Synthesis, structural and thermal studies of 3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-ethoxy-1H-indole (D2AAK1_3) as dopamine D2 receptor ligand

molecules Article Synthesis, Structural and Thermal Studies of 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5- ethoxy-1H-indole (D2AAK1_3) as Dopamine D2 Receptor Ligand Magda Kondej 1, Agata Bartyzel 2ID , Monika Pitucha 3, Tomasz M. Wróbel 1,4 ID , Andrea G. Silva 5, Dariusz Matosiuk 1, Marián Castro 5ID and Agnieszka A. Kaczor 1,6,* 1 Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Laboratory, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, 4A Chod´zki St., PL-20093 Lublin, Poland; [email protected] (M.K.); [email protected] (T.M.W.); [email protected] (D.M.) 2Department of General and Coordination Chemistry, Maria Curie-Skłodowska University, M. Curie-Skłodowskiej Sq. 2, PL-20031 Lublin, Poland; [email protected] 3 Independent Radiopharmacy Unit, Department of Organic Chemistry, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, 4A Chod´zki St., PL-20093 Lublin, Poland; [email protected] 4Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100 Copenhagen Ø, Denmark 5Department of Pharmacology, Universidade de Santiago de Compostela, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Avda de Barcelona, E-15782 Santiago de Compostela, Spain; andrea.gar[email protected] (A.G.S.); marian.castr[email protected] (M.C.) 6School of Pharmacy, University of Eastern Finland, Yliopistonranta 1, P.O. Box 1627, FI-70211 Kuopio, Finland *Correspondence: [email protected]; Tel.: +48-81-448-72-73 Received: 26 July 2018; Accepted: 31 August 2018; Published: 4 September 2018   Abstract: Compound D2AAK1_3 was designed as a modification of the lead structure D2AAK1 (an in vivo active multi-target compound with nanomolar affinity to a number of aminergic GPCRs) and synthesized in the reaction of 5-ethoxyindole and 1-benzyl-4-piperidone. This compound has an affinity to the human dopamine D 2 receptor with K i of 151 nM. The aim of these studies was the structural and thermal characterization of the compound D2AAK1_3. In particular; X-ray studies; molecular docking and molecular dynamics as well as thermal analysis were performed. The studied compound crystallizes in orthorhombic system; in chiral space group P2 1 2 1 2 1 . The compound has a non-planar conformation. The studied compound was docked to the novel X-ray structure of the human dopamine D 2 receptor in the inactive state (PDB ID: 6CM4) and established the main contact between its protonatable nitrogen atom and Asp (3.32) of the receptor. The obtained binding pose was stable in molecular dynamics simulations. Thermal stability of the compound was investigated using the TG-DSC technique in the air atmosphere, while TG-FTIR analyses in air and nitrogen atmospheres were also performed. The studied compound is characterized by good thermal stability. The main volatile products of combustion are the following gases: CO 2 ; H 2 O toluene and CO while in the case of pyrolysis process in the FTIR spectra; the characteristic bands of NH 3 ; piperidine and indole are additionally observed. Keywords: dopamine D 2 receptor; dopamine D 2 receptor antagonist; molecular modeling; thermal studies; X-ray studies Molecules 2018,23, 2249; doi:10.3390/molecules23092249 www.mdpi.com/journal/molecules Molecules 2018,23, 2249 2 of 17 1. Introduction Dopamine receptors belong to rhodopsin-like, aminergic G protein-coupled receptors (GPCRs) and play a crucial role in the central nervous system. There are five subtypes of dopamine receptors (D 1 , D 2 , D 3 , D 4 and D 5 ) which can be divided into the two groups D 1 -like (D 1 and D 5 ) and D 2 -like (D 2 , D 3 and D 4 ) depending on activation or inhibition of a secondary messenger, cyclic adenosine monophosphate (cAMP) [ 1 ]. Dopamine receptors are important for the pathomechanism of Parkinson’s disease [ 2 ], Huntington’s disease, schizophrenia [ 3 ], depression, bipolar disorder, sexual disorders, dementia etc. [ 4 ]. As a consequence, the dopaminergic system has been useful for over a half of century for the pharmaceutical industry, which resulted in a number of dopaminergic prodrugs, agonists, antagonists and enzyme inhibitors being involved in the synthesis or metabolism of dopamine. In particular, targeting the dopamine D 2 receptors is an established approach for the treatment of schizophrenia, which is in accordance with dopaminergic hypothesis of this disease [ 5 ]. First, second and third generation antipsychotics are currently available on the market and all of these are either dopamine D 2 receptor antagonists (with additional affinity to other dopamine receptor subtypes as well as to serotonin receptors, in particular 5-HT 2A receptors) or partial agonists/biased ligands of the dopamine D2receptor. Arylpiperazines [ 6 – 8 ] and to the