The quotient of both intensities for reactions made with eight different inhibitor concentrations was then analyzed using the Quattro Software Suite for IC50-determination. to the position analogous to afatinib (4), permitting the design of compounds 7a-m (Fig.?2). The election of the covalent reactive organizations was based on earlier works describing EGFR inhibition towards reversible and irreversible covalent relationship with cysteine residues35C38. Additionally, chemical reactivity studies and promiscuity profiles of the covalent reactive organizations were also regarded as39,40. Open in a separate window Number 2 Molecular conception of quinoxaline urea derivatives 7a-m designed as EGFR covalent inhibitors. Chemistry Synthesis of the derivatives 7a-m was performed through the synthetic strategy depicted in Fig.?3, employing 7-nitroquinoxaline-2-amine (8) as key intermediate. A simple multi-gram procedure to obtain 8 was developed, using the non-expensive and readily available dedication showed that or substituent in the phenyl group was deleterious for the EGFR inhibition, so attempts to elucidate the binding mode with the enzyme were only implemented with the non-substituted compounds 7h-7l, by means of molecular docking with Platinum 5.4 in the afatinib-containing wt-EGFR structure (PDB code: 4G5J). Compounds 7h, 7i and 7l have Michael acceptor organizations, whereas compounds 7j and 7k have chloride and cyanide in the -carbon to the carbonyl, respectively, which can act as leaving organizations, so that a covalent relationship can be probably created with the Cys797A sulfur atom by all compounds. Initially, simple and covalent docking of the three Micheal acceptor inhibitors were performed to identify possible binding modes that could help in the explanation of the loss of activity of compound 7i compared to the two additional compounds. The ChemPLP fitness function offered the best overall performance both in simple (RMSD equal to 2.81??) and covalent redocking studies (2.50??) based on the 4G5J [51] crystallographic structure. Simple docking studies confirmed the hypothesis that covalent ligands firstly form noncovalent adducts in the ATP binding site before the covalent relationship is formed. It was observed that all compounds possess the same binding mode before the covalent relationship is created (Figs?S1 and S2, supplementary material). Covalent docking studies were performed in the electrophilic -carbon of the carbonyl subunit (compounds 7j and 7k) and at the -carbon of the enone subunit (7h, 7i and 7l). Although molecular docking programs are effective in generating ligand-enzyme connection geometries, the respective scores do not match the experimental activity data so well. For this reason, for compounds 7j and 7k the generated enzyme-inhibitor complexes (Fig.?S3, supplementary material) were then used as input geometries for the calculation with the semi-empirical method PM7 [50] of the reaction enthalpies, which play a significant part in the enzyme-inhibitor complex stability. The results were analyzed from the point of view of the relative reaction enthalpies for the formation of a ligand-enzyme adduct, acquired from the nucleophilic substitution of the cysteine residue (Cys797) in the -carbon of carbonyl subunit (Fig.?4A). As can be seen in Table?2, the reaction enthalpy for the formation of the enzyme-inhibitor complex of 7j is much more favorable than that of 7k, in qualitative accordance with the greater activity of the past. Open in a separate Beta-Cortol window Number 4 Cysteine (Cys797) residue assault scheme in the electrophilic carbon of the -carbon of carbonyl subunit (A) and the enone subunit (B) of the quinoxaline urea derivatives. Table 2 Determined enzyme-inhibitor reaction relative enthalpies (kcal/mol) according to the reaction depicted in Fig.?6 (PM7 method, dielectric constant?=?78.4). 410.2 [M-1]-; 412.2 [M?+?2-1]-. 1-(7-nitroquinoxalin-2-yl)-3-(3-(trifluormethyl)phenyl)urea (9b) Compound 9b was synthetized via condensation of.Purity (HPLC at 254?nm; R.T.): 97.0%; 8.60?moments. 447.0 [M-1]-; 449.0 [M?