Discovery of Topoisomerase I Inhibitor Nitidine Derivatives with IL-10 Enhancing Activity for the Treatment of Sepsis

dose (0 – 4 μ mol/L), NC potentially prevented sepsis, and the underlying mechanisms may be the inhibition of TopoI, the induction of repairable DNA damage, and the subsequent activation of DNAdamage response and the promotion of Akt activation. This synergistically enhances the activation of the PI3K – Akt pathway and facilitates the transcription and expression of IL-10 in response to LPS stimulation. 11 However, the application of NC is limited due to its poor water solubility and low bioavailability. 12,18 Water solubility is an essential physicochemical property of an organic small-molecule drug. It is also an important issue during the drug discovery. High water solubility often results in good drug potency and an ideal pharmacokinetic pro ﬁ le. 19 Theoretically, structural modi ﬁ cation of a drug is an effective way to improve its water solubility


Introduction
Sepsis is a systemic inflammatory syndrome caused by the uncontrolled response of the organism to infection, which can lead to multiple organ dysfunction and death.It is currently one of the leading causes of death in patients in the surgical intensive care unit.It is also a common complication in patients suffering from severe burns that can lead to septic shock or death.][3] Excessive inflammatory response is a major cause of sepsis, and the release of pro-inflammatory factors ultimately causes circulatory dysfunction and the subsequent multiorgan failure, therefore, preventing excessive inflammatory response may be the focus of sepsis treatment. 4nterleukin-10 (IL-10) is reported to be an important antiinflammatory factor that blocks the release of pro-inflammatory factors by activated monocytes. 5,6A single injection of IL-10 reduces the mortality of an animal model of sepsis, which was induced by lipopolysaccharide (LPS; also known as endotoxin) in mice. 7,8Intraperitoneal injection of IL-10 attenuates inflammatory response and inhibits the activation of the coagulation system and fibrinolysis in a human endotoxin model. 9,10Therefore, IL-10 is essential in the negative regulation of endotoxin-induced host responses.
Natural product nitidine chloride (NC; structure shown in ►Fig. 1),derived from Zanthoxylum nitidum (Roxb.)2][13][14][15][16][17] In our previous study, we selected more than 500 monomeric compounds related to the development, differentiation, and functions of macrophages from the Chinese medicine compound library based on a screening platform. 11We found that at a noncytotoxic dose (0-4 μmol/L), NC potentially prevented sepsis, and the underlying mechanisms may be the inhibition of TopoI, the induction of repairable DNA damage, and the subsequent activation of DNA damage response and the promotion of Akt activation.This synergistically enhances the activation of the PI3K-Akt pathway and facilitates the transcription and expression of IL-10 in response to LPS stimulation. 11owever, the application of NC is limited due to its poor water solubility and low bioavailability. 12,18Water solubility is an essential physicochemical property of an organic smallmolecule drug.It is also an important issue during the drug discovery.High water solubility often results in good drug potency and an ideal pharmacokinetic profile. 19Theoretically, structural modification of a drug is an effective way to improve its water solubility, and this can be achieved by salt formation, introduction of polar groups, reduction of liposolubility, conformation optimization, as well as the incorporation of prodrugs.In this work, we attempted to simplify the structure of NC by removing the benzene ring to decrease its liposolubility and enhance its water solubility.We further assessed the activity of the NC derivatives obtained in increasing IL-10 and preventing lethality in an animal model of sepsis.
(Scheme 1).The route started with 5-nitro-2,3-naphthalenediol (1A).The phenolic hydroxyl groups of 1A were protected with dibromomethane to give 2A, and the nitro group of which was reduced to an amino group using hydrazine hydrate and palladium carbon.Compound (3A) undergoes a reductive amination reaction with 6-bromoveratraldehyde, followed by the subsequent protection of the amino group using benzyloxycarbonyl (Cbz) to obtain the intermediate 5A, which gives a cyclization product 6A via a free-radical mechanism under the action of the free-radical initiators (n-Bu 3 SnH and azodiisobutyronitrile [AIBN]).After removing the Cbz-protecting group of compound 6A, the oxygen in the air during the posttreatment process oxidizes the carbon and nitrogen single bonds to a double bond to generate the intermediate 7A without any additional oxidation.In the presence of methyl iodide, a nitrogen methylation of 7A was achieved to give the target product (8A).The total yield of the route was approximately 40%.
NC belongs to the benzophenanthridine group of compounds, in which four aromatic rings, A, B, C, and D, are linked together to form a conjugated planar structure, which is highly hydrophobic.Reducing the number of aromatic rings in the NC structure may be an effective strategy to increase its water solubility.According to the existing structureactivity relationships, the two methoxyl groups at C-8 and C-9 should be left unchanged, the methylenedioxy (E-ring), piperonyl ring (A-and E-ring), and A-ring can be simplified to improve the druggability.Therefore, NC derivatives 6B, 6C, and 6D were designed and synthesized.Their synthetic pathway is similar to that of NC, and the starting materials include 4-amino-1,3-benzodioxol (1B), naphthylamine (1C), and aniline (1D), respectively (Scheme 2).
Oxygen-containing groups were introduced at the paraposition of the N atom on 6D to improve the water solubility of the compound.The generated compounds included 6F (an ethyl carboxylate at the para-position of the N atom), 6H (an isopropoxy group at the para-position of the N atom), 7H (a phenolic hydroxyl at the para-position of the N atom), 8E (a hydroxyethyl group at the para-position of the N atom), and 9G (a hydroxymethyl group at the para-position of the N atom).The synthetic routes of 8E, 6F, and 6H are shown in Schemes 3, 4, and 5, respectively, which are similar to those of NC, and the starting materials include p-aminophenethyl alcohol (1E), ethyl 4-aminobenzoate (1F), and 4isopropoxy aniline (1H) with a total yield of 35 to 40%.Scheme 4 shows the synthetic route of 9G.The ester group in compound 5F was reduced to hydroxyl by DIBAL-H, and then underwent Dess-Martin oxidation, N methylation, and sodium borohydride reduction to obtain the target product (9G).As shown in Scheme 5, the isopropyl group in 6H was removed under the action of BCl 3 to obtain 7H.