lesser extent arylpiperidines [ 9 , 10 ] and aryltetrahydropyridines [ 9 ] have been reported as privileged structures for aminergic GPCRs, in particular serotonin and dopamine receptors. Moreover, aryltetrahydropyridines are known as ligands of other targets, e.g., σ receptor ligands [ 11 ] as well as peroxisome proliferators-activated receptors (PPARs, belonging to nuclear receptor family) agonists with antidiabetic activity and monoamine oxidase B (MAO-B) inhibitors [12,13]. In the search for novel potential antipsychotics, we performed structure-based virtual screening aimed at identifying dopamine D 2 receptor antagonists [ 14 ]. We identified 10 potentially interesting compounds and four of them, i.e., D2AAK1 possessing aryltetrahydropyridine scaffold [ 15 ], D2AAK2, D2AAK3 and D2AAK4 were subjected to detailed in vitro , in silico and in vivo investigation. In particular D2AAK1 (Figure 1) is a promising multi-target lead structure with nanomolar affinity to a number of dopamine and serotonin receptors with antipsychotic, anxiolytic and procognitive activity in vivo [ 15 ]. We designed a modification of D2AAK1, i.e., D2AAK1_3 (Figure 1) and performed detailed experimental and computational structural as well as thermal studies for this compound. The rationale of the presented study can be summarized as follows: (1) urgent need to design more efficient and safer antipsychotics; (2) promising in vitro and in vivo results for the lead structure D2AAK1; (3) describing ligand-receptor interactions at the molecular level to design more potent compounds; (4) significance of ligand X-ray structure to search for its bioactive conformation and (5) importance of thermal studies for characterization of the compound as a potential drug. Molecules 2018, 23, x 2 of 17 1. Introduction Dopamine receptors belong to rhodopsin-like, aminergic G protein-coupled receptors (GPCRs) and play a crucial role in the central nervous system. There are five subtypes of dopamine receptors (D 1 , D 2 , D 3 , D 4 and D 5 ) which can be divided into the two groups D 1 -like (D 1 and D 5 ) and D 2 -like (D 2 , D 3 and D 4 ) depending on activation or inhibition of a secondary messenger, cyclic adenosine monophosphate (cAMP) [1]. Dopamine receptors are important for the pathomechanism of Parkinson’s disease [2], Huntington’s disease, schizophrenia [3], depression, bipolar disorder, sexual disorders, dementia etc. [4]. As a consequence, the dopaminergic system has been useful for over a half of century for the pharmaceutical industry, which resulted in a number of dopaminergic prodrugs, agonists, antagonists and enzyme inhibitors being involved in the synthesis or metabolism of dopamine. In particular, targeting the dopamine D 2 receptors is an established approach for the treatment of schizophrenia, which is in accordance with dopaminergic hypothesis of this disease [5]. First, second and third generation antipsychotics are currently available on the market and all of these are either dopamine D 2 receptor antagonists (with additional affinity to other dopamine receptor subtypes as well as to serotonin receptors, in particular 5-HT 2A receptors) or partial agonists/biased ligands of the dopamine D 2 receptor. Arylpiperazines [6–8] and to the lesser extent arylpiperidines [9,10] and aryltetrahydropyridines [9] have been reported as privileged structures for aminergic GPCRs, in particular serotonin and dopamine receptors. Moreover, aryltetrahydropyridines are known as ligands of other targets, e.g., σ receptor ligands [11] as well as peroxisome proliferators-activated receptors (PPARs, belonging to nuclear receptor family) agonists with antidiabetic activity and monoamine oxidase B (MAO-B) inhibitors [12,13]. In the search for novel potential antipsychotics, we performed structure-based virtual screening aimed at identifying dopamine D 2 receptor antagonists [14]. We identified 10 potentially interesting compounds and four of them, i.e., D2AAK1 possessing aryltetrahydropyridine scaffold [15], D2AAK2, D2AAK3 and D2AAK4 were subjected to detailed in vitro, in silico and in vivo investigation. In particular D2AAK1 (Figure 1) is a promising multi-target lead structure with nanomolar affinity to a number of dopamine and serotonin receptors with antipsychotic, anxiolytic and procognitive activity in vivo [15]. We designed a modification of D2AAK1, i.e., D2AAK1_3 (Figure 1) and performed detailed experimental and computational structural as well as thermal studies for this compound. The rationale of the presented study can be summarized as follows: (1) urgent need to design more efficient and safer antipsychotics; (2) promising in vitro and in vivo results for the lead structure D2AAK1; (3) describing ligand-receptor interactions at the molecular level to design more potent compounds; (4) significance of ligand X-ray structure to