Synthesis and Evaluation of 3-Substituted-4-(quinoxalin-6-yl) Pyrazoles as TGF-β Type I Receptor Kinase Inhibitors

The transforming growth factor-β (TGF-β), in which overexpression has been associated with various diseases, has become an attractive molecular target for the treatment of cancers. Thirty-two quinoxaline-derivatives of 3-substituted-4-(quinoxalin-6-yl) pyrazoles 14a–d, 15a–d, 16a–d, 17a–d, 18a–d, 19a–d, 25a, 25b, 25d, 26a, 26b, 26d, 27b, and 27d were synthesized and evaluated for their activin TGF-β type I receptor kinase and p38α mitogen activated protein (MAP) kinase inhibitory activity in enzymatic assays. Among these compounds, the most active compound 19b inhibited TGF-β type I receptor kinase phosphorylation with an IC50 value of 0.28 µM, with 98% inhibition at 10 µM. Compound 19b also had good selectivity index of >35 against p38α MAP kinase, with 9.0-fold more selective than clinical candidate, compound 3 (LY-2157299). A molecular docking study was performed to identify the mechanism of action of the synthesized compounds and their good binding interactions were observed. ADMET prediction of good active compounds showed that these ones possess good pharmacokinetics and drug-likeness behavior.


Introduction
Transforming growth factor-β (TGF-β) superfamily members have a wide range of cellular functions, including cell proliferation, differentiation, adhesion, migration, and apoptosis [1]. Moreover, TGF-β superfamily members are proteins with similar structures, including TGF-βs, activins, bone morphogeneticproteins (BMPs), growth and differentiation factors. TGF-β plays a crucial role in initiation and progression of fibrosis in various tissues such as the heart [2], lung [3], liver [4], and kidney [5]. TGF-βs are composed of five homogeneous isomers with highly homologous amino acid sequences, TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5, though only the first three exist in humans. Among these isoforms, TGF-β1 is the prototype and major isoform of this family. TGF-β conducts signaling through two distinct serine and threonine kinase receptors as TGF-β type I (activin receptor-like kinase 5, ALK5) and type II receptors [6]. ALK5 is activated by the combination of TGF-β and the type II receptor in the juxtamembrane GS domain, stimulating its kinase activity. The activated ALK5 spread the signals through phosphorylation of Smad2 and Smad3, and followed by binding with Smad4 to form complexes. These Smad complexes will translocate into the nuclei, where they regulate target gene transcription such as cell differentiation, proliferation, apoptosis, migration, and extracellular matrix production [1]. Nevertheless, overexpression of TGF-β signaling was shown to result in various human diseases such as hematological malignancy [7], cancer [8], and pancreatic diseases [9].
Molecules 2018, 23, x FOR PEER REVIEW 2 of 20 migration, and extracellular matrix production [1]. Nevertheless, overexpression of TGF-β signaling was shown to result in various human diseases such as hematological malignancy [7], cancer [8], and pancreatic diseases [9]. For this reason, many small molecule ALK5 inhibitors, such as compounds 1 (SB-505124) [10], 2 (SD-208) [11], 3 (LY-2157299) [12], and 4 (EW-7197) [13] were synthesized at major research institutions ( Figure 1). These compounds inhibited ALK5 autophosphorylation and TGF-β-induced transcription of extracellular matrix genes at sub-micromolar concentrations in reporter assays, as shown in Figure 1. Among them, clinical candidates, compounds 3 and 4 have progressed to Phase II and Phase I trials as antitumor agents, respectively. We previously showed that a series of compounds, denoted as 5, containing the quinoxaline moiety, except for the 2,3-dimethyl substituted analogs, showed significant ALK5 inhibition in enzymatic assay [14]. P38α MAP kinase domain is among the most homologous to that of ALK5, so we selected p38α MAP kinase to evaluate the selectivity profile of these series of compounds. In addition, previous studies have found that compounds with high p38α MAP kinase selectivity are also highly selective to other kinases, such as compound 4, which showed high selectivity to 320 kinases in human body [13]. These series of compounds were selective for ALK5, compared with p38α MAP kinase. The most active compound inhibited ALK5 phosphorylation with an IC50 value of 0.013 µM and a selectivity index of >77 against p38α MAP kinase. We reported the effect of quinoxaline groups and length of side chains attached to pyrazole ring, and positional isomers on the activity of compounds containing pyrazole rings, but the effect of the pyridine portion attached to pyrazole ring on the activity of the compounds was not discussed. Tojo et al. described a novel class of ALK5 inhibitors possessing a thioamide linkage between the phenyl and pyrazole rings [15]. Among these, compound 6 (A-83-01) inhibited ALK5 with an IC50 value of 0.012 µM. Although including a thioamide linkage between the phenyl and pyrazole ring distinctly increased ALK5 inhibitory activity, as previously shown [16], the thioamide linkage was rather unstable and was slowly cleaved, to release a pyrazole ring, during long-term storage.
