Novel Potent Imidazo[1,2-a]pyridine-N-Glycinyl-Hydrazone Inhibitors of TNF-α Production: In Vitro and In Vivo Studies

In this work, we describe the design, synthesis and pharmacological evaluation of novel imidazo[1,2-a]pyridine-N-glycinyl-hydrazone derivatives (1a–k) intended for use as inhibitors of tumor necrosis factor alpha (TNF-α) production. The compounds were designed based on the orally active anti-inflammatory prototype LASSBio-1504 (2), which decreases the levels of the pro-inflammatory cytokine TNF-α in vitro and in vivo. The in vitro pharmacological evaluation of the imidazo[1,2-a]pyridine compounds (1) showed that substitution of the N-phenylpyrazole core present in prototype 2 by a bioisosteric imidazo[1,2-a]pyridine scaffold generated anti-TNF-α compounds that were more potent than the previously described N-phenylpyrazole derivative 2 and as potent as SB-203580, a p38 MAPK inhibitor. The most active derivative (E)-2-(2-tert-butylimidazo[1,2-a]pyridin-3-ylamino)-N’-(4-chlorobenzylidene) acetohydrazide, or LASSBio-1749 (1i) was orally active as an anti-inflammatory agent in a subcutaneous air pouch model, reducing expressively the levels in vivo of TNF-α and other pro-inflammatory cytokines at all of the tested doses.


Introduction
Tumor necrosis factor alpha (TNF-a) is a major pro-inflammatory cytokine that functions as a mediator of acute inflammation, platelet activation and participation in the genesis of fever and anemia. Increased production of this cytokine is associated with a number of autoimmune and inflammatory diseases, such as Crohn's disease, psoriasis, diabetes [1,2], multiple sclerosis [3], atherosclerosis and rheumatoid arthritis (RA) [4]. In these diseases, TNF-a modulates processes such as immune cell activation, proliferation, apoptosis and leukocyte migration. In RA, which is a chronic systemic inflammatory disease, TNF-a is involved in inflammation and in the mechanisms of cartilage and bone joint destruction. High levels of TNF-a have been detected in the synovial membranes of patients with acute and chronic RA [5].
The modulation of the biosynthesis of pro-inflammatory cytokines is an important strategy for the treatment of inflammatory diseases; the therapeutic potential of targeting TNF-a, for example, has been validated by the success of drugs such as infliximab and etanercept [6]. However, due to the various limitations of the chronic use of these drugs [7], such as the occurrence of serious side effects and high drug production costs, new small molecules capable of modulating the biosynthesis of TNF-a and other pleiotropic cytokines must be developed.
As part of a research program aimed at the identification of new anti-TNF-a lead compounds for the treatment of inflammatory diseases, our research group has recently described the discovery of LASSBio-1504 (2) [8], a structural analogue of BIRB-796 (3) (Figure 1) [9,10] with in vitro and in vivo anti-TNF-a activity and in vivo anti-inflammatory and antinociceptive properties.
In a continuing effort to identify potent and safe anti-TNF-a inhibitors, we report herein the design, synthesis and anti-TNF-a evaluation of several novel imidazo [1,2-a]pyridine-N-glycinylhydrazone derivatives (1a-k), which were produced via the structural modification of prototype 2. For the proposed derivatives (1a-k), we replaced the N-phenyl-pyrazole nucleus (A, Figure 1) of LASSBio-1504 (2) with the isosteric heterocycle imidazo[1,2-a]pyridine (B, Figure 1). We then performed a series of molecular simplifications in the functionalized naphthyl framework attached to the imine unit of the N-acylhydrazone (NAH) groups of compounds 1a and 1b to better understand the structure-activity relationship (Figure 1).