search for its bioactive conformation and (5) importance of thermal studies for characterization of the compound as a potential drug. Figure 1. The investigated compound D2AAK1_3 as a modification of the lead structure D2AAK1. Figure 1. The investigated compound D2AAK1_3 as a modification of the lead structure D2AAK1. Molecules 2018,23, 2249 3 of 17 2. Results and Discussion 2.1. Chemistry Compound D2AAK1_3 was synthesized from 5-ethoxyindole and 1-benzyl-4-piperidone in methanol/KOH (see Scheme 1) following previously reported methodology [16]. Molecules 2018, 23, x 3 of 17 2. Results and Discussion 2.1. Chemistry Compound D2AAK1_3 was synthesized from 5-ethoxyindole and 1-benzyl-4-piperidone in methanol/KOH (see Scheme 1) following previously reported methodology [16]. Scheme 1. Synthesis of D2AAK1_3. Compound D2AAK1_3 is a new compound and it is an analogue of known 3-(1-benzyl-1,2,3,6- tetrahydropyridin-4-yl)-1H-indole and 3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-methoxy-1H- indole. 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indole was reported as 5-HT 1A and 5-HT 2A receptor ligand (with pKi of 5.00 and 7.81, respectively) [17], intermediate in synthesis of N 1 -arylsulfonyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives as 5-HT 6 receptor antagonists [18], intermediate in synthesis of muscarinic receptor allosteric agents [19] and antimalarial compound [20]. 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-methoxy-1H-indole was reported as 5-HT 7 receptor ligand [21] and intermediate in synthesis of muscarinic receptor allosteric agents [19]. In the IR spectra of D2AAK1_3 the medium intense band at 3225 cm −1 can be assigned to ν (N-H) vibrations of the pyrrole ring [22]. The band is broad and shifted to lower frequencies which can indicate that those atoms are involved in formation of hydrogen bond. This was confirmed by structural analysis. The bands at 1344 cm −1 and 1206 cm −1 can be assigned to stretching and deformation vibration of the C-N group in the tetrahydropyridine ring system [23]. 2.2. Affinity to the Dopamine D 2 Receptor D2AAK1_3 fully displaced the specific radioligand binding at D 2 receptors in a concentration-dependent manner in competition binding experiments (Figure 2). The affinity of D2AAK1_3 to human dopamine D 2 receptor (K i ) resulting from these experiments was equal to 151 nM (pK i = 6.82 ± 0.05). Figure 2. Competition binding curve for D2AAK1_3 at human D 2 receptors. Displacement of the specific binding of the radioligand [ 3 H]Spiperone by D2AAK1_3 in membranes from CHO-K1 cells stably expressing human cloned D 2 receptors. Haloperidol was used as the reference compound in these assays. The graph shows the results (mean ± SEM) from a single experiment representative of 2 independent experiments performed in duplicate. Scheme 1. Synthesis of D2AAK1_3. Compound D2AAK1_3 is a new compound and it is an analogue of known 3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indole and 3-(1-benzyl-1,2,3,6-tetrahydropyridin- 4-yl)-5-methoxy-1H-indole. 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indole was reported as 5-HT 1A and 5-HT 2A receptor ligand (with pKi of 5.00 and 7.81, respectively) [ 17 ], intermediate in synthesis of N 1 -arylsulfonyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives as 5-HT 6 receptor antagonists [ 18 ], intermediate in synthesis of muscarinic receptor allosteric agents [ 19 ] and antimalarial compound [ 20 ]. 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-methoxy-1H-indole was reported as 5-HT7receptor ligand [21] and intermediate in synthesis of muscarinic receptor allosteric agents [19]. In the IR spectra of D2AAK1_3 the medium intense band at 3225 cm −1 can be assigned to ν (N-H) vibrations of the pyrrole ring [ 22 ]. The band is broad and shifted to lower frequencies which can indicate that those atoms are involved in formation of hydrogen bond. This was confirmed by structural analysis. The bands at 1344 cm −1 and 1206 cm −1 can be assigned to stretching and deformation vibration of the C-N group in the tetrahydropyridine ring system [23]. 2.2. Affinity to the Dopamine D2Receptor D2AAK1_3 fully displaced the specific radioligand binding at D 2 receptors in a concentration-dependent manner in competition binding experiments (Figure 2). The affinity of D2AAK1_3 to human dopamine D 2 receptor (K i ) resulting from these experiments was equal to 151 nM (pKi= 6.82 ±0.05). Molecules 2018, 23, x 3 of 17 2. Results and Discussion 2.1. Chemistry Compound D2AAK1_3 was synthesized from 5-ethoxyindole and 1-benzyl-4-piperidone in methanol/KOH (see Scheme 1) following previously reported methodology [16]. Scheme 1. Synthesis of D2AAK1_3. Compound D2AAK1_3 is a new compound and it is an analogue of known 3-(1-benzyl-1,2,3,6- tetrahydropyridin-4-yl)-1H-indole and 3-(1-benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-methoxy-1H- indole. 