Previously, we showed that the methyl group of 6-methylpyridine in compound 4 formed hydrophobic interactions with the aromatic ring of Tyr249 and the nitrogen atom of the same moiety formed a water-mediated hydrogen bonding network with the side chains of Tyr249 and Glu245 and the backbone of Asp351 [13]. In previous studies, we found that the introduction of electron-donating groups into quinoxaline or quinoline rings failed to increase ALK5 inhibition activity [14]. This is due to the steric hindrance of their rigid structures. Therefore, we assumed that the ALK5 inhibition activity will be enhanced by introducing the electron-donating groups or the structure with many binding sites into the non-rigid pyridine ring on the premise of maintaining the non-substituent We previously showed that a series of compounds, denoted as 5, containing the quinoxaline moiety, except for the 2,3-dimethyl substituted analogs, showed significant ALK5 inhibition in enzymatic assay [14]. P38α MAP kinase domain is among the most homologous to that of ALK5, so we selected p38α MAP kinase to evaluate the selectivity profile of these series of compounds. In addition, previous studies have found that compounds with high p38α MAP kinase selectivity are also highly selective to other kinases, such as compound 4, which showed high selectivity to 320 kinases in human body [13]. These series of compounds were selective for ALK5, compared with p38α MAP kinase. The most active compound inhibited ALK5 phosphorylation with an IC 50 value of 0.013 µM and a selectivity index of >77 against p38α MAP kinase. We reported the effect of quinoxaline groups and length of side chains attached to pyrazole ring, and positional isomers on the activity of compounds containing pyrazole rings, but the effect of the pyridine portion attached to pyrazole ring on the activity of the compounds was not discussed. Tojo et al. described a novel class of ALK5 inhibitors possessing a thioamide linkage between the phenyl and pyrazole rings [15]. Among these, compound 6 (A-83-01) inhibited ALK5 with an IC 50 value of 0.012 µM. Although including a thioamide linkage between the phenyl and pyrazole ring distinctly increased ALK5 inhibitory activity, as previously shown [16], the thioamide linkage was rather unstable and was slowly cleaved, to release a pyrazole ring, during long-term storage.
Previously, we showed that the methyl group of 6-methylpyridine in compound 4 formed hydrophobic interactions with the aromatic ring of Tyr249 and the nitrogen atom of the same moiety formed a water-mediated hydrogen bonding network with the side chains of Tyr249 and Glu245 and the backbone of Asp351 [13]. In previous studies, we found that the introduction of electron-donating groups into quinoxaline or quinoline rings failed to increase ALK5 inhibition activity [14]. This is due to the steric hindrance of their rigid structures. Therefore, we assumed that the ALK5 inhibition activity will be enhanced by introducing the electron-donating groups or the structure with many binding sites into the non-rigid pyridine ring on the premise of maintaining the non-substituent quinoxaline structure. To examine whether the capability of the nitrogen atom of the 6-methylpyridine moiety as an H-bond acceptor would be increased by other substitutions, we introduced 6-(dimethylamino)pyridin-2-yl, 4-methylthiazol-2-yl [17][18][19], and pyrimidin-4-yl groups [20], instead of the 6-methylpyridine moiety in 5 series compound. Based on this finding and previous research, we tried to replace the thioamide linkage with a chemically stable thioamidomethylene linkage and, designed compounds 18a-d, 19a-d, 27b, and 27d ( Figure 2). To compare the effects of the thioamidomethylene linkage in 18a-d, 19a-d, 27b, and 27d on ALK5 inhibitory activity, their counterpart derivatives 14a-d, 15a-d, 16a-d, 17a-d, 25a, 25b, 25d, 26a, 26b, and 26d possessing an amidomethylene linkage, were also designed. The target compounds 14b-d, 16b-d, 15b-d, 17b-d, 18b-d, 19b-d, 25b, 25b, 25d, 26b, 26b, 26d, 27b, and 27d each possess a substituent, either o-F, m-F or m-CN, in the phenyl ring because these were previously found to be most beneficial for ALK5 inhibitory activity and selectivity [13].
Compound 19b showed the most potent ALK5 inhibitory activity with an IC50 value of 0.28 µM in these three series of compounds. It was slightly less potent than compounds 3 (0.12 µM). Furthermore, all thioamides 18a-d, 19a-d, 27b, and 27d failed to inhibit p38α MAP kinase up to 10.0 µM. Compound 19b was the most selective in these three series, showing a selectivity index of >35, higher than that of positive control compound 3 (4). In this series of compounds (19a-d), the activity  14a-d, 15a-d, 16a-d, 17a-d, 18a-d, and 19a-d.
Compound 19b showed the most potent ALK5 inhibitory activity with an IC 50 value of 0.28 µM in these three series of compounds. It was slightly less potent than compounds 3 (0.12 µM). Furthermore, all thioamides 18a-d, 19a-d, 27b, and 27d failed to inhibit p38α MAP kinase up to 10.0 µM. Compound 19b was the most selective in these three series, showing a selectivity index of >35, higher than that of positive control compound 3 (4). In this series of compounds (19a-d), the activity of compounds with substituents is superior to that of unsubstituted one. Notably, 2-fluorine substituted compound 19b, which is two-fold more potent than unsubstituted compound 19a (IC 50 = 0.57 µM). 18a-d, 19a-d, 25a, 25b, 25d of compounds with substituents is superior to that of unsubstituted one. Notably, 2-fluorine substituted compound 19b, which is two-fold more potent than unsubstituted compound 19a (IC50 = 0.57 µM).

