Quinoline-based imidazole-fused heterocycles as new inhibitors of 15-lipoxygenase

Abstract

A series of 2-chloro-quinoline-based imidazopyridines 6a–l and imidazothiazoles 6m–o bearing a bulky alkylamine side chain were synthesized as soybean 15-LOX inhibitors. The target compounds 6a–o were prepared via one-pot reaction of 2-chloroquinoline-3-carbaldehyde (3), heteroaromatic amidine 4, and alkyl isocyanides 5, in the presence of NH₄Cl. All compounds showed significant anti-15-LOX activity (IC₅₀ values ≤40 μM). Among the title compounds, the imidazo[2,1-b]thiazole derivative 6n bearing a tert-butylamine moiety showed the highest activity against soybean 15-LOX enzyme.

Keywords:

• Docking study
• enzyme inhibitors
• 15-lipoxygenase
• imidazo[1,2-a]pyridine
• imidazo[2,1-b]thiazole

Introduction

Polyunsaturated fatty acids such as arachidonic acid are implicated in the control of several physiological processes. Lipoxygenases (LOXs) are a class of nonheme iron-containing enzymes which regio- and stereospecifically catalyze the hydroperoxidation of polyunsaturated fatty acids1. In humans, three major families of LOXs were found as 5-LOX, 12-LOX, and 15-LOX isoforms. These isoforms (5-, 12-, or 15-LOX) initiate the biosynthesis of leukotrienes, lipoxins, and other compounds by oxidizing the C-5, C-12, and C-15 positions of the key substrate, arachidonic acid2,3. For example, 15-LOX oxidizes arachidonic acid to produce mainly 15(S)-5Z,8Z,11Z,13E-hydroperoxyeicosatetraenic acid (15-HPETE). The bioactive metabolites of 15-LOX hydroperoxidation (e.g. HETE and leukotriene A4) are found to be potent signal transduction modifiers which affect the inflammatory processes4. Furthermore, 15-LOX has been implicated in neurodegenerative diseases5, atherosclerosis6,7, chronic obstructive pulmonary disease (COPD), and a variety of cancers8.

Consequently, small molecules affecting the 15-LOX pathway might be therapeutically useful in chronic inflammatory diseases, cardiovascular disorders, and some types of tumors. Thus, medicinal chemists have attended extensively to find new inhibitors of 15-LOX. A part of attentions has been focused on small molecules containing fused heterocyclic systems including indolizine9,10 and imidazo-fused heterocycles11,12 previously described (I, Figure 1) as a potential inhibitors of 15-LOX. In a study by Wisniewska et al., imidazo[1,2-a]pyridine-3-yl-amine analog (EP6, II) was evaluated as a 5-LOX inhibitor13. On the other hand, a series of quinoline-based compounds were reported as potent inhibitors of LOX14–18. These findings convinced us to design a new core containing quinoline and imidazole-fused system as a new 15-LOX inhibitors. Thus, we describe here, synthesis, in vitro anti-15-LOX activity and docking study of [2-(2-chloro-quinolin-3-yl)-imidazo[1,2-a]pyridin-3-yl]amines 6a–l and [6-(2-chloro-quinolin-3-yl)-imidazo[2,1-b]thiazol-5-yl]amines 6m–o (Figure 1). Following our interests on the chemistry aspects of this core meaning finding new catalysis for the synthesis of imidazo[1,2-a]pyridine and imidazo[2,1-b]thiazol-quinoline derivatives and their further coupling reaction19,20, herein, we report the synthesis of new quinolin imidazo[1,2-a]pyridine derivatives and also evaluate their soybean 15-LOX inhibitory activity.

Figure 1. Structures of reported LOX inhibitors I and II, and designed compounds 6a–o as new 15-LOX inhibitors.

Experimental

15-LOX inhibition assay

To evaluate 15-LOX (Lipoxidase from Glycine max, soybean) inhibitory activity of new synthesized compounds, the stock solution of the compounds were prepared by dissolving them in 1 mL of DMSO. To prepare substrate solution (stock concentration = 38 mM), 12 μL linoleic acid was dissolved in 988 μL ethanol. This solution should be used the same day it is made. The final concentration of substrate will be 122 μM. Five different concentrations of each compound were tested in triplicate to obtain the inhibition range between 20 and 80%. The test solution was a mixture of 3 mL phosphate buffer (0.1 M, pH = 8), 50 μL enzyme solution (final concentration: 167 U/mL), and 50 μL of target compound solution. Being incubated for 4 min, the substrate (Linoleic acid, final concentration: 122 μM) was added, and the change in absorbance was measured for 60s at 234 nm. A control test was done with the same volume of DMSO (50 μL) to eliminate the effect of DMSO on enzyme activity.