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indole was reported as 5-HT 1A and 5-HT 2A receptor ligand (with pKi of 5.00 and 7.81, respectively) [17], intermediate in synthesis of N 1 -arylsulfonyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole derivatives as 5-HT 6 receptor antagonists [18], intermediate in synthesis of muscarinic receptor allosteric agents [19] and antimalarial compound [20]. 3-(1-Benzyl-1,2,3,6-tetrahydropyridin-4-yl)-5-methoxy-1H-indole was reported as 5-HT 7 receptor ligand [21] and intermediate in synthesis of muscarinic receptor allosteric agents [19]. In the IR spectra of D2AAK1_3 the medium intense band at 3225 cm −1 can be assigned to ν (N-H) vibrations of the pyrrole ring [22]. The band is broad and shifted to lower frequencies which can indicate that those atoms are involved in formation of hydrogen bond. This was confirmed by structural analysis. The bands at 1344 cm −1 and 1206 cm −1 can be assigned to stretching and deformation vibration of the C-N group in the tetrahydropyridine ring system [23]. 2.2. Affinity to the Dopamine D 2 Receptor D2AAK1_3 fully displaced the specific radioligand binding at D 2 receptors in a concentration-dependent manner in competition binding experiments (Figure 2). The affinity of D2AAK1_3 to human dopamine D 2 receptor (K i ) resulting from these experiments was equal to 151 nM (pK i = 6.82 ± 0.05). Figure 2. Competition binding curve for D2AAK1_3 at human D 2 receptors. Displacement of the specific binding of the radioligand [ 3 H]Spiperone by D2AAK1_3 in membranes from CHO-K1 cells stably expressing human cloned D 2 receptors. Haloperidol was used as the reference compound in these assays. The graph shows the results (mean ± SEM) from a single experiment representative of 2 independent experiments performed in duplicate. Figure 2. Competition binding curve for D2AAK1_3 at human D 2 receptors. Displacement of the specific binding of the radioligand [ 3 H]Spiperone by D2AAK1_3 in membranes from CHO-K1 cells stably expressing human cloned D 2 receptors. Haloperidol was used as the reference compound in these assays. The graph shows the results (mean ± SEM) from a single experiment representative of 2 independent experiments performed in duplicate. Molecules 2018,23, 2249 4 of 17 2.3. X-ray Analysis The molecular structure of compound D2AAK1_3 is displayed in Figure 3. The crystallographic and refinement data for the compound are summarized in Table 1, and selected bond distances and angles are listed in Table 2, while hydrogen and π-ring interactions parameters are given in Table 3. Molecules 2018, 23, x 4 of 17 2.3. X-ray Analysis The molecular structure of compound D2AAK1_3 is displayed in Figure 3. The crystallographic and refinement data for the compound are summarized in Table 1, and selected bond distances and angles are listed in Table 2, while hydrogen and π-ring interactions parameters are given in Table 3. Figure 3. Molecular structures with atom numbering scheme of D2AAK1_3. Displacement ellipsoids are shown at the 30% probability level. Table 1. Crystal data and structure refinement of D2AAK1_3. Formula C22H24N2O Formula weight 332.43 Temperature K 293(2) Crystal system orthorhombic Space group P212121 a (Å) 5.9600(4) b (Å) 15.0986(9) c (Å) 16.1571(9) Volume (Å3) 1814.5(2) Z 4 Calculated density (g cm−3) 1.217 μ (mm−1) 0.075 Absorption correction multi-scan F(000) 712 Crystal size (mm) 0.50 × 0.20 × 0.20 θ range (°) 2.698 to 27.103 Index ranges −7 ≤ h ≤ 7 −19 ≤ k ≤ 19 −25 ≤ l ≤ 18 Reflections collected/unique 14,278/3993 Rint 0.0442 Data/restraints/parameters 3993/0/231 GooF on F2 1.000 Final R indices[I > 2σ(I)] R1 = 0.0437, wR2 = 0.0887 R indices(all data) R1 = 0.0745, wR2 = 0.1019 Largest diff. peak/hole, e Å−3 0.156/−0.111 Figure 3. Molecular structures with atom numbering scheme of D2AAK1_3. Displacement ellipsoids are shown at the 30% probability level. Table 1. Crystal data and structure refinement of D2AAK1_3. Formula C22H24N2O Formula weight 332.43 Temperature K 293(2) Crystal system orthorhombic Space group P212121 a (Å) 5.9600(4) b (Å) 15.0986(9) c (Å) 16.1571(9) Volume (Å3) 1814.5(2) Z 4 Calculated density (g cm−3) 1.217 µ(mm−1) 0.075 Absorption correction multi-scan F(000) 712 Crystal size (mm) 0.50 ×0.20 ×0.20 θrange (◦) 2.698 to 27.103 Index ranges −7≤h≤7−19 ≤k≤19−25 ≤l≤18 Reflections collected/unique 14,278/3993 Rint 0.0442 Data/restraints/parameters 3993/0/231 GooF on F21.000 Final R indices[I > 2σ(I)] R1= 0.0437, wR2= 0.0887 R indices(all data) R1= 0.0745, wR2= 0.1019 Largest diff. peak/hole, e Å−30.156/−0.111 Molecules 2018,23, 2249 5 of 17 Table 2. Interatomic distances and selected bond angles. Bond Lengths (Å) C1-N1 1.359(3) C10-C11 1.364(5) C1-C2 1.369(3) C11-C12 1.376(4) C2-C15 1.441(3) C13-N2 1.462(3) C2-C3 1.465(3) C13-C14 