Docking Study of 18b and 19b in the Alk5 Active Site
To rationalize the SAR shown in Tables 1 and 2, we examined the binding modes of two representative ligands (18b and 19b) using the semi-flexible molecular docking program DS CDOCKER [28]. Docking analyses were performed using the recently reported X-ray structure of ALK5 complexed to a pyrazole ALK5 inhibitor (PDB: 1RW8) [13] (Figure 4).
The sulfur atom of the thioamide in 19b contacted the hinge of ALK5, forming hydrogen bonds with the imidazole ring of His283, a residue previously reported to be important for inhibitory activity (C) [14]. The phenyl ring of 19b interacted with Lys232 via Pi-alkyl bond. The central pyrazole ring of 19b formed Pi-alkyl bond with the side chains of Leu340 and Val219. The thiazole N atom of 19b formed carbon-hydrogen bond with the backbone of Ser287 and the methyl group of 19b formed alkyl bond with the backbone of Lys337 and Pi-alkyl bond with the backbone of Phe289. Not only the calculated binding energy scores (CDOCKER_INTERATION_ENERGY) of these two compounds

Docking Study of 18b and 19b in the Alk5 Active Site
To rationalize the SAR shown in Tables 1 and 2, we examined the binding modes of two representative ligands (18b and 19b) using the semi-flexible molecular docking program DS CDOCKER [28]. Docking analyses were performed using the recently reported X-ray structure of ALK5 complexed to a pyrazole ALK5 inhibitor (PDB: 1RW8) [13] (Figure 4).
The sulfur atom of the thioamide in 19b contacted the hinge of ALK5, forming hydrogen bonds with the imidazole ring of His283, a residue previously reported to be important for inhibitory activity (C) [14]. The phenyl ring of 19b interacted with Lys232 via Pi-alkyl bond. The central pyrazole ring of 19b formed Pi-alkyl bond with the side chains of Leu340 and Val219. The thiazole N atom of 19b formed carbon-hydrogen bond with the backbone of Ser287 and the methyl group of 19b formed alkyl bond with the backbone of Lys337 and Pi-alkyl bond with the backbone of Phe289. Not only the calculated binding energy scores (CDOCKER_INTERATION_ENERGY) of these two compounds indicated that 19b (−56.18 kcal/mol) formed more stable complexes with ALK5 than did 18b (−54.81 kcal/mol), but also compound 19b (Lys232, His283, Ser287, Leu340, and Lys337) showed more bonding with previously reported key amino acids than did compound 18b (Lys232 and Leu340) (A) [29]. In particular, compound 18b did not form bond with the most important amino acid, His283. The 2-fluorophenyl ring of 18b and 19b was stretched to the backside hydrophobic pocket consisting of Lys232, Leu260, Leu278, Val231, and Ala230. Furthermore, compound 19b seemed to be more favorably accommodated in the binding pocket of ALK5 than compound 18b (B and D). Our docking results indicated that the most active compound, 19b, showed the more favorable intermolecular interactions in the ALK5 active site than compound 18b. This supported the conclusion that the substitution group size of the pyridine moiety and selection of a heterocycle in compound 5 may have been important for improving ALK5 inhibition.
bonding with previously reported key amino acids than did compound 18b (Lys232 and Leu340) (A) [29]. In particular, compound 18b did not form bond with the most important amino acid, His283. The 2-fluorophenyl ring of 18b and 19b was stretched to the backside hydrophobic pocket consisting of Lys232, Leu260, Leu278, Val231, and Ala230. Furthermore, compound 19b seemed to be more favorably accommodated in the binding pocket of ALK5 than compound 18b (B and D). Our docking results indicated that the most active compound, 19b, showed the more favorable intermolecular interactions in the ALK5 active site than compound 18b. This supported the conclusion that the substitution group size of the pyridine moiety and selection of a heterocycle in compound 5 may have been important for improving ALK5 inhibition.

ADMET Analysis
ADMET pharmacokinetics is a very important method in drug design and drug screening, which is responsible for drug failure [30,31]. The ADMET properties of the drug molecules are greatly influenced by the optimum value of the intestinal absorption, water solubility, blood-brain barrier (BBB) penetration, human cytochrome P450 2D6 (CYP2D6) inhibition, and plasma protein binding (PPB) level. The ADMET parameters of these good targeted compounds 19a-d was measured using Discovery Studio software as a drug reference was reported in Table 3.