Chemistry
To obtain the new imidazo [1,2-a]pyridine-N-glycinyl-hydrazone derivatives (1a-k), we performed multicomponent reactions (MCR) [11,12] between 2-aminopyridine (4), the appropriate aldehyde (5a or 5b) and ethyl 2-isocyanoacetate (6) (an isonitrile) ( Figure 2). MCRs represent rapid and efficient strategy for the generation of bioactive compounds [13,14] because the number of possible products increases with an increase in the number of components. In addition, the proposed strategy is step economical compared to the condensation of 2-aminopyridine with ahaloketones and subsequent functionalization [15][16][17][18]. To prepare the 3-amino-imidazo[1,2-a]pyridine esters, we used the MCR developed by Groebke [19], which is a variation of the classical Ugi [12] four-component reaction. In the MCR proposed by Ugi, the fourth component is often a carboxylic acid. The MCR developed by Groebke occurs between an aminoazine, aldehyde and isonitrile to form 3-amine-substituted heterocycles in one pot.
Thus, the first step for the production of imidazo[1,2-a]-pyridine-N-glycinyl-hydrazone derivatives 1a-k consisted of the preparation of imidazo[1,2-a]pyridine esters (7a-b) in 65-75% yield by an MCR in ethanol at room temperature ( Figure 2). Next, hydrazinolysis of the methyl esters (7a-b) with hydrazine hydrate in ethanol under reflux produced the corresponding hydrazide intermediates (8a-b) in 70-80% yield. The novel imidazo[1,2-a]pyridine-N-glycinyl-hydrazone derivatives 1a-k (Table 1) were prepared with satisfactory yields through the acid-catalyzed condensation of hydrazides (8a-b) with selected aromatic aldehydes at room temperature ( Figure 2). The structures of imidazo [1,2-a]pyridine-N-glycinyl-hydrazones1a-k were fully characterized using common spectroscopic methods, and the analytical results for C, H and N were within 60.4% of the calculated values. The purity of the imidazo[1,2-a]pyridine-Nglycinyl-hydrazones (1a-k) was greater than 97%, as determined by reversed-phase HPLC. With respect to the C = N double bond, N-acylhydrazones (NAHs) may exist as Z/E geometrical isomers and syn/anti amide conformers [20,21]. For most of the NAH derivatives described herein, the 1 H-NMR spectra were recorded at room temperature, and two species were clearly detected. In contrast, only one species was observed by reversed-phase HPLC analysis ( Figure S26 in File S1). In a study involving compounds 1f and 1 g, the 1 H-NMR spectra obtained in DMSO-d 6 at 90uC showed that the two species were in rapid equilibrium (Figures S12-S13 and S15-S16 in File S1). Interestingly, a complete coalescence of the signals was achieved at 90uC, and the reversibility of the changes was verified, indicating the presence of conformational isomers [22,23]. Indeed, a Monte Carlo conformational search performed on derivative 1 g followed by energy calculations of the selected conformers according to the Hartree-Fock 3-21G method indicated a slight difference in energy (DE = 219.83 kJ.mol -1 ) between the synperiplanar and antiperiplanar conformers in the favor of the former ( Figure 3). Therefore, we concluded that the novel derivatives 1 were obtained as single E geometrical isomers and that the observed duplication pattern was due to the presence of syn/anti amide conformers in DMSO ( Figure 3).