1.497(3) C3-C14 1.325(3) C15-C22 1.406(3) C3-C4 1.506(3) C15-C16 1.409(3) C4-C5 1.512(3) C16-C17 1.373(4) C5-N2 1.459(3) C17-O1 1.380(3) C6-N2 1.472(3) C17-C20 1.392(4) C6-C7 1.503(3) C18-O1 1.416(3) C7-C8 1.376(3) C18-C19 1.493(4) C7-C12 1.383(3) C20-C21 1.366(4) C8-C9 1.379(4) C21-C22 1.386(4) C9-C10 1.360(4) C22-N1 1.376(3) Bond angles (◦) C14-C3-C2 124.6(2) C3-C14-C13 124.1(2) C14-C3-C4 118.6(2) C16-C17-O1 124.3(2) C2-C3-C4 116.7(2) O1-C17-C20 114.6(2) C3-C4-C5 113.0 (2) C5-N2-C13 108.4(2) N2-C5-C4 111.0(2) C5-N2-C6 111.2(2) N2-C6-C7 114.1(2) C13-N2-C6 107.7(2) N2-C13-C14 113.1(2) C17-O1-C18 118.0(2) Torsion angles (◦) C4-C5-N2-C13 −65.0(3) C4-C3-C14-C13 0.6(4) C3-C4-C5-N2 47.6(3) N2-C13-C14-C3 −18.2(4) C14-C3-C4-C5 −15.0(3) C14-C13-N2-C5 49.2(3) Table 3. Hydrogen bonding and C-H···Cg interactions geometry. Hydrogen Bond [Å, ◦] D-H···A d(D-H) d(H···A) d(D···A) ∠DHA N1-H1N···N2 i0.88(3) 2.41(3) 3.217(3) 154(2) C-H···Cg interactions [Å, ◦] C-H···Cg d(D-H) d(H···Cg) d(C···Cg) ∠CHCg C4-H4B···Cg1ii 0.97 2.94 3.567(3) 124 C19-H19C···Cg4iii 0.96 3.00 3.799(3) 142 Symmetry codes: ( i ) x − 1/2, − y + 1/2, − z + 1; ( ii ) x − 1/2, − y + 1/2, − z + 1; ( iii ) x + 1, y, z; Cg1 and Cg4 are the centroids of the N1/C1/C2/C15/C22 and C15/C16/C17/C20/C21/C22 rings, respectively. The compound crystallizes in noncentrosymmetric space group P2 1 2 1 2 1 with one molecule in an asymmetric unit. The interatomic distances and angles agree with those described in the literature [ 24 ] and are comparable with those observed for the other closely related indole derivatives [ 25 – 27 ]. The molecule features a disubstituted indole molecule with an ethoxy group connected to a phenyl ring and a 1-benzyl-1,2,3,6-tetrahydropyridin-4yl group linked to a pyrrole ring. The indole ring of the compound is planar with a maximum deviation of 0.019(5) Å of C2 atom from the best plane. The plane formed by O-C-C atoms of ethoxy group is coplanar with the indole moiety (the angle between the two planes is only 0.34(12) ◦ ), while the tetrahydropyridine and phenyl rings are significantly rotated (interplanar angles equal 16.37(10)◦and 85.57(9)◦, respectively). The tetrahydropyridine ring system adopts a half-chair conformation with an N2 atom above (0.326(2) Å) and a C5 atom below ( − 0.317(3) Å) the mean plane defined by N2/C5/C4/C3/C14/C13 atoms. This is confirmed by the Molecules 2018,23, 2249 6 of 17 puckering parameters Q = 0.499(2) Å, θ = 49.6(2) ◦ and ϕ = 27.6(4) ◦ [ 28 – 31 ]. In the structure, the N1 atom participates in an intramolecular hydrogen bond (N1-H1N ··· N2 i , [symmetry code: (i) x − 1/2, − y + 1/2, − z + 1]), as shown in Table 3. As results of these interactions, one-dimensional columns lying parallel to the a-axis (Figure 4) are formed. The crystal structure is also stabilized by intermolecular C-H···πinteractions (Table 3). Molecules 2018, 23, x 6 of 17 N2/C5/C4/C3/C14/C13 atoms. This is confirmed by the puckering parameters Q = 0.499(2) Å, θ = 49.6(2)° and φ = 27.6(4)° [28–31]. In the structure, the N1 atom participates in an intramolecular hydrogen bond (N1-H1N···N2 i , [symmetry code: (i) x − 1/2, −y + 1/2, −z + 1]), as shown in Table 3. As results of these interactions, one-dimensional columns lying parallel to the a-axis (Figure 4) are formed. The crystal structure is also stabilized by intermolecular C-H···π interactions (Table 3). Figure 4. Fragment of the crystal structure of D2AAK1_3 showing formation of one-dimensional columns viewed along the a-axis. 2.4. Molecular Modeling In order to study ligand-receptor interactions on the molecular level, both the lead structure D2AAK1 and its derivative D2AAK1_3 were docked to the binding pocket of the human dopamine D 2 receptor (X-ray structure, PDB ID: 6CM4). The docking pose of the lead structure D2AAK1 (Figure 5A,B) is similar to the previously reported docking pose of this compound in the homology Figure 4. Fragment of the crystal structure of D2AAK1_3 showing formation of one-dimensional columns viewed along the a-axis. 2.4. Molecular Modeling In order to study ligand-receptor interactions on the molecular level, both the lead structure D2AAK1 and its derivative D2AAK1_3 were docked to the binding pocket of the human dopamine D 2 receptor (X-ray structure, PDB ID: 6CM4). The docking pose of the lead structure D2AAK1 (Figure 5A,B) is similar to the previously reported docking pose of this compound in the Molecules 2018,23, 2249 7 of 17 homology model of the D 2 receptor [ 14 , 15 ]. The main contact of D2AAK1 is the interaction between its protonatable nitrogen atom and Asp114 (3.32) [numbers in brackets are according to Ballesteros-Weinstein nomenclature] [ 32 ]. The ligand also forms π - π stacking interactions with Trp386 (6.48) and Phe390 (6.52). The investigated derivative of the lead structure, D2AAK1_3 adopts a similar binding pose to its parent