+?2C1]-. Conversation Molecular design of quinoxaline EGFR inhibitors The molecular design conception was based on the bioisosteric alternative of the quinazoline aromatic ring by a quinoxaline scaffold32, keeping sp2 nitrogen atoms for hydrogen relationship interactions to the hinge region33. Subsequently, the aniline moiety was replaced by a urea subunit. Aiming to explore an eventual covalent connection with EGFR cysteine 797 residue34, different electrophilic subunits were introduced to the position analogous to afatinib (4), permitting the design of compounds 7a-m (Fig.?2). The election of the covalent reactive organizations was based on earlier works describing EGFR inhibition towards reversible and irreversible covalent relationship with cysteine residues35C38. Additionally, chemical reactivity studies and promiscuity profiles of the covalent reactive organizations were also regarded as39,40. Open in a separate window Number 2 Molecular conception of quinoxaline urea derivatives 7a-m designed as EGFR covalent inhibitors. Chemistry Synthesis of the derivatives 7a-m was performed through the synthetic strategy depicted in Fig.?3, employing 7-nitroquinoxaline-2-amine (8) as key intermediate. A simple multi-gram procedure to obtain 8 was developed, using the non-expensive and readily available dedication showed that or substituent in the phenyl group was deleterious for the EGFR inhibition, so attempts to elucidate the binding mode with the enzyme were only implemented with the non-substituted compounds 7h-7l, by means of molecular docking with Platinum 5.4 in the afatinib-containing wt-EGFR structure (PDB code: 4G5J). Compounds 7h, 7i and 7l have Michael acceptor organizations, whereas compounds 7j and 7k have chloride and cyanide in the -carbon to the carbonyl, respectively, which can act as leaving organizations, so that a covalent relationship can be probably formed with the Cys797A sulfur atom by all compounds. Initially, simple and covalent docking of the three Micheal acceptor inhibitors were performed to identify Beta-Cortol possible binding modes that could help in the explanation of the loss of activity of compound 7i compared to the two additional compounds. The ChemPLP fitness function offered the best overall performance both in simple (RMSD equal to 2.81??) and covalent redocking studies (2.50??) based on the 4G5J [51] crystallographic structure. Simple docking Beta-Cortol studies confirmed the hypothesis that covalent ligands firstly form noncovalent adducts in the ATP binding site before the covalent bond is formed. It was observed that all compounds have the same binding mode before the covalent bond is formed (Figs?S1 and S2, supplementary material). Covalent docking studies were performed at the electrophilic -carbon of the carbonyl subunit (compounds 7j and 7k) and at the -carbon of the enone subunit (7h, 7i and 7l). Although molecular docking programs are effective in producing ligand-enzyme conversation geometries, the respective scores do not match the experimental activity data so well. For this reason, for compounds 7j and 7k the generated enzyme-inhibitor complexes (Fig.?S3, supplementary material) were then used as input geometries for the calculation with the semi-empirical method PM7 [50] of the reaction enthalpies, which play a significant role in the enzyme-inhibitor complex stability. The results were analyzed from the point of view of the relative reaction enthalpies for the formation of a ligand-enzyme adduct, obtained by the nucleophilic substitution of the cysteine residue (Cys797) at the -carbon of carbonyl subunit (Fig.?4A). As can be seen in Table?2, the reaction enthalpy for the formation of the enzyme-inhibitor complex of 7j is much more favorable than that of 7k, in qualitative accordance with the greater activity of the former. Open in a separate window Physique 4 Cysteine (Cys797) residue attack scheme at the electrophilic carbon of the -carbon of carbonyl subunit (A) and the enone subunit (B) of the quinoxaline urea derivatives. Table 2 Calculated enzyme-inhibitor reaction relative enthalpies (kcal/mol) according to the reaction depicted in Fig.?6 (PM7 method, dielectric constant?=?78.4). 410.2 [M-1]-; 412.2 [M?+?2-1]-. 1-(7-nitroquinoxalin-2-yl)-3-(3-(trifluormethyl)phenyl)urea (9b) Compound 9b was synthetized via condensation of 8 with 3-(trifluoromethyl)phenyl isocyanate resulting in a salmon powder with 65% yield. m.p..1H NMR (200?MHz, DMSO-d6) (ppm): 10.63 (1H, s), 10.59 (1H, s), 9.16 (1H, s), 8.83 (1H, d, 308.2 [M-1]-. 