Molecular modeling and docking stimulation

All docking simulations were performed using Autodock Vina (ver. 1.1.1). First, the 3D structure of soybean LOX in complex with 13(S)-hydroproxy-9(Z)-2,11(E)-octadecadienoic acid (code ID: 1IK3) was retrieved from protein databank (www.pdb.org). Then, the co-crystallized ligand and water molecules were removed, and the protein was converted to pdbqt format using Autodock Tools (1.5.4). To prepare the ligands for docking, the 2D chemical structure of ligands was sketched using MarvinSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and then converted to 3D format by Openbabel (ver 2.3.1). Finally, pdbqt format of ligands was prepared using an Autodock Tools python script, prepare_ligand4.py. The docking parameters were set as follow: size_x= 20; size_y = 20; size_z= 20; center_x =19.693; center_y = 0.054; center_z= 17.628. The exhaustiveness was set to 100, and the max number of retrieved final docked poses was set to 15 using num_modes parameter. The other docking parameters were left as default. Finally, the most favorable docked poses in terms of free binding energy were selected for analyzing of enzyme–inhibitor interactions.

Chemistry

Commercially available chemicals and reagents were purchased from Merck and Fluka Chemical Company and used without further purification. Melting points are measured with a Kofler hot stage apparatus and are uncorrected. ¹H and ¹³C NMR spectra were run on a Bruker FT-400 in CDCl₃, using TMS as an internal standard. IR spectra were recorded on a Shimadzu 470 spectrophotometer (KBr disks). MS were recorded with an Agilent Technology (HP) mass spectrometer operating at an ionization potential of 70 eV. Elemental analysis was performed with an Elementar Analysensysteme GmbH VarioELCHNS mode.

General procedure for the synthesis of compounds 6a–o

A mixture of 2-chloroquinolin-3-carbaldehyde 3 (1.0 mmol), heteroaromatic amidine 4a–f (1.0 mmol), appropriate alkyl isocyanide (1.2 mmol), and NH₄Cl (1.0 mmol) in toluene (5 mL) was heated under reflux for 12–24 h. After completion of the reaction, as indicated by TLC, the solvent was evaporated under reduced pressure, and the residue was recrystallized from petroleum ether–EtOAc to afford target compounds 6a–o in 65–93% yields.

[2-(2-Chloro-quinolin-3-yl)-imidazo[1,2-a]pyridin-3-yl]- (1,1,3,3-tetramethyl-butyl)-amine (6c)

Yield: 0.32 g (80%); pale yellow solid; mp 168–170 °C; IR (KBr): 3321, 2919, 2848, 1631, 1497, 754 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 0.94 (s, 9H, C(CH₃)₃), 1.29 (s, 6H, C(CH₃)₂), 1.42 (s, 2H, CH₂), 3.48 (s, 1H, NH), 6.94 (dd, J = 6.8, 4.0 Hz, 1H, H₆), 7.63 (t, J = 7.5 Hz, 1H, H_6′), 7.80 (t, J = 7.5 Hz, 1 H, H_7′), 7.93 (d, J = 8.0 Hz, 1H, H_5′), 8.09 (d, J = 8.4 Hz, 1H, H_8′), 8.52 (dd, J = 6.8, 1.6 Hz, 1H, H₇), 8.58–8.59 (m, 2H, H₈, H₅), 8.63 (s, 1H, H_4′); ¹³C NMR (100 MHz, CDCl₃): δ 29.0, 31.5, 31.7, 56.4, 59.4, 108.1, 112.0, 126.2, 127.2, 127.5, 127.9, 128.8, 129.0, 130.9, 131.2, 136.0, 141.8, 145.1, 148.0, 149.1, 150.0. MS: m/z (%) 408 (15, [M + 2]⁺), 406 (49, M⁺). Anal. Calcd for C₂₄H₂₇ClN₄: C, 70.83; H, 6.69; N, 13.77. Found: C, 70.97; H, 6.49; N, 13.84.