compound, with the benzyl moiety replacing 1,2-dihydroquinolin-2-one scaffold (Figure 5C,D). The positions of alkoxyindole moieties are slightly different for both compounds. The ligand forms the same contacts as the lead structure: Its protonatable nitrogen atom interacts with Asp114 (3.32) and there are π - π stacking interactions with Trp386 (6.48) and Phe390 (6.52). Importantly, the dopamine D 2 receptor binding site has enough space to accommodate 5-ethoxyindole moiety, which can be important for further optimization of the lead structure. Molecules 2018, 23, x 7 of 17 model of the D 2 receptor [14,15]. The main contact of D2AAK1 is the interaction between its protonatable nitrogen atom and Asp114 (3.32) [numbers in brackets are according to Ballesteros-Weinstein nomenclature] [32]. The ligand also forms π-π stacking interactions with Trp386 (6.48) and Phe390 (6.52). The investigated derivative of the lead structure, D2AAK1_3 adopts a similar binding pose to its parent compound, with the benzyl moiety replacing 1,2-dihydroquinolin-2-one scaffold (Figure 5C,D). The positions of alkoxyindole moieties are slightly different for both compounds. The ligand forms the same contacts as the lead structure: Its protonatable nitrogen atom interacts with Asp114 (3.32) and there are π-π stacking interactions with Trp386 (6.48) and Phe390 (6.52). Importantly, the dopamine D 2 receptor binding site has enough space to accommodate 5-ethoxyindole moiety, which can be important for further optimization of the lead structure. Figure 5. The lead structure D2AAK1 (A,B) and its derivative D2AAK1_3 (C,D) in the binding pocket of human dopamine D 2 receptor. (A,C) 3D view of the binding site. Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with grey carbon atoms, main interacting residues shown as sticks. Hydrogen bonds shown as yellow dashed lines. Non-polar hydrogen atoms not shown for clarity. (B,D) 2D view of the binding site. Figure 5. The lead structure D2AAK1 ( A , B ) and its derivative D2AAK1_3 ( C , D ) in the binding pocket of human dopamine D 2 receptor. ( A , C ) 3D view of the binding site. Ligands represented as sticks with magenta carbon atoms. Protein represented as wire with grey carbon atoms, main interacting residues shown as sticks. Hydrogen bonds shown as yellow dashed lines. Non-polar hydrogen atoms not shown for clarity. (B,D) 2D view of the binding site. Molecules 2018,23, 2249 8 of 17 100 ns molecular dynamics simulations were performed to investigate the stability of D2AAK1_3 complex with the human dopamine D 2 receptor. The decreasing value of potential energy (Figure 6A) and ligand RMSD below 2 Å (Figure 6B) indicate that the simulations were performed correctly and the ligand-receptor complex is stable. Molecules 2018, 23, x 8 of 17 100 ns molecular dynamics simulations were performed to investigate the stability of D2AAK1_3 complex with the human dopamine D 2 receptor. The decreasing value of potential energy (Figure 6A) and ligand RMSD below 2 Å (Figure 6B) indicate that the simulations were performed correctly and the ligand-receptor complex is stable. Figure 6. Changes in potential energy (A) and ligand RMSD (B) during 100 ns molecular dynamics simulations for D2AAK1_3 in complex with human dopamine D 2 receptor. In order to study D2AAK1_3 interactions with the human dopamine D 2 receptor during 100 ns molecular dynamics simulations, histogram of interactions was generated (Figure 7A). It can be seen that the ligand maintains the interactions with Asp114 (3.32) during 100% of simulations mainly through a hydrogen bond and to the lesser extent, ionic interaction and water-mediated contact. Moreover, hydrophobic contacts are kept between the investigated ligand and the receptor for Trp386 (6.48) during 100% of simulations, Phe389 (6.51) during 70% of simulations, Cys118 (3.36) for 65% of simulations, Phe390 (6.52) during 60% of simulations and Phe189 (5.39) for 40% of simulations. Ile184 from the third extracellular loop is also important for ligand-receptor interactions and maintains hydrophobic contact with the ligand for 30% of simulations and water-mediated interaction for 10% of simulations. Figure 7B summarizes the main ligand-receptor contacts during 100 ns simulations that occur for over 30% of simulations, i.e., Asp114 (3.32), Trp386 (6.48), Phe389 (6.51) and Phe390 (6.52). Figure 7. Molecular interactions of D2AAK1_3 with human dopamine D 2 receptor during 100 ns molecular dynamics simulations. (A) histogram of interactions. The stacked bar charts are normalized over the course of the trajectory: For example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. (B) summary of contacts. