C to allow the reaction to take place before removal of the culture medium, then the produced formazan blue was dissolved in dimethyl sulfoxide (DMSO). Cell viability was determined using a spectrophotometer and the absorbance was measured at 570 nm. The control group was considered 100%.

Measurement of pro-inflammatory mediators and cytokine productions.
The RAW 264.7 and DH82 cells were plated in a 96-well plate at a density of 5 × 10 4 cells/well and incubated for 24 h at 37 °C. The cells were pre-treated with various concentrations (50, 100, and 200 μM) of isolated compound 2 for 2 h before incubating with LPS (0.1 or 1.0 μg/mL) for 24 h at 37 °C. Nitric oxide production was determined by the reaction of a macrophages culture supernatant with a Griess reagent 27 . The culture supernatant (100 μL) was mixed with the Griess reagent (100 μL) at room temperature and shaken gently for 20 min. Finally, a microplate reader was used to measure the absorbance of the reactants at 548 nm. The levels of PGE 2 and cytokines were determined by ELISA using commercial reagent kits (BD Biosciences) according to the manufacturer's instructions.