1-(3-chloro-4-fluorophenyl)-3-(7-nitroquinoxalin-2-yl)urea (9d) Compound 9d was synthetized via condensation of 8 with 3-chloro-4-fluorophenyl isocyanate resulting in a salmon powder with 68% yield. around the bioisosteric replacement of the quinazoline aromatic ring by a quinoxaline scaffold32, maintaining sp2 nitrogen atoms for hydrogen bond interactions to the hinge region33. Subsequently, the aniline moiety was replaced by a urea subunit. Aiming to explore an eventual covalent conversation with EGFR cysteine 797 residue34, different electrophilic subunits were introduced to the position analogous to afatinib (4), allowing the design of compounds 7a-m (Fig.?2). The election of the covalent reactive groups was based on previous works describing EGFR inhibition towards reversible and irreversible covalent bond with cysteine residues35C38. Additionally, chemical reactivity studies and promiscuity profiles of the covalent reactive groups were also considered39,40. Open in a separate window Physique 2 Molecular conception of quinoxaline urea derivatives 7a-m designed as EGFR covalent inhibitors. Chemistry Synthesis of the derivatives 7a-m was performed through the synthetic methodology depicted in Fig.?3, employing 7-nitroquinoxaline-2-amine (8) as key intermediate. A simple multi-gram procedure to obtain 8 was developed, using the non-expensive and readily available determination showed that or substituent at the phenyl group was deleterious for the EGFR inhibition, so attempts to elucidate the binding mode with the enzyme were only implemented with the non-substituted compounds 7h-7l, by means of molecular docking with GOLD 5.4 in the afatinib-containing wt-EGFR structure (PDB code: 4G5J). Compounds 7h, 7i and 7l have Michael acceptor groups, whereas compounds 7j and 7k have chloride and cyanide at the -carbon to the carbonyl, respectively, which can act as leaving groups, so that a covalent bond can be possibly formed with the Cys797A sulfur atom by all compounds. Initially, simple and covalent docking of the three Micheal acceptor inhibitors were performed to identify possible binding modes that could help in the explanation of the loss of activity of compound 7i compared to the two other compounds. The ChemPLP fitness function presented the best performance both in simple (RMSD equal to 2.81??) and covalent redocking studies (2.50??) based on the 4G5J [51] crystallographic structure. Simple docking studies confirmed the hypothesis that covalent ligands firstly form noncovalent adducts in the ATP binding site before the covalent bond is formed. It was observed that all compounds have the same binding mode before the covalent bond is formed (Figs?S1 and S2, supplementary material). Covalent docking studies were performed at the electrophilic -carbon of the carbonyl subunit (compounds 7j and 7k) and at the -carbon of the enone subunit (7h, 7i and 7l). Although molecular docking programs are effective in producing ligand-enzyme conversation geometries, the respective scores do not match the experimental activity data so well. For this reason, for compounds 7j and 7k the generated enzyme-inhibitor complexes (Fig.?S3, supplementary material) were then used as input geometries for the calculation with the semi-empirical method PM7 [50] of the reaction enthalpies, which play a significant role in the enzyme-inhibitor complex stability. The results were analyzed from the point of view of the relative reaction enthalpies for the formation of a ligand-enzyme adduct, obtained by the nucleophilic substitution of the cysteine residue (Cys797) at the -carbon of carbonyl subunit (Fig.?4A). As can be seen in Table?2, the reaction enthalpy for the formation of the enzyme-inhibitor complex of 7j is much more favorable than that of 7k, in qualitative accordance with the greater activity of the former. Open in a separate window Physique 4 Cysteine (Cys797) residue attack scheme at the electrophilic carbon of the -carbon of carbonyl subunit (A) and the enone subunit (B) of the quinoxaline urea derivatives. Table 2 Calculated enzyme-inhibitor reaction relative enthalpies (kcal/mol) according to the reaction depicted in Fig.?6 (PM7 method, dielectric constant?=?78.4). 410.2 [M-1]-; COL4A3BP 412.2 [M?+?2-1]-. 1-(7-nitroquinoxalin-2-yl)-3-(3-(trifluormethyl)phenyl)urea (9b) Compound 9b was synthetized via condensation of 8 with 3-(trifluoromethyl)phenyl isocyanate resulting in a salmon powder with 65% yield. m.p. was 250C252?C. 1H NMR.