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. Figure 6. Changes in potential energy ( A ) and ligand RMSD ( B ) during 100 ns molecular dynamics simulations for D2AAK1_3 in complex with human dopamine D2receptor. In order to study D2AAK1_3 interactions with the human dopamine D 2 receptor during 100 ns molecular dynamics simulations, histogram of interactions was generated (Figure 7A). It can be seen that the ligand maintains the interactions with Asp114 (3.32) during 100% of simulations mainly through a hydrogen bond and to the lesser extent, ionic interaction and water-mediated contact. Moreover, hydrophobic contacts are kept between the investigated ligand and the receptor for Trp386 (6.48) during 100% of simulations, Phe389 (6.51) during 70% of simulations, Cys118 (3.36) for 65% of simulations, Phe390 (6.52) during 60% of simulations and Phe189 (5.39) for 40% of simulations. Ile184 from the third extracellular loop is also important for ligand-receptor interactions and maintains hydrophobic contact with the ligand for 30% of simulations and water-mediated interaction for 10% of simulations. Figure 7B summarizes the main ligand-receptor contacts during 100 ns simulations that occur for over 30% of simulations, i.e., Asp114 (3.32), Trp386 (6.48), Phe389 (6.51) and Phe390 (6.52). Molecules 2018, 23, x 8 of 17 100 ns molecular dynamics simulations were performed to investigate the stability of D2AAK1_3 complex with the human dopamine D 2 receptor. The decreasing value of potential energy (Figure 6A) and ligand RMSD below 2 Å (Figure 6B) indicate that the simulations were performed correctly and the ligand-receptor complex is stable. Figure 6. Changes in potential energy (A) and ligand RMSD (B) during 100 ns molecular dynamics simulations for D2AAK1_3 in complex with human dopamine D 2 receptor. In order to study D2AAK1_3 interactions with the human dopamine D 2 receptor during 100 ns molecular dynamics simulations, histogram of interactions was generated (Figure 7A). It can be seen that the ligand maintains the interactions with Asp114 (3.32) during 100% of simulations mainly through a hydrogen bond and to the lesser extent, ionic interaction and water-mediated contact. Moreover, hydrophobic contacts are kept between the investigated ligand and the receptor for Trp386 (6.48) during 100% of simulations, Phe389 (6.51) during 70% of simulations, Cys118 (3.36) for 65% of simulations, Phe390 (6.52) during 60% of simulations and Phe189 (5.39) for 40% of simulations. Ile184 from the third extracellular loop is also important for ligand-receptor interactions and maintains hydrophobic contact with the ligand for 30% of simulations and water-mediated interaction for 10% of simulations. Figure 7B summarizes the main ligand-receptor contacts during 100 ns simulations that occur for over 30% of simulations, i.e., Asp114 (3.32), Trp386 (6.48), Phe389 (6.51) and Phe390 (6.52). Figure 7. Molecular interactions of D2AAK1_3 with human dopamine D 2 receptor during 100 ns molecular dynamics simulations. (A) histogram of interactions. The stacked bar charts are normalized over the course of the trajectory: For example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. (B) summary of contacts. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. Figure 7. Molecular interactions of D2AAK1_3 with human dopamine D 2 receptor during 100 ns molecular dynamics simulations. ( A ) histogram of interactions. The stacked bar charts are normalized over the course of the trajectory: For example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. ( B ) summary of contacts. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. Molecules 2018,23, 2249 9 of 17 2.5. Thermal Analysis Thermal analyses of the investigated compound were performed using the TG-DSC (air) and TG-FTIR (air and nitrogen) techniques (Figures 8and 9). Molecules 2018, 23, x 9 of 17 2.5. Thermal Analysis Thermal analyses of the investigated compound were performed using the TG-DSC (air) and TG-FTIR (air and nitrogen) techniques (Figures 8 and 9). Figure 8. (A) TG, DTG and DSC curves of the studied compound recorded in air atmosphere using cylindrical shape crucible; (B) TG and DTG curves of the studied compound recorded in air atmosphere using used flat platinum plate (heating rate 10 °C min−1). Figure 9. TG and DTG curves of D2AAK1_3 recording during thermal analysis with heating rate 20 °C min−1 in air (A) and N2 (B) atmospheres. The TG-DSC curves providing information about the thermal properties of D2AAK1_3 are shown in Figure 8A. As it can be seen in the TG curves, the compound is stable at room temperature, which is a very important parameter for its potential application as a drug. During the heating, a first change is recorded on the DSC curve as an endothermic peak (Tonset = 151 °C, Tpeak = 154 °C). This peak is not accompanied by a mass loss and can be attributed to the melting process. It is sharp, indicating that the compound was synthesized as pure, crystalline substance [22,33]. The enthalpy of fusion calculated from the