Evaluation of the IL-10 Secretion-Promoting Activity
The activity of the derivatives and some intermediates on macrophage RAW264.7 was assessed to screen compounds with better IL-10 secretion-promoting activity and to explore the structure-activity relationship of NC in promoting IL-10 secretion.Based on the formula (NC)% ¼ (X -LPS)/(NC -LPS) (►Table 1). 20In the formula, X represents the average value of the three data sets obtained from the experimental group, where the LPS represents the average value of the control group in the three experiments, and NC represents the average value of the three experiments after treatment with LPS and NC.Unfortunately, the activity of IL-10 secretion of the selected derivatives and intermediates at a concentration of 2 μmol/L was weaker than that of NC.The activity of 8A on the secretion of IL-10 is approximately 65% of the NC, indicating that the anion may affect the secretion of IL-10.The activity of the derivatives and intermediates without A-ring (2B-6B), E-ring (2C-6C), and A-and E-rings (2D-6D) was also assessed with the maximum effect being seen in 6B (59.37%), 6C (57.08%), and 6D (46.92%), revealing that quaternary ammonium salt structures are important in promoting the secretion of IL-10.The A-and E-rings may be essential to maintain the IL-10 secretion-promoting activity and simplifying them will weaken their activities.In addition to simplifying the A-and E-rings, different oxygen-containing substituents at the para-position of N were designed.These compounds included ethyl carboxylate derivative (6E) and aldehyde derivative (8G), which showed better activity, as well as hydroxymethyl (9G) and isopropoxyl (6H), which were inactive, indicating that the oxygen-containing substituents affect the activity while the electron-withdrawing substituents might enhance the activity.The activity of a few derivatives with better activity was determined at 5 and 10 μmol/L.We found that when the concentration of NC and its derivatives were 5 and 10 μmol/L, their activities in promoting IL-10 secretion decreased compared with those at 2 μmol/L, suggesting better activities of the compound at lower concentrations.Meanwhile, fewer adherent and floating cells were found in the NC-treated group (5 μmol/L), which proved a stronger inhibitory effect of the compound on cell proliferation at the concentrations, subsequently leading to a decrease in the IL-10 secretionpromoting activity.