[2-(2-Chloro-quinolin-3-yl)-8-methyl-imidazo[1,2-a]pyridin-3-yl]-cyclohexyl-amine (6d)

Yield: 0.35 g (91%); pale yellow solid; mp 143–145 °C; IR (KBr): 3327, 2971, 1640, 1568, 781 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 1.01–1.69 (m, 10 H, 5CH₂, cyclohexyl), 2.41 (s, 3H, CH₃), 2.67 (s, 1H, NCH), 3.32 (s, 1H, NH), 6.79 (t, J = 6.4 Hz, 1H, H₆), 7.02 (d, J = 6.4 Hz, 1H, H₇), 7.61 (t, J = 7.6 Hz, 1H, H_6′), 7.78–7.82 (m, 1H, H_7′), 7.92 (d, J = 8.0 Hz, 1H, H_5′), 8.10 (d, J = 8.4 Hz, 1H, H_8′), 8.24 (d, J = 6.4 Hz, 1H, H₅), 8.61 (s, 1H, H_4′); ¹³C NMR (100 MHz, CDCl₃): δ 21.4, 24.6, 25.6, 33.8, 56.7, 111.6, 121.7, 123.4, 125.7, 127.0, 127.1, 127.3, 127.4, 128.4, 129.7, 130.5, 135.6, 140.8, 141.3, 147.1, 149.0. MS: m/z (%) 392 (19, [M + 2]⁺), 390 (58, M⁺). Anal. Calcd for C₂₃H₂₃ClN₄: C, 70.67; H, 5.93; N, 14.33. Found: C, 70.45; H, 6.13; N, 14.12.

[6-Chloro-2-(2-chloro-quinolin-3-yl)-imidazo[1,2-a]pyridin-3-yl]-(1,1,3,3-tetramethyl-butyl)-amine (6k)

Yield: 0.33 g (75%); yellow solid; mp 145–147 °C; IR (KBr): 3274, 2828, 1624, 1607, 1330, 772 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 0.91 (s, 6H, C(CH₃)₂), 0.96 (s, 9H, C(CH₃)₃), 1.48 (s, 2H, CH₂), 3.39 (s, 1H, NH), 7.19 (dd, J = 9.6, 2.0 Hz, 1H, H₇), 7.54 (dd, J = 9.6, 0.8 Hz, 1H, H₈), 7.61–7.65 (m, 1H, H_6′), 7.77–7.81 (m, 1H, H_7′), 7.93 (d, J = 8.0 Hz, 1H, H_5′), 8.11 (d, J = 8.0 Hz, 1H, H_8′), 8.24 (dd, J = 2.0, 0.8 Hz, 1 H, H₅), 8.51 (s, 1H, H_4′); ¹³C NMR (100 MHz, CDCl₃):δ 30.0, 31.5, 31.7, 56.5, 59.2, 118.6, 120.7, 121.0, 126.1, 127.4, 127.7, 127.9, 128.0, 128.5, 129.0, 131.1, 134.9, 140.7, 141.0, 147.4, 149.0. MS: m/z (%) 444 (10, [M + 4]⁺), 442 (58, [M + 2]⁺), 440 (90, M⁺). Anal. Calcd for C₂₄H₂₆Cl₂N₄: C, 65.31; H, 5.94; N, 12.69. Found: C, 65.24; H, 5.83; N, 12.76.

Results and discussion

Chemistry

In the synthetic route to target compounds 6a–o, initially aniline (1) was converted to N-phenylacetamide (2) via the acetylation reaction in the presence of acetyl chloride and potassium carbonate under mild condition (Scheme 1). Then, 2-chloroquinolin-3-carbaldehyde (3) was prepared using the Vilsmeier–Haak reaction in the presence of POCl₃ in DMF21. The final compounds 6a–o were synthesized via one-pot condensation reaction of aldehyde 3, an appropriate isocyanide 5a–c and various heteroaromatic amidine 4a–f, in the presence of catalytic amount of ammonium chloride in toluene. After recrystallization from petroleum ether–EtOAc, pure compounds 6a–o were obtained in 65–93% yields.

Scheme 1. Preparation of 2-chloro-quinoline-based imidazopyridines and imidazothiazoles.

15-LOX inhibitory activity

The inhibitory activity of synthesized compounds was determined22 against 15-LOX, and the obtained IC₅₀ values (IC₅₀ expressed as mean ± SD of three independent experiments) were listed in Table 1. All compounds showed significant inhibitory activity with the IC₅₀ values ≤40 μM. Among them, compound 6n possessing IC₅₀ value of 11.5 μM was the most potent compound. Furthermore, compounds 6g and 6i with IC₅₀ values of 15.3 and 14.1 μM were more active than remaining compounds. As seen in Table 1, compounds 6a–l were imidazopyridine derivatives, and compounds 6m–o had imidazothiazole substructure. The most potent compound 6n was imidazothiazole derivative. On the other hand, the potent compounds 6 g and 6i were imidazopyridine analogs. In the imidazopyridine series, introduction of bromo or chloro substituent at 6-position decreased the inhibitory activity. The effect of methyl group at 7 or 8 position of imidazopyridine ring depended on the alkyl side chain connected to the amine group. For example, while 7-methyl-imidazopyridine derivative 6i bearing a 1,1,3,3-tetramethyl-butylamine residue was more potent than 6c, but 7-methyl-imidazopyridine 6h containing a tert-butyl group found to be as potent as 6b. The comparison of 7- and 8-methyl regioisomers revealed that 7-methyl derivatives 6g and 6i exhibiting more potent activity in respect to their 8-methyl analogs 6d and 6f. In contrast, (7-methyl-imidazopyridin-3-yl)amine 6h was less potent than its 8-methyl regioisomer 6e.