DSC peak is 25.96 ± 0.93 kJ mol−1. Further heating leads to thermal decomposition of D2AAK1_3, which roughly occurs in two stages recorded on a TG curve. The first step of compound thermal degradation is observed at 212–390 °C and 54.43% of the sample is combusted. It is worth mentioning that on the DTG curve, two maxima (at 295 °C and 336 °C) are visible during this stage, but this is not clearly indicated on the TG curve. The formed products are unstable and immediately undergo further decomposition and combustion. The sample fully decomposes at a temperature of approximately 650 °C. In order to better understand the mechanism of the thermal decomposition process, the TG-FTIR analysis of D2AAK1_3 in oxidizing as well as inert atmosphere was performed (see Figure 9). As it can be seen from Figure 9A, the process performed at air atmosphere also occurs in two stages, but the different heating rate (20 °C min−1), a larger sample mass and a shape of the crucibles affected the temperature of the Figure 8. ( A ) TG, DTG and DSC curves of the studied compound recorded in air atmosphere using cylindrical shape crucible; ( B ) TG and DTG curves of the studied compound recorded in air atmosphere using used flat platinum plate (heating rate 10 ◦C min−1). Molecules 2018, 23, x 9 of 17 2.5. Thermal Analysis Thermal analyses of the investigated compound were performed using the TG-DSC (air) and TG-FTIR (air and nitrogen) techniques (Figures 8 and 9). Figure 8. (A) TG, DTG and DSC curves of the studied compound recorded in air atmosphere using cylindrical shape crucible; (B) TG and DTG curves of the studied compound recorded in air atmosphere using used flat platinum plate (heating rate 10 °C min−1). Figure 9. TG and DTG curves of D2AAK1_3 recording during thermal analysis with heating rate 20 °C min−1 in air (A) and N2 (B) atmospheres. The TG-DSC curves providing information about the thermal properties of D2AAK1_3 are shown in Figure 8A. As it can be seen in the TG curves, the compound is stable at room temperature, which is a very important parameter for its potential application as a drug. During the heating, a first change is recorded on the DSC curve as an endothermic peak (Tonset = 151 °C, Tpeak = 154 °C). This peak is not accompanied by a mass loss and can be attributed to the melting process. It is sharp, indicating that the compound was synthesized as pure, crystalline substance [22,33]. The enthalpy of fusion calculated from the DSC peak is 25.96 ± 0.93 kJ mol−1. Further heating leads to thermal decomposition of D2AAK1_3, which roughly occurs in two stages recorded on a TG curve. The first step of compound thermal degradation is observed at 212–390 °C and 54.43% of the sample is combusted. It is worth mentioning that on the DTG curve, two maxima (at 295 °C and 336 °C) are visible during this stage, but this is not clearly indicated on the TG curve. The formed products are unstable and immediately undergo further decomposition and combustion. The sample fully decomposes at a temperature of approximately 650 °C. In order to better understand the mechanism of the thermal decomposition process, the TG-FTIR analysis of D2AAK1_3 in oxidizing as well as inert atmosphere was performed (see Figure 9). As it can be seen from Figure 9A, the process performed at air atmosphere also occurs in two stages, but the different heating rate (20 °C min−1), a larger sample mass and a shape of the crucibles affected the temperature of the Figure 9. TG and DTG curves of D2AAK1_3 recording during thermal analysis with heating rate 20 ◦ C min−1in air (A) and N2(B) atmospheres. The TG-DSC curves providing information about the thermal properties of D2AAK1_3 are shown in Figure 8A. As it can be seen in the TG curves, the compound is stable at room temperature, which is a very important parameter for its potential application as a drug. During the heating, a first change is recorded on the DSC curve as an endothermic peak (Tonset = 151 ◦C, Tpeak = 154 ◦C). This peak is not accompanied by a mass loss and can be attributed to the melting process. It is sharp, indicating that the compound was synthesized as pure, crystalline substance [ 22 , 33 ]. The enthalpy of fusion calculated from the DSC peak is 25.96 ± 0.93 kJ mol −1 . Further heating leads to thermal decomposition of D2AAK1_3, which roughly occurs in two stages recorded on a TG curve. The first step of compound thermal degradation is observed at 212–390 ◦ C and 54.43% of the sample is combusted. It is worth mentioning that on the DTG curve, two maxima (at 295 ◦ C and 336 ◦ C) are visible during this stage, but this is not clearly indicated on the TG curve. The formed products are unstable and immediately undergo further decomposition and combustion. The sample fully decomposes at a temperature of approximately 650 ◦ C. 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