The preferred and most widely used route of drug is the oral route, and the mechanism of absorption from the gastrointestinal tract is passive diffusion through the intestinal epithelial cells. Hence, the absorption and solubility of the drug are two major factors for oral administration. All of the 3-substituted-4-(quinoxalin-6-yl) pyrazoles 19a-d showed good intestinal absorption. All compounds showed low or very low aqueous solubility at room temperature. However, the structure of these compounds contain thiazole and quinoxaline moiety, so it is easy to make a salt in stomach

ADMET Analysis
ADMET pharmacokinetics is a very important method in drug design and drug screening, which is responsible for drug failure [30,31]. The ADMET properties of the drug molecules are greatly influenced by the optimum value of the intestinal absorption, water solubility, blood-brain barrier (BBB) penetration, human cytochrome P450 2D6 (CYP2D6) inhibition, and plasma protein binding (PPB) level. The ADMET parameters of these good targeted compounds 19a-d was measured using Discovery Studio software as a drug reference was reported in Table 3. Predicted human intestinal absorption level (acceptable level: 0 is good, 1 is moderate and 2 is low). b Predicted aqueous solubility at room temperature (acceptable range: −6.0 < log (SW) < −4.1 is low and −4.1 < log (SW) < −2.0 is good). c Predicted blood-brain barrier (BBB) penetration level (acceptable range: 0 is very high, 1 is high, 2 is medium, 3 is low, 4 is very low). d Predicted human cytochrome P450 2D6 (CYP2D6) inhibitory ability (acceptable level: False is good). e Predicted plasma protein binding (PPB) possibility (acceptable level: True is good).
The preferred and most widely used route of drug is the oral route, and the mechanism of absorption from the gastrointestinal tract is passive diffusion through the intestinal epithelial cells. Hence, the absorption and solubility of the drug are two major factors for oral administration. All of the 3-substituted-4-(quinoxalin-6-yl) pyrazoles 19a-d showed good intestinal absorption. All compounds showed low or very low aqueous solubility at room temperature. However, the structure of these compounds contain thiazole and quinoxaline moiety, so it is easy to make a salt in stomach acid and dissolve in water. The BBB is an important organizational structure to maintain the stability of the central nervous system, which maintains the relative stability of the environment in the nervous system by restricting the entry of compounds into the central nervous system, and protects nerve cells from being invaded by harmful substances. All compounds, except compound 19b, showed BBB penetration in permissible level (1). These compounds are suitable for the treatment of systemic diseases. Compound 19b showed low BBB permeability and is suitable for non-brain diseases, which is characterized by cyano group at 3-position on the phenyl ring. In addition, the PPB binding ability of all compounds was good. CYP2D6 is an important drug metabolism enzyme in the family of cytochrome P450, and its catalysis is widely used. Over the years, the genes encoding CYP2D6 enzyme have been closely related to the genetic polymorphism, drug metabolism, production of adverse drug reactions, and activation of carcinogens. Also, all compounds did not inhibit CYP2D6, so they will be shown no or low side effects such as drug-drug interaction and wide metabolism. All of the parameters were within the acceptable range defined for human use and these good targeted compounds may exhibit significant pharmacokinetic and drug-likeliness properties.
The reaction cocktail was incubated at 30 • C for 60 min. The reaction was halted with 50 µL of 2% (v/v) H 3 PO 4 , plates were aspirated and washed two times with 200 µL 0.9% (w/v) NaCl. Incorporation of 33 Pi was established with a microplate scintillation counter (PerkinElmer, Boston, MA, USA). All assays were performed with a BeckmanCoulter/SAGIAN™ Core System.