In vitro TopoI Inhibition Assay
NC promotes IL-10 secretion from macrophages by inhibiting TopoI activity, thus, inhibitory activity of NC and its derivatives for TopoI was preliminary screened at the concentration of 10 μmol/L.Given that, the derivatives containing quaternary ammonium salt structures facilitated the secre- tion of IL-10.These derivatives were further screened for TopoI inhibitory activity at a concentration of 5 μmol/L.TopoI inhibitors act by stabilizing a covalent TopoI-DNA complex called the cleavable complex.The ability of derivatives to stabilize this complex was evaluated by incubating TopoI and supercoiled DNA pBR322 in the presence of drugs.Cleavable complexes were revealed by the appearance of short DNA fragments when the samples were analyzed by gel electrophoresis under denaturing conditions, and the results were shown in ►Fig. 2. Our data showed that the positive control drugs camptothecin (CPT) and NC, as well as NC derivatives 8A, 6B, and 7H, completely inhibited TopoI-mediated DNA supercoil relaxation at a concentration of 5 μmol/L.Derivatives 6C and 6D had no TopoI inhibitory activity, indicating the important effect of the A-and E-ring structures in maintaining the activity.Derivatives 6F and 6H partially inhibited TopoI activity at a concentration of 5 μmol/L, while 8E and 9G had no activity.This indicated that the paraoxygen substituent of N affects the activity.Subsequently, the TopoI inhibitory activity of 8A, 6B, 6F, and 6H was rescreened at 2 μmol/L, of which only 6H showed no effect.
The mode of binding between the derivatives and TopoI was investigated.The derivatives were analyzed using docking analysis with the Schrodinger software (10.6) (►Fig. 3).Arg364, Asp533, Asn722, and other critical amino acid residues were found at the active sites of TopoI. 21,22The 3D structure is shown in ►Fig.3A.The docking result showed that the methoxy groups in the NC (►Fig.3B) and 6B (►Fig.3C) form hydrogen bonds with Asn722, and the methylenedioxy groups form hydrogen bonds with Lys425 and Arg364, respectively.The planar structure allows their insertion into the DNA cleavage site, forming base-stacking interactions with upstream and downstream base pairs.Therefore, their TopoI inhibitory activities are relatively strong, showing partial inhibitory activity at a concentration of 2 μmol/L.Although the hydroxymethyl group in 9G can form hydrogen bonds with Thr718 (►Fig.3D), it cannot form hydrogen bonds with other critical residues, such as Arg364 and Asn722, nor can it embed in the DNA cracks via a π-π interaction.Therefore, the TopoI inhibitory activity of 9G was weak, with no activity at a concentration of 5 μmol/L.This can partially explain the strength of the inhibitory activity of TopoI in the derivatives.
Derivatives with structures similar to NC display consistency in promoting IL-10 secretion and inhibiting TopoI activities.For example, 6B and 6F have strong activity on both IL-10 secretion and TopoI inhibition.This consistency was not observed in the derivatives with structures different from NC, as they might promote the secretion of IL-10 via other mechanisms.Based on these results, we summarized the relationship between derivative structure and their activity on IL-10 secretion and TopoI inhibition as follows: • The quaternary ammonium salt structure is essential for the pharmacological effects.• The A-and E-rings are important for maintaining activity.
• The para-substituent of N influences the activity, for instance, phenolic hydroxyl groups and hydroxyethyl groups weaken the activity of IL-10 secretion and TopoI inhibition of the compound.

Water Solubility Determination
Water solubility of 6B and 6F was determined in phosphatebuffered saline (PBS).As presented in ►Table 2, compared with NC, the solubility of 6B and 6F in PBS was improved to varying degrees, especially 6F (10,600 μg/mL) was more than 500-fold higher than NC (18.707 μg/mL).Although the water solubility of 6B is worse than 6F, it is also a factor of 7 higher than NC.This suggests that reducing the number of aromatic rings, removing the A-ring for example, and introducing hydrophilic groups in the structure of NC can improve the water solubility while maintaining the activity of the compound.

In vitro Activity
The in vivo activities of 6B and 6F in a mouse sepsis model were evaluated.As shown in ►Fig. 4,NC (3 and 10 mg/kg)

Conclusion
NC prevents sepsis by inhibiting TopoI activity and promoting IL-10 secretion by macrophages.However, its development has been limited due to its poor water solubility and low oral bioavailability.In this study, we simplified the core structure of the NC to obtain 6B, 6C, and 6D.Then, oxygencontaining substituents were introduced at the para-position of the N atom of compound 6D.The activity of the derivatives and intermediates in IL-10 secretion and TopoI inhibition were evaluated to elucidate the structure-activity relationship.Among them, derivatives 6B and 6F showed consistent in promoting IL-10 secretion and inhibiting TopoI activity.In addition, their water solubility was 7-fold and 500-fold higher than that of NC.Moreover, 6F (3 mg/kg) significantly improved the survival rate of septic mice, which was comparable to that of NC (3 mg/kg) within 84 hours.6F can be used as a novel lead compound for further research.
The study provides new strategies and drug candidates for discovery of TopoI inhibitors with better activity and druggability for the treatment of sepsis.