Table 1. Used amidine (4) and isocyanide (5) derivatives for the one-pot synthesis of compounds 6a–o including their 15-Lox inhibitory activity.

Compound	2-amino azines 4	Isocyanide (R) 5	IC50 (μM)
6a	2-amino pyridine	cyclohexyl	21.2 ± 1.0
6b	2-amino pyridine	tert-butyl	27.9 ± 1.3
6c	2-amino pyridine	1,1,3,3-tetramethylbutyl	40.0 ± 1.8
6d	2-amino-3-methyl pyridine	cyclohexyl	26.0 ± 1.2
6e	2-amino-3-methyl pyridine	tert-butyl	21.8 ± 1.0
6f	2-amino-3-methyl pyridine	1,1,3,3-tetramethylbutyl	20.5 ± 0.9
6g	2-amino-4-methyl pyridine	cyclohexyl	15.3 ± 0.7
6h	2-amino-4-methyl pyridine	tert-butyl	28.9 ± 1.3
6i	2-amino-4-methyl pyridine	1,1,3,3-tetramethylbutyl	14.1 ± 0.6
6j	2-amino-5-chloro pyridine	cyclohexyl	44.3 ± 2.0
6k	2-amino-5-chloro pyridine	1,1,3,3-tetramethylbutyl	39.9 ± 1.8
6l	2-amino-5-bromo pyridine	tert-butyl	37.6 ± 1.9
6m	2-aminothiazole	cyclohexyl	40^a
6n	2-aminothiazole	tert-butyl	11.5 ± 0.5
6o	2-aminothiazole	1,1,3,3-tetramethylbutyl	19.8 ± 0.9
Quercetine	–	–	30

^aPercentage (%) of inhibition at 25 μM.

Docking study

The docking study was performed to clarify the binding mode of the target compounds in the active site of 15-LOX. For this purpose, the tested compounds were docked onto the active site of enzyme using Autodock Vina (ver. 1.1.1)23,24. Then, the best docked poses in terms of free binding energy were further analyzed to clarify interactions between ligands and the 15-LOX enzyme. Because of similar orientation of compounds in the active site of 15-LOX, further analysis was performed on the most active compound 6n. As shown in Figure 2, the target compound was laid near the Fe³⁺ion in the 15-LOX active site. In this position, the ligand interacted with Fe³⁺ion through a π-cation interaction via phenyl ring of its 2-chloroquinoline moiety. A careful inspection of the binding pocket indicated that this moiety oriented toward a hydrophobic cavity comprised of Trp519, Ile557, Leu560, Ala561, and Leu565. The ligand also established another remarkable hydrophobic interaction via the orientation of tert-butylamino group toward a hydrophobic pocket including side chains of Leu515, Val571, Ile572, Phe576, and Val769.

Figure 2. The best docked pose of compound 6n in the active site of 15-LOX.

Conclusions

We synthesized a series of 2-chloro-quinoline-based imidazopyridines 6a–l and imidazothiazoles 6m–o bearing a bulky alkylamine side chain as soybean 15-LOX inhibitors. The in vitro evaluation of title compounds against 15-LOX demonstrated that all compounds had significant inhibitory activity (IC₅₀ values ≤40 μM). The most potent compound 6n with IC₅₀ value of 11.5 μM was belong to the imidazo[2,1-b]thiazole series. However, the imidazopyridine derivatives 6g and 6i showed substantial inhibition against 15-LOX (IC₅₀ values ≤15.3 μM). The limited SAR study revealed that the effect of substituent on imidazopyridine ring depended on the attached alkylamine side chain. The docking study indicated that the target compound 6n was laid near the Fe³⁺ion in the 15-LOX active site. A π-cation interaction of 2-chloroquinoline with Fe³⁺ion and hydrophobic interactions had important roles in the favorable binding of the inhibitor with the enzyme active site.

Supporting information

Experimental details and ¹H and ¹³C NMR spectra are available, via the supplementary content section of this article’s web page.

Declaration of interest

The authors report no declarations of interest. This work was supported and funded by Research council of Tehran University of Medical Sciences (TUMS); Grant no: 95-01-92-31756; and Iran National Science Foundation (INSF).

Supplementary materials online only – For review only at proofing stage