Pharmacology
Next, we investigated the ability of NAH derivatives 1a-k to inhibit TNF-a production in vitro [24]. The prototype LASSBio-1504 (2) and the p38 MAPK inhibitor SB-203580 ( Figure S27 in File S1) were chosen as standards. As shown in Table 2, seven NAH derivatives, i.e., 1a, 1b, 1d, 1e and 1i-k, inhibited the in vitro LPS-induced production of TNF-a in cultured mouse peritoneal macrophages at a concentration of 10 mM. Among the NAH derivatives, 1a (IC 50 = 0.62 mM), 1b (IC 50 = 0.34 mM), 1i (IC 50 = 0.21 mM), 1j (IC 50 = 0.31 mM) and 1 k (IC 50 = 0.99 mM) showed the most potent inhibitory effects. Specifically, the inhibitory effects of 1a, 1b, 1i, 1j and 1k were superior to that of N-phenylpyrazole prototype 2 (IC 50 = 3.6 mM) and similar to that of SB-203580 (IC 50 = 0.22 mM). The in vitro anti-TNF-a inhibitory potency of N-acylhydrazone (NAH) derivatives (1) has increased with the addition of more lipophilic groups attached to  The analytical results for C, H and N were within 0.4% of the calculated values for all N-acylhydrazone derivatives 1a-k. The purity of N-acylhydrazone derivatives 1a-k was also determined by reversed-phase HPLC. b The yields refer to the condensation step of hydrazides 8a-b with the corresponding aromatic aldehydes (see Figure 2). the imine group of NAH moiety. This behavior could be seen through the comparison of the IC 50 of compounds 1e and 1i that presented para-chlorophenyl group (cLog P = 4.3) with the corresponding NAH presenting an unsubstituted phenyl ring, i.e. 1f (cLog P = 3.7) and 1 g (cLog P = 3.8). Additionally, similar profile was evidenced when we compared the inhibitory potencies of compounds 1a and 1b, presenting 4-(2-(naphthalen-1-yloxy)ethyl)morpholine unit (cLog P = 4.2) with those present 4-(2-(phen-1-yloxy)ethyl)morpholine group, i.e. 1 h and 1c (cLog P = 3.2). Because the novel N-acylhydrazone derivatives 1 were designed based on the p38a MAPK inhibitor BIRB-796 (3), some of them were evaluated for their in vitro capacity to inhibit p38a MAPK activity [25] at a concentration of 10 mM. Interestingly, as the prototype LASSBio-1504 (2) none of the new imidazo[1,2a]pyridine-N-glycinyl-hydrazone derivatives (1) showed to the able to inhibit p38a activity (Table S1 in File S1).
The cytotoxicity of the compounds was also evaluated using an MTT assay (the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, a yellow tetrazole, to form purple formazan by cellular mitochondrial enzymes) to test the macrophage viability [26]. The percentage of viable cells was determined by comparison to control conditions (100% cell viability). As shown in Table 2, few of the NAH compounds showed significant cytotoxicity at a concentration of 10 mM. Even derivatives that showed some degree of toxicity had higher IC 50 values for macrophage toxicity than for TNF-a inhibition. The observed selectivity index (SI) comparing macrophage toxicity and TNF-a inhibition demonstrated that the novel N-imidazo[1,2-a]pyridine-N-glycinyl-hydrazone derivatives 1 may be safer than prototype 2. In particular, the derivative LASSBio-1749 (1i) presented an improved potency/safety balance compared with the p38 MAPK inhibitor SB-203580.
Because compound 1i provided a better balance between the IC 50 value for TNF-a production and cellular toxicity in vitro, we evaluated its effects in the subcutaneous air pouch (SAP) model, an in vivo model of inflammation and TNF production [27,28]. In mice, oral pre-treatment with a solution of 1i significantly and dose dependently inhibited TNF-a production at all of the tested doses, and reductions greater than 90% were observed; i.e., 96.8%, 90.6%, 97.3% and 95.31% for 3, 10, 30 and 100 mmol/kg doses, respectively ( Figure 4).