Docking Assay
All molecular computation studies were carried out using Discovery Studio 2017 (Accelrys, San Diego, CA, USA). The X-ray crystal structure of ALK5 complexed with 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole inhibitor was obtained from protein data bank (PDB: 1RW8). The water molecules and heavy atom in protein were removed and the protein was prepared by adding hydrogen and correcting incomplete residues using Clean Protein tool of DS, then the protein was refined with CHARMm. The structures of compounds 18b and 19b were sketched in 2D and converted into 3D using the DS molecule editor. Automated docking studies were carried out to investigate the binding mode of compounds 18b and 19b in the crystal structure of 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole utilizing DS-CDOCKER protocol. The pose with the top CDOCKER_INTERACTION_ENERGY was chosen for analyzing the binding features of compounds 18b and 19b with ALK5.

Prediction of ADMET Properties
ADMET properties of good targeted compounds 19a-d as drug lead compound were predicted using ADMET descriptors in Discovery Studio 2017 (Accelrys, San Diego, CA, USA). It is a quick, easy and accurate method for prediction of absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. In this work, for the aforementioned compounds, human intestinal absorption level, aqueous solubility (log(SW)), blood-brain barrier (BBB) penetration level (AlogP98), human cytochrome P450 2D6 (CYP2D6) inhibitory ability, hepatotoxicity possibility, and plasma protein binding (PPB) levels were measured.

Conclusions
In our study, 32 quinoxaline-derivatives of 3-substituted-4-(quinoxalin-6-yl) pyrazoles 14a-d, 15a-d, 16a-d, 17a-d, 18a-d, 19a-d, 25a, 25b, 25d, 26a, 26b, 26d, 27b, and 27d were synthesized and evaluated for ALK5 and p38α MAP kinase inhibitory activities in enzymatic assays. We found that insertion of a 4-methylthiazol-2-yl moiety at the 3-position of the pyrazole ring showed more potent ALK5 inhibitory activity and selectivity than introduction of electron-donating groups into quinoxaline or quinoline ring. The most potent compound, 19b, inhibited ALK5 phosphorylation with an IC 50 value of 0.28 µM and showed 98% inhibition at 10 µM in the enzymatic assay. The selectivity index of 19b against p38α MAP kinase was >35, much higher than that of positive control compound 3 (4). Although compound 19b has slightly lower activity than the positive control compound 3, its selectivity is much higher than that of the positive control compound 3, so its side effects may be lower. The docking study described that compounds possessing 4-methylthazol-2-yl moiety was found to show better docking interaction than compounds possessing 6-(dimethylamino)pyridine-2-yl moiety on its active site. All good targeted compounds were subjected to ADMET prediction and the predicted ADMET parameters were within the acceptable range defined for human use. This result provides good data for the design of substituents for the 3-position introduction of the pyrazole ring, which may be a good choice if the lipophilic compounds are needed. In particular, compound 19b was the most promising and it could be considered worthwhile lead compound worthy of further investigation.