Synthesis of (8,9-Dimethoxyphenanthridin-2-yl) methanol (6G)
To a solution of 5F (311 mg, 1.00 mmol) in anhydrous DCM (10 mL) was added 1.0 mol/L diisobutylaluminum hydride (1.10 mL, solution in hexanes) dropwise at 0°C.The reaction mixture was warmed to room temperature and allowed to stir for 2 hours.The reaction was quenched by the addition of ammonium chloride solution, filtered through a pad of Celite, rinsed with DCM (10 mL Â2), washed with brine, dried over sodium sulfate, and concentrated to provide a crude, which was purified by silica gel column chromatography (PE:EA ¼ 30-40%) to give 6G (185 mg, 69%) as a white solid.

TopoI Inhibitory Activity Assay
TopoI inhibitory activity assay was conducted according to a reported study. 11The compound was dissolved in DMSO with a final concentration of 10, 5, and 2 μmol/L, respectively.The compound at a specific concentration was mixed with 10 Â DNA TopoI buffer (2 μL), 0.1% bovine serum albumin (2 μL), TopoI (0.5 U), pBR322 plasmid DNA (0.25 μg), and distilled water (varied as needed to bring the final volume to 20 μL) to achieve a final volume of 20 μL.The reactions were carried out for 15 minutes at 37°C and stopped by the addition of 2 μL of loading buffer Â 10.The samples were electrophoresed on a 0.8% agarose gel in TAE (Tris-acetate-EDTA) running buffer at 120 V for 40 minutes and then stained with 0.5 μg/mL of ethidium bromide for 10 minutes.DNA bands were visualized using a UV transilluminator (Syngene G:BOX F3, England).

Receptor Preparation
The receptor structures were prepared using Schrödinger 2018 (Schrödinger, United States) by importing Topo I-DNA and norindenoisoquinoline crystal structures (PDB:1TL8) and analyzing the protein structure using the Protein Preparation Wizard, removing water molecules and redundant structures, as well as hydrogenation, side chain hydroxylation, side chain repair, and main chain end processing.Using the Receptor Grid Generation module, the active cavity was defined by the ligand norindenoisoquinoline in the crystal complex, and the docking radius was set to 10 Å.

Ligand Preparation
For docking analysis, NC and its derivatives (6B and 9G) were imported into Schrodinger in an SDF format.These small molecules were conformationally optimized using Ligand Prepare and Minimize modules.

Molecular Docking
The receptor active cavity, as defined earlier, and the derivatives were docked using Glid-Ligand docking, with XP (extra precision) selected for docking precision and flexible mode for ligand.

Water Solubility Determination
The excess compounds were dissolved in 200 μL of PBS, respectively, sonicated, and allowed to stand at room temperature for 24 hours, then filtered through a microporous filter membrane (0.45 μm).Next, each of the drug solution (100 μL) was diluted to 1.5 mL with PBS, and 10 μL of which was injected into the column for high-performance liquid chromatography analysis, and the peak area was recorded.An Agilent 1100 HPLC system (Agilent Technologies, MA, United States), equipped with a quaternary pump, a vacuum degasser, an autosampler, and a column heater-cooler, was used.Separation was performed by an Agilent Zorbax extend-C18 column (250 mm Â 4.6 mm i.d., 5um, Agilent, United States).The column temperature was maintained at 25°C.Solvent A (water with 0.2% (v/v) acetic acid) and solvent B (acetonitrile) were used for gradient elution with the program as follows: 5 -95% B (0 -10 minutes); 95-95% B (10 -15 minutes).The flow rate was set at mL/min.The solubility data of each compound in PBS were calculated by an external standard method.