Conclusions
We described the design and synthesis of novel optimized NAH derivatives belonging to imidazo[1,2-a]pyridine-N-glycinyl-hydrazone series 1. These derivatives exhibited increased inhibitory  (1). LASSBio-1749 (1i) was determined to be the most active derivative of this series and was more potent than the prototype LASSBio-1504 (2) and equipotent to SB-203580 as an anti-TNF-a agent. Furthermore, the observed decrease in the cytotoxicity against macrophages indicated that the imidazo[1,2-a]pyridine-N-glycinyl-hydrazone derivatives are good candidates for safer anti-inflammatory drugs due to their potential low cytotoxicity profiles. The inhibition of the levels in vivo of IL-1b and IFN-c indicated that LASSBio-1749 could act as a blocker of a transcription factor cascade responsible for the signaling the biosynthesis of these proinflammatory cytokines. Nowadays, we are currently investigating the efficacy of LASSBio-1749 (1i) in chronic inflammatory disease models, as the adjuvant-induced arthritis in rats, in order to confirm their therapeutic potential and advance in the preclinical pharmacological studies as a safer, more accessible and less costly alternative to the biotech drugs usually exploited to treat these diseases.    General procedure for preparation of hydrazides (8a,b). A round-bottomed flask charged with 2 mmol of the respective ethyl ester (7a or 7b), 100% hydrazine hydrate (20 equiv.) and ethanol (5 mL) was stirred and heated at reflux for 2 hours. To the resulting mixture was added cold water and the precipitate formed was filtered out or the mixture was extracted with dichloromethane (3650 mL) to give the corresponding hydrazides as described next.  General procedure for the preparation of imidazo [1,2a]pyridine-N-glycinyl-N-acylhydrazones (1a-k). In a round flask containing hydrazide derivative 8a or 8b (1.6 mmol) in ethanol (10 mL), was added the respective aromatic aldehyde (1.68 mmol; 1.05 equiv.) and catalytic concentrated hydrochloric acid. The mixture was stirred for about 2 hours at room temperature. At the end of the reaction, the solvent was removed under reduced pressure and a mixture of crushed ice and saturated sodium bicarbonate solution was added to the obtained residue. The precipitate formed was filtered out or the mixture was extracted with dichloromethane (3650 mL) to furnish the title Nacylhydrazone compounds, as described next.  mented with 10% fetal bovine serum). The peritoneal macrophages were plated onto 96-wells plate (30.000 cells/well) for 1 hour at 37uC in a humidified 5% CO 2 atmosphere. Then, macrophages were incubated with the vehicle or compounds and 1 hour later stimulated with LPS(100 ng/mL) for 24 hours when the supernatants were collected to evaluate TNF-a production by ELISA kit (B&D Bioscience, USA). Cell viability by MTT assay. The peritoneal macrophage were obtained and plated as described above. The cells were incubated with the vehicle or compounds for 20 hours when was added 200 mL of RPMI medium containing 0.5 mg/mL MTT 20 mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) followed by 4 hour of incubation at 37uC in a 5% CO 2 atmosphere. After incubation supernatants were discarded and 200 mL DMSO (dimethylsulfoxide) were added to solubilize the MTT-formazan crystals. The optical density was measured in a microplate reader at 490 nm. The control groups consisted of cells with medium plus vehicle used to dissolve the substances and was considered as 100% of viable cells. Results are expressed as percentage of viable cells when compared with control groups.

Chemistry
Subcutaneous air pouch (SAP) model. The experimental protocol was similar to that described by Romano and colleagues (1997) [26] with several modifications described by Raymundo and colleagues (2011) [27]. The animals received a dorsal subcutaneous injection of sterile air (10 mL) on three alternate days to induce the SAP. On the sixth day, animals received a subcutaneous injection of sterile carrageenan suspension (1%; 1 mL). Mice were pre-treated with vehicle (Polysorbate 80), 1i (3, 10, 30 and 100 mmol/kg) or dexamethasone (1.5 mmol/kg, i.p.) 1 h before carrageenan injection into the SAP. The control group received an injection of sterile PBS (1 mL) into the SAP. Animals were sacrificed 24 h after carrageenan injection. The cavity was washed with 1 mL of sterile PBS. Exudates were collected, centrifuged at 11,000 rpm for 10 min at 4uC and TNF-a accumulated was quantified. The cytokines quantification was done by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (B&D, USA). The results are expressed as pg/mL of each cytokine. Animals were sacrificed after an exposition to a saturated carbon dioxide atmosphere in an appropriate gas chamber.
Statistical analysis. Each experimental group was composed by 6-8 mice. Data obtained from experiments were expressed as mean 6 S.E.M., compared with vehicle control and statistically analyzed by the Student's t test. p,0.05 was considered significant. When appropriate, the IC 50 values (i.e. the concentration able to inhibit 50% of the maximum effect observed) and the ED 50 dose (i.e. the dose able to inhibit 50% the effect in vivo) were determined by non-linear regression using GraphPad Prism software v. 5.0.