Animals
Male BALB/c mice (6-to 8-week-old) were obtained from Shanghai Laboratory Animal Company (Shanghai, China) and housed in the Experimental Animal House at the Second Military Medical University (Shanghai, China) in environmentally controlled conditions (22°C, a 12-hour light/dark cycle with the light cycle from 6:00 to 18:00 and the dark cycle from 18:00 to 6:00) with ad libitum access to standard laboratory chow.The study protocol was approved by the local institutional review board at the affiliated institutions.The animal experiments were conducted according to the established institutional guidelines for animal care and use at the Second Military Medical University.

Treatment
NC and its derivatives 6B and 6F were weighed (10 and 3 mg, respectively) and dissolved in 200 μL DMSO.Then, 5 mL of normal saline was added, and the mixture was sonicated for 20 minutes to dissolve the compounds completely.The mixture was diluted to 10 mL with normal saline to obtain sample solutions at 10 and 3 mg/kg doses.Male BALB/c mice were randomly divided into eight groups, including a control, a model, and treatment groups.Equal volumes of normal saline and NC, 6B, and 6F (3 and 10 mg/kg) were injected intraperitoneally into the mouse for 6 hours before modeling.An experimental mouse model of sepsis was established by intraperitoneally injecting LPS (15 mg/kg).Then, the mice in each group were returned to the cages with free access to food and water.The number of dead mice was recorded every 12 hours, and the dead mice were removed.The survival rate was observed within 84 hours.
Discovery of Topoisomerase I Inhibitor Nitidine Derivatives with IL-10 Enhancing Activity for Sepsis Treatment Liu et al. e50

Fig. 3
Fig.3Computer simulation of the combination between compounds and TopoI-DNA complex (PDB, 1BG1).(A) 3D structure of the TopoI-DNA and norindenoisoquinoline crystal complex (PDB: 1TL8); (B-D) Schematic representation of the proposed binding mode for compounds NC, 6B, and 9G in the TopoI-DNA complex, respectively.The figure was generated using Schrodinger (10.6).

Fig. 4
Fig. 4 Effect of 6B and 6F on the survival rate of septic mice induced by LPS.Male BALB/c mice were intraperitoneally injected with vehicle, NC, 6B, and 6F (dissolved in 1:9 mixtures of DMSO and PBS) for 6 hours.The sepsis model was established by intraperitoneally injecting LPS (15 mg/kg) into mice.n ¼ 6 in the model and 6F (10 mg/kg) groups; n ¼ 5 in the other groups.After the treatment, the survival rate was observed within 84 hours.A log-rank (Mantel-Cox) test was used for comparison with Ã p < 0.05; ÃÃ p < 0.01 being significant.LPS, lipopolysaccharide.

Table 2
Water solubility of NC, 6B, and 6F Compd.λmax (nm) Water solubility (μg/mL) Pharmaceutical Fronts Vol. 6 No. 1/2024 © 2024.The Author(s).Discovery of Topoisomerase I Inhibitor Nitidine Derivatives with IL-10 Enhancing Activity for Sepsis Treatment Liu et al. e53 could significantly improve the survival rate in comparison to the model group, the survival rate of NC (10 mg/kg) reaching 80% within 84 hours.However, 6B (3 and 10 mg/kg) as well as 6F (10 mg/kg) has no significantly effect while 6F (3 mg/kg) significantly enhanced the survival rate of septic mice.Given above, 6F was preferred for the further research.
1 hour.Then sodium cyanoborohydride (1.26 g, 0.02 mol) was added.After 8 hours, the solvent was removed under reduced pressure and extracted with dichloromethane (DCM; 20 mL Â 3).The combined organic layers were washed with brine (10 mL), dried over sodium sulfate, filtered, and concentrated to obtain the white solid product 4A (3.74 g, 90%). 1 H NMR (500 MHz, DMSO-d 6 ) d 7.68 (s, 1H), 7.16 (d, To a solution of 4A (700 mg, 1.68 mmol) and sodium carbonate (268 mg, 2.53 mmol) in DCM/H 2 O (12 mL/3 mL) was added benzyl chloroformate (0.48 mL, 3.37 mmol) dropwise.The reaction mixture was vigorously stirred at room temperature for 1 hour.After the completion of the reaction monitored by TLC, the mixture was extracted with DCM Shake the slurry under 50 psi of H 2 (g) pressure at room temperature overnight.The mixture was filtered through a pad of Celite and rinsed with DCM (100 mL Â 2).